Rapid Separation of Human Hemoglobin on a Large Scale From Non-clarified Bacterial Cell Homogenates Using Molecularly Imprinted Composite Cryogels

Abstract

The production of a macroporous hydrogel column, known as cryogel, has been scaled up (up to 150 mL) in this work for the purification of human hemoglobin from non-clarified bacterial homogenates. Composite cryogels were synthesized in the presence of adult hemoglobin (HbA) to form a molecularly imprinted polymer (MIP)network where the affinity sites for the targeted molecule were placed directly on an acrylamide cryogel by protein imprinting during the cryogelation. The MIP composite cryogel column was first evaluated in a well-defined protein mixture. It showed high selectivity toward HbA in spite of the presence of serum albumin. Also, when examined in complex non-clarified E. coli cell homogenates, the column showed excellent chromatographic behavior. The binding capacity of a 50 mL column was thus found to be 0.88 and 1.2 mg/g, from a protein mixture and non-clarified cell homogenate suspension, respectively. The recovery and purification of the 50 mL column for separation of HbA from cell suspension were evaluated to be 79 and 58%, respectively. The MIP affinity cryogel also displayed binding and selectivity toward fetal Hb (HbF) under the same operational conditions.

Molecularly Imprinted Polymers with Shape-Memorable Imprint Cavities for Efficient Separation of Hemoglobin from Blood

“…Like the MIPs of other proteins, previously in the preparation of MIPs of hemoglobin, usually the protein template was eluted under harsh conditions. Particularly, acetic acid/SDS-mixed solution was widely used as the eluent to remove hemoglobin. ,,,,,, In contrast, here, the template protein was removed under relatively mild conditions, that is, eluting using 20 mM pH 5 phosphate buffer with 154 mM NaCl. As shown in Table , for BHb-PC-MIP, 89.3% of BHb was extracted from the MIP in the first washing cycle.…”

“…In both cases, the protein should be separated and purified efficiently from complex biological fluids such as blood. 2,3 Currently, various methods have been developed for the purification of hemoglobin from biological samples, including liquid−solid extraction, 4 tangential flow filtration, 5 zinc precipitation, 6 anion exchange chromatography, 7 and immobilized metal affinity chromatography. 8,9 However, these methods either suffer from low selectivity or low throughput.…”

“…Among them, hemoglobin-based oxygen carriers (HBOCs) have demonstrated potential for therapeutic oxygenation. , The main component of HBOCs, hemoglobin, can be derived from human or bovine blood or produced by recombination technology. In both cases, the protein should be separated and purified efficiently from complex biological fluids such as blood. , Currently, various methods have been developed for the purification of hemoglobin from biological samples, including liquid–solid extraction, tangential flow filtration, zinc precipitation, anion exchange chromatography, and immobilized metal affinity chromatography. , However, these methods either suffer from low selectivity or low throughput. The massive, efficient separation and purification of hemoglobin still remains a big challenge.…”

“…Hemoglobin-imprinted polymers have also been developed for hemoglobin separation; ,− Molecularly imprinted polymers (MIPs) are artificial receptors synthesized by polymerization in the presence of template molecules. Removal of the template leaves imprint cavities complementary to the template in size, shape, and functional group orientation.…”

Interaction of haemin with albumin-based macroporous cryogel: Adsorption isotherm and fluorescence quenching studies

“…Since the introduction of cryogels in the 1970s, many different synthetic monomers/polymers, biological molecules and particles have been used to form cryogels ( Aragão Börner et al, 2014 ; Agarwal et al, 2021 ; Jones et al, 2021 ). These materials are found in many different applications ranging from separation and purification ( Hajizadeh et al, 2021 ) to biomedical applications ( Henderson et al, 2013 ) and environmental applications ( Jain and Kumar, 2013 ; Berillo et al, 2021 ).…”