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PR3145 > L4 - Assuring Quality of BMP > Flashcards

L4 - Assuring Quality of BMP Flashcards

1
Q

What are BMP?

A
  • 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

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2
Q

Recombinant DNA (rDNA) Technology (Bacterial Cells)

A
  • 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
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3
Q

Hybridoma Technology (Mammalian Cells)

A
  • 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
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4
Q

Characteristics of Mammalian Host Cells

A
  • 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
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5
Q

Characteristics of Microbial Host Cells

A
  • Cultivation: Fast
  • Disruption of cell needed (proteins secreted within the cells)
  • Lower yield
  • Simples proteins, non-glycosylated
  • Relatively safe
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6
Q

Safety concerns of using mammalian cells as host cells?

A

Transformed mammalian cells using hybridoma technology are “immortal” & potentially carcinogenic
–> Alright as long as purification process can reduce residual DNA to 10nanograms/dose

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7
Q

Critical QA & GMP issues of Biotechnology-derived Medicinal Products

A
  • 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

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8
Q

What is a Biosimilar product & what does it means to be “highly similar”?

A

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
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9
Q

Major steps in manufacturing of BMP

A
  • Cell banking
  • Cell cultivation
  • Harvesting
  • Purification
  • Viral clearance
  • Batching & storage of bulk biological API
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10
Q

Type of tests in Cell Banks

A

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
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11
Q

Type of cell banks & their controls

A

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

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12
Q

What happens during inspection of the Cell Bank System

A

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
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13
Q

Upstream processes of Cell Cultivation

A
  • Inoculum prep
  • Seed cultures
  • Production cultures
  • Harvest
  • Clarification
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14
Q

Downstream processes of Cell Cultivation (After cultivation & harvesting)

A
  • Pri purification (Affinity chromatography)
  • Virus inactivation (low pH, detergent)
  • Sec purification (column chromatography)
  • Virus filter (nanofiltration)
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15
Q

What is Cell Cultivation referred to as when using Microbial Cells

A

Fermentation

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16
Q

What is Cell Cultivation referred to as when using Mammalian Cells

A

Culturing

17
Q

Types of Fermentation (Bacterial cell growth)

A

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)

18
Q

Types of Culturing (Mammalian cell growth)

A

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
19
Q

What happens during inspection of the Cultivation Process

A

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

20
Q

What happens during inspection of the Cultivation Process

A

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

21
Q

Harvesting of Microbial Cell System

A

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

22
Q

Challenges of cell disruption

A
  • Heat denaturation of protein product
  • Oxidation of protein product
  • Uncontrolled release of other cell components
23
Q

Harvesting of Mammalian Cell System

A
  • Ab recovery process relatively simpler & do not require cell disruption
  • -> Ab produced are secreted extracellularly through cm
24
Q

What is the conventional harvest process in mammalian cell culture process

A

Centrifugation, Depth filtration, Membrane filtration

25
Q

Objective of Purification (of protein product)

A

To remove all known (chemical & biological) impurities to obtain pure protein product

26
Q

Methods of Purification (of protein product)

A

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

27
Q

What happens during inspection of the Purification Process

A
  • Control of chromatographic columns, buffers & other materials used for purification process
  • Level of purity depends on usage of product
28
Q

Impact of viral contamination

A
  • Product quality
  • -> Facility shutdowns
  • -> Disruption of medicine supply
  • -> Business impact
29
Q

Why is validation of viral clearance necessary?

A
  • 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

30
Q

List viral clearance methods

A

[Virus inactivation methods]
Chemical: Low pH incubation, sufactant/detergent
Physical: Heat treatment/UV

[Viral removal methods]

  • Ppt
  • Column chromatography
  • Membrane filtration
  • Nanofiltration
31
Q

Aim of validation study of viral clearance

A

To demonstrate that manufacturing/purification process can eliminate substantially more virus than what may potentially be present

32
Q

Points to note during inspection of Batching & Storage of Bulk Biological API

A
  • 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
33
Q

What are the factors to be considered when formulating, filling & packaging into the finished product?

A
  • 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

PR3145 (10 decks)

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