Please cite this article in press as: I. Stoji ´ cevi ´ c, et al., J. Chromatogr. B (2011), doi:10.1016/j.jchromb.2011.06.003 ARTICLE IN PRESS G Model CHROMB-17435; No. of Pages 7 Journal of Chromatography B, xxx (2011) xxx–xxx Contents lists available at ScienceDirect Journal of Chromatography B j ourna l ho me page: www.elsevier.com/locate/chromb Tetanus toxoid purification: Chromatographic procedures as an alternative to ammonium-sulphate precipitation Ivana Stoji ´ cevi ´ c a , Ljiljana Dimitrijevi ´ c a , Nebojˇ sa Dovezenski b , Irena ˇ Zivkovi ´ c a , Vladimir Petruˇ si´ c a , Emilija Marinkovi ´ c a , Aleksandra Ini ´ c-Kanada a , Marijana Stojanovi ´ c a,∗ a Institute of Virology, Vaccines and Sera – Torlak, Vojvode Stepe 458, 11152 Belgrade, Serbia b LKB Vertriebs GmbH (Belgrade office), Cviji´ ceva 115, 11120 Belgrade, Serbia a r t i c l e i n f o Article history: Received 17 February 2011 Accepted 3 June 2011 Available online xxx Keywords: Tetanus toxoid Hydrophobic chromatography Chelating chromatography Immobilized metal affinity chromatography Ammonium-sulphate precipitation a b s t r a c t Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More pre- cisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin’s biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromato- graphic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydropho- bic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium- sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the character- istics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatog- raphy generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Tetanus toxin (TTn) is an enormously potent neurotoxin secreted by the anaerobic soil bacterium Clostridium tetani [1]. As TTn induces death before an adaptive immunity could be gener- ated, active vaccine immunization is crucial for the prevention of death caused by tetanus [2]. Nowadays, vaccination against tetanus is obligatory during childhood, as it is a part of regular vaccination schedule [3]. Tetanus vaccine is produced by a multi-step process comprising C. tetani cultivation, inactivation of secreted TTn by formalde- hyde treatment, and purification of the obtained TTn derivatives. Formaldehyde-treated TTn, termed as tetanus toxoid (TTd), is devoid of toxicity, but is still highly immunogenic with a stabi- lized native conformation and it represents the main constituent of tetanus vaccine [4]. TTn-specific protective immune response ∗ Corresponding author. Tel.: +381 11 39 75 501. E-mail address: mazes@eunet.rs (M. Stojanovi ´ c). can be induced by application of vaccines composed of alum-based adjuvant and TTd as a sole antigen (monovalent vaccine), or within a mixture with additional bacterial/viral antigens (polyvalent vac- cine). More recently, we have been witnessing a raising demand to establish a process of vaccine production in a way that allows its complete validation and standardization. Therefore, several steps in traditional TTd production have to be modified. In this paper we focus on TTd purification. Specifically, we were looking at a possibility to replace the ammonium-sulphate precipitation with the chromatographic method. Although experiments performed in the laboratory imply better characteristics of TTd obtained by formaldehyde treatment of previously isolated TTn, this procedure is not widely implemented in large-scale production, primarily due to safety reasons but also because initial intensive purification is still performed at the level of TTd. Large-scale purification of proteins by ammonium-sulphate precipitation is a time consuming process and requires centrifuga- tion. In contrast, liquid chromatography-based purification would allow faster and fully controlled TTd purification process. Further- 1570-0232/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2011.06.003