Mass Spectrometry-Based Charge Heterogeneity Characterization of Therapeutic mAbs with Imaged Capillary Isoelectric Focusing and Ion-Exchange Chromatography as Separation Techniques

Wu, Gang; Yu, Chuanfei; Wang, Wenbo; Du, Jinyan; Fu, Zhihao; Xu, Gang; Li, Meng; Wang, Lan

doi:10.1021/acs.analchem.2c05071

Cited by 12 publications

(3 citation statements)

References 31 publications

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“…Although more time-consuming and labor-intensive, off-line fractionation is not limited to the separation of analytes requiring only MS-compatible components and may be necessary to fully characterize some highly complex proteins at the intact level. The new preparative iCIEF instrument allows simultaneous isolation and characterization of individual protein charge variants and can be directly connected to MS to deliver the fractionated protein samples for characterization [167][168][169][170]. During pressure mobilization, an electric field keeps the samples in the separation capillary focused.…”

Section: Cief and Imaged Ciefmentioning

confidence: 99%

“…The established methodology was employed for the charge heterogeneity characterization of pembrolizumab [169] and for commercial monoclonal antibodies (mAbs) and antibody-drug-conjugates [170]. Wu et al [168] recently compared this innovative iCIEF-MS approach for charge variant analysis of diverse mAbs to strong cation exchange-MS. It was found that iCIEF-MS outperformed strong cation exchange-MS in this study by demonstrating its outstanding sensitivity, low carryover, accurate protein identification, and higher separation resolution.…”

Section: Ce-ms Analysis Of Peptide-and Protein-based Therapeuticsmentioning

confidence: 99%

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Capillary electrophoresis‐mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018–March 2023)

Maráková

Opetová

Tomašovský

2023

J of Separation Science

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Capillary electrophoresis is recognized as a valued separation technique for its high separation efficiency, low sample consumption, good economic and ecological aspects, reproducibility, and complementarity to traditional liquid chromatography techniques. Capillary electrophoresis experiments are generally performed utilizing optical detection, such as ultraviolet or fluorescence detectors. However, in order to provide structural information, capillary electrophoresis hyphenated to highly sensitive and selective mass spectrometry has been developed to overcome the limitations of optical detections. Capillary electrophoresis‐mass spectrometry is increasingly popular in protein analysis, including biopharmaceutical and biomedical research. It is frequently applied for the determination of physicochemical and biochemical parameters of proteins, offers excellent performance for in‐depth characterizations of biopharmaceuticals at various levels of analysis, and has been also already proven as a promising tool in biomarker discovery. In this review, we focus on the possibilities and limitations of capillary electrophoresis‐mass spectrometry for protein analysis at their intact level. Various capillary electrophoresis modes and capillary electrophoresis‐mass spectrometry interfaces, as well as approaches to prevent protein adsorption and to enhance sample loading capacity, are discussed and the recent (2018–March 2023) developments and applications in the field of biopharmaceutical and biomedical analysis are summarized.

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Section: Cief and Imaged Ciefmentioning

confidence: 99%

Section: Ce-ms Analysis Of Peptide-and Protein-based Therapeuticsmentioning

confidence: 99%

Capillary electrophoresis‐mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018–March 2023)

Maráková

Opetová

Tomašovský

2023

J of Separation Science

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“…Capillary isoelectric focusing is a powerful analytical technique that provides efficient separation of zwitterionic compounds in accordance with their Isoelectric Points (pI). , The process of cIEF includes two steps; focusing and mobilization. Focusing involves the establishment of a pH gradient along the capillary and pI-controlled redistribution of the analytes, while mobilization provides delivery of the focused analytes to the point of detection.…”

Section: Introductionmentioning

confidence: 99%

Open Port Interface for Coupling Capillary Electrophoresis and Mass Spectrometry: Performance Evaluation for Capillary Isoelectric Focusing

Beloborodov,

Schneider,

Oleschuk

et al. 2023

J. Am. Soc. Mass Spectrom.

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Capillary electrophoresis (CE) combined with mass spectrometry (MS) is a powerful analytical technique that utilizes the resolving power of CE and the mass-detection capabilities of MS. In many cases, CE is coupled to MS via a sheath-flow interface (SFI). This interface has a simple design and can be easily constructed; however, it often suffers from issues such as MS signal suppression, interference of MS and CE electrical circuits, and the inability to set an optical point of detection close to the capillary end due to the specific design of the coupling union. In this paper, we describe a novel coupling of CE and MS based upon the open port interface (OPI). The OPI differs from classical sheath flow interfaces by operating at flow rates at least 1 order of magnitude higher. In addition to the flow rate difference, the OPI provides more efficient mixing of the capillary eluates with the transport fluid and thus minimizes MS signal suppression. In this work, we compared the performance of OPI and SFI in a series of capillary isoelectric focusing (cIEF) experiments with 5 pI markers, carbonic anhydrase II and NIST antibody. The evaluation criteria for the comparison of the OPI and SFI were analytical sensitivity, reproducibility, and pI marker linearity. Given the extent of sample dilution in the OPI, we also compared the peak resolution determined using an upstream UV detector to those determined by the downstream mass spectrometer. The results suggested that the OPI configuration reduced signal suppression, with no adverse effect on peak resolution. In addition, the OPI provided better decoupling of the CE and MS potentials as well as reduced signal dependence upon the sheath liquid composition. While these results are preliminary, they suggest that the OPI is a viable approach for CE-MS coupling.

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Imaged Capillary Isoelectric Focusing Coupled to High‐Resolution Mass Spectrometry (icIEF‐MS) for Cysteine‐Linked Antibody–Drug Conjugate (ADC) Heterogeneity Characterization Under Native Condition

Zhang,

Wu,

et al. 2024

Electrophoresis

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Native mass spectrometry (nMS) is a cutting‐edge technique that leverages electrospray ionization MS (ESI‐MS) to investigate large biomolecules and their complexes in solution. The goal of nMS is to retain the native structural features and interactions of the analytes during the transition to the gas phase, providing insights into their natural conformations. In biopharmaceutical development, nMS serves as a powerful tool for analyzing complex protein heterogeneity, allowing for the examination of non‐covalently bonded assemblies in a state that closely resembles their natural folded form. Herein, we present an imaged capillary isoelectric focusing–MS (icIEF–MS) workflow to characterize cysteine‐linked antibody–drug conjugate (ADC) under native conditions. Two ADCs were analyzed: a latest generation cysteine‐linked ADC polatuzumab vedotin and the first FDA‐approved cysteine‐linked ADC brentuximab vedotin. This workflow benefits from a recently developed icIEF system that is MS‐friendly and capable of directly coupling to a high‐sensitivity MS instrument. Results show that the icIEF separation is influenced by both drug payloads and the post‐translational modifications (PTMs), which are then promptly identified by MS. Overall, this native icIEF–MS method demonstrates the potential to understand and control the critical quality attributes (CQAs) that are essential for the safe and effective use of ADCs.

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Mass Spectrometry-Based Charge Heterogeneity Characterization of Therapeutic mAbs with Imaged Capillary Isoelectric Focusing and Ion-Exchange Chromatography as Separation Techniques

Cited by 12 publications

References 31 publications

Capillary electrophoresis‐mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018–March 2023)

Capillary electrophoresis‐mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018–March 2023)

Open Port Interface for Coupling Capillary Electrophoresis and Mass Spectrometry: Performance Evaluation for Capillary Isoelectric Focusing

Imaged Capillary Isoelectric Focusing Coupled to High‐Resolution Mass Spectrometry (icIEF‐MS) for Cysteine‐Linked Antibody–Drug Conjugate (ADC) Heterogeneity Characterization Under Native Condition

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