Dynamics of Protein Assemblies by Analytical Ultracentrifugation

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

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Abstract

In this project we seek to develop experimental methods for the study of macromolecular and particle size-distributions. This includes the observation of polymorphic dynamic multi-protein complexes that elude classical structural techniques. In previous years we have designed computational algorithms for the analysis of sedimentation velocity data with high hydrodynamic resolution, based on the deconvolution of diffusion and nonideality effects. These were implemented and distributed in the software SEDFIT, which is widely used in research and quality control applications in academic and biopharmaceutical laboratories. For example, SEDFIT analysis has become the de facto standard for the detection of oligomeric impurities of protein pharmaceuticals, as well as the characterization of viral vector preparations for vaccine and gene therapy applications. One of the drawbacks of sedimentation velocity analytical ultracentrifugation is the need for expert knowledge and the time-consuming data analysis. In order to address this limitation, in the reporting period we have developed a new automated programming interface for sedimentation velocity analysis in SEDFIT. This provides an opportunity to standardize and accelerate the data analysis through spawning SEDFIT analysis from a master program. Furthermore, it allows SEDFIT analyses to be incorporated into software that satisfies access control and documentation requirements in the GMP environment, thereby broadening application of sedimentation velocity in the biopharmaceutical production. The automated interface sets the stage for future development of expert systems or AI systems to carry out sedimentation analyses. In the reporting period, we have released a SEDFIT version incorporating this programming interface, along with a MATLAB script utilizing this interface, and a detailed description in a preprint currently under review for publication.