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This review provides an overview of the online hyphenation of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) with separation methods to date. The online coupling between separation techniques (gas and liquid chromatography, capillary electrophoresis) and FT-ICR MS essentially raises questions of compromise and is not look as straightforward as hyphenation with other analyzers (QTOF-MS for instance). FT-ICR MS requires time to reach its highest resolving power and accuracy in mass measurement capabilities whereas chromatographic and electrophoretic peaks are transient. In many applications, the strengths and the weaknesses of each technique are balanced by their hyphenation. Untargeted "Omics" (e.g. proteomics, metabolomics, petroleomics, …) is one of the main areas of application for FT-ICR MS hyphenated to online separation techniques because of the complexity of the sample. FT-ICR MS achieves the required high mass measurement accuracy to determine accurate molecular formulae and resolution for isobar distinction. Meanwhile separation techniques highlight isomers and reduce the ion suppression effects extending the dynamic range. Even if the implementation of FT-ICR MS hyphenated with online separation methods is a little trickier (the art of compromise), this review shows that it provides unparalleled results to the scientific community (the art of the possible), along with raising the issue of its future in the field with the relentless technological progress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

As part of RNA characterization, the identification of post-transcriptional modifications can be performed using hyphenation of separation methods with mass spectrometry. To identify RNA modifications with those methods, a first total digestion followed by a dephosphorylation step are usually required to reduce RNA to nucleosides. Even though effective digestion and dephosphorylation are essential to avoid further complications in analysis and data interpretation, to our knowledge, no standard protocol is yet referenced in the literature. Therefore, the aim of this work is to optimize the dephosphorylation step using a total extract of transfer RNA (tRNA) 1 from B. taurus as a model and to determine and fix two protocols, leading to complete dephosphorylation, based on time and bacterial alkaline phosphatase (BAP) 2 consumptions. Capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) was used to estimate the dephosphorylation efficiency of both protocols on many canonical and modified nucleotides. For a timesaving protocol, we established that full dephosphorylation was obtained after a 4-hour incubation at 37 °C with 7.5 U of BAP for 1 µg of tRNA. And for a BAP-saving protocol, we established that full dephosphorylation was obtained 3.0 U of BAP after an overnight incubation at 37 °C. Both protocols are suitable for quantitative analyses as no loss of analytes is expected. Moreover, they can be widely used for all other RNA classes, including messenger RNA or ribosomal RNA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Monoclonal antibodies (mAbs) and their related products as antibody-drug-conjugates (ADCs) or biosimilars represent a constantly growing class of molecules therapeutic proteins used as treatment against numerous diseases. These compounds can undergo several modifications which could alter the efficiency of treatments. In this context, several analytical methods were designed to deliver a comprehensive structural characterization and guarantee the quality of biotherapeutics. Capillary electrophoresis (CE) is considered today as a major technique for the analysis of biotherapeutics due to benefic characteristics as high resolution separation and miniaturized format. Different CE modes have been developed to characterize mAbs at different levels such as capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF), and capillary zone electrophoresis (CZE). Recent developments in CE-mass spectrometry (MS) coupling assessed this technology as a promising tool to obtain high level structural characterization of biopharmaceuticals. Moreover, upcoming techniques such as 2D CE-MS and microfluidic systems are now emerging to offer new possibilities beyond actual limits. This review will be dedicated to discuss the state-of-the-art CE-based methods for the characterization of mAbs and ADCs in the period 2016-2018., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Characterization of therapeutic proteins represents a major challenge for analytical sciences due to their heterogeneity caused by post-translational modifications (PTM). Among these PTM, glycosylation which is possibly the most prominent, require comprehensive identification because of their major influence on protein structure and effector functions of monoclonal antibodies (mAbs). As a consequence, glycosylation profiling must be deeply characterized. For this application, several analytical methods such as separation-based or MS-based methods, were evaluated. However, no CE-ESI-MS approach has been assessed and validated. Here, we illustrate how the use of CE-ESI-MS method permits the comprehensive characterization of mAbs N-glycosylation at the glycopeptide level to perform relative quantitation of N-glycan species. Validation of the CE-ESI-MS method in terms of robustness and reproducibility was demonstrated through the relative quantitation of glycosylation profiles for ten different mAbs produced in different cell lines. Glycosylation patterns obtained for each mAbs were compared to Hydrophilic Interaction Chromatography of 2-aminobenzamide labelled glycans with fluorescence detector (HILIC-FD) analysis considered as a reference method. Very similar glycoprofiling were obtained with the CE-ESI-MS and HILIC-FD demonstrating the attractiveness of CE-ESI-MS method to characterize and quantify the glycosylation heterogeneity of a wide range of therapeutic mAbs with high accuracy and precision., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The characterization of complex protein mixtures represents one of the biggest challenge in many research fields such as biological or biopharmaceutical sciences. Out of all categories, monoclonal antibodies (mAbs) and related products drawn the most interest due to their strong therapeutic potency and specificity. Because of their intrinsic complexity due to a large number of micro-heterogeneities, there is a crucial need for analytical methods to provide comprehensive in-depth characterization of these proteins. In this work, we developed a methodology using CE-UV/MALDI-MS to perform top-down or middle-down characterization after fraction collection enrichment applied to intact protein and mAbs samples. The performance of the method was evaluated with the rapid separation of three intact protein mixture. Good robustness of CZE separation and quality of MALDI-MS spectra and MALDI-ISD spectra of each protein confirms the usefulness of sample enrichment to obtain adequate quantity of deposed protein for top-down analysis and the proof of principle of the method. In a second step, the method was applied to the middle-down characterization of Fc/2 cetuximab variants. Identification of around 9% sequence coverage of Fc/2 cetuximab fragments allows to conclude on the feasibility of the strategy for middle-down characterization of Fc/2 cetuximab variants using CE-UV/MALDI-MS. Moreover, MALDI-ISD fragmentation of Fc/2 cetuximab variants confirm separation phenomenon based on the formation of Fc/2 dimers with and without C-terminal truncation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Out of all categories, monoclonal antibodies (mAbs), biosimilar, antibody-drug conjugates (ADCs) and Fc-fusion proteins attract the most interest due to their strong therapeutic potency and specificity. Because of their intrinsic complexity due to a large number of micro-heterogeneities, there is a crucial need of analytical methods to provide comprehensive in-depth characterization of these molecules. CE presents some obvious benefits as high resolution separation and miniaturized format to be widely applied to the analysis of biopharmaceuticals. CE is an effective method for the separation of proteins at different levels. capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF) and capillary zone electrophoresis (CZE) have been particularly relevant for the characterization of size and charge variants of intact and reduced mAbs, while CE-MS appears to be a promising analytical tool to assess the primary structure of mAbs and related products. This review will be dedicated to detail the current and state-of-the-art CE-based methods for the characterization of mAbs and related products., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Antibody-drug conjugates (ADCs) represent a fast growing class of biotherapeutic products. Their production leads to a distribution of species exhibiting different number of conjugated drugs overlaying the inherent complexity resulting from the monoclonal antibody format, such as glycoforms. ADCs require an additional level of characterization compared to first generation of biotherapeutics obtained through multiple analytical techniques for complete structure assessment. We report the development of complementary approaches implementing sheathless capillary electrophoresis-mass spectrometry (sheathless CE-MS) to characterize the different aspects defining the structure of brentuximab vedotin. Native MS using sheathless CE-MS instrument as a nanoESI infusion platform enabled accurate mass measurements and estimation of the average drug to antibody ratio alongside to drug load distribution. Middle-up analysis performed after limited IdeS proteolysis allowed to study independently the light chain, Fab and F(ab')2 subunits incorporating 1, 0 to 4 and 0 to 8 payloads respectively. Finally, a CZE-ESI-MS/MS methodology was developed in order to be compatible with hydrophobic drug composing ADCs. From a single injection, complete sequence coverage could be achieved. Using the same dataset, glycosylation and drug-loaded peptides could be simultaneously identified revealing robust information regarding their respective localization and abundance. Drug-loaded peptide fragmentation mass spectra study demonstrated drug specific fragments reinforcing identification confidence, undescribed so far. Results reveal the method ability to characterize ADCs primary structure in a comprehensive manner while reducing tremendously the number of experiments required. Data generated showed that sheathless CZE-ESI-MS/MS characteristics position the methodology developed as a relevant alternative for comprehensive multilevel characterization of these complex biomolecules., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Monoclonal antibody (mAb) therapeutics attract the largest concern due to their strong therapeutic potency and specificity. The Fc region of mAbs is common to many new biotherapeutics as biosimilar, antibody drug conjugate or fusion protein. Fc region has consequences for Fc-mediated effector functions that might be desirable for therapeutic applications. As a consequence, there is a continuous need for improvement of analytical methods to enable fast and accurate characterization of biotherapeutics. Capillary zone electrophoresis-Mass spectrometry couplings (CZE-MS) appear really attractive methods for the characterization of biological samples. In this report, we used CZE-MS systems developed in house and native MS infusion to allow precise middle-up characterization of Fc/2 variant of cetuximab. Molecular weights were measured for three Fc/2 charge variants detected in the CZE separation of cetuximab subunits. Two Fc/2 C-terminal lysine variants were identified and separated. As the aim is to understand the presence of three peaks in the CZE separation for two Fc/2 subunits, we developed a strategy using CZE-UV/MALDI-MS and CZE-UV/ESI-MS to evaluate the role of N-glycosylation and C-terminal lysine truncation on the CZE separation. The chemical structure of N-glycosylation expressed on the Fc region of cetuximab does not influence CZE separation while C-terminal lysine is significantly influencing separation. In addition, native MS infusion demonstrated the characterization of Fc/2 dimers at pH 5.7 and 6.8 and the first separation of these dimers using CZE-MS., (Copyright © 2016 Elsevier B.V. All rights reserved.)

An increased aldosterone concentration can lead to a progression of heart diseases and to myocardial fibrosis. These fatal processes can be prevented by e.g. inhibiting the mineralocorticoid receptor (MR), which is nowadays part of a commonly applied standard therapy. Moreover, selective inhibition of aldosterone synthase (CYP11B2) is a straightforward goal whereby CYP11B1, a key enzyme in glucocorticoid biosynthesis exhibiting a high structure identity with CYP11B2 should not be inhibited. Therefore, effective test systems have been developed and rather potent and selective CYP11B2 compounds like SIAS-1 have been identified by our group. In addition to finding new inhibitors, we investigated which proteins are directly influenced by aldosterone focussing on non-genomic effects. Schizosaccharomyces pombe was chosen as a model organism, since this yeast does not contain nuclear steroid receptors, but many genes and regulatory mechanisms that are close to those of mammals. Besides creating a reference map for this organism, protein spots affected by aldosterone as well as deoxycorticosterone (DOC) and corticosterone have been identified. In case of aldosterone, a regulatory effect of proteins that are connected with structural proteins, signal cascades, osmoregulation and calcium pathway as well as to general metabolism have been discovered. DOC causes overlapping but also different effects compared with aldosterone. As shown exemplarily for GAPDH, the aldosterone-mediated effects in S. pombe can also be verified in mammalian cells. These and further investigations contribute to a deeper understanding of so-called non-genomic aldosterone effects.