Process Biochemistry 46 (2011) 101–107 Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio Purification of rabbit polyclonal immunoglobulin G using anion exchangers Rattana Wongchuphan a,e , Beng Ti Tey b,d , Wen Siang Tan c,d , Senthil Kumar Subramanian c , Farah Saleena Taip a , Tau Chuan Ling a,∗ a Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia b Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia c Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia d Institute of Bioscience, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia e Department of Chemistry, Faculty of Science and Technology, Suratthani Rajabhat University, 84100, Surat Thani, Thailand article info Article history: Received 25 February 2010 Received in revised form 21 July 2010 Accepted 22 July 2010 Keywords: Polyclonal IgG STREAMLINE TM DEAE Rabbit serum Albumin removal Anion exchange adsorbents Negative chromatography antibody purification abstract Negative chromatography antibody purification (N-CAP) using the weak anion exchanger STREAMLINE TM DEAE to extract impurities while retaining the target antibody is proposed as an effective method for the recovery of antibody from rabbit serum. The effects of pH and initial protein concentration on the removal of albumin were investigated. The optimal pH and initial protein concentration for the efficient removal of albumin from rabbit serum were pH 8.0 and 0.5 mg/ml, respectively. Under optimal binding conditions, DEAE successfully removed more than 90% of the albumin from rabbit serum with less than 20% IgG loss. This process offered good polyclonal IgG yield of 80% with a purity of 83% and a purification factor of 5.5. The use of a strong anion exchanger like STREAMLINE TM Q XL for albumin removal was also explored. Under similar optimized conditions, albumin removal by Q XL was as high as 90%. However, IgG recovery and purity were reduced to about 70% and 62%, respectively. Thus, N-CAP using the anion exchanger DEAE removes albumin from rabbit serum and thereby offers an efficient means of purifying polyclonal antibodies. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Protein A affinity chromatography, which is used widely in anti- body purification, offers specificity and highly efficient product capture. Protein A has been the standard antibody manufactur- ing method during the past few decades due to its selectiveness, optimal throughput, and its stability to be cleaned and re-used [1,2]. In addition, it has been used for product capture in multistep platforms [3]. However, since advances in cell culture technol- ogy have been developed, this approach now has a number of disadvantages. As the antibody titer in cell culture increases, the downstream processes become a bottleneck and the product recov- ery cost increases in line with the production scale. In such a case, it may not be cost-effective to purify antibody using Protein A affinity chromatography. Moreover, the low stability of Protein A is a limi- tation in production scale antibody purification [2]. Therefore, due to its high cost and concerns over low ligand stability after many cycles of production, there is a genuine need for a replacement for Protein A affinity chromatography [1]. Hence, the development ∗ Corresponding author. Tel.: +60 603 89466447; fax: +60 603 89464440. E-mail address: ltc555@eng.upm.edu.my (T.C. Ling). of a cost-effective alternative method for antibody purification is needed to overcome these concerns. As recently recommended by some adsorbent manufacturers, negative chromatography antibody purification (N-CAP) can be used for antibody purification from animal serum. Typically, this requires passing the clarified antibody feedstock through a series of negative chromatography columns to remove all the impuri- ties. This approach could be an efficient way of overcoming the high cost and the physical capacity limits of Protein A affinity chro- matography. Ion exchange chromatography (IEC) is a simple, rapid technique used in protein separation that depends on the differen- tial ability of various proteins to be adsorbed onto the adsorbent [4]. IEC is used commonly in protein purification due to its high bind- ing capacity and cost-effectiveness [5–8]. Commercially available ionic matrices for protein purification (including DEAE sepharose and Q XL) are highly activated with a high density of ionic lig- ands such as diethylaminoethyl and quaternary amine groups [9]. These matrices have high adsorption capacity towards proteins even in the presence of Escherichia coli (E. coli) biomass and/or cell debris [4,10,11]. Recently, Pessela and colleagues successfully purified immunoglobulin G (IgG) from whey proteins concentrate (WPC) by eliminating BSA with DEAE-agarose adsorbent [9]. BSA was adsorbed strongly on highly activated DEAE-agarose, while 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.07.023