Heparin purification by expanded bed anion exchange in a countercurrent chromatography column

Abstract

In this study, an expanded bed anion exchange in a countercurrent chromatography column (EB-CCC) technique for separation and purification of heparin, an anticoagulant and antithrombotic glycosaminoglycan, is reported for the first time. A comparative evaluation of the EB-CCC technique with the conventional fixed bed column chromatography (FBCC) revealed its effectiveness in improving adsorption at high flow rates and reducing separation time. A significantly higher maximum adsorption (91.66%) was exhibited by EB-CCC in comparison with FBCC (45.16%) at the eluent flow rate of 1 mL·min^-1. Similarly, the experimental adsorption capacity of heparin was enhanced by 1.69, 2.06 and 2.58 times in the case of EB-CCC at the flow rates of 1, 2 and 5 mL·min^-1, respectively. Moreover, the directly proportional amplification of double loaded resin and double column volume was demonstrated at an EB-CCC rotational speed of 300 rpm and a flow rate of 2 mL·min^-1, and the experimental adsorption capacity was observed to increase from 66.42 to 136.48 mg·g^-1 after amplification. Heparin purified by EB-CCC displayed higher potency (216.09 ± 11.89 IU·mg^-1) as compared to FBCC (205.51 ± 7.90 IU·mg^-1) and the initial crude heparin 134.17 ± 4.12 IU·mg^-1. Furthermore, comparing to the purified heparin by FBCC, heparin purified by EB-CCC had low molecular weight, high FXa/FIIa, superior anticoagulation effect and enhanced suitability as an exogenous anticoagulant.

Keywords: Anticoagulation; Expanded bed anion exchange in a countercurrent chromatography column (EB-CCC); Heparin purification; Proportional amplification.