Cultivos Masivos de Células Animales (Upstream(

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1 Conceptos y Técnicas T de Biotecnología a I do cuatrimestre FBMC-FCEN FCEN-UBA Cultivos Masivos de Células Animales (Upstream( Upstream) Unidad de Transferencia Genética Instituto de Oncología Ángel H. Roffo Universidad de Buenos Aires

2 Bioprocessing

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5 Control de los parámetros p H F O 2 N O T ph 2 CO 2 Aire 2 T RPM Lectura Control

6 Optimización de los parámetros Temperatura ph Composición del medio Agitación Aireación Asepsia

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20 Microcarriers: A: DEAE-Dexttran; B & C: DEAE-Cellulose0

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22 Spin filter reactor. Combined perfusion (p) and aeration (A) spin filter of 65 um mesh stained steel used for high-density microcarrier culture.

23 Example of a 1000 L Bioreactor with an external spin filter

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29 These tanks are used for the holding of material from the bioreactors prior to product capture and initial chromatography.

30 Mammalian Antibody Production Cell Culture Media Prep HEAT COOL Media Pasteurizer Vial Thaw / Inoculum Expansion 50-Liter 500-Liter 5000-Liter 20,000-Liter

31 Biopharmaceuticals Over 140 on the market; over 400 in clinical trials Biopharmaceuticals include: Proteins (made in bacterial, fungal or mammalian cell culture) erythropoietin (EPO) insulin interferon (Intron A) granulocyte-colony stimulating factor (G-CSF) human growth hormone (HGH, human somatotropin) tissue plasminogenactivator (tpa) Monoclonal antibodies (made in mammalian cell culture) Vaccines live and inactivated viruses and bacteria subunit vaccines recombinant vaccines Gene Therapy Products (viral and non-viral)

32 FDA-Approved Antibodies Product Manufacturer Launch Indication OKT3 J&J 1986 Transplant rejection Murine ReoPro Eli Lilly 1994 Cardiovascular disease Chimeric Rituxan Idec 1997 Non-Hodgkin s lymphoma Chimeric Zenapax Roche 1997 Organ rejection Humanized Simulect Novartis 1998 Organ rejection Chimeric Remicade Centocor 1998 Rheumatoid arthritis Chimeric Herceptin Genentech 1998 Breast cancer Humanized Synagis MedImmune 1998 Respiratory syncytial virus Humanized Enbrel Immunex 1998 Rheumatoid arthritis Human Mylotarg Wyeth 2000 Leukemia Humanized Campath Millennium 2001 Lymphocytic leukaemia Humanized Zevalin Idec 2002 Non-Hodgkin s lymphoma Murine Humira Abbot 2002 Rheumatoid arthritis Human Xolair Genentech 2003 Asthma Humanized Bexxar Corixa/GSK 2003 Non-Hodgkin s lymphoma Murine Raptiva Xoma/Genen Psoriasis Humanized Erbitux Imclone 2004 Cancer (colorectal) Chimeric Avastin Genen Cancer (colon or rectum) Humanized

33 Recombinant Protein Production (including MAbs) Transfection Cell culture Purification Formulation/ Filling + Cells and plasmid Cell line Drug substance (crude) Drug substance (pure) Drug product - (sterile)

34 Bioprocess Engineering in Recombinant Protein Production Transfection Cell culture Purification Formulation/ Filling + Cells and plasmid Cell line Drug substance (crude) Drug substance (pure) Drug product - (sterile) Cell line manufacture Medium development Bioreactor process development & scale-up Downstream purification Analytical characterization

35 BIOPROCESS ENGINEERING Vaccines organism itself: attenuated or inactivated purified antigen recombinant antigen

36 Bioprocess Development Biological Assay Development and Support Cell Line/ Viral Vector/ Construction Cell/Virus Culture Development/ Media Optimization Purification Process Dev. Formulation Design/ Drug Delivery Design Facility/Equipment Design Technology Transfer Process Validation Pilot-Scale cgmp Production Commercial-Scale Production

37 Block Flow Diagram of a typical Production Process Innoculu m Seed Fermentati on Production Fermentati on Harvest UPSTREAM DOWN- STREAM Ultrafiltrati on 1 Chrom. 1 1 Ultrafiltrati on 2 Chrom. 2 Viral Filtration VIRAL NON-VIRAL Chrom. 3 Ultrafiltrati on 3 Final Formulation/ Sterile Filtration Sterile Fill

38 Mammalian Antibody Production Cell Culture Media Prep HEAT COOL Media Pasteurizer Vial Thaw / Inoculum Expansion 50-Liter 500-Liter 5000-Liter 20,000-Liter

39 Mammalian Process Flow- Upstream Diagram Transfer to Purification Suite Virus Inactivation rprotein A Dia: 1 meter CV: 236 L Bed: 30cm Depth Filter 75m 2 Disc-Stack Centrifuge

40 Mammalian Antibody Production - Downstream Processing Cation Exchange Dia: 1.6m CV: 600L Bed: 30cm Intermediate Storage Anion Exchange Dia: 1.6m CV: 600L Bed:30cm Intermediate Storage Viral Filtration 20m 2 I.B.I CryoPreservation System Bulk Filtration (BDS) 3m 2 UF/DF Step 80m 2 Intermediate Storage Hydrophobic Interaction Dia: 1.6m CV: 600L Bed: 30cm

41 Validation in Biomanufacturing A central concept in quality is that quality can not be tested for. Quality must be designed and built into the production process. Requires careful attention to raw material specifications, in process material specifications, and final product specifications.

42 Validation in Biomanufacturing Validating the performance of unit operations, analytical methods, and critical process points (sterilization, viral inactivation, cleaning procedures) is essential in insuring that the process generates a quality product.

43 Validation in Biomanufacturing Validation does not replace testing, but it does reduce the testing burden for raw materials, in-process materials, and final product

44 Validation in Biomanufacturing Validation itself is a process that evolves with the product. Validation requirements for production of pre-clinical material much less stringent then for phase III clinical material. Critical operations: raw materials, analytical methods, viral clearance, sterilization, cleaning.

45 An example of a facility / process validation Remicade (infliximab) is a chimeric mab * directed against TNF-α. Approved in 1998 (US) and 1999 (EU) to treat Crohn s disease, and RA. Produced by Centocor, Inc. in Malvern, PA * Contains mouse variable domains and human constant domains (IgG1)

46 Antibodies Proteins 2 heavy Chains 2 Light Chains Disulfide Bonds Variable region Recognizes antigen Constant region Effecter function Classes & subclasses Ig G class

47 Remicade Production

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