Significant growth of biopharmaceuticals requires powerful analytical methods to better understand their structure by establishing a complete characterization. To this end, a combination of bottom-up, middle-up and intact molecule levels with a capillary electrophoresis-mass spectrometry coupling has been performed to have a comprehensive picture of monoclonal antibodies. In this study, 7 worldwide health authorities approved mAbs have been analyzed to get information about their charge heterogeneity, the identification of post translational modifications (PTMs), their location and relative quantitation. Intact mAbs isoforms have been partially separated in less than 12 min and enabled to have a global illustration of mAbs heterogeneity and high masses PTMs characterization notably major N-glycosylation forms. Particularly, 2X-glycosylated and 1X-glycosylated forms have been partially separated. To deepen characterize PTMs carried by the backbone structure, advanced investigations at a middle-up level have been performed. Limited IdeS proteolysis allowed to study independently Fc/2 and F(ab)'2 fragments. Following the same separation conditions, isoforms of these fragments have been separated and data interpretation allowed to disclose additional PTMs as K-clip, oxidations or deamidations. A second intermediate level has been examined by adding a reduction step to establish a more precise assessment of PTMs and isoforms from the F(ab)'2 fragment. This reduction step released the light chains from the Fd fragment to get only 25 kDa fragments to analyze. CE-ESI-MS coupling allowed to get more information particularly about low masses PTMs. The precise location and relative quantitation of each PTM has been investigated at the peptidic level induced by a tryptic digestion of the studied mAbs. The concordance of the results shows the efficiency of the CE-ESI-MS coupling to characterize mAbs and highlight the need of the multi-level combination to get a comprehensive characterization of biotherapeutics.

中文翻译：

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完整，中上和下至上水平的组合，通过毛细管电泳-质谱表征7种治疗性单克隆抗体。

生物药物的显着增长需要强大的分析方法，才能通过建立完整的表征来更好地了解其结构。为此，已经进行了自下而上，中上和完整分子水平与毛细管电泳-质谱联用的结合，以全面了解单克隆抗体。在这项研究中，对7种全球卫生当局批准的mAb进行了分析，以获取有关其电荷异质性，翻译后修饰（PTM）鉴定，其位置和相对定量的信息。完整的mAb同工型已在不到12分钟的时间内被部分分离，并能够全面展示mAb的异质性和高质量的PTM，尤其是主要的N-糖基化形式。尤其，2X-糖基化和1X-糖基化形式已被部分分离。为了加深对骨干结构承载的PTM的表征，已经在中上级别进行了深入研究。有限的IdeS蛋白水解可以独立研究Fc / 2和F（ab）'2片段。在相同的分离条件下，已经分离了这些片段的同工型，并且数据解释允许披露其他PTM，例如K片段，氧化或脱酰胺。已通过添加还原步骤来建立第二中等水平，以建立更精确的F（ab）'2片段PTM和同工型评估。此还原步骤从Fd片段释放了轻链，仅得到25 kDa片段进行分析。CE-ESI-MS耦合可以获取更多信息，尤其是有关低质量PTM的信息。已通过胰蛋白酶消化研究的单克隆抗体诱导的肽水平研究了每个PTM的精确位置和相对定量。结果的一致性说明了CE-ESI-MS偶联用于表征mAb的效率，并强调了多级组合对生物治疗剂进行全面表征的需求。