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

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 p...

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Bibliographic Details
Published in:	Journal of pharmaceutical sciences Vol. 99; no. 2; p. 764
Main Authors:	Brych, Stephen R, Gokarn, Yatin R, Hultgen, Heather, Stevenson, Riki J, Rajan, Rahul, Matsumura, Masazumi
Format:	Journal Article
Language:	English
Published:	United States 01-02-2010
Subjects:	Antibodies - chemistry Chromatography, Gel Chromatography, High Pressure Liquid Cysteine - chemistry Disulfides - chemistry Electrophoresis, Polyacrylamide Gel Fluorescence Humans Hydrogen-Ion Concentration Immunoglobulin G - chemistry Microscopy, Electron, Scanning Nanoparticles Protein Conformation Protein Denaturation Spectrophotometry, Ultraviolet Spectroscopy, Fourier Transform Infrared Sulfhydryl Compounds - chemistry Surface-Active Agents - chemistry Temperature Ultracentrifugation
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Summary:	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 intermolecular disulfide bonds. The results presented in this study indicate the importance of the interactions between the antibody and the air-liquid interface during agitation in the formation of particles and suggests that reduced disulfide bonds may play a significant role in the particulation reaction. This phenomenon can be applicable to other proteins with similar free cysteine and structural characteristics.
ISSN:	1520-6017
DOI:	10.1002/jps.21868