Overview of Protein Therapeutics 1 What is Protein

Overview of Protein Therapeutics 1

What is Protein therapeutics? 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.

Why protein therapeutics? ØProteins cannot be mimicked by simple chemical compounds. ØThere is often less potential for protein therapeutics to interfere with normal biological processes and cause adverse effects. ØIt is often well tolerated and are less likely to elicit immune responses. ØProvide effective replacement treatment without the need for gene therapy ØTime of protein therapeutics may be faster

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

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-CD 3 approved 1993 CBER's Office of Therapeutics Research and Review (OTRR) formed 1997 First whole chimeric antibody, rituximab, and first humanized antibody, daclizumab, approved 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.

Classification • Protein therapeutics can be classified based upon their pharmacologic activity as drugs that: i) replace a protein that is deficient or abnormal, ii) augment an existing pathway, iii) provide a novel function or activity, iv) interfere with a molecule or organism, or iv) deliver a payload such as a radionuclide, cytotoxic drug, or protein effector 6

Classification of Protein therapeutics • • Group I: protein therapeutics with enzymatic or regulatory activity Ia: Replacing a protein that is deficient or abnormal Ib: Augmenting an existing pathway Ic: Providing a novel function or activity Group II : protein therapeutics with special targeting activity IIa: Interfering with a molecule or organism (TABLES 6, 7). IIb: Delivering other compounds or proteins • Group III : protein vaccines • Group IV : protein diagnostics • IIIa: Protecting against a deleterious foreign agent. • IIIb: Treating an autoimmune disease. • IIIc: Treating cancer.

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

Protein therapeutics augmenting an existing pathway (Group Ib)*

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

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

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

Protein vaccines (Group III )*

Protein diagnostics (Group IV )

Remaining Disadvantages 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.

Manufacturing of Recombinant Protein Therapeutics

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

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.

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

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

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 (v. CJD)

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

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

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 – preseroconversion 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 p. H -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.

“The risk of pathogen transmission through the use of human- or animalderived 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 1980 s – blood-derived • A recombinant product • Longer half-life • 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 animalderived 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