Measurement of Adenovirus-Based Vector Heterogeneity.
Measurement of Adenovirus-Based Vector Heterogeneity.
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DOI：
10.1016/j.xphs.2022.12.012
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发表时间：
2023-04
影响因子：
3.8
通讯作者：
Bracewell, Daniel G.
中科院分区：
文献类型：
--
作者：
Hickey, John M.;Jacob, Shaleem I.;Tait, Andrew S.;Vahid, Fatemeh Dastjerdi;Barritt, Joseph;Rouse, Sarah;Douglas, Alexander;Joshi, Sangeeta B.;Volkin, David B.;Bracewell, Daniel G.
关键词：
随着最近埃博拉和COVID-19产品的批准，腺病毒载体已成为一类重要的疫苗。在腺病毒载体制造过程中收集的过程质量属性数据主要集中在颗粒浓度和感染性比率（基于病毒基因组：基于细胞的感染性），数据表明最终疫苗产品中只有一小部分病毒颗粒是有效的。为了更好地了解这种产品的异质性，使用透射电镜（TEM）研究了两种腺病毒载体（黑猩猩腺病毒（ChAdOx1）和人类腺病毒5型（Ad5））的实验室规模制备。对不同的腺病毒形态进行了表征，并对空颗粒和满颗粒的比例进行了量化。这些比例与样品的传染性值表现出定性的相关性。采用液相色谱-质谱（LC-MS）多肽图谱鉴定参与病毒成熟的关键腺病毒蛋白。利用肽丰度分析，从ChAdOx1病毒的CsCl超离心制剂中提取的低（空）和高密度（满）馏分中观察到L1 52/55k丰度的变化约5倍。L1 52/55k病毒蛋白与DNA包装有关，并在病毒成熟过程中被切割，因此它可能是感染颗粒的标记物。作为腺病毒载体过程监测的一部分，TEM和LC-MS肽图谱是有希望的高分辨率分析表征工具，可以帮助区分空的、非传染性的和感染性的病毒颗粒的相对比例，这些结果是一个令人鼓舞的第一步，可以更好地区分不同的产品相关杂质。
Adenovirus vectors have become an important class of vaccines with the recent approval of Ebola and COVID-19 products. In-process quality attribute data collected during Adenovirus vector manufacturing has focused on particle concentration and infectivity ratios (based on viral genome: cell-based infectivity), and data suggest only a fraction of viral particles present in the final vaccine product are efficacious. To better understand this product heterogeneity, lab-scale preparations of two Adenovirus viral vectors, (Chimpanzee adenovirus (ChAdOx1) and Human adenovirus Type 5 (Ad5), were studied using transmission electron microscopy (TEM). Different adenovirus morphologies were characterized, and the proportion of empty and full viral particles were quantified. These proportions showed a qualitative correlation with the sample's infectivity values. Liquid chromatography-mass spectrometry (LC-MS) peptide mapping was used to identify key adenovirus proteins involved in viral maturation. Using peptide abundance analysis, a ∼5-fold change in L1 52/55k abundance was observed between low-(empty) and high-density (full) fractions taken from CsCl ultracentrifugation preparations of ChAdOx1 virus. The L1 52/55k viral protein is associated with DNA packaging and is cleaved during viral maturation, so it may be a marker for infective particles. TEM and LC-MS peptide mapping are promising higher-resolution analytical characterization tools to help differentiate between relative proportions of empty, non-infectious, and infectious viral particles as part of Adenovirus vector in-process monitoring, and these results are an encouraging initial step to better differentiate between the different product-related impurities.
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DOI：
10.3390/v13030388
发表时间：
2021-02-28
期刊：
Viruses
影响因子：
--
作者：
Kulanayake S;Tikoo SK
通讯作者：
Tikoo SK
DOI：
10.1016/j.omtm.2020.05.018
发表时间：
2020-09-11
影响因子：
4.7
作者：
Rumachik, Neil G.;Malaker, Stacy A.;Paulk, Nicole K.
通讯作者：
Paulk, Nicole K.
影响因子：
4.8
作者：
Benevento, Marco;Di Palma, Serena;Heck, Albert J. R.
通讯作者：
Heck, Albert J. R.
影响因子：
5.5
作者：
Fedosyuk, Sofiya;Merritt, Thomas;Douglas, Alexander D.
通讯作者：
Douglas, Alexander D.
影响因子：
7
作者：
Silva, JC;Gorenstein, MV;Geromanos, SJ
通讯作者：
Geromanos, SJ