Poly(4-vinylpyridine): a polymeric ligand for mixed-mode protein chromatography

• Poly(4-vinylpyridine) was coupled to Sepharose gel for use as a protein ligand.
• Both electrostatic and hydrophobic interactions worked on protein adsorption.
• Electrostatic interaction played the dominant role in protein adsorption.
• Bound protein could be recovered by elution with a buffer of pH 4.0 or 4.5.

Poly(4-vinylpyridine) (P4VP) was proposed for use as a polymeric ligand of mixed-mode chromatography (MMC) of proteins. P4VP has linear hydrophobic chains with ionizable pyridyl groups in its backbone. The polymer was coupled onto Sepharose FF gel at a pyridyl group density of 190 μmol/mL (FF-P4VP-190) by the substitution reaction of pyridyl amines with brominated Sepharose gel. Thereby the immobilized ligand possesses the intrinsic hydrophobic nature as well as the newly obtained electrostatic interaction properties endowed from the substituted positively charged pyridyl amines. The pore size distribution was measured by inverse size exclusion chromatography, and the results revealed that P4VP formed a three-dimensional layer on the matrix surface with a maximum layer depth of 4.2 nm. The adsorption isotherms of γ-globulin and bovine serum albumin (BSA) to FF-P4VP-190 were determined under varying pH values and salt concentrations to provide insights into the adsorption properties of the medium. It was found that the adsorption capacity of γ-globulin and BSA both presented an increase with pH increasing from 8.0 to 9.0. Moreover, FF-P4VP-190 exhibited stronger adsorption for BSA than γ-globulin. The higher affinity for BSA might be attributed to its more net negative charges. Protein adsorption capacities to FF-P4VP-190 decreased with increasing NaCl concentration, but still manifested moderate levels at high salt concentration such as 75 mg/mL for γ-globulin and 14 mg/mL for BSA at 0.5 mol/L NaCl and pH 9.0. The capacity decreases with increasing ionic strength, indicating the dominant role of electrostatic interactions, while the moderate capacity values at 0.5 mol/L NaCl confirmed the presence of salt-tolerant feature of FF-P4VP-190, making it function as an MMC material. Column chromatography was conducted to investigate protein elution behavior. Efficient protein recovery was achieved at mild elution conditions such as pH 4.0 for γ-globulin and pH 4.5 for BSA. The results indicate that the P4VP-based adsorbent would provide new possibilities for protein purification by MMC.