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FAQ about GMP rAAV

AAV design

Q: Does the pH measurement refer to the pH before or after concentration, and is a large amount of sample wasted for pH measurements?

A: The pH measurement is a mandatory assay for the release of rAAV Fast Services. A micro pH electrode may be used to save samples and thus the required sample volume is only 15uL-100uL.

Q: What is 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: How to interpret A260/A280 value?

A: A260/A280 is the measured sample absorbance at 260 and 280 nm. This ratio can be thought to represent the ratio of DNA to protein in a sample. For rAAV, A260/A280 can thought to reflect the full to empty shell rate or to identify other protein contamination. When A260/A280 is too small, the empty shell rate might be high. When A260/A280 is too high, protein contamination may be present be high. The greatest advantages of this measure are its convenience and speed.

Q: What are the considerations are made for detecting protein impurities separately?

A: SDS-PAGE is primarily used to identify the capsid protein band of rAAV. In addition, SDS-PAGE can directly identify any protein impurities. Possible protein impurities may include host proteins, BSA, and degraded AAV capsid proteins.

Q: How is infection titer measured for of viral vectors that do not integrate the host genome such as rAAV?

A: The current standard for testing is TCID50 (Median Tissue Culture Infectious Dose). IN this assay we test infection rates in H5 cells following exposure to various AAV sample dilutions. This test is then followed by qPCR detection.

Q: As If it has been determined that an rAAV DNA vector is capable of driving transgene expression in virto or in vivo before packaging, can it be assumed that rAAV generated by packing this same DNA vector will also drive transgene expression?

A: Successful transgene expression by rAAV infection is dependent on many processes independent of DNA vector functionality. It is therefore recommended that transgene expression is validated for all rAAV are independent of DNA vector functionality tests.

Q: Does rcAAV refer to double-stranded complementary AAV?

A: The term rcAAV refers to replication-competent AAV. rAAV should not be relication-compotenet and thus replication capability is also a key item required by regulations and should be negligible.

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 rules 0904, visible foreign particles refer to the insoluble matter that exists in injections, ophthalmic liquid preparations and sterile APIs and 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 naked eye and must therefore be detected by instrumentation – this is usually carried out with an insoluble particle analyzer. According to pharmacopoeia general rules 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: For mycoplasma and bacterial endotoxins, two batches of mouse experiments have been done before with good results. Can these two testings 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 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 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? Do specific primers need to be designed to determine GC/ml for a custom AAVs?

A: The terms genome copies per ml (GC/ml) and viral genomes per ml (vg/ml) are interchangeable and equal. At PackGene we can test GC by both qPCR and ddPCR. Testing by qPCR involves the use of a calibration standard while ddPCR may use optional reference products. qPCR is more likely to be influenced by facts associated with different laboratories and operators 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 are more often determined by ddPCR.

During the early development stage GC can be determined using primers directed at common vector elements such as polyA segments or ITRs. However, it is recommended that specific primers targeted at AAV vector transgenes are used for testing the GC of GMP products, and our final fast service titer test is designed to use such a specific primer.

Q: What method is used to determine the empty shell rate for AAV samples, and what is the expected empty shell rate PackGene AAV Fast Services?

A: Viral empty shell rate can be determined using several techniques including: Analytical Ultracentrifugation (AUC), Transmission Electron Microscopy (TEM), CyroTEM, or VG TITER/CAPSID TITER. Typically, AUC, TEM and CyroTEM are not suitable for quality control and thus PackGene’s standard method for empty shell rate determination is anion chromatography HPLC. PackGene can provide additional CyroTEM and AUC analytical services wich may serve to verify results derived from anion chromatography HPLC.

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 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: 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 the projects process 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%, the accepted 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 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 should is more than 20ul, and the sample delivery amount for empty shell rate testing is 5E+13vg.