High-Performance Native Separation of mAb Proteoforms by CZE-MS under Native and Denaturing nanoESI Conditions for...

Area of Science:

• Biopharmaceutical Analysis
• Analytical Chemistry
• Proteomics

Background:

• Monitoring charge variants in therapeutic antibodies is essential for ensuring drug efficacy and safety.
• Capillary zone electrophoresis (CZE) effectively separates charge variants but is incompatible with mass spectrometry (MS), hindering variant identification.
• Existing CZE methods for charge variant analysis often use electrolytes that prevent direct coupling to MS.

Purpose of the Study:

• To develop and present a novel CZE-MS method for detailed characterization of monoclonal antibody (mAb) charge variants and other proteoforms.
• To achieve separation performance comparable to existing non-MS-compatible methods while enabling direct MS identification.
• To assess the heterogeneity of therapeutic mAbs at the intact level with high sensitivity and flexibility.

Main Methods:

• A neutral static capillary coating was employed in conjunction with an ammonium acetate-based electrolyte at physiological pH.
• The CZE system was hyphenated to MS using a nanoflow sheath liquid (SL) interface for ultrasensitive ionization.
• Different SLs were tested under both denaturing and nondenaturing nanoESI conditions to optimize ionization and spectral quality.

Main Results:

• The developed CZE-MS method demonstrated high separation performance for mAb charge variants, similar to established non-MS-compatible techniques.
• The method enabled sensitive detection and identification of a large number of proteoforms: 115 for NISTmAb and 70 for trastuzumab.
• Several proteoforms were identified that were previously undetected at the intact level, providing deeper insights into mAb heterogeneity.

Conclusions:

• The presented CZE-MS method offers a fast and detailed approach for assessing therapeutic mAb heterogeneity at the intact level.
• This technique significantly advances the capability for proteoform-resolved structural and functional characterization of biopharmaceuticals.
• The method overcomes the limitations of traditional CZE by enabling direct MS identification of separated charge variants.