Biologics Flashcards
Examples of biologics
1) Hormones e.g. erythropoietin (EPO), human growth protein (HGH)
2) IgG
3) Monoclonal antibodies (MABs)
4) Insulin
Characteristics of biologics
Large molecules
Complex structures, difficult to characterize (and copy)
Heat sensitive
Susceptible to low pH, microbial contamination
How are the following produced:
1) Traditional biological medicinal products (TBMP)
2) Biotechnology-derived medicinal products (BDMP)
3) Advanced therapy medicinal products (ATMP) / Cell, tissue & gene therapy products (CTGTP)
1) Extracted directly from human/animal tissues
2) Produced in living cells via biotechnology
3) Based on gene, tissue/cells engineering
Examples of biotechnology
1) Recombinant DNA (rDNA) technology
2) Hybridoma technology
rDNA technology steps
1) Genetic engineering of plasmid to form recombinant plasmid
2) Recombinant plasmids added to test tube containing host cells (e.g. E.coli)
3) Subjected to heat shock to promote uptake of recombinant plasmids (transformation) to form transformed bacterium
4) Test tube contents poured into petri dish containing antibiotics. Antibiotics will kill off the cells that have not been transformed
5) Cultivation (fermentation)
6) Harvest protein of interest
What does the recombinant plasmid usually contain
1) Gene of interest / Target gene / Expression gene
2) Promoter
3) Antibiotic resistance gene
Types of host cells
1) Microbial cells (bacteria, yeast)
2) Mammalian cells (CHO cells commonly used)
Hybridoma technology steps
1) Mouse/Mammal immunized with specific antigen. Mouse/Mammal will develop antibody-producing immune cells
2) Isolation of antibody-producing cells
3) Antibody-producing cells fused with tumour cell (myeloma cell) to form hybrid cell line (hybridoma)
4) Hybridomas screened for production of desired antibody
5) Cultivation
6) Harvest MABs
Characteristics of hybridomas
1) Antibody-producing ability
2) Immortal
3) Able to replicate rapidly & continuously
Mammalian cells:
1) Application
2) Cultivation
3) Secretion of products
4) Yield
5) Type of protein produced
6) Safety
1) Usually used in hybridoma technology
2) Slower cultivation. More complicated cell culture (growth)
3) Protein products secreted extracellularly
4) Relatively higher yield (less complicated purification)
5) More complex proteins, may be glycosylated/undergo post-translational modification
6) Safety issues - Presence of endogenous virus, Residual tumour DNA (oncogenic)
Microbial cells:
1) Application
2) Cultivation
3) Secretion of products
4) Yield
5) Type of protein produced
6) Safety
1) Usually used in rDNA technology
2) Faster cultivation. Relatively straightforward fermentation
3) Protein products secreted intracellularly. Cell disruption/lysis needed
4) Relatively low yield (more difficult purification)
5) Simple proteins, non-glycosylated
6) Relatively safe
1987 WHO study: Mammalian cell lines may be used if purification process can _____
Reduce residual tumour DNA to < 10 ng per dose
US FDA Definition of biosimilars
A biological product that is ‘highly similar’ and has no significant clinical difference from an existing FDA approved reference product (usually the innovator product)
Comparative tests/studies to determine high degree of similarity between biosimilar & reference product include:
1) Molecular structure & potency (bioactivity)
2) Toxicity (non-human/animal study)
3) PK-PD studies (clinical/human study)
4) Immunogenicity (clinical/human study)
Critical GMP & QA Issues in Manufacture of BDMP
1) Assuring genetic stability of cell substrates with plasmid/vectors and gene of interest
2) Absence of chemical/biological impurities in nutrient media and starting materials
3) Absence of inherent endogenous viruses & adventitious viruses
4) Elimination of residual hybridoma DNA
5) Slight changes to manufacturing processes can have major impact on safety, efficacy & quality of product
6) Biological products are unique and difficult to replicate identically - the term biologic is used
How is inspection of biological products carried out
Follows flow of manufacturing:
1) Cell banking
2) Cell cultivation
3) Harvesting
4) Purification
5) Viral clearance
6) Batching & storage of bulk biological API
7) Formulation, packaging & release of final product
Control of master cell bank
1) Well characterized cells derived from specific cell lines, of a specific passage level
2) Stored under defined conditions
Control of working cell bank
1) WCB used to provide working cells for manufacturing
2) Derived from one or more containers of MCB
3) Must be tested before use
Testing of cell banks is a ___ activity that requires ____
May be outsourced to ____
Highly specialized activity Sophisticated equipment, infrastructure & expertise
Specialized contract testing laboratories
What to test for in cell banks
1) Genetic stability (using DNA fingerprinting, Southern Blot)
2) Endogenous viruses (in mammalian cell lines)
3) Adventitious viruses
4) Residual DNA (from hybridoma)
Inspection of cell banks - Items to inspect
1) Documentation of cell origin & history
- Evidence of well-characterized cell lines (via QC testing)
- Records of QC tests conducted on cell banks
2) Management of cell banks
- Appropriate storage conditions (-196oC, with liquid nitrogen)
- SOP & records on replacement of liquid nitrogen
- Stock control/Reconciliation of ampoules/vials of cells
- Restricted access to authorized personnel
- Cross-contamination measures during sampling, QC testing, cell expansion
- Maintenance & back-up arrangement
3) Contract testing laboratory
- Comprehensive written contract, which states roles & responsibilities of contract giver & acceptor
Cell culturing is used as a reference point to ____
Categorize biopharmaceutical processes into upstream & downstream processes
Upstream processes:
Cell banking
Cell expansion and scale up
Cultivation
Harvesting and clarification
Downstream processes:
Primary purification
Viral inactivation
Secondary purification
Viral removal
Batching and storage of bulk biological API
Formulation, filling and packaging, QC of finished biological product
