Distinct chemical degradation pathways of AAV1 and AAV8 under thermal stress conditions revealed by analytical anion exchange chromatography and LC-MS-based peptide mapping

• Published in: Journal of pharmaceutical and biomedical analysis 2024-12, Vol.251, p.116452, Article 116452
• Main Authors: Xing, Tao, Li, Shuai, Tang, Shuli, Huang, Yu, Liu, Gaoyuan, Yan, Yuetian, Liu, Dingjiang, Wang, Shunhai, Zhi, Li, Shameem, Mohammed, Li, Ning
• Format: Article
• Language: English
• Subjects: AAV; Anion exchange chromatography; Capsid - chemistry; Capsid Proteins - chemistry; Charge variants analysis; Chromatography, Ion Exchange - methods; Chromatography, Liquid - methods; Dependovirus - chemistry; Dependovirus - genetics; Genetic Therapy - methods; Genetic Vectors - chemistry; Hot Temperature; Humans; LC-MS; Liquid Chromatography-Mass Spectrometry; Peptide mapping; Peptide Mapping - methods
• ISSN: 0731-7085; 1873-264X; 1873-264X
• DOI: 10.1016/j.jpba.2024.116452

Adeno-associated virus (AAV)-based gene therapy is experiencing a rapid growth in the field of medicine and holds great promise in combating a wide range of human diseases. For successful development of AAV-based products, comprehensive thermal stability studies are often required to establish storage conditions and shelf life. However, as a relatively new modality, limited studies have been reported to elucidate the chemical degradation pathways of AAV products under thermal stress conditions. In this study, we first presented an intriguing difference in charge profile shift between thermally stressed AAV8 and AAV1 capsids when analyzed by anion exchange chromatography. Subsequently, a novel and robust peptide mapping protocol was developed and applied to elucidate the underlying chemical degradation pathways of thermally stressed AAV8 and AAV1. Compared to the conventional therapeutic proteins, the unique structure of AAV capsids also led to some key differences in how modifications at specific sites may impact the overall charge properties. Finally, despite the high sequency identity, the analysis revealed that the opposite charge profile shifts between thermally stressed AAV8 and AAV1 could be mainly attributed to a single modification unique to each serotype. •Opposite charge profile shifts observed for thermally stressed AAV8 and AAV1.•An optimized one-pot tryptic digestion protocol developed for AAV peptide mapping.•The location of modifications largely impacts the AAV retention on AEX.•A unique PTM specific to each AAV serotype is mainly responsible for the observed charge profile shift.