Automated Analytical Cation-Exchange Chromatography Fraction Collection and Structural Characterization of Monoclonal Antibody Charge Variants

Significance of the Topic

Cation-exchange charge variant profiling is a critical quality control measure for monoclonal antibody therapeutics. Variants arising from terminal lysine clipping, glycoform heterogeneity or deamidation can affect efficacy, stability and immunogenicity. A robust workflow that couples high-resolution separation with precise fraction collection and downstream structural analysis enables comprehensive characterization of these critical quality attributes.

Objectives and Study Overview

This study demonstrates an automated analytical-scale cation-exchange fraction collection workflow using a BioResolve SCX mAb column and Waters Fraction Manager-Analytical (WFM-A). Two marketed mAbs, infliximab and trastuzumab, were separated, fractions were collected, and each fraction was characterized by intact mass analysis, subunit analysis and peptide mapping to assign structural modifications underlying observed charge heterogeneity.

Used Instrumentation

• ACQUITY UPLC H-Class Bio System
• BioResolve SCX mAb Columns (4.6×50 mm and 4.6×100 mm)
• Waters Fraction Manager-Analytical (WFM-A)
• Xevo G2-XS QToF Mass Spectrometer
• Empower 3, MassLynx v4.1, UNIFI v1.9 software

Methodology

• Cation-exchange separations were performed using MES buffer gradients tailored to each mAb and column length. Loading studies established maximum sample capacity while preserving resolution.
• Fractions were automatically collected by WFM-A, minimizing dispersion for narrow UPLC peaks. Alternate fractions were pooled or simultaneously re-injected to increase throughput.
• Intact mass analysis: fractions were analyzed on a reversed-phase Polyphenyl column and deconvoluted to determine terminal lysine count and glycan composition.
• Subunit analysis: IdeS digestion generated scFc and F(ab')² fragments; DTT reduction further yielded light chain (LC) and Fd' fragments to confirm lysine variants and degrees of disulfide reduction.
• Peptide mapping: trypsin/Lys-C digestions of trastuzumab fractions identified deamidation sites by comparing extracted ion chromatograms and MS/MS fragmentation.

Key Results and Discussion

• Loading on the 4.6×100 mm column up to ~1–2 mg mAb maintained acceptable resolution, a 2.5× load increase relative to 50 mm length.
• Automated fraction collection yielded well-defined infliximab peaks; alternate fractions could be re-purified in batch without cross-contamination.
• Intact mass deconvolution of infliximab fractions 5, 6 and 10 confirmed 0, 1 or 2 C-terminal lysines, and glycoform variants differing by sialic acid or fucose content.
• Subunit LC-MS corroborated intact mass assignments and revealed partially reduced scFc species, indicating variable disulfide-bond reduction.
• Peptide mapping of trastuzumab acidic and main fractions pinpointed deamidation of the peptide ASQDVNTAVAWYQQKPGK, explaining the early-eluting acidic peak.

Practical Benefits and Applications

• This integrated workflow accelerates mAb charge variant purification at analytical scale for in-depth structural and functional assays.
• Precise low-volume fraction collection by WFM-A reduces sample loss and dispersion, facilitating downstream MS, glycan, oxidation or binding analyses.
• Batch re-purification of alternate fractions increases throughput and resource efficiency in CQA characterization.

Future Trends and Opportunities

Advances in column chemistries and miniaturized fraction collectors will improve resolution and reduce solvent and sample consumption. Coupling IEX fractionation with automated sample processing and high-throughput MS workflows will enable real-time monitoring of bioprocess variants. Integration of functional binding assays or cell-based readouts directly from collected fractions could streamline correlation of structure with activity.

Conclusion

The combination of BioResolve SCX mAb columns and Waters Fraction Manager-Analytical provides a reliable, high-resolution platform for mAb charge variant separation and fraction collection. Downstream intact mass, subunit and peptide mapping analyses accurately assign terminal lysine variants, glycoforms and deamidation modifications. This comprehensive workflow supports detailed CQA characterization to ensure biotherapeutic safety and efficacy.

References

• Khawli LA et al. Charge Variants in IgG1 mAbs. mAbs 2010, 2(6):613–624.
• Jablonski JM et al. Small Scale Peptide and Impurity Isolation Using the ACQUITY UPLC H-Class and Waters Fraction Manager-Analytical Systems. Waters App Note 720005500EN (2015).
• Lauber MA et al. Designing a new particle technology for robust charge variant analysis of mAbs. Waters App Note 720006475EN (2019).
• Jung SK et al. Physicochemical characterization of Remsima. mAbs 2014, 6(5):1163–1177.
• Sakae Y et al. Conformational effects of N-glycan core fucosylation of immunoglobulin G Fc region on its interaction with Fcγ receptor IIIa. Sci Rep 2017, 7:13780.