Drug Manufacturing

1. Drug Manufacturing BIT 230 Walsh Chapter 3

2. Drug Manufacturing • Most regulated of all manufacturing industries • Highest safety and quality standards • Parameters include: • Design and layout of facility • Raw materials • Process itself • Personnel • Regulatory framework

3. Pharmacopeias • Discussed before in other units and classes • Martindale- not a standards book • Gives information about drugs • Physiochemical properties • Pharmacokinetics • Uses and modes of administration • Side effects • Appropriate doses

4. GMP guidelines • Different publications world wide, but generally have similar information • Go over everything from raw materials to the facility • US guidelines issues publications called “Points to Consider” for additional guidelines for newer biotech products (will go over these later in semester)

5. Manufacturing facility • Most manufacturing facilities have requirements, but some specifics to biotech products, especially • Clean room • Water

6. Clean Rooms • Clean room views • Environmentally controlled areas • Critical steps for bio/injectable drugs are produced in clean rooms • Contain high efficiency particulate air (HEPA) filters in the ceiling • Figure 3.1 page 98 of chapter

7. Classification of Clean Roomsfor Pharma industry Class# microrganisms/m3 of air A <1 B 5 C 100 D 500 See table 3.5 page 100 of chapter

8. Other considerations • Exposed surfaces – smooth, sealed, non-penetrable surface • Chemically-resistant floors and walls • Fixtures (lights, chairs, etc.) minimum and easily cleaned • Proper entry of materials and personnel into clean room to reduce risk of contamination in clean room

9. Gowned person in Clean room

10. Clean Room clothing • Covers most of operators body • Change in a separate room and enter clean room via an air lock • Clothing made from non-shredding material • Number of people in a clean room at once limited to only necessary personnel (helps with automated processes)

11. CDS • Cleaning, decontamination and sanitization • C- removal or organic and inorganic material that may accumulate • D-inactivation and removal of undesired materials • S- destroying and removing viable microorganisms

12. CDS cont’d • Done on surfaces that either are direct or indirect contact with the product • Examples of surfaces in both categories?

13. CDS of process equipment • Of course trickier because comes in contact with the final product • Clean equipment, then rid equipment of cleaning solution • Last step involves exhaustive rinsing of equipment with pure water • WFI • Followed by autoclaving if possible • If possible use CIP (cleaning in place)

14. Examples of CIP agents used to clean chromatography columns • 0.5-2.0 M NaCl • Non-ionic detergents • 0.1-1.0 M NaOH • Acetic Acid • Ethanol • EDTA • Protease

15. Water • WFI- talked about this extensively before • 30,000 liters of WFI needed for 1kg of a recombinant protein • Use tap water just for non-critical tasks • Purified water – not as pure as WFI, but used for limited purposes (in cough medicines, etc.) • WFI used exclusively in downstream processing • Will not cover pages 105-112- water and documentation pages

16. Sources of Biopharmaceuticals • Genetic engineering of recombinant expression systems • Your talks will be about types of systems and how they are used- mammalian cells, yeast, bacteria etc. • Most approved products so far produced in E. coli or mammalian cell lines

17. E. coli • Cultured in large quantities • Inexpensive (relatively speaking) • Generation of quantities in a short time • Production facilities easy to construct anywhere in the world • Standard methods (fermentation) used

18. Current products from E. Coli • tPA (Ekokinase) • Insulin • Interferon  • Interleukin-2 • Human growth hormone • Tumor necrosis factor

19. Heterologous systems • Expression of recombinant proteins in cells where the proteins do not naturally occur • Insulin first in E. coli • Remember the drawbacks of expression in E. coli?

20. Other problems with E. coli • Most proteins in E. coli expressed intracellularly • Therefore, recombinant proteins expressed in E. coli accumulate in the cytoplasm • Requires extra primary processing steps (e.g. cellular homogenization) and more purification (chromatography)

21. Other problems with E. coli, cont’d • Inclusion bodies • Insoluble aggregates of partially folded product • Heterologous expressed proteins overload the normal protein-folding machinery • Advantage- inclusion bodies are very dense, so centrifugation can separate them from desired material

22. Preventing inclusion bodies • Lower growth temperature (from 37C to 30C) • Use a fusion protein (thioredoxin) - native in E. coli – protein expressed at high levels and remains soluble

23. Expression in animal cells • Major advantage- correct PT modifications • Naturally glycosylated proteins produced in: • CHO - Chinese hamster ovary • BHK - baby hamster kidney • HEK – human embryonic kidney

24. Current products from animal cells • tPA • FSH • Interferon - • Erythropoietin • FSH • Factor VIIa

25. Disadvantages of animal cells(compared to E. coli) • Complex nutritional requirements • Slower growth • More susceptible to damage • Increased costs • WILL NOT cover bottom of page 116 to page 124 (up to biopharmaceuticals)- you will cover these in your presentations

26. Final Product Production • Focus on E. coli and mammalian systems • Process starts with a single aliquot of the Master Cell Bank • Ends when final products is in labeled containers ready to be shipped to the customer

27. Production: Upstream and Downstream • Upstream: initial fermentation process; yields initial generation of product • Downstream: purification of initial product and generation of finished product, followed by sealing of final containers • biomanufacturing process overview

29. Upstream processing • Remove aliquot from MCB • Inoculate sterile medium and grow (starter culture) • Starter culture used to inoculate larger scale production culture • Production culture inoculates bioreactor • Bioreactors few to several thousand liters • See figure 3.13 of chapter (page 129)

30. Upstream cont’d • Pages 129-133 go over specific details for microbial fermentation • Pages 133-134 go over specific details for animal cell culture • Properties of animal cells • Anchorage dependent • Grow as a monolayer • Contact inhibited • Finite lifespan • Longer doubling times • Complex media requirements

31. Downstream processing • Diagram page 135 of chapter 3 • Detailed steps considered confidential • Clean room conditions for downstream

32. Downstream cont’d • Steps involved (intracellular products – E. coli.) – mammalian products secreted in media, so easier to isolate) • Centrifugation or filtration • Homogenization • Removal of cellular debris • Concentration of crude material (by precipitation or ultra filtration) • High resolution chromatography (HPLC) • Formulation into the final product

33. Downstream cont’d • Final product formulation • Chromatography yields 98-99% pure product • Add excipients (non active ingredients), which may stabilize the final product • Filtration of final product, to generate sterile product • Freeze drying (lyophilization) if product if to be sold as a powder (dictated by product stability)

34. Separation methods • Page 142,tables 3.18 and 3.19 • Familiar with: • Ion-exchange • Gel-filtration • Affinity chromatography • Protein A chromatography • Immunoaffinity chromatography

35. Factors that influence biological activity • Denature or modify proteins • Results in loss of/reduced protein activity • Need to minimize loss in downstream work • Problems can be chemical (e.g., oxidizing, detergents); physical (e.g., pH, temperature); or biological (e.g., proteolytic degradation) • Table 3.20 page 143

36. Proteolytic degradation • Hydrolysis of one or more peptide bonds • Results in loss of biological activity • Trace quantities of proteolytic enzymes or chemical influences • Several classes of proteases: • Serine • Cysteine • Aspartic • Metalloproteases (also in other ppt)

37. Protease inhibitors • PMSF – serine and cysteine proteases • Benzamidine – serine proteases • Pepstatin A – aspartic proteases • EDTA – metalloproteases • a.a residue known to be present at active site of protein, so disruption of it causes loss of activity

38. Others (mentioned before) • Deamidation – hydrolysis of side chain of asparagine and glutamine • Happens at high temp and extreme pH • Oxidation and disulphide exchange • Oxidation by air (met and cys in particular) • Alterations of glycosylation patterns in glycoproteins (more than one sugar) • Affect activity or immunological properties

39. Excipients • Substances added to final product to stabilize it • Serum albumin • Withstands low pH or elevated temps • Keeps final product from sticking to walls of container • Stabilize native conformation of protein

40. Excipients cont’d • Amino acids • Glycine – stabilizes interferon, factor VIII, stabilizes against heat • Alcohols (and other polyols) • Stabilize proteins in solution • Surfactants • Reduces surface tension; proteins don’t aggregate, so don’t denature

41. Final product fill • See figure 3.27 page 153 • Bulk product gets QC testing • Passage through 0.22 m filter for final sterility • Aceptically filled into final product containers • Uses automated liquid handling systems

42. Final product fill cont’d • Freeze drying (lyophilization) • Yields a powdered product • Reduces chemical and biological degradation of final product • Longer shelf life than products in solution • Storage for parenteral products (those administered intravenously or injected)

43. Freeze drying cont’d • Need to add cryoprotectors • Glucose or sucrose • Serum albumin • Amino acids • Polyols • Freeze drying can be done in many steps

44. Labeling and Packing • After sealed in final container, product quarantined • Samples are QC’d • Check potency, sterility and final volume • Detection and quantitation of excipients • Highly automated procedures • Labeling function critical- biggest error where many products are made

45. Label • Name and strength of product • Specific batch number • Date of manufacture and expiry date • Required storage conditions • Name of manufacturer • Excipients included • Correct mode of usage

46. Other final product items • Biopharmaceutical products undergo more testing than traditional pharma products • Products made in recombinant systems have more potential to be contaminated than synthetic chemical drugs • Larger, more complex molecules