Two-dimensional capillary zone electrophoresis–mass spectrometry for the characterization of intact monoclonal antibody charge variants, including deamidation products

Abstract

Capillary zone electrophoresis (CZE) is a powerful tool that is progressively being applied for the separation of monoclonal antibody (mAb) charge variants. Mass spectrometry (MS) is the desired detection method concerning identification of mAb variants. In biopharmaceutical applications, there exist optimized and validated electrolyte systems for mAb variant quantification. However, these electrolytes interfere greatly with the electrospray ionization (ESI) process. Here, a heart-cut CZE-CZE-MS setup with an implemented mechanical four-port valve interface was developed that used a generic ε-aminocaproic acid based background electrolyte in the first dimension and acetic acid in the second dimension. Interference-free, highly precise mass data (deviation less than 1 Da) of charge variants of trastuzumab, acting as model mAb system, were achieved. The mass accuracy obtained (low parts per million range) is discussed regarding both measured and calculated masses. Deamidation was detected for the intact model antibody, and related mass differences were significantly confirmed on the deglycosylated level. The CZE-CZE-MS setup is expected to be applicable to a variety of antibodies and electrolyte systems. Thus, it has the potential to become a compelling tool for MS characterization of antibody variants separated in ESI-interfering electrolytes. Graphical Abstract Two-dimensional capillary zone electrophoresis mass spectrometry for the characterization of intact monoclonal antibody (mAb) charge variants. A generic, but highly electrospray-interfering electrolyte system was used as first dimension for mAb charge variant separation and coupled to a volatile electrolyte system as second dimension via a four-port nanoliter valve. In this way, interference-free and precise mass spectrometric data of separated mAb charge variants, including deamidation products, were obtained.

Coupling imaged capillary isoelectric focusing with mass spectrometry using a nanoliter valve

“…A slightly higher mass compared with the main variant was observed (148 222.5 ± 0.2, n = 2), which corresponds to a shift of +1.4 Da. This charge variant can be most likely attributed to deamidation, which is a known modification for Trastuzumab and has been previously characterized by CZE‐CZE‐MS . In the last‐mentioned work the basic variant could not be characterized due to its lower abundance, similar to a recent work where CIEF is directly coupled with MS .…”

Insights from capillary electrophoresis approaches for characterization of monoclonal antibodies and antibody drug conjugates in the period 2016–2018

“…Indeed, in a comparison between reference CZE-UV and CZE-ESI-MS methods using different BGE, obtained electropherograms fitted well but lower resolutions were obtained for CZE-ESI-MS [103]. In 2017, Jooβ et al described a CZE-CZE-MS method for the characterization of intact mAbs charge variants [129]. Authors achieved interference-free, highly precise mass data (deviation less than 1 Da) of trastuzumab charge variants.…”

Separation methods hyphenated to mass spectrometry for the characterization of the protein glycosylation at the intact level

“…A neutral coating was thus necessary to completely suppress the EOF, preventing the introduction of high amounts of urea into the ESI source. Another option may be to carry out two-dimensional CZE-CZE-MS, with a four-port valve interface allowing the use of a non-volatile BGE in the first dimension leading to high resolution and a volatile BGE in the second dimension to favor the MS signal [68].…”