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

Material Selection n Original qualification of material vendor, specifications, safety, function, etc. n Lot-to-lot 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

QA Materials n n Start in R & D - in large companies, sometimes QA Materials n n 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

Technology n n n Technology groups (a. k. a. tech transfer) - transfer the Technology n n n 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

Engineering n n Keeps process going once a material is in use Need to Engineering n n 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

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

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

Suppliers n n n For start up biotech (and maybe large pharma too? ) Suppliers n n n 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

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

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

Nonproduct contact materials n Engineering chemicals – lubricants on equipment – coolants (freon) n 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 facilityshould not have a pest problem!

Nonproduct contact materials cont’d n Paints – solvents they emit- need ventilation (no longer 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 ironoxide red

Product Contact Materials n Two main types: – Basic chemicals (put into process intentionally) 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

Product Contact Materials cont’d n Basic chemicals – cell culture media – water - 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)

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

Process materials cont’d n Pumps – peristaltic pumps do not contain extractables so ideal 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

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

In-house testing n n Not just vendor specs, but need to establish in house In-house testing n n 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

Parameters to check n Basic chemicals” – appearance – identity (IR spec) – Assay 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)

Parameters to check cont’d n Toxicity - in vitro biological reactivity test – put 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

Testing of process materials n As is testing – Identity - IR – Reside 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)

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

In process containers n Stainless steel- has no toxic components n Glass - Type 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)

Final containers n Glass- same issues as with in-process containers- need to be aware 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 autoclaveddisposable)

Documentation n Departments involved in release of materials after validation: – Purchasing - request 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

Documentation cont’d n Microbiology - also initial and lot-to-lot tests n Toxicology - same 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

QA of analytical methods n Chapter 6 QA of analytical methods n Chapter 6

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

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

Proteins n n This chapter will summarize- more about proteins in next lecture Further Proteins n n 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

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

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

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

Identity tests n n n n Molecular weight retention times peptide mapping reaction with Identity tests n n n n 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

Assays n n n Quantify proteins Total protein content Amino acid composition Degradation products Assays n n n 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

Total protein assays n n n Lowry, Bradford, etc- (one we did) Just measure Total protein assays n n n 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

Native protein n Protein can lose its native form easily by fragmentation, aggregation, denaturation, Native protein n 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

Amino acid comp. analysis n n Quantitate amount of each a. a. in protein Amino acid comp. analysis n n 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

Carbohydrate analysis n n n No glycoproteins in bacteria; found in proteins produced in Carbohydrate analysis n n n 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

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

Purity tests n n Affected by contaminants and degradation products that co-purify with protein Purity tests n n 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

Added chemicals n n n Chemicals added during fermentation, cell culture and protein purification Added chemicals n n n 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

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

Other needs for purity n Residual DNA – reduce host cell DNA – use 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

Potency tests n n Measure dose-dependent biological activity Designed to mimic in-vivo activity of Potency tests n n 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

Potency tests cont’d n What is measured: – protection from viral infection – clot 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

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