Rapid SEC-UV Analysis of Monoclonal Antibodies Using Ammonium Acetate Mobile Phases

Importance of the Topic

Monoclonal antibody aggregation and fragmentation can compromise therapeutic safety and efficacy. Rapid, non-denaturing SEC-UV methods provide timely insights into protein size variants, accelerating biotherapeutic process development and quality control. Implementing a volatile, MS-friendly mobile phase further streamlines workflows by enabling shared instrumentation between SEC-UV and LC-MS analyses and reducing system maintenance.

Objectives and Study Overview

The primary goal was to develop a high-throughput SEC-UV method capable of resolving size variants in Protein A purified monoclonal antibodies within four minutes. Key aims included using a volatile ammonium acetate mobile phase compatible with LC-MS platforms, demonstrating method robustness over hundreds of injections, and verifying quantitative performance across multiple mAb samples and analytical systems.

Methodology and Instrumentation

SEC separations were performed on an ACQUITY Premier Protein SEC column (250 Å, 1.7 µm, 4.6×150 mm) using a 200 mM ammonium acetate mobile phase at pH ≈7.0 and 0.50 mL/min flow rate. Samples (0.5–2.0 mg/mL in PBS) were maintained at 6 °C and injected (5 µL) onto a 25 °C column. UV detection at 280 nm was carried out on an ACQUITY UPLC TUV detector. Method evaluations were conducted on stand-alone ACQUITY Premier UPLC systems and a BioAccord LC-MS (ESI-Tof) system. Automated Protein A purifications were executed with an Andrew+ pipetting robot, and data were processed using Empower 3 and UNIFI software.

Results and Discussion

Optimization of ammonium acetate concentration identified 200 mM as optimal for consistent resolution of high molecular weight species (HMWS1, HMWS2) and low molecular weight fragments (LMWS1, LMWS2) across seven mAbs. The method exhibited linear response and stable relative peak areas from 0.5 to 2.0 mg/mL. Mixtures of trastuzumab variants demonstrated accurate quantification of size variants down to sub-percent levels. Protein A-purified samples spiked into cell culture media achieved recoveries above 90%, with minor bias in HMW recovery. Column lifetime studies showed minimal performance loss after over 700 analyses in three months, and column-to-column reproducibility was confirmed across different packing batches.

Benefits and Practical Applications

• Fast four-minute analysis of mAb aggregation and fragmentation
• Compatibility with LC-MS instrumentation using a volatile mobile phase
• High throughput for upstream, purification, and formulation workflows
• Robust column performance over hundreds of injections
• Accurate quantification of low-level size variants

Future Trends and Potential Applications

Emerging developments include integration of inline SEC-MS workflows, expanded use of intrinsic fluorescence detection for improved sensitivity, automated sample preparation for higher throughput, and application to diverse biotherapeutic formats such as bispecific antibodies and fusion proteins. Potential advances in guard column technologies and mobile phase customization may further enhance method longevity and resolution.

Conclusion

This study established a rapid, high-resolution SEC-UV method using a 200 mM ammonium acetate mobile phase, delivering reliable size variant analysis of monoclonal antibodies in under four minutes. The approach offers seamless compatibility with LC-MS platforms, sustained column performance, and reproducible quantitative results, making it a valuable tool for biopharmaceutical development and quality control.

References

• Moussa EM et al. Immunogenicity of Therapeutic Protein Aggregates. J Pharm Sci. 2016;105(2):417–430.
• Koza SM, Yang H, Yu YQ. Expanding Size-Exclusion Chromatography Platform Method Versatility for Monoclonal Antibody Analysis. Waters Application Note. January 2022.
• Koza SM, Yu YQ. Rapid Size Variant Analysis of Monoclonal Antibodies Using UPLC and HPLC Compatible MaxPeak Premier Protein SEC Columns. Waters Application Note. March 2022.
• Pinhal S et al. Acetate Metabolism and the Inhibition of Bacterial Growth by Acetate. J Bacteriol. 2019;201(13):e00147–19.
• Koza SM et al. Impact of LC System Dispersion on the SEC Analysis of Monoclonal IgG Antibody Aggregates and Fragments. Waters Application Note. June 2019.
• Dunn ZD et al. Rapid two-dimensional Protein-A Size Exclusion Chromatography of Monoclonal Antibodies for Titer and Aggregation Measurements. MAbs. 2020;12(1):1702263.