Charge variants can profoundly influence biotherapeutic stability, biological activity, safety, and immunogenicity.
While imaged capillary isoelectric focusing (icIEF) is widely used to separate charge variants, additional characterization methods are needed to identify the molecular changes underlying these differences.
Mass Spectrometry (MS) characterization of the charge heterogeneity of biotherapeutics is frequently constrained by adsorption-driven sample loss, disproportionately affecting low abundance isoforms.
While surfactants and sugar additives found in formulation buffer can mitigate these losses, many are incompatible with MS analysis and contribute to ion suppression.
The MauriceFlex™ System enables fractionation of charge variants; however, substantial analyte depletion can occur post fraction elution and after frozen storage, impacting downstream MS characterization methods such as direct intact mass analysis, especially for low abundance species.
Join our talk on In-Depth Characterization of Low-Abundance ADC Charge Variants using icIEF Fractionation and Online Buffer Exchange Coupled Mass Spectrometry.
Will McElroy (Bio-Techne) and Dr.
Casey Vantucci (Andson Biotech) will describe a streamlined, integrated workflow that combines the MauriceFlex icIEF-fractions with the DynaChip X1™ automated online buffer exchange, enabling direct analysis with a Thermo Scientific™ UHMR mass spectrometer.
Using Herceptin and its antibody–drug conjugate (ADC) Kadcyla as model samples, the speakers will highlight candidate reagents for mobilizer additives that maximize recovery during storage of MauriceFlex isoform fractions and downstream sensitive MS characterization of variants.
This workflow highlights the importance of using formulation additives in the fraction mobilizer to prevent analyte depletion and the strength of rapid online buffer exchange combined MS in providing sensitive analysis of low abundance isoform fractions derived from a single icIEF-fractionation run.
This strategy combines high-resolution charge variant separation and fraction collection with an automated sample preparation step that reduces matrix effects while eliminating manual buffer exchange steps that exacerbate sample loss, improving the robustness and sensitivity of charge variant MS workflows.
Key topics:
- How combining the MauriceFlex fractions with the DynaChip X1’s rapid online buffer exchange step enables the analysis of low abundant isoforms
- How this workflow is effective for the analysis of both mAb charge isoforms and sticky ADC charge variants, particularly high DAR isoforms
- How an improved understanding of critical quality attributes will accelerate development timelines and strengthen regulatory submissions
