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GENETICALLY MODIFIED ORGANISMS FOR MEDICINES PRODUCTION

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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GENETICALLY MODIFIED ORGANISMS FOR MEDICINES PRODUCTION

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  1. GENETICALLY MODIFIED ORGANISMSFORMEDICINES PRODUCTION Antonio Moreira University of Maryland Baltimore County III SYMPOSIUMSINDUSFARMA-IPS/FIP-ANVISA New frontiers in manufacturing technology, regulatory sciences and pharmaceutical quality system Brasilia August 5, 2014

  2. 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

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

  5. 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

  6. Jones & Ecker. Pharmaceutical Processing, October 2013. 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.

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

  8. 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. 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

  10. NCE Versus Biopharm Comparison 11

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

  12. 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. 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

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

  15. 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. Clark, D. & Pazdernik, N. Biotechnology; Applying the Genetic Revolution. 2009 16

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

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

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

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

  20. 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). Carlson, ED, et al, Cell-Free Protein Synthesis: Applications Come of Age. Biotechnol Adv. (2011).

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

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

  23. Depth Filter Disc-StackCentrifuge Mammalian Antibody Production – Harvest Harvest from Production Bioreactor Transfer to Purification Suite Virus Inactivation rProtein A affinity chromatography

  24. Viral Filtration20m2 Ion (Cation) Exchange Chromatography Ion (Anion) Exchange Chromatography- Intermediate Storage Intermediate Storage Intermediate Storage Hydrophobic Interaction Chromatography UF/DF Step Bulk Filtration(BDS) I.B.ICryoPreservationSystem Mammalian Antibody Production – Downstream Processing API

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

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

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

  28. Bioreactor Operating Modes • Batch • Fed-Batch • Continuous • Perfusion 30

  29. Continuous Bioprocessing and Perfusion. E. Langer. Pharmaceutical Processing, July/August 2014, pg. 13. 31

  30. 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

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

  32. 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

  33. Production Scale Units

  34. Purity Goals • Residual host & contaminant proteins • ppm level • Nucleic acids • 100 pg/dose • Viruses • Below detection limits • Endotoxins • < 5EU/kg/hr for parenteral use; <0.25 EU/kg/hr for intrathecal use for drug products w/o a compendial limit • Microorganisms • None

  35. Regulation of New Technology Protein Production Issues • Expression Technology • Cell line selection and optimization • New production systems to increase yield • Bacteria – newer strains 10 g/L • Yeast – over 5 g/L • Mammalian Cells – achieving 2-5 g/L • Recovery & purification • Product aggregation • Solubility • Stability   • Characterization of complex structures • Glycosylation  • Pegylation • Process and Facility design • Automation • Cleaning 37

  36. Product Realization Design Space Determining What and How the Process Affects Product Characteristics • Process Control Strategy • Testing • Quality Systems • Past Experience • Preclinical Studies • Clinical Studies Ensuring Patient Receives the Expected Product Identification of Key Product Characteristics • Past Experience • Process Development • Product Characterization Target Product Profile Quality Assurance 38

  37. Discovery to Therapeutic Delivery Pathway and the Process Information Gap Phase Issues Discovery Product Characterization Non-Instrumented Fully Instrumented Process optimization Define Design Space Scale Production Quality Control Fundamental question: Can we predict manufacturing behavior of cell lines as early as in non-instrumented devices? Therapeutic delivery Discovery phase is not integrated with following phases. It lacks of the “know-why” process perspective. Based on Kirouac and Zandstra 2008, Cell Stem Cell 3:369-381

  38. PSDs Applications in Upstream Bioprocess Development and the Process Information Gap SCALE-UP STRAIN/CLONE SCREENING EARLY R&D STUDIES PROCESS OPTIMIZATION PRODUCTION Non-instrumented Fully instrumented Static + Stir Stir MCB vial Shake Roller Wide variety of PSDs. SCALE-DOWN

  39. Application of Process Analytical Technology for Extended Cell • Passaging: A Proof-of-Concept Revealing Study 3-Day passage scheme MCB vial Pi+1,j Expression system: Non-adherent SP2/0-based myeloma/ mouse (2055.5) Protein: IgG3 antibody specific for the Nisseria meningitides capsular-polysaccharide (MCPS). Media: CD Hybridoma GTTM 20mL CO2 Incubator 5% CO2 37OC Pi+3,j+1 P1,1, P2,1, P3,1 P4,2, P5,2, P6,2 Pi+n,j+n’ i: Number of passages j: Number of T-flasks i+n: Total number of passages j+n’: Total number of T-flasks Vallejos et al. (2010) “Dissolved oxygen and pH profile evolution after cryovial thaw and repeated cell passaging in a T-75 flask” Biotechnol & Bioeng. 105(6):1040-1047

  40. Application of Process Analytical Technology for Extended Cell • Passaging: A Proof-of-Concept Revealing Study • Cells are exposed periodically to sub-optimal DO levels (0%). • What happened at passages 18-20 and passages 27-29? Vallejos et al. (2010) “Dissolved oxygen and pH profile evolution after cryovial thaw and repeated cell passaging in a T-75 flask” Biotechnol & Bioeng. 105(6):1040-1047

  41. Application of Process Analytical Technology for Extended Cell • Passaging: A Proof-of-Concept Revealing Study Improving cell passaging techniques in T-flask at low cost! Vallejos et al. (2012) Biotechnol & Bioeng. 109 (9):2295-2305 • Sub-optimal DO and pH levels are avoided in rocking T-flasks. Vallejos et al. (2012) “Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools” Biotechnol & Bioeng. 109(9):2295-2305

  42. A Novel Scale-Down Paradigm for the Wave Bioreactor Vallejos et al. (2012) “Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools” Biotechnol & Bioeng. 109(9):2295-2305 44

  43. A Novel Scale-Down Paradigm for the Wave Bioreactor • At matched kLa both systems (rocking T-flasks and wave bioreactor) perform similar except for specific productivity Vallejos et al. (2012) “Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools” Biotechnol & Bioeng. 109(9):2295-2305 45

  44. Comparability Study 5L Vs. MB • Same seed in both systems; Passage 6-10 • DO 30% • pH 7.2, Control w/o base addition • Matched Kla • 3 replicates 46

  45. Difference in the Antibody Product Titer The antibody titer in 5L was about 50% higher than that in the minibioreactor 47

  46. Glutamine Profile in 5L Vs. Minibioreactor • Glutamine degradation was not significant • L- Glutamine levels reach zero when minibioreactors reach their stationary /peak cell density phase, while the 5L has sufficient amount of glutamine 48

  47. Glutamine Supplement Experiment Glut MB control Glut MB Supplemented Titer MB Control Titer MB Supplemented Titer 5L • Protein titers were similar to 5L when glutamine was supplemented • Identical amount of starting glutamine concentration Difference in the glutamine consumption 49

  48. Comparability Study WithCO2Monitoring ( Source: Ge et al., 2005) 50

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