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Trace adsorption of positively charged proteins onto Sepharose FF and Sepharose FF-based anion exchangers

Author:
Yu, Lin-Ling, Sun, Yan
Source:
Journal of chromatography 2012 v.1253 pp. 105-109
ISSN:
0021-9673
Subject:
adsorption, agarose, bovine serum albumin, cation exchange, cytochrome c, gels, ion exchange chromatography, ionic strength, lysozyme, protein products, sodium chloride
Abstract:
Agarose-based matrices have been widely used in ion exchange chromatography (IEC). We have herein observed that positively charged proteins (lysozyme and cytochrome c) are adsorbed on the agarose-based anion-exchangers (Q and DEAE Sepharose FF gels) in a capacity of 10–40μg/mL. In contrast, negatively charged protein (bovine serum albumin) is not adsorbed to Sepharose FF and SP Sepharose FF gels. Elemental analysis of the gel indicated that the residual anionic sulfate groups in agarose would have worked as the cation exchange groups for the positively charged proteins. The trace adsorption behavior of lysozyme onto Sepharose FF and Sepharose FF-based anion exchangers was studied and the effects of NaCl concentration and cation group density on the adsorption were examined for better understanding of the trace adsorption in chromatographic processes. At NaCl concentrations less than 0.05mol/L, which is the normal adsorption condition in IEC, the trace adsorption kept at a high level, so this trace adsorption cannot be avoided in the ionic strength range of routine IEC operations. Grafting poly(ethylenimine) (PEI) chain of 60kDa to a cation group density of 700mmol/L could reduce the adsorption capacity to about 20μg/mL, but further reduction was not possible by increasing the cation group density to 1200mmol/L. Therefore, attentions need to be paid to the phenomenon in protein purification practice using agarose-based matrices. The research is expected to call attentions to the trace adsorption on agarose-based matrices and to the importance in the selection of the suitable solid matrices in the production of high-purity protein products in large-scale bioprocesses.
Agid:
1129945