Journal of Chromatography A, 1107 (2006) 104–109 Purification of humanized monoclonal antibody by hydrophobic interaction membrane chromatography Raja Ghosh ∗ , Lu Wang Department of Chemical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ont., Canada L8S 4L7 Received 8 November 2005; received in revised form 6 December 2005; accepted 7 December 2005 Available online 18 January 2006 Abstract Humanized monoclonal antibodies (mAbs) hold significant promise as biopharmaceuticals. One of the main challenges faced in the purification of mAbs is their separation from bovine serum albumin, which is the main protein present in most mammalian cell culture media. This paper discusses the purification of humanized mAb hIgG1-CD4 from CHO cell culture media by hydrophobic interaction membrane chromatography using a stack of microporous synthetic membranes. The effects of solution conditions on mAb solubility and binding on the membrane were first studied. The separation of a simulated mixture of bovine albumin and the mAb was then carried out to examine the feasibility of mAb purification. Separation experiments carried out under optimized conditions demonstrated that this membrane-based technique could be used for mAb purification from cell culture media. High purity (97%) and recovery (in excess of 97%) were obtained. © 2005 Elsevier B.V. All rights reserved. Keywords: Membrane chromatography; Humanized monoclonal antibody; Hydrophobic interaction; Bioseparation; Purification; Bovine albumin; Biopharmaceuti- cals 1. Introduction Membrane chromatography is a fast growing bioseparation technique [1–5]. Porous membranes have some advantages over particulate chromatographic media, mainly faster separation, ease of scale-up and better process economics [6]. Membrane chromatography is mainly operated in the “bind and elute” mode of separation and its use has been reported for a wide range of protein purifications [7–9]. Membrane chromatography is par- ticularly attractive when the bound molecule has a relatively low concentration in the feed solution. Monoclonal antibodies (mAbs) are produced by mammalian cell culture and their con- centration in the culture supernatant rarely exceeds 0.5 mg/ml. Hence, the separation of mAbs using membrane chromatogra- phy is attractive [10–12]. Most serum-free media used for mammalian cell culture con- tain bovine albumin as key added ingredient. Therefore, the main protein–protein separation involved in mAb purification is its separation from bovine albumin. IgG1 type mAbs are usu- ∗ Corresponding author. Tel.: +1 905 525 9140x27415; fax: +1 905 521 1350. E-mail address: rghosh@mcmaster.ca (R. Ghosh). ally purified by affinity separation (using protein-A or protein-G based chromatographic media). This is expensive and relatively difficult to scale-up. Protein-A and protein-G are also known to leach out from affinity media and this can be problematic since these ligands are immunotoxic in nature [13]. Moreover, acidic buffers are used for elution of bound mAbs and this may result in adverse affects such as protein denaturation and dimeriza- tion [14]. Hydrophobic interaction chromatography gives good separation of IgG from most cell culture media constituents since the antibody binding on the chromatographic media at high anti-chaotropic salt concentrations is largely selective in nature. Hydrophobic interaction is also known to be “gentle” when compared to other types of chromatographic separations [15]. Column based purification of mAbs using hydrophobic inter- action chromatography has been widely reported (e.g. [16,17]). There are significantly fewer reports on hydrophobic interaction membrane chromatography of mAbs [18,19]. In a previous paper [18], the use of hydrophobic interaction membrane chromatography for the separation of a murine mAb (Campath-1G) from bovine albumin was discussed. Humanized mAbs are preferred as biopharmaceuticals and their develop- ment has opened up tremendous opportunities in the area of protein-based therapeutics. hIgG1-CD4 is a humanized IgG1 0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.12.035