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WO2015077317A1 - Compositions et procédés pour la purification et la production de butyrylcholinestérase - Google Patents

Compositions et procédés pour la purification et la production de butyrylcholinestérase Download PDF

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Publication number
WO2015077317A1
WO2015077317A1 PCT/US2014/066373 US2014066373W WO2015077317A1 WO 2015077317 A1 WO2015077317 A1 WO 2015077317A1 US 2014066373 W US2014066373 W US 2014066373W WO 2015077317 A1 WO2015077317 A1 WO 2015077317A1
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WIPO (PCT)
Prior art keywords
bche
polyproline
butyrylcholinesterase
peptide
tetramers
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2014/066373
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English (en)
Inventor
Oksana Lockridge
Marilynn A. Larson
Lawrence M. SCHOPFER
Steven H. Hinrichs
Florian Nachon
Xavier BRAZZOLOTTO
Cyril Ronco
Ludovic Jean
Emilie David
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universite de Rouen
Universite de Nice Sophia Antipolis UNSA
Service de Sante des Armees
University of Nebraska Lincoln
University of Nebraska System
Original Assignee
Universite de Rouen
Universite de Nice Sophia Antipolis UNSA
Service de Sante des Armees
University of Nebraska Lincoln
University of Nebraska System
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Publication of WO2015077317A1 publication Critical patent/WO2015077317A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01008—Cholinesterase (3.1.1.8), i.e. butyrylcholine-esterase

Definitions

  • the present invention relates to the fields of cholinesterases. More
  • the invention provides compositions and methods forthe improved production and purification of butyrylcholinesterase.
  • the human butyrylcholinesterase (BChE) protein is a tetramer of four identical subunits.
  • Amino acid sequencing of the mature BChE protein isolated from human plasma identified 574 amino acids and 9 N-linked carbohydrates per subunit of molecular weight 85,000 (Lockridge et al. (1987) J. Biol. Chem., 262:549-557).
  • the cDNA clone confirmed the amino acid sequence (McTiernan et al. (1987) Proc. Natl. Acad. Sci., 84:6682-6686).
  • Nothing in either the amino acid sequencing results or the cDNA clone indicated that the BChE structure included a partner produced from a different gene.
  • rBChE is useful in humans for, among other things, the protection against the toxicity of nerve agents.
  • BChE tetramers are stable in the circulation for long periods of time (half-life of 12 days in humans). A long residence time in the circulation is required because individuals will be pretreated with BChE - perhaps for several days - before they are sent to areas where they might be exposed to nerve agents. Accordingly, improved methods of generating and purifying BChE tetramers are needed.
  • butyrylcholinesterase tetramers are provided.
  • the methods comprise incubating butyrylcholinesterase (e.g., monomers and/or dimers) with a polyproline peptide.
  • the method is performed in a cell-free system.
  • the polyproline peptide may comprise at least about 80% proline residues.
  • the polyproline peptide is about 10 to about 250 amino acids in length.
  • the butyrylcholinesterase may be recombinant butyrylcholinesterase or
  • butyrylcholinesterase isolated from a natural source also encompasses the butyrylcholinesterase tetramer-polyproline complexes made by methods of the instant invention and compositions comprising the same.
  • methods of treating, inhibiting, and/or preventing a disease or disorder treatable with a cholinesterase comprises administering the
  • the disorder is nerve toxicity caused by exposure to an organophosphate agent.
  • the disorder is cocaine addiction and/or toxicity.
  • methods of purifying butyrylcholinesterase comprise purifying a Cohn Fraction IV over a huprine affinity column.
  • the huprine affinity column comprises Hup24 coupled to a resin.
  • the method comprises performing size exclusion chromatography on the eluted butyrylcholinesterase.
  • Figure 1 shows the determination of the size of the polyproline peptide required for assembly of BChE subunits into tetramers in cell-free incubations.
  • Figure 2 shows the determination of the size of the polyproline peptide required for assembly of BChE subunits into tetramers in the presence of
  • Figure 3 shows that polyproline of molecular weight >30,000 interacts with rBChE subunits.
  • Partially purified rBChE with an activity of 3200 U/ml was incubated with 0, 0.5, 2.5 and 25 mg/ml of big polyproline for 14 days at 4°C. Samples diluted to 2 U/ml were applied to the nondenaturing gel and stained for BChE activity.
  • Figures 4A and 4B show that rBChE treated in vitro with 5 mM polyproline assembles into tetramers.
  • Figure 4A nondenaturing gel stained for BChE activity.
  • Figure 4B the same gel counterstained with Coomassie blue.
  • Lane 1 5 ⁇ human plasma
  • lane 2 0.5 ⁇ rBChE with no addition incubated at 4 ° C for 35 days
  • lane 3 0.5 ⁇ BChE (29.3 U/ml) incubated with 5 mM polyproline P50 for 35 days at 4 ° C
  • lane 4 rBChE concentrated in UltraCULTURETM to 30 U/ml and stored 35 days at 4 ° C.
  • Figure 5 shows that rBChE treated in vitro with 0.1 mM polyproline P50 for
  • Lanes 1 and 2 human plasma; lanes 3 and 4: 4000 U/ml rBChE without and with polyproline; lanes 5 and 6: 3200 U/ml rBChE without and with polyproline; lanes 7 and 8: 220 U/ml rBChE without and with polyproline; lanes 9 and 10: 150 U/ml rBChE without and with polyproline; and lanes 1 1 and 12: 20 U/ml rBChE without and with polyproline.
  • Figure 6 shows the temperature and time dependence for assembly of rBChE into tetramers. 150 U/ml rBChEand 0.1 mM polyproline were used.
  • Figures 7 A and 7B show the structure of the BChE tetramer showing the location of the polyproline peptide within the core of the tetramerization domain.
  • Four subunits assemble through the tetramerization domain at the C-terminus.
  • the tetramerization domain has four parallel alpha helices wrapped around a single antiparallel polyproline helix.
  • the tetramer is a dimer of dimers. Dimers have an interchain disulfide bond at Cys571.
  • Figure 7A Viewed from the top, with the tetramerization domain and the polyproline peptide in the center. Two of the active sites are exposed to solvent, while two face the central cavity of the BChE tetramer.
  • Figure 7B Viewed from the side. Reproduced from Pan et al. (2009) J. Phys. Chem. B, 113:6543-6552).
  • Figure 8 shows an SDS gel stained with Coomassie blue.
  • BChE was purified from Cohn paste using ion exchange on Q-Sepharose® Fast Flow, followed by affinity chromatography on Huprine-Sepharose®.
  • the BChE recovered from huprine-Sepharose® is at least 98% pure, showing monomers and dimers.
  • Figure 9 shows a nondenaturing gel stained with Coomassie blue.
  • the samples from Figure 8 were applied to a nondenaturing gel.
  • the gel confirms that the huprine-purified BChE is at least 98% pure and consists entirely of tetramers.
  • the "pure BChE” was purified on procainamide affinity gel and ion exchange gel and shows a low amount of BChE trimers.
  • Figure 10 shows an SDS gel stained with Coomassie blue.
  • Lane 2 human plasma
  • lane 3 plasma proteins that did not bind to huprine-Sepharose® and includes albumin
  • lanes 4-9 BChE purified from plasma in a single step on Huprine- Sepharose®
  • lanes 10-1 1 pure BChE
  • lane 12 BChE purified from plasma on procainamide-Sepharose®.
  • Comparison of lanes 9 and 12 shows that
  • FIG. 7A is a top view of the BChE tetramer where each 574 amino acid subunit is represented by a different shading.
  • FIG. 7B is a side view of the BChE tetramer, showing the flexible tetramerization domain protruding from the globular BChE structure.
  • BChE dimers and monomers can be quantitatively converted to tetramers in vitro by incubation of a polyproline peptide with BChE (e.g., concentrated rBChE). This is the first demonstration that cellular machinery is not required for assembly of BChE subunits into tetramers.
  • culture medium contains proteases from lysed cells (Blonget al. (1997) Biochem. J., 327:747-757).
  • proteases from lysed cells Blonget al. (1997) Biochem. J., 327:747-757).
  • Experiments that used high concentrations of rBChE were successful when the rBChE was partially purified by passage through a procainamide affinity column. The formation of tetramers was less successful when culture medium was concentrated without a purification step.
  • the proteases in the concentrated culture medium cleaved off the C-terminal tetramerization domain of BChE, the site that is required for interaction with polyproline (Blonget al. (1997)
  • BChE activity is unaffected by loss of the 40 amino acids in the tetramerization domain, but deletion of the tetramerization domain yields BChE monomers.
  • BChE tetramers - but not dimers and monomers - have a long residence time in the circulation.
  • BChE tetramers are protected from proteases and from clearance by their sugar coating.
  • a total of 36 N-glycans coat the surface of the BChE tetramer, constituting 25% of the molecular weight of the protein.
  • BChE dimers and monomers have an exposed face that is unprotected by glycans, leading to clearance from the circulation in minutes.
  • Native plasma derived BChE consists of tetramers, but recombinant BChE consists predominantly of dimers and monomers.
  • pretreatment with human BChE provides protection from the toxicity of nerve agents.
  • the experiments reported herein used a mammalian expression vector containing glutamine synthetase as the selectable marker to create a stable cell line of Chinese hamster ovary (CHO) cells expressing human BChE.
  • the BChE was secreted into serum free culture medium and purified by procainamide affinity chromatography.
  • Synthetic polyproline peptides ranging in size from 8mers to 300mers were incubated with the recombinant BChE to test their effect on assembly into tetramers. Tetramers, dimers, and monomers of BChE were visualized on nondenaturing gels stained for BChE activity. Based on these results a plasmid expressing a 17mer polyproline peptide was selected and designed for transfection into the BChE-expressing cell line to determine the effect on tetramerization.
  • BChE protein concentrations 65 ⁇ (4000 U/ml) assembled into tetramers in the presence of 100 ⁇ polyproline in 1.5 hours at 25°C.
  • the polyproline construct transfected into a BChE expressing cell line did notprovide the level of proline needed to achieve optimal tetramerization based on the synthetic studies.
  • butyrylcholinesterase (BChE) tetramers are provided.
  • the methods comprise incubating BChE with a polyproline peptide.
  • the method is performed in the absence of cells (i.e., cell-free conditions).
  • the polyproline peptide is at least 10 amino acids in length, particularly at least 15 amino acids in length.
  • the polyproline peptide is about 10 to about 250 amino acids in length, particularly about 10 to about 200 amino acids, about 10 to about 100 amino acids, about 10 to about 75 amino acids, about 15 to about 60 amino acids, or about 15 to about 50 amino acids.
  • the polyproline peptide is a proline rich peptide comprising at least 50% proline, particularly at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% proline.
  • the non-proline amino acid is not a basic amino acid (e.g., lysine, arginine, histidine) nor an acidic amino acid (e.g., aspartic acid, glutamic acid).
  • the non-proline amino acid is a neutral amino acid (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, alanine, valine, leucine, isoleucine, phenylalanine, methionine, tryptophan), particularly a non- aromatic, neutral amino acid (e.g., glycine, asparagine, glutamine, serine, threonine, cysteine, alanine, valine, leucine, isoleucine, methionine).
  • a neutral amino acid e.g., glycine, asparagine, glutamine, serine, threonine, cysteine, alanine, valine, leucine, isoleucine, methionine.
  • the non-proline amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, and methionine.
  • the polyproline peptides of the instant invention may comprise a stretch of at least about 10 or at least about 15 proline residues in a row.
  • the BChE of the instant invention can be from any species.
  • the BChE is mammalian, particularly human.
  • Human BChE is described in GenBank Gene ID: 590 and GenBank Accession Nos. NM_000055.2 and NP_ 000046.1 provide examples of amino acid and nucleotide sequences for human BChE.
  • the BChE may be present at any concentration in the instant methods.
  • the BChE is concentrated.
  • the concentration of BChE may be at least 1 nM, at least 10 nM, at least 100 nM, at least ⁇ ⁇ , at least 10 ⁇ , at least 25 ⁇ , or at least 50 ⁇ .
  • the polyproline peptide may also be present at any concentration in the instant methods, particularly in excess of the concentration of BChE.
  • the ratio of the concentration of BChE to the concentration of the polyproline peptide is at least about 1 :0.1, at least about 1 :1, at least about 1 : 10, or at least about 1 : 100.
  • the ratio of the concentration of BChE to the concentration of the polyproline peptide is about 1 :0.1 to about 1 :10 6 , about 1 :0.1 to aboutl :10 5 , about 1 :0.1 to about 1 :10 4 , about 1 :0.1 to about 1 : 1000, about 1 :0.1 to about 1 : 100, about 1 : 1 to about 1 :10, about 1 : 1 to about 1 :5, or about 1 : 1 to about 1 :3.
  • the concentration of BChE is at least about 50 ⁇ and the concentration of the polyproline peptide is at least about 100 ⁇ .
  • the BChE may be purified and/or concentrated from natural sources (e.g., blood) or may be expressed recombinantly. In a particular embodiment, the BChE is expressed recombinantly. In a particular embodiment, the BChE is isolated. The BChE may be purified from other proteins, particularly proteases, that are present in the natural source or present upon recombinant expression. In a particular
  • the BChE is purified over a procainamide or huprine affinity column.
  • the BChE and the polyproline peptide may be incubated at any temperature for a desired length of time.
  • the BChE and the polyproline peptide are incubated at about 20°C or higher, about 24°C or higher, about 30°C or higher, or about 37°C or higher.
  • the BChE and the polyproline peptide are incubated together for at least about 30 minutes, at least about one hour, at least about 5 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 24 hours, at least about 36 hours, or at least about 48 hours.
  • methods of purifying BChE are provided. While the purification methods are exemplified with BChE, the methods can be used to purify other cholinesterases, such as
  • the methods comprise purifying BChE from recombinant preparation, blood or plasma, particularly Cohn Fraction IV.
  • the Cohn process is a five-step purification process for the extraction of albumin from blood plasma, where the alcohol (e.g., ethanol) concentration and pH is modulated with each step in sub 0°C conditions to result in different precipitates (i.e., the fractions).
  • Thealbumin is located in Fraction V.
  • Fraction I is precipitated under about 8% ethanol, pH -1.2; Fraction II is precipitated under about 25% ethanol, pH of -6.9; and Fraction III is precipitated under about 18% ethanol, pH -5.2.
  • Fraction IV the ethanol concentration is raised from 18 to about 40% and the pH is raised from -5.2 to -5.8.
  • red blood cell acetylcholinestrase is purified from a red blood cell sample or preparation.
  • the purification method comprises purifying the Cohn Fraction IV over a huprine affinity column such as a huprine-Sepharose® column.
  • Huprines are acetylcholinesterase (AChE) inhibitors that are hybrid compounds of tacrine and huperzine, particularly hybrid compounds that combine the carbobicyclic substructure of huperzine A with the 4-aminoquinoline substructure of tacrine.
  • the huprine or derivative thereof may be attached to any solid support or resin to generate an affinity column for purification.
  • Huprine derivatives are described, for example, in U.S. Patent Application Publication No. 2013/0029941 (e.g., Hup 24), Ronco et al. (2011) CHEM. Med.
  • the huprine is a huprine described in U.S. Patent Application Publication No. 2013/0029941, incorporated by reference herein, (e.g., huprine derivatives of formula I, la, Ia-1, Ia-2, Ia-3, lb, or Ib-1 or any one of Hupl-Hup42, particularly Hup24).
  • the huprine is Hup 24 (9-(2-aminoethyl)-3-chloro-6,7,10,l l-tetrahydro-7,11- methanocycloocta[b]quinoli -12-amine ditrifluoroacetic acid).
  • Hup 24 (9-(2-aminoethyl)-3-chloro-6,7,10,l l-tetrahydro-7,11- methanocycloocta[b]quinoli -12-amine ditrifluoroacetic acid).
  • the huprine is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the method comprises contacting the huprine with Sepharose® 4B resin (e.g., ECH-Sepharose® 4B resin or EAH-Sepharose® 4B resin).
  • Sepharose® 4B resin e.g., ECH-Sepharose® 4B resin or EAH-Sepharose® 4B resin.
  • the method may comprise contacting huprine (e.g., 90 ⁇ , dissolved in H 2 0/15% MeOH at pH 5.0) with Sepharose® 4B resin (GE-Healthcare; ⁇ 12-16 ⁇ ⁇ of active sites/ml drained matrix).
  • the coupling may be performed in accordance with the manufacturer's instructions for coupling compounds to the resin.
  • the coupling may be catalyzed with an excess of carbodiimide (e.g., 0.1 M final; e.g., N-(3-dimethylaminopropyl)-N'- ethylcarbodiimine (ED AC)).
  • carbodiimide e.g., 0.1 M final; e.g., N-(3-dimethylaminopropyl)-N'- ethylcarbodiimine (ED AC)
  • the re-suspended Cohn Fraction IV may then be passed through the column, which will retain the BChE.
  • the column may be subsequently washed to remove unbound proteins.
  • the BChE may be eluted with a cation which can bind the cation binding site of BChE, e.g.,tetramethylammonium (e.g., 0.5 M) and/or decamethonium (1 mM).
  • tetramethylammonium and decamethonium are used to elute the BChE.
  • the method may optionally comprise a further size exclusion chromatography to remove any residual contaminants or aggregates.
  • the methods comprise administering to a subject the BChE tetramers made by the methods of the instant invention.
  • the nerve toxicity is caused by exposure to an organophosphate agent (e.g., nerve agent, pesticide, insecticide, etc.).
  • organophosphate agents include without limitation, sarin, soman, VX, and tabun.
  • the BChE may be administered before or after exposure to the organophosphate agent, particularly within 24 hours, within 12, within 6, or within 1 hour of exposure.
  • the complex is administered at least one hour prior to potential exposure to the organophosphate agent.
  • compositions comprising the BChE tetramer of the instant invention and a pharmaceutically acceptable carrier.
  • Such compositions may be administered, in a therapeutically effective amount, to a patient in need thereof for the treatment and/or prevention of a disease or disorder, such as nerve toxicity due to exposure to an organophosphate agent.
  • compositions of the present invention can be administered by any suitable route, for example, by injection (e.g., for local (direct) or systemic administration (intravenous)), oral, pulmonary, topical, nasal or other modes of administration.
  • the composition may be administered by any suitable means, including parenteral, intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous, topical, inhalatory, transdermal, intraocular, intrapulmonary, intrarectal, and intranasal administration.
  • the composition is injected into the bloodstream (e.g., intravenously).
  • the pharmaceutically acceptable carrier of the composition is selected from the group of diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • the compositions can include diluents of various buffer content (e.g., TrisHCl, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., polysorbate 80), anti oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol).
  • buffer content e.g., TrisHCl, acetate, phosphate
  • pH and ionic strength e.g., TrisHCl, acetate, phosphate
  • additives e.g., polysorbate 80
  • anti oxidants e.g., ascor
  • compositions can also be incorporated into particulate preparations of polymeric compounds such as polyesters, polyamino acids, hydrogels, polylactide/glycolide copolymers, ethylenevinylacetate copolymers, polylactic acid, polyglycolic acid, etc., or into liposomes.
  • polymeric compounds such as polyesters, polyamino acids, hydrogels, polylactide/glycolide copolymers, ethylenevinylacetate copolymers, polylactic acid, polyglycolic acid, etc., or into liposomes.
  • Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of components of a pharmaceutical composition of the present invention. See, e.g., Remington's Pharmaceutical Sciences, (Mack Publishing Co., Easton, PA 18042).
  • the pharmaceutical composition of the present invention can be prepared, for example, in liquid form, or can be in dried powder form (e.g., lyophilized).
  • the composition of the instant invention may be administered for the treatment, inhibition, and/or prevention of neuron toxicity due to exposure to an organophosphate agent.
  • the dosage ranges for the administration of the composition of the invention are those large enough to produce the desired effect (e.g., treatment, inhibition, and/or preventing the neural toxicity).
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counter indications.
  • An effective amount of a drug is well known in the art and changes due to the age, weight, severity of a subject's condition, the particular compound in use, the strength of the preparation, and the mode of administration.
  • the determination of an effective amount is preferably left to the prudence of a treating physician, but may be determined using methods well known in the art.
  • the compositions of the invention may be prepared using methods known in the art.
  • treat refers to any type of treatment that imparts a benefit to a patient afflicted with a disease or disorder, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.
  • the term "prevent” refers to the prophylactic treatment of a subject who is at risk of developing a condition (e.g., neuron toxicity) resulting in a decrease in the probability that the subject will develop the condition.
  • a condition e.g., neuron toxicity
  • a “therapeutically effective amount” of a compound or a pharmaceutical composition refers to an amount effective to prevent, inhibit, or treat a particular disorder or disease and/or the symptoms thereof.
  • “therapeutically effective amount” may refer to an amount sufficient to reduce the effects of an organophosphate in a subject.
  • the term “subject” refers to an animal, particularly a mammal, particularly a human.
  • “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • a “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimerosal, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., TrisHCl, acetate, phosphate), bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered.
  • preservative e.g., Thimerosal, benzyl alcohol
  • anti-oxidant e.g., ascorbic acid, sodium metabisulfite
  • solubilizer e.g., polysorbate 80
  • emulsifier e.g., TrisHCl, acetate, phosphate
  • bulking substance e.g., lactose, mannitol
  • Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
  • the compositions can be incorporated into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes or micelles. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of components of a pharmaceutical composition of the present invention.
  • the pharmaceutical composition of the present invention can be prepared, for example, in liquid form, or can be in dried powder form (e.g., lyophilized). Suitable
  • isolated refers to the separation of a compound from other components present during its production. "Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not substantially interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.
  • isolated protein or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form. "Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.
  • substantially pure refers to a preparation comprising at least 50-
  • the compound of interest e.g., nucleic acid, oligonucleotide, protein, etc.
  • Purity may be measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).
  • solid support refers to any solid surface including, without limitation, any chip (for example, silica-based, glass, or gold chip), glass slide, membrane, plate, bead, resin, solid particle (for example, agarose, Sepharose®, polystyrene or magnetic bead), column (or column material), test tube, or microtiter dish/plate.
  • chip for example, silica-based, glass, or gold chip
  • membrane for example, glass slide, membrane, plate, bead, resin, solid particle (for example, agarose, Sepharose®, polystyrene or magnetic bead), column (or column material), test tube, or microtiter dish/plate.
  • PPPPPPPPPP (designated P8; SEQ ID NO: 1), PPPPPPPPPPPPPPPPPPPPPPPPP (designated P17; SEQ ID NO: 2), PPPPPPPPPPPPPPPPPLP(designated P15LP; SEQ ID NO: 3), and AS SP ALPTQP AEE AARKREVR (designated random peptide; SEQ ID NO: 4).
  • Poly-L-proline peptides containing about 50 or 300 consecutive prolines were from Sigma-Aldrich (catalog # P2254 and P3886).
  • L-Methionine sulfoximine was from Sigma (catalog M5379).
  • the mammalian expression vector pcDNA3 The mammalian expression vector pcDNA3, the
  • lipofectamine transfection reagent and G418 were from Invitrogen.
  • the plasmid vector containing glutamine synthetase as a selectable marker is similar to pcDNA3 but has the rat glutamine synthetase gene in place of the neomycin gene.
  • Chinese Hamster Ovary (CHO-K1) cells ATCC 61-CCL were from the American Type Culture Collection. Serum free culture medium, UltraCULTURETM without L- glutamine, was from BioWhittaker (Walkersville, MD, catalog 12-725F).
  • Procainamide-Sepharose® (Grunwaldet al. (1997) J. Biochem. Biophys. Methods 34:123-135) for purifying BChE was synthesized to contain 34 ⁇ procainamide bound per ml on a 6-carbon spacer arm.
  • Amicon Ultra- 15 centrifugal filters with 10,000 MW cut off membrane were from Millipore (Billerica, MA; catalog
  • BChE activity was measured with 1 mM butyrylthiocholine iodide in the presence of 0.5 mM 5,5'-dithiobis(2-nitrobenzoic acid) in 0.1 M potassium phosphate pH 7.0 at 25 °C.
  • the increase in absorbance at 412 nm was monitored on a Gilford spectrophotometer and recorded on a Macintosh computer interfaced to MacLab (AD Instruments).
  • Units of activity are micromoles hydrolyzed per minute at pH 7.0, 25°C.
  • a 1 mg/ml solution of pure human BChE has an activity of 720 units/ml and an absorbance of 1.8 at 280 nm.
  • a stable CHO Kl cell line expressing full-length human BChE was created by calcium phosphate transfection with a plasmid containing the glutamine synthetase as the selectable marker (Duysenet al. (2002) J.Pharmacol. Exp.Ther., 302:751-758).
  • the accession numbers are gi: 1280204 for the mRNA and P06276 for the BChE protein.
  • the mature human BChE contains 574 amino acids and 9 N-glycans for a molecular weight of 85 kDa per subunit. Cells were grown in UltraCULTURETM in the presence of 25 ⁇ methionine sulfoximine.
  • Culture medium from a T25 flask of confluent cells had a BChE activity of 1 U/ml.
  • Culture medium 100 ⁇ was incubated with 80 ⁇ g peptide for 2 hours at room temperature before 25% of the solution was loaded on the gel.
  • the final BChE and peptide concentrations were 16 nMBChE and 1 mM 8mer P8 for a 60,000 fold molar excess of polyproline over BChE; 16 nMBChE and 0.5 mM 17mer P17 for a 30,000 fold molar excess of polyproline over BChE: 16 nMBChE and 0.15 mM 50mer P50 for a 9,600 fold molar excess over BChE.
  • a polyproline gene encoding 17 L-proline residues and a 17 amino acid signal peptide was inserted into plasmid pcDNA3 for expression from the CMV promoter.
  • the polyproline plasmid was transfected with lipofectamine into CHO cells that already expressed BChE. Cells were grown in UltraCULTURETM containing 800 ⁇ g/ml G418 and 25 ⁇ methionine sulfoximine. Purification of rBChE
  • CHO cells secreting human rBChE were grown in roller bottles. The cells had been transfected with the human BChE plasmid, but not with the polyproline plasmid.
  • the serum-free culture medium collected in a period of 3 weeks had 3600 units of BChE activity (5 mg BChE protein) in a volume of 1.9 liters.
  • the culture medium was filtered to remove particulates, which clogged the chromatography media if left in place.
  • the filtered culture medium was applied to a 20 ml procainamide- Sepharose® affinity column packed in Pharmacia column CI 6/20. 96% of the starting BChE activity bound to the affinity column.
  • the column was washed with 500 ml of 20 mM TrisCl pH 7.5, 1 mM EDTA, 0.05% sodium azide.
  • BChE was eluted at room temperature with a gradient consisting of 250 ml of 20 mMTrisCl pH 7.5, 1 mM EDTA, 0.05% sodium azide versus 250 ml of 1 M NaCl in buffer. A total of 2700 units (75%) of 100-fold purified BChE were recovered.
  • the BChE was concentrated and desalted in an Amicon 10,000 Molecular Weight Cut off Centrifugal Filter. The final yield after desalting was 57% of the starting BChE activity.
  • rBChE was stored at 4 ° C in 20 mM TrisCl pH 7.5, 1 mM EDTA, 0.05% azide.
  • Concentrated rBChE samples with activities of 4000, 3200, 220, 150, and 20 U/ml were incubated with 100 ⁇ polyproline (Sigma 50mer, average MW 4800) for 1.5 hours at room temperature.
  • a 0.5 ⁇ solution of partially purified rBChE (29.3 U/ml) was incubated with 5 mM polyproline (Sigma 50mer) for 35 days at 4°C.
  • Samples were diluted to 2 U/ml in preparation for loading 0.02 units of rBChE per lane on a nondenaturing gel.
  • Product number P3886 (Sigma-Aldrich) CAS 25191-13-3 is a polyproline protein with a molecular weight greater than 30,000. This is an unusual protein because it is invisible on a gel stained with Coomassie blue and invisible in the spectrophotometer at 260 and 280 nm. It dissolves in cold water, but precipitates at room temperature. The lyophilized material is fluffy and occupies a large volume relative to its weight. Preparation of a 50 mg/ml solution required 3 days incubation at 4 ° C before the material dissolved in water. The 50 mg/ml stock solution of big polyproline was diluted with water to make 5 mg/ml and 1 mg/ml solutions.
  • Figure 1 (lanel) confirms that about 50-70% of the rBChE in the culture medium is a dimer, 15-40% is a monomer, and 10-30% is a tetramer (Blong et al. (1997) Biochem. J., 327:747-757).
  • the sample in lane 1 contained no added polyproline. However, a small amount of BChE tetramer is present, indicating that the CHO cells synthesized the polyproline peptides incorporated into the tetrameric rBChE in lane 1.
  • polyproline peptides present in native tetrameric BChE are assumed to derive from precursor proteins (Li et al. (2008) Biochem. J., 411 :425-432;
  • Concentrated rBChE was prepared after the culture medium was depleted of its major contaminating proteins by chromatography on a procainamide affinity column.
  • a rBChE solution with an activity of 76 U/ml was diluted with polyproline P50 to a BChE concentration of 0.5 ⁇ (29.3 u/ml) and a polyproline concentration of 5 mM in 20 mM TrisCl, 1 mM EDTA pH 7.5, 0.05% sodium azide.
  • the solution was incubated at 4°C for 35 days before the sample was diluted to 2 U/ml for analysis using gel electrophoresis.
  • Comparison of lanes 2 and 3 in Figure 4A shows that 5 mM polyproline converted more than 95% of the BChE to tetramers.
  • Lanes 2 and 3 in Figure 4B shows that partial purification of rBChE on procainamide affinity gel removed albumin.
  • Lane 4 in Figures 4A and 4B shows that culture medium concentrated to the same BChE activity as the samples in lanes 2 and 3, but without chromatography on procainamide affinity gel, resulted in degradation of the rBChE during storage.
  • the lower rBChE concentrations with 220 and 150 U/ml also showed conversion of dimers to tetramers but the conversion was less complete.
  • the 20 U/ml rBChE showed only a marginal increase in the intensity of the tetramer band following incubation with 0.1 mM polyproline P50 for 1.5 hours. It was concluded that assembly of rBChE subunits into tetramers is efficiently accomplished with high rBChE concentrations (e.g., in the 0.050 to 0.065 mM range and a polyproline concentration of 0.1 mM).
  • a stable CHO cell line that secretes human BChE was transfected with a plasmid that encodes a 17mer polyproline peptide.
  • the secreted BChE was visualized on a nondenaturing gel stained for BChE activity.
  • the proportion of BChE tetramers was not significantly different from that in Figure 1, lane 1. It was concluded that the cell line produced an inadequate supply of polyproline. However, increasing the expression of the polyproline would result in the formation of BChE tetramers.
  • BChE has been shown to be effective as a bioscavenger of nerve agents such as sarin.
  • BChE is a naturally occurring protein found in serum, but at very low concentrations.
  • BChE is found in a more concentrated form in a byproduct of the production of albumin from human serum called Conn Fraction IV paste.
  • Cohn Fraction IV paste is a rich source of human BChE.
  • Procedures for purification of BChE include filtration, procainamide affinity chromatography, and ion exchange chromatography (Lockridge et al. (2005) J. Med. Chem. Biol. Radiol. Def,
  • Procainamide affinity chromatography is an essential step in the purification method. However, the process yields high purity product only with multiple additional steps.
  • the identification of the ability of huprine to bind and purify recombinant BChE produced by cultured insect cells led to the investigation of whether the purification of BChE from human plasma and Cohn paste could be improved. Results demonstrate that the huprine purification resulted in an
  • the huprine affinity gel has two major advantages over the procainamide affinity gel. First, highly purified BChE is recovered from the huprine affinity gel. Second, the binding capacity of the huprine affinity gel is about 10-fold greater than that of the procainamide gel. This means 10- fold less affinity gel is required, thereby reducing the cost of the purification process.
  • the data provided herein demonstrates that the process can be used not only for purification of BChE or rBChE from Cohn Fraction IV paste but also plasma, human red blood cell preparations and recombinant expression systems. This significantly extends the importance of the approach as it can be used to study human red cell acetylcholinesterase, as well as recombinant BChE.

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Abstract

L'invention concerne des procédés et des compositions pour la production de tétramères de butyrylcholinestérase (BChE) et la purification de BChE.
PCT/US2014/066373 2013-11-19 2014-11-19 Compositions et procédés pour la purification et la production de butyrylcholinestérase Ceased WO2015077317A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3945092A1 (fr) 2020-07-27 2022-02-02 Etat Français représenté par la Direction Centrale Du Service de Santé des Armées Oximes de carbaldehydes en tant que réactivateurs de butyrylcholinestérase

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060253913A1 (en) * 2001-12-21 2006-11-09 Yue-Jin Huang Production of hSA-linked butyrylcholinesterases in transgenic mammals
US20130071394A1 (en) * 2011-09-16 2013-03-21 John K. Troyer Compositions and combinations of organophosphorus bioscavengers and hyaluronan-degrading enzymes, and methods of use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060253913A1 (en) * 2001-12-21 2006-11-09 Yue-Jin Huang Production of hSA-linked butyrylcholinesterases in transgenic mammals
US20130071394A1 (en) * 2011-09-16 2013-03-21 John K. Troyer Compositions and combinations of organophosphorus bioscavengers and hyaluronan-degrading enzymes, and methods of use

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MASSON ET AL.: "Butyrylcholinesterase for protection from organophosphorus poisons: catalytic complexities and hysteretic behavior.", ARCH BIOCHEM BIOPHYS., vol. 494, no. 2, 15 February 2010 (2010-02-15), pages 107 - 120, XP026883610, DOI: doi:10.1016/j.abb.2009.12.005 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3945092A1 (fr) 2020-07-27 2022-02-02 Etat Français représenté par la Direction Centrale Du Service de Santé des Armées Oximes de carbaldehydes en tant que réactivateurs de butyrylcholinestérase
WO2022023315A1 (fr) 2020-07-27 2022-02-03 État Français Représenté Par La Direction Centrale Du Service De Santé Des Armées Oximes de carbaldéhyde utilisés en tant que réactivateurs de la butyrylcholinestérase

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