School of Chemistry and Materials Science , Nanjing Normal University , Nanjing , China 210023.
Department of Chemistry , University of British Columbia , Vancouver , British Columbia , Canada V6T 1Z1.
Anal Chem. 2018 Aug 7;90(15):9495-9503. doi: 10.1021/acs.analchem.8b02175. Epub 2018 Jul 23.
Capillary isoelectric focusing directly coupled to high resolution mass spectrometry (cIEF-MS) provides information on amphoteric molecules, including isoelectric point and accurate mass, which enables structural interrogation of biopolymer pI variants. The coupling of cIEF with MS was facilitated by a flow-through microvial interface, made by stainless steel with high chemical resistance and mechanical robustness. Two on-column electrolyte configurations of cIEF-MS were demonstrated using peptide and protein pI markers. The pI resolution was 0.02 pH unit in the pH range of 5.5 to 7.0, with no anticonvective reagent (glycerol) added. High resolution Orbitrap detector provides mass spectra for midsized proteins (<30 kDa), enabling deconvolution with high accuracy for IEF-focused low abundance species. Charge heterogeneity of therapeutic monoclonal antibodies (mAb) is one of the most important attributes in the biopharmaceutical industry, and it is routinely monitored by IEF and fractionation-based methods. As a proof of concept, the commercial formulation of infliximab was directly analyzed using cIEF-MS for separation and online identification of mAb charge variants. The main intact antibody species along with two basic and one acidic variants were observed, and their accurate molecular weights ( M) recorded by MS detector readily revealed the structural differences of these variants. Variants with 0.1 unit in pI difference and 1 Da difference in molecular weight were readily resolved. The deconvoluted intact M values showed ppm level accuracy compared to theoretical predictions.
毛细等电聚焦直接与高分辨率质谱（cIEF-MS）联用，可提供两性分子的信息，包括等电点和精确质量，从而能够对生物聚合物 pI 变体进行结构分析。通过不锈钢制成的流通微管接口，实现了 cIEF 与 MS 的耦合，该接口具有高耐化学性和机械坚固性。使用肽和蛋白质 pI 标志物演示了 cIEF-MS 的两种柱上电解质配置。在 pH 5.5 至 7.0 的范围内，无需添加反对流试剂（甘油），pI 分辨率为 0.02 pH 单位。高分辨率 Orbitrap 检测器提供了中等大小蛋白质（<30 kDa）的质谱，可实现 IEF 聚焦低丰度物质的高精度解卷积。治疗性单克隆抗体（mAb）的电荷异质性是生物制药行业最重要的属性之一，通常通过 IEF 和基于分馏的方法进行监测。作为概念验证，直接使用 cIEF-MS 对英夫利昔单抗的商业制剂进行分析，以分离和在线鉴定 mAb 电荷变体。观察到主要的完整抗体物种以及两个碱性和一个酸性变体，并通过 MS 检测器记录它们的精确分子量（M），这很容易揭示了这些变体的结构差异。pI 差异为 0.1 单位且分子量差异为 1 Da 的变体很容易得到分辨。与理论预测相比，解卷积的完整 M 值具有 ppm 级的准确度。
