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B i o P r o c e s s Technical Metabolic Process Engineering A Novel Technology Platform Applied to Industrial Cell Culture Production Processes Bernhard M. Schilling, Susan Abu-Absi, and Patrick Thompson M etabolic process engineering (MPE) was developed at Bristol-Myers Squibb Company as a tool to effectively control and optimize industrial cell culture processes used for production of biological drugs. A fundamental need was identified to introduce manipulations to the metabolism of production cell lines without genetic engineering. Optimization goals for production cell line performance include, for example, volumetric productivity, control of product quality attributes and by-product formation, and improved process scalability. With MPE, we could achieve targeted changes to cellular metabolism through timed addition of chemicals to a production process. Here we describe the MPE concept and provide examples of its use. We made two further observations when applying it: Well-understood, scientifically based changes to the production process had the highest chance of meeting product Product Focus: Proteins Process Focus: Production Who Should Read: Process development, analytical, QA/QC, and manufacturing Keywords: Fed-batch, culture media supplementation, glycosylation, viability, lactate, optimization Level: Intermediate 42 BioProcess International 10(1) January 2012 A seed reactor at Bristol-Myers Squibb in Devens, MA comparability needs, thus facilitating regulatory approval. And poorly understood, empirical approaches carried the risk of introducing undesirable variability into process performance and changes to product quality attributes. In early product development, a wide array of activities are ongoing to define a cell culture production process: e.g., host cell choice, expression vector design, cell line development, clone screening and selection, media development, process development, and process scalability studies. In media development, improvements in the design of chemically defined media, taking advantage of spent media analysis, have advanced analytical capabilities bristol-myers squibb company (www.bms.com) and high-throughput technologies (1). However, empirical nutrient optimization is also continued, along with the use of complex raw materials (2). The opportunity to use the techniques described herein narrows when entering the commercialization phase of a biological drug. Furthermore, regulatory expectations are constantly increasing with regard to the level of expected process understanding, especially the required knowledge behind proposed changes. Our company therefore determined that a tool needed to be developed that would provide intrinsic process understanding while assisting process developers in meeting their desired optimization goals. Table 1: Examples of chemicals added to cell culture or microbial production processes Chemical and Reference Sodium butyrate (4) Cell Line CHO-K1 Sodium butyrate (5) Vanadate (6) CHO CHO, Vero, BHK, HeLa, COS, 293 CHO-K1, HEK-293 CHO Peptide-based chemical caspase inhibitors (7) Small-molecule enhancers: aromatic carboxylic acids, hydroxamic acids including hexanohydroxamic acid and acetamides (8) Rapamycin (9) Hybridoma Dimethyl sulfoxide (10) Dimethyl sulfoxide (11) Hybridoma CHO Pentanoic acid (12) Hydroxyurea (13) CHO Saccharomyces cerevisiae Conceptual MPE has similar goals as the wellknown concept of metabolic engineering (3), but it achieves them through timed addition of chemicals to a production process rather than the use of genetic engineering. Figure 1 presents the concept step by step. First, identify your goal for a desired production cell line performance change. Collect and analyze knowledge from key metabolic pathways, metabolic flux analysis, and scientific literature (including medical publications) to form a hypothesis for a potential mode of action. Next, attempt a targeted manipulation of your production cell line’s metabolism through addition of selected candidate chemicals to the production process. • MPE has similar goals as the well-known concept of metabolic engineering, but it achieves them through timed addition of chemicals rather than the use of genetic engineering. 44 BioProcess International 10(1) January 2012 Purpose/Observation Controlling sialic acid content of glycoproteins and cell-specific productivity Increasing synthesis of secreted proteins Enhancing cell survival so that recovery of a polypeptide of interest produced in the cells increases Increasing integrated cell densities and monoclonal antibody titers Increasing monoclonal antibody production Keeping cell viabilities high and delaying cell death; attaining higher maximum viable-cell densities and enhancing monoclonal antibody production Increasing specific monoclonal antibody and overall production Inducing growth arrest to reduce cell growth rate and achieve a high overall specific productivity Enhancing protein biosynthesis Controlling growth rate and cell division Finally, verify successful results through measurement of metabolic parameters using, for example, enzymatic or chemical analysis, metabolomics, and/or transcriptomics. Multiple published case studies report successful addition of chemicals to cell culture and microbial processes for the purpose of manipulating process performance. Table 1 lists some. Application Production cell lines usually have weaknesses that affect overall process performance. However, such cell lines have been used in manufacturing, where they define the biochemical and physicochemical characteristics of a biological drug. The goals for a change in cell-line performance may be • volumetric productivity (peak viable cell density, cell viability profile, cell-specific productivity) • product quality attributes (posttranslational modification, product isoforms, product aggregation) • by-product formation (lactate, ammonia, carbon dioxide), • process scalability. Areas that can be researched for potential targets of cell metabolism include bioenergetics, cell signaling, target protein expression, stress reduction, membrane renewal, and others. Enzymes, receptors, signaling compounds, and metabolic flux are all potential metabolic targets. Candidate chemicals may serve as effectors, precursors, traps, or Figure 1: Step-by-step description of metabolic process engineering (MPE) Identification of desired change in metabolic performance Analysis of relevant metabolic pathways (e.g., flux analysis, metabolomics, or transcriptomics) (large amount of information available in the public domain on mammalian cell metabolism, and from medical research literature re: cells, tissues, organs, organisms) Hypothesis formed on a possible metabolic target and mechanism Experiments with chemical agents executed for proof of concept If successful, verification of mechanism at metabolic level receptor blockers. Those chosen for evaluation need to fulfill certain criteria for use in industrial cell culture processes. They must not be nutrients, proteins or peptides, or animal derived. They must be nontoxic and economical in pricing. And they must not lead to performance trade-offs. Such tradeoffs are observed, for example, in the use of sodium butyrate (14), with which a higher observed cell specific productivity is tied to reduced cell growth. For MPE, high-throughput screening experiments are set up to evaluate candidate chemicals. Concentration and timing of additions to the culture also play a crucial role. To achieve a desired process performance change, several chemicals affecting different metabolic targets may be combined to improve overall effectiveness. Some Examples Here we present four examples of the MPE in application. These examples Figure 2: Impact of bioreactor scale on culture lactate formation and remetabolization address process optimization goals in the areas of • control of by-product formation • control of a product quality attribute, • volumetric productivity through cell-specific productivity, and • volumetric productivity based on cell viability profile. Example 1 — Control of By-Product Formation: Lactate formation is Lactate 5,000 L 75 L 5L generally regarded as detrimental to mammalian cell culture (14, 15), in which increases in culture osmolality can lead to cell death. We observed higher lactate formation was observed during scale-up of cell culture Figure 3: Activity in key-enzyme in electron transfer chain (ETC) increased after addition of chemical A (arrow marks time of addition). Enzyme Activity (mU/10 6 cells) 20,000 L With addition Control Time Time e le nc ilab e er a nf e Av ! o C ur ne h li oc on br April 16-18, 2012 • JW Marriott desert ridge resort • phoenix, arizona The Best Content in the Industry Conference Highlights Include: • Two Great Opening Plenary Topics: • Future Benefits for Patients: From Discovery to Commercial Products, Cellular and Gene Therapies, David Shanahan, President, Mary Crowley Research Center and President, CEO and Founder, Gradalis • The Future of Personalized Medicine – Challenges Ahead, Ted Love, MD, Executive Vice President, R&D and Technical Operations, Onyx Pharmaceuticals • Plenary Session Two: • The Future of the Biopharmaceutical Industry, David Urdal, Chief Scientific Officer, Dendreon • The Future of the Biopharmaceutical Regulatory Perspective, FDA Representative Invited • Student Call for Posters – Abstracts Due February 6, 2012 • Closing Plenary Topics: Also: • Manufacturing Opportunities and Post-Conference Challenges in the Next 10-20 Years, Workshop Matt Croughan, Professor, Keck Graduate on Single Use Institute of Applied Life Sciences Systems! • Emerging Regulatory Expectations, Emily Shacter, PhD, Chief, Laboratory of Biochemistry, CDER, FDA • NEW: Breakfast Sessions on: Career Development Strategies and the Quality and Regulatory Job Market Outlook 2012 • Networking Receptions & Events like the 6th Annual PDA Golf Tournament at the Wildfire Golf Club & the PDA 6th Annual Walk/Run (benefiting the Phoenix Children’s Hospital) • Post-Conference Workshop: PDA Single Use Systems Workshop on April 18-19 and eight courses hosted by PDA TRI on April 19-20 • Hotel activities for the entire family! www.pda.org/annual2012 Exhibition: April 16-17 | CArEEr FAir: April 16-17 | Post-ConFErEnCE WorKshoP: April 18-19 | CoursEs: April 19-20 Lactate Figure 4: Culture lactate levels decreased after addition of chemical A (arrow marks time of addition). Control Concentration 1 Concentration 2 efficiency of the pyruvate f lux and cellular energy charge. We hypothesized and confirmed that lactate can be remetabolized through LDH activity with achievement of a higher flux of Figure 5: Intracellular UDP-nucleotide concentration increased with addition of chemical B. Control Time Time Table 2: Decrease of monosaccharide #3 and associated reduction of serum half-life was observed during process scale-up; addition of chemical B restored monosaccharide #3 level and pharmacokinetic properties. Monosaccharides (normalized molar ratio) #1 #2 #3 #4 #5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.6 1.0 0.9 1.1 1.0 0.9 1.0 1.1 Development scale Industrial scale Industrial scale with addition of chemical B pyruvate into the tricarboxylic acid (TCA) cycle. We accomplished this by increasing the activity of a key enzyme in the electron transport chain (ETC). Figure 3 illustrates the increased activity of an ETC key enzyme when chemical A was added. Figure 4 describes the immediate reaction of cells to timed addition of chemical A and the resultant decrease in culture lactate levels. Example 2 — Control of Product Quality Attributes: A further With addition Concentration processes to industrial production scale (Figure 2). We considered an obvious metabolic target for manipulation: lactate dehydrogenase (LDH) activity. However, suppressing such activity involved significant performance trade-offs. So we focused instead on overall Serum half-life (normalized) 1.0 0.7 1.0 challenge during scale-up of highly glycosylated biological drugs is control of protein glycosylation characteristics (14). We observed a decrease of the content of a specific monosaccharide in the glycosylation pattern during scale-up to industrial production scale. This change affected the pharmacokinetic properties of the protein product. Several metabolic targets were identified. As Figure 5 shows, the timed addition of chemical B increased the level of a key nucleotide in the intracellular pool. That higher level then enabled comparability of BioProcess TM I N T E R N A T I O N A L POSTER HALL 2011-2012 A CompilAtion of proCess teChnologies And expertise presented in poster formAt AvAilAble At: www.bioprocessintl.com/posters Figure 6: Key-enzyme activity increased after addition of chemical C (level of control at 1.0). Figure 8: DNA laddering decreased with the addition of chemical D (bioreactor profiles from days 3 to 11; agarose gel after ethidium bromide staining). Enzyme Activity (mU/106 cells) 2 Control 1 Kb D3 With Addition D5 D7 D9 D11 D3 D5 D7 D9 D11 D4 D6 D8 D10 D4 D6 D8 D10 1 Kb 1 0 Time Cell Specific Productivity Figure 7: Cell-specific productivity increased after addition of chemical C (blue = control; red = with addition of chemical C) the protein glycosylation pattern from development to production scale (Table 2). Example 3 — Volumetric Productivity Increase through Cell-Specific Productivity: The physiological limit of a cell to produce, for example, monoclonal antibodies (MAbs) (qPmax) was estimated to be 175 pg per cell each day, based on an assumed ribosomal speed (RS max) of 8,000 MAbs/cell each second synthesized (16). We calculated qPmax by assuming an antibody molecular weight (MW) of 150 kDa and using the formula qPmax = RS max × MW ÷ Avogadro’s Number. Currently reported cellspecific productivities are below that theoretical maximum (17), so we focused on evaluating metabolic pathways that could improve target protein production. We identified a critical pathway connecting glycolysis and TCA and screened a number of candidate chemicals for their effectiveness. Figure 6 shows how a key enzyme connecting the two central metabolic pathways was elevated in its activity with chemical C present. And Figure 7 demonstrates a significant increase in cell-specific productivity when 48 BioProcess International 10(1) January 2012 Cell Viability Figure 9: Cell viability profile improved with addition of chemical D (arrow marks time of addition). With addition Control Time chemical C was added to the production process. Example 4 — Volumetric Productivity Increase Based on Cell Viability Profile: One of the easiest ways to increase volumetric productivity of a cell culture is to extend the viability of productproducing cells already present. This strategy offers other benefits, particularly in regard to maintaining similar viral and impurity levels at harvest as well as comparability of product quality attributes. We evaluated a number of candidate chemicals for their ability to delay cell apoptosis. Figure 8 demonstrates that timed addition of chemical D reduced the onset of DNA laddering, a measure of the apoptotic state of cultured cells (18). Figure 9 illustrates how timed addition of chemical D to the process significantly improved the cell viability profile. We have found MPE to be an effective tool for solving many typical process challenges encountered during late-stage and postcommercial product phases of development. Acknowledgments The authors thank Abhinav Shukla, PhD (KBI Biopharma); Siddhartha Jain, PhD, and Christoph Joosten PhD (Novartis AG); Jeff Savard, PhD (Merck); and Nancy Mackin, PhD, Tara Stanko, and Momina Andrabi (BMS) for their support. References 1 Zhang M, et al. Rapid Development and Optimization of Cell Culture Media. BioPharm Int. 21, 2008: 60–68. 2 Lu C, et al. A T-flask Based Screening Platform for Evaluating and Identifying Plant Hydrolysates for a Fed-batch Cell Culture Process. Cytotechnol. 55, 2007: 15–29. 3 Stephanopoulos GN, Aristidou AA, Nielsen J. Metabolic Engineering: Principles and Methodologies, 1st Edition. Academic Press: San Diego, CA, 1998. 4 Etcheverry T, et al. Process for Controlling Sialylation of Proteins Produced by Mammalian Cell Culture. Patent WO/1996/039488. 5 Dorner AJ, et al. Increased Synthesis of Secreted Proteins Induces Expression of Glucose-Regulated Proteins in ButyrateTreated Chinese Hamster Ovary Cells. J. Biol. Chem. 264(34) 1989: 20602–20607. 6 McGrew JT. Cell Culture Performance with Vanadate. US PTO 6,974,681. 7 Arden N, et al. Chemical Caspase Inhibitors Enhance Cell Culture Viabilities and Protein Titer. Biotechnol. Prog. 23, 2008: 506– 511. 8 Allen M, et al. Identification of Novel Small Molecule Enhancers of Protein Production by Cultured Mammalian Cells. Biotechnol. Bioeng. 100, 2008: 1193–1204. 9 Balcarcel RR, Stephanopoulos G. Rapamycin Reduces Hybridoma Cell Death and Enhances Monoclonal Antibody Production. Biotechnol. Bioeng. 76, 2001: 1–10. Continued from page 25 10 Ling WL, et al. Improvement of Monoclonal Antibody Production in Hybridoma Cells by Dimethyl Sulfoxide. Biotechnol Prog. 19, 2003: 158–62. 11 Liu CH, Chen LH. Promotion of Recombinant Macrophage Colony Stimulating Factor Production by Dimethyl Sulfoxide Addition in Chinese Hamster Ovary Cells. J. Biosci. Bioeng. 103, 2007: 45–49. 12 Liu CC, Hwang S. Pentanoic Acid, a Novel Protein Synthesis Stimulant for Chinese Hamster Ovary (CHO) Cells. J. Biosci. Bioeng. 91, 2001: 71–75. 13 Jagadish MN, Carter BLA. Effects of Temperature and Nutritional Conditions on the Mitotic Cell Cycle of Saccaromyces Cerevisiae. J. Cell Sci. 31, 1978: 71–71. 14 Sharfstein S. Advances in Cell Culture Process Development: Tools and Techniques for Improving Cell Line Development and Process Optimization. Biotechnol. Prog. 24, 2008: 727–734. 15 Ozturk SS, Riley MR, Palsson BO. Effect of Ammonia and Lactate on Hybridoma Growth, Metabolism, and Antibody Production. Biotechnol. Bioeng. 39, 1992: 418– 431. 16 Savinell JM, Lee GM, Palsson BO. On the Orders of Magnitude of Epigenic Dynamics and Monocolonal Antibody Production. BioProcess Eng. 4, 1989: 231–234. 17 Wurm FM. Production of Recombinant Protein Therapeutics in Cultivated Mammalian Cells. Nat. Biotechnol. 22, 2004: 1393–1398. 18 Thrift J, et al. Chapter One: Characterization of Apoptosis in a CHO Cell Line Cultivated in Batch and Continuous Culture: Effect of Medium, Specific Perfusion Rate and Chemical Inhibitors. Animal Cell Technology Meets Genomics: ESACT 2005 Proceedings, Volume 2. Springer Netherlands, 2005: 125–128. • Corresponding author Bernhard M. Schilling, PhD, is head of biologics technology transfer, Susan Abu-Absi, PhD, is head of biologics upstream process characterization, and Patrick Thompson is a scientist in biologics upstream process characterization at Bristol-Myers Squibb Company, 6000 Thompson Road, East Syracuse, NY 13057; [email protected]. To order reprints of this article, contact Rhonda Brown ([email protected]) 1-800-382-0808. Download a low-resolution PDF online at www.bioprocessintl.com. Effective PMP System LEX System technology offers several advantages over other systems (Figure 4). It minimizes the risk of accidental genetic release of product or contamination of food sources. Although nontoxic and edible, Lemna is not commonly used as a food source. A further advantage is that no pesticides, herbicides, or antibiotics are used in Lemna cultures. In addition, the single-use aspect of this production system has also nearly eliminated cleaning validation activities from upstream processes. The current single-use process has handed off materials and product validation responsibility to vendors and manufacturers of disposable products. This has provided additional benefits of reduced work-force requirements and documentation burden. Using disposable aseptic connectors eliminates the need for classified areas in upstream processes. This reduces facility and operating costs and process complexity. Aseptic connectors provided for a process that can specify and achieve low contamination levels, well within process requirements. In receiving a contaminant-free feed stream, downstream processing benefits as well. LEX System technology has merged plant-based pharmaceutical protein production with single-use materials. The benefits and advantages to this approach have been demonstrated through investor support and FDA acknowledgement of successful clinical trials. A crucial synergy has manifested itself in the careful adaptation of single-use products to the Lemna-based PMP technology. References 1 Office of the Press Secretary, The White House. Fact Sheet: Obama Administration Takes Action to Reduce Prescription Drug Shortages in the US; 31 October 2011; www.whitehouse.gov/thepress-office/2011/10/31/fact-sheet-obamaadministration-takes-action-reduce-prescriptiondrug-sh. 2 Molecular Farming: Plant Bioreactors. Bio Pro, Baden-Württemberg; www.bio-pro.de/ magazin/thema/00178/index.html?lang=en. 3 Thomson JA. Seeds for the Future: The Impact of Genetically Modified Crops on the Environment. Cornell University Press, Ithaca, NY; 2006. 4 Gasdaska JR, Spencer D, Dickey L. Advantages of Therapeutic Protein Production in the Aquatic Plant Lemna. BioProcessing J March–April 2003: 49–56. 5 Cox KM, et al. Glycan Optimization of a Human Monoclonal Antibody in the Aquatic Plant Lemna minor. Nature Biotech. 24 2006: 1591–1597. 6 Dzublyk I, et al. Phase 2a Study to Evaluate the Safety and Tolerability and Antiviral Effect of Four Doses of a Novel, Controlled-Release Interferon alfa-2b (LocteronTM) Given Every Two Weeks for 12 Weeks in Treatment-Naïve Patients with Chronic Hepatitis C (Genotype 1). J. Hepatology 46, 2007: 4S:232A. 7 Lawitz E, et al. Early Viral Response of Controlled-Release Interferon alpha2b and Ribavirin vs. Pegylated Interferon alpha 2b and Ribavirin in Treatment-Naïve Genotype1 Hepatitis C: 12 Week Results (SELECT-2 Trial). J. Hepatology 52, 2010: S114. 8 Long WA, et al. Timing and Frequency of Depression During HCV-Treatment with Controlled Release INFa2b (CR2b) vs. Pegylated IFNa2b (PEG2b): Results from SELECT-2, a Randomized Open-Label 72-Week Comparison in 116 Treatment-Naïve Patients with Genotype 1 HCV. J. Hepatology 54, 2011: S181–182. 9 Cox KM. Production of Antibodies in Plants. An Z and Strohl W, Eds. Therapeutic Antibodies: From Theory to Practice. Wiley, Inc: New York, NY; September 2009. 10 Private communication between Biolex and regulatory authorities 11 Biolex internal documents. • Corresponding author Keith Everett is director of process design and research at Biolex Therapeutics, Inc, 158 Credle St., Pittsboro, NC 27312; 1-919-542-9901, ext. 2032; [email protected]. Lynn Dickey, PhD is vice president of research and technology development, John Parsons is research scientist of plant biology, Rachel Loranger is upstream research coordinator, and Vincent Wingate is associate director of upstream process development and plant biology, all at Biolex Therapeutics. To order reprints of this article, contact Rhonda Brown ([email protected]) 1-800-382-0808. Download a low-resolution PDF online at www.bioprocessintl.com. January 2012 10(1) BioProcess International 49