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

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. Therapeutic 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 the total viral capsid number [the full-to-total (FTT) ratio]. This study reports on the interlaboratory and intertechnique variations of measurement methods for FTT among a cohort of 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, dual-wavelength ultraviolet spectrophotometry, and ELISA coupled with quantitative PCR (qPCR, dPCR, or ddPCR). FTT measurements for both AAV5 and AAV8 serotypes were similar, except for PCR-ELISA. The optical techniques (UV spectroscopy/SEC-MALS) showed <10% SD between laboratories, likely from the uniformity of existing industry protocols. AUC, while demonstrating good repeatability, had ≈25% SD interlaboratory, suggesting the need for standardized methods. PCR and ELISA had poor reproducibility due to variations in both PCR and ELISA protocols and instrumentation. The discussion presents intended future efforts to improve and harmonize these measurements to increase both the repeatability and reproducibility of AAV viral particle critical quality attributes such as FTT.

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Publication Details

Journal
Human Gene Therapy
Published
2024-12-26
DOI
https://doi.org/10.1089/hum.2024.124
Citations
2
Primary Topic
Virus-based gene therapy research
Type
article
Field-Weighted Citation Impact
1.10
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Interlaboratory Measurement of Adeno-Associated Virus: Comparative Quantification of Full and Empty Capsids

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

Tim Charlebois, Andrew Pla, Saurabh Singh, Austin Vogt, Diane McCarthy, Thomas W. Powers, Christopher M. Sims, Sharee Adams-Hall, Thomas E. Cleveland, Sanjay Ahuja, Brandon L. Doyle, Bashkim Kokona, Matthew W. Hyatt, Zhiwen Yang, Ruben G. Carbonell, Carsten Elger, Herbert A. Runnels, Ivan L. Budyak, Florian Semmelmann, Jean Richardson, B. C. Zhuang, Dean C. Ripple, James Z. Deng, Hoàng Văn Minh, Sean E. Lehman, Sandeep B. Kedia, Neal Whitaker, Jingzhong Guo, Wyatt N. Vreeland, David L. Duewer, Raphael Ruppert, Jeffrey A. Fagan, Amy Liu, Sambit R. Kar, Adnan Arnaout, Sabine Wenzel, Jorge F. Haller, Anna Błaszczyk, E. R. Noble, Jérôme Jacques, Li Ma, Luisa Desiree Hilgenfeld, Allison J. L. Huldin, Veronika Oettl, Hunter Balduf
article en
2 citations

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. Therapeutic 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 the total viral capsid number [the full-to-total (FTT) ratio]. This study reports on the interlaboratory and intertechnique variations of measurement methods for FTT among a cohort of 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, dual-wavelength ultraviolet spectrophotometry, and ELISA coupled with quantitative PCR (qPCR, dPCR, or ddPCR). FTT measurements for both AAV5 and AAV8 serotypes were similar, except for PCR-ELISA. The optical techniques (UV spectroscopy/SEC-MALS) showed <10% SD between laboratories, likely from the uniformity of existing industry protocols. AUC, while demonstrating good repeatability, had ≈25% SD interlaboratory, suggesting the need for standardized methods. PCR and ELISA had poor reproducibility due to variations in both PCR and ELISA protocols and instrumentation. The discussion presents intended future efforts to improve and harmonize these measurements to increase both the repeatability and reproducibility of AAV viral particle critical quality attributes such as FTT.

Human Gene TherapyVol. 36(1-2)
Merck & Co., Inc., Rahway, NJ, USA (United States) (US), National Institute of Standards and Technology (US), Eli Lilly (United States) (US), Pfizer (United States) (US), United States Pharmacopeial Convention (US), Roche Pharma AG (Germany) (DE), Spark Therapeutics (United States) (US), University of Delaware (US)
Zero hunger
Openalex Percentile: Top 17%
Virus-based gene therapy research
1.10
DOIOpen Access PDFOpenAlex
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