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Compliance & GMP > L4 > Flashcards

L4 Flashcards

1
Q

What is biotechnology?

A

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

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2
Q

Briefly describe the steps in Recombinant DNA Technology.

A
  1. Design a recombinant plasmid, containing the:
    - Target gene
    - Promoter for target gene
    - Abx resistance gene
  2. Transformation of host cell (e.g. via heat shock)
  3. Select cells that are successfully transformed by growing the cells in a petri dish containing Abx
    - Using Antibiotic Resistance Genes
  4. Fermentation / Cultivation of cells to produce proteins.
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3
Q

Briefly describe the steps in Hybridoma Technology.

A
  1. Specific antigen are injected into MURINE (spleen) or HAMSTER (ovary) cells. They produce specific cells which produce antibodies. These cells are harvested.
  2. As mammalian cells duplicate slowly, they are fused together with a myeloma (tumour cell) to form a hybridoma.
  3. Hybridoma are screened for the production of desired Antibodies/Protein
  4. Hybrid cells are cultivated to produce the proteins, undergoes clonal expansion to produce a large number of protein/Ab
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4
Q

What is the main safety concern with using mammalian host cells?

A

The possibility of contamination with potentially oncogenic host cell DNA.

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5
Q

WHO concluded that there is no reason to exclude continuous cell lines for biological production if the purification process can reduce residual DNA to what levels?

A

10 nanograms (ng) per dose

10ng/dose

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6
Q

What are the critical GMP & QA issues in the manufacture of biotechnology-driven medicinal products?
(Hint: Consider the starting materials and finished product)

A
  1. Genetic stability - assuring genetic stability of the host cells, plasmid & GOI
  2. Quality & yield - Assuring quality & yield through consistent manufacturing processes
  3. Absence of impurities - Absence of biological and chemical impurities from nutrient media and starting materials.
  4. Absence of viruses - Absence of inherent endogenous viruses and viruses introduces during production (e.g. EBV, Herpesvirus, Retroviruses, Adventitious Viruses)
  5. Elimination of residual DNA from hybridoma of mammalian cell (to 10ng/dose or below)
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7
Q

What is a biosimilar product?

A

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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8
Q

What are the major steps in the manufacture of biotechnology-driven medicinal products? (Hint: 7 steps from the cells to the finished product)

A
  1. Cell banking
  2. Cell cultivation
  3. Harvesting
  4. Purification
  5. Viral clearance
  6. Batching and storage of bulk biological API
  7. Formulation, packaging and release of finished product
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9
Q

What is inspected during inspection of Cell Bank System?

A
  1. Documentation of cell origin & history
  2. Management of cell banks (storage, contamination prevention, QC testing etc)
  3. Contract testing laboratories (Used to test the integrity of Cell bank cultures - genetic stability, presence of contaminants etc)
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10
Q

What are the important factors controlled during Cell Cultivation to achieve high quality and high yield product?

A

4M + 1P:

  • Manpower
  • Machine
  • Methods
  • Materials
  • Premises
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11
Q

What are some considerations during inspection of the Harvesting process?

A

Mammalian vs Bacterial cell cultures.

  • Mammalian cells: proteins are secreted into media, no lysis is needed.
  • Bacteria cells: Lysis and rapid purification is needed.

Lysis can be achieved through mechanical and non-mechanical means

For Mammalian Cells, Cell separation results in significant purification and no lysis is needed!
Therefore, Overall yield tends to be lower for bacteria cell system, as some product may be lost during the process of lysis and purification.

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12
Q

What is inspected during inspection of the Purification Process?

A
  1. Control of chromatographic columns, buffers & other materials used for the purification process.

The level of purity required depends on its usage (chronic use vs single use)

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13
Q

How is the validation study of viral clearance conducted?

A

It is conducted on a panel of at least 4 representative model viruses (with different physicochemical properties, size & resistance).
- Different characteristics of the viruses : e.g. Genome, Size (nM), Envelope Presence, Resistance profile

  • E.g. Reo-3, MMV, xMuLV, PrV are used
    Reo-3 = Reovirus Type 3 (Non-enveloped)
    MMV = Murine Minute Virus (Infects CHO cells)
    xMuLV = Xenotropic Murine Leukemia Virus (Found in CHO cells)
    PrV = Pseudorabies Virus (Found in CHO cells)

The test should demonstrate that the manufacturing/purification process can eliminate these viruses.

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14
Q

Why is validation of viral clearance necessary?

A

No single test is able to demonstrate the presence of all known viruses.

Additionally, it is not possible to have QbD in place to test for presence of viral contaminants

We cannot establish that a product is free from viruses particles just based on testing. We have to validate that the manufacturing process is capable of removing or inactivating the viruses through validated viral clearance methods

Demonstrate that purification process can eliminate substantially more virus than what may be potentially present in the unprocessed bulk material, to obtain the best possible assurance that the product is reasonably free from virus contamination.

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15
Q

What is a “batch”?

A

A specific quantity of a drug or other materials that is intended to have:

  1. UNIFORM CHARACTER and QUALITY
  2. Within specified limits
  3. Produced according to a single manufacturing order during the same cycle of manufacture.
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16
Q

Biological products may be manufactured in sub-lots and pooled as a bulk batch. T/F?

A

True.

Biologicals are usually more ‘carefully’ manufactured and tested thereafter, hence they are manufactured in sublots.

A bulk batch of biological APIs can be packed in different containers or packs in terms of volume. But they will all belong to the same batch of homogenous quality and characteristics belonging to the same manufacturing order and cycle

vs Medicinal Chemical products

17
Q

Biologics can be formulated into oral dosage formed. T/F?

A

False.

Biologics are formulated as sterile injections!

18
Q

What are the key performance test parameters that we need to verify during validation study of Bioanalytical Test Methods?
(Hint: There are 3 most important ones - S A R )

A
  1. Sensitivity
  2. Specificity
  3. Precision
  4. Repeatability
  5. Intermediate precision
  6. Reproducibility
  7. Accuracy
  8. Quantitation range
  9. Linearity
  10. Robustness

Most importantly: Specificity, Reproducibility, Accuracy

19
Q

What is the main biopharmaceutical manufactured in ASEAN currently?

A

Vaccines

20
Q

The ASEAN Mutual Recognition Arrangement (MRA) on GMP Inspection includes biopharmaceuticals. T/F?

A

False.

It currently excludes biopharmaceuticals, but the scope will be expanded by the end of this year (Nov 2020)

21
Q

One of the few (3) most important Key Performance Test Parameters Under Bioanalytical Test Validation is Specificity, Reproducibility and Accuracy. Define the terms.

A

Specificity: Ability to assess unequivocally the analyte in the presence of expected components such as impurities, degraded products and matrix

Reproducibility: Able to obtain the same results among different laboratories variations, different days, analysts, equipment etc.

Accuracy: Degree of closeness of determined value to the nominal or known true value under prescribed conditions (Trueness of Bioanalytical Test Methods)

22
Q

Viral Clearance methods include

A

Viral inactivation (Destroy)

  • Chemical Methods: Low pH incubation, surfactants/detergents
  • Physical methods: Heat treatment, UV
Viral Removal Method (remove)
- Precipitation with Ammonium Sulfate
- Column chromatography
(ion exchange, size exclusion, affinity, reverse phase etc)
- Membrane filtration
-Nanofiltration (remove viruses)
23
Q

What are the 2 most common Virus REMOVAL method? (Note: Referring to Physical Removal)

A

Column Chromatography and Nano-filtration

24
Q

Methods to purify protein product during Purification stage?

A

Precipitation, Adsorption, Ultrafiltration (not nanofiltration) and Chromatography

25
Q

What must inspectors note when inspecting the Cultivation Process? (Referring to cultivated Mammalian cells or fermented bacterial cells)

A
  1. Control of starting materials (e.g. Medium, Serums used etc.)
  2. Control of cell culture conditions (Temperature cycling, pH, oxygen levels etc.)
  3. Maintenance and Hygiene/Cleaning of Fermentors or Bioreactors
Fermenters = Bacteria
Bioreactors = Mammalian Cells
26
Q

Name the mechanical and non-mechanical methods used to harvest protein products during the harvesting stage

Hint: Required to produce cell lysis especially for proteins produced through Bacterial Fermentation (Mammalian cells usually don’t require cell lysis)

A

Mechanical: Ultrasonication, Milling, Homogenization, Oscillation

Non-mechanical: Surfactants, Alkalis, Solvents, Lysozymes, Osmotic Shock

27
Q

Upstream processes of Cell Cultivation/Fermentation?

A

Cell Banking, Expansion and Scale up, cultivation (including Harvesting and Clarification)
- Up to Harvesting and Clarification

  1. Inoculation preparation (Preparation of the inoculate, in MCB and WCB)
  2. Seed Culture (starting) - From Working Cell Bank (WCB)
  3. Production Culture
  4. Harvest
  5. Clarification
28
Q

Downstream processes of Cell cultivation/fermentation?

A

From upstream - After harvesting and clarification

(example of a possible downstream process)

  1. Affinity Chromatography
  2. Virus inactivation
  3. Chromatgraphy x2
  4. Virus Filter (Nanofiltration)
  5. Final Filtration of biological Drug substance
    …..
  6. Batching and storage of Bulk Biological API
  7. Formulation and Packaging into Finished Product
29
Q

What is the Master Cell Bank?

A

Contains well characterized cells derived from a specific cell line, at a specific passage level, where cells are frozen under defined conditions

Working Cell bank is derived FROM MCB

It is tested extensively for purity, consistency and especially genetic stability

30
Q

What is the Working Cell Bank?

A

Used to provide Working cells for manufacturing/production , derived from one or more containers of the Master Cell Bank

Must be tested extensively before use

31
Q

What are some tests conducted for Master Cell Banks/Working Cell Banks?
(Hint: Basically all of the GMP/QA issues in manufacture of biopharmaceutics except for one)

A
  1. Genetic Stability
  2. Test for endogenous viruses (INHERENT)
  3. Test for adventitious viruses (Introduced
    during sampling/cell expansion)
  4. Test for residual DNA

(except to test for purity and yield - irrelevant and no point)

Testing is often sophisticated ; hence may be outsourced to external contract testing laboratories

32
Q

Name and Define the two types of Mammalian Cell culturing

A

Anchored System - Mammalian cells are attached to solid support

Suspension system - Mammalian Cells suspended in liquid medium (nutrients)

33
Q

What are the three key differences between Mammalian Cell Cultures versus Bacterial Cell Cultures?

A
  1. Mammalian cells have NO lag, log, stationary or decline phases
  2. Mammalian cell cultures are considered more crucial and complex than Bacterial Cell cultures
    (Bovine serums are good for Mammalian Cell cultures, but risk of BSE Prion contamination)
  3. Mammalian cells are more fragile
    vs microbial cells (Larger size, lack of rigid cell wall, more complex nutrient requirements)
34
Q

Define the three types of Microbial Fermentation, and state which type is the most common

A
  1. Batch Fermentation (Closed System)
    - Only oxygen is continuously added, concentration of media decreases continuously and toxic metabolites accumulate
  2. Continuous Fermentation (Open System)
    - Open System of Bacterial Cells and Media, Sterile Media added continuously, waste products are continuously removed
    - Bacteria cells always in log phase
    - High yield (Advantage), but Chance of Contamination is higher (Disadvantage)
  3. Fed-Batch fermentation (Semi-closed, Most Common)
    - Media added and waste products removed at periodic intervals
    - Semi closed system
    - Most common

Compliance & GMP (7 decks)

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