Mechanisms of aggregate formation and carbohydrate excipient stabilization of lyophilized humanized monoclonal antibody formulations.

The purpose of this study was to evaluate the mechanisms of aggregate formation and excipient stabilization in freeze-dried formulations of a recombinant humanized monoclonal antibody. Protein degradation was measured using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and native size exclusion chromatography, and protein structure was studied using Fourier transform-infrared spectrometry and circular dichroism. The results showed that protein aggregates present following reconstitution were composed of native antibody structure and a reduced amount of free thiol when compared to protein monomer, which implied that intermolecular disulfides were involved in the aggregation mechanism. An excipient-free formulation resulted in reversible solid-state protein structural alteration and increased aggregation during storage. This correlated with dehydration to an extent that the amount of water was less than the estimated number of surface-accessible hydrogen-bonding sites on the protein. Improved native-like solid-state protein structure and reduced aggregation were obtained by formulation with enough carbohydrate to fulfill the hydrogen-bonding sites on the surface of the protein. Carbohydrate in excess of this concentration has less of an influence on protein aggregation. Reduced aggregation during storage was obtained by the addition of sufficient excipient to both stabilize solid-state protein structure and provide an environment that consisted of an amorphous glassy state matrix.