1. Overview of Protein Therapeutics 1

2. Contents Introduction 1 Production 2 Delivery 33 Future Direction 44

3. It is currently estimated that there are 25,000–40,000 different genes in the human genome, viewed from the perspective of disease mechanisms, as disease may result when any one of these proteins contains mutations or other abnormalities, so it gives a tremendous opportunity for Protein therapeutics to alleviate these disease. What is Protein therapeutics?

4. Why protein therapeutics?  Proteins cannot be mimicked by simple chemical compounds.  There is often less potential for protein therapeutics to interfere with normal biological processes and cause adverse effects.  It is often well tolerated and are less likely to elicit immune responses.  Provide effective replacement treatment without the need for gene therapy  Time of protein therapeutics may be faster

5. A STAR IS BORN Pre-1986 1986–1991 MORE BIOTECHNOLOGY SUCCESSES BIOTECHNOLOGY INDUSTRY 1992–1999 BIOTECHNOLOGY IMPROVEMENT 2002 and beyond History and Development

6. The Evolution of Protein Therapeutics : A Timeline 1953 First accurate model of DNA suggested 1982 Human insulin, created using recombinant DNA technology 1986 Interferon alfa and muromonab-CD3 approved 1997 First whole chimeric antibody, rituximab, and first humanized antibody, daclizumab, approved 1993 CBER's Office of Therapeutics Research and Review (OTRR) formed 2002 Market for biotechnology products represents approximately $30 billion of $400 billion in yearly worldwide pharmaceutical sales 2006 An inhaled form of insulin (Exubera) approved, expanding protein products into a new dosage form.

7. Group I: protein therapeutics with enzymatic or regulatory activity Group II : protein therapeutics with special targeting activity Group III : protein vaccines Group IV : protein diagnostics Classification of protein therapeutics

8. Protein therapeutics replacing a protein that is deficient or abnormal (Group Ia)*

9. Protein therapeutics augmenting an existing pathway (Group Ib)*

10. Protein therapeutics providing a novel function or activity (Group Ic)

11. Protein therapeutics that interfere with a molecule or organism (Group II a)*

12. Protein therapeutics that deliver other compounds or proteins (Group II b)

13. Protein vaccines (Group III )*

14. Protein diagnostics (Group IV )

15. Protein Therapeutics also have disadvantages that may limit their more widespread acceptance, include low oral and transdermal bioavailability, moreover, injections must be given frequently because the half-lives of proteins are short. Remaining Disadvantages

16. Manufacturing of Recombinant Protein Therapeutics

17. -Microorganisms -Plant cell cultures -Insect cell lines -Mammalian cell lines -Transgenic animals Types of Cell Factories:

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

19. Mammalian cells: Used for large-scale production of therapeutic proteins -Post-translational modifications -Proteins – natural form -60-70% of all recombinant therapeutic proteins produced in 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 -Pechia. Pastoris

20. 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

23. 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

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

25. 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

26. 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)

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

29. 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 -1998 – expansion of restricted countries -BSE known to exist -Department of Agriculture

30. 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

31. 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)

32. 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

33. 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.

35. “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.”

36. 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 serum-free formulation -PF, PEGylated recombinant – longer half life

37. 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.

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