The Sun Rises Over UHP‐SEC: μDAWN for Polymer Characterization by MALS

Importance of the Topic

Ultra-high-pressure size exclusion chromatography (UHP-SEC) combined with multi-angle light scattering (MALS) and dynamic light scattering (DLS) delivers rapid, high-resolution molecular characterization of proteins and polymers directly in solution without relying on calibration standards.
It reduces analysis time, solvent consumption, and sample requirements while improving sensitivity and data richness for research, quality control, and manufacturing applications.

Study Objectives and Overview

• Introduce µDAWN and UT-rEX detectors designed to minimize band broadening in UHP-SEC.
• Evaluate the performance of these detectors versus standard MALS and dRI systems in terms of peak dispersion and molecular weight accuracy.
• Demonstrate key applications: monoclonal antibody analysis, conjugate characterization, process monitoring of preparative HPLC, and advanced polymer chromatography (APC).

Instrumentation

• UHP-SEC platform: Waters Acquity system with BEH200 columns for proteins and APC XT columns for polymers.
• Detectors: µDAWN MALS detector and Optilab UT-rEX differential refractive index (dRI) detector from Wyatt Technology, plus UPLC TUV UV detector.
• Software: ASTRA for band-broadening correction and calculation of molecular weight, radius of gyration, and extinction coefficients.

Main Results and Discussion

• Band Broadening Reduction: µDAWN and UT-rEX flow cells feature ~10–20× smaller internal volumes than standard detectors, preserving narrow UHP-SEC peaks (protein peak FWHM: UV=44 µL, µDAWN=45 µL, standard MALS=55 µL, dRI=105 µL; polymer peaks: µDAWN=90 µL vs HELEOS=114 µL).
• Protein Characterization: Online MALS and DLS confirmed that four monoclonal antibodies are monomeric, identified IgG fragments by molecular weight and UV extinction coefficient, and used hydrodynamic radii to reveal column interactions.
• Conjugate Analysis: Glycoprotein fractions analyzed simultaneously by µDAWN, UV, and dRI yielded total, protein, and modifier mass distributions in a single run.
• Process Monitoring: UHP-SEC-MALS enabled real-time assessment of preparative HPLC fractions, detecting and quantifying aggregates and fragments in process streams.
• Polymer Analysis via APC: Advanced polymer chromatography of polydisperse polystyrene with µDAWN+UT-rEX provided molecular weight distributions (Mₙ, M_w, PDI, R_z) comparable to GPC-MALS-dRI but with significantly faster separations and no shear-induced degradation.

Benefits and Practical Applications

• Absolute measurement of molecular weight, size (R_g and R_h), conformation, and heterogeneity without calibration assumptions.
• Retention of UHP-SEC’s speed and resolution for high-throughput workflows.
• Versatile application across therapeutic proteins, bioconjugates, synthetic polymers, and process analytical technology (PAT).
• Simultaneous MALS and DLS capabilities to correlate size and conformation online.
• Integration into R&D, quality control, and manufacturing for detailed molecular insights.

Future Trends and Potential Applications

• Adoption of µSEC-MALS as part of automated, real-time PAT in biopharmaceutical and polymer production.
• Advancements in flow cell design and miniaturization to further reduce sample and solvent usage.
• Coupling UHP-SEC-MALS with orthogonal detectors (e.g., fluorescence, mass spectrometry) for multidimensional molecular characterization.

Conclusion

By minimizing dispersion and preserving chromatographic resolution, µDAWN and UT-rEX enable true ultra-high-pressure SEC-MALS analysis, delivering comprehensive, high-speed measurements of molecular weight, size, and conformation in proteins and polymers.
This approach expands the frontiers of molecular characterization in research, quality control, and process monitoring.