期刊
ANALYTICAL BIOCHEMISTRY
卷 550, 期 -, 页码 72-83出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2018.04.002
关键词
AUC - analytical ultracentrifugation; AU-FDS - Aviv fluorescence detection system; SV - sedimentation velocity; k(s) - hydrodynamic nonideality; 2BM(1) - thermodynamic nonideality
资金
- Boehringer-Ingelheim
- UMC AUC Facility
The preclinical characterization of biopharmaceuticals seeks to determine the stability, state of aggregation, and interaction of the antibody/drug with other macromolecules in serum. Analytical ultracentrifugation is the best experimental method to understand these factors. Sedimentation velocity experiments using the AU-FDS system were performed in order to quantitatively characterize the nonideality of fluorescently labeled therapeutic antibodies in high concentrations of human serum proteins. The two most ubiquitous serum proteins are human serum albumin, HSA, and gamma-globulins, predominantly IgG. Tracer experiments were done pairwise as a function of HSA, IgG, and therapeutic antibody concentration. The sedimentation coefficient for each fluorescently labeled component as a function of the concentration of the unlabeled component yields the hydrodynamic nonideality (k(s))This generates a 3x3 matrix of k(s) values that describe the nonideality of each pairwise interaction. The k(s), matrix is validated by fitting both 2:1 mixtures of HSA (1-40 mg/ml) and IgG (0.5-20 mg/ml) as serum mimics, and human serum dilutions (10-100%). The data are well described by SEDANAL global fitting with the k(s), nonideality matrix. The k(s) values for antibodies are smaller than expected and appear to'be masked by weak association. Global fitting to a k(s) and K-2 model significantly improves the fits.
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