AUC measurements of diffusion coefficients of monoclonal antibodies in the presence of human serum proteins

European Biophysics Journal : EBJ
Robert T WrightJohn J Correia

Abstract

The goal of this work is to develop a preclinical method for quantitative hydrodynamic and thermodynamic analysis of therapeutic proteins in crowded environments like human serum. The method utilizes tracer amounts of fluorescently labeled monoclonal antibodies and the Aviv AU-FDS optical system. We have performed sedimentation velocity experiments as a function of mAb, human serum albumin and human IgG concentration to extract self- and cross-term hydrodynamic nonideality effects. SV measurements are consistently complicated by weak mAb-mAb and mAb-IgG interactions (Wright et al. in Anal Biochem 550:72-83, 2018). In an attempt to explore different approaches we have investigated measurements of diffusion coefficients by traditional synthetic boundary experiments. Here we present a new technique incorporated into SEDANAL that can globally analyze the full time course of synthetic boundary experiments. This approach also utilizes F-mAb against a high concentration of unlabeled carrier protein (HSA or IgG). In principle both diffusion and sedimentation coefficient information can be extracted including hydrodynamic and thermodynamic nonideality. The method can be performed at a traditional low speed (5-7K rpm) or at high speeds. ...Continue Reading

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Citations

Sep 16, 2018·European Biophysics Journal : EBJ·Olwyn ByronTrushar R Patel
Nov 8, 2020·European Biophysics Journal : EBJ·J J CorreiaW F Stafford
Nov 20, 2019·The Journal of Physical Chemistry. B·Glenn M FerreiraChristopher J Roberts
Nov 23, 2021·Analytical Chemistry·Olenka Jibaja Valderrama, Ivo Nischang

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Methods Mentioned

BETA
dynamic light scattering
Fluorescence
FCS

Software Mentioned

mathop
FDS
Sednterp
WDA
SVEDBERG
SEDANAL
DCDT
Aviv

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