Multi-Angle Light Scattering goes micro

Significance of the Topic

Multi-Angle Light Scattering (MALS) is a cornerstone technique in analytical chemistry for absolute determination of molar mass, molecular size, and solution behavior of polymers and proteins. Unlike conventional methods that rely on calibration standards or assume ideal sample–column interactions, MALS delivers direct measurements without external references. Its non‐destructive nature preserves samples for further analysis and enables studies of aggregation, branching, and conformational changes.

Objectives and Study Overview

The study aimed to integrate MALS detection into Ultra High Performance Liquid Chromatography (UHPLC)‐based Size Exclusion Chromatography (SEC). Key goals included:

• Minimizing secondary band broadening associated with standard detectors.
• Maintaining absolute molar mass and size measurements at UHPLC flow rates.
• Demonstrating applicability to proteins, antibodies, and antibody–drug conjugates (ADCs).

Methodology and Instrumentation

The experimental setup combined:

• Waters Acquity UPLC system with BEH200 SEC columns (4.6×150 mm or 4.6×300 mm, 1.7 µm).
• Wyatt µDAWN MALS detector with reduced flow cell volume (10 µL) and low‐noise optics.
• Wyatt UT‐rEX differential refractive index (dRI) detector with band broadening <4 µL.
• Waters Ti UV detector for concentration measurement.
• ASTRA 6.1.2 software for data collection, band broadening correction, and molecular weight calculations.

Key Results and Discussion

Implementation of µDAWN and UT‐rEX significantly narrowed detector‐induced peak broadening compared to traditional miniDAWN/T‐rEX. Correction algorithms for band broadening and baseline subtraction enabled accurate molar mass profiles for:

• Bovine serum albumin (BSA), demonstrating retention of 21 kDa fragment resolution on a 150 mm UHPLC column.
• Immunoglobulin G (IgG) fragments separated on a 300 mm column, with distinct peaks at 142 kDa, 93 kDa, 61 kDa, and 27 kDa.
• Antibody–drug conjugates, yielding precise drug‐to‐antibody ratio (DAR) values (e.g., 10.1 and 6.5) by comparing modifier and antibody molar masses.

Benefits and Practical Applications

Integrating MALS with UHPLC‐SEC offers:

• High‐resolution separation of closely related species with minimal sample consumption.
• Absolute molar mass and size distributions in a single run.
• Rapid analysis times and reduced solvent use.
• Quantitative characterization of biopharmaceuticals, ADCs, and complex copolymers.

Future Trends and Possibilities

Anticipated developments include extension to alternative column chemistries (e.g., alkyl–polymer porous columns), further reductions in detector volume, and coupling with advanced fraction collectors or mass spectrometry. Applications may expand into polymer branching analysis, real‐time quality control in biomanufacturing, and screening of nanoparticle assemblies.

Conclusion

The µDAWN and UT‐rEX modules successfully overcome UHPLC detector limitations, preserving the fidelity of absolute MALS measurements while delivering UHPLC‐level resolution. This advancement paves the way for routine, high‐throughput characterization of macromolecules in research and industrial settings.

Reference

Astafieva A., Eberlein G., Nilsson L., Shortt D.W., Wyatt P.J. Multimeric conformation by multiangle laser light scattering and reversed‐phase high‐performance liquid chromatography. American Laboratory, March 1995, p. 30.