Demeler, Borries

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    Development of analytical ultracentrifugation standards
    (2023) Martin, Reece; Demeler, Borries
    This project entails the hydrodynamic study of biological macromolecules in the solution phase, specifically on double-stranded DNA molecules as a biological system that can be purified and prepared in colloidal, aqueous solutions suitable for study by analytical ultracentrifugation (AUC). The goal is to produce, purify, and analyze by AUC, preparations of double-stranded DNA molecules of defined length, intended for use as AUC standards for downstream applications in the biopharma industry to validate AUC results. The following sections will discuss the processes by which double-stranded DNA samples are produced, purified, and prepared for use in the AUC, and how these processes have been optimized. As such, once samples have been prepared, they are placed in an analytical ultracentrifuge under varying conditions, such as different rotor speeds, different temperatures, and in buffers with different ionic strengths. The results are then analyzed using the UltraScan software, which uses remote supercomputers to derive hydrodynamic parameters such as the sedimentation coefficient, the diffusion coefficient, and the partial concentrations of each solute in the mixture. From the hydrodynamic parameters, the partial specific volume and the anisotropy of the components in the mixtures can be derived. The data acquisition, processing, and refinement techniques used to derive the hydrodynamic parameters and extract meaningful results from them will be detailed as well. At the current stage of this project, the goal is to achieve batch-to-batch reproducibility in the generation and purification of double-stranded DNA fragments. Finally, the long-term expected outcome of this project is the development of Good Manufacturing Practice molecular standards for analytical ultracentrifugation.