An interlaboratory capillary zone electrophoresis-UV study of various monoclonal antibodies, instruments, and ε-aminocaproic acid lots.

Capillary zone electrophoresis ultraviolet (CZE-UV) has become increasingly popular for the charge heterogeneity determination of mAbs and vaccines. The ε-aminocaproic acid (eACA) CZE-UV method has been used as a rapid platform method. However, in the last years, several issues have been observed, for example, loss in electrophoretic resolution or baseline drifts. Evaluating the role of eACA on the reported issues, various laboratories were requested to provide their routinely used eACA CZE-UV methods, and background electrolyte compositions. Although every laboratory claimed to use the He et al. eACA CZE-UV method, most methods actually deviate from He's. Subsequently, a detailed interlaboratory study was designed wherein two commercially available mAbs (Waters' Mass Check Standard mAb [pI 7] and NISTmAb [pI 9]) were provided to each laboratory, along with two detailed eACA CZE-UV protocols for a short-end, high-speed, and a long-end, high-resolution method. Ten laboratories participated each using their own instruments, and commodities, showing excellence method performance (relative standard deviations [RSDs] of percent time-corrected main peak areas from 0.2% to 1.9%, and RSDs of migration times from 0.7% to 1.8% [n = 50 per laboratory], analysis times in some cases as short as 2.5 min). This study clarified that eACA is not the main reason for the abovementioned variations.

Two-Dimensional Capillary Zone Electrophoresis-Mass Spectrometry: Intact mAb Charge Variant Separation Followed by Peptide Level Analysis Using In-Capillary Digestion.

Characterization of charge heterogeneity is an essential pillar for pharmaceutical development and quality control of therapeutic monoclonal antibodies (mAbs). The highly selective and commonly applied capillary zone electrophoresis (CZE) method containing high amounts of ε-aminocaproic acid (EACA) provides a detailed and robust charge heterogeneity profile of intact mAb variants. Nevertheless, the exact location of protein modifications within these charge profiles remains ambiguous. Electrospray ionization mass spectrometry (ESI-MS) is a promising tool for this purpose; however, EACA is incompatible with electrospray. In this context, we present a two-dimensional CZE-CZE-MS system to combine efficient charge variant separation of intact mAbs with subsequent peptide analysis after in-capillary digestion of selected charge variants. The first dimension is based on a generic CZE(EACA) method in a fused silica capillary. In the second dimension, a neutral-coated capillary is used for in-capillary reduction and digestion with Tris(2-carboxyethyl)phosphine (TCEP) and pepsin, followed by CZE separation and MS/MS-characterization of the resulting peptides. The setup is demonstrated using stressed and nonstressed mAbs where peaks of basic, main, and acidic variants were transferred in a heart-cut fashion, digested, and characterized on the peptide level. Sequence coverages of more than 90% were obtained for heavy chain (HC) and light chain (LC) for four different mAbs, including low-abundance variants (<2% of the main peak). Frequently observed modifications (deamidation, oxidation, etc.) could be detected and localized. This study demonstrates a proof-of-concept for identification and localization of protein modifications from CZE charge heterogeneity profiles and, in this way, is expected to support the development and quality control testing of protein pharmaceuticals.

Characterization of therapeutic mAb charge heterogeneity by iCIEF coupled to mass spectrometry (iCIEF-MS).

Imaged capillary isoelectric focusing (iCIEF) has emerged as an important technique for therapeutic monoclonal antibody (mAb) charge heterogeneity analysis in the biopharmaceutical context, providing imaged detection and quantitation by UV without a mobilization step. Besides quantitation, the characterization of separated charge variants ideally directly by online electrospray ionization-mass spectrometry (ESI-MS) is crucial to ensure product quality, safety, and efficacy. Straightforward direct iCIEF-MS coupling combining high separation efficiency and quantitative results of iCIEF with the characterization power of MS enables deep characterization of mAb charge variants. A short technical setup and optimized methodical parameters (30 nl/min mobilization rate, 2%-4% ampholyte concentration, 0.5-2 mg/ml sample concentration) allow successful mAb charge variant peak assignment from iCIEF to MS. Despite a loss of separation resolution during the transfer, separated intact mAb charge variants, including deamidation as well as major and minor glycoforms even from low abundant charge variants, could be characterized by online ESI-MS with high precision. The presented setup provides a large potential for mAb charge heterogeneity characterization in biopharmaceutical applications.

nanoCEasy: An Easy, Flexible, and Robust Nanoflow Sheath Liquid Capillary Electrophoresis-Mass Spectrometry Interface Based on 3D Printed Parts.

Capillary electrophoresis-mass spectrometry (CE-MS) is a powerful tool in various fields including proteomics, metabolomics, and biopharmaceutical and environmental analysis. Nanoflow sheath liquid (SL) CE-MS interfaces provide sensitive ionization, required in these fields, but are still limited to a few research laboratories as handling is difficult and expertise is necessary. Here, we introduce nanoCEasy, a novel nanoflow SL interface based on 3D printed parts, including our previously reported two capillary approach. The customized plug-and-play design enables the introduction of capillaries and an emitter without any fittings in less than a minute. The transparency of the polymer enables visual inspection of the liquid flow inside the interface. Robust operation was systematically demonstrated regarding the electrospray voltage, the distance between the emitter and MS orifice, the distance between the separation capillary and emitter tip, and different individual emitters of the same type. For the first time, we evaluated the influence of high electroosmotic flow (EOF) separation conditions on a nanoflow SL interface. A high flow from the separation capillary can be outbalanced by increasing the electrospray voltage, leading to an overall increased electrospray flow, which enables stable operation under high-EOF conditions. Overall, the nanoCEasy interface allows easy, sensitive, and robust coupling of CE-MS. We aspire the use of this sensitive, easy-to-use interface in large-scale studies and by nonexperts.

Online mass spectrometry of CE (SDS)-separated proteins by two-dimensional capillary electrophoresis.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the fundamental technique for protein separation by size. Applying this technology in capillary format, gaining high separation efficiency in a more automated way, is a key technology for size separation of proteins in the biopharmaceutical industry. However, unequivocal identification by online mass spectrometry (MS) is impossible so far, due to strong interference in the electrospray process by SDS and other components of the SDS-MW separation gel buffer. Here, a heart-cut two-dimensional electrophoretic separation system applying an electrically isolated valve with an internal loop of 20 nL is presented. The peak of interest in the CE (SDS) separation is transferred to the CZE-MS, where electrospray-interfering substances of the SDS-MW gel are separated prior to online electrospray ionization mass spectrometry. An online SDS removal strategy for decomplexing the protein-SDS complex is implemented in the second dimension, consisting of the co-injection of organic solvent and cationic surfactant. This online CE (SDS)-CZE-MS system allows MS characterization of proteoforms separated in generic CE (SDS), gaining additional separation in the CZE and detailed MS information. In general, the system can be applied to all kinds of proteins separated by CE (SDS). Here, we present results of the CE (SDS)-CZE-MS system on the analysis of several biopharmaceutically relevant antibody impurities and fragments. Additionally, the versatile application spectrum of the system is demonstrated by the analysis of extracted proteins from soybean flour. The online hyphenation of CE (SDS) resolving power and MS identification capabilities will be a powerful tool for protein and mAb characterization. Graphical abstract Two-dimensional capillary electrophoresis system hyphenated with mass spectrometry for the characterization of CE (SDS)-separated proteins. As first dimension, a generic and high MS-interfering CE (SDS) separation is performed for size separation. After heart-cut transfer of the unknown CE (SDS) protein peak, via a four-port nanoliter valve to a volatile electrolyte system as second dimension, interference-free mass spectrometric data of separated mAb fragments and soybean proteins are obtained.

Recent advances in capillary electrophoresis-mass spectrometry: Instrumentation, methodology and applications.

Capillary electrophoresis (CE) offers fast and high-resolution separation of charged analytes from small injection volumes. Coupled to mass spectrometry (MS), it represents a powerful analytical technique providing (exact) mass information and enables molecular characterization based on fragmentation. Although hyphenation of CE and MS is not straightforward, much emphasis has been placed on enabling efficient ionization and user-friendly coupling. Though several interfaces are now commercially available, research on more efficient and robust interfacing with nano-electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI) and inductively coupled plasma mass spectrometry (ICP) continues with considerable results. At the same time, CE-MS has been used in many fields, predominantly for the analysis of proteins, peptides and metabolites. This review belongs to a series of regularly published articles, summarizing 248 articles covering the time between June 2016 and May 2018. Latest developments on hyphenation of CE with MS as well as instrumental developments such as two-dimensional separation systems with MS detection are mentioned. Furthermore, applications of various CE-modes including capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF) coupled to MS in biological, pharmaceutical and environmental research are summarized.

Two-dimensional capillary electrophoresis-mass spectrometry (CE-CE-MS): coupling MS-interfering capillary electromigration methods with mass spectrometry.

Electromigration separation techniques often demand certain compounds in the electrolyte to achieve the required selectivity and efficiency. These compounds, including the electrolyte itself, ampholytes, polymeric compounds for sieving, complexing agents, tensides, etc. are often non-volatile. Thus, interference with the electrospray ionization process is a common issue, impeding direct coupling of such electrolyte systems to mass spectrometry. Still, several options exist to obtain mass spectra after separation, including offline fractionation, alternative ionization, dilution, or the change to volatile constituents. In the first part of this article, these methods are discussed. However, all of these options are a compromise of separation performance and sensitivity of mass spectrometric detection. Two-dimensional capillary electrophoresis-mass spectrometry (CE-CE-MS) systems represent a promising alternative to the aforementioned challenges, as they allow the use of existing methods with best separation performance in combination with sensitive mass characterization. In this context, the second part of this article is dedicated to the advantages, limitations, and applications of this approach. Finally, an outlook towards future developments is given.