Mechanisms of Aggregate Formation and Carbohydrate Excipient Stabiliza-tion of Lyophilized Humanized Monoclonal Antibody Formulations - Monoclonal Antibodies

Mechanisms of Aggregate Formation and Carbohydrate Excipient Stabiliza-tion of Lyophilized Humanized Monoclonal Antibody Formulations
April 2003
James D. Andya1, Chung C. Hsu1, and Steven J. Shire1 1Department of Pharmaceutical Research and Development, Genentech, Inc., South San Francisco, CA 94080
AAPS PharmSci 2003; 5 (2) Article 10
Submitted: October 8, 2002; Accepted: February 5, 2003; Published: April 4, 2003
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Abstract
The purpose of this study was to evaluate the mecha-nisms of aggregate formation and excipient stabiliza-tion in freeze-dried formulations of a recombinant hu-manized monoclonal antibody. Protein degradation was measured using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and native size exclu-sion chromatography, and protein structure was studied using Fourier transform-infrared spectrometry and cir-cular dichroism. The results showed that protein aggre-gates 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 formula-tion 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 pro-tein. 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. Carbohy-drate in excess of this concentration has less of an in-fluence on protein aggregation. Reduced aggregation during storage was obtained by the addition of suffi-cient excipient to both stabilize solid-state protein structure and provide an environment that consisted of an amorphous glassy state matrix.