An Easy-to-Execute Direct-Connect 2D Protein A–SEC Method for the Analysis of Monoclonal Antibody Titer and Size Variants in Cell Culture

Importance of the Topic

Direct-connect two-dimensional Protein A–SEC chromatography provides a rapid and streamlined approach for monitoring monoclonal antibody titer and size variants in cell culture. By eliminating complex valve switching and additional pumps, this method supports timely decision making in bioprocess development and quality control.

Objectives and Overview

This application note presents a simplified ProA-SEC workflow that uses a single-pump UHPLC system to measure monoclonal antibody concentration, high molecular weight species (HMWS), and low molecular weight species (LMWS) directly in clarified cell culture samples. The study evaluates linearity, recovery, and comparative variant measurements for NISTmAb and trastuzumab spikes in CHO media.

Used Instrumentation

• UHPLC system: ACQUITY Premier QSM with tunable UV detector
• Protein A column: BioResolve Protein A Affinity Column, MaxPeak Premier, 3.5 µm, 2.1×20 mm
• SEC column: ACQUITY Premier Protein SEC Column, 250 Å, 1.7 µm, 4.6×150 mm
• Autosampler cooled to 6 °C, flow rate 0.30 mL/min, detection at 280 nm

Methodology

The method uses two sequential injections. First, the sample passes through the series-connected ProA and SEC columns: unbound matrix components elute through SEC, while target antibodies bind to Protein A. Second, a small volume injection of citric acid (pH 2) elutes the bound antibody and variants in a narrow peak volume (≤15 µL), which are then separated on SEC. Total cycle time is about 15 minutes per sample.

Main Results and Discussion

Recovery of monomer titer was high, averaging 98.6% for NISTmAb and 97.8% for trastuzumab, with calibration linearity over 1 to 10 µg loads. Comparative HMWS levels were lower for NISTmAb but higher for trastuzumab versus SEC alone, indicating some method-induced aggregation for molecules with additional ProA-binding sites. LMWS1 variants were detected for both antibodies, while LMWS2 (Fab fragments) appeared only for trastuzumab, consistent with its Fab domain affinity. Standard addition in CHO media showed linear response and minimal bias, with predicted endogenous titers matching measured values within 6% on average.

Benefits and Practical Applications

• Simplified two-dimensional setup without switching valves or dual pumps
• Rapid 15-minute analysis for both titer and size variant profiling
• High reproducibility and recovery for monomer quantitation
• Capability to detect ProA-binding low molecular weight fragments
• Applicability to diverse Fc-containing constructs including fusion proteins and multispecific antibodies

Future Trends and Opportunities

Further work may explore molecule-specific recovery assessments, minimize method-induced aggregation, and extend direct-connect ProA-SEC to emerging therapeutic formats. Integration with automated data analysis and inline process monitoring could enhance real-time control of biomanufacturing.

Conclusion

The direct-connect 2D ProA-SEC method offers an effective, easy-to-execute approach for simultaneous measurement of monoclonal antibody titer and size variant profiles in cell culture samples. Its straightforward configuration and rapid cycle time support streamlined workflows in bioprocess development and quality control.

References

• 1. Lemmerer et al Journal of Immunological Methods 2013
• 2. Gjoka et al Journal of Chromatography B 2014
• 3. Dunn et al MAbs 2020
• 4. Lavelay Kizekai et al Waters Application Note 2022