Title

Investigation of the Effect of Polyols on the Conformational and Solution Phase Stability of Aqueous Antibody Formulations

Date of Completion

January 2011

Keywords

Chemistry, Biochemistry|Chemistry, Physical|Health Sciences, Pharmacy

Degree

Ph.D.

Abstract

Polyols are widely used as excipients in protein formulations as they have been shown to increase the thermal/conformational stability of proteins. The mechanism of conformational stabilization of proteins by polyols has been primarily explained on the basis of the theory of preferential exclusion. However, besides affecting the conformational stability of proteins, polyols can also affect their solution phase stability as preferential exclusion leads to an increase in the chemical potential of a protein in the solution phase. ^ The tertiary structure of a monoclonal antibody-U (mAb-U) was analyzed using second derivative fluorescence spectroscopy under thermal and chemical stresses. This study established that changes in the intensity of the three spectral forms of tryptophan are indicative of subtle alterations in the tertiary structure of proteins. To investigate the origin of preferential exclusion of polyols, transfer free energies of the hydrophobic amino acid side chains from water to polyol solutions were determined. The results indicated that polyols can have favorable hydrophobic interactions with proteins as a parallel relationship was observed between increasing hydrophobicity of polyols and increasing solubility of the amino acid derivatives in polyol solutions. ^ Additionally, the effect of polyols on the physical stability of mAb-U was studied under thermal and mechanical stresses. It was observed that thermal stabilizers, which showed lower aggregation upon storage at high temperatures, increased aggregation of mAb-U under mechanical stress. To further investigate if the destabilizing effect of polyols can be generalized, mAb-P, relatively less hydrophobic than mAb-U, was studied. The results suggested that the effect of polyols on the physical stability of a protein is strongly dependent on its inherent nature (hydrophobic versus hydrophilic) and solution pH. Protein-polyol interactions were also characterized using static light scattering. Results from this study indicated that polyols, stabilizing or destabilizing, decrease attractive interactions between protein molecules by masking the hydrophobic sites. ^ The results demonstrated that polyols are not universal stabilizers as there are multiple routes to protein aggregation. An increase in the conformational stability of a protein by a polyol does not necessarily translate into an increase in its overall physical stability. ^

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