Abstract
The retention behavior of polyethylene glycol (PEG) on different types of hydrophobic interaction chromatography (HIC) resins containing butyl, octyl, and phenyl ligands was analyzed. An incomplete elution or splitting of the polymer peak into two parts was observed, where the first one was eluted at the dead time of the column, whereas the second one was strongly retained. The phenomenon was attributed to conformation changes of the polymer upon its adsorption on hydrophobic surface. The effect enhanced with increasing molecular weight of the polymer and hydrophobicity of the HIC media. Addition of PEG to the mobile phase reduced binding of proteins to HIC resins, which was demonstrated with two model systems: lysozyme (LYZ) and immunoglobulin G (IgG), and their mixtures. In case of LYZ, the presence of PEG caused reduction in the protein retention, whereas for IgG—a decrease in efficiency of the protein capture. The effect depended on the adsorption pattern of PEG; it was pronounced in the systems in which conformational changes of the polymer were suggested to occur.
Highlights
Production of proteins on an industrial scale is realized in fermentation processes, which output the target product contaminated with specific impurities present in the culture medium and coming from biological material
As mentioned above (“Introduction”), in this work we aimed at investigation of the influence of polyethylene glycol (PEG) on the hydrophobic interaction chromatography (HIC) process subsequent to aqueous twophase extraction (ATPE)
The experiments performed indicated that the influence of PEG on the protein retention can be explained by competitive adsorption between the protein and the polymer
Summary
Production of proteins on an industrial scale is realized in fermentation processes, which output the target product contaminated with specific impurities present in the culture medium and coming from biological material. Fermentation has to be followed by several downstream operations, where the protein can be isolated out of undesired side components to obtain the final product with high purity and preserved biological activity [1,2,3]. To satisfy these requirements, a series of different downstream operations is employed, in which precipitation or aqueous twophase extraction (ATPE) are often used in early purification stages, and followed by chromatographic separations. PEG residues can be present in the solvent environment of the protein processed
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