Characterization of Adeno-Associated Virus Capsid Proteins Using Hydrophilic Interaction Chromatography Coupled with Mass Spectrometry
Characterization of Adeno-Associated Virus Capsid Proteins Using Hydrophilic Interaction Chromatography Coupled with Mass Spectrometry
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DOI：
10.1016/j.jpba.2020.113481
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发表时间：
2020-09-10
影响因子：
3.4
通讯作者：
Li, Ning
中科院分区：
文献类型：
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作者：
Liu, Anita P.;Patel, Shailin K.;Li, Ning
为了支持基于腺相关病毒（腺相关病毒）的基因治疗开发，对重组腺相关病毒的三种衣壳病毒蛋白（VP; VP 1/VP 2/VP 3）进行表征可以提供有关衣壳身份、异质性以及产品和工艺一致性的见解。完整蛋白质质量分析是一种快速、可靠和灵敏的方法，用于基于组分衣壳蛋白的准确质量测量来确认AAV血清型。与常用的反相液相色谱（RPLC）方法相比，我们证明，使用宽孔酰胺键合柱，亲水相互作用色谱（HILIC）可以实现改进的分离VP从各种AAV血清型使用通用的方法之前，MS检测。此外，基于HILIC的分离显示在检测由不同翻译后修饰（PTM）（例如磷酸化和氧化）和蛋白质骨架剪切产生的衣壳蛋白变体方面特别敏感，使其非常适合衣壳异质性表征。为了克服与AAV样品的低蛋白浓度以及HILIC-MS分析期间三氟乙酸（TFA）诱导的离子抑制相关的挑战，实施不同的策略以提高方法灵敏度，包括增加HILIC柱负载和应用去溶剂化气体修饰装置。最后，我们证明了这种集成的HILIC-FLR-MS方法可以一般地应用于表征低浓度的各种AAV血清型样品，而无需任何样品处理，以实现明确的血清型鉴定、化学计量评估和PTM表征。(C)2020作者（S）由爱思唯尔公司出版
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. (C) 2020 The Author(s). Published by Elsevier B.V.