National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland.
National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland; School of Chemical and Bioprocess Engineering, University College Dublin, Dublin 4, Ireland.
J Pharm Biomed Anal. 2020 Jun 5;185:113218. doi: 10.1016/j.jpba.2020.113218. Epub 2020 Feb 29.
Charge variant profiling of therapeutic proteins is required by the International Council for Harmonisation guidelines and is traditionally performed by capillary electrophoresis or ion exchange chromatography. Recently, improvements in the hyphenation of capillary electrophoresis with mass spectrometry and the introduction of mass spectrometry compatible background electrolytes has allowed the implementation of native mass spectrometric determination of the charge variant profile obtained from the electrophoretic separation. The low flow operation of the microfluidic electrophoretic platform significantly boosts mass spectrometric sensitivity and increases the dynamic range, even when using sample amounts as low as 1 ng in capillary. In the current study, rituximab, trastuzumab and bevacizumab drug products were analysed using the ZipChip microfluidic CE-ESI-MS platform that facilitated confident identification of proteoforms with an average mass accuracy of <15 ppm. Up to 52 proteoforms were identified for trastuzumab drug product, while rituximab sample revealed the presence of fragments and sialylated N-glycans. Overall, the CE-ESI-MS platform proved to be a fast and robust tool for therapeutic protein charge variant profiling and facilitated efficient coupling with native mass spectrometry for the generation of highly informative characterisation data.
治疗性蛋白的电荷变异体分析是国际协调委员会指南所要求的，传统上采用毛细管电泳或离子交换色谱法进行。最近，毛细管电泳与质谱的联用技术得到了改进，并且引入了与质谱兼容的背景电解质，从而可以实现从电泳分离中获得的电荷变异体谱的Native Mass 直接测定。微流控电泳平台的低流速操作极大地提高了质谱的灵敏度，并增加了动态范围，即使在毛细管中使用低至 1ng 的样品量时也是如此。在本研究中，使用ZipChip 微流控 CE-ESI-MS 平台分析了利妥昔单抗、曲妥珠单抗和贝伐珠单抗药物产品，该平台能够对蛋白形式进行可靠鉴定，平均质量精度<15 ppm。曲妥珠单抗药物产品可鉴定多达 52 种蛋白形式，而利妥昔单抗样品则显示出片段和唾液酸化 N-糖基化的存在。总体而言，CE-ESI-MS 平台被证明是一种快速而强大的治疗性蛋白电荷变异体分析工具，并且能够与 Native Mass 高效耦合，生成高度信息丰富的特征化数据。
