Subunit charge variant characterization in the native state

Význam tématu

Monoclonal antibodies often exhibit charge heterogeneity due to post-translational modifications such as deamidation, glycation, oxidation and C-terminal lysine variants. Accurate and dynamic profiling of these charge variants is essential to ensure therapeutic efficacy and safety. Traditional methods like ion exchange chromatography and capillary isoelectric focusing require complex method development and orthogonal validation. Native capillary zone electrophoresis coupled with high-resolution mass spectrometry offers speed and resolution but lacks site-specific information at the intact level. Implementing a subunit approach adds domain specificity and deeper insight into modification localization.

Cíle a přehled studie / článku

This work presents a workflow integrating IdeS proteolysis with native CZE-UV separation to analyze mAb charge variants at the subunit (middle-up) level. The study demonstrates rapid (under 10 min) domain-specific profiling using the SCIEX Rapid Charge Variant Analysis Kit and peak identification via the ZipChip Native TOF system. Applications include monoclonal antibodies, antibody-drug conjugates and stress stability testing.

Použitá metodika a instrumentace

Sample Preparation

• Intact samples diluted to 1 mg/mL in CE-grade water for native CZE.
• Stress treatment: adalimumab incubated at 40 °C and pH 8.4 for up to 10 days; buffer exchange to pH 7.4 Tris prior to IdeS digestion.
• IdeS digestion: 2 h at 37 °C in pH 7.4 Tris; post-digest dilution to 1 mg/mL.

Instrumentation

• PA 800 Plus Pharmaceutical Analysis System with pre-assembled cartridge (SCIEX P/N A55625).
• CZE Rapid Charge Variant Analysis Kit (SCIEX P/N C44790) providing buffers and CE-grade water.
• Separation conditions: 15 kV (500 V/cm) over a 30 cm capillary, pressure injection at 0.5 psi for 10 s, UV detection at 214 nm.
• Peak identification: ZipChip microfluidic system coupled to SCIEX TripleTOF 6600 MS with Native CZE application and 32 Karat™ Software v10.

Hlavní výsledky a diskuse

Comparison of intact and IdeS middle-up CZE demonstrated enhanced resolution of charge variants in subunit analysis. Heat-stress studies on adalimumab revealed significant increases in acidic species after 10 days, with deamidation predominantly localized to the Fc domain while the Fab remained stable. IdeS cleavage preserved non-covalent interactions, enabling detection of Fc subunits rather than Fc/2 fragments. Analysis of Ado-trastuzumab emtansine (Kadcyla®) showed that subunit CZE resolves complex ADC charge heterogeneity in under 10 minutes with high reproducibility, overcoming resolution limitations at the intact level.

Přínosy a praktické využití metody

• Rapid domain-specific charge variant profiling of mAbs, ADCs and novel modalities in under 10 min.
• Improved separation resolution for challenging molecules compared to intact CZE.
• Ready-to-use kits and cartridges streamline training and method adoption.
• Unified workflow allows both intact and subunit analysis using the same reagents and conditions.
• Seamless peak assignment via ZipChip Native TOF integration.

Budoucí trendy a možnosti využití

Optimization of capillary length, separation voltage and temperature can further enhance resolution. Extending the subunit CZE-MS approach to diverse biotherapeutics and ADC formats will support comprehensive PTM mapping. Integration into high-throughput QC workflows and coupling with multi-attribute monitoring platforms promise broad adoption in biopharma development and manufacturing.

Závěr

The combination of IdeS subunit digestion with native CZE-UV and high-resolution MS provides a rapid, high-resolution and domain-specific workflow for charge variant characterization. This approach enhances PTM localization, streamlines peak identification and supports robust stability and heterogeneity assessments in biopharmaceutical analysis.

Reference

• He Y., Isele C., Hou W., Ruesch M. J. Sep. Sci. 2011;34:548–555.
• Yan An, Ying Zhang, Hans-Martin Mueller, Mohammed Shameem, Xiaoyu Chen. MAbs. 2014;6(4):879–893.
• SCIEX. Characterization of charge heterogeneity of monoclonal antibodies using CE-MS. RUO-MKT-02-10949-A.
• SCIEX. Charge variant liability analysis using the SCIEX flexible solution for MAM. RUO-MKT-02-11828-A.
• SCIEX. Application of SCIEX flexible solution for MAM in charge variant analysis. RUO-MKT-02-11862-A.