Characterization of Monoclonal Antibody Charge Variants under Near-Native Separation Conditions using Nanoflow Sheath Liquid Capillary Electrophoresis-Mass Spectrometry

03 May 2024, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


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 done using the nanoCEasy, a low-flow sheath liquid interface, and allowed the identification and quantitation of various low-abundance variants (<10% relative abundance with respect to the main compound). This method uses volatile buffers and operates at pH closer to non-denaturing conditions (pH 5.0), allowing for flexibility in hyphenation with MS. This method can be a useful tool for in-depth charge variants characterization of mAbs.


Monoclonal antibodies
Charge heterogeneity
Capillary zone electrophoresis
Mass spectrometry
Hydroxypropyl methylcellulose

Supplementary materials

Supplementary Information
Supplementary Information "Characterization of Monoclonal Antibody Charge Variants under Near-Native Separation Conditions using Nanoflow Sheath Liquid Capillary Electrophoresis-Mass Spectrometry"

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