Chen Yan, Kim Michael T, Zheng Laura, Deperalta Galahad, Jacobson Fred
Department of Protein Analytical Chemistry, Genentech, Inc. , 1 DNA way, South San Francisco, California 94080-4990, United States.
Bioconjug Chem. 2016 Sep 21;27(9):2037-47. doi: 10.1021/acs.bioconjchem.6b00316. Epub 2016 Aug 9.
The antibody-drug conjugate, trastuzumab emtansine (Kadcyla), is produced by attachment of the antitubulin drug, DM1, to lysine amines via a heterobifunctional linker, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). Following the reaction of the N-hydroxysuccinimide activated linker with antibody lysines to produce a linker-modified intermediate (Tmab-MCC), DM1 is added to yield the desired product. In addition to the expected distribution of drug-linked forms (from 0 to 8), mass spectrometry also demonstrates the presence of a second distribution shifted by about +222 Da. This series is consistent with the presence of a population containing a bound linker without DM1 ("unconjugated linker"). Extended characterization of trastuzumab emtansine was performed using capillary isoelectic focusing, CE-SDS, peptide mapping, and LC/MS following (18)O labeling of peptide digests to identify this family of product variants. These studies demonstrate that the presence of these +222 Da species is due to an unexpected reaction of the maleimide moiety in the MCC linker with antibody lysine residues to produce cross-linked species that cannot conjugate to DM1.
抗体药物偶联物曲妥珠单抗（赫赛汀）是通过将抗微管蛋白药物DM1经异双功能连接子SMCC（4-(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯）连接到赖氨酸胺上而产生的。在N-羟基琥珀酰亚胺活化的连接子与抗体赖氨酸反应生成连接子修饰的中间体（Tmab-MCC）后，加入DM1以得到所需产物。除了预期的药物连接形式分布（从0到8）外，质谱还显示存在第二个分布，其偏移约+222 Da。该系列与存在不含DM1的结合连接子（“未偶联连接子”）的群体一致。在肽段消化物进行(18)O标记后，使用毛细管等电聚焦、CE-SDS、肽图分析和LC/MS对曲妥珠单抗进行了扩展表征，以鉴定该产物变体家族。这些研究表明，这些+222 Da物种的存在是由于MCC连接子中的马来酰亚胺部分与抗体赖氨酸残基发生意外反应，产生了无法与DM1偶联的交联物种。
