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GENETICALLY MODIFIED ORGANISMS FOR MEDICINES PRODUCTION Antonio Moreira University of Maryland Baltimore County III SYMPOSIUM SINDUSFARMA-IPS/FIP-ANVISA New frontiers in manufacturing technology, regulatory sciences and pharmaceutical quality system Brasilia August 5, 2014

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Presentation Outline Primer on genetic engineering and biotherapeutics Main genetically modified organisms currently used in biomanufacturing Biomanufacturing processes Examples of bioprocess development studies Status of the biotech industry for therapeutic products

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How Do We Deliver & Maintain Our Protein Coding Sequences? Plasmids: Autonomously replicating, circular DNA molecules Circular plasmids can be transfected into cells directly or plasmids can be linearized. Selections used include antibiotics, puromycin, hygromycin, neomycin, presence of DHFR gene (MTX r ) Drug level can be used to select for increased numbers, or copies of gene. 4

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Clark & Pazdernik. Biotechnology: Applying the Genetic Revolution. 2009.

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Biologics As Therapeutics Natural Products Blood Products - Transfusions Vaccines Purified Proteins from natural sources / tissues Recombinant Proteins Microbial Cell Culture Monoclonal Antibodies Combination Products Cell Therapy Bone Marrow Replacement / Reconstruction Stem Cell Therapy 6

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Figure 1. Total sales in the US and Europe of traditional pharmaceuticals (blue) and biopharmaceuticals (green) are shown by year for the past decade. Sales information was obtained from company annual reports and other publically available sources. Jones & Ecker. Pharmaceutical Processing, October 2013. 7

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BioPharm International, February 2013 Figure 2: List of FDA-approved antibody therapeutics. 8

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BioPharm International, February 2013 Figure 1: Primary mechanism of action of antibody-drug conjugates: targeted delivery of a potent cytotoxic agent to cause cell death. 9

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Bioprocesses Mirror the Complexity of Biological Products Classical Pharmaceutical Drugs Defined Structure and Characteristics Basics of biopharmaceuticals Complex chemistry and structures Proteins, nucleic acids, lipids, polysaccharides Cells Inherently unstable (until purified or formulated) Degradative enzymes co-produced Temperature, pH, concentration Bio-safety constraints - complexity and purity Process design - control for productivity Process design for GMP vs. flexibility 10

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NCE Versus Biopharm Comparison 11

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Emerton, Duncan. Supplement to BioProcess International. June 2013. Figure 1: Global biosimilar approvals, 2006-2012 (in Europe, unless otherwise indicated. 12

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Thayer, C&EN Houston. cen.acs.org. October 7, 2013. KEY TARGETS Developers are trying to create functional replicas of leading biologic drugs.SOURCES: Company data, Biotechnology Information Institute 13

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Biomanufacturing Biomanufacturing involves three key processes: Controlled growth of microorganisms, cells, tissues or organisms Conversion of simple raw materials or complex molecules to desired product Isolation & purification of the product from complex mixtures 14

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Major Production Systems Bacterial Cells E. coli Yeast Cells Saccharomyces cerevisiae Pichia pastoris Mammalian Cells Chinese Hamster Ovary CHO Baby Hamster Kidney BHK NS0 or Sp2/0 (Mouse Myeloma) 15

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Clark, D. & Pazdernik, N. Biotechnology; Applying the Genetic Revolution. 2009 Subcellular Structure of Escherichia coli: Scanning electron micrograph of E. coli. The rod-shaped bacteria are approximately 0.6 microns by 1-2 microns. Courtesy of Rocky Mountain Laboratories, NIAID, NIH. 16

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Yeast Cells/Fungi Pichia, Saccharomyces, Kluyveromyces, Aspergillus Pichia spp.Saccharomyces spp. Aspergilus spp. Major Protein Expression Platforms

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Clark, D. & Pazdernik, N. Biotechnology; Applying the Genetic Revolution. 2009 18

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Clark & Pazdernik. Biotechnology: Applying the Genetic Revolution. 2009.

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Swartz, AIChE Journal, January 2012. Vol 58, No. 1. Figure 1: Industrial cell-free biology. 20

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Carlson, ED, et al, Cell-Free Protein Synthesis: Applications Come of Age. Biotechnol Adv. (2011). Fig. 2. Cartoon comparison of in vivo recombinant DNA protein expression with cell-free protein synthesis (CFPS). CFPS systems provide a more rapid process/product development timeline. Example proteins shown include a virus-like particle (VLP), single-chain antibody variable fragment (scFv), and a membrane bound protein (MBP).

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Swartz, AIChE Journal, January 2012. Vol 58, No. 1.

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Mammalian Antibody Production Cell Culture Vial Thaw / Inoculum Expansion 20,000-Liter 5000-Liter 500-Liter 50-Liter HEAT COOL Media Pasteurizer Media Prep Upstream Processes 23

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rProtein A affinity chromatography Virus Inactivation Disc-Stack Centrifuge Depth Filter Transfer to Purification Suite Harvest from Production Bioreactor Mammalian Antibody Production Harvest 24

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Bulk Filtration (BDS) Intermediate Storage UF/DF Step Intermediate Storage Intermediate Storage Viral Filtration 20m 2 Ion (Cation) Exchange Chromatography Ion ( Anion) Exchange Chromatography- Hydrophobic Interaction Chromatography I.B.I CryoPreservation System API Mammalian Antibody Production Downstream Processing 25

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Stirred-tank Reactor Most common type Cylindrical tank Agitator motor, shaft & impellors Air/gas inlet & exhaust Sampling & harvest ports 26

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Example of a Commercial Bioreactor Sandoz cell culture manufacturing facility Located in Schaftenau, Austria Facility contains two 13,000 L bioreactors 27

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Supplement to Pharmaceutical Engineering, 2013, p.10-16 29

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Bioreactor Operating Modes Batch Fed-Batch Continuous Perfusion 30

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Table 1: Selected Perfusion Operations Issues: PRODUCTMANUFACTURER IL-12/23 Mab (Stelara)**Janssen/J&J TNF Mab (Simponi)Janssen/J&J Glucosidase alfa (Myozyme)**Genzyme/Sanofi Galactosidase alfa (Fabrazyme)**Genzyme/Sanofi Protein C (Xigris)Lonza for Eli Lilly Factor VIII (Kogenate-FS)**Bayer Interferon beta (Rebif)**Merck-Serono IL-2 receptor Mab (Simulect)Novartis TNF mAb (Remicade)**Janssen/J&J FSH (Gonal-F)Merck-Serono Galactosidase. beta (Cerezyme)**Genzyme/Sanofi Platelet Mab Fab (Reopro)Janssen/J&J **Annual sales over 500 million Source: 11 th Annual Report and Survey on Biomanufacturing Capacity and Production, April 2014 Continuous Bioprocessing and Perfusion. E. Langer. Pharmaceutical Processing, July/August 2014, pg. 13. 31

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Selected Continuous Bioprocessing Benefits Reduction in facility size, manufacturing footprint, etc. Significant costs savings, particularly investment in facilities Increases in flexibility No scale-up of bioprocesses Increased process robustness Less manual interactions Less bulk fluid input Less sensor insertions and other incursions into the process Increased automation PAT and upfront bioprocess design using QbD can be easier to implement Continuous Bioprocessing and Perfusion. E. Langer. Pharmaceutical Processing, July/August 2014, pg. 13. 32

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Typical Downstream Processes Cell Lysis/Disruption (if needed) Chromatographic purification Product Concentration Sterile Filtration Formulation Fill & Finish 33

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Purification Sequence of steps, generally 3 to 4 Selective removal of contaminants Isolates molecules by physical or chemical characteristics Volume reduced at each step, ideally Initial coarse cuts Polishing steps 34

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Production Scale Units

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Purity Goals Residual host & contaminant proteins ppm level Nucleic acids 100 pg/dose Viruses Below detection limits Endotoxins < 5EU/kg/hr for parenteral use;