Interlaboratory Measurement of Adeno-Associated Virus: Comparative Quantification of Full and Empty Capsids

Published

December 26, 2024

Author(s)

Sean Lehman, Wyatt N. Vreeland, Jerome Jacques, Sharee Adams-Hall, Shreya Ahuja, Adnan Arnaout, Hunter Balduf, Ivan Budyak, Thomas Cleveland, Dennis Delgado, James Deng, Brandon Doyle, David L. Duewer, Carsten Elger, Jeffrey Fagan, Tim Guo, Jorge Haller, Luisa Hilgenfeld, Van Hoang, Allison Huldin, Matthew Hyatt, Sambit Kar, Sandeep Kedia, Bashkim Kokona, Amy Liu, Li Ma, Diane McCarthy, He Meng, Easton Noble, Veronika Oettle, Andrew Pla, Thomas Powers, Dean Ripple, Herbert Runnels, Raphael Ruppert, Florian Semmelmann, Christopher Sims, Saurabh Singh, Ronald Toth, Austin Vogt, Sabine Wenzel, Neal Whitaker, Zhiwen Yang, Brandon Zhuang

Abstract

Recombinant adeno-associated virus (AAV) is one of the main viral vector-based gene therapy platforms. AAV is a virus consisting of a ≈25 nm diameter capsid with a ≈4.7 kb cargo capacity. The safety and efficacy depend on the correct encapsidation of the DNA in individual virus particles, which is often characterized by the single scalar value of the ratio of full capsids with complete viral genomes to total viral capsid number (the Full-to-Total ratio, or FTT). This study reports on the interlaboratory and inter-technique variations of measurement methods for FTT among a cohort of interested organizations. The analytical methods used were sedimentation velocity-analytical ultracentrifugation (SV-AUC) with UV/Vis and/or Rayleigh interference optics, size exclusion chromatography (SEC) with multi-angle light scattering (MALS) and tandem UV/Vis and/or refractive index, cryogenic electron microscopy (cryo-EM), dual wavelength ultraviolet spectrophotometry, and ELISA coupled with quantitative PCR (qPCR, dPCR or ddPCR). Results for both AAV5 and AAV8 serotypes were similar, except for PCR-ELISA results. The optical techniques (UV spectroscopy/SEC-MALS) showed <10% SD between laboratories (for Full and Mix samples), likely from uniformity of existing industry protocols. AUC, while demonstrating good repeatability, had ≈25% SD interlaboratory reproducibility for mixed samples of 50% full and 50% empty capsids, suggesting the need for standardized methods. PCR and ELISA had poor reproducibility due to variations in how the values for PCR and ELISA were measured as the required inputs for FTT. The discussion presents intended future efforts to improve and harmonize these measurements to increase both the repeatability and reproducibility of AAV viral particle key parameters like FTT.

Citation

Human Gene Therapy

Pub Type

Journals

Download Paper

https://doi.org/10.1089/hum.2024.124

Local Download

Keywords

AAV, Full-To-Total Ratio, Gene Therapy

Citation

Lehman, S. , Vreeland, W. , Jacques, J. , Adams-Hall, S. , Ahuja, S. , Arnaout, A. , Balduf, H. , Budyak, I. , Cleveland, T. , Delgado, D. , Deng, J. , Doyle, B. , Duewer, D. , Elger, C. , Fagan, J. , Guo, T. , Haller, J. , Hilgenfeld, L. , Hoang, V. , Huldin, A. , Hyatt, M. , Kar, S. , Kedia, S. , Kokona, B. , Liu, A. , Ma, L. , McCarthy, D. , Meng, H. , Noble, E. , Oettle, V. , Pla, A. , Powers, T. , Ripple, D. , Runnels, H. , Ruppert, R. , Semmelmann, F. , Sims, C. , Singh, S. , Toth, R. , Vogt, A. , Wenzel, S. , Whitaker, N. , Yang, Z. and Zhuang, B. (2024), Interlaboratory Measurement of Adeno-Associated Virus: Comparative Quantification of Full and Empty Capsids, Human Gene Therapy, [online], https://doi.org/10.1089/hum.2024.124, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=957020 (Accessed January 13, 2025)