Epitope imprinted polymer on silica microparticle for heparin purification

Heparin, is a sulfated glycosaminoglycan (GAG) used as an anticoagulant and antithrombotic agent, is widely employed in hemodialysis and surgery. Heparin is extracted from animal source therefore, oversulfated chondroitin sulfate (OSCS) is impurity that may be present at low levels in pharmaceutical heparin products which, associated with side effects and death in some cases. Hence, it is important to purify heparin in order to reduce these side effects. For this purpose, molecule imprinted polymer (MIP) extraction approach may offer some advantages. A macromolecule such as heparin, an epitope imprinting is advantageous to increase the affinity of heparin separation and had been developed in this study. The heparin epitope imprinted polymer was employed via sol gel process, in which macroparticle silica gel was used as a binder. This work emphasized ftrstly on the investigation of the strength of interaction between the template (glucose sulfate) with three different functionalized silane as a functional monomer. A batch adsorption study shown that methylimidazole as a functional monomer had the highest binding capacity of 53.07 mg/g, whereas the amine possessed high imprinting factor (IF; 2.24). In the second part of the study, the epitope imprinting was implemented using low molecular weight heparin (LMWH) as a template. During the development of epitope imprinting, template bleeding was identified that interfered the binding performance. Thus, a mixture of MeOH:HCl solvent and either stirring or ultrasonic extraction techniques could overcome the template bleeding problems. Later, the optimization of epitope imprinting process were investigated for the development of heparin epitope imprinting. The significant binding capacity of heparin (19.48 mg/g) compared with LMWH (2.50 mg/g) suggested that the epitope surface-imprinted polymer using water as a porogenic solvent with the ratio 1:2 of template to functional monomer successfully adsorbed and enriched the heparin. The adsorption behavior of epitope imprinting indicated that Langmuir-Freundlich (single-site) was considered as the model with better ftting and kinetic batch studies showed pseudo-first-order kinetic model via physicochemical process. Final part of the study, an anticoagulant drug from local pharmaceutical industry was tested for the extraction and separation of heparin and OSCS on the molecularly imprinted solid phase extraction (MISPE). The mobile phase solvent necessary to perform the separation was optimized and results showed that ACN :H20 and MeOH:H20 of ratio 1:1, phosphate buffer pH 5, and ammonium chloride (NH4Cl) were selected as the suitable loading, washing, and modifier respectively in the elution solvent. Finally, the recovery of heparin in MISPE column was increased to 52.33% after increasing the solvent volume to two times of 1.0 mL eluting olvent. In conclusion, epitope surface-imprinted polymer was proven to be a promising resin for heparin purification in the pharmaceutical industry.