Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin.
BACKGROUND
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.


RESULTS
Secretion level of the fusion protein produced in vitro in BHK cells was approximately 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 approximately 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 (approximately 32 h) when compared with a transgenically produced huBChE preparation containing >70% tetramer (approximately 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.


CONCLUSIONS
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.
Huang
Yue-Jin
YJ
PharmAthene Canada Inc, (formally Nexia Biotechnologies Inc,), 7150 Alexander-Fleming, Montreal, QC H4S 2C8, Canada. yjhuang@videotron.ca
Lundy
Paul M
PM
Lazaris
Anthoula
A
Huang
Yue
Y
Baldassarre
Hernan
H
Wang
Bin
B
Turcotte
Carl
C
Côté
Mélanie
M
Bellemare
Annie
A
Bilodeau
Annie S
AS
Brouillard
Sandra
S
Touati
Madjid
M
Herskovits
Peter
P
Bégin
Isabelle
I
Neveu
Nathalie
N
Brochu
Eric
E
Pierson
Janice
J
Hockley
Duncan K
DK
Cerasoli
Douglas M
DM
Lenz
David E
DE
Wilgus
Harvey
H
Karatzas
Costas N
CN
Langermann
Solomon
S
D000818:Animals; D002091:Butyrylcholinesterase / Q000097:blood / Q000235:genetics / Q000493:pharmacokinetics*; D002460:Cell Line; D006224:Cricetinae; D006041:Goats; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D008657:Metabolic Clearance Rate; D051379:Mice; D008822:Mice, Transgenic; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics*; D012709:Serum Albumin / Q000235:genetics / Q000493:pharmacokinetics*; D013552:Swine
D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D002091:Butyrylcholinesterase