Development of a highly sensitive imaged cIEF immunoassay for studying AAV capsid protein charge heterogeneity.
adeno-associated virus
capillary immunoassay
capsid proteins
identity
isoelectric focusing
Journal
Electrophoresis
ISSN: 1522-2683
Titre abrégé: Electrophoresis
Pays: Germany
ID NLM: 8204476
Informations de publication
Date de publication:
08 2023
08 2023
Historique:
revised:
14
04
2023
received:
22
02
2023
accepted:
18
04
2023
medline:
17
8
2023
pubmed:
4
5
2023
entrez:
3
5
2023
Statut:
ppublish
Résumé
Post-translational modifications (PTMs) of adeno-associated virus (AAV) capsid proteins tune and regulate the AAV infective life cycle, which can impact the safety and efficacy of AAV gene therapy products. Many of these PTMs induce changes in protein charge heterogeneity, including deamidation, oxidation, glycation, and glycosylation. To characterize the charge heterogeneity of a protein, imaged capillary isoelectric focusing (icIEF) has become the gold standard method. We have previously reported an icIEF method with native fluorescence detection for denatured AAV capsid protein charge heterogeneity analysis. Although well suited for final products, the method does not have sufficient sensitivity for upstream, low-concentration AAV samples, and lacks the specificity for capsid protein detection in complex samples like cell culture supernatants and cell lysates. In contrast, the combination of icIEF, protein capture, and immunodetection affords significantly higher sensitivity and specificity, addressing the challenges of the icIEF method. By leveraging different primary antibodies, the icIEF immunoassay provides additional selectivity and affords a detailed characterization of individual AAV capsid proteins. In this study, we describe an icIEF immunoassay method for AAV analysis that is 90 times more sensitive than native fluorescence icIEF. This icIEF immunoassay provides AAV stability monitoring, where changes in individual capsid protein charge heterogeneity can be observed in response to heat stress. When applied to different AAV serotypes, this method also provides serotype identity with reproducible quantification of VP protein peak areas and apparent isoelectric point (pI). Overall, the described icIEF immunoassay is a sensitive, reproducible, quantitative, specific, and selective tool that can be used across the AAV biomanufacturing process, especially in upstream process development where complex sample types are often encountered.
Identifiants
pubmed: 37138377
doi: 10.1002/elps.202300039
doi:
Substances chimiques
Capsid Proteins
0
Antibodies, Monoclonal
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1258-1266Informations de copyright
© 2023 The Authors. Electrophoresis published by Wiley-VCH GmbH.
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