Antibodies are large proteins with a molecular weight of ~150 kDa. They are widely researched as potential therapeutic drugs, for the treatment of diseases like cancer and autoimmune disorders. Most commonly, these are IgG class antibodies.

The figure below illustrates the structure of an IgG antibody (left panel) and an exemplary Prometheus unfolding profile of an IgG (right panel). IgG molecules consist of four polypeptide chains: two heavy chains (blue) and two light chains (purple). The ‘arms’ of the Y-shaped molecule contain the variable antigen binding sites and are therefore commonly referred to as the Fab region (fragment, antigen-binding). The ‘foot’ of the Y is responsible for the antibody’s immunological properties and is called Fc region (fragment, crystallizable). The Fc region can be subdivided into the CH2 and CH3 domains (domains 2 and 3 of the constant parts of the heavy chains, respectively). In thermal unfolding experiments, three separate unfolding events are often visible in the unfolding profile, representing the three unfolding events of CH2 region, Fab region, and CH3 region. Typically, the CH2 region unfolds first (at the lowest temperature), followed by Fab and CH3.

The interpretation of an IgG unfolding profile is not always as straightforward as shown here. Depending on the exact molecular structure of the IgG, not all three regions may show as separate unfolding events. In some cases, two unfolding events may overlap or even occur simultaneously, precluding a clear separation. Some of the factors that influence the thermal stability of the separate antibody domains include: amino acid sequence and class of IgG, glycosylation pattern and other post-translational modifications, buffer/formulation conditions and excipients. Also, the height of the unfolding transition/peak does not necessarily allow conclusions about the corresponding domain.