What is biotechnology?
The use of living cells/organisms, or their products to modify human and animal health, mankind and his environment.
Briefly describe the steps in Recombinant DNA Technology.
- Design a recombinant plasmid, containing the:
- Target gene
- Promoter for target gene
- Abx resistance gene - Transformation of host cell (e.g. via heat shock)
- Select cells that are successfully transformed by growing the cells in a petri dish containing Abx
- Using Antibiotic Resistance Genes - Fermentation / Cultivation of cells to produce proteins.
Briefly describe the steps in Hybridoma Technology.
- Specific antigen are injected into MURINE (spleen) or HAMSTER (ovary) cells. They produce specific cells which produce antibodies. These cells are harvested.
- As mammalian cells duplicate slowly, they are fused together with a myeloma (tumour cell) to form a hybridoma.
- Hybridoma are screened for the production of desired Antibodies/Protein
- Hybrid cells are cultivated to produce the proteins, undergoes clonal expansion to produce a large number of protein/Ab
What is the main safety concern with using mammalian host cells?
The possibility of contamination with potentially oncogenic host cell DNA.
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?
10 nanograms (ng) per dose
10ng/dose
What are the critical GMP & QA issues in the manufacture of biotechnology-driven medicinal products?
(Hint: Consider the starting materials and finished product)
- Genetic stability - assuring genetic stability of the host cells, plasmid & GOI
- Quality & yield - Assuring quality & yield through consistent manufacturing processes
- Absence of impurities - Absence of biological and chemical impurities from nutrient media and starting materials.
- Absence of viruses - Absence of inherent endogenous viruses and viruses introduces during production (e.g. EBV, Herpesvirus, Retroviruses, Adventitious Viruses)
- Elimination of residual DNA from hybridoma of mammalian cell (to 10ng/dose or below)
What is a biosimilar product?
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.
What are the major steps in the manufacture of biotechnology-driven medicinal products? (Hint: 7 steps from the cells to the finished product)
- Cell banking
- Cell cultivation
- Harvesting
- Purification
- Viral clearance
- Batching and storage of bulk biological API
- Formulation, packaging and release of finished product
What is inspected during inspection of Cell Bank System?
- Documentation of cell origin & history
- Management of cell banks (storage, contamination prevention, QC testing etc)
- Contract testing laboratories (Used to test the integrity of Cell bank cultures - genetic stability, presence of contaminants etc)
What are the important factors controlled during Cell Cultivation to achieve high quality and high yield product?
4M + 1P:
- Manpower
- Machine
- Methods
- Materials
- Premises
What are some considerations during inspection of the Harvesting process?
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.
What is inspected during inspection of the Purification Process?
- 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)
How is the validation study of viral clearance conducted?
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.
Why is validation of viral clearance necessary?
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.
What is a “batch”?
A specific quantity of a drug or other materials that is intended to have:
- UNIFORM CHARACTER and QUALITY
- Within specified limits
- Produced according to a single manufacturing order during the same cycle of manufacture.
Biological products may be manufactured in sub-lots and pooled as a bulk batch. T/F?
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
Biologics can be formulated into oral dosage formed. T/F?
False.
Biologics are formulated as sterile injections!
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 )
- Sensitivity
- Specificity
- Precision
- Repeatability
- Intermediate precision
- Reproducibility
- Accuracy
- Quantitation range
- Linearity
- Robustness
Most importantly: Specificity, Reproducibility, Accuracy
What is the main biopharmaceutical manufactured in ASEAN currently?
Vaccines
The ASEAN Mutual Recognition Arrangement (MRA) on GMP Inspection includes biopharmaceuticals. T/F?
False.
It currently excludes biopharmaceuticals, but the scope will be expanded by the end of this year (Nov 2020)
One of the few (3) most important Key Performance Test Parameters Under Bioanalytical Test Validation is Specificity, Reproducibility and Accuracy. Define the terms.
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)
Viral Clearance methods include
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)
What are the 2 most common Virus REMOVAL method? (Note: Referring to Physical Removal)
Column Chromatography and Nano-filtration
Methods to purify protein product during Purification stage?
Precipitation, Adsorption, Ultrafiltration (not nanofiltration) and Chromatography
