adapting industry practice for rapid large scale manufacture of pharmaceutical proteins

1. Adapting Industry Practice for Rapid Large Scale Manufacture of Pharmaceutical Proteins David A. Estell, Ph.D. Genencor International

3. Genencor at a Glance History traced to 1982 - joint venture of Genentech and Corning $390 Million in total revenues during 2004 Among the world�s largest biotech companies 8 manufacturing sites; ~4 million liters of capacity

4. Pharmaceutical Protein Production Selling price > $1000/gram active protein Volumes < 10,000 gram active protein /month Full scale manufacturing more than 1 year after creation of final molecule

5. Genencor: Industrial Protein Production Selling price < $1/gram active protein Volumes > 30,000,000 gram active protein /month Full scale manufacturing within weeks of creation of final molecule

6. Rapid, Large Scale Pharmaceutical Protein Production The tools and methods of industrial biotechnology can be used to produce several million doses of a protein pharmaceutical within weeks of identification. Industrial costs and volumes enable topical, oral or inhaled delivery systems. THE TIME IS NOW.

7. Genencor Approach to Protein Production Identify protein scaffold Choose gene/host system Develop high yield fermentation process Design robust, rapid and efficient recovery process Create formulation and delivery system Engineer the scaffold to provide the desired properties

8. Protein Scaffolds Multi-Domain Fusion Proteins Monoclonal Antibody Viral or Bacterial Coat Protein Inhibitor Enzyme

9. Fusion Protein Scaffolds

10. Fusion Protein Scaffolds

11. Gene/Host System These are proven examples where GCI has used a variety of host systems for producing protein, metabolites, etc.. Product examples: E.coli � 1,3 propanediol; indigo; used as a screening host. Pantoea � AsA B. Subtilis � proteases (FNA, FN2, FN3, FN4); endoglucanase (BCE103); used as a screening host B. Licheniformis � amylases (LAT, low pH alpha FRED); pullulanase Strep lividans � endoglucanase (11AG8) Strep rubiginosus � glucose isomerase Pseudomonas � lipase (not commercial) These are proven examples where GCI has used a variety of host systems for producing protein, metabolites, etc.. Product examples: E.coli � 1,3 propanediol; indigo; used as a screening host. Pantoea � AsA B. Subtilis � proteases (FNA, FN2, FN3, FN4); endoglucanase (BCE103); used as a screening host B. Licheniformis � amylases (LAT, low pH alpha FRED); pullulanase Strep lividans � endoglucanase (11AG8) Strep rubiginosus � glucose isomerase Pseudomonas � lipase (not commercial)

12. Advantages of Microbial Systems Speed to construct stable production strains. Ability to screen for improvements in same host used for manufacturing. No animal products required during production. Short fermentation time, robust process up to very large scale. Reduced capital expenditure. Reduced cost of goods sold.

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14. Protein Recovery Processes Filtration Extraction Large Scale Chromatography Crystallization

15. High-Throughput Process Development for Purification of Recombinant Proteins High-throughput microtiter plate recovery process development Scalable screening technique Appropriate analytical methods to enable rapid analysis of screening results

16. Create formulation and delivery system Protein stable in formulation for months at > 40�C. Formulations may be solid or liquid. Release of product may be controlled. Formulations are food grade.

17. Typical Microbial Production Process Create Production Host 2-4 weeks Fermentation Process 3-20 days Recovery Process/Formulation 2-10 days

18. Rapid Protein Drug Production Expression system can be in place for each protein scaffold. Each scaffold can be engineered to have the basic properties required. Immunogenicity Stability Pharmakokinetics A high yield fermentation and recovery process can be put in place for each scaffold. Formulation and delivery system in place for scaffold protein.

19. Rapid Protein Drug Production For Protein drugs a small number of sequence changes in a protein scaffold will give the desired properties: Binding site in an antibody Epitopes in a viral or bacterial coat protein Enzyme/Receptor binding site in an inhibitor

20. Rapid Protein Drug Production Protein drug identified (e.g. by sequence of pathogen) Protein drug created through engineering one of the already developed protein scaffolds New protein drug is produced using processes put in place for the protein scaffold

21. Rapid Protein Drug Production Proteins for which gycosylation is not required for activity can be produced now. Proteins for which glycosylation is key may require additional host engineering or post production modification

22. Rapid Protein Drug Production Products with individual dose sizes of < 100 mg (e.g vaccines) can be made with existing technology and capacity. Products with dose sizes > 100mg (e.g. monoclonal antibodies) may require initial yield improvement for the scaffold protein

23. Fermentation capacity (8 weeks) for 100,000,000 doses @ 1mg/dose

24. Rapid Protein Drug Production Current production processes are capable of producing 100,000,000 g of protein in < 12 weeks. Yield drives fermentation capacity Yield needs to be at least 1 g/L to meet the timelines Fermentation and recovery processes need to be in place

25. Rapid Protein Drug Production Time and volume targets can best be achieved by developing robust processes for expression (> 1g/L), fermentation and recovery of the scaffold protein. These processes would then be used for the engineered final product.

26. Rapid Protein Drug Production Example: fungal production of a monoclonal antibody

27. Filamentous Fungi for Pharmaceutical Production More than $ 13 billion/year of injectable and oral antibiotics are produced through fungal fermentation. The published yields are 1-50 g/L Recovery processes can be used for proteins Sales prices are $1-$100/g

28. Strategy for Ab Production in Aspergillus

29. Assembly and Processing of Ab in Aspergillus at 1g/L

30. 1st Step Purification by HCIC

31. 2nd Step of Purification by SEC

32. Competition Binding Assay

33. CHO and Fungal Ab Display Similar Pharmacokinetics in Rat

34. Rapid, Large Scale Pharmaceutical Protein Production The tools and methods of industrial biotechnology can be used to produce several million doses of a protein pharmaceutical within weeks of identification. Industrial costs and volumes enable topical, oral or inhaled delivery systems. THE TIME IS NOW.