Characterization of Adeno-Associated Virus Capsid Proteins Using Hydrophilic Interaction Chromatography Coupled with Mass Spectrometry

作者：Anita P. Liu, Shailin K. Patel, Tao Xing, Yuetian Yan, Shunhai Wang, Ning Li · 发表于：Journal of Pharmaceutical and Biomedical Analysis · 年份：2020 · DOI：10.1016/j.jpba.2020.113481 · 被引用次数：60 · 研究领域：Virus-based gene therapy research、Viral Infectious Diseases and Gene Expression in Insects、Bacteriophages and microbial interactions

To support adeno-associated virus (AAV)-based gene therapy development, characterization of the three capsid viral proteins (VP; VP1/VP2/VP3) from recombinant AAV can offer insights on capsid identity, heterogeneity, and product and process consistency. Intact protein mass analysis is a rapid, reliable, and sensitive method to confirm AAV serotypes based on accurate mass measurement of the constituent capsid proteins. Compared to commonly applied reversed-phase liquid chromatography (RPLC) methods, we demonstrated that, using a wide-pore amide-bonded column, hydrophilic interaction chromatography (HILIC) could achieve improved separation of VPs from a variety of AAV serotypes using a generic method prior to MS detection. Moreover, HILIC-based separation was shown to be particularly sensitive in detecting capsid protein variants resulting from different post-translational modifications (PTMs) (e.g. phosphorylation and oxidation) and protein backbone clippings, making it ideally suited for capsid heterogeneity characterization. To overcome the challenges associated with low protein concentrations of AAV samples, as well as the trifluoroacetic acid (TFA)-induced ion suppression during HILIC-MS analysis, different strategies were implemented to improve method sensitivity, including increasing the HILIC column loading and the application of a desolvation gas modification device. Finally, we demonstrated that this integrated HILIC-FLR-MS method can be generically applied to charact...