Assessing production variability in empty and filled adeno-associated viruses by single molecule mass analyses

Eduard H T M Ebberink; Alisa Ruisinger; Markus Nuebel; Marco Thomann; Albert J R Heck

doi:10.1016/j.omtm.2022.11.003

Assessing production variability in empty and filled adeno-associated viruses by single molecule mass analyses

Mol Ther Methods Clin Dev. 2022 Nov 15:27:491-501. doi: 10.1016/j.omtm.2022.11.003. eCollection 2022 Dec 8.

Authors

Eduard H T M Ebberink^{1

2}, Alisa Ruisinger³, Markus Nuebel³, Marco Thomann³, Albert J R Heck^{1

2}

Affiliations

¹ Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.
² Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The Netherlands.
³ Gene Therapy Technical Development Analytics, Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.

Abstract

Adeno-associated viruses (AAVs) are useful vehicles for gene therapy because of their stability, low immunogenicity. and non-pathogenicity. However, disparity in AAV sample preparations (e.g., in capsid composition, DNA packaging, and impurities) gives rise to product heterogeneity, with possibly undesired effects on gene delivery. Ideally, AAV production should be with full control of AAV structure and genetic payload. Therefore, robust, efficient, and low material consuming methods are essential to characterize AAVs. Here, we use two emerging single-molecule techniques, mass photometry and Orbitrap-based charge-detection mass spectrometry, and show how they may efficiently and accurately characterize AAVs. We were able to resolve heterogeneous pools of particles, evaluating AAVs from two different serotypes (AAV8 and AAV2), produced by three independent production platforms, either lacking a genome or packed with a transgene. Together our data confirm that the different AAV production methods result in rather different and diverse AAV particle distributions. Especially for the packed AAVs, frequently additional subspecies were observed, next to the expected packed genome, mostly resulting from under- or overpackaging of genome material and/or residual empty particles. This work further establishes that both these single-particle techniques may become valuable tools in characterizing AAVs before they are used in gene therapy.

Keywords: AAV biomanufacturing; AAV2; AAV8; Adeno-associated virus; charge-detection mass spectrometry; empty-filled ratio; gene-delivery vector; mass photometry; native mass spectrometry; single molecule mass analyses.