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www.europeanpharmaceuticalreview.com Issue 5 · 2015 Combatting counterfeiters David Shore from Pfizer tells of his company’s progress in this illegal trade Dynamic vapour sorption of freeze-dried pharmaceuticals Claudia Kunz and Henning Gieseler, University of Erlangen, delve into this important drying technology Rapid Micro Methods In-depth articles from David Roesti and Erik Wilkens, Novartis, Hikma Pharmaceuticals’ Mostafa Eissa, and Hideharu Shintani, Chuo University

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Page 1: Rapid Micro - European Pharmaceutical Review › digital › epr...QC Insider Support – One-on-one guidance, software support, and more from our QC experts QC Insider™ Training

www.europeanpharmaceuticalreview.com Issue 5 · 2015

Combattingcounterfeiters

David Shore from Pfizer tells of hiscompany’s progress in this illegal trade

Dynamic vapour sorption offreeze-dried pharmaceuticals

Claudia Kunz and Henning Gieseler, University of Erlangen,delve into this important drying technology

Rapid MicroMethods In-depth articles from David Roesti and Erik Wilkens, Novartis, HikmaPharmaceuticals’ Mostafa Eissa, andHideharu Shintani, Chuo University

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Page 3: Rapid Micro - European Pharmaceutical Review › digital › epr...QC Insider Support – One-on-one guidance, software support, and more from our QC experts QC Insider™ Training

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 1

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 October 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–IME SPMatthew 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 Founder: Ian RussellManaging Director: Josh RussellEditor: Caroline RichardsEditorial Assistant: Stephanie AnthonyDigital Content Producer: Victoria WhiteProduction Manager: Brian ClokePublications Assistant: Sheila SkinnerPublisher: Graeme CathieSales Manager: Andrew JohnsonFront Cover Artwork: Steve Crisp

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

The pharmaceutical industry has long been blighted by drugs counterfeiters who not only riskpatients’ lives producing products that contain little or no active pharmaceutical ingredient,but threaten to undermine public trust in health systems. Thankfully, the industry is workingtirelessly with a wide range of stakeholders, including regulatory agencies, police andgovernments, to protect global drugs supplies. Pfizer summarises its efforts in the openingarticle of this issue of European Pharmaceutical Review on page 10; the company says it hasintercepted over 200 million spurious doses of its own medicines since 2004. If one (albeit thelargest) pharmaceutical company can report such startling figures, the statistics globallydon’t bear thinking about.

However, the World Health Organization believes that providing accurate statistics on theglobal scale of the problem is difficult. Sources of information vary, as do the methods usedto compile the data. Furthermore, counterfeiters are constantly adapting the methods theyuse to mimic drugs and finding new ways to evade detection. The incidence of fake medicinesalso varies hugely from country to country and even on a smaller scale, between rural andurban areas. It is therefore vital the industry stays one step ahead. “Cooperation is key”, Pfizerbelieves, and all those who play a part in getting drugs to patients must continue to find waysto intercept, deter and crack down on counterfeiters.

One way of doing so is by developing increasingly sophisticated techniques that exposecounterfeit drugs at their source. Using the top most counterfeited products (type 5phosphodiesterase inhibitors such as Viagra® (sildenafil)), as examples in her article in ourRaman In-Depth Focus on page 20, Sulaf Assi from Bournemouth University demonstrates theways in which handheld Raman techniques – using a combination of spectral evaluations – have been used to rapidly identify such products.

If you would like to get in touch, whether to suggest a feature article for a future issue ofEuropean Pharmaceutical Review or for anything else, you can contact me by email [email protected]. And to ensure you receive every issue of the magazine, you can subscribe at (www.europeanpharmaceuticalreview.com/subscribe); it’s free to do so. On our website, you can also find details of current and future issues, sector news and eventdetails. And find us on LinkedIn and Twitter – details are opposite.

Caroline RichardsEditor, European Pharmaceutical Review

Cracking downon counterfeits

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1 INTRODUCTIONCracking down on counterfeitsCaroline Richards, Editor, European Pharmaceutical Review

5 FOREWORDThe importance of good distribution practiceDavid Elder, GlaxoSmithKline and JPAG

6 NEWS9 EVENTS10 COUNTERFEIT DRUGS

Counterfeit medicines and the need for a global approachDavid Shore, Pfizer

14 REGULATORY INSIGHTNew third-party audit scheme for excipient suppliersIain Moore, President, EXCiPACT asbl

19 IN-DEPTH FOCUS: RAMANFeaturing articles from Sulaf Assi, BournemouthUniversity, Sulayman A. Oladepo, Hui Wang, David Barona and Reinhard Vehring, University of Alberta.Turn to page 34 for a Raman Roundtable

37 PAT SERIESContinuous manufacturing in pharma – an unstoppable trend?Bernhard Gutmann and Christian Oliver Kappe, University of Graz

43 SHOW PREVIEWFT Global Pharmaceutical andBiotechnology Conference

44 POLYMORPHSPromiscuous multicomponent drug crystalsMasataka Ito, Kiyohiko Sugano and Katsuhide Terada,Faculty of Pharmaceutical Sciences, Toho University

49 PRODUCT HUBMirus Bio

51 IN-DEPTH FOCUS: RMMFeaturing articles from David Roesti and Erik Wilkens,Novartis, Mostafa Eissa, Hikma Pharmaceuticals, andHideharu Shintani, Chuo University. A Product Profile with Eurofins Lancaster Labs can be found on page 67 and a RMM Roundtable starts on page 68

71 PARTICLE SIZINGMeeting biopharmaceutical analytical requirements for subvisibleparticle sizing and countingJohn Carpenter, University of Colorado, Amber Haynes Fradkin, KBI Biopharma, and Christina Vessely, Biologics Consulting Group Inc.

76 FREEZE DRYINGDynamic vapour sorption of freeze-dried pharmaceuticalsClaudia Kunz & Henning Gieseler, University of Erlangen

Contents

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 3

COMING UP IN THE NEXT ISSUE:

Published December 2015. Don’t miss out on your copy – subscribefor free today by visiting www.europeanpharmaceuticalreview.com

■ PAT In-Depth Focus■ Screening In-Depth Focus

■ Drug delivery series■ Single-use technology

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The Parenteral Drug Association Presents...

2015 PDA/FDAVaccines ConferenceThe New Vaccinology: Global Trends in Development, Manufacturing & Regulation

December 1-2, 2015 | Bethesda, MDBethesda North Marriott Hotel and Conference Center

Exhibition: December 1-2 | Courses: December 3-4

The 2015 PDA/FDA Vaccines Conference will deliver a global perspective on the rapidly evolving vaccine industry.

Come hear industry and regulatory experts address technical and regulatory challenges of development, showcase

innovative manufacturing approaches and how they are being applied, and explore how to effectively deliver new

vaccines to the global patient population.

NEW THIS YEAR! For the first time, PDA Europe will host the 2015 Europe Vaccines Conference concurrently,

December 1-2 in Berlin, Germany, and several presentations will be simulcast in real time between the two events. This

brand new, unique format will give participants a truly global experience.

Simulcast sessions include:

• The History and Future of Vaccines and their Impact will help participants understand how science and

technology have transformed research and the development of a wide array of new vaccines. In addition, the

impact new vaccines have on the patient population.

• Ebola Updates and Lessons Learned will present critical updates regarding Ebola and the current state of

developing a vaccine to address the epidemic. This session includes a must-hear presentation from the World Health

Organization and National Institutes of Health.

The Conferences in Berlin and Bethesda will also take an in-depth look at other “hot topics” in vaccines, including

technology transfer, analytical methods and vaccine manufacturing strategies (Bethesda); and fill/finish and drug

delivery systems, challenges in vaccines supply in developing countries and quality by design implementation (Berlin).

Be part of the global discussion about emerging trends in the development and manufacture of vaccines!

Register for the 2015 PDA/FDA Vaccines Conference by visiting pda.org/vaccines2015 or register for the

2015 Europe Vaccines Conference by visiting pda.org/vaccines.

Conference Theme: Focusing on Today’s Challenges to Deliver Tomorrow’s Vaccines

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One of the principle objectives of the Internal Conference onHarmonisation (ICH) initiatives was to introduce harmonisedapproaches, prevent duplication and eliminate wasteful andunnecessary testing. Although good progress was made initially, therewas evidence that certain countries, regions and trans-nationalorganisations were unhappy with some of the proposed harmonisedguidances, as exemplified by the withdrawal of ICH Q1F1.

Another area that has prompted concern within industry is theproliferation of importation testing or in-country testing. A recentarticle2 highlighted some of the practices and related issues. Based ona survey of six multi-national pharmaceutical companies under theauspices of International Federation of Pharmaceutical Manufacturersand Associations, a total of 184 data sets covering 149 countries werecollected over a three-month period in 20142. The survey assessedimport testing and other categories of country-specific re-testing, e.g., post-market surveillance or waivers. The participating companiesreported that 18,616 re-tests were requested per annum and this extratesting resulted in one additional batch failure, i.e., a rejection rate of0.005%2. This substantiates the view that this additional testing is notenhancing quality or safety benefits to the end user, provided that gooddistribution practice (GDP) and an appropriate control strategy isimplemented throughout the entire supply chain3,4.

It was estimated that the average delay caused by this additionaltesting was four weeks in duration, with a maximum delay of 22 weeksexperienced in China. Some countries, such as China, stipulate theminimum remaining shelf-life (RSL) should be 12 months5, hence a five-month-or-greater delay can significantly impact on the RSL,particularly for biologicals. In addition, the safety implications for theseadditional test samples need to be understood. Security/tamper-evident seals need to be breached to allow for additional testing, whichincreases the risk of batch contamination and/or samples being lost ordiverted (increasing counterfeit concerns). In contrast, all of these risksare significantly reduced when robust GDPs are in place6.

Re-testing typically covers the import of the product. However,testing can also cover the registration process (in conjunction with theoriginal approval, post-approval changes or license renewals) and theseare legal requirements in some countries. Registration testing oftenrequires the setting up of innovative analytical methodologies in aspecified country, which in turn can cause significant delays andadditional costs. Finally, surveillance testing is aimed at detectingfalsified or counterfeit medicines. Re-testing may be performed by localcompany subsidiaries, contract research organisations or governmentlaboratories. Additionally, the extra testing of a batch can often be

significant and extra local inventory/warehouse storage capacity isrequired. Increased costs (ca. €3000 for each imported batch) are also incurred. The estimated expense2 of local testing is about €20 million/annum. €38 million/annum is required for longer storage ofinventory at customs, so the total can reach €58 million/annum.

A survey conducted by the European Federation of PharmaceuticalIndustries and Associations in early 2015, which assessed the impact ofimport testing into the EU from the US, identified an impact on over8,000 product batches during 2014, equating to costs in excess of €25 million/annum2. Import testing can be waived4 when there is clearevidence that good manufacturing practice and GDP processes are inplace for the manufacture, packaging, testing, storage and distributionof pharmaceutical products, respectively. In addition, an effectivepharmaceutical quality system (as per ICH Q106), together with regularaudits/inspections by local and trans-national regulatory authoritiesshould be in place7,8.

To sum up, in-country testing potentially introduces additional risks tothe consumer, such as product contamination, reduced shelf-life due to delays and potential increased cost resulting in barriers to access.Additionally, there is no clear evidence to support the contention that import testing from a controlled distribution network plays anymeaningful role in detecting counterfeit products. Indeed, post-marketsurveillance is much better placed to provide an independent assessmentof the quality of medicines because it can pull samples directly from themarket and thus has greater relevance to the patient. Companiescomplying with GMP/GDP and using secure supply chains with justifiedcontrol strategies should be exempt from such import testing.

FOREWORD

Importation testing: an unnecessary burdenon industry?

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 5

Dave ElderGSK & JPAG

Karl EnnisGSK

1. Explanatory Note on the Withdrawal of ICH Q1F for the ICH Website.http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q1F/Q1F_Explanatory_Note.pdf. Accessed on 01st September, 2015

2. Garbe, J.H.O., Ennis, K., Furer, G.M., Jacobs, M.G., Roenninger, S.K. Import testing ofpharmaceurtical products has limited safety benefits and can add risk to patients.Pharmaceutical Technology Europe, Supplement to the August 2015 issue, S6-S20

3. European Commission, “Technical Barriers to Trade”, Initial EU TTIP Position Paper,16th July 2013

4. IFPMA. Appropriate Control Strategies Eliminate the Need for Redundant Testing ofPharmaceutical Products. 23rd April 2012 http://www.ifpma.org/uploads/media/IFPMA_Position_Paper_on_Redundant_Testing_05.pdf. Accessed on 01stSeptember, 2015

5. China State Food and Drug Administration (SFDA). Medical Product Import Regulation.Administration of import drug. SFDA Order No. 4. 2004

6. ICH Q10. Pharmaceutical quality systems. Current step 4, June 2008

7. IFPMA, 2010. Global GMP Inspection landscape – Industry point of view and the wayforward http://www.ifpma.org/fileadmin/content/Quality/Inspections/Foreign%20inspections%20IFPMA%20presentation%202010.pdf. Accessed on 02ndSeptember 2015.

8. EFPIA, 2014. Enhanced good manufacturing and good distribution practice (GMP/GDP)Inspection Efficiency http://www.efpia.eu/uploads/documents/EFPIA_Enhanced%20Inspection%20Practice%20-%20Final_v8a_19May2014.pdf. Accessed on 02ndSeptember 2015

References

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NEWS

6 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015 Catch up with daily news at www.europeanpharmaceuticalreview.com

WITEC

apyron – automated Raman imagingThe new intuitive WITec apyron automated

microscope system provides high Raman

signal sensitivity, outstanding spectral and

spatial resolution, and ultra-fast acquisition

times for 3D chemical imaging applications. It

features TruePower automated absolute laser

power determination and a new spectrometer

enabling challenging experiments at very low

light intensities surpassing any previous

standard of performance. The push-button

principle of the system has the advantage of

drastically reducing the time required to

become familiar with the operation of the

instrument, which accelerates the initiation of

measurements and increases the rate of sample

turnover greatly compared with conventional

systems. With the apyron’s straightforward

interface the user can concentrate entirely on

the experiment without being distracted by

complex operating procedures.

For the following groups of users, such a

system is particularly well suited:■ Multi-user laboratories with frequently

changing users or requirements;■ Industrial research laboratories with

frequently repeated experiments and

challenging requirements in terms of time-

critical implementation of measurements;■ Beginners in Raman microscopy with

demanding imaging tasks; and■ Raman specialists who rely on

high performance.

Confocal Raman Microscopy provides the ability

to non-invasively map chemical properties of

samples such as drug delivery systems or surface

coatings of medical devices at the highest

resolution. It is ideally suited to survey the

distribution of components within formulations,

characterise homogeneity of pharmaceutical

samples, determine the solid state of drug

substances and excipients, characterise contami -

nations and foreign particulates or analyse

polymorphism and crystallinity. With the apyronit is possible overcome the boundary between the

ease of use and ultimate capability in Raman

Imaging. The most intricate 3D Raman Imaging

experiments can be easily accomplished, leading

to a greatly improved data quality, ease of use

and productivity.

BRISTOL-MYERS SQUIBB

Bristol-Myers Squibb’s lung cancer drug,Opdivo, gains US approvalThe US Food and Drug Administration (FDA)

has approved Bristol-Myers Squibb’s Opdivo

(nivolumab) injection, for intravenous use in

patients with metastatic non-small cell lung

cancer (NSCLC) with progression on or after

platinum-based chemotherapy.

Opdivo targets the PD-1/PD-L1 pathway and

thus may help the body’s immune system fight

the cancer cells. In the CheckMate-057 Phase 3

trial, Opdivo demonstrated superior overall

survival (OS) in previously-treated metastatic

non-squamous NSCLC compared with chemo -

therapy, with a 27% reduction in the risk of death.

The median OS was 12.2 months in the Opdivo

arm and 9.4 months in the docetaxel arm. This

approval expands Opdivo’s indication for

previously-treated metastatic squamous NSCLC

to include the non-squamous patient population.

This approval is the third for Opdivo in the

United States this year. The drug is the only PD-

1 therapy to have been studied in a Phase 3 trial

of patients with previously-treated squamous

NSCLC and a separate Phase 3 trial of patients

with previously treated non-squamous NSCLC.

Biomarker testing is not required for Opdivo.

Merck’s drug Keytruda (pembrolizumab)

also targets the PD-1/PD-L1 pathway and was

recently granted accelerated approval for treating

NSCLC specifically for patients whose tumours

expressed PD-L1.

VERASTEM

Verastem halves its workforceVerastem has announced that it is to reduce its

workforce by approximately 50% to 20 full time

employees. As a result of the reduction in force

and associated costs, the firm estimates annual

savings of approximately $4.9 million USD in

cash operating expenses on a going-forward

basis, with estimated one-time severance and

related costs of approximately $825,000 over

the fourth quarter of 2015 and the first quarter

of 2016.

Verastem’s announcement comes just

weeks after the company had to abandon enrol -

ment in a Phase 2 study of its lead cancer drug,

VS-6063. The decision to stop enrolment

for futility followed a Data Safety Monitoring

Board (DSMB) review of a pre-planned int-

erim analysis. The results of the analysis

demonstrated that VS-6063 had a generally well

tolerated safety profile but that there was not a

sufficient level of efficacy to warrant

continuation of the study.

AMGEN

Amgen’s Proliabeats zoledronicacid in bonemineral density gain studyResults from a Phase 4 study of Amgen’s

Prolia (denosumab) showed that the

drug achieved greater gains in bone min-

eral density (BMD) than intravenous

bisphosphonate zoledronic acid in post -

menopausal women with osteoporosis

following previous treatment with oral

bisphosphonates.

The 12-month study included 643

women aged 55 years or older who had post -

menopausal osteoporosis and had been

taking oral bisphosphonate therapy

for two or more years. The women

were randomised 1:1 to receive either

subcutaneous denosumab every six months

plus intravenous placebo once yearly or

intravenous zoledronic acid once yearly plus

subcutaneous placebo every six months.

The change from baseline in lumbar spine

BMD at 12 months – the primary endpoint –

in the denosumab group was significantly

greater than that in the zoledronic acid

group. The denosumab group also had

significantly greater improvements than the

zoledronic acid group in secondary

and exploratory study endpoints, including

BMD changes in the total hip, femoral neck,

and 1/3 radius.

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Catch up with daily news at www.europeanpharmaceuticalreview.com VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 7

NEWS

TRACELINK

TraceLink Life Sciences Cloud now providesEU government reporting capabilities,enabling manufacturers to distributeserialised product in the European market

TraceLink is the world’s largest track and trace

network that connects the Life Sciences supply

chain and helps in eliminating counterfeit

prescription drugs from the global marketplace.

16 of the top 20 global pharma companies and

all of the top five global Biotech companies

trust TraceLink to improve the visibility and

traceability of pharmaceuticals from ingredient

to patient. A single point and click connec-

tion links any pharmaceutical company to

thousands of supply chain trading partners

to deliver the information, insight and

collaboration needed to comply with global

serialisation and regulatory requirements

anywhere in the world.

One of the latest innovations made to the

platform includes an EU Government

Reporting module that enables manu facturers

to interact with the European Medicines

Verification System hub. It supports specific

compliance workflows to generate EU

hub compliance reporting and allows cust -

omers to search and view submitted EU

compliance reports required by the EU hub. As

the EU finalises the specifics of the False

Medicines Directive (FMD) in the future, users

will be able to test the reporting functionality

against the latest EU requirements.

The latest advancements to the Life

Sciences Cloud expand the platform’s

integrated global serialisation and track and

trace capabilities, giving customers greater

flexibility in the way they meet different

compliance requirements in different countries.

It offers a fully integrated suite of applications

for commissioning and man aging serial

numbers, executing compliance workflows

for government reporting, generating data

reports for serial number analysis, and

utilising enterprise EPCIS data exchange –

eliminating the need for costly customisations.

TraceLink has engineered unprecedented serial

number read and write speeds of 75,000 per

second and 5,500 per second, respectively, all

while managing billions of serial numbers and

tens of billions of events annually, on a per-

company basis.

Contact us at +44 757 8800 989,

[email protected]

www.tracelink.com

ELI LILLY

Discontinued heart drug will cost Lilly up to $90 million Lilly’s expenses for the fourth quarter of 2015

will be hit by a research and development

expense of up to $90 million (pre-tax), after

it halted progression of the Phase III

cardiovascular drug, evacetrapid.

The firm was forced to accept the

recommendation of the independent data

monitoring committee to terminate the study

of the cholesterol esterase transfer protein

(CETP) inhibitor in patients with high-risk

atherosclerotic cardiovascular disease, due to

insufficient efficacy. The company will now

conclude other studies in the programme. The

independent data monitoring committee based

its recommendation on data from periodic data

reviews, which suggested there was a low

probability the study would achieve its primary

endpoint based on results to date.

“We’re obviously disappointed in this

outcome, as we hoped that evacetrapib would

offer an advance in treatment for people with

high-risk cardiovascular disease. We’ll be

working with investigators to appropriately

conclude these trials,” said David Ricks, Lilly

Senior Vice President and President of

Lilly Bio-Medicines. “We remain confident

in our pipeline as we prepare for launches in

other therapeutic areas with significant unmet

needs.” The Q4 charge works out as approxi -

mately $0.05 per share.

UNIVERSITY OF MANCHESTER

Eltrombopag shown to be effective in children with ITPA University of Manchester-led team of researchers have demonstrated the

relative safety and effectiveness of Promacta (eltrombo pag, Novartis) in

children with persistent or chronic immune thrombo cytopenia (ITP), an

autoimmune clotting disorder.

The GlaxoSmithKline-funded studies demonstrated that eltrombopag

was well tolerated and effective, consistently stabilising the platelet count

to over 50 X 109 per litre within two-to-six weeks for 40% of children

receiving the treatment, compared with no children on the placebo arm.

Four in every 100,000 children develop the disorder each year

globally. The symptoms of ITP include bleeding and bruising more easily.

Frequent nose bleeds and bleeding from the gums can be common, and

bruising often appears as purple patches or tiny red spots on the skin.

On rare occasions bleeding can be life threatening. The condition may

resolve by itself, but for one in every four of the affected children, it

becomes chronic, persisting after primary intervention and lasting for more

than twelve months.

Historically, second-line treatment options for children with ITP

have been scarce and one of the earliest options, surgical removal of the

spleen (splenectomy), was associated with a high risk of sepsis and

thrombosis. A better understanding about the underlying cause of ITP led

to the development of the use of newer immunosuppressant agents,

including rituximab.

The findings are published in The Lancet and The Lancet Haematology.

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ROCHE

Ocrelizumab signficantly reduces disability progression in MSRoche has announced positive data from three

Phase III studies of ocrelizumab in people with

relapsing multiple sclerosis (MS) and primary

progressive multiple sclerosis (PPMS).

Ocrelizumab is an investigational, human -

ised monoclonal antibody designed to selectively

target CD20-positive B cells. Data from the

OPERA I and OPERA II studies in people with

relapsing MS showed ocrelizumab was superior

to interferon beta-1a (Rebif) in reducing the three

major markers of disease activity over the two-

year controlled treatment period.

In a separate study (called ORATORIO) in

people with PPMS, ocrelizumab significantly

reduced the progression of clinical disability,

which was sustained for at least 12 weeks (the

primary endpoint) and 24 weeks (a second-

ary endpoint) compared with placebo.

Additionally, the study met other secondary

endpoints of reducing the time required to walk

25 feet, the volume of chronic inflammatory

brain lesions, and brain volume loss.

Roche plans to pursue marketing auth -

orisa tion for ocrelizumab in relapsing MS and

in PPMS. Data from the ocrelizumab OPERA I

and OPERA II studies and from the

ORATORIO study will be submitted to global

regulatory authorities in early 2016.

8 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015 Catch up with daily news at www.europeanpharmaceuticalreview.com

NEWS

NICE

NICE gives go-ahead for melanoma drug KeytrudaThe UK’s National Institute for Health and Care

Excellence (NICE) has recommended Merck’s

Keytruda (pembrolizumab) for treating advanced

skin cancer in final draft guidance.

This follows final guidance from NICE that

recommended Keytruda as an option for the

treatment of advanced melanoma following

disease progression with ipilimumab. This new

recommendation is for treating advanced

melanoma that has not been previously treated

with ipilimumab.

Professor Gillian Leng, Deputy Chief

Executive at NICE, said, “The incidence of

malignant melanoma has increased fivefold

since the mid-1970s in the UK, with around

37 new cases diagnosed every day. We are,

therefore, pleased to be able to recommend

pembrolizumab as an option for some people

with the disease that have not been previously

treated with ipilimumab, in final draft guidance.

I am sure this will be welcome news to

patients and healthcare professionals alike.”

Since the first approval in the United States just

more than a year ago, Keytruda has been

approved in 39 countries, including throughout

the European Union.

KAISER OPTICAL SYSTEMS, INC.

Analyses incontinuousmanufacturing – a solutions-based approachAn important consideration in successful

continuous manufacturing is integrating

analytical tools into the reaction flow.

In batch reaction monitoring, on-line and

at-line analyses enable Quality by Design

(QbD) and ensure stable operations. There is

a clear need for rugged and validated

analytical tools which will meet the specific

needs for continuous reaction monitoring.

Intense reaction conditions, non-traditional

chemistries and miniatur ised reactors of

continuous reactions are challenging

environments for analytical tools originally

developed for batch reaction monitor-

ing. With their complete solution-based

approach, Kaiser Optical Systems Inc. has

demonstrated success for in-flow Raman

analysis which en ables continuous reaction

monitoring, in situ solids analysis, and

precision in reaction control.

Kaiser’s process Raman solutions

feature a fiber-optic technology platform

which provides sampling versatility and

remote monitoring. Our solutions demon -

strate excellent scalability and model

transferability, and are compatible with

microreactor, laboratory, scale-up, pilot-plant

to production settings. Our systems are

deployed in GLP and GMP environments.

These features can be exploited to control

flow processes involving solids or liquids

in real time.

Our solutions-focused approach

provides information-rich data on both liquid

and solid phases in situ, via our suite of

phase-optimised sampling probes and optics.

We are proven leaders in monitor ing

reactions under intense conditions that are

used in continuous manufacturing. We

feature applications in high pressure and high

temperature reactions, exotic chemistries and

reactive chemical streams. We have also

addressed continuous solids monitoring and

solid phase unit operations including in situcontrol of crystallisation and polymorphism,

process-induced transformations, low-dose

formulations and tablet coating.

Kaiser process monitoring solutions

enable QbD and continuous process control,

which opens a new realm of opportuni-

ties for process developers. Our solutions,

featuring the RamanRXN2™ Hybrid, have set

the standard for quantitative and validated

analyses in laboratory and manufactur-

ing environments.

SMC

Scottish patients to benefit from a raft of new medicinesThe Scottish Medicines Consortium (SMC) has accepted six new drugs for routine use by NHS

Scotland. Four of these, Herceptin® (trastuzumab) for stomach cancer, Xofigo® (radium 223) and

Zytiga® (abiraterone) for prostate cancer, and Ofev® (nintedanib) for idiopathic pulmonary fibrosis,

were accepted after consideration under the SMC’s ‘Patient and Clinician Engagement’ process,

which aims to improve patient access to new medicines for the treatment of end-of-life and very

rare conditions.

The SMC also accepted Ikervis® (ciclosporin eye drops) for severe keratitis and Xultophy®

(insulin degludec / liraglutide) for diabetes. However, the SMC was unable to recommend

Afinitor® (everolimus) for breast cancer, which was also considered under the PACE process, due

to uncertainties surrounding the overall clinical benefit the medicine would provide for patients at

the end of their lives.

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NOVEMBER 2015Cancer Genomics 2015Date: 1 – 4 November Location: Heidelberg, Germanye: [email protected]: www.embl.de

Serialisation & TraceabilityDate: 2 – 4 November Location: Geneva, Switzerlandw: www.pharaserialisation.com

8th Euro Global Summit on Cancer TherapyDate: 3 – 5 November Location: Valencia, Spaine: [email protected]: www.cancer.global-summit.com/europe

Cell & Gene Therapy CongressDate: 9 – 10 November Location: London, UKe: [email protected]: www.celltherapy-congress.com/download-agenda-marketing

4th Annual Cell Culture & Bioprocessing CongressDate: 9 – 11 NovemberLocation: Londone: [email protected]: www.cellculture-congress.com

Genome Editing CongressDate: 12 – 13 November Location: Londone: [email protected]: www.genomeediting-congress.com

7th Annual Next GeneralSequencing ConferenceDate: 12 – 13 NovemberLocation: Londone: [email protected]: www.nextgenerationswquencing-congress.com

3rd Annual Single CellAnalysis Asia CongressDate: 12 – 13 NovemberLocation: Londone: [email protected]: www.singlecell-congress.com

Biotech & Money New YorkDate: 17 NovemberLocation: New York, USAe: [email protected]: www.biotechandmoney.com

DECEMBER 201510th Annual Cold ChainDistribution Conference & ExhibitionDate: 3 – 4 DecemberLocation: Victoria Park Plaza, Londone: [email protected]: www.coldchaing-distribution.com/EPR

JPAG symposium:maximising productivity in pharmaceutical QC and stability testingDate: 10 December Location: Royal Society of Chemistry, Londone: [email protected]: www.jpag.org/65

JANUARY 2016PharmaceuticalMicrobiology 2016Date: 20 – 21 January Location: London, UKe: [email protected]: www.pharma-microbiology.com/epr

SLAS 2016Date: 23 – 27 JanuaryLocation: San Diego, Californiaw: www.slas2016.org

FEBRUARY 201611th Annual Biomarkers CongressDate: 25 – 26 February Location: Manchester, UKe: [email protected]: www.biomarkers-congress.com

MARCH 2016Genetics in ForensicsDate: 14 – 15 March Location: London, UKe: [email protected]: www.forensicgenetics-congress.com

HPLC Congress 2016Date: 17 – 18 March 2016Location: London, UKe: [email protected]: www.hplc.conferenceseries.com

APRIL 20163rd Annual Peptides CongressDate: 18 – 19 AprilLocation: London, UKe: [email protected]: www.peptides-congress.com

POWTECH 2016Date: 19 – 21 April Location: Nuremberg, Germanyw: www.powtech.de

MAY 2016Analytica 2016Date: 10 – 13 MayLocation: Munich, Germanyw: www.analytica.de

PHARMA EVENTS

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 9

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

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Defining counterfeit medicinesThe World Health Organization (WHO) defines a counterfeit drug as “one which is deliberately and fraudulently mislabelled with respect toidentity and/or source. Counterfeiting can apply to both branded andgeneric products and counterfeit products may include products withthe correct ingredients or with the wrong ingredients, without activeingredients, with insufficient active ingredients or with fake packaging2.”

The manufacturing and distribution of counterfeit medication is acriminal activity in most countries and threatens the safety of theworld’s medicine supply. Medicines have become an attractive targetfor counterfeiters because they can be of high value and in strong

demand. Illegitimate medicinal products can also be producedrelatively cheaply. However, counterfeiters have shown to be negligentand make medications in unsanitary environments using cheap andtoxic substitutes. They keep their margins and profits significantly highby ignoring good manufacturing practices (GMP) standards. Theirproduction and distribution do not abide by regulatory authorityguidelines, which means that the adverse reactions from thesemedicines cannot be monitored. Most counterfeit medicines aremanufactured in unlicensed and unregulated sites, and their contentand quality is not regulated.

There are two main ways in which a counterfeit medicine can reach

Counterfeit medicines pose a serious risk to public health around the world. The trade in fake drugs is considerable;according to Pfizer’s own company records, more than 200 million counterfeit doses of Pfizer product have beenintercepted since 2004. Meanwhile, the issue of counterfeits has no single or simple solution1 and cannot beeliminated by any one individual, organisation or government. It is a global problem that needs а global,collaborative approach.

COUNTERFEIT DRUGS

Counterfeit medicinesand the need for a global approach

David ShorePfizer

10 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

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patients. They can purchase such drugs illegally from illicit sourcesincluding online pharmacies, or they can approach legitimate localpharmacies or online suppliers and unwittingly buy fake products from them3. Patients can avoid exposure to counterfeits if they knowtheir medicine. When they receive a refilled prescription, they shouldnote if there is any difference in colour, shape, size and taste, look for changes in the packaging or any evidence of tampering, and contact their physician or pharmacist if any of these counterfeitindicators are present4.

Counterfeit drugs have been shown to contain too much, too littleor none of the active ingredients of the legitimate medicine, withvarying consequences depending on the disease. They are thereforeinferior to the genuine medicines they copy in terms of safety, efficacyand quality, and their lack of active ingredients can negate any

therapeutic gains one can get from them. Some counterfeits – thosewith a low level of active ingredients – may be able to tackle weakerdiseases but the stronger strains may develop resistance to the drug.This means that the situation can be exacerbated as resistance togenuine medicines also increases. If the disease spreads across largepopulations and builds resistance as it progresses, this could reducethe efficacy of entire medicine classes.

On occasion, ingredients of counterfeit drugs have contributed topatient deaths5. Laboratory tests have shown them to contain toxicingredients, such as heavy metals, arsenic, boric acid and otherdangerous ingredients such as brick dust, floor wax, leaded paint, talcand cartridge ink. Pfizer takes the issues associated with falsified drugsvery seriously. The company has confirmed that its products have beencounterfeited in at least 109 countries and at least 78 Pfizer medicines,including treatments for erectile dysfunction (ED), pain, Alzheimer’sdisease, cardiovascular conditions and cancers have been detected asbeing fake. Many of these are lifesaving medicines.

Since 2004, working with enforcement authorities around the world, Pfizer Global Security has prevented almost 204 million illegiti -mate doses of Pfizer products from reaching patients, including over 108 million counterfeit finished doses (Pfizer tablets, capsules and vials) and enough active pharmaceutical ingredient to manufacturemore than 95.94 million additional tablets.

The global differences of an illicit marketEvidence of the impact of fake medicines has been seen in bothdeveloped and developing markets, although there are some broaddifferences in how each market operates. Counterfeiting has beenshown to be a concerning issue in developed markets6. Pfizer’s ownstatistics support this – during 2014, authorities from 22 Europeancountries reported the seizure of almost 1.36 million doses offraudulent Pfizer medicines. The illegitimate trade has made wide -

COUNTERFEIT DRUGS

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 11

The Viagra manufactured at this site in China was distributed over the Internet by a network that included hundreds of brokers in the US and EU.

In Operation Cross Ocean, a referral from Pfizer Global Security, Chinese authorities seized 10 lines of manufacturing equipment and counterfeit medicines

valued at $4.3million (USD)

Manufacturing site for production of counterfeit Lipitor in Pakistan.

An investigation initiated by Global Security identified a network responsible

for manufacturing and counterfeiting these tablets. The matter was referred

to authorities, who raided the manufacturing site and a second site where

counterfeit labels were produced. Lab analysis determined that the counterfeit

tablets contained no active pharmaceutical ingredient.

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spread use of the Internet to sell these drugs to patients and the WHOhas revealed that in over half of cases, medicines purchased over theInternet from illegal sites that conceal their physical address have beenfound to be copies of the real thing. While many countries seek tovalidate online pharmacies, the prevalence of unregulated web-basedservices remains. In the United States alone, where online pharmacieshave to be certified by the National Association of Boards of Pharmacy(NABP), a 2014 report revealed that from 10,750 online pharmacies,10,392 (97%) did not comply with NABP patient safety and pharmacypractice standards, or state and federal laws.

In emerging markets, while online trade exists, the ‘real world’ or‘street’ trade is often more apparent. Combined with a less welldeveloped trade and regulatory framework, the environment is at greater risk of falsified medicines reaching a broader population. For example, according to ‘International Policy Network’ research, 40 to 50% of the total supply of medicines to Pakistan and Nigeria could be counterfeit. Meanwhile, 36% of antibiotics and anti-malarialdrugs in Nigeria and Thailand have been reported as substandard. The same study reflected upon the prevalence of counterfeit medicinesin the Philippines, including anti-asthmatic drugs, anti-diarrhoeals,anti-hypertensive drugs, analgesics and vitamins7. According to the WTO, imitation anti-malaria medicine kills 100,000 Africans a year8.

Collaboration is keyCooperation between stakeholders including governments, healthprofessionals, regulatory authorities, police, customs services and thejudiciary is very important in combatting the counterfeit market andenforcing legislation. Governments should also be encouraged to introduce appropriatelegislations that form the basis for greaterregulation and can allow for proper control of medicines.

A minority of WHO member states havepassed deterrent laws that address the issue of counterfeiting andwhich penalise offenders appropriately6. Currently, only 20% of the 191 WHO member states seem to implement strong drug regulation,whilst 50% have drug regulations at rudimentary levels, and theremaining 30% have little to no drug regulation implemented9. Ensuringthat deterrent anti-counterfeiting legislation has been passed and isbeing enforced would help in decreasing the current proliferation ofcounterfeit medicines in the global market.

Others in the health system can help prevent the distribution ofbogus drugs. While some national pharmacy networks employ

electronic systems to track medicines and add integrity to the system,these are not universally available. More work can also be undertaken tosupport pharmacists, educating them further on how to recognisecounterfeits and manage suspect consignments when they arediscovered10. In addition, where possible, front-line professionals –pharmacists, doctors and others who have direct dealings with patientsand carers – should be equipped to educate the public on the dangersof acquiring medicines from illicit sources, and on the risks such drugsmay pose to health. There are numerous examples of public awarenessefforts taking place around the world; pharmacy trade associations,government agencies, consumer and patient groups, and otherstakeholders in public health have all contributed – but these are oftenlimited in duration and by geographic scope.

Equally important is the support of the pharmaceutical industry.The industry continuously works to improve thepresentation of a medicinal product to make it more challenging to counterfeit, usingtechnologies like tamper-evident packagingand holographic- or colour-shifting ink markers,all of which are intended to help supply chain

partners identify legitimate medicines. In addition, Pfizer employs an experienced network of security professionals to help identify illicit trading. This group provides intelligence to national and inter -national authorities and supports investigations and seizures ofcounterfeit product.

This long-term effort has led to some high-profile successes. For example, Pfizer worked with the UAE authorities to seize over601,000 counterfeit ED medications in the country through a series ofraids in 2014. The raids helped expose a counterfeiting network thatsupplied counterfeits from China for distribution in the Gulf States.Acting on information provided by Pfizer’s Global Security team,authorities in the UAE arrested the perpetrator who was first identifiedas a key member of the criminal enterprise in 2007, attempting tosmuggle counterfeit cardiovascular medicines from China to Dubai forfurther distribution throughout the Gulf States. This successfuloperation relied on evidence from separate investigations in China,Jordan, Romania, the UAE and the US.

In general, Pfizer has in place a focused and aggressive anti-counterfeiting program in which it develops leads and refers them toauthorities around the world for their action. In addition, Pfizer hasprovided training to customs and regulatory authorities in 149 countriesto assist law enforcement in preventing fake medicines from reaching

12 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

COUNTERFEIT DRUGS

Counterfeit Lipitor found in Pfizer’s supply chain in Libya contained an

incorrect statin, delivering a sub-therapeutic dosage to patients

Counterfeit Lipitor, found in Pfizer’s supply chain in the UAE, contained

primarily acetaminophen, an API found in pain relievers

Counterfeit drugs have been shown to contain too much, too little or

none of the active ingredients of thelegitimate medicine

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patients – and the company is committed to continuing its work in allaspects of this collaborative effort.

Future opportunity in emerging marketsThe supply and distribution of medicines is complex, and it is evident that counterfeiters have been able to breach the legitimatesupply chain on a number of occasions, as well as continue to supplysubstandard product via a black market in many countries. As developing economies mature it would be prudent to considerintroducing longer-term measures intended to stem the illicit trade.Cooperation between national bodies and their healthcareorganisations is key to this. The WHO has suggested a collaborativeapproach should include developing common strategies, timelyexchange of information and harmonisation of measures to prevent thespread of falsified drugs, and ultimately a more advanced plan couldinclude the development of an international convention to limit tradein counterfeit and substandard drugs11.

In order to support this, the pharmaceutical industry must continueto invest in improving access to medicines. Pfizer, for example, is

aggressively monitoring its supply chain, identifying and disrupting key suppliers of counterfeit medicines, and fostering collabora-tions with distributors, health professionals, law enforcement agencies and regulatory bodies to ensure integrity of its medicines is protected so that patients can benefit from the medical innova-tions it offers.

COUNTERFEIT DRUGS

1. http://www.reuters.com/article/2010/06/10/us-customs-drugs-idUSTRE65961U20100610

2. http://www.who.int/medicines/services/counterfeit/overview/en/

3. http://www.stimson.org/images/uploads/research-pdfs/

Full_-_Counterfeit_Drugs_and_National_Security.pdf

4. http://www.awarerx.org/get-informed/safe-acquisition/counterfeit-medications#how

5. Paul N. Newton et al., “Manslaughter by Fake Artesunate in Asia--Will Africa Be Next?”

PLoS Medicine 3, no. 6 (June 2006), available at http://dx.doi.org/10.1371/journal.

pmed.0030197 (accessed June 6, 2007).

6. Lancet, 2012, “Counterfeit Drugs: A Growing Global Threat”, 379(9817):685. Lybecker,

K.M., 2004, “Economics of Reimportation and Risks of Counterfeit Pharmaceuticals”,

Managed Care, 13(3):3-10.

7. http://counterfeiting.unicri.it/docs/Ctf%20medicines%20in%20less%20

developed%20countries.pdf

8. http://www.reuters.com/article/2010/06/10/us-customs-drugs-idUSTRE65961U20100610

9. http://www.who.int/medicines/services/counterfeit/overview/en/index1.html

10. https://www.fip.org/files/fip/news/Counterfeits_Chambliss_2012.pdf

11. http://counterfeiting.unicri.it/docs/Ctf%20medicines%20in%20less%20

developed%20countries.pdf

References

David Shore leads the Europe, Middle East and Africa regional Global

Security team for Pfizer, the world’s largest pharmaceutical company.

The remit of this team of security professionals is to protect the company’s

personnel; real and intellectual property; reputation; and the integrity of its

medicines. A primary concern is the threat that counterfeit medicines pose to

patient health and safety, and David oversees and directs the investigation

and resolution of criminal actsand allegations affecting Pfizer, including

halting the manufacture and distribution of counterfeit and illegal medicines.

Prior to joining Pfizer in 2005, David was employed for 20 years as

a detective on various units within the Metropolitan Police Service.

The majority of his police career was spent within dedicated teams investi -

gating major and organised crime. These included: the National Crime

Squad; the Regional Crime Squad; the Central London Crime Squad;

various Murder Squads; Drug Squads; and finally – Special Branch.

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In Europe, the Falsified Medicines Directive (FMD)1 was developed as aresult of product failures in legitimate supply chains in the early 2000sand had the objective of ensuring the availability of high quality andsafe medicines. Although many of the falsified medicines have involvedfinal dosage forms, starting materials have also been impacted, notablywith sub-standard active pharmaceutical ingredients (APIs) and someexcipients entering the supply chain. The notorious and tragic incidentsinvolving ethylene glycol substitution for glycerine and then adulteratedHeparin resulted in hundreds of patient deaths.

The FMD introduced the legal definition of a pharmaceuticalexcipient for the first time as being: “any constituent of a medicinalproduct other than the active substance and the packaging material”. It also requires that the manufacturer ensures the excipients aresuitable for use through the adoption of a risk assessment approach todetermine the required good manufacturing practices (GMP) for themanufacture of the excipient.

Some Guidelines2 on the risk assessment referred to in the FMDhave now been published by the European Commission and these detailthe methodology to be used to ascertain the GMP required forexcipients. The process involves an examination of the role of the

excipient in the drug product as well as the direct threats posed topatient safety. The route of administration and related patient safetyfactors are then used to determine an overall risk ranking. TheCommission recommends that the specific GMPs required to assure theexcipient is made with a high degree of assurance are then defined.

Once these steps are completed, existing suppliers can be assessedagainst the GMPs, noting their overall quality performance and anycertification held by the supplier. A key requirement is that the dataneeded to make the evaluation is obtained “through audit or frominformation received from the excipient manufacturer”. The guidelinegoes on to note that: “certification of quality systems and/or GMP heldby the excipient manufacturer and the standards against which thesehave been granted should be considered as such certification may fulfilthe requirements”. On conclusion of the process, the pharmaceuticalmanufacturer will then have to identify the residual risks and takemeasures to control them, for example, by increased testing or evenseeking another supplier.

So, the FMD and related guidelines now require that pharma -ceutical manufacturers have detailed knowledge about their startingmaterials, where they come from and the standards to which they are

There has always been a regulatory requirement for pharmaceutical manufacturers to audit their starting materialsuppliers, but the expectations are even clearer now that these audits, including those for excipients, have to be in vivo. With increasing requirements for physical audits, can all pharmaceutical companies address the number ofaudits within a realistic frequency? Equally, suppliers of excipients to their many pharmaceutical customers face anavalanche of audits. With both sides having limited resources is there a third-party audit solution which meetsregulatory expectations and is efficient in resources?

REGULATORY INSIGHT

New third-party audit schemefor excipient suppliers

Iain MoorePresident, EXCiPACT asbl

14 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

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made, with the expectation in law for the first time that excipients are made in accordance with GMP principles. The logical method toverify that the GMPs have been implemented is to conduct an on-siteaudit. However, current practices for the majority of pharmaceuticalmanufacturers are to qualify their excipient suppliers through othermeans (i.e., “information provided by the excipient manufacturer”).

In theory, the pharmaceutical manufacturer could increase theiraudit personnel to increase their audit frequency and coverage, but thesuppliers may not be able to accommodate all the additional audits.Excipients are often made for uses other than those of a pharma -ceutical nature, and the pharmaceutical use of excipients can therefore be very small in terms of the total quantity manufactured(e.g., cellulosics where around 0.2% of all that is made is used inpharmaceuticals). Where an excipient supplier has many pharma -ceutical customers, and hundreds would not be uncommon, then theincreased audit burden would quickly become intolerable. At that point,decisions on who comes to audit and who does not would most likelybe prioritised based on commercial considerations alone.

This scenario is well known by the International PharmaceuticalExcipients Council (IPEC) and its fellow associations: the European FineChemicals Group (ECFG), the Federation of European ChemicalDistributors (FECC) and the UK Pharmaceutical Quality Group (PQG).Well before the publication of the FMD and ascertaining GMPGuidelines, together they formed a consortium to design, develop andimplement a high quality, third-party audit Certification Scheme forpharmaceutical excipients. This Scheme is now called EXCiPACTTM and isowned by EXCiPACT asbl, a Brussels-based not-for-profit associationwho provide oversight for its correct use via registered Certifying Bodiesto meet the needs of pharmaceutical excipient suppliers and users.

Third-party certificationThe Guidelines allow for the use of third-party certification of thesupplier, however, what are the key criteria the pharmaceuticalmanufacturer should use to judge the value of the scheme andcertificate? In essence they are:� The standard used to assess the excipient supplier;� The competency of the auditors;� The oversight applied to the third-party certifying body to ensure

standards are upheld and maintained.

There has been much criticism of third-party Certification Schemes, for example, as with ISO 9001, and while these can be justified in manycases, many users of the certificates have not taken enough care tocheck on the three points above. Mindful of the reservations that manyin the pharmaceutical industry would have over third-party audits, theEXCiPACTTM Certification Scheme was designed and built to address allthree criteria and, therefore, to maximise the assurance of thecorresponding EXCiPACTTM certificates.

The EXCiPACTTM Scheme delivers certificates to the excipientsuppliers who pass the audits with the intention that the audit reportsare supplied to their customers: pharmaceutical manufacturers. This provides details of the scope of the audit, the findings and level ofconformance. Additionally, by following the classic ISO 9001 certificatethree-year audit cycle, a surveillance audit is conducted in years twoand three with a full recertification audit in year four. With such annual

audit reports, the pharmaceutical manufacturer can quickly build upthat knowledge and evidence as well as meeting the need for thatinformation to be based on current arrangements. Such information fitsnicely into the changes to Chapter 5.27 of the EU Part 1 GMP Guide whichcalls for the pharmaceutical manufacturer to have “supportingevidence for each supplier” and “a current knowledge of suppliers”.

Having regular audits is one matter but how can there beconfidence that the third-party Certifying Body is following the Schemerules, and using competent auditors? This is where EXCiPACT as anassociation comes into play. EXCiPACT asbl is a not-for-profitorganisation that was established in Belgium in 2014. It may only have asmembers those organisations that are “professional associationscomprised of natural or legal person as members, trade associations orsimilar organisations that represent groups of organisations who havean interest in manufacturing, distributing or using excipients”3. In thismanner no commercial organisation or individual has undue influenceover the EXCiPACTTM Certification Scheme.

In order to ensure the Certifying Bodies apply the EXCiPACTTM

Scheme rules, EXCiPACT asbl has a strict qualification process whichincludes a definition of the Scheme rules and a regular audit of theCertifying Bodies. It witnesses their audits once a year to ensure thesestandards are maintained and implemented. The Scheme rules forCertifying Bodies are based on the ISO 17021 standard “Conformityassessment requirements for bodies providing audit and certification ofmanagement systems”. This is the standard all credible ISO 9001Certifying Bodies should be accredited to by their national accreditationagency. In the case of the EXCiPACTTM Scheme it is EXCiPACT asbl whichtakes the role of the national accreditation body and ensures thatstandards are upheld. ISO 17021 also requires that Certifying Bodiesmaintain impartially and avoid conflicts of interest to ensure noconsultancy or similar involvement with the excipient supplier occurs byany of the EXCiPACT auditors.

Having established that the Certifying Body has fully implementedthe EXCiPACTTM Certification Scheme requirements into its managementsystems, and audited those systems to confirm it, EXCiPACT asbl takesan equally close view of the auditors themselves. Each auditor has toapply to be registered with the EXCiPACTTM Certification Scheme andprovide evidence of their audit skills, knowledge and recent audithistory. Auditors have to:� Be ISO 9001-registered auditors with good management syst-

ems knowledge;� Have some knowledge of pharmaceutical regulations and

requirements; � Attend a two-day EXCiPACTTM GMP and GDP training course designed

to concentrate on the GMP and GDP requirements in the EXCiPACTCertification Scheme that are not already covered in ISO 9001. The two-day course is only about the key GMP and GDP principlesand how they can be incorporated into an existing quality manage -ment system;

� Pass an end-of-course exam in order for them to progress their registration;

� Be witnessed in their first EXCiPACTTM audit, and then at three-yearlyintervals to ensure they have applied the learning points from thecourse. The person witnessing the auditor can be an EXCiPACT asblemployee (for example, as part of the Certifying Body registration or

REGULATORY INSIGHT

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 15

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re-registration process) or they can be a Certifying Body employeewith the same competency as the auditor. This auditor competencyregistration process ensures that not only are the EXCiPACTstandards communicated to the auditors, but they are alsoconfirmed as being implemented.

Each auditor is required to register with EXCiPACT asbl independently oftheir employer. This ensures their registration is transferable to otheremployers who may wish to use their services for EXCiPACTTM audits.

Excipient suppliers are held to two highly complementarystandards, one being GMP and the other, good distribution practices(GDP). The latter is aimed at distributors and other suppliers ofexcipients who do not manufacture or repack excipients. Thesestandards have been derived from the IPEC-PQG GMP and IPEC GDPGuides and have been fully updated with contemporary approaches,most notably the requirement for the supplier to conduct a number ofrisk assessments to identify how their excipients could be compromisedand then to apply suitable controls to minimise those risks. Such anapproach is exactly the same as elaborated in the more recentANSI/IPEC/NSF 363-2014 US National Standard, which can also be usedas the basis of an EXCiPACTTM audit for those suppliers who do not hold ISO 9001.

The EXCiPACT and ANSI 363 standards were predominantlydeveloped by the same individuals, with the objective of ensuring theyhad the same requirements. The parent IPEC-PQG GMP and IPEC GDPGuides have received very widespread acceptance as suitable standardsfor excipient suppliers, and with this pedigree the new EXCiPACT andANSI-363 standards are expected to be equally acceptable for themajority of excipients. Excipients with particular risks will require moreenhanced GMPs as is required in the EU Ascertaining GMP Guidelines,but these standards form the basis on which such enhancements could be built.

The audit processGiven qualified third-party Certifying Bodies and EXCiPACT-registeredauditors, how does the Scheme’s audit process work? The steps are as follows:1. The excipient supplier selects an approved third-party Certifying

Body from the list on the EXCiPACT asbl website. If they hold acurrent ISO 9001 Certificate, they can use that to have an audit tothe EXCiPACT GMP and/or GDP standard, and if not, they can beaudited against the NSF/IPEC/ANSI 363 standard which includes allthe ISO 9001 quality management system elements as well as GMPs.

2. The standard ISO audit certification process commences with aStage 1 audit, usually for one day. This is used to plan the Stage 2(full) certification audit by defining the scope of certification andthe audit man-days needed to complete the assessment.

3. Sometime later, the Stage 2 audit occurs, covering the quality andGMP Management Systems which are in scope for the audit. This isanother important aspect of the Scheme, since many excipientsuppliers can have other activities and products on site which arenot included in the audit process. It is essential that thepharmaceutical manufacturer checks that the scope of anyEXCiPACTTM certificate includes the excipients that they purchasefrom the supplier.

4. After the Stage 2 audit, for an EXCiPACT certificate to be issued theremust be no life-threatening, critical or major non-conformitiesopen. ‘Life-Threatening’ was a criteria requested by the US FDAwhich wanted to be informed immediately of any findings ofexcipients causing harm to patients. Note this is not situation inwhich there is a risk of harm, but that there is actual harm. In suchcases the EXCiPACT Scheme rules require the excipient supplier toinform the healthcare regulatory agencies immediately of thefinding and confirm it to the Certifying Body. ‘Critical’ non-conformities are where there is very high risk of patient safety beingcompromised and ‘majors’ would be significant gaps in the qualityand/or GMP management systems.

5. Once a Stage 2 audit is successfully completed, a certificate fee ispaid to EXCiPACT asbl and the supplier’s details are added to theEXCiPACT website. The fee covers the period the certificate is valid(three years) and is payable again on recertification.

6. The excipient supplier is now free to share the certificate and auditreport(s) on a confidential basis with their pharmaceuticalcustomers. Any issues noted in the audit reports would be down to the excipient supplier and their customers to resolve together.

7. The pharmaceutical manufacturer can then verify all aspects of thecertificate and audit report by checking if the Certifying Body andtheir auditor(s) are listed on the EXCiPACT website. If so, there is ahigh degree of assurance that the certificate and audit report hasbeen issued under the rules of the Scheme. The certificate andaudit report can be used with confidence to meet the require-ments in the European Commission Ascertaining GMP Guidelinesand therefore in full compliance with Chapter 5 requirements inPart 1 GMPs,

ConclusionA supplier with an EXCiPACTTM Certificate and who shares their auditreports with their customers proactively demonstrates that they haveimplemented current excipient GMP and/or GDPs. Pharmaceuticalmanufacturers can then be confident that they are purchasingmaterials from a reliable source. The Scheme meets all the legalrequirements and at the same time reduces the audit burden on allparties without compromising quality or patient safety.

To date the 22 EXCiPACTTM Certified suppliers and their customershave confirmed that these benefits can be realised by both sides whenusing the Scheme.

REGULATORY INSIGHT

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 17

1. Directive 2011/62/EU

2. Guidelines of 19 March 2015 on the formalised risk assessment for ascertaining the

appropriate good manufacturing practice for excipients of medicinal products for human

use (2015/C 95/02)

3. EXCiPACT asbl Articles of Association; www.excipact.org

References

Dr Iain Moore is chair of the EFfCI GMP Committee and

President of the Board EXiPACT asbl. After completing his

doctorate studies in organometallic chemistry he has been

with Croda International plc since 1987, and for the last 20

years in quality roles. He has helped define GMPs for

cosmetic and pharmaceutical ingredients and implemented

these requirements across Croda’s sites. He is currently

Global Head of Quality Assurance at Croda.

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You want to be sureThe new handheld Metrohm Instant Raman Analyzer (Mira) for chemical and pharmaceutical analysis. With 9000 spectra in its library, Mira gives you unparalleled power to identify what you are looking for – at the push of a button, anywhere!

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VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 19

20 Identification ofcounterfeit drugs usingdual laser handheld Raman Sulaf Assi, Bournemouth University

27 Low-frequency shift Raman spectroscopy of pharmaceuticalrespirable powdersSulayman A. Oladepo, Hui Wang, David Baronaand Reinhard Vehring, University of Alberta

34 Raman RoundtableModerated by Sulayman A. Oladepo, Assistant Professor of Analytical Chemistry,University of Alberta

Raman

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Type 5 phosphodiesterase inhibitors (PDE5is), including Cialis®(tadalafil), Levitra® (vardenafil) and Viagra® (sildenafil), are among themost counterfeited products3-5. Reported risks associated with sexualstimulants included a lack of active pharmaceutical ingredient (API), alow or excess amount of API and the presence of a toxic API orexcipient3. The World Health Organization reported in 2010 that 150 people were admitted to hospital with severe hypoglycemia

after taking a counterfeit copy of a PDE5i that was adulterated with glyburide2. Of these people, four died and seven suffered severebrain damage.

Although counterfeit versions of these products can beencountered anywhere in the legal supply chain6,7; they are mainly soldillegally via the Internet in developed countries or at street markets indeveloping countries. Subsequently, there is a need for developing a

Medicine counterfeiting represents an expanding global problem that accounts for 10% of the world market1. The use of fake medicines is of threat to the public health as their effects can range from treatment ineffectivenessto death. Counterfeiting can occur to any class of medicines, any formulation and at any dose. However, the mostcommonly reported counterfeit medicines include medicines used for lifestyle improvement and performance, suchas weight loss products, dietary supplements and sexual stimulants2.

IN-DEPTH FOCUS: RAMAN

Identification of counterfeitdrugs using dual laserhandheld Raman

Sulaf AssiBournemouth University

20 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

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rapid technique that can identify counter-feit sexual stimulants non-destructivelywherever they a encountered. Ramanspectroscopy offers this advantage.

Several studies have investigated theidentification of counterfeit PDE5is usinghandheld and conventional Raman instru -ments8-13. Studies using handheld Ramaninstruments mainly focused on discrimina -ting authentic from counterfeit products8,whereas those using laboratory-basedapparatus investigated the presence ofAPIs/excipients in counterfeit products9-13.For handheld instruments, discrimination ofcounterfeit PDE5is from authentic alterna -tives involved the construction of a library ofauthentic references and the comparison of the counterfeit PDE5is against that libraryusing various algorithms8.

The aforementioned algorithms involvedcorrelation-based methods, distance-basedmethods or principal component analysis(PCA). Additionally, for laboratory-basedinstruments, identifying the presence of the APIs/excipientsemphasised the presence of specific peaks relating to the medicine ofinterest9-11. This was made either with direct spectral comparison or theapplication of algorithms. Direct spectra comparison was successful in identifying counterfeit Cialis tablets that were shown to contain undeclared excipients10. In another case, lactose (Viagra’s major excipient) proved to be missing from counterfeit Viagra

tablets12. Furthermore, multivariate curve resolution algorithm applied to Raman spectra was able to characterise the API andexcipients present in counterfeit Cialis tablets9. Additionally,hierarchical clustering with PCA was successful in distinguishingauthentic from counterfeit Viagra tablets13. In another instance, PCA wasapplied in combination with both Raman and near-infraredspectroscopic techniques to authentic Cialis and Levitra tablets11.

Though the aforementioned studiescharacterised the medicines, their majorAPIs and major excipients, none of thestudies inspected thoroughly the individualconstituents in medicines. Thus, in theprevious studies handheld Raman onlycompared the counterfeit medicines’ spectraagainst the authentic versions, and lab -oratory-based Raman identified one or twomajor components in the medicines.Specifically with handheld Raman, the lack ofcharacterisation of individual componentswas encountered due to various reasons: (1) The presence of the API in a low con -centration which masked its Raman activity;(2) the complexity of the matrix measured;(3) the fluorescence of the excipients; and (4)the low spectral resolution which resulted inoverlapping peaks. One way of overcomingthese issues would be via the use of fluor -escence subtraction algorithm, which allowsthe characterisation of the API and differentexcipients in the medicine.

Susequently, this work aimed at non-destructively identifying counterfeit Cialis,

IN-DEPTH FOCUS: RAMAN

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 21

Figure 1: Raw Raman spectra of (a) authentic Cialis tablet, (b) counterfeit Cialis tablet, (c) authentic Levitra tablet and(d) counterfeit Levitra tablets measured with the Bruker BRAVO handheld Raman instrument

Figure 2: Raw Raman spectra of (a) authentic Viagra tablet, (b) counterfeit Viagra tablet, (c) sildenafil citrate and (d) lactose measured with the Bruker BRAVO handheld Raman instrument

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Levitra and Viagra tablets obtained worldwide usinghandheld Raman spectroscopy equipped with a duallaser power and fluorescence substraction algorithm.

ExperimentThree types of PDEIs were used in this study andincluded the branded, UK-purchased products: Cialis(15 authentic and eight counterfeit products), Levitra(10 authentic and 22 counterfeits) and Viagra (26authentic and 30 counterfeits) products. Meanwhile,fake medicines were collected worldwide across thesupply chain. Additionally, the APIs and mainexcipients present in the aforementioned threeproducts were used. These included: lactosemonohydrate, magnesium stearate, microcrystallinecellulose, sildenafil citrate, tadalafil, titanium dioxideand vardenafil hydrochloride. All products containedless than 20% m/m of API, whereas excipientsrepresented major components. Table 1 shows thedetails of the products and the percentage mass permass (% m/m) of API in each batch.

The products were measured ‘as received’ usingthe Bruker BRAVO handheld Raman instrumentequipped with dual laser power and charged coupled device detectorwith thermoelectric cooling. Twenty spectra were collected from 10 tablets in each batch and both sides of the tablets were measured.Each spectrum was the mean of one scan over the wavenumber range of 300 – 3200 cm-1. Additionally, APIs and excipients weremeasured through transparent glass vials. In this respect, 20 spectrawere taken per vial such that each vial was shaken and repositionedbefore each measurement.

Both the instrumental inbuilt algorithm and offline analysis wereused for comparing the counterfeit against the authentic products. The inbuilt identification utilised Hit Quality Index (HQI) algorithmwhich was based on correlation and compared the degree of similaritybetween the counterfeit and authentic product spectra. The thresholdvalue taken for HQI was 95%14. Subsequently, an HQI value above 95%was considered a similar product.

For offline analysis, spectra were exported into Matlab 2014a where principal component analysis (PCA) algorithm was applied.Where more than 20 authentic batches were present (as in the case

with Viagra tablets), the 95% equal frequency ellipses were constructedaround their PCA scores7. The location of the test batch inside theellipse indicated similarity.

Results and discussionSpectral evaluation and Raman activity of productsThe three measured types of medicines (Cialis, Levitra and Viagra) hada complex chemical make-up consisting of the API and more than eightexcipients in the core and coatings. The APIs were tadalafil, vardenafilhydrochloride and sildenafil citrate in authentic Cialis, Levitra andViagra tablets, respectively, and were present in quantities of 5.45, 11 and 16% m/m, respectively (Table 1). The excipients in theaforementioned products were present in higher than 70%m/m, whichmade their characterisation crucial for tablet authentication (Table 2;page 25). The inbuilt fluorescence subtraction algorithm enhanced the Raman activity of the excipients and thus improved the efficacy of identification.

Subsequently, the Raman activity of the authentic and counter-feit batches of each medicine were investigated and compared to their corresponding APIs and excipients. In this respect, the peakpositions and intensities were taken into account. Although the Raman signal is in arbitrary units, the intensity of Raman is stillproportional to the concentration of the Raman active substance14,15.This is effective in authenticating branded medicines where the matrices of these medicines are known and subsequently theconcentrations of their APIs14,16.

For Cialis, the authentic batches spectra showed characteristicpeaks for TiO2 (at 392, 514 and 638cm-1) and lactose (between 780 and2982cm-1). On the other hand, no peaks for tadalafil were observed inthe authentic Cialis tablets spectra (Figure 1; page 21). This could beattributed to tadalafil being present in a concentration of 5.45% m/m(Table 1) of the tablet which was below the detection limit of handheld

IN-DEPTH FOCUS: RAMAN

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 23

Figure 3: PCA scores plot of the raw Raman spectra of authentic (blue) and counterfeit (red) Cialis tabletsmeasured using the Bruker BRAVO handheld Raman spectrometer

Table 1: Active pharmaceutical ingredient (API) content in the authentic andcounterfeit Cialis, Levitra and Viagra tablets

API label Mean weight Mean API

Product N claim (mg) ± SD (mg) content ± SD

CB 15 20 367 ± 2.78 5.45 ± 0.04

UCB 8 20 438 ± 9.97 4.57 ± 0.1

LB 10 20 186 ± 2.85 11 ± 0.17

ULB 22 20 214 ± 3.42 9 ± 0.15

VB 26 100 627 ± 6.6 16 ± 0.17

UVB 30 100 662 ± 64.4 15 ± 1.33

%m/m: Percentage mass per mass, API: active pharmaceutical ingredient, CB: authentic

Cialis batches, UCB: counterfeit Cialis batches, LB: authentic Levitra batches, ULB:

counterfeit Levitra batches, VB: authentic Viagra batches, UVB: counterfeit Viagra

batches, MCC: microcrystalline cellulose, MgS: magnesium stearate, N: number of

batches, SD: Standard deviation, TiO2: titanium dioxide.

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Raman14,15. On the other hand, lactose was present ina concentration of 64.5% m/m which enhanced itsRaman scattering. However, the spectra for thebatches of copied Cialis showed characteristicfeatures for TiO2 (at 392, 514 and 638 cm-1) andsildenafil citrate (between 692 and 3000 cm-1). In thelatter spectra, no peaks for lactose were observed.This indicated that the counterfeit Cialis products didnot contain lactose and contained sildenafil that was not indicated on the label claim.

However, the spectra of authentic Levitra batchesshowed characteristic peaks to its corresponding API(vardenafil hydrochloride) as well as its mainexcipients (TiO2, hypromellose and MCC). This couldbe attributed to the fact that vardenafil hydro -chloride in Levitra was present in a higherconcentration (11% m/m) than tadalafil in Cialisbatches (5.45% m/m). Characteristics peakscorresponding to vardenafil hydrochloride were seenat 1236, 1594, 1700, 2900 cm-1. Similar to authenticCialis products, TiO2 showed characteristic peaks at392, 514 and 638 cm-1. Additionally, hypromelloseshowed characteristic peaks at 1376 and 1464 cm-1,whereas MCC showed characteristic peaks at 1092 and 2900 cm-1. Thecounterfeit Levitra batches showed peaks corresponding to TiO2 (392,514 and 638 cm-1), hypromellose (1376 and 1472 cm-1), MCC (1094 and 2890cm-1) and vardenafil hydrochloride (1596 and 2890 cm-1). It is noteworthyto mention that TiO2 had higher Raman intensity in counterfeit Levitrabatches than their authentic alternatives. This indicated that TiO2 waspresent in higher concentrations in the counterfeit Levitra batches.Additionally, the counterfeit Levitra batches showed significant Raman peaks between 700 and 3000 cm-1; which were not present

in the authentic Levitra tablets. Nonetheless, these peaks were of low intensities.

Unlike authentic Cialis and Levitra batches’ spectra, the legitimateViagra batches’ spectra did not show any significant peakscorresponding to its major excipient (lactose monohydrate). Thus, thespectra of authentic Viagra batches’ showed peaks corresponding to TiO2 (394, 516 and 638 cm-1) and sildenafil citrate (between 700 – 3000 cm-1); which was present in a concentration of 16% m/m(Figure 2; page 21). Yet, vardenafil hydrochloride in authentic Levitra

batches was present at a similar concentration (of 15%m/m) and did not have a strong Raman activity. Thisproved that sildenafil citrate was a stronger Ramanscatterer than vardenafil chloride. Moreover, the latterresult confirmed results from previous studies whichfound that the Raman activity of a pharmaceuticalproduct depended on both the Raman activity of theconstituents and their concentration14.

Remarkably, counterfeit Viagra tablets showedsimilar Raman spectra to authentic Viagra tablets inrelation to the peak positions of TiO2 and sildenafilcitrate (Figure 2; page 21). However, the counterfeitViagra tablets showed lower scattering for TiO2 andhigher for sildenafil citrate. This indicated that thecoating of the counterfeit Viagra tablets was of poorquality and they contained higher doses of sildenafilcitrate. Moreover, additional peaks were observed inthe regions between 700 and 1700 cm-1; and were notrelated to the API or excipients. Thus, the counterfeitViagra batches were adulterated by other substances.Also, counterfeit Viagra batches did not show peakscorresponding to lactose (the major API in Viagra).The latter result agreed with a study by Sacre et al.

24 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

IN-DEPTH FOCUS: RAMAN

Figure 4: PCA scores plot of the raw Raman spectra of authentic (blue) and counterfeit (red) Levitra tabletsmeasured using the Bruker BRAVO handheld Raman spectrometer

Figure 5: PCA scores plot of the raw Raman spectra of authentic (blue) and counterfeit (red) Viagra tabletsmeasured using the Bruker BRAVO handheld Raman spectrometer

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(2011) that did not detect lactose in counterfeit Viagra when measuredusing Raman imaging12.

Hit quality index identification algorithmTo confirm the authentication of tablets, further identificationalgorithms were applied. The first algorithm was the HQI; whichcompared the degree of similarity between the Raman spectra of thetest batches (authentic and counterfeit) and the spectra of their corresponding (1) API, (2) excipients and (3) reference batch (Table 3; page 26).

For the API and excipients, no threshold value was expected for HQI.Nonetheless, as the HQI is proportional to the concentration14, a highHQI value indicated that there was an excess amount of an API orexcipient. This was witnessed with counterfeit Viagra batches that hadmean HQI values of 82.7% against sildenafil citrate (Table 3; page 26). Onthe other hand, none of the authentic or counterfeit batches showed ahigh concentration against lactose, MCC or MgS. However, all thebatches (apart from the fake Cialis and Viagra batches) showed HQIvalues against TiO2 above 80%. This confirmed that counterfeit Cialisand Viagra batches had poor coating.

When comparing against the reference batch, the test batches wereinvestigated for type I and type II errors16. Since an HQI threshold of 95% was considered14, an authentic batch of HQI below 95% wasconsidered a type I error. Likewise, a counterfeit batch with an HQIabove 95% was considered a type II error. In this respect, type I errorwas not encountered with any of the authentic products since all gaveHQI values above 95% (Table 3; page 26). However, type 2 error wasobserved with counterfeit Levitra batches which gave a mean HQI valueof 94.6% against the authentic alternatives. The latter result wasencountered because the similarity between the authentic andcounterfeit Levitra spectral features was high; and thus HQI was notsensitive enough for their discrimination.

Offline analysisSubsequently, the PCA method was applied to the spectra of authenticand counterfeit Cialis, Levitra and Viagra tablets for furtherdiscrimination. Where more than 20 authentic batches were present6,95% equal frequency ellipses were applied to PCA scores. For all threemedicines, PCA was successful in discriminating the authentic fromcounterfeit batches.

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review

IN-DEPTH FOCUS: RAMAN

Table 2: Constituents of the authentic Cialis, Levitra and Viagra products

Product API Tablet core excipients Tablet coat excipients

Cialis tadalafil lactose monohydrate, lactose monohydrate,croscarmellose sodium, hypromellose, triacetin,hydroxypropylcellulose, TiO2 (E171), iron oxideMCC, sodium lauryl yellow (E172), talcsulfate, MgS

Levitra vardenafil Crospovidone, MgS, Macrogol 400,hydrochloride MCC, silica colloidal hypromellose, TiO2 (E171),

anhydrous ferric oxide yellow (E172),ferric oxide red (E172)

Viagra sildenafil MCC, calcium hydrogen hypromellose, TiO2 (E171),citrate phosphate (anhydrous), lactose monohydrate,

croscarmellose triacetin, indigo carminesodium, MgS aluminium lake (E132)

API: active pharmaceutical ingredient, MCC: microcrystalline cellulose,

MgS: magnesium stearate, TiO2: titanium

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However, type I error (misclassification of authentic batches) wasencountered with the authentic Cialis and Viagra medicines. Figure 3(page 23) shows the PCA score plots of authentic and counterfeit Viagrabatches. In this respect, PC1 accounted for 76.4%, whereas PC2explained 12.6% of the variance. When the PCA scores were interpreted,two of the authentic Cialis batches scores were not classified with theauthentic batches. This could be a type I error, yet was unassured dueto the lack of enough authentic samples for classification. Furthermore,when the spectra of these two products were compared to theremaining authentic batches, no significant difference was observed inthe peak positions. Hence, variation was only limited to the peakintensities in which several parameters play a role.

On the other hand, PCA could discriminate between the authenticand counterfeit Levitra batches with no type I or type II errors (Figure 4;page 24). The PC scores explained well the data as they accounted for98% of the variance among it. Yet there were not enough batches toevaluate accurately the classification algorithm.

Conversely, more than 20 authentic Viagra batches were available and hence PCA was applied along with the 95% equalfrequency ellipses. Figure 5 (page 24) shows the PCA scores plot ofauthentic and counterfeit Viagra tablets. The PC scores explained 94% of the variance among the data and were able to classify theauthentic from the counterfeit batches. However, only one authentic

batch (type I error) was misclassified. The spectra of the latter batchshowed differences in the peak intensities rather than position. This could be attributed to the sample measurement position or parameters.

ConclusionIn summary, handheld Raman spectroscopy offered a rapid and non-destructive method for the identification of counterfeit Cialis, Levitra and Viagra medicines. Since these drugs consisted of complexmatrices of APIs and excipients, no one spectral algorithm was enough for their identification; the combination of multiplespectral approaches including spectral evaluation, HQI and PCA offered a more accurate identification.

IN-DEPTH FOCUS: RAMAN

26 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

1. WHO Factsheet N˚275. Medicines: spurious/falsely-labelled/ falsified/counterfeit (SFFC)

medicines 2012. Available from: http://www.who.int/mediacentre/factsheets/fs275/en/

2. World Health Organization. (2010). Growing threat from counterfeit medicines. Bull World

Health Organ, 88(4), 247-248

3. PY Sacré, E Deconinck and L De Beer, 2013. Analytical strategies for the detection of

counterfeit erectile dysfunction drugs. Counterfeit Medicines Volume II: Detection,

Identification and Analysis

4. JK Maurin, F Pluciński, AP Mazurek and Z Fijałek, 2007. The usefulness of simple X-ray

powder diffraction analysis for counterfeit control – The Viagra® example. Journal of

Pharmaceutical and Biomedical Analysis, 43(4), 1514-1518

5. MJ Vredenbregt, L Blok-Tip, R Hoogerbrugge, DM Barends and D de Kaste, 2006.

Screening suspected counterfeit Viagra® and imitations of Viagra® with near-infrared

spectroscopy. Journal of Pharmaceutical and Biomedical Analysis, 40(4), 840-849

6. AC Moffat, S Assi RA and RA Watt, 2010. Identifying Counterfeit Medicines using

Near-infrared Spectroscopy. Journal of Near-infrared Spectroscopy, 18, 1-15

7. S Assi, RA Watt and AC Moffat, 2011. Identification of Counterfeit Medicines from the

Internet and the World Market Using Near-Infrared Spectroscopy. Analytical Methods,

3, 2231-2236

8. S Assi and S Halsey, 2014. Identification of counterfeit Viagra, Cialis and Levitra tablets

using handheld Raman spectroscopy, Euchem 2014, p. 8

9. K Kwok and LS Taylor, 2012. Analysis of counterfeit Cialis® tablets using Raman

microscopy and multivariate curve resolution. Journal of Pharmaceutical and Biomedical

Analysis, 66, 126-135

10. S Trefi, V Gilard, S Balayssac, M Malet-Martino and R Martino, 2009. The usefulness of 2D

DOSY and 3D DOSY-COSY 1H NMR for mixture analysis: application to genuine and fake

formulations of sildenafil (Viagra). Magnetic Resonance in Chemistry, 47(S1), S163-S173

11. PY Sacré, E Deconinck, T de Beer, P Courselle, R Vancauwenberghe, P Chiap and

JO de Beer, 2010. Comparison and combination of spectroscopic techniques for the

detection of counterfeit medicines. Journal of Pharmaceutical and Biomedical Analysis,

53(3), 445-453

12. PY Sacré, E Deconinck, M Daszykowski, P Courselle, R Vancauwenberghe, P Chiap and JO

De Beer, 2011. Impurity fingerprints for the identification of counterfeit medicines – a

feasibility study. Analytica Chimica Acta, 701(2), 224-231

13. M de Veij, A Deneckere, P Vandenabeele, D de Kaste and L Moens, 2008. Detection of

counterfeit Viagra® with Raman spectroscopy. Journal of Pharmaceutical and Biomedical

Analysis, 46(2), 303-309

14. S Assi, 2014. Investigating the quality of medicines using handheld Raman spectroscopy.

European Pharmaceutical Review, 19 (5), 55-60

15. S Assi, RA Watt and AC Moffat, 2011. On the quantification of ciprofloxacin in proprietary

Ciproxin tablets and generic ciprofloxacin tablets using handheld Raman spectroscopy.

Journal of Raman Spectroscopy, 43 (8), 1049-1057

16. S Assi, RA Watt and AC Moffat, 2011. Authentication of medicines using Raman

spectroscopy. European Pharmaceutical Review, 16 (1), 49-55

17. S Assi, 2013. Raw material identification using dual laser handheld Raman spectroscopy.

European Pharmaceutical Review, 18 (5), 25-31

References

Dr Sulaf Assi is an Associate Lecturer in Forensic Sciences

at the Faculty of Science and Technology, Bournemouth

University. She obtained her Bachelor and MSc in

Pharmacy from Beirut Arab University, Lebanon. Then, she

did her PhD in pharmaceutical analysis at the School of

Pharmacy, University of London, UK. She is involved in the

teaching and research of different aspects of forensic

sciences. Sulaf has a broad range of research interests including: counterfeit

medicines, alcohol, tobacco, drug abuse and misuse, novel psychoactive

substances, analytical techniques, handheld instruments and multivariate

data analysis.

Table 3: Hit Quality Index (HQI) values of authentic and counterfeit Cialis, Levitra and Viagra against their authentic reference, corresponding active pharmaceuticalingredients (APIs) and main excipients

Product Reference API lactose MCC MgS TiO2

CB 95.4 ± 10.5 54.4 ± 6.82 48.2 ± 7.35 57 ± 5.57 72.8 ± 3.79 81.4 ± 7.27

UCB 67.5 ± 13.1 63.6 ± 12.7 25.9 ± 17.5 39.4 ± 12.9 57.1 ± 11.8 65.1 ± 15.2

LB 97.3 ± 1.29 56 ± 0.99 46.3 ± 1.2 57.6 ± 0.96 73.3 ± 0.65 86.7 ± 1.31

ULB 94.6 ± 0.96 56.7 ± 1.77 46.2 ± 2 56 ± 1.38 73.4 ±1.04 87.1 ± 2.08

VB 95.1 ± 7.29 57.7 ± 6.56 47 ± 6.47 55.9 ± 5 72.6 ± 4.58 86.4 ± 7.48

UVB 64.1 ± 11.9 82.7 ± 10.7 15.3 ± 16.4 32.9 ± 11.2 51.1 ± 10.4 56.1 ± 12.4

API: active pharmaceutical ingredient, CB: authentic Cialis batches, UCB: counterfeit Cialis batches, LB: authentic Levitra batches, ULB: counterfeit Levitra batches, VB: authentic Viagra

batches, UVB: counterfeit Viagra batches, MCC: microcrystalline cellulose, MgS: magnesium stearate, Reference: reference batch, TiO2: titanium dioxide

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The development of pharmaceutical products, including respirableproducts, involves thorough characterisation of active ingredients andexcipients to ensure the finished products have the correct quality foroptimal performance. Particles present in respirable dosage blends canexist in crystalline or amorphous form, or a mixture of both. In addition,crystalline components may also exist in various polymorphs. Each ofthese forms results in different, and possibly, undesired characteristicsin the final product1. The possibility of inter-conversion betweencrystalline and amorphous forms of respirable powders, as well astransition between polymorphs of the same substance is of majorconcern for pharmaceutical respirable dosage forms2,3. Different phasesof active pharmaceutical ingredients and excipients in pharmaceuticalrespirable formulations can have implications on drug effectiveness

and potency4-7. Therefore, to ensure product integrity, expectedtherapeutic performance, consistent potency and regulatorycompliance, pharmaceutical respirable powders need to be accuratelycharacterised, from raw materials to finished products.

Several techniques may be used to characterise solid phasetransitions of pharmaceutical products. X-ray diffraction (XRD),calorimetry and terahertz pulsed spectroscopy (TPS) have all beendescribed3,8-10. These techniques may not be well suited for the analysisof crystalline or amorphous contents and polymorphism in respirabledosage forms3. For instance, TPS is only capable of covering awavenumber range of 3-120 cm-1, which is inadequate for probing higherenergy vibrations3.

Raman spectroscopy is a suitable tool for probing molecular

This article presents low-frequency shift Raman spectroscopy of pharmaceutical respirable powders. We show thatacquisition of this type of Raman spectra is possible with appropriate instrument design and choice of optics forRayleigh scattering rejection, enabling low wavenumber diagnostic bands to be observed. Our Raman spectra showunique spectral signatures for discriminating between different polymorphs of mannitol, and amorphous andcrystalline forms of glycopyrronium bromide. In addition, we present a deconvolution method to quantifyfluticasone propionate in three commercial pharmaceutical respirable powders.

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VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 27

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Low-frequency shiftRaman spectroscopy of pharmaceuticalrespirable powders

Sulayman A. Oladepo, Hui Wang, David Barona and Reinhard VehringUniversity of Alberta

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structures and solid phases11,12. The tech -nique is widely used in the pharmaceuticalindustry for material characterisation andanalysis, especially in the identification of chemical properties and solid phases ofdosage forms11,13. This is particularly import -ant for inhalable or respirable powders,which may undergo solid phase changesthroughout their shelf life14,15. The Ramantechnique is advantageous because it is non-destructive, water-compatible and does notrequire extensive sample preparation.

Low-frequency shift Raman spectro -scopy is especially suited for analysingcrystalline or amorphous contents ofrespirable pharmaceutical powders becausedifferent components and solid phasesproduce unique spectral signatures in thefingerprint (200cm-1 – 1800cm-1) and lowenergy (<200cm-1) regions of the Raman spectra. The low wavenumberregion provides a direct probe of crystal lattice structure and reveals thelevel of disorder in the lattice. Since poly -morphs of crystalline materials have a differentarrangement of the constituent moleculesrelative to the other forms, and given thesensitivity of low-frequency Raman spectro -scopy to inter-molecular lattice vibrations, different polymorphs of thesame crystalline substance can also be uniquely distinguished.

Such distinctive spectral features are less pronounced beyond the fingerprint region. Therefore, low-frequency shift Raman

spectroscopy constitutes a useful tool for solid state character isation of pharmaceuticaldosage forms.

Low-frequency shift Raman spectroscopy isnot a traditional form of Raman spectroscopy,

since it has inherent difficulty that stems from the intense Rayleigh line,which overshadows low-frequency vibrations. Rejection filters used in

IN-DEPTH FOCUS: RAMAN

Respirable powders may undergo solid phase changes

throughout their shelf life

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conventional dispersive Raman systems have largebandwidths and cannot reveal low vibrational modesthat are diagnostic of solid phases11,16. Low-frequencyshift Raman spectra can be recorded by using doubleor triple monochromators for Rayleigh rejection atthe expense of very low optical efficiency17-19,however, low-frequency vibrational modes as closeas 10cm-1 to the laser line can be measured withsingle-stage spectrographs by using new ultra-narrowband rejection filters20. This combination allows low-frequency shift (terahertz) Raman spectralacquisition with a compact Raman set up13.

In this paper, we present a low-frequency shiftRaman spectroscopy instrument that has been usedfor the characterisation and quantitative analysis ofseveral respirable powder samples. The Ramaninstrumentation makes use of multiple ultra-narrowrejection filters, which are arranged in series tomaximise Rayleigh line rejection, thereby enablinglow-frequency Raman measurements. In addition,the overall magnification of the Raman instrument isintentionally designed as low as 2.5, and the sampleholder is made large to enable macro-Ramansampling. This macro-Raman approach is significantbecause a larger sample area can be detectedsimultaneously, and higher laser flux density can beapplied without exceeding the limit of sample degradation.

Since Raman signals are recorded from a much larger sample area than in micro-Raman systems, spectral contributions from manyRaman scatterers are effectively averaged in real-time, making macro-Raman spectroscopy advantageous for the quantitative analysisof potentially heterogeneous bulk powders. Also, due to the largersampling area and higher allowable laser flux density, macro-Raman

spectroscopy permits faster data acquisition compared to micro-Ramansystems, in which several micrometer-scale spots have to be measuredseparately for a given sample area, leading to impractically longacquisition times13. This, in addition to the possibility of sample damagefrom a tightly-focused laser, makes macro-Raman spectroscopy moresuitable for measurement of bulk pharmaceutical respirable powders.

Materials and methodsMaterialsThe samples examined in this study are activepharmaceutical ingredients and excipients used inrespirable dosage forms for asthma and chronicobstructive pulmonary obstructive disease. D-mannitol was obtained from Sigma–Aldrich Corp.,(St. Louis, MO, USA). Glycopyrronium bromide (GP)was obtained from PCAS Oy (Turku, Finland).Crystalline GP was obtained from dry crystals of thissample following slow solvent evaporation andrecrystallisation in ethanol. Amorphous GP wasprepared by spray drying, the details of which havebeen described previously3. Three polymorphic formsof mannitol, α-, β- and δ-mannitol, were also meas -ured. The Raman spectrum of β-mannitol wasmeasured directly on the sample as received, whilethe other two polymorphic forms, α- and δ-mannitol,were prepared as described previously3,22. A set ofcommercial triple-component dry powder inhalerproducts, Seretide Accuhaler (GlaxoSmithKline, UK),consisting of 100, 250 and 500μg of fluticasone

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VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 29

Figure 1: Raman spectra of three polymorphs of mannitol. The inset is a blow-up version of the low-frequency (≤100 cm-1) region

Figure 2: Raman spectra of amorphous and crystalline glycopyrronium bromide

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propionate (FP) in the presence of a fixed 50μg amount of salmeterolxinafoate and lactose carrier up to a total mass of 12.5mg per dose, wereanalysed for the main active ingredient, FP, following measurements ofcalibration standards with known FP lactose compositions.

Raman instrumentThe Raman instrument used in this study has been described in detailpreviously3. Briefly, an argon ion laser at 514.5nm with maximum powerof 1700mW was used as the excitation source. Multiple notch filters withan overall optical density of 12 in combination with a single spectro -graph were used to detect low-frequency Raman signals.

Quantitative analysisWe used a deconvolution method to isolate and quantify differentcomponents of mixture samples. By assuming that the raw spectrum ofa mixture powder sample is composed of spectral contributions fromindividual components, we can describe the raw spectrum by:

(1)

where S represents the raw measured Raman spectrum, B is thebackground, Ii is the intensity contributed by component i, Si,N is the normalised reference spectrum of a given pure component, Δvis the Raman shift and bi is a minor wavenumber shift correction addedto the nominal band position to minimise the residual spectrum. The intensity factor, Ii, is the weighting factor used for each componentwhile iteratively subtracting its reference spectrum from the rawspectrum until the residuals are minimised. The final Ii obtained foreach component is then used to calculate the mass fraction of eachcomponent according to:

(2)

which relates the spectral intensity ratios of a given component and thereference component to its relative mass fraction, by means of acalibration factor, c.

Results and discussionThe unique feature of low-frequency shift macro-Raman spectroscopyin discriminating different polymorphs of the same substance is shown in Figure 1 (page 29). Different spectral signatures were obtainedfor the three polymorphic forms of mannitol in the low wavenumberregion. Their lattice modes, which represent unique distinguishingfeatures of these polymorphs, are thus revealed.

Our research team has been harnessing the discriminatory powerof low-frequency Raman in differentiating between crystalline andamorphous pharmaceutical dosage form3. This is also important formonitoring possible solid phase inter-conversions during production,storage, and even usage of inhaler products23. Figure 2 (page 29) showsthe Raman spectra of amorphous and crystalline forms of GP. Relativeto the sharp fingerprint bands indicating well-defined molecularvibrations of crystalline GP molecules, significantly broadened Ramanpeaks can be observed for amorphous forms of GP at correspondingRaman shifts, implying higher level of disorder. In comparison, moredistinct spectral differences are located in the low-frequency region.Crystalline GP has sharp and intense phonon peaks originating from

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Figure 3: Deconvolution of the Raman spectrum of FP-SX-lactose mixture; (a)normalized raw spectrum, reference spectra of FP and lactose, and normalizedresiduals spectrum that has been offset and blown up; (b) raw and reconstructedspectra of FP-SX-lactose mixture; (c) raw spectrum of FP-SX-lactose mixturecompared to the residuals obtained from the spectral deconvolution

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lattice vibrations, however, it is only a single broad peak envelope in thecase of amorphous GP due to lack of collective lattice vibrations. Thiskind of differentiation is significant for pharmaceutical respirablepowders because of the need to ascertain relative amounts ofamorphous and crystalline components, which may affect drugefficiency and potency6,24.

In addition to qualitatively identifying spectral signatures fordifferent substances and their solid states, it is desirable to determinethe amounts of different ingredients in a given pharmaceutical dosageform. To this end, our group has developed a deconvolution method forquantifying the mass fractions of different components constituting amixture3,11,13. Figure 3a (page 30) shows the deconvolution result of a Raman spectrum of a FP-SX-lactose triple-component mixture. In addition to the reference spectra of FP and lactose, the normalisedraw spectrum and normalised residuals are shown. FP, SX and lactose reference spectra are subtracted iteratively by adjusting their corresponding intensity factors until the normalised residualspectrum is minimised.

Once minimum residuals are obtained, the intensity factorscorrelate with the amount of each component. The normalisedresiduals in Figure 3a (page 30) have been offset and blown up tofacilitate clear viewing during the deconvolution process. Figure 3b(page 30) shows the raw spectrum of the FP-SX-lactose mixture and the corresponding spectrum that was reconstructed from the

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review

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deconvolution data. Other than the baseline andintensity offset observed in some regions of thespectra, there is generally a good match between the reconstructed and the raw spectra, implying gooddeconvolution. The offsets may be ascribed to thelimitation of equation 1 in accurately describing the raw spectrum across the entire wavenumberrange, perhaps because the raw spectrum andreference spectra have different level of back -grounds. Figure 3c (page 30) shows the flatness of the residuals compared to the raw spectrum ofFP-SX-lactose mixture.

The amounts of several components and theirsolid states present in a pharmaceutical dosage formcan be accurately determined following spectraldeconvolution3,11,13. This application has been shownwith the analysis of three commercial metered doseinhaler products; Seretide 100, 250 and 500. By usingthe dominant lactose carrier particles as internalstandard, and with the calibration factors obtainedvia deconvolution, we determined the mass fractionsof FP relative to the lactose excipient in thesesamples. We used equation 2 for the calibration curve(Figure 4, inset) and obtained a FP-to-lactosecalibration factor of 1.28, which was then used tocalculate FP mass fraction for each Seretide sample. The quantificationresults show good agreement between our measured mass fractionsand the nominal mass fractions (Figure 4). The observed discrepancy

between nominal and measured FP mass fractions falls withinexperimental error, thereby showing the accuracy of our deconvolutionapproach. Additionally, given the large sample area being measured

IN-DEPTH FOCUS: RAMAN

Figure 4: Mass fraction of FP in three commercial metered dose inhalers. Both nominal and measured massfractions are shown for each inhaler composition

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simultaneously, the Raman spectra presented here are therefore‘single-point’ measurements and are representative of the potentiallyheterogeneous samples used. This measurement approach is fast andeffective when compared to micro-Raman measurements.

ConclusionsWe have shown that low-frequency shift Raman spectroscopy is a goodtool for the characterisation and analysis of pharmaceutical respirablepowders. The unique spectral signatures obtained for three mannitolpolymorphs, as well as amorphous and crystalline GP demonstrate theutility of this method. We also showed the capability of low-frequency

shift Raman for quantitative analysis. The good agreement betweennominal and measured FP mass fractions validates our deconvolutionapproach and shows that low-frequency shift Raman spectroscopy iscapable of quantifying components of pharmaceutical respirablepowders. The macro-Raman platform of our instrument ensures largesample volume, which better suits heterogeneous powders and ensuresfast Raman measurements. The technique and deconvolution approachpresented here can be applied to a wide variety of other pharmaceuticalrespirable powders and can generally serve the pharmaceutical comm -unity in areas of raw material and product characterisation, qualitycontrol and in ensuring regulatory compliance.

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 33

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1. Strachan CJ, Rades T, Newnham DA, Gordon KC, Pepper M, Taday PF. Using terahertz

pulsed spectroscopy to study crystallinity of pharmaceutical materials. Chem. Phys. Lett.

2004;390(1-3):20-24

2. Willart JF, Descamps M, Caron V. Direct crystal to glass transformations of trehalose

induced by milling, dehydration, and annealing. AIP Conf. Proc. 2008;982:108-113

3. Wang H, Boraey MA, Williams L, Lechuga-Ballesteros D, Vehring R. Low-frequency

shift dispersive Raman spectroscopy for the analysis of respirable dosage forms.

Int. J. Pharmaceut. 2014;469(1):197-205

4. Hancock BC, Zograf G. Characteristics and significance of the amorphous state in

pharmaceutical systems. J. Pharm. Sci. 1997;86(1):1-12

5. Vippagunta SR, Brittain HG, Grant DJW. Crystalline solids. Adv. Drug. Deliver. Rev.

2001 ;48(1):3-26

6. Huang LF, Tong WQ. Impact of solid state properties on developability assessment of

drug candidates. Adv. Drug Deliver. Rev. 2004;56(3):321-334

7. York P. Solid-State Properties of Powders in the Formulation and Processing of Solid

Dosage Forms. Int. J. Pharmaceut. 1983;14(1):1-28

8. Brittain HG. X-ray diffraction III: Pharmaceutical applications of x-ray powder

diffraction. Spectroscopy. 2001;16(7):14-+

9. O’Neill MAA, Gaisford S. Application and use of isothermal calorimetry in

pharmaceutical development. Int. J. Pharmaceut. 2011;417(1-2):83-93

10. Zeitler JA, Taday PF, Newnham DA, Pepper M, Gordon KC, Rades T. Terahertz pulsed

spectroscopy and imaging in the pharmaceutical setting – a review. J. Pharm. Pharmacol.

2007;59(2):209-223

11. Vehring R. Red-excitation dispersive Raman spectroscopy is a suitable technique for

solid-state analysis of respirable pharmaceutical powders. Appl. Spectrosc.

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12. Boraey MA, Hoe S, Sharif H, Miller DP, Lechuga-Ballesteros D, Vehring R.

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ethanol-water cosolvent system. Powder Technol. 2013;236:171-178

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14. Willart JF, Descamps M. Solid State Amorphization of Pharmaceuticals. Mol.

Pharmaceut. 2008;5(6):905-20

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state study of polymorphic drugs: carbamazepine. J. Pharm. Biomed. Anal.

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16. Chen ZP, Li LM, Jin JW, Nordon A, Littlejohn D, Yang J, Zhang J, Yu, RQ. Quantitative

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its correction. Anal Chem. 2012;84(9):4088-4094

17. Deschamps T, Martinet C, de Ligny D, Bruneel JL, Champagnon B. Low-frequency

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ReferencesSulayman Oladepo* received his PhD in Chemistry from

the University of Alberta. He was a postdoctoral fellow

in the group of Prof. Reinhard Vehring at the University of

Alberta, where he worked on the development of low

frequency shift Raman instrumentation for pharmaceutical

respirable powders. He recently joined the Department of

Chemistry at King Fahd University of Petroleum and

Minerals, Dhahran, Saudi Arabia, as an Assistant Professor of Analytical

Chemistry. His current research involves the development of Raman

spectroscopic instrumentation for applications in pharmaceutical,

biomedical and oil and gas industries, as well as development of molecular

probes for biomedical applications. *Corresponding author. Email:

[email protected]

Hui Wang is currently a PhD student at the University of

Alberta, Edmonton, Alberta, Canada. He received his

Bachelor’s degree from Department of Materials Science

and Engineering, Southeast University, Nanjing, Jiangsu

Province, China, in 2012 and Master’s degree from the

Department of Mechanical Engineering, University of

Alberta, Edmonton, Alberta, Canada, in 2014. His research

has been about using macro-Raman spectroscopy for the characterisation of

pharmaceutical powders.

David Barona is a Project Manager & Research Associate

at the University of Alberta. Mr. Barona was awarded a BSc

in Aerospace Engineering from the Georgia Institute of

Technology (Atlanta, USA) in 2008. He has worked at the

University of Alberta as a Research Assistant on Sprayable

Superhydrophobic Surfaces (2008-2012), characterisation

of pharmaceutical powders (2013-present), as well as

Project Manager and Systems EIT for several space science R&D projects

(2010-present). Mr. Barona has specialised in the management of research

and technology development projects and has developed specific skills for

the effective management of research projects funded by national and

international agencies as well as industrial partners. His research interests

include micro/nanoparticle engineering and their applications, engineering

of multi-component sprayable solutions, surface engineering, lab-on-a-chip

devices and technology development projects.

Dr. Reinhard Vehring is a Professor in the Mechanical

Engineering Department at the University of Alberta and

holds the George Ford Chair in Materials Engineering. He

graduated with a diploma in Mechanical Engineering from

the Gerhard Merkator University in Duisburg, Germany,

and received a doctorate from the University of Bochum in

the field of molecular spectroscopy on microparticles.

Dr. Vehring has held positions in academia and industry advancing aerosol

science and particle technology for more than 25 years. Before returning to

academia, he worked on pulmonary delivery of peptides, proteins and small

molecules at Nektar Therapeutics and was part of the team developing

Exubera, the first inhalable insulin. Subsequently, he developed solid dosage

forms for virus vaccines, monoclonal antibodies and oncology therapeutics

at Medimmune, and supported FluMist, the first nasally administered live

attenuated influenza vaccine. Dr. Vehring was the lead inventor for the

cosuspension formulation technology which is used by Pearl Therapeutics to

develop metered dose inhaler based therapeutics for respiratory diseases.

At the University of Alberta, Dr. Vehring directs the Particle Engineering

facility focusing on advanced micro and nanoparticle design and analysis.

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Handheld Raman instruments are finding increasingacceptance and applications in the pharmaceuticalindustry. Would-be users will only embrace such portableinstruments if they provide comparable or betterperformance than the instruments they currently use.What are the current challenges you face in terms oftechnology development and wider acceptance of handheld Raman instruments?Armin: The challenge for performance in Raman is to find theappropriate laser excitation wavelength for materials to be analysed inorder to have best compromise between sensitivity and mitigation offluorescence. Now, with Sequentially Shifted ExcitationTM from Bruker,the first technology is on the market to mitigate the effects offluorescence while keeping high sensitivity. Other technologicaldevelopments to gain broader acceptance for handheld Ramaninstruments are the laser safety class and the ease of operation througha guided workflow. For instance, a laser class 1M does not requirerestricted areas, laser safety officers or safety glasses.

Valerian: Unlike traditional methods, our use of a unique 1064nmwavelength excitation laser in an ergonomic, handheld platformprovides users with a method to analyse the most comprehensive list ofmaterials at the time of receipt. We continue to educate the market onthe expanded capabilities of 1064nm Raman and the value gap it fillsover current processes where fluorescence can be problematic. The ability to analyse more incoming materials on-site outweighs thecommon challenge of a difficult and lengthy process to change existingworkflows and standard operating procedures. The benefits of superiormaterial coverage have become quickly evident in the bio-pharmaceutical segment.

Mark: Our current handheld Raman instruments are designed to answervery specific questions around material identification in waysequivalent to, or better than, benchtop instruments available today.However, material identification is only one of many applications thatRaman may be used for in the pharmaceutical industry. Providing aneffective response to the same analytical challenges that benchtop

IN-DEPTH FOCUS: RAMAN ROUNDTABLE

34 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

ModeratorSulayman Oladepo, King Fahd University of Petroleum and Minerals

Dr. Armin Gembus Global Business Unit Manager for Raman

and Gas Analytics, Bruker

Dr. Valerian Ciobotă Applications Scientist,

Europe, Rigaku

Mark Cabell Regional Sales Manager,

Thermo Fisher

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instruments face requires careful planning to ensure measurementquality in all potential testing environments, while maintaining thesimplicity of a handheld device.

Lasers that are used in handheld instruments are typically in the 532-1064nm range. Manufacturers andvendors have claimed the 1064nm wavelength helps toavoid sample fluorescence. However, given the inverserelationship between Raman intensity and the fourthpower of excitation wavelength, such longer wavelengthsmay lead to reduced Raman signal. Is this a concern in the application of longer wavelength handheld Raman instruments?Valerian: No, this is not of concern. Spectral quality is governed bysignal-to-noise ratio (SNR), not raw intensity. Handheld 1064nm systemsmaximise signal by using more powerful lasers than their counterparts.The more important part of the equation is noise, which has twosources: the instrument (combination of thermal, electronic, readout),and light returning from the sample (shot noise). 1064nm systemsminimise instrument noise through electronic design and detectorcooling. Therefore, all commercially-available instruments can reliablyidentify non-fluorescing samples. The practicalnoise driver for challenging samples comesfrom shot noise produced by fluorescence,which is minimised only by selecting a longerexcitation wavelength.

Armin: The approach of using a 1064nmwavelength for excitation in order to mitigatefluorescence has been taken with FT-Ramanspectrometers for a long time. The comparably low yield of Ramanscattering is compensated by the high throughput advantage of FT-spectrometers. In contrast, handheld Raman instruments, which arebased on dispersive technology, have to compensate the lowersensitivity either by high laser power and/or the measurement time. Forhandheld Raman instruments both options seem not to be well suited

since an increase in the laser power enhances the safety risk and anelongated measurement time exceeds the desired time for analysisusing handheld instruments.

Mark: Use of 1064nm lasers can provide multiple challenges for Raman analysis. Not only is the Raman effect 3.4 times less than with785nm lasers, but the detector technology lags significantly behindsilicon charge-couple devices with 785nm lasers in use today. The end result is lower signal and lower resolution that makes identifica-tion of pure materials difficult and detection of impurities oradulteration challenging.

A major advantage of handheld instruments is portabilityand field analysis. How crucial are slight discrepancies infield-to-field measurement conditions to the integrity ofresults obtained with such instruments? And how mightone correct for such field-to-field discrepancies?Mark: Thermo Scientific analysers are not only portable but alsoadaptive to the testing environment as it exists during the actualsample measurement. This is absolutely critical for handheld devicesoutside the well-controlled environment of a laboratory where

benchtop instruments have the benefit ofhighly reproducible sampling interfaces.

Armin: Depending on the impact of thediscrepancies, measures must be taken toensure unambiguous distinction of materials.Technical measures could be a thermalstabilisa tion management of at least the corecomponents such as the excitation laser and

detector and/or an inbuilt calibration source in combination withsoftware-implemented spectral performance tests for continuouscorrection and to prove the integrity of results.

Valerian: We improve the stability for field analysis by making theinstrument resilient to the types of circumstances that are encountered

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VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 35

Our current handheld Ramaninstruments are designed to answer very

specific questions around materialidentification in ways equivalent to, or

better than, benchtop instrumentsavailable today

Mark Cabell

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in the field. Because our Progeny handheld Raman features an activecooling detector and is sealed, it is stable across wide temperatureranges, high humidity, and is engineered to handle tough environ -mental conditions.

Nucleic acid-based drugs are now making their way tomarkets. Can handheld Raman instruments play some rolein the characterisation, analysis and quality assurance ofthese kind of drugs?Valerian: The benefit of handheld Raman in the manufacturing ofnucleic acid-based drugs is that it has the ability to analyse the rawmaterials used in manufacturing process.Raman does not have the ability to sequencenucleic acids, so it will not be possible to useRaman to look at the finished product.

Armin: As Raman instrumentation is becomingmore and more powerful, handheld Ramaninstruments may be used in the future for more demanding tasks such as the characterisation, analysis and quality assurance of nucleic acid-based drugs. Currently, limitations might not only be givenby the spectroscopic instrumentation but also by the availability ofsuitable commercial SERS substrates to reach the required levels of sensitivity.

Mark: High-performance handheld Raman instru -ments are capable of identifying and characterisinga percentage of biologic materials and mostcomponents of the excipient packages are easilyanalysed by Raman. Challenges exist in very dilute samples, prefilled syringes and other finaldosage forms. Innovation in sample preparationand SERS-type techniques will be required to more robustly address new biological materials inthe future.

Raman instruments in the laboratory have fixed or controlled distancesbetween sample and collection optics.Lack of such control may lead to lowquality spectra. Given the way portableinstruments are operated, how does this distance affect the quality of spectra and how can a typical user control this distance?Armin: Most handheld Raman instruments have aset of different measuring tips dedicated todifferent packing in which materials are presentedto the optics. To keep quality of spectra high thedistance to the material of interest is typically fixed. An adjustability of the distance as forscientific instruments with high flexibility is oftennot useful for handheld instruments since itcontradicts the ease of use required in fieldcompared with laboratories. Supporting users inapplying appropriate measuring tips for materials

is realised with IntelliTipTM by Bruker, where an assignment of tips for materials is possible.

Valerian: Limitations caused by a fixed focal point, especially foraccurate identification of materials contained in packaging, is a well-known obstacle for both generating high quality spectra and also obtaining an accurate match to the correct material spectra stored in the instrument’s database. To overcome this barrier, Progeny’s hardware, software and sampling accessories were designedto provide users with the ability to improve the laser signal per application by adjusting the focus. User-customised adjustable

focus optimises sampling sensitivity whichensures the acquisition of the most accurateand repeatable results from the broadest rangeof samples.

Mark: Our handheld Raman instruments have a very tightly controlled optical tolerance

and fixed optics to ensure user safety and the repeatability ofmeasurements. For material identification purposes, we maintain anaverage sample area of ~2mm. In special applications where SERS or where small beam spots are advantageous, our focal offsetaccessories take advantage of our optical geometry to maximise beamand signal performance.

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IN-DEPTH FOCUS: RAMAN ROUNDTABLE

The benefit of handheld Raman in the manufacturing of nucleic acid-

based drugs is that it has the ability toanalyse the raw materials used in

manufacturing process Dr. Valerian Ciobotă

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Drivers for the pharmaceutical industry to move production from batchto flow are much weaker than for the commodity chemical industry. Theproduction volumes of pharmaceuticals are considerably less than thoseof bulk chemicals and product lifecycles are often short. Furthermore,pharmaceuticals are significantly more complex than commoditychemicals and their production usually requires many – often widelydiverse – synthesis steps in addition to multiple rounds of isolation andpurification. The complexity and diversity of these molecules, and theconsequently involved and diverse process conditions required for theirsynthesis and isolation demand flexible, multipurpose reaction vessels

for their production. Stirred tank-reactors are easy to handle and can beemployed for a range of different operations. However, despite their longhistory and prevalence in synthesis laboratories, batch-type reactorshave some severe, well-recognised limitations. Most importantly,chemical reactions which release large amounts of energy or proceed viaunstable, highly toxic or explosive intermediates are difficult orimpossible to implement in tank reactors.

In the past few decades, technologies for continuous synthesis haveadvanced tremendously and a plethora of discrete flow components andmodules, including pumps, mixers, reactors and separation units, along

Continuous flow processes have many distinct advantages over discontinuous batch production and therefore, in the last century, continuous operation has become by far the most dominant form of production for high-volumeand low-cost materials such as petrochemical and commodity chemicals. The first applications of continuousprocesses in the pharmaceutical industry emerged only comparatively recently and the vast majority of productionis still undertaken in batch reactors. Herein, we highlight some of the advantages that continuous flow processingoffers for the synthesis of pharmaceuticals and fine chemicals.

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VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 37

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Continuousmanufacturing in thepharma industry – anunstoppable trend?

Bernhard Gutmann and Christian Oliver KappeUniversity of Graz

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with an increasing number of integrated standalone flow reactors, havebecome commercially available. The emergence of various reactordesigns, addressing the diverse physicochemical requirements ofchemical reactions, along with the emergence of technologies for feeddelivery, flow metering, continuous separation, etc., enables theassembly of specialised, high-performance flow systems by combiningthese operation units.

The modular approach provides the flexibility required for finechemical synthesis while the advantages inherent to continuousproduction are maintained. It is partly becauseof the commercial accessibility of flowequipment for organic synthesis on small tomedium scales that flow chemistry evolvedfrom a pure research topic in chemical engin -eer ing to an almost ubiquitously applicabletechnique in organic synthesis laboratories. Furthermore, thepharmaceutical industry is investing heavily in the development andadvancement of continuous manufacturing. In fact, virtually all largepharmaceutical companies now have dedicated groups working on thedevelopment and implementation of flow synthesis. Indeed, a 2005roundtable consisting of 15 pharmaceutical companies and contractmanufacturers identified continuous processing as one of the “keygreen engineering research areas for sustainable manufacturing”.1

MiniaturisationIt was estimated that around a half of the reactions currently run in thepharmaceutical industry could benefit from being performed in flow.Perhaps the foremost advantage of flow synthesis is the enhanced

safety profile.2 Since product is formed continuously, with no downtimeof the production for charging and discharging the reactor, largeamounts of material can be processed even in comparatively smallreactors. In fact, the actual reactor units typically have channeldiameters ranging from below 100μm (i.e., microreactors) to a fewmillimeters (meso/milli-reactors). Whereas tank reactors with volumesof tens of cubic meters are common in chemical companies, theinternal volumes of flow reactors typically range from below 1μL to acouple of liters. A smaller volume implies that there is less material in

the reactor and the amount of material orenergy released in a safety event is corre -spondingly reduced. In fact, the materialcontent in microreactors is often too small tocause serious damage to human health or theenvironment in the event of an accident. Thus,

continuous manufacturing enables reactions to be performed orreaction conditions to be applied which would be infeasible or nearlyimpossible in batch mode on larger scales.2

Short atom- and step- efficient synthetic routes frequently involvethe use of highly reactive or otherwise hazardous compounds, and, in the last decade, a large amount of work was directed towardsadapting these reactions for continuous processing. Continuous flowvariants have been developed for a plethora of notoriously hazardoustransformations, including nitration reactions, reactions with azides,phosgene, cyanides, isocyanides, diazo- and diazonium compounds.2

For instance, a chemist from DSM developed a pilot scale continuousflow nitration plant for the generation of the nitrate-building block inthe non-steroidal anti-inflammatory drug Naproxcinod (nitronaproxen ,

PAT SERIES

The pharmaceutical industry is investing heavily in the development

and advancement of continuousmanufacturing

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NicOx) (Scheme 1a).3 The reaction wasperformed with neat nitric acid in Corningglass reactors with an internal volume ofbelow 150mL. One reactor module deliveredabout 13kg/h of the product. Parallelisationof the modules afforded a production unitwith a capacity to produce 100kg/h ofproduct, corresponding to an annual prod -uctivity of 800t.3

Crucial for reactions involving veryreactive reagents or intermediates is theexceptionally fast heat and mass transferwhich can be accomplished in micro -structured devices.2 For fast or highlyexothermic reactions, mass and heattransfer become limiting and the reactionmixture has to be cooled or diluted to reducethe reaction rate and to keep the reaction asuniform as possible, or reagents have to bedosed slowly to the mixture to maintaintemperature at a constant level. Adequatemixing and dissipation of excess energybecomes increasingly difficult upon scale-upin stirred tank reactors and accumulation ofheat or formation of hot-spots mightdiminish reaction selectivity and can lead to serious risks of thermal runaways. In microstructured reactors, mixing can beachieved on time scales of milliseconds. Similarly, considerablyimproved heat exchange efficiency is obtained due to the small channeldimensions and the very large surface-to-volume ratios ofmicroreactors. Thus, fast reactions, such as reactions involvingorganometallic compounds like alkyl lithium or Grignard reagents, canbe run at significantly higher temperatures than usual, often near, oreven at, room temperature.2 The use of cryogenic temperatures is

rendered unnecessary and these reactions become economically andsubstantially more attractive.

Process intensificationOn the other hand, high temperatures might be applied deliberately toincrease the reaction rate.2 The reaction rate usually depends roughlyexponentially on temperature and increasing the reaction temperature

is often the easiest and most effective way toenhance reactivity. However, temperaturesabove around 200°C are largely outside theoperation range of most conventional tankreactors in the pharmaceutical industry.Furthermore, for batch reactions the wholeinstallation has to be heated to the targettemperature and cooled after the reaction isfinished. In contrast, for flow processes onlythe reaction mixture is heated while it iscarried through the channels of a flowreactor at constant temperature, and theprocessed mixture is subsequently cooled ina heat exchanger at constant temperature.

Combined with the rapid heat exchangecapability of microreactors, extremelyefficient and responsive heating is thusattained. Furthermore, the small dimensionsof microreactors entail high resistance topressure. As a result, temperatures far above

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VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 39

Scheme 1: Selected examples of continuous flow synthesis of active pharmaceutical ingredients

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the atmospheric boiling point of the solvent become accessible fairlyeasily and safely. Higher reaction rates, in turn, yield higher throughputsor allow a further reduction of the reactor size and consequentlyreactor cost. Since the advent of microwave reactors around 50 yearsago it became obvious that a wide range of diverse chemicaltransformations are very amenable to high temperature processing.Continuous flow microreactors now provide thetechnology necessary to scale these reactionsup to industrially-relevant scales.2

Reactions with gaseous reagentsContinuous flow microreactors present an idealplatform for reactions with gaseous reagents.Gases can be dosed into the flow system with precise stoichiometryusing mass flow controllers and intense mixing of the gaseous andliquid phase can be achieved. Furthermore, high pressure operationincreases the concentration of the gas in the liquid phase. Combustionand explosion hazards are reduced in channels of small diameter and,consequently, reactions can be performed under unusually harshprocess conditions in a safe and controllable manner. In particular,continuous flow hydrogenations have found extensive application in

the chemical industry, and, with the introduction of commercial bench-top high-pressure hydrogenators, it has been rapidly spread insynthesis laboratories.2

The synthesis of the penultimate intermediate of Eli Lilly’s product,LY500307, a potent, selective oestrogen receptor β agonist, required anasymmetric rhodium catalysed hydrogenation in the presence of a

Josiphos ligand (Scheme 1b; page 39).4

Pressures up to 70 bar were necessary to reducesubstrate-to-catalyst loading to economicallyviable levels. For the pilot-scale reaction, a 73L stainless steel tube reactor was employed.The asymmetric hydrogenation was coupledwith a continuous downstream work-up to

provide the desired product with more than 99% ee. In addition to safety benefits, the capital cost for the flow plant was significantlyless and it was considerably faster to build than a high pressurehydrogenation autoclave.4

Continuous multistep synthesisFor a flow process, multiple reactor and separation modules can bearranged consecutively to form a single continuous operation unit. For

continuous multistep synthesis, reagents arefed into the assembly at specified points andthe reaction stream is manipulated along thereaction path to suit the needs of individualtransformations. Reaction intermediates arethus generated and directly consumed in theclosed and, where necessary, pressurisedreaction environment of the microreactor. Inrecent years, considerable efforts have beenundertaken to devise uninterrupted multi -step flow reaction sequences, and elaborateprocesses for the direct synthesis of complexmolecules from simple starting materialshave been demonstrated. However, thoughimpressive, the benefits of these complexprocesses and the practicability for mediumto large synthesis have not yet beenestablished. Design and development ofmultistep continuous processes is certainlychallenging and requires a detailedunderstanding of the individual operations

40 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

PAT SERIES

Scheme 2: Continuous end-to-end production of aliskiren

Many challenges of photochemistry,including the exponential decrease of the

photochemical efficiency with the pathlength (Beer-Lambert law), are naturally

addressed by microreactors©

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and a careful, ‘holistic’ planning of the wholereaction sequence. An obvious advantagearises when a multistep sequence eliminatesthe need to isolate explosive or toxicintermediates.2

None-routine chemistryContinuous flow and microreactor tech -nology provides new opportunities for none-routine chemistry such as electro- andphotochemistry. These methods are trad -itionally hardly considered when a route to anew molecule is designed, even thoughtransformations are enabled which aredifficult to achieve by mainstream chemistry.In recent years research in continuous flowphotochemistry in particular has becomevery active. Many challenges of photo -chemistry, including the exponentialdecrease of the photochemical efficiencywith the path length (Beer-Lambert law), arenaturally addressed by microreactors. A striking example was demonstrated by chemists from the Max-Planck Institute for Colloids and Interfaces, Germany(Scheme 1c; page 39).5 The group demon -strated a fully continuous synthesis ofartemisinin, currently the most effective antimalarial drug. The key stepin the synthesis of artemisinin is an ene reaction of dihydroartemisinicacid with singlet-oxygen. The photo-generation of singlet oxygen in acontinuous flow photoreactor and the concomitant ene reaction wasdirectly coupled with the cleavage of the oxygen-oxygen bond as well assubsequent oxidation with triplet oxygen and condensation toartemisinin in a tube reactor. Pure artemisinin was obtained in 46%overall yield after a total residence time of only 12 minutes in thecontinuous flow system.5

Downstream processingThe actual chemical transformation in the synthesis of a pharma -ceutical frequently consumes only a fraction of the materials, time andenergy expended. About two to three separation operations arecommonly performed for every reaction step. The vast majority of theseseparation steps are currently performed in batch or semi-batchequipment. However, for flow processes to deliver the projected

benefits to the pharmaceutical industry, the whole generation of thefinal, purified pharmaceutical product from simple synthetic precursorsultimately needs to be continuous. Many work-up processes, such asdistillation, extraction or phase separations, are reasonably easilyadapted for continuous processing and are often considerably moreeffective when run continuously. Novel, continuous, and intensifiedtechniques are presently developed for challenging separationoperations including filtration, washing and drying. The Novartis–MITCenter for Continuous Manufacturing recently disclosed the continuousend-to-end production of Tekturna (aliskiren, Novartis) a drug used totreat high blood pressure (Scheme 2; page 40).6 The fully continuousprocess included all synthetic steps, separations, crystallisations,drying and formulation to produce the final tablets. The number of unitoperations was reduced from 21 to 14, and the total residence time was an order of magnitude shorter than the production time needed for the batch process.6

ConclusionIn 2011 the US Food and Drug Administration predicted that in the next25 years “continuous manufacturing will make current methodsobsolete”.7 However, even though continuous processing offers many rather clear advantages over traditional batch-wise operation,this technique has yet to become routinely used in the pharma-ceutical industry. The vast majority of operations in the pharmaceutical industry are still run in stirred tank reactors. The development of a continuous flow synthesis is engineering intensive and,additionally, requires a detailed understanding of the involved chemicalprocesses. Furthermore, although many of the presently performed

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VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 41

Continuous flow processing brings both advantages and new challenges

Advantages Challenges

■ Increased safety throughminiaturisation

■ Instantaneous heating/cooling

■ Fast mixing (< 1s)

■ Process intensification

■ Process integration

■ Automation

■ Straightforward scale-up

■ Novel not fully proven technology

■ Some technology missing or not fullymatured (handling of solids etc.)

■ Major investment in R&D still required

■ Regulatory uncertainty

■ Engineering-intensive

■ Lack of trained peoples

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Let Your Production Flow — RamanRxn2TM

Hybrid for Continuous Manufacturing of Your Liquids & Solids

• Optimized In-flow Liquids and Solids Analysis• Transferability from Laboratory, to Scale-up, to Pilot Plant• mm to micron Sampling• Meets GLP and GMP Requirements

Continuous LiquidsNeSSI (CPAC) Flow System Integrated with Kaiser’s Immersion Probe Technology• Reaction Analysis• Polymerization

Continuous Manufacturing of SolidsKaiser’s PhAT Probe for Solids in Unit Operations Production• Tablet Coating• Polymorphism / Crystallization• Process-induced Transformations• Extrusion Monitoring

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batch operations could be translated to continuous processes fairlyeasily, without major modifications or re-optimisation, there is littleincentive to do so in the presence of sufficient capacity in the existingbatch installations.

Perhaps the true potential of continuous manufacturing will berealised when a reaction sequence is planned from the very outset with

the unique opportunities enabled by this technology in mind.Chemistry which was impractical or simply impossible to run in batchreactors can be contemplated and entirely new synthesis routesbecome accessible. To fully establish continuous flow processes in thepharmaceutical industry, further development and maturation of the technology, as well as a continuous commitment of chemists and engineers to design ever more efficient and sustainable processes,will be required.8

PAT SERIES

Bernhard Gutmann studied Chemistry at the University of

Graz, Austria. In 2009 he obtained his diploma degree in the

field of microwave-assisted organic synthesis. He continued

with PhD work in the group of Professor C. Oliver Kappe on

projects related to continuous flow processing. After

receiving his PhD degree in 2013, he joined the Christian

Doppler Laboratory for Continuous Flow Chemistry at the

University of Graz and is currently Senior Researcher at the Research Center

Pharmaceutical Engineering (RCPE) where his research is focused on the

continuous-flow synthesis of APIs.

Christian Oliver Kappe is Professor of Chemistry at the

University of Graz and Key Researcher at the Research

Center Pharmaceutical Engineering (RCPE) in Graz,

Austria. He received his undergraduate and graduate

education at the University of Graz. After periods of

postdoctoral research work at the University of Queensland

and at Emory University, he moved back to the University

of Graz in 1996 to start his independent academic career. He became

Associate Professor in 1999 and was appointed Full Professor in 2011.

The co-author of ca. 350 publications, his main current research interests are

related to continuous flow chemistry, API manufacturing and process

intensification. He is Editor-in-Chief of the Journal of Flow Chemistry,

short-course instructor on continuous flow technology and a consultant for

the pharmaceutical industry.

1. (a) Poechlauer, P, Colberg, J, Fisher, E, et al. Pharmaceutical roundtable study

demonstrates the value of continuous manufacturing in the design of greener processes,

Org. Process Res. Dev. 2013; 17, 1472-1478; (b) Poechlauer, P, Manley, J, Broxterman,

R, et al. Continuous processing in the manufacture of active pharmaceutical ingredients

and finished dosage forms: an industry perspective, Org. Process Res. Dev. 2012; 16,

1586-1590; (c) Jiménez-González, C, Poechlauer, P, Broxterman, QB, et al. Key green

engineering research areas for sustainable manufacturing: a perspective from

pharmaceutical and fine chemicals manufacturers, Org. Process Res. Dev. 2011; 15, 900-

911

2. Gutmann, B.; Cantillo, D.; Kappe, C. O. Continuous-Flow Technology—A Tool for the

Safe Manufacturing of Active Pharmaceutical Ingredients, Angew. Chem. Int. Ed. 2015;

54, 6688–6728

3. Braune, S, Poechlauer, P, Reintjens, R, et al. Selective nitration in a microreactor for

pharmaceutical production under cGMP conditions, Chim. Oggi/Chem. Today. 2009;

27(1), 26-29

4. Johnson, M. D.; May, S. A.; Calvin, J. R.; et al. Development and scale-up of a

continuous, high-pressure, asymmetric hydrogenation reaction, workup, and isolation,

Org. Process Res.Dev. 2012; 16, 1017–1038

5. Kopetzki, D, Lévesque, F, Seeberger, PH. 2013; A continuous-flow process for the

synthesis of artemisinin, Chem. Eur. J., 19, 5450–5456

6. Heider, PL, Born, SC, Basak, S, et al. Development of a Multi-Step Synthesis and Workup

Sequence for an Integrated, Continuous Manufacturing Process of a Pharmaceutical, Org.

Process Res. Dev. 2014; 18, 402-409

7. http://www.in-pharmatechnologist.com/Processing/Continuous-manufacturing-will-

make-current-methods-obsolete-FDA-says

8. Baxendale, IR, Braatz, RD, Hodnett, B, et al. Achieving Continuous Manufacturing:

Technologies and Approaches for Synthesis, Workup, and Isolation of Drug

Substance May 20–21, 2014 Continuous Manufacturing Symposium. 2015; J. Pharm.

Sci. 104, 781-791

References

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As the global healthcare industry shifts and transforms, so too must thelife science industry. Life science companies will need to adjuststrategies to a world in which only true innovation and improved patientoutcomes become the key determinant of value in the healthcaresystem. They will also need to develop commercial models which takeaccount of the changing power balance between provider, payer andpatients; all the while keeping a watchful eye on the competitivelandscape for the incursion of non-traditional players with whom theywill increasingly compete (or partner) to deliver patient-centredproducts and services. Meanwhile, scrutiny of high prices for specialitydrugs and advanced technologies is increasing, impacting the ability oflife science companies to bring much needed innovation to patients.How will the industry’s future unfold?

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VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 43

Date: 16-17 November 2015Location: London, UKFor more information, please visit: live.ft.com/pharmabio

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Multi-component crystals are widely used to improve the physico -chemical properties of APIs, such as solubility1, stability2, powdercompaction3 and bioavailability4. When the drug possesses a dissociablegroup, salt formation is usually utilised. Recently, co-crystals have beenintensively investigated for their ability to improve the solubility ofundissociable drugs1,5. Although organic solvates have received lessattention compared with co-crystals, some have also been utilised asAPIs, including indinavir sulphate ethanolate, efonidipine hydrochlorideethanolate, metildigoxin acetonate, ledipasvir acetonate, anddapagliflozin propylene glycolate hydrate. Moreover, some drugs tend toform several organic solvates, which are collectively referred to as‘promiscuous solvate formers’. These promiscuous solvate formers offernew opportunities to improve the solubility of a drug; however, they could

also be problematic from the viewpoint of manufacturability. In thisreview, several examples of promiscuous solvate formers are discussed.

Examples of promiscuous pseudo-polymorphsInlyta Axitinib (axitinib, Pfizer) is a drug that targets vascular endothelialgrowth factor6. It is capable of forming five anhydrate polymorphs and66 pseudo-polymorphs. In its crystal form, it has a large polymorphspace7. During the later stage of the development of axitinib, a morestable low-energy polymorph emerged, indicating the need tointensively screen for polymorphs and pseudo-polymorphs. Severalscreening technologies were employed, such as fast evaporation, slowcooling of saturated solutions and slurry methods at different

The physicochemical properties of active pharmaceutical ingredients (APIs) are critical to the success of drugdevelopment. Most of the APIs in oral solid dosage forms are contained as drug crystals. Drug crystals can becategorised as single and multiple component systems, the latter of which include salts, co-crystals and solvates.When the solvent is water, the drug solvate is referred to as a hydrate. Salts formation is defined as proton transferfrom a drug molecule to a guest molecule and the guest molecule (i.e., a counter-ion) is bonded to the drug molecule(i.e., a drug) by one or several ionic bonds. In a co-crystal and a solvate, a guest molecule (a co-crystal co-former ora solvent) is bonded to the drug via inter-molecular interactions other than the ionic bond, such as with hydrogenbonds. Co-crystal co-formers are non-volatile molecules at ambient conditions. In most cases, the drug solvates ofAPIs are hydrates; however, they can be organic solvents.

POLYMORPHS

Improving solubility with promiscuous multi -component drug crystals

Masataka Ito, Kiyohiko Sugano and Katsuhide TeradaFaculty of Pharmaceutical Sciences, Toho University

44 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

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temperatures. These screening methodswere effective at thoroughly finding thepseudo-polymorphs of axitinib.

SulphathiazoleThe crystal structure of the host molecules inmulti-component crystals is sometimesclassable. Sulphathiazole is an antibacterialdrug that is known to crystallise over 100solvates and co-crystals, and shows fivepolymorphs8. Two types of crystal structureshave been identified9, as determined by theassemblage between the guest molecule andsulphathiazole: a) clathrates, in which thehost molecules have a channel, layer, orthree-dimensional framework structure andthe guest molecule fills the cavity; b) co-crystals, in which the guest molecule is anessential part of the hydrogen-bondedframework. Salt formation was observed inboth cases.

FurosemideThe flexibility of a molecule is important when considering crystalstructure classification. Methylene carbon chains make a moleculeflexible, increase the number of structure classes, and make thestructure classes more continuous. Forexample, furosemide (Lasix, Sanofi) forms fourpseudo-polymorphs: tetrahydrofuran solvate(1:1), 1,4-dioxane solvate (1:1), N,N-dimethylformamide solvate (1:1), and dimethyl sulph -oxide solvate (1:1)10. These pseudo-polymorphsshow four different conformations originating from the methylenecarbon. Diverse solvents can be included in the crystal because of theflexibility of the molecule.

OlanzapineIn contrast to flexible molecules, structurally rigid molecules show alower number of conformations. However, even these molecules caninclude diverse solvents in spite of lesser flexibility, as is the case witholanzapine. Olanzapine (originally branded Zyprexa by Eli Lilly) forms

pseudo polymorphs with diverse solvents11,12.Two hydrates (2 hydrate, 2.5 hydrate), adichloromethane solvate (2:1), and a methanol(1:1) are found in the Cambridge structuredatabase. Triple component pseudo poly -morphs are also found: olanzapine-water-

ethanol (2:2:1), olanzapine-water-butanol (1:1:1) and olanzapine-water-dimethyl sulphoxide (1:1:1). When comparing the crystalstructures of these pseudo-polymorphs, the olanzapine molecule

forms a mirror image pair in all crystals, and thesolvent molecules are positioned between the pairsof olanzapine molecules. Most of the solventmolecules form four hydrogen bonds with the twopairs of olanzapine molecules. The tendency ofolanzapine to form a mirror image pair is alsoobserved in anhydrate crystals. It is likely that thepair of olanzapine molecules is the key factor forsupramolecular building blocks. The building blockwould be capable of generating diverse crystal formsbecause of the adjustable space between theolanzapine molecules.

PranlukastSome solvent molecules in pseudo-polymorphs arecapable of being exchanged with different solventmolecules in the vapour phase. Pranlukast (Onon,Azlaire), a leukotriene receptor antagonist, formseight pseudo-polymorphs with seven solvents, i.e.,water, methanol, ethanol, 1-propanol, N,N-dimethyl

POLYMORPHS

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 45

Figure 1: Dissolution profiles of Pranlukast (PRS)/ethanol solvate (solid line), PRS/urea cocrystal (dashed line) andPRS hemihydrate (dotted line) (mean ± S.D, n = 3). Adapted from13 with permission

Recently, co-crystals have been intensively investigated for their

ability to improve the solubility ofundissociable drugs

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formamide, dimethyl sulphoxide, and propylene glycol13. Their crystalstructures are classified as a sheet-like and channel-like pattern.Pranlukast hemihydrate, N,N-dimethyl formamide solvate, dimethylsulphoxide solvate and water-methanol solvate form the sheet-likepattern. The sheet-like pattern includes asolvent in the space between the sheetstructures of pranlukast molecules. In contrast,pranlukast methanol solvate, ethanol solvate,and 1-propanol solvate form the channel-likepattern. Single crystal X-ray diffraction analysisrevealed that the channel-like pattern onlyexists in the pseudo-polymorphs with alcohols. The pseudo-polymorphs of the channel-like pattern solvates can transform into ahydrate with the sheet-like pattern in a humidified environment.Solvent exchange can also occur from alcohols to water. However, it isnot known whether or not the solvent exchange reaction is invertible.

Structural features and advantagesThe existence of a space that can expand or contract to fit the solventsin the crystal structure appears to be important for the formation ofpseudo-polymorphs with diverse solvents, regardless of whether thespace is intramolecular or between molecular complexes.

In terms of the advantages of promiscuous pseudo-polymorphs,their use can promote effective drug development. Pranlukast is apoorly soluble compound of the biopharmaceutical classificationsystem class IV. In previous studies, pranlukast was formulated as ananosuspension14 and high-pressure homogenised formulation15 toimprove the dissolution behaviour and oral absorption. Recently, wefound that the dissolution behaviour of pranlukast can be improved byusing an ethanol solvate13 (Figure 1; page 45). In addition, desorption ofa solvent from a pseudo-polymorph can be a method for producing aspecific polymorph. For example, the four pseudo-polymorphs offurosemide transform to different anhydrate crystals by solventdesorption. Furosemide-tetrahydrofuran (1:1) transforms to anhydrateform III, whereas furosemide-dimethyl sulphoxide (1:1) transforms toanhydrate form I10. In general, severe control of temperature andconcentration is necessary to generate a specific polymorph byrecrystallisation. Solvent desorption of a pseudo-polymorph mayprovide a new approach for generating specific polymorphs.

OutlookIn the future, it should become standard practice to study pseudo-polymorphs so as to improve the physical property of drugs in development. Pseudo-polymorph formation does not require

chemical structure modification and con -version to a pseudo-polymorph can improvethe dissolu tion behaviour of poorly solublecompounds. The use of pseudo-polymorphscan provide an effective method to generatespecific poly morphs. These cases indicate thatpseudo-polymorphs can be a solution to the

problems often encountered in drug development. However, there issome difficulty in manufacturing pseudo-polymorphs. When a pseudo-polymorph is being applied as an API, it is important to pay sufficientattention to the desolvation and transformation processes in drug development.

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 47

POLYMORPHS

1. Good DJ, Rodriguez-Hornedo N. Solubility Advantage of Pharmaceutical Cocrystals. Cryst

Growth Des. 2008;9(5):2252-64

2. Bolton O, Matzger AJ. Improved Stability and Smart-Material Functionality Realized in an

Energetic Cocrystal. Angew Chem Int Ed Engl. 2011;50(38):8960-3.

3. Sun CC, Hou H. Improving Mechanical Properties of Caffeine and Methyl Gallate Crystals

by Cocrystallization. Cryst Growth Des. 2008;8(5):1575-9.

4. McNamara DP, Childs SL, Giordano J, Iarriccio A, Cassidy J, Shet MS, et al. Use of a

Glutaric Acid Cocrystal to Improve Oral Bioavailability of a Low Solubility API. Pharm

Res. 2006;23(8):1888-97

5. Babu NJ, Nangia A. Solubility Advantage of Amorphous Drugs and Pharmaceutical

Cocrystals. Cryst Growth Des. 2011;11(7):2662-79

6. Campeta AM, Chekal BP, Abramov YA, Meenan PA, Henson MJ, Shi B, et al. Development

of a Targeted Polymorph Screening Approach for a Complex Polymorphic and Highly

Solvating API. J Pharm Sci. 2010;99(9):3874-86

7. Chekal BP, Campeta AM, Abramov YA, Feeder N, Glynn PP, McLaughlin RW, et al.

The Challenges of Developing an API Crystallization Process for a Complex Polymorphic

and Highly Solvating System. Part I. Org Process Res Dev. 2009;13(6):1327-37

8. Price CP, Glick GD, Matzger AJ. Dissecting the Behavior of a Promiscuous Solvate Former.

Angew Chem Int Ed Engl. 2006;45(13):2062-6

9. Bingham AL, Hughes DS, Hursthouse MB, Lancaster RW, Tavener S, Threlfall TL.

Over One Hundred Solvates of Sulfathiazole. Chem Commun. 2001(7):603-4.

10. Minkov VS, Beloborodova AA, Drebushchak VA, Boldyreva EV. Furosemide Solvates: Can

They Serve As Precursors to Different Polymorphs of Furosemide? Cryst Growth Des.

2014;14(2):513-22

11. Cavallari C, Santos BP, Fini A. Olanzapine Solvates. J Pharm Sci. 2013;102(11):4046-56

12. Clarke HD, Hickey MB, Moulton B, Perman JA, Peterson ML, Wojtas Ł, et al. Crystal

Engineering of Isostructural Quaternary Multicomponent Crystal Forms of Olanzapine.

Cryst Growth Des. 2012;12(8):4194-201

13. Furuta H, Mori S, Yoshihashi Y, Yonemochi E, Uekusa H, Sugano K, et al. Physicochemical

and Crystal Structure Analysis of Pranlukast Pseudo-polymorphs II: Solvate and Cocrystal.

J Pharm Biomed Anal. 2015;111:44-50

14. Baek IH, Kim JS, Ha ES, Choo GH, Cho W, Hwang SJ, et al. Dissolution and Oral

Absorption of Pranlukast Nanosuspensions Stabilized by Hydroxypropylmethyl Cellulose.

Int J Biol Macromol. 2014;67:53-7

References

Masataka Ito is Assistant of Pharmaceutics in the Faculty

of Pharmaceutical Science, Toho University, Japan.

His research focuses on the physical characterisation

of pharmaceutical solid and drug formulation design. He

conducted research at TOWA Pharmaceutical Co. Ltd.

during 2012 until 2015. He received his BS in 2012 at

Toho University. Contact Masataka at: masataka.itou@

phar.toho-u.ac.jp

Kiyohiko (Kiyo) Sugano is Associate Professor of Toho

University. He has over 18 years of experience working

within the pharmaceutical industries in Japan and the UK

(Chugai, Pfizer and Asahi Kasei Pharma). He received his

Bachelors and Masters degrees in Chemistry from

Waseda University. He received his PhD degree in

Pharmaceutical Sciences from Toho University.

Dr. Katsuhide Terada is Professor of Pharmaceutics in the

Faculty of Pharmaceutical Sciences, Toho University in

Japan. His research interests include the physical

characterisation of pharmaceutical solids and the quality

control in the manufacturing process using many kinds

of analytical methods. He is the author and or co-author of

more than 210 research papers, 50 reviews and 50 book

chapters. He is an active member of several professional organisations,

president of Japan chapter of PDA, and former president of Japanese Society

of Pharmaceutical Machinery and Engineering (JSPME). He received his

BS in 1975 and MS in 1977 at Chiba University and received a PhD in 1983

at the University of Tokyo.

Solvent desorption of a pseudo-polymorph may provide

a new approach for generating specific polymorphs

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Laura Juckem, PhD, R&D Group Leader at Mirus Bio, discusses the company’ssystems for optimising suspensionChinese hamster ovary (CHO) cells

During preclinical development, identification of promising candidateproteins is vital to maintaining a robust product pipeline, Mirus Biobelieves. Laura Juckem, PhD, R&D Group Leader at Mirus Bio tellsEuropean Pharmaceutical Review: “Obtaining sufficient quantities ofprotein early in this process allows for testing functionality and criticalquality parameters like glycosylation and aggregation which greatly aidsin making the go or no-go decision for a particular target.”

However, expression of relevant protein targets in CHO cells – oftenthe cell line of choice – has been hampered by low transienttransfection efficiencies making them an impractical host for early-stage screening, she believes. Higher titers can often be obtained fromHEK 293 derived cell types; however, the use of different host cellsbetween early transient and later stable protein production is aconcern. “Material obtained from 293 cells is not always consistent withCHO-derived material causing false-positive candidates to be advanced.Protein produced transiently in CHO cells better represents the finalbiologic that will be produced from the stable CHO clones, and withrecent advancements in transient transfection of CHO cells, high titersare can now be realised in both CHO and 293 cell types,” Dr. Juckem says.

Significant improvements have been made in serum-free,chemically-defined growth media for suspension CHO cells; however,the majority of media formulated for robust stable cell line growth donot support chemical transfection methods. Dr. Juckem explains thatknown inhibitors are polyanions, such as dextran sulfate or heparin, butcommercial media compositions are frequently proprietary making the assessment of transient chemical transfection compatibility relianton empirical testing. “The most stringent test for evaluating media compatibility is to form transfection complexes in the culturemedium and assay for expression; this will determine if the plasmidDNA can be adequately condensed and formed into a stabletransfection complex.”

She adds: “We have created a pre-optimised system that takesadvantage of synergy between the transfection reagent (TransIT-PRO®Reagent) and growth medium (CHOgro® Expression Medium). Thesecomponents work in concert to create a unique system allowingsuspension CHO cells to grow to elevated densities. A high percentageof the cells are transfected and more antibodies are secreted per cell.High density growth is sustained with suspension CHO cells grown in

CHOgro® Expression Medium leading to high viability and minimalclumping post-transfection. Happier cells post-transfection leads tomore target protein and less protease release through cell lysis whichultimately leads to a higher quality product.”

Dr Juckem explains that basic polymeric transfection methods, suchas 25 kDa linear PEI, are commonly employed in biotherapeutics largelydue to the inexpensive material cost. However, the actual incurred costsare much higher considering lower protein yields, additional labourcommitment and hidden cost of materials. “In head-to-headcomparisons with PEI, the CHOgro® Expression System yields three-to-four-fold greater antibody levels validating the CHOgro® system as acost-effective platform. Additionally, with lower total culture volumesrequired, precious incubator space can be freed up to increase overallproductivity when using the CHOgro® Expression System. Use of theTransIT-PRO® Also, Transfection Reagent and CHOgro® ExpressionMedium is hassle-free since no licensing is required for furthermanufacturing use.”

Dr. Juckem describes the CHOgro® Expression System as being“robust, easy-to-use and designed to require little or no optimisation toobtain high protein titers”. “No additional boosts or supplements areadded to the culture post-transfection, minimising the contaminationrisk and allowing increased flexibility in the timing of experiments. An optional step of moving the cultures to mild hypothermic conditions24 hours post-transfection can lead to a two-fold increase in titers.”

She explains that researchers can quickly obtain protein viatransient transfection because suspension CHO cells, e.g., FreeStyle™CHO-S, seamlessly adapt to the CHOgro® Expression Medium. A simplefull media exchange to CHOgro® Expression Medium 24 hours prior totransfection can yield 10-fold increases in protein titers in CHO-S cells.All components are free of animal origin components and worksynergistically to give high levels of usable protein in seven-to-14 dayspost-transfection. “The CHOgro® Expression System, which consists ofcomplete growth medium, transfection reagent, complex formationbuffer and supplements, allows all researchers to obtain high transienttiters in suspension CHO cells with this turn-key platform.”

Mirus Bio is an expert in providing transfection services globally, using its experience in nucleic acid chemistry,cellular and molecular biology to establish innovative technologies and products for use in life science research.

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VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 51

52 Implementation of a rapid methods portfolio at a pharmaceuticalmanufacturing site David Roesti and Erik Wilkens, Novartis

58 Use of RMMs in qualitycontrol: challenges and benefitsMostafa Eissa, Hikma Pharmaceuticals

61 Rapid assay for bioburdenand other contaminations Hideharu Shintani, Chuo University

67 Product ProfileEurofins Lancaster Laboratories discusses itsbio/pharmaceutical laboratory testing services

68 RMM RoundtableModerated by David Roesti, Senior Facilitator for RMM and Global Support,Novartis Pharma Stein AG

RMM

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In a 2013 PDA survey1 of the pharmaceutical, over-the-counter, medicaldevices and cosmetics industries, 20% of respondents answered that theywere using RMMs in routine quality control. So why are the vast majority ofcompanies still relying on conventional methods for microbiologicalquality control? It is largely down to multiple hurdles such as:� Immediate return on investment not being evident if inventory cost

reductions are not taken into account. Actual running costs ofRMMs are often higher than conventional methods (e.g., reagents,maintenance costs, more hands-on time);

� Special technology requiring additional and sustainable expertise;� RMM technology potentially not being applied 1:1 to the comp-

anies’ process or products and needing further improvement or adaptation;

� A more complicated and/or lengthy regulatory approval process;� Company management reluctance to take a risk or their focusing on

short-term financial objectives;� Compendial or regulatory guidelines that are generally requiring

that limits or results be obtained with growth-based methods.

Rapid microbiological methods (RMMs) can offer the benefit of significantly reducing the time to result ofmicrobiological tests and therefore have the potential to shorten throughput time for drug product release. Otherpotential advantages of RMMs include: a reduction in inventory costs, faster stop or go decisions duringmanufacturing, the decreased risk of stock-outs and supply bottlenecks, improved data integrity, automation andthe potential for paperless laboratories. More importantly, RMMs may become essential for assuring patient safetyof novel product types with very low shelf lives (e.g., cell based therapeutics). This article reports on actual RMMsthat have been validated since 2010 at the Novartis Pharma Stein manufacturing site. It contains a brief descriptionof six methods that have been evaluated or implemented so far. The article also develops notions such as non-inferiority tests to demonstrate equivalence as well as challenges related to the implementation of RMMs.

IN-DEPTH FOCUS: RMM

Implementation of a rapid methods portfolio at a pharmaceuticalmanufacturing site

David Roesti and Erik WilkensNovartis Pharma Stein

52 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

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Especially regarding the latter point, the limitations of theconventional methods are increasingly being recognised byhealth authority and compendia experts setting thestandards and guidelines. Actually, the new USP <1223>chapter on validation of alternative microbiological methods2

describes the history and limitations of the colony formingunit (CFU) that “cannot be considered the only unit ofmicrobiological enumeration.” The US Food and DrugAdministration’s (FDA’s) strategic plan from 20113 also writesthat in order to reduce the risk of microbial contamination of products, RMMs should be developed when assessingproduct sterility.

For RMMs to express their full potential, all conventionalmicrobiological quality control (QC) methods shouldeventually be replaced. Since there is no universal RMMcurrently available on the market, it is advisable that systemsare evaluated to determine which one would be the mostsuitable to replace a particular conventional method.

If the RMM is foreseen to substitute a conventionalmicrobiological test, it should be validated according to Ph.Eur. 5.1.64, USP<1223>5 and PDA TR 336. Within this validation it must yieldequivalent (or at least non-inferior) results or performance ascompared with the compendial method.

In the new USP 38 NF 33 S2 <1223>2, more flexibility in terms ofdemonstrating equivalence of an alternative microbiological method isprovided. For example, on:� Acceptable procedures where a comparison to an official

compendial method is not mandatory;� Performance equivalence to the compendial method;� Results equivalence to the compendial method; � Decision equivalence to the compendial method.

For RMM validations, the relevant question is that of “non-inferiority”,i.e., the validation aims to demonstrate that the RMM is not inferior tothe reference method with respect to a predefined lower boundary(Figure 1). If the RMM method is slightly superior to the conventionalmethod, this would not impact the microbiological safety risk. If theRMM is highly superior (e.g., in the case of non-growth based methods),then the limits based on CFU counts may have to be adapted or newlimits would need to be created and based, for instance, onexperimental studies and risk to microbiological safety.

The Rapid Methods team established at Novartis Pharma in Stein,Switzerland, serves as a center of excellence for all Novartis divisions

and has the task of evaluating, validating and implementing RMMs formicrobiological QC testing.

Examples of methods that have been validated or are underevaluation by the team will be presented briefly below (Figure 2).

Sterility testing Filterable samplesThe rapid sterility test (RST) using the Merck Millipore Milliflex® RapidSystem reduced the incubation time for filterable samples to only fiveinstead of 14 days in the compendial method7. In contrast to thecompendial sterility test, the RST requires use of solid nutrient media(modified, gamma-irradiated Schaedler blood agar). The underlyingdetection principle of the Milliflex® Rapid System is based onadenosine triphosphate (ATP) bioluminescence.

Since completion of method validation in 2010, the rapid sterilitytest is used routinely for several drug products and has beentransferred to other sites and divisions within the Novartis Group. It hasgained approval of many health authorities, including but not limited tothe US FDA CBER and CDER, EMA, PMDA, MHRA, ANVISA and TGA.Furthermore, an independent study conducted by FDA CBER8 confirmedthat the RST based on the Milliflex® Rapid System represents a suitablealternative to the compendial method.

The greatest challenges were selecting the appropriate growthmedium and the fact that it was the first RMM evervalidated in the laboratory meaning that expertisehad to be built from scrap. The pathway to approvalfor the RST depended on the health authoritytargeted. For instance, a comparability protocolapproach was used for the FDA who accepted theprotocol within four months. After acceptance it took another one-and-a-half years to completevalidation according to plan. For other health auth -orities a formal change procedure with postauthorisation variation for major changes wasfollowed (e.g., type II variation for the EMA). The time

IN-DEPTH FOCUS: RMM

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 53

Figure 1: Mean recovery and the 95% confidence interval of a method under evaluation in relationto a reference method. A, B, C: Non-inferiority regarding a lower boundary of 70% is not shown.D, E: Non-inferiority successfully demonstrated, because the lower limit of the 95 % confidenceinterval is above 70 %. F: Since the lower limit of the 95% confidence interval of the method underevaluation is above the recovery of the reference method, statistical superiority is shown.

Figure 2: Overview of methods for which RMM methods have been evaluated or implemented at NovartisPharma Stein Switzerland. Numbers are described in the corresponding paragraphs in the main text.

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Save Days in Sterility Testing.

— Closed Loop Sample Prep

— Positive Results in Hours

— Final Results in 7 days vs. 14

— Non-destructive

The Growth DirectTM System – Rapid Sterility Testing

Sterility testing with the Growth Direct™ System accelerates sterility testing, often a gating test for product release. The non-destructive test can provide positive results in hours and final results in half the time of traditional methods. The test includes both anaerobic and aerobic test cassettes and uses no reagents for testing. Sample preparation mirrors the existing method, simplifying training. System alerts notify users immediately via email or text message when samples are out of conformance.

Visit www.rapidmicrobio.com to learn how to automate your Sterility testing.

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from submission until approval lasted about sixmonths for the EMA and two years for the PMDA.

Non-filterable samplesA Rapid Sterility Test for Direct Inoculation (RST-DI)was validated using the Celsis AdvanceTM System(Celsis is a Charles River company) with theAMPiScreenTM reagent kit, reducing the incubationtime for non-filterable samples to only seven days.

The quantity of product tested, the growthmedium and incubation conditions (Soybean–CaseinDigest Medium at 20-25°C and Fluid ThioglycollateMedium at 30-35°C) remain unchanged as comparedto compendial requirements. The used reagent kitamplifies the ATP bioluminescence reaction byaddition of ADP, which is subsequently converted toATP by the cellular enzyme adenylate kinase. The system detects microbial growth based on an ATPbioluminescence signal translated into relative lightunits (RLU). A sample is declared as positive (microbial growthdetected) if the RLU number exceeds a pre-defined threshold.

The main technical challenges were relatedto slow growing microorganisms in highvolumes of growth medium used for primarypackaging of external sterility tests thatextended the incubation growth period. Also, aglass bead treatment was added afterincubation to break mould colonies and disperse mycelia to enablereliable detection when pipetting an aliquot for the Celsis AdvanceTM

reading step.

In-process bioburden For in-process controls such as testing of bioburden prior tosterilisation, a wide variety of RMMs may be used. For example, the RMMteam has validated a rapid bioburden method with a reduced

incubation time of three days using Merck Millipore’s Milliflex®Quantum system. The detection principle relies on fluorescent staining

of metabolically-active microorganisms. Otheralternative methods under evaluation are, forinstance, automated enumeration such as theGrowth Direct™ System or direct enumerationusing solid phase cytometry or flow cytometry9.The latter systems are especially interesting

for stop or go decisions during manufacturing since the results are available within a few hours.

Water testingFor the testing of water for injection, an automated, rapid water testusing the Growth DirectTM System (Rapid Micro Biosystems) wasvalidated10. The method now used in routine testing is based onmembrane filtration and requires 90 hours of incubation. The Growth

DirectTM counts microbial colonies by de -tecting their autofluorescence using highlysensitive CCD imaging. As this setup offers afar superior resolution compared to thehuman eye, detection of much smallercolony sizes is possible. Stacking andevaluation of consecutive images therebyenables the software to automaticallydistinguish between growing microbialcolonies and other fluorescent particlespotentially present on the membrane.

The system has been presented at manyhealth authority inspections (e.g., the FDAand Swissmedic) and has been accepted byall. The main issue for the validation wasrelated to the technical complexity of thefirst-generation machine. Based on the experience gained and on the directfeedback from Novartis and other pharma -ceutical companies, a second-generation

IN-DEPTH FOCUS: RMM

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 55

Figure 3: Example of a feasibility experiment covering active air monitoring in a non-sterile facilitycleanroom where the Growth DirectTM counts are compared with visual enumeration by a qualified analyst.Automatic reading was performed three days before the visual enumeration and yielded significantly betterresults than visual enumeration of plates. This is probably due to better discrimination of merging colonies.Significant differences in CFU counts were calculated with the Mann-Whitney paired test using theGraphPad Prism 6 software (*** p<0.001). Data was evaluated for statistical equivalence using the non-inferiority test (negative binomial distribution) with the R 2.8.1 software.

For RMMs to express their fullpotential, all conventional micro -biological quality control methodsshould eventually be replaced

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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.

machine was developed by Rapid Micro Biosystems. This secondgeneration system has, among other things, an improved hardwaretechnology and expanded application and is now being evaluated for anenvironmental monitoring application (see point under environmentalmonitoring sub-heading) in addition to water testing.

An interesting application of RMMs to water testing would becontinuous loop monitoring to detect almost instantly adverse trendsusing online water bioburden analysers that may enumerate viableparticles. Such systems are now under evaluation by thepharmaceutical industry as described by Cundell et al.11.

Microbiological examination of non-sterile productsA further alternative method to the compendial microbiologicalexamination of non-sterile products has been validated with anincubation time of three days based on the Celsis AdvanceTM Systemwith the AMPiScreenTM reagent kit. Because it isa presence/absence test, the rapid testmethod will only be implemented for drugproducts/excipients which are of excellentmicrobiological quality. If no microbial growthcan be detected with the rapid test method,presence of any specified microorganisms inthe product tested can also be excluded.

In case of a detected contamination, further tests have to beperformed: on the one hand, identification of the contaminant(s) isnecessary, e.g., using the remainder of the sample enrichment broth.On the other hand, re-testing according to the compendial method hasto be performed to provide a quantitative result. It is of note, however,

that this does not represent a re-test in a sense that the initial findingis invalidated, but provides a count estimate to the detected microbialcontamination.

To provide optimal growth conditions for bacteria as well as yeastsand moulds, two different incubation temperatures are applied (20-25°C and 30-35°C), although the same medium (Soybean–Casein DigestMedium + Lecithin + Polysorbate) is used in both cases. This rapidmethod has been accepted for a pilot drug product testing by severalhealth authorities (e.g., the EMA and TGA).

Environmental monitoringThe Growth DirectTM System has also been designed for environmentalmonitoring (as reported by Sage et al.12). Our feasibility study iscompleted and has shown that the system is suitable for the purpose of environmental monitoring of total aerobic microbial counts

in cleanroom environments. Among theexperiments performed for the study, acomparison of environmental monitoringsamples in cleanroom environments of a non-sterile facility demonstrated that the GrowthDirectTM automatic enumeration of total aerobicmicrobial counts was non-inferior as comparedto conventional media and manual enumera -

tion (example shown in Figure 3; page 55). The validation of the environmental monitoring method

demonstrating equivalence of the Growth DirectTM with the conventionalmethod actually used at Novartis Pharma Stein AG is planned for thefinal quarter of 2016.

IN-DEPTH FOCUS: RMM

The selection and implementationof RMMs is accompanied by a variety ofchallenges and there is no ‘universally

applicable’ RMM

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An additional approach for environmental monitoring is continuousmonitoring of viable particles that would allow for instantaneousdetection of adverse trends and loss of microbiological control incleanroom air. One of the main challenges with such systems is thecorrelation between the guideline’s microbiological action levels whichare based on CFU counts and the counts obtained with the viableparticle counter.

Viable particle counts are expected to be much higher or differentas compared with CFU counts, one of the reasons being that viable butnon-cultivable microorganisms represent probably 90-99% ofmicroorganisms present in the environment13,14. Determination of actionor alert levels based on a baseline of viable particle counts might thenbe necessary for these systems. One approach could be to calculate theaction or alert level based on the percentiles of historical data. Sincemicrobiological data are generally not normally distributed, otherstatistical models may be taken into consideration as written in Gordonet al.15. In addition, interfering factors that would be signalled as ‘falsepositive’ (e.g., Isopropyl alcohol droplets) need to be evaluated.

Even if viable particle counters may not directly replace active airmonitoring or settle plates for routine environmental monitoring due tolack of experimental data and regulatory guidance, these systems maybe used for investigation purposes to find sources of contamination, foraseptic training of operators, evaluation of manual interventions, re-organisation of production lines, etc., not only for aseptic areas butalso for non-sterile production facilities.

ConclusionThe selection and implementation of RMMs is accompanied by a variety of challenges and there is no ‘universally applicable’ RMM. Depending on the intended application, different parametershave to be considered, such as time-to-result, frequency of testing,required sensitivity, destructive/non-destructive method, need toidentify contaminants, and others. It is therefore essential to test (proof of concept/feasibility study) each system with your materials,process and requirements before taking a final purchasing decision and validation.

In this context, the RMM expert team in Stein strives to acquire alarge portfolio of validated RMMs in order to provide the most

appropriate alternative solutions to replace a particular conventionalmicrobiological test method. A full understanding of the advantagesand limitations of the RMM systems strengthened by continuouscollaboration with the companies developing these rapid methodshelps achieve a sustainable implementation of RMMs within thecompany. Also, by avoiding unnecessary duplication of validationefforts, a centralised approach allows for a simplified, efficient andmore cost-effective way to introduce rapid methods.

Finally, a change in paradigm in microbiological quality controlshould be expected as long as all actors (industry, regulators, researchinstitutes and system developers) continue cooperating openly toscientifically comprehend the results obtained with RMM methods andtranscribe them into new standards.

AcknowledgmentsThe authors would like to thank Melanie Nast and Sabrina Scheper fortheir continuous and excellent technical support in evaluating andvalidating RMMs in the laboratory. We also thank Marcel Goverde and Alexandra Staerk for their critical review of the manuscript.

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VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 57

1. PDA Survey. 2013 PDA Objectionable Microorganisms for Nonsterile Pharmaceutical,

Consumer Health, Medical Devices, Dietary Supplement and Cosmetic Products. 2014 Jan.

Available from: www.pda.org/bookstore

2. USP 38 NF 33 Second Supplement chapter <1223>. Validation of alternative micro -

biological methods

3. U.S. Food and Drug Administration. A Strategic Plan – Advancing Regulatory Science at

FDA [Internet]. 2011 August [cited 2015 Sep 14]. Available from: http://www.fda.gov/

ScienceResearch/SpecialTopics/RegulatoryScience/ucm267719.htm

4. Ph. Eur 8th Edition chapter 5.1.6. Alternative methods for control of micro-

biological quality

5. USP 38 NF 33 chapter <1223>. Validation of alternative microbiological methods

6. PDA Technical Report No. 33, Evaluation, Validation and Implementation of Alternative

and Rapid Microbiological Methods. Revised 2013

7. Gray JC, Staerk A, Berchtold M, Mercier M, Neuhaus G, Wirth A. Introduction of a rapid

microbiological method as an alternative to the pharmacopeial method for the sterility test.

American Pharmaceutical Review. 2010; 13(6) 88-94

8. Parveen S, Kaur S, David SA, Kenney JL, McCormick WM, Gupta RK. Evaluation of

growth based rapid microbiological methods for sterility testing of vaccines and other

biological products. Vaccine. 2011; 29(45): 8012-8023

9. Bhusari PK, Tabor DE, Yamagata R, Galinski MS. Application of Flow Cytometry for Rapid

Bioburden Screening in Vaccine Virus Production. PDA Journal of Pharmaceutical Science

and Technology. 2012; 66, 445-452

10. Gordon O, Gray JC, Anders H, Staerk A, Schlaefli O, Neuhaus G. Overview of rapid

microbiological methods evaluated, validated and implemented for microbiological quality

control. European Pharmaceutical Review. 2011; Volume 16, Issue 2, 9-11

11. Cundell A, Gordon O, Haycocks N, Johnston J, Luebke M, Lewis N, Mateffy J, Weber JW.

Novel Concept for Online Water Bioburden Analysis: Key Considerations, Applications,

and Business Benefits for Microbiological Risk Reduction. American Pharmaceutical

Review. 2013; Volume 16, Issue 4. 26-31

12. Sage A, Timas N, Jones D. Determining incubation regime and time to results for automated

rapid microbiology EM methods. European Journal of Parenteral & Pharmaceutical

Sciences. 2014; 19(2), 45-55

13. Nannipieri et al. Microbial diversity and soil functions. European Journal of Soils Science.

2013; 54:655-670

14. Amman R et al. Phylogenetic identifications and in-situ detection of individual microbial

cells without cultivation. Microbiological Reviews. 1995; 59:143-169

15. Gordon O, Goverde M, Pazdan J, Staerk A, Roesti D. Comparison of Different Calculation

Approaches for Defining Microbiological Control Levels Based on Historical Data. PDA

Journal of Pharmaceutical Science and Technology. 2015; 69, 383-398

References

Dr. David Roesti is a global expert in microbiology for

Novartis and is currently leading the microbiology network

and the Rapid Microbiological Methods team at Novartis

Pharma AG in Stein, Switzerland. Prior to this assignment,

David was the laboratory supervisor for the microbiological

testing of non-sterile drug products at Novartis Pharma

Stein AG. David Roesti holds a PhD in Microbial Ecology

from the University of Neuchâtel, Switzerland, and has 15 years of

experience in the field of microbiology within various domains (drug

product manufacturing, food microbiology, biogaz production and microbial

interactions in the rhizosphere).

Dr. Erik Wilkens joined Novartis Pharma Stein AG,

Switzerland, in 2012 as a QA Specialist in the Rapid

Microbiological Methods Team of the QA/QC micro -

biology department. In this function he is involved in

evaluation and validation of Rapid Microbiological

Methods and their implementation in routine use, including

method transfer to interested sites within the Novartis

Group. He studied Biotechnology at the Technical University of

Braunschweig, Germany. After completion of his studies he earned his

doctorate at the Institute of Agricultural Technology of the Johann Heinrich

von Thünen Institute in Braunschweig, Germany, in the field bio -

technological conversion of renewable resources.

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The often overlooked benefits of RMMs are: reduced cycle time of aproduct’s release, reduction in downtime, lower inventory hold timeand space, and reduced investigation time with the associated retest-ing and rework, in addition to increased yield of products sold withlower costs. However, making a rational decision on whether to proceed on an RMMs project needs a careful scientific approach with the aid offinance experts1.

It should be noted that the value of RMMs can be maximised if therange of quality control tests are broadened. RMM techniques can be

useful when applied to the following tests/studies:� Preservative efficacy test;� Disinfectant and sanitiser validation study;� Antibiotic potency determination assay;� Method suitability or validation study;� Validation of microbiological cleaning efficiency for machines

and equipment;� Cleaning and disinfection efficacy verification on microorganisms of

classified area in situ.

Over the past few decades, different rapid microbiological methods (RMMs) that are useful in microbial bioburdenidentification (characterisation), qualification (detection) and quantitation (enumeration) have been developed.Although these technologies are a real breakthrough in the field of biological sciences, especially pharmaceuticalmicrobiology, many drugs manufacturers are still hesitating and resisting the application of the new technologies intheir facilities. This problem is notably evident in the developing countries. The primary barrier against theirimplementation is financial: they cost more than conventional microbiological methods. This can be attributed tocapital expenses through the initial investment and the subsequent maintenance charges, with the associatedoutlays arising from the training on the novel techniques, qualification and validation.

IN-DEPTH FOCUS: RMM

Use of RMMs in qualitycontrol: challenges and benefits

Mostafa EissaHikma Pharmaceuticals

58 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

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The main systems that are of interest in pharmaceutical andbiopharmaceutical industries include automated biochemical reactions(metabolic fingerprinting), fluorescent labelling assays, impedance/conductivity, gas consumption or production, ELISA, polymerase chainreaction (PCR), fatty acid analysis using gas chromatography, adenosinetriphosphate (ATP) bioluminescence, riboprinting and analysis ofbiomolecules using mass spectrometry2. The primary technologicalplatforms of RMMs fall mainly into four categories3,4,5:� Growth-based methods: e.g., carbon assimilation, enzymatic

reactions, carbon dioxide generation or ATP bioluminescence;� Artifact-based methods, e.g., fatty acid analysis using gas

chromatography, ELISA and MALDI-TOF mass spectrometry of cellcomponents such as nucleic acids and proteins;

� Nucleic acid-based methods: e.g., PCR and automated South-ern blotting;

� Viability-based methods: e.g., fluorescent labelling methods, suchas flow fluorescence cytometry; immunofluorescence andfluorescent nucleic acid stains used as a viability marker along withpropidium iodide as a membrane-compromised cell marker.

Each technique has its advantages and limitations and it is the duty ofthe experts in each industry to find and select the most appropriateinstruments that fit their product’s type and activities.

Superiority of RMMs over conventional methodsAs touched upon already in this article, rapid microbiologicaltechniques and technologies offer many benefits over conventionalmethods. These are examined in turn, below.

Accuracy and reproducibilitySince RMMs are based on automated tech -nology, there is low variability and highprecision in the analysis compared with theusual microbiological methods which are highly dependent on theoperators’ skills.

High samples processing rateMost of the new technologies can manipulate many samplessimultaneously with high throughput, while conventional micro -biological methods rely on the skills, capability and the number ofoperators available.

Fast data generation and control chartsSince new technologies are linked with computer systems, results are produced swiftly and consequently data can be trendedsimultaneously. This process facilitates the application of lean conceptand six sigma tools. This is rarely applicable for manual procedureswhere several steps and personnel need to be involved to facilitate the process.

Short time to almost instantaneous analysisThe fast results of assay allow for rapid release, lower product cycle timeand enable quick decisions to be made on solid bases when judgingcritical situations during investigation. Traditional microbiologicaltechniques require long incubation times where any corrective or

investigational actions will be delayed long enough such that anyfurther steps may complicate and extend the time for decisions andsometimes may be inconclusive.

Detection abilityOne of the major drawbacks of the conventional culture media is thatits ability to detect microorganisms is limited. Even the most generalculture media cannot recover the whole spectrum of microorganisms inaddition to those subjected to stress and injury. Viable butnonculturable (VBNC) microorganisms have the potential risk to causeinfection in the host, while they do not show any growth in ordinaryculture media. Many advanced techniques in RMMs are sensitiveenough to detect very low contamination levels even from injured andVBNC microorganisms.

Success storiesSelected examples that illustrate the challenges that could be resolvedif RMMs were used to solve the microbiological dilemma in thepharmaceutical industry are given below.

Unpalatable antimalarialsA pilot batch of antimalarial tablets was tested microbiologically using conventional microbiological culture methods and showedunexpectedly high total viable aerobic count results (>1500 CFU/g of theproduct). The contaminating microorganism was identified asStenotrophomonas maltophilia after a miniaturised biochemicalidentification system was used. The test was done in triplicate but when

it was repeated again just two days after theinitial testing, the culture plates showed nogrowth with results being reported as <100 CFU/g. In this case, a complete investiga -tion could not be accomplished because therewas doubt about whether this microorganism

had declined in number, died off or become VBNC. It is thereforedifficult to make accurate decisions for the product based on scientificbackground due to the lack of sufficient reliable data.

Production area challengeEnvironmental monitoring samples from a production area showed anexcursion. An active air sample was taken from an area where solid oraltablets were produced and was found to be out of specification (OOS)after an incubation period of seven days. The main contaminate of thissample was from the family Staphylococcaceae. One of the actionstaken was to put a hold on the manufacturing of products during thisperiod, then to quarantine the area and undertake testing. Theexcessive efforts that were made could possibly have been avoided ifrapid microbiological testing techniques had been applied so thatprompt actions could be taken in real time.

Water alertPharmaceutical water is a key ingredient in drug manufacturing andother processes in production areas. In one case, an ‘out of alert’ resultwas observed in the water treatment station for the production ofpurified water from raw city water using conventional culture media byfiltration techniques after three days from the testing. The main source

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VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 59

Making a rational decision onwhether to proceed on an RMMs projectneeds a careful scientific approach with

the aid of finance experts

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of this excursion was from Gram-negative rods such as Acinetobacterbaumannii, A. lwoffii and others, which were identified usingbiochemical identification kits. The actions that were taken includedthe review of all activities related to the affected loop and thesubsequent microbiological investigation of them with consequentstoppage of all production activities in the manufacturing area until thesituation had been resolved. This decision resulted in the delay ofproduction schedule beyond the previously planned deadline.

Why the resistance to change?Human nature is such that the majority of people resist changes to theirhabits and show a certain degree of resistance even if these changes willbe really beneficial for them. Thus, being in thecomfort zone of traditional simple and slowmicrobiological methods is more favourablethan facing new challenging situations of noveltechnologies that may impose risk to thebusiness of a company. There are also obstaclesin relation to company culture, the level of acceptance from regulatorybodies for the alternative techniques and the fear of technicallimitations. However, Europe has begun to implement RMMs in thepharmaceutical field; for example, in the UK, the European MedicinesAgency and the Medicines and Healthcare products Regulatory Agencygranted the use of Chemunex® rapid monitoring equipment in 20036. Onthe other hand, the developing countries, where the balance is usuallyshifted towards the lower cost processes at the expense of time, are veryslow in acquiring the rapid technologies. In addition, the regulatoryauthorities in most of these countries have not adopted specific systemsand requirements for alternative technologies monitoring.

The application of the six sigmaThe application of new, high-throughput technologies with accuracyand reproducibility, coupled with computer systems for instantaneousdata gathering and processing, allows for continuous monitoring of the microbiological processes in a timely manner. This enables thegeneration of precise trends and control charts that can demonstratethe efficacy of the overall processes over a long time period. In suchcases, the behaviour and the pattern of the chart can show the degreeof compliance of each process or product, give a prediction of thechanges and its relation to other variables, measure the efficiency ofprocesses, determine specification limits and measure processcapabilities/performances. Thus, rapid microbiological techniques cantake the analysis much beyond routine testing.

A significant challenge in the pharmaceutical industry is ensuringthat the eventual consumption of the drug product by the consumerwill not carry any microbial risks to that patient. Safety advisories andproduct recalls issued by the US Food and Drug Administration’s SafetyInformation and Adverse Event Reporting System (AERS) have listedmany cases of product contamination by microorganisms that have ledto their withdrawal from that market. This problem highlights thecriticality of the tools that must be used for microbiological qualitycontrol of the medicinal products before the release to the market.Using appropriate tools for assessing the microbial risk by both the conventional and the advanced selected technique will aid in thedecisions concerning the choice of RMMs.

Potential of lab-on-a-chip to reform RMMsA lab-on-a-chip (LOC) is a device that integrates one or severallaboratory functions on a single chip of only millimeters to a few squarecentimeters to achieve automation and high-throughput screening andon-site testing7. This technology platform is an extension of RMMs butis still in its infancy and it is much less common in the pharmaceuticalindustry field than in other areas. This is unfortunate because thepossibility to perform sample collection, sample preparation, andsimultaneous testing of various samples in one miniaturised devicewould be of great benefit to the pharmaceutical microbiologist. LOCs would be especially beneficial if used for antimicrobialeffectiveness testing, disinfectant/sanitiser efficacy evaluations, for

testing in support of equipment and facilitycleaning validation, and during the collection ofthe multitude of samples when trouble -shooting a microbial contamination event.However as with RMMs, LOC is a new technologythat was not really developed with pharma -

ceutical testing in mind; there are still some challenges that need to be overcome before it can be used to the benefit of pharma-ceutical microbiologists6.

ConclusionAlthough new microbiological laboratory technologies have becomeimplemented in the pharmaceutical industry field, they are still laggingbehind other industries which take the lead with these techniques andrealise the importance of the continuous innovation and developmentof new methods and technologies for microbiological analysis. This situation is especially pronounced in the developing countrieswhere the barriers are significantly retarding.

60 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

IN-DEPTH FOCUS: RMM

1. Return on Investment (ROI) – Rapid Microbiology and Rapid Microbiological Methods;

Cited 2015 Aug 19. Available from: http://rapidmicromethods.com/files/roi.php

2. Alexander R, Clements JA, Guest R, et al. Rapid methods in Microbiology.

New Technologies Forum 6. 2003; I:\Judith C\Science Committee\WP_New

Technologies\Forum 6 Rapid Micro\Forum_6 Report_FINAL.doc

3. Miller M. Encyclopedia of Rapid Microbiological Methods. 2006; Volume 1–3,

River Grove, IL: DHI Publishing

4. Fung. The Handbook on Rapid Methods and Automation in Microbiology Workshop.

2000; Kansas State University, Manhattan

5. Murray PR, Baron EJ, Jorgensen JH, et al. Manual of Clinical Microbiology. 2003;

8th ed. Washington, DC, USA: ASM Press

6. Clontz L. Microbial limit and bioburden tests: validation approaches and global

requirements. 2008; 2nd ed. New York: CRC Press

7. Volpatti LR, Yetisen AK. Commercialization of microfluidic devices. Trends Biotechnol.

2014;32 (7):347–350

References

Mostafa Essam Ahmed Eissa, MSc, is Microbiology

Section Head at Hikma Pharmaceutical Company (formerly

Alkan Pharma). Prior to this, he was Microbiology

Supervisor in 2010, Senior Microbiologist in 2009, and

Quality Control Microbiologist from 2004 to 2009, also at

Hikma. He was a research specialist in the Biotechnology

department at El-Nile Pharmaceuticals & Chemical Industry

Company from 2001 to 2004. He also worked as Inspector at the Central

Administration of Pharmaceutical Affairs (CAPA) of the Ministry of Health

& Population from 1999 to 2001. Mr Eissa is author of several cultural and

scientific articles that have been published in Dubai Cultural magazine.

He has published eight scientific manuscripts and one book.

Rapid microbiological techniques can take the analysis much

beyond routine testing

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The pharmaceutical industry is lagging behind the food industry andclinical microbiology laboratories in terms of its take-up of RMMmethods, despite their many proven business and quality benefits andthe fact that the FDA’s initiative to promote the use of process analyticaltechnology (PAT) includes rapid microbial methods. Their use bypharma companies would certainly offer them faster test turnaroundtimes to accommodate the aggressive deadlines for manufacturingprocesses and product release. Some rapid methods also offer thepossibility for real-time microbial analyses, enabling management torespond to microbial contamination events in a more timely fashion,and can provide cost savings and higher efficiencies in quality controltesting laboratories.

There are several reasons why pharmaceutical and biopharma -ceutical industries have been somewhat slow to embrace alternativemicrobial methodologies. The major factor is that the bioburden countsdetected by the incubation method and rapid assay are greatlydivergent. Yet, the use of rapid methods is a dynamic field in appliedmicrobiology and one that has gained increased attention nationallyand internationally over time. Recently, the use of alternative methods

for control of the microbiological quality of pharmaceutical productsand materials used in pharmaceutical production has been addressedby the compendia in an attempt to facilitate implementation of thesetechnologies by pharmaceutical companies.

The US Food and Drug Administration has, over the past few years,encouraged pharmaceutical manufacturers to develop and implementeffective, efficient and innovative approaches to provide high-qualitypharmaceuticals to the public1. Meanwhile, the linkage of RMMs to PATis largely based on real-time release, which is the ability to evaluate andensure the acceptable quality of the in-process and/or final product onthe basis of the collection and analysis of in-process data. The PATcomponent of real-time release typically includes a valid combinationof assessed material attributes and process controls. Materialattributes such as bioburden, endotoxin content and sterility can beassessed using direct and/or indirect process analytical methods. Thecombined process measurements and other test data gathered duringthe manufacturing process can serve as the basis for real-time releaseof the final product and would demonstrate that each batch conformsto established regulatory quality attributes. In real-time release,

Microbial testing performed in support of pharmaceutical and biopharmaceutical production falls into three maincategories: detection (qualitative), enumeration (quantitative) and characterisation/identification. Traditionalmicrobiological methods are labour-intensive and time-consuming and such tests require several days ofincubation for microbial contamination (bioburden) to be detected. Managers are, therefore, seldom able to takeproactive corrective measures. Furthermore, microbial growth is limited by the growth medium used and incubationconditions, thus impacting on testing sensitivity, accuracy and reproducibility. However, various technologies forrapid microbiological methods (RMM) have now been developed. Herein, the author will attempt to define the roleof RMM in PAT and discuss the application of RMM in aseptic filling, biopharmaceutical upstream and downstreamprocessing, environmental monitoring and control in clean rooms, before describing the selection, development,validation and implementation of RMM for PAT applications. Regulatory and compendial guidelines for RMM and thefuture of rapid methods in pharmaceutical and biopharmaceutical manufacturing will also be discussed.

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VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 61

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Rapid assay for bioburdenand other contaminations

Hideharu ShintaniChuo University

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Your products. Our passion. When it comes to endotoxin testing, you need precision and speed. Our revolutionary FDA-licensed LAL cartridge technology has made accurate 15-minute endotoxin testing a reality for labs around the world. From our handheld, point-of-sample-collection PTS™ system to our high-throughput, fully automated Nexus™ instrument, our Endosafe® innovations continue to decrease testing time and accelerate product manufacturing. Learn more about our endotoxin testing solutions at www.criver.com/emd.

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material attributes such as formulation, bioburden, container size andload pattern, as well as process parameters such as sterilisationparameters, are measured and controlled.

Traditional techniques Traditional microbial testing methods rely on the growth ofmicroorganisms in culture media for detection, enumeration andselective isolation. These time-honoured methods continue to be usedbecause of simplicity, effectiveness, the low cost and their suitability foruse in all microbiological testing laboratories. However, serious questionsneed to be raised as to whether the continued use of these traditionalmethods is the right strategy to improve quality and efficiency in thepharmaceutical industry. Such methods were originally designed for the detection of human pathogens and not for the microbiological quality control of pharmaceutical processes and products.

The drivers of microbial testing should be the criticalmicrobiological quality attributes associated with a specific drugproduct and the risk assessment of the potential for microbialcontamination of that drug product and resulting patient infection. The inferiority of the traditional method is due to the relatively longperiod of cultivation. Examples of where conventional methods fallshort are given below.

Bioburden testing Nonsterile drug substances, pharmaceutical excipients and drugproducts are evaluated for bioburden using microbial limit ormicrobiological examination tests. The traditional test procedures are clearly unsuitable for PAT applications due to their extendedincubation times, relative insensitivity and low precision, and even havelimitations as release test methods since they may not detect allobjectionable microorganisms that could be present in a nonsteriledrug product.

Sterility testing Sterility testing can be conducted by inoculating a microbiological brothwith an aliquot of the test material and scoring growth by the detectionof turbidity. The compendial sterility tests have been harmonised interms of media, growth-promotion requirements, suitability tests,incubation conditions, number of containers and amounts of materialtested, and observation and interpretation of the results. Themembrane filtration test is the preferred test over the direct inoculationtest because it has the capacity to test the entire contents of a productcontainer and inhibitory substances may be rinsed from the membrane.The incubation period for the test is at least 14 days, making itunsuitable for a PAT application.

Types of RMMsAn RMM is an alternate microbiological test that is completed in ashorter time than the classical tests that depend on incubation formicrobial growth to detect microorganisms as either colonies on a plateor turbidity in a broth. It may involve reducing the incubation time forthe plate count by at least half, processing a sample to obtain a resultin two to three hours, or a direct analytical method. The latter twoapproaches are typically not growth-based, and hence move towardreal-time analysis.

As pharmaceutical microbiologists, our primary objectives are todetermine which microorganisms, if any, are present in pharmaceuticalingredients, intermediates, plant environments or drug products and, ifpresent, their quantity, type and potential impact, to help quality units,make decisions to proceed with the manufacturing and release of theproduct to the market. The test methods are classified as detection,screening, enumeration and identification2. Examples from thecompendial microbial tests are: sterility testing (detection/qualitative),absence of specified microorganisms (screening/qualitative) andmicrobial count (enumeration/quantitative). In addition, there is thenoncompendial microbial identification test (identification/qualitative).

The classification systems for rapid methods proposed in the PDATechnical Report No. 33 are based on what the technology involves, forexample, the growth of microorganisms, viability of microorganisms,presence/absence of cellular components or artifacts, nucleic acidmethods, traditional methods combined with computer-aided imaging,and combination methods3,4 (explained in more detail below). Similar,but slightly different classifications may be found in compendialsections discussing the validation of alternative microbiological testmethods (USP 12235).

Growth-based technologies These methods are based on measurement of biochemical orphysiological parameters other than turbidity or colony formation usedin classical methods that reflect the growth of the microorganisms.Examples include adenosine triphosphate (ATP) bioluminescence,colorimetric detection of carbon dioxide production and measurementof change in head-space pressure, impedance, advanced imaging andbiochemical assays.

Viability-based technologies These types of technologies do not require growth of microorganismsfor detection. Differing methods, including vital staining andfluorogenic substrates, are used to determine if the cell is viable ornonviable, and, if viable cells are detected, they can be enumerated.Examples of this technology include solid-phase cytometry and flowfluorescence cytometry.

Cellular component or artifact-based technologies These technologies look for a specific cellular component or artifactwithin the cell for detection and/or microbial identification. Examplesinclude fatty acid profiles, matrix-assisted desorption ionised-time offlight (MALDI-TOF) mass spectrometry, enzyme-linked immunosorbentassay (ELISA), fluorescent probe detection and bacterial endotoxin LAL test.

Nucleic acid-based technologies These technologies use nucleic acid methods as the basis of opera-tion for detection, enumeration and/or identification. Examplesinclude DNA probes, ribotyping polymerase chain reaction (PCR) andribosomal DNA-based sequencing.

A survey of RMMsIn most cases, RMMs may be divided according to their principle ofdetection. In a survey of RMMs, a ranking was made on the basis of

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VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 63

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Get to know the latest generation of air samplers!The MAS-100® family extended. The microbial air samplers of the MAS-100® family are designed for your individual air monitoring needs.

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successful implementation in the pharmaceutical industry. It can beconcluded from the survey that some of the most successful RMMsolutions are the ChemScan®, AkuScreen®, and BacTI ALERT® systems.More companies use these RMMs for in-process controls than productrelease. The latter is often product-dictated. The rate of success of theimplementation is determined by the ability to focus and reservemanpower in the qualification and validation work.

Real-time methodsIn general, decision makers (i.e., physicians, production managers and quality units) claim that the microbiological testing laboratories in the hospitals, food production sector and pharmaceutical industryare the rate-limiting steps for patient treatment and product release. As microbiologists, we recognised the truth in their criticismthat microbial tests are imprecise with long incubation times; indeed,micro biology laboratories count the time in days or even weeks toobtain a result. Furthermore, the results may need to be interpreted,reviewed and approved before they can be reported. That is not all. The time to ship the samples to the laboratory must be considered. It boils down to a simple addition calculation: time to report = time toship the sample to the laboratory + administrative time + analysis time+ incubation time + verification time + approval time + time to reportthe result. Product release cycle times are protracted and are the sumof all these sequential activities. That means seven (EuropeanPharmacopoeia 5.1.6, 2006) items to speed up the overall testingprocess. With RMMs, in most cases only the analysis time andincubation time is considered. It is important not to forget the othertime-consuming factors in the analytical process when consideringRMM implementation.

Other important differences that we recognise lie within the in-

process RMM testing for production process control, RMM testing fortroubleshooting and RMM testing for product release. All three mayhave different goals. To give some background informatioin, the normalway we perform microbial analysis (when the sample is taken to themicrobiology laboratory) is called off-line testing; if an analysis takesplace near the production line but the sample is taken out of theproduction process, it is called at-line testing, and the last type is in-line testing, where there is a continuous analysis ongoing in theproduction process.

Conventional microbial testing, in most cases, is off-line, with a fewcases of at-line testing (depending on the manufacturing infrastructure).If we examine RMMs, they also belong to these two categories with someexceptions that have the potential to be used in-line.

What determines whether an RMM can be used off-line, at-line, orin-line? In most cases, it is the underlying principle of the tech-nique. For this discussion, RMMs can be subdivided into differentcategories on the basis of their detection principle: (1) detection of early growth, (2) viability-based testing, and (3) detection of microbialcell components. RMMs based on the early detection of growth are the slowest; the other two will be faster depending on the kind of application. As an example, detection of CO2 production is a growth-dependent technique that may be used for sterility testing. This application is unlikely to be an in-line application because of the aseptic handling that is inherent to the sterility test. In the best case, it could be an at-line application. Detection via flow cytometry has a viability-based detection principle. Although it is not on the market, we can imagine that an in-line application could be used to detect and count microorganisms via a laser detection principle. In fact, there are some techniques available that are potential in-line detection systems based on viable cell

IN-DEPTH FOCUS: RMM

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detection. In the last category, detection of cell components has many applications: detection of DNA, fatty acids, ATP, etc. In most cases, it requires a sample preparation that automatically converts it to an off-line application. There are several examples of at-linedetection of bacterial endotoxins that may be used in pharma-ceutical manufacturing.

We can conclude that the only real-time RMM is a system that isbased on viability cell detection in an in-line PAT application. However,in most cases, it is not possible to use the viability cell detectionprinciple in-line. The best possible and most practical option for theproduction departments and microbiology laboratories that serve themwould be at-line testing with a viability-based cell detection principle.However, because the viability-based cell detection systems aretechnically limited in their lack of sensitivity (limit of detection/quantification) and specificity (differentiating between cells andparticulates), we end up with an at-line testing option of detection of early growth or cell components to eliminate ambiguity (time toreport: 24-to-48 hours).

It must be emphasised that with RMMs, the objective of the testing

determines which kind of system is needed. For RMM testing forproduct release for the market, an off-line testing system is the rightchoice because there is no need for testing at the production floor.RMM testing for troubleshooting, in contrast to that for product release,can be both at-line and off-line. RMM testing for in-process testingwould preferably be done at-line. With the latter, the difficulty andworkability of a test method determines the at-line or off-lineapplication of a test.

The future of RMMs in parenteral medication manufacturingSo in conclusion, the major RMMs trends at present are: (1) the moveaway from traditional growth-based methods to RMMs on the basis ofvital cell staining, ATP, or nucleic acid concentration; (2) the move fromthe microbiology laboratory to the production floor as the site of themicrobial testing; and (3) the use of RMM for PAT applications by the real-time testing of in-process samples.

IN-DEPTH FOCUS: RMM

1. FDA. (2004) Guidance for Industry-PAT A Framework for Innovative Pharmaceutical

Development, Tl Manufacture and Quality Assurance. Rockville, MD: Government

Printing Office, 2004

2. Cundell, A.M. (2005) Risk-Based Approach to Pharmaceutical Microbiology.

In Encyclopedia w of Rapid Microbiological Methods. Miller MJ, ed, Bethesda, MD:

Davis Horwood/PDA

3. Parenteral Drug Association. (2000) Technical Report 33, evaluation, validation and

implementation of new as microbiological testing methods. J. Pharm. Sci. Technol, 54, 35

4. Moldenhauer, J. (2005) Rapid microbiological methods and the PAT initiative. Biopharm.

Int., 18, 31-46

5. European Pharmacopoeia 5.1.6. Alternative Methods for Control of Microbiological

Quality. European Directorate for the Quality of Medicines, Strasbourg, France

References

Hideharu Shintani, PhD, is Guest Professor at Chuo University’s School

of Science. Previously, he was a Researcher at the National Institute of

Health Sciences (NIHS) in Tokyo (19977-87) and Director of Department

from 1992 until 2009. He was also a Research Associate at Texas Tech

University, Department of Chemistry, Lubbock in 1986-87. He is Director of

Antibacterial and Antifungal Society Japan and a Chairman of the Japanese

Government Committee entitled ‘Sterilization and inactivation of endotoxin

and prion by gas plasma exposure’. He is author of the following books:

Bioinstrumentation and Biosensors (1990), Ohio Science Workbook

Polymers (1994), Analytical Applications of Immobilized Enzyme Reactors

(1994), Sterilization Systems (1995) and Adsorption and its Application in

Industry and Environmental Protection (1998). He is also Editor of the

following publications: Handbook of Analytical Application of Capillary

Electrophoresis (1996) and Sterilization and Disinfection by Plasma

–Sterilization Mechanisms, Biological and Medical Applications (2011).

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Recent innovative therapies transforming and multiplyingsomatic cells ex vivo and reintroducing them back intopatients have been developed. Considering the very lowshelf life of some of these products, what would youpropose as an alternative to the compendial sterility test,and which in-process controls would you suggest for themonitoring of adventitious agents?David: Such therapies create a tremendous potential for patients andnew challenges for quality control. Short shelf-life products are difficultto adequately test for sterility, because the test takes 14 days. Inaddition, many of these products cannot be filtered, making traditionalsterility testing difficult. The use of a rapid microbiological method(RMM) creates options. First, an RMM can be selected that allows directinoculation of the sample; an alternative option would be the use of anRMM as described in USP <1223> and EP 5.1.6. With technologies on themarket that can cut the final testing time in half, but start to reportorganism growth in hours, aligning to these innovative therapiesbecomes simpler.Marcus: Often, a classic procedure simply cannot be used to sterility-test pharmaceuticals that have a very low shelf life, which frequentlyinclude radiopharmaceuticals. In such cases, lots can be released onlyretrospectively. Small-volume batches can also pose problems.

To improve this situation, 21 CFR 610.12 has already described a fewflexible options for testing. RMMs, such as adenosine triphosphate,polymerase chain reaction (PCR) or colorimetric methods, can certainlybe used after careful validation, in particular for cell therapy products.A suitable method should be selected according to the specific processor risk involved. Meanwhile, for detection of mycoplasms inadventitious agents, excellent rapid PCR methods are now available.

What are your thoughts on the four options demonstratingthe equivalence of an alternative microbiological methoddescribed in the new USP <1223> chapter?David: The new guidance on equivalence is an interesting progression.There is a need for further clarification by examples of how the industryshould interpret the technology and application to the option selectionprovided. Option 4 is probably clear, however the decision betweenOption 1, 2 and 3 may lead to extensive discussions. It is also a concernthat this has moved away from the harmonisation to TR33 and the new EP 5.1.6.

Marcus: Undoubtedly, it is important to define criteria for qualityassurance. Each method certainly also calls for compromises. Often youwill find that the contaminating microorganisms’ pharmacopeias that

IN-DEPTH FOCUS: RMM ROUNDTABLE

68 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

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ModeratorDavid Roesti, Senior Facilitator for RMM and Global Support, Novartis Pharma Stein AG

David Jones Director of Technical Services,

Rapid Micro Biosystems

Marcus Schütte Product Manager, Microbiology,

Sartorius Lab Instruments GmbH & Co.KG

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are required to be detected for validation are thesame, critical ones in many applications, in-processcontrols and final quality control tests. So it definitelymakes sense for all other methods to also be capableof detecting these microbes and additional ones, asnecessary. The limit of detection for sterility testing isspecified as one viable microbe. Whether two culturemedia are, overall, sufficient for testing at twodifferent incubation temperatures is questionable inmy opinion.

Based on your experience, do you observe that RMMs are increasingly being used within the industry orgovernments nowadays as compared withprevious years? If yes, for which test arethey applied and what is the main trigger for implementation?David: There is a definite increase in the interest forrapid methods in the pharma, personal care products,and medical device industries. The interest is across the board ofapplications from water and raw material testing to final productrelease as well as environmental monitoring. The driver depends on theindustry. For PCP a shorter product release time to the market is the driver, while other industries desire improved quality throughautomation and error reduction. Often more efficient environmentalmonitoring processes to improve testing throughput and reducenonconformance drive the decisions.

Marcus: Currently, I see that RMMs are most advantageous for twoindustries: pharma, and food and beverage. In the pharma industry,such methods are highly useful for IPCs and products with low shelflives or small volumes. RMMs for IPCs and final product release testingare conceivable in the food and beverage sector. The reason is that thesensitivity does not have to be as high as for sterility testing since acertain basic bioburden is present due to the nature of the material tobe tested. Moreover, RMMs enable certain tendencies to be identifiedquickly, and critical organisms in particular can be detected by PCR.Analyses confirm that predominantly PCR-based mycoplasma testingfor determination of the cell line purity is well accepted on the market.

Do you envision the future of microbiology using growth-based methods and the colony-forming unit as a gold standard? If not which alternative would be most appropriate and how long do you think it would take to make this change?David: In the long term, with continued innovation in the RMM space,the use of the colony-forming unit (CFU) as the gold standard will likelyend. The CFU and traditional growth will remain the mainstay of theconservative pharma micro testing for at least the next 10 years. Fastersingle cell methods will increase in popularity as their validation andimpact on the quality of production get fully realised and regulators buyinto the capabilities of the products available. But for the near term, thegold standard is accepted by regulatory authorities worldwide, makingthe use of CFU a critical path to a more streamlined validation.

Marcus: I think it remains to be seen which method will prevail in thecoming years. In fact, I believe that one method will not necessarily ruleout the other. It appears to be legitimate to require an application-specific choice of method. Naturally, this will also strongly depend onwhether the current regulatory standards will remain in force or on theextent to which they are liberalised. Whether it will continue to bemandatory to benchmark each new method against the classic sterilitytesting procedure will also be of relevance. Ultimately, this raises thesimple and basic question of which method will provide the degree ofprocess reliability needed.

Recently there have been a series of fusion/acquisitions in the RMM world. Which business strategy and companystructure would enable RMM systems to be economicallyviable in the long term and what are the main challengesthat might prevent this from happening?David: Rapid methods provide significant benefits to the lab and thebusiness as a whole. Companies which provide rapid methods must befocused as much on the validation and ongoing support of theirtechnology as they are on the method and science. Being a partnerthrough the validation process will ensure routine use of the methodand long term economic viability of the provider.

Marcus: This has primarily been driven by the importance of focusingon the objectives and advantages of RMMs, which should not be takento mean only economic interests. We all, as consumers and patients,expect that each new method will cover our needs for safety at least tothe same or even to a much better degree. The extent to which RMMswill be viable in the long term naturally depends on new developmentsand new approvals, for example, in cell therapy. In addition, a decisivechallenge will be posed by how far standard tests run up against theirlimits in these young markets. A business strategy tailored to thesespecialised markets can certainly be successful. I think that anotherfactor favouring the success of RMM is, however, to offer a largeportfolio of different products and methods.

IN-DEPTH FOCUS: RMM ROUNDTABLE

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Analytical instrumentation for the biopharmaceutical industrycontinues to develop to meet evolving needs, and new tools are beingcommercialised on a regular basis. Such innovations allow researchersto use orthogonal techniques for subvisible particle characterisa-tion and to gain the insight needed to formulate safe, stable andeffective products.

Investigating the impact of subvisible particlesSubvisible particles are usually defined as being in the size range 0.1μmto 100μm, often above the upper size limit for measurement by size-

exclusion chromatography but not sufficiently large to be detectable byeye. Such particles may be proteinaceous (aggregates or proteinsadsorbed onto other contaminants), silicone oil from the syringes,devices and stoppers used during drug delivery, or other contaminantsarising from either the manufacturing or drug delivery process.

Aggregation is a common degradation route for proteins, and is acommon source of particles in the subvisible range that must beinvestigated for all products. The formation of protein particles canhave the undesirable effect of reducing product efficacy and also hassignificant potential to cause adverse side effects. Such effects depend

Quantifying and sizing subvisible particles in biopharmaceutical products are crucial aspects of formulationdevelopment, stability studies, process development, product release and extended characterisation of the finaldrug product. The particles, which may consist of aggregated proteins, and/or components shed from processmaterials or container closure systems, can directly impact on the efficacy and immunogenicity of a drug product.Also, they often act as nucleation sites for further protein aggregation and/or lead to the development of largerparticles by agglomeration. Measuring the size and concentration of subvisible particles within a formulation is anessential precursor to their effective control, and of growing importance as the industry works towards ‘zero defect’and ‘essentially particle-free’ products. In this article we consider requirements for subvisible particlemeasurement within the context of current regulatory expectations, and more broadly, review the technologyavailable to meet them.

PARTICLE SIZING

Meeting biopharmaceuticalanalytical requirements for subvisible particle sizing and counting

John Carpenter University of Colorado � Amber Haynes Fradkin KBI BiopharmaChristina Vessely Biologics Consulting Group Inc.

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on the drug delivery method and the size and type of aggregatesinvolved, but may include: local reaction, embolism and/orimmunogenicity. Immunogenicity is considered a particular problemsince it may lead to a fraction of patients becoming ‘secondary non-responders’ due to the formation of neutralising antibodies. This effectcan threaten the success of ‘miracle drugs’ and can pose a severe threatto patient treatability.

Current US Pharmacopoeia (USP) specifications include numericallimits for particles >10μm and >25μm in size. These limits are associatedwith concerns about particles blocking capillaries (average diameteraround 7μm) upon injection and causing other health issues1. However,as the technology to characterise and quantify particles in the micronand submicron size ranges has become available, it is clear that largenumbers of such particles are routinely delivered to patients.

High subvisible particle counts can result from shedding by processequipment or particles that may be present in components used fordrug delivery – IV bags or syringes – or may even be produced bymechanical stress if the product is mishandled during transport. Theseparticles are not routinely filtered out as part of the drug administrationprocess but rather delivered along with the drug. Even with the 0.22 micron in-line filters used for intravenous administration, a largefraction of the submicron particles can readily pass through and bedelivered to the patient.

In summary then, developing knowledge of the provenance andimpact of subvisible particles is a vital strategy for engineering stabilityand safety into a product. Indeed, better characterisation andunderstanding of the role of subvisible particles has the potential to bea cost-effective strategy for developing stable and effective productswith the added advantage of offering opportunities for interventionsearly in the development process.

The regulatory perspectiveEvolving understanding of the importance of subvisible particles andtheir role in triggering immunogenic responses is evident from therequirements of pharmacopoeial chapters USP <788>, Ph. Eur. 2.9.19, andthe more recently released USP <787>, which came into effect in 20142,3,4.The latter relates specifically to protein therapeutics and is associatedwith general information chapter 17875.

The latest guidance reflects the growing concern about particles inthe 0.1μm-to-10μm size range and recommends the determination ofparticle concentrations for the >2μm and >5μm fraction, in addition tothe enumeration of particulates in the >10μm and >25μm size ranges. It also calls for assessment of the range and levels of particles in the

2μm-to-10μm range both initially and over the course of a product’sshelf life. Particle concentration limits are included for the larger sizeranges, but the new guidance also permits the establishment of product-specific limits. In addition, there is a useful reduction in required testingvolumes, with the new chapter allowing for volumes as low as 0.2mL. This not only saves on product costs but also enables the determinationof particle count variability from vial to vial or syringe to syringe.

However, beyond these specific requirements it is clear that theregulatory drive is for sufficient subvisible particle characterisation todemonstrate the necessary understanding and control of productquality, within the constraints of current analytical capabilities. Forexample, the new FDA guidance highlights the need to distinguishsilicone oil from proteinaceous, inherent or intrinsic particles, and inaddition states that “as more methods become available, sponsorsshould strive to characterise particles in smaller (0.1-to-2 microns) sizeranges.”6. This wider investigative requirement is echoed by the EMAGuidelines7 which state that “[the] formation of aggregates, subvisibleand visible particulates in the drug product is important and should beinvestigated and closely monitored on batch release and duringstability studies. In addition to the pharmacopoeial test for particulatematter, other orthogonal analytical methods may be necessary todetermine the level and nature of particles.”

So while formulators and quality control groups may understandablylook for clarity with regard to regulatory requirements, the reality is thatthe issue of subvisible particles requires rigorous systematicinvestigation. Subvisible particle characterisation requirements extendto particle size, number, shape, optical properties and chemicalidentification, and increasingly include submicron particles.

Establishing the analytical toolkitBoth USP <787> and <788> recommend the technique of lightobscuration (LO) for the sizing and enumeration of subvisible particles.LO instrumentation measures particle size and number on the basis ofthe blockage of light by individual particles passing through a light-sensing zone.

More widely used for subvisible particle assessment than any othertechnique, LO nevertheless suffers from certain limitations when setagainst the evolving list of requirements in this field. The mostimportant of these limitations is a lack of detailed information aboutthe nature of the subvisible particles it detects. Also, LO has a tendency

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72 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

Figure 1: Flow imaging microscopy systems capture images of individual particlesenabling detailed characterisation, most especially for particles >5 microns

Figure 2: Resonant mass measurements quantify particle size and shape in the 50nm to 5µm size range and are able to directly differentiate silicone oil from protein -aceous particles

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to undercount translucent particles, such as thosecomposed of protein and polysorbate, and has anupper detection limit of around 18,000 to 38,000particles/mL, depending on the instrumentationemployed. The orthogonal techniques that are nowbeginning to be used widely tend to be those thatdirectly address these limitations and, in addition,extend information-gathering capabilities in theareas of most importance, creating a more completepicture of a sample’s particle profile.

Flow imaging microscopyFlow imaging microscopy measures the size, shape and transparency ofparticles using bright-field images captured as an unfiltered solutionpasses through a flow cell. The resulting data enable the robustclassification and differentiation of particles in solution on the basis ofa number of different metrics. The ability to measure directly in thesolution of interest is important because studies suggest that filtrationcan significantly impact results of particle characterisation studies8.

A key advantage of flow microscopy is that images of each particlecan be obtained, which allows for further particle characterisation (see Figure 1; page 72). For example, with digital filtering methods, thetechnique can distinguish certain relatively larger size (e.g., >5 micron)silicone oil droplets from proteinaceous particles. This is due to thespherical nature of silicone oil particles. Other insights into particlecharacteristics obtained from imaging include their density andwhether they are fibrillar or compact. In addition, with samplescontaining silicone oil one can observe if particles are composed ofagglomerates of protein aggregates and silicone oil droplets.

For these reasons flow microscopy has become a popularworkhorse widely used alongside LO; however, it too has an importantlimitation: for particles smaller than 5μm, the method begins to loseresolution and the ability to accurately differentiate types of particles.

Resonant mass measurementResonant mass measurement (RMM) sizes and counts particles in the50nm-to-5μm size range, on the basis of particle buoyant mass, bymeasuring changes in resonant frequency as individual particles flow

through the measurement zone (see Figure 2; page 72). The datagenerated include buoyant mass, dry mass and size. This technique istherefore able to differentiate between silicone oil, which is less densethan water, and proteinaceous particles, in the size range where flowmicroscopy loses resolution; most silicone oil droplets are smaller than5 micron. Furthermore, RMM has the advantage of being able to directlydistinguish silicone oil droplets from protein particles, and does notrely on post-measurement data manipulations. A caveat is that someparticles composed of a combination of protein and silicone oil mayhappen to be neutrally buoyant and are therefore not detected.

These unique advantages make RMM a valued addition to theanalytical toolkit, especially for samples that include silicone oil, due tothe use of prefilled syringes or other primary packaging componentsthat are siliconised or otherwise treated to improve performance. When used in combination, RMM and MFI allow for the robust differ -entia tion and characterisation of both silicone oil and proteinaceousparticles across the wide submicron-to-10μm size range.

Raman spectroscopyFor certain particles in the subvisible region, the preceding techniquesfail to provide specific chemical identification. For example, excipientssuch as polysorbate can degrade and form insoluble particles that aresimilar in shape and transparency to proteinaceous particles. Imagingtechniques augmented by Raman spectroscopy offer effectivedifferentiation in such situations by providing the verification of particlechemistry needed for detailed assessment of the provenance andidentity of specific populations.

Raman is well known for its high chemical specificity, and, unlikeFourier transform infrared spectroscopy, is insensitive to the presenceof water, enabling the characterisation of aqueous solutions. Unknownmaterials are typically identified through interrogation of libraries ofreference spectra, to which product-specific spectra may be added asrequired. Automated Raman spectroscopy systems provide efficientparticle enumeration, characterisation and sample-dependentidentification down into the 3μm-to-5μm size range, even for particlespecies with closely similar morphology. As a result, although it is not ashigh throughput as flow microscopy or LO, the technique has provenvalue for its ability to answer the ‘what is it?’ question that arises sofrequently in development, manufacturing and stability programs (see Figure 3).

Nanoparticle tracking analysisAs the sub-micron fraction of the subvisible particle size range comes

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VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 73

Figure 3: Particle images and representative spectra collected from a filtered lysozyme sample: membranefilter background (black), lysozyme aggregate (blue), polystyrene sphere (red) and, inset, (green) thespectrum of a rubber particle that was found in the solution

Figure 4: Nanoparticle Tracking Analysis measures particle size and count in the 50-to-1000nm range, which is growing in interest

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under greater scrutiny, techniques that enable the enumeration andsizing of nanoparticles are also increasingly vital additions to thecharacterisation toolkit. Nanoparticle tracking analysis (NTA) is onesuch technique (see Figure 4; page 73).

NTA uses a high-resolution digital camera, a laser and speciallydesigned software to track the Brownian motion of individual particlesunder a microscope. The resulting measurements are used to generateparticle size distributions for particles in the size range of about 50nmto 1000nm, in solution. Each particle is sized individually and within aknown sample volume; so the concentration of particles, or particlecount, can also be determined. These capabilities provide animmediate solution for the growing requirement to understand thecontribution of all size fractions of the subvisible particle size range.

The practicalities of measurementThe preceding discussion highlights how the strengths of a range ofanalytical techniques are being exploited and combined to meet the analytical requirements of the industry. However, it is important torecognise that many of these methods are relatively new and requiresubstantial expert analytical input at every stage: from methoddevelopment, through sample measurement, and up to and includingthe process of data interpretation.

The extended characterisation (as opposed to release) methods associated with subvisible particle characterisation are not currently subject to any regulatory requirement for validation.However, analytical data must always be robust to be meaningful andrelevant. (Note that even if particle counts are not related to safety and efficacy in patients, the data may still be required for qualityassessments and process control). Furthermore, to fully realise theirvalue, techniques must be easy to apply at every stage of the drug development process. For example, if subvisible particles are notbeing examined in sufficient detail in the early stage of processdevelopment, how will later-stage changes that indicate a problem withscale-up be detected?

This practicality and application of methods is an area whereprogress is urgently required to address issues associated with:� Data variability – from analyst to analyst and site to site.� Sample handling – how should the sample be prepared for analysis

to produce results that are as relevant as possible to formulationstudies, particularly, for example, in the presence of silicone oil?

� The transfer of particle size standards from one protein to another– a standard developed for one protein is not necessarilymeaningful for another.

� Sample size – minimising the volume required for analysis is helpfulthroughout the formulation workflow, but this must be balancedagainst the requirement to measure representatively.

� Data interpretation – many techniques produce substantialquantities of information but this raises the question of whichmetrics are critical in terms of defining performance.

� Data archiving – is it necessary to save every digital image obtainedfor a sample during analysis by flow microscopy?

Addressing these issues is essential for the development of qualified,validated methods that can be used throughout the drug developmentprocess and potentially in quality control.

Looking aheadDeveloping a rigorous understanding of the provenance of subvisibleparticles and their role in product quality, as well as their potential rolein initiating or causing harm or loss of efficacy is an important and ongoing goal for the biopharmaceutical industry. Being able torobustly characterise such particles is a prerequisite for progress, andthere are a number of techniques that have proven value in this field.The need for researchers to delve deeper into the nature and source ofsubvisible particles and to develop secure control strategies that mitigate risk creates an ongoing push for advancement inanalytical technology. By developing robust methods that can be usedacross the development cycle, analytical scientists and instrumentmanufactures can help to accelerate drug development as well asincrease product safety.

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 75

PARTICLE SIZING

1. Christie, M, Torres, RM, Kedl, RM, Randolph, TW, Carpenter, JF. ‘Recombinant Murine

Growth Hormone Particles are More Immunogenic with Intravenous than Subcutaneous

Administration.’ Journal of Pharmaceutical Sciences. 2014; Vol 103, pp128 – 139

2. United States Pharmacopeia, <788>: Particulate Matter in Injections

3. United States Pharmacopeia, <787>: Subvisible Particulate Matter in Therapeutic

Protein Injections

4. European Pharmacopeia, 2.9.19 Particulate Contamination: Subvisible particles, Method

1. Light obscuration particle count test

5. United States Pharmacopeia general information chapter 1787: Measurement of

Subvisible Particulate Matter in Therapeutic Protein Injections

6. Food and Drug Administration (US) Guidance for Industry: Immunogenicity Assessment

for Therapeutic Protein Products. August 2014

7. European Medicine Agency Guideline on Development, Production, Characterisation

and Specifications for Monoclonal Antibodies and Related products. Dec 2008

8. ‘Characterization of protein aggregates in suspension and on a filter membrane using

Morphologically Directed Raman Spectroscopy (MDRS).’ Malvern Instruments

application note. Available for download at: http://www.malvern.com/en/support/

resource-center/application-notes/AN150722G3-IDProteinSuspensionAnalysis.aspx

References

John F. Carpenter, PhD, is Professor of Pharmaceutical

Sciences at the University of Colorado School of Pharmacy,

and a Co-Founder and Co-Director of the University of

Colorado Center for Pharmaceutical Biotechnology.

His research interests include mechanisms for protein

degradation and stabilisation in pharmaceutical formula -

tions during bioprocessing and in delivery systems, and

development of advanced analytical methods for therapeutic proteins.

He has worked for several years on defining rational strategies for stabilising

proteins and vaccines during freeze drying and storage in the dried solid.

He has published more than 230 peer-reviewed papers and is an inventor on

30 issued patents. He has received several teaching awards and The Ebert

Prize. He is a Fellow of the American Association for Advancement of

Science and the American Association of Pharmaceutical Scientists (AAPS)

and has received the AAPS Research Achievement Award in Biotechnology.

He also is the Organiser for the Colorado Protein Stability Conferences.

Amber H. Fradkin, PhD, received her PhD in Chemical

Engineering at the University of Colorado at Boulder. Amber

currently holds the position of Associate Director at KBI

Biopharma where she manages the Particle Characterization

Core which specialises in analytical methods for quantifying,

characterising and identifying submicron, subvisible and

visible particulates.

Christina Vessely has over 17 years’ experience in

analytical/formulation development, with products ranging

from peptides to monoclonal antibodies, therapeutic

proteins to vaccines, and novels to biosimilars. Areas of

expertise include method development, qualification

and validation, the development of reference standards,

stability strategy and evaluation, and establishment of

comparability and/or similarity.

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Both formulation and process parameters affect the degree ofcrystallinity of excipients. For example, a high total or relativeconcentration of the bulking agent facilitates its crystallisation.5

Meanwhile, the freezing step is important, since freezing conditionsstrongly influence the crystallisation tendency of formulationcompounds. A slow freezing rate and/or exposure to a temperaturebelow the freezing point but well above the glass transitiontemperature of the maximally freeze-concentrated solute, Tg′, supportcrystallisation.2,5,6 In addition, during secondary drying, crystallisationmay be finalised for several excipients, such as mannitol, and hydratescan be eliminated.3,7

Accurate determination of the degree of crystallinity is imperativein drawing a reliable conclusion about process and formulation

performance. In the field of freeze-dried products, differential scanningcalorimetry (DSC) and X-ray powder diffraction (XRPD) are establishedmethods for this purpose.8 However, dynamic vapour sorption (DVS)offers a new opportunity to measure the degree of crystallinity of theformulation components in a more representative way.

Crystallinity of excipients In general, a material may exist in two distinct physicochemical states:crystalline or amorphous1,9. In a crystalline material, the constituentsare arranged in an orderly repeating pattern extending in all threespatial dimensions10. An amorphous material lacks this long range orderand consists of molecules that are randomly oriented11. Heating a frozencrystalline material above the eutectic temperature, Teut, results in melt.

Freeze drying is gaining in importance as the number of biopharmaceuticals that are unstable in a solutionincreases1. According to recent reports, a growth of 10% may be expected for freeze-dried products in the next 10 years. The technique offers the opportunity to gently dry temperature-sensitive drugs such as proteins orpeptides. Since freeze drying is a rather expensive drying technology, formulation and process optimisation stronglyfocus on the design of a robust and fast cycle2. Interestingly, partially crystalline systems offer advantages withregard to processability as well as product appearance3,4.

FREEZE DRYING

76 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

Dynamic vapour sorption of freeze-driedpharmaceuticals

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Claudia Kunz and Henning GieselerDepartment of Pharmaceutics, University of Erlangen

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Tg′ applies for the conversion of an amorphous material froma rigid glass into a rubber-like state. Both conversions relateto the critical formulation temperature of a system. Exceedingthis temperature during primary drying may result in loss ofcake structure and, thus, possible impairment of cakeelegancy and/or loss of activity of the active pharmaceuticalingredient (API)12-14.

Stabilisers and bulking agents are frequently added to an API to improve final product characteristics15. Thephysicochemical state essentially influences ability tosuccessfully perform their desired assignment. Chatterjee etal. demonstrated that a well-defined partially crystallinesystem of glycine and a carbohydrate ensured short processtimes and virtually complete recovery of the model proteinlactate dehydrogenase16. Kim et al. showed that crystallisationof the bulking agent, such as mannitol, in the freezing phasesupports formation of a robust cake structure and typicallyresults in an elegant product appearance with fastreconstitution times5. In contrast, insufficient crystallisationduring freezing bears the risk of vial breakage during the earlyphase of primary drying; furthermore, elevation of the residual moisturecontent in the product due to release of crystal water during long-termstorage may compromise storage stability of some APIs3,17. It is wellknown that the eutectic temperature of amaterial is much higher than Tg′18. Bulkingagents that remain amorphous can significantlyreduce the critical formulation temperature. Ma et al. reported that complete crystallisationof glycine significantly reduced process time offreeze drying of a monoclonal antibody19.Therefore, a high degree of crystallinity helps to avoid optical defectsand may reduce process length. On the other hand, if an excipient isadded to stabilise the active ingredient, it must remain amorphous andcrystallisation must be avoided. A stabiliser has to remain in theamorphous phase in order to be able to form hydrogen bonds with the API1,18. Accordingly, it has been reported that mannitol crystallisationmay compromise protein recovery due to loss of the stabilising effect20,21.

Historical and modern application of DVSDVS is a technique used to investigate the effect of humidity onmaterials. The concept is based on gravimetric determination of the

amount of vapour sorbed by a sample at acertain given humidity and temperature.Historically, sorption experiments were time-and labour-intensive since a certain humiditywas generated by using defined saturated saltsolutions in desiccators and samples wererepeatedly weighed until a constant mass was

reached17. Moisture sorption behaviour of pharmaceuticals became aresearch area of interest in the early eighties. Ahlneck et al. reportedthat disorder in crystalline excipients in freeze-dried products mayincrease tendency towards water vapour absorption which is connectedwith an increase in chemical degradation of drugs22.

Likewise, Hageman et al. demonstrated that the choice of excipientsinfluences vapour sorption behaviour and, thus, protein degradation in

freeze-dried preparations23. When the first DVS was developedin 1991, the obstacles of continuous humidity generation wereovercome. These modern instruments allow for an auto-mated variation in vapour concentration and consist of amicrobalance that constantly records change in sample mass.Hence, the determination of rapidity of water sorption ispossible which provides further valuable information. In 1994,Marshall et al. collected information about water sorptionbehaviour of a series of pharmaceutical compounds for thefirst time with the automated instrument24.

Today, the technology is routinely used in the pharma -ceutical industry for numerous applications25. Duringpharmaceutical development, possible drug substances andexcipients may be analysed regarding stability, processingand application performance. Consequently, required storageconditions may be derived from sorption analysis at a giventemperature and relative humidity. DVS analysis also plays arole in the analysis of packaging material with regard to

VOLUME 20 ISSUE 5 2015 European Pharmaceutical Review 77

Figure 1: XRPD patterns of freeze-dried mannitol products spiked with an internal standard,lithium fluoride (shows a characteristic peak from 38.4° to 39.0° 2θ)32

Figure 2: DSC heating diagram of 10 mg/mL freeze-dried mannitol (melting point at 165.33°C)

Accurate determination of the degree of crystallinity is

imperative in drawing a reliableconclusion about process andformulation performance

FREEZE DRYING

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permeability and effective protection of the pharma -ceutical26,27. As a side note, several instruments also permitthe use of a wide range of organic solvents besides the use of water17.

A decade ago, a few first specific fields of use of DVS havebeen reported in the literature. For example, investigationsincluded determination of the surface area calculation usingthe BET equation for either water or organic vapours28-30.

Although DVS is widely used for the investigation ofsolids, detailed data in the field of freeze-dried pharma -ceuticals is lacking. In fact, analysis of freeze-dried productshas a high demand for rational method development andthus requires a systematic approach. However, investigationof vapour-induced physicochemical changes of samples ishighly promising17.

Established methods to investigate the degree of crystallinityFrequently, XRPD is used in pharmaceutical development forthe determination of the degree of crystallinity. Thephysicochemical state can be differentiated since crystalline phaseslead to sharp peaks and amorphous phases to diffuse hills17. In order toestimate the amorphous and crystalline fraction of a substance theEuropean Pharmacopeia advises to set the integrated area of the peaksarising from the crystalline fraction of the sample in relation to the totalarea of the graph. However, the diffraction pattern is characteristic for a defined individual substance, and total peak intensity stronglyvaries between different substances for the same amount of sample. In addition, XRPD analysis may be prone to errors since differentpolymorphs provide a different x-ray diffraction pattern. XRPD is a veryvaluable tool in order to qualitatively determine a substance but showsmajor weaknesses in quantitative phase analysis31. Use of an internalstandard, for example lithium fluoride, mayimprove results. This method encompasses thespiking of each sample with a defined amountof an internal standard. Next, area of specificpeaks is set in relation to the peak area of theinternal standard (Figure 1; page 77). In thisinstance, it is necessary to determine a calibration line32,33. However, thelimit of detection of this method has been determined at 10%34.

Furthermore, analysis of DSC thermograms offers various optionsfor quantification of the amorphous portion. DSC heating profiles ofpartially crystalline systems may show a glass transition endotherm,crystallisation exotherm and melting endotherm. Saleki-Gerhardt et al.propose correlating the amorphous content to the heat of crystalli sa -tion. Here complete crystallisation of the amorphous fraction isrequired33. Similarly Grisedale et al. compare the quantity of theamorphous fraction with the ratio of recrystallisation enthalpy to the melting enthalpy of the crystalline material. Both techniques fail ifcalibration is performed with different polymorphs. Additionally, they

suggest that the quantification should be based on the assumption thatheat capacity at the glass transition temperature is proportional to theamorphous content35. Finally, quantification of the amorphous phase ispossible through the connection with the melting enthalpy of thecrystalline fraction (Figure 2; page 77)36. It has been shown that DSCmethods also fail to quantify amorphous content lower than 5% inpredetermined mixtures34.

The concept: measurement of amorphous contents Quantification of the amorphous content by DVS relies on the principlethat amorphous material absorbs significantly more water vapourcompared with their corresponding crystalline phases.34 Since

water may act as a plasticiser, the glasstransition temperature can be lowered toambient temperature leading to crystallisationof the amorphous substance, which can beseen from a distinct release of water (Figure 3).

Prior to the analysis, calibration is generallyrequired between the amount of water absorbed by the sample inequilibrium and the amorphous content37. In the literature, a moreprecise method is described that can be applied for a material thatreadily crystallises upon exposure to elevated relative humidities38. As a first step, sorption profiles of the material are analysed thoroughlyin order to build up a sound understanding of recrystallisationbehaviour. The information obtained is then utilised for thedevelopment of the measuring method. The main experiment consistsof three main stages (Figure 4; page 79). Firstly, the amount of adsorbedwater is measured by the partially crystalline sample at a relativehumidity below the onset of re crystallisation. Secondly, recrystallisationis induced at elevated relative humidity, for example at 70% relative

78 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

FREEZE DRYING

DVS is based on gravimetricdetermination of the amount of vapoursorbed by a sample at a certain given

humidity and temperature

Table 1: Results of the determination of the amorphous content by XRPD (internal standard method)32, DSC (melting point method)36 and DVS analysis38

Sample X-Ray Powder Diffraction (XRPD) Differential Scanning Calorimetry (DSC) Dynamic Vapor Sorption (DVS)

Mannitol 10 mg/mL 96.20% ± 2.05% 93.60% ± 3.46% 99.96% ± 0.07%

Mannitol 100 mg/mL 98.94% ± 0.35% 95.82% ± 3.72% 99.90% ± 0.18%

Figure 3: DVS analysis of freeze-dried product consisting of mannitol + sucrose 1:3, 50 mg/mLsolid content, shows release of adsorbed water upon recrystallisation of the amorphous fraction at60% relative humidity and 25°C

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humidity. Finally, the amount of adsorbed water vapour bythe fully crystalline sample is determined at the relativehumidity already applied in the first step. The differencebetween the first and second gain in mass at the relativehumidity below the onset of recrystallisation solely accountsfor the amorphous content. For the determination of acalibration curve, about five known mixtures of the crystallineand amorphous material ranging from zero to one hundredpercent of amorphous content are analysed with the above-described method. Subsequently, the actual sample analysisis performed in the same way and the amorphous content iscalculated by the correlation.

The direct comparisonTable 1 (page 78) illustrates the results during a laboratoryexperiment evaluating the influence of solute concentrationon the degree of mannitol crystallinity. For this purpose,freeze-dried cakes consisting of either 10mg/mL or 100mg/mLmannitol solution (28 vials, 20 mL, 5 mL fill volume) were freeze dried ina laboratory freeze dryer (Lyostar II, SP Scientific, Stone Ridge, NY, USA).The samples were frozen with a cooling rate of 1°C/min to -45°C.Primary drying was then performed at a shelf temperature of -37°C anda chamber pressure of 44 mTorr. Secondary drying was performed at ashelf temperature of 40°C and a chamber pressure of 44 mTorr.

The results obtained by XRPD (Philips X’pert MPD, PANalytical,Eindhoven, Netherlands), DSC (DSC822e, Mettler Toledo, Greifensee,Switzerland) and DVS (DVS Intrinsic, Surface Measurement Systems,London, UK) analysis are in good agreement and show that both

concentrations result in approximately 100% crystallinity of mannitol(Table 1; page 78). When taking a closer look at feasibility of alltechniques the most notable difference is that DVS is able to detect alower amorphous content compared with XRPD or DSC. According to theliterature, DVS is able to detect 0.5% amorphous content in a crystallinematerial34. All three techniques require calibration. Generally,calibration is performed in a certain range of crystallinity covering theexpected result, before the operator analyses the sample andinterpolates the unknown crystallinity.

It is rather difficult to utilise the same polymorph or mixture of

FREEZE DRYING

Figure 4: DVS change in mass profiles for a 10 mg/mL sample of freeze-dried mannitol

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polymorphs for the calibration for XRPD analysis that is present in the final freeze-dried product. It can even be observed that thecomposition of polymorphs in one batch of a freeze drying cycle differs. Furthermore, integration of the diffraction pattern is demand-ing since placement of the baseline for peak integration cannot berealised in a standard operating procedure and thus is rathersubjective. A merit of XRPD is that the average sample analysis in therange 0-40° 2θ takes only about 45 minutes, but requires samplemasses of approximately 200mg. In contrast, DVS has the advantagethat a small sample mass of about 5-50mg is sufficient for analysis.Finally, it can be seen from the obtained results that DVS providesvalues that are more precise with a smaller standard deviationcompared to XRPD or DSC method.

ConclusionsDVS has already proved to be a valuable tool in pharmaceuticaldevelopment. Considering determination of the amorphous content, itcan be concluded that DVS experiments are one of the most sensitivemethods. In the area of freeze-dried products, accurate determinationof small amounts of amorphous material is essential in order torationally optimise process and formulation parameters and ensurefinal product quality. Although further research is needed, preliminary

results suggest that DVS even succeeds in detecting subtle changes inamorphous content.

80 European Pharmaceutical Review VOLUME 20 ISSUE 5 2015

FREEZE DRYING

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3. MJ Pikal. Freeze-Drying. Encycloppedia of Pharmaceutical Technology. 2007; p. 1807-33

4. EY Shalaev, W Wang, LA Gatlin. Protein Formulation and Delivery, 2nd Edition. Rational

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6. T Oguchi, K Terada, K Yamamoto, N Yoshinobu. Freeze-Drying of Drug – Additive Binary

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8. K Izutsu, S Yoshioka, T Terao. Effect of mannitol crystallinity on the stabilization of

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References

Claudia Kunz studied pharmacy at the University of

Wuerzburg and received her license as a pharmacist in

Germany in 2011. After first gaining professional

experience in retail pharmacy, she joined the Freeze Drying

Focus Group within the Division of Pharmaceutics at the

University of Erlangen in 2013 as a graduate student.

Her research interest is currently focused on Dynamic

Vapour Sorption of freeze-dried products as well as freeze drying from

organic co-solvent systems. More information about Claudia Kunz can be

found on the group’s official website: www.freeze-drying.eu.

Henning Gieseler studied pharmacy at the University of

Wuerzburg and has been a licensed pharmacist in Germany

since 2000. He received his PhD in pharmaceutics at the

Department of Pharmaceutics, University of Erlangen, in

2004. After a post-doctoral research period at the School of

Pharmacy, University of Connecticut (Prof. Michael

J. Pikal, “UConn Freeze Drying”) until early 2006, he

returned to his former department at the University of Erlangen where he

founded the Freeze Drying Focus Group (FDFG) within the Division of

Pharmaceutics. In 2010, Dr. Gieseler obtained his habilitation and venia

legendi in Pharmaceutical Sciences from the University of Erlangen with the

thesis “Quality by Design (QbD) in Freeze-Drying Using Advanced Process

Analytical Technology”. In the same year, he also founded GILYOS in

Wuerzburg, a contract service provider for pharmaceutical freeze drying.

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