Characterization of antibody aggregation: role of buried, unpaired cysteines in particle formation

Journal of Pharmaceutical Sciences
Stephen R BrychMasazumi Matsumura

Abstract

Proteins are susceptible to degradation upon exposure to a variety of stresses during product manufacturing, transportation and storage. In this study, we investigated the aggregation properties of a monoclonal antibody during agitation stress. Agitation exclusively led to insoluble aggregates, or particle formation. Removal or modification of the air-liquid interface with a surfactant (e.g., polysorbate) abrogated particle formation. The supernatant postagitation was analyzed using SE-HPLC, FTIR, and AUC analyses and revealed no changes in conformation and aggregation profile when compared to the nonagitated antibody sample. The antibody particles were comprised of a combination of nonnative intermolecular disulfide-linked covalent as well as noncovalent interactions. Analysis of the antibody's unpaired cysteines revealed that the nonnative intermolecular disulfide bonds were formed through buried cysteines, which suggested at least partial unfolding of the antibody domains. FTIR analysis indicated that the particulated antibody maintained significant native-like secondary structure suggesting that particle formation led to minimal structure changes, but capable of exposing free cysteines to solvent to form the nonnative inter...Continue Reading

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