Journal of Chromatography B, 889–890 (2012) 95–102 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B j ourna l ho me page: www.elsevier.com/locate/chromb Molecular imprinting based composite cryogel membranes for purification of anti-hepatitis B surface antibody by fast protein liquid chromatography Sevgi Asliyuce a , Lokman Uzun a,∗ , Abbas Yousefi Rad b , Serhat Unal c , Ridvan Say d , Adil Denizli a a Hacettepe University, Department of Chemistry, Biochemistry Division, Ankara, Turkey b TOBB ETU Hospital, Ankara, Turkey c Hacettepe University, Faculty of Medicine, Ankara, Turkey d Anadolu University, Department of Chemistry, Eskis ¸ ehir, Turkey a r t i c l e i n f o Article history: Received 7 October 2011 Accepted 3 February 2012 Available online 9 February 2012 Key words: Molecular imprinted polymers Anti-hepatitis B surface antibody Composite cryogel membrane Fast protein liquid chromatography Affinity purification a b s t r a c t In the present study, we have focused our attention to prepare molecular imprinted composite cryogel membranes for purification of hepatitis B surface antibody (anti-HBs) by fast protein liquid chromatogra- phy. Before the preparation of the molecular imprinted composite cryogel membranes (MI-CMs) by free radical polymerization at sub-zero temperature, we have synthesized and characterized the anti-HBs imprinted particles. Then, the cryogel membranes (CMs) were characterized by swelling test, scanning electron microscopy and Fourier transform infrared spectroscopy. Prior to chromatographic purification studies, the effective parameters on the anti-HBs adsorption process were evaluated by investigating the dependency of the adsorption capacity on flow-rate, anti-HBs concentration, contact time and ionic strength. The maximum anti-HBs adsorption capacity was calculated as 701.4 mIU/g CM. The selectivity of the MI-CMs was shown by competitive adsorption of anti-HBs, total anti-hepatitis A antibody (anti-HAV) and total immunoglobulin E (IgE) adsorption studies. The MI-CMs have relative selectivity coefficients as 5.45 for anti-HBs/total anti-HAV and 9.05 for anti-HBs/total IgE, respectively. The phosphate buffer solution (pH 7.4) containing 1.0 M NaCl was used for elution, almost completely, of adsorbed anti-HBs molecules. The MI-CMs could be used many times without any significant decrease in the adsorption capacity. The chromatographic purification performances of the MI-CMs were also investigated. The chromatographic parameters such as capacity and separation factors, the theoretical plate number and resolution of the MI-CMs were calculated as 5.48, 6.02, 1153.9, and 1.72 for anti-HBs molecules, respec- tively. As a conclusion, we can say that the MI-CMs could be used for specific purification of anti-HBs from anti-HBs positive human plasma. © 2012 Elsevier B.V. All rights reserved. 1. Introduction The prevalence of hepatitis B virus (HBV) infection is getting wider as the day goes on although an extensive vaccination has been applied [1]. Due to 75% of the world’s population living in areas having high infection level and more than 350 million people are chronic carriers, the governments have to apply and set a huge bud- get for the preventive vaccination [2,3]. Not only diseases directly related to acute HBV infection but also associated with diseases such as liver cirrhosis and hepatocellular carcinoma depending on chronic HBV development are important global health problem concerns [4]. Especially in case of infants born to hepatitis B posi- tive mothers, the fatal chronic disease development is potentially occurred [5]. For prevention of these infants, they are immunized passively by giving high titer neutralizing antibodies, anti-hepatitis ∗ Corresponding author. Tel.: +90 312 297 7963; fax: +90 312 299 2163. E-mail address: lokman@hacettepe.edu.tr (L. Uzun). B surface antibodies (anti-HBs), against to antigenic determinants on HB viruses immediately after birth. In the following 2–5 months, HBs vaccines should be repeated several times [6]. In addition to the infants, a considerable numbers of people at high risk of HBV infection are required to be immunized passively against to HBV infection because they are not protected during the first month after vaccination. An immediate protection should be introduced to these people such as patients and personals in dialysis unit, close contacts of chronic HBV carriers, tourist gone to where high HBV prevalence etc. after accidental inoculation with infectious materials, needle stick or other means [7]. The antibodies trans- ferred passively prepared from the sera of anti-HBs positive donors according to regulations assured by strict product standard, pre- vents the infection during the period of development of an active immune response [8]. Anti-HBs containing sera are prepared not only from anti- HBs positive donors but also from immunized animal plasma and transgenic plants [4]. The purification system was mainly based on protein A affinity chromatography used widely for antibody 1570-0232/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2012.02.001