Calorimetric and spectroscopic evaluation of humanized monoclonal antibodies in amino acid formulations.

摘要

Protein stability is a major concern for protein pharmaceuticals and this problem remains unsolved for protein chemists and the pharmaceutical industry. Freeze-drying is a widely accepted approach for the manufacture of protein pharmaceutical and diagnostic agents that are physically or chemically unstable in aqueous solution. Minimization of damage to proteins due to stresses arising from the freeze-drying process, however, remains a major challenge in the development and manufacture of such products. Addition of protective excipients in protein formulations significantly improves protein stability during the freeze-drying process and subsequent storage. The studies outlined in this dissertation investigate protein/amino acid interactions in the solid state and evaluate the stabilization effects of amino acid formulations on the structural changes of antibodies in both solid and solution states. Two monoclonal humanized antibodies were freeze-dried with L-histidine, L-arginine, glycine and L-aspartic acid at pH 6.0, respectively. Protein concentrations ranging from 0% to 100% (w/w) were employed. Isoperibol calorimetry, differential scanning calorimetry and solid-state NMR were utilized to explore interactions between proteins and amino acids. Deviations from linearity in heats of solution and decreased and/or loss of melting temperatures of amino acid indicate the presence of protein/amino acid interactions. Moreover, solid-state NMR showed that the weak interactions occurred on the side chain of arginine in the lyophilized protein formulation. Diffuse reflectance FTIR was used to monitor changes in the secondary structure of proteins in the solid state. The addition of amino acids inhibited protein secondary structure alteration in a concentration-dependent manner. The efficacy of amino acids in preventing these secondary structural changes was demonstrated as L-arginine ≥ L-histidine > L-aspartic acid > glycine. Circular dichroism and UV-Vis were performed to detect the secondary and tertiary structural stability of proteins upon heat-induced perturbation. The effects of amino acids on protein aggregation were also illustrated. Overall results suggest that direct interactions occur between proteins and histidine (or arginine) upon freeze-drying and these interactions may preserve protein native conformation in the dried state. In addition, histidine and arginine suppressed thermally induced protein aggregation in solution.