Banking of master and working cell banks (MCB, WCB) is performed in a separate and dedicated GMP facility. PackGene has an experience team capable of preparing and banking cell lines suited for your specific projects. Our team has extensive experience in the preparation of both adherent and suspension HEK293/293T cell lines and will perform high yield screening and identification of candidate cell lines to ensure that high yield cell lines are obtained prior to cell banking. Clients may also provide their own cell lines for banking at PackGene.
PackGene’s cell banks for AAV gene and cell therapy are structured to ensure quality and consistency across the production lifetime of a biological product. We take a two-tiered approach to preserves the integrity of therapeutic producing cells. First, we create a master cell bank that is derived from a single clone and is expanded and extensively characterized for use in future production. This master cell bank can then be divided into dozens of working cell banks to ensure the stability of cell passages while maintaining the stability of the master cell bank. Working cell banks can be used for fast service production without fear of contaminating, or otherwise compromising, the integrity of the master cell bank.
Final deliverable cells are subject to a rigorous assay procedure to confirm the suitability of the cells as well as to rule out detrimental mutations and to ensure that cells do not produce carcinogenic substances.
Cells are resuscitated and passaged from the working cell bank, transferred to the bioreactor for passaged digestion, cultured according agreed upon specifications, and then digested for lyophilization or sent for testing. All cell resuscitation and passaging methods are carried out according to the production process SOP.
The establishment of cell banks at PackGene adheres to the regulatory requirements laid out in the pharmacopoeia. Tests include, but are not limited to:
- Identification tests: cell morphology, species, identification, insertion gene identification, large T antigen nucleic acid sequence determination
- Bacterial and fungal tests
- Mycoplasma testing: culture method, DNA fluorescent staining
- Bifidobacterium examination
- Intracellular and extracellular virulence factor examination
- Integrity, copy number determination, transgene sequence determination
- Quantitative tumorigenic examination
In addition to the minimum testing requirements for cell banking several additional testes are recommended in the Pharmacopoeia. PackGene offers these tests as addon services for cell banking, including:
- Cell identification
- Purity
- Genotyping/phenotype
- Genetic stability
- Tumorigenicity/carcinogenicity
- Identification, integrity and copy number of the introduced sequence
- Endogenous/exogenous viral factors
- Sterility testing
- Mycoplasma detection
- Fungus detection
- Spiroplasma detection
Resources
Are pH measurements required, and is a large amount of sample wasted to carry out pH measurements?
Measurement of pH is a mandatory for the release of rAAV Fast Service deliverables. A micro pH electrode may be used to save sample and thus the required sample volume to perform pH measurements is only ~15uL-100uL.
What is loading?
In accordance with the Pharmacopoeia General Rules 0942, we use the minimum filling quantity inspection method for detecting sample loading quantity.
How to interpret A260/A280 value?
A260/A280 is the ratio of sample absorbance measured at wavelengths of 260nm and 280nm. This measure is commonly thought to represent the ratio of DNA to protein in a sample. For rAAV, A260/A280 can used as a measure of the full to empty shell rate and to identify protein contamination. Low A260/A280 levels may suggest that the empty shell rate is high. Alternatively, high A260/A280 may suggest that the sample has been contaminated with proteins that are not incorporated into the AAV capsid shell. The greatest advantages of this measure are its convenience and speed.
What tests are performed to differentiate rAAV capsid proteins from specific protein impurities?
SDS-PAGE is used to identify rAAV capsid proteins. In addition, SDS-PAGE can be used to directly identify specific protein impurities including the presence of host proteins, BSA, or degraded AAV capsid proteins.
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