Measuring Antibody Molecular Weight by SEC-MALS
In this application note, a purified polyclonal antibody (IgG) is separated using Size Exclusion Chromatography (SEC) and characterized using the Viscotek SEC-MALS 20, allowing calculation of molecular weight and radius of gyration (Rg).
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Figure 1: Molecular structure of an Immunoglobulin
Therapeutic recombinant antibodies, primarily of the Immunoglobulin G isotype (IgG) (figure 1), represent a growing proportion of biopharmaceuticals. There is an ever increasing number of approved antibody therapies available to treat a variety of modern-day diseases such as cancer, rheumatoid arthritis and diabetes. With many more candidate IgG's in the biopharmaceutical development pipeline, the pressure to accurately understand and develop these candidates becomes more and more important.
Protein aggregation has long been established as a major concern in the biopharmaceutical industry. Proteins have a limited shelf-life and precipitate over time, with strong evidence that the presence of protein aggregates in the bloodstream can regularly stimulate the active/adaptive immune response. Size-exclusion chromatography (SEC) is an established and powerful tool that is commonly implemented to assess the aggregation content of protein formulations.
SEC separates proteins by size, and is frequently employed to measure molecular weight and identify aggregate sample proportions. While most SEC systems use a single detector (such as an ultraviolet absorbance detector), the addition of a light scattering detector allows direct measurement of protein molecular weights and is used to differentiate between monomer, dimer and aggregate independent of retention volume. Light scattering is substantially more sensitive to aggregate formation than absorbance measurements and allows detection of aggregates that may otherwise be missed. If the light scattering detector employed is a multi angle light scattering (MALS), then it can also be used to measure the radius of gyration (Rg) of the large aggregates that scatter light anisotropically (unevenly with respect to angle), which offers further structural/shape information.
The Viscotek SEC-MALS 20 system (figure 2) is a 20 angle light scattering device, capable of making measurements of protein and protein aggregate molecular weight, independent of elution volume.