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Ion exchange chromatography (IEX) is a common chemical separation technique that is used to separate ionizable target molecules from a liquid mixture mobile phase by adsorption of the molecule to a stationary phase. In chromatography, the stationary phase is a porous solid that the target molecule and mobile phase flows through, and is commonly referred to as the resin. Resins are commonly packed into hollow cylindrical columns. The mobile phase is the liquid solvent which flows through the column containing the stationary phase. The target molecule will have an affinity for the resin and adsorb to it, while impurities in the mixture may not have any affinity to the resin and pass through the column to be discarded. IEX is classified into four broad categories, which are based off the chemistry of the resin. The categories are strong cation exchange, strong anion exchange, weak cation exchange, and weak anion exchange. The ability of IEX to separate target analytes depends on the chemical composition of the mobile phases and stationary phase, as well as the properties of the analyte. IEX has many practical purposes, including purification of biomolecules from mixtures, water softening, and other applications.
This study was completed at Vestaron Corporation, an industrial biotechnology company that specializes in the development of insecticidal peptides for use in the agricultural industry. One proprietary peptide, codenamed VST-6700, is a potential active ingredient in Vestaron’s next insecticidal product. The resin that Vestaron currently uses to separate peptides, SP Sephadex C-25, is no longer in large-scale production, and therefore must be replaced with a more modern commercially available resin. The purpose of this study was to evaluate, optimize, and compare four commercial cation exchange resins of different chemical and physical properties for the small-scale purification of VST-6700 from cell-free fermentation beer aliquots. The four resins optimized were Capto S and SP-Sephadex C-25 from GE Healthcare, and Macro-Prep CM and Nuvia HR-S from Bio-Rad. An optimized IEX process for a chosen resin balanced minimal process time, maximum VST-6700 retention on the resin, and maximum VST-6700 recovery from the column.
The IEX process for each of the resins were optimized in a trial-by-trial basis. In each trial, buffer mobile phase composition and pH, NaCl concentration in the buffers, peptide loading mass, and mobile phase flow rates were all considered as variables. The most optimal commercial resin for VST-6700 purification was determined to be Capto S, from GE Healthcare. While Nuvia HR-S and SP Sephadex C-25 resins could load more VST-6700 before saturation, 100% of VST-6700 could be consistently recovered from Capto S. Furthermore, the IEX process time with Capto S was found to be smaller than both Nuvia HR-S and Macro-Prep CM. This study shows a benchtop-scale IEX optimization of four cation exchange resins for VST-6700 IEX purification at Vestaron, the results of which be used for further optimization of IEX purification of VST-6700 and similar peptides.
Willoughby, Jamin, "Optimization of Cation Exchange Chromatography Purification of a Small Peptide: An Industrial and Practical Approach" (2020). Honors Theses. 3438.
Honors Thesis-Open Access