Giorgetti Jérémie, Beck Alain, Leize-Wagner Emmanuelle, François Yannis-Nicolas
Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS) UMR 7140 (Unistra-CNRS), Université de Strasbourg, France.
Centre d'Immunologie Pierre Fabre, Saint-Julien-en-Genevois, France.
J Pharm Biomed Anal. 2020 Apr 15;182:113107. doi: 10.1016/j.jpba.2020.113107. Epub 2020 Jan 23.
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.
生物制药的显著增长需要强大的分析方法，通过建立完整的表征来更好地了解其结构。为此，已将自下而上、中间向上和完整分子水平与毛细管电泳-质谱联用相结合，以全面了解单克隆抗体。在本研究中，对7种全球卫生当局批准的单克隆抗体进行了分析，以获取有关其电荷异质性、翻译后修饰（PTM）的鉴定、其位置和相对定量的信息。完整的单克隆抗体异构体在不到12分钟内得到了部分分离，能够全面展示单克隆抗体的异质性以及高质量PTM的表征，尤其是主要的N-糖基化形式。特别是，2X-糖基化和1X-糖基化形式得到了部分分离。为了更深入地表征主链结构所携带的PTM，已在中间向上水平进行了深入研究。有限的IdeS蛋白酶解允许独立研究Fc/2和F(ab)'2片段。在相同的分离条件下，这些片段的异构体得到了分离，数据解释揭示了额外的PTM，如K-剪辑、氧化或脱酰胺。通过添加还原步骤来检查第二个中间水平，以更精确地评估F(ab)'2片段的PTM和异构体。该还原步骤将轻链从Fd片段中释放出来，仅得到25 kDa的片段进行分析。CE-ESI-MS联用能够获得更多信息，特别是关于低质量PTM的信息。在对所研究的单克隆抗体进行胰蛋白酶消化诱导的肽段水平上，研究了每个PTM的精确位置和相对定量。结果的一致性表明了CE-ESI-MS联用在表征单克隆抗体方面的有效性，并突出了多层次组合对于全面表征生物治疗药物的必要性。
