A batch technique for connecting multimodal ligand chemistry to chromatographic separability
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Authors
Denbaum, Eric
Issue Date
2024-08
Type
Electronic thesis
Thesis
Thesis
Language
en_US
Keywords
Biochemistry and biophysics
Alternative Title
Abstract
This dissertation describes the development and utilization of a high-throughput parallel batch adsorption screen with sequential salt step increases to rapidly generate protein elution profiles for multiple resins at different pHs using a protein library. The chromatographic ligand libraries screened using this technique include commercial resins, Bio-Rad prototype small molecule resins, custom synthesized peptide-based ligands, and Solventum prototype membrane adsorbers. The chromatographic sets used in this work includes single, multimodal anion-exchange (MMA), and multimodal cation-exchange resins (MMC). The protein library consists of proteins with isoelectric points ranging from 3.4-11.4 with varying hydrophobicities as determined by their retention on hydrophobic interaction chromatography. The batch sequential experiments are carried out using one protein at a time with a wide set of resins at multiple pH conditions, thus enabling simple microtiter plate detection. A mathematical formulation is then used to determine the first moment of the distributions from each chromatogram (sequential step elution) generated in the parallel batch experiments. Batch data first moments (expressed in salt concentration) were compared to results obtained from column linear salt gradient elution, and the techniques are shown to be consistent. In addition, first moment data was used to calculate one-resin separability scores, which are a measure of a resin’s ability, at a specified pH, to separate the entire set of proteins in the library from one another. Again, the results from the batch and column experiments were shown to be comparable. The first moment data sets were then employed to calculate the two-resin separability scores, which are a measure of the ability of two resins to synergistically separate the entire set of proteins in the library. Importantly, these results based on the two-resin separability performances derived from the batch and column experiments were again shown to be consistent. This analytical approach was applied to all the screened ligand libraries and connections between ligand chemistry/stereochemistry and chromatographic behavior were identified. This approach for rapidly screening large numbers of chromatographic resins and mobile phase conditions for their elution behavior may prove useful for enabling the rapid discovery of new chromatographic ligands and resins.
Description
August 2024
School of Science
School of Science
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY