Comparative removal of solvent and detergent viral inactivating agents from human intravenous immunoglobulin G preparations using SDR HyperD and C18 sorbents Thierry Burnouf a, * , Makram A. Sayed b , Miryana Radosevich a , Magdy El-Ekiaby c a Research Department, Human Protein Process Sciences (HPPS), 59800 Lille, France b Fayoum University, 63514 Fayoum, Egypt c Shabrawishi Hospital Blood Bank, 12311 Giza, Egypt article info Article history: Received 9 February 2009 Available online 19 March 2009 Keywords: Solvent/Detergent SDR HyperD C18 Immunoglobulin G abstract The capacity of hydrophobic octadecyl (C18) and SDR HyperD materials to remove the combination of 1% (v/v) solvent (tri-n-butyl phosphate, TnBP) with 1% (v/v) nonionic detergents (Triton X-100 and Triton X- 45) used for viral inactivation of plasma-derived polyvalent intravenous immunoglobulin G (IVIG) prep- aration has been evaluated. Efficient removal of TnBP (<10 ppm in IVIG preparation) was found at ratios of 0.5 g of C18/7 ml of IVIG and 0.22 g of dry SDR HyperD/7 ml of IVIG. Binding capacities of TnBP were greater than 140 mg/g of C18 and greater than 318 mg/g of dry SDR HyperD. Complete removal of Triton X-45 (<2 ppm) was obtained at ratios of 1 g of C18/7 ml of IVIG and 0.44 g of dry SDR HyperD/7 ml of IVIG or above, corresponding to binding capacities in excess of 70 mg/g of C18 and in excess of 159 mg/g of dry SDR HyperD. Residual Triton X-100 was less than 30 ppm at a ratio of 4 g/14 ml of immunoglobulin G (IgG) for the C18 sorbent. Triton X-100 was less than 10 ppm when using SDR HyperD at a ratio of 0.66 g/7 ml of IgG, corresponding to a binding capacity of approximately 106 mg of Triton X-100/g of dry SDR HyperD. Good recoveries of IVIG were achieved in the effluent from both sorbents. Ó 2009 Elsevier Inc. All rights reserved. Polyvalent intravenous immunoglobulins G (IVIGs) 1 are cur- rently the driving protein fraction behind the need of fractionation plasma [1]. IVIGs are used to protect immunodeficient patients from recurrent bacterial or viral infections [2,3] and to treat an increasing number of autoimmune, inflammatory [4], and neurological [5] dis- orders. Like other biological products (e.g., a wide range of human plasma-derived proteins, monoclonal antibodies, and recombinant proteins) produced from human or animal sources or using mamma- lian cells, IVIGs must be subjected to viral inactivation and/or re- moval procedures to ensure its viral safety for therapeutic use in humans [6]. Solvent/Detergent (S/D) is a robust, well-established viral inac- tivation treatment applied to numerous biological products on the market [7,8], including IVIG. The S/D viral inactivation procedures often involve incubating protein solutions for 1 to 6 h in the pres- ence of 0.3 to 1% of an organic solvent, typically tri-n-butyl phos- phate (TnBP), and 0.2 to 1% of one or several detergents, usually Tween 80 and (increasingly) Triton X-100 [9,10]. More recently, the application of Triton X-45 and Tween 20 combined with TnBP has also been described for the viral inactivation of plasma, cryo- precipitate, and factor VIII [11–13]. One of the most crucial difficulties associated with S/D treat- ment is the removal of the solvent and detergent subsequent to the viral inactivation step. Three distinct procedures have been de- scribed for the removal of the S/D chemicals. One approach is to make use of ion exchangers or affinity resins in chromatographic processes designed to eliminate the S/D agents in the flow-through (unadsorbed) fraction while target proteins are bound and then selectively eluted from the chromatographic material [14]. Other methods include phase extractions using vegetable oil—typically soybean or castor oil—followed by decantation to separate the oily and aqueous phases and to deplete the proteins from both TnBP and some oil-soluble detergents such as Triton X-45 [7,11,12]. Fi- nally, hydrophobic interaction chromatography (HIC) may be used to capture the S/D agents while the hydrophilic proteins are di- rectly recovered in the chromatographic flow-through [7]. This HIC process can be applied either directly on the S/D-rich protein fraction or, as done for large-pool plasma for transfusion [15,16] or fibrin sealant, after one oil extraction step introduced to reduce the concentration in S/D agents. The advantage of HIC is that it is fast and it induces minimal impact on protein composition. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.03.016 * Corresponding author. Fax: +33 3 2042 1953/2838 1930. E-mail address: tburnou@attglobal.net (T. Burnouf). 1 Abbreviations used: IVIG, intravenous immunoglobulin G; S/D, solvent/detergent; TnBP, tri-n-butyl phosphate; HIC, hydrophobic interaction chromatography; LFB, Laboratoire Français du Fractionnement et des Biotechnologies; IgG, immunoglobulin G; IgA, immunoglobulin A; IgM, immunoglobulin M; HIV, human immunodeficiency virus; HBV, hepatitis B virus; HCV, hepatitis C virus. Analytical Biochemistry 389 (2009) 69–73 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio