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
What is the source of PackGene cell bank?
PackGene’s h293 cell bank is officially authorized for commercial use.
What is the AAV output for a single batch fermentation?
PackGene offers single batch fermentation at several volumes, including: 2L, 7L, 25L, 50L, and 200L. AAV yields for each of these production volumes varies across AAV serotypes. As an example, AAV9 is a medium to high-yielding serotype, and expected yields for AAV9 are as follows:
| Expected yield for AAV9 | |
| Volume | Yield |
| 2L | 1E+14GC |
| 50L | 1E+16GC |
| 100L | 2E+16GC |
What method is used to determine the empty shell rate for AAV samples?
Viral empty shell rate can be determined using several techniques including anion chromatography HPLC, Analytical Ultracentrifugation (AUC), Transmission Electron Microscopy (TEM), CyroTEM, or VG Titer/Capsid titer. AUC, TEM and CyroTEM are typically not suitable for quantitative quality control determinations and thus PackGene’s standard method for empty shell rate determination is anion chromatography HPLC. PackGene can provide additional CyroTEM and AUC analytical services to serve as a secondary verification of the results derived from anion chromatography HPLC.
How are GC/ml and vg/ml related to one another, and how does PackGene determine GC/ml for AAV products?
The terms genome copies per ml (GC/ml) and viral genomes per ml (vg/ml) are interchangeable and equal in most cases. At PackGene we may test GC by both qPCR and ddPCR. Testing by qPCR involves the use of a calibration standard while ddPCR may use optional reference products. Measurements by qPCR are more likely to be influenced by inter-lab and inter-operator variables, and ddPCR generally shows lower %RSD precision. Typically, GC is determined by qPCR during process exploration phase and for intermediate products while the GC of final products is more often determined by ddPCR.
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