Proteins exist in a highly ordered, folded state. This highly ordered structure of a protein is integral to the efficacy and safety of a protein-based biotherapeutic. Protein unfolding and refolding are an indicator of protein stability. As a protein begins to unfold, the hydrodynamic radii of the protein species change.

Denaturation of proteins may range from slight and reversible conformational changes to a drastic loss of solubility, leading to irreversible aggregation. Monitoring the stability of the different protein conformations is important in protein purification. A variety of denaturants are used during different stages of purification. Denaturation of proteins through the use of concentrated guanidine hydrochloride (Gdn HCl) is one of the primary techniques in evaluating protein structure, folded vs. unfolded or partially folded structures.

A change in the conformation of a protein can be detected by the change in the intrinsic fluorescence of the tryptophan-containing proteins. Here we report the use of a 3 μm particle size, 30 nm pore size, TSKgel® UltraSW Aggregate size exclusion chromatography (SEC) column for monitoring folded (native) and unfolded (denatured) states of a monoclonal antibody using fluorescence
detection (FLD).