Preparation and characterization of pH-responsive polyacrylamide molecularly imprinted polymer: Application to isolation of recombinant and wild type human serum albumin from biological sources.

Abstract

In this work pH-responsive neutral and cationic polyacrylamide molecularly imprinted polymers (nMIP and cMIP, respectively) were prepared for separation of recombinant and wild type human serum albumin (HSA, pI 4.7) using mixture of polymerization initiators. The effect of pH during preparation and adsorption stages at pI(HSA)±2.0 on binding capacity and selectivity; imprinting factor (IF) was thoroughly investigated. SE-HPLC and RP-HPLC were employed for thorough evaluation of the stability of HSA at the studied experimental conditions and for simultaneous determination of HSA and erythropoietin (EPO) in their mixtures, respectively. Results showed that nMIP were generally superior to cMIP, where nMIP prepared at pH 2.7 and tested at pH 6.7 showed superior binding characteristics (IF 42.91). The pH at the preparation stage imposed minimal effect on the stability of HSA owing to entrapment of HSA within the polymer network. Adsorption experiments carried out at pH 2.7, regardless of polymer type and pH of preparation revealed poor selectivity. Adsorption of HSA onto MIP followed Sips model with pseudo second-order kinetics. Scanning electron microscopy (SEM) revealed a rough surface for MIP and a smooth one with wider pore diameter for non-imprinted polymer (NIP). Successful separation of recombinant HSA from its binary mixture with EPO and wild type HSA from crude plasma was demonstrated using RP-HPLC. This suggested that MIP should be applicable for downstream purification of therapeutic grade HSA at scale either from plasma or recombinant sources and isolation of HSA from plasma for diagnostic purposes.