School of Pharmaceutical Sciences, University of Geneva, CMU - Rue Michel-Servet, 1, 1206 Geneva, Switzerland.
Advanced Materials Technology, 3521 Silverside road, Suite 1-K, DE 19810, Wilmington, USA.
J Chromatogr A. 2020 May 10;1618:460901. doi: 10.1016/j.chroma.2020.460901. Epub 2020 Jan 21.
A novel column-coupling approach is suggested to improve both the selectivity and efficiency of protein separations in liquid chromatography. Protein separations often suffer from limited selectivity or not appropriate resolving power. For a new biopharmaceutical product, the identification of the main and minor variant species is required. For that purpose, often offline collection fractioning is applied which is time consuming and regularly dilute the samples to an unacceptable extent. By serially coupling columns in the order of their increasing retentivity and applying "multi-isocratic" elution mode, indeed any (arbitrary) selectivity can be attained. Moreover, if a protein peak is trapped at the inlet of a later column segment - of a coupled system -, its band will be refocused and elute in unprecedented sharp peak. Furthermore, it becomes possible to perform online on-column fractioning of protein species within a very short analysis time (∼ 1 min) and without sample dilution. Two-, three- or multiple column systems can be developed and applied for complex sample separations (such as antibody mixtures). This new methodology can be particularly useful to improve the analysis (and therefore, safety and quality) of therapeutic mAbs and related products and offers benefits compared to offline fractionating. It is also demonstrated in this proof of concept study, that methyl (C1) modified RP phase has a great potential for protein separations despite it is not commercially available in state-of-the-art wide pore superficially porous particle format..
一种新的柱联方法被提出，以提高液相色谱中蛋白质分离的选择性和效率。蛋白质分离往往受到选择性有限或分辨率不合适的影响。对于一种新的生物制药产品，需要鉴定主要和次要变异体。为此，通常应用离线收集分级，这既耗时又经常将样品稀释到不可接受的程度。通过按其保留能力的顺序串联柱，并应用“多等度”洗脱模式，可以获得任何（任意）选择性。此外，如果蛋白质峰被捕获在串联系统中后续柱段的入口处 - ，其峰带将被重新聚焦并以前所未有的尖锐峰洗脱。此外，还可以在非常短的分析时间（约 1 分钟）内在线进行柱上蛋白质物种的分级，而无需样品稀释。可以开发和应用二、三或多柱系统进行复杂样品分离（如抗体混合物）。这种新方法对于改善治疗性 mAb 和相关产品的分析（因此，安全性和质量）特别有用，并与离线分级相比具有优势。在本概念验证研究中还证明，尽管甲基（C1）修饰的反相相在商业上没有可用于最新的宽孔径表面多孔颗粒形式，但它在蛋白质分离方面具有很大的潜力。
