11th protein expression in animal cells kananaskis canada ...claes gustafsson - dna2.0 systematic...
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11th Protein Expression in Animal Cells
KananaskisCanada
September 22 - 26, 2013
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Table of conTenTS
A Message from the Organizers 4
Sponsors 5
Organizing Committees 7
Conference schedule 9
Oral presentations :
MABNET SyMpOSiuM 21
Session 1: CEll ENgiNEEriNg i: TrANSCripTiON & TrANSlATiON 59
Session 2: CEll ENgiNEEriNg ii: phySiOlOgy ANd METABOliSM 71
Session 3: TrANSiENT prOTEiN ExprESSiON 85
iNduSTriAl WOrkShOp 93
Session 4: ANTiBOdy ExprESSiON 101
Session 5: BiOprOCESSiNg & ANAlyTiCAl TEChNOlOgiES 119
Session 6: STEM CEllS: ThE FuTurE 133
poster presentations : 139
Authors index 252
participants list 254
For Research Use Only. Not for use in diagnostic procedures. ©2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. CO25498 0912
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Message from the Chair - pEACe
dear participants
This year (2013) brings forward the pEACe – protein Expression in Animal Cells. The non-profit conference is formatted as a 5-day event to discuss the latest developments in animal cell technology with the strong emphasis on biopharmaceutical production but also including technologies such as stem cells associated with cell therapies. The global conference locations are carefully chosen for their interesting and relaxing environment, conducive to both a social and scientific interaction between participants. Past locations have included Crete, Brazil, Wyoming and portugal. The location of the present meeting at kananaskis in the heart of the Canadian rocky Mountains follows this tradition and positions our conference in a site of outstanding natural beauty.
The conference organizing committee has worked hard at identifying and inviting speakers who have made a significant contribution to the field over the last 2 or 3 years. We feel that the selection of leading-edge scientists as speakers is key to the success of our meetings. The speakers have been allocated to six scientific sessions that are planned to cover a full range of animal cell technologies. As in previous conferences we have organized a specialized symposium for the first day (Sunday) which this time will be on the theme “Monoclonal Antibody Expression Technologies”. For this we are especially grateful to the Natural Science and Engineering research Council (NSErC) of Canada who provided substantial funding for this symposium under their program Strategic Network Enhancement initiative (SNEi). This has been used to fund travel and conference registration for around 30 graduate students who are working in the Canadian network (MabNet). Further funding has been provided by the pEiS organization for a limited number of student bursaries. These funding initiatives have allowed the attendance of far more graduate students than we have had in the past. This is a welcome development which we feel is important for the future of our scientific area.
We would of course like to thank all our corporate sponsors for their generosity, without which we would be unable to hold a conference of this type. As well as thanking the combined efforts of the organizing committee in putting together this scientific meeting, I would like to extend my special gratitude to Bruno Begin who has been the conference manager since 2003. his expertise and hard work has maintained a strong organization which bodes well for the continuing future of the society and this series of pEACe meetings.
As Conference Chair i would like to wish you all a successful and enjoyable pEACe meeting in kananaskis.
M.ButlerChair of pEACe and director of MabNet
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Organizing Committee PEACe 2013
Chairman,Michael Butler University of Manitoba, Canada
Amine Kamen National Research Council
Canada Canada
Tom Kost GlaxoSmithKline R & D, USA
Christopher KempKempbio, Inc.
USA
Kenneth Lundstrom PanTherapeutics, Switzerland
Bruno Bégin
Conferium Conference Services
Canada
Advisory Committee Members
Michael KallosUniversity of AlbertaCanada
Linda Lua
The University of QueenslandAustralia
Devender Sandhu Therapure Biopharma Inc., Canada
Georg Schmid F. Hoffmann-La Roche Ltd
Switzerland
Girish Shah Consultant UK
Mark SmalesUniversity of Kent,
UK
Kim. J. Stutzman-Engwall ConsultantUSA
SUPPORTING & MEDIA SPONSORS
NEWSLETTER of the European Society for Animal Cell Technology
March 2010
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ESACT Newsletter March 2010
News
Proceedings Dresden and Dublin
The Book of Proceedings from 20th ESACT Meeting - Dresden is coming out soon. The Dublin Scientific Committee Chair, Professor Nigel Jenkins, is now inviting all the 21th ESACT poster presenters to contribute with a extended abstract (3 pages max) to the Dublin proceedings book. The deadline is April 9, 2010. For more information please contact ESACT Dublin Secretariat, Ms Bernadette Douglas ([email protected]).
2010 Elmer Gaden Award
E. Terry Papoutsakis and former group member Ryan Senger (now an Assistant Professor at Virginia Tech, are honored with the 2010 Elmer Gaden Award by Biotechnology & Bioengineering and John Wiley & Sons, Inc. for their paper:"Genome-Scale Model for Clostridium acetobutylicum: Part I. Metabolic Network Resolution and Analysis".
Biotechnology and Bioengineering, Volume 101, Issue 5, 2008, Pages 1036–1052 DOI 10.1002/bit.22010.
Synthetic Biology - what is it?
In cell culture technology we hope to make useful products and we think that key to this is a complete and “happy” cell. When our cells are happy then they work well for us, i.e. they work for example as protein production machines. However and maybe one day, this could be done in an entirely different way. The “black-box” - our cells - could be replaced by a true biological machine where we know every part involved and could modify or optimize it for our purposes to fit better to the final product intended. So, are we able to design such a machine? At least some attempts have been made in the past towards this goal. As Martin Fussenegger, our highly respected chairman said, small successes have been sold with some (too much?) hype.
In any case, if you want to read more about this highly fascinating and maybe still very provocative new field, please read the commentary Professor Fussenegger has recently published in Nature (Nature 463, 301-302 (21 January 2010))
JIN - our service offer to the industry and interested cell culture professionals for finding matching interests. The JIN is apparently becoming more and more successful and the website is heavily utilized. We are still looking, however, for sponsor companies who would help us to pay for the costly program that ESACT has supported since its establishment several years ago. In return for a sponsor contribution the logo of the sponsor company will be displayed on the web site.
http://www.jin-esact.org
Contents March 2010
News 1
ESACT 2009 Workshops 2
9th PEACe Conference 4
5th ETW Meeting 7
JAACT 2010 8
New members 10
Contributions should be sent to eletter esact.org
© 2010 ESACT - The European Society for Animal Cell Technolgy
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PROGRAM 11TH PEACESUNDAY-SEPTEMBER 22, 2013
MabNet Symposium
Registration
Michael Butler - University of ManitobaIntroduction/ Progress in MabNet
Session 1: Chair: Katrin Braasch
Calvin D’Eall - University of OttawaMabNet 1:Characterization of the Antitumor Activity of the Fc-Engineered Human:Camelid Chimeric Heavy Chain Antibody EG2-hFc
Nathalie Okun - University of ManitobaMabNet 2: Effect of N-glycosylation on the structure and function of a heavy chain monoclonal antibody
July Dorion-Thibaudeau - Ecole Polytechnique of MontrealMabNet 3: Towards the development of a surface plasmon resonance assay to evaluate glycosylation pattern of antibodies
Alan Dickson - University of ManchesterExternal 1: Novel format antibody expression in CHO cells
Martina Stützle - University of Applied Science BiberachExternal 2: Production of an anti-Insulin-receptor Fab fragment from hybridoma cells
Coffee Break
Session 2: Chair: Celine Raymond
Shantha Raju - Janssen R&DExternal 3: Glycosylation of antibodies
Eric Blondeel - University of WaterlooMabNet 4: Loading the inoculant – indirect feeding of nucleotide sugar precursors to affect glycosylation
Cécile Toussaint - Université de MontréalMabNet 5: Expression of recombinant cytoplasmic yeast pyruvate carboxylase alters primary metabolism of CHO cells
Navid Ghaffari - University of British ColumbiaMabNet 6: Varied CHO cell responses to amino acid limitations in cultures
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Lunch
Session 3: Chair: Gerry Drouilard and Ed Bodnar
Hengameh Aghamohseni - University of WaterlooMabNet 7: The Impact of Culture pH and Glutamine Level on the Glycoform Distribution of Eg2-hFc
Adolfo Castillo - Center of Molecular ImmunologyExternal 4: Study of relationship between Oxidative Metabolism and Antibody Specific Production Rate in insulin and glucose supplemented continuous cell cultures of Recombinant NS0 cell line
Katrin Braasch - University of ManitobaThe Changing Dielectric Properties of CHO Cells Can be Used to Determine Early Apoptotic Events in a Bioprocess.
Venkata Tayi - University of ManitobaSynergetic effect of glucose-dependent metabolic pathways on productivity and glycosylation of a heavy chain monoclonal antibody in Chinese hamster ovary cells
Volker Sandig - ProBioGen AGAntibody effector functions - impact on product development and strategies for adjustment.
Katharina Hassel - University of WaterlooCapture and Recovery of Immunoglobulin G with Weak Cation Exchange Membranes
Panel Discussion for MabNet topics
MabNet Poster presentation
PEACe opening reception
Presentation on Kananaskis Park
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OpeningChair: Michael Butler
PEACe Lecture- Ron WeissSynthetic biology: from parts to modules to therapeutic systems
Session 1 – Cell Engineering ITranscription & TranslationChairs: Kim Strutzman-Engwall, Kenneth Lundstrom
Claes Gustafsson - DNA2.0Systematic analysis and quantification of gene design variables and their effect on heterologous protein expression Johannes Grillari - BOKU-VIBTSmall non-coding RNAs pathway engineering of CHO cell factories that avoids translational burdening
Coffee Break
Mahvash Tavassoli - GUY’s HospitalEfficient expression and delivery of tumour selective therapeutic proteins
Joseph Shiloah - NIDDK, NIHLarge-scale screening identifies a novel miRNA, miRNA-15a-3p induces apoptosis in human cancer cell lines
Andrew Dean - Univeristy of KentEngineering of mTOR signalling in CHO cells for enhanced recombiant protein production
Lunch
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PROGRAM 11TH PEACEMONDAY-SEPTEMBER 23, 2013
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GERMANY
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Session 2 – Cell Engineering IIPhysiology & MetabolismChairs: Georg Schmid, Mark Smales
Alan Dickson - University of ManchesterMetabolic profiling: Moving from design of media and feeds to strategies for cell
engineering and synthetic biology
Volker Sandig - ProBioGen AG
Cell lines for Biosimilars and Biobetters: Closest match and improved performance by
selection and molecular design.
Lars Kober - Cellca GmbHDevelopment of a novel ER stress based selection system for the isolation of highly
productive clones
Coffee Break
Sampath Kumar - Biogen IdecGamma-Glutamyl Carboxylation of Vitamin K-Dependent Proteins: Strategies to Address
the Post-Translational Bottleneck
Nicola Beauchamp - Roche Diagnostics GmbH; Omit semicolonDiving into the metabolism of a Chinese hamster ovary cell
Tatiane Maldonado Coelho - University of Sao PauloComparative study of recombinant equine Chorionic Gonadotropin (eCG) expressed in
three different cell lineages: correlation between glycosylation and biological activity
Cocktails / Poster Session
Dinner
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Session 3 – Transient Protein ExpressionChairs: Linda Lua, Girish Shah
Georg Schmid - Hoffmann-La RocheParallelized 24-well-plate based transient expression of secreted and membrane protein constructs
Yves Durocher - National Research CouncilStable CHO pools vs large-scale CHO transfection.
Bernie Sweeney - UCB New MedsTransient Platform for Protein Expression
Brian MurphyDeveloping Scalable and Controllable Unit Operations for Allogeneic Cellular Products
Session 4 – Industrial WorkshopChair : Tom Kost
Henry ChiouLife Technologies Any Bass - CellectisSequence-specific nucleases to engineer bioproduction cell lines: achievements and perspectives
Henry George - SAFCNew CHO cell line hosts engineered using zinc finger nuclease technology
Sightseeing-Dinner at Boundary Ranch
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PROGRAM 11TH PEACETUESDAY-SEPTEMBER 24, 2013
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Session 5 – Antibody ExpressionChairs: Michael Butler, Devender Sandhu
Roger Mackenzie - National Research Council CanadaExpression, Engineering and Utility of Single-Domain Antibodies
James Pan - University of TorontoStrategies for rapid identification of functional monoclonal antibodies targeting surface receptors
Shantha Raju - Janssen Research & DevelopmentTerminal Sugars and Their Impact on Antibody Functions
Coffee Break
Adolfo Castillo - Center of Molecular ImmunologyStudy of relationship between Oxidative Metabolism and Antibody Specific Production Rate in insulin and glucose supplemented continuous cell cultures of Recombinant NS0 cell line
Andrew J. Racher - Lonza BiologicsUtilizing Chinese Hamster Ovary Cell Population Genetic/Epigenetic Heterogeneity for the Isolation of New Host Cell Lines with Enhanced Performance
Celine Raymond - Université de MontréalProduction of sialylated antibodies in CHO cells
Meredith Kolpak - PfizerLate Stage Clinical Process Development – Change From NS0 to CHO Host Using Glucose Limitation Technology
Karen Kiplinger - Late Stage Cell Culture - Pharma Technical Development, Genentech, A Member of the Roche GroupImpact of Manganese on Galactosylation of a Recombinant Humanized Monoclonal Antibody Produced in CHO Cell Culture
Lunch
PROGRAM 11TH PEACEWEDNESDAY-SEPTEMBER 25, 2013
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Session 6 – Bioprocessing & Analytical TechnologiesChairs: Amine Kamen, Chris KempLife Technologies Manon Cox - Protein Sciences CorpFlublok, the first FDA approved recombinant influenza vaccineSAFC
James Piret - University of British ColumbiaRaman and microfluidic single cell analysis
Jude Samulski - University of North Carolina at Chapel HillAAV production, WAVE BAG, suspension HEK293 cells and transient transfection: a recipe for success
Masaru Shiratori - Genentech a Member of the Roche GroupProduct quality lessons learned from developing and implementing a chemically-defined CHO platform cell culture process
Linda Lua -The University of QueenslandPurifying Secretory Glycoproteins: Overcoming Common Challenges
Christine Thompson - National Research CouncilDeveloping a Production Process for Influenza VLPs: A Comparison Between HEK 293SF and Sf9 Production Platforms
General Assembly PEIS (Protein Expression International Society)
Cocktails/Poster Session
Gala Dinner
USA
CANADA
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AUSTRALIA
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PROGRAM 11TH PEACETHURSDAY-SEPTEMBER 26, 2013
Session 7 – Stem Cells the FutureChair: Mike Kallos - University of Calgary
Jacques Galipeau - Emory UniversityAutologous mesenchymal stromal cells engineered to secrete therapeutic proteins as part of a synthetic endocrine device: pre-clinical development and roadmap to clinical use
Mick Bhatia - McMaster UniversityHuman Reprogramming of Cell Fate:Implications and Applications
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MONOCLONAL ANTIBODY EXPRESSION
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S-1-1characterization of the antitumor activity of the fc-engineered Human:camelid chimeric Heavy chain antibody eG2-hfc
Calvin D’Eall1,2, Robert Pon2, Maureen Spearman3, Michael Butler3, and Jamshid Tanha1,2
1University of Ottawa, Faculty of Medicine, BMI, Ontario, Canada2National Research Council Canada, Human Health Therapeutics, Ontario, Canada
3University of Manitoba, Faculty of Science, Microbiology, Manitoba, Canada
Eg2-hFc is a small (80 kda) chimeric heavy chain antibody (chCAb), comprised of a human igg1-derived frag-ment crystallisable (Fc) region bivalently linked to EG2: a camelid-derived variable heavy chain (VHH) specific for the tumor-associated epithelial growth factor receptor (EgFr). in-vivo imaging studies have shown Eg2-hFc to demonstrate improved tumor binding and penetrance compared to current therapeutic Abs; however its capacity to mediate tumor-cell lysis is unknown. Through use of the 51Chromium release assay, the lytic capac-ity of Eg2-hFc towards EgFr-expressing tumor cells will be assessed by comparison against the gold standard anti-EgFr antibody Cetuximab. Additionally, conserved Fc associated oligosaccharides will be engineered at key residues that have been linked to augmentation of antibody-dependent cell-mediated cytotoxicity (AdCC) of tumor cells. In this way, we aim to establish optimized glycan profiles for anti-tumor effector function of a camelid:human chCAb. To date, dose response and effector to target ratio studies using several glyco-engi-neered EG2-hFc variants have shown minimal specific lysis (2-20%) of various tumor cells, compared to Cetux-imab (50-95%). The poor lysis observed thus far with the EG2-hFc variants may reflect a reduced capacity to mediate ADCC, and may be correlative to intrinsic affinity, overall avidity, or Fc impairment of the EG2-hFc chCAb. Subsequent characterization of novel Fc-engineered Eg2-hFc variants will help to resolve this issue.
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S-1-2 effect of n-glycosylation on the Structure and function of a Heavy chain Monoclonal antibody
Natalie Okun2, Maureen Spearman1, Trushar Patel2, Markus Meier2, July Dorion-Thibaudeau3, Gregory De Crescenzo3, Jörg Stetefeld2, and Michael Butler1
1 Department of Microbiology, University of Manitoba, Canada, 2 Department of Chemistry, University of Manitoba, Canada, 3 Department of Chemical Engineering, École Polytechnique de Montréal, Canada
Monoclonal antibodies (mAbs) are commonly used as biotherapeutic treatments in cancer and other autoimmune diseases. Engineering these mAbs to have a high affinity for their receptor as well as an increased ADCC (antibody-dependent cell-mediated cytotoxicity) response is crucial for effective biotherapeutic use. One method to increase the affinity and efficacy of the antibody is by altering the N-linked glycosylation pattern in the Fc region (Asn297) using various glycoprotein processing inhibitors in the growth media. These inhibitors include kifunensine, swainsonine, castanospermine and fucosyl transferase 8 inhibitor. The difference in the N-glycosylation pattern can also induce a change in the structure of the antibody and hence impart on it unique characteristics. The antibody used for analysis is an engineered 80 kda chimeric antibody designed with a camelid Fv attached to a human Fc region (Eg2-hFc). This antibody’s small size and lack of light chains makes it an ideal candidate for therapeutic use as it can probe areas too small for the current igg (150 kda) biotherapeutics on the market. This unique structure was viewed by producing a structural model generated by SAxS (small angle x-ray scattering), threading, and homology modelling which was compared to a fully formed igg1 (dp12). due to the position of the glycosylation site, the structural model of the antibody showed how important altering the sugar can affect both the structure and subsequently the affinity of the antibody. This structural effect was analyzed via DLS (dynamic light scattering) and Cd (circular dichroism) with some preliminary functional analysis using iTC (isothermal titration calorimetry) to analyze the change in the affinity that the variously glycosylated antibody have to the Fcγ receptors. it was found that upon alteration of the glycan by glycoprotein processing inhibitors, the secondary structure of the antibodies (Eg2-hFc and dp12) was affected. however, swainsonine had a more prominent effect on the secondary structure of Eg2-hFc while kifunensine had a more prominent effect on the secondary structure of dp12.
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S-1-3 Towards the development of a surface plasmon resonance assay to evaluate glycosylation pattern of antibodies
July Dorion-Thibaudeau1, Céline Raymond2, Yves Durocher2, Gregory DeCrescenzo1
1Department of chemical Engineering, Ecole Polytechnique de Montreal, Canada , 2 NRC Human Health Thera-peutics Portfolio, National Research Council, Montreal, Canada
in the treatment of cancer or autoimmune diseases, monoclonal antibodies (Mabs) injected in the patient must interact with FcY receptors (FcYRs). It has been shown that the glycosylation of Mabs directly influence the antibody-receptor interactions. in order to get a functional drug for the patient, it is necessary to produce Mabs with an appropriate glycosylation pattern. The goal of the project is to evaluate the antibodies according to their glycosylation pattern by their interactions with Fcyrs. First, all four Fcyrs and Mabs were produced by mam-malian cell culture and purified via affinity chromatography. The focus of the presentation will be the development of a routine assay using surface plasmon resonance (Spr) in order to characterize Mab-Fcyr interaction. in order to develop the assay, different approaches have been tested with Cd16a and Cd64 such as the immobilization of the receptors by affinity with His-tag and covalent binding on the dextran surface. Also, the immobilization of the Mab on the surface has been studied. Since the goal is to test different antibodies with the Fcyrs, the immobiliza-tion of the receptor on the surface was prefered. While developing the SPR assay, it was shown that 2% aggrega-tion in the analyte sample will influence the kinetics obtained. Finally, non-glycosylated and high-galactose Mabs were analyzed with Cd16a and have shown different kinetics compared to the model antibody used to develop the assay. As expected, non-glycosylated Mabs do not interact with Fcyr and high-galactose antibodies have shown to dissociate slower than the model antibody.
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S-1-4 novel format antibody expression in cHo cells
Alan DicksonUniversity of Manchester, UK
Antibodies have proven to be commercial successes with exciting therapeutic uses. The next generation of anti-body-related molecules (third generation biopharmaceuticals) are being developed as synthetic molecules with engineering and modifications that generate novel format molecules designed ti have improved functions. More that 50 novel format antibody types are under development. In this presentation, I will briefly discuss the develop-ment and expected advantages of novel formats, explain their manufacturing processes and discuss the challenges faced in turning potential into therapeutic and clinical success.
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S-1-5 Production of an anti-Insulin-Receptor fab fragment from Hybridoma cells
Martina Stützle1, Alina Moll1, René Handrick1 and Katharina Zimmermann1
1 Institute of Applied Biotechnology, University of Applied Science Biberach, Germany
The targeted drug delivery for intranasal applications of CNS active antibodies can be improved by minimizing the antibody size. As a proof of concept an agonistic anti-insulin-receptor specific Fab fragment is generated that may allow modulation of insulin mediated effects on cognition and memory in the brain.
For this purpose we optimized a fermentation process for antibody producing hybridoma cells - which are still of particular interest for diagnostics and research - to exploit fast and sufficient antibody titer. Employing Design of Experiment (doE), the inoculum conditions could be improved by lowering fetal bovine serum (FBS) concentra-tion and supplementing human insulin-like growth factor (igF) and the surfactant pluronic F68. Compared to the original basal medium an improvement in cell growth, viability and antibody titer was achieved and the optimized inoculum conditions were used for subsequent bioreactor fermentations. A novel rapid purification protocol was established for the generation of the corresponding Fab by evaluating different novel anti-kappa light chain affin-ity resins. Initially, the full IgG was captured by Protein A affinity chromatography and processed with papain into two Fabs and the Fc part, testing various antibody, papain and incubation times. Subsequently, undigested antibody and Fc fragments were removed using a second protein A capture step. The Fab fragment was polished by compar-ing Capto L affinity medium (GE) and CaptureSelect anti-kappa (mur, BAC). Employment of the CaptureSelect anti-kappa affinity matrix revealed high yield at highest specificity resulting in >98 % pure protein. Finally, the functional binding of the purified Fab to its antigen (human insulin receptor) was studied by flow cytometry using fluorescently labeled anti-Fab antibodies.
This paper presents DoE as a powerful and efficient tool saving time in process optimization as well as a novel and easy to use protocol for generation and purification of Fabs from full IgGs.
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S-1-6 Glycosylation of antibodies
Shanta Raju, Janssen R&D, USA
Antibodies are serum glycoproteins that play a significant role in the defense mechanism of the immune system. Among antibodies iggs are the major components that are N-glycosylated in the Fc region and these Fc glycans are important for antibody effector functions. however, the N-glycans in the Fc region are highly heterogeneous and their heterogeneity greatly impacts the antibody effector functions. Since recombinantly produced iggs are the major human therapeutics, understanding the structure and func-tions of antibody Fc glycans is very important. This presentation will discuss the importance of analytical methods to evaluate the structure and functions of antibody glycans.
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S-1-7 loading the Inoculant – Indirect feeding of nucleotide Sugar Precursors to affect Glyco-sylation without negative Growth effects
Eric J.M. Blondeel, Marc G. AucoinUniversity of Waterloo
The glycosylation of biopharmaceuticals such as monoclonal antibodies (MAbs) is recognized as a major critical quality attribute (CQA) affecting final therapeutic efficacy. MAb glycans exhibit heterogeneity influenced by perturbations in the substrate supply-chain during cultivation. The direct feeding of nucleotide-sugar precursors in growth medium as a method for influencing glycan patterns has yielded conflicting results and in some cases negative growth effects. leveraging metabolomics and unique feeding strategies, relationships between precursors such as uridine, glucosamine and galactose, as well as their interaction with cells, can provide a clearer perspective of the mechanisms leading to resulting glycoform shifts, as well as the negative growth effects. A design of experiments approach including factorial designs was applied to Chinese hamster ovary (ChO) cells producing the chimeric camelid MAb, Eg2hFc. intracellular and extracellular metabolites were analyzed during and after nucleotide sugar precursor treatment via targeted profiling of 1D-1H-Nuclear Magnetic Resonance (NMR) spectra. This was compared to a detailed glycan analysis of the Eg2hFc MAb.
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S-1-8 expression of recombinant cytoplasmic yeast pyruvate carboxylase alters primary metabolism of cHo cells
Cécile Toussaint1,2, Olivier henry3 and yves durocher 1,2
1département de Biochimie, université de Montréal, Montréal (Qc) h3C 3J7, Canada, 2life Sciences NrC human health Therapeutics portfolio, Building Montreal-royalmount, National research Council Canada,
Montréal (Qc) h4p 2r2, Canada,3département de génie Chimique, École polytechnique de Montréal, Montréal (Qc) h3T 1J4, Canada
ChO cells are widely used to produce therapeutic monoclonal antibody using fed-batch process. in most fed-batch processes lactate and ammonium accumulate in the culture over the time inhibiting cell growth and affecting the product quality. The analysis of the metabolic rates of glucose consumption and lactate formation show that in the presence of non limiting glucose concentration cells consume glucose at a rate much higher than what would be required to maintain cell growth. The use of metabolic flux analysis (MFA) has helped in the understanding of the limitation in the metabolic pathways of continuous cell lines. indeed, MFA showed that most of the pyruvate obtained from the glucose metabolization is diverted to lactate. Therefore, only a small percentage of pyruvate is incorporated into the TCA cycle. One of the enzymes responsible of the connection between the glycolysis and the TCA cycle is the mitochondrial pyruvate carboxylase which converts pyruvate into oxaloacetate. in ChO cells only 5 to 10% of the pyruvate is metabolized through the pyruvate carboxylase. In this paper we propose to modify genetically a ChO cell line producing an antibody with a cytoplasmic yeast pyruvate carboxylase gene. Following selection, survivors are transferred as single cells, according limiting dilution technique, to a second cultivation vessel. Then cultures are expanded and individual clones are evaluated for cytoplasmic yeast pyruvate carboxylase (pyC2) expression. Clones over expressing the enzyme are retained for further cultivation and metabolic analysis. The analysis of the clones overexpressing pyC2 shows that all clones present an altered lactate metabolism compared to the parental cell line. The modifying metabolism obtained may be further exploited to develop a highly performing fed-batch process capable of improving the cell productivity and product quality.
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S-1-9 Varied cHo cell Responses to amino acid limitations in cultures
Navid GhaffariUniversity of British Columbia
ChO cells are widely used to produce therapeutic monoclonal antibody using fed-batch process. in most fed-batch processes lactate and ammonium accumulate in the culture over the time inhibiting cell growth and affecting the product quality. The analysis of the metabolic rates of glucose consumption and lactate formation show that in the presence of non limiting glucose concentration cells consume glucose at a rate much higher than what would be required to maintain cell growth. The use of metabolic flux analysis (MFA) has helped in the understanding of the limitation in the metabolic pathways of continuous cell lines. indeed, MFA showed that most of the pyruvate obtained from the glucose metabolization is diverted to lactate. Therefore, only a small percentage of pyruvate is incorporated into the TCA cycle. One of the enzymes responsible of the connection between the glycolysis and the TCA cycle is the mitochondrial pyruvate carboxylase which converts pyruvate into oxaloacetate. in ChO cells only 5 to 10% of the pyruvate is metabolized through the pyruvate carboxylase. In this paper we propose to modify genetically a ChO cell line producing an antibody with a cytoplasmic yeast pyruvate carboxylase gene. Following selection, survivors are transferred as single cells, according limiting dilution technique, to a second cultivation vessel. Then cultures are expanded and individual clones are evaluated for cytoplasmic yeast pyruvate carboxylase (pyC2) expression. Clones over expressing the enzyme are retained for further cultivation and metabolic analysis. The analysis of the clones overexpressing pyC2 shows that all clones present an altered lactate metabolism compared to the parental cell line. The modifying metabolism obtained may be further exploited to develop a highly performing fed-batch process capable of improving the cell productivity and product quality.
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S-1-10 The Impact of culture pH and Glutamine level on the Glycoform Distribution of eg2-hfc
Hengameh Aghamohseni *, Kaveh Ohadi Hector M. Budman, Jeno M. Scharer, Murray Moo-Young, Raymond L. Legge,
The effect of operating conditions on the glycosylation pattern of secreted human camelid monoclonal antibody (mAb) Eg2-hFc has been evaluated. The experimental results as well as modeled observations are presented.The Chinese hamster ovary cell line, ChO-dxB11 cell line were cultivated at four different levels of initial gluta-mine (0, 2, 4 and 8 mM ) at optimum glucose level of 25- mM in the 500 ml polycarbonate shake-flasks with a 200 ml working volume and an initial cell density of 0.2 x 106 cell/ml. The environmental ph was changed only in the culture with 25 mM glucose and 4 mM glutamine concentrations that were found to be optimal for growth. The ph was maintained at 6.7±0.2 employing two strategies: i) reducing the ph at the start of the batch culture and ii) shift-ing the ph to 6.7±0.2 at time period of peak cell density. glycan structures and distributions were monitored by hydrophilic interaction chromatography (hiliC) followed by exoglycosidase enzyme array digestion. To facilitate the assessment of glycosylation, the relative abundance of glycan structures was grouped together and quantified by glycosylation indices: the galactosylation index (gi), the sialyation index (Si), and the fucosylation index (Fi).The complex biantennary fucosylated glycans with different levels of terminal galactose and sialic acid were observed as the main primary glycan structures in Eg2-hFc. in comparison to the other indices, the gi changed most significantly with culture time. Both the GI and SI levels were higher in cultures with lower initial level of glutamine but the cell growth and mAb concentration were lower. reducing the ph initially resulted in higher Si and gi levels. These results highlight the importance of initial glutamine level on both the rate of glucose con-sumption and the availability of internal nucleotide sugars pool. reducing the culture ph can be a useful method to compensate for the elevated undissociated (toxic) ammonia concentration. it seems that there is an optimal tradeoff between growth and mAb concentration and the desirable glycan profile. The developed comprehensive model was able to predict the glycan profiles of Mab successfully in term of glycosylation indices.
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S-1-11 Study of relationship between Oxidative Metabolism and Antibody Specific Production Rate in insulin and glucose supplemented continuous cell cultures of Recombinant nS0 cell line
Svieta Víctores1, Luis E. Diez2, Adanys Fleites1, Yulier Alvarez1, Rosmery Santana1, Julio Dustet3, Fabiola Pazos2, Adolfo Castillo1
1Early Stage Product Development Direction, Center of Molecular Immunology, Havana, Cuba, 2Faculty of Biol-ogy, Havana University, Cuba, 3Faculty of Chemical Engineering, Technical High School “ Jose. A Echeverría”,
Havana, Cuba.
Among mammalian cell lines, NS0 is one of the most used for monoclonal antibodies (mAb) production. Culture medium optimization and characterization of cell metabolism had contributed to increase cell growth, reaching higher specific products concentration and gross productivity. In particular, strategies based on manipulation of signal pathways that influence cell growth and survival, energy-consumption and protein synthesis have been addressed. Use of continuous cell culture allows the establishment of well-defined steady states that is determinant to evaluate the relationship between the concentration of substances inside the bioreactor and biological reaction rates. The goal of this work is to study the relationship between the oxidative metabolism and antibody specific production rate in insulin and glucose supplemented continuous cultures of NS0/hr3h7 cell line expressing anti-EgFr igg1 humanized mAb (Nimotuzumab).
Cells were cultured in 2-4 l stirred tank bioreactors using a basal medium developed in-house. glucose concentrations in the feed ranged from 17 to 50 mmol /L, with or without insulin supplement. Levels of mTOR, Akt, eIF2α and gluT1, gluT4 were detected by FACS and western blotting. Enzymatic activities of hk, g6pdh, pFkFB3, ldh, pyk, glutaminase and AlAT were measured. glucose and lactate concentrations were determined by using an ySi analyzer. Adp/ATp ratio was measured by bioluminescence assay. The extracellular igg was assayed by sandwich EliSA. intracellular igg and apoptosis levels were detected by FACS.
Obtained results showed that NS0 cells increase twice specific mAb production rate and reduce the fraction of apoptotic cells in glucose and insulin supplemented medium. An enhancement in glucose transporters, mTOr, Akt and eIF2α expression, as well as increase activity of G6PDH, PYK and ALAT is detected. The addition of insulin induces a 2-fold increase in glucose uptake; meanwhile the residual lactate remains constant and ldh activity is reduced. A mechanistic model that explains the effect of insulin and glucose additions in modifications of energetic metabolism and expression capacity of recombinant mAbs in NS0 cell line involving cellular pathways for cell survival, protein biosynthesis and glucose metabolism will be discussed.
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S-1-12 The changing Dielectric Properties of cHo cells can be Used to Determine early apoptotic events in a bioprocess.
Katrin Braasch1, Marija Nikolic-Jaric2,3, Tim Cabel2, Elham Salimi2, Ashlesha Bhidea2, Bahareh Saboktakin Rizi2, Kaveh Mohammad2, Douglas J. Thomson2, Gregory E. Bridges2 and Michael Butler1
1 Department of Microbiology, University of Manitoba, 45 Chancellor Circle, Winnipeg2 Department of Electrical and Computer Engineering, University of Manitoba,
3 HIECO Ltd., May-Ruben Technologies
Cell density and viability are two important parameters that are monitored during a mammalian cell bioprocess. The predominant method used to determine the viability of a cell population is trypan blue exclusion. This marks the loss of viability by the inability of cells to exclude the dye as a result of membrane damage. however, the loss in cell membrane integrity can be regarded as a late-stage event in the demise of cells and often marks the end of apoptosis. Early indicators of apoptosis are desirable in cell bioprocesses because the initial stages are reversible by appropriate intervention such as nutrient feeding. This paper presents a prototype dielectrophoretic (dEp) cytometer developed at the university of Manitoba to analyze individual ChO cells subjected to a radiofrequency actuator and detector in a narrow bore capillary. The focus of this paper is the study and comparison of the dEp cytometer and four different and independent methods for the determination of viable and/or total cell density as well as cell viability. These included the particle counter (Coulter Counter), image analyser (CEdEx with trypan blue exclusion), an on-line capacitance probe (Aber-Instruments) and an off-line flow cytometer (Guava Millipore). The multiple techniques of cell monitoring were applied to the batch and perfusion culture of a novel ChO cell line producing a human-llama chimeric antibody (Eg2) grown in a bench-top bioreactor. Our results show that cell samples during the course of a bioreactor run show distinct shifts in the dielectric properties corresponding to loss of cell viability. Discrete cell sub-populations can be identified during apoptosis and can be correlated with alternative measurements by fluorescent markers, a cell population-based capacitance probe and adenylate energy charge. This opens up the possibility of monitoring the incremental changes that occur during cell growth and death in bioreactors to allow better and more in depth process monitoring as well as process control.
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S-1-13 Synergetic effect of glucose-dependent metabolic pathways on productivity and glycosylation of a heavy chain monoclonal antibody in chinese hamster ovary cells
Venkata S. Tayi, Carina Villacrés Barragán, Bo Liu and Michael Butler
Department of Microbiology, University of Manitoba,
objective: To investigate the synergetic role of glucose-regulated energy metabolism, nucleotide sugars synthesis and glycan precursors’ assembly on productivity and glycosylation of a heavy chain monoclonal antibody in Chinese hamster ovary cells.
Methods: Cells were cultured in in 3l Applikon bioreactor in continuous mode at a dilution rate of 0.6/day. The media of different glucose concentrations were fed and effluent is removed with a pump controlled by liquid level sensor to reach different steady states. The reactor was maintained at 37oC, pH 7.2 and 40% DO throughout the run. The samples were taken every day and/or at each steady state for the analyses.
Results: For the four steady states examined, the residual glucose concentrations in the bioreactor were maintained at 4.25, 0.45, 0.1 and 0.06 mM (corresponding glucose uptake rates are 5.9, 4.7, 2.1 and 1.2 µmoles/106 cells.day, respectively). The 5-fold decrease in available glucose made the cells to shift towards energy efficient metabolism by reducing their lactate production by 2.5-fold. however, ammonia production rate was increased by 2-fold. The increase in ammonia was resulted from 5-fold reduction in alanine production while maintaining similar glutamine and asparagine consumption rates. The cell specific productivity of mAb was reduced by 35% and the galactosylation index was decreased from 0.72 to 0.51. The 5-fold reduction in glucose uptake rate resulted in 2.5-fold reduction in intracellular levels of gdp-mannose and corresponding decrease in dol-p-glcNAc2-Man5 lipid-linked oligosacharides. But the intracellular udp-galactose levels were not affected indicating that the availability of udp-galactose was not the limiting factor for the reduction in galactosylation. The increase in intracellular ammonia might have decreased the galactosylation process by mis-translocating the galactosyltransferases in golgi apparatus.
conclusion: Though limited glucose supplementation is needed to reduce the lactate accumulation, care should be taken to provide optimum glucose amounts for the production of mAbs with high galactosylation. The optimum glucose level should facilitate the synthesis of sufficient amounts of nucleotide sugars and lower the NH3 generation from amino acid metabolism while producing acceptable level of lactate.
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S-1-14 antibody effector functions - impact on product development and strategies for adjustment.
Volker Sandig, ProBioGen AG
Antibody effector functions, mainly antibody dependent cellular cytotoxicity (AdCC) and Complement dependent Cytotoxicity (CdC) contribute to the therapeutic effect and can mediate side effects of therapeutic antibodies
Since it has been discovered that antibody effector functions not only depend on the immunoglobulin class but also on the cell type used for manufacturing and finally the Fc glycan was identified as the primary structural course a broad range of technologies has evolved to control and modulate this mechanism.
We have not only introduced enzymes to enhance galactosylation and sialalyation but also identified enzymes that interferes with a natural synthesis pathway and blocks synthesis of the monosaccharide fucose.
This presentation will give an overview and historic perspective about the impact and ways to control antibody effector functions. it will also discuss metabolic intervention in detail.
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S-1-15 capture and Recovery of Immunoglobulin G withWeak cation exchange Membranes
Katharina J. Hassel1 and Christine Moresoli 1
1Department of Chemical Engineering, University of Waterloo, Canada
The objective of our work is to evaluate a novel high binding capacity weak cation exchange membrane material produced by NATrix SeparationsTM for the capture and recovery of monoclonal antibodies (mAbs). MAbs are of great interest, as pharmaceuticals in cancer therapy, due to their high affinity towards specific antigens.
improving mAb downstream processes is of major importance to reduce mAb production costs. Traditionally, Protein-A affinity resin chromatography has been used as the initial mAb capture step from the cell culture medium. The Protein-A ligand has an excellent specificity for mAbs but is costly and may be subject to leaching. Resin chromatography is well established but limited by pore diffusion mass transfer and significant pressure drop. Alternative ligand materials and alternative purification systems are desired.
A weak cation exchange membrane material, with a functionalized carboxylic acid polymer matrix, represents a promising alternative to conventional protein-A resin chromatography. Membrane material properties such as ionization, swelling and pore size can be controlled according to ionic strength and ph conditions.
in this presentation, membrane characteristics according to different ph and ionic strength conditions will be reported. Our emphasis has concentrated on membrane charge characteristics obtained via streaming potential and swelling potential. Results show that pH has a significant influence while ionic strength has a moderate influence on these membrane material characteristics. Static binding capacity and elution recovery behavior of a model antibody, Immunoglobulin G (IgG), will also be discussed according to specificity and the effect of process ionic strength and ph required for maximum capture. Our results indicate that ph and ionic strength significantly influenced binding whereas elution is primarily regulated by pH.
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Keynote: Synthetic biology: from parts to modules to therapeutic systems
Ron Weiss, Massachusset Institute of Technology, USA
Synthetic biology is revolutionizing how we conceptualize and approach the engineering of biological systems. Recent advances in the field are allowing us to expand beyond the construction and analysis of small gene networks towards the implementation of complex multicellular systems with a variety of applications. in this talk i will describe our integrated computational / experimental approach to engineering complex behavior in mammalian cells. in our research, we appropriate design principles from electrical engineering and other established fields. These principles include abstraction, standardization, modularity, and computer aided design. But we also spend considerable effort towards understanding what makes synthetic biology different from all other existing engineering disciplines and discovering new design and construction rules that are effective for this unique discipline. We will briefly describe the implementation of genetic circuits and modules with finely-tuned digital and analog behavior and the use of artificial cell-cell communication to coordinate the behavior of cell populations. The first system to be presented is a genetic circuit that can detect and destroy specific cancer cells based on the presence or absence or specific biomarkers in the cell. We will also discuss preliminary experimental results for obtaining precise spatiotemporal control over stem cell differentiation for tissue engineering applications. We will conclude by discussing the design and preliminary results for creating an artificial tissue homeostasis system where genetically engineered stem cells maintain indefi-nitely a desired level of pancreatic beta cells despite attacks by the autoimmune response, relevant for diabetes.
PEACe Lecture
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cell enGIneeRInG I:TRanScRIPTIon & TRanSlaTIon
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T-1-1 Systematic Analysis and Quantification of Gene Design Variables and their Effect on Heter-ologous Protein expression
Claes Gustafsson and Mark WelchDNA2.0 Inc.
Due to the degeneracy of the genetic code a protein can be encoded by >10100 different nucleotide sequences. The dNA sequence used to encode a polypeptide can have dramatic effects on its expression (up to 1,000 fold in mammalian cells). until recently researchers have been limited either to using naturally occurring sequences, or to make a single new version in which an alternative dNA sequence is used to encode the same amino acid sequence. Accordingly, evidence to support any variable-specific hypothesis has been sparse, anecdotal and biased in favor of experiments which yielded positive (and therefore publishable) data. We here will illustrate how modern syn-thetic biology tools, design of Experiment (doE), cloud computing and big data analysis software can be used to model, predict, and experimentally validate the sequence-function correlation between a gene’s primary nucleotide sequence and key quality attributes (e.g. desired function, expressibility, solubility) thereby enabling Quality by design (Qbd) very far upstream of the manufacturing process. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches and highlight some of the surprising and promising results emerging from the developing field of gene engineering. Several unpublished case studies will be used to illustrate the technology.
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T-1-2 Small non-coding Rnas – pathway engineering of cHo cell factories that avoids translational burdening
Matthias Hackl1, Vaibhav Jadhav1, Nicole Borth1,2, Johannes Grillari1,3
1Department of Biotechnology, BOKU – VIBT University of Natural Resources and Life Sciences Vienna, Austria2 ACIB GmbH,Austrian Center of Industrial Biotechnology , Graz, Austria.
3 Evercyte GmbH, Vienna, Austria
Chinese hamster ovary cells are among the most important cell factories for the production of biopharmaceuticals. Only recently genomic, mrNA, as well as mirNA sequence information has become public. MirNAs are regulating translation of a wide range of mrNAs, whereby any individual mirNA targets up to several hundred mrNAs and thus globally regulates cellular behavior similar to transcription factors. however, since mirNAs are not translated they do not add to the translational burden of the cell factories. Therefore, we propose that mirNAs are of high interest for cell engineering (‘engimirs’).We have recently sequenced and annotated the ChO mirNome by next generation sequencing and alignement to currently available ChO genomes, have developed a medium-throughput screening system to identify engimirs and have given proof-of-principle that mirNAs indeed are able to improve biotechnologically relevant characteristics of CHO cells. Finally, we will suggest that other small non-coding RNAs, specifically piRNAs, might be used in biotechnological applications as well.
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T-1-3 Efficient expression and delivery of tumour selective therapeutic proteins
Mahvash Tavassoli1, Jessica Bullenkamp1, Joop Gaken2, Jie Jiang2
1Department of Molecular oncology and 2Haematolgical Medicine, King’s College London.
despite major advances in our understanding of the molecular pathogenesis of cancer and intensive efforts on developing more effective drugs, many cancer types remain hard to treat with standard modalities such as chemo- and radiotherapy. Additionally, standard chemo-radiation often results in collateral damage to healthy tissue, causing unwanted side effects. Therefore researchers have been working diligently to develop drugs that target the processes of cancer cells specifically and leave healthy cells unharmed. The best target for therapy is a molecule or pathway that is present in cancer cells and absent in normal cells to ensure that the therapy will only attack cancer cells. However, it is often difficult to find targets that are present only in cancer cells, partly because cancer cells evolve from normal cells.
In recent years a class of cellular or viral derived proteins have been identified which show tumour selective cytotoxicity independent of what pathways and targets cause the cancer. Although the mechanism for such a biased effect is currently unknown, this inherent selectivity offers important potential for improved cancer therapeutics.
For the past 10 years we have been investigating the therapeutic potential of the CAV (chicken anemia virus) derived protein, Apoptin, which has the ability to induce p53-independent apoptosis in a variety of human cancer cells while leaving normal cells unharmed. We have achieved effective killing in many cell lines and tumour xenografts by expressing the protein from lenti and adenovirus vectors. To circumvent the potential harmful effects of viral delivery we generated recombinant Apoptin protein and delivered this into cells using protein transduction peptide (pTd) of TAT, which penetrate the cell membrane. Although the protein was delivered and was functional, the majority of the proteins delivered by this means were trapped and subsequently degraded in the endosomes-lysosomes of recipient cells limiting its clinical potential. We therefore developed a TAT-Apoptin secretable strategy where the resistant cells produced Apoptin which was then taken up by the sensitive cancer cells in a co-culture assay. One of the main clinical applications of this strategy is for purging the bone-marrow (BM) from leukemic cells in autologous BM transplantation used for the treatment of multiple myelomas.
Furthermore we have constructed a synthetic, codon optimised Apoptin derived from a recently discovered human variant of CAV and were able to show that this protein, although less than 50% homologous shows a similar tumour specific induction of apoptosis as CAV Apoptin.
In addition we have identified domain/s of the Apoptin protein which, while small enough to be efficiently deliverable by nano-particles or by fusion to TAT-pTd, would maintain the tumour selective, anticancer function. The rationale for such drugable region/s arose from our discovery that Apoptin binds to an important cellular kinase called protein kinase C (pkC) (Jiang et al., Cancer research 2010). We have therefore focused on the regions which are important for Apoptin/pkC interaction. A panel of Apoptin deletion mutants were expressed and tested by immuno-precipitation, proximity ligation assay (plA) and Fluorescence resonance Energy Transfer (FrET), life imaging technique, for their ability to interact with pkC. The apoptin fragments which are able to interact have been tested for the ability to induce cell death.
data on the in vitro effect of these cytotoxic fragments will be presented.
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T-1-4 engineering of mToR signalling in cHo cells for enhanced Recombinant Protein Production
Andrew Dean, Mark SmalesDepartment of Biosciences, University of Kent, Kent, UK
here, we address a key area that underpins recombinant protein synthesis yields from mammalian cells in culture: investigating how a global regulator of protein synthesis, mTOr (mammalian target of rapamycin), contributes to recombinant protein synthesis and devising new ways to manipulate this process to enhance recombinant protein yields. Mammalian target of rapamycin (or mTOr) is a master regulator of cell growth, proliferation (and hence biomass) and of protein synthesis, but the contribution of this master regulator to recombinant protein synthesis, and whether it can be manipulated to enhance growth and product levels, remains to be fully established. during the process of polypeptide elongation, elongation factor 2 (eEF2) is a key control point that regulates protein synthesis via its de/phosphorylation. phosphorylation of eEF2 results in its inactivation; slowing or halting elongation and hence attenuating protein synthesis. phosphorylation of eEF2 is regulated by a number of mechanisms, but particularly via the mTOr (mammalian target of rapamycin) signalling pathway. mTOr consists of two complexes, mTOrC1 and mTOrC2; of these mTOrC1 is responsible for eEF2 regulation. mTOrC1 responds to a number of environmental stimuli and signals including shear stress, amino acid depletion, and oxidative stress. its kinase activity activates S6k1 which in turn modulates elongation factor 2 kinase (eEF2k) activity. eEF2k directly regulates eEF2 by phosphorylating Thr56 of eEF2 deactivating it. To date we have constructed stable Chinese hamster ovary (ChO) cell lines over expressing eEF2 and mutated variants: Thr56 Ala and Thr56glu, all with and without a V5 tag. We have also engineered ChO cells by knockdown of the kinase, eEF2k, which phosphorylates eEF2. In each of these cell lines we have assessed cell growth and recombinant protein expression. By profiling these cell lines we are defining the role of eEF2 in determining recombinant protein production and the regulation of eEF2 via mTOr. These studies have also been extended to the manipulation of eiF2s regulators, to determine the influence upon rP production and cell growth.
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T-1-5 Large-scale screening identifies a novel microRNA, miR-15a-3p, which induces apoptosis in human cancer cell lines
Aliaksandr Druza,b, Yu-Chi Chenc, Rajarshi Guhac, Michael Betenbaughb, Scott Martinc and Joseph Shiloacha*
a Biotechnology Core Laboratory NIDDK, NIH Bldg 14A Bethesda, Maryland 20892b Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore Maryland
21218c NIH Chemical Genomics Center, National Center for Advancing Translational Sciences
MicrorNAs (mirNAs) have been found to be involved in cancer initiation, progression and metastasis and as such have been suggested as tools for cancer detection and therapy. in this work a large-scale screening of the complete mirNA mimics library demonstrated that hsa-mir-15a-3p had a pro-apoptotic role in the following human cancer cells: hela, Aspc-1, MdA-MB-231, kB3, ME180, hCT-116, and A549. Mir-15a-3p is a novel member of the pro-apoptotic mirNA cluster, mir-15a/16, which was found to activate Caspase-3/7 and to cause viability loss in B/CMBA cells during preliminary screening. Subsequent microarrays and bioinformatics analyzes identified the following four anti-apoptotic genes: bcl2l1, naip5, fgfr2, mybl2 as possible targets for the mmu-miR-15a-3p in B/CMBA cells. Follow-up studies confirmed the pro-apoptotic role of hsa-miR-15a-3p in human cells by its ability to activate Caspase-3/7, to reduce cell viability and to inhibit the expression of bcl2l1 (bcl-xL)
in hela and Aspc-1 cells. Mir-15-3p was also found to reduce viability in hEk293, MdA-MB-231, kB3, ME180, hCT-116, and A549 cell lines and, therefore, may be considered for apoptosis modulating therapies in cancers associated with high Bcl-xl expression (cervical, pancreatic, breast, lung, and colorectal carcinomas). The capability of hsa-mir-15a-3p to induce apoptosis in these carcinomas may be dependent on the levels of Bcl-xl
expression. The use of endogenous inhibitors of bcl-xL and other anti-apoptotic genes such as hsa-mir-15a-3p, may provide improved options for apoptosis-modulating therapies in cancer treatment compared with the use of artificial antisense oligonucleotides.
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cell enGIneeRInG II: PHySIoloGy & MeTabolISM
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T-2-2 Metabolic profiling: Moving from design of media and feeds to strategies for cell engineer-ing and synthetic biology
Alan Dickson, The MIchael Smith Building, Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK
Tremendous strides have been made in development of platform processes for using Chinese hamster Ovary (ChO) cell lines to produce recombinant therapeutic proteins. Much of this was achieved be development of improved media and feeds, alongside careful selection of ChO cell clones. The details of the biology that underlie enhanced production - through increased cell numbers, more prolonged (efficient) survival in culture or enhancement of specific productivity - remain unclear. We selected to interrogate the metabolic profiles associated with effective production yield, using batch culture phase, medium formulation and feed regimes as models to develop a system-level understanding of regulatory events that enhance product yield. This has involved development of approaches to harvest physiologically-valid intracellular metabolites and analytical methodss for wide-scale metabolite assessment. This approach, in particular changes in response to feeding regimes, has identified metabolic reactions associated with determination of product yield. By assessment of the timing and composition of feed addition, it is possible to rationalise how cells might be engineered - metabolically or genetically - to generate improved performances in bioprocessing. Whilst such engineering may have relevance for determination of recombinant protein yield, consideration also need to be given to the consequences of engineering to the cell system (e.g. changing glycosylation or use of other metabolically-derived substrates for post-translational modifications. As we move towards the potential for synthetic biology and development of "designer ChO host" cells, we need to take heed of the functional information that arises from metabolic profiling (and other "omics approaches) to ensure that the synthetic biology approach takes into account the multi-level interactions within the cell. i will discuss how our specific data, and its interpretation can be used and why metabolic profiling presents a very powerful approach to dissect cell functional relationships.
The work presented was funded by the Biotechnology and Biological Sciences Research Council (BBSRC, through the Bioprocessing Research Industry Club, BRIC; BB/E005985/1), the European Regional Development Fund (ERDF) and the Norwest Regional Development Agency (NWDA). We thank Lonza Biologics for cell lines.
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T-2-3 cell lines for biosimilars and biobetters: closest match and improved performance by selection and molecular design.
Karsten Winkler1, Hans Henning von Horsten1, 2, Thomas Rose1, Susanne Seitz1 and Volker Sandig1
1ProBioGen AG, Berlin, Germany, and 2Hochschule für Technik und Wirtschaft Berlin, Germany
With increasing information on genome, transcriptome and metabolome of commonly used production cell lines, engineering becomes an increasingly popular approach to achieve desired product attributes, growth behavior and nutrient consumption. Tools range from feeding intermediate metabolites, overexpression or deregulation of key enzymes of a pathway to knock-out and rNA silencing. While conceptionally simple, the latter approaches are either labor intensive or costly to apply at large scale.
Aiming at glycan modulation we added another principle to this toolbox: enzymatic deflection of a biochemical pathway. A bacterial enzyme is used to redirect synthesis towards a heterologous activated hexose that cannot be utilized by the cell resulting in depletion of the natural pathway. To our surprise, even lowest level expression of the enzyme completely abolishes fucose synthesis in stably modified cells. We will provide further insights into the mechanism that explain this effect.
Besides cell lines for new high potency drugs cell engineering approaches have also merit for biosimilar development. We will show that modulation of glycans and secretion apparatus can overcome the gap between the narrow range of structural and functional requirements associated with the original molecule and a modern, more efficient production process.
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T-2-4 Development of a novel eR Stress based Selection System for the Isolation of Highly Productive clones
Lars Kober1, Christoph Zehe1 and Juergen Bode2
1Cellca GmbH, Uhlmannstrasse 28, 88471 Laupheim, Germany2Experimental Hematology, Hannover Medical School (Hans-Borst Center for Heart and Stem Cell Research),
Hannover, Germany
Most biotherapeutic drugs are recombinant monoclonal antibodies which are mostly produced in monoclonal cell lines derived from Chinese hamster ovary (ChO) cells. Various stably transfected clones expressing a monoclonal recombinant antibody were subjected to a fed-batch experiment and analyzed, in parallel, by real time rT-pCr. Thereby a correlation of the antibody concentration and the relative mrNA level of calreticulin (CAlr), glucose-regulated protein 78 kda and 94 kda (grp78, grp94) was observed. By means of these results we were motivated to establish a novel selection system based on endoplasmic reticulum (Er) stress, which allows the rapid identification and isolation of high-expressing clones out of a pool mainly consisting of low- and medium-producing cells. For this purpose, Er stress promoter reporter constructs were introduced by recombinase mediated cassette exchange (rMCE) in antibody expressing “reporter” and “Mock” cell lines. unexpected at first, only the GRP78 reporter construct revealed a strong stimulation upon antibody expression. Using ER stress inducing reagents it could be demonstrated that the grp78 Er stress reporter was not yet maximally stimulated by the expression of 1.6–1.9 g/l antibody. hence, it can be anticipated that it should be possible to detect clones with significantly higher productivities with this construct. Furthermore clones, isolated after a standard cell line development procedure employing an antibody expression construct, showed a strong correlation between the amount of secreted antibody and the measured GFP fluorescence. Based on these results, it is concluded, that the novel Er stress based selection system is suitable to identify and isolate clones with a high level of antibody expression.
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T-2-5 Gamma-Glutamyl carboxylation of Vitamin K-Dependent Proteins: Strategies to address the Post-Translational bottleneck
Sampath Kumar, John Kulman, Bob Pape, Lily Zhu, Kai Chen, Baisong Mei and Robert Peters
Biogen Idec Hemophilia, 9 4th Avenue, Waltham MA, USA
Vitamin K dependent (VKD) proteins are a small group of proteins with fewer than twenty members identified to date. While these proteins play roles in diverse physiological functions several of them play crucial role in hemostasis and have been, or are being, currently developed as human therapeutics. The Vkd proteins are characterized by a unique post-translational modification, gamma-glutamyl carboxylation, which converts specific Glu residues into gamma-carboxylated Glu, or Gla. Cellular capacity for this modification has frequently been found to be limited while expressing Vkd proteins in recombinant form. Thus glutamyl carboxylation is a bottleneck to achieving high productivity bioprocesses for VKD therapeutic proteins. The modification involves multiple enzymes, of which two have been identified thus far. Cell engineering efforts have conferred cells a moderately enhanced capacity for the modification but the extent of the benefit is apparently limited by unknown protein(s) involved in the glutamyl carboxylation process.
The present study is a prelude to our long term efforts to better understand glutamyl carboxylation, and engineer improved host cell lines for the production of Vkd proteins. The objective of this study was to identify a host cell line that possesses relatively high glutamyl carboxylation capacity. We employed the novel protein Z-luciferase reporter protein method developed by Souri, et al. (2009) and compared the glutamyl carboxylation capacities of ChO.k1SV, hEk293, Bhk, hepg2 and Sk-hep1 cell lines. Among these cell lines, hEk293 showed the highest capacity followed by CHO and BHK in line with the findings of Berkner, 1993. The potential molecular mechanisms that underlie these differences, such as mrNA abundance and gene copy number, are currently being investigated.
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T-2-6 Diving into the metabolism of a chinese hamster ovary cell
Nicola Beaucamp, Oliver Popp, Wolfgang Paul and Ingo Gorr
Roche Diagnostics GmbH, Pharma Research and Early Development (pRED), Large Molecule ResearchDepartment of Cell Culture Research, Nonnenwald 2, 82372 Penzberg
Mammalian ChO (Chinese hamster ovary) cells are widely used for the biotechnical production of therapeutic proteins. Early identification of robust CHO high producer clones that fit to the respective production platforms is prerequisite for a fast technical development phase and a subsequent successful market supply with therapeutic proteins.
Roche developed a proprietary, chemically defined, fully up- and downwards compatible media/feed platform for ChO manufacturing cell lines. A major goal was to overcome well known lot-to-lot variations of complex media components. Special focus was laid on the consistency of excellent protein quality throughout process phases and on high expression titer. The new platform was planned to be suitable for standard antibody molecules as well as for more complex antibody derived molecules e.g. bispecific antibodies.
during development of the platform we further improved our knowledge on cell metabolism by comprehensive and high throughput analysis of medium components and cellular metabolites. in depth metabolic cell analysis was enabled by the design of an in silico metabolic network model and multivariate statistical analysis of experimental results. Benefits from these approaches for better understanding metabolism of high producer cell lines were translated into the concept and generation of the platform. The success of the platform and our metabolic approach was proven in several of our portfolio projects already by developing complex antibody and antibody-like bispecific formats.
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T-2-7 comparative study of recombinant equine chorionic Gonadotropin (ecG) expressed in three different mammalian cell lineages: correlation between glycosylation and biological activity
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Coelho, TM1; Carreira, ACO1; Butler, M2 and Sogayar, MC1.1- Cellular and Molecular Therapy Centre - Biochemistry Department, Chemistry Institute, University of Sao
Paulo – SP – Brazil
2- Animal Cell Technology - Microbiology Department, University of Manitoba – MB - Canada
The gonadotropins constitute the most structurally complex hormone family, composed by heterodimeric proteins with a common subunit and a hormone-specific subunit. The equine chorionic gonadotropin (eCG) b subunit is coded by the same gene as the equine lutheinizing hormone (elh). Although these gonadotropins share the same aminoacid sequence, they differ in their biological activity as well as in the types and complexity of their glycans. While eCG is highly glycosylated, with 45% of its molecular weight corresponding to oligosaccharide chains terminated by sialic acid, elh has a lower amount of less complex glycans, terminated mainly by sulfated N-acetylgalactosamine. Although mammalian expression systems can be used to produce glycosylated proteins, great variability is found in the glycosylation patterns of the recombinant proteins they generate, since the enzymatic machinery for oligosaccharides addition differs from one cell type to the other, generating specific glycoisoforms according to the cell lineage. We present a comparative study of cloning and expression of the eCg cdNA in three different mammalian cell lines in order to assess their expression levels and attached glycans structure and biological activity. Methodology: in order to perform this study, we have cloned the cdNAs corresponding to both chains of eCg gene in an expression vector which contains a selective mark. plasmid preparations of this vector were transfected into the three cell lines and cell clones and populations were isolated. The reCg expression was determined and the in vitro and in vivo biological activities of the supernatants containing reCg were assayed. The purification of the reCG from the supernatants as well as glycosylation analysis of the obtained gonadotropins is underway. results: several cellular clones and populations were obtained for the three cellular lineages and it was possible to compare these lineages in terms of facility of clones obtaining, survival after the selective process, recombinant protein expression and biological activity. glycan analysis is being performed in order to establish a parallel between the biological activity and the complexity and amount of glycans attached by each cell line. Conclusions: The cell line A expresses larger amounts (iu/ml) of reCg, with high in vivo biological activity. The cell line B allows the isolation of a larger number of cell clones, with average reCg expression. The cell line C is the hardest to transfect and generates lower amounts of cell clonesdisplaying low reCg expression.
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T-3-1 Parallelized 24-well-plate based transient expression of secreted and membrane protein constructs
Georg Schmid, Hoffmann-La Roche, pRED Pharma Research & Early Development, Discovery Technologies, CH-4070 Basel, Switzerland
We have established a parallelized 24-well-plate based expression platform for performing construct screening and evaluating expression conditions for secreted and membrane proteins in transient transfections.
The platform’s throughput is suitable for handling several 10s to 100s of constructs or conditions. 24-well-plate cultures are compared to spin tubes, shake flasks and bioreactor expressions. Coupling the system to a Guava FACS analyzer equipped for handling 96-well-plates allows for quantification of secreted proteins via bead-based technology and of membrane proteins via gFp tag or antibody labeling. Solubilized membrane protein quality is then additionally analyzed by fluorescent SEC (FSEC).
Selected results and solutions to technical challenges will be presented for cell culture and FACS analysis including the assessment of the following factors (general expression parameters for deepwell plates, post-transfection conditions, transfection reagent, host cells, different vectors, overall reproducibility, cell aggregation issues, etc.).
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T-3-2 Stable cHo pools vs large-scale cHo transfection.
Yves Durocher, National Research Council, Canada
We will present data describing our new platform that allows the generation of stable ChO pools capable
of producing up to 200 mg/l (9 days batch) or 700 mg/l (14 days fed-batch) of monoclonal antibodies
in less than 40 days post-transfection. This platform represents a viable alternative to large-scale ChO
transfection at providing gram quantities of r-protein candidates needed for therapeutics development
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T-3-3 Transient Platform for Protein expression
Bernie Sweeney, UCB New Meds, UK
development of antibody therapeutics, from early-stage research through to preclinical and clinical
development, requires ever-increasing amounts of antibody. in order to meet this challenge, here at uCB
we have developed a transient system capable of delivering reagents for all our in-house preclinical
requirements using an engineered ChO host cell line and optimized transfection methodology, media
and culture conditions. My talk will be based around the development of tour platform and the on-going
work to optimize the system further to achieve even higher titres and throughput.
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T-3-4 Developing Scalable and controllable Unit operations for allogeneic cellular Products
Brian Murphy, Director, Celgene Cellular Therapeutics, Warren, NJ 07059Protein Expression in Animal Cells Conference, September, 2013
Successful commercialization of cell therapy products will require development of robust and reproducible processes that yield consistent, high-quality products in a cost-effective manner. in order to avoid process failures, products that don’t meet release criteria, and inconsistent product quality, it is essential to invest, throughout the product develop-ment life cycle, in intense process characterization efforts to understand the effects of input parameters on product quality attributes. Selection of unit operations that enable process control and product homogeneity will facilitate process development towards these goals. These types of unit operations are typically amenable to scale up instead of scale out, which also reduces cost of goods and increases overall process capacity. Some unit operations that the cell therapy field is moving towards for the production of allogeneic cellular products include microcarrier-based bioreactors for cell expansion, tangential flow filtration and continuous centrifugation for cell harvest and buffer exchange, and cryopreservable vials for final product storage. Examples will be given as to how to apply solid engi-neering principles to properly develop and scale up these unit operations and how a deep understanding of the pro-cess science behind these unit operations can help with process characterization efforts and ultimate process success. Selection of unit operations that enable process control and product homogeneity will facilitate development of robust, reproducible processes yielding consistent, high-quality products in a cost-effective manner. Examples will be given as to how to apply solid engineering principles to properly develop and scale up unit operations such as microcarrier-based bioreactors, tangential flow filtration, and filling/freezing in cryopreservable vials.
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InDUSTRIal WoRKSHoP
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T-4-2 Expression, Engineering and Utility of Single-Domain Antibodies
Andy BassCellectis
recombinant protein production is a multiple step process involving transcription, translation, post-translational modification pathways, and finally secretion. Targeting these pathways to increase recombinant protein expression is the core principle of engineering high producing cells. Though cell engineering yielded some results in improving cell productivity, this approach was so far limited by the lack of control over gene manipulation. The recent ability to produce sequence-specific nucleases now opens unprecedented possibilities to manipulate enzymatic, transport and regulatory functions of the cells by specifically modifying genes at the single-copy level. This session will present the current and future applications of the sequence-specific nucleases TALEN? and Meganucleases to engineer improved production traits of mammalian cell lines.
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T-4-3 Expression, Engineering and Utility of Single-Domain Antibodies
George HenrySAFC
Zinc Finger Nucleases (ZFNs) are a class of engineered dNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations. Double-strand breaks are used to facilitate site-specific mutagenesis by stimulating the cell’s natural DNA-repair processes. These cellular repair processes can be harnessed to generate precisely targeted genomic edits resulting in cell lines, including somatic cell lines, with targeted gene deletions (knock-outs), integrations (knock-ins), or modifications.
We have conducted significant research and development work to deploy ZFN technology across biopharmaceutical applications. SAFC has created several new Chinese Hamster Ovary (CHO) cell lines with modifications in specific genes of interest. Example cell lines include CHO GS-/- and CHO dhfr-/- modifications that allow for more efficient selection of high r-protein producing cell lines. This talk will open with an overview of the ZFN technology and uses for ChO cell line engineering applications. in addition, we will focus on current research activities to create CHO cell lines with additional gene specific modifications within the N and O linked glycosylation pathways. Such cell lines lead to the production r-proteins with a specific and tailored glycan profiles. in one such example, we have created a gS/Mgat1 disrupted (ChO) cell line for the production of recombinant glycoproteins with mannose as the predominant (>95%) N-linked glycosylation species.
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anTIboDy exPReSSIon
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T-5-1 Expression, Engineering and Utility of Single-Domain Antibodies
Roger MacKenzie
Human Health Therapeutics, National Research Council Canada, Ottawa, Ontario K1A 0R6Email: [email protected]
Single-domain antibodies (sdAbs) derived from the variable domains of camelid heavy chain antibodies and from the variable light and variable heavy chain domains of conventional antibodies are among the smallest known antigen-binding fragments and offer many advantages over larger antibody molecules in drug development. These advantages include high expression yield, excellent thermostability and high refolding efficiencies after treatment at high temperatures, capacity to access epitopes that are inaccessible to conventional antibodies and little tendency to aggregate. The minimal aggregation tendency of the molecules makes them excellent candidates for fusion to other proteins or nanoparticles.
We have isolated non-immune and immune camelid sdAbs which bind to CEACAM6 and which show promise as tumor-targeting molecules. CEACAM6 is a cell adhesion molecule which is overexpressed in many tumor types. Characterization and engineering of the anti-CEACAM6 sdAbs highlighted many of the advantages of sdAbs noted above. We fused a non-immune anti-CEACAM6 sdAb to a pentamerization molecule to confer avidity and showed that, compared to conventional antibodies against CEACAM6, it was highly tumour specific, possibly due to recognition of an epitope that is inaccessible to conventional antibodies. This sdAb has been fused to a therapeutic molecule by helix Biopharma and has entered clinical trials in this format for the treatment of lung cancer. As an Fc fusion, an immune anti-CEACAM6 sdAb showed good imaging of pancreatic cancer in a mouse xenograft study.
Single-domain antibodies seem well positioned to contribute significantly to the next generation of antibody-based therapeutics.
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T-5-2 Strategies for rapid identification of functional monoclonal antibodies targeting surface receptors
James pandonnelly Center, university of Toronto, Toronto, Canada
Currently, over 30 monoclonal antibodies have been approved and marketed for clinical use and several hundreds more are in clinical development. However, identification of functional antibodies against receptors, especially integral membrane proteins including gpCrs, channels and transporters, that can be utilized to elucidate target biology and/or further developed for therapeutic applications, remains challenging. We have now established a phage-display based antibody discovery platform that may be used to rapidly identify and validate functional antibodies against families of surface receptors by using purified soluble antigens as well as antigens directly expressed on mammalian cells. The platform covers antigen design and production, antibody generation, biochemical and biological characterization and testing in animal models. We have applied these strategies to a large number of potential drug target antigens, including some of the traditionally hard to target integral membrane proteins. Identification and characterization of the functional antibodies against these receptors will be discussed.
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T-5-3 functional Diversity of antibody Sialylation in the fc
T. Shantha RajuBiologics Research, Biotechnology Center of Excellence, Janssen Research & Development, LLC, 1400 McKean
Road, Spring House, PA 19477, USA
Sialic acid as a terminal sugar residue of N-glycans exhibits remarkable functional as well as structural roles for glycoproteins. Terminal sialic acid residues have been shown to increase biological activity, serum half-life as well as structural stability of glycoproteins. Alternatively, sialic acid residues may act as mediators for pathogens to invade host systems. Such properties of terminal sialic acid residues of N-glycans of glycoproteins have been extensively studied. in many of these cases N-glycans of glycoproteins are surface exposed and hence easily accessible for interactions with receptors, enzymes and other molecules along with many pathogenic organisms. however, N-glycans present in the Ch2 domain of the Fc region of igg molecules are sequestered within the cavity of two Ch2 domains of Fc and exhibit a high degree of heterogeneity due to the presence or the absence of terminal sugars including sialic acid residues. These Fc glycans are required for antibody effector functions including binding to various Fc receptors and also to C1q protein. Over the last several decades biological significance of Fc glycans has been extensively explored and importance of terminal galactose, bisecting glcNAc and core fucose has been realized. Recently biological significance of terminal sialic acid residues of Fc glycans has been explored. This presentation will review the functional effects of sialic acid residues on antibody functions.
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T-5-4 Development of an anti-idiotypic Mab as cancer vaccine: from discovery to approval
Adolfo Castillo1, Kathya de la Luz1, Julio Santo-Tomás1, Yoan Machado1, Yadira Aguilar1, Svieta Víctores1, Yadira Prieto1, Orlando Morales1, Amparo Macías2, María C. Barroso2, Tania Crombet2, Eduardo Ojito3,
Yudisleydis Fortunato3, Yanitzia Crespo3, Dasha Fuentes4, Yamilet Rabasa1 , Blanca Pacheco1, Yulier Alvárez1 and Ana M. Vázquez1
1Early Stage Product Development Direction, Center of Molecular Immunology, Havana, Cuba; 2Clinical Direction, CIM, Havana, Cuba; 3EPOVAC Manufacturing Facility, CIM, Havana, Cuba, 4National Center for
Production of Laboratory Animals, Bejucal, Mayabeque, Cuba.
racotumomab (1E10) is a murine anti-idiotypic antibody that mimics N-glycolyl-gM3 gangliosides. This antibody has been tested in some countries as an anti-idiotypic vaccine adjuvated in Al(Oh)3 in several clinical trials phase ii and iii for NSClC, melanoma and breast cancer. recently this product was approved for treatment for NSlC by regulatory authorities in Cuba and Argentina. in this paper we describe the development strategy allowed to take the product from the original idea in the laboratory to test the concept in advanced clinical trials and approval, including its scale-up.Initially the product was obtained from mice ascites fluid (AF), however a new pilot scale cGMP process based in stirred tank continuous mode cell culture using protein free medium was developed (ST) and further scaled up to 1000 l bioreactor. Bioequivalence between vaccine products obtained from ascites and different stirred tank scales through a comparability tests was studied. different characteristics as primary structure, microheterogeneity, identity, purity and biological activity of obtained Mab, as well as vaccine formulation characteristics as stability, absorption to AlOh3, immunogenicity and antitumor activity “in vivo” were analyzed. Some differences were observed like charge heterogeneity and glycans attached to Fc-γ, mainly due to varying amount of sialylated species, asparagine deamination and oxidation in each condition. These characteristics didn’t affect the immune response elicited in chicken and antitumor effect on F3ii carcinoma model. Moreover optimization of product formulation significantly increased the percentage of adsorbed protein to alumina and stability. Influence of pH, ionic strength and phosphate concentration of buffer on the adsorption of the Mab to the aluminum gel was investigated. Formulations with different adsorption percentages and antibody/adjuvant ratios were characterized by dlS. in vivo experiments using leghorn chickens were performed to evaluate effects on immunogenicity of the different vaccine formulations. However no influence of the adsorption and the racotumomab/Alumina ratio on the immunogenicity of the vaccine was observed. in summary, the introduction of a well-established process platform for the production of monoclonal antibodies, which led to increased levels of vaccine safety and reducing production costs, yielded a product very similar in each case, enabling the scaling and clinical development of the product until its health record.
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T-5-5 Utilizing chinese Hamster ovary cell Population Genetic/epigenetic Heterogeneity for the Isolation of new Host cell lines with enhanced Performance
Peter M O’Callaghan1†, Andrew J Racher2, Robert J Young1
1New Expression Technologies Group, Lonza Biologics Plc2Process Development Sciences, Lonza Biologics Plc
† Contact email: [email protected]
genetic/epigenetic variation within host Chinese hamster Ovary (ChO) cell lines impacts on key biomanufacturing characteristics such as growth rate, recombinant monoclonal antibody (Mab) production yield and product quality. however, few studies have attempted to systematically analyze such heterogeneity or to utilize this property to isolate new host cell lines to function as generic Mab production vehicles. in this project we systematically analyze a series of lonza ChOk1SV clonal derivatives* for their suitability as Mab production vehicles and interrogate their inherent biological capabilities as cell factories by assessment of key cellular attributes such as growth rate, mitochondria and endoplasmic reticulum (ER) content (determined by flow cytometry and microscopy), and glycosylation pathway gene expression (determined by glycosylation qpCr array). Across the panel of ChOk1SV clonal derivatives variation was observed in cellular mitochondria content (between 0 – 40% deviation from the ChOk1SV parent), although interestingly this did not appear to correlate with clone growth rate or transient Mab production yield. Substantial variation in clone cellular ER content was also observed (up to 50% of CHOK1SV parent), although Er content was not a direct predictor of clone performance as a transient host cell line, suggesting that Er quality may be more important than content per se. The performance of the clones as stable Mab production vehicles was also assessed and it was shown that transient Mab concentration is a poor predictor of a clone’s performance as a stable host cell line for Mab production. This work demonstrates the utility of leveraging genetic/epigenetic diversity within existing cell lines to identify new production vehicles with improved characteristics.
*davies Sl, lovelady CS, grainger rk, racher AJ, young rJ, James dC. (2012). Functional heterogeneity and heritability in ChO Cell populations. Biotechnol Bioeng. 110:260-74.
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T-5-6 Production of sialylated antibodies in cHo cells
Céline Raymond1,2,4, Erika Lattová3,4, Hélène Perreault3,4 and Yves Durocher1,2,4
1Department of Biochemistry, Université de Montréal, Canada, 2 National Research Council Canada (Montréal), Canada, 3Department of Chemistry, University of Manitoba, Winnipeg, Canada, 4Mabnet Network
Antibody-dependent cellular cytotoxicity (AdCC) and complement-dependent cytotoxicity (CdC) are two major effector functions of monoclonal antibodies (mabs). They are mediated by the interaction of the fragment crystallisable region (Fc) of the antibody with the Fc receptors of the immune cells and the molecules of the complement. The Fc N-glycans are necessary for Fc binding to their receptors and the subsequent activation of cytotoxicity. Therapeutic monoclonal antibodies (mabs) produced in Chinese hamster ovary (ChO) cells contain low level (2%) of terminal α2,3-linked sialic acids (α2,3SA) on the Fc glycan. In sharp contrast, ~15% of circulating human antibodies are sialylated on their Fc and most of the linkages are of the α2,6 type. While the impact of the Fc glycans composition on effector functions is being extensively studied, the effect of terminal SA on these and other biological functions is still less understood. To better understand the role of Fc glycan sialylation on antibodies’ biological functions, sialylated antibodies are needed in large quantities. This study presents a method for the rapid and efficient production of α2,6SA enriched mabs by transient co-expression of glycosyltransferases in CHO cells. The glycosylation profiles of an IgG1 and a F243A mutant were analyzed by lectin-blotting, isoelectric focusing and mass spectrometry. The impact of the co-expression of various glycosyltransferases on these profiles has been explored. The kinetic and extent of sialylation were also examined at two cultivation temperatures in order to better define the conditions for the efficient production of mab with low to high levels of Fc-sialylation.
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T-5-7 late Stage clinical Process Development – change from nS0 to cHo Host Using Glucose limitation Technology
Meredith Kolpak and Gregory Hiller
Pfizer, Andover, MA, USA
Clone selection is a critical decision in cell culture process development. ideally, the clone chosen for phase 1 process development continues through to be used in the final commercial process. Late stage clinical development with a new cell line is considered a significant process change that requires demonstration of sufficient product quality comparability. however, to improve productivity and align with platform strategies, the decision was made to switch from gS-NS0 to a gS-ChO host cell line for the late stage process development of a therapeutic monoclonal antibody. Following transfection, the lead gS-ChO clone was evaluated in 2-l bioreactors in the fed-batch platform process and a 3-fold increase in titer was achieved. however, the metabolic shift to lactic acid consumption lacked robustness and adversely affected viability and productivity. Therefore, hipdOg (hi-end ph-controlled delivery Of glucose), a glucose limiting strategy, was employed. An increase in the integrated viable cell density was achieved, leading to a 4-fold increase in titer over the gS-NS0 process. By optimizing several key parameters, including the glucose concentration in the base media and the day to end glucose limitation, the culture reached a peak viable cell density of 50 x 106 cells/ml. With process development nearing completion, the discovery of an unexpected sequence variant on a small percentage of product molecules required that a new transfection be performed in the gS-ChO host cell line. under a compressed timeline to meet the original deliverable dates, several clones were evaluated in the fed-batch platform process and hipdOg process. The production of lactate and subsequent metabolic shift varied for the clones and the effect of utilizing the hipdOg technology was observed to be clone specific. The final lead clone in the optimized HIPDOG process resulted in a 3.5-fold increase in productivity and product quality sufficiently comparable to that produced using the original gS-NS0 cell line process.
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T-5-8 Impact of Manganese on Galactosylation of a Recombinant Humanized Monoclonal antibody Produced in cHo cell culture
karen kiplinger, kellye Adkins and Brian horvathlate Stage Cell Culture - pharma Technical development,
genentech, A Member of the roche groupSouth San Francisco, CA
Modification of a CHO cell culture monoclonal antibody process from one chemically-defined media approach to a state-of-the-art chemically-defined media approach resulted in the expected outcome of increased biomass and product titer, but also resulted in a 20-30% decrease in galactosylation of the Fc N-linked glycan species. Initial investigational studies showed that the batch feed composition and concentrations and cupric sulfate concentration were correlated with antibody galactosylation. Manganese is a co-factor for galactosyltransferase-1 (gal-T1), and its concentration was held constant between the old and new processes. it was hypothesized that increased manganese concentration could support increased gal-T1 activity. Therefore, studies were carried out to evaluate varying levels of manganese in both processes. The results from this work showed that increased concentrations of manganese in the culture medium substantially increased galactosylation in all conditions tested.
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bIoPRoceSSInG & analyTIcal TecHnoloGIeS
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T-6-1Flublok, the first FDA approved recombinant influenza vaccine
Manon CoxProtein Sciences Corp, USA
Flublok is a recombinant hemagglutinin influenza vaccine produced in this production platform and provides an attractive alternative to the current egg-based influenza vaccine (TIV) manufacturing process. Protein Sciences Corporation was awarded a contract in June 2009 from the u.S. department of health and human Services to further develop this technology for the production of recombinant influenza vaccines for pandemic preparedness and FDA granted approval of Flublok for the prevention of influenza in adults (18 - 49 Years) on January 16, 2013.
The speaker will discuss the challenges on the path to approval and the manufacturing process for Flublok/panblok, in particular how the annual changes in vaccine composition are being handled.
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T-6-2 Raman and Microfluidic Single Cell Analysis
James PiretUniversity of British Columbia, Canada
raman microspectroscopy offers a non-invasive and label-free approach to monitor the chemical composition of living cells. Spectral markers, such as the ratio of nucleic acid to tyrosine associated band intensities, can discriminate between pluripotent stem cells and their differentiated progeny. This provides a rapid, noninvasive approach to distinguish stem cells from differentiated cells using changes in cell composition. Cellular responses to nutrient deprivation also can be analyzed by raman microspectroscopy, such as based on a phospholipid-associated raman band intensity found to increase upon glutamine limitation. This may be characteristic of the autophagic response that involves membrane formation. This analysis was used to monitor the recovery dynamics when glutamine levels were restored. Thus, raman spectral indicators can be used to non-destructively monitor cellular processes including the dynamics of cellular physiological changes and stem cell differentiation.
Emerging microfluidic technology provides an alternative approach to the analysis of single cells. This technology allows combined automated medium exchange, live-cell immunostaining and recovery of selected cells. inoculating cells into 4 nl volume culture chambers provides conventional 250,000 cells/ml seeding concentrations for subsequent clonal culture. The robustness, flexibility and scalability of microfluidic platforms provide unique advantages for the rapid analysis and generation of clonal cell lines.
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T-6-3 AAV production, WAVE BAG, suspension HEK293 cells and transient transfection: a recipe for success
R. Jude Samulski and Josh GriegerGene Therapy Center, University of North Carolina at Chapel Hill, U.S.A.
Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of pre-clinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high titer, highly pure and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. using the triple transfection method, the suspension hEk293 cell line generates greater than 1x105 vector genome containing particles (vg)/cell or greater than 1x1014 total vg/l of cell culture. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high purity vector preps of all AAV serotypes and various chimeric capsids tested. This user-friendly process can be completed within one week, results in high full to empty particle ratios (>90% full particles), provides post-purification yields (>1x1013 total vg/l of culture) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles established to date. This manufacturing technology has been utilized to manufacture 2 phase i clinical AAV vectors, which have been administered into patients. This and summary comparison to alternative AAV production approaches (hSV and BAC) will be discussed.
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T-6-4 Product Quality lessons learned from developing and implementing a chemically-defined cHo platform cell culture process
Masaru Shiratori1
1Department of Late Stage Cell Culture, Genentech,
Key WordsCell culture platform, chemically defined media, product quality
objectivesMany biopharmaceutical companies have developed cell culture platform processes for the production of recombinant monoclonal antibodies in mammalian cells. The use of platform processes for the production of clinical material has several advantages including lower costs for process development and faster generation of clinical material, thus enabling a reduced timeline to entry into clinical studies. Considerable efforts in the industry have been directed towards the development of chemically defined media for industrial cell culture processes. The use of such media reduces or eliminates some of the risks associated with the use of hydrolysate containing media, including inconsistent performance due to lot to lot variability, potential to introduce adventitious agents, and raw material sourcing. During the development and implementation of a chemically-defined CHO cell culture process platform, we observed some changes in mAb product attribute profiles compared to results obtained in an older platform process using hydrolysate-containing media.
Cell culture experiments aimed at identifying media components that could be responsible for the observed changes in product attributes (charge variants, drug substance color) were conducted using several cell lines expressing different mAbs. different concentrations of certain media components or component groups were evaluated and product attributes were assessed.
Our studies led to the identification of several media components that can have a significant effect on several product attributes. Copper and zinc were identified as key components that can affect the presence of basic variants (proline-amidation, C-terminal lysine). Certain vitamins and iron were identified as levers that can be used to manipulate drug substance color. This presentation highlights that the use of chemically-defined media enables improved process understanding, and will ultimately lead to better process control.
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T-6-5 Purifying Secretory Glycoproteins: overcoming common challenges
Christopher A. Munro, Emilyn Tan, Cindy Chang, linda h.l. lua
The university of Queensland, protein Expression Facility, St lucia, Qld 4072, Australia
Secretory glycoproteins are required for diverse research applications and are critical for therapeutic and vaccine development. glycoproteins require eukaryotic protein processing and are commonly generated recombinantly in yeast, insect and mammalian expression systems. The baculovirus-insect cell expression system combines high expression levels with the ability to facilitate complex post-translational modifications, thus making it an attractive option for the production of glycoproteins. recombinant glycoproteins are often expressed with a fusion tag to aid downstream recovery. A widely used tag is poly-histidine, which interacts with metal ions immobilised on a chromatography matrix. however, purifying histidine-tagged glycoproteins from spent culture supernatant presents a number of challenges. The presence of interfering components in some cell culture media hinders direct loading of spent culture supernatant onto chromatography column. immobilised metal ions are ‘stripped’ from the purification media during sample loading, thus preventing the capture of histidine-tagged proteins. Low secretion efficiency, large volumes of spent culture supernatant and endogenous host proteins can add further complexity to the recovery of secretory glycoproteins. here we present practical solutions for overcoming challenges commonly encountered in purifying recombinant secretory glycoproteins from eukaryotic expression systems.
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T-6-6 Developing a Production Process for Influenza VLPs: A Comparison Between HEK 293SF and Sf9 Production Platforms
Christine M Thompson1, 2, Emma petiot1, Marc g Aucoin3, Olivier henry2, Amine A kamen 1,2
1human health Therapeutics, Vaccine program, NrC, Canada2department of Chemical Engineering, École polytechnique de Montréal Canada
3department of Chemical Engineering, university of Waterloo, Canada
Influenza virus-like particle (VLP) vaccines are one of the most promising approaches to respond to the constant threat of the emergence of pandemic strains, as they possess the potential for higher production capabilities compared to traditional vaccines made in egg-based technology. Vlps are particles produced in cell culture utilizing recombinant protein technology composed of viral antigens that can elicit immune response but lack viral genetic material. Past influenza VLP studies have been mainly performed in the baculovirus-insect cell system and report contamination with recombinant baculovirus. Baculovirus with mammalian promoters (Bacmam) have been shown to efficiently transduce mammalian cells and further express genes but are unable to replicate, efficiently repressing baculovirus production. In order to address the issue of baculovirus contamination, VLP production was performed in hEk 293SF cells using the Bacmam gene delivery system. The proposed system was assessed for its ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix protein (M1) and compared to Vlps produced in Sf9 cells through the lens of bioprocessing. Vlps from both systems were characterized using currently available influenza quantification techniques such as single radial immunodiffusion assay (Srid), hA assay, negative staining electron microscopy (NSEM) and western blot. Vlps were found to be associated with the cell pellet in hEk 293SF production in addition to in the supernatant. it was found that Vlp production in Sf9 cells produces 1.5 logs more Vlps than in hEk 293SF. Sf9-Vlps had higher hA activity and were generally more homogeneous in morphology and size. however, Sf9 Vlp samples contained 20x more baculovirus than Vlps, which can contribute to hA activity in both the hA assay and Srid which has to be acknowledged during process development stages. This study shows the strength of the insect-cell baculovirus system for Vlp production when compared to proposed alternatives in hEk 293SF cells from the bioprocessing point of view but also highlights that there is a major need for baculovirus removal to properly characterize and consider them as vaccine candidates.
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STeM cellS: THe fUTURe
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T-7-1 autologous mesenchymal stromal cells engineered to secrete therapeutic proteins as part of a synthetic endocrine device: pre-clinical development and roadmap to clinical use.
Jacques Galipeau, MD FRCP(C)Emory University, Atlanta, GA 30322
Over the course of the last decade, mesenchymal stromal cells (MSC) made a remarkable entry into the field of cellular therapy. Their ability to differentiate into several mesenchymal lineages, as well as their role in hematopoiesis, provided the basis for several clinical investigations in the field of regenerative medicine, ranging from heart repair to hematopoietic support in patients undergoing hematopoietic peripheral blood progenitor cells transplantation. in addition, results from animal disease models and clinical trials in humans have proven the potential of MSC as immunosuppressive as well as anti-inflammatory agents. MSCs are readily amenable to genetic engineering so as to produce cytokines and growth factors for therapeutic use. Engineered MSCs can be utilized as synthetic endocrine cells when implanted subcutaneously in experimental mice as an “organoid” leading to sustained pharmacological release of proteins such as erythropoietin and igF for treatment of anemia and blood related disorders. The use of autologous and “universal donor” allogeneic MSC cellular products will be discussed as well as the potential pitfalls of immune response to random donor MSCs and how this informs the development of manufactured stem cells for use as an engineered protein delivery cellular pharmaceutical.
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T-7-2Human Reprogramming of cell fate: Implications and applications
Mick BathiaStem Cell and Cancer Institute, McMaster University, Canada
The ability to reprogram human skin cells to pluripotent stem cell fate, akin to embryonic stem cells, has transformed the views, approaches and attitudes towards regenerative medicine. however, differentiation into cell types required for drug discovery or cell replacement therapies from these pluripotent stem cells remains a major limitation.
recent advancements in cell reprogramming beyond pluripotency, and holding pluripotency in vitro using different methods and sources of human tissue, may circumvent these limitations. The evolution of this field, and these recent advancements will be discussed.
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POSTER PRESENTATIONS
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P.1 Increasing Production Capacity of a Legacy Product through Medium EnrichmentYuval Shimoni, Volker Moehrle and Venkatesh Srinivasan
P.2 Development & Quantification of a Magnetic nanoparticle Enrichment Strategy for glycopeptides using Tandem Mass TagsE.D. Bodnar H. Perreault
P.3 Establishment of Amplified Fragment Length Polymorphism (AFLP) to quantify genomic rear-rangements in Chinese Hamster Ovary (CHO) Cells Sabine Vcelar, Inmaculada Hernandez-Lopez, Norbert Auer, Martina Baumann, Angelika Zotter, Nicole Borth,
P.4 Application of Advanced Methods for Process Optimization to Tuning Lonza’s Platform Process for the GS Xceed™ Cell LineColin Jaques and Danni LegaP.5 Impact of a Toxic Single-Use Bio-Process Container Film Leachable on Chinese Hamster Ovary CellsHeather Nunn and Matthew Hammond
P.6 .A comparison between the oligosaccharide structures of native and recombinant glycosylated hu-man prolactin obtained from CHO cells Capone MVN, Suzuki MF, Oliveira JE, Bartolini P and Soares CRJ
P.7 An unstructured kinetic model for growth and production of recombinant protein in S cell line.Barral MF(), Aguiar MA(), Piccoli RAM(), Augusto EFP(,,*)
P.8 Evaluation of Homogeneity and Genetic Stability of REOLYSIN® (pelareorep) After Large Scale Production by Massively Parallel SequencingRomit Chakrabarty, Hue Tran, John Kolman, David Onions, Allison Hagerman and Matt Coffey
P.9 Host cell protein analysis in REOLYSIN® (pelareorep), an oncolytic viral therapyHue Tran, Romit Chakrabarty, Rosa Tran, Julia Transfiguracion, Kelly Fulton, Allison Hagerman, Su-san Twine, Robert Voyer, Amine Kamen, and Matt Coffey
P.10 Performance Evaluation of Critical Raw Materials: Working Cell and Virus Bank CharacterizationAllison Hagerman, Romit Chakrabarty, Hue Tran, Julia Transfiguracion, Rosa Tran, Pocheng Liu, Bobby Fredin, Mia Garrett, Mehrdad Malaz, Mary Carter, Jacquelyn Davidson, Tiffany Hawkins, Jing Shi, Wolf Klump, Robert Voyer, Amine Kamen, Matt Coffey
P.11 Developing a Scalable, High Performance Bio-Production Process in Minimal TimeStacy Holdread*; Kaci Conaway; Emily Gramiccioni; Kirti Chaturvedi; James W. Brooks, PhD, Kathie Fritchman
P.12 Using on-line capacitance monitoring as an in-process quality control tool of the REOLYSIN® (pelareorep) production processRobert Voyer, Sven Ansorge,, Stéphane Lanthier, Rosa Tran, Julia Transfiguracion, Allison Hagerman, Matt Coffey and Amine Kamen
Poster Presentations
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P.13 Optimization of a Humanized Monoclonal Antibody Productivity using Single Use BioreactorsDevender Sandhu, Houssam Alosta, Srinivasa Rokkam, Katherine Lu, Nishi Singh, Tanya Sheinin, Kathryn Matthews, David Bell.
P.14 Synthetic transposons for the generation of stable CHO cell pools expressing recombinant proteinsSowmya Balasubramanian, David L. Hacker, Lucia Baldi, Florian M. Wurm
P.15 Utilization of Recombinase Mediated Cassette Exchange (RMCE) for the generation of recombi-nant CHO cell lines with minimal clonal variationMartina Baumann, Elisabeth Gludovacz, Nicole Borth,
P.16 Stable miR-7 engineering enhance recombinant protein production in rCHO cellsVaibhav Jadhav , Matthias Hackl, Gerald Klanert,,Juan A. Hernandez Bort,, Nicole Borth,, Johannes Grillari
P.17 Efficient Production of High Titer Lentiviral Vectors With CaffeineAna Paula Costa de Pontes, Marcela Cristina Corrêa de Freitas, Luiza Cunha Junqueira Reis, Daianne Maciely Alves de Carvalho, Dimas Tadeu Covas and Virginia Picanço e Castro1
P.18 Characterization of human cell lines for production of gama-carboxilation proteinsMarcela Cristina Corrêa de Freitas,; Aline de Sousa Bomfim,; Virgínia Picanço Castro; Kamilla Swiech,; Elisa Maria de Sousa Russo , and Dimas Tadeu Covas,
P.19 Characterization of human cell lines producing recombinant Factor IXAline de Sousa Bomfim,; Virgínia Picanço Castro; Marcela Cristina Corrêa de Freitas,; Mário Soares de Abreu Neto; Kamilla Swiech,; Dimas Tadeu Covas, and Elisa Maria de Sousa Russo ,
P.20 Engineering a Mammalian Cell Line ToolboxShelly Martin, Mark Tié, John Follit, Chapman Wright, Brian Majors, Scott Estes
P.21 Large scale microRNA screen in Chinese hamster ovary cells reveals great potential for novel cell line engineering strategiesSimon Ficher,, René Handrick, Andreas Wagner and Kerstin Otte.
P.22 Adaptation of mammalian cell to Serum Free MediaSandra Fernanda Suárez; Thaissa Bernardino; Polyana Ataliba; Carlos Augusto Pereira; Renato Astray and Soraia Jorge.
P.23 Engineering the CHO Cell LineBjørn Voldborg, Henrik Claussen, Maicheal J Betenbaugh, Bernhard Parlsson
P.24 A CHO-M Cell Combinatorial Library for the Improved Selection of Recombinant Protein Produc-tion ClonesPierre-Alain Girod
P.25 A Novel CHO Host Cell Line Using HPRT as an Amplifiable Selectable Marker for Therapeutic Protein ProductionNatalie Sealover, Kenji Kobayashi, Henry George, Kevin Kayser, and Nan Lin
P.26 In-vitro modification of N-glycans using N-acetylglucosaminyltransferase III (GnT)Jann Catherine Ang, Marc G Aucoin, Guy Guillemette
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P.27 An in silico platform enabling optimization of fed-batch cultures of CHO cells producing a mono-clonal antibodyJulien Robitaille and Mario Jolicoeur
P.29 Shortened Timeline for Cell Line Development – Adaption of Recombinase-Mediated Cassette Exchange (RMCE) to Suspension CHO CellsElisabeth Bludau, Nathalie Veith , Volker Hecht and Holger Ziehr
P.30 Reduction of Fucosylation of Monoclonal Antibody Glycans Using Several Glycosylation Inhibi-torsMaureen Spearman, Natalie Okun, Sarah Chan and Michael Butler
P.31 Effect of redox potential on antibody glycosylation in mammalian cell cultures Ben Dionne, Michael Butler
P.32 Effects of Glucose and Glutamine Concentrations on the Glycosylation of CHO EG-hFc monoclo-nal antibody. Carina Villacrés Barragán, Erika Lattová, Michael Butler
P.33 High-Level Recombinant Protein Production in CHO Cells Using the Cumate Gene-Switch in Combination with Lentiviral Vectors or Optimized Pool Selection ProtocolNasha Nassoury, Bruno Gaillet, Adeline Poulain, Louis Bisson Félix Malenfant, Sylvie Perret , Rénald Gilbert, Alaka Mullick, , Yves Durocher, , Bernard Massie,
P.34 Differential affects of low glucose on the macroheterogeneity and microheterogeneity of glycosyl-ation in CHO-EG camelid monoclonal antibodiesLiu Bo, Erika Lattova, Maureen Spearman and Michael Butler
P.35 Towards tailored insect cell lines to improve recombinant protein production in both stable and transient baculovirus infected cell linesMaren Bleckmann, Steffen Meyer, Bahar Baser and Joop van den Heuvel
P.36 A Stirred Tank Bioreactor for Human Mesenchymal Stromal/Stem Cell Expansion Maintains De-sired Cell CharacteristicsKimberly Mann, Sandhya Punreddy, Anjali Verma, Manjula Aysola, Neethu Sunil, Donghui Jing, Dan-iel Kehoe, Knut Niss, Martha Rook, Julie Murrell
P.37 SweetBac: The way for better pharmaceuticals in Trichoplusia ni cells Dieter Palmberger, Kazem Ashjaei, Imre Berger, Gary Blissard, Karin Hoffmann-Sommergruber, Rein-gard Grabherr
P.38 CAP: An Efficient Platform for Transient and Stable Protein Production based on Immortalized Human AmniocytesSilke Wissing, Corinna Bialek, Sabine Hertel, Helmut Kewes, Tanja Spörk, Markus Ribbert, Michael Schomberg, Jens Wölfel, Gudrun Schiedner, Nicole Faust
P.39 Construction of an antiviral recombinant protein using a baculovirus/ SF-9 cells systemR.Z. Mendonça, D.N.S. Giovanni, R.M.Z. Mendonça, Figueiredo, C. A. ; Oliveira, M. I. ; Silva, P.E; Curti, S. P ; R.H.P. Moraes A.C.V. Carmo
P.40 Co-expression of protein phosphatases in insect cells affects phosphorylation status andexpression levels of proteinsEilleen McCall, Rebecca Williams, Mark Abbott
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P.41 Improving Protein Secretion in the Baculovirus-Insect Cell Expression SystemCindy Chang, Emilyn Tan, Linda H.L. Lua
P.42 Use of mammalian expression systems for secreted proteins at AstraZeneca; FAMB PANDER a new type of signaling moleculeLovisa Holmberg Schiavone, Jenny Gunnarsson and Patrik Johansson
P.43 Protein Expression at the National Institute of Environmental Health Sciences: Assisting the sci-entists with protein expression and purification needs with a multiple system approach.Molly E. Cook, Gary K. Powell, Lori L. Edwards, Christine C. Malone, Robert M. Petrovich
P.44 Production of viral particle for the NS-HCV expression in mammalian cells Lemos, MAN
P.45 Transfection of HEK9 and CHO Mammalian Cells using Polyethylenimine-based PolyplexesLaurence Delafosse and Yves Durocher
P.46 Is the low cell specific yield of Baculovirus infected Insect cells at high cell densities due to a limi-tation of transcription rather than translation?S. Reid, H. Huynh, T. Tran, L. Nielsen and L. Chan
P.47 Uncovering Methods for the Prevention of Protein Aggregation and Improvement of Product Quality in a Transient Expression SystemBram Estes, Raj Haldankar, Yueh-Rong Hsu, Lei-Ting Tam, and Jackie Sheng
P.48 Large-scale Production and Purification of the Mouse Serotonin 5-HTA Receptor and Generation of Functional Antibody FragmentsGherici Hassaine, Cédric Deluz, Hugues Nury, Horst Vogel and Kenneth Lundstrom
P.49 Characterization of a novel protein antiviral from Lonomia obliqua using bioinformatics tools and activity analysis by real time. Carmo, A.C.V., Giovanni, D.N.S., Yamasaki, L. H. T., Figueiredo, C. A., Oliveira, M. I., Santos, F. C. P., Curti, S. P., Tonelotto, M., Rahal, P., 5Moraes, D., Mendonça, R.Z.
P.50 High Throughput Production of Complex Recombinant Proteins in Mammalian CellsJens Jacob Hansen, Thomas Egebjerg, Anette Amstrup Pedersen, Mats Åkesson, Bjarne Rask Poulsen, Michael Skjøt, Laust Bruun Johnsen, Claus Kristensen
P.51 Targeted transfection using E/Kcoil system for recombinant protein productionElodie Louvier, and Yves Durocher ,
P.52 Exploring endosomal pathways using novel nanoparticle polymers as transfection reagentsMarianne Gillard, Nghia P. Truong, Zhongfan Jia, Michael J Monteiro and Trent P Munro.
P.53 Mechanistic Engineering of Polyplex Mediated Transfection of CHO CellsOlivia L Mozley, Ben C Thompson and David C James
P.54 Bioproduction Using Flow Electroporation Systems: Gram/L Antibody Production via Transient Gene Expression (TGE) Through Rapid, High Yield Stable Cell Line Generation.Weili Wang, James Brady, Rama Shivakumar, Krista Steger, Karen Donato, and Madhusudan Peshwa
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P.1 Increasing Production capacity of a legacy Product through Medium enrichment
Yuval Shimoni1, Volker Moehrle2 and Venkatesh Srinivasan1
1Manufacturing Sciences, Bayer HealthCare, USA, 2 Bayer Technology Services, Germany
The implementation of post-licensure improvements to legacy processes can benefit patients and biopharmaceutical drug manufacturers alike. This talk will present our approach to this challenge and consist of two parts. First we will describe the identification of changes to production medium that yielded ~30% higher titers than the current legacy process and demonstration of the concept in 1L and 15L scale perfusion bioreactor runs (see figure 1). In the second part, we will illustrate the scope and complexity of validating and obtaining global regulatory approval to implement the change in commercial production.
Analytical and cell culture platforms were established, allowing a reasonable balance between throughput (parallel testing of different experimental conditions) and scale (which is required for a comprehensive analytical testing). Following spent media analyses, design of experiments (doE) were conducted at bench scale (repeated batch mode) to evaluate media enriched for various components, with titer being the primary output. The formulation with the highest titer consisted of higher levels of a sugar and a metal ion (which acts as a stabilizer of the protein product) and was further tested in 1l followed by 15l perfusion-mode bioreactors. productivity gains, product quality attributes and process performance (cell growth rate and metabolism), were reproducible and consistent between the perfusion bioreactor scales and titer gains were maintained over several weeks of perfusion cell culture.
We further describe the road map leading to implementation in commercial operations: from business case justification through key milestones; emphasizing the required timely activities performed by stakeholders including r&d, Quality, Manufacturing and regulatory Affairs. The approach presented is general in demonstrating how an idea can be taken from proof of concept to demonstration of feasibility, including risk assessment and validation.
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P.2 Development & Quantification of a Magnetic nanoparticle Enrichment Strategy for glycopeptides using Tandem Mass Tags
E.D. Bodnar, H. Perreault University of Manitoba
Studying glycosylation of glycoproteins at the tryptic glycopeptide level offers advantages; it allows to determine stie-specific heterogeneity, and circumvents the difficulties of identifying hindered glycoforms when other peptides are present. A method using magnetic nanoparticles (MNp) is presented, aiming to enrich glycopeptides for downstream mass spectrometric (MS) analysis. This method presents minimal sample handling. Glycoproteins (α1 acid, Eg2-mAb, fetuin, IgG) were digested with trypsin to generate mixtures containing peptides and glycopeptides. MNp were synthesized as previously reported1, and used to enrich each standard glycoprotein based on hydrophilic liquid interaction (hiliC). This protocol was compared with other established glycopeptide enrichment workflows e.g. the popular EMD-Millipore HILIC kit method2. Enriched samples were labeled with tandem mass tag reagents according to manufactures protocols (TMT™, Thermo inc.) for relative quantification. Measurements were achieved through matrix assisted laser desorption ionization (MALDI-MS), electrospray ionization (ESI-MS) as well as tandem MS (MS/MS) in positive scan mode. At first, TMT-labeled acidic glycopeptide fractions had to be characterized by linear mode on a Bruker UltrafleXtreme MALDI-TOF/TOF to enable detection of highly sialylated species. The efficiency of each sequestration method could be determined in qualitative terms using this approach, which showed the superiority of the MNps for sialylated glycopeptides. Subsequently, samples were injected on an AbSciex 5600 Triple ToF™ mass spectrometer, followed by quantification using the TMT protocol of comparing the abundances of specific reporter ions. Through quantitative and semi-quantitative comparison for different glycopeptides, this presentation will show several advantages of the magnetic nanoparticle protocol described here.
1 Adv. Funct. Mater. (2011), 21, 1457-1464.2 http://www.emdmillipore.com
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P.3 Establishment of Amplified Fragment Length Polymorphism (AFLP) to quantify genomic rearrangements in chinese Hamster ovary (cHo) cells
Sabine Vcelar1, Inmaculada Hernandez-Lopez1, Norbert Auer2, Martina Baumann1, Angelika Zotter1, Nicole Borth1,2
1Austrian Centre of Industrial Biotechnology, Austria; 2Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria
ChO cells are the most commonly used mammalian expression system and are the preferred host cells for the production of therapeutic proteins. Amenities such as an easy cultivation, fast growth, complex protein folding and human-like post-translational modifications are in contradiction to the frequently occurring genomic rearrangements. These alterations lead to a declined cell line and process development and problems concerning screening and reproducibility. despite the frequent occurrence of genomic instability, no methods are currently available to rapidly assess large scale chromosomal rearrangements, apart from FISH (fluorescence in-situ hybridization) and karyotyping.
The focus of the present work is to allow simple detection and quantification of chromosomal changes using standard laboratory equipment by AFlp. The approach which is already established in cancer research and plant breeding was modified for use in CHO cells. The principle of AFLP is the digestion of genomic DNA with different restriction enzymes, followed by the ligation of these fragments to adapters with a predefined sequence. DNA amplification of restriction fragments is performed using selective AFLP primers complementary to the annealed adapter sequence containing one or two extra nucleotides. resulting bands are analyzed using a Bioanalyzer.
The method defines an initial pattern of bands of digested genomic DNA and therefore enables the detection and quantification of chromosomal changes over time or differences between subclones and parental cell lines. Results indicate that by addition of two additional nucleotides to the pCr primers, the number of fragments could be reduced to 1/256 of the initial number, leading to clearly structured patterns for specific primer pair combinations. We propose the use of AFlp both for assessment of the rate of genomic changes in a given cell line with time in culture and for identification of subclones or cell lines characterized by genomic differences.
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P.4 application of advanced Methods for Process optimization to Tuning lonza’s Platform Pro-cess for the GS xceed™ cell line
Colin Jaques and Danni LegaLonza Biologics, colin.jaques@lonza .com
When a new host cell line is introduced into a mature platform process, can substantial performance improvements be expected if the process is tuned to fit the new cell line’s growth and metabolic characteristics more closely? Transfectant cell lines made from lonza’s ChOk1SV gS-kO host cell line host display satisfactory growth, productivity and scale-up in lonza’s version 8 gS-ChO platform process. But could they do better in a ChOk1SV GS-KO specific process rather than one developed for CHOK1SV?
principal components analysis was used to summarize 240 variables from 22 bioreactor cultures. The analysis indicated that improvement to culture metabolism leading to decreased lactate metabolism and increased culture viability at harvest might be possible.
Five inorganic constituents and 4 amino acids were identified as having potential to improve growth, metabolism and productivity. A response surface experiment with 11 factors was run in 96 miniature bioreactors. Numerical optimization was performed on the data for 20 responses simultaneously targeting improved metabolism, improved manufacturability, increased robustness and increased productivity. The conditions identified were tested on two gS xceed™ cell lines in 10 liter bioreactors. As predicted substantial improvements in growth, metabolism and productivity were achieved.
We have demonstrated that, even for a mature mammalian cell culture process, advanced process optimization techniques can enable substantial improvements to be achieved for multiple process outputs in a single round of experimentation.
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P.5 Impact of a Toxic Single-Use bio-Process container film leachable on chinese Hamster ovary cells
Heather Nunn1 and Matthew Hammond2
1Cell Sciences and Technology, Amgen Inc. Seattle, U.S.A., 2Product Attribute Sciences, Amgen Inc. Thousand Oaks, U.S.A.
A high-throughput cell-based assay (HT-CBA) was developed to investigate the impacts of the polymer films used to construct single-use bio-processing equipment such as bio-process containers (BpCs) and single-use bioreactors (SuBs) on the growth and health of Chinese hamster ovary (ChO) cells. Cell growth media were incubated in BPCs constructed from several different films, and the media thus treated was used to culture CHO cells in 24 deep-well plates. Multiple films negatively impacted culture growth and viability. The compound bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) which leaches at low levels from these films was shown to be highly detrimental to cell growth. The toxic compound is derived from the breakdown of tris(2,4-di-tert-butylphenyl)phosphite (trade name irgafos 168®), a common antioxidant additive present in many formulations of polyethylene (one of the polymers commonly used as the fluid contact layer of BPCs). Cell growth experiments with multiple recombinant protein-expressing CHO cell lines show a 50% reduction in culture growth at bDtBPP concentrations below the parts-per-million range for all cell lines. Cell specific productivity was not impacted. Cellular response to bDtBPP is rapid with a significant decrease in mitochondrial membrane potential within minutes of compound spiking. The ChO cells recover growth and health when returned to culture medium free of bdtBpp.
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P.6 .a comparison between the oligosaccharide structures of native and recombinant glycosylated human prolactin obtained from cHo cells
Capone MVN, Suzuki MF, Oliveira JE, Bartolini P and Soares CRJBiotechnology Department, IPEN-CNEN/SP, São Paulo - SP, Brazil
human prolactin (hprl) is a pituitary polypeptide hormone mainly involved in mammary gland development and lactation. It has a unique N-glycosylation site, only partly occupied (~10%), being therefore possible to separate non-glycosylated (Ng-hprl) from glycosylated prolactin (g-hprl). The goal of the present work was, therefore, to study, in comparison, the N-glycan structures present in the native, pituitary form (provided by the National hormone and pituitary program of the uS) and in the recombinant, ChO-derived form, synthesized and purified in our laboratory. Recombinant G-hPRL was obtained in spinner flasks from suspension-adapted CHO cell cultures, to which 0.6 μg/ml cycloheximide were added, a medium modification favoring glycosylation and that provided ~50% of G-hPRL on the total amount of the hormone. This artifact greatly facilitated purification, which was carried out via a two-step chromatographic purification process: a cationic exchanger (SP-Sepharose fast flow) and a preparative reversed-phase HPLC (RP-HPLC). The overall yield was approximately 37,5%, the purity of recombinant G-hPRL >95%, as confirmed by Western blotting, SDS-PAGE, RP-HPLC and high-performance size-exclusion chromatography (hpSEC), while its biological activity, determined via the classical Nb2 in vitro bioassay was, as expected, ~17 IU/mg. N-glycosylation profiling, determined via mass spectrometric techniques, identified 28 different N-glycan structures in pituitary and 14 N-glycan structures in recombinant G-hPRL. The most abundant structures were biantennary, in both cases: N2gN2F2SO4 (33.8%) and N2GN2F1(SO4)2 (28.1%) in the former and N2G2S2F1 (56%) and N2G2S1G1 (12%) in the latter. The difference in sialic acid content was even greater than expected: 0.03 moles/mole of g-hprl in the pituitary and 1.4 moles/mole in the recombinant. This is quite relevant since sialic acid is known to influence in vivo bioactivity. Considering the abundance of each N-glycan and the well-known molecular mass for G-hPRL of 25,000 d, a total carbohydrate content of 7.13% (pituitary G-hPRL) and of 8.5% (recombinant G-hPRL) was obtained by stoichiometric calculations. These data were found in very good agreement with the values of 7.7% and 8.3% obtained, respectively, via MALDI-TOF-MS.
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P.7 an unstructured kinetic model for growth and production of recombinant protein in S2 cell line.
Barral MF(1), Aguiar MA(2), Piccoli RAM(2), Augusto EFP(2,3,*)
(1)Santo André Foudation, Brazil; (2)Laboratory of Industrial Biotechnology, IPT, Brazil; (3)Federal University of São Paulo, UNIFESP, S. José dos Campos, SP, Brazil.
Drosophila melanogaster Schneider 2 (S2) cells have been used as a suitable expression system for the production of different recombinant proteins. in this study, an unstructured kinetic model was proposed to describe the growth of a transfected S2 cell line and the associated recombinant membrane rabies virus glycoprotein (rVgp) synthesis. The data were obtained from eight runs conducted in a 1.5l bubble free bioreactor, in batch mode, using supplemented TC100 serum free medium, under different conditions of: dissolved oxygen tension (pO2) - 5 to 80% (air saturation); initial glucose concentration (GLC) – 1 to 10g/L; and initial glutamine concentration (GLN) - 0,6-3.5 g/l. The proposed model formulation considers eight state variables, nineteen parameters, and represents the following phenomena: a) growth limited by glucose, glutamine, and inhibited by ammonium; b) death limited by ammonium and inhibited by glucose and glutamine; c) Alanine formation partially associated to growth and limited by glucose; d) ammonium formation associated to growth; e) rVgp synthesis associated to growth and inhibited by ammonium; and f) RVGP degradation. In order to compensate the extreme flexibility of cells to environmental condition (initial or changing during process), correction factor, relating maximum specific growth rates, were applied. Model could be validated through statistical analysis (F test, a=0.05), for non-limiting glucose and glutamine conditions, i.e., for concentration above 0,1 g/l.
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P.8 evaluation of Homogeneity and Genetic Stability of ReolySIn® (pelareorep) after large Scale Production by Massively Parallel Sequencing
Romit Chakrabarty1, Hue Tran1, John Kolman2, David Onions2, Allison Hagerman1 and Matt Coffey1
1 Oncolytics Biotech Inc., Canada 2 BioReliance, USA
rEOlySiN® (pelareorep) is a proprietary isolate of reovirus Type 3 dearing strain that is currently being tested in several clinical trials as an anticancer therapeutic agent. The reovirus genome is composed of 10 segments of double stranded rNA. These segments are characterized by genome size; large (l1 to l3), medium (M1 to M3) and small (S1 to S4). The objective of this work was to evaluate the homogeneity and genetic stability of this investigational new drug after large scale production. Massively parallel Sequencing (MpS) was performed using a roche-454 genome Sequencer Flx instrument. previous analysis on the rEOlySiN virus bank was performed using Sanger sequencing and a number of SNps (Single Nucleotide polymorphism) were detected. MpS is performed without virus cloning and this method surveys and reports the sequences of a vast population of genome molecules. The greatest challenge was sequencing the genome ends, as the 10 genome segments of reovirus offered twenty genome ends. roche-454 sequencing resulted in 69,821,115 bases and an average of 335 bases per read. Twenty-nine high confidence differences relative to the GenBank reference sequences were identified by MPS. Of those, 27 were previously characterized by Sanger sequencing and now confirmed by MPS. Of the remaining 2 nucleotide differences, one was predicted by MpS to be silent (l3 genome-T3163C, codon 1054) at the amino acid level. This was previously identified by Sanger sequencing as a mutant and now by MPS to be a mixed SNP (86% of the population was wild-type: “T” and 13% of the population was reported as mutant: “C”). The other mutation was non-coding (M1 genome-A2284G) and was present in 97% of the population. In conclusion, the results obtained from MpS and previous results from Sanger sequencing were comparable and demonstrated a high level of homogeneity at the amino acid level and genetic stability for rEOlySiN.
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P.9 Host cell protein analysis in ReolySIn® (pelareorep), an oncolytic viral therapy
Hue Tran1, Romit Chakrabarty1, Rosa Tran2, Julia Transfiguracion2, Kelly Fulton3, Allison Hagerman1, Susan Twine3, Robert Voyer2, Amine Kamen2, and Matt Coffey1
1Oncolytics Biotech Inc., Calgary, Canada, 2National Research Council, Montreal, Canada, 3National Research Council, Ottawa, Canada
rEOlySiN® (pelareorep), a proprietary isolate of the replication competent reovirus Type 3 dearing strain, is currently being tested in phase i to phase iii clinical trials in a variety of indications. reovirus is a ubiquitous, naturally occurring oncolytic virus, which specifically lyses cells possessing an activated Ras-signaling pathway. REOLYSIN is manufactured and purified from human HEK293 cells, and has undergone host cell protein (HCP) analysis to quantify residual hCp. EliSA-based assays are a common method to quantify hCp, however this analysis is variable and dependent on the specificity of the antibody for the antigen. We performed ELISA assays using a commercially available HEK293 HCP kit with varying results. This led us to investigate the specificity of the anti-hEk293 antibody provided in the commercial kit. Western blot analysis using the anti-hEk293 and anti-rEOlySiN antibodies suggests that: (1) the reoviral proteins and some hCp may co-migrate on SdS-pAgE or (2) the anti-HEK293 antibody is non-specifically detecting the reoviral proteins. Three of the most abundant HEK293 HCP: HSP90, HSP70 and actin, identified in REOLYSIN by mass spectrometry (LC-MS/MS) were used to further the investigation. The co-migration of reoviral proteins with hCp may be due to (1) their similarity in Mw, or (2) the reovirus proteins interacting with specific HCPs. Currently, we are using 2D-PAGE and native gel analysis to investigate both hypotheses as well as the specificity of the anti-HEK293 antibody. Our preliminary data indicates that reovirus interacts with HCP proteins, specifically actin and HSP90.
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P.10 Performance evaluation of critical Raw Materials: Working cell and Virus bank characterization
Allison Hagerman1, Romit Chakrabarty1, Hue Tran1, Julia Transfiguracion2, Rosa Tran2, Pocheng Liu3, Bobby Fredin3, Mia Garrett3, Mehrdad Malaz3, Mary Carter4, Jacquelyn Davidson4, Tiffany Hawkins4, Jing Shi4, Wolf
Klump3, Robert Voyer2, Amine Kamen2, Matt Coffey1
1Oncolytics Biotech Inc., Canada, 2National Research Council, Canada, 3SAFC Carlsbad Inc., USA, 4BioReliance Corporation, USA
rEOlySiN® (pelareorep), a proprietary formulation of the human reovirus Type 3 dearing strain, is currently in late stage clinical testing in a broad range of cancer indications. A formal risk assessment was conducted on the pelareorep drug substance (dS) production process using standard project management risk tools. This assessment identified the HEK293 Working Cell Bank (WCB) and Reovirus Working Virus Bank (WVB) as critical raw materials. Analysis of data obtained during continued verification of the manufacturing process showed variation in the cell growth kinetics post-infection. in depth characterization of both the WVB and WCB was undertaken to evaluate the source of this variability.
The effective Multiplicity of Infection (MOI) of the viral stock used during infection was confirmed by assessing the vial-to-vial variability of the recoverable viral titer. As multiple freeze/thaw cycles were used to generate the virus bank material, the resulting vials contained variable amounts of a liquid fraction and an insoluble pellet fraction. Therefore, digestion of the insoluble fraction was performed and the recoverable viral titre assessed in both fractions. high performance liquid Chromatography (hplC) was used to measure the total number of viral particles, and Median Tissue Culture infectious dose (TCid50) to assay the number of infectious viral particles.
Concurrently, a cell-based assay utilizing resazurin indicator dye was established to characterize the cell bank for virus production. The approach provides easy assessment of the cell bank performance. in parallel, the apoptotic cell death induced by the reovirus infection was evaluated by measuring caspase-3 activation in hEk293 cells using flow cytometry. Analysis of post-infection growth patterns between cell banks using the cell-based and caspase assays allows for selection of the most appropriate cell bank to be used in the large scale production process.
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P.11 Developing a Scalable, High Performance Bio-Production Process in Minimal Time
Stacy Holdread*; Kaci Conaway; Emily Gramiccioni; Kirti Chaturvedi; James W. Brooks, PhD, Kathie Fritch-man
*Stacy Holdread, BD Advanced Bioprocessing
With the tight timelines associated with the biotherapeutic product development and launch process, process development groups are challenged with developing robust, scalable processes in minimal time. Cell culture media and feeding strategies are critical factors, so it is essential to quickly identify a high performance medium that is easily adaptable and scalable. One strategy is to perform media optimization and feed studies to design a process specific for a particular cell line, but this can be costly and time consuming. An alternative approach is to screen commercially available production media and feeds to quickly identify a high performance medium and feed supplement that can be used as part of a platform process. This strategy speeds time to market, which is critical in launching any new product.
A series of studies were performed using scale-down models to quickly identify a base medium that demonstrated high performance with ChO lines. The evaluation included the use of a microbioreactor system to model the conditions that would be observed in a scaled up bioreactor system. Feed studies were also conducted using the microbioreactor system to further enhance the performance of the base medium.
By using predictive scaled down screening methods, a large number of conditions were screened in minimal time using minimal resources. As a result, a optimized base and feed media were quickly identified as high performing candidates for use in a scaled up process. d
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P.12 Using on-line capacitance monitoring as an in-process quality control tool of the Reoly-SIn® (pelareorep) production process
Robert Voyer1, Sven Ansorge1,2, Stéphane Lanthier1, Rosa Tran1, Julia Transfiguracion1, Allison Hagerman3, Matt Coffey3 and Amine Kamen1
1National Research Council, Canada, 2currently GSK Vaccines, Belgium 3Oncolytics Biotech Inc., Canada
rEOlySiN® (pelareorep), a proprietary isolate of the replication competent reovirus Type 3 dearing strain, is currently being tested in phase i to phase iii clinical trials. The rEOlySiN manufacturing process uses human hEk293 cells that are diluted with fresh serum-free medium prior to infection once the viable cell density (VCd) falls within a specific range. Production harvest is initiated at the end of the infection phase, typically once one of the viability criteria is reached. On-line capacitance monitoring of cell culture was demonstrated to correlate well with viable cell biovolume and was identified as a promising in-process control tool as it generally correlates well with the VCd content of cultures during growth phase due to limited observed variation in cell diameter and viability. Here, we explore the potential to use the on-line capacitance monitoring profile within the REOLYSIN manufacturing process (1) to confirm when the viable cell density window of infection is reached, (2) to monitor the quality of growth and infection phases and (3) to monitor the drop in viability and identify when any of the har-vest viability criteria is reached. preliminary results show linear corelation between VCd and capacitance during the growth phase with an r2 of 0.95 and a capacitance to viability correlation during the virus release phase with an r2 of 0.84. A full characterization of the infection phase allowed us to correlate the on-line capacitance monitoring signal with the onset of cell lysis and viral release in the culture. Variation in the capacitance profile of the manu-facturing process under variable process parameter conditions such as agitation rate, ph, dO, and multiplicity of infection (MOi) is also discussed.
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P.13 optimization of a Humanized Monoclonal antibody Productivity using Single Use bioreac-tors
Devender Sandhu, Houssam Alosta, Srinivasa Rokkam, Katherine Lu, Nishi Singh, Tanya Sheinin, Kathryn Matthews, David Bell.
Therapure Biopharma Inc, Canada
recombinant antibody and protein-drug conjugates are increasingly being used as therapeutics for the targeted delivery and improved safety of therapeutic drug molecules. Therapure has generated an antibody to human Cd163, a cysteine-rich scavenger receptor expressed on peripheral blood monocytes, some tissue macrophages and on a small percentage of Cd34+ hematopoietic progenitor cells (Matthews et al, 2006). We have demonstrated that incubation of the antibody (TBi 304) with Cd34+ cells and subsequent plating on colony-forming assays allows for the generation of larger, more hemoglobinized erythroid colonies (BFue) indicating an increase in erythroid proliferation and differentiation.
Background: improving the productivity of ChO cell lines is of critical importance for the manufacturing of therapeutic antibody. Following the successful creation of a stable cell line, an optimized upstream process is required to improve yields of the product of interest. A fed-batch process producing a humanized antibody is presented herein as a case study. The original process in shaker flasks had suitable yields at the end of a 9 day process. however, the same cell growth and productivity was not maintained upon scale-up to a small scale bioreactor.
Objectives: Therapure employs well-defined criteria to develop an optimized upstream process to meet titer goals. These parameters include: reactor throughput, specific production rate (SPR), growth rate (seed vs. production), maximum viable cell concentration, and maintenance of high viability. This work focuses on the optimization of culture medium, feeds, feeding schedule, and operational parameters (dO, ph, agitation, etc). Material and Method: The media and feeds are serum-free proprietary formulation purchased from media suppliers. The small-scale reactors (2.5 l working volume) are single use, the bioreactor controllers provide maintenance and control for agitation, temperature, dissolved oxygen, and ph. upstream samples collected were analyzed for 17 different parameters which included igg titers, cell density, viability, and metabolites evaluation using the BioProfile Flex analyzer (Nova Biomedical). The parameters which were targeted for optimization were grouped in three and a design of experiment approach was used to identify optimum results.
results: preliminary results show an increase of 2-3-fold over the original process.
Impact: This work utilizes a combination of well-defined parameters, single-use technology, and fast analysis to meet the project timeline.
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P.14 Universal, Fully Scalable Transfection Platform for Production of Complex or Difficult-to-Express Proteins: Superior Performance over Other Transfection Methods & Expression Systems
Mark Schmeizl, Weili Wang, Karen Donato, Rama Shivakumar, Krista Steger, James Brady, and Madhusudan
Peshwa
Maxcyte, USA
Biotherapeutic development often requires the production of gram level quantities of recombinant proteins. Transient transfection offers a means of rapidly expressing a variety of proteins including antibodies, antibody-like molecules, biochemical targets of interest, vaccines, viral vectors and virus-like particles (VLPs). Although a variety of tran-sient transfection methods are available, most do not meet the requirements of scalability, consistency and cell type flexibility. MaxCyte’s proprietary flow electroporation technology produces proteins from a variety of adherent and suspension cell types faster than creation of stable cell lines. In this poster we describe large scale electroporation us-ing the MaxCyte STX Transfection System for the production of several antibodies, including bi-specific antibodies, VLPs and a lentiviral vector. Data will be presented for high efficiency transfection of cells commonly used in pro-tein production including CHO, HEK293 and insect cells. Results from comparisons to other transient transfection technologies such as lipid reagents and PEI demonstrate the superior utility and quality of MaxCyte electroporation.
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P.15 Utilization of Recombinase Mediated cassette exchange (RMce) for the generation of recombinant cHo cell lines with minimal clonal variation
Martina Baumann1, Elisabeth Gludovacz2, Nicole Borth1,2
1Austrian Centre of Industrial Biotechnology (ACIB), Austria, Graz2University of Natural Resources and Life Sciences, Austria, Vienna
Metabolic engineering is mostly achieved by random transgene integration into the genome of host cells. The unpredictable effects of random integration, however, make it difficult to assess the influence of the gene of interest (gOi) in engineering applications, as observed variations in clone behaviour may be caused by overexpression of the gene to be tested or by genetic rearrangement following stable transfection. To circumvent this problem, the utilization of site-specific Flippase recombinases opened up the possibility to transfer GOIs into pre-selected genomic locations with defined properties, thus minimising clonal variation between the recombinant and the parental clone. in this study we successfully established a strategy for generating recombinant Chinese hamster Ovary (ChO) cell lines suitable for recombinase mediated cassette exchange (rMCE), exchanging a reporter gene for up to two transgenes and thereby integrating it at a pre-defined genomic locus. A sortable marker gene (CD4) with a leaky start codon, and flanked by two heterospecific Flippase Recognition Targets (FRTs), was stably transfected into CHO cells. The leaky start codon reduces the efficiency of protein translation and allows sorting for the highest productivities, while the FrT sites enable subsequent exchange of the expression cassette. After two rounds of rMCE using different reporter genes and FACS sorting for top producers, the stability of transgene expression was tested by removal of selection pressure, followed by re-sorting and final sub-cloning. The established clones showing stable Cd4 expression serve as reference cell lines into which the gOi(s) can be inserted without major genetic alterations. A combination of chemical selection using alternative selection markers and FACS sorting for absence of CD4 expression as the criterion for successful gene exchange enables efficient establishment of recombinant cell lines for multiple genes within one month. By choosing among different start codons (ATG, CTG, TTG) the expression of the GOI can be controlled at 100%, 10% or 1% of the maximum expression level to adjust for the desired concentration of the engineered genes.
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P.16 Stable miR-17 engineering enhance recombinant protein production in rcHo cells
Vaibhav Jadhav 1, Matthias Hackl
1, Gerald Klanert
1,2,Juan A. Hernandez Bort
1,2, Nicole Borth
1,2, Johannes
Grillari1
1Deprtment of biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
2Austrian Centre of Industrial Biotechnology GmBH, Graz, Austria.
MicrorNAs are key regulators of gene expression at the post-transcriptional level, controlling a broad range of physiological functions such as development, regulation of cell proliferation and metabolism. microrNA engineering for optimization of the efficiency of Chinese Hamster Ovary (CHO) cells is a promising new strategy for recombinant protein production. The identification and functional analysis of miRNAs can lead to knowledge-based mirNA mediated ChO cell engineering of process relevant cellular properties. Currently there are 365 ChO-mirNA genes known, thus the functional analysis of the physiological effect of such a large number requires an efficient screening method. In the current paper we present a method to perform functional characterization of mirNA effects in ChO cells. The screening assay was developed by cloning chimeric mirNA genes into a small rNA hairpin expression vector containing an emgFp cassette. initial transient screening was performed by transfection of recombinant ChO (rChO) cells and subsequent analysis of growth and productivity over a 4-day period. in this transient screening assay, mir-17 increased cell proliferation without negatively impacting productivity, providing proof of principle that the method enables the identification of miRNA engineering candidates. To further characterize the effect of mir-17, we established stable mir-17 overexpressing rChO cells. In these, miR-17 resulted in enhanced growth by significantly increasing the viable cell densities and further lead to 2 fold increase in recombinant protein production. Further analysis of stable engineered ChO cells can provide insight into mirNAs-mrNA interaction to understand the molecular basis of this effect. This can ultimately lead to more refined cell engineering strategies to improve the bioindustrially relevant characteristics of CHO cells.
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P.17 Efficient Production of High Titer Lentiviral Vectors With Caffeine
Ana Paula Costa de Pontes1,2, Marcela Cristina Corrêa de Freitas1,2, Luiza Cunha Junqueira Reis1,3, Daianne Maciely Alves de Carvalho1,2, Dimas Tadeu Covas1,2 and Virginia Picanço e Castro1
1 Fundação Hemocentro de Ribeirão Preto, Brazil, 2 Faculdade de Medicina de Ribeirão Preto, Uni-versidade de São Paulo, Brazil, 3 Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São
Paulo, Brazil
The use of lentiviral vectors has become a fundamental tool in the modification of cell lines, as well as in the production of recombinant proteins, expanding into clinical trials for gene therapy. This wide application of lenti-viral vectors is due to their great capacity to integrate into the genome of the host cells, offering a long term gene expression. in addition, these vectors also possess wide tropism and are able to transduce quiescent cells. Since the interest in using and applying these vectors is increasing, many methodologies for the production of lentivi-ral vectors in large scale have been standardized. however many of them are expensive and laborious, thus it is necessary to develop a simpler and cheaper production generating high viral titers. This work has the objective to increase the production of lentiviral vectors. We used as transfection reagent a cationic polymer, polyethylamine (pEi), in combination with caffeine. Caffeine can work as a possible viral transcription activator. We used caffeine at concentrations of 2mM and 4mM after transfection in 293FT cell line. We were able to demonstrate viral titer eight times higher using the 2mM Caffeine concentration than in the experiments without caffeine, showing that caffeine exerts an important effect on the transcription activation. The combination of pEi method and caffeine has offered us a simpler and cheaper methodology which allowed us to increase lentiviral vector titers.
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P.18 characterization of human cell lines for production of gama-carboxilation proteins
Marcela Cristina Corrêa de Freitas2,3; Aline de Sousa Bomfim1,2; Virgínia Picanço Castro2; Kamilla Swiech1,2; Elisa Maria de Sousa Russo 1,2 and Dimas Tadeu Covas2,3
1Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil, 2Fundação Hemo-centro de Ribeirão Preto, Brazil, 3Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil
There is a constant need to improve the existing expression systems, and also developing novel approaches for production of recombinant therapeutic proteins. human cell lines have emerged as a new and powerful alternative for the production of human therapeutic proteins because it is expected to produce recombinant proteins with post translation modifications more similar to human pattern. The production of the most recombinant proteins vitamin k-dependent, has as main problem an incomplete g-carboxylation performed by murine cells. The g-carboxylation involves the enzymes g-carboxylase and 2,3 epoxide reductase (VkOr) and calumenina (CAlu) that acts as cycle regulator. The aim was to select a human cell line able to produce more efficiently g-carboxylated recom-binant proteins. Three cell lines were chosen (hepg2, Sk-hep-1, hkB-11) and mrNA expression of the genes g-carboxylase and CAlu were measured before and after transduction of strains with a gene related to a recombi-nant human protein. We carried out the extraction of total rNA from each cell lines, followed by cdNA synthesis and analysis of gene expression by real time PCR. In addition to determining the efficiency of production of the recombinant protein we performed a chromogenic assay biological activity. Our data showed hepg2 cell presents as the best alternative for the production of g-carboxylated proteins, since the ratio of inhibitor CAlu compared to g-carboxylase gene was only 0.79. however, after transduction, there was an increase in the expression of the inhibitor CAlu, which made the reason CAlu / g-carboxylase became equated in the three cells: hepg2 (6.68), Sk-hep-1 (7.03) and hkB-11 (6.47) suggesting that the recombinant protein induces the expression of CAlu. The evaluation of the recombinant protein biological activity showed a production of 600 miu / 106 cels in Sk-hep-1, 300 miu / 106 cels in hepg2 and 70 miu / 106 cels in hkB-11, indicating that despite a similar reason between genes related to g-carboxylation, Sk-hep-1 cells has the best machinery for producing g-carboxylated recombinant proteins.
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P.19 characterization of human cell lines producing recombinant factor Ix
Aline de Sousa Bomfim1,2; Virgínia Picanço Castro2; Marcela Cristina Corrêa de Freitas2,3; Mário Soares de Abreu Neto2; Kamilla Swiech1,2; Dimas Tadeu Covas2,3 and Elisa Maria de Sousa Russo 1,2
1Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil, 2Fundação Hemocentro de Ribeirão Preto, Brazil, 3Faculdade de Medicina de Ribeirão Preto, Universidade de São
Paulo, Brazil
The production of vitamin k-dependent recombinant proteins in the last decades has as main impairment the low yields of functional proteins. Several factors influence this performance as the complexity of the protein structure and characteristics of the cells used as expression system. in this study we characterized two human cell lines producing recombinant human Factor ix (rhFix) according to gene expression of key proteins of the vitamin K cycle, evaluated the growth kinetics of these genetically modified cells as well as the FIX productivity. The human cell lines 293T and Sk-hep-1 were transduced with a third generation lentiviral vector loaded with Fix complementary DNA (cDNA). These cells were grown in static bottles with Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and vitamin K. The extraction of total RNA from each transduced cell line was performed, followed by cdNA synthesis and analysis of gene expression by real time polymerase Chain Reaction. The studied genes codify for proteins involved in the γ-carboxylation process: γ carboxylase, vitamin k 2,3 -epoxide reductase (VkOrC1) and the inhibitory gene calumenin. Concentrations of rhFix in supernatants were determined as rhFix antigen by an enzyme-linked immunosorbent assay and biological activity was measured by chromogenic assay. The kinetics of cell lines growth was carried out for a period of 7 days and the cell viability was assessed by trypan blue exclusion. The 293T cells presented higher expression of genes related to the γ-carboxylation process than SK-Hep-1 cells. The levels of rhFIX antigen were 28,5 mUI/mL/106
cells for 293T and 45 mui/ml/106 cells for Sk-hep-1. The biological activity of rhFix secreted by Sk-hep-1 cells was 10 times higher than 293T. The growth pattern of the cell lines presented difference between transduced and non-transduced cells. Among the transduced cells, 293T-FIX-1054 cells exhibited a maximum specific cell growth (μmax) of 0.0362 h-1, whereas SK-Hep-FIX-1054 cells showed μmax of 0.0264 h-1. The data obtained suggest that liver Sk-hep-1 cells are more promising in producing rhFix.
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P.20 engineering a Mammalian cell line Toolbox
Shelly Martin, Mark Tié, John Follit, Chapman Wright, Brian Majors, Scott EstesCell Culture Development, Biogen Idec, U.S. A.
Although the simplicity of having a single, well-characterized host upon which to initiate cell line development has many advantages, a one size fits all approach does have drawbacks. The range of product quality attributes achievable will be limited by the intrinsic genotype and phenotype of the host. unfortunately, these product attri-butes may not overlap with the optimum profile for a given therapeutic. The host proteome, particularly as it relates to folding and trafficking pathways, may differ in a manner that could influence the ability to effectively express some therapeutics. With this in mind, we have engineered a “toolbox” of different ChO and hEk293 cell lines as suitable hosts for the manufacturing of the variety of therapeutic proteins.
Using precise genome-editing technologies, we created and characterized modified mammalian hosts with tailor made phenotypes. This work describes the process in which new candidate hosts were i) adapted and evolved for enhanced performance in a platform process, ii) engineered for effective selection and amplification, and iii) sub-sequently auditioned with model proteins to assess the resulting diversity of the hosts. particular interest was paid to individual engineered host growth rate, productivity, and product quality attributes.
Across the different host cell lines generated and characterized, we saw variations in the ability to express the model proteins, frequency of potentially immunogenic glycans such as alpha-galactose and hydroxylated sialic acid, and different propensities for protein aggregation. This supports our original premise that each engineered host could contribute unique product quality attributes resulting in a “cell line toolbox”. We believe that moving forward this host cell toolbox affords us far greater flexibility and capabilities than a single host system could provide.
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P.21 large scale microRna screen in chinese hamster ovary cells reveals great potential for novel cell line engineering strategies
Simon Ficher1,2, René Handrick1, Andreas Wagner1 and Kerstin Otte.1Institute of Applied Biotechnology, University of Applied Sciences Biberach, Germany, 2Faculty of Medecine,
University of Ulm, Germany
Although the simplicity of having a single, well-characterized host upon which to initiate cell line development has many advantages, a one size fits all approach does have drawbacks. The range of product quality attributes
achievable will be limited by the intrinsic genotype and phenotype of the host. unfortunately, these product attributes may not overlap with the optimum profile for a given therapeutic. The host proteome, particularly as it relates to folding and trafficking pathways, may differ in a manner that could influence the ability to effectively express some therapeutics. With this in mind, we have engineered a “toolbox” of different ChO and hEk293
cell lines as suitable hosts for the manufacturing of the variety of therapeutic proteins.
Using precise genome-editing technologies, we created and characterized modified mammalian hosts with tailor made phenotypes. This work describes the process in which new candidate hosts were i) adapted and evolved for enhanced performance in a platform process, ii) engineered for effective selection and amplification, and iii) sub-sequently auditioned with model proteins to assess the resulting diversity of the hosts. particular interest was paid to individual engineered host growth rate, productivity, and product quality attributes.
Across the different host cell lines generated and characterized, we saw variations in the ability to express the model proteins, frequency of potentially immunogenic glycans such as alpha-galactose and hydroxylated sialic acid, and different propensities for protein aggregation. This supports our original premise that each engineered host could contribute unique product quality attributes resulting in a “cell line toolbox”. We believe that moving forward this host cell toolbox affords us far greater flexibility and capabilities than a single host system could provide.
Figure 1: Schematic overview on the non-viral miRNA delivery into CHO DG44 cells used for screening of miRNA mimics.
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P.22 adaptation of mammalian cell to Serum free Media
Sandra Fernanda Suárez; Thaissa Bernardino; Polyana Ataliba; Carlos Augusto Pereira; Renato Astray and Soraia Jorge.
Laboratorio de Imunologia Viral, Instituto Butantan, Brazil
Supplementation of culture media with fetal bovine serum (FBS) has been necessary for cell growth in vitro. however, FBS, used more often, presents some disadvantages, such as the potential to induce hypersensitivity, the variability of serum batches and the risk for contamination, besides the high cost of a good quality serum. For these reasons, current biotechnological approaches of cell culture need to avoid the use of serum. Our aim was to establish and to study mammalian cell lines adapted to serum free medium for the expression of recombinant proteins. Thus, we assessed cell growth, nutrient consumption and metabolite production kinetics in the cell culture media tested. in this work, Bhk-21 (adherent) and Bhk-21 C13 (suspension) cells were adapted to different serum free media (SFM) using sequential adaptation approach. Bhk-21 (adherent) cells were adapted to 5 different SFM: Vp®-SFM, hybridoma®-SFM, MAb® medium and ChO®-S-SFM ii; while Bhk-21 C13 (suspension) cells were adapted to 2 SFM: hybridoma®-SFM and pro293a®. kinetics parameters of adherent cells were analyzed in duplicate for 5 days in 6 well plates with 2 ml of medium. Suspension cells were analyzed in 25 cm2 T-flasks with 7 mL of medium and spinner flasks with 50 mL of medium. The initial cell concentration in all experiments was 2 × 105 cells/ml. Maximum cell concentrations of adherent cell cultures were reached after 96 hours of cultivation: 2×106 cells/ml; 2.83×106 cells/ml; 2.37×106 cells/ml; 1.36×106 cells/ml; 2.49×106 cells/ml for Hybridoma, MAb, CHO-SFM II, VP-SFM and Control (DMEM with 10% of FBS), respectively. For suspension cells in T-flasks, the maximum cell concentrations reached after 96 hours were 1.76×106 cells/ml; 1.18×106 cells/ml and 1.77×106 cells/mL for Hybridoma, Pro293a and Control (DMEM+IMDM+5%FBS) respectively. Whereas for suspension cells in spinner flasks the cell concentration achieved were 5.10×106 cells/ml; 2.20×106 cells/ml; 4.78×106 cells/ml, respectively. Metabolic analysis showed differences in the consumption of nutrients and production of metabolites among adherent and suspension lineages. in conclusion, the cell concentrations achieved in all SFM were similar to those obtained in the media containing serum, suggesting a good adaptation of Bhk-21 to the serum free media.
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P.23 Synthetic transposons for the generation of stable cHo cell pools expressing recombinant proteins
Sowmya Balasubramanian, David L. Hacker, Lucia Baldi, Florian M. Wurm
Laboratory of Cellular Biotechnology, Faculty of Life Sciences, École Polytechnique Fédérale de Lausanne, Switzerland
The generation of stable pools of recombinant cells is an alternative strategy to the establishment of stable clonal cell populations or to transient gene expression for the production of recombinant proteins. Although the generation of cell pools is rapid and scalable, they appear limited in volumetric productivity and possibly expression stability. dNA transposons (Tp) are natural genetic elements that have been engineered to catalyze the integration of a transgene into the genome of the cell. piggyBac (pB) and Tol2 are two Tps commonly used in mammalian cells. The objective of this study was to assess the efficiency of these two TPs for the generation of stable cell pools using Chinese hamster ovary (ChO) cells. Cells were transfected with two expression vectors, one vector containing the Tp repeats and, enclosed by these repeats, the gene of interest and a selection gene (donor vector) and the other encoding the transposase enzyme (helper vector). The model protein used was a fusion molecule consisting of the extracellular domain of the human tumor necrosis factor receptor 2 (TNFr) and the Fc part of a human igg1 (TNFr:Fc). Transfected cells were selected with antibiotic. The volumetric productivity of the pools was then assessed over a period of 4-6 weeks in the absence of selective pressure. The integrated TNFr-Fc gene copy number was measured by qpCr. using both pB- and Tol2-based transposons, cell pools were obtained by exposure to selective pressure for just 10 days. The volumetric productivity of the Tp-based pools was 3-4 times higher than that of pools generated by transfection with the donor vector alone. however, the average integrated gene copy number (gCN) per cell was the same for the two pools generated in the presence and absence of the pB transposase. The productivity of the pB-based pools was stable for up to 6 weeks in the absence of selection. gCN and stability studies for the Tol2-based pools are currently in progress. Overall, these studies indicate that Tps offer a more efficient approach than standard plasmid transfection for the generation of recombinant cell pools.
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P.24 engineering the cHo cell line
Bjørn Voldborg1, Henrik Claussen2, Maicheal J Betenbaugh3, Bernhard Parlsson4
1Technical University of Denmark, 2University of Copenhagen, 3 Johns Hopkins University ,4UCSD
Over the past two decades, the ChO Cell has become increasingly used as the expression host of choice in the industrial production of therapeutic proteins. despite this fact, the ChO cell as production host is poorly understood and most of the development within this area has been done to optimise the expression of a specific protein product as the end goal. As the three main drivers that have been driving the cell factory development in the microbial field, (i) whole genome sequences and analytical ”omics”, (ii) efficient genetic modification tools and (iii) genome-scale in silico models, have now become available for the ChO cells, it is now possible to adress the ChO cells as cell factories for the production of therapeutic proteins in a completely novel and more general way.
At the NNF-Center for Biosustainability (CFB), we are launching a 8-year project based on genome scale science to develop optimised ChO cell lines, with the distinct goal of creating ChO cell lines optimised for the industrial production of therapeutic proteins. using full scale metabolic models and cutting edge genetic engineering tools, we will systematically engineer ChO cell lines to improve expression, secretion, growth, glycosylation, metabolism, etc… to obtain a panel of optimised ChO cell lines specialised for high productivity of therapeutic proteins with custom-designed homogenous glycosylation, made for optimal performance under large scale bioprocessing conditions.
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P.25 a cHo-M cell combinatorial library for the Improved Selection of Recombinant Protein Production clones
Pierre-Alain GirodSelexis SA
With some proteins, optimal productivity requires more than elevated transcription. Metabolic limita-tions, trafficking backlogs, improper folding and altered post-translational modifications can all effect output. utilizing the genome and transcriptome of our ChO host (ChO-M), we have engineered com-binatorial ChO-M cell libraries over-expressing auxiliary proteins to address a broad range of these production bottlenecks. data will be presented on the use of the ChO-M libraries in generating cell lines with improved biopharmaceutical production.
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P.26 A Novel CHO Host Cell Line Using HPRT as an Amplifiable Selectable Marker for Thera-peutic Protein Production
Natalie Sealover1, Kenji Kobayashi2, Henry George1, Kevin Kayser1, and Nan Lin1
1Cell Sciences and Development, SAFC/Sigma-Aldrich, U.S.A., 2Washington University, U.S.A.
hypoxanthine-guanine phosphoribosyltransferase (hprT) is a key enzyme in the purine salvage pathway in eukaryotic cells, and it is widely used as a selectable marker in the hybridoma technology for monoclonal antibody generation. HPRT enables gene amplification that is similar to glutamine synthase (GS) and dihydrofolate reductase (dhFr). unlike gS and dhFr, use of hprT as a selectable marker has encountered a major obstacle for biotherapeutic protein production. The hprt locus has been reported to have a high mutation rate that leads to phenotype reversion in hprt (0/-) cell lines obtained by spontaneous and chemical mutagenesis. in order to overcome this obstacle, we used a novel Zinc-Finger Nuclease (ZFN) mediated genome editing technique and successfully deleted a larger portion of the hprt locus in the ChOZN® gS host cell line [gS (-/-)]. To maximize the deletion size and effectively prevent reversion, we used a single-strand oligonucleotide donor method coupled with ZFNs. After 6-thioguanine (6-Tg) selection for loss of function for hprT and clone selection, we isolated a clone with a 3.4kb deletion in the hprT gene, including the complete deletion of exon 3. The absence of hprT protein was also confirmed by immunoblot analysis. This GS/HPRT double knock-out clone was then utilized to study recombinant protein expression via gS and hprT selection methods. hAT (hypoxanthine, aminopterin, thymidine) selection was optimized to create “bulk” stable pools producing a human igg (anti-rabies). igg-producing clones from the gS and hprT selection pools were isolated and current studies are evaluating the use of both selection methods simultaneously. This is part of our ongoing effort of host cell engineering to achieve high expression levels of recombinant therapeutics. A novel amplifiable selectable marker in addition to DHFR and gS may provide additional tools, and hence versatility, in future work for producing multi-subunit recombinant proteins, or even multiple recombinant therapeutic proteins in the same production cell line.
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P.27 In-vitro modification of N-glycans using n-acetylglucosaminyltransferase III (GnT3)
Jann Catherine Ang1, Marc G Aucoin2, Guy Guillemette1
1Department of Chemistry, University of Waterloo, Canada2Department of Chemical Engineering, University of Waterloo, Canada
Post-translational modifications such as glycosylation are involved in the attachment of sugar moieties to proteins. For monoclonal antibodies (MAbs), N-glycans that attach to asparagine residues have been shown to influence Fc-receptor mediated effector functions. Of these, glycans with a bisecting N-acetylglucosamine show promising characteristics in the enhancement of therapeutic efficacy. Different strategies have been implemented to intro-duce various non-human glycosylation patterns, and in vitro modification has the potential to be developed into a scalable remodeling process that allows precise glycan remodeling. in the present study, recombinant human N-acetylglucosaminyltransferase iii (hgnT-iii) is produced in the baculovirus expression system (BEVS) in the Sf9 insect cell line and used to modify the glycan structure of the camelid MAb (EG2hFc). Specifically, EG2 anti-bodies were reacted with ß-galactosidase, sialidase and gnT-iii at 24, 48 and 72 hours incubation times to get a uniform bisecting glcNAc population in the pool. These pools of antibodies were analyzed using glycan analysis and the activity of the commercial gnT-iii enzyme was tested using a glycosyltransferase assay. The optimization of the reaction conditions and challenges are being determined to create a process that will be applicable for large scale therapeutic protein production.
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P.28 an in silico platform enabling optimization of fed-batch cultures of cHo cells producing a monoclonal antibody
Julien Robitaille and Mario JolicoeurDepartment of Chemical Engineering, École polytechnique de Montréal, Canada
Complementary to intuition, the use of an in silico platform capable to simulate hypothesis on fed-batch culture management strategies can be more than helpful. Such predictive mathematical model can contribute, on the one hand, to a better understanding of cell behaviour, and on the other hand, save time and efforts while seeking for optimal culture strategy. Since the fed-batch mode allows to favor specific culture parameter(s) by defined actions on the cell environment, i.e. composition and quality, we propose here the use of a dynamic model linking the system’s inputs (culture conditions) to outputs (ChO cells behavior). Our model thus describes ChO cell metabolic states, including intracellular fluxes and concentrations, in order to precisely describe cell behavior. in the present case, we have adapted a model to the ChO-dg44 cell line producing the Eg2 antibody, and specifically modified the model to simulate fed-batch cultures. The model, calibrated with experimental data, shows to simulate adequately the evolution of cell growth and respiration, and mAb production, with time. Moreover, culture medium concentrations of glucose, lactate, glutamine, ammonium and up to fourteen amino acids are also simulated. interestingly, it takes into account several intracellular metabolites involved in glycolysis, pentose phosphate pathway, tricarboxylic acid (TCA) cycle and energetic and redox shuttles, all simulated by the model. Model simulations agree with literature that the glycolysis is a highly regulated pathway in ChO cells. The data suggest that metabolites involved in glycolysis are at steady-state for the duration of the culture, even during the stationary phase when glucose is nearly depleted. Moreover, there are no significant changes in the levels of both ATP and ADP. On the other hand, the stationary phase is characterized by a decrease of the specific oxygen uptake rate and a decrease in the intracellular concentrations of metabolites of the TCA cycle. We also observed reverse reactions leading to both aspartate and alanine consumption. data suggest that there is a shift in the cell metabolism at glucose depletion, a phenomenon simulated by the model. At this stage of development, our model shows to be a valuable in silicon tool to assist in defining fed-batch culture strategies.
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P.29 Shortened Timeline for cell line Development – adaption of Recombinase-Mediated cassette exchange (RMce) to Suspension cHo cells
Elisabeth Bludau1, Nathalie Veith 1, Volker Hecht1 and Holger Ziehr1
1Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Department of Pharmaceutical Biotechnology, Braunschweig, Germany
The first step in manufacturing recombinant glycosylated therapeutic proteins is the search for a high and stable expressing cell clone. in contrast to traditional approaches for cell line development Fraunhofer iTEM developed an alternative strategy called recombinase-mediated cassette exchange (rMCE). Traditional concepts for the development of cell lines are based on random integration of the expression vector containing the gene-of-interest (gOi) which makes the screening for an appropriate cell clone very time and labor intensive. The application of the rMCE-strategy based on the site-directed integration of a single copy of the gene-of-interest into a transcriptional, exchangeable and known genomic locus dramatically shortens the timeline for the cell line development. in order to mark the genomic locus, ChO-cells were initially transfected with either a lentiviral or plasmid based Tagging vector containing a selection and a fluorescent marker flanked by two heterospecific recombination target sites. The transfected cell pool was subsequently screened for highly expressing, stable cell clones with an exchangeable locus. The following exchange of the Tagging versus the Targeting cassette, carrying the gene-of-interest (gOi), was catalysed by Flp-recombinase. Enrichment of the gOi-producing cell population was made without drug selection. The correct exchange of the Tagging against the Targeting vector was confirmed via PCR. Targeted clones were further analyzed concerning their productivity, growth and long-time stability. Both lentiviral and plasmid tagged ChO suspension cells were targetable and showed high gOi expression after two rounds of selection without antibiotics. Targeted clones were able to be cultivated in suspension again which allows a potential scale-up. PCR analysis further verified correct exchange and integration of the Targeting vector. Targeted clones showed stable expression without application of selection pressure. Additional experiments are in progress to examine whether a certain genomic locus is suitable to express only certain protein classes.
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P.30 Reduction of fucosylation of Monoclonal antibody Glycans Using Several Glycosylation Inhibitors
Maureen Spearman, Natalie Okun, Sarah Chan and Michael ButlerDept. of Microbiology, University of Manitoba, Canada
Biotherapeutic monoclonal antibodies (Mabs) are now commonly used as treatments in cancer, autoimmune diseases and other conditions. Although commercialization requires high productivity of Mabs in recombinant cell lines, product quality, with respect to posttranslational modification is equally important. N-linked glycosylation of the Fc region (Asn297) in Mabs plays a critical role in their ability to elicit effector functions. Oligosaccharides with low fucosylation allow the Fc region to interact more efficiently with Fc receptors allowing greater effector activation, such as antibody-dependent cell-mediated cytotoxicity (AdCC). reduction of core fucosylation and an increase in bisecting glcNAc of Asn297 oligosaccharides has been the focus of several strategies using sirNA and genetic engineering. An alternative method is to use media additives that modify glycans at discrete points during the glycoprotein processing reactions in the golgi. The objective of this study was to produce Mabs with a wide range of glycan microheterogeneity in order to study the effect of glycosylation on stability and function. We have compared two recombinant ChO cell lines: one produces a chimeric camelid-human Mab (ChO-Eg2) that lacks light chains, and the second is a humanized murine igg (ChO-dp12 clone 1934). Several glycoprotein processing inhibitors (kifunensine, swainsonine, castanopermine) have been used to alter glycosylation and reduce fucosylation. Mabs can be efficiently produced in the presence of these inhibitors with some effect on growth or productivity of the cells. glycosylation of the Mabs was determined with hiliC (hydrophilic interaction liquid chromatography) analysis showing the modified glycan chains. We have also used a new fucosylation inhibitor, a fluorinated analogue of fucose (2F-peracetyl-fucose), which is incorporated as GDP-F-fucose and inhibits the FUT 8 fucosyltransferase. With this inhibitor we are able to produce ChO-Eg2 with variable core fucosylation, while maintaining the remainder of the glycan structure.
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P.31 effect of redox potential on antibody glycosylation in mammalian cell cultures
Ben dionne, Michael Butler university of Manitoba, dept. Of Microbiology, Canada
Consistent and proper glycosylation is very important in ensuring Mabs (monoclonal antibodies) efficacy and effectiveness. The consensus glycan in an immunoglobulin has often been related to its position in the interstitial space between the disulfide bonded CH2 domains of two heavy chains. This can reduce the accessibility of glycosyltransferase enzymes particularly the galactosyltranfersase that adds the terminal galactose to form the g1 or G2 structures. Lowering culture redox potentials (CRP) may disrupt Mab interchain disulfide bonds and lead to different glycan profiles. Using CHO and NS0 cell cultures, several Mabs were analysed for their glycan profiles; including 2 humanized igg1s and a humanized camelid ig in the presence of a reducing agent. Cell cultures of ChO and NS0 were subjected to various concentrations of reducing agent (dithiothreitol;dTT) and monitored daily for growth parameters and redox potential. using hiliC-hplC methods, shifts in the gi (galactosylation Index) were monitored in the presence of DTT. In addition to glycan profiling, IgG1 from NS0 cultures were radiolabelled to determine the assembly pathway and changes in pathway intermediates in the presence of the reducing agent. The GI was decreased in NS0-IgG1 cultures by as much as 35% in those cultures with lower CRP. In contrast, CHO-IgG1 cultures had no change in GI and the camelid Ig exhibited a 16% increase in GI. The autoradiographs of the protein A purified intracellular NS0-IgG1 verified an assembly pathway of HC →HC2→LCHC2→LC2HC2. Densitometry analysis of assembly intermediates showed that the ratio of heavy chain dimer to heavy chain monomer increased over time within the reducing agent cultures. A correlation between redox potential, gi shifts and assembly intermediates is suggested by the data for this particular igg1. This could have wide ranging implications for process development activities and lead to control mechanisms that influence glycan profiles of Mabs.
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P.32 effects of Glucose and Glutamine concentrations on the Glycosylation of cHo eG2-hfc monoclonal antibody.
Carina Villacrés Barragán1, Erika Lattová2, Michael Butler1
1Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada; 2Chemistry Department, University of Manitoba, Canada,
Biological systems are capable of reacting to minimal changes in the environment. These changes can perturb normal cell function and metabolism, which in turn may affect pathways involved in glycosylation. Thus, different strategies in media and feed development have been implemented not only to maximize production rates from available nutrients and minimal byproduct formation, but also to improve product quality. The objective of this study was to determine whether low concentrations of glucose and glutamine had an effect on the glycosylation of a human-camelid monoclonal antibody (ChO Eg2-hFc mAb) synthesized during batch culture. For this purpose, cells were cultured for 18 hours in an adaptation media and then were split in different shaker flasks with a matrix of various ratios of glucose (3mM to 25mM) and glutamine (0mM to 4mM). Cell growth, substrate consumption, by-product formation and antibody concentration were measured. The chosen range of glutamine did not affect cell growth and productivity. However, as glucose became a limiting nutrient, specific cell growth and specific cell productivity declined. glycosylation was analyzed using hydrophillic interaction liquid chromatography (HILIC). The mAbs glycan profile showed similarities in the oligosaccharide compositions, bearing biantennary core fucosylated glycans associated with igg1 molecule. however, an increase in the presence of biantennary fucosylated glycan with zero galactose residues was observed in mAb produced under low glucose concentrations. A difference in sialylated glycans was also observed; they were higher in mAb produced in media containing 25mM glucose in comparison to mAb produced under low glucose concentrations. A galactosylation index of 0.6 was obtained in mAb produced under low glucose concentrations (3 and 5.5mM), where cells were glucose-deprived for 48 and 24hours respectively, compared to 0.7 under normal glucose concentrations (25mM). Non-glycosylated forms were detected only under low glucose concentrations, results that were confirmed by mass spectrometry analysis, indicating a change in the macroheterogeneity. Those results altogether indicate that glucose affected cell growth, productivity and glycosylation of the Eg2-hFc mAb, which can be a result of changes in the nucleotide sugar availability during the glycosylation process.
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P.33 High-level Recombinant Protein Production in cHo cells Using the cumate Gene-Switch in combination with lentiviral Vectors or optimized Pool Selection Protocol
Nasha Nassoury1, Bruno Gaillet3, Adeline Poulain2, Louis Bisson1 Félix Malenfant1, Sylvie Perret 1, Rénald Gilbert1, Alaka Mullick1, 2, Yves Durocher1, 4, Bernard Massie1, 2
1Human Health and Therapeutics Portfolio, National Research Council Canada, Canada. 2Départment de microbiologie et immunologie, Université de Montréal, Canada. 3Chemical Engineering Department, Université
Laval, Québec, Canada. 4Départment de biochimie, Université de Montréal, Canada
in order to deal with the growing demand of large quantities of therapeutic proteins in a timely fashion, expression systems are being optimized to improve the time of generation of stable clones as well as the levels of protein secretion. This can be achieved by a combination of expression cassette optimization, cell engineering and selection process. We have previously developed the cumate gene-switch, which is a very efficient expression system for protein production, and we have shown that the cumate-inducible promoter (Cr5) is the strongest promoter we have tested so far in ChO cells. We were able to generate stable pools capable of producing 160-235 mg/l of therapeutic proteins using the cumate gene-switch in combination with lentiviral vector (lV) transduction. The best subclones are showing an increased productivity of only 1.5-1.8 fold, indicating that the ChO pools generated by lV transduction are fairly homogenous.recently, we have developed a new ChOBri/Cum2 platform for generating stable pools expressing high levels of recombinant proteins. Following an optimized transfection and selection protocol these pools outperform those generated by lV transduction. Our ChOBri/Cum2 platform allows generation of CHO pools stably expressing high-level of recombinant proteins (>200 mg/L) in less than 6 weeks. The best ChOBri/Cum2 clones isolated from the pools generated by transfection show increased productivity of up to 4-fold (2.5 times more than lV pools ), yielding volumetric productivity of up to 1.5 g/l (in batch). This ChOBri/Cum2 platform is thus not only more efficient than LV transduction for rapid production of therapeutic proteins in ChO cells but is also readily amenable to biomanufacturing under gMp as it is based on an established and documented cell line.
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P.34 Differential affects of low glucose on the macroheterogeneity and microheterogeneity of gly-cosylation in cHo-eG2 camelid monoclonal antibodies
Liu Bo, Erika Lattova, Maureen Spearman and Michael Butler
The objective of this study was to determine how variable concentrations of glucose affect the glycosylation pat-terns of a chimeric heavy chain antibody (Eg2), with a camelid Fab domain fused to a human Fc region, produced by stable expression from CHO cells. The cells were inoculated at high density (>106 /ml) into growth media containing 0-25 mM glucose for 24 h. during the 24 h incubation glucose was completely depleted in cultures at ≤15 mM initial glucose concentration. A proportion of the Protein A-purified Mab was non-glycosylated in the cultures in which glucose was depleted. The fraction of the non-glycosylated Mab was directly correlated to the exposure time of cells in media depleted of glucose. Deprivation of glucose for the full 24h resulted in a 45% non-glycosylated Mab fraction.
Steady state levels of lipid-linked oligosaccharides (llOs) were analyzed by organic solvent extraction from the cells after the incubation. glucose depletion resulted in a reduction in the amount of full length llO (glcNac-2Man9glc3; glcNAc: N-Acetylglucosamine; Man: mannose; glc: glucose), with a concomitant increase in the production of smaller mannosyl-glycans (glcNAc2Man2-5).
The microheterogeneity of the glycans of purified Mabs accumulated over the 24 h incubation was manifest as variable galactosylation and sialylation. Expression of these parameters as galactosylation and sialylation indices (GI and SI) showed that the values were directly correlated to the cell specific glucose uptake over the incubation period. The high level of galactosylation of this antibody may be due to its overall low molecular weight chimeric structure or to 3 amino acid changes that were observed in the human Fc region.
This work is important because it relates to the variable levels of glucose that may occur in large-scale fed-batch cultures for therapeutic Mab production.
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P.35 Towards tailored insect cell lines to improve recombinant protein production in both stable and transient baculovirus infected cell lines
Maren Bleckmann, Steffen Meyer, Bahar Baser and Joop van den HeuvelHelmholtz Center for Infection Research, Inhoffenstraße 7, 38124 Braunschweig (Germany)
The Baculovirus Expression Vector System (BEVS) in insect cell lines is a widely used expression system for recombinant protein expression as it offers high yields of recombinant target proteins, posttranslational modifications and cost-efficient cultivation. However, being a lytic system it may only be used for batch production. Moreover, virus infection compromises the secretory pathway resulting in low yields of recombinant expression of membrane-bound and secreted proteins.
Therefore, stable expression in insect cells is desired for both productions of target proteins as well as for the expression of auxiliary proteins that enhance the post-translational modification and secretion of difficult to express target proteins introduced by recombinant baculoviruses.
here we present a versatile recombinase-mediated Cassette Exchange (rMCE) platform for targeted integration into the host cell genome that provides reduced epigenetic effects on recombinant gene expression by avoiding random integration. The system is based on a highly flexible exchange vector for expression in both insect and mammalian cell lines. during rMCE the Flipase recombinase (Flp) precisely excises a marker gene of the master cell line and introduces the gene of interest. Several improvements of the rMCE system like an insect cell opti-mized Flp and the combination of positive and negative selection traps aim to increase the efficiency of the cassette exchange in our insect master cell lines to provide a highly flexible and fast insect protein expression platform.
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P.36 a Stirred Tank bioreactor for Human Mesenchymal Stromal/Stem cell expansion Main-tains Desired cell characteristics
Kimberly Mann, Sandhya Punreddy, Anjali Verma, Manjula Aysola, Neethu Sunil, Donghui Jing, Daniel Kehoe, Knut Niss, Martha Rook, Julie Murrell
EMD Millipore Corporation,
human mesenchymal stromal/stem cells (hMSCs) are being used extensively in clinical studies as therapeutic agents and as drug discovery tools. Traditional culture methods involve using cumbersome multilayer flatbed culture systems. in this work we present an expansion paradigm that uses a scalable, single use, stirred tank bioreactor with microcarrier scaffold for hMSC expansion. A bioreactor system enables direct monitoring for the specific characteristics of hMSCs at any point during the expansion, thus assuring product quality and consistency. in addition, a bioreactor provides ease of use in handling and lower medium volume requirements. hMSCs cultured in both systems (flatbed and bioreactor) were evaluated using flow cytometry to assess a variety of markers that denote identity and purity. No differences were observed in the positive or negative cell surface markers between the expansion schemes. in addition to protein expression, we examined genetic integrity using comparative genomic hybridization technology. While no differences were seen at the 500kb or 50kb levels we observed differences in a small subset of the targets at the 15kb resolution levels. Differentiation potential was then confirmed using traditional protocols. In this work, we verified that the cells expanded in the single use stirred tank bioreactor were identical in phenotypic and genotypic profile in comparison to flat culture and maintained the desired cell characteristics of hMSCs making this a viable alternative to the traditional flatbed expansion systems.
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P.37 Sweetbac: The way for better pharmaceuticals in Trichoplusia ni cells dieter palmberger1, kazem Ashjaei2, imre Berger3, gary Blissard4, karin hoffmann-Sommergruber2, reingard
grabherr1
1VIBT - Vienna Institute of Biotechnology, University of Natural Resources and Life Sciences, Austria2Dept. of Pathophysiology and Allery research, Medical University of Vienna, Austria
3EMBL – European Molecular Biology Laboratoy, France4Boyce Thompson Institute at Cornell University, USA
The baculovirus/insect cell system has proven to be a powerful tool for the expression of eukaryotic proteins. Nevertheless, these proteins sometimes suffer from reduced biological activity. Several studies have demonstrated that glycosylation can greatly influence the structure, function, antigenicity, and immunogenicity of various, especially viral, glycoproteins. yet, the glycosylation pattern of insect cell-derived products is not favourable for many applications. We therefore investigated flexible and efficient methods for improving immunogenicity of Trichoplusia ni expressed Influenza A Virus-Like particles and at the same time for reducing negative side effects.
dendritic cells (dC) have been shown to play a key role in the initiation of immune responses by binding and up taking antigen via mannose-receptor mediated endocytosis. We therefore, generated a SweetBac backbone, based on the recently sequenced transcriptome of Trichoplusia ni, allowing for the gene knock-down of α-mannosidase i. The resulting oligomannosidic Vlps were tested for enhanced binding and uptake characteristics to naive dCs.
The presence of specific carbohydrate structures, mainly core α1,3-linked fucose, has been shown to cause allergic reactions in some patients. To diminish the fucose de novo systhesis pathway, we integrated the enzyme gdp-6-deoxy-d-lyxo-4-hexulose reductase from Pseudomonas aeruginosa in the SweetBac backbone. This setup gives the possibility to produce Vlps almost free of fucose. Engineered Vlps were assessed for a reduced allergenic potential.
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P.38 CAP: An Efficient Platform for Transient and Stable Protein Production based on Immortalized Human amniocytes
Silke Wissing, Corinna Bialek, Sabine Hertel, Helmut Kewes, Tanja Spörk, Markus Ribbert, Michael Schomberg, Jens Wölfel, Gudrun Schiedner, Nicole Faust
CEVEC Pharmaceuticals, Germany
production of biotherapeutic proteins encounters many challenges concerning protein yield and quality. We have found that the CAp system, based on immortalized human amniocytes, represents a protein production platform that can efficiently address some of these challenges. While the system was specifically designed for successful production of complex glycosylated proteins, it has also proven useful for expressing antibodies of different IgG classes and for vaccine production. A modified version of CAP cells (CAP-T) which is very similar to the CAp cells is highly suitable for transient protein expression and yields proteins with the same human-like post-translational modifications (PTM) as CAP. Taken together, the CAp technology presents a system enabling transient and stable protein expression of a wide variety of biotherapeutic proteins.
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P.39 construction of an antiviral recombinant protein using a baculovirus/ Sf-9 cells system
R.Z. Mendonça1, D.N.S. Giovanni1, R.M.Z. Mendonça2, Figueiredo, C. A.3 ; Oliveira, M. I.3 ; Silva, P.E3; Curti, S. P 3; R.H.P. Moraes 1 A.C.V. Carmo1
1Laboratory of Parasitology and Entomology, Instituto Butantan, São Paulo/SP, 2Laboratory of Virology, Instituto Butantan, São Paulo/SP, 3Núcleo de Doenças Respiratórias, Centro de
Virologia, Instituto Adolfo Lutz, Brazil
The control of human and animals virus infections is of great interest in public health. Effective control of viral infection has remained an unachieved goal, due the limited availability of anti viral drugs, the intracellular replication characteristic of viruses and high possibility of genome mutation. Several studies have shown the presence of antiviral activity in compounds obtained from plants. Antiviral active properties in the hemolymph of arthropods had been described, some of which are of interest for the development of new pharmacological drugs. recently, we have demonstrated the existence of a potent antiviral property in the hemolymph of Lonomia obliqua caterpillars. Experiments with the whole hemolymph led to a 157-fold reduction in measles virus production, 91-fold reduction in picornavirus virus production and 64-fold reduction in Influenza virus (H1N1) replication. in this study, our aim was to produce a recombinant protein of this protein in a baculovirus/Sf9 cell system. The resulting bacmid contains the sequence coding for the antiviral protein previously described by our group. Total rNA from L. obliqua caterpillars were extracted with Trizol and used in the reverse transcription assay with oligo(d)T primer followed by polymerase chain reactions (RT-PCR) with specific primers for the cDNA coding for the antiviral protein, based on the sequence deposited in the genBank database. restriction sites were inserted in the cdNA for ligation in the donor plasmid pFastBac1™. The recombinant plasmid was selected in Escherichia coli dh5a and subsequently used in the transformation of E. coli dh10Bac for the construction of the recombinant bacmid. This bacmid was used for the expression of the antiviral protein in the baculovirus/Sf9 cell system. After identifying the protein by western blot, activity tests were performed, showing that the recombinant protein was able to reduced the viral replication. When the whole antiviral recombinant protein was tested, a reduction in picornavirus production was of 1.069 fold. After recombinant protein purification, the virus reduction titers were of 106 times to herpes virus and 10,000 folds to rubella or picornavirus, showing that the recombinant antiviral protein remained fully active and that this product is a strong candidate for use as an antiviral agent. At the moment, we are performed studies for optimization of the production of this recombinant product.
financial support: FApESp (08/57263-5), CApES.
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P.40 co-expression of protein phosphatases in insect cells affects phosphorylation status andexpression levels of proteins
Eilleen McCall1, Rebecca Williams1, Mark Abbott1AstraZeneca,
The activity of kinases is regulated by phosphorylation on Ser, Thr or Tyr residues within the activation loop. The ability to produce these enzymes recombinantly with a specific phosphorylation status is essential in order to understand structure and function. This poster describes a screening approach to co-express different phosphatases together with a kinase in the baculovirus expression system. This enables the testing of different phosphatases as well as different levels of both phosphatase and kinase by varying the multiplicity of infection (MOi) of the different baculoviruses. This approach translates well to a larger scale. An unexpected observation was that co-expression of the phosphatase could have profound effects on expression levels even of heterologous target proteins that would not be a substrate for the phosphatase. This was most apparent with lambda phosphatase, an enzyme that removes phosphorylation from Ser and Thr residues, where expression was almost completely abolished for all proteins even at modest MOis. The effect of lambda phosphatase was observed irrespective of whether co-expression was from two separate baculoviruses or from two genes on the same vector. The effect was shown to be due, in part at least, to a decrease in transcription.
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P.41 Improving Protein Secretion in the baculovirus-Insect cell expression System
Cindy Chang, Emilyn Tan, Linda H.L. LuaThe University of Queensland, Protein Expression Facility, Australia
The baculovirus – insect cell expression system is emerging as a favourable system for the production of recombi-nant glycoproteins due to its eukaryotic protein processing capabilities. Low secretion efficiency is often a bottle-neck for this system. To improve protein secretion using the baculovirus-insect cell expression system, varying expression conditions were tested on three glycoproteins in a high-throughput expression platform. parameters include different baculovirus systems (Bac-to-Bac® and flashBAC™), signal peptides (honeybee melittin signal sequence, glycoprotein gp67 signal sequence and novel signal sequence), insect cell lines (high Five™ and Sf9) and culture temperatures. Overall, higher protein secretion is observed in high FiveTM cell line. interestingly, no notable enhanced secretion is observed in the modified FlashBACGOLD™ system, which contains a dual gene deletion (chitinase chiA and cathepsin v-cath) postulated to improve protein secretion. The effect of the different signal peptides on secretion efficiency varies amongst target proteins. Lowering the culture temperature has an impact on the stability and secretion efficiency. Optimisation of the expression parameters is imperative to achieve better secretion efficiency and quality of glycoproteins.
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P.42 Use of mammalian expression systems for secreted proteins at astraZeneca; faM3b PanDeR a new type of signaling molecule
Lovisa Holmberg Schiavone1, Jenny Gunnarsson1 and Patrik Johansson2
Reagents and Assay Development (1) and Structure and Biophysics (2) Discovery Sciences, AstraZeneca, Mölndal, Sweden
in reagents and Assay development we work with a considerable number of secreted protein targets with the aim to be able to establish robust gene-to-protein supply for all. To increase our success we have recently modified our processes for two mammalian systems; hEk293-6E (1) and a proprietary ChO-based system (unpublished results). The purpose of these changes was to make robust and economic protocols that could be used at many different scales with minimal change. improvements included different transfection protocols, culture conditions, feed of cultures, temperature shifts to maximize yields and protein engineering techniques to increase expression levels and purified protein yields. One recent example is production of FAM3B, PANDER which has been shown to regulate glucose homeostasis and beta cell function (2). Earlier studies describe production of pANdEr in Pichia pastoris with production of multiple truncated variants starting at different N-terminal amino acids (3). in the case of pANdEr we explored several different expression systems before establishing a robust protocol with hEk293-6E cells. Changing of signal peptide from the native sequence to Cd33 increased yields up to 100 mg / l and also secured production of full-length uniformly processed pANdEr. Access to larger quantities of pANdEr protein enabled us to do follow up structural studies to elucidate the structure-function relationship which has been elusive. The structure of murine pANdEr shows that the protein exhibits a novel fold which shares no relation to the predicted four-helix cytokines but is conserved throughout the superfamily (4). We are now continuing the work, and are aiming to identify the receptor for pANdEr and related FAM3C ilEi and to elucidate the biology of this exciting class of signaling molecules.
1. durocher et al 2002. Nucleic Acids research 30; E9.2. Wilson et al 2011. FEBS letters 585, 21373. Zhu et al 2002. genomics 80, 1444. Johansson et al 2013. Structure 21, 306
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P.43 Protein expression at the national Institute of environmental Health Sciences: assisting the scientists with protein expression and purification needs with a multiple system approach.
Molly E. Cook, Gary K. Powell, Lori L. Edwards, Christine C. Malone, Robert M. PetrovichLaboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, USA
The protein Expression Core Facility (pECF) was created in Feb. 2003 to support intramural investigators at the National institute of Environmental health Science (NiEhS) by meeting their protein expression needs for projects that range from cell-based assays to structure studies. The Core does this by implementing multiple expression approaches that include bacterial, insect and mammalian expression systems. This is primarily achieved using invitrogen’s gateway® technology which allows for the placement of a single gene of interest into multiple expression systems without the need to subclone. depending on the expression system and the investigators needs, a variety of constructs are created via the gateway® system using tags such as hisx6, gFp, gST, MBp and NusA. These tags also function downstream in improving solubility and/or purification of the targeted proteins.
With the E. Coli expression, the pECF initially uses rosetta2TM (dE3) placl cells. if further optimization is required, Bl21 (dE3), Bl21 (dE3) Codon plus, Bl21 Ai, and Arctic Express lines are all available for screening. Sf9 is the primary insect cell used for protein expression with the Baculovirus expression system (BES). Two additional technologies the pECF uses with the BES are the use of Sf-9ET cells for determining viral titer and the TipS/BiiC technology for long-term storage of baculovirus viral stocks. A transient expression system, BacMam, merges the Baculovirus and Mammalian expression system by using virus produced by the transfected insect cells to transduce and express genes in a variety of mammalian cell lines. COS7, hEk293-f, and ChO-s cells are the primary cells used for protein expression in mammalian cell culture.
in summary, through the use of a variety of expression systems, the pECF aids NiEhS investigators in achieving their protein expression goals and introduces novel technologies and systems to optimize protein expression levels and purification yields.
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P.44 Production of viral particle for the nS3-HcV expression in mammalian cells
Lemos, MAN; Paschoal, JFB; Rezende, AG; Pereira, CA; Jorge, SAC.
One of the major health problems in the world is hepatitis caused by hepatitis C virus (hCV), given that the current treatment provides satisfactory immune response only to some patients. Multiple approaches have been tested in order to develop a vaccine to prevent hepatitis C. dNA vaccines and virus-like particles, carrying portions of hCV genome are under development. Within this perspective, our work aims to produce two viral pseudoparticles. One is based on the Semliki Forest Virus (SFV-hCV) system and the other on chimeric particles of Murine leukemia Virus (MlV) and hCV (pphCV). Both systems carry the gene for a portion of the nonstructural protein 3 (NS3p) of hCV. These pseudoparticles have been produced and used for “infection” of mammalian cells in minimal medium (supplemented with 10% FBS) or serum free media (SFM). Our results show a production of 1.2E4 of SFV-NS3p particles per microliter and 4E2 of pphCV-NS3p particles per microliter. in addition to the production of the viral pseudoparticles, we performed the adaptation of human hepatoma cells (huh7), in SFM (ChO®, Vp® and prO293®). infection the cells with the pseudoparticles produced is under investigation in order to compare the expresion the NS3p expression in different systems and conditions. The protein production is expected to higher in cells grown in SFM thar can promote a higher entry of particles in cells.
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P.45 Transfection of HeK293 and cHo Mammalian cells using Polyethylenimine-based Poly-plexes
Laurence Delafosse and Yves DurocherNational Research Council Canada
Mammalian transient protein expression platforms allow the production of biogically active proteins within a few days following transfection. polyethylenimines (pEi) are cationic polymers which are synthesized as either linear (l-PEI) or branched (b-PEI) forms and shown to be among the most efficient non-viral DNA carrier for in vitro gene delivery. Once added to DNA, PEIs associate with and efficiently condense DNA, forming PEI-DNA nanoparticles termed “polyplexes”. despite optimization of transient gene expression in mammalian cells, protein expression efficiencies varies among cell host system. Polyethylenimine formulations and medium composition contribute as well to gene expression variability. Thus, efforts must be strengthened on clarifying the pEi-based transfection mechanisms. in order to identify differences that may explain the variability in transfection poten-cies reported, we looked at some biological parameters in human Embryonic kidney 293 (hEk293) and Chinese hamster Ovary (ChO) cells transfected with the linear and branched 25 kda pEi, 40kda pEimax and pEipro. Both cell types were grown in suspension in FreeStyle™ F17 serum-free medium. We evaluate the binding and internalization kinetics of polyplexes using fluorescently-labeled DNA as well as transfection efficiencies (BFP) and secreted protein expression levels (herceptin). localization of polyplexes within the cell was monitored by confocal microscopy.
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P.46 Is the low cell specific yield of Baculovirus infected Insect cells at high cell densities due to a limitation of transcription rather than translation?
S. Reid, H. Huynh, T. Tran, L. Nielsen and L. ChanAustralian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, Brisbane
4072, Australia.
The baculovirus-insect cell system has been extensively studied for the production of recombinant proteins, vaccines and vectors for gene therapy, as well as biopesticides to control various insect pests. For low cost applications (animal vaccines and biopesticides), yield improvements are required to make this production platform more cost competitive against alternatives. The cell density effect is a well-established constraint in the baculovirus-insect cell expression platform, in which cell-specific productivity declines with increasing cell density, hence limiting the maximum achievable volumetric yield of protein product. A deeper elucidation of this phenomenon is sought in this study, by tracking the peak production of viral DNA (vDNA), recombinant β-galactosidase (β-gal) mRNA, and β-gal protein, over a wide range of cell densities for both Sf9 and Hi5 cells. In addition similar studies were conducted to investigate occlusion body (OB) production following the infection of hzea cells using a wild type hearNpV baculovirus. Sf9 and hzea suspension cell cultures were propagated in Sf-900 iii serum-free medium, and synchronously-infected with rβgal-AcMNPV or HearNPV respectively, at multiple infection cell densities of between 0.5-8×106 cells/mL. The related Hi5 cell studies were conducted with the same rβgal-AcMNPV virus used for the Sf9 cells using Express Five® serum-free medium. As expected there was a strong negative linear correlation between the specific β-gal/OB yield and the peak cell density (PCD) post infection. However there also was also a corresponding strong negative linear correlation between the specific vDNA or β-gal/OB mRNA yield, and the PCD. These findings suggest that protein yield deterioration with increasing cell density originated from limitations during upstream events such as virus gene replication or transcription, rather than during the translational phase. Such limitations may be largely nutritional, but a more complex mechanism may be implicated. For all three cell/virus systems the high MOi infections used resulted in minimal cell growth post infection and similar infection kinetics regardless of the cell density at the time of infection. This suggests that the infections at high cell densities proceed as normal but the production of viral DNA, mRNA and protein is less efficient than is the case at lower cell densities.
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P.47 Uncovering Methods for the Prevention of Protein aggregation and Improvement of Product
Quality in a Transient expression System
Bram Estes1, Raj Haldankar2, Yueh-Rong Hsu1, Lei-Ting Tam1, and Jackie Sheng1
1Biologics, Amgen, Inc., Thousand Oaks, CA, USA, 2 Protein Science, NGM Biopharmaceuticals, Inc., USA
Mammalian expression systems are routinely used for the production of recombinant proteins as therapeutic mol-ecules as well as research tools. Transient expression has become increasingly popular in recent years due to its rapid timeline and improvements in expression level. While improvements to transient expression systems have focused mainly on the level of protein expression, the aspect of protein quality has received little attention. The removal of undesirable products, such as aggregation, depends primarily on purification, requiring additional cum-bersome steps which can lead to a lower product yield and longer timelines. An observed discrepancy between the aggregation of an ECd-Fc protein generated in transient versus stable platforms compelled us to explore issues of product quality, which frequently arise among recombinant proteins expressed in transient systems. in this study, we have identified expression conditions that can be used to effectively improve the quality of molecules prone to aggregation. Specifically, expression under mildly hypothermic conditions and performing transfections at a lower gene dose were identified as advantageous approaches. When used in combination, these approaches had a complementary effect. The observed improvements not only minimize aggregation levels to mimic that observed from stable cells, but also generate product with overall superior quality such as more homogeneous signal peptide cleavage, N-linked glycosylation profiles, and preserved biological activity. These techniques have since been applied to a variety of other molecules and have produced a similar improvement in product quality, suggesting that this may be a general approach to enhance product quality from transient expression systems.
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P.48 Large-scale Production and Purification of the Mouse Serotonin 5-HT3A Receptor and Generation of functional antibody fragments
Gherici Hassaine1, 2, Cédric Deluz2, Hugues Nury2, Horst Vogel2 and Kenneth Lundstrom3
1Theranyx, Marseille, France, 2Swiss Federal Institute of Technology, Lausanne, Switzerland, 3PanTherapeutics, Lutry, Switzerland
ion channels constitute therapeutic targets for treating a number of human diseases such as emesis, epilepsy, depression, analgesia, sleep disorders, parkinson’s disease and Alzheimer’s disease. however, the existing ion channel-based drugs have been hampered by shortcomings such as poor selectivity and dose-limiting side effects due to drug screening procedures lacking established robust high throughput assays and methods for evaluation of functional activity. We have recently cloned the mouse 5-hT3A receptor cdNA with an N-terminal Strep-tag into the pcdNA5 TO vector and generated stable T-rex ChO cell lines. induced ChO cells were subjected to large-scale 5-hT3 receptor production resulting in 6-8 mg receptor per liter culture and followed by solubilization in presence of the C12E9 detergent. A single step affinity purification using a strep tag provided pure protein in active form at milligram scale. Purified 5-HT3A receptor was used for immunization in camelids. Generated antibodies selected by phage display comprised efficient binders for the 5-HT3 receptor. Among 21 antibodies, specifically the nVhh7, nVhh15 and nVhh16 antibodies were demonstrated to possess biochemical and functional activity. The characterized antibodies and other antibodies to be discovered in the future might be applied in drug discovery, target validation, pharmacology, diagnostics, and as chaperons for receptor crystallization.
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P.49 characterization of a novel protein antiviral from Lonomia obliqua using bioinformatics tools and activity analysis by real time. 1Carmo, A.C.V.,
1Giovanni, D.N.S., 2Yamasaki, L. H. T., 3Figueiredo, C. A., 3Oliveira, M. I., 3Santos, F. C. P., 4Curti, S. P., 1Tonelotto, M., 2Rahal, P., 5Moraes, D., 1Mendonça, R.Z.
1Laboratory of Parasitology and Entomology, Instituto Butantan, Brazil2Laboratory of Genomic Studies, IBILCE-Unesp, Brazil
3Center for Respiratory Diseases, Center for Virology, Instituto Adolfo Lutz, Brazil 4Center for Vector Transmitted Diseases, Center for Virology, Instituto Adolfo Lutz, Brazil
5Faculdade Oswaldo Cruz, Brazil
The control of viruses is of great interest to the public health area. Several studies have been conducted that show the presence of pharmacologically active substances in the hemolymph of insects. recently we have demonstrated the existence of a potent antiviral in the hemolymph of Lonomia obliqua caterpillar. This protein was able to reduce at 106 times the replication of herpes virus and in 10,000 fold the rubella virus. Assays using rT-pCr to determine viral rNA present in no treated and rAVlO treated infected cells also showed a reduction in the same scale. The analysis of this protein by bioinformatics suggests that this protein is globular, secreted with a signal peptide which is cleaved between amino acids 16 and 17. The studies also allows us to infer that this antiviral protein has the ability to bind to MhC class i. it was found that there are several protein binding sites on the weak and strong bases with various HLA. The bioinformatic analysis also shows a strong presence of α-helices in the N-terminal region and allowed to classify the antiviral protein as α/β type of structure, as we detected the presence of more than 30% α-helix and 20 % of β-sheet found separately along the protein chain. In the BLAST sequence analysis of cdNA antiviral protein, no sequence similarity was found in genbank, suggesting that it is from a novel protein family. it can be inferred by an analysis of this region that the possible antigenicity region would be between the 70-110 amino acids, showing high accessibility. This high antigenic region on the surface, can be a possible region to interaction with other proteins.
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P.50 High Throughput Production of complex Recombinant Proteins in Mammalian cells
Jens Jacob Hansen1, Thomas Egebjerg1, Anette Amstrup Pedersen1, Mats Åkesson2, Bjarne Rask Poulsen2, Michael Skjøt3, Laust Bruun Johnsen4, Claus Kristensen1
1Mammalian Cell Technology, Novo NordiskDenmark2Cell Culture Technology, Novo Nordisk Denmark
3Molecular Automation, Novo Nordisk Denmark4Automation Technology, Novo Nordisk A/S, Denmark
introduction of automated approaches for high throughput (hTp) generation of gene variants puts pressure on the capacity for protein expression and purification. This is in particular true when it comes to expression of complex recombinant proteins in mammalian cells. For low yielding proteins, neither the expensive transient transfection procedures, nor the very time consuming generation of stable cell lines is appealing for high-throughput production.
in order to solve the bottleneck in the capacity for expression of low yielding recombinant proteins, we have successfully integrated the semi-stable episomal QMCF expression system, developed in ChO cells by icosagen, in our panel of expression platforms.
The QMCF expression system is cost effective and sufficiently stable to allow scale-up to 10+ ltr. scale, at yields and timelines comparable or superior to conventional lipid-based transient transfection methods performed in hek293 cells. The QMCF expression system allows seamless integration with hTp variant generation, small-scale dNA preparation, automated scale-up procedures and production in wave bags or in stirred tank fermenters. We present an overview of how the QMCF system has been integrated, with the automated molecular biology and hTp transfection and scale-up platforms, in the production train at Novo Nordisk. data on stability of expression, scalability and timelines for production of proteins will be presented, along with selected data on quality of the protein produced using the system.
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P.51 Targeted transfection using e/Kcoil system for recombinant protein production
Elodie Louvier1,2 and Yves Durocher 1,2
1 Département de Biochimie, Université de Montréal, Canada.2 Life Sciences, NRC Human Health Therapeutics Portfolio, National Research Council of Canada, Canada.
polyplexes are widely used to transiently transfect mammalian cells for recombinant protein production. While most of the cells uptake polyplexes, not all of them are able to express the transgene. Since only 5 to 10% of captured plasmid dNA (pdNA) reach the nucleus, this suggests that polyplexes have a limited ability to route and translocate to the nucleus and thus large amounts of polyplexes have to be used to achieve high expression levels. In contrast, many viruses can efficiently transduce cells with only one or a few viral genome copy. Exploitation of receptor’s internalization and trafficking functions could optimize the transfection process by reducing quantities of pdNA and transfection reagent needed. in this study, we evaluated the usefulness of the E/kcoil system to target a cell surface receptor for pdNA delivery.
The E- and K-coil are two repeated heptapeptides which interact together with high affinity and specificity to form a coiled-coil structure. We fused the Ecoil to a dNA binding protein (dBp). We postulate that the dNA:dBp-Ecoil complex would be able to target a receptor bearing the kcoil fused to its extracellular domain. A stable cell line over-expressing the kcoil-receptor was established in suspension for scalability. Our results showed that the presence of the Ecoil did not interfere with pdNA binding capacity of the dBp, and the kcoil did not affect membrane localization of the receptor. The system was able to deliver pdNA inside hEk293 cells and reducing sulfation of the cell surface heparan sulfate proteoglycans was required to reduce non-specific binding of the complexes. however, dNA:dBp complexes were sequestrated inside endosomes. using chloroquine treatment to enhance endosomal escape, transgene expression was achieved. Furthers improvements to the approach will be discussed in this presentation.
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P.52 exploring endosomal pathways using novel nanoparticle polymers as transfection reagents
Marianne Gillard, Nghia P. Truong, Zhongfan Jia, Michael J Monteiro and Trent P Munro.University of Queensland, Australian Institute for Bioengineering and Nanotechnolog, Australia
Non-viral gene delivery systems are widely used for the transfer of nucleic acids into mammalian cells. Currently, there is a need to developed cheap and efficient transfection reagents for use in scalable production of therapeutic proteins via transient gene expression (TgE). One of the key barriers of non-viral gene delivery systems is the ability to escape the endosomal pathway and this requires a more detailed understanding of mechanisms involved in cellular uptake and intracellular trafficking, in order to develop highly efficient transfection reagents. This study investigated the utility of three novel nanoparticle polymers series as transfection reagents and specifically their ability to escape the endosomal pathway using two commonly used cell lines, Chinese hamster Ovary (ChO) and human Embryonic kidney (hEk). All three polymer series contains the same poly(2-dimethylaminoethyl acrylate) (pdMAEA) polymer backbone however, each series differs by their functional side groups. The E series polymers contain protonatable amine side groups, h series polymers contain protonatable amine side groups and alkylated carboxylic acid side groups and i series contain only the alkylated carboxylic acid side groups. Endo-some escape was measured with the aid of chloroquine which is known to cause endosomes and lysosomes to swell and burst. Transfection rates were measured using GFP fluorescence via flow cytometry. Our results indicate that the h series, which contains both side groups, displayed the highest rates of transfection and endosome escape. The i series were the next best performers, followed by E series. Cellular dNA-polyplex uptake was also investigated though the use of specific inhibitors to block endocytosis pathways (dynasore, chlorpromazine and filipin III). Our results indicate that internalisation pathway is both dependent on cell type and polymer type with no one pathway mediating successful transfection. These results highlight that further investigations into the mechanisms behind transfection processes will result in the development of highly efficient transfection reagents.
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P.53 Mechanistic engineering of Polyplex Mediated Transfection of cHo cells
Olivia L Mozley ([email protected]), Ben C Thompson and David C JamesCBE, University of Sheffield,, UK
Polyethylenimine (PEI) mediated transfection is used for early supply of recombinant proteins through transient gene expression (TGE). We present data exploring the mechanism of transfection and offer strategies for improv-
ing CHO based TGE systems. Experiments utilised CHO-S cells, CD CHO medium and 25kDa linear PEI.
polyplex-cell surface bio-molecular interactions were explored. The kinetics of transfection, with respect to cyto-association and cyto-internalization of polyplex were analysed using fluoro-labelled plasmid DNA and flow cytometry. Alongside polyplex-cell surface association and cyto-internalization, anionic cell surface heparan sulphate proteoglycans (hSpgs), previously implicated in pEi mediated transfection mechanics, were found to deplete from the cell surface (>80%). However, validated enzymatic depletion of cell surface HSPGs was found to have no effect on polyplex-cell surface binding. Although, sequestration of cholesterol from the cell surface and thus perturbation of lipid raft functionality, was found to dramatically reduce polyplex-cell surface binding.
Bio-physical polyplex-cell surface interactions were then investigated. inhibition of hydrophobic interac-tions using a non-ionic surfactant was found to reduce polyplex-cell surface binding. Culture media and additives were found to have a dramatic effect on the surface properties of pEi-dNA polyplexes. under optimized condi-tions, the surface of pEi-dNA polyplexes in Cd ChO media was charge-neutral. Culture additive, ferric iii citrate, known to inhibit pEi mediated TgE, was found to inhibit polyplex-cell surface binding and confer a negative charge on the polyplex.
Clones were isolated with inherently superior and heritable properties for TgE relative to the parental and were found to vary with respect to polyplex-cyto-association and cyto-internalization capacity. Furthermore, as utilized for in vivo applications, a selection of pEis, chemically derivatized for enhanced hydrophobicity, were tested for transfection of CHO-S cells in a synthetic culture environment. Propyl-PEI was found to be a more effi-cient gene delivery vehicle, with respect to plasmid DNA input, compared to unmodified PEI. A two fold increase in efficiency was achieved through combined use of a clonal derivative of the parental cell line and propyl-PEI, relative to transfection of the parental cell line with unmodified PEI.
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P.54 bioproduction Using flow electroporation Systems: Gram/l antibody Production via Tran-sient Gene Expression (TGE) Through Rapid, High Yield Stable Cell Line Generation.
Weili Wang, James Brady, Rama Shivakumar, Krista Steger, Karen Donato, and Madhusudan PeshwaMaxcyte, USA
Flow electroporation technology greatly shortens the timeline of antibody development by enabling large scale transient gene expression (TgE) directly within ChO cells, thus reducing the creation of stable cells early in the development process and eliminating the need to change cell backgrounds during scale up to biomanufacturing. MaxCyte offers two flow electroporation-based systems: the MaxCyte STX® Scalable Transfection System and the MaxCyte Vlx® large Scale Transfection System. These systems offer a universal means of fully scalable,
high efficiency TGE and are capable of producing multiple grams of antibodies and bi-specific antibodies follow-ing a single ChO transient transfection. We will present data demonstrating the reproducibility and scalability
of MaxCyte flow electroporation as well as its capacity to produce antibody titers >1g/L using TGE. In addition, we will present data showing the use of MaxCyte electroporation for creation of stable ChO pools and the rapid
generation of high-yield stable cell lines within 6-8 weeks of transfection.
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AUTHORS INDEXAbbott, Mark P-40Adkins, Kellye T-5-8Aghamohseni, Hengameh S-1-10Aguiar, Marcelo P-7Åkesson, Mats P-50Alexandre, Rezende P-44Alosta, Houssam P-13Alves de Carvalho, Daianny Maciely P-17Amstrup Pedersen, Anette P-50Ang, Jann Catherine P-27Ansorge, Sven P-12Astray , Renato P-22Ataliba, Polyana P-22Aucoin, Marc P-27, S-1-7, T-6-6Auer, Norbert P-3Augusto, Elisabeth P-7Aysola, Manjula P-36Balasubramanian, Sowmya P-23Baldi, Lucia P-23Barral, Manuel P-7Bartolini, Paolo P-6Baser, Bahar P-35Bass, Andy T-4-2Bathia, Mick T-7-2Baumann, Martina P-15, P-3Beaucamp, Nicola T-2-6Bell, David P-13Bernardino, Thaissa P-22Betenbaugh, Michael J P-24Bhidea, Ashlesha S-1-12Bialek, Corinna P-38Bjarne Rask Poulsen, Bjarne P-50Bleckmann, Maren P-35Blondeel, Eric S-1-7Bludau, Elisabeth P-29Bodnar, Edward P-2Borth, Nicole P-15, P-3, T-1-2Braasch, Katrin S-1-12Brady, James P-54, P-14Bridges, Gregory S-1-12Bruun Johnsen, Laust P-50Budman, Hector M. S-1-10Bullenkamp, Jessica T-1-3Butler, Michael S-1-2, T-2-7, S-1-12, P-30, S-1-1, P-31, P-32, S-1-13, P-34Cabel, Tim S-1-12Capone, Marcos P-6Carmo, Ana P-49Carreira, Ana Claudia T-2-7Carter, Mary P-10Castillo-Vitlloch, Adolfo S-1-11, T-5-4Chakrabarty, Romit P-8, P-9, P-10
Chan, Leslie P-46Chan, Sarah P-30Chang, Cindy P-41, T-6-5Chen, Kai T-2-5Chiou, Henry T-4-1Clausen, Henrik P-24Coffey, Matt P-8, P-9, P-12Coffey, Matthew P-10Cook, Molly P-43Correa de Freitas, Marcela Cristina P-18, P-17, P-19Costa de Pontes, Ana Paula P-17Cox, Manon T-6-1Cunha Junqueira Reis, Luiza P-17Curti, Sueli P-49D’Eall, Calvin S-1-1Davidson, Jacquelyn P-10de Abreu Soares Neto, Mario P-19De Crescenzo, Gregory S-1-2, S-1-3de la Luz-Hernandez, Kathya T-5-4de Sousa Bomfim, Aline P-19Dean, Andrew T-1-4Delafosse, Laurence P-45Deluz, Cédric P-48Dickson, Alan S-1-4, T-2-2Dionne, Ben P-31, P-14Dorion-Thibaudeau, July S-1-2, S-1-3Durocher, Yves S-1-8, S-1-3, P-45, T-5-6, T-3-2, P-51, P-33Egebjerg, Thomas P-50Estes, Bram P-47Estes, Scott P-20Faust, Nicole P-38Figueiredo, Cristina P-49Fischer, Simon P-21Follit, John P-20Fredin, Bobby P-10Fritchman, Kathie P-11Fulton, Kelly P-9Gaillet, Bruno P-33Gaken, Joop T-1-3Galipeau, Jacques T-7-1Garrett, Mia P-10George, Henry P-26, T-4-3Ghaffari, Navid S-1-9Gillard, Marianne P-52Giovanni, Dalton P-49, P-39GIROD, PIERRE-ALAIN P-25Gludovacz, Elisabeth P-15Gorr, Ingo T-2-6Grieger, Joshua T-6-3Grillari, Johannes T-1-2
Guillemette, Guy P-27Gunnarsson, Jenny P-42Gustafsson, Claes T-1-1Hacker, David P-23Hackl, Matthias T-1-2Hagerman, Allison P-8, P-9, P-10, P-12Hammond, Matthew P-5Handrick, René S-1-5, P-21Hansen, Jens Jacob P-50Hassaine, Gherici P-48Hassel, Katharina J. S-1-15Hawkins, Tiffany P-10Henry, Olivier S-1-8, T-6-6Hernandez-Lopez, Inmaculada P-3Hertel, Sabine P-38Hiller, Gregory T-5-7Holmberg Schiavone, Lovisa P-42Horvath, Brian T-5-8Huynh, Hoai P-46Jadhav, Vaibhav T-1-2, P-16James, David P-53Jaques, Colin P-4Jia, Zhongfan P-52Jiang, Jie T-1-3Jing, Donghui P-36Johansson, Patrik P-42Jolicoeur, Mario P-28Jorge, Soraia P-22, P-44Kallos, Mike Kamen, Amine T-6-6, P-9, P-10, P-12, P-26Kehoe, Daniel P-36Kemp, Chris Kewes, Helmut P-38Kiplinger, Karen T-5-8Klump, Wolf P-10Kobayashi, Kenji P-26Kober, Lars T-2-4Kolman, John P-8Kolpak, Meredith T-5-7Kost, Tom Kristensen, Claus P-50Kulman, John T-2-5Kumar, Sampath T-2-5Lanthier, Stéphane P-12Lattova, Erika P-34, T-5-6, P-32Lega, Danniela P-4Legge, Raymond L S-1-10Lemos, Marcos P-44, P-22Lin, Nan P-26Liu, Bo S-1-13, P-34Liu, Pocheng P-10
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Louvier, Élodie P-51Lu, Katherine P-13Lua, Linda P-41, T-6-5Lundstrom, Kenneth P-48 MacKenzie, Roger T-5-1Majors, Brian P-20Malaz, Mehrdad P-10Maldonado Coelho, Tatiane T-2-7Mann, Kimberly P-36Maria de Sousa Russo, Elisa P-19Martin, Shelly P-20Massie, Bernard P-33Matthews, Kathryn P-13Mccall, Eileen P-40Mei, Baisong T-2-5Meier, Markus S-1-2Mendonça, Rita P-39Mendonça, Ronaldo P-49Mendonça, Ronaldo Zucatelli P-39Meyer, Steffen P-35Moehrle, Volker P-1Mohammad, Kaveh S-1-12Moll, Alina S-1-5Monteiro , Michael J P-52Moo-Young , Murray S-1-10Moraes, Dante P-49Moresoli, Christine S-1-15Mozley, Olivia P-53Munro, Christopher T-6-5Munro, Trent P P-52Murphy, Brian T-3-4Murrell , Julie P-36Nassoury, Nasha P-33Nielsen, Lars P-46Nikolic-Jaric, Marija S-1-12Niss, Knut P-36Nunn, Heather P-5Nury, Hugues P-48O’Callaghan, Peter T-5-5Ohadi, Kaveh S-1-10Okun, Natalie S-1-2, P-30Oliveira, João P-6Oliveira, Maria P-49Onions, David P-8Otte, Kerstin P-21Palmberger, Dieter P-37Palsson, Bernhard P-24Pan, James T-5-2Pape, Robert T-2-5Paschoal, Juliana P-44Patel, Trushar S-1-2Paul, Wolfgang T-2-6Pereira, Carlos P-44
Pereira, Carlos Augusto P-22Perreault, Helene P-2, T-5-6Peshwa, Madhusudan P-54, P-14Peters, Robert T-2-5Petiot, Emma T-6-6Picanço Castro, Virginia P-18, P-19, P-17Piccoli, Rosane P-7Piret, James T-6-2Pon, Robert S-1-1Popp, Oliver T-2-6Poulain, Adeline P-33Punreddy, Sandhya P-36Racher, Andrew T-5-5Rahal, Paula P-49Raju, Shantha S-1-6Raju, T. Shantha T-5-3Raymond, Céline T-5-6, S-1-3Reid, Steven P-46Ribbert, Markus P-38Robitaille, Julien P-28Rokkam, Srinivasa P-13Rook, Martha P-36Rose, Thomas T-2-3Russo, Elisa P-18Saboktakin Rizi, Bahareh S-1-12Salimi, Elham S-1-12Samulski, R. Jude T-6-3 Sandhu, Devender P-13Sandig, Volker S-1-14, T-2-3Santos, Fabiana P-49Scharer, Jeno M. S-1-10Schiedner, Gudrun P-38Schmeizl, Mark P-14Schmid, Georg T-3-1Schomberg, Michael P-38Sealover, Natalie P-26Seitz, Susanne T-2-3Sheinin, Tanya P-13Shi, Jing P-10Shiloach, Joseph T-1-5Shimoni, Yuval P-1Shiratori, Masaru T-6-4Shivakumar, Rama P-54, P-14Singh, Nishi P-13Skjøt, Michael P-50Smales, Mark T-1-4Soares, Carlos P-6Sogayar, Mari T-2-7Sousa Bomfim, Aline P-18Spearman, Maureen S-1-2, P-30, S-1-1, P-34Spörk, Tanja P-38
Srinivasan, Venkatesh P-1Steger, Krista P-54, P-14Stetefeld, Jörg S-1-2Stützle, Martina S-1-5Stutzman-Engwall, Kim. J. Suárez Patiño, Sandra Fernanda P-22Sunil, Neethu P-36Suzuki, Miriam P-6Sweeney, Bernie T-3-3Swiech, Kamilla P-18, P-19Tadeu Covas, Dimas P-17, P-18, P-19Tan, Emilyn P-41, T-6-5Tanha, Jamshid S-1-1Tavassoli, Mahvash T-1-3Tayi, Venkata S-1-13Thompson, Ben P-53Thompson, Christine T-6-6Thomson, Douglas S-1-12Tié, Mark P-20Tonelotto, Mariana P-49Toussaint, Cécile S-1-8Tran, Hue P-8, P-9, P-10Tran, Rosa P-9, P-10, P-12Tran, Trinh P-46Transfiguracion, Julia P-9, P-10, P-12Truong, Nghia P. P-52Twine, Susan P-9Van den Heuvel, Joop P-35Vcelar, Sabine P-3Verma, Anjali P-36Victores, Svieta S-1-11Villacrés Barragán, Carina P-32, S-1-13Vogel, Horst P-48Voldborg, Bjørn P-24von Horsten, Hans Henning T-2-3Voyer, Robert P-9, P-10, P-12Wagner, Andreas P-21Wang, Weili P-54, P-14Weiss, Ron KeynoteWelch, Mark T-1-1Williams, Rebecca P-40Winkler, Karsten T-2-3Wissing, Silke P-38Wölfel, Jens P-38Wright, Chapman P-20Wurm, Florian P-23Yamasaki, Lilian P-49Young, Robert T-5-5Zhu, Lily T-2-5Zimmermann, Katharina S-1-5Zotter, Angelika P-3
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Aghamohseni, Hengameh University of Waterloo CanadaAmes, Robert GlaxoSmithKline USAAndersen, Christina Rottbøll Symphogen DenmarkAng, Jann Catherine University of Waterloo CanadaAucoin, Marc University of Waterloo CanadaBalasubramanian, Sowmya Ecole Polytechnique Fédérale de Lausanne SwitzerlandBass, Andy Cellectis bioresearch FranceBaumann, Martina ACIB - Austrian Centre of Industrial Biotechnology AustriaBeaucamp, Nicola Roche Diagnostics GmbH GermanyBégin, Bruno Conferium CanadaBhatia, Mickie McMaster University” CanadaBirch, April Kempbio, Inc. USABleckmann, Maren Helmholtz Zentrum für infektionsforschung GermanyBlondeel, Eric University of Waterloo CanadaBludau, Elisabeth Fraunhofer ITEM - Pharmaceutical Biotechnology GermanyBodnar, Edward University of Manitoba CanadaBraasch, Katrin University of Manitoba CanadaBruse, Shannon Mirus Bio LLC USABudman, Hector University of Waterloo CanadaButler, Michael University of Manitoba CanadaCarmo, Ana Faculdade Oswaldo Cruz BrasilCastillo-Vitlloch, Adolfo Center of Molecular Immunology CubaChakrabarty, Romit Oncolytics Biotech Inc. CanadaChan, Sarah U Manitoba CanadaChang, David Chenomx Inc. CanadaChen, Yaoqi Bayer USAChiou, Henry Life Technologies USACoffey, Matt Oncolytics Biotech Inc. CanadaCook, Molly National Institute of Environmental Health Sciences USACorrea De Freitas, Marcela Cristina Faculdade de Medicina de Ribeirão Preto-USP BrazilCox, Manon Protein Sciences Corp USAD’eall, Calvin University of Ottawa/National Research Council Canada CanadaDean, Andrew Univeristy of Kent UKDelafosse, Laurence University of Montréal, Canada National Research Council CanadaDickson, Alan University of Manchester UKDionne, Ben University of Manitoba CanadaDorion-Thibaudeau, July Ecole Polytechnique de Montreal Canada
List of participants
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Drouillard, Gerry University of Guelph - Graduate Student CanadaDurocher, Yves National Research Council Canada CanadaEstes, Bram Amgen USAFischer, Simon Institute of Applied Biotechnology Biberach GermanyFleischanderl, Daniel Baxter AustriaFritchman, Kathie BD Biosciences, Advanced Bioprocessing USAGalipeau, Jacques Emory University USAGately, Dennis Ambrx, Inc. USAGeorge, Henry Sigma Aldrich USAGhaffari, Navid University of British Columbia CanadaGillard, Marianne AIBN - UQ AustraliaGirod, Pierre-Alain SELEXIS S.A. SwitzerlandGrieger, Joshua University of North Carolina USAGrillari, Johannes BOKU - Universität für Bodenkultur Vienna AustriaGustafsson, Claes DNA2.0 USAHagerman, Allison Oncolytics Biotech Inc. CanadaHannas, Zahia MERIAL, Development Process, Virology EU FranceHansen, Jens Jacob Novo Nordisk A/S DenmarkHardy, Matthew CSL Limited AustraliaHassel, Katharina University of Waterloo CanadaHayes, Scott Mirus Bio LLC USAHeine, Holger Novartis Pharma AG SwitzerlandHirate, Maki Taisho Pharmaceutical Co.,LTD. JapanHubert, Rene Agensys USAJaques, Colin lonza Biologics UKJuckem, Laura Mirus Bio LLC USAKallos, Michael University of Calgary CanadaKamen, Amine NRC, Canada CanadaKaseko, Galina The Stephen Sanig Research Institute AustraliaKemp, Chris Kempbio, Inc. USAKiplinger, Karen Genentech USAKober, Lars Consultant MexicoKolpak, Meredith Pfizer USAKost, Tom GlaxoSmithKline USAKumar, Sampath Biogen Idec USAKurz, Dorothee Luise University of Waterloo CanadaLemos, Marcos Instituto Butantan Brasil
Lim, Ben ABBVIE, Inc. USALiu, Bo University of Manitoba CanadaLouvier, Elodie Université de Montréal CanadaLovisa, Holmberg Schiavone AstraZeneca SwedenLua, Linda The University of Queensland AustraliaLundstrom, Kenneth PanTherapeutics SwitzerlandLynch, Tom Aldevron USAMacKenzie, Roger National Research Council Canada CanadaMahaworasilpa, Tohsak The Stephen Sanig Research Institute AustraliaMahmoud, Wafaa protein Sciences Corporation USAMaldonado Coelho, Tatiane University of Sao Paulo BrazilMann, Kimberly EMD Millipore USAMartin, Shelly BiogenIdec United StatesMcCall, Eileen AstraZeneca UKMendonça, Ronaldo Instituto Butantan BrasilMishra, Neha University of Manitoba CanadaMonteil, Dominique École Polytechnique Fédérale de Lausanne SwitzerlandMoo-Young, Murrray univ of waterloo CanadaMozley, Olivia University of Sheffield UKNunn, Heather Amgen Inc. USAO’Callaghan, Peter Lonza Biologics UKOhadi, Seyed Kaveh University of Waterloo CanadaOkun, Natalie University of Manitoba CanadaOverton, Laurie GlaxoSmithKline USAPalmberger, Dieter University of Natural Resources and Life Sciences AustriaPan, James University of Toronto, Donnelly Centre CanadaPiret, James University of British Columbia CanadaPoltilove, Bryan Life Technologies USAPoulain, Adeline Université de Montréal CanadaQuinsey, Noelene MOnash University AustraliaRaju, T. Shantha Janssen Research & Development, LLC, USARaymond, Céline Université de Montréal CanadaReid, Steven The University of Queensland AustraliaRheault, Patrick GSK Vaccines CanadaRobitaille, Julien École polytechnique de Montréal CanadaRogers, Jeff Life Technologies USASadhukhan, Ramkrishna (Ramu) AbbVie USASafly, Teresa BD Biosciences - Advanced Bioprocessing USASandhu, Devender Therapurebio CanadaSandig, Volker ProBioGen AG Germany
Sanford, James Saint-Gobain Life Sciences USAScharer, Jeno University of Waterloo CanadaSchmeizl, Mark MaxCyte, Inc. USASchmid, Georg F. Hoffmann-La Roche AG SwitzerlandSealover, Natalie SAFC, Sigma-Aldrich USAShah, Girish Consultant UKShiloach, Joseph NIDDK/NIH USAShimoni, Yuval Bayer HealthCare USAShiratori, Masaru Genentech, a member of the Roche group USASimeonova, Katya Bulgarian Academy of Sciences BulgariaSmales, Mark University of Kent UKSmithee, Shane TAP Biosystems USASoares, Carlos Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP BrazilSpearman, Maureen Dept of Microbiology, University of Manitoba CanadaStützle, Martina Institute of Applied Biotechnology GermanyStutzman-Engwall, Kim Consultant USASweeney, Berni UCB Celltech Ltd UKTalukdar, Sadhan JANATA DAL UNITED IndiaTanha, Jamshid National Research Council Canada CanadaTavassoli, Mahvash King’s College London UKTayi, Venkata University of Manitoba CanadaThompson, Christine National Research Council of Canada CanadaToussaint, Cécile Université de Montréal CanadaTran, Hue Oncolytics Biotech Inc. CanadaVaibhav, Jadhav University of Natural Resources and Life Sciences AustriaVasantha Kumar, Shyam Charan Life Technologies United StatesVcelar, Sabine ACIB GmbH - Austrian Centre of Industrial Biotechnology AustriaVillacrés Barragán, Carina Department of Microbiology. University of Manitoba CanadaVoldborg, Bjørn Technical University of Denmark DenmarkVoyer, Robert National Research Council Canada CanadaWalker, Matthew TAP Biosystems UKWang, Weili MaxCyte, Inc. United StatesWeiss, Ron MIT USAWissing, Silke CEVEC Pharmaceuticals GmbH GermanyWu, Xue Bayer Healthcare LLC USAZhang, John R&D Systems USAZhao, Xiaojian Wuhan YZY Biopharma CO., Ltd ChinaZhu, Ying Bayer Healthcare USA
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