High-efficiency purification of divergent AAV serotypes using AAVX affinity chromatography

Michael Florea; Fotini Nicolaou; Simon Pacouret; Eric M Zinn; Julio Sanmiguel; Eva Andres-Mateos; Carmen Unzu; Amy J Wagers; Luk H Vandenberghe

doi:10.1016/j.omtm.2022.12.009

High-efficiency purification of divergent AAV serotypes using AAVX affinity chromatography

Mol Ther Methods Clin Dev. 2022 Dec 16:28:146-159. doi: 10.1016/j.omtm.2022.12.009. eCollection 2023 Mar 9.

Authors

Michael Florea^{1

2

3}, Fotini Nicolaou¹, Simon Pacouret¹, Eric M Zinn¹, Julio Sanmiguel¹, Eva Andres-Mateos¹, Carmen Unzu¹, Amy J Wagers^{3

4

5

6}, Luk H Vandenberghe^{1

7

8

4}

Affiliations

¹ Grousbeck Gene Therapy Center, Schepens Eye Research Institute and Massachusetts Eye and Ear Infirmary, Boston, MA, USA.
² Harvard Ph.D. Program in Biological and Biomedical Sciences, Division of Medical Sciences, Harvard University, Boston, MA, USA.
³ Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.
⁴ Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA.
⁵ Paul F. Glenn Center for the Biology of Aging, Harvard Medical School, Boston, MA, USA.
⁶ Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA, USA.
⁷ Ocular Genomics Institute, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA.
⁸ The Broad Institute of Harvard and MIT, Boston, MA, USA.

Abstract

The adeno-associated viral vector (AAV) provides a safe and efficient gene therapy platform with several approved products that have marked therapeutic impact for patients. However, a major bottleneck in the development and commercialization of AAV remains the efficiency, cost, and scalability of AAV production. Chromatographic methods have the potential to allow purification at increased scales and lower cost but often require optimization specific to each serotype. Here, we demonstrate that the POROS CaptureSelect AAVX affinity resin efficiently captures a panel of 15 divergent AAV serotypes, including the commonly used AAV2, AAV8, AAV9, PHP.B, and Anc80. We also find that AAVX resin can be regenerated repeatedly without loss of efficiency or carry-over contamination. While AAV preps purified with AAVX showed a higher fraction of empty capsids than preps purified using iodixanol ultracentrifugation, the potency of the AAVX purified vectors was comparable with that of iodixanol purified vectors both in vitro and in vivo. Finally, optimization of the purification protocol resulted in a process with an overall efficiency of 65%-80% across all scales and AAV serotypes tested. These data establish AAVX affinity chromatography as a versatile and efficient method for purification of a broad range of AAV serotypes.

Keywords: AAV; AAVX; adeno-associated; affinity chromatography; downstream; gene therapy; manufacturing; purification.

Grants and funding

DP1 AG063419/AG/NIA NIH HHS/United States