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

• Published in: Journal of pharmaceutical and biomedical analysis 2020-09, Vol.189, p.113481-113481, Article 113481
• Main Authors: Liu, Anita P., Patel, Shailin K., Xing, Tao, Yan, Yuetian, Wang, Shunhai, Li, Ning
• Format: Article
• Language: English
• Subjects: AAV serotypes; Adeno-Associated Virus; capsid proteins; HILIC-MS; hydrophilic interaction chromatography; PTMs
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2020.113481

[Display omitted] •HILIC shows excellent separation of capsid proteins from various AAV serotypes.•HILIC is sensitive to AAV phosphorylation, oxidation and protein backbone clipping.•The MS sensitivity was enhanced by a desolvation gas modification strategy.•The method is generally applicable to various AAV serotypes at low concentrations.•The method is suitable for assessing serotype identity, stoichiometry and PTMs. 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 characterize a variety of AAV serotype samples at low concentrations without any sample treatment to achieve unambiguous serotype identification, stoichiometry assessment, and PTM characterization.