L4 - Assuring Quality of BMP Flashcards
What are BMP?
- Large molecules produced/extracted from living cells or systems
- Complex structures, difficult to characterise
- Heat-sensitive & susceptible to microbial contamination
i) Traditional BMP
- BP extracted directed from human/animal tissues
ii) Biotechnology-derived MP
- Produced in living (mammalian/microbial) cells
Recombinant DNA (rDNA) Technology (Bacterial Cells)
- Recombinant plasmid + Host cell (eg E.coli) -> Transformed cell
- > Recombinant plasmid: Promoter, GOI, Antibiotic resistance gene
- -> Apply heat shock to facilitate take up of recombinant plasmid (DNA from ligation)
- -> Antibiotic in colony bed is to kill any E.coli that has not been successfully transformed
Hybridoma Technology (Mammalian Cells)
- Specific antigen injected in murine (spleen) or hamster (ovary)
- Immune cells isolated -> Ab-forming cells -> Fusion with myeloma cell to create Hybridoma (Hybrid cell line) which multiplies rapidly & continuously
- Hybridoma can be stored in cell bank until it is ready to be cultivated/expanded to get specific glycosylated protein
- Hybridomas screened for production of desired antibody
- Cultivation & clonal expansion to monoclonal antibodies
Characteristics of Mammalian Host Cells
- Cultivation: Slow (more complicated cell culture for growth)
- No cell disruption needed
- Higher yield
- More complex proteins produced with glycosylation & post-translational modification
- Many safety issues
Characteristics of Microbial Host Cells
- Cultivation: Fast
- Disruption of cell needed (proteins secreted within the cells)
- Lower yield
- Simples proteins, non-glycosylated
- Relatively safe
Safety concerns of using mammalian cells as host cells?
Transformed mammalian cells using hybridoma technology are “immortal” & potentially carcinogenic
–> Alright as long as purification process can reduce residual DNA to 10nanograms/dose
Critical QA & GMP issues of Biotechnology-derived Medicinal Products
- Assuring genetic stability
- Absence of (biological & chemical) impurities
- Assuring quality & yield
- Absence of (inherent) endogenous viruses & adventitious viruses
- Elimination of residual DNA
Note: Slight changes to manufacturing processes can have a major impact on quality, safety & efficacy of product
What is a Biosimilar product & what does it means to be “highly similar”?
Biosimilar: Biological pdct which “highly similar” to… no clinically significant differences from reference product (eg. innovator product)
“Highly Similar”
- Molecular structure & potency (bioactivity)
- Toxicity
- Pharmacodynamics
- Pharmacokinetics
- Immunogenicity
- -> Minor diff in terms of clinically-inactive components are acceptable
Major steps in manufacturing of BMP
- Cell banking
- Cell cultivation
- Harvesting
- Purification
- Viral clearance
- Batching & storage of bulk biological API
Type of tests in Cell Banks
Highly specialised activity requiring sophisticated equipment, infrastructure & expertise (sometimes outsource to specialised contract testing laboratories)
- Tests for genetic stability
- Tests for endogenous viruses
- Tests for adventitious viruses
- Tests for residual DNA
Type of cell banks & their controls
1) Master cell bank
- cells to be stored frozen under defined conditions
- has > tests than working cell bank
2) Working cell bank
- used to produce “working” cells for manufacturing
- WCB must be tested before use
What happens during inspection of the Cell Bank System
Documentation of cell origin & history
- Evidence of well characterised cell line (via QC tests)
- Records of QC tests conducted
Management of Cell Banks
- appropriate storage & condition
- SOP & records on replacement of liquid nitrogen
- Stock control/reconciliation
- Cross-contamination measures
- QC testing & cell expansion
- Restricted access to authorised personnel
- Maintenance & back-up arrangements
Contract testing laboratories
- Availability of comprehensive written contract
- Roles & responsibilities of contract acceptor & giver
Upstream processes of Cell Cultivation
- Inoculum prep
- Seed cultures
- Production cultures
- Harvest
- Clarification
Downstream processes of Cell Cultivation (After cultivation & harvesting)
- Pri purification (Affinity chromatography)
- Virus inactivation (low pH, detergent)
- Sec purification (column chromatography)
- Virus filter (nanofiltration)
What is Cell Cultivation referred to as when using Microbial Cells
Fermentation
What is Cell Cultivation referred to as when using Mammalian Cells
Culturing
Types of Fermentation (Bacterial cell growth)
1) Batch fermentation
- Closed system (Only O2 continuously added)
- Minimal contamination
- Low yield (not economical)
2) Continuous fermentation
- Open system (fresh sterile media added & waste products removed continuously)
- Always in Log phase (Steady state growth)
- Higher chance of contamination
3) Fed-Batch fermentation (balance)
Types of Culturing (Mammalian cell growth)
1) Anchored system
- Mammalian cells attached to solid support
2) Suspension system
- Mammalian cells suspended in liquid medium
Note:
- Nutrient media composition crucial to consistent quality & yield
- Serum-free nutrient media for safety & $ reasons
- Mammalian cells more fragile, larger size & lack of rigid cell wall
- Optimal stirring speed, circulation & shearing dynamics in bioreactors are crucial parameters
What happens during inspection of the Cultivation Process
Control of starting materials
- Supplier, specifications, test results of starting materials
- Water purification system
Control of cell culture conditions
- Formulation of nutrient media
- Temp, O2, pH
- Fermentation/Culture Time
- Growth rate, culture purity & fatty acid profile
Maintenance & cleaning of Fermentors/Bioreactors
What happens during inspection of the Cultivation Process
Control of starting materials
- Supplier, specifications, test results of starting materials
- Water purification system
Control of cell culture conditions
- Formulation of nutrient media
- Temp, O2, pH
- Fermentation/Culture Time
- Growth rate, culture purity & fatty acid profile
Maintenance & cleaning of Fermentors/Bioreactors
- SOPs & records of cleaning & maintenance
Harvesting of Microbial Cell System
Recombinant proteins are contained intracellularly within microbial cells
- Need to undergo disruption (lysis) to release proteins
- Disruption may be mechanical/non-mechanical
Mechanical: Ultrasonification, Milling, Homogenization, Oscillation
Challenges of cell disruption
- Heat denaturation of protein product
- Oxidation of protein product
- Uncontrolled release of other cell components
Harvesting of Mammalian Cell System
- Ab recovery process relatively simpler & do not require cell disruption
- -> Ab produced are secreted extracellularly through cm
What is the conventional harvest process in mammalian cell culture process
Centrifugation, Depth filtration, Membrane filtration
Objective of Purification (of protein product)
To remove all known (chemical & biological) impurities to obtain pure protein product
Methods of Purification (of protein product)
Ultra-filtration, Chromatography, Precipitation, Adsorption
Chromatography
- Diff mol in stationary phase are separated from one another while moving in a mobile phase
- Separates/isolates desired protein product from a complex mixture
Note: Rapid purification needed for bacterial system to prevent cleavage by lysed bacteria
What happens during inspection of the Purification Process
- Control of chromatographic columns, buffers & other materials used for purification process
- Level of purity depends on usage of product
Impact of viral contamination
- Product quality
- -> Facility shutdowns
- -> Disruption of medicine supply
- -> Business impact
Why is validation of viral clearance necessary?
- No single test able to demonstrate the presence of all known virus
- All test require min level of viral contamination to produce +ve result
- Limited by statistical consideration
- Impt in establishing product safety
–> Need to also demostrate that manufacturing process is capable or removing/inactivating them
List viral clearance methods
[Virus inactivation methods]
Chemical: Low pH incubation, sufactant/detergent
Physical: Heat treatment/UV
[Viral removal methods]
- Ppt
- Column chromatography
- Membrane filtration
- Nanofiltration
Aim of validation study of viral clearance
To demonstrate that manufacturing/purification process can eliminate substantially more virus than what may potentially be present
Points to note during inspection of Batching & Storage of Bulk Biological API
- Biological pdcts can be manufactured in sub-lots & pooled as batch
- Containers of biological APIs should be of appropriate material, inert & compatible with API
- Bulk biological API should be stable in containers - stability testing studies
What are the factors to be considered when formulating, filling & packaging into the finished product?
- Choice of excipients
- Water purification system
- Design & construction of facility
- Environmental/Microbial Monitoring
- Validation of aseptic filling
- Validation of autoclaving of packaging components
- Validation of final container-closure integrity
