Zhou, Yu and Wang, Yueju (2023) Direct Deamidation Analysis of Intact Adeno-Associated Virus Serotype 9 (AAV9) Capsid Proteins using Reversed-Phase Liquid Chromatography (RPLC)- Tandem Mass Spectrometry (MS) or RPLC-Fluorescence Detector (FLD). Analytical biochemistry, 668 (115099). pp. 1-9. ISSN Print ISSN: 0003-2697; Online ISSN: 1096-0309

Abstract

Recombinant adeno-associated viral (AAV) vectors with nonpathogenic nature and ability to provide long-term gene expression have taken center stage as gene delivery vehicles for gene therapy. AAV capsid proteins (VP) are the major components that determine the tissue specificity, immunogenicity, and in vivo transduction performance of the vector. Asparagine deamidation of AAV capsid proteins has been reported to alter vector function, reduce vector stability and potency of AAV gene therapy products. Deamidation of asparagine residue is a common post-translational modification (PTM) of proteins that is readily detected and quantified by liquid chromatography-tandem mass spectrometry (LC-MS)-based peptide mapping. However, artificial deamidation can be spontaneously induced during sample preparation for peptide mapping prior to LC-MS analysis. We have developed an optimized sample preparation method to reduce and minimize deamidation artifacts induced during sample preparation for peptide mapping, which typically takes several hours to complete. To shorten turnaround time of deamidation results and to avoid artificial deamidation, we developed orthogonal RPLC-MS and RPLC-fluorescence detection (FLD) methods for direct deamidation analysis at the intact AAV9 capsid protein level to routinely support downstream purification, formulation development, and stability testing. Similar trends of increasing deamidation of AAV9 capsid proteins in stability samples were observed at the intact protein level and peptide level, indicating that the developed direct deamidation analysis of intact AAV9 capsid protein is comparable to the peptide mapping-based deamidation analysis and both methods are suitable/alternative for deamidation monitoring of AAV9 capsid proteins.

Item Type:	Article
Keywords:	AAV9, capsid proteins, deamidation, intact protein, RPLC-MS, RPLC-fluorescence detector
Date Deposited:	07 Oct 2023 00:45
Last Modified:	07 Oct 2023 00:45
URI:	https://oak.novartis.com/id/eprint/49371