Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, de Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
Department of Drug Sciences, University of Pavia, via Taramelli 12, I-27100 Pavia, Italy.
Talanta. 2018 Jul 1;184:375-381. doi: 10.1016/j.talanta.2018.03.015. Epub 2018 Mar 8.
Glycosylation is considered a critical quality attribute of therapeutic proteins. Protein heterogeneity introduced by glycosylation includes differences in the nature, number and position of the glycans. Whereas analysis of released glycans and glycopeptides provides information about the composition and/or position of the glycan, intact glycoprotein analysis allows assignment of individual proteoforms and co-occurring modifications. Yet, resolving protein glycoforms at the intact level is challenging. We have explored the capacity of hydrophilic liquid chromatography-mass spectrometry (HILIC-MS) for assessing glycosylation patterns of intact pharmaceutical proteins by analyzing the complex glycoproteins interferon-beta-1a (rhIFN-β - 1a) and recombinant human erythropoietin (rhEPO). Efficient glycoform separation was achieved using a superficially-porous amide HILIC stationary phase and trifluoroacetic acid (TFA) as eluent additive. In-source collision-induced dissociation proved to be very useful to minimize protein-signal suppression effects by TFA. Direct injection of therapeutic proteins in aqueous formulation was possible without causing extra band dispersion, provided that the sample injection volume was not larger than 2 μL. HILIC-MS of rhIFN-β - 1a and rhEPO allowed the assignment of, respectively, 15 and 51 glycoform compositions, next to a variety of posttranslational modifications, such as succinimide, oxidation and N-terminal methionine-loss products. MS-based assignments showed that neutral glycan units significantly contributed to glycoform separation, whereas terminal sialic acids only had a marginal effect on HILIC retention. Comparisons of HILIC-MS with the selectivity provided by capillary electrophoresis-MS for the same glycoproteins, revealed a remarkable complementarity of the techniques. Finally it was demonstrated that by replacing TFA for difluoroacetic acid, peak resolution somewhat decreased, but rhEPO glycoforms with relative abundances below 1% could be detected by HILIC-MS, increasing the overall rhEPO glycoform coverage to 72.
糖基化被认为是治疗性蛋白的关键质量属性。糖基化引入的蛋白质不均一性包括聚糖的性质、数量和位置的差异。虽然释放的聚糖和糖肽的分析提供了关于聚糖组成和/或位置的信息，但完整糖蛋白分析允许分配单个蛋白亚型和共存的修饰。然而，在完整水平上解析蛋白糖型具有挑战性。我们探索了亲水液相色谱-质谱（HILIC-MS）通过分析复杂的糖蛋白干扰素-β-1a（rhIFN-β-1a）和重组人促红细胞生成素（rhEPO）来评估完整药物蛋白糖基化模式的能力。使用表面多孔酰胺 HILIC 固定相和三氟乙酸（TFA）作为洗脱添加剂，实现了高效的糖型分离。在源内碰撞诱导解离被证明非常有用，可以最小化 TFA 对蛋白质信号抑制的影响。只要样品进样体积不超过 2μL，就可以直接将治疗性蛋白注入水制剂中，而不会引起额外的带弥散。rhIFN-β-1a 和 rhEPO 的 HILIC-MS 允许分别分配 15 和 51 种糖型组成，以及各种翻译后修饰，如琥珀酰亚胺、氧化和 N 端甲硫氨酸缺失产物。基于 MS 的分配表明，中性糖单元对糖型分离有显著贡献，而末端唾液酸仅对 HILIC 保留有微小影响。将 HILIC-MS 与毛细管电泳-MS 对相同糖蛋白提供的选择性进行比较，显示出两种技术的显著互补性。最后证明，用二氟乙酸代替 TFA，峰分辨率略有下降，但通过 HILIC-MS 可以检测到相对丰度低于 1%的 rhEPO 糖型，从而将 rhEPO 糖型总覆盖率提高到 72%。
