Q: Are pH measurements required, and is a large amount of sample wasted to carry out pH measurements?
A: 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.
Q: What is loading?
A: In accordance with the Pharmacopoeia General Rules 0942, we use the minimum filling quantity inspection method for detecting sample loading quantity.
Q: How to interpret A260/A280 value?
A: 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.
Q: What tests are performed to differentiate rAAV capsid proteins from specific protein impurities?
A: 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.
Q: How is rAAV infection titer measured, given that rAAV does not integrate the host genome?
A: The current standard for determining infection titer is TCID50 (Median Tissue Culture Infectious Dose). In this assay we test the infection rates of rAAV sample serial dilutions in H5 cells. This initial test is then followed by qPCR detection of the rAAV genome.
Q: If I have determined that a rAAV DNA vector reliably drives transgene expression in host cells prior to rAAV packaging, can I assume that this DNA vector will also drive transgene expression after it is packaged into an rAAV that is then used to infect cells?
A: Successful transgene expression via rAAV infection relies on several factors beyond the functionality of the DNA vector. We therefore recommend that independent validation of transgene expression is performed for all packaged rAAV.
Q: What does rcAAV refer to, and are there regulations regarding rcAAV content in GMP rAAV samples?
A: The term rcAAV stands for replication-competent AAV. In most cases rAAVs are designed to be replication incompetent. Thus, rAAV samples should not contain rcAAV and regulations require negligible replication capability for GMP rAAV samples.
Q: What is the difference between visible foreign matter and insoluble particles?
A: Visible foreign matter can be identified by visual inspection. According to pharmacopoeia general rule 0904, visible foreign matter in injections, ophthalmic liquid preparations, and sterile APIs can be visually observed under specified conditions . Visible foreign matter is usually derived from insoluble particles of a size or length is greater than 50μm.
Insoluble particles between 10μm and 50μm cannot be seen with the unaided eye and must therefore be detected by instrumentation. Insoluble particles identification is commonly carried out with an insoluble particle analyzer. According to pharmacopoeia general rule 0903, evaluation of the size and quantity of insoluble particles within materials to be delivered by intravenous injection (solution injection, sterile powder for injection, concentrated solution for injection) can be made according to the project nature.
Q: If I have successfully carried out several in vivo experiments in mice may I assume that mycoplasma and bacterial endotoxin levels are within acceptable ranges and forgo direct testing for these contaminants?
A: Reagents used for Gene Therapies should abide by all domestic regulations in the “Guiding Principles for Quality Control of Human Recombinant DNA Products” by the EDC. Under these standards Mycoplasma and endotoxin testing is required.
Q: What is the source of PackGene cell bank?
A: PackGene’s h293 cell bank is officially authorized for commercial use.
Q: What is the AAV output for a single batch fermentation?
A: 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 |
Q: How are GC/ml and vg/ml related to one another, and how does PackGene determine GC/ml for AAV products?
A: 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.
Q: Do specific primers need to be designed to determine GC/ml for a custom AAVs?
A: Genome copies per ml (GC/ml) can be determined using primers directed at common vector elements during the early development stage, and thus we use common features such polyA segments or ITRs to determine GC/ml during this stage. However, it is recommended that specific primers targeted at AAV vector transgenes are used for testing the GC of GMP products. Our final fast service titer test is designed to use transgenes specific primers.
Q: What method is used to determine the empty shell rate for AAV samples?
A: 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.
Q: What is the expected empty shell rate PackGene AAV Fast Services?
A: The expected empty shell rate for our AAV Fast Services varies across AAV serotypes. As one example, the expected empty shell rate for AAV9 generated through our AAV Fast Service is lower than 10%.
Q: Is verification of cell banks in accordance with the Chinese Pharmacopoeia 2015 Three General Principles "The Preparation and Verification Regulations of Animal Cell Matrix for the Verification of Biological Products"?
A: Yes.
Q: HIV, HBV, HCV testing, pyrogen-free tests, and wild virus tests are not in the standard quality control services list. Do these tests need to be applied?
A: The harvest liquid generated through PackGene’s AAV fast service will be broadly tested for exogenous viral elements as necessary following an evaluation of project characteristics. Both the original harvest liquid and final deliverables for the AAV Fast service will be devoid of such exogenous viral elements.
Q: What does PackGene consider acceptable error or deviation ranges for standard QC analysis methods?
A: The %RSD of general biochemical methods is usually 15%-25%, and the accepted standard of ddPCR is 10%. In most cases the %RSD for PackGene’s QC ddPCR verification data is lower than 5%, and the %RSD of TCID50 is lower than 10%.
Q: Are there any requirements for the number of samples sent in batches?
A: It is recommended that the sample volume of a single commissioned express delivery is not less than 50uL to avoid the effects of freezing and thawing, evaporation, and tube wall adhesion. The recommended sample delivery volume for genome titer, plasmid DNA residue, rcAAV, etc. is more than 10ul. The recommended sample delivery volume for infection titer is more than 20ul, and the sample delivery amount for empty shell rate testing is 5E+13vg.