Faculty of Chemistry, Aalen University, Beethovenstraße 1, Aalen 73430, Germany; Faculty of Science, University of Tübingen, Auf der Morgenstelle 8, Tübingen 72076, Germany.
Rentschler Biopharma SE, Erwin-Rentschler-Straße 21, Laupheim 88471, Germany.
J Pharm Biomed Anal. 2025 Jan 1;252:116495. doi: 10.1016/j.jpba.2024.116495. Epub 2024 Oct 1.
The analysis of product-related substances and impurities is a critical step in the biopharmaceutical quality control of multiattribute monoclonal antibodies (mAbs), as posttranslational modifications or other variants can influence the product's biological activity. Many approaches are available for variant analysis; however, they are either variant-specific, mAb-specific, time-consuming, or require expensive equipment. Here, we present a generic capillary electrophoretic method based on a neutral-coated capillary which was coupled to mass spectrometry (MS) via the nanoCEasy interface for mAb variant analysis at the subunit level (enzymatically digested and reduced mAb). The method enabled the separation of several (i) size variants (e.g. glycosylation variants) and (ii) charge variants (e.g. c-terminal lysin clipping) as well as (iii) multiple other proteoforms (e.g. additional glycation) and (iv) incompletely reduced subunits. Separated variants were confirmed by MS/MS fragmentation even for small mass deviations like deamidation or open disulfide bridges. The system, initially developed for one mAb, was tested with nine other IgG1s to show the general applicability of the system. The presented multiattribute method enables fast and detailed characterization of mAb variants with little sample preparation and relatively simple separation equipment enabling the separation of a large set of mAb variants.
分析产品相关物质和杂质是多属性单克隆抗体（mAb）生物制药质量控制的关键步骤，因为翻译后修饰或其他变体可能会影响产品的生物活性。有许多方法可用于变体分析；然而，它们要么是变体特异性的，要么是 mAb 特异性的，要么耗时，要么需要昂贵的设备。在这里，我们提出了一种基于中性涂层毛细管的通用毛细管电泳方法，该方法通过 nanoCEasy 接口与质谱（MS）耦合，用于亚基水平（酶消化和还原的 mAb）的 mAb 变体分析。该方法能够分离多种（i）大小变体（例如糖基化变体）和（ii）电荷变体（例如 C 末端赖氨酸切割）以及（iii）多种其他蛋白形式（例如额外的糖化）和（iv）不完全还原的亚基。通过 MS/MS 碎片分析对分离的变体进行了确认，即使对于小质量偏差（如脱酰胺或打开的二硫键）也是如此。该系统最初是为一种 mAb 开发的，然后用其他 9 种 IgG1 进行了测试，以显示该系统的通用性。所提出的多属性方法能够快速、详细地表征 mAb 变体，只需进行少量样品制备，且分离设备相对简单，能够分离大量 mAb 变体。
