Synthesis and characterization of chitosan-polyacrylamide cryogels for the purification of human IgG by IMAC

Abstract

The performance of polyacrylamide-chitosan monolithic cryogels functionalized with iminodiacetic acid (IDA) was evaluated for the chromatographic separation of Immunoglobulin G (hIgG) from human serum using immobilized metal ion affinity chromatography (IMAC). Monoliths were prepared using the cryopolimerization technique, varying the type of chitosan (CS) used, and final concentrations of allyl glycidyl ether (AGE) and glutaraldehyde (GA), keeping the amount of acrylamide (AAm) and bisacrylamide (MBAm) constant. Cryogels were characterized by their surface area, SEM, FTIR, porosity, ligand density, mechanical resistance, and flow rate. Purification of hIgG was investigated using the PAAm-CS-GA-AGE-IDA cryogel chelated with Cu(II) and Ni(II) ions. The analysis demonstrated isolation of hIgG from human serum in a sodium phosphate buffer at pH 7.0 containing 2 mmol/L imidazole on PAAm-CS-GA-AGE-IDA-Ni(II) cryogel, with purity of 96% and purification factor of 12.4 (based on total protein concentration and radial immunodiffusion). The hIgG adsorption equilibrium data on the IDA-Ni(II) cryogel followed the Langmuir–Freundlich isotherm model, showing maximum hIgG binding capacity of 31.19 mg hIgG/g dry cryogel and a dissociation constant in the order of 10−7 to 10−8 mol/L. These results demonstrate the potential of PAAm/CS-based cryogels with chelated Ni(II) ions for the purification of hIgG from human serum using IMAC.