AUC measurements of diffusion coefficients of monoclonal antibodies in the presence of human serum proteins
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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. The high speed method can also be used to measure D and s for small molecules like fluorescein (often contaminants of F-HSA and F-mAb). The advantage of synthetic boundary over the standard sedimentation velocity method is that it allows for higher precision determination of diffusion coefficients. The concentration dependence of D can be corrected for hydrodynamic nonideality effects by plotting D * (1 + kijcj) vs total carrier concentration. The slope of the fitted data allows an alternate approach to determine self- and cross-term thermodynamic nonideality. This method can also explore cross-term diffusion coefficient effects. These results are compared to dynamic light scattering approaches which are limited to kD determinations for solutions of pure protein.
KeywordsSecond virial coefficient Diffusion Hydrodynamic nonideality Sedimentation velocity Synthetic boundary Thermodynamic nonideality
Supported by Boehringer-Ingelheim and UMC AUC Facility. This work was presented at the 23rd International AUC Workshop and Symposium, Glasgow, Scotland. We thank Dave Bain, Tom Laue and Sharon Lobert for constructive comments.
RTW and JJC designed and performed the experiments, analyzed and interpreted the data, and wrote the manuscript. WFS and PJS wrote the SEDANAL software modifications. DH provided materials and discussion. All authors did see and agreed to the final version of the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
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