A Trap & Elute Style 2D Protein A–SEC Heart-Cut Method for the Analysis of Monoclonal Antibody Titer and Size Variants in Cell Culture

Significance of the Topic

Monoclonal antibodies (mAbs) are central to modern biopharmaceutical production, with rigorous monitoring of titer and size variants essential for ensuring product efficacy, safety, and consistency. Integrating affinity and size‐exclusion chromatography into a streamlined two‐dimensional (2D) workflow enhances analytical throughput and offers precise control over protein purification and characterization.

Objectives and Overview of the Study

This application note describes the development and evaluation of two simplified trap‐and‐elute, heart‐cut 2D Protein A–size exclusion chromatography (ProA‐SEC) methods for simultaneous determination of mAb titer and size variants directly in CHO cell conditioned media (CM). The high‐throughput (HT) version targets rapid assessment of high molecular weight species (HMWS), while the high‐resolution (HR) version extends analysis to low molecular weight species (LMWS) with Protein A affinity.

Methodology and Instrumentation

Both 2D methods employ compact, high‐pressure BioResolve Protein A columns (3.5 µm, 2.1 × 20 mm and 3.5 µm, 3.9 × 5 mm) and direct trap‐and‐elute transfer onto high‐performance SEC columns (1.7 µm, 4.6 × 300 mm for HR; 2.5 µm, 7.8 × 150 mm for HT). A single six‐port valve and two UPLC/UHPLC pumps control flow paths, using compatible mobile phases (pH 7.4 loading buffer, pH 3.0 elution buffer, and phosphate‐buffered SEC eluent).

Used Instrumentation

• ACQUITY UPLC H-Class System with Column Manager and six‐port valve
• ACQUITY UPLC I-Class Binary Solvent Manager
• ACQUITY Tunable UV Detector (280 nm and 214 nm)
• BioResolve Protein A Affinity Columns (2.1 × 20 mm and 3.9 × 5 mm)
• ACQUITY Premier Protein SEC Column (4.6 × 300 mm, 1.7 µm)
• XBridge Premier Protein SEC Column (7.8 × 150 mm, 2.5 µm)
• Empower Chromatography Data System

Main Results and Discussion

• Protein A columns delivered sharper, more concentrated mAb elution peaks (up to 3× higher height and reduced volume), enabling direct loading onto SEC without holding loops.
• Both 2D configurations achieved >97% monomer recovery; method-induced HMWS formation was minimal (<0.2%), near the limit of quantification (LOQ). Overall HMWS recovery was estimated at ~85%.
• HR method resolved HMWS and LMWS (100 kDa fragment) effectively; HT method provided rapid HMWS monitoring.
• Calibration using NISTmAb spiked into CM demonstrated linear titer assessment (R2 > 0.997) and accurate recovery of CM titer (slopes 0.94–0.97).
• LOQ for titer determination was ~0.2 µg mAb; estimated minimum sample loads for 90% confidence were 0.06 µg HMWS (HT) and 0.04 µg HMWS or 0.004 µg LMWS (HR).

Benefits and Practical Applications

• Streamlined 2D ProA-SEC setup with minimal hardware complexity and no holding loops.
• Rapid analysis: ~5 min for mAb titer and HMWS (HT) and < 20 min for titer, HMWS, and LMWS (HR).
• Enhanced SEC resolution and reduced protein degradation via immediate neutralization of acidic eluent.
• Applicable to diverse Fc-containing constructs including fusion proteins and multi-specific antibodies.

Future Trends and Applications

• Broader adoption of high-pressure, low-volume affinity columns to extend 2D workflows to novel biotherapeutics.
• Integration with mass spectrometry for enhanced variant identification and glycosylation profiling.
• Automated method optimization leveraging real-time feedback for adaptive control of heart-cut timing.
• Expansion into continuous bioprocess monitoring and in-line quality control platforms.

Conclusion

The simplified trap-and-elute 2D ProA-SEC heart-cut methods deliver robust, high-throughput analyses of mAb titer and size variants directly in cell culture media. By minimizing dispersion, reducing hardware complexity, and ensuring accurate variant recovery, these approaches support efficient process development, quality control, and comparative screening of biotherapeutic candidates.

References

1. Lemmerer M., London A.S., Panicucci A., Gutierrez-Vargas C., Lihon M., Dreier P. Coupled affinity and sizing chromatography: a novel in-process analytical tool to measure titer and trend Fc-protein aggregation. Journal of Immunological Methods. 2013;393(1–2):81–85.
2. Gjoka X., Schofield M., Cvetkovic A., Gantier R. Combined Protein A and size exclusion high performance liquid chromatography for the single-step measurement of mAb, aggregates and host cell proteins. Journal of Chromatography B. 2014;972:48–52.
3. Dunn Z.D., Desai J., Leme G.M., Stoll D.R., Richardson D.D. Rapid two-dimensional Protein-A size exclusion chromatography of monoclonal antibodies for titer and aggregation measurements from harvested cell culture fluid samples. mAbs. 2020;12(1):1702263.
4. Kizekai L., Shiner S.J., Lauber M.A. Waters ACQUITY and XBridge Premier Protein SEC 250 Å Columns: A New Benchmark in Inert SEC Column Design. Waters Application Note 720007493; 2022.
5. Koza S.M., Yang H., Yu Y.Q. Expanding Size-Exclusion Chromatography Platform Method Versatility for Monoclonal Antibody Analysis Using Waters XBridge Premier Protein SEC Columns. Waters Application Note 720007500; 2022.