Removal of tightly bound endotoxin from biological products

The method for endotoxin removal described in this paper is useful for sepa ration of tightly bound endotoxin from biological products, particularly th ose produced in Escherichia coli in the form of inclusion bodies for which a denaturation step is required to solubilise the product. We employed guan idine hydrochloride and ammonium sulphate in combination with hydrophobic i nteraction chromatography (HIC). These conditions enable binding of the end otoxin to the matrix, giving unbound product in the column flow-through. Th is makes the method generally applicable to biological products. An endotox in reduction of about 3.7 logs was achieved: from as much as 1 100 000 EU m g-l in the solubilised material to about 200 EU mg(-1) in the product purif ied by this method. The method was developed for a cervical dysplasia vacci ne, a fusion protein comprising L2, E7 and E6 from Human Papilloma Virus ty pe 16, because both conventional and commercially available methods of endo toxin removal were ineffective in removing the tightly bound endotoxin from this product. (C) 2001 Elsevier Science B.V. All rights reserved.