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PR3145 > Lecture 4 Assuring Quality of BMP > Flashcards

Lecture 4 Assuring Quality of BMP Flashcards

1
Q

Compare and Contrast Pharmaceuticals vs Biopharmaceuticals

A

Pharmaceuticals are:

  • smaller
  • produced through chemical synthesis
  • defined (simpler) and easier to characterize
  • relatively heat, pH stable and less prone to microbial contamination

Biologicals:

  • large molecules, extracted from living cells/systems
  • complex structures, more difficult to characterise (& to copy)
  • heat-sensitive and susceptible to microbial contamination
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2
Q

How are traditional biological medicinal products (TBMP) derived?

A

Historically extracted directly from human and animal tissues

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3
Q

What are biotechnology-derived medicinal products (BDMP)?

A

Newer biological products produced in living cells , e.g. microbial , mammalian , insect, rodent and plant cells via biotechnology

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4
Q

What are some examples of biotechnology-derived medicinal products (BDMP)?

A 
Examples of BDMP include human insulins, HGH and EPO made via
recombinant DNA (rDNA) and monoclonal antibodies made via hybridoma technology.
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5
Q

What are some examples of traditional biological medicinal products (TBMP)?

A

Examples of TBMP include blood components albumin, plasma, platelets (human) and rabies vaccine (dogs), heparin (pigs) and antivenoms (snakes)

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6
Q

What is biotechnology?

A

Use of living cells/organisms, or their products to modify human and animal health, mankind and his environment

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7
Q

What are the steps involved in Recombinant DNA technology?

A
  • Transformation (Heat shock –> selection –> cultivation in cell bank)
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8
Q

What are the steps involved in Hybridoma technology?

A
  • Immunization
  • Isolate B cells
  • Fuse B cells with myeloma cells
  • screening and selection (of the hybrid cell line)
  • cultivation (clonal expansion)
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9
Q

What are the advantages of Microbial cells over Mammalian cells for use in biotechnology?

A
  • Faster cultivation (relatively straightforward fermentation)
  • Relatively safe product
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10
Q

What are the advantages of Mammalian cells over Microbial cells for use in biotechnology?

A
  • No cell disruption (product secreted outside cells)
  • Higher yield due to less complicated purification
  • More complex proteins with post-translational modifications
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11
Q

What was a key safety concern initially for hybridoma technology?

A

Possible Contamination of Protein Product with potentially oncogenic Host Cell (Hybridoma) DNA delayed use of Mammalian Cells for many years

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12
Q

Which study report helped enable the use of mammalian cells for biological production?

A

1987 WHO Study Report
- Concluded No Reason to exclude Continuous (Mammalian) Cell Lines for Biological Production if Purification process can reduce Residual DNA to <10 nanogram per dose

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13
Q

Why is the term biosimilar used instead of generics for biologics that refer from the innovator biologic?

A

Each biotech derived medicinal product is unique and difficult to replicate identically

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14
Q

What are some key GMP and QA issues in manufacture of biotechnology derived medicinal products?

A
  • Assuring genetic stability (of plasmid and GOI)
  • Absence of impurities, endogenous (inherent) viruses and adventitious viruses (introduced during production) and residual DNA (from hybridoma of mammalian cell)
  • Assuring quality and yield through consistent manufacturing process
  • absence of (biological & chemical) impurities from nutrient media & starting materials
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15
Q

Is it acceptable to introduce small changes to manufacturing processes for biotechnology derived medicinal products?

A

No. As cell substrates are “living factories”, slight changes to manufacturing processes can have major impact on quality, (safety and efficacy) of product

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16
Q

What are biosimilars?

A

According to the US FDA, a biosimilar is a biological product which is “ highly similar ” to, and has no clinically significant differences from an existing FDA approved reference product, often the innovator product

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17
Q

What are the comparative studies required to determine the high degree of similarity between biosimilar and reference product?

A
  • Molecular Structure and Potency (Bioactivity)
  • Toxicity Study (Non-Clinical/Animal Study)
  • Pharmacodynamics (Clinical/Human Study)
  • Pharmacokinetics (Clinical/Human Study)
  • Immunogenicity (Clinical/Human Study)
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18
Q

In the production of biosimilars, are minor differences in terms of clinically inactive components, e.g. buffers and stabilizers acceptable?

A

Yes

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19
Q

Why are biosimilars often lower cost than innovator products?

A

Lower R and D/Regulatory Costs

- 65 - 85% cost

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20
Q

Which PICS GMP Annex helps us in inspection of biotechnology medicinal products?

A

Annex 2 of PICS GMP guide for Medicinal Products

Annex 2: Manufacture of Biological Products

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21
Q

What are the major steps in the manufacture of biotechnology medicinal product?

A

(1) Cell Banking
(2) Cell Cultivation
(3) Harvesting
(4) Purification
(5) Viral Clearance (Inactivation/Removal)
(6) Batching and Storage of Bulk Biological API
(7) Formulation, Packaging and Release of Finished Product (injection)

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22
Q

Where does inspection start for biotechnology medicinal product manufacture?

A

We can start inspection at the cell bank as we would start at the warehouse for a conventional inspection of a pharmaceutical facility

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23
Q

What does the master cell bank (MCB) contain?

A
  • Contains well characterized cells derived from specific cell line, at a specific passage level
  • Cells are stored frozen under defined conditions, e.g. liquid nitrogen (-196 degrees Celsius)
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24
Q

What is a working cell bank (WCB) for and what needs to done prior to its use?

A
  • Used to provide “working” cells for manufacturing
  • WCB is derived from one or more containers of MCB
  • A WCB must be tested before use
  • Tests are usually less than those conducted at MCB
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25
What are the types of tests conducted for cell banks?
Tests for: - Genetic Stability - Endogenous viruses - Adventitious viruses - Residual DNA
26
How are Adventitious viruses introduced into a cell bank?
During Sampling, cell expansion or transfer from MCB to WCB
27
Why are some companies outsourcing Testing of Cell banks to Sometimes Outsourced to Specialized Contract Testing Laboratories?
The tests are a highly specialized activity that requires sophisticated equipment, infrastructure and expertise
28
What is inspected during an inspection of Cell Bank System?
1. Documentation of Cell Origin and History (via QC Tests records) 2. Management of Cell Banks (MCB and WCB) 3. Contract Testing Laboratories
29
How should the Cell Banks be managed and documented?
- Appropriate storage condition - SOPs and records for liquid nitrogen replacement - Stock control/reconciliation of cell vials - Cross-contamination preventative measures - Restricted access - Maintenance and back-up arrangements
30
What do inspectors look at when inspecting contract test laboratories?
- Availability of comprehensive written contract, clearly showing the Roles and Responsibilities of Contract Acceptor and Giver
31
What is the difference between "Fermentation" and "Cell Culturing"?
- “Fermentation” used when Microbial Cells e.g. E. coli (Bacteria); S. cerivisiae (Yeast) are cultivated in Fermentor - “Cell Culturing” used when Mammalian Cells , e.g. CHO Cells, Murine Myeloma Cells are cultivated in Bioreactor
32
What are the Upstream Processes of biotechnology manufacturing?
1. Cell Banking 2. Cell Expansion and Scale Up 3. Cultivation 4. Harvesting Inoculum Prep --> Seed cultures --> production culture --> harvest --> clarification
33
What are the Downstream Processes of biotechnology manufacturing?
1. Primary Purification (Affinity Chromatography) 2. Viral Inactivation (Low pH, detergent) 3. Secondary Purification (Column chromatography) 4. Viral Removal (Nano filtration) 5. Batching and Storage of Bulk Biological API 6. Formulation, Filling and Packaging, QC of Finished Biological Product (Annex 1)
34
What is the mean generation time for E. Coli?
20 minutes
35
What is the mean generation time for CHO cells via mitosis?
20-24 hours
36
What are the physical factors required for growth of bacteria?
- Temperature - pH - Osmotic Pressure
37
What are the chemical factors required for growth of bacteria?
Water, O, C, S, N, P, Trace elements, Organic growth factors
38
What are the 4 phases of a typical Bacterial growth curve?
1. Lag Phase 2. Log Phase 3. Stationary Phase 4. Death Phase
39
What is the Batch Fermentation Method?
- Closed System of Bacterial Cells and Media; Only Oxygen is Continuously Added - Concentration of Media Decreases Continuously; Toxic Metabolites Accumulate - Characteristic (Bacterial Cell) Growth Curve: Lag, Log Stationary and Decline Phases
40
What is the Continuous Fermentation Method?
- Open System of Bacterial Cells and Media; Fresh Sterile Media Added Continuously - Waste Products Removed Continuously - Bacterial Cells are always growing in Log Phase Steady State Growth
41
What is/are advantage(s) of the Batch Fermentation Method over the Continuous Fermentation Method?
Chance of Contamination is Minimal for the Batch Fermentation Method (Due to Closed System)
42
What is/are diadvantage(s) of the Batch Fermentation Method over the Continuous Fermentation Method?
Lower Product Yield for the Batch Fermentation Method (Not economical)
43
What is the Fed-Batch Fermentation Method?
- Media added and Waste Products removed at period intervals | - Semi Closed System
44
Why is bovine serum not used for nutrient media?
Concerns with BSE Prions
45
Why is there a move towards serum-free nutrient media?
Safety and cost reasons
46
What are the 2 types of Cell culturing Methods for Mammalian Cell Growth?
1. Anchored System | 2. Suspension System
47
What is the Lag, Log, Stationary and Decline phases like for Cell culturing methods of mammalian cell growth?
There are no such phases
48
What are crucial parameters for production in bioreactors? (in cell culturing)
Optimal stirring speed , circulation and shearing dynamics in bioreactor
49
What are the things we should note about the nutrient media used for Cell culturing?
* Nutrient media composition is crucial to consistent quality and yield * Minor deficiencies in nutrients have major effect on cell growth and protein production
50
What are the factors affecting Product Quality in Cell cultivation?
4M and 1 P - Materials - Method - Machine - Man - Premises
51
What are the main points to note for inspection for Cultivation Process?
1. Control of Starting Materials (cell line, water, nutrient media) 2. Control of cell Culture conditions 3. Maintenance, Cleaning of fermenters and Bioreactors Note: increasing trend towards SUT (Single use technology), including single use columns &bioreactors
52
What are some challenges of cell disruption?
- Heat denaturation pf protein pdt - Oxidation of protein pdt - Uncontrolled release of other cell components, together w protein pdt, during disruption
53
Does the mammalian cell system require cell disruption to collect products?
No
54
What are the steps in conventional *harvest* process in mammalian cell culture process?
Bioreactor >> Centrifugation >> Depth Filtration >> Membrane filtration >> Harvest vessel Centrifugation: remove intact cells & large debris (micron size) Depth filtration: remove cell debris (micron and sub-micron size) Membrane filtration: aseptic boundary Centrifugation + filtration suitable for cell culture with less than approx 10 million of cell density
55
Why is rapid purification necessary for bacterial harvest?
To prevent cleavage by protease released by lysed bacteria
56
What are some methods of Purification?
- Precipitation - Adsorption - Ultra Filtration - Chromatography
57
How do we control chromatographic columns, buffers and materials used for the purification process?
Inspectors must look at: - Supplier approval - Specifications - Maintenance and storage - Effective cleaning - *use of single use, disposable columns
58
Which product (chronic use or single use) needs to be of a higher purity level?
Chronic use products i.e. insulin need to be of higher purity Single use products (i.e. vaccines need not be as pure)
59
What are the virus inactivation methods?
- Chemical (low pH incubation and surfactant/detergent) | - Physical (heat and UV treatment)
60
What are the virus removal methods?
- Precipitation (using ammonium sulfate) - Column Chromatography - Membrane filtration - Nanofiltration
61
Why is validation of Viral Clearance necessary?
1. No single test is able to demonstrate the presence of all known viruses (Any Single Test has limited viral species detection) 2. All test systems require a minimum level of viral contamination to record a positive result (Test systems are not very sensitive) 3. Tests are also limited by Statistical considerations in sampling
62
How can we demonstrate a biological product is free from virus?
Testing AND Demonstrate manufacturing process is capable of removing/inactivating viruses
63
What is the aim of validation studies of viral clearance?
- Demonstrate manufacturing process eliminate more virus than may potentially be present - Obtain the best reasonable assurance that product is virus-free - impt role in establishing product safety
64
What is the virus panel used for viral clearance studies?
- 4 representative model viruses with different physicochemical properties, size, chemical resistance to demonstrate robustness of viral clearance capability - Include members from each of the 4 major classes of virus (env, non-env, DNA, RNA)
65
What are things to note for Batching and storage for bulk biological product API?
* Biological products can be manufactured in sub lots and pooled as batch * A bulk batch of biological API can be packed in different containers or packs in terms of volume * Containers of biological APIs should be made of appropriate material, inert and compatible with API * Bulk biological API should be stable in containers stability testing studies submitted in DMF for MA), can be verified during inspection
66
What are the storage considerations for Bulk Biological API?
- Product temperature to be maintained (as defined in stability study) - Adequate size of storage space to prevent overcrowding - Storage in refrigerators/freezers with temperature recording and Alarm systems
67
Why are biologics commonly formulated into sterile dosage forms?
Biologics are very susceptible to microbial growth
68
What kinds of validation studies need to be done for formulation of sterile dosage forms?
- Aseptic filling - Autoclaving - Final Container Closure integrity
69
What are factors to consider in the formulation of sterile dosage forms?
- Excipient choice (heat resistant & not prone to microbial growth) - Water purification system - Design and construction of the facility - Environmental/microbiological monitoring - validation of aseptic filling - validation of autoclaving of packaging components - Validation of final container-closure integrity (to assure no ingress of MO)
70
What are the QC methods in biotechnology?
- DNA Sequencing/Hybridization - PCR - SDS-PAGE and Immunoassays - Reverse Transcriptase, electron microscopy, antibody production tests - Peptide mapping, IEF, MS - Microbial Limit test and Sterility Test - Pyrogen test - Modified 21 CFR Method (mycoplasma)
71
What does specificity mean?
Ability to assess unequivocally the analyte in the presence of expected components such as impurities, degraded products and matrix
72
What does reproducibility mean?
Ability to obtain the same results among different laboratories variations, different days, analysts, equipment
73
What does accuracy mean?
The degree of closeness of determined value to known true value under prescribed conditions
74
What is Minute Virus of Mice (MMV)? is it ss or ds DNA and is it enveloped?
ssDNA and non-enveloped model rodent virus infect CHO cells
75
What is retrovirus type 3 (reo-3)? is it ss or ds DNA and is it enveloped?
dsDNA and non-enveloped ability to infect both human and animal cells
76
What is murine leukiaemia virus (MuLV)? is it ss or ds DNA and is it enveloped?
ssDNA and enveloped retrovirus-like particles found in CHO cells
77
What is pseudorabies virus? is it ss or ds DNA and is it enveloped?
dsDNA and enveloped retrovirus-like particles found in herpesvirus
78
How are recombinant proteins harvested from a microbial cell system?
- have to undergo disruption (lysis) as recombinant protein are contained intracellularly - cell envelope physically broken - mechanical or non-mechanical disruption can be done mechanical: ultrasonification, milling, homogenisaition, oscillation Non-mechanical: surfactants, solvents, lysozyme, osmotic shock
79
for purification, what does chromatography mean?
ultra-filtration and chromatography are the MOST common methods of protein purification - different molecules in a mixture (stationary phase) are separated from one another while moving in a mobile phase - in affinity chromatography: protein product interacts or bond specifically with *stationary phase*, and bound protein is subject to further purification

PR3145 (10 decks)

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