Viscotek SEC-MALS 20 to measure protein aggregation

Introduction

Protein aggregation is recognized as a major issue in the biopharmaceutical industry.  Proteins have a tendency to aggregate over time and the risk for biologic drugs is that the presence of aggregates will stimulate an immune response.  Size-exclusion chromatography (SEC) is a powerful tool that is commonly used to look at the aggregation of proteins.

SEC separates proteins by size.  It is commonly used to measure their molecular weight and characterize aggregation. While most SEC systems use a single concentration detector such as ultraviolet (UV), the addition of light scattering allows the measurement of the molecular weight of the protein independent of its retention volume.  The molecular weight of the protein monomer, oligomers and aggregates in a sample can then be measured to understand its behavior.  Multi angle light scattering (MALS) can also be used to measure the radius of gyration (Rg) of molecules that scatter light anisotropically (unevenly with respect to angle).

The Viscotek SEC-MALS 20 system (figure 1) is a 20 angle light scattering device capable of measuring the molecular weight of protein and protein aggregates independent of elution volume.  The addition of further detectors such as concentration detectors (RI, UV), intrinsic viscosity (IV) and dynamic light scattering (DLS) can greatly increase the amount of information available from a single SEC measurement.  In this application note, a selection of proteins are separated using SEC.  The molecular weights of their oligomers and aggregates are measured by SEC-MALS and the results discussed.