Size exclusion chromatography (SEC) is a technique for separating proteins, oligonucleotides, and other complex biopolymers by size using aqueous eluents. In particular, it is an essential tool for quantification of aggregates present in protein biotherapeutics. Manufacture of a biopharmaceutical such as a monoclonal antibody is a complex process and aggregation of the protein is an issue that can arise during cell culture, isolation, purification, and formulation. The presence of dimers and higher aggregates can affect both efficacy and safety of the final product; quantification of aggregate content must be carried out during process development to establish the product’s critical quality attributes (CQA) as well as during final product characterization to ensure the extent of aggregation is minimized and controlled at safe levels.
The size, type, and content of aggregates present in protein biopharmaceuticals can affect both efficacy and formulation – or worse, induce an immunogenic response. Aggregate formation occurs through a variety of mechanisms, including disulfide bond formation and noncovalent interactions.
Because the size of protein aggregates, including dimers, is sufficiently different from the protein monomer, you can separate the various forms using SEC. In fact, SEC with UV or light scattering is a standard technique for quantifying protein aggregation.