BIOSEPARATIONS - APPLICATIONS NOTEBOOK

Significance of the Topic

Resolving and quantifying charge variants of monoclonal antibodies is a critical quality attribute in biopharmaceutical manufacturing. Non‐enzymatic modifications, including C-terminal lysine clipping, deamidation, and glycan heterogeneity, alter net protein charge and can impact safety, efficacy, and shelf life. Ion-exchange chromatography (IEX) under non-denaturing conditions is widely used for charge variant analysis, but method development often involves time-consuming buffer preparation and screening.

Objectives and Overview of the Study

This study demonstrates how Auto•Blend Plus Technology, integrated with the ACQUITY UPLC H-Class Bio System, simplifies and accelerates IEX method development. Key aims include:

• Automating pH and salt gradient preparation from concentrated stocks.
• Optimizing separation of C-terminal lysine truncation variants of a chimeric monoclonal antibody.
• Evaluating robustness across pH range, ionic strength, and sample load.

Methodology and Instrumentation

• Column: Protein-Pak Hi Res SP (strong cation exchange), 4.6×100 mm, 7 µm.
• System: ACQUITY UPLC H-Class Bio with Auto•Blend Plus to create four-solvent gradients: acidic reservoir (MES or phosphate), basic reservoir, NaCl, and water.
• Detection: UV at 280 nm.
• Sample: Infliximab (1.25 mg/mL) charged variants generated via carboxypeptidase B.
• Screening: pH gradients (5.2–7.9) and ionic strength (20–100 mM NaCl) evaluated automatically.

Main Results and Discussion

1. Automated pH gradients: Auto•Blend Plus blended buffers to deliver extended linear pH profiles (pH 5.2–7.9) with excellent fidelity (<0.05 pH units deviation) even under load.
2. Robust pH optimization: Separation of C-terminal lysine variants improved markedly between pH 6.4–6.6, enabling clear resolution of des-Lys, mono-Lys, and di-Lys species.
3. Ionic strength screening: A series of pH 6.6 separations with NaCl from 20 to 100 mM revealed optimum resolution at 40 mM, reducing iterative buffer dilutions.
4. Method robustness: Automated 40-run stability study yielded <12% RSD in individual peak areas and <9% RSD in total area, confirming system stability.
5. Sample load effects: Retention times and quantitation remained consistent over a 9-fold mass increase (20–180 µg), demonstrating reliable quantification.

Benefits and Practical Applications

• Time and cost savings by eliminating multiple buffer preparations and manual pH adjustment.
• Rapid identification of optimum pH and salt conditions using one stock solution set.
• High method reproducibility facilitates transfer between analysts and labs.
• Capability to monitor charge variants throughout development and quality control.

Future Trends and Applications

• Integration with mass spectrometry for on-line confirmation of variant identity.
• Expansion to two-dimensional IEX–RP separations for deeper proteoform characterization.
• Automated feedback loops for real-time method optimization.

Conclusion

Auto•Blend Plus Technology, in combination with the ACQUITY UPLC H-Class Bio System and Protein-Pak Hi Res IEX column, provides a streamlined, robust, and reproducible platform for pH-based charge variant method development. This approach dramatically reduces buffer preparation effort and accelerates optimal condition discovery, ensuring reliable quantification and resolution of monoclonal antibody charge variants.

References

1. Liu H et al. mAbs. 2012;4(5):578–85.
2. Rosenberg AS. AAPS J. 2006;8(3):E501–07.
3. Vlasak J, Ionescu R. Curr Pharm Biotechnol. 2008;9(6):468–81.
4. Wheat TE et al. J Chromatogr. 1990;512:13–22.