QA Production Material and Analytical Methods

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

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

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

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

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

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

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

8. Suppliers • For start up biotech (and maybe large pharma too?) use biggest, most experienced, technically sound suppliers • Don’t want to find a guinea pig here • 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 • Meet timing needs (both material and information requests about a material) • History with supplier • Technical knowledge • had product for year or more • Follow GMP guidelines • Size of supplier company • Good documentation with material

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

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

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

13. Product Contact Materials • 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 • Basic chemicals • cell culture media • water - most important raw material • glass • Process materials • containers - glass or plastic (plastic needed FDA approval)

15. Process materials cont’d • Closures • Hoses and piping • recommended: hard-piped stainless steel • other types also acceptable (silicone) • 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 • Pumps • peristaltic pumps do not contain extractables so ideal to use • Gaskets, O-rings etc. • rubber problematic in biotech production • problems with sticking • need to verify integrity with toxicity testing

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

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

19. Parameters to check • 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 • Toxicity - in vitro biological reactivity test • put material in fermentor with cells, look for the material to damage or kill the cells • Biological purity • pyrogens • viruses • mycoplasma • bacteria

21. Testing of process materials • 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 • 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 • Stainless steel- has no toxic components • Glass - Type 1 accepted standard • careful with high levels of aluminum in glass • Plastics- good properties - especially polypropylene (what we use here)

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

25. Documentation • 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 • Microbiology - also initial and lot-to-lot tests • Toxicology - same as micro for tox tests • Project Engineering - how exactly a material is used in a process

27. QA of analytical methods • Chapter 6

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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