1. QA Production Material and Analytical Methods BIT 230 Chapters 5 and 6 (Huxsoll)

2. Material Selection n Original qualification of material - vendor, specifications, safety, function, etc. n Lot-to-lot testing for release for production use n This chapter about original qualification

3. QA Materials n Start in R & D - in large companies, sometimes R & D personnel don’t know that QA group should be aware of their materials n Need to know QA materials need testing and approval

4. Technology n Technology groups (a.k.a. tech transfer)- transfer the process from research to development to production (manufacturing technology or process development n Perform scale-up procedures n Don’t always use initial research materials in production

5. Engineering n Keeps process going once a material is in use n Need to know specs of required materials (equipment parts, e.g.) n Approve new materials or parts before installation n Work from approved vendors

6. Qualification of Materials n Safety first! –Toxicity of extractables –Qualification request: material to be approved vendor identification use process conditions general information

7. Personnel involved n Requestor n Chemist n Biologist n Toxicologist n Safety Qualifications Manager n If pass test, the material can be released to production

8. Suppliers n For start up biotech (and maybe large pharma too?) use biggest, most experienced, technically sound suppliers n Don’t want to find a guinea pig here n Small co. can’t do a lot of their own testing, so have to reply on tried and true vendor

9. Considerations when choosing a Supplier n Meet timing needs (both material and information requests about a material) n History with supplier n Technical knowledge –had product for year or more n Follow GMP guidelines n Size of supplier company n Good documentation with material

10. Type of Material Uses n Nonproduct contact n Product contact

11. Nonproduct contact materials n Engineering chemicals –lubricants on equipment –coolants (freon) n Sterilants –steam - most common one used - use WFI for the steam n Pesticides - not used in GMP facility- should not have a pest problem!

12. Nonproduct contact materials cont’d n Paints –solvents they emit- need ventilation (no longer lead or mercury paints) n Packaging –not a big problem with nonproduct contact n Colorants –low toxicity colors such as white and iron- oxide red

13. Product Contact Materials n Two main types: –Basic chemicals (put into process intentionally) –Process materials (may be by-products of the process)- does not bind up product

14. Product Contact Materials cont’d n Basic chemicals –cell culture media –water - most important raw material –glass n Process materials –containers - glass or plastic (plastic needed FDA approval)

15. Process materials cont’d n Closures n Hoses and piping –recommended: hard-piped stainless steel –other types also acceptable (silicone) n Affinity antibodies –non-intentional components of products- may leach from column, even though should be tightly bound –frequent washing of columns necessary

16. Process materials cont’d n Pumps –peristaltic pumps do not contain extractables so ideal to use n Gaskets, O-rings etc. –rubber problematic in biotech production –problems with sticking –need to verify integrity with toxicity testing

17. Testing of materials n Plastics –material must pass USP testing n Closure

18. In-house testing n Not just vendor specs, but need to establish in house specs for raw materials n USP the basis n Other tests - chemical, physical, n Chemistry, toxicology and microbiology involved in determining tests

19. Parameters to check n Basic chemicals” –appearance –identity (IR spec) –Assay (HPLC) –Purity - need to define impurities first; also use HPLC, or TLC (thin layer chromatography)

20. Parameters to check cont’d n Toxicity - in vitro biological reactivity test –put material in fermentor with cells, look for the material to damage or kill the cells n Biological purity –pyrogens –viruses –mycoplasma –bacteria

21. Testing of process materials n As is testing –Identity - IR –Reside on Ignition (ROI) - solid is ashed at 600°C; ensures supplier is using same inorganic raw materials –Emission Spectrographic Analysis (ESA) - residue from ROI tested for heavy metals (cadmium or lead, for example)

22. Testing of process materials cont’d n Extracts testing –Distilled water (DW) extractant for testing pH changes oxidizable substances heavy metals nonvolatile residue UV scan the DW extract

23. In process containers n Stainless steel- has no toxic components n Glass - Type 1 accepted standard –careful with high levels of aluminum in glass n Plastics- good properties - especially polypropylene (what we use here)

24. Final containers n Glass- same issues as with in-process containers- need to be aware of aluminum levels n Plastics- low levels of extractables, toxicity and aluminum, esp. PP (as long as not repeatedly autoclaved- disposable)

25. Documentation n Departments involved in release of materials after validation: –Purchasing - request vendor audits –Inspection and Receiving - know about arrival of material and how to handle –Safety and Environmental –Chemistry - choose tests for initial evaluation and lot specific tests

26. Documentation cont’d n Microbiology - also initial and lot-to-lot tests n Toxicology - same as micro for tox tests n Project Engineering - how exactly a material is used in a process

27. QA of analytical methods n Chapter 6

28. Biotech products n Produced by either fermentation or cell culture n Uses genetically engineered bacteria or eukaryotic cells n Monoclonal antibodies - from hybridoma cells

29. Proteins n Similar properties - therefore need to develop methods to characterize them n High molecular weight –10,000-20,000 Daltons (insulin and interferon) –> 950kD (IgM monoclonal antibodies)

30. Proteins n This chapter will summarize- more about proteins in next lecture n Further testing parameters for proteins: –primary, secondary, tertiary & quaternary –disulfide bonding –multiple chains –carbohydrate content

31. Uses of biotech products n Therapeutic drug products n In vivo diagnostic testing n In vitro diagnostic testing n Medical devices n Agricultural products n Products for animals

32. Sterile injectable drugs n This chapter’s focus n Same 4 parameters - identity, quality, purity and potency n More tests on this type than others- make sure they don’t pose a health risk

33. Physical tests n Gross appearance –color –appearance –pH n Make sure there is no particulate matter formed and precipitated –provides stability information

34. Identity tests n Molecular weight n retention times n peptide mapping n reaction with an antibody n biological activity in assays n N- and C- terminal sequence analysis n Review each one pages 78-80- will go over more in next lecture

35. Assays n Quantify proteins n Total protein content n Amino acid composition n Degradation products n Measure of glycosylation (carbohydrate content) n HELPS measure lot-to-lot consistency of a biotech product

36. Total protein assays n Lowry, Bradford, etc- (one we did) n Just measure total protein (not specific) n Need external reference for each test n Bradford uses Coomassie blue dye (one we used- the darker the blue color the greater amount of protein) n Each assay requires spectrophotometry measurements

37. Native protein n Protein can lose its native form easily - by fragmentation, aggregation, denaturation, or chemical modifications n Use separation methods to remove from native proteins n Use HPLC, SDS-PAGE

38. Amino acid comp. analysis n Quantitate amount of each a.a. in protein n Used especially to identify after a manufacturing change occurs n Routine control test, too n Digest protein into a.a. componments

39. Carbohydrate analysis n No glycoproteins in bacteria; found in proteins produced in eukaryotic cells n Sugars found include galactose, glucose and mannose n Individual sugar content determined by HPLC or GC

40. Immunoassays n Determines identity (by reacting with specific antibody) n Determines specific activity (when uses as part of total protein measurements) –RIA –ELIZA –IRMA (immunoradiometric assays)

41. Purity tests n Affected by contaminants and degradation products that co-purify with protein during production n WCB can be source of contaminants n Purification accomplished by chromatography n See page 84 Table 6.1 - test with sensitivity ability

42. Added chemicals n Chemicals added during fermentation, cell culture and protein purification n How do you then get rid of the chemicals from the final product? n End product testing required - many methods can be used to test absence of added chemicals in final product

43. Added chemicals cont’d n GC n NMR n HPLC n Immunoassay n Remove chemicals near or below detection limits

44. Other needs for purity n Residual DNA –reduce host cell DNA –use hybridization assays n Endotoxins –use LAL test - see accompanying presentation n Mycoplasma –broth and agar cultures

45. Potency tests n Measure dose-dependent biological activity n Designed to mimic in-vivo activity of the biological product n Performed in animals or in cellular assays n Cellular assays ideal over animal assays

46. Potency tests cont’d n What is measured: –protection from viral infection –clot dissolving properties –binding to specific antigens –inhibition of protein synthesis –inhibition of DNA replication

47. Potency tests cont’d n Correlated results to WHO, NIH or USP standards n measure in IU (international units) n Need consistent reagents n Well characterized reference standard materials n Numerous replicates