Concluding Remarks

The percentage of full adeno-associated virus (AAV) vector particles is quantified using the gold standard analytical ultracentrifugation (AUC) method. However, alternative techniques using (i) the capsid titer (using capsid ELSIA) and vector genome titer (using ddPCR/Q-PCR), (ii) transmission electron microscopy, and (iii) electrophoresis have been employed for % full determination [1]. These techniques can be expensive, laborious, reagent intensive, time-consuming, and not amenable to in-line or at-line monitoring. Owing to the wide variability of samples in terms of titer, buffer matrix, and % full, it is crucial to develop a quicker and easier characterization technique to quantify the titer and empty/full ratio for (i) more rapid, reliable characterization for quick decision making, and (ii) act as an orthogonal technique to supplement the existing techniques. In this work, we developed a method for quantifying AAV9 percent full using a simple, direct, and rapid UV-Vis method based on prior art demonstrating a similar approach for AAV2 [2]. The absorbance measurement of the samples at 260 nm (A₂₆₀), 280 nm (A₂₈₀), and the A₂₆₀/A₂₈₀ ratio enable the accurate prediction of % full capsids of process intermediates that were orthogonally verified by AUC and ddPCR / capsid ELISA. Highly pure full and empty AAV9 preparations were used to generate this serotype-specific model. These were produced using a hybrid sample preparation method, comprising ultracentrifugation and automated, UV-based fractionation. The developed model enables rapid, online/at-line, and accurate estimation of the fraction of full AAV9 vector particles using UV-Vis absorbance, circumventing time-consuming and manually intensive offline assays.

References

[1] Xiaotong Fu, Wei-Chiang Chen, Christopher Argento, Peter Clarner, Vinay Bhatt, Ryan Dickerson, George Bou-Assaf, Meisam Bakhshayeshi, Xiaohui Lu, Svetlana Bergelson, and John Pieracci. Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development. Human Gene Therapy Methods, 2019, 30, 4, 144.

[2] Jurg Sommer, Peter Smith, Sumathy Parthasarathy, Jesse Isaacs, Sharmila Vijay, Jane Kieran, Sharon Powell, Alan McClelland, and Fraser Wright. Quantification of adeno-associated virus particles and empty capsids by optical density measurement. Molecular Therapy, 2003, 7, 1, 122.