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Regulation > Biologics > Flashcards

Biologics Flashcards

1
Q

Examples of biologics

A

1) Hormones e.g. erythropoietin (EPO), human growth protein (HGH)
2) IgG
3) Monoclonal antibodies (MABs)
4) Insulin

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2
Q

Characteristics of biologics

A

Large molecules
Complex structures, difficult to characterize (and copy)
Heat sensitive
Susceptible to low pH, microbial contamination

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3
Q

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)

A

1) Extracted directly from human/animal tissues
2) Produced in living cells via biotechnology
3) Based on gene, tissue/cells engineering

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4
Q

Examples of biotechnology

A

1) Recombinant DNA (rDNA) technology

2) Hybridoma technology

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5
Q

rDNA technology steps

A

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

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6
Q

What does the recombinant plasmid usually contain

A

1) Gene of interest / Target gene / Expression gene
2) Promoter
3) Antibiotic resistance gene

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7
Q

Types of host cells

A

1) Microbial cells (bacteria, yeast)

2) Mammalian cells (CHO cells commonly used)

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8
Q

Hybridoma technology steps

A

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

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9
Q

Characteristics of hybridomas

A

1) Antibody-producing ability
2) Immortal
3) Able to replicate rapidly & continuously

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10
Q

Mammalian cells:

1) Application
2) Cultivation
3) Secretion of products
4) Yield
5) Type of protein produced
6) Safety

A

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)

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11
Q

Microbial cells:

1) Application
2) Cultivation
3) Secretion of products
4) Yield
5) Type of protein produced
6) Safety

A

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

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12
Q

1987 WHO study: Mammalian cell lines may be used if purification process can _____

A

Reduce residual tumour DNA to < 10 ng per dose

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13
Q

US FDA Definition of biosimilars

A

A biological product that is ‘highly similar’ and has no significant clinical difference from an existing FDA approved reference product (usually the innovator product)

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14
Q

Comparative tests/studies to determine high degree of similarity between biosimilar & reference product include:

A

1) Molecular structure & potency (bioactivity)
2) Toxicity (non-human/animal study)
3) PK-PD studies (clinical/human study)
4) Immunogenicity (clinical/human study)

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15
Q

Critical GMP & QA Issues in Manufacture of BDMP

A

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

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16
Q

How is inspection of biological products carried out

A

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

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17
Q

Control of master cell bank

A

1) Well characterized cells derived from specific cell lines, of a specific passage level
2) Stored under defined conditions

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18
Q

Control of working cell bank

A

1) WCB used to provide working cells for manufacturing
2) Derived from one or more containers of MCB
3) Must be tested before use

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19
Q

Testing of cell banks is a ___ activity that requires ____

May be outsourced to ____

A

Highly specialized activity Sophisticated equipment, infrastructure & expertise
Specialized contract testing laboratories

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20
Q

What to test for in cell banks

A

1) Genetic stability (using DNA fingerprinting, Southern Blot)
2) Endogenous viruses (in mammalian cell lines)
3) Adventitious viruses
4) Residual DNA (from hybridoma)

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21
Q

Inspection of cell banks - Items to inspect

A

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

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22
Q

Cell culturing is used as a reference point to ____

A

Categorize biopharmaceutical processes into upstream & downstream processes

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23
Q

Upstream processes:

A

Cell banking
Cell expansion and scale up
Cultivation
Harvesting and clarification

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24
Q

Downstream processes:

A

Primary purification
Viral inactivation
Secondary purification
Viral removal
Batching and storage of bulk biological API
Formulation, filling and packaging, QC of finished biological product

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25
Q

Growth requirements for bacteria

A

1) Physical: Temperature, pH, osmotic pressure

2) Chemical: Water, oxygen, C, N, S, P, Trace elements, organic growth factors

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26
Q

Types of fermentation

A

1) Batch fermentation
2) Continuous fermentation
3) Fed-batch fermentation

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27
Q

Batch fermentation

A

Closed system
Only oxygen continuously added
Concentration of media decreases continuously; Toxic metabolites accumulate
Growth phase: Lag, log, stationary, decline
Advantages: Low chance of contamination
Disadvantages: Low yield

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28
Q

Continuous fermentation

A

Open system
Sterile fresh media media added continuously; Waste products removed continuously
Growth phase: Log phase
Advantages: High yield
Disadvantages: High chance of contamination

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29
Q

Fed-batch fermentation

A

Semi-open/Semi-closed system

Fresh sterile media added & waste products removed at periodic intervals

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30
Q

Types of cell culturing

A

1) Anchored system

2) Suspension system

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31
Q

Nutrient media composition needed for mammalian cells

A

Composition is crucial for consistent yield & quality
More sophisticated/richer than that needed for bacterial cells
Any minor deficiencies will have major impacts on cell growth & protein production

32
Q

Types of nutrient media preferred for mammalian cells

A

Move towards serum-free media due to safety and cost concerns (e.g. bovine serum might contain BSE prions)

33
Q

Crucial parameters in bioreactor

A

Optimal stirring speed, circulation & shear dynamics

34
Q

Cell culture factors affecting product quality

A

1) Materials - Cell line, nutrient media, buffers, reagents, water
2) Method - Culture process and conditions
3) Machine - Bioreactors/Fermenters, sensors, computer software & hardware
4) Manpower - Aseptic processing techniques, restricted access
5) Premises - Clean rooms, microbiological monitoring program

35
Q

Control of:

1) Starting materials in cell culture
2) Cell culture conditions
3) Maintenance and cleaning of fermenters/bioreactors

A

1) Reliable suppliers, specification & test results of starting materials, water purification system
2) Formulation of nutrient media (BSE free), growth requirements (temperature, oxygen, pH), fermentation/culture time, growth rate, culture purity, fatty acid profile
3) SOP & records of maintenance & cleaning, move towards SUTs

36
Q

Harvesting in microbial cells: Protein products are secreted ____, thus microbial cells have to undergo ____ to release proteins

A 
Intracellularly;
Cell disruption (lysis)
37
Q

Cell disruption methods

A

Mechanical: Homogenization, milling, oscillation, ultra-sonification
Non-mechanical: Surfactants, alkali, solvents, lysozyme, osmotic shock

38
Q

____ cell disruption methods are more damaging

A

Mechanical

39
Q

Disadvantages of cell disruption

A

1) Heat denaturation of product
2) Oxidation of product
3) Uncontrolled release of other cell components together with protein product

40
Q

Conventional harvesting process in mammalian cell system

A

Cell separation using centrifugation & filtration (depth filtration, membrane filtration)

41
Q

Purpose of the following processes in harvesting:

1) Centrifugation
2) Depth filtration
3) Membrane filtration

A

1) Removes whole cells and large debris (micron size)
2) Removes cell debris (micron and sub-micron size)
3) Serves as an aseptic boundary

42
Q

Objective of purification

A

Removal of all known impurities to obtain pure protein product

43
Q

Purification methods: ____

Most common methods: ___

A

Chromatography, Ultrafiltration, Adsorption, Precipitation

Chromatography, Ultrafiltration

44
Q

How chromatography works

A

Different molecules in a mixture (mobile phase) are separated while moving through a stationary phase

45
Q

Chromatography in purification of biological products

A

Affinity chromatography: Protein product binds/interacts specifically with stationary phase
Bound protein is further purified

46
Q

What to inspect in purification process

A

1) Control of chromatographic columns, buffers, other materials used in purification process
- Supplier approval
- Specifications for columns/buffers
- Maintenance and storage of columns/buffers
- Effective cleaning of columns
- Use of single-use, disposable columns

2) Level of purity
- Depends on use of products (chronic VS single use)
- Higher purity level needed for products for chronic diseases

47
Q

Virus contamination may arise from

A

1) Original source of cell lines (inherent endogenous virus)

2) Adventitious virus introduced during production

48
Q

Viral contamination can lead to

A

1) Decrease in product quality
2) Facility shutdown
3) Disruption of medicine supply
4) Business impact

49
Q

Types of viral clearance methods

A

1) Inactivation methods

2) Removal methods

50
Q

Viral inactivation methods

A

1) Chemical methods
- Low pH
- Surfactants / Detergents
2) Physical methods
- Heat treatment
- UV

51
Q

Viral removal methods

A

1) Chromatography
- Size exclusion
- Ion exchange
- Affinity
- Revere phase
- Hydrophobic interaction
2) Precipitation (e.g. ammonium sulfate)
3) Membrane filtration
4) Nanofiltration

52
Q

Establishing freedom of biological product from virus requires both:

A

1) Virus testing

2) Validation of viral clearance

53
Q

Importance of viral clearance validation

A

1) Plays an essential and important role in establishing viral safety
2) Testing for viral contamination alone is insufficient to conclude product safety - due to limitations of viral tests

54
Q

Limitations of viral contamination tests

A

1) No single test is able to demonstrate presence of all known viruses
2) Minimum level of contamination required before test gives positive result
3) Tests limited by statistical considerations in sampling

55
Q

Aim of validation of viral clearance

A

1) Demonstrate that manufacturing/purification process is able to eliminate or substantially reduce virus that what may potentially be present in unprocessed bulk material
2) To obtain best reasonable assurance that biological product is free of viral contamination

56
Q

A virus panel consists of __ representative virus models with different ____ to demonstrate ___ of viral clearance capability

A

4
Physiochemical properties, size, chemical resistance
Robustness

57
Q

Virus models / Virus panel chosen for viral clearance studies:

A

1) MMV (minute virus of mice)
2) Reo-3 (retrovirus type-3)
3) MuLV (murine leukemia virus)
4) PRV (pseudorabies virus)

58
Q

Characteristics of 4 virus models chosen

A

1) MMV - ssDNA, non-enveloped, very high resistance
2) Reo-3 - dsRNA, non-enveloped, high resistance,
3) MuLV - ssRNA, enveloped, low resistance
4) PRV - dsDNA, enveloped, low-medium resistance

59
Q

Definition of a batch

A

A specific quantity of drug/other materials that is intended to have uniform quality and character, within specified limits, and is produced according to the same manufacturing order during the same manufacturing cycle

60
Q

Inspection of batching & storage of bulk biological API - Items/Areas to inspect

A

1) Storage conditions in building and facilities
- Must maintain the product temperature within limits, as defined on product label (based on stability tests)
- Warehousing, storage and/or holding of product areas should be of adequate size for their intended purpose i.e. no overcrowding
2) Storage in refrigerators and/or freezers with recording & alarm systems
3) Stability testing studies (submitted in DMF for MA) can be verified during inspection

61
Q

Biological products are formulated into ___

A

Sterile dosage forms (injections)

62
Q

Control of formulation, filling & packaging

A

1) Choice of excipients - should be heat stable and not prone to microbial growth
2) Water purification system
3) Environmental & microbiological monitoring (on a regular basis)
4) Design & construction of facility (certified clean rooms)
5) Validation of aseptic filling (sterile media fills)
6) Validation of autoclaving of packaging material (bioburden monitoring)
6) Validation of final container-closure integrity (to prevent ingress of microorganisms)

63
Q

Bioanalytical test methods (QC test methods)

A

1) DNA sequencing - Test for genetic stability in cell banks
2) DNA hybridization - Test for residual DNA impurities
3) Reverse transcriptase (RT) & MAP/HAP tests, Electron microscopy - Test for viral contamination
4) PCR - Test for specific viral sequence
5) SDS-PAGE & Immunoassay - Test for media protein impurities
6) Peptide mapping, IEF, MS - Test for mutant proteins
7) Microbial limits test, Sterility testing - Test for bacteria, yeast, fungi
8) Pyrogen test - Test for endotoxins produced by host cell
9) Modified 21 CFR method - Test for mycoplasma contamination

64
Q

Importance of validating bioanalytical test methods

A

Assures data reliability

65
Q

Validation of bioanalytical test methods is based on the following key performance test parameters:

A

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

66
Q

Sensitivity

A

Lower limit of detection

Lowest concentration of analyte that an analytical procedure can reliably differentiate from background noise

67
Q

Specificity

A

Ability to assess unequivocally the analyte in the presence of other expected components (e.g. impurities, degraded products, matrix)

68
Q

Precision

A

Closeness of agreement among measurements obtained from multiple samples under defined conditions

69
Q

Repeatability

A

Ability to repeat under the same operating conditions, within a short period of time

70
Q

Intermediate precision

A

Able to obtain the same results within laboratory variations, different days, analysts, equipment etc.

71
Q

Reproducibility

A

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

72
Q

Accuracy

A

Degree of closeness between determined value and nominal/known true value under prescribed conditions

73
Q

Quantitation range

A

Range of concentration, including ULOQ and LLOQ, that may be reliably and reproducibly quantified using a concentration-response relationship

74
Q

Linearity

A

Extent to which relationship between experimental response value & concentration of analyte approximates a straight line

75
Q

Robustness

A

Ability of method to give accurate, precise results under normal operating condition variations

Regulation (6 decks)

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