Cell Culture Technology for Pharmaceutical and Cellular Therapies Sadettin S. Ozturk, Ph.D. Centocor Inc. 200 Great Valley Parkway, Malvern, PA 19355 Outline 1. Introduction to Monoclonal Antibodies 2. Production of Monoclonal Antibodies 3. Introduction of Centocor 4. Autoimmune disorders 5. Development of Monoclonal Antibody based Pharmaceutical Therapies 6. Case study: Fed-batch Process development for Monoclonal Antibodies 1
Immune Response as First Line of Defense When a pathogen (bacteria, foreign proteins, virus,.) enters the blood stream it is recognized, attacked, and eliminated by a sophisticated defense mechanism: Body s Immune Response Immune System: The Body's First Line of Defense Lymphatic vessels form a circulatory system that operates in close partnership with blood circulation. Organs and tissues of the immune system dot the body in a protective network of barriers to infection. 2
The antibody a.k.a Immunoglobulin Antibodies are produced by B-cells as part of immune response. Each antibody is specific to a specific antigen -The Variable region is different for each antibody and determines its specificity. -The Constant region is identical for each type of antibody and allows recognition by your immune cells. Evolution of Antibody Technology: Development of Antibody Based Medicines Utilization of antibodies as medicines took some time: B-cells cannot be expanded in vitro for practical purposes It is difficult to find out which B-cell makes a specific antibody Kohler and Milstein discovered Hybridoma Technology and cloning in 1975: A revolution in antibody technology Hybridoma cells (a fusion of B-cell and myeloma cell) can be expanded indefinitely Utilization of cloning techniques allows to isolate cells that make a specific antibody: These antibodies are called Monoclonal Antibodies (Mab) Expansion of hybridoma cells in vivo (mouse) or in vitro (bioreactors) allowed the development of first MAbs Over the last 30 years antibody technology was further developed Engineering of antibodies to make them more human To use cell lines other than hybridoma cells (CHO, NS0, etc) The use of antibody fragments and fusion proteins The use of antibodies for targeted drug delivery 3
Engineering of Antibodies Fab F c Murine IgG Chimeric IgG Humanized IgG Humanization of antibody minimizes/eliminates immune reaction when injected to the patients Fully Human IgG Current Products in Development 4
Monoclonal Antibodies as Medicines There are 18 approved antibody treatments in the market for: Autoimmune disorders Cancer Asthma Organ rejection Sales of antibodies is expected to be $13 Billion in 2005 There are 500 new antibody products in development There are 75 new antibodies in clinical trials Sales of antibodies is expected to be $26 Billion in 2010 Some of the indications require as high as 2,000 kg/year product These antibodies are produced in large (20,000L) bioreactors Therapeutic Antibodies Approved to Date * Nature Biotechnology, Sept. 2005, 23(9), p.1075 5
Production of Monoclonal Antibodies Seed Bioreactor 50L Production Bioreactors 300L, 1500L, 5000L, 20,000L Monoclonal Antibody Production Process Bioreactor and Product Capture Inoculum Bioreactor Production Bioreactor 6
Monoclonal Antibody Production Process Monoclonal Antibody Production Process Shipment to Fill and Finish Site 7
Production Bioreactors: Continuous Perfusion Operation Cells are retained in the bioreactor by physical means Cells grow, stay in the bioreactor, and produce proteins Media is added and harvest is collected continuously Can be operated for months Usually compact bioreactors (1000L) Production Bioreactors: Batch Operation Inoculate with media and cells Cells grow and produce proteins All of the contents are harvested after typically 2 weeks Usually very large bioreactors (20,000L) 8
Purification of Monoclonal Antibodies using Column Chromatography Concentration and Diafiltration of Monoclonal Antibodies 9
Centocor : An Antibody Company 10
Centocor Timelines Merger with Centocor Founded Leiden Mfg. 1982 Plant Opens 1993 Launched ReoPro 1998 1999 REMICADE Launched in ERA & AS 2005 1979 IPO Raised 1987 Marketing $21 Million; Alliance with 1995 First Diag. Product Lilly for ReoPro Approved by FDA RETAVASE Approved; REMICADE Launched in Crohn s 1999 REMICADE Launched in Rheumatoid Arthritis 2004 REMICADE launches in psoriasis (OUS), ulcerative colitis (U.S.) and PsA (U.S.) In the past five years, sales have grown from $500 million to over $3 billion. Centocor Products Launched 1998 Approved in over 80 countries World Wide 2004 WW sales $2.63 Billion Indications: RA / CD / AS / PsA/UC Launched 1995 Approved in over 50 countries World Wide 2004 WW sales $363Million Indications: PCI 11
U.S. Crohn s Disease Rheumatoid Arthritis signs and symptoms Rheumatoid Arthritis structural damage REMICADE Approvals Rheumatoid Arthritis physical function in failed methotrexate patients Crohn s Disease luminal CD Crohn s Disease fistulizing Rheumatoid Arthritis signs & symptoms of RA, inhibiting x-ray diseases progression, and improving physical functioning in patients not previously treated with methotrexate Ankylosing Spondylitis Psoriatic Arthritis Ulcerative Colitis 1998 1999 2000 2001 2002 2003 2004 2005 2006 EU Crohn s Disease fistulizing Rheumatoid Arthritis structural damage Rheumatoid Arthritis physical function in failed methotrexate patients Crohn s Disease luminal CD Ankylosing Spondylitis Crohn s Disease fistulizing Psoriatic Arthritis Psoriasis (OUS) Number of Patients Treated Worldwide With REMICADE 12
Autoimmune disorders: What Are They? Disorders caused by an immune response against the body's own tissues. Immune system disorders occur when the immune response is inappropriate, excessive, or lacking. Rheumatoid arthritis Crohn s Disease Psoriasis Multiple sclerosis (MS) Systemic lupus erythematosus Autoimmune disorders: Rheumatoid Arthritis inflammation begins in the tissue lining your joints and then spreads to the whole joint (hand joints are the most common site, but it can affect most joints in the body) muscle pain deformed joints Weakness Fatigue loss of appetite weight loss becoming confined to bed in severe cases 13
Autoimmune disorders: Crohn's Disease Chronic autoimmune disease where immune cells attack any part of the gastrointestinal tract The lining of the intestine may ulcerate and form channels of infection, called fistulas Ulcerative colitis is a similar inflammation of the colon, or large intestine Autoimmune disorders: Psoriasis Immune-mediated, genetic disease manifesting in the skin and/or the joints Psoriasis and psoriatic arthritis affect more than 4.5 million people in the United States A person's quality of life including emotional health can be seriously jeopardized 14
Autoimmune disorders: Multiple sclerosis (MS) weakness and trouble with coordination, balance, speaking, and walking paralysis Tremors numbness and tingling feeling in arms, legs, hands, and feet Lupus swelling and damage to the joints, skin, kidneys, heart, lungs, blood vessels, and brain butterfly rash across the nose and cheeks rashes on other parts of the body painful and swollen joints sensitivity to the sun Autoimmune disorders: A lot of things can go wrong in the immune system to result in autoimmune disorders T cell proliferation and interferon production Differentiation of T-cells Cytokine production Cytokine, receptor binding B-cell differentiation Antibody production Migration of cells to the tissue Antibodies can be used to intercept or block these events 15
The use of antibody based treatment for Psoriasis Psoriasis activity before and after treatment with a specific antibody: 0.1 mg/kg dose (1 week post-treatment [baseline not available] and 16 weeks post-treatment); 1.0 mg/kg dose (baseline and 16 weeks post-treatment) J Invest Dermatol. 2004 Dec;123(6):1037-44. Development of Antibodies for Pharmaceutical Therapies 16
Antibody Development and Commercialization Process Early Development Clinical Development Late Development Submit BLA Target Research Preclinical Studies Phase I / II Clinical Trials Phase III Clinical Trials Reg. Filing Review Approval Drug Development Cell Line Selection and Purif./Form. Dev. Clinical Manufacturing Process Validation Launch Preparation Process Development Tech. Transfer Manufacturing Investment Decision Assay Validation Year -3 Year 1 Year 2 Year 3 Year 4 Year 5 Steps in Drug Development Pharmaceutical Development Discovery Research Cell Line Development Identify Molecular Target Clinical Initiate Clinical Trials Create New Molecular Entity Media Development Bioreactor Process Development Develop Purification Process Make Clinical Supplies Develop Formulation 17
Cell Line Development Clone product gene cdna Develop expression vector -Gene -Promoter -Enhancer -Selective marker Insert into expression plasmids Host Cell Transfection Evaluate in Bioreactors Master Cell bank (MCB) Master working Cell bank (MWCB) Development Cell bank (DCB) Amplify, Clone, Select, Amplify, Clone, Select, Clone selection for high producing cells Media Development Cell culture medium contains: Salts, trace elements, glucose, amino acids, other nutrients, vitamins, buffers, etc. Early media formulations used serum or other animal derived proteins (albumin) Issues related to safety (BSE), availability, and cost became driving force to eliminate serum and to develop animal product free (APF) (safer and economical) Today chemically defined medium (CDM) is a reality for many cell culture based processes (consistent and traceable) Most of the companies use specially formulated in-house proprietary media formulations for their processes (independence) 18
Bioreactor Process Development Advances in biochemical engineering made it possible to grow animal cells in conventional bioreactors (no need for specialized systems) Today stirred-tank based bioreactors are in operation at sizes up to 20,000L Batch, fed-batch, and perfusion process options are in use for commercial production It is possible to get 5 g/l titers in fedbatch and about 50 MM cells/ml in perfusion Bioreactor process development involves Optimization of culture environment (ph, temperature, DO, CO2) Optimization of media exchange rates Development of feeding solutions and feeding strategies Process Development Options Process/Media Perfusion Commercial Batch Fed-batch Proprietary Selection System gpt/mhx GS/MSX DHFR/MTX Neo/G418 Therapeutic Monoclonal Antibody Sp2/0 CHO-dhfr - Host cell NS0 CHOK1SV 19
Case Study: Development of a Fedbatch Process for Monoclonal Antibody Production Cell line: CHOK1SV Glutamine Synthetase selection system Animal product free medium Fed-batch process in stirred tank bioreactors Optimized ph, temperature Optimized feeding schedule Process Scale-up Consistency Cell Line Development: Introduction/Selection System Glutamine Synthetase (GS) catalyzes the biosynthesis of glutamine from glutamate and ammonia, providing the only pathway for L-glutamine formation in the cell ATP Glutamate + NH 3 GS ADP + P i L-glutamine MSX MSX = L-Methionine Sulfoximine In the absence of glutamine, the GS enzyme is essential for cell survival 20
Cell Line Development: Schematic Overview of Plasmid Construction hcmv Amplification by PCR V H mrna HC Clone CH1 CH2 CH3 hcmv GS LC cdna V L C k - Start with a research cell line (expressing < 20 mg/l) - Isolate RNA, reverse transcribe to generate cdna - Use sequence information from genomic constructs to design PCR primers to isolate specific HC and LC cdnas - Clone cdnas into Lonza GS vectors, pee 6.4 and pee 12.4 - Construct a GS double-gene plasmid hcmv-ie promoter and intron CNTO X LC Kozak sequence GS cdna SV40 promoter SV40 poly A Not I GS CNTO X Double Gene 11479 bp Pvu I hcmv-ie promoter and intron Kozak sequence SV40 poly A Sal I CNTO X HC b-lactamase (Amp resistance) Cell Line Development: Process of Developing High Producing GS-CHO Cell Lines STATIC CULTURE 96w plates 24w plates Transfections 3-6 weeks 200-300 transfectants 1 week Rank order clones by 96w single-point ELISA 60-100 transfectants Rank order clones by 2 weeks 24w Neph. (overgrow) 3-10 parental cell lines 8 weeks 3 weeks 6 weeks 12-16 weeks SUSPENSION (shake flask culture) Adapt to CD-CHO 3-10 parental cell lines Perform shake flask 1-3 parental cell lines 1-3 parental cell lines growth profiles Prepare and test DCBs Bioreactor process development SUBCLONES 3 weeks 3 weeks 8 weeks Subclone 30-100 subclone cell lines 3-10 subclone cell lines Rank 24w (Neph.) Adapt to CD-CHO 3-10 subclone cell lines 3 weeks Perform shake flask growth profiles ~ 4.5 months ~ 6 months 21
Cell Line Development: Clone Selection Immuno-precipation method for rapid selection of high expression/ secretion clones: Patent: WO 2005 / 020924 A2 (Publication date 10 March 2005) Automated Halo-Colony Picking ClonePix interior HEPA filtration CCD camera 1µm encoders Wash and sterilise Stacker for microplates Holder for 5 Culture dishes 22
Cell Line Development: Transfection and Colony Screening Rank Order Clones: 96w ELISA / 24w Nephelometry Clone # 96w ELISA Titer 24w Neph Titer 3 21 26.6 mg/l 76.0 mg/l 4 23 20.3 mg/l 52.9 mg/l 6 36 18.7 mg/l 61.8 mg/l 16 113 37.1 mg/l 67.5 mg/l 20 123 25.3 mg/l 93.6 mg/l 21 127 4.1 mg/l 59.1 mg/l 22 134 25.3 mg/l 79.7 mg/l Cell Line Development: RESULTS - Expand in 24w, T-flasks, and cryopreserve - Adapt highest expressing cell lines to APF medium (CD-CHO) - Adapt to suspension culture in shake flasks - Perform 10-passage stability study - Perform growth profiles in shake flasks in APF medium - Transfer cell line(s) to bioreactor process development group VCD vs. Days Antibody Titer (mg/l) Specific Productivity y = 18.055x R 2 = 0.9474 y = 17.049x R 2 = 0.9445 400 300 200 100 0 0 5 10 15 20 25 Integral of Viable Cells (E6/mL * days) VCD (x 10^6 cells/ml) Antibody Titer (mg/l) 5 4 3 2 1 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 400 300 200 100 Days Titer Accumulation vs. Days 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Days 23
Fed-batch Process Development: VCC Temperature and ph Optimization in Batch C1180A Batch Cultures Temp and ph DOE 4.500 4.000 J006 36.5 C 7.0 H010 36.5 C 7.0 H006 35 C 7.2 H008 35 C 7.2 3.500 VCC 3.000 2.500 2.000 1.500 1.000 H009 38 C 7.2 0.500 J003 38 C 7.2 0.000 0 2 4 6 8 10 12 14 16 18 20 Day H005 35 C 6.8 J004 35 C 6.8 H007 38 C 6.8 J005 38 C 6.8 H005 H006 H007 H008 H009 H010 J003 J004 J005 J006 % Viability Fed-batch Process Development: Temperature and ph C1180A Batch Optimization Temp in and Batch ph DOE Cultures % Viable 120 100 80 60 40 20 H005 35 C 6.8 H009 38 C 7.2 H007 38 C 6.8 H006 35 C 7.2 H010 36.5 C 7.0 J004 35 C 6.8 J003 38 C 7.2 J005 38 C 6.8 H008 35 C 7.2 J006 36.5 C 7.0 H005 H006 H007 H008 H009 H010 J003 J004 J005 J006 0 0 5 10 15 20 25 Day 24
C1180A Fed-batch Process Development: Temperature and ph Temp and ph DOE Optimization Titer in Comparison Batch Cultures 600.00 500.00 400.00 300.00 200.00 100.00 H010 36.5 C 7.0 H005 35 C 6.8 H007 38 C 6.8 H006 35 C 7.2 H008 35 C 7.2 H009 38 C 7.2 J006 36.5C 7.0 J004 35 C 6.8 J005 38 C 6.8 J003 38 C 7.2 Titer H005 Titer H006 Titer H007 Titer H008 Titer H009 Titer H010 Titer J003 Titer J004 Titer J005 Titer J006 0.00 0 2 4 6 8 10 12 14 16 18 20 Day Fed-Batch Process Development: Feeding Strategies GS-CHO Cell line producing a fully human antibody Animal Product Free Medium ph, DO, and temperature set-points from batch optimization study Feeding solutions include Glucose, plant hydrolysate, MEM, NEAM, Vitamins, Specially formulated cocktails Feeding strategies include daily additions of predetermined amounts to the bioreactor 25
9.00 Fed-Batch Process Development: Feeding Strategies CD CHO Fed Batch Viable Cell Density Comparison 8.00 7.00 Glu, BRX, Nucleosides, PHyd BRX=MEM+NEM+Vitamins PHyd=Plant Hydrosylate Viable Cell Density (x10e6 cells/ml) 6.00 5.00 4.00 3.00 2.00 Glu, PHyd MEM, NEAM, GS Glu, PHyd MEM Glu, PHyd BioGro 1.00 Glu, PHyd Batch 0.00 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Day M04E001 M04E002 M04E003 M04E004 MO4E007 M04E008 M04D014 M04D015 M04F038 M04F039 M04E031 M04F032 1600 Fed-Batch Process Development: Feeding Strategies CD CHO Fed Batch Antibody Comparison 1400 Glu, BRX, Nucleosides, PHyd Antibody (mg/l) 1200 1000 800 600 Glu, Phyd, MEM, NEAM, GS Glu, Phyd, MEM Glu, Phyd, BioGro 400 Glu, PHyd 200 Batch BRX=MEM+NEM+Vitamins PHyd=Plant Hydrosylate 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Day E001 E002 E003 E004 E007 E008 D014 D015 M04F038 M04F039 M04E031 M04F032 26
Fed-Batch Process Development: Results 8 Maximum Viable cells, MM/mL 7 6 5 4 3 2 1 0 Batch Glu, soy Glu,soy,MEM Glu, soy, BioGro Glu, soy, MEM, NEM, GS Supp Glu, BRX, soy, nucleosides Fed-Batch Process Development: Results 1600 1400 1200 IgG, mg/l 1000 800 600 400 200 0 Batch Glu, soy Glu,soy,MEM Glu, soy, BioGro Glu, soy, MEM, NEM, GS Supp Glu, BRX, soy, nucleosides 27
In-process Testing : Agilent 2100 Bioanalyzer Non-reduced Reduced Process Scale-up and Commercialization 28
Process Scale-up and Consistency 10 9 8 VCC (x10e6 cells/ml) 7 6 5 4 3 2 1 0 0 2 4 6 8 10 12 14 Days Process Scale-up and Consistency 2500 2000 Titer (mg/l) 1500 1000 500 0 0 2 4 6 8 10 12 14 Days 29
Process Consistency: SDS-PAGE Reduced Non-Reduced Process Consistency: IEF 8.25 8.10 7.89 7.74 30
Process Consistency: Tryptic Peptide Maps Ref Std Batch-1 Batch-2 Batch-3 Conclusions 1. Monoclonal Antibodies evolved over the years to become an essential part of biotechnology 2. Monoclonal Antibodies can be used as an effective therapy for immune disorders 3. There are several processing options for the manufacture of Monoclonal Antibodies. The final choice may depend on a variety of reasons 4. Development and manufacturing of Monoclonal Antibodies require extensive optimization, consistency, and comparability studies 5. It can be tedious, frustrating, costly, and very risky, but making a drug that can help people s life is worth it. 31