Multi Dimensional HPLC Separation of Monoclonal Antibody Mixtures from Cell Culture Supernatants

Significance of the Topic

A robust and automated platform for multi-dimensional liquid chromatography coupled to mass spectrometry (2D-LC/MS) addresses critical challenges in therapeutic monoclonal antibody (mAb) analysis. The structural heterogeneity of mAbs demands simultaneous assessment of multiple quality attributes for development, production and quality control in biopharmaceutical workflows.

Aims and Overview of the Study

This application note presents an integrated 2D-LC/MS workflow combining Protein A affinity capture with secondary separations by size-exclusion (SEC), cation-exchange (CEX) and hydrophobic interaction (HIC) chromatography. The goal is to purify mAbs directly from cell culture supernatants and perform multi-attribute analysis (MAA), including titer, size/charge/hydrophobic variants, molecular weight, sequence verification and post-translational modifications.

Methodology and Sample Preparation

• Capture mAbs from supernatant using a Protein A affinity column in the first dimension.
• Perform heart-cutting into a second dimension employing SEC, CEX or HIC as required.
• Desalt and analyze glycan structures via SEC-MS.
• Sample filtration using PES syringe filters and use of deactivated glass or polypropylene vials to minimize nonspecific adsorption.
• Buffer preparation with HPLC-grade reagents and routine pH calibration.
• Column storage protocols for Protein A, SEC and HIC phases to maintain performance.

Used Instrumentation

• Automated Agilent InfinityLab 2D-LC ProtA-SEC multi-attribute analyzer.
• Protein A, AdvanceBio SEC and AdvanceBio HIC columns.
• Mass spectrometer configured for desalting SEC-MS glycan analysis.

Main Results and Discussion

The system enabled rapid purification and attribute mapping of trastuzumab originator and a CHO cell clone sample. High resolution in both dimensions was maintained during automated switching between SEC, CEX and HIC. Deconvoluted mass spectra confirmed primary sequence and glycoform distributions (G0F, G1F, G2F), while charge and hydrophobic variants were resolved in respective second dimensions. This approach streamlines the measurement of titer and multiple quality attributes in a single automated run.

Benefits and Practical Applications

• Consolidates capture, separation and MS analysis on one platform.
• Reduces hands-on time and manual buffer exchange.
• Supports high-throughput screening of clones and process samples.
• Improves data consistency across size, charge, hydrophobicity and glycosylation assessments.

Future Trends and Possible Applications

Integration of real-time data evaluation and machine learning algorithms could enable predictive process control. Miniaturization and higher-throughput column formats may further accelerate clone selection. Expanding the platform to subunit or peptide mapping could broaden coverage of critical quality attributes.

Conclusion

An automated 2D-LC/MS system combining Protein A capture with SEC, CEX and HIC heart-cutting delivers a powerful multi-attribute analysis workflow for monoclonal antibodies. It enhances throughput, data quality and operational efficiency for biologics development and QC.

References

1. Deamidation, oxidation, fragmentation and aggregation analysis by MAA multi-attribute analyzer.
2. Protein A affinity chromatography with multimethod second dimension (SEC, CEX, HIC).
3. Application note 5994-3521EN: glycan analysis by SEC-MS.
4. Automated subunit analysis by online reduction using 2D-LC/MS (5994-4309EN).
5. Heart-cutting 2D-LC of innovator vs. biosimilar mAbs (5991-6673EN).
6. Agilent 1290 Infinity 2D-LC Solution user guide (G2198-90001).