The multi-attribute method (MAM) has garnered attention as a new quality control method of therapeutic monoclonal antibodies (mAbs). MAM analysis allows multiple relative quantifications of several structural attributes of therapeutic mAbs; however, some issues remain to be addressed in its procedures especially for sample preparation. The goal of this study was to optimize the sample preparation method for MAM analysis of mAbs. Using a model mAb, we compared five sample preparation methods based on sequence coverage, peptide redundancy, missed cleavage and chemical deamidation. It was found that low pH buffer and short digestion time reduced artificial deamidation. The desalting process after carboxymethylation was essential to obtaining high sequence coverage by a short digestion time. The generation of missed cleavage peptides was also improved by using a trypsin/lysyl endopeptidase (Lys-C) mixture. Next, we evaluated the usefulness of our method as a part of MAM analysis. Finally, 17 glycopeptides, 2 deamidated peptides and N- and C-terminal peptides of the heavy chain were successfully monitored with acceptable mass accuracy and coefficient of variation (CV, %) of the relative peak area. On the other hand, 4 oxidated peptides indicated the unavoidable slightly higher inter-assay CV (%) of the peak area ratio due to the instability in the MS sample solution. Collectively, we demonstrated that our method was applicable as an easy and reliable sample preparation method for MAM analysis, and the variation in the relative peak area could be influenced by the modification type rather than by the amount of each peptide.

多属性方法 (MAM) 作为治疗性单克隆抗体 (mAb) 的一种新的质量控制方法而受到关注。MAM 分析允许对治疗性 mAb 的几种结构属性进行多次相对量化；然而，在其程序中仍有一些问题有待解决，特别是在样品制备方面。本研究的目标是优化用于 mAb 的 MAM 分析的样品制备方法。使用模型 mAb，我们比较了基于序列覆盖率、肽冗余、漏切和化学脱酰胺的五种样品制备方法。发现低 pH 缓冲液和短消化时间可减少人工脱酰胺。羧甲基化后的脱盐过程对于通过短消化时间获得高序列覆盖率至关重要。通过使用胰蛋白酶/赖氨酰内肽酶 (Lys-C) 混合物，还可以改善漏切肽的产生。接下来，我们评估了我们的方法作为 MAM 分析的一部分的有用性。最后，以可接受的质量精度和相对峰面积的变异系数 (CV,%) 成功监测了 17 种糖肽、2 种脱酰胺肽以及重链的 N 和 C 末端肽。另一方面，由于 MS 样品溶液的不稳定性，4 个氧化肽表明峰面积比不可避免地略高。总的来说，我们证明了我们的方法可用作 MAM 分析的一种简单可靠的样品制备方法，并且相对峰面积的变化可能受修饰类型而不是每种肽的量的影响。