Cytogenetics and Cell Genetics · cell culture medium was found to be the most significant parameter affecting the mAb C-K level. Further, the correlations between media copper and

Sunday, April 22, 2012 Opening Keynote

FROM GENETIC ENGINEERING TECHNOLOGY (GENENTECH) TO EPIGENETICS

Arthur Riggs, Ph.D., City of Hope National Medical Center 1500 East Duarte Road, Duarte, CA, 91010, USA

T: 1-626-256-4673, [email protected]

I will give a short personal view of the somatostatin/insulin project that helped start Genentech and the biotech industry. This project also led me to some pioneering studies in the field of epigenetics. I will give an introduction to epigenetics and then describe some recent results on epigenetic stability, adaptability, and reversibility in human embryonic stem cells.

Arthur D. Riggs, Ph.D., professor of biology, is chair of the Department of Diabetes and Metabolic Diseases Research at City of Hope. Formerly the director of Beckman Research Institute of City of Hope, Riggs is widely known for his contributions to the field of epigenetics, a branch of genetics that seeks to understand the biological processes that underlie inherited genetic characteristics that do not result from changes or mutations in the primary chemical base sequence of DNA. In 1975, Riggs published a landmark theoretical paper in the journal Cytogenetics and Cell Genetics that correctly postulated the first known epigenetic mechanism, essentially founding a new branch of genetics. He was the first to identify DNA methylation as the mechanism of epigenetic regulation and inheritance. Methylation is the process by which certain gene bases exchange chemicals with another compound. The chemical exchange acts as a kind of key that “locks” targeted genes so they will not be able to begin the production of proteins. Riggs was the first to propose that methylation influenced DNA-protein interactions and was essential to an organism’s development. The Institute for Scientific Information declared Riggs’ paper a “Citation Classic” owing to the number of times other scientists referred to it in subsequent papers. Riggs, along with City of Hope’s Keiichi Itakura, Ph.D., and University of California, San Francisco’s Herbert Boyer, Ph.D., also helped launch the genetic engineering revolution and the biotechnology industry. Their work in the late 1970s and early 1980s led to recombinant DNA technology that was used by Genentech to produce the first biotechnology product approved by the Food and Drug Administration, a type of synthetic insulin called Humulin that is now used by millions of people with diabetes worldwide. Through his work with the production and engineering of monoclonal antibodies, Riggs has contributed to the development of effective cancer therapies, including Herceptin (breast cancer), Avastin (colon cancer) and Rituxan (lymphoma), and medicines targeting other diseases. Elected to the National Academy of Sciences in 2006, Riggs also was honored with the Juvenile Diabetes Foundation Research Award. He has received distinguished alumni awards from the University of California, Riverside, where he earned his bachelor’s degree in chemistry, and the California Institute of Technology, where he received his doctoral degree in biochemistry.

Monday, April 23, 2012 Session I: Impact of Process on Product Quality

CONTROLLING HIGH MANNOSE GLYCAN LEVEL AND OPTIMIZING TITER THROUGH A BALANCED MODULATION OF CELL CULTURE PROCESS AND MEDIUM CHANGES

Nicole Le, Amgen, Inc One Amgen Center Drive, Thousand Oaks, CA 93012

T: 1-805-447-6539, F: 1-805-499-6819, [email protected] Henry Lin, Amgen, Inc

Jonathan Lull, Amgen, Inc Hedieh Barkhordarian, Amgen, Inc

Amanda Kano, Amgen, Inc

High mannose glycoform may be an important quality attribute that can affect the efficacy of therapeutic monoclonal antibodies. Meeting comparable high mannose glycan level (%HM) is often important when developing a commercial process to replace the early phase clinical process. Strategies were developed to tune the %HM in order to meet the comparability range while at the same time increasing titer for the commercial process. The strategies utilized a combination of process optimization and medium modification in order to produce the desirable %HM profile. In several process instances, titer had to be sacrificed in order to meet the desirable %HM level; however, this was overcome by modulation of certain medium components. Yet, precise balance between process and medium needed to be made in order to achieve the most desirable state for both titer and %HM. Overall strategies and learning on taking this dual approach to controlling the %HM while improving titer will be discussed.


UNDERSTANDING INCREASED C-TERMINAL LYSINE IN A RECOMBINANT MONOCLONAL ANTIBODY PRODUCTION USING CHINESE HAMSTER OVARY CELLS WITH CHEMICALLY

DEFINED MEDIA

Jun Luo, Genentech 1000 New Horizons Way, Vacaville, CA 95688, USA

T: 1-707-454-5483, F: 1-707-454-5483, [email protected] Jian Zhang, Genentech

Diya Ren, Gilead Sciences Wen-Lin Tsai, La Jolla Biologics

Terry Hudson, Genentech

C-terminal lysine (C-K) variants are commonly observed in therapeutic monoclonal antibodies and recombinant proteins. Heterogeneity of C-K residues is believed to result from varying degree of proteolysis by endogenous carboxypeptidase(s) during cell culture production. The achievement of batch-to-batch culture performance and product quality reproducibility is a key cell culture development criterion. In this study, atypical high variation (from ~5% to ~31%) of C-K level at different scales and process conditions was observed during development of a monoclonal antibody produced by CHO cell line X. The specific CHO cell line was selected as the model cell line due to the exhibited sensitivity of its C-K level to the process conditions. A weak cation exchange chromatography (WCX) method with or without carboxypeptidase B (CpB) treatment was developed to monitor the C-K level for in-process samples. The heterogeneity of basic variant was contributed to cell culture process and no selective removal effect was observed through purification steps. Then, the three different parameters to generate high and low C-K levels of mAb X, were evaluated. They are a medium component (copper) and operation conditions (temperature and culture duration). Of those tested, the copper concentration in the cell culture medium was found to be the most significant parameter affecting the mAb C-K level. Further, the correlations between media copper and zinc concentrations, zinc/copper ratio, and carboxypeptidase(s) activity were examined in both shake flask and bioreactor cultures. The opposite effect of copper and zinc on C-terminal processing was observed. The existence of intracellular carboxypeptidase(s) in CHO was confirmed by Western blot. A hypothesis on C-K processing was proposed based on the analyses of C-K levels in bioreactor cultures with cell culture fluid (CCF) and harvested cell culture fluid (HCCF) samples by WCX and the Western blot results. Understanding the biological cause of the lysine removal and the process parameters affecting the extent of C-K may provide valuable insights to cell culture process development and control.


MODULATING PRODUCT QUALITY THROUGH CELL LINE AND PROCESS MODIFICATIONS

Anne Kantardjieff, Alexion Pharmaceuticals 352 Knotter Drive, Cheshire, CT 06410, USA

T: 1-203-271-8351, F: 1-203-271-8197, [email protected] Adam Lucka, Alexion Pharmaceuticals Praik Jaluria, Alexion Pharmaceuticals

Protein glycosylation is an inherently variable process and understanding the factors which contribute to this variability is critical to developing robust cell culture processes. This study details the product quality changes observed when adapting a non-antibody CHO cell culture process developed by a third party manufacturer to our CHO platform expression system and process. Further improvements to the process were also characterized for their impact on the glycosylation profile of this recombinant fusion protein.

In-house characterization of the original process identified several limitations, including poor cell growth and insufficient productivity levels. Efforts were therefore initiated to develop a new cell line using our GS-CHO platform expression system. Early characterization of GS-CHO material revealed that the host cell change had resulted in differences in terminal sialic acid content and had also impacted the amount of Man6 species detected.

We next sought to identify process parameters which could modulate similar changes in glycosylation profiles. One potential parameter was viability at harvest. The original process contained a narrow harvest criterion based on cell viability, and we wanted to better characterize the impact of this parameter on glycosylation profiles. We found that sialic acid content decreased with decreasing cell viability, although this trend was more pronounced in the original cell line than in GS-CHO cells. The impact of additional process parameters on glycosylation, including pH and temperature shifts, as well as alternative basal media and feeds, was also investigated. To complement these studies, we also examined the product quality changes observed between primary and secondary clones.

These investigations provided an early characterization of the process design space and allowed us to assess the impact of key process parameters on product quality. The final process was successfully scaled-up and transferred for clinical material production.


CONTROLLING ACIDIC VARIANT FORMATION AND GLYCAN PROFILE THROUGH MANIPULATION OF CULTURE TEMPERATURE PROFILE AND MEDIA COMPOSITION

Nathan McKnight, Genentech 1 DNA Way, South San Francisco, CA 94080, USA

T: 1-650 467 4292, F: 1-650 467 5477, [email protected] Lauren Feeney, Genentech

Thomas DiRocco, Genentech Robel Tezare, Genentech

Veronica Carvalhal, Genentech

This case study will describe development of a fed-batch CHO culture with improved monoclonal antibody yield, but with particular emphasis on modulating formation of acidic variants and glycan profile through changes in process parameter setpoints and media composition. The goal of the project was to improve process yield, but maintain product quality comparable to the existing process. Yield improvement was realized through screening peptones and peptone blends, supplementation of media with copper and optimization of the culture temperature profile, with increased titer achieved through increased culture growth. However, acidic variant formation and glycan profile, primarily G0 content, were initially outside or near the edge of their targeted ranges for comparability. Correlations between these product quality attributes and process parameter values, peptones evaluated during screening, and key performance indicators were evaluated. Higher final culture viability was observed to correlate with positive product quality outcomes (i.e., better alignment with comparability ranges). Further process development focused on maintaining increased culture growth, and the resulting increase in titer, while also increasing final culture viability and desired product quality. Ultimately, a multiple temperature shift profile was tailored to promote growth through most of the culture then reduce metabolic activity and maintain higher viability at later stages, and enabled the final process to successfully meet the goals of improved yield and comparable product quality.


BI-HEX® –OPTIMISING PRODUCT QUALITY ATTRIBUTES THROUGH HOST CELL ENGINEERING AND UPSTREAM PROCESS OPTIMIZATION

Anurag Khetan, Boehringer Ingelheim Process Science Germany, Cell Culture, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der

Riss, Germany T: +49-7351-54-141430, F: +49-7351-83-92159, [email protected]

Barbara Enenkel, Stefan Schlatter, Harald Bradl, Jochen Schaub, Till Wenger, Anne B. Tolstrup and Hitto Kaufmann, Boehringer Ingelheim

Boehringer Ingelheim´s Biopharma Process Science is dedicated to developing cell lines and manufacturing processes both to a diverse panel of costumer novel biological entities (NBEs) as well as for internal products including biosimilars. Our CHO-based BI-HEX® platform combines state-of-the-art technologies within vector design, cell line generation, process and media optimization in one concept enabling us not only to meet the growing demands for fast CMC development times but importantly also to manipulate molecule properties and product quality attributes. The aspect of tailoring product quality through CMC process optimization is getting increasingly important, not only for development of efficacious NBE functionalities, but also to allow fast and successful development of biosimilars with the adequate properties.

Product mechanism of action and efficacy are at least for monoclonal antibodies frequently linked to the glyco-profile of the molecule. We will show how key platform tools such as expression vector elements, use of genetically modified CHO subclones as well as DoE-based approaches to media and process optimization have been used at BI to manipulate and obtain distinct glyco-patterns. High-throughput glycoanalysis technologies have been established as very important tools in this regard and will be described. Furthermore, the presentation will focus on molecule property manipulation through the use of host cell evolution to obtain subclones with different glyco-profiles, and we will also show data from the combined use of BI-HEX tools in conjunction with the GlymaxX glyco-engineering technology.


EFFECT OF A MEDIA REDUCING AGENT ON MONOCLONAL ANTIBODY ASSEMBLY AND GLYCOSYLATION IN NS0 CELL CULTURE

Ben Dionne, University of Manitoba 45 Chancellor Circle, Winnipeg, Manitoba, R3T 2N2, Canada

T: 1-204-474-8782, [email protected] Michael Butler, University of Manitoba

Therapeutic and market values of monoclonal antibodies (Mabs) have been dramatically increased over the past few years. The intracellular assembly and glycosylation of Mabs is very important in ensuring consistent glycan profiles which are essential for efficacy and effectiveness. Differing theories have been proposed for how Mabs are assembled and this assembly mechanism may play a role in glycosylation events. The two main models have secondary intermediates of either a heavy chain dimer (100kDa) or heavy chain-light chain (75kDa). The research here highlights the association between IgG1 intracellular intermediates and glycan profiles by examining the temporal relationship between glycosylation and disulfide bond formation between the individual chains of a Mob produced from a murine cell line (NS0). Using a non-cholesterol dependent NS0 cell line in serum free media containing radioactive isotopes of 35S labeled cysteine and methionine, Mabs were labeled, produced and examined at various time points under varying reducing media conditions to extract information regarding this association. Using HILIC-HPLC methods a 33% downward shift in GI (Galactosylation Index) was observed when reducing agents were introduced. The autoradiographs of the protein A purified intracellular IgG1 and its fragments provided two results. Firstly, the assembly pathway of this IgG1 followed published reports that low galactosylation was favored in situations where heavy chain dimers (100kDa) formed as opposed to heavy chain-light chain(75kDa) intermediates. Secondly, the ratio of heavy chain dimer to heavy chain monomer increased over time within the reducing agent cultures. The increase in heavy chain dimers and lower GI appear to be correlated, possibly due to disruption of the disulfide bonds at the higher levels of assembly. A change in the assembly pathway may alter the final IgG glycan pattern and possibly lead to control mechanisms that influence glycan profiles of monoclonal antibodies.

Monday, April 23, 2012 Session II: Application of ‘Omics in Biotherapeutic Process Development and Control

GENOME-SCALE ANALYSIS OF CHINESE HAMSTER OVARIAN CELL LINES

Bernhard O. Palsson, CHOmics, Inc P.O. Box 927153, San Diego, CA, 92192-7153, USA

T: 1-858-534-5668, F: 1-858-822-3120, [email protected] Nathan E. Lewis, CHOmics, Inc. Aarash Bordbar, CHOmics, Inc.

Deniz Baycin, Johns Hopkins University Michael J. Betenbaugh, Johns Hopkins University

Over the past 15 years, microbe-based engineering has advanced through three major innovations: 1) genome sequencing, 2) genome-scale metabolic models, and 3) tools for genome editing. These have allowed engineers to identify cellular parts, simulate product synthesis, and manipulate host genomes to enhance production. Similar advances are now upon us in the engineering of Chinese hamster ovarian (CHO) cell lines for bioprocessing. Over the past few years several groups have developed zinc-finger nucleases to facilitate targeted genome editing. Here we present progress in the other two major innovation areas that will enhance metabolic engineering in these cell lines. First, as part of an international team, we recently published the genome sequence for the ancestral CHO-K1 cell line this past year, and demonstrated that many previously reported “missing” genes seem to still be in the genome, but not expressed. We also have sequenced six additional CHO cell lines and we will highlight the genomic differences between these here. Lastly, we have integrated -omics data to construct a genome-scale model of CHO-K1 metabolism and have used this to study how metabolic pathways can modulate the production of glycosylated biotherapeutics. Thus, with these tools, we are now entering an era of genome-scale engineering of CHO cell lines.


USING CHO SEQUENCE DATABASES FOR MICRO-RNA ENGINEERING

Nicole Borth, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria

Muthgasse 18, Vienna, 1190, Austria T: +43 1 47654 6232, F: +43 1 47654 6675, [email protected]

Matthias Hackl, Vaibhav Jadhav, Juan A. Hernandez Bort, Johannes Grillari

The main impact of the now published genome, transcriptome and miRNome of CHO cells is that it enables researchers to begin to understand the molecular mechanisms of how these cells perform their tasks of efficient growth, high productivity and safe product quality. As an example this presentation will focus on how this information has improved our ability to use microRNAs for phenotypic engineering of cells.

MicroRNAs are master regulators that control, in a co-ordinated fashion, the gene activity of entire pathways on a post-transcriptional level. The active sequence of microRNAs is very short, consisting of approximately 22 nucleotides, and is highly conserved across species, as are the target sequences in the respective mRNAs. However, due to the complex processing of pre-microRNAs in the nucleus, the sequence of the primary transcript and demonstration of the typical folding patterns are required for inclusion in miRbase. Extraction of these sequences from the genome and prediction of their folding structure revealed that the cross-species-homology of the entire hairpin loop was significantly lower, raising the question whether there may be species specific differences in the biogenesis machinery that lead to better processing of autologous miRs in a given cell line. This is especially important in the case of entire miR clusters that are expressed by polycistronic promoters. For the identification of the genomic sequences of the pre-mir an independently obtained genomic sequence, although of low coverage, was used in addition to the published one, and yielded additional pre-miR sequences that were not found in the other genome, and vice versa. In addition, some SNPs were detected between the two genomes. Thus, especially in view of the genomic diversity known to exist between CHO cell lines, it will be necessary to obtain additional sequences of entire genomes to finally arrive at a comprehensive picture of CHO cells.

Comparing the miR expression pattern of cells cultivated with serum and adapted to protein free media, an overall reduction in expressed miRs was observed. Our results show that the expression of important enzymes in this pathway, including Drosha and Dicer is positively correlated to the growth rate, indicating that a higher level of translational repression as enforced by miRs is an important strategy of cells to ensure efficient use of translation and resources. Engineering cells to achieve specific phenotypes will thus be dependent on modifying the expression levels of specific miRs, both toward overexpression or reduction, for instance by miR sponges. The obvious path forward now is analysis of the effect of each of the now known 350 miRs expressed in CHO cells both on a phenotypic and molecular level. For this an efficient testing system was developed that allows assessment of miR effects on process relevant properties at medium throughput. Using this test system and analyzing the transcriptome and proteome of cells after transfection will help us to identify the targets of miRs, which, in combination with all available sequence information, will enable the development of better algorithms for miR target prediction, as well as for optimization of vectors for recombinant products, to take full advantage of the miR machinery for improved productivity.


A MOLECULAR PROFILE OF INDUSTRIAL CELL CULTURE: EXAMINING THE TRANSCRIPTOME DYNAMICS OF RECOMBINANT PROTEIN PRODUCING FED-BATCH AND PERFUSION CULTURES

Karthik P. Jayapal, Bayer HealthCare 800 Dwight Way, B28A-309A, Berkeley, CA, 94710, USA

T: 1-510-705-5056, F: 1-510-705-4720, [email protected] Kathryn C. Johnson, Chemical Engineering and Materials Science, University of Minnesota, Minneapolis,

MN Wei-Shou Hu, Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN

Chetan T. Goudar, Bayer HealthCare

The past two decades have witnessed the rise of mammalian cells as hosts for biomanufacturing of complex therapeutic glycoproteins. Large stirred tank vessels continue to remain as the industry’s preferred workhorse for culturing these cells, primarily in fed-batch or perfusion modes of operation. Yet, even in this era of genomics, knowledge of such processes from a biomolecular viewpoint remains scarce. The limited set of studies on this subject mostly pertain to non-producing, small-scale cultures and/or exponentially growing mouse and human cell lines which may not directly relate to the vast majority of commercial recombinant protein producing processes, particularly those employing hamster-derived cell lines. In this study, we employed DNA microarrays to examine the transcriptome dynamics of two industrial recombinant protein producing processes employing hamster-derived cell lines. In the first case, we examined the dynamics of a non-steady state CHO fed-batch culture in which initial lactate accumulation was followed by subsequent lactate consumption. Clusters of genes correlating with this lactate profile as well as those potentially relating to growth and stationary (protein-producing) phases were identified. In the second study, we profiled the long-term dynamics of a presumably ‘steady-state’ BHK perfusion process. We show that gene expression profiling can identify candidate genes that correlate with certain subtle changes in cell behavior attributable to cell age and other important process variables. In addition, we also demonstrate the utility of such tools in comprehensive process comparisons – in this example, for validation of scale-down process models.


DECIPHERING CHO CELLS AND BIOPROCESS PERFORMANCE THROUGH METABOLITE PROFILING

Alan Dickson, University of Manchester The Faculty of Life Sciences, Michael Smith Building, Oxford Road, Manchester, ., M13 9PT, UK

[email protected] Alexandra Croxford, Arfa Maqsood, Christopher Sellick, The Faculty of Life Sciences, Michael Smith

Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK Gillian Stephens, School of Chemical Engineering & Analytical Sciences, The Manchester

Interdisciplinary Biocentre, University of Manchester, Manchester, UK Hans Westerhoff, School of Chemical Engineering & Analytical Sciences, The Manchester

Interdisciplinary Biocentre, University of Manchester, Manchester, UK Roy Goodacre, School of Chemistry, Manchester Interdisciplinary Biocentre, University of Manchester,

Manchester UK

Metabolite profiling of intra- and extra-cellular metabolites offers the potential for sensitive read-outs of cellular status and response to environmental conditions – read-outs that may be interpreted to define approaches for optimization of product quality and yield. As with all ‘omics technologies, metabolite profiling (metabolomics) generates vast amounts of data and data quality is paramount for meaningful interpretation. We have developed a quenching/extraction protocol for suspension-cultured mammalian cells that enables the isolation of intracellular metabolites in physiological concentrations1. Multiple intracellular samples can be processed simultaneously and, along with extracellular metabolite profiles, a balanced interpretation of overall cellular metabolism during CHO cell cultures can be obtained and this forms a verified starting point for meaningful analysis of cell performance. A series of studies have performed on suspension CHO cells in chemically-defined culture medium and these have illuminated general characteristics of CHO cell metabolism. Studies include (a) batch cultures in which metabolic reactions have been identified as limiting culture performance (b) feed responsiveness identifying the metabolic events the define growth and stationary phases of culture and (c) metabolic responsiveness to external growth regulators associated with decreased cell growth. Phasic handling/redirection of metabolites linked with pyruvate metabolism has been highlighted from all model systems. Interpretation of co-ordinated changes across multiple metabolites/pathways has been facilitated by in-house data-handling and visualization packages. As outputs amalgamated information from our studies has allowed design (minimal components and optimal time of addition) of a simple 4-component feed that doubles secreted product yield. Fundamental elucidation of cellular metabolism under industrially-relevant conditions of culture has highlighted potential sites for cell engineering to generate improved CHO cell performance in relation to existing and developing culture processes. We have extended our experimental analyses of metabolite profiling to development of a model for predictive assessment of CHO cell performance in relation to medium and feed. This model, developed through convenience kinetic modeling on the COPASI platform, has been tested with several of the experimental datasets obtained in our experimental approaches. The model is highly predictive of the fates of the majority of core metabolites and has indicated specific metabolic nodes, with potential for regulatory intervention, that require further detailed experimental assessment. In summary, we have shown metabolite profiling to be a powerful “stand-alone” ‘omics approach for interrogating and enhancing CHO cell performance in varied culture conditions. Moving ahead to merge information from metabolite profiling with data from other ‘omics will provide a fascinating vision of whole cell function – a status that will provide powerful future insights towards bioprocess design.

Sellick, C.A., Hansen, R., Stephens, G.M., Goodacre, R. & Dickson, A.J. (2011) “Metabolite extraction protocol for global metabolite profiling of suspension-cultured mammalian cells” Nature Protocols. 6: 1241-1249.

Tuesday, April 24, 2012 Session III: Rapid Material Supply for R&D, Toxicology, Early Clinical Manufacturing, and Biodefense

VECTOR AND CELL ENGINEERING FOR RAPID PRODUCTION OF MABS IN CHO CELLS.

Trent Munro, Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland

Cnr College and Cooper Rd, St Lucia, QLD, 4072, Australia T: +61-7-334-643-947, F: +61-7-334-643-973, [email protected]

Jongwei Wooh, Joe Codamo, Jeff Hou, Ben Hughes and Peter Gray, Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland

Rapid production of high quality recombinant proteins is critical for early stage product development. Many groups utilize HEK based transient expression systems, however commercial manufacturing remains dominated by CHO host cells. Development of efficient and scalable CHO transient expression systems is required. We have previously reported the EpiCHO episomal-based transient expression system for CHO cells. The EpiCHO system enables elevated and prolonged recombinant protein expression through the utilization of elements from the Polyoma (Py) and Epstein-Barr (EBV) viruses to promote the replication and maintenance of plasmid DNA.

In this work we show a 5-fold improvement in monoclonal antibody (mAb) volumetric productivity by examining key parameters including transfection medium, cell density, transfection reagent, DNA:reagent ratio, the time of transfer to mild hypothermia and feeding strategy post transfection. The EpiCHO system allowed for a 6-fold expansion in culture volume post-transfection without significantly effecting specific productivity. This system generates 400% more mAb/μg of plasmid DNA when compared to a non-episomal system. In addition we have created a second-generation system, in which we created a cell line – eCHO-T - that stably expresses both EBNA-1 and Polyoma virus large-T antigen. Using this line we have demonstrated further enhancement of mAb production while simplifying the expression construct and incorporating a simple fed batch production procedure.

In summary we have demonstrated the utility of CHO based transient gene expression for rapid production of mAbs.


USE OF AN ANTI-APOPTOTIC HOST CELL LINE FOR HIGH THROUGHPUT TRANSIENT TRANSFECTIONS

Athena Wong, Genentech Inc 1 DNA Way, S San Francisco, CA, 94080, USA

T: 1-650-467-4767, F: 1-650-467-5477, [email protected]

Transient transfection of mammalian cells is used to rapidly produce recombinant proteins for research, manufacturability assessments and proof of concept tox and PK studies. This presentation will discuss advances in our PEI-mediated transient transfection system, in particular, the transfection process development for an anti-apoptotic CHO host cell line. Pro-apoptotic proteins, Bax and Bak, induce apoptosis by permeabilizing the mitochondrial membrane and activating the caspase proteolytic cascade resulting in cell death. An anti-apoptotic CHO cell line, referred to as the Bax Bak double knockout (DKO), was generated by deleting Bax and Bak in a CHO-K1 cell line using zinc finger nuclease mediated gene disruption. Parameters investigated to optimize transfection of the Bax Bak DKO cell line include: seed train and production media, DNA:PEI ratios, temperature set point and shift timing as well as feeding strategy. The Bax Bak DKO cells maintained high viability in the seed train and during transfection, and appeared more resistant to transfection reagent-induced viability declines. Transfected Bax Bak DKO cells also yielded ~1.5-2 fold higher levels of protein (i.e. human and murine antibodies, Fc fusion) than standard CHO cells. To decipher the possible mechanism for the increased transfection productivity, we compared DNA uptake levels, nuclear plasmid copy number and mRNA expression of transfected Bax Bak DKO vs standard CHO cells. Bax Bak DKO cells internalized ~2-3 fold higher amounts of transfected DNA than standard CHO cells. Cell surface heparan sulfate has been demonstrated to mediate uptake of DNA:PEI transfection complexes. FACS analysis revealed that the Bax Bak DKO cells had higher levels of cell surface heparan sulfate. In summary, utilizing an anti-apoptotic CHO host cell line which has enhanced DNA:PEI uptake capacity and high viability in seed train and post-transfection, we developed a new serum free, high yield transient transfection process.


DEVELOPMENT OF PREDICTIVE METHODS FOR CELL LINE SELECTION AND PROCESS DEVELOPMENT

Matthieu Stettler, Merck Serono Zone Industrielle B, Fenil-sur-Corsier, N/A, 1809, Switzerland

T: +41 21 923 26 18, [email protected] Arnaud Périlleux, Merck Serono Milène Marsaut, Merck Serono Martin Jordan, Merck Serono

Cell line selection programs and early stage process development are currently undergoing major changes. Newly designed methods and tools are more predictive and aim to reduce both timelines and costs. Efforts to further increase recombinant protein expression levels from mammalian cells heavily rely on such improved methods. The evaluation of a larger number of cell lines in a predictive system increases access to diversity and the probability of identifying high producers.

This study focuses on the development of a robust fed-batch platform process in a 96-deepwell plate system for cell line screening and process development. The purpose of the system is to screen hundreds of cell lines in suspension with a feeding system and run duration equivalent to the platform bioreactor process used at Merck Serono. We highlight some of the key process parameters that impact cell growth and culture viability of fed-batch suspension cultures performed in shaking 96-deepwell plates. We also describe the workflow that was implemented to transfer the cell lines from static conditions (clone picking) and adaptation to suspension conditions in such a system, prior to the initiation of a fed-batch screening. Additionally, we show how the cell line stability can be assessed in the 96-deepwell plate system.

Challenges such as robustness of the high throughput 96-deepwell plate system, integration of robotic liquid handling systems and implementation of high throughput analytics will be discussed. The screening system demonstrated cell line specific culture performances which are similar to those of cultures carried out at larger scale in bioreactors and predictive in terms of final titer. The implementation of such tools improved cell line generation programs in terms of timelines and intrinsic performance of the manufacturing cell lines. Additionally, the 96-deepwell plate cultivation system can be efficiently used to complement existing development tools with increased throughput and enhanced potential for Design of Experiment approaches.


DEVELOPMENT PIPELINE DEBOTTLENECKING FOR INCREASED SPEED AND THROUGHPUT OF THERAPEUTIC ANTIBODY OPPORTUNITIES

Kevin Bailey, Regeneron Pharmaceuticals, Inc. 777 Old Saw Mill River Road Tarrytown, NY, 10591, USA


Getting novel therapeutic antibodies to patients with unmet medical needs necessitates the implementation of strategies to allow rapid preclinical development. Attaining a robust, highly efficient process for commercialization requires totally separate goals and strategies. Regeneron's unique approach aimed at meeting these competing objectives will be described along with the technologies and strategies used to carry this out. Increased reliance on Quality by Design (QbD) principles has been incorporated into our approach to provide sound scientific rationale for changes in cell lines and processes. Regeneron's VelociSuite™ technologies that drive this approach will also be described.


DISCOVERY AND THERAPEUTIC OPTIMIZATION OF THE NEXT GENERATION OF ANTIBODY DRUGS

George Georgiou, University of Texas, Austin CPE Building, Austin, TX 78712, USA


This presentation will focus on two strategies we have recently developed for changing the paradigms of antibody pharmacological optimization and discovery. 1. Aglycosylated antibodies: The function of many antibody drugs is critically dependent on the engagement of innate immune cells and lymphocytes via the interaction of the Fc domain of IgG with Fc receptors. The binding of the Fc to the effector Fc receptors is modulated by the glycan at N297 and is essentially abolished in antibodies that are not glycosylated. However using HT screening approaches we have generated a set of Fc mutants that bind to one or more pro-inflammatory Fc receptors but not to the inhibitory receptor FcRIIb. We have shown that these engineered aglycosylated antibodies elicit significantly improved ADCC and ADCP relatively to their glycosylated counterparts and also importantly, can display novel effector functions not normally elicited by antibodies. Thus engineered aglycosylated antibodies have the potential to significantly accelerate bioprocess development and improve pharmacological action. 2. Discovery: So far antibody discovery has relied on the isolation of binders to known molecules associated with disease. We are developing a radically different approach, for IgG discovery, whereby we mine the serological responses in patients that have overcome the disease. For this we have developed a proteomic strategy for the molecular deconvolution of the monoclonal antibodies in the patient's serum and then we express these antibodies to evaluate cytotoxicity and therapeutic potential in vitro, in the absence of any preconceived notions about the identity/nature of the antigen.

Tuesday, April 24, 2012 Session IV: Business and Regulatory Considerations for Managing the Lifecycle of Commercial Biologics

BIOSIMILARS AND INNOVATION

Barry C. Buckland, BiologicB.LLC 5911 Carversville Road, Doylestown, PA 18902, USA


A major new cycle of events is about to begin. After a golden era of therapeutic protein development resulting in sales of tens of billions of dollars and a wonderful impact on human health we are about to experience an extraordinary wave of patent expiration in the USA between the years of 2014 and 2020.

The race has begun! The original innovators are looking for ways to protect their franchise perhaps by introducing improved versions of the original product. The competition are looking for ways to introduce Biosimilar versions of the original product. For this latter group innovation will come in the form of production processes and approaches that result in lower cost of goods, novel business approaches and partnerships, and innovative approaches for addressing Regulatory issues.

The market in the USA is at the same time both the most lucrative and also one of the hardest to penetrate. This talk will present an overview of the opportunities and also the challenges not only in the USA but globally.

Companies attempting to create and license Biologics vary from leading Pharmaceutical companies to major Generic companies, new virtual companies and also companies in Emerging Markets. There are a diverse range of approaches being taken and these will be described. The talk will highlight the significant barriers to entry and how this will limit the number of major players. An overview will be given of the evolving Regulatory environment.

It is likely that significant progress will occur in the Emerging markets where the need for accessibility to these fantastic products is acute and the market is currently significantly underserved. If the vaccine industry is a guide, this will often result in local manufacturing and product development strategies in order to meet the constraints of the pricing environment. Examples will be given from the vaccine industry to illustrate this point.


MANAGING DECISIONS ACROSS BIOPHARMACEUTICAL LIFECYCLES FROM DEVELOPMENT THROUGH TO COMMERCIAL SUPPLY

Suzanne S. Farid, University College London The Advanced Centre for Biochemical Engineering, Dept. of Biochemical Engineering, Torrington Place,

London, WC1E 7JE, UK T: +44 20 7679 4415, F: +44 20 7916 3943, [email protected]

The biotech sector has come under increasing economic and regulatory pressures for continuous improvement on both drug development and manufacturing fronts. As a result, assessing the value potential of alternative strategies has become critical for decision-making in areas such as the design of bioprocesses and facilities, tech transfer as well as capacity sourcing for long-term commercial supply. UCL’s Decisional Tools team have developed advanced decision-support tools that effectively integrate concepts from bioprocess economics, dynamic simulation, risk analysis, combinatorial optimization and operations research to address such challenges. This presentation will show practical applications of such models to industrially-relevant problems faced during a biopharmaceutical’s lifecycle from collaborative projects between UCL’s Decisional Tools team and industrial collaborators such as Pfizer, Lonza Biologics and MedImmune.

The first case study focuses on cell culture technology decisions for the commercial manufacture of monoclonal antibodies. Specifically it compares the current and future potential of single-use fed-batch cell culture to two continuous perfusion technologies, spin-filter and alternating tangential flow (ATF) perfusion, across a range of titers, scales and failure rates. The second case study evaluates the potential of alternative purification platforms for future cell culture antibody titers using stochastic evolutionary optimization algorithms to facilitate prioritization of optimal ratios of upstream to downstream trains, purification sequences and equipment sizing strategies. The third case study focuses on rapid prediction of facility fit issues upon tech transfer by linking uncertainty analysis with multivariate statistical methods so as to determine the likelihood of unwanted events such as product loss and their root causes. Finally, the fourth case study addresses the challenge of long-term production planning for a portfolio of commercial candidates with different product stabilities and hence different cell culture modes (fed-batch versus perfusion). Mathematical programming is used to optimize the capacity plans across several multiproduct and multisuite facilities globally as well as third party contract manufacturers. Methods for visualizing the financial implications and robustness of alternative solutions will be illustrated for each case study so as to facilitate meaningful dialogue to priorities solutions.


OPTIMIZING PRODUCTION AND DEVELOPMENT WORK FLOWS USING REAL DATA, SIMULATIONS, AND DESIGN OF EXPERIMENTS

Rick Johnston, Ph.D., Bio-G 1250 Addison St, Suite 107, Berkeley, CA, 94704, USA T: 1-510-704-1803, F: 1-510-704-0569, [email protected]

We outline methods for optimizing facility capability based on real data, simulations and design-of-experiments. With rapidly evolving titers and process platforms, biomanufacturers need to understand how existing and new products will optimally 'fit' into facilities. We show how a more comprehensive approach - including variability, resource constraints, and supply confidence levels - can create a more accurate picture of facility capacity. We also show how this concept extends to optimizing work flows in process development, clinical manufacturing and tech transfer. Several case studies will be presented.


A NOVEL BACTERIAL CONTAMINATION IN CELL CULTURE MANUFACTURING

Robert Kiss, Genentech, Inc. 1 DNA Way, MS 96A, So. San Francisco, CA, 94080, USA

T: 6502255135, F: 6504675477, [email protected] Jesse Bergevin, David Peers, Joseph Chen, Harry Lam, Patricia Lufburrow, Anders Vinther, Genentech,

Inc.

A Genentech CHO cell culture manufacturing facility recently experienced bacterial contamination events with a novel organism never previously observed within the manufacturing network. This organism presents unique challenges to both prevention (e.g, ability to penetrate 0.1 um filtration) and detection (undetectable via standard microbiology methods). Thus, this experience warrants disclosure to the industry at large. This presentation will include discussion of the approaches taken to enhance prevention and detection, as well as the assessment of the potential for impact to drug product, should a contamination remain undetected.


REGULATORY CONSIDERATIONS FOR MANAGING LIFECYCLE OF BIOLOGICS

Art Blum, Biomarin Pharmaceutical Inc. 105 Digital Dr., Novato, CA, 94949, USA

T: 1-415-506-6181, F: 1-415-506-6941, [email protected] Terry Milby, Biomarin Pharmaceutical Inc., USA

Regulatory considerations play a prominent role in managing the lifecycle of commercial biologics. This talk will explore philosophies and decision points as manufacturing processes are developed from research through clinical trials and commercialization. The “do it fast” and “do it right” approaches must be balanced and risk/benefits identified to assure success. This presentation will illustrate examples where aggressive and well thought through development strategies have been successful in bringing products to market meeting applicable regulatory requirements.

Wednesday, April 25, 2012 Session V: Challenges and innovation in late stage process development and manufacturing sciences

IMPLEMENTATION OF INTEGRATED CONTINUOUS BIOPROCESSING FOR THE PRODUCTION OF VARIOUS TYPES OF THERAPEUTIC PROTEINS

Konstantin Konstantinov, Genzyme Corp. 45 New York Ave., Framingham, MA, 01701, USA

T: 1-508-685-5356, F: 1-508-271-3452, [email protected] Tim Johnson, Chris Hwang, Jean McLarty, Betsy Simmons, Megan Blewis, Jason Walther, Marcella Yu, Ben Wright, Weichang Zhou, Veena Warikoo, Rahul Godawat, Kevin Brower, Dan Cummings, Frank Riske, Ken

Karey, Genzyme Corp.

At the 2010 Cell Culture Engineering Conference, we presented Genzyme’s vision of an integrated bioprocessing platform, along with the lessons biotech can learn from other industries in terms of continuous manufacturing. Since then, we have established the required technology platforms, which are now fully integrated and implemented at development scale. The streamlined process train, which eliminates multiple non-value added unit operations (hold steps, clarification, etc.), is reduced to a high density perfusion bioreactor directly linked to a continuous chromatography capture step. We will present recent results with the long-term steady state production of both stable (MAb) and unstable (enzyme) proteins. The bioreactors are operated at a cell density of 50e6 cells/ml, and perfused with chemically defined media. The continuous chromatography step is based on periodic countercurrent technology, utilizing Protein A or high capacity mixed-mode resins. Strategic advantages of the integrated continuous platform are minimal residence and cycle times, dramatically reduced equipment footprint, steady-state operation (consistent product quality), high productivity, minimal scale-up requirements (small bioreactors and chromatography columns), flexibility, and modularity. The elimination of multiple non-value added steps and the order-of-magnitude decrease in equipment size results in a minimalistic manufacturing facility design and significantly reduced capital investment. Based on these attributes, integrated continuous bioprocessing is being explored as a universal platform for the production of various therapeutic proteins at Genzyme.


IMPROVING OUR UNDERSTANDING OF RAW MATERIALS AND THEIR IMPACT ON CELL CULTURE PROCESSES

Gregg Nyberg, Amgen 4000 Nelson Road, Longmont, CO, 80234, USA


When faced with a technology transfer of a recombinant cell culture manufacturing process to a new site and/or scale, cell culture engineers/scientists often direct their attention to factors potentially impacting mass transfer or shear. Factors such as reactor geometry, scale and sparger design are often obvious differences that are easily identified. However, reflecting back on over fourteen years of clinical and commercial process technology transfer/scale-up experience, only three out of fourteen significant issues encountered by one cell culture engineer could be categorized as directly related to mass transfer/shear. The remaining issues could be categorized as related to raw material lot-to-lot variability (five issues), trace elements (four issues) or media chemistry (3 issues). The relatively limited number of mass transfer/shear related problems should not be taken to imply these issues are not important; rather, adequate tools and awareness exist such that an experienced cell culture engineer can usually anticipate and avoid issues. In contrast, the relative awareness and sophistication of our tools to address issues related to raw material lot variability and/or media chemistry are less developed. This talk will present case study examples of issues encountered due to inadequate understanding of raw materials and media chemistry, and discuss approaches to avoid such problems in the future. The importance of sensitive, representative scale-down models will be emphasized, as will comprehensive analysis of trace elements and application of tools such as multivariate statistical process monitoring.


THE IMPACT OF LOT-TO-LOT VARIABILITY OF A DISPOSABLE CELL CULTURE BAGS ON CELL GROWTH DURING THE SCALE-UP OF A MAMMALIAN PRODUCTION CELL LINE: ROOT CAUSE

ANALYSIS AND LESSONS LEARNED FOR THE PIPELINE.

Patrick Gammell, Pfizer Grange Castle, Dublin, Dublin, Ireland

T: 1-353-1-469-6910, [email protected] Catriona Crawford, Mary Heenan, Neysi Ibarra, Carmel Jennings, Anne Marie Molloy,

Enda Moran, Pfizer

Disposables technology is an important component of many therapeutic protein manufacturing processes due to the lower capital outlay required for implementation and reduced labor all of which lead to improved cycle times, manufacturing flexibility and lower cost of goods. The advantages of manufacturing strategies that incorporate disposable technologies are well documented and are evidenced by the rapid adoption of disposable devices throughout the biotech industry. In this presentation however, the authors will highlight the potential pitfalls associated with rigid adherence to a specific manufacturing approach leading to increased exposure to the risks associated with a dependence on a single, critical raw material. This is a potentially significant issue for the biotech industry and will be illustrated using data from a specific example involving disposable cell culture bags.

This presentation will describe lab studies that linked growth stasis noted in the early scale up train for a particular cell line to lot-lot variability within the disposable cell culture bags used for that process step. The detailed experimental program used to identify the root cause for the reduced growth during bag culture will be presented, specifically the process employed to eliminate twenty nine other process-related variables including cells, raw materials, medium components and operator influence. The potential for leachables/extractables from the plastic to interact with the cell culture process was examined in the experimental designs. Nutrient adsorption to culture bags was additionally considered in the experimental plan. The evolution of the root cause analysis will be presented for what evolved to be a very complex and intermittent cause of failure that on occasion, was confounded by un-related external influences such as filter variability and mechanical issues. The authors will present the substantial data gathered over the course of this investigation that enabled elucidation of the root cause of this phenomenon. During this part of the presentation, possible scenarios relating to bag manufacturing processes such as the impact of gamma irradiation on polymer chemistry will be presented for consideration.

The authors will finish by describing the responses to this observation to (i) initially circumvent and (ii) subsequently resolve the poor growth during scale up for this particular cell line. The overall network response will also be described with specific reference to the impact of the stage in the product lifecycle on the response adopted. Recommendations and proposed strategic approaches developed as a consequence of this investigation will be outlined to describe best practices to minimize risk to robust manufacturing for future pipeline products.


ON THE CHALLENGES ASSOCIATED WITH ESTABLISHING PRODUCT QUALITY COMPARABILITY WHILE TRANSITIONING FROM A PEPTONE CONTAINING TO A CHEMICALLY DEFINED PROCESS.

Natarajan Vijayasankaran, Genentech, Inc. One DNA Way, South San Francisco, South San Francisco, 94080, United States

T: 1-650-225-5654, F: 1-612-238-53575, [email protected] Sharat Varma, Genentech, Inc. Steven Meier, Genentech, Inc.

Due to the various advantages associated with the use of chemically defined media, there is an industry wide trend to move away from serum containing and peptone containing processes. This presentation will focus on a case study of the development of a chemically defined cell culture process for production of a monoclonal antibody and challenges associated with matching product quality attributes to protein produced from an earlier, peptone containing version of the process. Specifically, the new chemically defined process initially led to the production of protein with higher glycation and with an altered charge variant distribution. The modeling and experimental approaches used to modify the cell culture process to establish product quality comparability will be discussed. We identified that the altered charge variant distributions correlated with altered concentrations of several components in the culture media. Accordingly, the culture medium was modified to achieve a charge variant distribution comparable to the earlier cell culture process. Glycation is the non-enzymatic addition of reducing sugars such as glucose to specific lysine resides, and it can be elevated in specific monoclonal antibodies due to the presence or absence of a 'glycation hot spot”. A dynamic mathematical model was developed to simulate the relationship between certain process parameters and observed glycation levels. The model simulated the growth of cells, substrate consumption, protein synthesis and glycation. After it was confirmed that previously collected experimental data were in general agreement with the results of these simulations, the simulation was used to guide the design of subsequent experiments. Using this approach, glycation levels were reduced from greater than 30% to as low as 15% by lowering the glucose concentration in the nutrient feeds as well as altering the overall glucose feeding strategy. Through this example we aim to show that, in spite of the limitations of using mathematical models to describe biological processes, carefully selected modeling techniques are useful thought-experiment tools that aid in practical process development by summarizing knowledge, formalizing hypotheses and elucidating relationship between process parameters.


THE CLINICAL AND SCIENTIFIC BASIS BEHIND POLYCLONAL ANTIBODY THERAPY

Jennifer Maynard, University of Texas at Austin, USA

Wednesday, April 25, 2012 Session VI: Designing Proteins, Vectors and Cells for Enhanced Biotherapeutic Production

ABERRANT RNA SPLICING IN THERAPEUTIC ANTIBODIES

Dennis P. Gately, Applied Molecular Evolution, Eli Lilly 10300 Campus Point Drive, Suite 200, San Diego, CA, 92121, USA

T: 1-858-638-8660, F: 1-858-638-8601, [email protected] Luhong He, Christal Ann Winterrowd, Bioproduct Research and Development, Lilly Research Laboratories,

Eli Lilly Melinda Ann LaCerte, Lisa Bafetti, Ying Tang, Scott Wooden, Applied Molecular Evolution, Eli Lilly

The use of human and humanized monoclonal antibodies as therapeutic drugs has been well established. However, a subset of antibodies have been identified which have the desired biological activity, but cannot be converted into pharmaceutical drugs due to poor expression levels or unacceptable levels of protein aggregation. In a number of cases, aberrant alternative RNA splicing is the cause of the observed poor expression and protein aggregation. We created CHO cell lines expressing affinity optimized humanized antibodies where the expression levels of the antibody were far below the expected values and/or there were unacceptably high levels of aggregated antibody after purification. Analysis of the heavy and light chain coding regions of these antibodies by RT-PCR produced multiple transcripts suggesting aberrant alternative RNA splicing. Sequence analysis confirmed that these extra transcripts were caused by alternative RNA splicing. The putative splice acceptors and donors were identified, modified and new expression cell lines were created. RT-PCR analysis confirmed that the modifications of the donor and acceptor abolished the alternative splicing. Moreover, protein expression and aggregation levels were restored to acceptable levels in CHO cell lines created using these modified coding sequences. To further investigate this phenomenon, we developed an RT-PCR based RNA stability test using RNA from transiently transfected CHO cells which utilizes the distinctive mobility of spliced products. We demonstrate that this method is sensitive and robust enough to detect one spliced message in a background of 100 non spliced copies.


PRECISE CONTROL OF RECOMBINANT PROTEIN PRODUCTION BY ENGINEERING TRANSLATION INITIATION SITES

Clifford L. Wang, Stanford University Department of Chemical Engineering, Stanford, CA, 94305, USA T: 1-650-736-1807, F: 1-650-725-7294, [email protected]

Joshua P. Ferreira, Stanford University

With current genetic tools, it is generally straightforward to engineer and select for cells that over-express their recombinant genes at the highest levels. Yet the highest achievable expression does not necessarily make for the optimal production or the most desirable cell line. Previous methods have been able to specify the level of gene expression through use of transcriptional promoters of varying strengths. However, in practice the use of different promoters can present challenges since transcription is not only affected by the promoter itself but also by the genomic integration site of its vector. One way to avoid problems due to genomic integration effects is to control expression not at the transcriptional level but at the translational level. We have developed a new method to precisely control expression by manipulating the rate of protein translation initiation. Although it is generally the rule that only the first open reading frame (ORF) is translated from eukaryotic mRNA transcripts, we have been able to take advantage of exceptions to this rule and control translation initiation by engineering regulatory ORFs upstream of the ORF encoding the recombinant protein of interest. While promoter-based methods have been able to specify expression over a 10-40 fold range, by manipulating translation initiation, we have been able to specify protein production over a 300-600 fold range in various cell lines including leukemia cell lines, HEK-293, and CHO. We will demonstrate the use of translation initiation control to optimize growth signaling pathways and cell-cycle regulation.


ENGINEERING CHINESE HAMSTER OVARY (CHO) CELLS FOR PRODUCING RECOMBINANT PROTEINS WITH SIMPLE GLYCOFORMS BY ZINC-FINGER NUCLEASE (ZFN) -MEDIATED GENE

KNOCKOUT OF N-ACETYLGLUCOSAMINYLTRANSFERASE I (MGAT1)

Natalie Sealover, SAFC/Sigma-Aldrich 2909 Laclede Ave., St. Louis, MO, 63103, USA

T: 1-314-771-5765 x3814, F: 1-314-286-7645, [email protected] Jeanne Brooks, SAFC/Sigma-Aldrich

Henry J. George, SAFC/Sigma-Aldrich Kevin Kayser, SAFC/Sigma-Aldrich

Nan Lin, SAFC/Sigma-Aldrich

While complex bi-antennary sialylated glycans are often desired for improved half-life of protein therapeutics, recombinant proteins with simple glycoforms are advantageous for X-ray crystallography studies. Therapeutic proteins such as glucocerebrosidase (Cerezyme®) target the mannose receptor of antigen presenting cells. Simple glycoforms with only terminal mannose residues may lead to increased efficacy by facilitating mannose receptor-mediated update for these therapeutic proteins. Terminal mannose species may also have applications in recombinant vaccine production.

Several strategies have been used to produce recombinant proteins with increased terminal mannose residues. These methods, while effective, have limitations. Non-mammalian expression systems (i.e. insect, yeast) may introduce non-human glycans and pose an increased risk of immunogenicity. Chemical glycoengineering by addition of glycosylation inhibitors to cell culture media can affect cell growth and productivity, or even raise regulatory concerns. In mammalian host cell engineering, researchers have targeted N-acetylglucosaminyltransferase I (GlcNAc-TI) encoded by Mgat1. GlcNAc-TI is responsible for the addition of the first N-acetylglucosamine (GlcNAc) to the oligomannose core of N-linked oligosaccharides, the first step in complex N-glycan synthesis. It is well established that GlcNAc-TI deficient CHO Lec1 cells produce predominantly high mannose (Man5-9) species. However, the Lec1 cell line has been observed to have increased complex glycoforms over time. The Lec1 line may also lack other essential characteristics of a CHO host cell line for bioprocessing, such as robust growth, high transfectability and productivity, and chemically-defined suspension culture adaptability.

A method for engineering high producing CHO cell lines secreting recombinant proteins with terminal mannose residues is presented here. Mgat1 knockout cells were created in an anti-Rabies human IgG producing SAFC CHOZN (gs -/-) cell line. ZFN expression vectors targeting CHO Mgat1 were designed and used to create a transfected pool. After confirming ZFN activity in the pool, Ricinus communis agglutinin-I (RCA-I), a cyctotoxic lectin that does not bind Man5GlcNAc, was used to enrich the ZFN transfected pool. The RCA-I enrichment led to an increase in ZFN activity from 3.5% to 35%. The RCA-I enriched pool displays significantly increased levels of the Man5 glycoform (mock transfected cells: 0.3%, Mgat1 ZFN transfected unselected pool: 13.7%, post-RCA enrichment: 92.8%) and these elevated Man5 levels have been confirmed in Mgat1 (-/-) single-cell clones. Potential effects of Mgat1 stable overexpression on increasing complex glycan formation were studied. Ongoing work also includes expressing recombinant proteins targeting mannose receptors in selected Mgat1 (-/-) clones.

In summary, ZFN-mediated knockout of Mgat1 has been successfully employed to create CHO host cell lines for producing high levels of therapeutic proteins with terminal mannose residues. These cell lines may be efficacious for producing mannose receptor-targeted therapeutics and may have the additional benefit of allowing X-ray crystallography studies of recombinant protein therapeutics to be conducted in the same cell line used for production.


ENGINEERING CHO CELLS FOR IMPROVED PRODUCTIVITY BY OVEREXPRESSING KEY ENZYMES OF THE GALACTOSE METABOLISM

Ziomara P. Gerdtzen, Centre for Biochemical Engineering and Biotechnology, Department of Chemical Engineering and Biotechnology, Millennium Institute for Cell Dynamics and Biotechnology: a Centre for

Systems Biology, University of Chile Beauchef 850, Santiago, Santiago, 8370448, Chile

T: 56-2-978-4712, F: 56-2-699-1084, [email protected] Natalia Eugenia Jiménez, Centre for Biochemical Engineering and Biotechnology, Department of Chemical

Engineering and Biotechnology, Millennium Institute for Cell Dynamics and Biotechnology: a Centre for Systems Biology, University of Chile

Camila Alejandra Wilkens, Centre for Biochemical Engineering and Biotechnology, Department of Chemical Engineering and Biotechnology, Millennium Institute for Cell Dynamics and Biotechnology: a Centre for

Systems Biology, University of Chile

It has been observed that CHO cells in culture consume more glucose than required for cell growth, which leads to the accumulation of metabolic-end products such as lactate. Reducing the accumulation of these metabolites has been one of the main research targets towards the improvement of cultures' yield.

Alternative carbon sources which are metabolized more slowly than glucose have been used, as a strategy for reducing lactate production in culture. In cultures with a mixture of glucose and galactose, it has been observed that in a galactose consumption phase, the flux towards pyruvate production was decreased compared to the culture in glucose, causing a metabolic shift towards lactate consumption to supply pyruvate for energy production. However, the specific growth rate of these cultures is slightly lower than that of cells grown in glucose as the only carbon source.

An alternative approach is the use of cell engineering to improve cellular metabolism. In a previous work cells grown in fructose, which is also metabolized slower than glucose leading to the behavior previously mentioned, were transfected with a fructose transporter. Transfected cells are able to achieve higher cell densities than the wild type. Overexpression of limiting step enzymes can lead to increased fluxes in their corresponding metabolic pathways. This has been applied to reduce pyruvate availability for lactate production by the overexpression of the PYC gene and MDH II. These changes enhanced energy metabolism, enabling higher fluxes through the TCA cycle and as a consequence, reduced lactate accumulation.

In this work we target the improvement of the metabolism of CHO cells growing in galactose as an alternative carbon source, by introducing multiple changes in key steps of galactose metabolism. We have targeted the following genes: galactokinase (GALK1) and a galactose transporter (Slc2a8) since there is evidence indicating that these reactions are limiting steps of this pathway. t-PA producing CHO TF 70R cells were transfected with these genes, and the best clone was selected. Selected clones show reduced lactate production and undergo a metabolic shift where they remetabolize lactate. They are also capable of growing in the presence of galactose at a higher specific rate than control cells. Specifically, CHO-Galk1 cells maintain viability in media with galactose as their only carbon source during 100 hours, in which they show almost no lactate accumulation.

This strategy of cell engineering, where multiple changes in key points of the metabolism are targeted, enables cells to grow in alternative carbon sources such as galactose, while reducing lactate production. The engineered cells exhibit higher cell growth rates than the control, extended lifespan and higher recombinant protein production yields.

Thursday, April 26, 2012 Session VII: Emerging Technologies and Novel Applications

A HIGH-THROUGHPUT ASSAY TO ASSESS ENZYME ACTIVITY IN CENTRAL METABOLISM OF PRODUCTION CELL LINES

Robert Janke, Max Planck Institute for Dynamics of Complex Technical Systems Sandtorstr. 1, Magdeburg, Sachsen-Anhalt, 39106, Germany

T: 49-391-611-0217, F: 49-391-611-0203, [email protected] Yvonne Genzel, Max Planck Institute for Dynamics of Complex Technical Systems

Udo Reichl, Max Planck Institute for Dynamics of Complex Technical Systems and Chair for Bioprocess Engineering at the Otto von Guericke University

Mammalian cells are widely used for the production of a variety of biopharmaceuticals, such as monoclonal antibodies, recombinant proteins, and viral vaccines. However, during growth in glutamine-containing media, large amounts of toxic by-products, such as lactate and ammonia, are secreted into the culture medium that often not only affects cell viability, productivity and product quality but also can prevent growth to high cell densities. Many different analytical methods, such as liquid chromatography-mass spectrometry for measuring intracellular metabolites, were used to characterize and to better understand the inefficient metabolism of production cells. The objective of the present study to further assess cell metabolism was the development of an efficient assay system based on 96-well microplates for central metabolic enzyme activities, which allowed the grouping of various enzymes in modules that share a common detection method. The enzyme platform consists of four sensitive cycling assays to quantify low amounts of NAD+(H), NADP+(H), glycerol 3-phosphate or dihydroxyacetone phosphate, and glutamate or 2-oxoglutarate. The sensitivity limit of all cycling assays was between 0.025 and 0.4 nmol product. Cell extracts could, therefore, be highly diluted, which reduced eventual interferences caused by other components in the extract and additionally minimized under- or overestimates of actual enzyme activity. Furthermore, possible enzyme inhibition by high concentration of a product was prevented since substrate concentrations could be maintained at a near constant level throughout the assay.

Adherent Madin-Darby canine kidney (MDCK) cells were grown to stationary and exponential phases in 6-well plates in GMEM supplemented with glutamine or pyruvate, and key metabolic enzyme activities of cell extracts were analyzed. Significant differences were found in maximum enzyme activities from cells grown with pyruvate-containing medium compared to glutamine-containing medium. The overall activity of the pentose phosphate pathway was up-regulated during exponential cell growth in pyruvate-containing medium, which suggests that more glucose 6-phosphate was channeled into the oxidative branch. Furthermore, the anaplerotic enzymes pyruvate carboxylase and pyruvate dehydrogenase showed higher cell specific activities with pyruvate. Increased specific activities were also found for NAD+-dependent isocitrate dehydrogenase, glutamate dehydrogenase and glutamine synthetase in MDCK cells grown with pyruvate. It can be assumed that the increase in enzyme activities was most likely required to compensate for the energy demand and to replenish the glutamine pool. On the other hand, the activities of the glutaminolytic enzymes aspartate transaminase, alanine transaminase, malic enzyme (ME) and phosphoenolpyruvate carboxykinase were decreased in cells grown with pyruvate, which seems to be related to a decreased glutamine metabolism.

When MDCK cells were infected with an influenza A (H1N1) virus at a high multiplicity of infection, infected cells showed an up-regulation of some key enzymes producing the reduction equivalent NADPH (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and ME) and acetyl-CoA (citrate lyase and acetate-CoA ligase), a precursor needed for lipid and cholesterol biosynthesis. It seems that the synthesis of fatty acids and cholesterol plays a crucial role for the replication of influenza viruses in adherent MDCK cells as they acquire lipid envelopes from their host cells.

Based on the established enzyme assays the metabolic states of production cell lines can now be further characterized. This can then be used to improve our understanding of metabolic pathways relevant for cell line and media optimization. Furthermore, it will support the validation of mathematical models of cellular metabolism in systems biology approaches.


TECHNOLOGY IMPROVEMENTS TO ACCELERATE PROCESS DEVELOPMENT OF BIOLOGICS

Krista Alvin, Merck & Co., Inc RY80Y-115, 126 E. Lincoln Ave, PO Box 2000, Rahway, NJ, 07065, USA

T: 1-732-594-0521, F: 1-732-594-4400, [email protected] Rachel Bareither, Merck & Co., Inc.

David Pollard, Merck & Co., Inc

Pressures continue to reduce the time from discovery to product launch and minimize the costs of not only manufacturing but also process development. This is particularly difficult for upstream development where large DOE designs in lab scale reactors provide a significant equipment and resource constraint.

This presentation will show how innovation through automation and single-use technology has led to more efficient process development. This includes the creation of multi use tools to handle both cell culture and microbial expression platforms. Improvements will be shown for end to end development focusing on upstream processing followed by purification and analytics. Examples include the use of cell line development automation for the elimination of manual shakeflask stages and increase throughput of clonal evaluation. For more advanced process development a novel small scale single use prototype bioreactor is evaluated. This system is designed for automated multi tank experimentation with robotic sampling, feeding and control. This is integrated with high throughput purification and analytics using a systematic approach of statistical design of experiments in combination with 'omics' technologies. This allows for rapid end to end process development and builds a fundamental understanding of the impact of process operations to control process consistency and product quality.


LARGE SCALE MANUFACTURING EXPERIENCE IN SINGLE USE BIOREACTORS, SPECIFICS FOR ANCHORAGE DEPENDENT CELL LINES AND VACCINES

Jean-Marc Guillaume, Sanofi Pasteur 1541 Avenue Marcel Merieux, Marcy L'etoile, Rhone Alpes, 69280, France

T: 33 4 37 37 08 74, F: 33 4 37 37 37 48, [email protected] Eric Calvosa, Sanofi Pasteur Nicolas Seve, Sanofi Pasteur

Caroline Sellin, Sanofi Pasteur

Several data have been reported on the benefits of disposable bioreactors for suspension cell culture such as CHO for recombinant protein production; however fewer applications have so far been reported for vaccine manufacturing and anchorage dependent cell line like Vero. Although and taking into account general shear and mixing design specificities associated with the development of anchorage dependent cell cultures, current range of vaccine manufacturing scale makes disposable bioreactors a very attractive solution. Prospect and commitment of vaccine industry for long term and affordable supply of products, makes risk analysis and thorough procurement study a prerequisite over process justifications and such requires multidisciplinary team review for industrial implementation. Within the prospect of a new rabies vaccine, manufacturing process, we have developed serum protein free media formulations allowing achieving comparable results than what obtained with serum supplemented culture. Further media development and additives selection based on design of experiments, metabolic studies , FACS analysis, gene profiling of cells prior and after infection have resulted on average two fold viral productivity improvement. In parallel, disposable bioreactor process evaluation was combined leading to industrialization up to the 1000L scale with PG-ATMI Nucleo bioreactors series, being so far largest scale reported for such vaccine manufacturing application. Efficiency of mixing and transfer (Kla), scale-down/scale-up is investigated across scales including 20, 200 and 500L scale. When compared to stainless steel bioreactor, comparable cell growth is obtained at all scales, with however higher viral productivity, between 50 to 100%, and attributed to better gas transfer and a series of incremental equipment improvement. Further we have established new conditions where cells can be amplified within the same bioreactor at 200L scale and starting from initial frozen vial, so called ‘’All in One Process’’ . This improvement and reduction of needs for multiple bioreactor seed train before production scale, has several advantages, in reducing risk of contamination, limited capital investment, improved cycle time... In summary, we have demonstrated the feasibility and higher productivity of Vero cells for viral vaccine manufacturing within disposable bioreactors and up to an industrial scale of 1000L, Preliminary operating cost evaluations indicate favorable figures for implementation.


ENGINEERING CELLULAR FUNCTION FOR ENHANCING CELL AND GENE THERAPY PRODUCT POTENCY FOR ONCOLOGY AND REGENERATIVE MEDICINE APPLICATIONS

Madhusudan V. Peshwa, MaxCyte, Inc. 22 Firstfield Road, Suite 110, Gaithersburg, MD, 20878, United States of America

T: 1-301-944-1641, F: 1-301-944-1703, [email protected] Cornell Allen, MaxCyte, Inc.

Pachai Natarajan, MaxCyte, Inc. Angelia Viley, Rama Shivakumar, MaxCyte, Inc.

Linhong Li, MaxCyte, Inc.

The ability to specifically enhance biological function and control cell fate decisions without genetic modification represents a desired approach for generation of 'enhanced' potency of immune and stem cells; as well as for 'pharmaceutical grade' primary cell / iPS cells derived tools for drug screening or therapeutics development applications.

We have developed a rapid, automated, cGMP and regulatory compliant, scalable platform to process 200 billion cells in under 30 minutes by loading them with small molecule, protein, siRNA, miRNA, mRNA or plasmid DNA; singly or in combinations that permit targeted modulation of intracellular pathways resulting in augmentation of desired cellular function per defined kinetics that maps to therapeutic window for improved efficacy. The platform technology utilizes a cGMP compliant closed system for transient, non-viral approaches to modulation of intracellular signaling pathways and is supported by an FDA Master File and a CE Mark. The platform has also been used for manufacture of viral vectors and delivery of components leading to chromosomal engineering of cells intended for therapeutic use (gene-modified cell therapy) and permits identification of critical process variables, control of manufacturing process, and delivery of cellular therapeutic products meeting regulatory requirements of viability, integrity, purity, identity, and exhibiting ‘enhanced’ potency and efficacy.

Our experiences in enabling development and scale-up for clinical / commercial delivery of non-virally engineered cellular therapies encompass an autologous cellular vaccine [marketed as an Oncology therapy in Japan], chimeric antigen receptor engineered T- & NK-cell immunotherapies [in human clinical trials in US], and multiple other 'engineered' immune- and stem-cell therapies is various stages of clinical and pre-clinical development. In each of these instances, the primary objective of the design and delivery of the therapeutic product is to specifically ‘enhance’ the desired biological activity of cells resulting in improved product efficacy profile, while permitting rapid, robust, seamless, cGMP and regulatory compliant scale-up for cost-effective clinical / commercial delivery.

This presentation will summarize data on how modulation of biological activity leads to development of enhanced potency immune and stem cell therapy products for use as clinical / commercial treatment of oncology and in regenerative medicine applications.


HUMAN STEM CELLS AND PRIMARY CULTURES FOR DRUG DISCOVERY AND CELL THERAPY: BIOPROCESSING CHALLENGES

Paula M. Alves, ITQB-UNL/iBET Apartado 12, Oeiras, 2780-901, Portugal

T: +351 21 4469361, F: +351 21 4421161, [email protected] Catarina Brito, Margarida Serra, Rui M. Tostões,

Sofia B. Leite, Cláudia Correia, Daniel Simão, Marcos F. Sousa, Manuel J.T. Carrondo, Animal Cell Technology Unit, ITQB-UNL/iBET, Portugal

Janne Jansen, Petter Bjorquist, Cellartis AB, Sweden

Human stem cells (hSCs), with their ability for extensive proliferation and multi-lineage differentiation, are renewable cellular resources with outstanding potential for cell therapy, tissue engineering, drug screening and in vitro toxicology. An imperative pre-requisite for the transition of hSCs to these fields is the establishment of effective and robust cell culture and cryopreservation protocols for their large-scale expansion, differentiation, storage and distribution.

The laborious and time consuming 2D-cultures are difficult to control, present poor yields and often lack the required cell functionality. Additionally, these 2D settings are simplified and do not recapitulate the in vivo complexity. These characteristics can have severe consequences on robustness, reproducibility, scalability and relevance of the cell systems, hampering their possible application in Cell Therapy and Drug Discovery.

Our work has focused on the development of 3D-culture systems for expansion, differentiation and/or maturation of clinically relevant cells, namely human pluripotent (embryonic and induced) and multipotent (neural, mesenchymal) stem cells and hepatocytes, exploiting the potential of bioreactor technology. These 3D settings provide a cellular context closer to what actually occurs in vivo, as cells integrate external signals, including those from cell-cell and cell-ECM direct interactions, as well as secretion/exchange of soluble factors and/or metabolites.

Results concerning novel culture and cryopreservation systems will be presented and their advantages and applicability in the production of high quality advanced therapeutic products or functional screening tools for preclinical research will be discussed.

Thursday, April 26, 2012 Session VIII: Control and optimization of cell metabolism in culture

PHYSIOLOGY OF METABOLIC SHIFTS IN CULTURED MAMMALIAN CELLS - A MECHANISTIC ANALYSIS AND A SCHEME FOR METABOLIC CONTROL

Bhanu Chandra Mulukutla, University of Minnesota 421 Washington Ave SE, Minneapolis, Minnesota, 55455, United States

T: 1-612-205-9590, F: 1-612-626-7246, [email protected] Nandita Vishwanathan, University of Minnesota

Huong Le, University of Minnesota Wei-Shou Hu, University of Minnesota

Cultured mammalian cells consume large amounts of glucose and divert most of it towards lactate, whose accumulation is inhibitory to growth and product synthesis. In fedbatch cultures, fortuitous metabolic shifts to low lactate production or lactate consumption lead to sustained viability and higher productivity of recombinant proteins. The mechanisms governing this phenomenon are still not clearly understood. Analysis of such shifts through a systems biology approach revealed that various pathways, both metabolic and signaling, play key roles in inducing such shifts.

Our experimental data as well as the process data of over two hundred manufacturing runs revealed that lactate consumption occurs in slow growth stage and is accompanied by low glucose uptake rates. Using a mechanistic model for the central metabolic network, high lactate concentration, low glucose consumption rate and growth regulation were identified as key factors for such a metabolic shift. However, overwhelming experimental evidence demonstrate that under the same set of glucose and lactate concentrations, a culture could be in a lactate production state or in a lactate consumption state, strongly suggesting that the concentrations of lactate and glucose are not the only controlling variables.

The glycolysis pathway is subject to complex allosteric regulations that impart non-linear behavior to its activity. Model analysis demonstrates that multiplicity of steady states is present under some culture conditions. The results also imply that the metabolism of the culture is affected by its history in addition to its present state.

The model was employed to simulate the transient behavior of lactate metabolism under different culture conditions, and it indeed predicted vastly different lactate profiles even under the same culture conditions. In the metabolically shifted culture, lactate consumption may proceed to its exhaustion as observed experimentally.

Our model prediction suggested that robust lactate consumption can be attained by restricting glucose consumption while the growth rate is decreasing. Such shift to lactate consumption was experimentally illustrated, both by diminishing sugar consumption in late stage of fedbatch culture and by the modulation of signaling pathways regulating glycolysis, such as AKT-mTOR, in the late stages of culture.

The insights from the mechanistic model enhances our understanding of the physiology of metabolic shifts in mammalian cells and is likely to contribute to the design of strategies for process enhancement through manipulation of cellular energy metabolism.


PRESERVATION OF A BALANCED CELL CULTURE ENVIRONMENT FOR FED-BATCH PROCESSES

Yen-Tung Luan, Pfizer Inc. 1 Burtt Road, Andover, MA, 01810, USA

T: 1-978-247-2232, F: 1-978-247-2602, [email protected] Wenge Wang, Pfizer Inc. Ryan Nolan, Pfizer Inc.

During the biosynthesis of recombinant proteins in mammalian cell culture, maintaining well-balanced intracellular and extracellular environments are essential for achieving optimal productivity and consistent product quality. At the beginning of a fed-batch culture, cells are incubated in an environment with suitable pH, dissolved oxygen, temperature, nutrients, osmolarity, and redox potential. However, as time progresses, the operational characteristics of fed-batch culture systems result in accumulation of wasteful byproducts and depletion of certain nutrients, thereby creating an imbalanced culture environment.

The imbalanced environment includes high osmolarity, high level of inhibitors, nutrient depletion or excess and low or high CO2 levels in culture medium. High osmolarity is indicative of inappropriate nutrient feeding and excess base addition which is introduced by high lactate and CO2 levels. High osmolarity, high lactate, or high CO2 alone or in combination would have a negative impact on cell culture performance. Nutrient depletion or excess is generally caused by inadequate feeding methods which would include feed medium composition, how to feed, when to feed, and how much to feed. Nutrient depletion would slow down cell growth, decrease productivity, and even cause cell death. Conversely, excess nutrient feed will increase culture osmolarity and may raise the production of byproducts, which also impacts cell growth and longevity of the culture. The challenge of preserving the environmental balance in cell culture processes can be achieved through a thorough understanding and manipulation of certain process parameters.

This presentation will demonstrate with examples the adverse effects of cell culture imbalance on cell growth and productivity, as well as specific solutions to remedy these problems. A simple solution for recovering the drop in cell viability observed during the late stage of cell culture will also be discussed. Finally, a systematic approach in medium feeding to achieve high cell density, high viability, and high titer processes will also be described.


PCO2 CONTROL IN CHO FERMENTATIONS: BIOPROCESS AND METABOLIC ENGINEERING APPROACHES

Thomas Noll, University of Bielefeld Universitaetsstrasse 25, Bielefeld, NRW, 33615, Germany

T: +49-521-106-8700, F: +49-521-106-6328, [email protected] Alexander Jockwer, Christian Klinger, Research Center Jülich; now at Roche Diagnostics GmbH

Betina Ribeiro, University of Bielefeld; now at Novartis Pharma AG Jochem Gätgens, Research Center Jülich Raimund Hoffrogge, University of Bielefeld

In recent years dissolved carbon dioxide (pCO2) has been identified as an important process parameter affecting cell growth, productivity and product quality (e.g. glycosylation) of recombinant proteins when exceeding critical levels, occurring especially in industrial large-scale cell culture processes due to the increased hydrostatic pressure. As CO2 can easily pass the cellular membrane and thereby influence intracellular pH (pHi), important cellular processes (e.g. cell cycle regulation, enzymes of TCA cycle) are directly influenced by pCO2 and dependent bicarbonate concentration. Consequently, process control strategies attend to keep pCO2 within physiological range. Using a pCO2-control strategy that allows constant pCO2-levels independently from pO2 and pH-control we have compared Na2CO3 and NaOH as base for pH control and found that Na2CO3 resulted in increased viable cell density, culture time and space-time-yield in a CHO culture producing a recombinant fusion protein. Based on a sampling device that maintains the bioreactors’ (hydrostatic) pressure in the sample, we found that stripping strategies, used to reduce pCO2 in bioreactors, strongly influence the intracellular pH of the cells resulting in a biphasic re-alkalization of the cytoplasm. A recombinant CHO cell line producing human GM-CSF and stably expressing pyruvate carboxylase 2 from S.cerevisiae was cultivated at different controlled pCO2-levels and under uncontrolled pCO2-conditions. pCO2-control positively influenced viability and fermentation time resulting in up to 100% increased product concentration. Surprisingly a pCO2 of 15% was superior to 5%. Lactate formation was reduced by 30% at pCO2 of 15%, but no metabolic shift towards lactate re-metabolisation was observed. In a second metabolic engineering approach an antibody producing CHO cell line stably expressing human carbonic anhydrase (hCAII), the enzyme that catalyzes the equilibrium of CO2 in aqueous solutions was generated and used to characterize CO2 effects in simulated CO2 acid load and high CO2 levels as they occur in large scale mammalian cell culture. The cell line expressing active hCAII showed more rapid initial re-alkalinization of cytoplasm after induced CO2 acid load. Results also suggest that cellular pHi fine tuning was performed by the Cl-/HCO-3 exchanger (AE) and Na--dependent Cl-/HCO-3 exchanger (NCBE) instead of the Na+/H+ exchanger (NHE1). In general, increased CO2 profile triggered the quicker progress of G0/G1 cell cycle phase for both transfected and control cell lines. Neither hCAII expression nor CO2 profile had strong negative impact on product glycosylation in the investigated range. In a global proteomic analysis significant differences in protein expression profile was observed for the hCAII expressing cell.


ELUCIDATING THE DYNAMICS OF METABOLIC FLUXES IN CHO CELL CULTURES USING 13C-DYNAMIC METABOLIC FLUX ANALYSIS

Maciek R. Antoniewicz, University of Delaware 150 Academy St, Newark, DE, 19716, USA

T: 1-302-831-8960, F: 1-302-831-1048, [email protected] Woo Suk Ahn, University of Delaware

Chinese hamster ovary (CHO) cells are the most widely used mammalian cell line for the production of bio-therapeutics. In the pharmaceutical industry CHO cells are grown in fed-batch culture, where cellular metabolism is characterized by high glucose and glutamine uptake rates combined with high rates of ammonium and lactate secretion. The metabolism of CHO cells changes dramatically during a fed-batch culture as the cells adapt to a changing environment and transition from exponential growth phase to stationary phase. Thus far, it has been challenging to study metabolic flux dynamics in CHO cell cultures using conventional metabolic flux analysis techniques that were developed for systems at metabolic steady state. Here, we describe a novel technique that we have developed and applied to elucidate flux dynamics in CHO cells cultures. The 13C-dynamic metabolic flux analysis (13C-DMFA) methodology is based on integrating time-series of metabolite measurements and non-stationary 13C-isotopic labeling data to quantify in vivo flux dynamics. Three key advantages of our 13C-DMFA method are: 1) time-series of metabolite concentration data can be applied directly for estimating dynamic fluxes, making data smoothing and estimation of average extracellular rates unnecessary; 2) characteristic metabolic phases during a culture are identified automatically by the algorithm, rather than selected manually/arbitrarily; 3) non-stationary 13C-labeling data provides information on parallel and cyclic pathways, and allows transients in metabolic fluxes to be determined. Here, we demonstrate the application of our new 13C-DMFA methodology by elucidating the dynamics of intracellular metabolic fluxes in fed-batch CHO cell culture. CHO K1 cells were grown over 6 days on an enriched medium containing specifically labeled 13C-glucose and 13C-glutamine tracers that were selected by a new framework for a priori 13C-tracer experiment design based on the elementary metabolite units (EMU) framework. Medium samples were collected daily to quantify extracellular metabolite concentrations and measure 13C-labeling of extracellular metabolites that were exchanged with intracellular metabolites (e.g. lactate, pyruvate, glutamate). In addition, 13C-labeling measurements of intracellular metabolites (by GC-MS) were collected at strategically placed measurement time points during the culture, i.e. more frequently during the transition from the growth phase to stationary phase. The combined data set was analyzed with the DMFA algorithm that we developed to quantify detailed transitions in extracellular and intracellular metabolic fluxes. The flux results revealed significant rewiring of metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. Surprisingly, the fluxes of pyruvate dehydrogenase and TCA cycle were very similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.

Thursday, April 26, 2012 Chair Select Session: Understanding CHO biology with application to bioprocessing

INTRACELLULAR TARGETING AND ROLE OF BCL-XL IN CHINESE HAMSTER OVARY CELLS

Abasha Lewis, Johns Hopkins University 3400 North Charles Street, Baltimore, MD, 21218, USA

T: 1-443-740-2064, F: 1-410-516-5510, [email protected] Teruo Hayashi, NIDA, NIH Tsung-Ping Su, NIDA, NIH

Michael J. Betenbaugh, Johns Hopkins University

The survival-promoting Bcl-2 family of proteins is generally believed to exist at the mitochondria to block cytochrome c release. However, Bcl-2 family proteins have emerging roles in other cellular processes acting at various subcellular locations including the endoplasmic reticulum (ER) and outer nuclear membranes. Thus, it is possible that the localization of Bcl-2 family proteins affects their function. In this project, we investigate Bcl-xL, an anti-apoptotic homologue of Bcl-2, and its cellular localization and potential roles. We observed by confocal microscopic examinations that heterologous Bcl-xL exists in Chinese hamster ovary (CHO) cells in three distinct patterns: (1) some Bcl-xL distributes throughout the outer mitochondrial membrane (OMM); (2) about 43% of Bcl-xL clusters on the OMM adjacent to the ER-mitochondrial interface; and (3) approximately 20% of Bcl-xL is in the cytosolic region but juxtaposed to the mitochondria. Here Bcl-xL colocalizes with proteins specific to the mitochondrion-associated ER membrane (MAM), such as sigma-1 receptor, BiP (binding immunoglobulin protein), IP3R3 (type 3 IP3 receptor), and mitofusion-2. The MAM is a specialized ER subdomain physically associated with mitochondria to regulate intracellular transport between the two compartments. A cell fractionation study with standard markers confirms that about 45% of Bcl-xL localizes to the mitochondria with 15% of Bcl-xL at MAM-enriched membranes whereas a small fraction of Bcl-xL resides in the bulk ER. The BH4 domain of Bcl-xL was also shown to physically interact with IP3R3 residing at the MAM. Upon cellular stress induced by thapsigargin, more Bcl-xL translocates from the OMM to the MAM and interacts with IP3R3. Since IP3R3 regulates ER Ca2+ efflux at the MAM, it is possible that Bcl-xL plays a role in Ca2+ transport. Indeed, preliminary data from our Ca2+ signaling study found that overexpression of Bcl-xL alters thapsigargin-induced Ca2+ efflux from the ER into the cytosol and into the mitochondria. When taken together, the above results suggest that MAM-localized Bcl-xL may alter communication between the mitochondria and the ER membrane via interactions with type 3 IP3 receptors including a potential role in Ca2+ signaling and bioenergetics. Future efforts will explore the effects of Bcl-xL overexpression on cellular energetic processes with the end goal of altering cell productivity.


A KINETIC STUDY OF ENDOGENOUS UNFOLDED PROTEIN RESPONSE AND ITS APPLICATIONS IN CHO PRODUCTION CULTURE

Zhimei Du, Amgen Inc. 1201 Amgen Ct W, Seattle, WA, 98119, U.S.A.

T: 1-206-265-7367, F: 1-206-217-4692, [email protected] Dave Treiber, Amgen Inc Becca McCoy, Amgen Inc

Pranhitha Reddy, Amgen Inc

Unfolded protein response (UPR) is the primary signaling network activated in response to the accumulation of unfolded and/or misfolded protein in the endoplasmic reticulum (ER). The expression of high levels of recombinant proteins in mammalian cell cultures have been linked to increased UPR. However, the kinetics of different UPR –mediated events and their impact on cell performance and recombinant protein secretion during production are ill defined. We created an UPR-responsive, fluorescence-based reporter system to detect and quantify specific UPR-mediated transcriptional activation of different intracellular signaling pathways. We generated stable antibody-expressing clones containing this UPR responsive system and established FACS-based methods for continuous, real-time monitoring of endogenous UPR activation in cell cultures. We found that clones differed in their UPR induction pattern; both the timing and the degree of UPR-induced transcriptional activation were linked to the growth, viability, and productivity of the cells. In addition, endogenous UPR activation was significantly impacted by the cell culture environment, i.e. amino acid levels and osmolarity. We will discuss the role of UPR-mediated transcriptional activation of different signaling pathways on cell performance during recombinant protein production, and the use of an inducible system and UPR monitoring to engineer or improve control of recombinant protein production.


EXPLORING THE TRANSCRIPTOME SPACE OF RECOMBINANT BHK CELL THROUGH NEXT GENERATION SEQUENCING

Kathryn C. Johnson, University of Minnesota 421 Washington Ave SE, Minneapolis, MN, 55455, USA

T: 1-612-625-3051, F: 1-612-626-7246, [email protected] Nitya M. Jacob, Nandita Vishwanathan, Andrew Yongky, Wei-Shou Hu, University of Minnesota

Karthik P. Jayapal, Chetan T. Goudar, Global Biological Development, Bayer HealthCare Pharmaceutical

Baby hamster kidney (BHK) cell lines, as well as the Syrian hamster from which they are derived, have for decades served as valuable model systems in many areas of biomedical research ranging from virus production to studies of prion diseases, cardiomyopathy, and metabolism. BHK cells are also employed in the pharmaceutical industry to produce several important recombinant protein therapeutics. BHK-specific resources would facilitate more comprehensive assessment of characteristic transcriptome profiles for these cells. We therefore set out to assemble and annotate a complete BHK transcriptome, starting with 40 Gbp of paired-end 90-100 bp reads obtained using Illumina high-throughput sequencing. This resource allowed us to establish a baseline profile against which future studies can more confidently identify and evaluate transcriptome dynamics to answer many physiological questions.

The cDNA libraries sequenced represented a recombinant protein-producing BHK cell line as well as two transcriptionally diverse Syrian hamster tissues, liver and brain, which were included to increase the number of genes detected. De novo assembly was performed using the Oases transcriptome assembler, followed by long-read assembly in which 6,000 previously obtained BHK Sanger ESTs were incorporated. The final assembly is comprised of ~221,000 contigs with an average length of 577 bp. Annotation performed by BLAST alignment yielded over 50,000 unique gene IDs representing more than 15,000 unique Ensembl mouse gene IDs. In particular, we were able to annotate 85% of contigs of at least 600 bp, and 94% of contigs at least 1000 bp in length. Comparison of the BHK assembly to an in-house CHO transcriptome revealed 94% sequence identity, while only 91% identity exists between BHK and Ensembl mouse transcripts.

Transcript abundance levels, determined by alignment and frequency computation for each library against assembled transcripts, revealed a wide dynamic range spanning five orders of magnitude. Visualization in a pathway context enables one to conjecture possible physiologically active pathways in these cultured cells. Comparative transcriptome analyses between different libraries provide insight into gene expression changes, such as isozyme preferences, which may occur in high-producing cell lines. The diversity of libraries sequenced also permitted exploration of single nucleotide variants in transcripts between cell line and tissue sources as well as within a single library. We also investigated more broadly how such variants can be distinguished from sequencing errors. These genomic resources should serve as valuable process diagnostic tools by providing further opportunities to fingerprint, engineer and enhance BHK cells in their role as recombinant protein producers.


DETAIL ANALYSIS OF CHROMOSOME REARRANGEMENTS IN CHO CELLS USING BAC-BASED PHYSICAL MAP

Takeshi Omasa, Institute of Technology and Science; The University of Tokushima 2-1 Minamijosanjima-cho, Tokushima, 770-8506, Japan

T: +81-88-656-7408, F: +81-88-656-9148, [email protected] Yihua Cao, (Osaka University)

Shuichi Kimura, (The University of Tokushima) Takayuki Itoi, (Osaka University)

CHO (Chinese hamster ovary) cells have frequently been used in biotechnology for many years as a mammalian host cell platform for cloning and expressing genes of interest. A detailed physical chromosomal map of the CHO DG44 cell line was constructed by fluorescence in situ hybridization (FISH) imaging using randomly selected 303 BAC clones as hybridization probes (BAC-FISH) [1,2]. The two longest chromosomes were completely paired chromosomes; other chromosomes were partly deleted or rearranged. The end sequences of 624 BAC clones, including 287 mapped BAC clones, were analyzed and 1,119 informative BAC end sequences were obtained. Among@303 mapped BAC clones, 185 clones were used for BAC-FISH analysis of CHO K1 chromosomes and 94 clones for primary Chinese hamster lung cells. Based on this constructed physical map and end sequences, the chromosome rearrangements between CHO DG44, CHO K1, and primary Chinese hamster cells were investigated. Among 20 CHO chromosomes, 8 were conserved without large rearrangement in CHO DG44, CHO K1, and primary Chinese hamster cells. The longest two chromosomes are only conserved paired chromosomes in both the CHO DG44 and CHO K1 cell lines. We compared these chromosomes with the mouse genome for further detailed analysis. Eighty-two BAC clones were mapped on these two chromosomes. The end sequences of 48 BAC clones showed homology with mouse genome sequences. Twenty BAC clones had a 70 - 140 kb homology region with mouse genome contigs. It was estimated that these clones covered about 2.14 Mb of hamster genomes and were homologous with mouse genomes among 11 mouse chromosomes.

REFERENCE [1]Omasa T, Cao YH, Park JY, Takagi Y, Kimura S, Yano H, Honda K, Asakawa S, Shimizu N, Ohtake H. Bacterial artificial chromosome library for genome-wide analysis of Chinese hamster ovary cells, Biotechnol Bioeng, 104, 986-994 (2009) [2]Cao YH, Kimura S, Itoi T, Honda K, Ohtake H, Omasa T. Fluorescence in situ hybridization using bacterial artificial chromosome (BAC) clones for the analysis of chromosome rearrangement in Chinese hamster ovary cells, Methods (in press) (DOI:10.1016/j.ymeth.2011.11.002) [3]Cao YH, Kimura S, Itoi T, Honda K, Ohtake H, Omasa T. Construction of BAC-based physical map and analysis of chromosome rearrangement in Chinese hamster ovary cell lines, Biotechnol Bioeng, (in press) (DOI 10.1002/bit.24347)


MECHANISTIC STUDIES ON THE IMPACT OF PGAM1 AND OTHER KEY GENES IN GLYCOLYSIS ON ENERGY METABOLISM AND PROTEIN GLYCOSYLATION IN IgG PRODUCING CHINESE HAMSTER

OVARY (CHO) CELLS

Joaquina Mascarenhas, SAFC/Sigma Aldrich 2909 Laclede Avenue, Saint Louis, Missouri, 63103, USA

T: 1-314-771-5765 Ext 3767, F: 1-314-286-7645, [email protected] Henry George, SAFC/Sigma Aldrich Kevin Kayser, SAFC/Sigma Aldrich

Nan Lin, SAFC/Sigma Aldrich

Energy metabolism in recombinant protein producing mammalian cells can profoundly affect productivity and product quality of secreted therapeutic proteins. Researchers aim to control glycolysis and direct pyruvate flux into the TCA cycle, thus increasing the net ATP generated in order to improve recombinant protein productivity. Our particular interest lies in understanding the potential role of energy metabolism in recombinant protein post-translational modification specifically N-linked glycosylation. In the present study, we focus on characterizing and manipulating four genes in the glycolytic pathway: Phosphoglycerate mutase 1 (Pgam1), Phosphofructokinase (Pfk), Phosphoglycerate kinase 1 (Pgk1) and Pyruvate Kinase (Pk) to study the impact on productivity and glycosylation.

Pgam1 has been reported to contribute to an alternative glycolytic pathway in rapidly proliferating cells via the conversion of phosphoenolpyruvate to pyruvate, independent of enolase activity. Transcriptional profiling studies using cDNA microarrays on a recombinant IgG producing CHO cell line, comparing non-targeting control (higher IgG expression) vs. an IgG Heavy and Light Chain siRNA knockdown culture (lower IgG expression levels), showed that Pgam1 was moderately down-regulated, Pfk significantly down-regulated and Pk moderately down-regulated in the higher IgG expressing cells. Additionally, differential gel electrophoresis (DIGE) studies on two IgG producing CHO (dhfr -/-) lines with different %Man5 glycans (indicating incomplete glycosylation) showed that Pgam1 protein level was down-regulated 1.66 fold in the cell line with lower %Man5. In order to further explore the link between energy metabolism and glycosylation efficiency, we conducted qRT-PCR differential expression studies of Pfk, Pgk1 and Pk on a panel of IgG producing SAFC CHOZN (gs -/-) cell lines with varying growth, productivity and glycosylation phenotypes.

Quantitative RT-PCR for expression levels of Pgam1 showed an inverse correlation between Pgam1 relative expression and IgG productivity. Furthermore, shRNA stable knock down of Pgam1 in a cell line from the panel with highest Pgam1 expression and lowest productivity indicated a mild increase in IgG productivity. The effect of Pgam1 on glycosylation was studied by the stable shRNA knockdown of Pgam1 in another model CHO cell line (dhfr -/-) producing antibodies with higher %Man5. Pfk, Pgk1 and Pk were also stably knocked down in selected IgG producing CHO cell lines, and the effects on N-glycan profiles, growth, IgG productivity, lactate production and ATP generation were studied.

The results from this study may suggest methods for engineering CHO host cell lines towards better energy metabolism and potentially resultant improved glycosylation profiles.

Thursday, April 26, 2012 Chair Select Session: Cell line development

ENGINEERING CHO CELLS AND VECTORS FOR IMPROVED TRANSGENE INTEGRATION AND ANTIBODY PRODUCTION

Nic Mermod, University of Lausanne Station 6, Lab of Molecular Biotechnology, EPFL, Lausanne, 1015, Switzerland

T: +41 21 693 6151, F: +41 21 693 7610, [email protected]

Epigenetic regulatory DNA elements can be incorporated in expression vectors to yield stable and very high specific transgene expression from CHO cells and increased antibody production in the bioreactor. However, extremely high specific productivities reveal new cellular bottlenecks from CHO cell lines, encompassing transgene genomic integration, protein secretion and cell physiology. We have determined the sequence of the genome and trasncriptome of a CHO sub-line used for the production of pharmaceuticals and have identified genes involved in transgene genomic integration and in recombinant protein processing and secretion. Cell engineering methods for increased genomic integration of the transgene by homologous recombination will be presented. Proper protein secretion and modifications is another bottleneck met with high level expression, especially for some difficult to express immunoglobulin variants. We will show how the faulty steps can be identified at the molecular level and how the screening and expression of a number of protein-folding, transport, secretion or modification pathway proteins can be used to solve processing and secretion limitations. This presentation will illustrate how the use of a systematic and multi-level approach can be used to generate improved gene transfer methods and recipient cells for more efficient expression of pharmaceutical proteins.


UTILIZING A GFP TOOL TO MONITOR EFFORTS AT IMPROVING GS-CHO CELL LINE GENERATION EFFICIENCY AND PRODUCTIVITY THROUGH HIGHLY STRINGENT SELECTION SYSTEM

Jeffrey L Larson, Eli Lilly and Company Lilly Corporate Center, Indianapolis, IN, 46285, USA

T: 1-317-276-1138, F: 1-317-276-8838, [email protected] Lianchun Fan, Eli Lilly and Company

Lara Krebs, Eli Lilly and Company Margaret Shaw, Eli Lilly and Company

Christopher Frye, Eli Lilly and Company

Eli Lilly and Company utilizes the GS-CHO expression technology and has developed a rapid and efficient process for the generation of clonally-derived cell lines in support of therapeutic protein clinical development. Recently, efforts have been undertaken in our lab to improve cell line generation efficiency and bulk culture productivity through alternative selection schemes, focusing primarily on selection stringency. These approaches include modifications to the selection procedure itself, as well as manipulation of the expression plasmids and expression host cell engineering. A GFP expression cassette has been developed that enables the monitoring of these changes on the selected bulk population for multiple therapeutic antibodies. The GFP profile of a bulk culture as related to its productivity has provided an insight into the selection process. The GFP profile has enabled the development of processes and molecular tools to increase the efficiency of improved cell productivity.


USE OF HOMOLOGOUS RECOMBINATION BASED GENOME EDITING FOR CHO CELL LINE ENGINEERING

Joshua Kapp, Horizon Discovery Ltd. Building 7100 Cambridge Research Park, Cambridge, Cambridge, CB25 9TL, UK

T: +44-12-236-55583, F: +44-12-238-62240, [email protected]

Since the sequencing and draft genome assembly of the CHO-K1 cell line was published in Nature Biotechnology in July 2011, it has set the stage for routinely modifying the CHO genome to improve the production of recombinant antibodies. Certain key genes such as FUT8, which encodes α 1,6 fucosyltransferase, an enzyme that catalyzes the post-translational fucosylation of expressed proteins, have already been targeted using gene engineering techniques to prevent fucosylation of recombinant antibodies. Many other proteins encoded by the CHO genome, however, have yet to be explored for their potential impact on the efficacy, safety and half-life of recombinant proteins. Horizon Discovery’s proprietary gene targeting technology utilizes recombinant adeno-associated virus vectors (rAAV) to exclusively stimulate homologous recombination, a natural high-fidelity repair mechanism that exists to maintain the integrity of the genome during mitosis. This process can be easily piggy-backed to create targeted, in-frame and error free genome alterations, by providing DNA vectors with stretches of homology to a target locus, which at a certain frequency are seamlessly recombined with the endogenous sequence and can be used to insert, delete or substitute specific sequences with single nucleotide resolution. No DNA breaks are created by rAAV and there are no additional off-target integrations within targeted cell lines. The only drawback is that the actual targeting process can be slower, principally if one wants to create bi-allelic alterations, since alleles have to be targeted sequentially. In stark contrast, alternative endonuclease technologies i.e. Zinc-finger Nucleases (ZFNs) use a fundamentally difference process to perform gene editing. Firstly, the engineering process to target your locus of interest is a complex task involving the alteration of the binding specificity of a protein, and thus in practice is often less than optimal. Even on well validated ZFN’s there are a large number of additional ‘off-target’ cuts as well as the intended cut, which means the process is inherently not a clean one. Secondly, dsDNA breaks are predominantly repaired by the ‘non-homolgous recombination end-joining’ (NHEJ) pathway. NHEJ simply rejoins and ligates the DNA ends, usually after some chewing back of the free endes, frequently creating null alleles. The important thing to note is that none of these events involve targeted, in-frame, splicing events, which can only be achieved by eliciting homologous recombination. I will describe how Horizon’s technology has already been successfully applied to reconstitute hundreds of oncogenes in human cell lines, and how, due to its accuracy, rAAV based genome editing is well placed both to decipher the CHO genome sequence, and to modify the genomes of CHO cell lines and other host cell lines used for bioproduction. I will draw upon examples from Horizon’s bioproduction development program to demonstrate the applicability of the technology to the field of bioproduction. These include projects to knock-out metabolic genes (Glutamine Synthetase) and to generate ‘landing pad’ CHO cell lines that contain an antibody back-bone ‘constant region’, wherewith variable regions can be ‘knocked-in’ using an rAAV vectors to facilitate rapid product of antibody isoforms.


CELL LINE GENERATION, MANUFACTURING, RELEASE AND CHARACTERIZATION OF RECOMBINANT ANTIBODY MIXTURES

Søren K. Rasmussen, Symphogen A/S Electrovej, Building 375, Lyngby, 4000, Denmark

T: 45-24-78-75-63, F: 45-45-26-50-60, [email protected] Frank Nygaard, Symphogen A/S Christian Müller, Symphogen A/S

Torben P. Frandsen, Symphogen A/S

Recombinant antibody mixtures represent an important new class of antibody therapeutics where combinations of two or more antibodies show superiority compared to monoclonal antibodies for treatment of e.g. cancer and infectious diseases. Recombinant antibody mixtures can in principle be made in three different ways, i.e. as individual drug products that are simply administered to the patients as a combination, as individual drug substances that are mixed as one drug product and finally using a single batch manufacturing of drug substance and subsequently drug product. Simple mixtures, containing 2-3 antibodies, are typically produced, released, and characterized as individual drug substances and subsequently mixed as one drug product. One example of such a product is Sym004, composed of two antibodies targeting non-overlapping epitopes of the epidermal growth factor receptor (EGFR), that act in a synergistic manner to induce an efficient internalization of EGFR leading to subsequent receptor degradation. Sym004 exhibits superior anticancer efficacy as demonstrated in several preclinical in vivo models. At Symphogen A/S, we have developed an expression platform, Sympress™, for cost-efficient production of antibody mixtures. Rozrolimupab, composed of 25 anti RhD antibodies is like the vast majority of recombinant antibodies produced by a CHO expression platform, based on site specific integration using the Flp-In system in CHO-K1 cells. The Sympress™ technology has subsequently been optimized to achieve higher titers and the currently employed expression technology is based on expression in the ECHO cell line, a genetically modified version of the dihydrofolate reductase (DHFR) negative Chinese Hamster Ovary (CHO) cell line DG44. ECHO parental cells are transfected separately with each of the individual antibody expression vectors using standard transfection technology, after which cells are subjected to a methotrexate (MTX) selection schedule. The selected stable pools are single-cell cloned by FACS and high-expressing clones are expanded and frozen, still as individual research cell banks. The antibody mixtures are produced using a single-batch manufacturing approach where a polyclonal working cell bank (pWCB) prepared by mixing the individual stable cell lines producing all the desired antibodies is used as seed material for a bioreactor process. This technology has certain challenges in terms of cell banking strategy, manufacturing approach and strategies for the release and characterization of such types of products and this will be addressed in the presentations and compared to individual manufacturing approaches. Furthermore, a comparison in terms of development timelines, preclinical developmental costs, and manufacturing COGS between the two manufacturing approaches will be made in the presentation.


ADAPTATIONS OF MONOCLONAL ANTIBODY-PRODUCING CHO CELL LINES: PERSPECTIVES FROM GENOMICS, TRANSCRIPTOME, GLYCOMICS AND METABOLOMICS

Bernard Loo*, Bioprocessing Technology Institute 20 Biopolis Way #06-01, Centros, Singapore, N/A, 138668, Singapore T: +65-64070908, F: +65-64789561, [email protected]

Ying Swan Ho*, Faraaz Yusufi*, Terk Shuen Lee, Yuan Sheng Yang, Dong Yup Lee, Bioprocessing Technology Institute

Niranjan Nagarajan, Ruan Xiaoan, Ken Sung Wing Kin, Genome Institute of Singapore Wei-Shou Hu, Department of Chemical Engineering and Materials Science, University of Minnesota

Miranda GS Yap and Muriel Bardor, Bioprocessing Technology Institute

Recombinant CHO cell lines are the workhorses for the production of protein therapeutics. The generation of these stable cells lines typically involves random integration of the genes into the genome and a selection of stable high producers follows. These processes typically take at least 6 months using high through-put methods. There is a lack of understanding of the physiological adaptive mechanisms that occur during cell line development. To address these questions, we compared the genomic, transcriptome, glycomic and metabolic profiles of parental CHO-K1 and SH87, a recombinant CHO-K1 anti-Her 2 producing cell line. The recombinant clone was generated using an in-house tricistronic vector using standard cell-culture transfection methods and was isolated within two months (Ho et al., 2011). The high expressing clone was then adapted back into suspension in protein-free medium with G418 selection. Productivity and growth characterization of the parental and recombinant cell lines were perfomed in shake-flasks.

The genomic profile and transcriptome profile of the CHO-K1 and SH87 cell lines were obtained using next-generation sequencing technology. The CHO-K1 genome was reassembled and compared with the recently published genomic data (Xun et al., 2011). As expected, high degree of similarity has been found between the two genomes. The genome of CHO-K1 and SH87 were scanned for structural variances, point mutations and integration sites. Interestingly, initial results show multi-copy integration of vector into the recombinant genome. RNA-Seq and full-length RNA-PET analysis of the clone reveal potential transcriptome hotspots, functionally enriched pathways and all possible transcript isoforms present in CHO-K1 and SH87.

Liquid chromatography-mass spectrometry (LC-MS) based metabolomics analysis was also carried out to identify key metabolic differences between the two cell lines. Preliminary results suggest these differences include molecules involved in lipid metabolism and the removal of reactive oxygen species. Additionally, the glycosylation profiles of the two cell lines are currently under investigation to evaluate whether the development of such recombinant cell line result in a different N- and O-linked glycosylation capabilities.

This comprehensive study yields valuable insights into the cellular physiological mechanisms of recombinant cell line development and suggest strategies to optimize future cell line development work.

References Ho SC, Bardor M, Feng H, Mariati, Tong YW, Song Z, Yap MG and Yang Y. (2011) IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J. Biotechnol. 2011 Oct 17. [Epub ahead of print] Xun Xu, Harish Nagarajan, Nathan E Lewis et al. (2011) The genomic sequence of the Chinese hamster overy (CHO)-K1 cell line. Nature Biotechnology 29:735-741. *All authors have contributed equally to this work

Thursday, April 26, 2012 Chair Select Session: Process Characterization and Quality Control

EVALUATION OF CELL METABOLISM AS A HIGH THROUGHPUT INDICATOR OF THE IMPACT OF MEDIUM COMPONENTS ON AUTOLOGOUS CELLULAR IMMUNOTHERAPY PRODUCT ATTRIBUTES

Pascal R Beauchesne, Dendreon Corporation 1301 2nd Avenue, Suite 3200, Seattle, WA, 98101, USA

T: 1-206-455-2301, F: 1-206-829-1650, [email protected] Christopher Ramsborg, Dendreon Corporation

Kien Khuu-Duong, Dendreon Corporation Lisa Joslin, Dendreon Corporation

Mickey Emde, Dendreon Corporation

In 2010 Provenge® (sipuleucel-T), an autologous cellular immunotherapy (ACI), was approved by the FDA for the treatment of asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer. Sipuleucel-T consists of a patient’s own peripheral blood mononuclear cells (PBMC) collected by leukapheresis and cultured in the presence of a recombinant human fusion protein consisting of prostatic acid phosphatase, a prostate-specific antigen, and granulocyte-macrophage colony stimulating factor, an immune cell activator. The resulting cellular product contains antigen presenting cells (APC’s) that are infused back into the patient 3 days later.

Ex vivo cell culture is a key unit operation in the manufacturing of ACI; cell culture conditions can strongly influence APC activity. An investigation of the effects of cell culture medium components on process performance and robustness was undertaken. Given the high variability among healthy human donors, a scale-down model was developed to allow the screening of multiple culture conditions using PBMC from a single donor. In order to support increasing sample numbers, higher throughput assays are required. APC activity has traditionally been assessed in cell-based antigen presentation assays using HLA-restricted responder cell lines. Assay complexity limits throughput while HLA specificity restricts the size of the donor pool. It was hypothesized that the metabolic state of the cells could be used as a predictor of APC activity. A metabolic flow cytometry-based approach using C12-resazurin was investigated as an early high throughput development tool for cell culture medium optimization. Within the cell, C12-resazurin is reduced into fluorescent C12-resorufin which can be readily detected by flow cytometry. The C12-resazurin assay was combined with immunophenotyping, allowing the assessment of the impact of different medium formulations on specific cell populations including monocytes, B cells, and T cells. Within a common medium base and initial culture parameters, cell culture supplement combinations leading to increased APC metabolism were found to be a good predictor of APC activity measured by the low throughput cell-based bioassay. This method can be performed in less than 3 hours and is not limited by HLA haplotype-restrictions, thus allowing for near real-time results and access to a large eligible donor pool. This metabolic approach will facilitate the high throughput screening of media formulations and selection of promising candidates for additional APC activity analysis. While not a replacement for the APC-responder assay, this metabolic strategy has the potential to accelerate cell culture medium development for autologous cellular immunotherapies.


THE METABOLIC LOAD OF HETEROLOGOUS PROTEIN EXPRESSION IN CHO CELLS

Olivier Henry, Chemical Engineering, Ecole Polytechnique de Montreal P.O. Box 6079, Centre-Ville Station, Montreal, Quebec, H3C 3A7, Canada

T: 1-514-340-4711 ext:2191, F: 1-514-340-4159, [email protected] Zahra Sheikoleslami, Chemical Engineering, Ecole Polytechnique de Montreal

Mario Jolicoeur, Chemical Engineering, Ecole Polytechnique de Montreal Patrick Daoust, Viropro International Inc. Patrick Benoist, Viropro International Inc.

Recombinant mammalian cell lines typically exhibit reduced growth and higher rates of nutrient utilization compared to parental cells, presumably to meet the increased demand for energy and precursors needed for heterologous protein synthesis, folding, modification and secretion. In order to quantitatively assess the impact of recombinant protein expression on the primary metabolism of CHO cells in culture, we have employed an efficient inducible expression system (named the 'cumate gene-switch') and performed an extensive metabolic characterization of the on and off states.

To this end, a comparative 13C-metabolic flux analysis was conducted, whereby cells were grown in parallel semi-continuous cultures containing various labeled glucose and glutamine tracers and the resulting mass isotopomer distributions of extracellular metabolites (three secreted amino acids and lactate) were measured by LC-QTOF-MS. This approach allowed us to obtain reliable estimates for the main intracellular fluxes, including pathways that cannot be observed from external rate measurements (e.g. the pentose phosphate pathway).

Upon addition of a non-toxic concentration of cumate and under mild-hypothermic conditions (30°C), the cell specific productivity was 23 pg/cell.d, corresponding to an on/off induction ratio of approximately 20. From this measured cellular productivity, it was estimated that recombinant proteins accounted for at most 15 % of the total cellular protein mass. Accordingly, our study revealed that recombinant protein expression is correlated with small but significant variations in a number of key intracellular pathways related to ATP and NADPH formation, including the pentose phosphate pathway, the malic enzyme reaction and the TCA cycle. When expressing the recombinant antibody, the cells notably exhibited a more efficient utilization of glucose, characterized by a higher fraction of pyruvate entering the TCA cycle. Conversely, the catabolic rates of most amino acids, including glutamine, remained unaffected by the onset of protein expression.

Elucidating the alterations in central carbon metabolism caused by protein production is instrumental for the establishment and optimization of a productive mammalian cell expression platform. Such analysis can help guide the identification of robust biomarkers of productivity, the selection of a proper induction time, as well as rationalize the development of improved medium formulations and feeding strategies for biphasic processes.


IMPACT OF RAW MATERIALS AND MANUFACTURING PROCESSES ON DRY POWDER CELL CULTURE MEDIA PERFORMANCE.

Aline Zimmer, Merck KGaA Frankfurter Strasse 250, Darmstadt, Hessen, 64283, Germany

T: 49-6151-72-54276, F: 49-6151-72-53632, [email protected] Joachim Eichner, Merck KGaA Frank de Groot, Merck KGaA

Maria Wehsling, Ronja Müller, Merck KGaA Jörg von Hagen, Merck KGaA

Mammalian cell culture media have a huge impact not only on cell growth and protein yield, but also on quality and robustness of the biopharmaceutical molecules. The biological performance of any medium is closely linked to the optimization of the formulation, but also to the quality of raw materials and the manufacturing process.

We demonstrate that the quality of raw materials and parameters used in the production process of dry powder media have a direct impact on cell growth and protein production by CHO-S cells. The quality of raw materials was analyzed by benchmarking dry powder cell media from various suppliers for several parameters like pH, osmolality, turbidity or moisture content. The impact of powder milling technologies on raw material particle size distribution, cell growth and quality attributes of the final molecule was evaluated. Finally, analytical methods such as near infrared resonance spectrometry were used to determine unique fingerprints for cell culture media resulting in the study of potential contaminants, degradation products or missing components. Multivariate data analysis allowed the clustering of commercial media while the chemical fingerprinting could be correlated to cellular performance, resulting in a fast and efficient prediction of media performance.

Finally, this study demonstrates that the combination of raw materials quality and robust manufacturing processes is mandatory for excellent dry powder media performance. Besides known benefits of understanding raw material quality and consistency, analytical methods could also lead to the identification of correlations between cell culture media characteristics and cell growth or quality of expressed recombinant proteins. The establishment of such correlations could contribute to the rational development of new cell culture media and to improve robustness in media engineering and manufacturing.


RESOLVING PROCESS VARIABILITY WITH AN INCREASED UNDERSTANDING OF CELL METABOLISM

Rashmi Kshirsagar, Biogen Idec 14 Cambridge Center, Cambridge, MA, 02142, US

T: 1-617-679-2571, F: 1-617-679-3415, [email protected] Alan Gilbert, Biogen Idec

Kyle McElearney, Biogen Idec Marty Sinacore, Biogen Idec

Thomas Ryll, Biogen Idec

We have developed a robust, scalable process platform using chemically defined medium that delivers over 3 g/L in monoclonal antibody concentration. Recently, large variations in metabolism were observed at extended cultivation times when applied to an antibody program. This variability is undesirable and limits process options. We investigated the causes of the metabolic variability and have identified several triggers which ultimately result in variation in metabolism. We have used lactate production rate as a marker of the metabolic variability as it demonstrates this metabolic variability most effectively. By implementing an intracellular fluorescent staining protocol, a likely mechanism inducing the variability was elucidated. Risk mitigation techniques were implemented taking direct advantage of the mechanistic knowledge, and these mitigation steps will also be discussed.


DEVELOPMENT OF A METHOD TO MODEL THE CELL METABOLISM IN VARYING ENVIRONMENTAL CONDITIONS BASED ON EXTRACELLULAR COMPONENT MEASUREMENTS

Véronique Chotteau, School of Biotechnology, Royal Institute of Technology, KTH Roslagstullsbacken 21, Stockholm, SE-10691, Sweden

T: +46 8 55 37 84 78, F: +46 8 5537 8481, [email protected] Joan Gonzalez Hosta, School of Electrical Engineering, Automatic Control, Royal Institute of Technology, KTH

Antonio Aliaga, Ye Zhang, Andreas Andersson, Erika Hagrot, School of Biotechnology, Royal Institute of Technology, KTH

Elling W Jacobsen, School of Electrical Engineering, Automatic Control, Royal Institute of Technology, KTH

The information, which can potentially arise from metabolic pathway modeling, is a better understanding of the pathways effectively used by the cells and a tool to force the cells to use more favorable pathways, e.g. leading to less toxic by-product production, given that the way to influence which pathways the cells are using, is known. For this purpose a model has to identify all the reactions that the cells can potentially use as well as their kinetics. One way to tackle this difficult task is to trigger all the reactions used by the cells by systematically varying the cell states. This can be achieved by varying the environmental conditions for instance by varying the availability of the nutrients, the growth factors, the precursors, or varying the environmental parameters.

The purpose of the present study was to develop a method generating a mathematical model able to predict the behavior of a cell culture system in varying environmental conditions while measuring the extracellular components only.

In the present study, in order to reduce the complexity of the task, a simple model including the reactions involving the glycolysis, the TCA cycle and the amino acid metabolism was considered. An antibody producing CHO cell line was cultured in defined medium. The amino acid concentrations in the cultivation medium were varied in pseudo-continuous cultivation systems and the resulting concentrations of the extracellular components, glucose, lactate and amino acids, were measured. A quasi steady-state assumption was adopted. A reduced model of macro-reactions was generated by elementary flux analysis approach using software Metatool 5.1 to algebraically eliminate the intracellular components. Michaelis-Menten models were adopted for the macro-reaction rate kinetics in which the maximal kinetic rates were unknown constants. The maximal kinetic rates were determined by non-negative least squares algorithm minimizing the error between the modeled macro-reaction rate kinetics and the measured fluxes of consumption or production of the extracellular components. Single values of the maximal kinetic rates were determined for the whole set of experimental data, i.e. in presence of varied concentration of amino acids resulting in a unique model valid for varying amino acid concentrations. Finally an excellent fitting was observed between the measured fluxes of consumption or production of the extracellular components and the values estimated by this model.

Thursday, April 26, 2012 Chair Select Session: Hydrodynamics in Industrial Cell Culture

SCALE-DOWN STUDIES OF THE EFFECT OF HYDRODYNAMIC FORCES ON CHO CELLS; IMPLICATIONS FOR INDUSTRIAL PRODUCTION CONDITIONS

Steven Meier, Genentech, A Member of the Roche Group Mailstop 96A, 1 DNA Way, South San Francisco, CA, 94080, USA

T: 1-650-225-7460, F: 1-650-467-5477, [email protected] William Scott, University of Birmingham, UK

Robert Kiss, Genentech, A Member of the Roche Group Ashraf Amanullah, Gilead, Inc.

Alvin Nienow, University of Birmingham, UK

The effect of hydrodynamic forces on CHO cells used for the production of biopharmaceuticals has been studied by multiple academic and industrial researchers. Such studies have included evaluation of lethal forces, as well as non-lethal forces, for their effect(s) on cell growth and productivity as well as recombinant protein quality attributes. In this work, two different CHO production cell lines were studied in a repetitive defined elongational laminar shear field device, and in high turbulent shear field bioreactor cultures. Specific energy dissipation rates in the laminar flow in the shear device were tested at levels comparable to peak turbulent levels for typical bioreactor operating conditions for successful industrial operation and at significantly higher levels. Even specific energy dissipation rates much higher than those practically required do not show any adverse effects on performance or protein quality for both cell lines, consistent with previous results showing no significant effects on growth or productivity with multiple cell lines. This work will be summarized in the context of practical conditions in typical stirred tank bioreactors, and the likelihood that such hydrodynamic forces would significantly impact process performance or protein quality attributes.


EFFECT OF HYDRODYNAMIC CONDITIONS ON EXPRESSION OF STRESS PROTEINS, CELL CYCLE AND RECOMBINANT PROTEIN PRODUCTIVITY

Claudia Berdugo, William G. Lowrie Chemical Engineering Department 140 W 19th Ave, Columbus, Ohio, 43210, USA

T: 1-614-292-2727, F: 1-614-292-3769, [email protected] Oscar Lara-Velasco, GlaxoSmithKline

Jeffrey Chalmers,

Stress proteins are expressed in response to different environmental stresses such as heat, nutritional deficiency, oxidative stress and inhibitory chemicals. While it has been speculated for years, to our knowledge, the expression of stress proteins in response to hydrodynamic stress in bioreactors has not been demonstrated. Complementary to stress proteins, speculation exists with respect to a link between production of recombinant proteins by mammalian cells with a particular cell cycle growth phase. Understanding the relationship between cell cycle phase and productivity could contribute to optimization of large scale processes. In this work we explored the expression of stress proteins under different hydrodynamic conditions in different culture vessels, including static cultures, spinners and bioreactors. Cell cycle profiles were evaluated in 2 L working volume bioreactors with different impeller/sparger configuration in order to determine the potential effect of different hydrodynamic conditions on cell cycle profile and recombinant protein production. Significant difference in stress protein expression between bioreactors and T-Flasks was observed. In contrast, with cell cycle studies, specific productivity seems to be associated with G1 phase and no significant differences were observed when culturing environments (vessels) were changed.


MIXING ISSUES IN CELL CULTURE BIOREACTORS USING MICROCARRIERS

Alvin W Nienow, Centre for Biochemical Engineering University of Birmingham, Birmingham, B15 2JJ, UK

T: +44-12-1440-2344, F: +44-12-1414-5324, [email protected]

Though in the early 1980s animal cell culture was generally based on attaching cells to microcarriers, little work was done to optimize their suspension. Partly this was because, around this time, the ability to grow cells in free suspension was established under which conditions, cells were much less likely to be damaged by fluid dynamic stresses. For example, Croughan et al (1987) showed that mean specific energy dissipation rates, W/kg , had to be < ~1 x 10-3 W/kg to prevent damage with 180 mm microcarriers. However, work was not undertaken to search for vessel/agitator configurations that would achieve this end. Indeed, some geometric recommendations (for example, the use of hemispherical bioreactor bases to aid suspension (van Wezel, 1985)) were counter to the general findings on particle suspension (Nienow, 1985). Now with the increasing importance of regenerative medicine and the need to grow stem cells on microcarriers at large scale for allogeneic usage, it is essential to establish efficient bioreactor geometries for microcarrier suspension. Clearly, it is essential for the microcarriers to be suspended if the important well-mixed features of the stirred bioreactor are to be achieved. The minimum speed, NJS, and mean specific energy dissipation rate at that speed, W/kg, has been measured for many geometries but a review of the literature still shows little work has been done on microcarriers. The most relevant study (Ibrahim and Nienow, 2004) investigated these parameters using Cytodex 3 microcarrier beads using a range of different diameter Chemineer HE-3 hydrofoils, a pair of Ekato InterMIG impellers and a six-blade, 45-pitch turbine impeller in a baffled vessel of 19.2 L operating volume containing phosphate buffer saline solution. Flat and modified tank bases were used and NJS values were observed to be in the range of 50 to 90 rpm. The use of Zwietering’s correlation using existing literature geometric suspension parameters, S, to predict NJS would have given values up to 50% higher. The low NJS values obtained were attributed to the very small particle–liquid density difference (40 kg/m3), which eased the lifting of the particles from the tank bottom, compared to those used in non-microcarrier studies. With these microcarriers, the three-blade hydrofoil HE-3 impeller of D/T = 0.39 in a cone-and-fillet based tank was marginally the most efficient; that is, it had the lowest W/kg, ~ 0.5 x 10-3 W/kg. However, mean specific energy dissipation rate was < ~1 x 10-3 W/kg in most cases with the different size HE-3 hydrofoils, which implies that these geometries would probably be suitable for application in shear-sensitive cell culture systems using such microcarriers. If cells have to be attached to microcarriers, then damage due to this energy dissipation rate might occur to cells from impeller-microcarrier, microcarrier-microcarrier and microcarrier-vessel internals impacts or due to cells being stripped off them; plus direct stress from turbulent eddies, so it is important to minimize it. However, it must also be remembered that mean specific energy dissipation rate is a critical parameter and needs to be sufficient to achieve the required rate of oxygen and carbon dioxide transfer and give an adequate quality of homogenization. Thus, it is important to consider all these parameters which are dependent on W/kg when developing the stirred bioreactor


IMPACT OF BIOREACTOR DESIGN ON THE PERFORMANCE OF MICROCARRIER CELL CULTURES

Manuel J. T. Carrondo, IBET Ap. 12, Oeiras, 2781-901, Portugal

T: +351.21.446.93.62, F: +351.21.442.11.61, [email protected] Marcos Sousa, IBET João Clemente, IBET

Paula Alves, IBET

Gaining from the pioneer work on microcarrier cell culture hydrodynamics of Matt Croughan and Danny Wang in the ’80, at IBET we have been optimizing use ofMC for processes involving industrial cell lines used for protein or vaccine production (BHK, VERO, MRC-5,..), cells for viral vector production for gene therapy (A549, TeFLY...) or primary and stem cells for cell therapy or preclinical/discovery studies. Hydrodynamics being of special concern, we have been evaluating some recently available disposable bioreactors, as they are becoming the option of choice in many processes, from small, hospital environmental or research lab scale up to thousand liters, industrial scale. Data obtained from tests with the low shear Air-Wheel PBS Biotech bioreactor using MC cell culture will be presented, showing superior cell attachment and growth on microcarriers; when these are used to grow viruses, high viral productivities are also obtained.


A METHOD FOR ASSESSING CELL LYSIS-MEDIATED MONOCLONAL ANTIBODY REDUCTION IN INDUSTRIAL CELL CULTURE PROCESSES

Brian Horvath, Genentech 1 DNA Way, S. San Francisco, CA, 94080, USA

T: 1-650-225-4041, F: 1-650-225-2006, [email protected] Donald Lee, Genenetech

Michael W. Laird, Genentech

The potential for monoclonal antibody (MAb) disulfide reduction, and eventual product loss, during harvest operations warrants a lab-scale method to assess the risk associated with cell culture production processes. A cell line’s susceptibility to lysis has become an important characteristic as cell lysis in high cell density processes can lead to MAb reduction in harvested cell culture fluid. A working hypothesis is that as cells lyse they release cellular components including macromolecules (e.g. reducing enzymes) and active proton carriers (e.g. NADPH) into the cell culture fluid (CCF). Released cellular components in turn partner to hydrolyze the interchain disulfide bonds of the MAb. Lysis susceptibility can be screened by subjecting cultures to a flow contraction device (FCD) in which variable degrees of lysis between cell lines are achieved at known energy dissipation rates (EDR). The EDRs generated by the FCD are between 10E5 – 10E8 W/m3 across flow rates of 10 – 100mL/min. These EDRs are sufficient to cause lysis in CHO cells. In the complete method shown here, lysis susceptibility screening is integrated with analysis of MAb reduction in the resulting lysates. Part of this method includes screening for the effect of cell-size on lysis where cell-size is modulated through the use of a salt-shock technique just prior to the lysis event. The method has been used to characterize lysis susceptibility and MAb reduction risk in numerous cell lines and processes at Genentech.

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