Direct and Rapid SEC Analysis of Monoclonal Antibody Titer and Aggregation in Cell- Culture

Significance of the Topic

A rapid and reliable assessment of monoclonal antibody (mAb) concentration and aggregation directly in cell-culture harvests accelerates bioprocess development and quality control. Traditional workflows use Protein A purification followed by size-exclusion chromatography (SEC), which can introduce artifacts and prolong analysis. A direct SEC approach with fluorescence detection reduces sample preparation, improves throughput, and maintains native protein states.

Aims and Overview

This study demonstrates a 2.4-minute SEC method employing intrinsic protein fluorescence (FLR) to quantify mAb titer and high molecular weight species (HMWS) directly in clarified CHO cell-culture samples. Key goals include minimizing injection volume, preserving column performance over extended use, and comparing UV (280 nm) vs. FLR detection for sensitivity and specificity.

Methodology and Instrumentation

The method uses an XBridge Premier Protein SEC 250 Å, 2.5 µm, 4.6×150 mm column packed with BEH-PEO particles and hydrophilic MaxPeak HPS hardware. A 200 mM ammonium acetate mobile phase at 1.0 mL/min provides anti-microbial properties and LC-MS compatibility. Intrinsic FLR detection (ex 280 nm, em 350 nm) enables injection volumes as low as 0.1 µL. Detection alternates an autozero event at 0.6 min to prepare for subsequent runs.

Instrumentation

• ACQUITY Premier UPLC with Quaternary Solvent Manager and CH-A column heater
• ACQUITY UPLC Tunable UV Detector, 5 mm titanium flow cell, 280 nm
• ACQUITY UPLC FLR Detector (ex 280 nm, em 350 nm)
• XBridge Premier Protein SEC column, 250 Å, 2.5 µm, 4.6×150 mm
• Empower 3 chromatography software

Results and Discussion

FLR detection provided over tenfold higher signal-to-noise vs. UV 280 nm, eliminating interference from DNA/RNA present in cell-culture medium. mAb titers from 0.25 to 4.0 mg/mL were accurately quantified with <3.2% bias using 0.1 µL injections. FLR also enabled reliable detection of dimeric HMWS1 at ≥1% abundance, while UV detection failed at low volumes. Stress-induced HMWS2 was quantified linearly down to ~1.5% levels. Column robustness testing over 500 consecutive cell-culture injections projected baseline resolution after ≥1000 analyses. Sample dilution (1:1 with buffer) further reduced fouling without compromising sensitivity.

Benefits and Practical Applications

• High-throughput analysis with 2.4 min cycle times
• Direct injection of clarified cell-culture samples—no Protein A cleanup
• Sensitive FLR detection allows low nanogram sample loads
• Extended column lifetime (>1000 injections) with minimal maintenance
• Real-time monitoring of mAb titer and aggregation during cell-line or process development

Future Trends and Potential Applications

Advances may include integration with online sampling for real-time process monitoring, miniaturized columns for further reduced sample consumption, and coupling to mass spectrometry for detailed variant profiling. Incorporating guard columns or in-line filters and exploring alternative benign mobile phases could further extend column life and broaden applicability to other protein therapeutics.

Conclusion

The described SEC-FLR method enables direct, rapid, and sensitive quantification of mAb titer and aggregation in clarified cell-culture samples. By minimizing sample handling and injection volume and leveraging a robust column and mobile phase, the workflow supports high-throughput bioprocess development with consistent performance over extended use.

References

1. Dunn ZD, Desai J, Leme GM, Stoll DR, Richardson DD. 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.
2. Paul AJ, Schwab K, Hesse F. Direct analysis of mAb aggregates in mammalian cell culture supernatant. BMC Biotechnology. 2014;14:11.
3. Koza SM, Jiang AHW, Yu YQ. Rapid SEC-UV Analysis of Monoclonal Antibodies Using Ammonium Acetate Mobile Phases. Waters Application Note. 2023.