CE-MS and CE-MS/MS for the multiattribute analysis of monoclonal antibody variants at the subunit level.

The analysis of product-related substances and impurities is a critical step in the biopharmaceutical quality control of multiattribute monoclonal antibodies (mAbs), as posttranslational modifications or other variants can influence the product's biological activity. Many approaches are available for variant analysis; however, they are either variant-specific, mAb-specific, time-consuming, or require expensive equipment. Here, we present a generic capillary electrophoretic method based on a neutral-coated capillary which was coupled to mass spectrometry (MS) via the nanoCEasy interface for mAb variant analysis at the subunit level (enzymatically digested and reduced mAb). The method enabled the separation of several (i) size variants (e.g. glycosylation variants) and (ii) charge variants (e.g. c-terminal lysin clipping) as well as (iii) multiple other proteoforms (e.g. additional glycation) and (iv) incompletely reduced subunits. Separated variants were confirmed by MS/MS fragmentation even for small mass deviations like deamidation or open disulfide bridges. The system, initially developed for one mAb, was tested with nine other IgG1s to show the general applicability of the system. The presented multiattribute method enables fast and detailed characterization of mAb variants with little sample preparation and relatively simple separation equipment enabling the separation of a large set of mAb variants.

Getting the Best out of Capillary Electrophoresis and Capillary Electrophoresis-Mass Spectrometry by Quantifying Sources of Peak Broadening for Proteins Using Polyelectrolyte Multilayer Coated Fused Silica Capillaries.

Capillary electrophoresis (CE) has emerged as a relevant technique for protein and biopharmaceutical analysis, as it combines high separation efficiency, sensitivity, and versatility. The use of capillary coatings, including successive multiple ionic-polymer layers (SMILs), reduces interactions between analytes and the capillary, further improving the CE performance. Nevertheless, separations done on SMIL coatings rarely surpass 500 × 103 plates/m. To obtain the best out of the CE, it is interesting to have a detailed look at the sources of peak dispersion. Separations of a mix of model proteins were performed on (poly(diallyldimethylammonium chloride)/poly(styrenesulfonate))2.5-coated capillaries at different electrical field strengths, leading to plate height H against migration velocity u plots that enabled a quantitative analysis of each contribution. Using this model, capillary lengths and injected volumes were systematically varied. For the first time, the contribution of sample electrophoretic heterogeneity to the total peak dispersion was deciphered for model proteins and a monoclonal antibody. Dispersion due to electromigration was seen to have an impact on plate heights in the case of triangular peaks of small molecules but not for proteins under the present conditions. UV and mass spectrometry detections were compared on the same capillary, providing valuable information on the impact of the detection type on separation efficiency. Close to 1 million plates/m were reached in the best conditions.

Charge variant analysis of monoclonal antibodies by CZE-MS using a successive multiple ionic-polymer layer coating based on diethylaminoethyl-dextran.

The characterization of the impurities of pharmaceutical monoclonal antibodies (mAbs) is crucial for their function and safety. Capillary zone electrophoresis (CZE) is one of the most efficient tools to separate charge variants of mAbs; however, peak characterization remains difficult, since the hereby used background electrolytes (BGEs) are not compatible with electrospray ionization-mass spectrometry (ESI-MS). Here, a method that allows the separation of intact mAb charge variants is presented using CZE-ESI-MS, combining a cationic capillary coating and an acidic BGE. Therefore, a successive multiple ionic-polymer layer coating was developed based on diethylaminoethyl-dextran-poly(sodium styrene sulfonate). This coating leads to a relatively low reversed electroosmotic flow (EOF) with an absolute mobility slightly higher than that of antibodies, enabling the separation of variants with slightly different mobilities. The potential of the coating is demonstrated using USP mAb003, where it was possible to separate C-terminal lysine variants from the main form, as well as several acidic variants and monoglycosylated mAb forms. The presented CZE-MS method can be applied to separate charge variants of a range of other antibodies such as infliximab, NISTmAB (Reference Material from the National Institute of Standards and Technology), adalimumab, and trastuzumab, demonstrating the general applicability for the separation of proteoforms of mAbs.

Chemical Mobilization-Based Capillary Isoelectric Focusing-Mass Spectrometry Using the nanoCEasy Interface for Pharmaceutical Protein Analysis.

Capillary isoelectric focusing (CIEF) coupled with electrospray ionization mass spectrometry (ESI-MS) is regarded as an outstanding approach for protein and proteoform analysis, combining a high-resolution separation technique and an enhanced detection technique. The few so-far developed CIEF-ESI-MS approaches exhibit limitations regarding sensitivity and separation performance. Here, we report a new generic methodology for CIEF-ESI-MS based on chemical mobilization, leading to highly efficient separation. This new integrated methodology relies on exchanging catholyte, initially introduced in the nanoCEasy interface in the focusing step, with sheath liquid (SL) in order to chemically mobilize the analytes into the ESI-MS system. The CIEF-MS method is evaluated by separation of a peptide set, model proteins, and monoclonal antibody charge variants. The effect of various parameters including master mixture composition, field strength, catholyte, SL composition, focusing time, and capillary conditions is optimized and discussed. Excellent separation performance can be achieved with a pI resolution down to 0.1 pH unit. The mobilization reproducibility is demonstrated with "migration time" RSDs below 10%. Additionally, the chemical mobilization is compared with the pressure assistance-chemical mobilization method, demonstrating that even a small pressure causes a strong decrease in separation performance, which clearly indicates the benefit of the chemical mobilization-based method. The applicability and separation power of the developed method are further exhibited by separation of Fc-conjugated insulins (mass = 62 kDa) differing in only one amino acid.

Dual Contributions of Analyte Adsorption and Electroosmotic Inhomogeneity to Separation Efficiency in Capillary Electrophoresis of Proteins.

Improving separation efficiency in capillary electrophoresis (CE) requires systematic study of the influence of the electric field (or solute linear velocity) on plate height for a better understanding of the critical parameters controlling peak broadening. Even for poly(diallyldimethylammonium chloride) (PDADMAC)/poly(sodium styrenesulfonate) (PSS) successive multiple ionic-polymer layer (SMIL) coatings, which lead to efficient and reproducible separations of proteins, plate height increases with migration velocity, limiting the use of high electric fields in CE. Solute adsorption onto the capillary wall was generally considered as the main source of peak dispersion, explaining this plate height increase. However, experiments done with Taylor dispersion analysis and CE in the same conditions indicate that other phenomena may come into play. Protein adsorption with slow kinetics and few adsorption sites was established as a source of peak broadening for specific proteins. Surface charge inhomogeneity was also identified as a contribution to plate height due to local electroosmotic fluctuations. A model was proposed and applied to partial PDADMAC/poly(ethylene oxide) capillary coatings as well as PDADMAC/PSS SMIL coatings. Atomic force microscopy with topography and recognition imaging enabled the determination of roughness and charge distribution of the PDADMAC/PSS SMIL surface.

Stable overexpression of native and artificial miRNAs for the production of differentially fucosylated antibodies in CHO cells.

Cell engineering strategies typically rely on energy-consuming overexpression of genes or radical gene-knock out. Both strategies are not particularly convenient for the generation of slightly modulated phenotypes, as needed in biosimilar development of for example differentially fucosylated monoclonal antibodies (mAbs). Recently, transiently transfected small noncoding microRNAs (miRNAs), known to be regulators of entire gene networks, have emerged as potent fucosylation modulators in Chinese hamster ovary (CHO) production cells. Here, we demonstrate the applicability of stable miRNA overexpression in CHO production cells to adjust the fucosylation pattern of mAbs as a model phenotype. For this purpose, we applied a miRNA chaining strategy to achieve adjustability of fucosylation in stable cell pools. In addition, we were able to implement recently developed artificial miRNAs (amiRNAs) based on native miRNA sequences into a stable CHO expression system to even further fine-tune fucosylation regulation. Our results demonstrate the potential of miRNAs as a versatile tool to control mAb fucosylation in CHO production cells without adverse side effects on important process parameters.

Investigating cationic and zwitterionic successive multiple ionic-polymer layer coatings for protein separation by capillary electrophoresis.

Successive multiple ionic-polymer layers (SMILs) have long since proved their worth in capillary electrophoresis as they ensure stable electroosmotic flow (EOF) and relatively high separation efficiency. Recently, we demonstrated that plotting the plate height (H) against the solute migration velocity (u) enabled a reliable quantitative evaluation of the coating performances in terms of separation efficiency. In this work, various physicochemical and chemical parameters of the SMIL coating were studied and optimized in order to decrease the slope of the ascending part of the H vs u curve, which is known to be controlled by the homogeneity in charge of the coating surface and by the possible residual solute adsorption onto the coating surface. SMILs based on poly(diallyldimethylammonium chloride) (PDADMAC) and poly(sodium styrene sulfonate) (PSS) were formed and the effect of each polyelectrolyte molar mass and of the number of polyelectrolyte layers (up to 21 layers) was studied. The use of polyethylene imine as an anchoring first layer was considered. More polyelectrolyte couples based on PDADMAC, polybrene, PSS, poly(vinyl sulfate), and poly(acrylic acid) were tested. Finally, zwitterionic polymers based on the poly(α-l-lysine) scaffold were synthesized and used as the last layer of SMILs, illustrating their ability to finetune the EOF, while maintaining good separation efficiency.

CE-MS/MS and CE-timsTOF to separate and characterize intramolecular disulfide bridges of monoclonal antibody subunits and their application for the assessment of subunit reduction protocols.

Characterization at the subunit level enables detailed mass spectrometric characterization of posttranslational modifications (PTMs) of monoclonal antibodies (mAbs). The implemented reduction often leaves the intramolecular disulfide bridges intact. Here, we present a capillary electrophoretic (CE) method based on a neutral-coated capillary for the separation of immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) digested and reduced mAb subunits followed by mass spectrometry (MS), MS/MS identification, and trapped ion mobility mass spectrometry (timsTOF). Our CE approach enables the separation of (i) different subunit moieties, (ii) various reduction states, and (iii) positional isomers of these partly reduced subunit moieties. The location of the remaining disulfide bridges can be determined by middle-down electron transfer higher energy collisional dissociation (EThcD) experiments. All these CE-separated variants show differences in ion mobility in the timsTOF measurements. Applying the presented CE-MS/MS method, reduction parameters such as the use of chaotropic salts were studied. For the investigated antibodies, urea improved the subunit reduction significantly, whereas guanidine hydrochloride (GuHCl) leads to multiple signals of the same subunit in the CE separation. The presented CE-MS method is a powerful tool for the disulfide-variant characterization of mAbs on the subunit level. It enables understanding disulfide bridge reduction processes in antibodies and potentially other proteins.

Developing microRNAs as engineering tools to modulate monoclonal antibody galactosylation.

N-linked glycosylation is one of the most important post-translational modifications of monoclonal antibodies (mAbs) and is considered to be a critical quality attribute (CQA), as the glycan composition often has immunomodulatory effects. Since terminal galactose residues of mAbs can affect antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytolysis (CDC) activation, serum half-life, and antiviral activity it has to be monitored, controlled and modulated to ensure therapeutic effects. The ability of small noncoding microRNAs (miRNAs) to modulate glycosylation in Chinese hamster ovary (CHO) production cells was recently reported establishing miRNAs as engineering tools for modulation of protein glycosylation. In this study, we report the characterization and validation of miRNAs as engineering tools for increased (mmu-miR-452-5p, mmu-miR-193b-3p) or decreased (mmu-miR-7646-5p, mmu-miR-7243-3p, mmu-miR-1668, mmu-let-7c-1-3p, mmu-miR-7665-3p, mmu-miR-6403) degree of galactosylation. Furthermore, the biological mode of action regulating gene expression of the galactosylation pathway was characterized as well as their influence on bioprocess-related parameters. Most important, stable plasmid-based overexpression of these miRNAs represents a versatile tool for engineering N-linked galactosylation to achieve favorable phenotypes in cell lines for biopharmaceutical production.

Capillary electrophoresis-mass spectrometry for protein analyses under native conditions: Current progress and perspectives.

Native mass spectrometry is a rapidly emerging technique for fast and sensitive structural analysis of protein constructs, maintaining the protein higher order structure. The coupling with electromigration separation techniques under native conditions enables the characterization of proteoforms and highly complex protein mixtures. In this review, we present an overview of current native CE-MS technology. First, the status of native separation conditions is described for capillary zone electrophoresis (CZE), affinity capillary electrophoresis (ACE), and capillary isoelectric focusing (CIEF), as well as their chip-based formats, including essential parameters such as electrolyte composition and capillary coatings. Further, conditions required for native ESI-MS of (large) protein constructs, including instrumental parameters of QTOF and Orbitrap systems, as well as requirements for native CE-MS interfacing are presented. On this basis, methods and applications of the different modes of native CE-MS are summarized and discussed in the context of biological, medical, and biopharmaceutical questions. Finally, key achievements are highlighted and concluded, while remaining challenges are pointed out.

Ion mobility in gas and liquid phases: How much orthogonality is obtained in capillary electrophoresis-ion mobility-mass spectrometry?

Ion mobility-mass spectrometry (IM-MS) is an ever-evolving tool to separate ions in the gas phase according to electrophoretic mobility with subsequent mass determination. CE is rarely coupled to IM-MS, possibly due to similar separation mechanisms based on electrophoretic mobility. Here, we investigate the orthogonality of CE and ion mobility (IM) by analyzing a complex peptide mixture (tryptic digest of HeLa proteins) with trapped ion mobility mass spectrometry (TIMS-MS). Using the nanoCEasy interface, excellent sensitivity was achieved by identifying thousands of peptides and achieving a peak capacity of 7500 (CE: 203-323 in a 150 cm long capillary, IM: 27-31). Plotting CE versus mass and CE versus (inverse) mobility, a clear grouping in curved striped patterns is observed according to the charge-to-size and mass-to-charge ratios. The peptide charge in the acidic background electrolyte can be estimated from the number of basic amino acids, with a few exceptions where neighboring effects reduce the positive charge. A surprisingly high orthogonality of CE and IM is observed, which is obviously caused by solvation effects leading to different charges and sizes in the liquid phase compared to the gas phase. A high orthogonality of CE and ion mobility is expected to be observed for other peptide samples as well as other substance classes, making CE-IM-MS a promising tool for various applications.

Augmented region of interest for untargeted metabolomics mass spectrometry (AriumMS) of multi-platform-based CE-MS and LC-MS data.

In mass spectrometry (MS)-based metabolomics, there is a great need to combine different analytical separation techniques to cover metabolites of different polarities and apply appropriate multi-platform data processing. Here, we introduce AriumMS (augmented region of interest for untargeted metabolomics mass spectrometry) as a reliable toolbox for multi-platform metabolomics. AriumMS offers augmented data analysis of several separation techniques utilizing a region-of-interest algorithm. To demonstrate the capabilities of AriumMS, five datasets were combined. This includes three newly developed capillary electrophoresis (CE)-Orbitrap MS methods using the recently introduced nanoCEasy CE-MS interface and two hydrophilic interaction liquid chromatography (HILIC)-Orbitrap MS methods. AriumMS provides a novel mid-level data fusion approach for multi-platform data analysis to simplify and speed up multi-platform data processing and evaluation. The key feature of AriumMS lies in the optimized data processing strategy, including parallel processing of datasets and flexible parameterization for processing of individual separation methods with different peak characteristics. As a case study, Saccharomyces cerevisiae (yeast) was treated with a growth inhibitor, and AriumMS successfully differentiated the metabolome based on the augmented multi-platform CE-MS and HILIC-MS investigation. As a result, AriumMS is proposed as a powerful tool to improve the accuracy and selectivity of metabolome analysis through the integration of several HILIC-MS/CE-MS techniques.

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.

Critical parameters for highly efficient and reproducible polyelectrolyte multilayer coatings for protein separation by capillary electrophoresis.

Since the introduction of polyelectrolyte multilayers to protein separation in capillary electrophoresis (CE), some progress has been made to improve separation efficiency by varying different parameters, such as buffer ionic strength and pH, polyelectrolyte nature and number of deposited layers. However, CE is often overlooked as it lacks robustness compared to other separation techniques. In this work, critical parameters for the construction of efficient and reproducible Successive multiple ionic-polymer layers (SMIL) coatings were investigated, focusing on experimental conditions, such as vial preparation and sample conservation which were shown to have a significant impact on separation performances. In addition to repeatability, intra- and inter-capillary precision were assessed, demonstrating the improved capability of poly(diallyldimethylammonium chloride) / poly(sodium styrene sulfonate) (PDADMAC / PSS) coated capillaries to separate model proteins in a 2 M acetic acid background electrolyte when all the correct precautions are put in place (with run to run%RSD(tm) < 1.8%, day to day%RSD(tm) < 3.2% and cap to cap%RSD(tm) < 4.6%). The approach recently introduced to calculate retention factors was used to quantify residual protein adsorption onto the capillary wall and to assess capillary coating performances. 5-layer PDADAMAC / PSS coatings led to average retention factors for the five model proteins of ∼4×10-2. These values suggest a relatively low residual protein adsorption leading to reasonably flat plate height vs linear velocity curves, obtained by performing electrophoretic separations at different electrical voltages (-10 to -25 kV).

A novel system for glycosylation engineering by natural and artificial miRNAs.

N-linked glycosylation is a crucial post-translational modification of many biopharmaceuticals, including monoclonal antibodies (mAbs), capable of modifying their biological effect in patients and thus considered as a critical quality attribute (CQA). However, expression of desired and consistent glycosylation patterns remains a constant challenge for the biopharmaceutical industry and constitutes the need for tools to engineer glycosylation. Small non-coding microRNAs (miRNAs) are known regulators of entire gene networks and have therefore the potential of being used as tools for modulation of glycosylation pathways and for glycoengineering. Here, we demonstrate that novel identified natural miRNAs are capable of altering N-linked glycosylation patterns on mAbs expressed in Chinese hamster ovary (CHO) cells. We established a workflow for a functional high-throughput screening of a complete miRNA mimic library and identified 82 miRNA sequences affecting various moieties including galactosylation, sialylation, and α-1,6 linked core-fucosylation, an important glycan feature influencing antibody-dependent cytotoxicity (ADCC). Subsequent validation shed light on the intra-cellular mode of action and the impact on the cellular fucosylation pathway of miRNAs reducing core-fucosylation. While multiplex approaches increased phenotypic effects on the glycan structure, a synthetic biology approach utilizing rational design of artificial miRNAs further enhanced the potential of miRNAs as novel, versatile and tune-able tools for engineering of N-linked glycosylation pathways and expressed glycosylation patterns towards favourable phenotypes.

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.

Concepts and recent advances in microchip electrophoresis coupled to mass spectrometry: Technologies and applications.

The online coupling of microchip electrophoresis (ME) as a fast, highly efficient, and low-cost miniaturized separation technique to mass spectrometry (MS) as an information-rich and sensitive characterization technique results in ME-MS an attractive tool for various applications. In this paper, we review the basic concepts and latest advances in technology for ME coupled to MS during the period of 2016-2021, covering microchip materials, structures, fabrication techniques, and interfacing to electrospray ionization (ESI)-MS and matrix-assisted laser desorption/ionization-MS. Two critical issues in coupling ME and ESI-MS include the electrical connection used to define the electrophoretic field strength along the separation channel and the generation of the electrospray for MS detection, as well as, a miniaturized ESI-tip. The recent commercialization of ME-MS in zone electrophoresis and isoelectric focusing modes has led to the widespread application of these techniques in academia and industry. Here we summarize recent applications of ME-MS for the separation and detection of antibodies, proteins, peptides, carbohydrates, metabolites, and so on. Throughout the paper these applications are discussed in the context of benefits and limitations of ME-MS in comparison to alternative techniques.

Successive Multiple Ionic-Polymer Layer Coatings for Intact Protein Analysis by Capillary Zone Electrophoresis-Mass Spectrometry: Application to Hemoglobin Analysis.

Adsorption of analytes, e.g., proteins, often interfere with separation in CE, due to the relatively large surface of the narrow capillary. Coatings often are applied to prevent adsorption and to determine the electroosmotic flow (EOF), which is of major importance for the separation in CE. Successive multiple ionic-polymer layer (SMIL) coatings are frequently used for protein analysis in capillary electrophoresis resulting in high separation efficiency and repeatability. Here, the coating procedure of a five-layer SMIL coating is described using quaternized diethylaminoethyl dextran (DEAEDq) as polycation and poly(methacrylic acid) (PMA) as polyanion. Depending on the analyte, different polyions may be used to increase separation efficiency. However, the coating procedure remains the same.To demonstrate the applicability of SMIL coatings in CE-MS, human hemoglobin was measured in a BGE containing 2 M acetic acid. DEAEDq-PMA coating was found to be the most suitable for hemoglobin analysis due to relatively low reversed electroosmotic mobility leading to increased electrophoretic resolution of closely related proteoforms. Thereby, not only alpha and beta subunit of the hemoglobin could be separated, but also positional isoforms of glycated and carbamylated species were separated within 24 min.

High-throughput glycosylation analysis of intact monoclonal antibodies by mass spectrometry coupled with capillary electrophoresis and liquid chromatography.

The analysis of monoclonal antibodies glycosylation is a crucial quality control attribute of biopharmaceutical drugs. High throughput screening approaches for antibody glycoform analysis are required in various stages of process optimization. Here, we present high throughput screening suitable mass spectrometry-based workflows for the analysis of intact antibody glycosylation out of cell supernatants. Capillary electrophoresis and liquid chromatography were coupled with quadrupole time-of-flight mass spectrometry or Orbitrap mass spectrometry. Both separation methods offer fast separation (10-15 min) and the capability to prevent the separated cell supernatant matrix to enter the mass spectrometry by post-separation valving. Both mass spectrometry instruments provide comparable results and both are sufficient to determine the glycosylation pattern of the five major glycoforms of the measured antibodies. However, the Orbitrap yields higher sensitivity of 25 µg/mL (CE-nanoCEasy-Orbitrap mass spectrometry) and 5 µg/mL (liquid chromatography-Orbitrap mass spectrometry). Data processing was optimized for a faster processing and easier detection of low abundant glycoforms based on averaged charge-deconvoluted mass spectra. This approach combines a non-target glycoform analysis while yielding the same glycosylation pattern as the traditional approach based on extracted ion traces. The presented methods enable the high throughput screening of the glycosylation pattern of antibodies down to low µg/mL-range out of cell supernatant without any sample preparation.