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“Eukaryotic Protein Expression for Pharmaceutical Research”

Date: September 20, 2010 Time: 08:00 – 18:15 Location: Congress Center Basel, Hall Sydney Organizers: Lorenz M. Mayr, Novartis Pharma (Basel, CH) Florian M. Wurm, EPFL (Lausanne, CH) Stefan R. Schmidt, Erabiotech (Barcelona, ES) Session I & II: Mammalian Expression Technologies Chairs: S. Schmidt, Erabiotech (Barcelona, ES)

F. Wurm, EPFL (Lausanne, CH) 08:00 – 08:15 Welcome L. M. Mayr

Novartis Pharma AG (Basel, CH) 08:15 – 09:00 Keynote Lecture

Protein diversity panels as a discovery platform: Mining the mammalian extracellular proteome S. Lesley Genomics Institute of the Novartis Research Foundation (La Jolla, USA)

09:00 – 09:30 Large scale transient transfection in CHO and HEK293 Cells

Y. Durocher National Research Council Canada (Montreal, CA) 09:30 – 10:00 Recombinant protein expression in HEK293 vs. CHO cells:

Does it make a difference? S. Geisse Novartis Pharma AG (Basel, CH)

10:00 – 10:30 Coffee Break (sponsored by Icosagen Inc.) 10:30 – 11:00 Development of the extrachromosomal expression system for

production of therapeutic proteins and for cell-based assays M. Ustav University of Tartu (Tartu, EE) 11:00 – 11:30 Protein expression using engineered mammalian cell lines

and vectors N. Mermod University of Lausanne (Lausanne, CH)

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11:30 – 12:00 Fast establishment of insect and mammalian production cell lines

for structural biology by site-specific recombination J. van den Heuvel

Helmholtz Centre Infectious Biology (Braunschweig, DE) 12: 00 – 12:30 Rapid and high yield production of proteins for preclinical and

basic research in cultivated mammalian cells M. de Jesus

ExcellGene SA (Monthey, CH) Sponsored Lunch Seminars 12:45 – 12:55 Optimized genes: Tools for expression enhancement &

functional genomics M. Graf Geneart AG (Regensburg, DE)

12:55 – 13:05 Selexis Genetic Elements (SGE) applications for rapid screening

and generation of highly productive stable mammalian clones D. Huber Selexis SA (Geneva, CH)

13:05 – 13:15 Lonza: Your global Life Science Partner right next door J. White Lonza (Basel, CH)

13:15 – 13:25 High titer processes for early development - High, consistent

quality, 5 – 10 fold yield increase compared to standard Fed-Batch processes G. Zijlstra

DSM Biologics (Groningen, NL) 13:25 – 13:35 Simplifying protein expression with new platform technologies and

integrated services R. Hickey Life Technologies (Carlsbad, USA)

13:35 – 13:45 Leveraging the Pfenex expression technology platform to enable

protein expression for drug discovery D. Retallack Pfenex Inc. (San Diego, USA)

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Session III: Baculovirus / Insect Cell Expression Technologies Chair: R. Assenberg, Novartis Pharma AG (Basel, CH) 14:00 – 14:30 Optimisation of gene expression using baculovirus vectors in

insect cells L. King Oxford Brooks University (Oxford, UK) 14:30 – 15:00 BacMam gene delivery in drug discovery T. Kost Glaxo Smith Kline (Raleigh-Durham, USA) 15:00 – 15:30 Freedom of expression: Delivering the genome S. P. Chambers AB-Pro Laboratories (Cambridge, USA) 15:30 – 16:00 Coffee Break (sponsored by Icosagen Inc.) Session IV: High – Throughput Technologies Chair: B. Gerhartz, Novartis Pharma AG (Basel, CH) 16:00 – 16:30 New HT Baculovirus expression tools for multiprotein complex

research I. Berger EMBL (Grenoble, FR) 16:30 – 17:00 ESPRIT: Library-based construct screening for difficult-to-express

proteins D. Hart European Molecular Biology Laboratory (Grenoble, FR) 17:00 – 17:30 Challenges and opportunities for high-throughput systems with

suspension cultures of animal cells D. Hacker EPFL (Lausanne, CH) 17:30 – 18:00 The structural proteomics of glycoproteins R. J. Owens University of Oxford (Oxford, UK) 18:00 – 18:15 Closing Remarks L. M. Mayr

Novartis Pharma AG (Basel, CH)

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Abstracts and Contact Details I & II: Mammalian Expression Technologies Protein diversity panels as a discovery platform: Mining the mammalian extracellular proteome S. Lesley…………………………………………………………………………………………………..p.5 Large ccale transient transfection in CHO and HEK293 Cells Y. Durocher ………………………………………………………………………………………............p.6 Recombinant protein expression in HEK293 vs. CHO cells: Does it make difference? S. Geisse………………………………………………………………………………………………….p.7 Development of the extrachromosomal expression system for production of therapeutic proteins and for cell-based assays M. Ustav…………………………………………………………………………………………………..p.8 Protein expression using engineered mammalian cell lines and vectors N. Mermod………………………………………………………………………………………………...p.9 Fast establishment of insect and mammalian production cell lines for structural biology by site-specific recombination J. van den Heuvel………………………………………………………………………………………...p.10 Rapid and high yield production of proteins for preclinical and basic research in cultivated mammalian cells M. de Jesus………………………………………………………………………………………………p.11 III: Baculovirus / Insect Cell Expression Technologies Optimisation of gene expression using baculovirus vectors in insect cells L. King…………………………………………………………………………………………………...p.12 BacMam gene delivery in drug discovery T. Kost…………………………………………………………………………………………………...p.13 Freedom of expression: Delivering the genome S. P. Chambers………………………………………………………………………………….............p.14 IV: High – Throughput Technologies New HT Baculovirus expression tools for multiprotein complex research I. Berger………………………………………………………………………………………………….p.15 ESPRIT: Library-based construct screening for difficult-to-express proteins D. Hart………………………………………………………………………………………………......p.16 Challenges and opportunities for high-throughput systems with suspension cultures of animal cells D. Hacker………………………………………………………………………………………………...p.17 The structural proteomics of glycoproteins R. J. Owens………………………………………………………………………………………………p.18 Sponsors…………………………………………………………………………………………p.19 - 26

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Protein diversity panels as a discovery platform: Mining the mammalian extracellular proteome

Scott Lesley Genomics Institute of the Novartis Research Foundation, San Diego, California, USA Diverse small molecule libraries have long-served as a principal resource for new drug leads. More recently, protein diversity panels have emerged as a valuable screening source for biological discovery and biotherapeutic leads. Such diversity panels can provide a more straightforward translation of screen to pathway biology. The human extracellular proteome is a means of cell to cell communication. A vast array of cellular pathways are influenced and regulated via secreted proteins making them a rich source of targets. To begin analyzing the function of the human extracellular proteome, we have developed a large scale high-throughput mammalian protein expression and purification platform to create protein diversity panels that are screened in a dose-dependent way to identify novel activities. We have shown that, even within a small prioritized protein diversity panel, new and interesting biology can be discovered. ____________________________________________________________________________

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Contact Details Dr Scott Lesley Phone: +1 858 812 1551 Director of Protein Sciences Email: slesley@gnf.org Genomics Institute of the Novartis Research Foundation 10675 John Jay Hopkins Dr San Diego, CA 92121 USA

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Large scale transient transfection in CHO and HEK293 Cells Yves Durocher Biotechnology Research Institute, National Research Council, Montreal, CA The rapid generation of sufficient quantities of recombinant proteins, antibodies and membrane receptors is required for structure/function analyses, animal studies and HTS campaigns. Mammalian cells are the host of choice to express those proteins as they allow post-translational modifications that are often essential for r-protein’s function and/or activity. The large-scale transfection of HEK293 and CHO cells has become a mature technology that meets these needs, with productivities of up to 300 mg/L achieved in CHO cells. We will illustrate some potential advantages of using both cell lines in parallel to evaluate expression of r-proteins. ____________________________________________________________________________

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Contact Details Dr Yves Durocher Phone: +1 514 496 6192 Project Leader, Mammalian Cell Technology Email: Yves.Durocher@cnrc-nrc.gc.ca Biotechnology Research Institute National Research Council Canada 6100 avenue Royalmount, Montréal (Québec) Canada H4P 2R2

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Recombinant protein expression in HEK293 vs. CHO cells: does it make a difference?

Sabine Geisse Novartis Institutes for BioMedical Research, Basel, CH Three characteristics are of paramount importance to the outcome of recombinant protein expression in mammalian cells: the efficiency, the reproducibility and the robustness of the process. We describe our efforts to meet these prerequisites by developing “research-friendly” expression platforms for HEK293 and CHO cells in purely transient and stable episomal fashion. This includes expression optimization on small, medium and large scale to cover the full spectrum of multi-parallel expression trials in the context of e.g. secretomics approaches up to the generation of gram quantities of proteins and antibodies. In addition, the question of superiority of one approach over the other in view of quantity and quality of the product will be addressed. ____________________________________________________________________________

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Contact Details Dr Sabine Geisse Phone: +41 61 324 8274 Sr. Res.Investigator II Fax: +41 61 324 6303 Novartis Institutes for BioMedical Research Email: sabine.geisse@novartis.com NBC/PPA WSJ-506.3.04 4056 Basel Switzerland

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Development of the extrachromosomal expression system for production of therapeutic proteins and for cell-based assays Andres Tover2, Urve Toots2, Radi Tegova2, Anne Kalling2, Toomas Silla 1 and Mart Ustav1,2

1Department of Biomedical Technology, Institute of Technology, University of Tartu, Tartu, EE 2Icosagen Cell Factory OÜ, Nooruse 9, Tartu, EE The number of suspension and adherent cell lines (QMCF cells) supporting stable high copy-number extrachromosomal replication of the expression vectors carrying hybrid viral origins has been constructed. The replication of the vector in these cells is directed in S-phase by mouse polyomavirus enhancerless minimal origin and largeT-antigen. Maintenence of the episomal vector in mitosis is achieved by the BPV1 E2 multimeric binding sites in the plasmid and E2 protein or by the EBNA1 multimeric binding sites in the plasmid and by the EBNA1 protein. The replication factor and maintenance proteins are produced from the integrated expression cassettes in these cell lines. The suspension QMCF cell lines (CHO-S and HEK293-S) were optimized for the high level protein production like antibodies, growth factors, cytokines and different kind of virus-like particles pseudotyped with membrane-bound proteins. Adherent U2OS, CHO K1 and HEK293 QMCF cells were used to develop cell-based assays for screening drug-candidates. ____________________________________________________________________________

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Contact Details Prof Mart Ustav, Ph.D. Phone: +37 273 75047 Professor of Biomedical Technology +37 273 77050 Institute of Technology, University of Tartu Email: mart.ustav@ut.ee And CEO Icosagen AS and Icosagen Cell Factory OÜ mart.ustav@icosagen.ee 1 Nooruse Street 50411 Tartu Estonia

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Protein expression using engineered mammalian cell lines and vectors Nicolas Mermod Institute of Biotechnology, University of Lausanne, CH DNA vectors incorporating epigenetic regulatory elements such as MAR can be used to prevent transgene silencing and increase transcription rates, yielding high and stable transgene expression in the laboratory and in the bioreactor. These vectors allow very high specific productivities, such that new production bottlenecks have emerged, encompassing transgene integration, protein secretion and cell physiology. Methods to increase transgene integration using a gene transfer process based on homologous recombination will be presented, opening new avenues to engineer cells to achieve more efficient recombinant protein expression. ____________________________________________________________________________

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Contact Details Prof Nicolas Mermod, PhD Phone: +41 21 693 76 16 Director of Institute of Biotechnology Fax: +41 21 693 76 10 University of Lausanne Email: nicolas.mermod@unil.ch 1015 Lausanne www.unil.ch/biotech Switzerland

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Fast establishment of insect and mammalian production cell lines for structural biology by site-specific recombination Steffen Meyer, Sonja Wilke, Balaji Sinhadri, Volker Jäger, Konrad Büssow and Joop van den Heuvel

Protein Sample Production Facility (PSPF), Department of Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, DE Insect (baculoviral) and mammalian cell culture techniques are becoming more and more important for the production of large amounts of ultra-pure recombinant protein for 3-dimensional structural analysis. Due to the long and expensive generation process of stable producer cell lines, new strategies involving preparative FACS and site-specific recombination were developed to solve this bottle-neck. We established GFP-positive master cell lines derived from a glycosylation mutant CHO cell line by selecting stable, high-level GFP expressing cells by preparative cell sorting. Target genes were introduced into the optimal GFP expression loci of master cell lines by recombinase mediated cassette exchange (RMCE). Establishing a production cell line from a master cell line by RMCE took about two to four months, which is considerably faster than traditional approaches. Expression of mammalian proteins like the Toll-like receptor proteins in the baculoviral/insect expression system is often compromised by the degeneration of the host cell’s secretion machinery following viral infection. Therefore, a system compatible to the mammalian RMCE method was established for creating new stable master insect cell lines for the production of challenging secreted glycoproteins. Our results demonstrate that the combination of FACS and site-specific recombination enables fast and reproducible cloning of protein producer cell lines that are stable without antibiotics. ____________________________________________________________________________

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Contact Details Dr Joop van den Heuvel Phone: +49 531 6181 7046 Structural Biology, PSPF Email: Joop.vandenHeuvel@helmholtz-hzi.de Helmholtz Centre for Infection Research GmbH www.helmholtz-hzi.de Inhoffenstrasse 7 www.pspf.de 38124 Braunschweig Germany

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Rapid and high yield production of proteins for preclinical and basic research in cultivated mammalian cells Maria DeJesus ExcellGene SA, Monthey, Switzerland The pipeline of pharmaceutical and biotech companies are full of interesting and potentially highly promising therapeutic candidates, but also basic research into structure-function relationships of proteins and their targets is a most active area of interest. In both cases, rapid translation of a DNA concept into the reality of its protein target is a challenge and limits progress. Insights into the molecular biology of DNA transfer to cells, but also into the physiology of cells in bioreactors, together with the required physico-chemical principles of reactor engineering have provided opportunities of unprecedented impact to deliver high quality proteins in milligram to gram quantities from mammalian cells, just days after the availability of appropriately constructed vectors. The talk will summarize and discuss our approaches to deliver proteins to both academic and industrial clients. ____________________________________________________________________________

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Contact Details Dr Maria de Jesus Phone: +41 24 471 96 60 COO Fax: +41 24 471 96 61 VP Process Sciences Email: Maria.DeJesus@excellgene.com ExcellGene SA Rte de l'Ile au Bois 1A 1870 Monthey Switzerland

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Optimisation of gene expression using baculovirus vectors in insect cells Linda King Oxford Brookes University and Oxford Expression Technologies Ltd, Oxford, UK Gene expression in insect cells using baculovirus expression vectors is often optimised by varying a number of parameters including cell type, multiplicity of infection and time to harvest. This presentation will discuss how genetic modification of the baculovirus genome may also be used to optimise production of specific types of proteins, particularly the so-called ‘difficult to express’ membrane and secreted proteins. Such modifications include the deletion of non-essential virus genes, the over-expression of chaperones or enzymes that facilitate the processing of complex glycoproteins or multi-subunit proteins, and the use of a variety of gene promoters to control the levels and timing of gene expression. ____________________________________________________________________________

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Contact Details Prof Linda King Phone: +44 (0)1865 483241 Dean, School of Life Sciences Email: laking@brookes.ac.uk Head, Insect Virus Research Group Oxford Brookes University Gipsy Lane Oxford OX3 0BP United Kingdom

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BacMam gene delivery in drug discovery Tom Kost Molecular Discovery Research, GlaxoSmithKline, Research Triangle Park, North Carolina, USA Baculoviruses containing mammalian cell expression elements (BacMam viruses) have proven to be a powerful tool for developing cell based assays and producing recombinant proteins. The viruses provide a flexible alternative to transient transfection approaches and stable cell line development. Viruses are readily produced, have a wide host cell range, can carry large and/or multiple gene inserts and have a good biosafety profile. Transduction, even at high virus to cell ratios, results in little to no obvious cytotoxicity. Importantly virus transduction is amenable to automated systems. The approach has proven particularly valuable for expressing membrane proteins such as seven transmembrane receptors, ion channels and transporters. In addition to assay development applications, BacMam gene delivery is also proving to be useful for the production of recombinant proteins. Continual vector enhancements and host cell engineering approaches will serve to expand the host cell range and expression levels of BacMam transduction. ____________________________________________________________________________

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Contact Details Tom Kost Phone: +1 919 483 3218 Molecular Discovery Research Email: tom.a.kost@gsk.com GlaxoSmithKline www.gsk.com 5 Moore Drive Research Triangle Park North Carolina 27709 USA

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Freedom of expression: Delivering the genome Stephen P. Chambers With less than 5% of the proteins encoded by the human genome commercially available: the lack of protein content is seen as a major obstacle in drug research and discovery. Typically protein production is a highly customized, low-throughput process, which while successful was not designed for generating a large number of proteins. It is only relatively recently with the availability of the human genome that high-throughput approaches to protein production have been developed in the desire for greater efficiencies. We have designed an industrialized automated expression and purification platform capable of rapidly delivering thousands of highly purified proteins. At the center of the technology are a series of proprietary expression vectors, which allow protein production to take place in multiple expression systems. This ability to utilize different expression systems has been shown to greatly increase the probability of success: particularly when attempting to make the complex, hard to produce, proteins. When coupled with the huge processing power of the platform, large-scale experiments exploring expression and purification conditions can be used to optimize and model protein production. The technology and processes involved in the creation of this unique platform will be discussed. ____________________________________________________________________________

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____________________________________________________________________________ Contact Details Dr Stephen P. Chambers, PhD Email: SChambers@abpro-labs.com Abpro www.abpro-labs.com 1 Kendall Sq, Building 200 Cambridge, MA 02139 USA

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New HT Baculovirus expression tools for multiprotein complex research Imre Berger European Molecular Biology Laboratory (EMBL), Structural biology of eukaryotic complexes, Joint appointment EMBL Genome Biology Programme Heidelberg, EMB Grenoble, FR Most eukaryotic proteins, including many of pharmaceutical interest, exert their physiological function as parts of large multisubunit complexes in the cell. Typically, these complexes exist in very scarce amounts, impeding their extraction from native source material, and necessitating recombinant overproduction for molecular level studies. We have developed MultiBac, an advanced baculovirus system which facilitates eukaryotic multiprotein complex production using insect cell culture. We also addressed parallelization and automation of gene assembly for multiprotein complex expression by developing robotic routines for multigene vector generation. Here, we focus on several improvements of baculovirus expression system performance which we introduced: the modifications of the transfer plasmids to enable HT applications, the methods for generation of composite multigene baculoviral DNA, and the simplified and standardized expression procedures which we delineated using our MultiBac BEVS system.

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Contact Details Dr Imre Berger Phone: +33 47 620 7061 Group Leader, Structural biology of eukaryotic Fax: +33 47 620 7786 Complexes; Joint appointment EMBL Genome Email: iberger@embl.fr Biology Programme Heidelberg www.embl.fr EMBL Grenoble Polygone Scientifique 6 Rue Jules Horowitz BP181 38042 Grenoble CEDEX 9 France

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ESPRIT: Library-based construct screening for difficult-to-express proteins Darren Hart European Molecular Biology Laboratory (EMBL), High Throughput Protein Lab, Grenoble, FR Expression of sufficient quantities of soluble protein for structural biology and other applications can be a difficult task, especially when multimilligram quantities are required. To improve yield, solubility or crystallisability of a protein, it is common to subclone shorter genetic constructs corresponding to single or multidomain fragments. However, it is not always clear where domain boundaries are located, especially when working on novel targets with little or no sequence similarity to other proteins. The ESPRIT (Expression of Soluble Proteins by Random Incremental Truncation) construct screening technology has been developed at EMBL to identify soluble constructs of “difficult-to-express” protein targets that resist the classical approach of bioinformatics and PCR cloning. In each experiment, 30,000 individual constructs are assayed in E. coli for yield and solubility using a highly automated colony array format. Here we review several datasets (e.g. influenza polymerase, human kinases) to provide a view on its capabilities. ____________________________________________________________________________

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Dr Darren Hart Phone: +33 47 620 7768 Team Leader Fax: +33 47 620 7199 High Throughput Protein Lab Email: hart@embl.fr Grenoble Outstation www.embl.fr/research/unit/hart/index.html EMBL Grenoble 6 Rue Jules Horowitz BP181 38042 Grenoble CEDEX 9 France

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Challenges and opportunities for high-throughput systems with suspension cultures of animal cells David L. Hacker, S. Tissot, Divor Kiseljak, Yashas Rajendra, Lucia Baldi, Florian M. Wurm EPFL (Swiss Federal Institute of Technology Lausanne), Laboratory of Cellular Biotechnology (LBTC), Life Science and Basic Science Faculties, Lausanne, CH Large-scale suspension cultures of CHO cells provide the basis for the production of high value protein products. The cell culture processes for these products need to be highly complex in order to maximize product yield. In the past, the optimization of large-scale bioprocesses with CHO cells have been performed in small spinner flasks. However, they are not an ideal cell culture vessel due to limitations in mixing. Therefore, more performant small-scale cell culture systems, hopefully reflecting the environment at large scale, are needed to facilitate large-scale bioprocess optimization. This presentation will provide a perspective on using Tubespin bioreactors as a scale-down system with orbital shaking as a mixing principle. This system has the potential to be developed for high-throughput approaches to bioprocess optimization. The engineering principles for oxygen supply and pH control in orbitally shaken bioreactors will also be discussed. ____________________________________________________________________________

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Contact Details Dr David Hacker, PhD Phone: +41 21 693 6142 Laboratory of Cellular Biotechnology and Fax: +41 21 693 6140 Protein Expression Core Facility Email: david.hacker@epfl.ch EPFL CH J2-496, Station 6 1015 Lausanne Switzerland

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The structural proteomics of glycoproteins

Ray J. Owens OPPF-UK, The Research Complex at Harwell and Division of Structural Biology, Welcome Trust Centre for Human Genetics, University of Oxford, UK It is estimated that approximately half of all proteins encoded by the human genome have attached sugars. Many of these molecules are important cell surface receptors and the target of disease modifying antibodies. Obtaining structural information from glycoproteins presents significant technical challenges due to the effect of glycosylation on crystallization. We have developed methods to address these issues and have assembled a pipeline for producing and crystallizing glycoproteins in the Oxford Protein Production Facility. The pipeline includes parallelized ligation independent cloning using the Infusion™ system; semi-automated small scale transient expression screening in HEK 293 cells; scale-up of protein production in HEK cells; automated protein purification; quality assessment by mass spectroscopy; and nanodrop crystallization. The application of these processes to solving the structure of a number of glycoprotein complexes will be presented. ____________________________________________________________________________

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____________________________________________________________________________ Contact Details Dr Ray J. Owens Phone: +44 1235 567727 OPPF-UK Email: ray@strubi.ox.ac.uk The Research Complex at Harwell R92 Rutherford Appleton Laboratory Harwell Science and Innovation Campus Didcot, Oxon OX11 0FA United Kingdom

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We would like to thank the following Sponsors for their support:

Optimized genes: Tools for expression enhancement & functional genomics Efficient recombinant expression of human recombinant proteins is indispensable for biomedical research and product development. Often human proteins are difficult to express outside their natural context and differ from their native form, limiting the application range. Accordingly, autologous expression in ideally human cells would be highly desirable, but expression might be hampered by the message encoding the transgene, while low yields are limiting structural and functional analysis. Recent advances in geneoptimization combined with de novo gene synthesis address this problem. Here we report the first large-scale study addressing the influence of multiparameter optimization on autologous human protein expression. Following RNA- and codonoptimization, 50 human candidate genes representing the 5 most important classes of scientific and pharmaceutical interest – transcription factors, ribosomal and polymerase subunits, protein kinases, membrane proteins and a selection of chemokines and cytokines – showed reliable expression and elevated protein levels in 86% of modified genes with no detrimental effect on protein function as exemplarily proven for kinase JNK1 and CDC2. Expression enhancement has been consistently proven for HEK293T-, CHO- and insect cells and thus does not seem to be restricted to a distinct mammalian cell system. Molecular analysis of the representative sequence-optimized cytokine mip1α revealed enhanced transgene expression based on a cumulative effect of enhanced transcriptional activity, increased mRNA stability and improved translational efficiency. Noteworthy optimized gene constructs represent powerful tools in functional genomics as demonstrated by the successful rescue of a siRNA-mediated knock-down of endogenous cdc2-gene following transient transfection with a sequence-optimized counterpart. Contact Details Dr Marcus Graf Email: marcus.graf@geneart.com Geneart AG www.geneart.com Im Gewerbepark B35 93059 Regensburg Germany

Selexis Genetic Elements (SGE) applications for rapid screening and generation of highly productive stable mammalian clones High titers, short timelines and stability are challenging criteria in the development of therapeutic protein producing cell lines. Selexis developed efficient technologies using engineered epigenetic-based DNAs and proprietary SURE tech vectors designed to maximize the potency of these elements. Selexis has different cell line platforms adapted to different needs. The D2 platform is specifically dedicated to the screening of protein variants based on titer/activity, while developing the stable mammalian cell line expressing the lead candidate.

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This innovative platform accelerates the pharmaceutical research allowing to bring the selected lead straight from discovery to preclinical and clinical studies. Contact Details Dr Denise Huber Phone: +41(0)223089322 Director, Business Development Fax: +41 223089361 Selexis SA Email: denise.huber@selecis.com Chemin des Aulx, 18 www.selexis.com 1228 Plan-Les-Ouates Geneva Switzerland

Lonza : Your global Life Science Partner, right next door Lonza is one of the world's leading suppliers to the pharmaceutical, healthcare and life science industries. Its products and services span its customers’ needs from research to final product manufacture. Lonza is the global leader in the production and support of active pharmaceutical ingredients both chemically as well as biotechnologically. Lonza has strong capabilities in large and small molecules, peptides, amino acids and niche bioproducts which play an important role in the development of novel medicines and healthcare products. Lonza is a leader in cell-based research, endotoxin detection and cell therapy manufacturing. Lonza is also a leading provider of value chemical and biotech ingredients to the nutrition, hygiene, preservation, agro and personal care markets. Lonza is headquartered in Basel, Switzerland and is listed on the SIX Swiss Exchange. In 2009, Lonza had sales of CHF 2.690 billion. Further information can be found at www.lonza.com. Contact Details Marc R. Comer, MBA Phone : +1 201 316 9200 Global Marketing Communications Manager Fax : +1 201 312 8639 Custom Manufacturing Email: marc.comer@lonza.com Lonza Inc. www.lonza.com 90 Boroline Road Allendale, NJ 07401 USA

High titer processes for early development – High, consistent quality, 5-10 fold Yield increase compared to standard Fed-Batch processes Major economic drivers for recombinant therapeutics are time to market, cost of goods, and reduction of the financial risks associated with the huge capital investments for dedicated production facilities.One of the strategies to achieve these goals is to improve the manufacturing processes by process intensification.DSM Biologics has developed a highly intensified cell culture process termed XD® in which, similarly to fed-batch, both cells and

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product are retained in the bioreactor while feed is supplied, but, in contrast to fed-batch, waste by-products are removed. As result, viable cell densities over 150 million cells/mL have been achieved with CHO and other cell lines. Product titers up to 27 g/L have been obtained.The XD® technology has been developed to the level that it is virtually clone- and product-independent, through progressive process and media optimization. As such minimal process development is required, thereby reducing development times.Product quality tends to be better in XD® than fed-batch due to the improved metabolic state of the cells and high viability condition. As result, profiles of relative impurities (DNA, HCP per product) have also been showed to be comparable or even better than fed-batch.The scale-up principles behind operating at these extreme cell densities and product titers have been elucidated and excellent scale-up results have been demonstrated in disposable bioreactors. Highly intensified purification steps able to cope with the XD® process output have been developed.Here we present the latest advancements in the development and scale-up of the XD® process and achievements improving productivity and product quality of mammalian cell lines. Contact Details Dr Gerben Zijlstra Email: Gerben.Zijlstra@DSM.com Senior Scientist www.dsmbiologics.com DSM Biologics Zuiderweg 72/2 9744 AP Groningen The Netherlands

Simplifying protein expression with new platform technologies and integrated services Protein expression is perceived as a means to an end to ultimately analyze protein function and identify novel therapeutics. However, there is a significant amount of optimization with labor intensive steps involved to obtain the protein of interest, especially for difficult to express or complex target proteins. Over the years Life Technologies has been providing researchers with tools to facilitate protein expression using a diverse range of host systems. Our collection of expression platforms has evolved into cohesive protein expression services capability initiating with custom gene synthesis progressing through the work flow to purification. We also continue to introduce new technologies to address current limitations with studying important drug target classes including membrane proteins. We have recently developed a mammalian based expression system, MembranePro™ Functional Protein Expression (FPE) System, to produce lipoparticles containing functional G protein-coupled receptors (GPCRs) captured from the cell plasma membrane. MembranePro™ particles offer higher receptor density than crude membrane fractions while maintaining pharmacological equivalence with corresponding native cellular receptors. This balance of technology and custom services addresses a critical bottleneck in protein expression for supporting applications in basic research and drug discovery. Contact Details Reed Hickey Phone: 760 822 9498 Product Manager Fax: 760 476 6878 Molecular Biology Systems Email: Reed.Hickey@lifetech.com 5791 Van Allen Way www.lifetechnologies.com Carlsbad, CA 92008 USA

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Leveraging the Pfenex expression technology platform to enable protein expression for drug discovery Pfēnex Expression Technology™ has been developed specifically as a protein production platform to enable rapid development of expression strains capable of expressing high titers of soluble, active protein and reflects the additional sophistication required of engineered proteins being developed today. This P. fluorescens based system represents a new paradigm of microbial strain development that overcomes today’s slow, iterative and error-prone process through the use of a novel, streamlined, high throughput platform. The combination of an off the shelf toolbox of expression plasmids and host strains, along with high throughput methods for growth and analysis of expression strain candidates, enables the rapid production of proteins for discovery and development. Pfenex’s unprecedented success rate at expressing highly complex proteins combined with an ability to screen up to 1,000 unique expression strains in parallel make it a powerful tool enabling discovery research. Contact Details Dr Diane Retallack Phone: + 1-858-352-4416 Director, Molecular Biology Fax: +1-858-352-4557 Pfenex Inc. Email: dretallack@pfenex.com 5501 Oberlin Dr www.pfenex.com San Diego, CA 92121 USA

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Furthermore we thank the following Sponsors for their kind support:

www.icosagen.com www.coulterflow.com

www.oetltd.com www.kuhner.com

www.atg-biosynthetics.com www.gelifesciences.com

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www.phynexus.com www.bucher.ch

www.wyatt.eu www.roche-diagnostics.ch

www.sartorius-stedim.com www.iba-bioTAGnology.com

www.scilproteins.com www.bio-rad.com

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www.genetix.com www.blueskybiotech.com

www.clontech.com www.promega.com/ch

www.proqinase.com www.dna20.com

www.sigma-aldrich.com www.infors-ht.com

www.plasmidfactory.com www.proteros.com

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www.qiagen.com www.thermofisher.com

www.mn-net.com www.calipers.com

www.aldevron.com www.agilent.com

www.maxcyte.com