stemcells outsourcing focus advice · handheld raman spectrometer, the bruker bravo. for more than...

www.europeanpharmaceuticalreview.com Issue 2 · 2015

Stem cellsfocusWith articles from the University ofCambridge’s Centre for MolecularInformatics & MRC Centre forRegenerative Medicine, University of Edinburgh

Real-time biological particle counting

Tim Sandle, Head of Microbiology at Bio Products Laboratory, discusses the latest innovations in bio-air sampling

Outsourcingadvice

Roger A. Stroud, R Stroud Pharmaceutical

Quality Solutions Ltd.

Bruker Optik GmbH

Rudolf-Plank-Str. 2776275 Ettlingen

Tel. +49 7243 504 2000 Fax. +49 7243 504 2050E-Mail: [email protected] us for more details www.bruker.com/bravo

Dedicated Raw Material Analyzer

BRAVO - A new era of

Handheld Raman Spectroscopy

SSETM – Sequentially Shifted Excitation

mitigates fl uorescence

Duo LASERTM provides the highest

sensitivity throughout a large spectral range

including the CH-stretching region

IntelliTipTM – automated recognition of

measurement tips

Class 1M Laser safe in all modes of operation

Intuitive and guided touch screen operation

BRAVO makes Raman analysis accessible to everybody. New technologies especially designed for BRAVO provide an effi cient verifi cation of the widest range of materials. BRAVO supports the complete manufacturing process from raw material verifi cation to fi nished product inspection with functionality that meets your requirements.

RAMANInnovation with Integrity

“The Bruker BRAVO (Bruker RAman Verification Optics) opens a new erain handheld Raman spectroscopy because it addresses all of the so farpresent limitations,” says Armin Gembus, Global Business Unit Managerfor Raman and Gas Analytics at Bruker. “Patented Sequentially ShiftedExcitation (SSETM) technique takes care of fluorescent samples while stillkeeping signal-to-noise ratio very high and hence allows successfulmeasuring of a much wider range of raw materials compared to anyother existing handheld Raman devices,” explains Gembus.

Bruker is well known to be a very innovative company and thus alsothe BRAVO is fully packed with outstanding unique features which makeit a superior design. The combination of two lasers called Duo LASERTM

excitation allows accessing a large spectral range including informationon the CH-stretching region. “Spectral quality is most crucial for anunambiguous verification,” Gembus confirms and adds that “BRAVOfeatures IntelliTipTM, an automatic measuring tip recognition whichextends the capabilities into this direction and also prevents the Ramanlaser being fired without having a measuring tip attached. Even better,BRAVO is a class 1M laser product in all modes of operation enabling fora safe operation without the need of laser safety protection devices oraccess restrictions for third.”

Automatically included in the library, IntelliTipTM informs userswhich measuring tip needs to be used in order to get best spectralquality. Usage of the proper sample tip is always ensured. “Beingfocused on customer experiences and needs, the innovation of BRAVOcomprises an intuitive and guided workflow on a large touch screen

which is especially dedicated for the pharmaceutical manufactures. At any time all required information is clearly displayed and possibleactions can easily executed like on a cell phone. This enables everybodyto get used to BRAVO within a very short time and will speed up allworkflows at a maximum,” Gembus highlights.

Data and report transfer between the BRAVO and a PC or network is enabled either wirelessly or via a docking station depending on user-specific demands. Handling of data is done from Bruker’s OPUSspectroscopy software. “OPUS is the leading software for measurement,processing and evaluation of IR, NIR and Raman spectra,” Gembus says and continues: “BRAVO and its software suite complies with CRF 21 part 11 of the Code of Federal Regulations and meets cGMPprocedures as well.”

Discussing the further potential of BRAVO, Gembus states: “The outstanding performance of BRAVO is competitive to high perform -ance benchtop Raman spectrometers which might make BRAVO aninteresting solution for many more applications and markets in future.”

“We are never satisfied with the common market standards andcommit ourselves to always offering our customers the highest qualitysolutions including extraordinary support that ranges from training,method development to validation consultation and service. At Bruker,customers are welcome to benefit from our know-how and worldwidesupport that sets new standards on the market when it comes toprecision and efficiency, ergonomics and ease of operation, consultingand services.”

PRODUCT HUB

Armin Gembus, Global Business Unit Manager for Raman and Gas Analytics at Bruker looks at the new generation ofhandheld Raman spectrometer, the Bruker BRAVO. For more than 50 years, Bruker has enabled scientists to makebreak through discoveries and develop new applications that improve the quality of human life. Bruker’s high-performance scientific research instruments and high-value analytical solutions enable scientists to explore life andmaterials at molecular, cellular and microscopic levels. In close cooperation with their customers, Bruker is enablinginnovation, productivity and customer success in life science molecular research, in applied and pharmaapplications, in microscopy, nano-analysis and industrial applications, as well as in cell biology, preclinical imaging,clinical research, microbiology and molecular diagnostics.

VOLUME 20 ISSUE 2 2015 European Pharmaceutical Review 1

Seamless transition from clinical research to commercialization. Use the same product with tailored quality attributes for your evolving regulatory requirements - preclinical to GMP grade

• Stringent quality control: ADCF/serum-free policy, stability tests, proven batch to batch reproducibility

• Comprehensive documentation FDA Drug Master Files, data sheets, CoA, on-site audits

From Research to ATMP Your steady partner for cell therapy

preclinical cytokines & media

GMP cytokines & media

Research

Preclinical

Clinical trialsphase I / II / III

Manufacturing/Commerzialisation

CellGenix GmbH – www.cellgenix.comHQ: Am Flughafen 16 | 79108 Freiburg | Germany US: One New Hamsphire Avenue, Suite 125 | Portsmouth, NH 03801 | USACellGro® is a registered trademark of CellGenix in several global markets. In North America and a few other countries CellGro® reagents are marketed under CellGenix™.

ChondrocytesMesenchymal Stromal Cells

ESC and iPS Cells NK CellsT CellsDendritic Cell

Hematopoietic Stem Cells

Cytokines, serum-free media & more


INTRODUCTION

Free print and digital subscriptions are available formembers of JPAG – subscribe now by visitingwww.europeanpharmaceuticalreview.com

Registered Office as above.Russell Publishing Ltd, is registered as a Limited Company in England, Number 2709148VAT Number GB 577 8978 47

Follow us on Twitter:http://twitter.com/PharmaReview

No responsibility can be accepted by Russell Publishing Limited, the editor, staff or any contributors for action taken as a result of the information and other materials contained in our publications. Readers should take specific advice whendealing with specific situations. In addition, the views expressed in our publi cations by any contributor are not necessarily those of the editor, staff or Russell Publishing Ltd. As such, our publications are not intended to amount to advice onwhich reliance should be placed. We therefore disclaim all liability and responsibility arising from any reliance placed on such materials by any reader, or by anyone who may be informed of any of its contents. Published April 2015

Independent auditwatchdog service forprinted publications

European Pharmaceutical Review can guarantee its circulation is 11,999 (for the 6 issues distributed between 1 January 2014 and 31 December 2014). The publication is ABC audited.This is an independent verification that our circulation is genuine.

Join us on LinkedIn:http://linkd.in/PharmaReview

EDITORIAL BOARDSheraz GulVice President and Head of Biology, Fraunhofer Institute for Molecular Biology and Applied Ecology IMEMatthew MoranDirector, PharmaChemical IrelandDon ClarkPfizer Global SupplyMichael J. MillerPresident, Microbiology ConsultantsMichael H. ElliottCEO, Atrium Research & ConsultingDavid ElderDirector Externalisation Group, GlaxoSmithKlineAndrew TeasdalePrincipal Scientist – Chair of Impurities Advisory Group, AstraZeneca

RUSSELL PUBLISHING LTD Managing Director: Josh RussellEditor: Caroline RichardsPublisher: Graeme CathieSales Manager: Andrew JohnsonProduction Manager: Brian ClokePublications Assistant: Caroline MeagerFront Cover Artwork: Steve CrispFounder: Ian Russell

European Pharmaceutical Review (ISSN No: 1360-8606, USPS No: 023-422) is published bi-monthly by Russell Publishing Ltd, GBR and distributed in the USA by Asendia, 17B S Middlesex Ave, Monroe NJ 08831. Periodicals postage paid New Brunswick, NJ and additional mailing offices.POSTMASTER: send address changes to European PharmaceuticalReview, 701C Ashland Ave, Folcroft PA 19032.

European Pharmaceutical Review is published bi-monthly (six times per annum) in print and digital formats and circulated on a free-of-charge subscription membership. EuropeanPharmaceutical Review is available for pharmaceutical industryprofessionals and you can subscribe now by visitingwww.europeanpharmaceuticalreview.com

European Pharmaceutical Review: Published by Russell Publishing Ltd, Court Lodge, Hogtrough Hill, Brasted, Kent, TN16 1NU, UK Tel: +44 (0) 1959 563311 Fax: +44 (0) 1959 563123 Email: [email protected]

ISSN 1360 – 8606Copyright rests with the publishers.All rights reserved©2015 Russell Publishing Limited

Very soon, the Congress Center Messe Frankfurt in Germany will be opening its doors todelegates keen to network and further their knowledge at ACHEMA 2015, the leading processindustry event of the year. Over the course of a week in June, some 3,800 exhibitors from allaround the globe will showcase a wide range of innovative products, new developments andsystem solutions in the chemical and pharmaceutical industries. As I’m sure many EuropeanPharmaceutical Review readers will be attending ACHEMA, we decided to include acomprehensive Show Preview detailing the event, which starts on page 61. The floor planshould come in handy when navigating through the 16 hall levels covering 140,000m2 ofexhibition space!

Topics covered at this year’s ACHEMA include laboratory and analytical techniques, hand-held spectrometers, pharmaceutical production, formulation and continuous processing.Related to this, one hot topic at the moment is real-time process analytics. Pharmaceuticalproduction has to comply with increasingly higher standards in terms of quality control,individualisation and process efficiency. Highlighting how real-time process control canbecome a reality with the methods and tools available today, Ravendra Singh, MarianthiIerapetritou and Rohit Ramachandran of Rutgers University describe how they havesuccessfully employed process analytical technology (PAT) methods in a combined feed-forward/feed-back control system of a continuous tablet manufacturing process in thisissue’s PAT Series (page 76).

Another interesting article in our Spring edition is Roger A. Stroud’s contribution onOutsourcing (page 12), which should prove an invaluable guide to any researcher consider-ing outsourcing analytical and microbiological testing services. Whatever the reason forseeking the expertise of another company, and, indeed, whatever those tasks that need to beoutsourced are, the helpful advice here will help direct you through the entire process.

As always, there are two In-Depth Focus supplements in this issue as well, both on verydifferent areas: the first, on the hugely topical area of stem cells, starts on page 21. This should whet your appetite for ISSCR (International Society for Stem Cell Research) 2015, which takes place in June (see Show Preview on page 32). The second In-Depth Focus,on LC-MS (page 43) contains articles on the interesting areas of high-performance liquidchromatography and nanoLC-MS.

Feel free to contact me by email on [email protected] if you would beinterested in contributing to a future issue of European Pharmaceutical Review. And don’tforget to bookmark our website at www.europeanpharmaceuticalreview.com where you canfind details of current and future issues, sector news and event details. We are also onLinkedIn and Twitter – details are opposite.

Caroline RichardsEditor, European Pharmaceutical Review

An eventfulsummer ahead

Pharma&Biotech

Shaping the Present and Future of Endotoxin Testing

©2015 Lonza.

1-3 June, 2015

Endotoxin Testing Summit

You are invited to attend Lonza’s Global Endotoxin Testing Summit. Interact with peers and regulators to discuss the current status of industry hot topics, including Low Endotoxin Recovery and recent updates on regulatory guide-lines related to endotoxin testing. The event will be held 1 – 3 June 2015 in Annapolis, MD.

Join our growing panel of industry experts eager to share their latest views on these topics, including:

Glenn Gauvry – Founder, Ecological Research and Development Group (ERDG)Johannes Reich – University of Regensburg, Germany/Hyglos, GmbHKevin Williams – Lonza, Inc. Mick Dawson – Associates of Cape Cod, Inc. Allen Burgenson – Lonza, Inc.

Included with registration is a trip to a Horseshoe Crab Sanctuary Beach in the Delaware Bay to help save horseshoe crabs that have been stranded during spawning.

Join Us at the 1st Global Endotoxin Testing Summit

For registration details and complete agenda visit: www.lonza.com/endosummit

Beautiful downtown, Annapolis, MD.

1 PRODUCT HUBArmin Gembus, Global Business Unit Manager of Ramanand Gas Analytics at Bruker looks at the new generationof handheld Raman spectrometer, the Bruker BRAVO

3 INTRODUCTIONAn eventful summer aheadCaroline Richards, Editor, European Pharmaceutical Review

7 FOREWORDPrecompetitive collaborations in the pharmaceutical industryDavid Elder, GlaxoSmithKline and JPAG

8 NEWS11 EVENTS12 OUTSOURCING

Outsourcing in the analytical and microbiology areaRoger A. Stroud, R Stroud Pharmaceutical Quality Solutions Limited

17 UNDER THE MICROSCOPEBeth DiPaolo, President of Eurofins LancasterLaboratories Professional Scientific ServicesSM, talks about Eurofins’ insourcing solutions

18 REGULATORY INSIGHTDrug pricing reforms: the Danish experienceUlrich Kaiser, University of Zurich, Susan Mendez,Melbourne Institute of Applied Economic and SocialResearch, Thomas Rønde, Copenhagen Business Schooland Hannes Ullrich, DIW Berlin and University of Zurich

21 IN-DEPTH FOCUS: STEM CELLSFeaturing articles from Yu Wang and David Hay, University of Edinburgh, and Andreas Bender andYasaman Kalantar Motamedi, University of Cambridge,Maryam Peymani and Mohammad Hossein Nasr Esfahani,Royan Institute, Isfahan. David Hay is also moderator of a Roundtable on stem cells on page 29.

32 SHOW PREVIEWISSCR 2015

35 MICROBIOLOGY SERIESReducing microbial contamination via sterile risk assessment Guenther Gapp, Lachman Consulting Ltd/Independent Consultant

39 ENVIRONMENTAL MONITORINGReal-time biological particle countingin environmental monitoringTim Sandle, Bio Products Laboratory

43 IN-DEPTH FOCUS: LC-MSFeaturing articles from David Elder, Phil Borman andGeorge Okafo, GSK, William McDowell, PolyTherics Ltd andHanne Røberg-Larsen, University of Oslo. A Roundtableon LC-MS, on page 58, is moderated by David Elder.

61 SHOW PREVIEWACHEMA 2015

69 MANUFACTURING SOLUTIONSIvo Backx of Siemens discusses continuous processing in the context of the company’s Simatic PCS 7system and PAT software

70 WORKSHOP REVIEW76 PAT SERIES

The scope of PAT in real-time advancedcontrol of tablet qualityRavendra Singh, Marianthi Ierapetritou and RohitRamachandran, Rutgers University

Contents


COMING UP IN THE NEXT ISSUE:

■ Microbiology In-Depth Focus■ PCR In-Depth Focus

■ Proteomics■ Microfluidics

Published June 2015. Don’t miss out on your copy – subscribe for free today by visitingwww.europeanpharmaceuticalreview.com

www.mt.com/ta-promotions

50 Years of Innovation in Thermal Analysis by METTLER TOLEDOGet the full picture thanks to the extremely wide range of heating and cooling rates.

Flash DSC 1 – DSC 2 Package at an Exceptional Price

Bingham and Ekins4 go further and advocate the benefits of competitivecollaboration. Also known as co-opetition, this may be defined as the‘strategy embodying simultaneous cooperation and competitionbetween firms’. Co-opetition is significantly more critical within hightechnology businesses such as the pharmaceutical industry because ofthe challenges described above. However, the rewards of suchcollaboration can be immeasurable; since competitors possess alignedresources and face similar challenges, collaboration betweencompetitors allows companies to share relevant information that is notonly beneficial to all parties concerned, but more importantly, isbeneficial to the patient.

Generally, companies in collaborations will require ‘freedom-to-operate’ within the sphere of the collaboration and they will want theability to publish and otherwise disseminate the newly-acquiredinformation without significant impediments, such as the requirementto pay royalty or licensing fees.

There appear to be several different ways for potential collabor -ators to share their work. Just do It (JDI) and Formalized Collaborationsare initiatives where the research is carried out by all of the participantsand the data are shared for mutual benefit. The only differencebetween the two is that the latter approach typically involves a signed agreement.

Some pharmaceutical companies choose to arrange mutuallybeneficial research via independent third parties, for example, theymay become members of the Product Quality Research Institute (PQRI),International Society for Pharmaceutical Engineering (IPSE), Innova-tion and Quality Consortium (IQ) and Lhasa Ltd., among others. Theobjective here is to demonstrate that the activities/findings have notbeen overly influenced by the pharmaceutical collaborators (i.e., theyhave conducted independent research) and/or allow the third party toprovide a ‘consensus voice’ on behalf of the industry collaborators.5

With the use of the ‘honest broker’ approach, a formal entity can becreated on behalf of the collaboration consortium, for example, theConsortium for the Investigation of Genotoxicity of Aromatic Amines isone such consortium6. Other honest brokers include joint ventureinitiatives; for example, ViiV Healthcare7 is a joint venture between GSK,Pfizer and Shionogi where all of the collaborators have transferred theirHIV assets into one entity. Additional programs may be initiated after the inception of the initial undertaking(s).

In terms of academic collaborations, an Academic Consortiumenables a group of laboratories to collaborate with a governmentalagency on areas of mutual interest; the funding is primarily drawn fromthe governmental agency. In an Industry/Academic Consortium, a groupof academic laboratories collaborates with trade organisations such asthe European Federation of Pharmaceutical Industry Associations(EFPIA), or multiple industry members, on areas of mutual interest. This is primarily funded by a national or supra-national agency.

Additional precompetitive partnerships include the InnovativeMedicines Initiative8 (IMI), a joint undertaking between the EuropeanUnion (EU) and the EFPIA, and Europe’s largest public-private initiative(PPI) aimed at accelerating the development of improved and safermedicines for patients. IMI supports ‘collaborative research projectsand builds networks of industrial and academic experts in order toboost pharmaceutical innovation in Europe’. In the US, the FDA’s CriticalPath9 and Advancing Regulatory Science10 initiatives highlight how theagency is assessing and utilising new technologies to develop the ‘tools,standards, and approaches required to assess the safety, efficacy,quality, and performance of innovative products’.

In today’s challenging pharmaceutical environment, the FDA’sbelief that ‘bringing regulatory science into the 21st century requires thecollaborative efforts of all stakeholders - including academia, industry,and other governmental agencies’ is clearly not to be ignored.

FOREWORD

Precompetitivecollaborations in thepharmaceutical industryDave Elder

GlaxoSmithKline and JPAG


1. Gnyawali, D.R.; Park, B-J.R.; Co-opetition between giants: Collaboration with competitors for

technological innovation. Research Policy 40, 2011, 650-663

2. Woodcock, J. Precompetitive research: A new prescription for drug development?

Clin. Pharmacol. Ther. 87(5), 2010, 521-523

3. Welch, C.J.; Hawkins, J.M.; Tom, J. Precompetitive intelligence on enabling technologies for the

pharmaceutical industry. Org. Proc. Res. Dev. 18, 2014, 481-487

4. Bingham, A.; Ekins, S. Competitive collaboration in the pharmaceutical and biotechnology

industry. Drug Discovery Today, 14 (23/24), 2009, 1079-1081

5. Thomson, N.M.; Seibert, K.D.; Tummala, S.; Bordewekar, S.; Kiesman, W.F.; Irdam, E.A.;

Phenix, B.; Kumke. D. Case Studies in the Applicability of Drug Substance Design Spaces

Developed on the Laboratory Scale to Commercial Manufacturing Org. Proc. Res. Dev. 2014,

DOI: 10.1021/op500187u

6. Lhasa. Consortium for the Investigation of Genotoxicity of Aromatic Amines (CIGAA).

http://www.lhasalimited.org/research-and-collaboration/collaboration.htm. Accessed on 30th

January, 2015

7. ViiV. http://www.viivhealthcare.com/what-we-do/we-collaborate-to-innovate.aspx. Accessed on

30th January, 2015

8. Innovative Medicines Initiative http://www.imi.europa.eu/ Accessed on 14th February 2015

9. FDA Critical Path Initiative http://www.fda.gov/ScienceResearch/SpecialTopics/

CriticalPathInitiative/. Accessed on 14th February 2015

10. FDA Advancing Regulatory Science Initiative http://www.fda.gov/ScienceResearch/

SpecialTopics/RegulatoryScience/default.htm?utm_campaign=Goo. Accessed on 14th

February 2015

References

Increasing research and development costs, low productivity, reduced product life cycles, governmental pricingcontainment, convergence of technologies and increasing regulatory oversight1 are challenges that increasingly provokepharmaceutical companies into making precompetitive collaborations with other organisations. Organisations that wouldtypically compete with one another are now realising the benefits of working together to share information. JanetWoodcock, Acting Director of the US Food and Drug Administration’s Office of Pharmaceutical Quality has definedprecompetitive research as ‘a subset of translational research that is focussed on improving the tools and techniquesneeded for successful translation, and not on development of a specific product’2. Enhanced cross-industry collaborationsincrease efficiency, innovation, sustainability and ultimately patient benefit.3

NEWS

8 European Pharmaceutical Review VOLUME 20 ISSUE 2 2015 Catch up with daily news at www.europeanpharmaceuticalreview.com

DAIICHI SANKYO

Swissmedic approves Lixiana for theprevention of stroke and systemic embolismSwissmedic, the regulatory authority of Switzerland, has granted approval of Daiichi Sankyo’s

Lixiana® (edoxaban), an oral, once-daily selective factor Xa inhibitor, for the prevention of stroke

and systemic embolism in adult patients with non-valvular atrial fibrillation (NVAF).

Simultaneously, Lixiana has received marketing authorisation in Switzerland for the treatment

of adult patients with venous thromboembolism (VTE), including deep vein thrombosis and

pulmonary embolism, following previous treatment with fractionated or unfractionated heparin for

five days, as well as for the prevention of recurrent VTE.

The approved indications in Switzerland for Lixiana are based on data from the Phase 3

ENGAGE AF-TIMI 48 and Hokusai-VTE studies, the largest and longest single comparative

global trials of a novel oral anticoagulant in patients with NVAF or acute VTE, involving 21,105

and 8,292 patients, respectively.

INCYTE

Incyte to establish European headquarters in GenevaIncyte Corporation, a biopharmaceutical

company focused on the discovery, develop-

ment and commercialisation of proprietary

therapeutics, primarily for oncology, will

establish the new headquarters of Incyte Europe

in Geneva, Switzerland.

Incyte Europe will be the base from which the

Company will conduct its European clinical

development operations. The Company expects to

occupy the 9,000 sq. ft. facility by mid-2015. “The

establishment of Incyte Europe in Geneva is a

natural step in our company’s evolution,” said

Hervé Hoppenot, President and CEO of the

Company. “Incyte has a broad and growing

pipeline of proprietary, wholly-owned products,

and we expect that this new facility in the centre of

Europe will enable us to create the infrastructure

needed to support our global drug development

programs, and to bring additional, potentially life-

changing medicines to patients with cancer.”

OMEROS

Compassionate use of OMS721 for approvedthrombotic microangiopathyOmeros’ investigational product, OMS721, has been approved in Europe for compassionate use.

OMS721 is Omeros’ lead human monoclonal antibody in its mannan-binding lectin-associated

serine protease-2 (MASP-2) program for the treatment of thrombotic microangiopathies (TMAs),

including atypical hemolytic uremic syndrome (aHUS). TMAs are a family of rare, debilitating

and life-threatening disorders characterised by excessive thrombi (clots) in the microcirculation of

the body’s organs, most commonly the kidney and brain. The two patients in the compassionate-

use study suffer from aHUS and were enrolled in the first cohort of the Phase 2 clinical trial. Based

on improvements across markers of disease activity in that cohort, the investigator requested that

Omeros continue to provide OMS721 so that the patients could extend their treatments.

UMC UTRECHT

UMC Utrecht’s new liver cancer treatmentapproved for use in EuropeA new treatment for liver cancer developed by the

University Medical Center (UMC) Utrecht has

received the European CE mark for quality and

safety. The innovative treatment uses radioactive

holmium microspheres to attack liver tumours.

It involves injecting radioactive beads into the

hepatic artery, which then join the blood flow and

become trapped in the tiniest blood vessels

located in and around the liver tumours.

They therefore emit their radiation close to the

tumour. This type of radiation treatment is also

called radioembolisation.

The treatment is being marketed by Quirem

Medical, a spin-off company of the UMC

Utrecht. “We consider our holmium microspheres

as ‘the next generation of microspheres’’’ says

Dr. Frank Nijsen, founder of Quirem. “Treating

liver tumours with yttrium microspheres

is already a proven and valued cancer

therapy using radioembolisation. The new

holmium microspheres constitute the next

step in the development of this technology.

Because they show up on MRI scans and

SPECT-CT, these microspheres can be tracked,

allowing customised treatment for each

individual patient.”

WHO

WHO calls for thedisclosure of resultsfrom all clinical trialsThe World Health Organization (WHO) has

issued a public statement calling for the

disclosure of results from clinical trials for

medical products, whatever the result.

The move aims to ensure that decisions

related to the safety and efficacy of vaccines,

drugs and medical devices for use by

populations are supported by the best

available evidence.

“Our intention is to promote the sharing

of scientific knowledge in order to advance

public health,” said Dr Marie-Paule Kieny,

WHO Assistant Director-General for Health

Systems and Innovation. “It underpins the

principal goal of medical research: to serve

the betterment of humanity.” “Failure to

publicly disclose trial results engenders

misinformation, leading to skewed priorities

for both R&D and public health inter -

ventions,” said Dr Kieny. “It creates indirect

costs for public and private entities, including

patients themselves, who pay for suboptimal

or harmful treatments.”

MOTIF BIO

FDA agrees to Phase 3clinical developmentprogramme ofbreakthroughantibiotic iclaprimThe US Food and Drug Administration has

agreed to the proposed Phase 3 clinical

development programme for Motif’s lead

product candidate, iclaprim. The Phase 3

programme is designed to obtain marketing

approval for an intravenous formulation of

iclaprim in the treatment of acute bacterial

skin and skin structure infections (ABSSSI)

and hospital acquired bacterial pneumonia

(HABP) caused by Gram-positive pathogens,

including resistant strains such as methicillin-

resistant Staphylococcus aureus (MRSA)

and MDRSP (multi-drug resistant Strepto -

coccus pneumoniae).

The FDA confirmed that two ABSSSI

trials or one ABSSSI trial plus one

HABP trial meeting their pre-specified

primary endpoints are required for approval

of iclaprim. Motif is working to determine

the costs and timeline of these options.

Assuming that funds can be raised or a

partnership can be entered into, the first

Phase 3 trial for ABSSSI is expected to

commence in the second half of 2015.

Catch up with daily news at www.europeanpharmaceuticalreview.com VOLUME 20 ISSUE 2 2015 European Pharmaceutical Review 9

NEWS

THERMO SCIENTIFIC

Universal UHPLC detection in an integrated solutionCharged aerosol detection (CAD) is a well-

established, near universal liquid chromatography

detection technology that provides a consistent

response that is independent of the analyte’s

chemical structure. This is especially useful for

detecting substances without chromophores and

the quantification of unknowns without a

comparable chemical standard. The technology is

now available as a detection module for the

Thermo Scientific™ Vanquish™ UHPLC system.

Compounds separated by chromatography

have such a diverse nature that, often, no single

detector sees them all. The Vanquish Charged

Aerosol and Diode Array detectors are very

powerful instruments and when combined, offer

superior detection abilities addressing the broadest

range of analytes, including:■ Pharmaceutical drugs and impurities■ Excipients■ Biomolecules■ Natural products, supplements & botanicals■ Foods and beverages■ Surfactants and polymers.

Analytes without chromophore are hard to detect

by a diode array detector but will typically be

revealed by a charged aerosol detector. With both,

effectively, no sample component will be missed.

Analysts can be assured of the best data for

verification and reporting by choosing the most

appropriate results.

The Vanquish UHPLC System with fully

integrated Charged Aerosol and Diode Array

Detector modules provides the most powerful

UHPLC platform on the market. Scientists can

now address the widest range of challenging

analytical applications with speed, resolution,

sensitivity and confidence.

Learn more about the Vanquish UHPLC

system with charged aerosol detection at:

thermoscientific.com/Vanquish

Interested to learn more on our

HPLC trade in program? Please contact

www.thermoscientific.com/lcpromotion

for more details.

RENEURON

ReNeuron files IND for its eye disease stem cell therapy candidateReNeuron has announced that it has filed an

Investigational New Drug (IND) application

with the FDA to commence a Phase I/II clinical

trial with its human Retinal Progenitor Cell

(hRPC) therapy candidate for retinitis

pigmentosa (RP).

RP is a group of hereditary diseases of the

eye that lead to progressive loss of sight due to

cells in the retina becoming damaged and

eventually dying.

Preclinical studies carried out in disease

models by the company’s academic collab -

orators have demonstrated that, when

transplanted into the retina, ReNeuron’s

retinal progenitor cell technology has the

potential to preserve existing photo-

receptors, potentially reducing or halting

further deterioration of vision. In addition,

the progenitor cells have been shown to mature

into functional photo receptors that engraft into

the photoreceptor layer, bringing the possibility

of restored vision.

The proposed Phase I/II clinical trial will be

conducted at Massachusetts Eye and Ear,

Boston. The trial design is an open-label, dose

escalation study to evaluate the safety,

tolerability and preliminary efficacy of the

hRPC stem cell therapy candidate in up to

15 patients with advanced RP. The method of

administration of the hRPCs will be a single

sub-retinal injection. The primary endpoint of

the study is safety, with patients being foll-

owed up for 12 months post-treatment with

monitoring including measurements of visual

acuity. ReNeuron expects to be able to

commence the clinical trial in the second half

of this year.

ReNeuron’s candidate has been granted

Orphan Drug Designation in Europe.

MERCK

Merck CapacityAdvancementProgram toimprove diabetesand hypertensionhealthcarecapacity in Africaand AsiaMerck, in collaboration with Maharashtra

University of Health Sciences (MUHS), is

introducing European accredited clinical

diabetes management training for more

than 5,000 medical students in 18 medical

colleges of MUHS as part of the Merck

Capacity Advancement Program in Asia.

Dr. Stefan Oschmann, Vice Chairman

and Deputy CEO of Merck, said, “Merck

is pleased to collaborate with Maharashtra

University of Health Sciences and

Directorate of Medical Education &

Research as part of our commitment to

building healthcare capacity and providing

sustainable access to high-quality health

solutions and safe medicines in India. It

marks another step in our commitment to

working with governments and other

stakeholders in building healthcare

capacity with a focus on non-comm -

unicable diseases in various countries in

Asia-Pacific, Middle East Africa and

Latin America.”

Prof. Dr Arun Jamkar, Vice Chanc -

ellor of MUHS, Nashik, added, “It gives us

immense pleasure to invite the

stakeholders in the field of medicine and

diabetes in Maharashtra. In joint coll -

aboration with DMER and Merck, this

diabetes education course aims to provide

guidelines and clinical practice for

prevention, diagnosis and management of

diabetes and its complications for medical

undergraduates of the 18 medical colleges

in Maharashtra University.”

10 European Pharmaceutical Review VOLUME 20 ISSUE 2 2015 Catch up with daily news at www.europeanpharmaceuticalreview.com

NEWS

ABIVAX

Mechanism ofaction of first-in-class anti-HIVdrug, ABX464,publishedABIVAX, a clinical phase II stage

biotechnology company developing

first-in-class anti-viral drugs and

vaccines, has announced publication of

the mechanism of action of ABX464 in

Retrovirology. ABX464 is a novel small

molecule in phase II clinical trial that

inhibits HIV replication through an

entirely new mechanism.

The research described in the

publication was conducted by six

research institutions from France,

Canada and Switzerland, in addition to

the ABIVAX laboratories, under the

leadership of Professor Jamal Tazi of the

Molecular Genetics Institute at CNRS in

Montpellier, France.

The research team was able to

demonstrate that: ■ ABX464 blocks viral replication by

preventing the export of viral RNA

from the nucleus to the cytoplasm in

infected cells. This transport is

normally mediated by a viral protein

called Rev, and the activity of Rev is

efficiently inhibited by ABX464.

Never targeted before, Rev has been

postulated of potential interest for

HIV treatment for some time, but

ABX464 is the first molecule under

development aimed at inhibiting it. ■ ABX464 does not affect the

physiological cellular RNA-pro -

cessing in humans. This suggests that

ABX464 is specific for HIV RNAs

and does not influence the synthesis of

human proteins. ■ ABX464 does not lead to HIV

mutants that become resistant to

treatment. In contrast to all other anti-

HIV drugs, ABX464 may be effective

as a monotherapy.

Bruker recently introduced Bruker

FUSION-SV™, a new software solution

for comprehensive, fully automated

small molecule structure verification.

Building on Bruker’s leading nuclear

magnetic resonance (NMR) spectro -

scopy and mass spectrometry (MS), two

fundamental techniques for small

molecule structure verification, this new

software simplifies and accelerates the

drug design and discovery phases in

the pharmaceutical industry significantly.

Bruker experts have developed new

proprietary algorithms to integrate and

process NMR and MS measurements into one result. The new Bruker FUSION-SV™ is a software

solution integrating high resolution accurate mass (HRAM) data and complementary NMR data,

therefore increasing significantly the specificity for small organic molecule structure verification.

The complex proprietary algorithms which deliver this improved performance have been

embedded in a streamlined workflow concept and user-friendly interface. Unique auto-analysis

algorithms based on complex human logic emulation interpret NMR spectra to ensure structure

verification with high confidence. Bruker’s well established and further enhanced SmartFormula™

algorithm is utilised for automated molecular weight determination by combining accurate mass data

with True Isotopic Pattern (TIP) analysis.

By using Bruker FUSION™ synthesis chemists are guided straight to meaningful and clearly

understandable results with minimal interaction. Structures and data derived from NMR and MS

analysis can be loaded in batches for automatic verification and thus throughput will be significantly

improved. Synthesis chemists will get a prompt result without being an expert spectroscopist.

Bruker FUSION-SV is ideal for both academic and industry laboratories, proving particularly

useful for synthetic chemists in the pharmaceutical industry. For more information, see:

www.bruker.com/FUSION-SV

ICHOR

Ichor and Janssen to collaborate on immunotherapies for chronic Hepatitis BIchor Medical Systems has announced that it has entered into a product

development collaboration and worldwide license agreement with Janssen

Pharmaceuticals. Under the agreement, which was facilitated by Johnson

& Johnson Innovation, the parties will work together to develop and

commercialise DNA-based vaccine products for the treatment of

chronic hepatitis B using Ichor’s TriGrid™ electroporation technology

for clinical administration.

Ichor will receive an upfront payment, R&D support, and

development and sales milestone payments up to a potential total of

approximately $85 million USD, as well as royalty payments on any

future licensed product sales. Janssen will assume responsibility for

certain development costs and all commercialisation costs associated with

the program, including manufacturing and distribution expense for Ichor’s

TriGrid Delivery System.

Hepatitis B is a potentially life-threatening liver infection caused by

the hepatitis B virus (HBV). More than two billion people alive today

have been infected with HBV. Presently, there are over 240 million people

worldwide that carry the virus and remain chronically infected. Current

treatments seldom eliminate the virus and are often associated with severe

adverse reactions.

MEDIMMUNE

MEDI8897 gets fast track status for theprevention of RSV-induced respiratory diseaseMedImmune has announced that it has receivedfast track designation from the US Food and Drug Administration (FDA) for the development of MEDI8897.

This high-potency, extended half-life mono -clonal antibody (mAb) is being investigated for theprevention of lower respiratory tract illness (LRTI)caused by respiratory syncytial virus (RSV) ininfants and young children.

RSV is the most prevalent cause of lowerrespiratory tract infections among infants andyoung children, resulting in annual epidemicsworldwide. In children younger than one year of

age, RSV is the most common cause ofbronchiolitis, an inflammation of the small airwaysin the lung, and pneumonia, an infection of thelungs. There is currently no treatment for RSV onceit is contracted, nor is there an approved preventa -tive therapy for healthy populations.

MedImmune previously discovered,developed and currently markets a mAb for severeRSV disease. The company aims to increase thenumber of infants who are protected from LRTIcaused by RSV through its development ofMEDI8897 for the passive immunisation of allinfants, term and preterm.

BRUKER

Bruker rolls out new FUSION-SV™ software solution

MAY 2015

Biomarkers & Diagnostics World CongressDate: 2 – 7 MayLocation: Philadelphia, PA, USAe: [email protected]: http://www.biomarkerworldcongress.com/

GCC PharmaceuticalCongressDate: 3 – 5 MayLocation: Dubai, UAEe: [email protected]: http://www.gccpharmacongress.com/

ISPE 2015 Europe Annual ConferenceDate: 4 – 7 May Location: Frankfurt, Germanye: [email protected]: http://ispe.org/2015-europe-annual-conference

Nordic Chemical Biology MeetingDate: 5 – 6 MayLocation: Stockholm, Swedene: [email protected]: http://europe-slas.org/conference-Nordic-Chemicals-Biology-Meeting.htm

GPCMDate: 5 – 7 MayLocation: Berlin, Germanye: [email protected]: http://www.gpcmevent.com

The RDD 2015 Scientific ConferenceDate: 5 – 8 May Location: Palais des Congrès d’Antibes, Nice, Francee: [email protected]: www.rddonline.com/rddeurope2015

Pharma Lab Expo 2015Date: 13 – 15 MayLocation: Mumbai, Indiae: [email protected]: http://www.pharmalabexpo.in/index.aspx

JPAG symposium:Antibody drug conjugates:analytical efforts andexpectationsDate: 14 MayLocation: Royal Society of Chemistry, London, UKe: [email protected]: www.jpag.org/63

The Health IndustrySummit (tHIS)Date: 15 – 18 MayLocation: National Exhibition and ConventionCenter, Shanghai, Chinae: [email protected]: http://www.thishealthsummit.com/

Formulation & DrugDelivery CongressDate: 18 – 19 MayLocation: London, UKe: [email protected]: www.formulation-congress.com/download-agenda-marketing

ADC SummitDate: 18 – 19 MayLocation: Holiday Inn Bloomsbury, London, UKe: [email protected]: http://www.smi-online.co.uk/2015conjugates73.asp

Advances in qPCR & dPCRDate: 21 – 22 MayLocation: Singaporee: [email protected]: https://selectbiosciences

3rd Annual NextGeneration SequencingData CongressDate: 24 – 25 MayLocation: Dortmund, Germanye: [email protected]: http://www.europe-slas.org/conference-Annual-Compound-Management-Conference-Industry-Academia.htm

JUNE 2015

Preclinical Form &Formulation for DrugDiscoveryDate: 7 – 12 JuneLocation: Waterville, USAw: https://www.grc.org/programs.aspx?id=15849

16th Annual DrugDiscovery Summit 2015Date: 8 – 9 June Location: Berlin, Germanye: [email protected]: www.drugdiscovery-summit.com

3rd Annual DiscoveryChemistry & Drug Design Congress 2015Date: 8 – 9 June Location: Berlin, Germanye: [email protected] http://www.discoverychemistry-congress1.com

Stem Cells: from basic research to bioprocessingDate: 9 – 11 JuneLocation: London, UKe: [email protected]: www.regonline.co.uk/builder/site/Default.aspx?EventID=1563016#

DIA 2015: 50th Annual MeetingDate: 14 – 18 JuneLocation: Washington D.C., USAe: [email protected]: http://www.diahome.org.en-US/Flagship-Meetings?DIA2015.aspx

3rd Annual NextGeneration SequencingData CongressDate: 15 – 16 JuneLocation: London, UKw: www.nextgenerationsequencingdata-congress.com

ACHEMA 2015Date: 15 – 19 JuneLocation: Frankfurt am Main, Germany t: +49 69 7564 100w: www.achema.de

2nd Annual Microbiology andInfectious Diseases Asia CongressDate: 23 – 24 JuneLocation: Singaporee: [email protected]: http://www.microbiologyasia-congress.com/

ISSCR 2015Date: 24 – 27 JuneLocation: Stockholm, Swedene: [email protected]: http://www.isscr.org/home/annual-meeting/isscr2015

Challenges in current GMP includingcleaning validationDate: 25 June 2015Location: Royal Society of Chemistry, London, UKe: [email protected]: www.jpag.org/62

PHARMA EVENTS


If you have a diary event youwish to publicise, send details to:[email protected]

There are a number of reasons for outsourcing. Virtual companies, witha small number of experts from different areas of pharmaceuticaldevelopment and manufacture, by definition don’t have ‘hands-on’analysts or suitable laboratories. Meanwhile, established companies,whether small firms or multinationals, have efficiency and costconsiderations, which means they try to ensure that personnel are fullyoccupied at all times. They outsource aspects of their work to cope withlogjams and peaks and troughs in their workload. Another reason foroutsourcing is that companies may not have the expertise or theequipment to undertake specific pieces of work, but in these economictimes it may be difficult to justify recruiting a suitably qualified ‘hands-

on’ analyst with the necessary expertise or to justify the purchase ofequipment, especially if it is costly and required for a few specific tasks.Space is another consideration, with companies perhaps not havingenough space in their facility. A good example of this is a GMP compliantformal ICH stability storage facility, which may already be full.

Tasks that can be effectively outsourcedRelease testing of incoming ‘raw materials’, including activepharmaceutical ingredients (APIs) and excipients to pharmacopoeiamonographs, can be quite complicated, requires particular expertiseand comprehension of pharmacopeias and their monographs, tends

Increasing numbers of pharmaceutical companies are considering outsourcing in the area of analytical andmicrobiological testing, from the very smallest start-up, virtual, or small company up to the largest multinational.This article will examine the factors that lead to a company deciding to outsource, and provides invaluable guidanceon the important considerations that should be made before, during and after the outsourcing process in order toensure it runs as smoothly as possible.

OUTSOURCING

Outsourcing in the analytical andmicrobiology area

Roger A. StroudR Stroud Pharmaceutical Quality Solutions Limited

12 European Pharmaceutical Review VOLUME 20 ISSUE 2 2015

© J

ane0

606

/ Shu

tter

stoc

k.co

m

to need a wide range of reagents and is quite time consuming. This should be one of the first tasks that is outsourced to a verycompetent contractor.

Other tasks that can be outsourced include: development andvalidation of analytical methods; release testing of product, especiallydosage forms that are new to the company e.g., inhalation products;and microbial testing aspects of environmental monitoring ofmanufacturing cleanrooms and water for injection ring mains.

Microbiological testing of incoming APIs and excipients and theprocess of formally justifying the decision not to test microbiologicallyappropriate ‘raw materials’ is quite a time consuming processcompared to testing of chemicals; a significant percentage of APIs andexcipients do not support microbial growth. Regulatory authorities arevery keen for companies to adopt this approach, but many firms do nothave the confidence or in-house expertise to take this approach so testall materials microbiologically which can be very time consuming andexpensive. This should be one of the first tasks to hand over tocompetent microbiology testing contractors who can evaluate allmaterials and where appropriate prepare suitable justifications for nottesting materials, only testing those that do have the potential tosupport microbial growth.

Risks and challengesThere are potentially many risks and challenges involved in the variousphases of outsourcing. To be successful in outsourcing there are twokey points to remember.

Firstly, and this is very important for start-ups, virtual and smallcompanies, it is vital to identify and acknowledge the areas where the company does not have the expertise andexperience. Several of these potential areas areidentified in this article. It is important in thesesituations that the company uses a consultantwith expertise who has successfully carried outthis role many times. In the long term, thisapproach is a good investment.

Secondly, in a number of areas it is vital to use risk assessment andmitigation as a tool to identify the high risks and mitigate them toreduce them to risks of an acceptable level.

Criteria for the ideal contractor are as follows:� Really understands your needs� Has the exact expertise that you require� Has the exact equipment with the correct documentation

needed for your work� Operates to quality standards appropriate to your needs� Has an excellent record in relation to regulatory inspections� Has all the appropriate documented systems and procedures

in place� Has all the required licences, permits and certification to

meet your needs� Charges reasonable costs for undertaking the work� Has suitably qualified and trained staff � Keeps you informed� Always submits the results on time� Has a good location.

Process of identification, evaluation and selection of outsource contractorsThe steps and tasks relating to the identification of contractors include:� Defining and listing potential tasks that need to be outsourced,

company requirements and selection criteria� Researching the market� Using the Internet to check Regulatory Authority websites for lists of

certified testing laboratories and to undertake searches� Searching journals and directories� Consulting known professionals in their fields of expertise� Producing a list of potential outsource companies.

Steps and tasks relating to evaluation include:� Documenting all the relevant information found for each

potential contractor� Studying contractor websites; these contain much relevant

infor mation, often including lists of equipment, available expertiseand copies of inspection outcomes.

In the ‘First Level Evaluation’ one selects a shortlist of three to fivepotentially suitable companies in relation to requirements and theselection criteria. The ‘Second Level Evaluation’ involves circulating aquality questionnaire for completion by each shortlisted company.

Make a site visitDuring the discussions the company should outline work that contractorsare needed to undertake and using which quality standards. Looking tothe future, it should outline other work that needs to be undertaken at

later stages of the project. To aid the contractor’spreparation for the meeting outline plans shouldbe forwarded in advance of the meeting.

During a site tour, it is important to look out for notices such as copies of inspec -tion outcomes, awards and trophies in meetingrooms – companies tend to proudly exhibit

awards that satisfied customers have presented. Look for tidy desks andclean, well-maintained laboratories and welcoming laboratory staffwearing properly fastened lab coats.

From all the information gleaned about the short-listed contractorsincluding the quality questionnaire and site visit, the company should select a final short list of two (three at most) contractors for thefinal selection process.

Final selection processListed below are some reasons for and against using a specificcontractor. These make common sense but are worth noting.

Reasons to use a specific contractor:� Professional� Personal recommendation� Price is ‘right and fair’� Worked on similar projects� Regulatory inspected/approved� Suitable location� Ease of travelling to their site.

OUTSOURCING


During a site tour, it is important to look out for notices such as copies of inspec tion outcomes, awards and

trophies in meeting rooms

Reasons against using a specific contractor:� Difficulty in travelling to their site� Negative about being audited� Non-existent quality systems� Badly equipped� Inexperienced temporary staff� ‘Promise the world’� Price just too good to be true.

Before starting the last phase of the selection process, the final veryimportant tasks to undertake are firstly, requesting that visitedcontractors submit quotations for the early phases of the proposedwork. Secondly, undertaking GMP audits of each contractor isimportant. A company should use a competent consultant if no-one inthe company has expertise and experience in the area of GMP audits ofanalytical and microbiology testing facilities.

The final selection process should consider the following:� Completed quality questionnaire and quality audit findings � Regulatory Inspections outcomes� Information gathered during meetings with potential contractors;

the identification and evaluation process; provided by knownprofessionals in their fields of expertise and any of the contractorsknown current customers

� ‘Gut’ feelings� Ballpark costings of proposed work.

Ideally the outcome of this exercise leads toselection of the best contractor and a very goodsecond contractor who can be a back-up andtake on other projects.

Required documentation CDASome contractors require a one-way agreement and others a two-wayagreement. It is often quicker to agree to use the contractor’s standardCDA with perhaps one or two changes that will meet the company’sspecific needs.

Technical/quality agreementBe warned: getting this finalised and signed by both parties may takemuch longer than originally anticipated. The level of complexity of thedocument is dependent on where the company is in terms of the development or commercial phase of the project. In realitymultinational companies require a more detailed agreement coveringevery possible eventuality.

Sensible companies aim to keep the length of the agreement to aminimum and to use a tabulated format with a column detailing eachaspect/task of the agreement with additional columns for each party toindicate who is responsible for each aspect/task listed. For one or twoaspects/tasks it is likely that both parties have a responsibility.

Lists of contacts (individuals or in bigger companies, departments)In reality, if you have selected the correct outsource company and

you are a good customer this agreement will probably not be needed. There is no need to refer to the majority of the documentunless things go wrong.

Managing outsource companiesMost companies tend to use a project manager to manage their chosencontractor. For the most effective management of the contractor and contract, the nominee should have a really good understanding of analysis and microbiology. If this is not the case, there is a realdanger of the work turning out to be more expensive, not scientificallysound, not acceptable in the longer term by regulatory authorities andlengthy to complete. It is important to remember that one week’sunnecessary delay to a project will result in one week’s delay in gettingthe final product to market which equates to a one week delay in theproduct making money.

If nobody in the company is suitably experienced and qualified totake on this role, the solution is to use a consultant with expertise inthis area who has successfully carried out this role many times.

Working with a partner can be a serious challenge! It is advisable to establish a good rapport, have regular meetings, including tech-nical staff in such meetings, and make regular site visits. Trying to meet up now and again with the actual staff undertaking the practicalwork and taking time to be friendly and talk to them, remembering to

thank them for all their hard work, are alsothings to remember.

Other advice is to pay invoices on time,keep on top of the paperwork and keep thedoors of communication open so that eitherparty tells each other if they are unhappy. Usingmore than one contractor is also advisable.

Potential issues that must be consideredWhen companies use contractors, they do expect things to go wrongand for problems to arise from time to time. However there are otherissues that most companies do not think about until they do actuallyoccur. These include:

Contractor moving to new facilityEven if the move is only over a short distance, equipment has to berelocated and requalified, contractors may decide to purchase newequipment which needs to be installed and qualified, with standardoperating procedures produced and laboratory staff trained. If duringthe selection process a contractor tells you they are moving orsubsequently they decide to move, it is very sensible to ask for a copyof their detailed plan for achieving this, ask any pertinent questions andseek assurances that it will not affect the work being undertaken. One must feel reassured. If the contractor is mentioned on anyMarketing Licence Authorisation (MLA) there is a need to initiate theprocess for informing the Agency and updating the MLA.

Interaction of contractors It may be that you have two contractors, where there is possibly the chance of some direct interaction with regards to your comp-any’s project. Even if your company has an excellent interaction with them individually and you allow them to interact directly, there


OUTSOURCING

Keep the doors of communicationopen so that either party tells each

other if they are unhappy

is the possibility of things going wrong, so do not withdraw and leave them to it.

Merger and acquisitions In this day and age there are many mergers and acquisitions (M&As). Ifa contractor is involved in this process, their staff may be unhappy andthere are always worries at this time, so it is very likely that yourcontractor’s time lines may lengthen and you may well have to copewith a situation where they are merging or being acquired by a similarcontractor who was on your long list of ‘possibles’ but who waseliminated for good reason!

If an M&A happens to your own company, this can cause difficulties.For a start the company name could change which means there aremany types of documents where the name needs to change. Oneexample of where this can cause confusion is when samples andpaperwork arrive at your contractor and the new company name is notrecognised. You need to tell them as soon as possible after thecompany name changes.

In times of rumours, speculation, facts about acquisition,rationalisation, consolidation, downsizing, globalisation etc. there ismuch concern, fear and worry. The key to preventing slippage duringthese times is remaining totally focussed.

ConclusionsOutsourcing in the analytical and microbiology area can be quite a

challenging exercise, with many potential pitfalls firstly in identifying,evaluating and selecting the right contractor and secondly in managingthem. It is likely that some issues will arise. Prepare for the fact thatcertain of these issues that occur may be totally unexpected.

In those situations where the company does not have personnelwith the required expertise and experience it is important that thecompany uses a consultant with the expertise and who has successfullycarried out this role many times. Risk assessment and mitigation is atool that should be employed to reduce high risks to an acceptable levelof risk. Build a good relationship with your contractor and rememberyou are on the same side: both of you want the project to be on time, on budget and successful. Good two way communication and afocused mind are key.

OUTSOURCING


Roger A. Stroud is a director at R Stroud Pharmaceutical

Quality Solutions Ltd and has worked in the pharmaceutical

industry since 1970. During this time he has worked for

virtual companies, small companies and large multinationals

before setting up his own consultancy. An analyst by

training, he has for many years been involved with all

aspects of outsourcing including identifying, evaluating,

selecting, risk assessment and litigation and managing contractors in a wide

range of pharmaceutical disciplines. He has also run training courses within

companies on a wide range of aspects of outsourcing and spoken on the

subject at conferences. He has carried out many audits in a large number of

countries. In addition he spends much time undertaking work in the area

of quality including preparing companies for inspection by regulatory

authorities including the US FDA and a number of European agencies.

© S

tuar

t Mile

s / S

hutt

erst

ock.

com

Eliminate the ups and downs of continually replacing

and retraining temps by retaining our scientists at your

site. Hired, trained and managed by us, our award-

winning Professional Scientific ServicesSM (PSS):

Eliminates headcount, temporary worker and project

management worries

Avoids Temp turnover rate with managed insourcing

Costs you less than your own full-time employees

Delivers a 50-year history of regulatory compliant

technical expertise in your lab

Holds numerous client awards as the top insourcing

service provider for the past 10 years

Choose the PSS Insourcing solution that enables us to

keep staff grounded.

Tired of YourTemps Bouncing?

Partner and prosper with Professional Scientific ServicesSM

www.eurofins.com/pss

BioPharmaProduct Testing

The largest network of harmonized bio/

pharmaceutical GMP product testing labs worldwide

What is the PSS programme and how would you describe a typical customer?Put simply, PSS, or Professional Scientific Services, provides insourcinglaboratory services at our client’s facility using scientists and managersthat we hire, train and manage. Unlike with temporary staffing, staffmembers are full-time employees of Eurofins with comprehensivebenefits packages, as well as training, development and careeradvancement opportunities. In addition, we run and manage laboratoryoperations, either on client scientific teams or with distinct bodies ofservices in client environments. Our PSS insourcing solution is anextension of our laboratory capabilities as a part of Eurofins BiopharmProduct Testing group – the largest global network of harmonised GMP laboratories.

Our typical PSS customer is a bio/pharmaceutical manufacturerwho prefers to keep their laboratory testing in-house while alsominimising headcount for their operations. We can accommodategroups of any size, and we adjust the length of term to meet the client’sneeds, ranging anywhere from one to five years, or more.

Why and when did you decide to include the PSS programme in your business model?We developed this service model over 13 years ago for clients who havethe space, instrumentation and business need, but are either unable tomeet workload demands with their own internal resources or prefer to get their work done with a strategic partner who provides bothoutsourcing and insourcing. Our clients were facing challenges withtemporary staffing, such as time restrictions, quality of resources andtraining burdens and so wanted a partner to delegate laboratoryservices to so that they could focus their resources on other priorities.Our PSS model allows them to meet their workload demandscontinuously without these challenges.

Which countries do you serve and how do you ensureharmonisation between markets? We currently have approximately 1,000 Eurofins employees servingclients in the US, Ireland, UK, France, Italy, Belgium, Netherlands,Switzerland, Spain, Germany, Norway, Sweden, and Denmark. And withour global Eurofins laboratory network, we are rapidly expanding ourPSS services beyond this existing footprint.

We infuse our 50-year-plus track record of scientific expertise and

HR best practices to recruit, hire, train and manage highly qualifiedscientists to perform laboratory services throughout our client sites.These consistent processes and procedures allow us to harmonise thisservice model no matter where it is being applied throughout the world.We recognise cultural preferences and ensure that we develop solutionsthat are culturally and legally compliant with the same uniformapproach of delighting our clients.

How do laboratories benefit from insourcing theirrequirements rather than outsourcing? There are benefits to both, actually. Insourcing allows the client to keepthe testing at their facility using their laboratory equipment. Yet they donot have the overhead costs associated with staffing these teams sincePSS sits under outsourcing/insourcing costs that aren’t consideredclients’ labour costs and internal processes. This is beneficial when theywant the scientific support and exchange or dedicated servicesimmediately on site, or when they have short hold times. Conversely,outsourcing is good for a specific project or test, allowing the client touse methods and instrumentation not available at the client’s facilityand providing the benefit of expertise from a third party contract lab.Outsourcing is also beneficial if the client has space constraints andneeds dedicated resources. The FTE model in our laboratories forcontinuous work of six months or more benefit from dedicatedresources working under our Quality systems.

Most of our clients strategically decide how to best meet theirbusiness needs using a combination of insourcing and outsourcingsolutions. The decision is often driven by duration of needs, the type of work, space, turn-around time, the level of scientific collabora-tion needed.

How are you different than other companies that provide PSS services? Our PSS program has been recognised seven times in the last sevenyears with strategic partner awards from our clients for our abilities toprovide scientific insourcing – the only insourcing solution with such a designation in the pharmaceutical industry. We don’t just supply people, we provide laboratory services. We have been providingthese services successfully for the past 13 years and have achievedsignificant growth, which demonstrates our success in exceeding ourclients’ expectations.

UNDER THE MICROSOPE


© i3

d / S

hutt

erst

ock.

com

In an exclusive interview, Beth DiPaolo, Presidentof Eurofins Lancaster Laboratories Professional

Scientific ServicesSM, tells European PharmaceuticalReview about its PSS Insourcing Solutions

To give a brief overview of a few of the factors that shape the Danishhealthcare and pharmaceutical markets, Denmark maintains a universal health care system that is financed through general taxrevenues. As in many European countries, there are rules on adver -tising, and doing so for prescription drugs directly to patients isprohibited. However, unlike in many other European countries, inDenmark pharmacists must in the first instance offer the patient thecheapest product within a group of substitutes (mandatorysubstitution). Patients then decide themselves whether or not to buythat cheapest product or whether to buy a more expensive product.

Meanwhile, product pricing is unregulated in Denmark. Sellers mustnotify the governmental body, the Danish Medicines Agency, ofpharmacy wholesale prices. The agency updates prices every 14 daysand makes them publicly available online. It also ensures that prices are identical nationwide and classifies products into substitutiongroups based on active substance, administration form, strength, andpackage size.

Reference pricing in DenmarkGovernments and regulators around the world are facing increasingcosts of medical treatments driven by a steadily growing life expectancy,aging populations, and often broad public health insurance coverage. In an attempt to reduce the expenditure growth, various cost

containment tools have been introduced. Among those tools,restricting patients’ reimbursements using drug-specific referenceprices appears to be a particularly widely-embraced approach.Reference pricing makes patients more price-conscious as their out-of-pocket payment – the co-payment – increases in the retail priceof the drug chosen.

Patient co-payments are determined as the pharmacy retail priceminus the reimbursement, which in turn constitutes a fraction of adrug’s reference price. Reference prices are either functions (often theaverage or minimum) of prices of substitute products in other countriesor a function of prices of domestic substitutes. While the former isreferred to as an ‘external’ reference price, the latter is termed an‘internal’ reference price. While it is well documented that referencepricing leads to reduced prices for drugs, much less is known about thedesign of such systems. To overcome this research gap, we studied the switch from external to internal reference pricing in Denmark thattook place in April 2005 (documented in our recent paper1). Denmarkused to base reference prices on the European average price ofsubstitute products before the reference pricing reform and has basedit on the price of the cheapest domestic substitute since the reform.

There are several reasons why we would expect the switch frominternal to external reference pricing to strengthen competition and toreduce prices. Firstly, firms now faced demand that was more

Reference price systems for prescription drugs have found widespread use as cost containment tools. Under suchregulatory regimes, patients co-pay a fraction of the difference between pharmacy retail price of the drug and areference price. Reference prices are either externally (based on drug prices in other countries) or internally (basedon domestic drug prices) determined. In a recent study, we analysed the effects of a change from external to internalreference pricing in Denmark in 2005, finding that the reform led to substantial reductions in prices, producerrevenues, and expenditures for patients and the health insurance system. We also estimated an increase inconsumer welfare but the size effect depends on whether or not perceived quality differences between branded andother drugs are taken into account.

REGULATORY INSIGHT

Drug pricing reforms:the Danish experience

Ulrich Kaiser University of Zurich � Susan Mendez Melbourne Institute of Applied Economic and Social ResearchThomas Rønde Copenhagen Business School � Hannes Ullrich DIW Berlin and University of Zurich


© am

enic181 / Shutterstock.com

REGULATORY INSIGHT


price-elastic above the external reference price than below it, prior tothe reform, introducing a tendency for prices to cluster at the referenceprice. Since Danish prices were comparatively low in Europe, this keptthe Danish price level up. Secondly, since the reform, Danish patientshave had to pay the full difference between the retail price and the reference price ‘out-of-pocket’ when buying a product that is not the least expensive one. This makes patients more price-sensitiveand leads to tougher competition among firms in the market.

Our analysis focuses on cholesterol-lowering drugs statins. Toestimate the reform’s effects on price, demand and economic welfarewe would ideally observe the market for statins simultaneously withboth internal and external reference pricing. Given that we can onlyobserve the market either with one or the other at any one time, theempirical challenge we face is how best to construct an artificial,‘counter-factual’ world in which the reform had taken place in the pre-reform period already. The empirical questions we specifically askare, (i) how high would list prices have been had the reform taken placepre-reform, and (ii) how would demand have adjusted to a change inco-payments? By constructing such a counter-factual world, we canfilter out factors other than the reform that may have affected post-reform market outcomes. The details about the methodology weemploy are given in Kaiser et al (2014).

A mixed bagThe key finding of our analysis is that the reference price reform hassubstantially different effects on producer revenues, governmentexpenditures and patients’ co-payments. Our estimated pricedecreases are most substantial for generics where list prices decline by 44%, while retail prices for branded drugs change comparativelylittle. Even though we estimate a decrease in patient co-payments on average, they increase for branded drugs. This is the case sincereference prices for branded drugs decrease relatively more than list prices.

These changes in absolute and relative price terms have largeeffects on the demand for statins. Overall demand for statins increasesby 13%. Growth in demand is strongest for generic drugs where itincreases by 20%. Parallel imported drugs witness a demand increase of62%, up from a pre-reform market share of 13%. Demand for brandeddrugs declines by 21%. Combining price and demand effects, weestimate overall decreases in reimbursements and consumerexpenditures of around 10%.

Turning to the effects of the reform on firms, we find that parallelimporters benefit most from the reform. Their overall revenues increaseby 42%, while the revenues of firms selling generic and branded drugsincrease by 20% and decrease by 29%, respectively. Overall, producerrevenues decrease by 10%.

Finally, we calculate consumer welfare under two polarassumptions: (i) any consumer preference for branded drugs is ‘real’and should be included in the calculation of consumer surplus and (ii) any preference for branded drugs is ‘artificial’, for example, based onan unsubstantiated belief that branded drugs have superior therapeuticeffects, and should not be included in consumer surplus. Ourestimations reveal that consumers prefer branded drugs over parallelimports and generics. If we treat the perceived quality differencesbetween these different types of drugs as real, the increase in

consumer surplus is lower because consumers substitute away frombranded drugs (for which they have a preference) to generic drugs.Hence, we estimate a modest reform-induced increase in consumersurplus of 7% under this assumption. Estimated consumer welfare ismuch higher if we treat the perceived quality differences as artificial,resulting in an increase in consumer surplus of 36% due to the reform.

ConclusionDespite the specific features of the regulatory setup, we believe that theDanish experience provides some general insights into the optimaldesign of reference price systems. Under the internal reference pricingregime adopted in Denmark, patients always pay the full pricedifference between the drug bought and the cheapest substitute. This makes patients more sensitive to relative prices and strengthenscompetition. Also, policy makers do not have to find ways of settingreference prices close to the competitive level in order to mimic acompetitive market. This constitutes an important advantage overexternal reference price systems that often effectively turn thereference price into the market price.

There is no doubt that reference pricing must be complementedwith other rules and regulations for generic competition to work well. In that vein, we believe that mandatory substitution spurred genericcompetition and helped realise the gains from the switch from externalto internal reference pricing.

1. Kaiser, U., S.J. Mendez, T. Rønde, H. Ullrich (2014): Regulation of pharmaceutical

prices: Evidence from a reference price reform in Denmark. Journal of Health

Economics 36, p. 174-187.

Reference

Ulrich Kaiser is a chaired professor of Entrepreneurship at

University of Zurich (Switzerland) and holds a part-time

position at Copenhagen Business School (Denmark). His

research focuses on empirical analysis of innovation,

regulation and business strategy. He studied economics at

University of Konstanz (Germany) where he also gained his

PhD. He is presently affiliated with Centre for European

Economic Research (Mannheim, Germany) and Institute for the Study of

Labor (Bonn, Germany).

Susan Mendez is a Research Fellow at Melbourne Institute

of Applied Economic and Social Research (Australia).

Before joining the Institute, Susan was a Research

Associate at University of Zurich (Switzerland) from where

she also has her PhD. Her research interests are applied

industrial organisation, applied microeconomics, health

economics and labour economics.

Thomas Rønde holds a professorship in innovation and

entrepreneurship at Copenhagen Business School

(Denmark). He did his PhD in economics at University

Pompeu Fabra (Barcelona, Spain). His primary research

interests are innovation, health economics, regulation, and

competition policy. He is affiliated with Centre for

Economic Policy Research (London, UK) and serves as

Chief Economist at the Danish Competition and Consumer Authority.

Hannes Ullrich is a senior research associate at DIW

Berlin (Germany) and University of Zurich (Switzerland).

His research focuses on empirical analysis of product

market competition and regulation with an emphasis on the

pharmaceutical industry. He studied economics at

University of Mannheim (Germany) and Toulouse School

of Economics (France) and completed his PhD in

economics at University of Zurich.


22 The differentiation ofpluripotent stem cells tohepatocyte-like cells Yu Wang and David Hay, MRC Centre for Regenerative Medicine,University of Edinburgh

25 Computational methods forsmall molecule selection instem cell differentiation Andreas Bender and Yasaman KalantarMotamedi, University of Cambridge; Maryam Peymani and Mohammad HosseinNasr-Esfahani, Royan Institute

29 Stem Cells Roundtable Moderated by Dr David Hay, PrincipalInvestigator, MRC Centre for RegenerativeMedicine, University of Edinburgh

Stem Cells

© C

reat

ions

/ Sh

utte

rsto

ck.c

om

SPONSORS

Human embryonic stem cells (hESCs) were first isolated and maintainedin culture by Thomson and colleagues2. Later, in 2006, Takahashi andYamanaka first obtained PSCs from murine fibroblasts. In theseexperiments, somatic cells were reprogrammed to pluripotency usingretroviral-mediated introduction of four transcription factors: SOX2, Krϋppel-like factor 4 (KLF4), octamer binding protein 4 (OCT4) andc-MYC3. The derivative PSCs were termed induced pluripotent stem cells(iPSCs). Following on from this seminal work, the first human iPSCs weregenerated from the same laboratory using a similar methodology4.Notably, iPSCs and human embryonic stem cells (hESCs) display manysimilarities5, however, the ability to generate iPSCs from specificindividuals promises a new paradigm, where models or therapies canbe tailor-made6.

Derivation of iPSCsiPSCs are most commonly generated from somatic cells by ectopicexpression of defined transcription factors. Previously, the most widelypracticed methods of cell reprogramming employed retroviral- or

lentiviral-mediated delivery of the reprogramming factor cocktail3,4,7.While these strategies were highly efficient, the use of integrating virusto deliver exogenous materials came at a price. This included transgenereactivation, genome disruption or inefficient silencing, which are knownto be deleterious to cell stability and negatively affect differentiation8. In an attempt to bypass those issues, researchers in the field developedalternative strategies9 including, doxocycline-inducible lentiviruses fortunable transgene expression10 or Cre-deletable lentiviral vectors toefficiently excise integrated transgenes11.

While these approaches have been useful in developing thereprogramming technology, the use of targeted or random integration,as well as transgene deletion, may damage genetic code leading toissues with cellular differentiation and/or malignant mutagenesis12. As a result, there has been a major focus in the field on non-integrativeand virus-free reprogramming methods. Replication-defectiveadenoviral vectors have been employed as they are maintainedepisomally and without integrating into host chromosomal DNA13. While this approach was promising, the requirement to repeatedly

Pluripotent stem cells (PSCs) can give rise to all cell types found in the human body1. In theory, the self-renewal anddifferentiation properties of PSCs offer the prospect of unlimited amounts of human somatic cells for application.PSC usage is now common place throughout the world, with promising models of human ‘in a dish’ and cell-basedtherapies likely to have significant impact on modern medicine.

IN-DEPTH FOCUS: STEM CELLS

The differentiation ofpluripotent stem cells to hepatocyte-like cells

Yu Wang and David HayMRC Centre for Regenerative Medicine, University of Edinburgh


© S

pect

ral-D

esig

n / S

hutt

erst

ock.

com

Advancing the field of stem cell - based therapiesStem cell isolation, expansion and differentiation:complete xeno-free culture system - from research to clinical applications

BIOLOGICAL INDUSTRIES ISRAEL BEIT HAEMEK LTD.Kibbutz Beit Haemek 2511500, Israel. Tel. 972-4-9960595Email: info bioind.com | www.bioind.com

Come see as at ISSCR booth #B07:33

ES ISRAEL BEIT HAEMEK LTD.

infect cells to efficiently reprogramme them was deemed a signifi-cant disadvantage.

One of the most successful approaches to date has employed theSendai virus (SeV) to deliver the four Yamanaka factors which displayssimilar efficiency to retrovirus-mediated reprogramming, but withoutthe requirement for transgene integration14. In addition to non-integrative virus delivery, there has also been a focus on the use ofvirus-free strategies such as the polycistronic expression of Yamanakafactors from plasmid DNA, to generate iPSCs15.

The piggyBac transposon-transposase has also been used toproduce transgene-free iPSCs16, as has the oriP/EBNA1 episomal vector, derived from the Epstein-Barr virus17. More recently, synthetic -ally modified mRNAs have been used toreprogramme somatic cells18, however therequirement to repeatedly transfect cells has proved less efficient than other methods.In addition to nucleic acid delivery, cellpermeable recombinant proteins (Oct3/4, Sox2,Klf4 and c-Myc) have been used to repro -gramme cells, albeit it with low efficiency19,20. A more recentbreakthrough was the generation of chemically-induced pluripotentstem cells using a combination of small molecules with murineembryonic fibroblasts21. While this approach is promising, chemically-induced pluripotency has yet to be achieved in human cells.

Generation of hepatocyte-like cellsThe ability to generate renewable sources of human somatic cells fromdefined genetic backgrounds has enormous potential for modernmedicine. The likely short term impact will be the production of more sophisticated cell-based models of human biology ‘in a dish’.

Whereas the delivery of new cell-based therapies for degenerativedisorders is likely to follow. Our particular interest lies in the delivery ofhuman hepatocytes from PSCs. Numerous human hepatocytedifferentiation protocols have been established using two- and three-dimensional cell culture22.

Encouragingly, stem cell derived hepatocyte-like cells (HLCs) exhibittypical hepatocyte characteristics, which include hepatocyte markerexpression and function23,24; the capacity to model human disease25;supportiveness of hepatitis virus26-30 and parasite lifecycles31; and theypermit modelling of human drug metabolism32,33 and drug-induced liverinjury34,35. While these attributes are promising, the persistentexpression and secretion of alpha fetoprotein (AFP) still implies a

foetal-like status. To try to address the issue ofimmature phenotype, research groups haveemployed numerous strategies, which includethe use of different combinations of cytokinesor chemicals and bio-engineered scaffolds,amongst others, to mimic the liver niche or thethree dimensional architecture36-42.

Most recently, the generation of vascularised and functional humanliver tissue from PSC-derived HLCs and primary cells was achieved. iPSC-derived HLCs were co-cultured with human umbilical veinendothelial cells and human mesenchymal stem cells, and allowed toself-assemble into macroscopically visible 3D cell clusters. Importantly,the iPSC-HLCs containing liver buds were mechanically stable and ableto function in vivo after transplantation40. It is evident that tissueengineering, using the relevant cell types of the organ of interest, isgoing to be an important part of the incremental process in deliveringbona fide tissue from PSCs. While significant progress has been made,there is still room for improvement. Issues relating to cell maturity,


One of the most successfulapproaches to date has employed theSendai virus (SeV) to deliver the four

Yamanaka factors

stability and manufacture at scale, among others, still persist andremain key areas of focus.

ConclusionThese days, PSCs are an essential part of the biologist’s tool box. Theability to derive renewable somatic cells in limitless amounts providesthe field with a vital resource that overcomes many of the limitations ofassociated with the use of transformed or primary cells. The coupling of efficient differentiation and tissue engineering promises that in thefuture it will be able to manufacture human tissue ‘in a dish’ whichdisplay many of the attributes observed in vivo. With this in mind it istempting to speculate that somatic cells, from defined genetics, willfacilitate the development of cutting edge and tailor-made drug- andcell-based therapies of the future.

AcknowledgementsDr. Hay was funded by the UK Regenerative Medicine Platform. Ms. YuWang was funded by a China Scholarship.



1. Sterneckert, J. L., Reinhardt, P. & Scholer, H. R. Investigating human disease using stem cell

models. Nature reviews. Genetics 15, 625-639, doi:10.1038/nrg3764 (2014).

2. Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science

282, 1145-1147 (1998).

3. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic

and adult fibroblast cultures by defined factors. Cell 126, 663-676, doi:10.1016/

j.cell.2006.07.024 (2006).

4. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by

defined factors. Cell 131, 861-872, doi:10.1016/j.cell.2007.11.019 (2007).

5. Yamanaka, S. Induced pluripotent stem cells: past, present, and future. Cell stem cell 10,

678-684, doi:10.1016/j.stem.2012.05.005 (2012).

6. Grskovic, M., Javaherian, A., Strulovici, B. & Daley, G. Q. Induced pluripotent stem cells--

opportunities for disease modelling and drug discovery. Nature reviews. Drug discovery 10,

915-929, doi:10.1038/nrd3577 (2011).

7. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science

318, 1917-1920, doi:10.1126/science.1151526 (2007).

8. Toivonen, S. et al. Comparative analysis of targeted differentiation of human induced

pluripotent stem cells (hiPSCs) and human embryonic stem cells reveals variability

associated with incomplete transgene silencing in retrovirally derived hiPSC lines. Stem

cells translational medicine 2, 83-93, doi:10.5966/sctm.2012-0047 (2013).

9. Gonzalez, F., Boue, S. & Izpisua Belmonte, J. C. Methods for making induced pluripotent

stem cells: reprogramming a la carte. Nature reviews. Genetics 12, 231-242,

doi:10.1038/nrg2937 (2011).

10. Brambrink, T. et al. Sequential expression of pluripotency markers during direct

reprogramming of mouse somatic cells. Cell stem cell 2, 151-159, doi:10.1016/

j.stem.2008.01.004 (2008).

11. Hanna, J. et al. Treatment of sickle cell anemia mouse model with iPS cells generated from

autologous skin. Science 318, 1920-1923, doi:10.1126/science.1152092 (2007).

12. Revilla, A. et al. Current advances in the generation of human iPS cells: implications in cell-

based regenerative medicine. Journal of tissue engineering and regenerative medicine,

doi:10.1002/term.2021 (2015).

13. Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G. & Hochedlinger, K. Induced pluripotent stem

cells generated without viral integration. Science 322, 945-949, doi:10.1126/

science.1162494 (2008).

14. Fusaki, N., Ban, H., Nishiyama, A., Saeki, K. & Hasegawa, M. Efficient induction of

transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA

virus that does not integrate into the host genome. Proceedings of the Japan Academy. Series

B, Physical and biological sciences 85, 348-362 (2009).

15. Gonzalez, F. et al. Generation of mouse-induced pluripotent stem cells by transient

expression of a single nonviral polycistronic vector. Proceedings of the National

Academy of Sciences of the United States of America 106, 8918-8922, doi:10.1073/

pnas.0901471106 (2009).

16. Woltjen, K. et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem

cells. Nature 458, 766-770, doi:10.1038/nature07863 (2009).

17. Yu, J. et al. Human induced pluripotent stem cells free of vector and transgene sequences.

Science 324, 797-801, doi:10.1126/science.1172482 (2009).

18. Warren, L. et al. Highly efficient reprogramming to pluripotency and directed differentiation

of human cells with synthetic modified mRNA. Cell stem cell 7, 618-630,

doi:10.1016/j.stem.2010.08.012 (2010).

19. Kim, D. et al. Generation of human induced pluripotent stem cells by direct delivery of

reprogramming proteins. Cell stem cell 4, 472-476, doi:10.1016/j.stem.2009.05.005 (2009).

20. Zhou, H. et al. Generation of induced pluripotent stem cells using recombinant proteins.

Cell stem cell 4, 381-384, doi:10.1016/j.stem.2009.04.005 (2009).

21. Hou, P. et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule

compounds. Science 341, 651-654, doi:10.1126/science.1239278 (2013).

22. Szkolnicka, D., Zhou, W., Lucendo-Villarin, B. & Hay, D. C. Pluripotent stem cell-derived

hepatocytes: potential and challenges in pharmacology. Annual review of pharmacology and

toxicology 53, 147-159, doi:10.1146/annurev-pharmtox-011112-140306 (2013).

23. Cai, J. et al. Directed differentiation of human embryonic stem cells into functional hepatic

cells. Hepatology 45, 1229-1239, doi:10.1002/hep.21582 (2007).

24. Hay, D. C. et al. Highly efficient differentiation of hESCs to functional hepatic endoderm

requires ActivinA and Wnt3a signaling. Proceedings of the National Academy of Sciences

of the United States of America 105, 12301-12306, doi:10.1073/pnas.0806522105 (2008).

25. Rashid, S. T. et al. Modeling inherited metabolic disorders of the liver using human induced

pluripotent stem cells. The Journal of clinical investigation 120, 3127-3136,

doi:10.1172/JCI43122 (2010).

26. Roelandt, P. et al. Human pluripotent stem cell-derived hepatocytes support complete

replication of hepatitis C virus. Journal of hepatology 57, 246-251, doi:10.1016/

j.jhep.2012.03.030 (2012).

27. Schwartz, R. E. et al. Modeling hepatitis C virus infection using human induced pluripotent

stem cells. Proceedings of the National Academy of Sciences of the United States of

America 109, 2544-2548, doi:10.1073/pnas.1121400109 (2012).

28. Shlomai, A. et al. Modeling host interactions with hepatitis B virus using primary and

induced pluripotent stem cell-derived hepatocellular systems. Proceedings of the National

Academy of Sciences of the United States of America 111, 12193-12198,

doi:10.1073/pnas.1412631111 (2014).

29. Wu, X. et al. Productive hepatitis C virus infection of stem cell-derived hepatocytes reveals

a critical transition to viral permissiveness during differentiation. PLoS pathogens 8,

e1002617, doi:10.1371/journal.ppat.1002617 (2012).

30. Zhou, X. et al. Modulating innate immunity improves hepatitis C virus infection and

replication in stem cell-derived hepatocytes. Stem cell reports 3, 204-214,

doi:10.1016/j.stemcr.2014.04.018 (2014).

31. Ng, S. et al. Human iPSC-Derived Hepatocyte-like Cells Support Plasmodium Liver-Stage

Infection In Vitro. Stem cell reports 4, 348-359, doi:10.1016/j.stemcr.2015.01.002 (2015).

32. Medine, C. N. et al. Developing high-fidelity hepatotoxicity models from pluripotent stem

cells. Stem cells translational medicine 2, 505-509, doi:10.5966/sctm.2012-0138 (2013).

33. Takayama, K. et al. Prediction of interindividual differences in hepatic functions and drug

sensitivity by using human iPS-derived hepatocytes. Proceedings of the National Academy

of Sciences of the United States of America 111, 16772-16777, doi:10.1073/

pnas.1413481111 (2014).

34. Szkolnicka, D. et al. Accurate prediction of drug-induced liver injury using stem cell-

derived populations. Stem cells translational medicine 3, 141-148, doi:10.5966/sctm.2013-

0146 (2014).

35. Holmgren, G. et al. Long-term chronic toxicity testing using human pluripotent stem cell-

derived hepatocytes. Drug metabolism and disposition: the biological fate of chemicals 42,

1401-1406, doi:10.1124/dmd.114.059154 (2014).

36. Takayama, K. et al. 3D spheroid culture of hESC/hiPSC-derived hepatocyte-like cells for

drug toxicity testing. Biomaterials 34, 1781-1789, doi:10.1016/j.biomaterials.2012.11.029

(2013).

37. Sivertsson, L., Synnergren, J., Jensen, J., Bjorquist, P. & Ingelman-Sundberg, M. Hepatic

differentiation and maturation of human embryonic stem cells cultured in a perfused three-

dimensional bioreactor. Stem cells and development 22, 581-594, doi:10.1089

/scd.2012.0202 (2013).

38. Farzaneh, Z., Pournasr, B., Ebrahimi, M., Aghdami, N. & Baharvand, H. Enhanced

functions of human embryonic stem cell-derived hepatocyte-like cells on three-

dimensional nanofibrillar surfaces. Stem cell reviews 6, 601-610, doi:10.1007/s12015-010-

9179-5 (2010).

39. Vosough, M. et al. Generation of functional hepatocyte-like cells from human pluripotent

stem cells in a scalable suspension culture. Stem cells and development 22, 2693-2705,

doi:10.1089/scd.2013.0088 (2013).

40. Takebe, T. et al. Vascularized and functional human liver from an iPSC-derived organ bud

transplant. Nature 499, 481-484, doi:10.1038/nature12271 (2013).

41. Gieseck, R. L., 3rd et al. Maturation of induced pluripotent stem cell derived hepatocytes by

3D-culture. PloS one 9, e86372, doi:10.1371/journal.pone.0086372 (2014).

42. Du, C., Narayanan, K., Leong, M. F. & Wan, A. C. Induced pluripotent stem cell-derived

hepatocytes and endothelial cells in multi-component hydrogel fibers for liver tissue

engineering. Biomaterials 35, 6006-6014, doi:10.1016/j.biomaterials.2014.04.011 (2014).

References

Dr David Hay is a Principal Investigator at the University

of Edinburgh’s MRC Centre for Regenerative Medicine.

David has worked in the field of stem cell biology and

differentiation over the last decade. David and his team have

highlighted the important role that pluripotent stem cells

have to play in modelling human liver biology ‘in a dish’.

Yu Wang is currently a PhD student at the University of

Edinburgh’s MRC Centre for Regenerative Medicine.

Yu has been researching somatic cell reprogramming and

hepatocyte differentiation over the last five years.

Since the concept of ‘stem cells’ has been discovered and refined manytimes, biological processes leading to cellular differentiation intovarious cell types and tissues have been under much investigation, andhence our understanding of what can (and cannot) be done with stemcells has advanced significantly. A concrete application of stem cellswhich is of significant current interest is their use in regenerativemedicine. Given that patient-specific differentiated cells can begenerated to address a given physiological need this paves the way toreplace tissues whose functionality has been lost due to a particulardisease, or due to ageing.1,2

However, we are currently unlikely to be close to therapeuticapplication in many areas, and the main reasons for this involve

obtaining suitable stem cells in the first place; directed differentiationinto the desired cell type or tissue (at sufficient purity etc.); as well asapplying this process in the more complex system, namely the humanbody. In this review, we will outline current approaches to address thesecond of the above points: How to differentiate stem cells into the desired target cell type in a more efficient manner, and to do so inparticular by utilising small molecules3,4 which in many cases can beconsidered to be more suitable than biologics (at least at the currentstage) for therapy.

Stem cells can be classified according to their ability to differentiateinto partiular cell types (e.g., omnipotent vs. pluripotent stem cells), aswell as the source from which they are derived (ie, embryonic vs. foetal

The utilisation of stem cells for regenerative medicine has gained significant interest in recent years, in diseaseareas as diverse as cardiovascular, diabetes, central nervous system and ophthalmology. However, while geneticmethods such as transcription factors are able to differenciate pluripotent stem cells into a variety of cell types andtissues, processes are often still rather unspecific and inefficient. The utilisation of small molecules has helpedadvance the field considerably; however, the question remains of how to rationally select small molecules to steerspecificity and yield of differentation. In this review, we will illustrate how computational (‘in silico’) methods canhelp to rationally choose bioactive small molecules to improve stem cell differentiation in the desired way.



© n

obea

stso

fierc

e / S

hutt

erst

ock.

com

Computational methods for small molecule selectionin stem cell differentiation

Andreas Bender and Yasaman Kalantar Motamedi University of CambridgeMaryam Peymani and Mohammad Hossein Nasr-Esfahani Royan Institute, Isfahan

Avoid spontaneous differentiation of your hiPS cellsGenerate a standardized source of hiPS cells. Cellartis® DEF-CS™ is a complete, feeder-free and defi ned system for increased expansionand scale up of your hiPS cell culture with up to 99.4% purity.

• Single cell passaging

• Effi cient expansion

• High reproducibility

• Extreme purity

• Complete system (coating included)

15

Growth rate of cultures grown in DEF-CS compared to other stem cell culture vendors. Cells were cultured for 3 weeks prior to testing.

Comparison of growth rate

100

1000

10

10 Day

Num

ber o

f cel

ls x

106

5 10

Cellartis DEF-CS

Reference feeder culture

Vendor 1-4

Pluripotency of cells grown for 5 weeks in DEF-CS stem cell culture media as compared to other available culture systems. DEF-CS revealed the highest proportion and intensity of TRA1-60 (A), and SSEA4 (B).

Quantitative pluripotent marker analysis

On feederA

Vendor 3

Vendor 1

Vendor 4

Vendor 2

Cellartis DEF-CS

TRA

1-60

95.9%

95.1%

88.5%

95.8%

74.6%

98.3%

TRA

1-60

B On feeder

Vendor 3

Vendor 1

Vendor 4

Vendor 2

Cellartis DEF-CS

SS

EA4

99.0%

99.8%

98.7%

98.8%

92.4%

99.4%

SS

EA4

Learn more at www.clontech.com/stemcells

Sample size available

vs. adult, or somatic, stem cells). Usually – and also in man via naturalprocesses – cellular differentiation is achieved by expression ofdifferent transcription factors at different time points. However, theutilisation of transcription factors in therapy is difficult due to multiplereasons (ease of production, administration, stability, distribution inthe body, etc.). In addition, it has been found that for multipleobjectives, including the induction of pluripotent stem cells,5 theadministration of small molecules3-5 can significantly improve efficiencyof differentiation. Hence, methods for the directed selection of suchsmall molecules to support cell differentiation gains importance.

However, this now evokes the question of how to select a smallmolecule which will give rise to a particular cell type – andcomprehensive, well-validated approaches to picking small moleculesto more efficiently differentiate stem cell are scarce. Previous studiessuch as CellNet6 were focused on identifying important transcriptionfactors for the differentiation of stem cells to 16 human cells andtissues, and to direct trans-differentiation between them. While this isvery insightful work from the biological side, and suitable for choosingbiological matter such as transcription factors for differentiation, thesmall molecule side is not considered in this approach. On the otherhand, systematic approaches were focused on identifying micro-RNAsfor inducing the differentiation of stem cells.7,8 MicroRNAs are shortRNAs that play a crucial role in a wide variety of biological processes,including differentiation, and they can direct messenger RNAdegradation or disrupt mRNA translation. With those properties theycan, for example, upregulate important cardiac transcription factors topromote the differentiation of stem cells to cardiomyocytes.7 Thoselineage commitments during stem cell differentiation can also occur by

epigenetic events such as histone deacetylation and promoter DNAmethylation. In this regard, DNA methylation inhibition agents such asZebularine and AzadC are known to enhance differentiation of stemcells to cardiomyocytes.9 However, to the best of our knowledge nostudies yet have focused on the prediction, or informed selection, of small molecules for promoting stem cell differentiation in thedesired way.

Hence, while transcription factors can be picked upon biologicalunderstanding of differentiation processes (e.g., by measuringexpression of genes in particular stages along differentiation), decidingon how to pick small molecules to support this process is less obvious.An approach that has been explored in our lab is that of using geneexpression data for small molecule treatments of particular cell lines(which, due to practical reasons, may often be different cell lines thanthat of current interest), and then selecting small molecules fordifferentiation which exhibit the desired change in gene expression inthe test sytem.

This process is outlined in Figure 1 (page 27), which illustrates thecombination of gene expression-based and target-based approachesfor the selection of small molecules for modulating a particularbiological process - such as cancer, or the differentiation of stem cellsinto particular cell lines. The process is generally applicable, so for anytwo different biological states where gene expression data for both isavailable (e.g., differentiated vs. stem cells, or diseased vs healthytissue, etc.) this algorithm can be applied to select suitable smallmolecule modulators. In this particular example focused on cancer thegene signature of the disease is matched to each of the compounds inthe database, in order to select a small molecule most likely able to


modulate the disease of interest (or, here, to differentiate stem cellsinto the desired target tissue). On the other hand, protein targets forthe small molecule under investigation are considered, in order to gaina second type of insight into its biological activities, which can henceconfer more confidence for its selection for experiment. This processhas been validated by our group for the selection of selectively cytotoxiccompounds in cancer, as well as for the directed differentiation of stemcells into cardiomycytes, as well as other cell types.

Those approaches use on the one hand small molecule-geneexpression information (e.g., from ConnectivityMap10 or LINCS), and onthe other hand gene expression information about origin and desiredtarget state (e.g., from GEO or ArrayExpress, or custom-madeexperiments). Similar approaches of mapping small molecule todisease space via their respective gene expression readouts have beenapplied by multiple groups and validated in disease areas such ascancer and inflammatory bowel disease.11 However, their application tothe selecting of small molecules to induce the differentiation of stemcells has not been performed before to the best of our knowledge.

As a concrete successful example, recent research taking place inour group was focused on predicting small molecules for promotingdifferentiation of stem cells to cardiomyocytes. In this study, geneexpression data of adult ventricular heart was compared to

gene expression data derived from stem cells, and a set of differentiallyexpressed genes between both states were identified. On the otherhand, we employed the databases mentioned above – namely,ConnectivityMap and LINCS – in order to identify suitable smallmolecules which are able to either over- or under-express those specificgenes required for differentiation. While ConnectivityMap, as the firstdatabase of its type, covered relatively small chemical space (of 1,309compounds), its successor LINCS already contains gene expression dataof 200,000 compound treatments covering 20,000 compounds appliedto 77 cell lines, hence increasing coverage in both chemical space as wellas biological space (cell lines, doses, time points) considerably. As a result, four novel compounds were selected for experimentalvalidation with regard to their ability to differentiate stem cells into cardiomyocytes.

The compounds were added to the medium either in the first 7 days, during cardiac proginator cells formation, or during days 7-14,post cardiac proginator cells formation, in order to see whether qualityof the cardiomyocytes would be different. Qualitative assessment wasperformed by comparing morphological features of the beating cells and the strength of beating cells to control groups, and bothgenetic and proteomic readouts were employed additionally to verifythe generation of cardiomyocytes on the molecular level.

It was found in this study that all four selected compounds led tooverexpression of cardiac and cardiac precursor markers, as predictedby the algorithm. Also on the protein level intensity and percentage ofcardiac markers, in particular when embryoid bodies were treated. The result for a sample compound is visualised in Figure 2, where it canbe seen that the application of compound led to better cardio-myocytes on the phenotypic level (visible as darker and more clearlyseparated cells). This outcome was also confirmed by genomic andproteomic level experiments, and we are currently also extending this concept to other areas of selecting small molecules for thedifferentiation of stem cells.

In summary, small molecules are able to lead to a higher efficiencyin stem cell differentiation, as well as possessing con siderable



Figure 1: Combination of gene expression-based and target-based approaches for theselection of small molecules for modulating a particular biological process (such ascancer, or the differentiation of stem cells into particular cell lines). In this particularexample focused on cancer the gene signature of the disease is matched to each of thecompounds in the database, in order to select a small molecule most likely able tomodulate the disease of interest (or, here, to differentiation stem cells into the desiredtarget tissue). On the other hand, protein targets for the small molecule underinvestigation are considered, in order to gain a second type of insight into itsbiological activities, which can hence confer more confidence for its selection forexperiment. This process has been validated by or group for the selection ofselectively cytotoxic compounds in cancer, as well as for the directed differentiationof stem cells into cardiomycytes, as well as other cell types.

Figure 2: The application of compound (bottom, a and b) led to bettercardiomyocytes on the phenotypic level (visible as darker and more clearly separatedcells) than control+DMSO (top, a and b) both on day 10 and day 12 of thedifferentiation protool . This outcome was also confirmed by genomic and proteomiclevel experiments, and we are currently extending this concept also to other areas ofselecting small molecules for the differentiation of stem cells.

Custom Engineered Cells from Sigma® Life Science

Cell Design Studio™

Bioelevate.No matter how obscure or exact your specifications, our custom-engineered cell lines are tailored using CRISPR/Cas9, CompoZr® ZFN and MISSION® shRNA technologies to fit your application perfectly. Leverage our expertise with iPSC gene manipulation to create cell lines that more accurately predict disease biology.

Custom engineered cell lines from CDSsigma.com/CDS

©2015 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA and SIGMA-ALDRICH are trademarks of Sigma-Aldrich Co. LLC, registered in the US and other countries. CompoZr and MISSION are registered trademark of Sigma-Aldrich Co. LLC. Cell Design Studio and Where bio begins are trademarks of Sigma-Aldrich Co. LLC. 83498

therapeutic potential. Given the relative ease and costeffectiveness of using gene expression data to characterisesmall molecules, we are now in the position to select smallmolecules in an informed manner, in order to supportstem cell differentiation with regard to both quality andefficiency of the resulting differ entiated cells.


Andreas Bender is a Lecturer for Molecular Informatics with the Centrefor Molecular Science Informatics at the Department of Chemistry of theUniversity of Cambridge, leading a group of about 20 postdocs, PhD andgraduate students and academic visitors. In his work, Andreas is involvedwith the integration and analysis of chemical and biological data, aimed atunderstanding phenotypic compound action (such as cellular readouts, andalso organism-level effects) on a mechanistic level, ranging from

compound efficacy to toxicity. He received various awards (such as the Bayer EarlyExcellence in Science Award 2011) as well as an ERC Starting Grant 2013, on the topic ofmodelling the effects of combinations of bioactive compounds. Andreas worked in theLead Discovery Informatics group at Novartis in Cambridge/MA as well as at LeidenUniversity in the Netherlands before his current post.

Yasaman Kalantar Motamedi became a PhD candidate in theChemistry department of Cambridge University in January 2012 in the group of Dr. Andreas Bender. Her work combines Bioinformatics andCheminformatics approaches to predict novel therapies for diseases aswell as to promote differentiation of stem cells to different organs.Yasaman works closely with experimental groups to validate herpredictions. Her predictions in pancreatic cancer, breast cancer andleukaemia as well as differentiation of stem cells to cardiomyocytes are experimentallyvalidated so far. She obtained her MSc in Machine Learning from University CollegeLondon and BSc. in Computer Engineering from University of Isfahan.

Maryam Peymani is a Research Assistant at the Royan Institute ofIsfahan, Iran, as well as a faculty member in Shahrekord Azad University.During her education in Molecular Genetics, she became very interested instem cell biology and has been involved in several research projects in thisfield. She graduated with a PhD in molecular genetics.

Mohammad Hossein Nasr-Esfahani received his PhD from theUniversity of Cambridge, UK, in 1991 and is currently an academicmember of the Royan Institute in Tehran, Iran. He has been working asLaboratory Director of the Isfahan Fertility and Infertility Centre since1992 and has especial interests on male infertility. He is also the head ofRoyan Institute for biotechnology in Isfahan, Iran. The main researchareas of the groups with which he works are stem cells with interest onneuro-regeneration, animal cloning, recombinant protein and male infertility. He has over256 publications in international and national journals. The project of the first Iraniancloned sheep and transgenic animal was carried out under his supervision.

1. Bajada S, Mzakova I, Richardson JB, Ashammakhi N. Updates on stemcells and their applications in regenerative medicine. J. Tissue Eng.Regenerative Med. 2008 Jun; 2(4):169-183.

2. Caplan AI. Adult Mesenchymal Stem Cells for Tissue EngineeringVersus Regenerative Medicine. J. Cell. Physiol. 2007; 213: 341-347.

3. Zhang Y, Li W, Laurent T, Ding S. Small molecules, big roles -- thechemical manipulation of stem cell fate and somatic cellreprogramming. J. Cell Sci. 2012 Dec; 125(Pt 23): 5609-5620.

4. Ling D, Kumar S, Munusamy MA, Alarfaj AA, Chang Y, Kao SH, LinKC, et al. Generation of pluripotent stem cells without the use of geneticmaterial. Laboratory Investigation 2015; 95: 26–42.

5. Huangfu D, Maehr R, Guo W, Eijkelenboom A, Snitow M, Chen AE,Melton DA. Induction of pluripotent stem cells by defined factors isgreatly improved by small-molecule compounds. Nature Biotech. 2008; 26: 795 – 797.

6. Cahan, P et al. CellNet: Network Biology Applied to Stem CellEngineering. Cell 2014; 158: 903–915.

7. Wilson, KD et al. Dynamic microRNA expression programs duringcardiac differentiation of human embryonic stem cells: role for miR-499. Circ. Cardiovasc. Genet. 2010; 3: 426–435.

8. Tay Y, Zhang J, Thomson AM, Lim B, Rigoutsos I. MicroRNAs toNanog, Oct4 and Sox2 coding regions modulate embryonic stem celldifferentiation. Nature 2008; 455:1124–1128.

9. Horrillo, A. et al. Zebularine regulates early stages of mESC differ -entiation: effect on cardiac commitment. Cell Death Dis. 2013; 4: e570.

10. Lamb J, Crawford ED, Peck D, Modell JW, Blat IC, Wrobel MJ, et al.The Connectivity Map: Using Gene-Expression Signatures to ConnectSmall Molecules, Genes, and Disease. Science 2006 Sep; 313 (5795):1929-1935.

11. Sirota M, Dudley JT, Kim J, Chiang AP, Morgan AA, Sweet-Cordero A,et al. Discovery and preclinical validation of drug indications usingcompendia of public gene expression data. Sci. Transl. Med. 2011 Aug17; 3(96): 96ra77.

References

Pluripotent stem cells offer an unprecedentedopportunity to model human physiology and disease.What do you think are the major challenges that facepluripotent stem cell biologists in their quest to model human biology ‘in a dish’?Runeberg: Pluripotent cells have the potential to differentiate towardsall cell types of the human body. Culturing pluripotent cells in a robustmanner, one that maintains pluripotency and prevents differentiationand one that ultimately will allow reproducible differentiation intosought-after cell types, is a clear challenge. To accomplish this, youneed an advanced culture system, such as our DEF-CS™ system, toensure that you start the differentiation protocol with a homogenouspopulation of single cells. Having this type of reliable starting materialfacilitates reproducible differentiation into your cell type of interest,providing a robust model to study human biology ‘in a dish’.

Jones: Stem cell technology enables the direct study of human cells byallowing researchers to ostensibly produce any cell type at will. The most significant challenge facing the field is the development ofprotocols for differentiating cell type(s) of interest with consistentpurity and quantity to ensure data collection is not obscured byuncontrolled changes in the cells themselves. These differentiationprotocols also need to yield large quantities of high quality cells(independent of donor starting material) in order to maximisereproducibility and ensure that the fidelity of the data collected fromthese models reflects human, rather than individual, biology.

Gerber: Replicating a true microenvironment or niche in an in vitrosystem is extremely challenging. As with most model systems, we canonly prognosticate using the observations we make in largely artificialsettings. We must continue to rely on multiple lines of evidence in order

IN-DEPTH FOCUS: STEM CELLS ROUNDTABLE


ModeratorDr David Hay, Principal Investigator, MRC Centre for Regenerative Medicine, University of Edinburgh

Kristina Runeberg Site Head/Senior Director, Business

Development, Takara-Clontech

Eugenia Jones PhD, MyCell® Products Manager,

Cellular Dynamics

Mark A. Gerber, Jr. PhD, Principal R&D Scientist, Cell

Design Studio, Sigma-Aldrich

© n

obea

stso

fierc

e / S

hutt

erst

ock.

com

to advance our understanding of human biology and associated diseaseprocesses. The promise of some types of adult stem cell therapies hasbeen realised to various degrees, and it is nearly certain thatpluripotent stem cells will advance medicine accordingly, albeit at aslower rate of approval and acceptance.

Pluripotent stem cells hold great promise to provide stemand somatic cells populations to regenerate failing humantissue. What are the major obstacles you envisage as thetechnology moves from the lab to the clinic?Jones: Stem cell-derived regenerative therapies promise to end thescarcity of donor material and reduce the need for immunosuppressivetherapies – both hallmarks of current regenerative therapy approaches.The most significant obstacles for stem cell-derived regenerative therapiesinvolve immunogenicity management, treatment costs and reimburse -ment issues. Autologous therapies are optimal for alleviating rejection risk,but are costly and time-consuming; an alternative approach involvesgenerating banks of induced pluripotent stem cell (iPSC) lines fromindividuals homozygous for the most common HLA haplotypes. Suchbanks would be more cost efficient, solve the scarcity issue, reduce theneed for immunosuppressive therapy, and enable rapid treatment.

Gerber: Many of these hurdles will be regulatory in nature. Whetherthese cell-based therapies rely on personalised medicine or a‘matched’ donor will obviously have an impact on the ease and speedto clinic, but thorough testing to ensure that transplanted material willnot only be effective, but also safe is paramount. Adoption of thesekinds of therapies has been slow thus far, and for good reason. The inability to control cell fates post-transplantation and the potential health hazards that may result are very real concerns. Carefulcharacterisation and a thorough understanding of the regenerativecapacity of differentiated or progenitor cell populations must be part of the process.

Runeberg: The first challenge is obtaining a gross manufacturingproduct cell line with proper donor consent―consent that allows forcommercial therapeutic use. Today, iPSCs and embryonic stem (ES) celllines are obtained under donor consent allowing only for in vitroresearch, and these cell lines are often derived using reagents thatcontain animal and/or human components. Secondly, converting toclinical-grade culture reagents is not always an easy task. We can workwith researchers to generate GMP iPS and ES cell lines, and we haveextensive experience in converting differentiation protocols to meetclinical standards.

Following on from this, scaling up laboratory basedsystems to levels which facilitate large-scale screeningand/or clinical deployment can prove problematic. Canyou provide an overview of the major areas that scientistsmust consider when designing prototype models?Gerber: Quality control has long been a linchpin for manufacturing. The ability to maintain quality as one scales a process or system iscritical. As with many areas of research, the scientists that developmethods and techniques are rarely familiar with the hurdles present inquality assurance/control, and the partnering of research, develop -

ment and manufacturing expertise will be essential to advance many ofthese laboratory systems to the clinic. With cell-based therapies,careful control of raw materials will be necessary to minimise batch-to-batch and lot variability. Furthermore, when considering human cells,features such as passage number and population doublings can havean impact on viability and robustness.

Runeberg: For both large-scale screening and clinical protocols, youneed to start with a large amount of quality-controlled, undiffer -entiated cells. A therapeutic dose may range from 108 to 1010 cells, whichmeans that you need a lot of cells. To obtain these cells, you need arobust, clinical-grade culture system for pluripotent cells. We aredeveloping a clinical-grade version of the DEF-CS culture system that is totally free from human- and animal-derived components, iscompletely chemically defined, and contains material of clinical qualitywith traceable production processes. This system is based on rigoroussafety standards for the sourcing of the components.

Jones: iPS cells enable study of a great variety of human disorders andprovide a clear path forward for regenerative therapies. The mostnotable hurdles to moving laboratory based methodologies to large-scale production are: quality of the starting material and robustness ofthe methodology. The best methodology for producing high quality iPScell starting material combines episomal, footprint free technology withculturing in defined media. Translatability of differentiation protocols,





from lab-to-lab and iPSC line-to-iPSC line, is also important; thoseprotocols most amenable to large scale production will work across awide variety of cell lines and function robustly and predictably.

Cost of goods is an important consideration when movingtechnology from the laboratory to industry. Can youprovide suggestions as to how stem cell scale-up anddifferentiation can be delivered at an acceptable cost?Runeberg: You first need to develop cost-effective protocols, generatinglarge amounts of pluripotent stem cells in a cost-efficient and reliablemanner, and you need a differentiation protocol that is based on cost-effective reagents. Currently, most groups are culturing pluripotent cellsin cell culture flasks. We have concluded that to lower both the labourcost and the cost for culture media and other reagents, cells should becultured in a 3D environment. We are developing a culture system forsuspension culture of pluripotent cells and downstream differentiation.In addition to a cost savings benefit, 3D culture better represents theway cells grow in vivo.

Jones: Cost ‘acceptability’ must consider end product value. If newscreening methodologies generate better data quality than currentmodels, their relative cost is insignificant. For example, cardiotoxicitystudies performed using iPS cell-derived cardiomyocytes (instead ofhERG-expressing immortalised cells) more strongly predict clinical drugbehaviour, thereby improving decision making and reducing cost.

iPSC-derived cell models may actually decrease costs in screeningand cell therapy applications that require large-scale production. Whilecurrent manufacturing methods/scale can supply adequate dosages forPhase III and later trials for some disease indications, clinical trials for large tissue patches or whole organs will require greater scale and efficiency.

Gerber: I think collaborative effort is an important thing to rememberwith regard to this issue. When multiple partners collaborate to achievea singular goal, cost of investment is reduced and mitigation of risk ismaximised for each. Many of the entities that possess the acumen andresources to commercialise a therapy do not have direct access to rawmaterial supply chains and must rely on secondary providers. If strongpartnerships can be built between these two groups, the potential forreduced cost of goods may be more readily realised.

Genome editing in combination with stem cell biologyallows the scientist to create cell lines which possess theappropriate wild type or mutant gene on the same geneticbackground. Can you describe the implications of this inyour research and the opportunities provided by thisenabling technology?Jones: Gene editing at the iPSC stage enables researchers to study thenatural history of a disease in any cell type. By genetically engineeringiPSCs (line derived from an apparently healthy donor carrying a disease-associated allele and/or line derived from a disease carriermodified to wild-type allele), we can study the role that individualgenotypes play in determining disease and gauge the utility of genetherapy. iPSC genome editing also enables production of rare genotypesin different genetic backgrounds, yielding reagents from rare disorders.Lastly, gene editing can be used to produce isogenic controls for use indiscovery applications.

Gerber: It is clear that the most promising applications of genomeediting will be realised in the context of stem cell biology. The ability togenerate isogenic stem cells, create or reverse disease-relatedmutations, and then differentiate such cells into appropriate lineagesand somatic cells for focused analysis is a powerful indication that these two fields of research are converging. Indeed, manypharmaceutical companies have recognised the potential of thismarriage, and have partnered with genome editing service providers togenerate systems that will hopefully be more predictive models ofdisease and extending this marriage into cell-based therapies hasalready begun.

Runeberg: I agree: the combination of genome engineering andpluripotent stem cells offers infinite possibilities. It provides theopportunity to really ‘design’ cells. We can remove or introducemutations in the cells, or we can introduce fluorescent ‘beacons’ so wecan follow a specific cell type or a specific process in living cells, thusincreasing our knowledge around specific cellular processes. Moreover,if genome modification is performed in pluripotent stem cells, you canalways go back to the same starting material and avoid batch-to-batchvariations – this is in sharp contrast with adult stem cells where there’slimited cell life span and supply.

© L

eigh

Pra

ther

/ Sh

utte

rsto

ck.c

om

“The ISSCR is excited to bring its annualmeeting to Stockholm, a city that shares ourpassion and reputation for great scientificresearch and collaboration,” said ISSCRPresident Rudolf Jaenisch, Managing Director,Whitehead Institute for Biomedical Research.“We look forward to learning more about thestrong work being done in Sweden and across Europe.”

The meeting will open with the Presi -dential Symposium on 24 June from 13:15-15:15local time. The symposium sets the stage forthe meeting with world renowned speak-ers, including Nobel Prize winner ShinyaYamanaka. It is also the platform for theformal recognition of the 2015 recipients of the McEwen Award for Innovation and the ISSCR Public Service Award. Anotherprestigious award, the ISSCR-BD BiosciencesOutstanding Young Investigator Award, will bepresented during Plenary VI on 27 June from9:00-11:20 and followed by an award lecture.

“I look forward to the PresidentialSymposium setting the tone for the entireprogram,” Jaenisch said. “A thread throughoutwill be the use of stem cells to drive ourunderstanding of development and disease,as we explore disease modeling, gene andtissue engineering technologies and otherimportant advances that are bringing stemcells into the clinic.”

Presidential Symposium speakers will include:� Fred H. Gage, PhD, Salk Institute for

Biological Sciences, US� Jürgen Knoblich, PhD, Institute of

Molecular Biotechnology, Austria

� Shinya Yamanaka, Managing Director, PhD, Center for iPS Cell Research &Application, Japan

� Jeannie Lee, M.D., PhD, MassachusettsGeneral Hospital, US

The McEwen Award for Innovation award winners(Presidential Symposium):� Irving Weissman, Managing Director,

Stanford School of Medicine, US� Hans Clevers, Managing Director, PhD,

Hubrecht Institute, Netherlands

The ISSCR Public Service Awardwinner (Presidential Symposium):� Alan Trounson, Ph., MIMR-PHI Institute of

Medical Research, Australia

The ISSCR-BD BiosciencesOutstanding Young InvestigatorAward winner (Plenary VI):� Paul Tesar, PhD, Case Western Reserve

University School of Medicine, U.S.

“The ISSCR is thrilled to present theprestigious McEwen Award for Innovation toIrving Weissman and Hans Clevers, who haveeach made enormous contributions to stem cell science,” said Hans Schöler, PhD, Max Planck Institute for Molecular Bio -medicine, chair of the ISSCR’s McEwen Awardsselection committee. “Working in the bloodand gut systems, respectively, and extendingtheir findings in different tissues, they have defined the concepts and technologiesthat underpin many avenues of research. Each has made pioneering conceptual

advances in disease modeling and re -generative medicine.”

“We are privileged to present our ISSCR-BD Biosciences Outstanding YoungInvestigator Award to Paul Tesar,” Jaenischsaid. “He has leveraged his understanding ofmammalian development to create trans -formative stem cell-based technologies and toenable access to new areas of biology. Hisaccomplishments advance our understandingof human health and would be impressive atany stage, but they are remarkable forsomeone so early in his career.”

The 2015 Annual Meeting ProgramCommittee is chaired by Leonard Zon, M.D.,Harvard Medical School, Boston Children’sHospital. Zon and the committee have workedtogether over the past year to assemble adiverse program and an internationalcontingent of stem cell researchers, cliniciansand industry professionals to share thenewest research, technologies and clinicaladvancements.

“When the idea for the InternationalSociety for Stem Cell Research was born,nearly 13 years ago, I hoped the stem cell fieldwould have an exponential impact on clinicalmedicine,” Zon said. “This process has begun,and the field has a bright future as evidencedby this year’s ISSCR meeting program.”

“The basic biology of stem cells has led tobrilliant discoveries and served as afoundation for burgeoning therapies,” Zonsaid. “Attendees of ISSCR 2015 will see anincreasing clinical focus as the meeting movestoward Friday and Saturday, and we learn ofcutting edge approaches now entering the clinic.”

The International Society for Stem Cell Research’s 13th annual meeting will take place at the StockholmsmässanExhibition and Convention Center in Stockholm, Sweden. The meeting will bring together approximately 4,000 stemcell scientists, bioethicists, clinicians and industry professionals from over 50 countries to present and discuss thelatest discoveries and technologies within the field.

SHOW PREVIEW: ISSCR


Clevers, winner of the McEwen Award forInnovation, will present during the “DiseaseModeling” plenary, sharing research publishedin two recent papers in the scientific journalCell. The papers describe the development ofa culturing system for human liver stem cells,as well as stem cells from pancreatic cancer,discoveries with the potential to revolutioniseliver transplantation and aid in the fightagainst pancreatic cancer, respectively.

The “Therapy with Stem Cells” plenarysession includes a talk by Douglas Melton,PhD, Harvard University, who led recent workto produce human insulin-producing betacells in the massive quantities needed for cell transplantation and pharmaceuticalpurposes. This work represents a leap forwardin the quest to find a truly effective treatmentfor type 1 diabetes. Allan Robbins, PhD,Viacyte, will discuss the development of hiscompany’s stem cell-derived encapsulatedcell replacement therapy and the initiation oftrials to evaluate safety and efficacy.

Saturday opens with the “Stem Cells andImmunology” plenary. In this session, CarlJune, Managing Director, University ofPennsylvania School of Medicine, will presenthis group’s groundbreaking research,developing ways to enhance the body’s own immune system to recognise and killcancer cells.

The final plenary of the meeting, “MakingTissues and Organs,” will feature a talk byMasayo Takahashi, Managing Director, PhD,RIKEN Center for Developmental Biology.Takahashi is leading the first-ever clinical trialusing induced pluripotent stem cells (iPSCs).In 2014, she and her team at the RIKEN Centerfor Developmental Biology transplanted aretinal pigment epithelium (RPE) cell sheetderived from iPSCs into a patient sufferingfrom age-related macular degeneration toassess the safety of the transplantation ofiPSC-derived RPE sheets.

Concurrent topics include control andinduction of pluripotency, disease model-ing, epigenetics, tissue engineering and organ development, neural degeneration,stem cells and cancer, hematopoiesis andclinical translation.

Robert Langer, Scientific Director,Massachusetts Institute of Technology, aleader in the field of biotechnology and one ofthe most cited engineers in recent history, willdeliver the closing keynote address. Langerholds nearly 1,080 patents worldwide which have been sublicensed to over 250pharmaceutical, chemical, biotechnology andmedical device companies.

The ISSCR received over 2,000 abstractsfor the 2015 meeting, resulting in the additionof 20 new speaking slots. A total of 100

speakers will be identified from outstandingabstracts and will present their research in theconcurrent tracks. Three poster receptionsallow further opportunities for researchers topresent and discuss their work.

“The posters are always the genesis forsome of the meeting’s greatest scientificconversations and collaborations,” Zon said.“Year after year, we hear that networking withpeers is one of the most valuable componentsof the meeting.”

ISSCR 2015 registration dates and costs are as follows:� Advance registration deadline: 6 May (USD

840 member/USD 1,140 nonmember)� Regular registration deadline: 27 June

(USD 965 member/USD 1,265 nonmember)

This year’s public symposium, “Stem Cells andthe Ageing Brain,” will take place on 23 June,17:00-20:00 in the Aula Medica lecture hall atthe Karolinska Institute. The symposium isopen to the public and free of charge.

The ISSCR 2015 Annual Meeting is co-sponsored by the Wallenberg Institute forRegenerative Medicine.

SHOW PREVIEW: ISSCR


Date: 24 – 27 June 2015Location: Stockholm, SwedenMore information: http://www.isscr.org/

Cour

tesy

of O

la E

ricso

n m

edia

bank

.vis

itsto

ckho

lm.c

om

Your products. Our passion. For decades, our customers have trusted Charles River to listen and respond to the unique manufacturing challenges of bringing drugs to market safely and efficiently. The result is our extensive offering of microbial risk management solutions designed to fulfill the unmet demands of the industry. With innovations like our Endosafe® endotoxin testing, rapid bioburden detection, and Accugenix® microbial identifications, we continue to offer vanguard technologies that help ensure your facility’s state of control. Learn more about our micro QC solutions at www.criver.com/emd.

A PASSION FOR INNOVATION I www.criver.com

A P A S S I O NF O R I N N O V A

T I O N

Rapid Endotoxin TestingMicrobial Identification and Strain TypingMicrobial Bioburden Detection

© a

nyai

vano

va /

Shut

ters

tock

.com

Microorganisms are omnipresent in the environment, making sterilemanufacturing plants, especially those with aseptic filling operations,highly vulnerable to microbial contamination. There are a variety ofpotential root causes of non-sterile results. These may includeweaknesses of design, insufficient maintenance of the production plantor poor aseptic working practices of the cleanroom operators, to namebut a few. Also, it is often unclear as to whether a lack of sterility is dueto a systematic weakness of the process or if it has been caused by theoften-cited ‘single incidence’ resulting from an individual human error.As the non-homogenous distribution of contaminants within the

product gives rise to low reproducibility when testing, it makes thesearch for an assignable cause even more complex and hides the impact it may have.

Since only a small number of random samples are examined forsterility testing, the statistical significance of the results is hardlysufficient to draw conclusions about the sterility of the whole batch. Thus,neglecting appropriate precautions would ultimately create a very highrisk for the patient. Therefore, it is imperative to understand all possiblemicrobial entry points and to implement quality risk management (QRM) strategies that aim to prevent microbial contamination.

Several years ago, microbiologist Guenther Gapp created a new sterile risk assessment tool (based on a hazardoperability analysis [HAZOP] approach) to identify and reduce the microbial contamination and compliance risk ofaseptically-produced sterile products and production plants. The following article describes the operating principleof three risk analysis tools with a special focus on the revised 2015 edition. This latest edition incorporates the improvements that have been implemented in recent years, which make the tool more applicable and thusvaluable for the user.

MICROBIOLOGY SERIES


Guenther GappLachman Consulting Ltd/Independent Consultant

Reducing microbialcontamination via sterilerisk assessment

The European Pharmaceutical Review's Microbiology Series isbrought to you in association with Charles River Laboratories

It is highly recommended that a systematic approach be followedboth when developing a proactive microbial control strategy and whenperforming an investigation of a microbial contamination deviation.Accordingly, each sterile manufacturing production site is required tohave a risk analysis tool in use.1 It allows for a comprehensive riskassessment that uncovers potentially hazardous process steps andlacking controls, and provides information about potential microbialcontamination root causes.2 Thus, in sterile production, risk manage -ment and the performance of risk analysis take on a very importantrole, serving both as investigative tools in case of deviations (for rootcause analysis and batch disposition decisions) and as proactive tools for preventing non-compliance during regulatory audits and of thefinal product.

These tools, such as the ones described below, incorporate aprofound understanding of the manufacturing process and productattributes. They are simple, usable and have been relied on for several years.

Managing risk effectively using process-specific risk analysis toolsStarting nearly a decade ago, the author began compiling first-handinformation on technical experience and knowledge derived fromeveryday practice in the field of quality assurance (QA)/quality control(QC) microbiology, sterile production and aseptic processing andgenerating from this a register of questions. By further incorporatingquestions relating to regulatory audit requirements and findings and then ranking all questions according to their criticality, thequestionnaire evolved into a new risk assessment tool based on a

HAZOP methodology. Over many years this tool has been continuouslyadapted, refined and extended. Meanwhile, three specific sterileproduct compliance risk analysis tools have been successfullyemployed in numerous sterile active product ingredient (API) andfinished dosage form (FDF) plants worldwide.

To reflect the variable contamination risk inherent in the differenttypes of production plants, the production steps are classified intoindividual units according to their process flow. For example, an APIplant is a complex multi-step processing system with relation totechnology, associated aseptic operations and control mechanisms.Consequently, the risk analysis tool for a sterile API plant consists of fiveunits (Figure 1). On the other hand, a sterile FDF filling line for liquid orsolid product filling with or without sterile filtration (SF) incorporatesfewer production steps. Hence, the risk analysis tools for sterile FDFplants are made up of either two (FDF without SF) or four (FDF with SF)units, depending on the particular manufacturing process employed, asillustrated in Figure 1.

For each unit, a multitude of specific questions are asked,encompassing all areas of risk involved in aseptic processing (for example, raw material, cleanroom operator qualification and training, QA/QC microbiological controls, packaging, etc.). Each question can be answered on a scale from 1 (excellent) to 5 (very poor or missing) (Figure 2).

The sum of all answered questions from one unit is then averagedto give the Unit Average Risk Factor. The smaller the Unit Average RiskFactor, the lower the evaluated risk to the production plant with regardto the quality of its sterile product. To provide instant visual informationabout overall risk and potential problem areas, all answers andcalculated results are colour-coded from green to red. A risk analysistarget and optimum situation would ultimately be low numbers andgreen colours.

In addition to tailoring each risk analysis tool to fit the underlyingprocess by using distinct units, it is also necessary to weigh these unitsaccording to the impact they have on the overall sterility. To correctlyaccount for these differences, a unit-specific multiplication factor isintroduced, termed Risk Emphasis Factor (REF). The unit REF can takeon the value 1 (low), 3 (medium) or 5 (high), depending on the inherentcontamination risk and the standard of equipment of the respectiveunit. For example the ‘Raw Material Unit’, which bears a low risk forcontamination, is assigned REF 1. Production units that utilise


MICROBIOLOGY SERIES

Figure 1: Schematic overview of process units and related risk emphasis factors (REFs)

Figure 2: Example of the questionnaire showing real-life data

Figure 3: Schematic overview of the sterile product compliance risk analysis tool foran FDF plant with SF combined with real-life data

Microsart®@media

Innovation in Microbial Enumeration

www.sartorius.com/microsart

significant pressure differences, such as vacuum driers orlyophilisers, are considered especially risky. Similarly, primarypackaging materials, such as aluminium cans, harbour an inherentvulnerability for microbial contamination. Therefore, ‘APIProduction Unit’ and ‘API Packaging/Transportation Unit’ arealways assigned the maximum value of REF 5. In contrast, smallpackaging sizes, such as small vials used in FDF filling lines are lessprone to pressure-drop related contamination, thus the allocatedREF is 3 (Figure 1, page 36).

Each Unit Average Risk Factor is multiplied by itscorresponding unit REF to achieve the Unit Risk Factor as in thesimple equation below:

Unit Risk Factor = Unit Average Risk Factor x Unit REF

The QRM analysis is finally concluded by calculating the Total RiskFactor (TRF), which is the sum of all Unit Risk Factors (Figure 3,page 36):

TRF = ∑ Unit Risk Factors

The TRF provides definitive information about the overall risk of microbial contamination (sterility/endotoxins) for allproduction steps of an aseptic processing operation. Further -more, it allows the user to estimate the compliance status, as well as possible observations of future regulatory audits of thesterile plant. By providing simple numerical and colour-codedanswers in each unit questionnaire, as well as by incorporating aunit REF, the risk analysis tool additionally serves to uncoverpotential weaknesses of the process and enables proactivecorrective and preventive actions (CAPAs) for further systematicimprovement. Any uncovered weaknesses provide solidarguments for investments in improving the compliance status and, finally, the TRF provides managers with a conclusivescore for benchmarking of sterile production plant performance(Figure 3, page 36)

Amendments, updates and real-life examplesVariable Unit REFsIn the previous guidance, each unit was assigned a single specificREF (low/medium/high) according to the inherent risk for non-compliance and/or microbial contamination. However, tocorrectly represent lower contamination risks associated withusing advanced aseptic technologies and to create pressure forinvestments, a variable unit REF was introduced. For example, the‘Aseptic Filling Unit’ was initially assigned a REF of 3 (medium),3 butnow has a variable REF according to the standard of equipmentand filling technology used. A variable REF thus ensures that anadvanced filling line is rewarded a significantly lower TRF than aconventional filling line, even if the respective individual UnitAverage Risk Factors were identical.

Knock-out questionsIn the initial tool, a five-point scale was applied to all answers in the questionnaire. After some years of practical

VOLUME 20 ISSUE 2 2015 European Pharmaceutical Review

MICROBIOLOGY SERIES

experience it became apparent that certain questions had adisproportionately high impact on overall sterility assurance compared to others and that they represented deficiencies that couldlead to product non-sterility or a US Food and Drug Administration form483 finding. To ensure that a negative answer in these cases isappropriately penalised, a value of 100 is assigned and these questionsare thus referred to as ‘knock-out questions’ (KO-questions) (see Example 1).

Example 1:Q401: Is the sterilisation of primary packaging material validated(autoclave/dry heat/gamma-irradiation) (1) or not (100).

As described above, assigning a value of 100 upon negatively answering this question prevents a severe risk factor being dilutedwhen averaging all unit questions. Although the impact of KO-questionson the Unit Risk Factor score is greater for units comprising fewer questions, a negative answer will in any case push the TRF fromLOW to at least MODERATE, which should be considered unacceptablefor a production plant.

Ultimately, the purpose of KO-questions is to illuminate criticalparameters that allow effective measures to be set and to get the seniormanagement’s attention if resources or investments are required forremediation. Typically, in a well-controlled sterile manufacturing plant anegative answer to a KO-question does not, however, necessitate thatthe plant is shut down.

Latest release (2015)Three different risk assessment tools are available (refer to Figure 1,page 36) in the most recent 2015 release:� Sterile API plant: Units 1/2/3/4/5 (238 questions)� Sterile FDF plant (with SF): Units 1/2/4/5 (203 questions)� Sterile FDF plant (without SF): Units 4/5 (175 questions)

36 knock-out questions have been defined in total (5 units): � Raw material: none� Sterile filtration: 4 KO-questions � Sterile production: 6 KO-questions� Aseptic filling: 23 KO-questions� Packaging/ transportation: 3 KO-questions

Illustrative example The following example demonstrates the operating principle of theproduct compliance risk analysis tool for a FDF filling plant with SFconsisting of four units (raw material/SF/aseptic filling/packaging)(refer to Figure 2, page 36 and Figure 4). Figure 2 (page 36) shows partof the questionnaire for the ‘Aseptic Filling Unit’, where some of theanswers have uncovered specific weaknesses in the system, asillustrated by the score 5 in red, leading to an Unit Average Risk Factorof 1.9 (risk is still LOW). The bar chart in Figure 4 shows all four UnitAverage Risk Factors, whereof only one (SF) displays a MODERATE risk(yellow colour). The summary table (bottom left corner in Figure 4)shows that following multiplication with the respective unit REFs resultsin a TRF that exhibits a MODERATE risk for product non-sterility andregulatory non-compliance.

SummaryHAZOP approaches are effective and useful measures for reducingmicrobiological contamination risks and for complying with regulatoryrequirements in the pharmaceutical industry to assure safety for thepatient. The sterile product compliance risk analysis tools describedhere are characterised by simplicity, usability and the option to add newquestions based on recent experiences, audit findings and newlypublished guidelines, which, taken together, ensure a high level offunctionality and performance. By enabling a comprehensive and in-depth assessment it serves at a senior management level to helpcontrol the GMP compliance status and to monitor the risk of non-complying products, whilst at an executive management level itprovides solid arguments for investments. However, the successful useof the tool requires both expertise and honesty in answering thequestions, which necessitates a competent and highly professionalmultidisciplinary risk assessment team. If these requirements arelacking, or if there is not sufficient time available to dig deeper, thebenefit and outcome will certainly be poor.

MICROBIOLOGY SERIES


1. US Food and Drug Administration Department of Health and Human Services:

Pharmaceutical cGMPS for the 21st Century – A Risk Based Approach. Final Report –

Fall 2004 [Internet]. 2004 Sept. Available from: http://www.fda.gov/Drugs/Development

ApprovalProcess/Manufacturing/QuestionsandAnswersonCurrentGoodManufacturing

PracticescGMPforDrugs/ucm137175.htm

2. Gapp G. How to deal with non-sterile results in aseptic processing. European

Pharmaceutical Review. 2014;19(3):33-36.

3. Gapp G., Holzknecht P. Risk analysis of sterile production plants: a new and simple,

workable approach. PDA J Pharm Sci Technol. 2011 May-Jun;65(3):217-26.

References

In 2013 microbiologist Dr. Guenther Gapp founded the

consulting company Gapp Quality GmbH (Austria). Today

he divides his time between working as a Senior Associate

for Lachman Consulting Ltd. and as an independent

consultant. Guenther spent the first 20 years of his career at

Biochemie Kundl (Sandoz GmbH), Austria, where he

became Head of QA/QC Microbiology. He gained in-depth

experience in media fill practices of finished dosage forms and bulk

products, environmental monitoring, rapid testing methods, aseptic

operations practices and regulatory agency audits. He has been a subject

matter expert in more than 20 FDA audits. Several years ago he created a

new sterile risk assessment tool (based on a HAZOP approach) to identify

and reduce the microbial contamination and compliance risk of sterile

products and production plants and subsequently won the 2011 PDA Journal

of Pharmaceutical Science and Technology Award. His risk assessment tool

has been used worldwide by sterile manufacturing plants.

Figure 4: Example showing actual data of a risk assessment outcome in an FDF plant(with SF)-5

The new technologies to be developed are optical spectroscopycounting instruments designed to simultaneously detect the numberand size of particles from a volume of air (and thus enable ‘real-time’microbiological assessment.) Such systems operate in a similar way toconventional particle counters, which measure inert particles as part ofthe GMP assessment of cleanrooms (and comply with such cleanroomstandards as ISO 14644). Here the instruments count airborne particlesby measuring light scattering. The difference with the spectro -photometric counters over traditional counters is that the instrumentsare capable of determining if the detected particles are biological orinert. Thus the key innovation is that such devices can differentiatebetween non-viable particles and biological material, which mayindicate if microorganisms are present in the sample of air.

The implementation of such technologies is in keeping with theindustry and regulatory drive, particularly from the US Food and DrugAdministration1, for rapid methods and for pharmaceuticalmanufacturers to utilise the concept of process analytical technology(PAT). PAT is a set of mechanisms to design, analyse, and controlpharmaceutical manufacturing processes through the measurement ofcritical process parameters. With the PAT philosophy the ideal is tomake such assessments in ‘real-time’, so that quality issues can beaddressed during batch manufacture.

In terms of advantages to microbiologists and cleanroommanagers, the new generation of optical counting instruments will notonly provide a real time assessment of the probable numbers ofmicroorganisms in the air but will also detect microorganisms that

The methods for monitoring air in cleanroom environments: viable counting techniques (settle plates and biologicalair samplers) and particle counters, are long established technologies and have been widely used in pharmaceuticalmanufacturing environments for decades. Although innovations have taken place with both particle counters andbiological air samplers, primarily in relation to the size of the instruments and in reducing the time taken to collecta fixed volume of air, the essential technology has remained unchanged. However, recently, some new technologiesfor biological air sampling have been developed (bio-air systems), which will be discussed in this article.

ENVIRONMENTAL MONITORING


© T

atia

na S

hepe

leva

/ Sh

utte

rsto

ck.c

om

Real-time biologicalparticle counting inenvironmental monitoring

Tim SandleBio Products Laboratory

cannot be recovered using conventional culture-based microbiological methods. The reason whyconventional cultural-based environmentalmonitoring methods are limited is multifaceted. Onereason is because only small samples sizes are taken.A second reason is that there is no universal culturemedium or set of incubation conditions which willgrow all microorganisms2. Moreover, there are many‘viable but non-culturable microorganisms’3 found incleanrooms. The term ‘viable but non-culturablemicroorganisms’ describes bacterial cells which,whilst maintaining certain features of viable cells(measurable metabolic activity), will not culture onagar4. Theoretically real-time technologies will beable to detect these non-culturable microorganisms.A further factor is that drawing a correlation betweennon-viable and viable particles from a given volumeof air is problematic5.

Bio-air systems and their applicationThere are different bio-air systems on the market.They tend to function in similar ways, although thereare commercial differences. They are relativelyimmature technologies, having been commerciallyavailable for the past five years, and the application ofthe measurement of biologic particle concentrationsusing a fluorescent particle is relatively novel6.

The different systems are similar in terms of theirdesign and biophotometry technology6. In terms of design, they areinstruments housed in stainless boxes and the air is drawn in throughan isokinetic probe powered by an air pump. In terms of the technology,the core part of the systems consists of an optical laser. When a particlepasses through the laser it intersects the laser. A particle size detectoris then used to measure individual particle sizes (similar to establishedparticle counters, based on the Lorenz–Mie solution which formulatesthe scattering of electro magnetic radiation by a particle. Here theamount of light scattered can be used to calculate the size of the particle)7. In addition, there is a paralleloptical assembly (photo detector) to detect anultra violet laser-induced fluorescence signal,which is triggered by certain metabolites insidemicroorganisms. The collected data is analysedby computer software which is able to differ -entiate between inert particles and microorganisms.

The viable counting technology of the instruments functions on thebasis that microorganisms contain metabolites. Certain metabolites are‘excited’ by a specific wavelength of light. The metabolites looked forare the co-enzyme nicotinamide adenine dinucleotide (NADH),dipicolinic acid and the micronutrient riboflavin (vitamin B2). These metabolites are found within all cells. The excitation of thesemetabolites, using ultra violet photon energy (at a wavelength of405nm), causes the emission of auto-fluorescence light, which theinstrument then detects. Fluorescence detection is well-suited fordetection of microbial contamination as it has a high sensitivity andrequires only a short collection time. The design of the instruments

allows them to scan large volumes and to express results per cubicmetre of air. The systems use different preparatory units ofmeasurement, although they are similar in that each unit of measure -ment is said to be equivalent to one colony forming unit (as would be recorded from a conventional culture based environmentalmonitoring method)8.

The instruments are being marketed for different applications. One such use is for aseptic filling operations with the idea thatthose involved with sterile filling will be able to detect not only

particles, but also biological activity. This would allow cleanroom operators to stop theprocess if microorganisms are suspected to bepresent in the air. Action can then be taken to address the contamination or to segre-gate a portion of the batch. The ability to

instantaneously detect microorganisms is a significant advance asconventional methods require several days to culture bacteria andfungi. In such scenarios, the systems can be used for continuous orepisodic monitoring.

Another application is using the instrumentation to set a benchmarkand then taking measurements in the event of an incident to determineif a suitable recovery has occurred, for example, following an air handlingsystem failure. A further use is collecting data about cleanrooms, such asassessing the maximum number of personnel allowed within acleanroom with a given air-exchange rate9. Another advantage is theability to trend and the possibility of correlating airborne particles withmicroorganisms. Indeed the technology may possibly be able to show



The reason why conventionalcultural-based environmental monitoringmethods are limited is multifaceted

© M

icha

elTa

ylor

3d /

Shut

ters

tock

.com

Our IMD-A® systems monitor controlled areas in real time, detecting microbes instantaneously so you can act. Our systems utilize advanced optics and signal processing, requiring no staining, no reagents, and little human intervention. Let us help shed light on what may be lurking in your environment.

Our Instantaneous Microbial Detection™ Solutions Can Help Shed Light.

that a rise in concentration of ≥0.5μm particles above a certain thresholdwill provide an early warning of a rise in microorganisms.

Points for considerationThere are, however, some important points to consider with regard tothe future application of the technology.

The technologies require considerable validation and methoddevelopment before they can be used for batch release. As with particlecounters, the validation relies upon the instrument being able tocalculate and recover known particles. Criteria such as precision,robustness and limit of detection should eachbe assessed. The systems must also be able todistinguish between inert particles and viableparticles and not react to any other substancewhich might trigger fluorescence. One studyshowed a vulnerability, in terms of reacting todisinfectant sprays and clothing fibres10.

Moreover, it is important and indeedessential with viable microorganisms that the limit of detection andlimit of quantification is assessed, since regulatory standards requiremonitoring systems to be able to detect one colony forming unit in acubic metre of air.

A more difficult aspect is the method development. Since the levelof microorganisms present in cleanroom air but not detected byconventional methods cannot be quantified, direct parallels cannot bemade. Furthermore, suppose within a Grade A/ISO class 5 clean zonethere is one microorganism and a real-time counting device is runalongside a settle plate. The single organism may be captured by the

real-time device or it may fall onto the settle plate. It cannot bedetected in both and in such a situation there is no directcomparability. Comparative analysis can be undertaken using abioaerosol chamber and a single, mixed population of microorganisms.However, the problem of assessing the instrument in situ remains.

The next important point is that real-time technology is adestructive method. It will indicate that a microorganism is present butit will not determine the species. Although some would argue that withsterile manufacturing the species is less relevant because anymicroorganism presents a risk, knowing the species allows the origin

of the contamination to be assessed (aStaphylococus spp., for example, indicates anassociation with human skin) and forcomparisons to be made with differentenvironments (if the same microorganism isrecovered at Grade A/ISO class 5 as is recoveredat Grade B/ISO class 7, then they may be aconnection). This means that conventional

methods would be required to be run alongside real-time technology.A final consideration rests on the logistics of using real-time. If a

suspected microbial event occurs, what is the appropriate response? Isit to suspend filling? What happens if further events occur on re-start:is this batch rejection? If no further events are detected is a portion ofa batch to be rejected? Or is there, as with conventional methods, nodirect link between detection of contamination and product risk?Furthermore, what is the appropriate response if conventional methodsdetect microorganisms and real-time methods do not, or vice versa? Finally, what does this mean for established alert and action


Fluorescence detection is well-suited for detection of microbial

contamination as it has a high sensitivity and requires only a

short collection time

Network online with your industry peers, visit:

http://linkd.in/PharmaReviewMembers and non-members are welcome to join the discussions

LET’S CONNECT!

Exchange information, ideas and opportunities

levels? These questions pose dilemmas for the pharmaceuticalmanufacturer to resolve.

If these points may appear to dampen the potential of suchtechnologies, they are not intended to do so. Nonetheless, these arequestions which require unravelling as real-time technologies becomeestablished. There is little doubt, not least given the considerabledevelopment costs, that these systems will become acceptedmethodologies.

SummaryReal-time instantaneous monitoring systems, capable of detecting anddifferentiating between non-viable and viable particles in the air, areprogressing and several systems are being trialled in pharmaceuticalorganisations. Such systems offer advantages in providing moredetailed information about the status of clean zones and canpotentially act as useful investigational tools. In the longer-term thetechnology has the potential to replace conventional methods entirelyonce some of the points posed in this paper are resolved. The mostsignificant of these is the speciation question: how can microbiologistsreliably determine the species of microorganism and relate this to apossible source or location?

These issues notwithstanding at some future point real-timemonitoring systems will probably become part of the acceptedmethods used for assessing non-viable and viable particulates withinpharmaceutical grade cleanrooms.


1. FDA (2004). Guidance for industry: PAT – A framework for innovative pharmaceutical

development, manufacturing and quality assurance”. Food and Drug Administration,

Rockville: MD, USA.

2. Sandle, T. (2014) Examination of the Order of Incubation for the Recovery of Bacteria

and Fungi from Pharmaceutical Cleanrooms, International Journal of Pharmaceutical

Compounding, 18 (3): 242 – 247

3. Weichart, D. (1999). "Stability and survival of VBNC cells – conceptual and practical

implications". In Bell, C.B-G. (Ed.), Proceedings of the 8th Symposium on Microbial

Ecology, Atlantic Canada Society for Microbial Ecology, Halifax: Canada.

4. Tidswell, E. (2011). "Sterility". In Saghee, M. Sandle, T. and Tidswell, E. Microbiology

and Sterility Assurance in Pharmaceuticals and Medical Devices, Business Horizons,

New Delhi: India.

5. Raval JS, Koch E, Donnenberg AD. (2012) Real-time monitoring of non-viable airborne

particles correlates with airborne colonies and represents an acceptable surrogate for daily

assessment of cell-processing cleanroom performance, Cytotherapy. 14(9):1144-50

6. Dai C, Zhang Y, Ma X, Yin M, Zheng H, Gu X, Xie S, Jia H, Zhang L, Zhang W (2015)

Real-time measurements of airborne biologic particles using fluorescent particle counter

to evaluate microbial contamination: Results of a comparative study in an operating

theatre, Am J Infect Control. 43(1):78-81

7. Miller, M. J.; Lindsay, H.; Valverde-Ventura, R.; O’Conner, M. J. Evaluation of the

BioVigilant IMD-A, a novel optical spectroscopy technology for the continuous and real-

time environmental monitoring of viable and nonviable particles. Part I. Review of the

technology and comparative studies with conventional methods. PDA J. Pharm. Sci.

Technol. 2009, Vol. 63, No. 3, pp244 –257

8. Sandle, T.; Leavy, C.; Jindal, H.; Rhodes, R. Application of Rapid Microbiological

Methods for the Risk Assessment of Controlled Biopharmaceutical Environments.

Journal of Applied Microbiology. 116(6): 1495-1505

9. Sandle, T., Leavy, C. and Rhodes, R. (2015) Assessing airborne contamination using a

novel rapid microbiological method, European Journal of Parenteral & Pharmaceutical

Sciences, 19(4): 131-142

10. Eaton, T.; Wardle, C.; Whyte, W. Use of a Real-Time Microbial Air Sampler for

Operational Cleanroom Monitoring. PDA Journal of Pharmaceutical Science and

Technology. 68(2): 172-184

References

Dr. Tim Sandle’s primary role is Head of Microbiology at

Bio Products Laboratory, a sterile products manufacturer.

In addition, he is a tutor with the School of Pharmacy and

Pharmaceutical Sciences, University of Manchester, for the

university’s pharmaceutical microbiology MSc course and a

longstanding committee member of the pharmaceutical

microbiology society Pharmig.


45 HPLC mass spectral analysis of glycans David Elder, Phil Borman and George Okafo, GlaxoSmithKline, and William McDowell, PolyTherics Ltd

53 Analysis of limited andcomplex samples with nanoLC-MS Hanne Røberg-Larsen, University of Oslo

58 LC-MS Roundtable Moderated by David Elder, GlaxoSmithKline

LC-MS

© S

oul w

ind1

23 /

Shut

ters

tock

.com SPONSORS

The structural complexity of glycans is far greater than that of eitherproteins or nucleic acids and this in turn allows them to separatelyencode molecular information vital for molecular recognition, and todefine protein folding (defines secondary or tertiary structure), stabilityand the pharmacokinetic/pharmacodynamic properties of the intactprotein. Incorrect folding can lead to serious clinical consequences.Therefore, the fingerprinting and sequencing of oligosaccharides arehugely important. As such, chromatographic (size exclusion (SEC),supercritical fluid chromatography (SFC), ion exchange chromatography(IEX), high performance liquid chromatography (HPLC) and capillaryelectrophoresis (CE)) separation methods are very important. However,accurate structural insight is highly challenging when just separationmethods (with well characterised standards) are used. Thus, HPLC (or

CE) is often linked with nuclear magnetic resonance (NMR), massspectroscopy (MS) detectors or the output used in sequencing studies1.However, the analysis of carbohydrates has always been amongst themost challenging areas of analytical chemistry, which arises from the many isomeric forms, as a result of the many possible configura -tions of the monosaccharides in the resulting oligosaccharides2.

Chromatographic approaches and trendsHistorically, gas chromatography-mass spectroscopy (GC-MS) usingpermethylated alditol acetate derivatives was the standard approach.Similarly, normal phase chromatography of 2-aminobenzamidederivatives of oligosaccharides coupled with exo-glycosidase digestioncould be used to elucidate structure in combination with matrix-

The terms glycan and oligosaccharide are defined by the International Union of Pure and Applied Chemistry (IUPAC)as ‘compounds consisting of a large number of monosaccharides linked glycosidically’. However, in common usagethe term glycan has come to mean the carbohydrate section of a glycoconjugate, for example, a glycoprotein,glycolipid, or a proteoglycan, even if the carbohydrate is only an oligosaccharide. Glycans usually consist solely ofO-glycosidic linkages of monosaccharides. For example, cellulose is a glycan composed of β-1,4-linked D-glucose,whereas chitin is a glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can be either homo orheteropolymers of monosaccharide residues, and structurally they can be linear or branched in nature. Glycans canbe further characterised as either N-linked (connected to the conjugate via a nitrogen atom in the sidechain of anamino acid like asparagine) or O-linked (where the bond is via the oxygen atom in serine or threonine residues).

IN-DEPTH FOCUS: LC-MS


© m

olek

uul.b

e / S

hutt

erst

ock.

com

HPLC mass spectralanalysis of glycans

David Elder, Phil Borman and George Okafo GSK � William McDowell PolyTherics Ltd

The Fused-Core® Advantage for Glycan Separations

BIOshell™ Glycan HPLC Columns are

specifically engineered to deliver fast, high

resolution, reproducible glycan identification

using HILIC chromatography. The Fused-Core

columns provide improved resolution when

compared to conventional LC methods,

thus allowing more reliable quantitation and

confident identification of glycans.

To learn more, visit

sigma-aldrich.com/bioshell12294

1045

©2015 Sigma-Aldrich Co. LLC. All rights reserved. SAFC, SIGMA-ALDRICH and

SUPELCO are trademarks of Sigma-Aldrich Co. LLC, registered in the US and other

countries. Ascentis is a registered trademark of Sigma-Aldrich Co. LLC. Solutions

within and BIOshell are trademarks of Sigma-Aldrich Co. LLC. Fused-Core is a

registered trademark of Advanced Materials Technology, Inc.

Empowering the Analysis of Glycoproteins

with Exceptional Reproducibility

assisted laser desorption/ionisation (MALDI-MS) to confirm theassignments by mass. HPLC-MS typically utilised reverse phasechromatography using five micron stationary phases.

Although this approach is still prevalent, there is a trend towardssmaller particle size allied with the use of ultra-high pressure liquidchromatography (UPLC or UHPLC) that affords better specificity,decreased analysis times and improved electrospray ionisation (ESI)sensitivity3. In addition, to lower particle sizes, the column technology isalso improving4. HPLC-MS using porous graphitic columns has beenreported to yield rapid and detailed structural assignments ofimmunoglobulin oligosaccharaides5. Gradient elution has become themost typical approach for high throughput HPLC-MS. There is also agreater usage of other separation modes, i.e., monolithic phases,hydrophilic interaction liquid chromatography (HILIC) and super criticalfluid chromatography.

HILIC is a reverse/reverse phase or an aqueous version of normalphase chromatography, and is much superior to reverse phasechromatography for highly polar molecules. Operationally, it is muchsimpler than the standard normal phase chromatography when used inconjunction with MS. As HILIC typically uses high volumes of organicsolvents in the mobile phase, increased HPLC-MS sensitivity is affordedbecause of the enhanced desolvation properties. A recent Waters®application note6 describes the rapid preparation of released N-glycansfor HILIC analysis using a novel fluorescence and MS-active labellingreagent. According to the note, a N-hydroxysuccinimidy (NHS)carbamate ‘tagging’ group coupled to a quinolinylfluorophore and atertiary amine charge tag, gives HPLC-MS analysis which is 1000x moresensitive than standard labelled oligosaccharides.

As quantitative HPLC-MS often requires both high throughput andenhanced sensitivity, separations that are more efficient are preferable.Therefore, monolithic stationary phases and ballistic gradients (thesetypically refer to fast or ultra-fast gradients (often with gradient times of0.5 to five minutes), which use large ranges of organic modifier (often 5-95% v/v or 0-100% v/v), narrow 2.1 mm ID columns, and relatively highflow rates for that diameter (1.0 mL/min), with short column lengths,are now widely used7,8. Superficially porous columns afford improvedpeak capacity using standard HPLC operating conditions9.

Detection approachesThe vast majority of all HPLC-MS analyses utilise two different ionisationmodes; either electrospray ionisation (ESI) or atmospheric pressure


chemical ionisation (APCI). However, APCI has less utility for largermolecules such as glycans, due to the requirement for analytevolatilisation prior to ionisation10.

The great utility of ESI is due to the robustness afforded byoperating ionisation at standard atmospheric pressures. Thus thesample introduction/desolvation of the analyte occurs in anatmospheric chamber, separated from thestandard low pressure regions via an aperture(pin hole or capillary). In the ESI mode, thespray is generated using a needle-like sprayunit which is then charged by applying highelectrical fields of different polarity (eitherpositive or negative). The resulting ions retain the same charge as theapplied field (positive or negative ions). Rayleigh Dissociation thenoccurs, i.e., as the droplets shrink via evaporation, the excess chargedestabilises them and they shrink into smaller charged droplets. Hence,gas phase ions are generated by ion desorption. For larger molecules,such as glycans, there is believed to be a second, competitivemechanism, whereby gas phase ions are produced via completedesolvation (charged residue model). In summary, the popularity of ESI

is attributable to the ease of interfacing with the HPLC system, the widerange of analytes that can be ionised (both small and large bio-molecules) and of course the high sensitivity.

There are several different type of mass detector available to theanalyst. Choice depends on several factors: mass resolution, massrange, scan speed, duty cycle, cost and ease of use. The various

types and their pros and cons are summarisedin Table 1.

However, it is sometimes erroneouslyassumed that mass spectral (MS) analysis with allof its myriad variants can overcome any and all problems that an analyte will present. This

is simply not the case. For instance, with glycans, intact oligo saccharidesare poorly ionised by soft MS ionisation methods, for example, ESI, fast atom bombardment (FAB) or MALDI. This is because the oligo -saccharides are very polar, relatively non-volatile and thermally labile.

Derivatisation approachesDerivatisation can occur pre- or post-column, but is necessary prior to MS analysis11. Derivatisation is appropriate for any functional moiety, numerous different classes of analytes and to a broad range of instrumentation. Thus it can both enhance sensitivity and improve specificity. For example, reagents with the 5-(dimethylamino)naphthalene-1-sulfonyl (aka) moiety, which inducesfluorescence via the naphthalene group, are also suited for highsensitivity HPLC-MS analyses and can be used as specific derivatisingagents for many different functional groups. DANSYL-hydrazine reactswith carbonyls, whereas DANSYL-Cl reacts with amino and hydroxylgroups. In addition, the tert. amine moiety within the DANSYL derivativeis ionised at acidic pHs, thereby allowing the derivative to enter the ionsource of the MS already ionised. The increase in sensitivity can bethree orders of magnitude12.

Whilst improvements in specificity and sensitivity are important,derivatives also introduce lipophilicity into the analyte, making themeasier to isolate. This is particularly important for sugars which areinvariably lipophobic. It is therefore an important adjunct for detection,even with high-end MS detection12. Several variables need to beoptimised to deliver the best derivative for subsequent analysis andidentification; these include molecular size of the oligosaccharide, thesolubility of the oligosaccharide, the derivative in the solvent of choice,


Table 1: The different types of mass detector and their pros and cons

MS Type Pros Cons

Single Quadrapole Most common, durable and Limited resolution and cost effective detector less sensitive for full

scan applications

Triple Quadrapole Very high selectivity using Cost and ease of useSRM (selected reactionmonitoring) make thisdetector useful for traceanalysis of complexbiomolecules

Ion Trap Full scan sensitivity with the Cost and ease of useoption to perform tandem MS(i.e., MS-MS). Linear 2D iontraps have resulted in highersensitivity

High End Ion Trap High mass resolution, with a Cost and ease of usehigh duty cycle. Orbitrap trapsions in an electrostatic field andis less expensive than FT-MS.

Time of Flight High mass resolution allowing Cost and ease of usefor exact mass determination,very high acquisition rates (50µs/spectrum), Q-ToF variant is very useful for structural elucidation in complex mixtures

The structural complexity of glycans is far greater than that of either

proteins or nucleic acids


© m

olek

uul.b

e / S

hutt

erst

ock.

com

PHARMACEUTICAL • HEALTH SCIENCES • FOOD & ENVIRONMENTAL • CHEMICAL MATERIALS

T H E

P R E S S E S O N .

The ACQUITY QDa® Detector from Waters. SEPARATING BEYOND QUESTION.™ Imagine a lab where

all analytical chemists can acquire high-quality mass spectral data. On their own. Within their existing

workflow. Across every sample. A lab where uncertainty about compounds is replaced by fast,

efficient confirmation and the confidence that comes with crossing the LC/MS divide like never before.

Now imagine all of this happening at the push of a button. This is the ACQUITY QDa Detector.

To turn on the future, visit waters.com/separate

©2015 Waters Corporation. Waters, ACQUITY QDa and The Science of What’s Possible are registered trademarks of Waters Corporation. Separating Beyond Question is a trademark of Waters Corporation.

Sitting proudly and powerfully on top of your instrument

stack, this 14” X 26” X 8” small wonder ushers in a new

era of separation science.

Gain confidence in peptide, glycan and

oligonucleotide analysis with mass detection.

the desired hydrophobicity and molecular weight of the resultantderivative and the need to introduce basic groups for optimal MS analysis.

There are several reported approaches for oligosaccharides. Table 2summarises typical chemical reactions for the common glycanderivatisation methods.

Schiff base A Schiff base is formed between the amino function of thederivatisation agent and the reducing terminal of the carbohydrate, i.e.,carbonyl group of the reducing sugar, in its open chain form. Although,these derivatives are not very stable, they have still found commonusage in carbohydrate chemistry. Caesar et al.13 used the pentafluroderivative of 4-aminobenzoic acid (PFAB) to impart fluorescence as wellas facilitating negative ion CI MS, as substituted phenyl ring system is agood electron capturing moiety.

Reductive aminationThe Schiff-base is acid labile and is subsequently reduced to a stable,secondary amine – with one derivative per saccharide. Sialic acidsubstituted oligosaccharides are particularly susceptible to acidichydrolysis. The two most common dervatives are 2-aminobenzamide (2-AB) in the EU/US and 2-aminopyridine (2-AP) in Japan. Excessderivatisation agent can also present problems in downstreamseparation or in the subsequent MS analysis and is typically removed byHILIC using SPE (solid phase extraction).

Esters of 4-aminobenzoic acid (4-ABA) are routinely used toenhance the UV chromophore and to enhance the lipophilicity of theanalyte to facilitate FAB MS. The optimisation of the alkyl chain lengthhas been assessed and the octyl derivative is optimal for FAB MSsensitivity14. In contrast, the ethyl (4-ABAEE) and butyl ester (4-ABABE)were best for HPLC/MALDI MS or HPLC ESI15. Tert. amine derivatives of 4-ABA have been used to improve detection in MALDI MS based on thehigher proton affinity of the tert. amino group16.

In both positive and negative ion MS, the 2-AB derivative reducesthe number of MS fragments. The structurally similar derivative, 2-aminobenzoic acid (2-AA), readily forms [M-H]- ions in MS, whichaffords greater sensitivity; however, the charge localisation of thederivative can suppress the formation of many diagnostic ions, whichmakes negative ion MS/MS such a useful method for structuralevaluation of N-linked glycans17.

2-Aminoacridone (2-AMAC) derivatives have been used inconjunction with both normal-phase and reverse-phase HPLC and thenby MALDI-TOF-MS, ESI, and nano-spray MS1,18,19.

GlycosylaminationThe reaction of an amino compound with the carbonyl of a reducing

sugar in the absence of reducing agents yields glycosylamines.Derivatisation of oligosaccharides with 4-aminobenzoic acid butylester(4-ABBE) and 2-AP allowed the assessment of the inter-glycosidiclinkages of the oligosaccharide with negative ion FAB-MS20.

Carbonyl Condensation ReactionsSubstituted OximesThe O- and N-substituted 7-hydroxycoumaryl- and 3-methoxybenzyl-hydroxylamines gave quantitative yields of oxime derivatives of typicalcarbohydrates under very mild aqueous conditions. These derivativeswere HPLC/MS-compatible21.

HydrazonesHydrazine or hydrazine derivatives react with reducing sugars to producehydrazones. Classically, phenylhydrazine has been used to form the corresponding hydrazine which impart increased sensitivity for HPLC-MS. Analysis was by HPLC-EI or by MALDI MS22,23). Girard’s T reagent


Table 2: Summarising typical chemical reactions for the common glycanderivatisation methods

Derivatisation Typical Chemical Reaction

Approach (usually occurs with the ketone/aldehyde tautomer)

Schiff’s Base

Reductive Amination

Glycosylamination

Carbonyl condensation reactions – Substituted Oximes

Carbonyl condensation reactions – Hydrazones Derivatives

Carbonyl condensation reactions – Pyrazolone Derivatives

Derivatives at Carboxylic Acid group e.g., sialic acid

Derivatives at Amino Group

IN-DEPTH FOCUS: LC-MS©

mol

ekuu

l.be

/ Shu

tter

stoc

k.co

m

The Vanquish System reveals the full picture. • Discover more at thermoscientific.com/Vanquish

See what others are missing.The Thermo Scientific™ Vanquish™ UHPLC System was built around the column and the user to deliver better

separations, more results, and easier interaction. Now we add near-universal detection capabilities by adding

the Thermo Scientific Vanquish Charged Aerosol Detector to the Vanquish portfolio. Whether your detection

and quantification challenges are in pharmaceutical, biopharma, food, or environmental analysis, the Vanquish

system will help you get more results by separating and revealing peaks that would otherwise remain hidden.

© 2

015 T

her

mo F

isher

Sci

entifi

c In

c. A

ll rig

hts

res

erve

d. A

ll trad

emar

ks a

re

the

pro

per

ty o

f Ther

mo F

isher

Sci

entifi

c an

d it

s su

bsi

dia

ries.

Thermo Scientific AppsLab

Library of Analytical Applications

Cross-technique application expertise

in a single online location

thermoscientific.com/appslab

Thermo Scientific Vanquish

Charged Aerosol Detector

Unique and novel complement to

UV detection that delivers a sensitive,

universal response

Are you interested to learn more

about our HPLC trade in program?

Request more information on

thermoscientific.com/lcpromotion

(2-hydrazino-N,N,N- trimethyl-2-oxo-ethanaminium chloride) and other Girard’s Reagents possess a quarternary amine group at all pHvalues, which introduces a pre-charged ion into the ion source12. Thisenhances ESI and MALDI-MS sensitivity by approximately 10X24. This reagent has been used to analyse N-glycans. While Girard’s T reagentis specific for ketone and aldehyde groups, glycidyltrimethyl ammoniumchloride reacts with sugar alcohols. Two other related derivativesprepared from 4-phenyl-pyridine and the tripropylamino analogue ofGirard’s T reagent were assessed by HILIC nano HPLC-MS and showedincreased sensitivity24.

Pyrazolone derivativesPMP (1-phenyl-3-methyl-2-pyrazolin-5-one) derivatives are well retainedon C18 columns and are suitable for both ESI26 and MALDI27 detection.The 4-methoxy analogue (MPMP: (1-(4-methoxy)phenyl-3-methyl-2-pyrazolin-5-one)shows higher reactivity towards carbohydratesgiving higher UV absorbances17. Due to therelatively benign reaction conditions, thesederivatives are suitable for sialic acid con -taining glycans. Due to enhanced lipophilicity and recoveries, PPMP (1-(4-isopropyl)-3-methyl-5-pyrazolone) was used to analyse 12 monosaccharides by HPLC-MS28.

Derivatives at carboxylic acid group, e.g., sialic acidsSialic acids are extremely unstable under MS conditions, readilyliberating free sialic acid. However, derivatisation of the α-keto-carboxymoiety of sialic acids using 1,2-Diamino-4,5-methylenedioxybenzene

(1,2-DMB) produces quinoxaline derivatives that have proven to beuseful for separations of sialic acids using HPLC ESI29.

Derivatives at amino groupThe reagent 3-(4-carboxybenzoyl-)-2-quinoline carboxyladehyde (CBCQA)has been utilised for the dervatisation of amino sugars. Alternatively,using a two-step derivatisation process, amino groups can be introducedas the alditol derivatives, which can then be derivatised using 5-carboxytetra methylrhodamine succinimidyl ester. This derivatisationapproach was utilised in the analysis of six different oligosaccharides30.

ConclusionThe analysis of structurally complex glycans (oligosaccharides) is verychallenging. Although the vast majority of HPLC-MS analyses of

oligosaccharides utilise ESI, oligosaccharidesare still very poorly ionised by soft MSionisation methods, for example, ESI, FAB orMALDI. This is because the oligosaccharides arevery polar, relatively non-volatile and thermallylabile. Derivatisation can facilitate ionisation.

Oligosaccharides can form with derivatives via their carbonyl, amine orcarboxyl groups. However, by far and away the most common approachis derivatisation with the carbonyl group (reducing sugars). Similarly,the most common reaction approach is reductive amination because of the high reaction yields, the availability of large numbers of aminocompounds with variable physical properties (charge or hydrophobicityor both) and absorbance in UV/visible allowing dual detectionapproaches (HPLC-UV and/or HPLC-MS).


There is a trend towards smaller particle size allied with the

use of ultra-high pressure liquid chromatography



1. Okafo, G., Burrow, L., Carr, S.A., Roberts, G.D., Johnson, W., Camilleri, P. A coordinated

high-performance liquid chromatographic, capillary electrophoretic, and mass spectrometric

approach for the analysis of oligosaccharide mixtures derivatized with 2-aminoacridone.

Anal. Chem. 68, 1996, 4424-4430.

2. Momenbeik, F., Khorasani, J.H. Separation and determination of sugars by reverse-phase

liquid-chromatography after pre-column micro-wave assisted derivatization. Anal. Bioanal.

Chem. 384, 2006, 844-850.

3. Majors, R.E. Current trends in HPLC column usage. LC-GC North America. 2007.

4. Kavakevich, Y., LoBrutto, R. Stationary Phases, in: HPLC for pharmaceutical scientists, Ed.

Kavakevich, Y., LoBrutto, R. 3: 75-138, 2007, New York, Wiley.

5. Stadlmann, J., Pabst, M., Kolarich, D., Kunert, R., Altmann, F. Analysis of immunoglobulin

glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides. Proteomics 2008, 8,

2858-2871

6. Waters. Glycoworks RapiFluor-MS N-Glycan Kit. http://www.waters.com/webassets/cms/

support/docs/715004793.pdf.

7. Romanyshyn, L.A., Tiller, P.R. Ultra-short columns and ballistic gradients: considerations

for ultra-fast chromatographic liquid chromatographic-tandem mass spectrometric analysis.

J. Chromatographr. A., 928, 2001, 41-51.

8. Wu, J.T., Zeng, H., Deng, Y., Unger, S.E. High speed liquid chromatography/tandem mass

spectrometry using monolithic column for high throughput bioanalysis. Rapid Commun.

Mass Spectrom. 15, 2001, 1113-1119.

9. Kirkland, J.J., Truskowski, F.A., Dilks, C.H., Engal, G.S. Superficially porous silica

microspheres for fast high-performance liquid chromatography of macromolecules.

J. Chromatographr. A. 890, 2000, 3-13.

10. Ackermann, B.L., Berna, M.J., Eckstein, J.A., Ott, L.W., Chaudhary, A.K. Current

applications of liquid chromatography/mass spectrometry in pharmaceutical discovery after

a decade of innovation. Annul. Rev. Anal. Chem. 1, 2008, 357-396.,

11. Lamari, F.N., Kuhn. R., Karamanos, N.K. Derivatization ofcarbohydrates for

chromographic, electrophoretic and mass spectrometric structure analysis. J.

Chromatographr. B. 793, 2003, 15-36.

12. Rosenfeld, J. Enhancement of analysis by analytical derivatization. J. Chromatographr. B.

879, 2011, 1157-1158.

13. Caeser, J.P.J., Sheeley, D.M., Reinhold, V.M., Anal. Biochem., 191, 1990, 247.

14. Poulter, L., Karrer, Burlingame, A.L. n-Alkyl p-aminobenzoates as derivatizing agents in the

isolation, separation, and characterization of submicrogram quantities of oligosaccharides

by liquid secondary ion mass spectrometry. Anal. Biochem. 195, 1991, 1-13.

15. Schmid, D., Behnke, B., Metzger, J., Kuhn, R. Nano-HPLC-mass spectrometry and MEKC

for the analysis of oligosaccharides from human milk. Biomed. Chromatographr. 16,

002, 151-156.

16. Takoa, T., Tambara, Y., Nakamura, A., Yoshino, K-I., Fukuda, H., Fukuda, M., Shimonishi,

Y. Sensitive Analysis of Oligosaccharides Derivatized with 4-Aminobenzoic Acid 2-

(Diethylamino)ethyl Ester by Matrix-assisted Laser Desorption/Ionization Mass

Spectrometry. Rapid Commun. Mass Spectrom. 10, 1996, 637-640.

17. Harvey, D.J. Derivatization of carbohydrates for analysis by chromatography;

electrophoresis and mass spectrometry. J. Chromatographr. B. 879, 2011, 1196-1225.

18. Grimm, R., Birrell, H., Ross, G., Charlwood, J., Camilleri, P. Capillary liquid-

chromatographic and automated MALDI-TOF mass spectrometry analysis of complex

carbohydrate mixtures. Anal. Chem. 70, 1998, 3840-3844.

19. Birrell, H., Charlwood, J., Lynch, I., North, S., Camilleri, P. A dual-detection strategy in the

chromatographic analysis of 2-aminoacridone-derivatized oligosaccharides. Anal. Chem.

71, 1999, 102-108.

20. Li, D.T., Her, G.R. Linkage analysis of chromophore-labelled disaccharides and linear

oligosaccharides by negative ion fast atom bombardment ionization and collisional induced

dissociations with B/E scanning. Anal. Biochem. 211, 1993, 250.

21. Ramsay, S.L., Freeman, C., Grace, P.B., Redmond, J.W., MacLeod, J.K. Mild tagging

procedures for the structural analysis of glycans. Carbohydr. Res. 333, 2001, 59-71.

22. Lattová, E., and Perreault, H. Labelling saccharides with phenylhydrazine for electrospray

and matrix-assisted laser desorption-ionization mass spectrometry. J. Chromatographr. B.

793, 2003, 167-179.

23. Lattová, E., and Perreault, H. Method for investigation of oligosaccharides using

phenylhydrazine derivatization. Methods Mol. Biol. 534, 2009, 65.

24. Naven, T.J.P., Harvey, D.J. Cationic Derivatization of Oligosaccharides with Girard's T

Reagent for Improved Performance in Matrix-assisted Laser Desorption/Ionization and

Electrospray Mass Spectrometry. Rapid Commun. Mass spectrum. 10, 1996, 829-834.

25. Bereman, M.S., Comins, D.L., Muddiman, D.C. Increasing the hydrophobicity and

electrospray response of glycans through derivatization with novel cationic hydrazides.

Chem. Commun. 46, 2010, 237.

26. Pitt, J.J., Gorman, J.J. Oligosaccharide characterization and quantitation using 1-phenyl-3-

methyl-5-pyrazolone derivatization and matrix-assisted laser desorption/ionization time-of-

flight mass spectrometry. Anal. Biochem. 248, 1997, 63-75.

27. Shen, X.D., Perreault, H. Electrospray ionization mass spectrometry of 1-phenyl-3-methyl-

5-pyrazolone derivatives of neutral and N-acetylated oligosaccharides. J. Mass Spectrom.

34, 1999, 502-510.

28. Zhang, P, Wang, Z, Xie, M., Nie, W., Huang, L. J. Detection of carbohydrates using a pre-

column derivatization reagent 1-(4-isopropyl) phenyl-3-methyl-5-pyrazolone by high-

performance liquid chromatography coupled with electrospray ionization mass

spectrometry. Chromatographr. B. 878, 2010, 1135-1144.

29. Morimoto N, Nakano, M., Kinoshito, M., Kawabata, A., Morita, M., Oda, Y., Kuroda, R.,

Kakehi, K. Specific distribution of sialic acids in animal tissues as examined by LC-ESI-MS

after derivatization with 1,2-diamino-4,5-methylenedioxybenzene. Anal. Chem. 73, 2001,

5422-5448.

30. Le, X., Scaman, C., Zhang, Y., Zhang, J., Dovichi, N.J., Hindsgaul, O., Palcic, M.M.

Analysis by capillary electrophoresis-laser-induced fluorescence detection of

oligosaccharides produced from enzyme reactions. J. Chromatographr. A. 716, 1995,

215-220.

References

William McDowell has BSc and PhD degrees in Bio -

chemistry from Heriot-Watt University, Edinburgh, UK,

and is well versed in carbohydrate biochemistry and

glycobiology research through working on inhibitors of

protein glycosylation and structure-function relationships

of glycoprotein oligosaccharides. He spent 20 years at

GlaxoSmithKline (GSK) and its legacy companies Glaxo

and Glaxo Wellcome supporting a variety of anti-

inflammatory, respiratory, anti-viral, anti-tuberculosis and anti-malarial

research programmes. Dr McDowell is currently at PolyTherics where he is

leading a team of bioconjugation and analytical chemists making antibody

drug conjugates.

David Elder has BSc and MSc degrees in chemistry

from Newcastle upon Tyne, before he moved to Edinburgh

to study for a PhD in Crystallography. He is a visiting

professor (King’s College, London). Dr Elder has 37 years’

experience at a variety of different pharmaceutical

companies (Sterling, Syntex and GSK). He is currently

a director within the product development group in

GSK R&D. Dr Elder is a member of the British

Pharmacapoeia (Expert Advisory Group PCY: Pharmacy), a council

member of the Analytical Division, Royal Society of Chemistry (RSC),

UK and a council member of the Joint Pharmaceutical Analysis Group, UK.

He is a fellow of the RSC (FRSC) and a member of the Royal Pharma-

ceutical Society (SRPharmS). He has co-edited one book on the

Analytical Characterisation and Separation of Oligonucleotides and

their Impurities.

George Okafo has a BSc (Joint Honours) in Chemistry and

Biochemistry and a PhD in Chemical Carcinogenesis from at

Imperial College of Science, Technology and Medicine,

London. Dr Okafo completed a Postdoctoral Research

Fellowship at the University of Toronto in nitrosamine-

induced chemical carcinogenesis. Dr Okafo has 25 years’

experience at GSK, where he is a Consultancy Director and

Drug Discovery Project Leader. He has led numerous drug development

projects, co-authored regulatory documents, been an invited speaker at

international conferences and has published 45 papers, authored three book

chapters and is co-inventor of two patents covering a wide range of analytical

and separation sciences areas applied to small molecules, proteins,

oligosaccharides and DNA. He has co-edited one book on the Analytical

Characterisation and Separation of Oligonucleotides and their Impurities.

Dr Okafo is a Member of the Royal Society of Chemistry (MRSC), an

Associate of the Royal School of Chemistry (ARCS) and Chair of the

Separation Science and Technology Group at the Society for Chemistry in

Industry (SSTG/SCI).

Phil Borman is a Chartered Chemist with more than

18 years of experience in the pharmaceutical industry, having

obtained a Masters in Chemistry from UMIST (Manchester)

University and a Masters in Industrial Data Modelling from

De Montfort (Leicester) University. Phil is currently account -

able for the provision of QbD Support (with an emphasis on

Analytical QbD) across multiple late-phase project teams at

GSK. Phil pioneered the adaptation of QbD principles to Analytical methods

and has published widely in the field of Analytical Chemistry.

INNOVATION DELIVERED

See what you’ve been missing

CESI 8000 HIGH PERFORMANCE SEPARATION - ESI MODULE

© 2014 AB SCIEX. For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of AB SCIEX Pte. Ltd. or their respective owners.

Capillary Electrophoresis

Heterogeneity, stability, and purity from a SINGLE enzyme in a SINGLE run

CESI-MS: Robust ultra-low flow electrospray ionization technology that minimizes ion suppression, maximizes ionization efficiency and sensitivity, eliminates sample carryover, and increases throughput.

Biologics primary sequence heterogeneity determination through extensive glycopeptide coverage and quantitation

Stability indicating degradative hotspot information like aspargine- deamidation, methionine-oxidation, and glutamic-acid-cyclization

Identification and purity by full peptide mapping coverage

See more of what you’ve been missing at www.sciex.com/cesi-gen or contact your local sales representative www.sciex.com/contact

An LC-MS system consists of a pump which forces the mobile phasethrough an autosampler/injector, transferring the sample through theanalytical column where the different sample components areseparated. After separation, the sample components are transferredfurther on to a detector, in this case a mass spectrometer, for detection.Adjusting parameters such as the stationary phases, mobile phases,stationary-phase particle sizes or column inner diameters combinedwith different MS technologies can result in increased selectivity and/orsensitivity. In this way, identification of almost all analytes of interest ispossible, making LC-MS a miscellaneous technique.

Today LC-MS is used in clinical laboratories as well as in research,becoming the workhorse for most analytical chemists. However, eventhough the technique is highly popular, some pitfalls are to be aware of,

such as ion suppression and other matrix effects. Overall though, thecapability of the LC to separate components and reduce samplecomplexity makes the combination of LC and MS a versatile tool.

History of LC-MSThe coupling of LC with MS was for a long time seen as almostimpossible due to the fact that the LC uses liquids and MS demands avacuum. However, the potential of combining LC with MS was large, asthis would allow analytes not volatile enough for gas chromatography(GC) to be analysed with the increased specificity the MS detector gives.Also, the need for complete base line separation of eluting analytepeaks would not be necessary as long as the analytes had different m/z,as these would be separated in the detector.

Liquid chromatography-mass spectrometry (LC-MS) is a diverse technique used in analysis of substances such asbiomolecules and environmental targets. The analytes are separated on a stationary phase inside an LC column,before they are separated based on their mass-to-charge ratio (m/z) in a mass spectrometer and detected. This technique has great potential in both qualitative and quantitative analysis using either accurate mass foridentification, external standards for quantification or a combination of both. Today, these techniques can be usedto identify biomarkers and environmental pollutants and to diagnose disease among other uses. This article aims todemonstrate just how versatile LC-MS is: how LC can be combined with a variety of MS systems and the increasingpopularity of nanoLC.



© D

erm

ot M

cBrie

rty

/ Shu

tter

stoc

k.co

m

Analysis of limited and complex sampleswith nanoLC-MS

Hanne Røberg-LarsenUniversity of Oslo

The first attempt to couple LC with MS was published in1968 by Victor Tal’rose in the Russian Journal of PhysicalChemistry. Victor and colleagues used a traditional GC-MS ionsource, electron impact ionisation, to transfer a small amountof liquid into gas phase ions. However, these ion sources wereunusable in combination with normal mobile phase flows dueto the fact that a liquid produced large amounts of gas whenthe pressure was reduced.

In subsequent years, several attempts to combine LC withMS were conducted. Some of them, such as Thermospray,were commercialised. However, the versatile technique as we know it today would not have happened without thedevelopment of atmospheric pressure ionisation sources. The invention of electrospray ionisation (ESI), atmosphericpressure chemical ionisation (APCI) and matrix-assisted laserdesorption (MALDI) made routine LC-MS possible in the waywe are familiar with it today.

Electrospray ionisationElectrospray ionisation (ESI) is possibly the most usedionisation source in LC-MS. ESI transfers charged ions from the liquid in mobile phase into charged gas phase ions. The liquid flow from the column goes through a needle and ahigh voltage (typically 3,000-to-4,500 V) is applied. The liquid forms aTaylor cone, and if the voltage is high enough, the liquid stream willbreak down to small highly charged droplets (aerosol). Solventevaporation will shrink these droplets and create repulsive forcesamong the ions. When these repulsive forces are large enough, the droplets will be break up into smaller highly charged droplets. This process repeats itself, resulting in even smaller charged droplets. In the end, the droplet will be so smalland the repulsive forces so large that ions canescape the droplets (as in the ion evaporationmodel for small molecules) or one ion will beleft as the solvent evaporates, creating onecharged species in gas form (charged residuemodel for large molecules).

The combination LC-ESI-MS functions for almost all LC-separationprinciples, as long as the mobile phase with additives is volatile.However, one should be aware of the possible matrix effects when usingLC-ESI-MS that can enhance or suppress the signal from your analyte.The possible reason for these effects is the presence of co-elutingmatrix compounds. If these compounds are more easily ionised in ESIthan the analyte of interest, they can compete for surface in thedroplets with the analytes, giving fewer analyte ions. The competingions can also make neutral complexes with your analytes. As the MSmeasures m/z, these neutral species will not be visible. These matrixeffects can give an over- or under-estimation of the concentration ofthe analyte in the sample and highlight the need for a separation infront of the MS analysis in addition to a good sample clean up beforethe analysis.

Mass spectrometry systemsDifferent mass spectrometry systems can be used in combination withLC. The most commonly used instrument for quantification is the

quadrupole instrument (Q), or more often triple quadrupole (QqQ). As the name implies, the Q consist of four parallel rods and mass-filtersthe ions based on their trajectories’ stabilities when different voltagesare applied. Only ions of a certain m/z will reach the detector – allothers will collide in the rods.

When combining three QqQs using the first as a mass filter, thesecond as a fragmentation source and the last one as a mass filter,

tandem MS (also known as MS/MS) is possible.MS/MS techniques increase specificity andsecure identification of one or more analytes bysingle reaction monitoring (SRM). Only one m/zresulting in one or more known fragments willreach the detector, giving little backgroundnoise and increased signal-to-noise ratio, thus

lowering detection limits. QqQ is therefore the most obvious choice inLC-MS analysis for quantification of known analytes and the technologyhas become particularly useful in the diagnoses of specific disordersthat involve chemical elevations.

LC-MS also enables unknown analytes to be identified throughseparation for less sample complexity and accurate mass detectionusing instrument such as Time of Flight (ToF) and Orbitrap with highmass accuracy. These instruments can also be combined with a collisioncell for MS/MS analysis. Both the ToF and Orbitrap instruments areideally used for discovery analysis in metabolomics and proteomics,and are frequently used in research and clinical laboratories around the world.

nanoLC-MS In nanoLC, narrow columns and low flow gives less chromatographicdilution during the analysis, resulting in more concentrated peakseluting from the column. If the system is equipped with a concentra -tion-sensitive detector, such as ESI-MS, this will lead to lower detection



The coupling of LC with MS was for along time seen as almost impossible dueto the fact that the LC uses liquids and

MS demands a vacuum

© V

ILev

i / S

hutt

erst

ock.

com

Please find further information: www.bruker.com/FUSION-SV

For research use only. Not for use in diagnostic procedures.

FUSION-SVBruker

Two techniques - one decisionSmall molecule structure verification with Bruker FUSION-SV: fully integrating HRAM-MS and NMR data for increased specificity

NMR Spectroscopy and Mass Spectrometry are two fundamental techniques for small molecule structure verification. Bruker has developed a new software solution with sophisticated proprietary algorithms to synergize NMR and MS measurements into one result.

• Fully automatic structure verification for small organic molecules synergizing NMR and MS data into one result• Synthesis chemist will get a prompt result without being an expert spectroscopist

NMR & MSInnovation with Integrity

limits; hence a more sensitive method is achieved. The columns can beless than 100μm in inner diameter (ID) and the mobile phase flow aretypically below 500nL/min. All tubing and connections must be scaleddown as well. Combined with nanospray-ESI, the droplets created willbe smaller, thus the evaporation process will be faster. In theory, fewerions will compete for droplet surface area, giving a more efficienttransition from liquid to gas phase ions. In addition, a more narrowspray will be created, directing more ions into the MS instead of outsidethe ion source, giving a higher signal and possible lower detectionlimits. Hence, the nanoLC-ESI-MS systems are ideal when sample iscomplex and limited.

A common misconception when using nanoLC-ESI-MS is thatlowering the column ID will give better separation efficiency, resulting inbetter separation of the sample components. This, however, is not the case since lowering the column ID will not affect the efficiency. If samples are very complex and increased efficiency is needed,increasing column length or decreasing particle size is required, thelatter being more effective. Thus, both actions come with a cost. Increasing column length will give better resolution, though it will increase analysis time. Decreasing particle size will more effectivelyincrease column efficiency and resolution; however it will also give increased back pressure. NanoLC normallyuses the same particle size as regular LC,operating in the same pressure area, hence hasapproximately the same separation conditions.

NanoLC comes with both benefits andchallenges. The reduced ID of the column leadsto lower mobile phase consumption, providingboth economic and environmental benefits; however, special LC pumpsare needed since regular LC pumps are usually unstable at the low flowrates used in nanoLC. In addition, the pump’s mixing chamber, togetherwith all connective tubing, needs to be of appropriate size that largedead volume and gradient dwell times are prevented. In general, deadvolumes in nanoLC are more critical than regular LC due to the extremelow flow rate. Increasingly, zero-dead-volume couplings are becomingavailable, making nanoLC much more easily operated.

Another challenge with nanoLC is injection. To gain the potentialincreased sensitivity, the same amount of analyte must be injected onthe column. However, the low mobile phase flow and ID of the columnmakes this difficult. For example, a 10μL injection would use 20 minutesto leave the sample injector at a flow rate of 500nL/min. Furthermore,since a 100μm ID, 10cm long column typically has a volume of 0.4μL, a 10μL injection volume will result in overloading of the column. Directinjection of sample on nanoLC columns should therefore be in the nLarea, demanding high concentration of the analyte in the sample toaccomplish detection.

Since nanoLC is often used for limited samples with lowconcentration of the analytes, the low injection volume is a mismatch.To overcome these problems, injections in nanoLC are most oftenperformed using column switching systems (Figure 1). In a columnswitching system, the sample is loaded on a reusable solid phaseextraction (SPE) column with high flow with non-eluting conditions.Ideally, the sample is up-concentrated and focused on the SPE-column.When a valve is switched, eluting mobile phase goes through the SPE-column, transferring the sample in a narrow band to the

analytical column for separation. Hence large volume injections can beperformed on narrow columns without loss of column efficiency, inaddition to an online sample clean up, as all non-retaining componentsin sample are washed out into waste during the loading of the sample.

Use of nanoLC-MS: ProteomicsDespite all of the physical challenges in nanoLC systems, the increasedsensitivity it affords for limited samples with low concentrations ofanalytes makes nanoLC a highly used technique in research. Indeed, inproteomics, nanoLC is almost exclusively used. Most commonly, the proteins in the samples are digested into peptides by an enzyme,since peptides are more compatible with reversed phase chromato -graphy and will give more easily interpretable mass spectrometry data. Long narrow columns together with long gradients give high separa-tion power, allowing separation of more peptides and there is probablyalso less ion suppression. The peptides are separated and amino acid sequences are identified in MS/MS mode. Specially designedsoftware that searches the data against databases is then used to put the pieces back together and identify the original proteins. The versatility of the technique allows for short targeted analysislooking for, for example, biomarkers, as well as long comprehensive

analysis to map the whole proteome.

NanoLC-MS in other analytical fieldsIn other fields of analytical chemistry such aslipidomics, metabolomics and environmentalanalysis, the use of nanoLC has not yet gainedthe same popularity as in proteomics. The

reason for this might be that at this time point the sensitivity gained inregular LC and ultra-high-performance liquid chromatography (UHPLC)is good enough for the sample size and higher throughput is moreimportant. In any case, the use of nanoLC in fields such asmetabolomics is possible when looking at cell sub-populations andsub-cellular research. As the technique continues evolving, thepopularity of nanoLC-MS in these fields will probably grow as well.

ConclusionThe versatility of LC-MS makes the technique suitable for almost allapplications in biological analysis, since secure identification ispossible due to the specificity of the MS. Due to the improvedtechnological development of nanoLC systems, these techniques aregaining more popularity as previous problems such as robustness are minimalised. This makes nanoLC-MS a technology which willcontinue to increase in popularity and become an even morefrequently-used technique in the future as sample complexity increasesand sample size decreases.



Hanne Røberg-Larsen is a PhD candidate in the group for

bioanalytical chemistry at the Department of Chemistry,

University of Oslo. Her main fields of interest are

development of new automated analytical methods and

technology for metabolomics studies. Through her short

academic career she has won both waters innovation prize

and a prize for youth scientists. Her expertise areas are high

sensitive LC-MS and cancer metabolomics for early diagnosis and response

to drug treatment.

MS/MS techniques increasespecificity and secure identification of one or more analytes by single

reaction monitoring (SRM)

Test a publisher’s statement

of circulation. In today’s business

climate you can’t afford not to.

Our ABC Certificate provides

accurate, independently

verified circulation figures,

giving you confidence in

your advertising investment.

For more information visit www.abc.org.uk

or email [email protected]

Be wisewhen youadvertise

If you want to reach 11,9991

total average net circulation, then call Andrew Johnson on +44 (0) 1959 563 311 or email [email protected]

1. Circulation figure relates to period from 01/01/2013 to 31/12/2013

IN-DEPTH FOCUS: LC-MS ROUNDTABLE


© sc

ienc

e ph

oto

/ Shu

tter

stoc

k.co

m

ModeratorDavid Elder, GlaxoSmithKline and JPAG

Kevin Ray PhD, Senior Manager,Analytical Research

& Development, Sigma-Aldrich

Stéphane Moreau Product manager

MS range, ShimadzuEuropa GmbH

Iain Gibb Market Development

Manager EMEAPharmaceutical,

Sciex

Sean M. McCarthy Senior Scientific

Operations Manager,BiopharmaceuticalBusiness, Waters

Corporation

With an increasing biopharmaceutical portfolio, can we move away from reagents being the limiting factor and move to high resolution LC-MS? Is the selectivity sufficient?Ray: Liquid chromatography-tandem mass spectrometry (LC−MS/MS)methods can be a viable alternative to ligand binding assays formonoclonal antibody (mAb) quantification in biological matrixes. This isparticularly attractive early on in a program when target-specific assayreagents are often not available. LC−MS/MS has been demonstrated toprovide excellent specificity in a variety of matrices. Recently,

researchers have identified a set of conserved peptides liberated fromthe Fc region of antibodies which represent a generic LC-MS assay to quantify human mAbs in non-human ‘matrices. This ‘universal’approach offers great potential value to the biopharmaceuticaldevelopment process particularly during early preclinical studies.

Moreau: Biopharmaceutical science is a complex and resource-intensive enterprise and the scientific progress in the bioanalysis ofpeptide and proteins with MS-based techniques has been a keytechnology to analyse large molecules and make a meaningful impact

on biopharmaceutical development. However, as novel scientificstrategies consider a broad range of new scientific approachesincluding antisense RNAi interference (RNAi) therapy, therapeuticcancer vaccines, cell therapy and gene therapy, it is likely that reagentsand LC-MS/MS will both play a significant role in analysis.

Gibb: We believe that the near- and medium-term future lies with somereagents in the workflow. Making their usage easier and, viaautomation, very reproducible makes LC-MS able to cover mostquestions in the biopharmaceutical market. If you are looking to seeevery protein in plasma and want to do thatwithout some form of reagent fractionation ordigestion, the instruments are not there yet interms of dynamic range. Proteases, protein A orG enrichment, SISCAPA, or other enrichmentand fractionation technologies are going to bearound for a while. With those, however, themove away from ELISA or other more specific reagent-requiringtechniques is well underway and technologically feasible.

McCarthy: There is no simple answer, other than that it depends on thetarget or goal of the assay. We do see a move towards using LC-MS as ageneric platform for protein characterisation, but there will always bechallenging molecules that require differing levels of selectivity. In suchcases, you could use an orthogonal assay such as LC/UV. It also dependson the complexity of the molecular entities. There are instances whereyou can achieve a selective assay by using high resolution LC-MS,however it is challenging to find an authentic standard, which is why theindustry seeks certified reference materials.

We currently use ELISA for some biopharmaceuticalproducts due to excellent sensitivity but methoddevelopments are long, labour intensive and expensive,requiring experienced molecular biologists. What are thepanel’s views on the applicability of LC-MS to test allprotein related residuals in a single analysis? Is sensitivity still the big challenge?Moreau: As the therapeutic space is continually evolving, thebiopharmaceutical pipeline offers great hope for patients. The reality isthat the biology of many diseases is complicated and makes thebiopharma discovery process challenging and uncertain. As therapeuticproteins are complex analytes due to microheterogeneity and multiplein vivo binding partners, the biological question will drive thetechnology selection and the future outlook may well consider a hybrid approach using both LBA and LC-MS/MS to help characteriseactive isoforms.

Gibb: The choice of technique depends as much on the desire foranalytical efficiency and productivity as other factors. Techniques likeMALDI, FAB and CAD, might work, but they may have drawbacks by notbeing usable in routine environments, having lower dynamic range, orrequiring a separate instrument than one being used for other defaulttechniques such as peptide mapping and intact molecular weight.Therefore for an organisation under pressure to develop robust androutine techniques, having one choice may make more economic

sense. As vendors of instrumentation, we have to cater for all areas,including a drive to efficiency.

McCarthy: The trend we see, driven somewhat by regulatory agencies, isto use high resolution MS to provide more concrete proof of what is andis not there. As such, it can be used to guide the development ofappropriate ELISA assays: You can use MS to identify the specificproteins within a product, and design a targeted ELISA assay for those proteins. There is also a trend to characterise using TOF-MS tounderstand which proteins are present, and then migrate those assays

to a tandem quadrupole MS for monitoring. So LC-MS has a lot of potential in development,if not on the release side.

Ray: The potential use of LC-MS/MS for theanalysis of host cell proteins (HCPs) inbiopharmaceutical products has been demon -

strated very recently, but detecting ppm levels of HCPs in abiotherapeutic background presents a major challenge to the dynamicrange of such methods. Two-dimensional chromatography and HCPenrichment steps have been employed to provide comprehensive andaccurate HCP characterisation of biological drug substances.

Some analytes, like oligosaccharides, are still very poorly ionised by soft MS ionisation methods, e.g., ESI, FAB or MALDI, necessitating pre- or post-columnderivatisation. Do the panel envisage any futuredevelopments in this field?Gibb: In the past, ionisation was a limitation in ESI, but this is now lessof a problem. The issue has been addressed in a number of ways,including source efficiency, new inlet techniques such as CESI, 2D chromatography, ion mobility selectivity improvements, and ofcourse column and labelling techniques for released glycans.Increasingly, customers are switching to working on GlycoPeptideanalysis to maintain the site occupancy information whilst concurrentlyperforming their peptide maps. Additionally, the issue is increasinglyabout managing the mounds of raw data with automated software,which is why we have developed software that copes with thefragmentation of labile modifications.

McCarthy: We’re really excited to be leading new technologyadvancements in this area. In January, we introduced a new rapidlabelling reagent, RapiFluor-MS, which solves the problem of MSionisation for glycans and improves sample preparation productivity.We’ve shown that you can move through deglycosylation, N-glycanlabelling, and cleanup in three straightforward steps in as little as 30 minutes. Our reagent also has unique characteristics that haveshown MS sensitivity improvements of up to 1000x. With this newreagent, scientists can now use what’s not been traditionally viewed ashaving the necessary sensitivity: a single quadrupole, electrospray MS that’s conducive to downstream monitoring.

Ray: The lability of some biomolecular bonds such as glycosidic bondsnecessitates a gentle ionisation approach, which will likely continue toutilise ESI. Thus for poorly ionising biomolecules, labelling will be an



As the therapeutic space is continually evolving, the

biopharmaceutical pipeline offers great hope for patients

Stéphane Moreau



essential part of their analysis workflow. Judicious choice of labellingmolecules also enables sensitive spectrophotometric detection, whichis beneficial to many LC-MS systems that have inline UV-Vis orfluorescence detectors. At Sigma-Aldrich we are engaged in making and improving reagents for pre- and post-column derivatisation. In addition, we envision online derivatisation will play a more importantrole in the future from the perspectives of automation, throughput, and sensitivity.

Moreau: The development of LC or MALDI MS has evolved as ourunderstanding of the physical processes involved in ionisation and iontransport has increased. However, despite advances in improvingionisation efficiencies and better ion transport of gas phase ions intothe mass analyser, radically new modes of ionisation have not beendiscovered. The techniques of electron and chemical ionisation thatdominated the early 1990s have become routine tools which are stillbroadly recognisable today but to create a game changer with thepower to be considered as a universal detector is still elusive.

HILIC-MS is becoming increasingly used in LC-MS, owing tothe greater sensitivity afforded by the enhanced desolva -tion properties of the HILIC mobile phase. What otheradvances in column technology do the panel envisage?McCarthy: There are many improvements that can be made in HILIC-MS.We do know that having wider pore particles is going to be helpful in theresolution of glycoforms. HILIC has been applied very well to releasedglycans, however customers we work with also want to have equivalentseparations for glycopeptides or intact proteins or protein subunits todetermine site-specific heterogeneity. These types of separations willmost likely require stationary phases with larger pores. This issomething we’ve been actively investigating and we envision columntechnology coming onto the market to address this within the next year.

Ray: Orthogonal separations allow chromatographers to overcome a keychallenge in method development, namely insufficient resolution.While columns that exhibit orthogonalretention patterns are widely available forreversed-phase chromatography, they are stillquite uncommon when it comes to HILIC mode.Thus, a wider variety of HILIC phases will likelyemerge in order to address this methoddevelopment concern. In addition, a tighter integration between theseparation technology, or column, and the instrument hardware can beexpected. Operation with an integrated source/column technologyprovides benefits of improved ESI response, reduced matrix effects anddecreased solvent consumption.

Moreau: HILIC-MS has become an accepted tool for the analysis of polarmolecules and recent commercial products have generated highlyreproducible HILIC phases. Increasing the chemical space is still anunmet need for many applications and creating a highly selectiveseparation for LC-MS mobile phases is still challenging. However, thistechnology has seen significant changes over the decades and recentdevelopments in biphenyl and C18 have made a major impact onroutine LC-MS analyses.

Gibb: There will always be work on new column technologies going on. Each of those new technologies will have its unique niche and provide value to a subset of the market. Our focus has been onslightly orthogonal technologies such as CESI-MS. The abilities in theareas of protein purity determination, charge heterogeneity analysis,and carbohydrate or other post-translational modification profilingopen up a large range of possibilities for both discovery and qualitycontrol groups.

The advent of ICH M7 has increased the popularity of using LC-MS to test mutagenic impurities at ppm levels on active pharmaceutical ingredient (API) specifications.Will these sophisticated methods become part of a quality control (QC) environment in the future? Ray: The structure of the potential genotoxic impurities (PGIs) and genotoxic impurities (GIs) and required limit of detection will define the appropriate method to be used to control levels of suchsubstances in APIs and drug products. While many PGIs/GIs can besufficiently monitored with techniques such as GC-FID and RPLC-UV,more challenging analyses will likely require the routineimplementation of GC-MS, RPLC-MS, and even 2D-LC-MS in particularlychallenging scenarios.

Moreau: The International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human Use (ICH) M7 guidelines for mutagenic, specifically genotoxic, impurities could, potentially, significant impact on drug develop-ment. One challenge is to accurately assess the rate of formation of potential mutagenic impurities at lower levels than those listed in Q3A and Q3B guidelines. Analysis of mutagenic impurities is complex, the reactive nature of the impurity makes sampling difficult and analysis methods need to be highly sensitive and selective. The decision to migrate such methods into a QC environ-ment will be influenced by regulatory needs but could well become the future norm.

Gibb: With the introduction of more sensi-tive LC-MS methods and total workflowsolutions there will be a move into this space.There are workflow solutions in this space thatallow the routine and simultaneous quanti -

fication of mutagenic impurities at ppm levels but at the same time allowing qualitative confirmation that the correct impurity is being monitored.

McCarthy: I am not aware of discussion of these aspects in relation tobiopharmaceuticals. However, I believe in small molecules there is atrend towards monitoring these impurities and it is likely that they willmigrate into QC if, for certain products, there is a significant risk thatthese impurities will be present either by the nature of the molecule orby degradation of that molecule. I have only heard of this analysis inupstream labs, but it is not out of the realm of possibility that they could end up in QC for lot release, likely as an MS assay, andpotentially a tandem quadrupole MS at that given the low levelsrequired for detection.

Orthogonal separations allowchromatographers to overcome a key

challenge in method development Kevin Ray


▲

Welcome toEuropean Pharmaceutical Review’sShow Preview of:

European Pharmaceutical Review is alsopleased to announce the following showpartners who will be exhibiting at ACHEMA:Atris ........................ Hall 11, Booth A33GEA ......................... Hall 4, Booth F46Ocean Optics......... Hall 4.2, Booth B78Sentronic ............... Hall 4.2, Booth E80Siemens................. Hall 11, Booth C3

15 – 19 June 2015Frankfurt am Main, Germany


SHOW PREVIEW: ACHEMA 2015

Challenges in lab automation and formulationWhat’s hot in the process industries? The answers might differdepending on who you ask, but some topics are on top of the globalagenda for sure, such as advances in process analytical technology orthe handling of ‘big data’. The organisers of ACHEMA 2015 have set out toinvestigate the latest trends in the laboratory in advance of the globalforum for the process industries in June in Frankfurt.

Long gone are the days when an under graduate spent his dayspreparing and measuring sample by sample and copying the data fromsome cryptic output format to a self-created spreadsheet. Today’s labsare highly automated and able to process literally millions of samples.Especially in drug development, the number of substances that areavailable to be screened is stunning. Substance libraries made up ofpotential active ingredient molecules for drug develop ment currentlycontain well over a hundred thousand active substances. Lab robotsbuild these libraries based on a pre-defined set of synthesis rules. Withthe aid of lab automation systems, the lab teams use high-throughputscreening to process in the region of 10,000 samples per day.

Ultra high-throughput screening was developed at the beginning ofthe 1990’s by Evotec in collaboration with international pharmaceuticalproducers such as Novartis and SmithKlineBeecham. Using this tech -nique, more than 100,000 samples can be processed on a daily basis inactive ingredient research for the development of new pharmaceuticalproducts. Each microtitration plate can contain up to 3,456 wells tofacilitate efficient handling and archiving by automated systems. Lab information and management systems (LIMS) have been in usesince the 1980’s to integrate lab workflows into the IT landscape, buthigher levels of automation greatly increase the complexity of thedemands placed on lab IT. This is particularly the case when automatedworkflows extend beyond PC and microcontroller based control on asingle piece of equipment. Attention is currently focused primarily onprocess and data management as well as inclusive lab management.

The big data challenge The term ‘big data’ is used to describe the enormous amount of datawhich is generated during automated processing and extends well into



ACHEMA is the world forum for chemical engineering, process engineering and biotechnology. Every three years theworld’s major fair for the process industry attracts around 4,000 exhibitors and 167,000 participants from over 100 different countries to present new products, processes and services to professionals from all over the world. The spectrum ranges from laboratory equipment, pumps and analytical devices to packaging machinery, boilers andstirrers through to safety technology, materials and software, thus covering the entire needs of the chemical,pharmaceutical and food production industries. The accompanying congress, featuring 800 scientific lectures andnumerous guest and partner events, complements the wide range of themes of the exhibition.

the terabyte and petabyte range. Data volumes worldwide are expectedto double every two years or so. The enormous increase in dataresources for genome sequencing is one example from the LifeSciences. The abundance of data also creates huge potential for

development of new drugs and life-sustainingmedical analysis. Companies including BoehringerIngelheim, CHDI, Evotec, Genentech, MedImmune/AstraZeneca, Ono Pharmaceutical and UCB haveforged research alliances to develop new path-ways for treating Alzheimer’s, diabetes and cancerand for palliative medicine. Insilico Biotechnologyoperates one of the world’s leading system biologyplatforms which draws together proprietary data bases, cell models and computer-basedanalysis. The goals are validation of activeingredients and produc tion of biochemicals and biopharmaceuticals.

Data management designed for the long term isessential for labs which use automation systems.The data must be available in a variety of formats tosupport data exchange with other labs as well as for

distributed access to the lab’s own data. The researchers should be ableto use the data which is captured in the lab for automatic analysis andgraphic representation. Without the need to invest significant amountsof time and money, the results of completed test series can be retrieved



Atris Information Systems GmbHAtris Information Systems is a software solution and cloud provider for the

pharmaceutical and chemical industry.

The solutions of Atris Information Systems support

■ Quality reporting to FDA and EMA

■ Process monitoring and improvement

■ Statistical analysis and data mining

■ Big data management

The systems facilitate the fulfilling of the requirements of Process Validation

and Continued Process Verification.

Products PANDA (Process ANalysis DAtabase)The data-integrating and consolidating-analysis and reporting system

PANDA is designed by Atris Information Systems especially for standard

compliant Process Validation according to the US Food and Drug

Administration and European Medicines Agency in pharmaceutical

production. PANDA provides and evaluates information from all relevant

source systems. With PANDA, raw material and drug substance data, as well

as development and process data, can be structured, linked, aggregated and

statistically analysed.

GECCO (GEneriC Communicator) GECCO is a web-based system for manual capturing and reporting of batch

related data (e.g., batch process data or quality data) in the intranet and the

extranet which can be used among others by external companies

and laboratories. The webpages for data capture can be individually

designed, generated, tested and released quickly and easily by authorised

users. A standardised approval work flow (with electronical signature) for the

entered data is integrated in the system. An interface to the system PANDA is

provided.

BAT (BAtch Tracebility) The web based BAT provides the grafical visualisation of the batch genesis. The

data for the visualised batch tree is imported from ERP systems like SAP or BPCS.

In combination with PANDA and GECCO all process data and quality data of

each batch can be presented directly in the web interface on demand.

for future experiments and analyses, and theresults can be also made available to otherlabs. Data analysis in an interdisciplinarycontext, for example using special datamining algorithms, is gaining momentum inthe biosciences. Data mining techniquescreate new opportunities to detect medicalrisks and can also produce tangible value-add in practical healthcare delivery.

SiLA initiative to create uniform standardsA heterogeneous assortment of specialisedequipment is currently installed in manybiotechnology, pharmaceutical and clinicaldiagnostics labs. The IT infrastructure hasdeveloped over time, and it tends to bedisjointed or poorly coordinated. Devicedrivers and platforms, which are based on uniform standards and canbe addressed by products from any manufacturer, create theopportunity to integrate this heterogeneous equipment. To developsustainable IT solutions for automated lab environments, systemsmanufacturers, software service providers, system integrators andpharma ceutical and biotechnology companies have joined forces in theSiLA Initiative (StandardiSation in Lab Automation) to createauthoritative standards. The goal of the initiative is seamlessintegration of lab equipment and IT systems made by differentmanufacturers based on uniform comm unication interfaces, device

drivers and lab consumables. Highly specialised experts are assigned bymembers of the non-profit consortium to technical working groups forjoint development of authoritative standards. Further information onSiLA and participating companies is available at http://www.sila-standard.org.

Lab automation has many benefits, but it can involve cost-intensivemodifications depending on the lab’s size and requirements profile.Cost-conscious lab operators who intend to introduce automation step-by- step could well benefit from modular systems which can beexpanded as needed. To make the systems as future-proof as possible,


© J

org

Hac

kem

ann

/ Shu

tter

stoc

k.co

m



the modules should conform to current inter national standards andinclude standardised interfaces.

The next step: formulationAn active ingredient alone does not make a drug. Formulation is an artin its own right, making sure the pharmaceutical is delivered at the righttime in the right amount to the right place in an organism. Efficientsystems for automated formulation develop ment save valuableresearch time, and they can also reduce material consumption while producing better out-comes. These systems are used in the development of coatings in the chemical industry, healthcare

products and drugs in the pharmaceuticalindustry and in bio-technology.

In drug discovery, high-performanceformulation development systems play apivotal role in the advancement of medicalscience. According to management consult -ants CRA International, companies canexpect to spend up to $1.6 billion for marketintroduction of a drug containing a newactive ingredient. Novartis, one of theworld’s leading pharmaceutical producers,reports that the drug development processfrom conception to approval takes onaverage 12 years. The Novartis Group inGermany currently has around 200 in-houseclinical development projects underway.

Drug research in Germany is financedalmost exclusively from private investment,special initiatives and funding programs.

As a result, time and cost efficiency are crucial factors in medicalprogress. During develop ment of a new drug, researchers evaluate up totwo million compounds. Only a single relevant active ingredient willeventually be selected and submitted for approval.

Up to 60% time and cost savingsInformation provided by Bosch indicates that lab process automation inthe chemical industry can increase lab efficiency by a factor of 2.5, producing time and cost savings of up to 60%. The differentindustries set very high standards for cost-efficient time to market anddependable product quality. Manu facturers as well as product

© C

hris

tian

Mue

ller /

Shu

tter

stoc

k.co

m

Next-generation miniature spectrometer from Ocean Optics

Ocean Optics has launched a spectrometer range that combines decades of

miniature spectrometer design expertise with industry-leading manufacturing

techniques. The Flame spectrometer delivers high thermal stability and low

unit-to-unit variation without compromising the flexibility and configurability

that are the hallmarks of modular, miniature spectro meters. Features such

as interchangeable slits, indicator LEDs and simpler device connectors

provide great flexibility for a wide range of UV-Vis applications including

OEM integration and lab, industrial and field use.

The Flame is fully configurable across the 190-1100nm wavelength range

for use in absorbance, transmission, reflectance, irradiance and colour

applications. Spectrometers come preconfigured or custom configured, with

interchangeable slits that enable users to adjust resolution and throughput on

demand. For example, the user can reconfigure the same spectrometer

from high resolution for absorbance to high throughput for fluorescence in

seconds. To further increase measurement power, the Flame works seamlessly

with Ocean Optics’ range of light sources, optical fibres, sampling accessories

and software.

Optical bench improvements and high precision alignment methods

elevate Flame performance for demanding applications in industrial and field

environments. The Flame has thermal stability of 0.05nm/°C from 200-850nm

and highly consistent unit-to-unit performance that meets the needs of

OEMs and other high-volume customers. Its small footprint (89 x 63 x 34 mm)

and low weight (265g) make Flame ideal for OEM integration.

Flame operation is plug and play. A choice of USB or RS-232

communications, supported by drivers and software, make it easy to integrate

into almost any system. Indicator LEDs show continuous power and data

transfer status.

To learn more about the Flame, please contact an Ocean Optics Applications Scientist at [email protected]

or visit the website at www.OceanOptics.com

Flame builds on 20 years of expertise from the producers of the first miniature spectrometer

and system developers respond to these challenges at various levels.Ongoing technological advances in automated processing and comp -anies with highly specialised lab structures which have establishedpartnerships with the pharmaceutical, chemical and biotechnologyindustries are at the forefront of current developments. Comparisonsbetween trials based on manual and automated formulationdevelopment show that researchers arrive at different outcomesdepending on which option they choose. In a manual process, certainexcipients could possibly be deter mined to be unsuitable for a product.However in automated trials, the very same compounds could prove tobe beneficial when tried in combination with other substances.

Development platforms for the entire productdevelopment processSpecial development platforms are designed to run simple or complexautomatable development processes. Various suppliers offer modularsystems or complete robotic platforms to suit a variety of needsthrough out the development process, ranging from design ofexperiments to sample selection and processing as well as evaluation,analysis and data mining. Flexible, modular solutions for formulationdevelopment can be adapted to individual needs and process flows.The various modules feature a uniform technology base and can becombined as needed. Workflow managers help regu- late the labprocesses using standardised software. Specialised systems which canbe integrated into automated lab systems also support efficientworkflows in highly specialised research.

Solutions provided by companies such as Chemspeed Technologiesand Zinsser Analytic can be used for selective optimisation of labprocesses during the search for active ingredients as well as incombinatorial chemistry, screening and synthesis. In the case ofZinsser, this includes a high-throughput synthesiser capable of performing more than 800 liquid and solid syntheses in parallel anda peptide synthesiser for peptide libraries which the company claims

can process 864 peptides in 30 hours. One of the latest innovations informulation develop ment is a pipetting tool for liquid handling systems,which includes precision dosing capability for high viscosity substances.This feature is needed for sample preparation during the developmentof healthcare products, lubricants and polymers.

Particle size determination for multi-phase systemsA BASF robotic high-throughput screening system can be used for auto -mated particle size determination which is needed for development ofmulti-phase systems (emulsions and dispersions). The system supportsfully automatic screening of formulations including the constituentswhich characterise them. The objective is targeted, time-savingstatistical analysis of the recipe and product characteristics to supportefficient formulation development. The manufacturer claims thatintegrated particle size measurement on the system provides the basisfor highly accurate qualitative formula tion assessment. The process isas follows.

After experimental design, the dosing robots on the systemdispense the required amounts of selected compounds. The comp -ounds are homogenised by shaking, stirring, ultrasound or ultra-turraxin homogenisation stations. The samples are then in an optimal state for rheology (flow characteristics), stability and particle sizeanalysis. For particle size determination, a Beckman Coulter laserdiffraction analyser with patented PIDS technology (polarisationintensity differential scattering) is used, which is capable of very highresolution particle detection down to the submicron range. The dataobtained is automatically transferred to the lab information system(LIMS) where it is available for further analysis.



Date: 15 – 19 June 2015Location: Frankfurt am Main, GermanyMore information: www.achema.de

The SentroPAT series to be presented at AchemaAchema, the world leading exhibition for the chemical and pharmaceutical

industry, will once more become a platform where Sentronic will present the

SentroPAT series of NIR analysers as comprehensive solutions for process

analytical technology (PAT) and Quality by Design.

The SentroPAT system series is dedicated to solid dose manufacturing in

batch or continuous production lines. The application of PAT to typical

processes such as blending, granulation, drying, compression, capsule filling,

or other unit operations is realised from early R&D to routine operation and

real-time release testing.

All Sentronic solutions are fully dedicated to the needs of PAT in

pharmaceutical operations. From a GMP compliant design to validated

software, the SentroPAT series provides turnkey solutions. A variety of

possibilities for interfacing these systems (e.g., to SiPAT, PharmaMV and

SynTQ) and the ATEX certified sample interfaces enable the seamless

integration into new or existing production lines.

Due to years of innovation and continuous improvement processes,

today’s SentroPAT systems provide the following benefits:

■ Outstanding with respect to performance, reliability, and stability

■ Available with a unique multi-channel operation

■ Highest measurement rates and intelligent monitoring of data quality based

on diode array spectrometer technology

■ Advanced self-monitoring for longest possible maintenance free operation

■ Completed by a full life cycle documentation.

Sentronic was established in 1993 and is today a recognised partner for PAT

solutions in the pharmaceutical industry. With a wide range of experience and

know-how for all aspects of the implementation of process analytical

equipment in a regulated industry, Sentronic successfully serves many

pharmaceutical companies all over the world.

Creating Innovations

E20001-F40-T114-X-7600_AZ_Pharma_210x297_EN.indd 1 30.03.15 14:38

The main arguments in favour of continuous processing, according toJanet Woodcock, Director of the FDA’s Center for Drug Evaluation andResearch (CDER), are agility, flexibility, geography, quality, costs, and societal benefits1. Continuous manufacturing will permit increasedproduction volume without the current problems related to scale-upand also helps accelerate clinical development. Additionally, continu -ous manufacturing facilitates regional or in-country manufacture andscalable capacity in case of emergencies. It also improves uniformity ofproducts so that higher-quality drugs can be produced with less waste.Less waste and a better utilisation of resources also reduces theenvironmental impact.

With a view to these opportunities, experts expect that continuousprocesses will grow from 5% of business today to as much as 30% overthe next 10 years. At the same time, continuous manufacturing requiresa shift in mind-set on different levels within the pharmaceuticalindustry. It creates the need for a tighter integration between process,PAT, and control systems.

From proof of concept to production scaleAt the 2013 Continuous Bioprocessing Conference in Barcelona, Spain,an industry consortium initiated by Bayer Technology Servicespresented a proof of concept for a complete continuous bioprocess atlab scale. Since then, the concept has been developed further to cometo a production-scale application, including the matching systems forprocess control and PAT. The findings so far illustrate the significantpayoff of establishing continuous manufacturing in bioprocessing:continuous processes work as a closed production system, so there isno need to enclose equipment units in clean rooms and physicallyseparate the different processing steps from the general productionenvironment. This could result in savings of up to 70%2.

Upgrading processes and systems for continuous processingAs part of a consortium, Siemens provides the process controltechnology (based on the Simatic PCS 7 process control system) and thePAT software (based on SIPAT). Currently, Siemens is working with the partners in the project to create the required standardised

architecture for an integrated process control system for upstream,fermenter, and downstream processing. In continuous processing, theintegration of unit operations requires global coordination of the entireprocess flow. Continuous systems have to be equipped with a second-level software control system that supervises and aligns the operationsof the individual units. Both hardware and software should provide ahigh degree of automation, requiring minimal operator involvement.Another focus is on defining and developing a standard interface foreasy and efficient integration of equipment units from multiplemanufacturers, which is a prerequisite for modular continuous-production processes.

Maybe the biggest challenge is in developing suitable analysers tomonitor critical-to-quality data in process and in real time. To create a working solution while the available technology is developed toprovide adequate options for monitoring biological processes, theintegration of Siemens’ SIPAT software with rapid at-line, on-linesampling and analysis with integrated advanced modelling tools willprovide a basis for continuous quality verification and, ultimately, real-time release capability.

Continuous manufacturing: The next stepAlthough a large amount of research and development is still needed,continuous manufacturing is an essential part of meeting the growingneed for agile, efficient, local and high-quality biological production inthe pharmaceutical industry. It will help the industry achieveoperational excellence through a higher degree of automation,streamline drug development by offering a uniform platform for clinicaldevelopment and commercial production, and significantly reduceproduction footprint and capital expenditures.

MANUFACTURING SOLUTIONS

The pharmaceutical industry is currently dominated by batch processes. However, a continuous process offerssubstantial benefits – so substantial, in fact, that the US Food and Drug Administration (FDA) actively promotes thedevelopment and introduction of continuous process concepts in pharmaceutical manufacturing. Recently,companies have presented the first concepts for continuous production plants for commercial application in OralSolids Dosing Manufacturing. Now, concepts are being developed for Bioprocessing. A key factor for implementingsuch concepts in an actual production environment are integrated automation solutions and process analyticaltechnology (PAT) systems for in-line quality control.


© to

toja

ng19

77 /

Shut

ters

tock

.com

1. Janet Woodcock, “Modernizing Pharmaceutical Manufacturing – Continuous Manu -

facturing as a Key Enabler” (paper presented at MIT-CMAC International Symposium on

Continuous Manufacturing of Pharmaceuticals, Cambridge, MA, May 20, 2014)

2. Thomas Daszkowski, “Continuous Processing in Biotech Production: An Alternative to a

Modern Single Use, Batch, Facility?” (paper presented at Integrated Continuous

Biomanufacturing Conference, Castelldefels, Spain, October 20–24, 2013)

References

Ivo BackxManager, Business & Project Development for the Pharmaceutical Industry, Siemens

Workshop conceptRecent years have witnessed an expansion in the disciplinesencompassing drug discovery outside of the pharmaceutical industry. Asignificant number of universities worldwide now host infrastructuresuch as compound libraries and automated screening centres1-3. Anarchetypal small molecule drug discovery project will aim to identifychemical starting points that modify the functions of genes, cells, orbiochemical pathways. In some but not all instances, these functionsmay be linked to disease processes, and an opportunity therefore existsto further develop the chemical starting points into novel therapeuticagents. In small molecule drug discovery, the ultimate aim is to identifynew therapeutics, an activity that for reasons of high risk and cost hashistorically been conducted within the commercial sectors.

We are now witnessing a resurgence of cell-based assays includingphenotypic assays where a particular cellular change is monitored, insome cases without knowledge of the underlying target(s) upon whichcompounds are acting. There are some great successes from using theseapproaches for drug discovery4. It is interesting to note that in somecases where an efficacious compound was identified using a cell-basedassay, the target(s) it acts upon was successfully and subsequentlyidentified.4 However, compound efficacy may still be due to a poly-pharmacological effect whereby it acts upon additional targets that maystill be unidentified. Advances in cell-based assays are also being made,for example using human-induced pluripotent stem (iPS) cell-derivedcells that better recapitulate normal human biology compared totransformed cell lines and non-human primary cells. Some of these wereexplored in the workshop by way of lectures and practical work usingassay technologies from Biolog, PerkinElmer and Tecan.

Participant profile, learning objectives and contentThe workshop was designed for scientists at all levels (undergraduates,postgraduates and laboratory-based scientists within academic andindustrial research organisations) engaged in early-stage drug discoveryand with an interest in the development, validation and utilisation ofcell-based assays for screening against small molecule libraries. It wasequally well suited to technically-focused staff from core facilities orcontract research organisations who may wish to extend theirexpertise. The dinner on the first day offered an opportunity for theparticipants to network and establish relationships of mutual benefit.

The main learning objectives were to examine, by way of practical

sessions and lectures, the design and application of cell-based assaysfor small molecule screening campaigns in drug discovery. Allparticipants took part in the practical sessions, which involved thedevelopment of screening compatible assays, primary screening using asmall molecule library and the profiling of compounds in dose-response experiments. Important aspects of the course were:1) To discuss and demonstrate practically the appropriate steps in

selecting suitable assays in light of the fact that a multitude ofassay technologies are currently available for a given target.

2) How to select an appropriate assay technology and which criteriashould be examined during the drug discovery process.

3) Evaluating whether a generic, flexible set of assay methodologies orcustomised solutions should be applied to the targets beinginvestigated in small molecule cell-based screening campaigns.

Learning outcomes Upon completion of the course, participants gained an insight into thekey parameters to be considered when developing cell-based assaysand performing small molecule screening campaigns, associated dataanalysis and validation of Hits in suitable secondary assays.

The cell-based assays for screening workshop was approved by theSociety of Biology for purposes of Continuing Professional Development(CPD) and may be counted as 72 CPD credits if registered on the Societyof Biology CPD Scheme.

This workshop, organised by European Pharmaceutical Review, took place on 9-11 December 2014 at Fraunhofer-IME SP in Hamburg, Germany and had support from Biolog, Tecan and PerkinElmer.

WORKSHOP REVIEW

Cell-based assays for screening:a practical workshop


1. Frearson JA and Collie IT. HTS and hit finding in academia – from chemical genomics to

drug discovery. Drug Discov Today 2009;14:1150

2. Frye S, et al. US academic drug discovery. Drug Discov Today 2011;10:409

3. Baker M. Academic screening goes high-throughput. Nat Meth 2010;7:787

4. Swinney DC and Anthony J. How were new medicines discovered? Nat Rev Drug Discov

2011;10:507

References

Sheraz Gul is Head of Biology at Fraunhofer-IME SP,

Hamburg, Germany where he manages the assay develop -

ment and screening of academic targets. He has worked in

Big Pharma and academia and has experience in developing

biochemical and cellular assays for high-throughput

screening (HTS). His research interests are directed towards

maximising the impact of HTS for drug discovery. He is

responsible for all scientific aspects of the workshop. To contact Sheraz,

e-mail him at: [email protected]

UPCOMING WORKSHOPSDon’t miss the next two workshops taking part this year:

1. Cell-based assays for screening workshop (26-28 May 2015)2. Biochemical assays for screening workshop (24-26 November 2015)

If you would like to sponsor or attend this or another European Pharmaceutical Review workshop,

contact Andrew Johnson on +44 (0) 1959 563 311 or email [email protected]


WORKSHOP REVIEW

Biolog is a world leader in cell-based phenotypic testing technologiesand assays. Its technologies and products cost-effectively testmetabolic properties of cells (phenotypes) very simply and efficiently.The OmniLog® Phenotype MicroArray™ System’s custom cell-basedassays in five categories can determine up to 1400 metabolic andchemical sensitivity phenotypes of mammalian cells. Our products arecurrently in use world-wide by biotech and pharmaceutical companies;private, academic and government research institutions; as well asbioprocess and nutritional industries.

OMNILOG® Automated Incubator-ReaderThe OmniLog® Automated Incubator-Reader, when used with Biolog’sPhenotype MicroArray™ plates, offers low cost, simple and rapidcharacterisation of metabolically-related phenotypes. Its proprietarysoftware offers unique capabilities beyond conventional ‘readers’ toautomate the kinetic analysis and display of our cell-based metabolicassays. It is a true ‘random-access’ instrument permitting automatedincubation of up to 50 microplates (from multiple users and differentstart times and programs) at a user-specified temperature, withcontinuous collection of colorimetric assay data over time.

For phenotypic drug discovery, cell line quality control andauthentication, bioprocess improvement, liver and mitochondrialtoxicity, stem cells and differentiation, and many other applications, theOmniLog Phenotype MicoArray System offers a unique and cost-effective complement to other metabolomic technologies.

For more information visit: www.biolog.comand see page 72 for Biolog’s contribution to the workshop

Tecan offers a wide range of solutions to streamline cell-biologyworkflows, from standalone readers and microplate washers to fully-integrated platforms offering walkaway automation of cell-basedassays. From colony picking to 3D cell culture maintenance, Tecan workswith a number of expert partners to offer automated solutions to fitvirtually any workflow.

The Fluent workstation is a unique automation concept builtaround the application-specific needs of laboratories, delivering morecapacity and increased speed. Designed to take the complexity out ofcell biology research, Fluent brings together all the modules anddevices required for automation of these assays in a single efficient and easy-to-use platform. Even complex cell-based assays can be

automated using the system’s FluentControl™ software, freeingresearchers to focus on other tasks.

Tecan has partnered with a number of experts in cell-based assays –including HTRF® technology supplier Cisbio Bioassays, market leader inGPCR assays DiscoveRx, and leading contract research organisationFraunhofer IME ScreeningPort – to ensure exceptional versatility,walkaway operation and robust data. The result is a compact system thatoffers higher throughput, greater precision and excellent reliability,allowing researchers to concentrate on what really matters – the results.

Find out more at: www.tecan.com/fluentand see page 73 for Tecan’s contribution to the workshop

With a growing emphasis on translational insight, it is more importantthan ever to be able to examine the molecular mechanisms of diseaseand translate your in vitro models into in vivo results. PerkinElmer offersleading solutions and renowned expertise in assays, such asAlphaScreen®, multimode detection and imaging as well as informaticsthat will help you bring it all together. Whether working in a well, cells,tissue or small animals, now you can focus on your science, gain insightsooner and succeed faster.

EnSpire® Multimode Plate Reader is a high performance bench-topinstrument for biochemical and cell-based assays. It is the onlymultimode reader that provides a wide range of industry-leadinglabelled technologies and Corning® Epic® label-free technology for atruly versatile detection system.

EnSpire instrument offers best performance AlphaLISA®/AlphaScreen® detection for a no-wash, homogeneous format that isideal for cellular and protein:protein interaction assays. Ultra-sensitiveluminescence enables detection of just a few cells for when workingwith primary, stem or difficult-to-transfect cells. Quad-monochromatorfluorescence and absorbance offer detection with any wavelength forthe best possible signal to background. Time-resolved fluorescenceprovides high sensitivity for biological and cellular assays when sampleis at a premium or in low concentration. Industry standard Corning®Epic® label-free technology provides physiologically relevantinformation for a more complete picture.

EnSpire Multimode Plate Reader is the ideal solution for multi-userenvironments seeking best-in-class sensitivity, flexibility and ease-of-use in multimode detection.

Find out more at: www.perkinelmer.com/enspireand see page 74 for PerkinElmer’s contribution to the workshop

Workshop partners and products

The OmniLog® Phenotype MicroArray™ System employs cell-basedassay panels in a 96-well format to determine nearly 1500 metabolic andchemical sensitivity phenotypes of mammalian cells. Using acolorimetric redox dye assay, the technology measures pathway fluxesin living cells, enabling sensitive measurement of cellular responses toa genetic alteration or a chemical/drug challenge. This integratedsystem of assays, instrumentation and bioinformatics software revealsunique and insightful information on specific metabolic pathwayactivities and cellular sensitivities to nutrients, ions, hormones,cytokines and anti-cancer agents

Two complementary capabilities of the Phenotype MicroArraySystem were performed by workshop participants: (1) multiplexed dose-response assays to assess the toxicity of a mitochondrial inhibitor anda cytotoxic anti-cancer drug on multiple energy substrate pathways and (2) label-free multiplexed screening of a chemical library to detectcellular toxicity. Chemical toxicity is sensitively detected usingPhenotypic MicroArrays™ by measuring inhibition of dye reduction thatoccurs after cells are exposed to chemicals in different media.

Participants in the Workshop evaluated the effect of themitochondrial uncoupler FCCP and the anti-cancer drug daunorubicinon C3A liver cells while incubating in eight different media generated byBiolog PM-M TOX1 MicroPlates. The two chemicals displayed verydistinct patterns of inhibition as shown by examining the IC50concentrations in each of the eight media. The mitochondrial inhibitoris much more toxic to cells using mitochondria-requiring substratessuch as pyruvate and α-ketoglutarate compared to glucose and

inosine which utilise the glycolytic and pentose phosphate pathways(Figure 1 and 2). By contrast, the cytotoxic anti-cancer drug daunomycinshows similar chemical sensitivity profiles with all eight substrates(Figure 3). The simplicity of this assay format underscores the ease ingenerating an extensive database in which chemicals are characterisedfor their effects on cellular energetics, especially those targetingmitochondrial function. This can be done, not only for liver cells, butalso for cells representing other important organs or tissues.

In the second experiment (data not shown), C3A liver cells wereagain utilised to screen a compound library for cytotoxicity. Cells were harvested after trypsinisation, suspended in glucose-supplemented Biolog IF-M1 medium and dispensed into a 384-wellplate already containing a chemical library. After incubating for 20 hoursat 37°C under 5% CO2-95% air, Biolog Redox Dye MA was added and theplate incubated for 2 hours more before an endpoint measurement ofthe reduced tetrazolium was made by absorbance at 590 nm (lessabsorbance at 750 nm to correct for well-dependent light dispersions).A variety of chemicals that lowered dye reduction due to toxicity werepresumptively identified for three plates tested.

The workshop study with Biolog’s PM-M MicroPlates demonstrateda simple and highly sensitive assay technology to easily and cost-effectively screen and detect chemicals that are cytotoxic, as well asthose which specifically exhibit mitochondrial toxicity. Using Biolog’sproprietary redox dye chemistry that gives a simple colorimetricreadout in conjunction with a novel liver cell line with greater metabolicversatility than HepG2, one can profile the response of energygeneration pathways driven by 8 diverse carbon energy/substrates. Useof the OmniLog® Phenotype MicroArray™ Platform and kinetic analysissoftware to incubate read and record 50 MicroPlates at a time permitsaccurate high throughput quantitation of metabolic rates for detecting


WORKSHOP REVIEW

For more information, please visit:www.biolog.com

High content cell-based phenotypic assay technologyBiolog is a world leader in cell-based phenotypic testing technologies and assays. The company has focused itsefforts on developing technologies and products to cost-effectively test hundreds of properties of cells(phenotypes) very simply and efficiently and with a high degree of sensitivity.

Figure 1

Figure 2

Figure 3

Percent Rate of Redox Dye MB reduced by C3A cells after 12000 weredispensed in IF-M1 + 2mM GIn + PS + 1% DMSO + Indicated Supplement

and exposed to FCCP for 20h before adding Dye with 30mM Glucose

Percent Rate of Redox Dye MB reduced by C3A cells after 12000 weredispensed in IF-M1 + 2mM GIn + PS + 1% DMSO + Indicated Supplement

and exposed to Daunorubicin for 20h before adding Dye with 30mM Glucose


WORKSHOP REVIEW

Materials and methodsTwo cell lines (A549 and HepG2) were chosen to determine the toxicityof four compounds and demonstrate the capabilities of thisexperimental set-up for screening anti-cancer agents. The CellTiter-GloLuminescent Cell Viability Assay (Promega, USA) was used to determinethe effect of the compounds on the viability of the cells. The assay wasperformed in accordance with the manufacturer’s protocol.

The cell viability assay was run in a 384-well plate format. Testcompounds were serially diluted on the Fluent using the MultipleChannel Arm to create a dose-response stock plate, from which anintermediate plate was created, with compounds diluted in media toreduce DMSO levels in the final assay. The 384-well cell culture plates,pre-seeded just prior to the compound addition, were then transferredfrom the CO2 incubator to the Dynamic Deck by the Robotic Gripper Armand compounds are added by the Multiple Channel Arm.

After 24 hours incubation in the CO2 incubator, the CellTiter-Gloreagent was added to the assay plate by the Multiple Channel Arm,mixed and incubated in the dark for 10 minutes before reading theluminescent signal on the plate using the integrated Infinite® M1000PRO reader from Tecan.

Z prime and cell viability were then determined and dose responsecurves plotted.

ResultsZ prime was very high, especially for a cell-based assay, with valuesranging from 0.65 to 0.88.

Compounds 1 and 3 represent typical dose response curves withlower viability at high compound concentrations and high viability at low compound concentrations showing the cytotoxic effect of thesecompounds. Compounds 2 and 4, on the other hand, show an overallhigh viability at all concentrations, demonstrating that the compoundsare not toxic, but have a cytostatic effect (Figure 2).

ConclusionsAn industry standard assay format, CellTiter-Glo, was used for the cellviability measurement. Using the Fluent to perform cell-based assays in384-well plate format is a straightforward exercise. The workshopparticipants could easily start the pre-programmed assay fromFluentControl™ software through the integrated touch screen. Very high Z prime values confirm that this assay is running very stableon the Fluent platform. Furthermore, workshop participants could see that the Fluent with the integrated Infinite M1000PRO is perfectlysuited to run cell-based assays such as dose response experiments for screening.

It was shown that the rapid speed and low volume liquid handlingcapability of the Fluent are perfectly aligned with modern throughputand miniaturisation requirements.

For more information, please visit:www.tecan.com

Automated cell viability assay on theFluent™ cell-based assay workstationFluent is the latest in Tecan’s successful family of liquid handling automation platforms. The Fluent cell-based assaysolution offers rapid, high definition pipetting for both the eight-channel Flexible Channel Arm (FCA) and theMultiple Channel ArmTM 384 (MCA384). Its patented Dynamic DeckTM increases the worktable capacity and boostsproductivity by allowing integration of a wide range of Tecan modules – including a CarouselTM, for storage of various consumables; a HydroSpeed™ plate washer (not required for this assay); an Infinite® M1000 PRO platereader; and carriers for troughs, stacked disposable tips and microplates – as well as a third-party high capacity CO2 incubator (Figure 1).

Figure 1: The Fluent cell-based assay workstation. A Fluent 780 is shown,equipped with an eight-channel Flexible Channel Arm, a Multiple ChannelArm 384 and a Robotic Gripper Arm. A Carousel is integrated onto the right hand side of the instrument, along with the latest generation of CO2 incubator. An Infinite M1000 PRO microplate reader is located below theDynamic Deck. The dimensions of the compact system are indicated.

Figure 2: Cell viability (%) versus compound concentration for A549 andHepG2 cell lines. Cells were exposed to compounds for 24 hours.

260 cm / 102 in

190 cm / 75 in

WORKSHOP REVIEW


Simultaneous Detection of IL-6 and IL-8 Secretion by Cell Lines using AlphaPlex TechnologyCells react to various stimuli in the body by secreting modulator proteins called cytokines. These proteins bind tospecific receptors to generate a response from the targeted cell. These responses range from cell growth, mobility,to alterations in differentiation and function, and even cell death. Cytokines are involved in many pathologicalpathways, including inflammation and cancer. As such, they are interesting research targets.

Immunoassays are the primary method used to measure productionand modulation of cytokines by cells. However, the majority of thesetechnologies are work-intensive, require large amounts of sample andcan only analyse one cytokine per assay.

In the assays used in the workshop we showed how the AlphaPlex™Technology could be used to perform the analysis of two differentcytokines in the same sample. The combination of beads based oneuropium (AlphaLISA) and terbium (AlphaPlex 545) chemiluminescenceallows for a fast, homogenous assay using as little as 5μL of cellsupernatant. We demonstrated the value of this assay for the detection of both IL-6 and 8, key cancer biomarkers, from two cell linesstimulated by IL-1b.

Materials and methodsThe AlphaPlex assay for IL-6 and IL-8 contains the followingcomponents: Acceptor beads (europium) coated with an antihuman IL-8 antibody, Acceptor beads (terbium) coated with an anti-human IL-6antibody, biotinylated anti-IL-6 and anti-IL-8 antibodies, StreptavidinDonor beads, recombinant IL-6 and IL-8 and AlphaLISA immunoassaybuffer. The cell experiments used human colorectal adenocarcinomaHT-29 or human cervical cancer HeLa cells, DMEM culture media (with orwithout FBS) and human IL-1b for stimulation. Additional materialsincluded AlphaLISA immunoassay buffer.

For the cellular assay, the cells were plated in 96-well cell cultureplates at 80,000 to 625 cells per well. Each concentration was made intriplicate. The cells were left to adhere overnight. The cells were thenstarved by washing with PBS and replacing the media with serum-freeculture media overnight. Next, they were stimulated by washing withPBS then supplementing with serum-free culture media containing5ng/mL of IL-1b and overnight incubation. Finally, the cell supernatantwas harvested for the immunoassay.

ProtocolStock solutions of all the required reagents (analyte, Acceptor beads,biotinylated antibody, Donor beads) were prepared.

The following were mixed in final 1X AlphaLISA immunoassay bufferin three wells of a 384-well plate:� 5 μLof analyte (cell supernatant or recombinant IL-x) with 10μL of

AlphaLISA and AlphaPlex 545 anti-analyte Acceptor beads (25 μg/mL)

� 10μL of biotinylated anti-analyte antibody (2.5nM) (incubate for 60 minutes at 23°C)

� 25 μL Streptavidin Donor beads in each well (80 μg/mL (incubate for30 minutes at 23°C in the subdued light conditions)

� Read using EnVision® Multilabel Plate Reader (with appropri-ate filters)

ResultsData showed that the HT-29 cell line can produce high amounts of IL-8 even with a very low number of cells, but not any measurable IL-6.HeLa cells produce both cytokines at high levels with detectableamounts at 500 cells per well or below. Both cell lines were starved thenstimulated for 24 hours with 5ng/mL of IL-1b. Each cell line was testedwith its own standard curve to insure that the matrix of the standardcurve and the samples were the same.

SummaryHere, we demonstrate the feasibility and value of using AlphaLISA andAlphaPlex 545 beads to create a duplex immunoassay in a complexmatrix. This allows for more meaningful experiments with moreanalytes tested per well and more accurate quantification of the analytes as well as significant savings in time and cost (amounts of reagents and plates used). With this newly-developed assay, we have created standard curves for both target cytokines in a single wellwith high sensitivity, dynamic range and specificity.

The IL-6/IL-8 duplex assay identified specific cytokines generatedby two different cell lines, showing a very different secretion pattern for IL-6 between HT-29 (no measurable amounts) and HeLa (ng/mL amounts). IL-8 was secreted in large, comparable amounts byboth cell-lines.

Finally, the assay showed very high sensitivity, with the capacity towork at 500 cells per well or less, while detecting amounts of cytokineswell within the standard curve.

These various attributes make AlphaPlex technology a powerful andversatile method for measuring a variety of molecules and mediators ina complex biological setup.

For more information, please visit:www.perkinelmer.com

Extensive development on PAT in recent years provides a suitableplatform for a paradigm shift of QbD-based pharmaceuticalmanufacturing1-2. Today, commercially-available methods and toolsmake the application of PAT possible for real-time pharmaceuticalprocess control, to enable real-time product release within a highlyregulated environment. Moreover, recently developed continuouspharmaceutical manufacturing techniques involving solid dosage forms

catalytically accelerate this revolutionary shift by enabling both FF andFB control strategies.

The FF controller takes into account the effect of processdisturbances and raw material variability proactively while the FBcontrol system ensures consistency in end product quality. For FF control, the sensor (e.g., near infrared; NIR) should be placedupstream to measure the process disturbances and the measured

Continuous pharmaceutical manufacturing together with process analytical technology (PAT) provides a suitableplatform for automatic feed-forward/feed-back (FF/FB) control of the end product quality as desired by quality bydesign (QbD)-based efficient manufacturing. The precise control of the quality of the pharmaceutical product requiresproactive, corrective actions on the process/raw material variability. Therefore, PAT tools are necessary to monitorthe FF as well as FB process variables that need to be sent to the automatic real-time control system. This articlehighlights the scope of PAT in a combined FF/FB control system of a continuous tablet manufacturing process.

PAT SERIES

The scope of PAT in real-time advancedcontrol of tablet quality

Ravendra Singh, Marianthi Ierapetritou and Rohit RamachandranRutgers University


© T

riff /

Shu

tter

stoc

k.co

m

NIR process analyzer for GMP use

From R&D to RTRT

Robust and compact design

Proven Diode Array technology

Up to 4 channels fast multiplexed

Highest performance and stability

SentroPAT NIRfor batch and continuous processing

PAT-Solutionsfor Solid DoseManufacturing

For more information:

www.sentronic.euTel.: +49 351 871 8653 - [email protected]

Visit us at:

ACHEMA 2015 - Hall 4.2, Booth E80

signal should be transmitted in real time to downstream unitoperation to take the compensative actions. Variations in raw materialproperties (e.g., particle size), feeder hopper level, amount oflubrication and milling and blending action, applied shear in differentprocessing stages, can affect the blend density significantly andthereby also affect tablet weight, dissolution and hardness. Therefore,the inline real-time monitoring of the blend density and itsincorporation into the control system so that it does not affect the endproduct quality is highly desired3. For FB control, the sensor should beplaced at downstream unit operations to measure the critical qualityattributes (CQAs) and critical process parameters (CPPs) and based onthat the process parameters need to be manipulated to control themeasured variables.

In the past few years, very few attempts have been made tocontrol a tablet manu facturing process by utilising a FB controlalgorithm along with PAT tools4-5. But when such methods are used, itis important to note that FF control systems need to be coupled withthe FB control system for it to work effectively. The coupled FF/FBcontrol system ensures minimum variability in the final productquality irrespective of process and raw material variations and it is verysuccessful in different manufacturing industries. The FF control loop isbased on real-time monitoring of the powder bulk density while the FBcontrol loops are based on the drug concentration, powder level,tablet weight and hardness. We propose an NIR-based real-timemonitoring of the blend density for FF control.

The processA continuous direct compaction tablet manufacturing pilot-plant hasbeen installed and situated at C-SOPS, Rutgers University5. The processflowsheet model is shown in Figure 1 (page 78). To describe the set-up,there are three gravimetric feeders, a co-mill, a continuous blenderand a tablet press. The NIR sensor for inline monitoring of powderblend uniformity, powder blend composition and powder blenddensity has been integrated through a chute placed in between tabletpress and blender. The local level controllers are inbuilt in each feederin order to control the powder flow rate. A ratio controller has beenadded that provides the flow rate set points of API, Excipient andLubricant feeders for a given total flow rate and API composition. Six supervisory control loops have been then added.

The first loop is for PAT based FF control (FFC) which takes thecorrective action on variations in powder bulk density. Powder blend isfed to the tablet press die through a feed frame. For a specific filldepth and punch displacement settings, the variation in blend densitycan lead to variations in the tablet weight, hardness and dissolution.The powder blend density during continuous tablet manufacturingoperation can change at different processing units for several reasons.For example, the powder hopper level can change the powder bulkdensity. Powder particle size has significant effects on the powder bulk density. So if the raw material specification has been changed,then it can have significant effects on the final product quality if asuitable control strategy has not been implemented.

The shear level can also change the powder bulk density.Furthermore, the powder bulk density can alter during feedingoperation because of shear introduced by helix. Milling operation canalso change the powder bulk density because of change in particle size

VOLUME 20 ISSUE 2 2015 European Pharmaceutical Review

PAT SERIES

and applied shear force, while the blending operation changes the bulkdensity because of shear force. The bulk density can also change duringthe powder flow for several reasons; for example segregation andcompression. An NIR sensor together with chemometric tools can be used for real-time inline monitoring of the powder blend density. The signal of powder blend density is then sent to the feed-forward controller that manipulates the fill cam depth of the tablet press proactively.

The second loop has been added to control the main compression force of the tablet press. This control loop is in cascade arrangement with amaster controller (loop 3) specifically designed to control the tablet weight. The input of this mastercontroller is weight and it generates the set point of main compression force. Loop four has beendesigned to control the tablet hardness by manipu -lating the punch displacement. Checkmaster (Fette)has been used for real-time monitoring of tabletweight and hardness. Loop 5 has been added tocontrol the powder level in instrumented hopper anda webcam has been used for online monitoring of thepowder level. The drug concentration is controlledthrough the PAT-based 6th control loop, while the NIRsensor has been used for inline real-time monitoringof drug concentration. The powder bulk density anddrug concentration can be monitored using a single

NIR probe. Two PLS models have been used to predict the powder bulk density and drug concentration separately.

In order to implement the designed FF/FB control into the pilot-plant, the signal from the NIR sensor (raw spectrums) has to be sent to a chemometric tool that utilises the NIR calibrationmodels for blend density and drug concentration and a real-timeprediction tool generates the signals for the control variables in realtime. The generated signals are then sent to a commercially-available


PAT SERIES

Figure 1: Continuous tablet manufacturing process integrated with PAT and combined FF/FB control strategy. MPC: Model predictive control, PID: Proportional IntegralDerivative, FFC: Feed-forward control.

Figure 2: In-line monitoring of powder bulk density using NIR

control platform via an OPC (OLE process control)where the combined FF/FB control loop has beenimplemented. The blend density is the input for theFF controller while the blend composition togetherwith powder level, tablet weight and hardness are theinputs of the FB control system.

Real-time measurement of powder bulk density, drug concentration and blend uniformityNIR has in the past been used for the real-timemonitoring of powder bulk density, drug con -centration and blend uniformity, the latter two ofwhich have been previously reported4-6. Here, wedescribe real-time monitoring of powder bulk densityusing an NIR sensor. The first step in the process is to calibrate the NIR. In order to develop the NIRcalibration model, the spectrums need to be collected for powdersamples of different densities. The powder has been filled in agraduated cylinder placed on the top of atapping machine. Then the taps have beenapplied on the graduated cylinder.

Tapping changes the bulk density of thepowder and thereby the powder volume. Basedon the change in the volume, the referencevalues of the density can be calculated. The highest changes in densityare obtained for the first 20 taps. The spectrums have been collected foreach density. The PLS-based calibration model for density is developed

using a chemometric tool and an NIR sensor is integrated with thedirect compaction tablet manufacturing pilot-plant through a chute

interface. The calibration models for densityand drug concentration are then integratedwith a chamometric tool for real-timeprediction of density and concentration signals,respectively. Blend uniformity (relativestandard deviation) can then be calculated

from the drug concentration signals. A proprietary PAT data management tool (synTQ) is used for the

management of raw data and predicted signals. The data across

PAT SERIES

In order to develop the NIRcalibration model, the spectrums need

to be collected for powder samples of different densities

Figure 3: Effect of powder bulk density on tablet weight

different software tools communicate via OPC interface. In gravimetric mode of the operation of lose-in-weightfeeder, the powder flow rate is controlled by manipulatingthe feeder screw speed. So if there is variation in the powderbulk density then the screw speed needs to be adjustedautomatically in order to deliver the same mass flow rate ofthe powder. Therefore, the feeder screw speed is the primaryindication of the change in the powder bulk density.

Results and discussionThe real-time monitoring of powder bulk density along withfeeder screw speed is shown in Figure 2 (page 78). As shown inthe figure, the screw rotational speed first decreases and thenincreases indicating that the powder density slightly risesbefore dropping non-linearly. The figure demonstrates howthe change in bulk density affects the feeder screw rotationalspeed. The density measurement response is delayed incomparison to feeder screw speed response. This resultdemonstrates the proof of concept that NIR can be used forreal-time monitoring of powder bulk density.

The effect of powder bulk density on tablet weight isshown in Figure 3 (page 79). Step change in the powder bulkdensity has been introduced by switching the excipient fromAvicel 101 to Avicel 301 and back to Avicel 301. Avicel 301 is known to bedenser than Avicel 101. API (APAP) and lubricant (MgSt) remains same.

Figure 3 (page 79) shows that powder bulk density has a significantimpact on tablet weight. Similar effects have been observed on tablethardness as well. Therefore, the powder bulk density is critical tomonitor for real-time feed-forward control. It can be proposed that theeffect of density variation is compensated by adjustment of the filldepth and the results do demonstrate that the fill depth can bemanipulated to compensate the variation in density so that aconsistent tablet weight and hardness can be achieved.

ConclusionThis review highlights the scope of PAT for FF/FB control to obtain aprecise pre-defined end-product quality of a pharmaceutical product,as mandated by regulatory authorities. NIR has been used for real-timemonitoring of powder bulk density, drug concentration and blenduniformity. Future work includes the full implementation of an FF/FBcontrol system into our pilot-plant.

AcknowledgementsThis work is supported by the National Science Foundation EngineeringResearch Center on Structured Organic Particulate Systems, throughGrant NSF-ECC 0540855.


PAT SERIES

1. FDA. (2004). PAT—A framework for innovative pharmaceutical development,manufacturing, and quality assurance. http://www.fda.gov/downloads/Drugs/Guidances/ucm070305.pdf

2. FDA. (2007). Guidance for industry, Q8 (R2) pharmaceutical development.http://www.fda.gov/downloads/Drugs/Guidances/ucm073507.pdf

3. García-Munoz , S. Dolph, H. W. Ward II. Handling uncertainty in the establishment of adesign space for the manufacture of a pharmaceutical product. Computers and ChemicalEngineering 2010, 34, 1098-1107.

4. Singh, R.; Sahay, A.; Karry, K. M.; Muzzio, F.; Ierapetritou, M.; Ramachandran, R.Implementation of a hybrid MPC-PID control strategy using PAT tools into a directcompaction continuous pharmaceutical tablet manufacturing pilot-plant. InternationalJournal of Pharmaceutics 2014, 473, 38–54.

5. Singh, R.; Sahay, A.; Muzzio, F.; Ierapetritou, M.; Ramachandran, R. Systematicframework for onsite design and implementation of the control system in continuous tablet manufacturing process. Computers & Chemical Engineering Journal2014, 66, 186-200.

6. Vanarase, A.; Alcal, M.; Rozo, J.; Muzzio, F.; Romaach, R. Real-time monitoring of drugconcentration in a continuous powder mixing process using NIR spectroscopy. Chem.Eng. Sci. 2010, 65 (21), 5728 – 5733.

References

Ravendra Singh is Assistant Research Professor at the

Department of Chemical and Biochemical Engineering,

Rutgers University, USA. He is the recipient of a prestigious

EFCE Excellence Award given in Recognition of an

Outstanding PhD Thesis, from European Federation of

Chemical Engineering. He is a recognised scientist in the

field of continuous pharmaceutical manufacturing, PAT and

process control, and has published 40 research articles in international

journals and magazines, written two book chapters, has given several invited

industrial lectures and presented at over 50 international conferences. He is

lead guest editor of Journal of Chemistry special issue for PAT.

Marianthi Ierapetritou is a Professor and Department

chair of Chemical and Biochemical Engineering at Rutgers

University in Piscataway, New Jersey. She obtained her BS

from National Technical University in Athens, Greece

(magma cum laude), her PhD from Imperial College

(London, UK) in 1995 and subsequently completed post-

doctoral research at Princeton University (Princeton, NJ)

before joining Rutgers University in 1998. Among her accomplishments are

the Outstanding Faculty Award in 2012, the Rutgers Board of Trustees

Research Fellowship for Scholarly Excellence in 2004, and the prestigious

NSF CAREER award in 2000.

Rohit Ramachandran is currently an Assistant Professor at

the Department of Chemical & Biochemical Engineering,

Rutgers University, New Jersey, USA. He completed his

bachelors and masters in Chemical Engineering at the

National University of Singapore. This was followed by his

PhD in Chemical Engineering at the Centre of Process

Systems Engineering, Imperial College London. He then

undertook postdoctoral work at MIT in the Process Systems Engineering

Laboratory and the Novartis-MIT Center for Continuous Manufacturing.

His research interests include modeling, control and optimisation of

pharmaceutical and chemical processes.

© T

riff /

Shu

tter

stoc

k.co

m

GEA Process Engineering

Maximise bio-availability with spray drying

Throughout the global pharmaceutical industry, spray drying is recognised as an

exceptionally efficient and effective way to maximise bio-availability and minimise

production costs. And no one is further ahead of the game than GEA Process Engineering.

Our GEA Niro spray drying technology gives you unmatched control and flexibility over

particle engineering, with a single step continuous process that produces stable powders

with high purity profiles and specific properties, such as solubility, flowability and

dispersability. For better results on any scale, look to GEA Process Engineering.

engineering for a better world

GEA Process Engineering A/SGladsaxevej 305, DK-2860 Soeborg, Denmark

Phone: +45 39 54 54 54, Fax: +45 39 54 58 00

[email protected], www.gea.com

Frankfurt, Germany June 15-19 2015

Meet us at stand F46

in hall 4.0

2015

For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2015 BDXEUR9640-02

Reveal Stem Cell Diversity

BD [email protected]/eu

• Evaluate the stemness of cell cultures

• Assess the expression of differentiation markers

• Run functional assays on cell populations

• Isolate your preferred cell types using cell sorting

BD Biosciences supports stem cell researchers in obtaining results at single cell level using microscopy and fl ow cytometry by delivering best-in-class reagents and instruments..

For more information, visit bdbiosciences.com/eu/applications/research/stem-cell-research/m/1505552

stemcells outsourcing focus advice · handheld raman spectrometer, the bruker bravo. for more than...

Documents