Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin
1 PharmAthene Canada Inc. (formally Nexia Biotechnologies Inc.), 7150 Alexander-Fleming, Montreal, QC H4S 2C8, Canada
2 Chemical Biological Defence Section, Defence Research and Development Canada-Suffield, Box 4000, Medicine Hat, AB T1B 8K6, Canada
3 US Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, Aberdeen Proving Ground, MD 21010-5400, USA
4 Quebec Transgenic Research Network, McGill University, 3655 Promenade Sir William Osler, Montreal, QC H3G 1Y6, Canada
5 NewLife Fertility Centre, 4250 Sherwoodtowne Boulevard, Mississauga, ON L4Z 2G6, Canada
6 CNKonsulting and Aegis Bioresearch, 251 Sherwood Road, Beaconsfield, QC H9W 2H4, Canada
BMC Biotechnology 2008, 8:50 doi:10.1186/1472-6750-8-50Published: 16 May 2008
Human butyrylcholinesterase (huBChE) has been shown to be an effective antidote against multiple LD50 of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA) and characterize the fusion protein.
Secretion level of the fusion protein produced in vitro in BHK cells was ~30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04–1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC)-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of ~150 kDa by Western blot. The purified fusion protein produced in vitro was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (~32 h) when compared with a transgenically produced huBChE preparation containing >70% tetramer (~3 h). In vitro nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested.
Both the pharmacokinetic study and the in vitro nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced in vitro and in vivo. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.