Human Gene Therapy - April 2023 - 325

METHODS
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Application of Size Exclusion Chromatography with Multiangle
Light Scattering in the Analytical Development of a Preclinical
Stage Gene Therapy Program
Bryan Troxell,1,2,* I-Wei Tsai,1 Kinjal Shah,1 Christopher I. Knuckles,1 Sarah Shelton,1 Kate Lindsey,1
Selene M. Barbosa Cardenas,1 and Taylor Roberts1
1Analytical Development and Quality Control, StrideBio, Research Triangle Park, North Carolina, USA.
2AjaxBio, LLC, Holly Springs, North Carolina, USA.
To provide safe recombinant adeno-associated viruses (rAAV) to patients, scalable manufacturing processes are
required. However, these processes may introduce impurities that impact the performance and quality of the final drug
product. Empty rAAV capsids are product-related impurities. Regulatory guidance requires that accurate analytical
methods be implemented early in product development to measure the level of empty capsids. A process confirmation
vector, produced from 200 L production, was used to develop and optimize a size exclusion chromatography with UV
and multiangle light scattering (SEC-MALS) method. Vector produced from a 500 L production was used to assess the
full-to-empty ratio using the following analytical methods: sedimentation velocity analytical ultracentrifugation (SVAUC),
droplet digital PCR (ddPCR) with capsid enzyme-linked immunosorbent assay (ELISA), bulk absorbance at
260/280 nm, cryogenic electron microscopy, and SEC-MALS. This test article was used for a 30-day, non-Good
Laboratory Practices animal study that assessed biodistribution of the product (STRX-330). SEC-MALS outperformed
the other methods and correlated well with SV-AUC values of full-to-empty particles. In addition, SEC-MALS agreed
with ddPCR and ELISA measurements for vector genomes/mL and capsid particles/mL, respectively. SEC-MALS was
linear, accurate, and precise while achieving chromatography quality control (QC) recommendations. Compared to
other stability-indicating assays, SEC-MALS performed similarly to ddPCR, capsid ELISA, and infectivity assays in
accelerated stress studies. In response to alkaline, but not acidic stress, SEC-MALS indicated distinct changes in
the DNA content of the monomer Adeno-associated viruses (AAV) peak for STRX-330, which was supported by
ddPCR data. Conversely, acidic treatment resulted in more aggregated vector, but did not impact the DNA content.
This work indicates that SEC-MALS is a valuable analytical tool in the analytical development and QC testing of
AAV. In addition, this work suggests that SEC-MALS can provide fundamental understanding of AAV in response
to environmental stress. This may impact steps of the manufacturing process to minimize conditions that reduce
performance.
Keywords: SEC-MALS, ddPCR, gene therapy, SV-AUC, cryo-EM, AAV
*Correspondence: Dr. Bryan Troxell, Analytical Development and Quality Control, StrideBio, 5 Laboratory Drive, Suite 1200, Research Triangle Park, NC27709, USA. E-mail:
b.troxell@ajaxbio.com
ª Bryan Troxell et al. 2023; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License [CC-BY] (http://
creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
HUMAN GENE THERAPY, VOLUME 34, NUMBERS 7 and 8
Mary Ann Liebert, Inc.
DOI: 10.1089/hum.2022.218 j 325

http://creativecommons.org/licenses/by/4.0 http://creativecommons.org/licenses/by/4.0

Human Gene Therapy - April 2023

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