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Rapid Characterization of AAV gene therapy vectors by Mass Photometry

View ORCID ProfileDi Wu, Philsang Hwang, Tiansen Li, View ORCID ProfileGrzegorz Piszczek
doi: https://doi.org/10.1101/2021.02.18.431916
Di Wu
1Biophysics Core Facility, National Heart, Lung, and Blood Institute, 50 South Drive, Bethesda, MD 20892-8012, USA
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Philsang Hwang
2Ocular Gene Therapy Core Facility, National Eye Institute, 6 Center Drive, Bethesda, Maryland 20892, USA
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Tiansen Li
2Ocular Gene Therapy Core Facility, National Eye Institute, 6 Center Drive, Bethesda, Maryland 20892, USA
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Grzegorz Piszczek
1Biophysics Core Facility, National Heart, Lung, and Blood Institute, 50 South Drive, Bethesda, MD 20892-8012, USA
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  • For correspondence: grzegorz_piszczek@nih.gov
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Abstract

Recombinant adeno-associated viruses (rAAV) are extensively used as gene delivery vectors in clinical studies, and several rAAV based treatments have already been approved. Significant progress has been made in rAAV manufacturing, and large-scale vector production and purification methods have been developed. However, a better and more precise capsid characterization techniques are still needed to guarantee the purity and safety of the rAAV preparations. A recently developed single-molecule technique, mass photometry (MP), measures mass distributions of biomolecules with high resolution and sensitivity. Here we explore applications of MP for the characterization of capsid fractions. We demonstrate that MP is able to resolve and quantify not only empty and full-genome containing capsid populations, but also identify the partially packaged capsid impurities. MP data accurately measures full and empty capsid ratios, and can be used to estimate the size of the encapsidated genome. MP distributions provide information on sample heterogeneity and on the presence of aggregates. Current analytical techniques used to characterize rAAV preparations are susceptible to background signals, have limited accuracy, or are time-consuming and require a large amount of material. MP can analyze sub-picomole quantities of sample, and data can be obtained and analyzed within minutes. This method provides a simple, robust, and effective tool to monitor physical attributes of rAAV vectors.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.
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Posted February 19, 2021.
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Rapid Characterization of AAV gene therapy vectors by Mass Photometry
Di Wu, Philsang Hwang, Tiansen Li, Grzegorz Piszczek
bioRxiv 2021.02.18.431916; doi: https://doi.org/10.1101/2021.02.18.431916
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Rapid Characterization of AAV gene therapy vectors by Mass Photometry
Di Wu, Philsang Hwang, Tiansen Li, Grzegorz Piszczek
bioRxiv 2021.02.18.431916; doi: https://doi.org/10.1101/2021.02.18.431916

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