Kevin Jooß

The analytical determination of critical quality attributes (CQAs) of monoclonal antibodies (mAbs) is often time-consuming. Hence, increasing analysis throughput can shorten biopharmaceutical development timelines and enables rapid decision-making. Microfluidic capillary electrophoresis coupled to mass spectrometry (MCE-MS) allows fast analysis of charge variants and glycoforms of mAb samples. In a recently published MCE-MS method, we used a microfluidic chip with a 22-cm separation channel length, permitting analysis of 96 samples per day. To further increase the throughput of the method and better meet the needs of the biopharmaceutical industry, we propose the use of a chip with a distinctly shorter channel length (10 cm). However, a substantial drop in electrophoretic resolution was observed due to the increased laminar flow caused by the pressure put on the shortened channel during analysis. To mitigate this phenomenon, the MCE setup was modified to allow independent adjustment of the gas pressure on the separation channel inlet. The mAb charge-variant resolution significantly improved by reducing the inlet pressure from 2.0 to 0.75 psi. The performance of the modified setup was assessed by analysis of mAb samples from cell lines, mimicking a typical biosimilar clone screening. The original (22-cm channel, 2.0 psi on inlet) and modified (10-cm channel, 0.75 psi on inlet) setups yielded comparable separation profiles and number of MS-identified proteoforms across all clone mAbs. Overall, the new MCE-MS method reduces total analysis time per sample by 3.3-fold, which conceptually facilitates the analysis of more than 300 samples per day with minimal loss in separation performance. Graphical abstract

Mass spectrometry (MS)‐based top–down proteomics (TDP) has emerged as a powerful tool for characterizing proteoforms to advance both fundamental and translational research. TDP requires high‐efficiency liquid‐phase separation, high‐resolution MS, and tandem MS. Capillary zone electrophoresis (CZE)‐MS has been proposed as a promising analytical technique for protein analysis decades ago because of its unique and valuable features, including high separation efficiency and high detection sensitivity. However, CZE‐MS has not been widely adopted by the proteomics community, mainly due to concerns with its robustness and reproducibility. Here, we hypothesized that CZE‐MS is sufficiently robust and reproducible for broad adoption due to the continued efforts of the community over the last three decades. In this work, for the first time, research teams from around the world validated the robustness, repeatability, and reproducibility of CZE‐MS for TDP in both simple and complex model proteoform mixtures employing a full spectrum of commercially available capillary electrophoresis (CE)‐MS interfaces, instrumentation, and compared CZE‐MS performance with state‐of‐the‐art liquid chromatography (LC)‐MS methods. This study offers the research community an informative resource of ready‐to‐use experimental CE‐MS techniques and a better understanding of the CZE‐MS approach and its potential in TDP, accelerating the broad adoption of CZE‐MS in proteoform research.

G protein-coupled receptors (GPCRs) are the largest class of integral membrane receptors and responsible for transmitting diverse signals in response to extracellular stimuli. Post-translational modifications serve to dictate the subcellular trafficking and function of a GPCR across space and time. Despite significant interest in mapping the diversity of GPCR modification states (proteoforms), technical challenges have hindered this characterization. While advancements in membrane mimetics and mass spectrometry instrumentation have improved analysis, current workflows require large amounts of homogenous protein, limiting the study of many GPCRs from mammalian sources. Here, we present capillary zone electrophoresis (CZE) as an inline separatory tool for characterizing proteoforms of both intact and partially digested GPCRs. This method allowed for the characterization of multiple proteoforms of both the beta-2-adrenergic receptor and metabotropic glutamate receptor 2 using low sample volumes and without buffer optimization. Notably, in the case of smaller phosphorylated analytes, CZE was able to readily separate positional phosphorylation isomers, and provide superior fragmentation coverage to conventional reverse phase-liquid chromatography (RP-LC).

Monoclonal antibodies (mAbs) undergo multiple post-translational modifications (PTMs), e.g., charge variants, oxidation, etc., during production and storage, necessitating evaluation of the resulting PTMs as critical quality attributes (CQA) for protein quality and safety. Charge variants PTMs can be separated using capillary zone electrophoresis (CZE). The CZE EACA method developed by He et al. (2011) with UV detection is applied routinely in the pharmaceutical industry for analyzing charge variants. However, the method cannot be directly hyphenated with mass spectrometry (MS), preventing direct identification of separated charge variants due to the non-volatile background electrolyte (BGE), which hinders reliable charge variant identification. This study presents a CZE-UV/MS method using a neutral static capillary coating of hydroxypropyl methylcellulose combined with a volatile BGE at pH 5.0 to allow MS-compatible mAb charge variant separations. The effect of several parameters, including pH and concentration of BGE, voltage, and injected mAb concentrations in terms of separation performance on a panel of mAbs was investigated. The final method was tested with mAbs of IgG subclasses (IgG1 and IgG4) with different pI (7.4-9.2), and degrees of heterogeneity. Basic and acidic variants were separated from the parent mAb using a BGE of 50 mM acetic acid adjusted at pH 5.0. A linear correlation in relative charge variant abundance was obtained between our method and the EACA method. CZE-MS coupling was accomplished using the nanoCEasy, a low-flow sheath liquid interface, which enabled the identification and quantitation of basic, acidic, and incomplete pyroglutamate variants, and glycoforms of the tested mAbs.This method can be a useful tool for in-depth charge variants characterization of mAbs.