Recombinant therapeutic proteins

1. RECOMBINANT THERAPEUTIC PROTEINS

2. ATRACTIVE FEATURES Therapeutic proteins are used for the treatment of abnormal health conditions. Replacing chemically synthesized drugs Highly specific function less interference with normal biological processes faster clinical development and FDA approval time less likely to elicit immune responses.

3. Evolution of Protein Therapeutic 1982 Human insulin, created using recombinant DNA technology 1986 Interferon alfa and muromonab-CD3 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 $ 30 billion share of biotechnological drugs of $400 billion in yearly worldwide pharmaceutical sales 2006 An inhaled form of insulin ( Exubera ) approved

4. Classification of Protein therapeutics 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

5. 5 Process of Drug Production Cells and plasmid + Cell line Transfection Cell culture Purification Drug substance (crude) Drug substance (pure) Drug product - (sterile) Formulation/ Filling Cell line manufacture Medium development Bioreactor process development & scale-up Downstream purification Analytical characterization

6. World market EPO alone : ~ $ 11 Billion per year - $ 50 Billion (2007)  $ 190 Billion (2015) - Antibodies > 50 % - Intensive investment in monoclonal antibodies : Therapeutic proteins will form the back-born of future medicinal therapy

7. PRODUCTION SYSTEMS FOR PRODUCING THERAPEUTIC PROTEINS : simple physiology short generation times, as bacteria grow and multiply rapidly large yields of product - up to 10 % of mass (low cost) The expressed proteins often do not fold properly and so are biologically inactive. The synthesised protein is often toxic to bacteria preventing the cell cultures from reaching high densities BACTERIAL CELLS

8. Yeast cells Yeast is a simple eukaryote and performs many of the post-translational modifications required for human proteins Presence of active proteases that degrade foreign (expressed) proteins, thereby reducing their yield (a solution to this problem is the construction of yeast strains from which the protease genes have been deleted ).  

9. Bacillus Sp. Actinomycetes Sp . Eschericia coli ( Yeast) MICROBES USED FOR PROTEIN PRODUCTION

10. High level of expression Correct folding More difficult to work with Expensive Slow generation time Not suitable for proteins with repetitive sequences INSECT CELLS competitive cost the availability of established practices for their efficient harvesting, transporting, sorting and processing PLANTS

11. Transgenic animals cheap method produce the desired proteins in vast quantities when using larger animals like cows. long lead time to generate a herd of transgenic animals concerns about the health of the animal. cause serious negative health effects

12. In vitro systems E. coli extract; plant wheatgerm extract; and mammalian sources, rabbit reticulocyte lysate. lack both the genomic material and the cellular boundary system contain the cellular components required for transcription and/or translation of genes.

13. Comparison of Recombinant Protein Expression

14. PURIFICATION STEPS

15. Some recombinant proteins approved for human use Protein Company Disorder Factor VIII Baxter, Bayer Hemophilia A Factor IX Genetics Institute Hemophilia B Tissue plasminogen activator (TPA) Genetech Acute myocardial infarction Insulin Eli Lilly, Novo Nordisk Diabetes mellitus Human growth hormone Eli Lilly, Genetech , Upjohn, Novo Nordisk GH deficiency in children (dwarfism) Erythropoietin Amgen, Ortho Biotech Anemia DNase I Genetech Cystic fibrosis Various interferons (IFN) Schering, Biogen , Chiron, Genetech Hepatitis B and C, multiple sclerosis

16. PRODUCTION OF RTPs

17. DRUG ANIMAL USED GENETIC MODIFICIATION WHO/WHERE PRODUCED Anti-Cancer Drugs (currently in the process of making). Chickens The anti-cancer drug is produced in the chickens eggs. Roslin Institute in the United Kingdom is LACTOFERRIN (Breast Milk Supplement) COWS Recombinant DNA targets lysosome from the breast milk and modifies it in the cow which gives a more nutritional boost for infants Hermitech , Inc. in Sioux Falls, South Dakota. & China Drugs in transgenic animals

18. Production of Recombinant Therapeutic Proteins in the Milk of Transgenic Animals

19. Schematic representation of the process used to purify ATryn from the milk of transgenic goats.

20. Recombinant Hepatitis vaccine The hepatitis B virus (HBV) vaccine surface antigen purified from the blood of infected individuals. Due to safety concerns, the HBV vaccine became the first to be produced using recombinant DNA technology (1986) Produced in bakers’ yeast (Saccharomyces cerevisiae

21. to respond to a human influenza pandemic. to respond to a human influenza pandemic.                                                                              Vaccine Production at industry level

22. FOLLICLE STIMULATING HORMONE (FSH) single vector containing the coding sequences for both subunit genes . After transfection, a genetically stable transformant producing biologically active recombinant FSH was isolated 150–450 gene copies were present in mammalian cells. FSH products from cell culture supernatants. collected from a perfusion-type bioreactor downstream purification

23. rhDNase I

24. CANCER VACCINE

25. Bio therapeutics are delicate drugs Much larger and more complex than traditional pharmaceuticals Composed of unstable proteins with a precise structure Easily damaged by unfavorable temperature history during storage