Journal
ANALYTICAL BIOCHEMISTRY
Volume 388, Issue 2, Pages 279-287Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.03.012
Keywords
Xolair; rhuMabE25; Omalilzumab; Biopharmaceutics; Serum; Characterization; IgE; Ex vivo; Labeling; Analytical ultracentrifugation; Complex; Protein; Fluorescence; Sedimentation velocity
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During the development of protein therapeutics, characterization of the active pharmaceutical Ingredient is performed extensively to ensure the stability, safety, and efficacy of the drug Little is known, however, about the characteristics of protein drugs circulating in the blood. The recent availability of a fluorescence detection system (FDS) in analytical Ultracentrifugation (AUC) instruments enables the characterization of fluorescently labeled proteins in biological fluids. AUC provides information about protein size, shape. self-association, and binding while avoiding many limitations associated With size exclusion chromatography. Furthermore, With the specificity and sensitivity of FDS, measurements call be performed at physiological concentrations directly in serum. In the current study, we used omalizumab, an anti-immunoglobulin E (IgE) monoclonal antibody, to demonstrate the potential of using AUC-FDS for the Study of a monoclonal antibody and its complexes directly in human serum. Omalizumab properties were essentially unaltered after labeling with the fluorescent dye Alexa Fluor 488. In addition, omalizumab and IgE formed different complexes in serum than in phosphate-buffered saline in terms of both size and affinity. (c) 2009 Elsevier Inc. All rights reserved.
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