The influence of the degree of cross-linking, type of ligand and support on the chemical stability of chromatography media intended for protein purification

Abstract

The release of organic compounds from different liquid chromatography media in static conditions has been analysed with a total organic carbon (TOC) analyser. TOC results show that chemical stability increases with the degree of cross-linking in agarose beaded chromatography media and thus extend the working pH-range of the media. Of the unsubstituted chromatography media investigated, Sepharose® 6B, Sepharose CL-6B, Sepharose 4 Fast Flow, Sepharose 6 Fast Flow and Sepharose High Performance, the latter was the most stable medium. Sepharose High Performance releases only about 0·06% of its total carbon content after 1 week in 0·01 m HCl. Agarose beads are more stable to basic conditions (pH 14) compared with acidic conditions (pH 2). From UV spectroscopic and gel filtration results it was found that all Sepharose media release low amounts of 5-(hydroxymethyl)-2-furaldehyde and agarose fragments in acidic conditions. To investigate the effect of different ligands on chemical stability Q Sepharose 6 Fast Flow, DEAE Sepharose 6 Fast Flow, SP Sepharose 6 Fast Flow, CM Sepharose 6 Fast Flow, Phenyl Sepharose 6 Fast Flow, Octyl Sepharose 4 Fast Flow media were also studied under static conditions. In basic conditions it was found that all these chromatography media release carbon compounds to a higher extent than the unsubstituted Sepharose support. In addition, Hofmann elimination of Q and DEAE groups contributes to the decrease in the carbon content of the corresponding anion exchangers. During exposure to acidic conditions (pH 2) the release of carbon compounds was lower than the release from the support to which the ligands were coupled. The exceptions are Octyl Sepharose 4 Fast Flow and SP Sepharose 6 Fast Flow. In the case of Octyl Sepharose 4 Fast Flow, the ligand did not seem to influence chemical stability, whereas the SP group increases the degradation of the Sepharose support. In the case of SP Sepharose 6 Fast Flow the stability in acidic conditions can be improved by increasing the ionic strength. Anion exchangers based on different support polymers (agarose-, polystyrene-, methacrylate- and polyvinyl-based matrixes) were studied under static conditions. Agarose-based anion exchanger was the most stable in basic conditions (pH 14). In acidic conditions (pH 2) the chemical stability was about the same for many different anion exchangers.