Identification of a monoclonal antibody clipping variant by cross-validation using capillary electrophoresis – sodium dodecyl sulfate, capillary zone electrophoresis – mass spectrometry and capillary isoelectric focusing – mass spectrometry

Proteomic analysis plays an important role in basic biological studies and precision medicine. However, real samples contain numerous proteins with a wide dynamic distribution range. Such high complexity of the samples has a drastic effect on the identification coverage of proteins. Consequently, with advancements in mass spectrometry (MS) technology, concomitant improvements in separation technologies for simplifying the sample should be critical. With the advantages of small sample loading volume, high separation efficiency, and high speed, capillary electrophoresis (CE) coupled to MS has been gained much attention in the field of proteomics research. A nanoflow sheath liquid interface and a sheathless interface have been developed and commercialized, boosting the development of the CE-MS technology. Capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and capillary electrochromatography (CEC) have been successfully combined with MS, and CZE-MS has widespread application. In proteomic research, the "bottom-up" strategy, which is based on the separation and identification of enzymatic peptides, is widely applied. With the limit of detection as low as 1 zmol for peptides, CE-MS has been successfully applied to single-cell proteomic analysis. Besides, CE is complementary to reversed-phase liquid chromatography (RPLC), providing a new approach for the separation and identification of peptides with similar hydrophobic properties (especially, post-translational peptides). The "top-down" strategy, which is based on the separation and identification of intact proteins, can directly provide more accurate and complete information about proteins. For protein separation, CE is advantageous in terms of the high separation resolution and high protein recovery, thereby improving the sensitivity and coverage of protein identification. Native MS enables successful identification and characterization of protein complexes under nondenaturing conditions. Because of the good compatibility of CE with MS, attempts have been made to use CE coupled with native MS for the separation and identification of protein complexes. In this review, the development of the CE-MS technology is first reported, including a robust and sensitive CE-MS interface, and a separation mode coupled to MS. Then, the application of the CE-MS technology to "bottom-up", "top-down" and native MS analysis is discussed. The superiority of CE-MS in proteomic analysis is also emphasized. Finally, the promising future prospects of CE-MS are discussed.

Identification of a Monoclonal Antibody Clipping Variant by Cross-Validation Using Capillary Electrophoresis – Sodium Dodecyl Sulfate, Capillary Zone Electrophoresis – Mass Spectrometry and Capillary Isoelectric Focusing – Mass Spectrometry

Capillary electrophoresis–mass spectrometry for the analysis of intact proteins

15 Capillary electrophoresis and bioanalysis

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), a high-resolution and high-sensitivity analytical technique, is an essential tool for analyzing the critical quality attributes (CQAs) of monoclonal antibodies (mAbs) and their derivatives, including antibody-drug conjugates (ADCs) and bispecific antibodies (bsAbs). This study systematically reviews the applications of CE-SDS in analyzing the purity and fragments of mAbs, characterizing positional isomers of ADCs, and identifying mismatch impurities in bsAbs. Focusing on the core technical challenge that CE-SDS cannot be directly coupled with mass spectrometry (MS) for fragment structure identification, the study summarizes technical solutions based on indirect identification approaches and offline/online coupling strategies with Capillary Zone Electrophoresis-Mass Spectrometry (CZE-MS). In addition, from a regulatory science perspective, this study details the key considerations for method validation, establishment of quality standards, and preparation of regulatory submissions for CE-SDS. This study aims to provide a systematic reference for the development and quality control of related biopharmaceuticals, highlighting future development directions, including high-throughput analysis, coupling techniques, and degradation prediction.

The aspiration of the multi-attribute method (MAM) is to utilize a single mass spectrometry-based method that can measure multiple attributes simultaneously, thus enabling data-driven decisions more quickly and efficiently. However, challenges associated with identifying and quantitating critical quality attributes such as asparagine deamidation and isoaspartic acid using conventional ultrahigh-pressure liquid chromatography (UHPLC) coupled to mass spectrometry have necessitated long gradients to ensure sufficient separation for quantitation. Microfluidic chip-based capillary zone electrophoresis mass spectrometry (CZE-MS) shows potential to enable rapid charge-based separation of peptide mixtures, and this approach was evaluated using multipeptide mixtures of synthetic peptides as well as digested protein therapeutics. In these experiments, repeatability, linearity, and peak-to-peak resolution of several peptide families containing asparagine deamidation and/or isoaspartic acid were demonstrated. In addition, a comparison of peptide map results acquired with both UHPLC-MS and CZE-MS for two enzymatically digested biological therapeutics showed comparable sequence coverage and quantitation results between the two approaches. As MAM becomes increasingly utilized for analysis of biological therapeutics, MS instrument demand will rapidly increase, resulting in a bottleneck. A CZE-based separation shows potential to alleviate this bottleneck by drastically increasing MAM throughput while providing results comparable to those acquired using conventional UHPLC separations.

Capillary electrophoresis (CE) mass spectrometry (MS), with its ability to separate compounds present in extremely small volume samples rapidly, with high separation efficiency, and with compound identification capability based on molecular weight, is an extremely valuable analytical technique for the analysis of complex biological mixtures. The highest sensitivities and separation efficiencies are usually achieved by using narrow capillaries (5-50 micro m i.d.) and by using sheathless CE-to-MS interfaces. The difficulties in CE-to-MS interfacing and the limited loadability of these narrow columns, however, have prevented CE-MS from becoming a widely used analytical technique. To remedy these limitations, several CE-MS interfacing techniques have recently been introduced. While electrospray ionization is the most commonly used ionization technique for interfacing CE-to-MS, matrix assisted laser desorption ionization has also been used, using both on-line and off-line techniques. Moreover, the high concentration detection limit of CE has been addressed by development of several sample concentration and sample focusing methods. In addition, a wide variety of techniques such as capillary zone electrophoresis, capillary isoelectric focusing, and on-column transient isotachophoresis have now been interfaced to MS. These advances have resulted in a rapid increase in the use of CE-MS in the analysis of complex biological mixtures. CE-MS has now been successfully applied to the analysis of a wide variety of compounds including amino acids, protein digests, protein mixtures, single cells, oligonucleotides, and various small molecules relevant to the pharmaceutical industry.

该文为2020年毛细管电泳(capillary electrophoresis, CE)技术年度回顾。归纳总结了以“capillary electrophoresis-mass spectrometry”或“capillary isoelectric focusing”或“micellar electrokinetic chromatography”或“capillary electrophoresis”为关键词在ISI Web of Science数据库中进行主题检索得到的2020年CE技术相关研究论文222篇,以及中文期刊《分析化学》和《色谱》中CE技术相关的研究论文37篇。对2020年影响因子(IF)≥5.0的Analytical Chemistry, Food Chemistry, Analytica Chimica Acta和Talanta等13本期刊的38篇文章报道的科研工作作了逐一介绍;对IF<5.0的期刊中CE技术报道较为集中的Journal of Chromatography A和Electrophoresis两本分析化学类期刊发表40篇文章中的代表性内容作了综合介绍;对重要的中文期刊《分析化学》出版的“核酸适配体专刊”和《色谱》出版的2期CE技术专刊所收录的37篇文章中的工作作了总体介绍。总体来说,2020年CE技术发展趋势仍以毛细管电泳-质谱(CE-MS)的新方法和新应用最为突出,主要集中在CE-MS与电化学检测、固相萃取以及多种毛细管电泳模式的联用方面,CE-MS接口相关的报道较前几年有所减少;常规CE技术则以胶束电动毛细管色谱(MEKC)在复杂样本分析、浓缩富集应用为主,尤其在食品和药品等复杂基质样本分析方面的报道较为集中;此外,我国CE相关领域专家学者的科研成果涵盖了CE在生命科学、临床医学、医药研发、环境科学、天然产物、食品分析等领域的应用,代表了国内CE科研应用水平和现状。

Analyses of proteins in complex mixtures such as cell lyzates are presently performed mainly by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. For structural analysis, each protein in a spot is digested with proteases and the fragment peptides are subjected to Edman sequencing and/or mass spectrometry. These works aim at the total analysis of proteins in a complex mixture and reconstruction of their cooperative functions. Genomic studies are now being combined with these proteomic studies. This review article focuses on the application of capillary electrophoresis aiming at the total analysis of complex protein systems or structural analysis of each separated protein. From this viewpoint, articles on capillary zone electrophoresis, capillary isoelectric focusing, and sieving SDS capillary electrophoresis are reviewed. Since these techniques of capillary electrophoresis have been thoroughly reviewed previously, papers published in 1997 and 1998 are mainly covered.

High-resolution capillary zone electrophoresis - mass spectrometry (CZE-MS) has been of increasing interest for the analysis of biopharmaceuticals. In this work, a combination of middle-down and intact CZE-MS analyses has been implemented for the characterization of a biotherapeutic monoclonal antibody (mAb) with a variety of post-translational modifications (PTMs) and glycosylation structures. Middle-down and intact CZE separations were performed in an acidified methanol-water background electrolyte on a capillary with a positively charged coating (M7C4I) coupled to an Orbitrap mass spectrometer using a commercial sheathless interface (CESI). Middle-down analysis of the IdeS-digested mAb provided characterization of PTMs of digestion fragments. High resolution CZE enabled separation of charge variants corresponding to 2X-deamidated, 1X-deamidated, and non-deamidated forms at baseline resolution. In the course of the middle-down CZE-MS analysis, separation of glycoforms of the FC /2 fragment was accomplished due to hydrodynamic volume differences. Several identified PTMs were confirmed by CZE-MS2 . Incorporation of TCEP-HCl reducing agent in the sample solvent resulted in successful analysis of reduced forms without the need for alkylation. CZE-MS studies on the intact mAb under denaturing conditions enabled baseline separation of the 2X-glycosylated, 1X-glycosylated, and aglycosylated populations as a result of hydrodynamic volume differences. The presence of a trace quantity of dissociated light chain was also detected in the intact protein analysis. Characterization of the mAb under native conditions verified identifications achieved via intact analysis and allowed for quantitative confirmation of proteoforms. Analysis of mAbs using CZE-MS represents a complementary approach to the more conventional liquid-chromatography - mass spectrometry-based approaches.

Examination of capillary zone electrophoresis, capillary isoelectric focusing and sodium dodecyl sulfate capillary electrophoresis for the analysis of recombinant tissue plasminogen activator

Capillary electrophoresis is an electric field‐mediated microseparation technique that utilises narrow‐bore fused‐silica capillaries (10–100 μm) and high applied electric fields (100–1000 V/cm) enabling short analysis times with high separation efficiency and excellent resolution, whereas the narrow bore capillary readily dissipates the generated Joule heat. The main separation modes of capillary electrophoresis are capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), capillary isoelectric focusing (cIEF), capillary gel electrophoresis (CGE) and capillary electrochromatography (CEC). As a truly orthogonal method to high‐performance liquid chromatography (HPLC), capillary electrophoresis is recently gaining high importance in the pharmaceutical, biotechnology and biomedical industries, especially for the analysis of protein therapeutics. Key Concepts: Capillary electrophoresis (CE) is a truly orthogonal separation method to chromatography‐based techniques. CE is a fully automated approach to electrophoresis. The narrow bore capillaries used in CE enable application of high separation voltages without significant heat generation. Electroendoosmotic flow (EOF) generates a bulk fluid flow within the narrow bore capillary and therefore one of the driving forces of CE analysis. The main separation modes of CE are: Capillary zone electrophoresis (CZE) separation is based on free solution mobility; micellar electrokinetic chromatography (MEKC) utilises partitioning of hydrophobic analytes within charged micelles; capillary gel electrophoresis (CGE) applies sieving polymers for size separation of macromolecules; and capillary isoelectric focusing (cIEF) separates amphoteric analytes based on their charge states; and capillary electrochromatography (CEC) based on the interplay between electric field and chromatography‐mediated separations. CE is one of the emerging separation techniques in the biotechnology, biopharmaceutical and biomedical industries. Microfluidics devices integrate the concept of CE with fluidics sample manipulation such as on chip sample preparation.

Strategy for selecting separation solutions in capillary electrophoresis–mass spectrometry

The article discusses capillary electrophoresis (CE) as an advanced technique used for the separation and detection of various pharmaceutical drugs. CE involves the application of high voltages across buffer-filled capillaries to produce separation based on various separation theories, including capillary zone electrophoresis (CZE), micellar electrokinetic capillary chromatography (MEKC), capillary gel electrophoresis, and capillary isoelectric focusing. While traditional CZE is not suitable for the separation of neutral substances, MEKC was developed by Shigeru Terabe in the early 1990s to expand the use of CE to neutral analytes that cannot be separated using straightforward free solution CE. MEKC employs an ionic micellar solution that interacts with the analytes through partitioning processes like a chromatographic technique. To create a pseudostationary phase, a surfactant such as sodium dodecyl sulfate (SDS) is added to the buffer solution at a concentration higher than its critical micellar concentration. The anionic SDS micelles are electrostatically drawn towards the anode, while the electro-osmotic flow carries the bulk solution toward the negative electrode due to the negative charge on the inside surface of the silica capillaries. When a neutral analyte is introduced into the micellar solution, a portion is integrated into the micelle, while the remaining fraction of the analyte migrates with the electroosmotic velocity. Separation depends on the individual partitioning equilibrium of the various analytes between the micellar and the aqueous phase. The bigger percentage of analyte dispersed inside the micelle, the slower it will travel. This article provides an in-depth understanding of the separation principle and the mechanism involved in MEKC, highlighting its usefulness in separating neutral analytes that cannot be separated using traditional CZE.

In the last few years capillary electrophoresis (CE) has become established alongside high performance liquid chromatography (HPLC) as a complementary, powerful separation technique for peptides and proteins. Along with the high speed, low sample requirement and overall lower running costs, a major advantage of CE is its flexibility. On one hand, typical HPLC separation modes, like reversed phase HPLC using differences in the hydrophobicity of components or ion-exchange chromatography using differences in the net charge of components can also be performed by micellar electrokinetic chromatography (MEKC) or capillary zone electrophoresis (CZE), respectively. On the other hand, typical slab gel electrophoresis separation modes like SDS (sodium dodecyl sulfate)—polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing (IEF) can also be carried out by CE as capillary gel electrophoresis (CGE) or capillary isoelectric focusing (CIEF).