Characterisation and Comparability Laboratory, The National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, A94 X099, Co. Dublin, Ireland.
908 Devices Inc., 645 Summer Street #201, Boston, MA, 02210, USA.
Anal Bioanal Chem. 2024 Feb;416(4):1069-1084. doi: 10.1007/s00216-023-05097-5. Epub 2023 Dec 15.
Adeno-associated viruses (AAVs) are viral vectors used as delivery systems for gene therapies. Intact protein characterization of AAV viral capsid proteins (VPs) and their post-translational modifications is critical to ensuring product quality. In this study, microchip-based ZipChip capillary electrophoresis-mass spectrometry (CE-MS) was applied for the rapid characterization of AAV intact VPs, specifically full and empty viral capsids of serotypes AAV6, AAV8 and AAV9, which was accomplished using 5 min of analysis time. Low levels of dimethyl sulfoxide (4%) in the background electrolyte (BGE) improved MS signal quality and component detection. A sensitivity evaluation revealed consistent detection of VP proteoforms when as little as 2.64 × 10 viral particles (≈26.4 picograms) were injected. Besides the traditional VP proteoforms used for serotype identification, multiple VP3 variants were detected, including truncated VP3 variants most likely generated by leaky scanning as well as unacetylated and un-cleaved VP3 proteoforms. Phosphorylation, known to impact AAV transduction efficiency, was also seen in all serotypes analysed. Additionally, low abundant fragments originating from either N- or C-terminus truncation were detected. As the aforementioned VP components can impact product quality and efficacy, the ZipChip's ability to rapidly characterize them illustrates its strength in monitoring product quality during AAV production.
腺相关病毒（AAV）是用作基因治疗传递系统的病毒载体。AAV 病毒衣壳蛋白（VP）的完整蛋白特性及其翻译后修饰对于确保产品质量至关重要。在这项研究中，基于微芯片的 ZipChip 毛细管电泳-质谱联用（CE-MS）被应用于快速表征 AAV 完整 VP，特别是 AAV6、AAV8 和 AAV9 血清型的完整和空病毒衣壳，分析时间仅需 5 分钟。背景电解质（BGE）中低水平的二甲基亚砜（4%）改善了 MS 信号质量和组分检测。当注射量低至 2.64×10 个病毒颗粒（约 26.4 皮克）时，仍能一致检测到 VP 蛋白变体，表明该方法具有较高的灵敏度。除了用于血清型鉴定的传统 VP 蛋白变体外，还检测到多个 VP3 变体，包括可能由漏扫描产生的截短 VP3 变体，以及未乙酰化和未切割的 VP3 蛋白变体。所有分析的血清型都存在已知会影响 AAV 转导效率的磷酸化。此外，还检测到源自 N 端或 C 端截断的低丰度片段。由于上述 VP 成分会影响产品质量和功效，因此 ZipChip 快速表征它们的能力说明了其在监测 AAV 生产过程中产品质量方面的优势。
