Bayne Elizabeth F, Gao Xiong-Zhuo, Jiang Yuan, Bennett Raffeal, Regalado Erik L, Pirrone Gregory F, Makarov Alexey A
Discovery Analytical Research, Analytical R&D, MRL, Merck & Co., Inc., Boston, MA, 02115, USA.
Data Rich Measurements, Analytical R&D, MRL, Merck & Co., Inc., Rahway, NJ, 07065, USA.
Anal Chim Acta. 2025 Sep 22;1368:344312. doi: 10.1016/j.aca.2025.344312. Epub 2025 Jun 14.
Biotherapeutics are a prominent class of molecules used to treat a wide range of diseases, including cancer, infections, inflammatory and immune disorders. The discovery workflow involves high-throughput screening of critical quality attributes using liquid chromatography (LC) to optimize cell line development and facilitate drug candidate selection. However, several LC techniques, such as size exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC), are not directly compatible with electrospray ionization (ESI), hindering mass spectrometry (MS)-based identification of impurity peaks. Consequently, significant time and effort are often required to adapt these method conditions to ESI-MS, which slows down discovery efforts.
Matrix-assisted laser desorption ionization (MALDI) is a surface-based ionization technique that offers an attractive alternative to ESI for high-throughput screening due to its rapid analysis times and resistance to buffer/matrix interferences. MALDI-MS is highly compatible with automated liquid handling tools for sample transfer and requires minimal sample volume. To address the increasingly complex needs and challenging timelines of next-generation biologics development, we have developed an LC-MALDI-MS offline approach for rapid peak identification using an LC-fraction collector and MALDI-TOF-MS. This streamlined approach is adaptable to multiple chromatographic techniques, regardless of their inherent MS compatibility, and is suitable for automation. The workflow is versatile and can be applied universally in combination with various chromatographic techniques and analytes, including monoclonal and bispecific antibodies, fusion proteins, and peptides. Importantly, fraction collection and MS data acquisition can be completed within hours without the need for additional method development or sample preparation for MS-based measurements.
Herein, we systematically demonstrate the use of an automated LC-MALDI-MS workflow for rapid peak identification involving SEC, HIC, and Reversed Phase LC for proteins and peptides, as well as a use case with a bispecific antibody and fusion protein using SEC.
生物治疗药物是一类重要的分子，用于治疗多种疾病，包括癌症、感染、炎症和免疫紊乱。发现工作流程涉及使用液相色谱（LC）对关键质量属性进行高通量筛选，以优化细胞系开发并促进候选药物的选择。然而，一些LC技术，如尺寸排阻色谱（SEC）和疏水相互作用色谱（HIC），与电喷雾电离（ESI）不直接兼容，这阻碍了基于质谱（MS）的杂质峰鉴定。因此，通常需要大量时间和精力来使这些方法条件适应ESI-MS，这减缓了发现工作的进度。
基质辅助激光解吸电离（MALDI）是一种基于表面的电离技术，由于其分析速度快且对缓冲液/基质干扰具有抗性，为高通量筛选提供了一种有吸引力的ESI替代方法。MALDI-MS与用于样品转移的自动化液体处理工具高度兼容，并且所需样品体积最小。为了满足下一代生物制品开发日益复杂的需求和具有挑战性的时间线，我们开发了一种LC-MALDI-MS离线方法，使用LC馏分收集器和MALDI-TOF-MS进行快速峰鉴定。这种简化的方法适用于多种色谱技术，无论其固有的MS兼容性如何，并且适用于自动化。该工作流程具有通用性，可普遍应用于与各种色谱技术和分析物的组合，包括单克隆和双特异性抗体、融合蛋白和肽。重要的是，馏分收集和MS数据采集可以在数小时内完成，而无需进行额外的方法开发或基于MS测量的样品制备。
在此，我们系统地展示了使用自动化LC-MALDI-MS工作流程进行快速峰鉴定，涉及用于蛋白质和肽的SEC、HIC和反相LC，以及使用SEC的双特异性抗体和融合蛋白的应用案例。
