Emerging Trends in Plasma-free Manufacturing of Recombinant Protein Therapeutics Expressed in Mammalian Cells Yiben Wang 11/16/11 Leopold Grillberger, Thomas R. Kreil, Sonia Nasr, and Manfred Reiter

Recombinant Therapeutic Proteins: -Microorganisms -Plant cell cultures -Insect cell lines -Mammalian cell lines -Transgenic animals Over 165 biopharmaceutical products – globally -Majority are proteins

Recombinant protein benefit – a platform for developing more advanced products: -Enhanced safety -Lower immunogenicity -Increased half-life -Improved bioavailability Production: -Established microbial expression systems using bacteria or yeast. Problem: -Unable to perform necessary modifications (glycosylation) – needed for large, complex proteins.

Mammanlian cells: -Used for large-scale production of therapeutic proteins -Post-translational modifications -Proteins – natural form % of all recombinant therapeutic proteins – mammalian cells, Chinese hamster ovary (CHO). CHO: -Ease of manipulation -Proven safety profile in humans -Similar glycosylation patterns Alternative, non-mammalian cell system: -Advances in modulating the glycosylation patterns in certain yeast strains -P. pastoris

Hemophilia A: -X-linked coagulation disorder -Mutations in the coagulation factor VIII (FVIII) gene. FVIII replacement therapy: -Plasma-derived purified FVIII concentrates (1970s) -Recombinant FVIII concentrates (1992) -Animal and human plasma free recombinant FVIII (2003) -Eliminated the risk of blood-borne infections during therapy

Additives: -Derived from human or animal sources: -Blood -Milk -Bones -Hides -Tendons -Hair -Skin -Pancreas

Serum: -Production of therapeutic proteins on a commercial scale -Main threat – serum-derived proteins -Risk of pathogen transmission -Viral outbreaks -Mad cow disease -High protein content and variability -Increase in immunogenicity

Threats of infectious diseases: -Risk of using human or animal component -Serum: albumin and gelatin – stabilizers in formation

Risks: Amplified: -Multiple steps in manufacturing -Repeated administrations Virus transmission: -Blood-borne infectious agents -long-lasting, silent carrier states – no noticeable symptoms; highly infectious blood and plasma -Solvent/detergent and nanofiltration – not 100% efficient

Transmissible spongiform encephalpathies (TSEs): -Prions – self-replicating infectious proteins -Highly resistant -Physical/Chemical inactivation -Virus-removal methods can’t target -No detection method in plasma donors – early stages/pre-symptomatic of infection -Bovine spongiform encephalpathies (BSE) -Variant Creutxfeldt-Jacob disease (vCJD)

Plasma-free production process: 1.Development 1.Selection of a cell line that can yield high protein output in serum-free medium 2.Upstream processing 1.Production of protein that is stable in animal-free cell culture medium 3.Downstream processing 1.Purification without the addition of other plasma proteins 4.Final formulation 1.Formulation without animal-derived additives 5.Testing 1.Assure safety of product

Response From Regulatory Agencies & Physicians’ Organizations

Measures to assure product safety: -Controlling the source -Test raw material -Implement virus-inactivation and removal -Test end products BSE outbreak: -Strict requirements regarding bovine-derived materials’ country of origin – expansion of restricted countries -BSE known to exist -Department of Agriculture

Center for Biologics Evaluation and Research (CBER) -Manufacturers - products: -Cell culture history -Isolation -Media -Identity and pathogen testing of cell lines Politics: -Safety regulations -Donor screening policies

US Centers for Disease Control and Prevention (CDC): -Single greatest risk of transfusion-transmitted viral infections -Failure of screening – infected donors – pre- seroconversion phase of infection More sensitive tests: -PCR-based nucleic acid amplification testing (NAT) -Minipool NAT -Single donor testing (ID NAT)

NAT: -Shorten the lag time – no detection of infection -HIV: 22 days  12 days -HCV: 70 days  14 days -No complete elimination of lag time Pathogens: -HBV -HCV -HIV-1 and HIV-2 -HTLV-I and HTLV-II -Syphilis -WNV

Methods – Inactivation and Removal of Viruses: -Pasteurization -Vapor heating -Low pH -Solvent/detergent treatment -Separation/purification techniques -Ion-exchange -Immunogenicity chromatography -Nanofiltration FDA & The International Conference on Harmonisation: -Documents guiding the sourcing, characterization, testing of raw materials, and evaluating of therapeutic proteins for virus.

Discussion

Recombinant Therapeutic Proteins: -Blood factors -Anticoagulants -Growth factors -Cytokines -Hormones -Vaccines -Therapeutic enzymes -Monoclonal antibodies Evolution in production: -Enhanced safety -Lower immunogenicity -Increased half-life -Improved bioavailability -Alternative routes of administration

“The risk of pathogen transmission through the use of human- or animal- derived raw materials in the manufacture of pharmaceuticals was the major driver behind the development of PF technology.”

Erythropoesis-stimulating agents: -Manage anemia – chronic kidney disease -Good example of evolution -Introduced in 1980s – blood-derived -A recombinant product -Longer half-life -Conversion to an HAS-free formulation -Conversion to serum-free formulation -PF, PEGylated recombinant – longer half life

Complete Elimination of Risk of Transmission: Recombinant Therapeutic Proteins: -Production: cell lines free of human- or animal- derived proteins -Processing: strict pathogen removal and/or inactivation -Testing: lipid- and non-lipid-enveloped viruses -Packaging: in absence of human- or animal-derived proteins Average cost for developing a biopharmaceutical product exceeding $1 billion.

Future: -False sense of security -PF technology – prevention -Area of research: -Different culture, formulation, and storage conditions -Physical stability of proteins