Issue link: https://resources.nanotempertech.com/i/1267034
11 Going to extremes: mAb unfolding Antibodies are composed of 6 to 70 immunoglobulin (Ig) fold domains, o en connected by inter-domain disulfide bonds. Separated single immunoglobulin fold domains can be refolded through complex methods consisting of solubilization with detergents, removal of solubilizing reagents and, for disulfide-containing proteins such as antibody fragments, oxidation of sulfhydryl groups. Low concentrations of aggregation suppressors or folding-assisting agents are included in the refolding solvent to enhance refolding yield 11 . However, when pairs of domains become denatured, this is irreversible 12 . Having an understanding of the unfolding, solubilization, and aggregation properties of target candidates leads to better development of mAb biotherapeutics. Each Ig domain starts unfolding at different temperatures. For example, the constant heavy chain 3 (CH3) domain of IgGs— such as monoclonal antibodies used as therapeutics—unfolds at a higher temperature than its CH2 domain. At certain temperatures, antibody solutions have a mixture of folded and unfolded structures. Co-existence of folded and unfolded domains in a single polypeptide chain may increase the tendency to aggregate, which inactivates the antibody 13 . IgG denaturation becomes irreversible at temperatures higher than 65 °C, and IgG almost completely loses its antigen-binding activity a er heat treatment at 90 °C for several minutes 13 . Forced degradation studies Forced degradation demonstrates the specificity of stability-indicating methods and also provides insight into degradation pathways and degradation products of the drug substance. The assays also help elucidate the structures of the degradation products and show the chemical behavior of the molecule. This, in turn, helps in developing formulation and packaging 12 . Forced degradation studies are performed on the drug substance (DS)—the active ingredient that is intended to furnish pharmacological activity or other direct effects in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the human body, but does not include intermediates used in the synthesis of such ingredient [21 CFR 314.3]. They are also performed on the drug product (DP)—a finished dosage form, for example, tablet,