Protein Resolution in Elution Chromatography Using Novel Cation-Exchange Polymer-Brush-Immobilized Particles

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A sulfonic acid group as a cation-exchange group was introduced into a polymer brush grafted onto a polyethylene particle with an average diameter of 35 µm. A bed charged with the resulting cation-exchange polymer-brush-immobilized particles, SS bed, had a sulfonic acid group density of 0.36 mmol/mL-bed. A mixture of α-chymotripsinogen A (chy), cytochrome C (cyt), and lysozyme (lys) was loaded to the SS bed. The subsequent linear gradient elution performance of the proteins adsorbed by the SS bed with 20 mM phosphate buffer (pH 6.0) and 1 M NaCl was compared with those by commercially available cation-exchange-bead-packed beds. The resolutions of the SS bed of both the chy/cyt and cyt/lys pairs at a linear velocity of 600 cm/h exceeded 2.0 at a loading amount of 1 mg/mL-bed, which was not achieved using the beds charged with SOURCE® 30S, POROS® 50HS, or Fractogel® EMD SO3- (s). Even at a loading amount of 12 mg/mL-bed, the resolutions of the SS bed for the chy/cyt and cyt/lys pairs at a linear velocity of 300 cm/h were 1.4 and 2.7, respectively.