Determination of protein charge variants with (imaged) capillary isoelectric focusing and capillary zone electrophoresis

Abstract

Biopharmaceuticals are increasingly present in pharmaceutical therapy. These highly complex molecules are still challenging for development and quality control. Charge heterogeneity is next to size heterogeneity, glycan analysis, or peptide mapping one main aspect of their thorough characterization. This review focuses on the charge-based analysis of biopharmaceuticals through CE. An overview is given about the three separation techniques cIEF, imaged cIEF and CZE for the analyses of charge variants, emphasizing assays and their precision. Especially the developments in the experimental conditions have been given a high priority: sample preparation, capillary properties, anolytes and catholytes, carrier ampholytes, sacrificing agents, solubility enhancement, isoelectric point marker, mobilization techniques, and detection modes have been discussed. CIEF shows a particularly high heterogeneity in its methods. Therefore, a design space is suggested that states the most important adjustable parameters and their range. This enables an adequate design of experiments for individual sample separations, in order to accelerate and simplify method developments.

Peptidomics and proteomics based on CE‐MS as a robust tool in clinical application: The past, the present, and the future

“…applied CE using coated capillary coupled via a sheathless interface to MS in parallel to other methods for analysis of a therapeutic peptide, aviptadil, and showed that CE‐MS is complementary to LC‐MS, especially with respect to detecting impurities. In total, these examples and additional published studies on CE‐MS in biopharmaceutical analysis indicate substantial potential in the context of manufacturing and quality control of biopharmaceuticals, as also outlined in detail in an excellent recent review .…”

Insights from capillary electrophoresis approaches for characterization of monoclonal antibodies and antibody drug conjugates in the period 2016–2018

“…For this purpose, neutral capillaries using dynamic or permanent coating have been implemented. In cIEF, the most described coatings are LPA [54,57,59], hydroxypropyl methylcellulose (HPMC) [60,61] and PVA [62]. Concerning icIEF, a majority of reports in the literature described the use of a fluorocarbon capillary due to manufacturer's near monopoly (Proteinsimple) [55,[63][64][65].…”

“…Authors concluded that their method used to separate native mAbs offers complementary results but extend five folds the analysis time and elicits a loss of sensitivity. The same year, Kahle et al realized a comparison of protein charge variants separated by CZE and cIEF to upgrade the quality control of batches [59]. Another approach on the development of innovative coating was performed by Xiao et al They reported an easy-to-use coating which gave a good separation of cetuximab (Erbitux®) and rituximab (Mabthera®) with respectively nine and five separated variants [88].…”

Application of affinity capillary electrophoresis for charge heterogeneity profiling of biopharmaceuticals

“…For this purpose, a CZE–CZE–MS approach was published recently . Due to the individual properties of different biopharmaceuticals, various modifications of the initial method from He and colleagues have been published . Goyon et al.…”

“…For this purpose, a CZE-CZE-MS approach was published recently [10]. Due to the individual properties of different biopharmaceuticals, various modifications of the initial method from He and colleagues [8] have been published [11][12][13]. Goyon et al demonstrated an alternative approach for the analysis of charge variants of mAbs using a BGE containing polyethylene oxide for dynamic coating and Bis-tris as a buffering agent.…”