Method for the production of pure virally inactivated butyrylcholinesterase

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.

1 . A method for producing purified, virally inactivated butyrylcholinesterase comprising:
 a. contacting a butyrylcholinesterase-containing biological fluid with a carboxymethyl-agarose cation exchange chromatography material;   b. concentrating the fall through from the cation exchange chromatography step by ultrafiltration;   c. contacting the ultrafiltration concentrate with a procainamide affinity chromatography material to bind butyrylcholinesterase;   d. eluting butyrylcholinesterase from the procainamide affinity chromatography material;   e. solvent-detergent treating the eluate from the procainamide affinity chromatography material with about 1% (v/v) Tween-80 and about 0.3% (v/v) TnBP;   f. contacting the solvent-detergent treated eluate with a procainamide affinity chromatography material to bind butyrylcholinesterase;   g. eluting butyrylcholinesterase from the procainamide affinity chromatography material; and   h. pasteurizing the eluted butyrylcholinesterase at about +60° C. for at least about 8 hours.   
   
   
       2 . The method of  claim 1 , wherein the butyrylcholinesterase containing biological fluid comprises a plasma fraction selected from the group consisting of Cohn Fraction IV-4, Cohn Fraction IV-1, PPT. IV from a Kistler-Nitschmann fractionation, and combinations thereof. 
   
   
       3 . The method of  claim 1 , wherein the butyrylcholinesterase containing biological fluid comprises recombinantly produced butyrylcholinesterase. 
   
   
       4 . A method for producing purified, virally inactivated butyrylcholinesterase comprising:
 a. contacting a butyrylcholinesterase-containing biological fluid with a carboxymethyl-agarose cation exchange chromatography material;   b. solvent-detergent treating the biological fluid with about 1% (v/v) Tween-80 and about 0.3% (v/v) TnBP;   c. contacting the solvent-detergent treated fall through with a procainamide affinity chromatography material to bind butyrylcholinesterase;   d. recovering butyrylcholinesterase from the procainamide affinity chromatography material; and   e. pasteurizing the recovered butyrylcholinesterase at about +60° C. for at least about 8 hours.   
   
   
       5 . The method of  claim 4 , wherein the butyrylcholinesterase containing biological fluid comprises a plasma fraction selected from the group consisting of Cohn Fraction IV-4, Cohn Fraction IV-1, PPT. IV from a Kistler-Nitschmann fractionation, and combinations thereof. 
   
   
       6 . The method of  claim 4 , wherein the butyrylcholinesterase-containing biological fluid comprises recombinantly produced butyrylcholinesterase. 
   
   
       7 . A method for producing purified, virally inactivated butyrylcholinesterase comprising:
 a. contacting a butyrylcholinesterase-containing biological fluid with a cation exchange chromatography material;   b. subjecting the biological fluid to affinity purification using a peptide ligand affinity chromatography material that binds butyrylcholinesterase;   c. recovering butyrylcholinesterase from the peptide ligand affinity chromatography material;   d. solvent-detergent treating the biological fluid;   e. contacting the solvent-detergent treated biological fluid with a peptide ligand affinity chromatography material;   f. recovering butyrylcholinesterase from the affinity chromatography material; and   g. pasteurizing the recovered butyrylcholinesterase.   
   
   
       8 . The method of  claim 7 , wherein said butyrylcholinesterase containing biological fluid comprises a plasma fraction selected from the group consisting of Cohn Fraction IV-4, Cohn Fraction IV-1, PPT. IV from a Kistler-Nitschmann fractionation, and combinations thereof. 
   
   
       9 . The method of  claim 7 , wherein said butyrylcholinesterase-containing biological fluid comprises recombinantly produced butyrylcholinesterase. 
   
   
       10 . The method of  claim 7 , wherein said pasteurized butyrylcholinesterase is formulated in about 0.1 to about 0.15 M NaCl and about 0.02 M sodium phosphate. 
   
   
       11 . The method of  claim 7 , wherein said peptide ligand is selected from the group consisting of:
 a. alanine, lysine, aspartic acid, glutamine, isoleucine, and proline;   b. alanine, lysine, glycine, aspartic acid, glutamine, and asparagine;   c. tryptophan, lysine, aspartic acid, alanine, valine, and glutamine;   d. glycine, phenylalanine, valine, glycine, 2-naphthylalanine, and alanine;   e. glycine, phenylalanine, histidine, glycine, 2-naphthylalanine, and isoleucine;   f. alanine, phenylalanine, threonine, asparagine, glycine, and glutamic acid;   g. alanine, phenylalanine, threonine, asparagine, glutamine, and glutamic acid;   h. glycine, threonine, asparagine, tyrosine, histidine, glutamine; and   i. alanine, glutamic acid, valine, aspartic acid, proline, glycine.   
   
   
       12 . A method for producing purified, virally inactivated butyrylcholinesterase comprising:
 a. contacting a butyrylcholinesterase-containing biological fluid with a cation exchange chromatography material;   b. solvent-detergent treating the biological fluid;   c. contacting the biological fluid with at least one peptide ligand affinity chromatography material that binds butyrylcholinesterase;   d. recovering butyrylcholinesterase from the peptide ligand affinity chromatography material; and   e. pasteurizing the eluted butyrylcholinesterase.   
   
   
       13 . The method of  claim 12 , wherein said butyrylcholinesterase containing biological fluid comprises a plasma fraction selected from the group consisting of Cohn Fraction IV-4, Cohn Fraction IV-1, PPT. IV from a Kistler-Nitschmann fractionation, and combinations thereof. 
   
   
       14 . The method of  claim 12 , wherein said butyrylcholinesterase-containing biological fluid comprises recombinantly produced butyrylcholinesterase. 
   
   
       15 . The method of  claim 12 , wherein said pasteurized butyrylcholinesterase is formulated in about 0.1 to about 0.15 M NaCl and about 0.02 M sodium phosphate. 
   
   
       16 . The method of  claim 12 , wherein said at least one peptide ligand is a peptide selected from the group consisting of:
 a. alanine, lysine, aspartic acid, glutamine, isoleucine, and proline;   b. alanine, lysine, glycine, aspartic acid, glutamine, and asparagine;   c. tryptophan, lysine, aspartic acid, alanine, valine, and glutamine;   d. glycine, phenylalanine, valine, glycine, 2-naphthylalanine, and alanine;   e. glycine, phenylalanine, histidine, glycine, 2-naphthylalanine, and isoleucine;   f. alanine, phenylalanine, threonine, asparagine, glycine, and glutamic acid;   g. alanine, phenylalanine, threonine, asparagine, glutamine, and glutamic acid;   h. glycine, threonine, asparagine, tyrosine, histidine, glutamine; and   i. alanine, glutamic acid, valine, aspartic acid, proline, glycine.   
   
   
       17 . A method for isolating butyrylcholinesterase comprising contacting a butyrylcholinesterase-containing biological fluid with a peptide ligand affinity chromatography material wherein the peptide ligand is selected from the group consisting of:
 a. alanine, lysine, aspartic acid, glutamine, isoleucine, and proline;   b. alanine, lysine, glycine, aspartic acid, glutamine, and asparagine;   c. tryptophan, lysine, aspartic acid, alanine, valine, and glutamine;   d. glycine, phenylalanine, valine, glycine, 2-naphthylalanine, and alanine;   e. glycine, phenylalanine, histidine, glycine, 2-naphthylalanine, and isoleucine;   f. alanine, phenylalanine, threonine, asparagine, glycine, and glutamic acid;   g. alanine, phenylalanine, threonine, asparagine, glutamine, and glutamic acid;   h. glycine, threonine, asparagine, tyrosine, histidine, glutamine; and   i. alanine, glutamic acid, valine, aspartic acid, proline, glycine;   and recovering butyrylcholinesterase bound to said peptide ligand chromatography material.   
   
   
       18 . The method of  claim 17 , wherein the peptide ligand is covalently bound to a support selected from the group consisting of agarose, dextran, cellulose, polyacrylamide, polystyrene, acrylic polymers, vinyl polymers, silica, and cross-linked versions thereof. 
   
   
       19 . The method of  claim 17 , further comprising contacting the biological fluid with a cation exchange chromatography material. 
   
   
       20 . The method of  claim 17 , further comprising subjecting the biological fluid to a solvent-detergent treatment step. 
   
   
       21 . The method of  claim 17 , further comprising subjecting the biological fluid to a pasteurization step.