An international study was conducted to evaluate the performance and reliability of an online multi-dimensional (mD)-LC-MS/MS approach for the characterization of antibody charge variants. The characterization of antibody charge variants is traditionally performed by time-consuming, offline isolation of charge variant fractions by ion exchange chromatography (IEC) that are subsequently subjected individually to LC-MS/MS peptide mapping. This newly developed mD-LC-MS/MS approach enables automated and rapid characterization of charge variants using much lower sample requirements. This online workflow includes sample reduction, digestion, peptide mapping, and subsequent mass spectrometric analysis within a single, fully-automated procedure. The benefits of using online mD-LC-MS/MS for variant characterization include fewer handling steps, a more than 10-fold reduction in required sample amount, reduced sample hold time as well as a shortening of the overall turnaround time from weeks to few days compared to standard offline procedures. In this site-to-site comparison study, we evaluated the online peptide mapping data collected from charge variants of trastuzumab (Herceptin®) across three international laboratories. The purpose of this study was to compare the overall performance of the online mD-LC-MS/MS approach for antibody charge variant characterization, with all participating sites employing different mD-LC-MS/MS setups (e.g., instrument vendors, modules, columns, CDS software). The high sequence coverage (95%–97%) obtained in each laboratory, enabled a reproducible generation of tryptic peptides and the comparison of values of the charge variants. Results obtained at all three participating sites were in good agreement, highlighting the reliability and performance of this approach, and correspond with data gained by the standard offline procedure. Overall, our results underscore of the benefit mD-LC-MS/MS technology for therapeutic antibody characterization, confirming its potential to become an important tool in the toolbox of protein characterization scientists.

进行了一项国际研究，以评估用于表征抗体电荷变体的在线多维 (mD)-LC-MS/MS 方法的性能和可靠性。抗体电荷变体的表征传统上是通过离子交换色谱 (IEC) 耗时、离线分离电荷变体部分来进行的，然后单独进行 LC-MS/MS 肽图分析。这种新开发的 mD-LC-MS/MS 方法能够使用低得多的样品要求自动快速表征电荷变体。此在线工作流程包括在单个全自动程序中进行样品还原、消化、肽图分析和随后的质谱分析。使用在线 mD-LC-MS/MS 进行变异表征的好处包括更少的处理步骤、与标准离线程序相比，所需样品量减少了 10 倍以上，减少了样品保持时间，并将总周转时间从几周缩短到几天。在这项站点间比较研究中，我们评估了从三个国际实验室的曲妥珠单抗 (Herceptin®) 电荷变体收集的在线肽图数据。本研究的目的是比较在线 mD-LC-MS/MS 方法在抗体电荷变体表征方面的整体性能，所有参与站点采用不同的 mD-LC-MS/MS 设置（例如，仪器供应商、模块、列、CDS 软件）。在每个实验室中获得的高序列覆盖率 (95%–97%)，使得胰蛋白酶肽的可重复生成和电荷变体值的比较成为可能。在所有三个参与站点获得的结果都非常一致，突出了这种方法的可靠性和性能，并且与标准离线程序获得的数据相符。总体而言，我们的结果强调了 mD-LC-MS/MS 技术在治疗性抗体表征方面的优势，证实了其成为蛋白质表征科学家工具箱中重要工具的潜力。