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FAQ for GMP rAAV

Q: Does the pH measurement refer to the pH before or after concentration? A large amount of sample is required for pH measurement and will it lead to waste?

A: The pH measurement is a mandatory assay for the release of rAAV products. If you want to save samples, it is recommended to use a micro pH electrode. The sample volume needs only 15uL-100uL.

Q: How to understand loading?
A: In accordance with the Pharmacopoeia General Rules 0942, the minimum filling quantity inspection method is used to carry out the detection of the loading quantity. If there is any change, it is recommended to communicate with CDE.
Q: A260/A280 reflects the relative ratio of DNA/protein. What is its specific meaning?
A: A260/A280 is an intuitive manifestation of DNA/protein. For rAAV, this indicator can reflect the empty shell rate or other pollution. When A260/A280 is too small, the empty shell rate might be high. When A260/A280 is too high, other pollution might be high. The advantages of this method are convenient and quick.
Q: Generally speaking, protein impurities come from host cells and culture media. If host protein residues and BSA have been tested, what are the considerations for detecting protein impurities separately?
A: SDS-PAGE is to identify the capsid protein band of rAAV, and it can also directly reflect the protein impurity. In addition to host protein and BSA, protein impurities may also be a capsid degradation product.
Q: How to detect the infection titer for viral vectors that do not integrate the host genome like rAAV?
A: The current standard for testing is TCID50, and the basic principle is based on the helper virus adenovirus H5 cell experiment and the following qPCR detection.
Q: As for gene expression and function, considering that the same plasmid package rAAV has been applied for in vitro experiments and mouse experiments, these two items have been completed. Can they be omitted as long as the DNA sequence is correct?

A: Gene expression is related to process stability and is also an important item of potency. As for functional testing, it is recommended to communicate with CDE and carry out testing from a scientific perspective.

Q: Does rcAAV refer to double-stranded complementary AAV? My AAV is not self-complementary and I wonder the reason for this testing.

A: rcAAV refers to the replication competent AAV, which limits the residues of AAV with replication capability and is also a key item required by regulations.

Q: What is the difference between insoluble particles and visible foreign matter?
A: The inspection method for visible foreign particles is visual inspection. The main criterion is that there is no foreign particles larger than 50μm. According to pharmacopoeia general rules 0904, visible foreign particles refers to the insoluble matter that exists in injections, ophthalmic liquid preparations and sterile APIs and can be visually observed under specified conditions. Its particle size or length is usually greater than 50μm.

The inspection method for insoluble particles usually is an insoluble particle analyzer, and the range of insoluble particles is 10μm and above. According to pharmacopoeia general rules 0903, in order to check the size and quantity of insoluble particles in intravenous injections (solution injection, sterile powder for injection, concentrated solution for injection) and sterile bulk drugs for intravenous injection, evaluation can be made according to the project nature.

Q: For mycoplasma and bacterial endotoxins, two batches of mouse experiments have been done before with good results. Can these two testing be omitted?

A: Gene therapy should refer to domestic regulation “Guiding Principles for Quality Control of Human Recombinant DNA Products” by EDC. Mycoplasma and endotoxin should be tested. If you have further questions, it’s recommended to communicate with the CDE.

Q: What is the source of PackGene cell bank?

A: PackGene h293 cell bank is officially authorized for commercial use.

Q: What is the production volume of a single batch fermentation and the corresponding AAV output?

A: the volume of a single batch attached fermentation include 2L, 7L, 25L, 50L, 200L. AAV yields may be different for various AAV serotypes. Take AAV9 (a medium to high yielding serotype) as an example. The yield of 2L, 50L and 100L are 1E+14GC, 1E+16GC and 2E+16GC.

Q: Dose the unit GC/ml and vg/ml share a conversion relationship? How does PackGene test the GC/ml of AAV? Provided with qPCR, should it apply virus-specific primers or insert sequence-specific primers?
A: GC/ml=vg/ml. qPCR and ddPCR are two methods to test GC. qPCR involves the standard calibration and ddPCR can set reference products, which is optional. ddPCR shows lower %RSD precision, especially the intermediate precision. qPCR is more likely to be affected laboratories and operators. qPCR is usually applied during process exploration and intermediate products, while ddPCR are usually applied in final products.

GC method development can use specific primers for GOI target genes, and elements such as polyA or ITR can also be used as universal detection in the early development stage. It is recommended to use specific primers for GMP products, and the final product titer test is based on specific primer.

Q: Has the empty shell rate been measured for AAV products? What method is used to maintain the empty shell rate at what level?

A: Usually AUC, TEM, CyroTEM, or VG TITER/CAPSID TITER is applied, but AUC, TEM, CyroTEM are not suitable for QC. Currently, anion chromatography HPLC is used for process development. PackGene can provide analytical method development services and compare and verify with CyroTEM and AUC test results. The empty shell rate obtained by column chromatography for each serotype is different. PackGene Biotechnology produces AAV9 with empty shell rate lower than 10%.

Q: Is the verification of the cell bank 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: There are no HIV, HBV, HCV testing, pyrogen-free tests, and wild virus testing in the general inspection items. Do these tests need to be applied?

A: The product harvest liquid will be tested for exogenous viral factors according to the process characteristics and will not be included in the standard of the original liquid and final product.

Q: The acceptable error or deviation range of the current analysis method?

A: The %RSD of general biochemical methods is usually 15%-25%, the acceptance standard of ddPCR is 10%, but usually the verification data is lower than 5%, and the %RSD of TCID50 is lower than 100%.

Q: Are there any requirements for the amount 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. should be more than 10ul. the recommended sample delivery volume for infection titer should be more than 20ul, and the sample delivery volume for testing the empty shell rate is recommended to reach 5E+13vg.