A Comprehensive Workflow to Optimize and Execute Protein Aggregate Studies

Importance of the Topic

Protein aggregation poses significant challenges in the development and quality control of biotherapeutics. Aggregates can reduce drug efficacy, trigger immunogenic responses, and affect stability. Reliable analytical workflows for aggregate detection, quantitation, and characterization are essential to ensure product safety and performance.

Objectives and Study Overview

This study demonstrates a comprehensive workflow to optimize high-performance size exclusion chromatography (SEC) conditions for monoclonal antibodies, accurately quantify monomers, dimers, and higher-order aggregates, and augment UV-based quantitation with light scattering detection. Key goals include automating mobile phase scouting using Buffer Advisor software and characterizing aggregate profiles of rituximab innovator and biosimilar samples.

Methodology and Instrumentation

Aquous mobile phases spanning phosphate buffers and sodium chloride at varying pH (6.2–7.4) were prepared automatically in real time by the Agilent Buffer Advisor and delivered by the bio-inert quaternary pump of the Agilent 1260 Infinity II Bio-inert LC System. A short AdvanceBio SEC 15 cm column enabled rapid screening (<10 min per run), while UV detection at 220 and 280 nm quantified monomer and aggregate peaks. The Agilent AdvanceBio SEC Multi Detector Suite (Bio-MDS) provided dynamic light scattering (DLS) and light scattering (LS) data for absolute molecular weight and hydrodynamic radius determination.

Used Instrumentation

• Agilent 1260 Infinity II Bio-inert Quaternary Pump
• Agilent 1260 Infinity II Bio-inert Multisampler with cooler
• Agilent 1260 Infinity II Multicolumn Thermostat
• Agilent 1260 Infinity II Diode Array Detector WR
• Agilent AdvanceBio SEC 300 Å columns (150 × 7.8 mm or 300 × 7.8 mm)
• Agilent AdvanceBio SEC Multi Detector System (G7805AA) including UV, LS, and DLS detectors

Main Results and Discussion

Systematic scouting of twelve buffer compositions revealed that pH 7.0 conditions with either 150 mM phosphate or 100 mM phosphate plus 150 mM NaCl delivered the best peak shape and reproducible aggregate quantitation. Low-salt conditions induced peak tailing and reduced sensitivity. UV integration showed similar aggregate levels for innovator and biosimilar at optimal conditions. LS data confirmed monomer molecular weights (~147 kDa) and highlighted differences in higher-order aggregate formation between the two samples. DLS analysis provided hydrodynamic radii (5.1 nm for biosimilar monomer) and enabled detection of subvisible aggregates not evident by UV alone.

Benefits and Practical Applications

• Automated buffer mixing accelerates method development and reduces manual preparation errors.
• Rapid SEC screening (<10 min) increases laboratory throughput.
• Bio-inert flow path and advanced coatings minimize secondary interactions, improving resolution.
• Multidetector capability (UV, LS, DLS) provides comprehensive aggregate size, weight, and conformation data in a single run.

Future Trends and Applications

Emerging approaches include multiplexed SEC columns for enhanced resolution, fully integrated high-throughput screening platforms, and coupling with mass spectrometry for structural insights. Advances in detector sensitivity and data analysis software will further streamline aggregate profiling, support subvisible particle characterization, and enable real-time control in bioprocess monitoring.

Conclusion

The Agilent 1260 Infinity II Bio-inert LC System combined with Buffer Advisor and the Bio-MDS suite offers a robust, automated workflow for SEC-based aggregate analysis of monoclonal antibodies. Optimized mobile phase conditions and multidetector data yield accurate quantitation and detailed characterization of protein aggregates, enhancing method development efficiency and analytical confidence.

References

• U.S. Department of Health and Human Services Food and Drug Administration. Guidance for Industry Immunogenicity Assessment for Therapeutic Protein Products. CDER/CBER, 2014.
• Mahler H-C, Friess W, Grauschopf U, Kiese S. Protein Aggregation: Pathways, Induction Factors and Analysis. Journal of Pharmaceutical Sciences. 2008;98(9).
• Schneider S. 2D-LC/MS Characterization of Charge Variants Using Ion Exchange and Reversed-Phase Chromatography. Agilent Technologies Application Note 5991-6673EN, 2016.