Chromatographic separation of hemoglobin variants using robust molecularly imprinted polymers

Devising a robust, efficient and cost effective hemoglobin (Hb) purification strategy is one of the key challenges in the development of Hb-based blood substitutes. The aim of this study was to use molecularly imprinted polymers (MIPs) as a novel and efficient chromatographic resin to selectively recognize and purify different Hb variants. The results showed that the Hb-MIP material developed here could selectively recognize and purify various Hb directly from either crude E. coli extracts or human body fluids, such as blood plasma and cerebrospinal fluid (CSF), in one-step. The dynamic binding capacity at 10% breakthrough was around 7.4 mg mL(-1) resin for adult lib (HbA) and fetal lib (HbF). This chromatographic material also allowed identification of changes related to amino acid substitutions on the Hb protein surface. For instance, when an additional lysine residue was introduced, the HbA alpha Y42K mutant eluted later in an Hb-MIP column than wildtype HbA. Additional negative charges on the protein surface, such as aspartate, mitigated the interaction between the protein and imprinted polymers, and therefore an alpha A19D-alpha A12D HbF mutant eluted earlier, at -2.7 column volumes compared to wildtype HbF.