Luo Quanzhou, Chung Hyo Helen, Borths Christopher, Janson Matthew, Wen Jie, Joubert Marisa K, Wypych Jette
Department of Process Development, Amgen Inc. , Thousand Oaks, California 91320, United States.
Anal Chem. 2016 Jan 5;88(1):695-702. doi: 10.1021/acs.analchem.5b03709. Epub 2015 Dec 14.
Structural characterization was performed on an antibody-drug conjugate (ADC), composed of an IgG1 monoclonal antibody (mAb), mertansine drug (DM1), and a noncleavable linker. The DM1 molecules were conjugated through nonspecific modification of the mAb at solvent-exposed lysine residues. Due to the nature of the lysine conjugation process, the ADC molecules are heterogeneous, containing a range of species that differ with respect to the number of DM1 per antibody molecule. The DM1 distribution profile of the ADC was characterized by electrospray ionization mass spectrometry (ESI-MS) and capillary isoelectric focusing (cIEF), which showed that 0-8 DM1s were conjugated to an antibody molecule. By taking advantage of the high-quality MS/MS spectra and the accurate mass detection of diagnostic DM1 fragment ions generated from the higher-energy collisional dissociation (HCD) approach, we were able to identify 76 conjugation sites in the ADC, which covered approximately 83% of all the putative conjugation sites. The diagnostic DM1 fragment ions discovered in this study can be readily used for the characterization of other ADCs with maytansinoid derivatives as payload. Differential scanning calorimetric (DSC) analysis of the ADC indicated that the conjugation of DM1 destabilized the C(H)2 domain of the molecule, which is likely due to conjugation of DM1 on lysine residues in the C(H)2 domain. As a result, methionine at position 258 of the heavy chain, which is located in the C(H)2 domain of the antibody, is more susceptible to oxidation in thermally stressed ADC samples when compared to that of the naked antibody.
对一种抗体药物偶联物（ADC）进行了结构表征，该偶联物由IgG1单克隆抗体（mAb）、美登素药物（DM1）和一种不可裂解的连接子组成。DM1分子通过mAb在溶剂暴露的赖氨酸残基处的非特异性修饰进行偶联。由于赖氨酸偶联过程的性质，ADC分子是异质的，包含一系列在每个抗体分子上DM1数量不同的物种。通过电喷雾电离质谱（ESI-MS）和毛细管等电聚焦（cIEF）对ADC的DM1分布图谱进行了表征，结果表明0至8个DM1与一个抗体分子偶联。利用高质量的MS/MS谱图以及通过高能碰撞解离（HCD）方法产生的诊断性DM1碎片离子的精确质量检测，我们能够在ADC中鉴定出76个偶联位点，这些位点覆盖了所有推定偶联位点的约83%。本研究中发现的诊断性DM1碎片离子可很容易地用于表征其他以美登素衍生物作为有效载荷的ADC。对ADC的差示扫描量热法（DSC）分析表明，DM1的偶联使分子的C(H)2结构域不稳定，这可能是由于DM1在C(H)2结构域的赖氨酸残基上偶联所致。因此，与裸抗体相比，位于抗体C(H)2结构域的重链第258位的甲硫氨酸在热应激的ADC样品中更容易被氧化。
