Titer is not a single indicator. vg/mL describes the amplifiable vector genomic signal, capsid particles/mL describes total capsids, and infectivity or transduction units reflect the functional output in a specific system. Different primers, standards, lysis and cell-free DNA removal conditions will change the qPCR/ddPCR results, so cross-laboratory comparisons should first check methods instead of just comparing numbers. [6-1 to 6-5]
Explanation of three levels of quality
|
Level |
Representative indicators |
Questions that cannot be answered individually |
|
Quantity |
vg/mL, total vg, total capsids |
Whether the genome is complete and whether the particles can be transduced |
|
Composition |
Empty/full shell, partially loaded, aggregate, residual DNA/protein |
Expression efficacy in target cells |
|
Function |
Infectivity, transduction efficiency, transgene expression or biological activity |
Direct comparability across cell lines and species |
The empty/real shell ratio should be interpreted by AUC, AEX, mass spectrometry, or other orthogonal means; "real shells" may still contain partial genomes or anomalous fragments. Research in 2023-2024 will continue to promote multi-attribute detection of genome integrity and capsid identity. Therefore, QC configuration should be based on uses and risks rather than pursuing the optimization of a single indicator. [6-3 to 6-5]
References for this chapter
[6-1] Gimpel AL, et al. Analytical methods for process and product characterization of recombinant AAV-based gene therapies. Mol Ther Methods Clin Dev. 2021;20:740-754. DOI:10.1016/j.omtm.2021.02.010. PMID:33738328. [H]
[6-2] Werle AK, et al. Comparison of analytical techniques to quantitate the capsid content of AAV vectors. Mol Ther Methods Clin Dev. 2021;23:254-262. DOI:10.1016/j.omtm.2021.08.009. PMID:34703846. [H]
[6-3] Richter K, et al. Purity and DNA content of AAV capsids assessed by analytical ultracentrifugation and orthogonal biophysical techniques. Eur J Pharm Biopharm. 2023. PMID:37196871. [N]
[6-4] Smith J, et al. Rapid AAV identity testing through intact mass analysis of full-length capsid proteins. J Proteome Res. 2023. DOI:10.1021/acs.jproteome.3c00513. PMID:38123456. [N]
[6-5] Park S, et al. Enhancing AAV2 and AAV9 production with high full-capsid ratio by design-of-experiment optimization. Biotechnol J. 2024;19:e2300667. DOI:10.1002/biot.202300667. PMID:38479987. [N]