Charge variant analysis is a widely used tool to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs). Although it is a powerful technique for revealing mAb heterogeneity, an unexpected outcome, for example the appearance of previously undetected isoforms, requires further, time-consuming analysis. The process of identifying these unknowns can also result in unwanted changes to the molecule that are not attributable to the manufacturing process. To overcome this, we recently reported a method combining highly selective cation exchange chromatography-based charge variant analysis with on-line mass spectrometric (MS) detection. We further explored and adapted the chromatographic buffer system to expand the application range. Moreover, we observed no salt adducts on the native protein, also supported by the optimal choice of MS parameters, resulting in increased data quality and mass accuracy. Here, we demonstrate the utility of this improved method by performing an in-depth analysis of adalimumab before and after forced degradation. By combining molecular mass and retention time information, we were able to identify multiple modifications on adalimumab, including lysine truncation, glycation, deamidation, succinimide formation, isomerisation, N-terminal aspartic acid loss or C-terminal proline amidation and fragmentation along with the N-glycan distribution of each of these identified proteoforms. Host cell protein (HCP) analysis was performed using liquid chromatography-mass spectrometry that verified the presence of the protease Cathepsin L. Based on the presence of trace HCPs with catalytic activity, it can be questioned if fragmentation is solely driven by spontaneous hydrolysis or possibly also by enzymatic degradation.

电荷变异分析是一种广泛使用的工具，用于监控单克隆抗体（mAb）制造过程中产品质量的变化。尽管这是揭示mAb异质性的强大技术，但出乎意料的结果（例如，以前未检测到的同工型的出现）需要进一步且费时的分析。识别这些未知物的过程也可能导致分子的不需要的变化，而不是归因于制造过程。为了克服这个问题，我们最近报道了一种将基于高选择性阳离子交换色谱的电荷变异分析与在线质谱（MS）检测相结合的方法。我们进一步探索和调整了色谱缓冲系统，以扩大应用范围。此外，我们没有观察到天然蛋白质上的盐加合物，质谱参数的最佳选择也支持该方法，从而提高了数据质量和质量准确性。在这里，我们通过对强制降解前后的阿达木单抗进行深入分析，证明了这种改进方法的实用性。通过结合分子量和保留时间信息，我们能够鉴定出对阿达木单抗的多种修饰，包括赖氨酸截短，糖基化，脱酰胺基，琥珀酰亚胺形成，异构化，N末端天冬氨酸丢失或C末端脯氨酸酰胺化和片段化以及N这些鉴定出的蛋白形式的β-聚糖分布。宿主细胞蛋白（HCP）分析是使用液相色谱-质谱法进行的，该方法验证了蛋白酶组织蛋白酶L的存在。基于痕量具有催化活性的HCP的存在，