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Method for the production of pure virally inactivated butyrylcholinesterase

a technology of butyrylcholinesterase and production method, which is applied in the field of isolation and purification of butyrylcholinesterase, can solve the problems of not being able to commercially viable, easily performed methods to efficiently and economically produce large quantities of purified butyrylcholinesterase, and achieve high yield and purity

Inactive Publication Date: 2009-04-09
AMERICAN NAT RED CROSS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006]To satisfy the need in the art, we have developed a versatile, commercially viable method for producing purified, virally inactivated butyrylcholinesterase at high yields and purity. Specifically, the present invention provides a novel method for recovering purified, virally inactivated butyrylcholinesterase from a biological fluid such as plasma, Cohn plasma Fraction IV-4 or IV-1, analogous blood fractions, or fluids from bioreactors. The various embodiments of the method comprise subjecting a butyrylcholinesterase-containing fluid to a series of steps including cationic exchange chromatography, affinity chromatography, solvent detergent treatment, and pasteurization. Those steps need not be performed in that order, and the methods can be successfully performed with duplication of one or more of those steps.

Problems solved by technology

Until now, there have been no commercially viable, easily performed methods to efficiently and economically produce large quantities of purified, virally inactivated butyrylcholinesterase from biological fluids.

Method used

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Examples

Experimental program
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Effect test

example i

Purification and Viral Inactivation of Butyrylcholinesterase

[0036]Materials: Cohn Fraction IV-4 paste obtained by the Cohn cold ethanol fractionation process of pooled human plasma was used in this example. The cation exchange materials used (CM-SEPHAROSE7, FAST FLOW7 OR SP-SEPHAROSE7, FAST FLOW7) are commercially available resins.

[0037]The affinity resin was prepared by coupling procainamide covalently to ECH-SEPHAROSE 4B7 (Pharmacia) using EDC as a coupling reagent. Procainamide was added in 5-fold molar excess per gram of swollen gel relative to the resin ligands. EDC was then added to a final concentration of about 0.1 M. The coupling procedure was performed in distilled water, adjusted to a pH ranging from about 4.5 to about 5.5 with HCl. The procainamide / EDC mixture was rotated gently for about 24 hours at room temperature, and the resin was subsequently washed with several cycles of high and low pH solutions (0.1 M acetate buffer, 0.5 M NaCl pH 4.0; 0.1 M Tris-HCl buffer; 0.5...

example ii

Affinity Purification of Butyrylcholinesterase Using Peptide Ligands

[0046]Identification of positive peptides: Solid phase combinatorial peptide libraries were synthesized on polymethacrylate beads (Buettner et al, Int. J. Pep Prot. Res., 47, 70-83 (1996)) and screened for the binding of butyrylcholinesterase using the radiolabeled technique of Jentoft et al. (Methods in Enzymology, 91:570-79 (1983)) to radiolabel butyrylcholinesterase. To detect positive ligands that bind butyrylcholinesterase, the screening method of Mondorf et al. (J. Peptide Res., 52(6):526-36 (1998)) combined with an activity assay using the substrate butyrylthiocholine and DTNB (5,5-Dithiobis-2-Nitrobenzoic Acid) were used. This stained the beads that bound active butyrylcholinesterase yellow.

[0047]We have identified the following peptides as preferred peptide affinity ligands for the concentration, separation, and purification of butyrylcholinesterase: AKDQIP (SEQ ID NO: 1) (alanine, lysine, aspartic acid, gl...

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Abstract

The present invention provides a method for purifying butyrylcholinesterase from various biological fluids. Biological fluids include, e.g., blood, blood fractions, plasma, and bioreactor broths, and other such mixtures containing butyrylcholinesterase. In one embodiment, the invention provides a method for the production of purified, virally inactivated butyrylcholinesterase by contacting a biological fluid containing butyrylcholinesterase with a cationic exchange chromatography material, with an affinity chromatography material, and treating the fluid with solvent detergent. The resulting purified butyrylcholinesterase can also be subjected to a pasteurization step, and formulated in a sodium chloride / sodium phosphate solution for storage or lyophilization.

Description

[0001]This application is a divisional application of Ser. No. 10 / 10 / 061,233, filed Feb. 4, 2002, the entire content of which is hereby incorporated by reference.FIELD OF THE INVENTION[0002]The present invention provides a method for the isolation and purification of butyrylcholinesterase from complex biological fluids such as plasma. More specifically, the present invention provides a method for purifying and disinfecting butyrylcholinesterase from butyrylcholinesterase-containing biological fluids, e.g., Cohn Fraction IV-4 or IV-1, comprising subjecting such fluids to cationic exchange chromatography, affinity chromatography, a solvent detergent treatment, and pasteurization. As discussed more fully below, the order of those steps can be varied.BACKGROUND OF THE INVENTION[0003]Butyrylcholinesterase is an enzyme found mainly in plasma. Although the normal physiological role of butyrylcholinesterase is unknown, butyrylcholinesterase has been shown to metabolize acetylcholine, degrad...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/99C12N9/14C12N9/18
CPCC12N9/18
Inventor RALSTON, ANNEMARIE H.KOLEN, BILLY L.HAMMOND, DAVID JOHN
Owner AMERICAN NAT RED CROSS
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