High-Speed Process Monitoring with UHP-SEC-MALS

Importance of the Topic

At-line process monitoring of biotherapeutics enables rapid decision-making during purification and stability testing, reducing reliance on time-consuming offline analyses. Ultra-high-performance SEC coupled with multi-angle and dynamic light scattering delivers absolute molar mass, size, and concentration data in under five minutes, making real-time characterization of aggregates and fragments feasible.

Study Objectives and Overview

This application note assesses UHP-SEC-MALS for high-speed monitoring of monoclonal antibody samples under purification and stability conditions. A model antibody for purification fractions (mAb1), a second antibody for accelerated stability tests (mAb2), and a BSA control were analyzed to evaluate chromatographic resolution, mass accuracy, and throughput at different flow rates.

Methodology and Instrumentation

• Chromatography: Waters Acquity UPLC system with BEH SEC column (200 Å, 1.7 µm, 4.6 × 150 mm), phosphate buffered saline mobile phase, flow rates of 0.3–0.5 mL/min, 5-minute runs, 2–5 µL injections.
• Detection: Inline UV/Vis; Wyatt µDAWN three-angle MALS; Optilab UT-rEX differential refractive index; integrated WyattQELS dynamic light scattering.
• Data Analysis: ASTRA software for peak integration, weight-average molar mass, hydrodynamic radius, and mass fraction calculations.

Main Results and Discussion

• BSA Control: No loss of resolution or change in monomer/dimer/trimer fractions when increasing flow rate from 0.3 to 0.5 mL/min; consistent molar masses confirmed robustness.
• mAb1 Purification: Five species defined; monomer fraction increased from ~70% in crude to >95% in final product within a 5-minute analysis, demonstrating effective real-time monitoring of aggregate removal.
• mAb2 Stability Testing: Three stress conditions produced distinct monomer, dimer, and fragment profiles. Conditions 1 and 2 showed similar ratios; condition 3 exhibited shifted elution time with identical molar mass, indicating altered column interactions rather than degradation; fragment mass differences suggested different cleavage pathways.
• Hydrodynamic Radius: DLS measurements yielded Rh ≈ 5.1 ± 0.1 nm for mAb2 monomer across all conditions, confirming structural integrity despite retention shifts.

Benefits and Practical Applications of the Method

• Real-time, at-line bioprocess monitoring minimizes sample handling and material loss.
• High-throughput screening of hundreds of stability or purification samples.
• Accurate identification and quantification of co-eluting or low-abundance aggregates and fragments.
• Enhanced process development, formulation optimization, and quality control workflows.

Future Trends and Opportunities

• Integration of inline UHP-SEC-MALS with automated process control for real-time decision making.
• Extension to other biotherapeutic formats, including fusion proteins and complex formulations.
• Advances in detector technology and miniaturization for true inline continuous monitoring.
• Hybrid analytical platforms combining light scattering with mass spectrometry or other spectroscopic methods for comprehensive characterization.

Conclusion

UHP-SEC-MALS offers a fast, reliable platform for process-line characterization of biotherapeutic aggregates, fragments, and monomeric species. Its five-minute runtime and robust molar mass and size determinations support efficient purification monitoring and high-throughput stability screening.

Reference

1. Mou X, et al. Pharm. Bioprocess. 2014;2(2):141–156.