QA Production Materialand Analytical Methods

BIT 230

Chapters 5 and 6 (Huxsoll)

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

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

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

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

Qualification of Materials

Safety first!– Toxicity of extractables– Qualification request:

• material to be approved• vendor identification• use• process conditions• general information

Personnel involved

Requestor Chemist Biologist Toxicologist Safety Qualifications Manager

If pass test, the material can be released to production

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

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

Type of Material Uses

Nonproduct contact

Product contact

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!

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

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

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)

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

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

Testing of materials

Plastics

–material must pass USP testing

Closure

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

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)

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

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)

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

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)

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)

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

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

QA of analytical methods

Chapter 6

Biotech products

Produced by either fermentation or cell culture

Uses genetically engineered bacteria or eukaryotic cells

Monoclonal antibodies - from hybridoma cells

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)

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

Uses of biotech products

Therapeutic drug products In vivo diagnostic testing In vitro diagnostic testing Medical devices Agricultural products Products for animals

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

Physical tests

Gross appearance– color– appearance– pH

Make sure there is no particulate matter formed and precipitated– provides stability information

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

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

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

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

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

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

Immunoassays

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

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

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

GC NMR HPLC Immunoassay Remove chemicals near or below

detection limits

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

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

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

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