MassIVE MSV000094521

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

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Description

Monoclonal antibodies (mAbs) can undergo post-translational modifications (PTMs) in the production process, these are part of the critical quality attributes (CQA) to ensure protein safety. Charge variants are important PTMs in mAbs and can be separated by using capillary zone electrophoresis (CZE). The EACA method, developed by He et al. (2011), is a popular method for analyzing charge variants in pharmaceutical industries. However, it requires optical detection (e.g., UV) due to non-volatile buffers and does not allow for MS coupling and the identification of the separated charge variants. This study presents a CZE-UV/MS method that uses a coated neutral static hydroxypropyl methylcellulose (HPMC) with a background electrolyte (BGE) at pH 5 using a nanoflow sheath liquid MS interface (nanoCEasy). Here we describe the effect of several parameters, including pH of BGE, ionic strength, applied voltage, and concentration of mAb in terms of separation performance. Our final method was tested with mAbs of different pIs (7.4-9.2), IgG subclasses (IgG1 and IgG4), and degrees of heterogeneity. Basic and acidic variants were separated from the main variant using 50 mM acetic acid at pH 5, which was adjusted using ammonium hydroxide to the appropriate pH. A linear correlation was obtained in relative abundance of charge variants between our method and the EACA method of He et al. (2011). Coupling the method with the low-flow sheath liquid nanoCEasy interface allowed the identification of low-abundance species (< 10 % relative abundance with respect to the main variant). This method uses volatile buffers and operates at pH closer to non-denaturing conditions (pH 5), allowing for flexibility in hyphenation with MS. It is an open platform method in which no commercial capillaries and interfaces are required. This method can be a useful tool for in-depth charge variants characterization of mAbs. [doi:10.25345/C5377662N] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: mAbs, charge variants, CZE-MS

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Principal Investigators:
(in alphabetical order) 		Annika van der Zon, University of Amsterdam, The Netherlands
Submitting User: AZON
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