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Anal Biochem. 2018 Jun 1;550:72-83. doi: 10.1016/j.ab.2018.04.002. Epub 2018 Apr 11.

Characterization of therapeutic antibodies in the presence of human serum proteins by AU-FDS analytical ultracentrifugation.

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Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS, 39216, USA.
Biotherapeutics Discovery Research, Boehringer Ingelheim, Ridgefield, CT, 06877, USA.
Department of Systems Biology, Harvard Medical School, Boston, MA, 02115, USA.
Interactive Technology, Oakland, CA, 94602, USA.
Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS, 39216, USA. Electronic address:


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 γ-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 (ks). This generates a 3x3 matrix of ks values that describe the nonideality of each pairwise interaction. The ks 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 ks nonideality matrix. The ks values for antibodies are smaller than expected and appear to be masked by weak association. Global fitting to a ks and K2 model significantly improves the fits.


2BM(1) – thermodynamic nonideality; AU-FDS – Aviv fluorescence detection system; AUC – analytical ultracentrifugation; SV – sedimentation velocity; k(s) - hydrodynamic nonideality

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