Charge variant analysis of cysteine-linked antibody-drug conjugates using an online multiple heart-cut 2D- SCX-SEC-LC HRAM MS approach

Importance of the topic

The detailed characterization of charge heterogeneity in antibody-drug conjugates (ADCs) is critical to ensure product quality, safety, stability and efficacy. ADCs combine the specificity of monoclonal antibodies with cytotoxic drugs to target diseased cells. Post-translational modifications and the conjugation chemistry introduce multiple charge variants that must be resolved and quantified as part of regulatory quality control.

Objectives and study overview

This study aimed to develop and optimize an online multiple heart-cut two-dimensional liquid chromatography (2D-LC) method combining strong cation exchange (SCX) and size exclusion chromatography (SEC) with high-resolution accurate-mass (HRAM) mass spectrometry for native charge variant analysis of the cysteine-linked ADC polatuzumab vedotin. The goal was to separate up to five SCX fractions per injection, desalt them inline via SEC and identify charge variant populations by intact mass analysis.

Methodology and instrumentation

• A Thermo Scientific Vanquish Online 2D-LC System coupled to an Orbitrap Exploris 240 mass spectrometer was employed.
• First dimension: Thermo Scientific ProPac 3R SCX-1 column (100×4 mm, 3 μm) with a linear pH gradient using CX-1 buffers.
• Second dimension: Thermo Scientific MAbPac SEC-1 column (300×4 mm, 5 μm) operated in ammonium acetate to maintain native conditions.
• Fraction transfer: Six 250 μL MP35N loops under backflush mode to sharpen SEC peaks and improve sensitivity.
• MS settings: Positive ion mode, 60 000 resolution, profile acquisition and ReSpect deconvolution in BioPharma Finder.

Main results and discussion

• SCX separation resolved eighteen charge variant peaks, corresponding to DAR0, DAR2, DAR4 and DAR8 species modified by deamidation, oxidation and C-terminal lysine truncation.
• Backflush transfer markedly sharpened SEC peaks and increased signal-to-noise versus forward flush.
• Fraction volumes up to 210 μL provided complete peak transfer without excessive SEC band broadening.
• HRAM MS deconvolution identified low-abundance variants (e.g. ~0.4%) with high confidence, revealing combinations of payload number, glycoform and Lys truncation.
• Deamidation and oxidation generated acidic variants eluting earlier, while lysine truncation yielded basic variants eluting later.

Benefits and practical applications

• Integrated SCX-SEC-MS workflow simplifies sample handling by inline desalting, eliminating offline buffer exchange.
• Backflush fraction transfer improves chromatographic resolution and mass spectrometric sensitivity.
• Intact mass analysis allows rapid screening of known charge variants and confirmation of DAR distribution under native conditions.
• Method supports detailed quality assessment of ADC batches and monitoring of critical quality attributes.

Future trends and potential applications

• Extension to multi-attribute methods combining hydrophobic interaction and ion mobility separations.
• Automation of method development for pH gradient optimization across diverse ADC platforms.
• Integration with peptide mapping to correlate intact mass variants with site-specific modifications.
• Application to other biotherapeutic modalities such as bispecific antibodies and fusion proteins.

Conclusion

The online multiple heart-cut 2D-SCX-SEC-HRAM MS approach provides a powerful platform for in-depth characterization of ADC charge heterogeneity in native conditions. The combination of efficient fraction transfer, high-resolution separation and sensitive mass detection enables identification and quantification of principal and low-abundance variants, supporting robust quality control and accelerating ADC development.

Reference

1. Lin S et al. A novel pH gradient separation platform for monoclonal antibody charge variant analysis. Thermo Fisher Scientific Application Note 20784.
2. Millán-Martín S et al. Optimisation of the use of sliding window deconvolution for comprehensive characterisation of trastuzumab and adalimumab charge variants by native high resolution mass spectrometry. Eur J Pharm Biopharm 2021.
3. Grübner M et al. Intact mass analysis of monoclonal antibody charge variants by multi heart-cut 2D-LC/MS coupling ion-exchange and reversed-phase chromatography. Thermo Fisher Scientific Application Note 001938.
4. Ehkirch A et al. An online four-dimensional HIC×SEC-IM×MS methodology for proof-of-concept characterization of antibody drug conjugates. Anal Chem 2018.