Thermal and Chemical Unfolding of a recombinant monoclonal IgG1 antibody: Application of the Multi-State Zimm-Bragg Theory

Abstract

The thermal unfolding of a recombinant monoclonal antibody IgG1 (mAb) was measured with differential scanning calorimetry (DSC). The DSC thermograms reveal a pre-transition at 72{degrees}C with an unfolding enthalpy of {Delta}Hcal [~] 200-300 kcal/mol and a main transition at 85 {degrees}C with an enthalpy of [~]900 - 1000 kcal/mol. In contrast to single-domain molecules, mAb unfolding is a complex reaction that is analysed with the multi-state Zimm-Bragg theory. For the investigated mAb, unfolding is characterised by a cooperativity parameter {sigma} [~]10-4 and a Gibbs free energy of unfolding of gnu [~]100 cal/mol per amino acid. The enthalpy of unfolding provides the number of amino acid residues v participating in the unfolding reaction. On average, v[~]220{+/-}50 amino acids are involved in the pre-transition and v[~]850{+/-}30 in the main transition, accounting for [~]90% of all amino acids. Thermal unfolding was further studied in the presence of guanidineHCl. The chemical denaturant reduces the unfolding enthalpy {Delta}Hcal and lowers the midpoint temperature T0. Both parameters depend linearly on the concentration of denaturant. The guanidineHCl concentrations needed to unfold mAb at 25 {degrees}C are predicted to be 2-3 M for the pre-transition and 5-7 M for the main transition, varying with pH. GuanidineHCl binds to mAb with an exothermic binding enthalpy, which partially compensates the endothermic mAb unfolding enthalpy. The number of guanidineHCL molecules bound upon unfolding is deduced from the DSC thermograms. The bound guanidineHCl-to-unfolded amino acid ratio is 0.79 for the pre-transition and 0.55 for the main transition. The pre-transition binds more denaturant molecules and is more easily destabilised than the main transition.\n\nOverall, the current study shows the strength of the Zimm-Bragg model for the quantitative description of unfolding events of large, therapeutic proteins, such as a monoclonal antibody.\n\nStatement of significanceFirst quantitative thermodynamic study of an antibody with differential scanning calorimetry and analyzed with the multi-state Zimm-Bragg theory.