Fully Automated Characterization of Monoclonal Antibody Charge Variants Using 4D-LC/MS

Significance of the topic

The characterization of monoclonal antibody (mAb) charge variants is critical for ensuring product safety, efficacy, and consistency in biopharmaceutical development and quality control. Post-translational modifications such as deamidation, lysine truncation, and glycosylation can alter the charge profile of mAbs, affecting their biological activity and stability. Streamlined, high-resolution analysis methods that integrate chromatography and mass spectrometry are therefore essential for comprehensive profiling of these variants.

Study Objectives and Overview

This work presents a fully automated four-dimensional liquid chromatography/mass spectrometry (4D-LC/MS) platform for in-depth profiling of mAb charge variants. The main goals are to separate charge isoforms via cation-exchange chromatography (CEX), perform sequential online desalting, denaturation, reduction, and tryptic digestion, and achieve detailed peptide mapping in a single, continuous workflow. The method is demonstrated using the clinically relevant mAb trastuzumab and its high-pH stressed variant.

Methodology and Instrumentation

The 4D-LC/MS system comprises:

• First dimension (1D): CEX on a nonporous column (Agilent Bio MAb) at pH 7.65 with a NaCl gradient to resolve charge variants.
• Multiple heart-cuts: Collection of individual peaks and a blank fraction into 40 µL loops via 2D-LC valve.
• Second dimension (2D): Reversed-phase desalting, denaturation, and reduction on a polymeric cartridge with formic acid, acetonitrile, and DTT buffer.
  
• Third dimension (3D): On-column tryptic digestion in an Orachrom StyrosZyme TPCK-trypsin PEEK column at 40 °C, with inline mixing of digestion buffer.
• Fourth dimension (4D): Peptide mapping on an Agilent AdvanceBio peptide mapping column at 60 °C, followed by Q-TOF MS detection (m/z 100–3200).

Instrumentation includes the Agilent InfinityLab 2D-LC Solution (1290 Infinity II pumps, valves, DAD detectors) and the Agilent 6545 LC/Q-TOF with Jet Stream ESI. Data acquisition and control were performed using Agilent OpenLab CDS and MassHunter software with BioConfirm for peptide identification.

Key Results and Discussion

The automated workflow achieved over 90% sequence coverage for trastuzumab peptide mapping within a single 110-minute cycle. Three principal CEX peaks (pre-peak, main peak, post-peak) were successfully characterized:

• The pre-peak contained a singly deamidated variant (Asn30→Asp30) on one light chain.
• The main peak matched the unmodified mAb.
• The post-peak featured isoaspartate formation at Asp102 in one heavy chain.

In high-pH stressed samples, four acidic variants were resolved, revealing double and single deamidation events at light-chain Asn30 and heavy-chain Asn387 sites. Extracted ion chromatograms confirmed site-specific modifications and cleavage differences between variants.

Benefits and Practical Applications

• Fully online and automated processing reduces manual intervention and sample loss.
• High-resolution separation and MS detection enable unambiguous identification of charge modifications.
• Streamlined workflow supports rapid screening of multiple charge variants in biopharmaceutical R&D and QC.

Future Trends and Applications

Advancements may include replacing CEX with other first-dimension separations (e.g., Protein A, SEC, HIC) to profile additional attributes. Integration with higher-throughput MS platforms and machine-learning data analysis could further accelerate variant identification and quantification. The approach could be expanded to antibody-drug conjugates and complex fusion proteins for comprehensive biotherapeutic characterization.

Conclusion

This study demonstrates a robust, multidimensional LC/MS strategy for automated, high-resolution characterization of mAb charge variants. The 4D-LC/MS platform provides detailed peptide-level insights into post-translational modifications, supporting critical quality attribute monitoring in biopharmaceutical workflows.

References

1. Sandra, K.; Vanhoenacker, G.; Sandra, P.; Sandra, K. Modern Chromatographic and Mass Spectrometric Techniques for Protein Biopharmaceutical Characterization. J. Chromatogr. A 2014, 1335, 81–103.
2. Fekete, S.; Gritti, F.; Sebastiao, A.; Veuthey, J.-L.; Carr, P. W. Chromatographic, Electrophoretic and Mass Spectrometric Methods for the Analytical Characterization of Protein Biopharmaceuticals. Anal. Chem. 2016, 88, 480–507.
3. Walsh, G. Biopharmaceutical Benchmarks 2018. Nat. Biotechnol. 2018, 32, 992–1000.
4. Harris, R. J.; Poon, T. K.; Hossler, P.; Hu, W.-S. Identification of Multiple Sources of Charge Heterogeneity in a Recombinant Antibody. J. Chromatogr. B 2001, 752, 233–245.
5. Stoll, D. R.; Qin, Z.; Carr, P. W. Characterization of Therapeutic Antibodies and Related Products by Two-Dimensional Liquid Chromatography Coupled with UV Absorbance and Mass Spectrometric Detection. J. Chromatogr. B 2016, 1032, 51–60.
6. Sandra, K.; Balcaen, L.; Sandra, P.; et al. Characterizing Monoclonal Antibodies and Antibody-Drug Conjugates Using 2D-LC-MS. LCGC Europe 2017, 30, 149–157.
7. Stoll, D. R.; Carr, P. W.; Avdalovic, N. Direct Identification of Rituximab Main Isoforms and Subunit Analysis by Online Selective Comprehensive Two-Dimensional Liquid Chromatography–Mass Spectrometry. Anal. Chem. 2015, 87, 8307–8315.
8. Sandra, K.; Balcaen, L.; Sandra, P.; et al. Multiple Heart-Cutting and Comprehensive Two-Dimensional Liquid Chromatography Hyphenated to Mass Spectrometry for the Characterization of the Antibody-Drug Conjugate Ado-Trastuzumab Emtansine. J. Chromatogr. B 2016, 1032, 119–130.
9. Schneider, S. 2D-LC/MS Characterization of Charge Variants Using Ion Exchange and Reversed-Phase Chromatography. Agilent Technologies Application Note 5991-6673EN, 2016.
10. Gstöttner, C.; et al. Fast and Automated Characterization of Antibody Variants with 4D HPLC/MS. Anal. Chem. 2018, 90, 2119–2125.
11. Goyon, A.; et al. Streamlined Characterization of an Antibody-Drug Conjugate by Two-Dimensional and Four-Dimensional Liquid Chromatography/Mass Spectrometry. Anal. Chem. 2019, 91, 14896–14903.
12. Goyon, A.; et al. From Proof of Concept to the Routine Use of an Automated and Robust Multi-Dimensional Liquid Chromatography Mass Spectrometry Workflow Applied for the Charge Variant Characterization of Therapeutic Antibodies. J. Chromatogr. A 2020, doi:10.1016/j.chroma.2019.460740.
13. Vandenheede, I.; et al. Characterize mAb Charge Variants by Cation-Exchange Chromatography. Agilent Technologies Application Note 5991-5273EN, 2014.