Removal of tightly bound endotoxin from biological products.
The method for endotoxin removal described in this paper is useful for separation of tightly bound endotoxin from biological products, particularly those produced in Escherichia coli in the form of inclusion bodies for which a denaturation step is required to solubilise the product. We employed guanidine hydrochloride and ammonium sulphate in combination with hydrophobic interaction chromatography (HIC). These conditions enable binding of the endotoxin to the matrix, giving unbound product in the column flow-through. This makes the method generally applicable to biological products. An endotoxin reduction of about 3.7 logs was achieved; from as much as 1,100,000 EU mg(-1) in the solubilised material to about 200 EU mg(-1) in the product purified by this method. The method was developed for a cervical dysplasia vaccine, a fusion protein comprising L2, E7 and E6 from Human Papilloma Virus type 16, because both conventional and commercially available methods of endotoxin removal were ineffective in removing the tightly bound endotoxin from this product.
Wilson
M J
MJ
Downstream Process Development, Xenova, 310 Cambridge Science Park, Milton Road, CB4 0WG, Cambridge, UK. mwilson@cantab.co.uk
Haggart
C L
CL
Gallagher
S P
SP
Walsh
D
D
D002845:Chromatography / Q000295:instrumentation / Q000379:methods*; D004731:Endotoxins / Q000302:isolation & purification*; D002479:Inclusion Bodies / Q000737:chemistry; D009856:Oncogene Proteins, Viral / Q000235:genetics; D050725:Papillomavirus E7 Proteins; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012097:Repressor Proteins; D014614:Vaccines, Synthetic / Q000302:isolation & purification*
C068651:E6 protein, Human papillomavirus type 16; D004731:Endotoxins; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014614:Vaccines, Synthetic; C059731:oncogene protein E7, Human papillomavirus type 16; C044101:endotoxin, Escherichia coli