WO1992008790A1 - Conjugates of poly(vinylsaccharide) with proteins for the stabilization of proteins - Google Patents
Conjugates of poly(vinylsaccharide) with proteins for the stabilization of proteins Download PDFInfo
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- WO1992008790A1 WO1992008790A1 PCT/US1991/008493 US9108493W WO9208790A1 WO 1992008790 A1 WO1992008790 A1 WO 1992008790A1 US 9108493 W US9108493 W US 9108493W WO 9208790 A1 WO9208790 A1 WO 9208790A1
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- 0 CCC(C)(*)N=NC Chemical compound CCC(C)(*)N=NC 0.000 description 6
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
- C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
- C07H5/06—Aminosugars
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6815—Enzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6883—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/12—Acyclic radicals, not substituted by cyclic structures attached to a nitrogen atom of the saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1292—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Actinomyces; from Streptomyces (G)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/34—Introducing sulfur atoms or sulfur-containing groups
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/06—Enzymes or microbial cells immobilised on or in an organic carrier attached to the carrier via a bridging agent
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/545—Synthetic resin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
- Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
- Y10S530/815—Carrier is a synthetic polymer
- Y10S530/816—Attached to the carrier via a bridging agent
Definitions
- This invention herein relates to polymer protein compounds or protein polymer conjugates, a method of making such compounds and a method of stabilizing proteins in the form of the polymer protein conjugates of the invention in a hostile environment.
- Proteins are used in a variety of applications. Whether the proteins, such as enzymes, antibodies, and the like, are used in diagnostics, the food industry, the bioconversion of organic compounds in either water or organic solutions etc., a common problem with using proteins in all of these applications is the instability of the protein itself. Enzymes, antibodies, and the like would be more useful if they were stable in a soluble or an insoluble form at elevated temperatures, in nonoptimal aqueous solutions or in organic solutions while retaining the ability to recognize and bind molecules, and in some cases, exhibit catalytic activity. The protection of proteins against proteolytic attack is also highly desirable.
- Proteins including enzymes are quite sensitive to their environment. Such environmental conditions include the pH of the solution, temperature, shear forces, the presence of organic solvents, buffer
- the stability of a protein is in reference to its conformational stability and biological activity. The effect of temperature, organic solvent, pH, and storage on the stability of the protein is largely determined by the conformational stability of the
- Inactivation of the protein is primarily due to denaturation of the protein; however, changes in the protein (such as chemical modification) can also occur, resulting in inactivation without denaturation of the protein.
- changes in the protein such as chemical modification
- the protein may be partially or fully denatured which results in a decrease or loss of activity.
- immobilization can give enzymes that have enhanced, diminished, or unchanged activity relative to the native enzymes. Examples of each effect exist (For a review of the effect of immobilization on the stability of enzymes, see: Zaborsky Immobilized Enzymes CRC Press: Boca Raton at 49-165 (1978)). For example, one possible way to stabilize the structure and reactivity of a protein such as an enzyme is by immobilization on a soluble or
- insoluble support In addition to the possible stabilization of the protein, immobilization on insoluble supports is desirable because of the ease of recovery of the protein and the prevalent use of equipment for applications which use insoluble material.
- Advantages of immobilization of proteins on a soluble support include higher rates of transfer of substrate to the protein and greater accessibility of the substrates to the protein.
- Another advantage to the use of a soluble support is that the solubility of the system allows the use of reactor configurations such as
- difficulties associated with the immobilization of proteins including enzymes using known methodologies include: necessary prior activation of the polymeric support, relatively harsh chemical treatments, which destroy or reduce the activity of the protein, necessary for the immobilization process, and the number of functional groups, or the density of functional groups available for the attachment on the polymeric support.
- the methods do not offer a general solution to the stabilization and immobilization of protein structure.
- Another object of this invention is to provide a method for stabilizing a protein in an environment which would reduce an intended functional property of the protein.
- Still another object of this invention is to provide a method for making the polymer protein compounds of the invention.
- Another object of the invention is to provide for a method of synthesizing peptides.
- Still another object of the invention is to provide for a method of assaying using the polymer protein compounds of the invention.
- the invention herein includes a method of stabilizing proteins, is a polymer protein compound and a method for making the polymer protein compounds of the invention.
- the invention also includes the use of the stabilized protein in assays and provides a method of synthesizing peptide bonds.
- the protein is stabilized by covalently bonding it to a particular polymer structure which includes hydroxyl groups in an amount effective for stabilizing the protein.
- the hydroxyl groups and polymer cooperate with other polar groups such as polar groups in the region of the protein to provide the stabilization of the protein.
- the polymer protein compound of the invention provides stabilization to the structure of the protein which is part of the compound of the invention.
- composition of the invention In order to achieve protein stabilization, there are three primary portions of the composition of the invention regardless of the route of synthesis.
- compositions are: a protein, a linker group, and a polymer.
- the protein is bonded to the linker group which in turn is bonded to the polymer.
- the polymer and/or linker provide hydroxyl groups in an amount effective for stabilizing the protein, as
- the polymer must have or permit substitution to provide for one or more pendent groups extending from and along the main chain which pendent groups will be
- the polymer is selected from the group consisting of a polyvinyl polymer, an acrylic polymer, a polyester polymer, a polyamide polymer and mixtures thereof.
- the polymer has a main chain which has the pendant groups. These pendant groups may form part or all of the linker group.
- the linker group is covalently bonded to the polymer main chain through an ester, amide, ether, thioether, thioester, peptide or a carbon-carbon linkage.
- the linker group is covalently bonded to the protein (using methods mild enough that the catalytic and
- the linker group can be derived from linkers which are carbohydrates or polyols.
- the carbohydrate portion of a glycoprotein may be a linker and be bonded directly to the polymer pendant group which is reactive with the glycoprotein.
- the proteins can be enzymes, antibodies, and the like. Forming the protein to linker bond (if the linker is already on the polymer) or the linker to polymer bond (if the protein is already bonded to the linker) or polymerization bonds (if the protein and linker are already attached to a polymerizable group) is done under sufficiently mild conditions so that the catalytic and functional activity of the protein is not destroyed.
- a linker can be incorporated into a monomer which then is polymerized, which polymer is subsequently bonded to the protein; the linker may be bonded to a polymer, with the polymer-linker combination subsequently being bonded to the protein; the linker may be bonded to the protein and the protein-linker
- a linker with a polymerizable group may be bonded to a protein and subsequently polymerized to a protein-linkerpolymer.
- an intended functional property of the protein is stabilized such that the functional property is either maintained to be at least equal to the native protein (the protein in its natural state) or it is enhanced over a reduced activity that the protein would have in a hostile
- the linker group includes all of the atoms between the polymer main chain (which is made of the atoms bonded to each other to form the length and longitudinal axis of the polymer) and the amino acid residue of the protein attached to the linker and will separate the main chain of the polymer from the protein by two or more carbon atoms and include at least three hydroxyl groups.
- the linker group will be a saccharide group which includes the residue of a monosaccharide, a disaccharide and a trisaccharide.
- the polymer is an acrylic polymer combined with a saccharide linker which forms a linker group which links the protein to the acrylic polymer.
- the acrylic polymer has a molecular weight of at least 500, preferably at least about 5,000 and most preferably 300,000 or greater.
- Another important aspect of the invention includes polymer protein compounds whereby solubility of the compounds in aqueous and organic solvents can be controlled by cross-linking the polymer protein compounds and by polymerizing the compounds of the invention into gels.
- the polymer protein compound of the invention can be made insoluble which will permit the composition of the invention to be utilized in currently known processing equipment.
- the polymer protein compound or conjugated protein is stable and can be employed under adverse conditions which are hostile to the protein and would reduce an intended functional property of the protein.
- an important aspect of the invention is that new and improved methods for assaying using the compounds of invention are provided.
- the invention herein could be used in a wide variety of industrial,
- trypsin-polymer conjugate and native trypsin in buffer and water.
- Figure 3 describes the activity over time of trypsin, poly(2-N-methacrylamido-2-deoxy-D-glucose)-trypsin conjugate and poly(6-N-methacrylamido-6-deoxy-D-glucose)-trypsin conjugate at 55oC.
- Figure 4 describes the activity of the poly(6-N-methacrylamido-6-deoxy-D-glucose)-chymotrypsin
- FIG. 5 describes the enzyme immunoassay rate (EIA) of M ab B11C2-1 antibody.
- FIG. 6 describes the enzyme immunoassay rate (EIA) of M ab B11C2-1 antibody-poly(2-N-methacrylamido-2-deoxy-D-glucose) conjugate.
- Figure 7 describes the enzyme immunoassay rate (EIA) on plates coated with Ive-Con antigen-poly(2-N-methacrylamido-2-deoxy-D-glucose) conjugate.
- Figure 8 describes the use of M ab B11C2-1 antibody-polymer conjugate and Ive-Con antigen-poly(2-N-methacrylamido-2-deoxy-D-glucose) conjugate in a
- Figure 9 illustrates the stability over time at 60°C of the conjugated monoclonal antibody 8H11 as compared to the native monoclonal antibody 8H11.
- Figure 10 illustrates that the denaturation of both the conjugated monoclonal antibody 8H11 and native monoclonal antibody 8H11 is a first order kinetic
- Figure 11 illustrates the stability of the conjugated monoclonal antibody 8H11 in methanol as compared to the native monoclonal antibody 8H11.
- horseradish peroxidase at different temperatures was measured. Squares represent the activity HRPO which was not conjugated to the polymer. Circles represent the activity of HRPO which was conjugated to the polymer, and dA/dt represents the change in absorbance/minute. In the upper panel, the raw data is shown. In the lower panel, the activity of the conjugated enzyme was standardized to be identical to the untreated enzyme at 25oC.
- Figure 13 illustrates the thermo competence of lactic dehydrogenase (LDH). The activity of lactic dehydrogenase at different temperatures was measured.
- LDH lactic dehydrogenase
- Circles represent the activity of LDH which was not conjugated to the polymer. Circles represent the
- dA/dt represents the change in absorbance/minute.
- the raw data is shown.
- the activity of the conjugated enzyme was standardized to be identical to the untreated enzyme at 25oC.
- Figure 14 illustrates the cleavage patterns of ⁇ DNA digested by conjugated EcoRI in lane 2 and native EcoR1 in lane 1.
- Figure 15 illustrates the cleavage patterns of PBR322 plasmid digested by the conjugated EcoR1 and native EcoR121.
- Figure 16 illustrates the digestion of bovine serum albumin by conjugated trypsin in lane 1 and native trypsin in lane 2. Note that lanes 3, 4 and 5 are molecular weight markers.
- Enzymes, antibodies, and the like are complex proteins each with a specific sequence of amino acids.
- the structure of the protein is critical to the activity of the protein such as the catalytic activity of an enzyme and the capacity of an antibody to recognize ligands.
- the method and compounds of the invention described herein provide a means for stabilizing proteins by protecting or maintaining the structure of the protein by using effective amounts of hydroxyl groups, the structure of a polymer, and in some instances polar groups covalently bonded in the compounds of the
- proteins are stabilized such that if put into a hostile environment, intended function is maintained to be at least equal to the proteins in their natural states or is enhanced over a reduced activity that the proteins would have in the hostile environment.
- Acrylic polymer means a polymer or copolymers of
- alkyls having 1 to 12 carbons
- triethyleneglycol 7-hydroxyheptyl, 3,4- dihydroxybutyl, 5-hydroxypentyl, 6- hydroxyhexyl, 1,3-dimethyl-3-hydroxybutyl, 5,6- dihydroxyhexyl, 7-hydroxyheptyl, a residue of a mono-, di-, and trisaccharide, such as 2-deoxy- D-glucose;
- n 2 to 7.
- Polyacids or “polyester polymer” means the polymerized reaction product of polyacids and polyols; polyacids include diacids such as isophthalic,
- the diacids or polyols must have or permit substitution to provide for one or more pendent groups along the main chain which will be reactive with other compounds to form a linker group or which pendent group itself will form a linker group which will bond the protein to the polymer through the linker group.
- hydroxyl group or ester groups may be such pendent groups extending from the main chain of the polyester.
- the polyols which react to form the main chain of the polyester include linear diols such as
- cycloaliphatic diols such as hydroquinone, 1,4-dihydroxymethyl-cyclohexane and "hydrogenated bisphenol A”
- diol ethers such as diethylene glycol, triethylene glycol and dipropylene glycol
- polyols such as glycerol, pentaerythritol, trimethylol propane,
- Polyamide or “polyamide polymer” means a polymer in which the structural units are linked by an amide or thioamide groupings such as the reaction product of a diamine and dibasic acid.
- Polyvinyl polymer means polymers
- the polymers have a molecular weight of at least about 500, preferably at least about 5,000 and most preferably at least about 300,000. It is believed that molecular weights of the polymers may go as high as 100,000,000 or more.
- Protein means proteins, including proteins modified to include additional amino groups such as lysine groups, polypeptides, enzymes, antibodies, and the like, which are composed of a specific sequence of amino acids.
- Poly group means any group which has an affinity for or an ability to bind with water, such as
- hostile environment means an environment which will reduce a functional property or activity of a protein over a native protein or that which the protein has in its natural state.
- a hostile environment can include temperatures which create adverse thermal environments which could be elevated or reduced
- the compounds of the invention comprise a protein covalently bonded to a polymer through a linker group.
- the protein of the compounds is stabilized so that under hostile conditions an intended functional property of the protein is either maintained to be at least equal to the native protein or it is enhanced over a reduced activity that the protein would have in hostile conditions by providing an environment which includes hydroxyl groups in the region where the polymer, linker group and protein are covalently bonded.
- the hydroxyl groups may be on the polymer or on the linker group and are in an effective amount and close enough to the covalently bonded combination of the polymer, linker group and protein to stabilize the protein as described.
- the hydroxyl groups and polymer cooperate with other polar groups such as polar groups in the region of the protein to provide the stabilization of the protein.
- the linker group will have hydroxyl groups, and as such, in a preferred aspect of the invention, will be a polyol, a carbohydrate or carbohydrate-like.
- the polymer is selected from the group consisting of a polyvinyl
- the polymer has a main chain which has pendent groups. These pendent groups may form part or all of the linker group.
- the main chain and/or the pendent group include hydroxyl groups which stabilize the protein.
- the linker group is covalently bonded to the polymer main chain through an ester, amide, ether, thioether, thioester, peptide (one or more amide linkages), or a carbon to carbon linkage. If the pendent group on the polymer forms all of the linker group, the pendent group should be reactive such that it will covalently bond to the protein.
- the pendent group on the polymer main chain forms part of the linker group between the polymer main chain and the protein, it should be reactive with a linker to permit covalent bonding between the pendent group and the linker. After bonding with the protein, the linker group is completed.
- the linker group is covalently bonded to the protein through an ester, amide, ether, thioether, thioester, peptide (one or more amide linkages) or amine linkage.
- this is synthetically achieved by using methods mild enough that the catalytic and
- the linker group includes all atoms between the polymer main chain and the amino acid residue of the protein attached to the linker group and will separate the main chain of the polymer from the protein by two or more carbon atoms and have at least three hydroxyl groups.
- a linker is a precursor to a linker group.
- the linker group is a saccharide group which includes the residue of a monosaccharide, a disaccharide and a trisaccharide.
- a linker group which includes hydroxyl groups stabilizes the protein; and while there is not necessarily a defined limited as to the exact number of carbon atoms in a linker group, steric and kinetic considerations limit the size of the linker group to a total of about 60 carbon atoms.
- the polymer, linker group and protein can be attached to one another, such as shown below:
- Polymer main chain of a polymer selected from the group consisting of a polyvinyl polymer, a
- polyacrylic polymer a polyester polymer and a polyamide polymer
- Protein residue of a protein
- R 1 and R 2 H, OH, acetoxy, alkoxy, phenylene and all of the substituents defined for R in the
- acrylic polymer halogen and phosphate
- R 3 all of the substituents defined for R in the definition of acrylic polymer; having at least three hydroxyl
- n at least 2
- R 1 , R 2 , R 4 and R 5 are selected from the group consisting of H, OH and alkyl having 1 to 4 carbons.
- R 3 H.
- the polymer is covalently bound to and substituted with at least about 1 weight percent of the linker groups.
- the protein will be bonded to the polymer linker group combination at least at one point on the protein, however, stabilization of the protein will be increased as the number of bonding points between the protein with a linker polymer combination increases.
- polymer there may be more than one protein bonded to one polymer and one protein may be bonded to more than one polymer to create a matrix of polymer chains which are linked by covalent bonds.
- the protein has functional groups, which on interaction with the linker, will provide the residue B when subjected to an appropriate reaction with the linker.
- a linker which includes a saccharide proteins that have pendant amino functionality are preferred, such as those proteins which contain lysine and arginine groups.
- the protein contains more than one functionality such as a e-lysine residue for attachment to the linker and preferably, the protein does not contain ⁇ -lysine residues in the active site.
- the protein contains at least one lysine residue and preferably greater than 3 surface lysine residues.
- the pendant amino functionality on the protein readily attaches a saccharide linker which is in turn attached to the polymer.
- the linker group can include a saccharide based compound which will have aldehyde or lactone functionality capable of forming covalent bonds with the amino functionality of the protein molecule. This pendant aldehyde or lactone functionality on the
- saccharide may be protected as hereafter described.
- An advantage of the invention is that in connection with the saccharide linker when there is an aldehyde precursor, the linked protein may be protonated at the nitrogen of the protein (which nitrogen is incorporated into B) at a pH similar to the pH of the protein. This protonation is not permitted with the lactone precursor.
- R 1 and R 2 are independently alkyls having 1 to 4 carbons or H group
- R 7 all of the substituents defined for R in the definition of acrylic polymer; wherein A is defined above, B is defined above, L is defined above,
- n 0 to 50,000
- T 10 to 50,000; the total repeating units represented by p, n and T is not more than 50,000.
- the protein is bonded to the polymer by at least one covalent bond, but may be covalently bonded at multiple points along the protein.
- the above formula does not necessarily represent a .protein as being repeated in the parenthesis, but more than one protein may be represented by the above formula as being bonded to the same polymer.
- the protein can be linked or bonded to another polymer chain to provide cross- linking of the compounds of the invention through a protein, as will be more fully described infra.
- the polymer is an acrylic polymer and where the linker group includes residues of
- these linkers are residues of monosaccharides or
- the monosaccharides are preferably 2-amino-2-deoxy-D-glucose, 3-amino-3-deoxy-D-glucose, or 6-amino-6-deoxy-D-glucose.
- the linker is most preferably 6-amino-6-deoxy-D-glucose.
- the polydispersity of the polymer materials is not critical, although it is highly
- the polymer may also be in the form of either random, regular, or block copolymers. For example, the copolymerization of 18 with methacrylamide (19) gives (20).
- the polymers can be block, random, or homopolymers although a random or homopolymer is desirable, a homopolymer is the most desirable.
- a polymer with n values of 1 - 100 and p values of 1 - 5 and the total molecular weight greater than 500,000 is highly desirable.
- linker (20) and (21) could also be in the form of a di- or
- trisaccharide or could be a five-membered ring or a six-membered ring joined to a five-membered ring.
- the monomer or polymerizable group as defined in the polymer above in Formula III, the group in the parentheses with the repeating integer n is used to form the polymer chain above or along with the comonomer if so desired.
- the monomer group can be monomers which provide the acrylic polymer as defined above.
- a very important aspect of the invention is where a protein is an enzyme or antibody, or a mixture of enzymes and antibodies, especially when linked to the polymer through a residue which includes a saccharide.
- Enzymes which may be used in the invention include enzymes used in bioprocessing industries, in medical tests and in quick tests for toxins. Enzymes important to the invention include but are not limited to
- transferases such as cycloglucanotransferases (CGTase); proteases such as metalprotease, Staph V8 protease, rennet, papain, subtilisin, ficin, rennin, pronase E, bromelain and [neutral] proteases such as pepsin;
- CGTase cycloglucanotransferases
- proteases such as metalprotease, Staph V8 protease, rennet, papain, subtilisin, ficin, rennin, pronase E, bromelain and [neutral] proteases such as pepsin;
- oxgenases such as lipooxygenase; hydrolases such as lipase, [phospho]-lipase, cellulase hemicellulase, ⁇ -glucanase, ⁇ -glucanase, trypsin, pectinase,
- cellobiohydrolase [alpha-beta-]-[gluco-galacto-]-sidases, transglucosidase, pullanase, cellobiase,
- sucrases [dextran-levan] sucrases; isomerases such as xylose isomerase and glucose isomerase; amylases such as alpha amylase, beta amylase, fungal alpha amylase and
- glucoamylase oxidases such as glucose oxidase, glucose (1,2)-oxidase, peroxidase, catalase and horseradish peroxidase; esterases such as cholestrol esterase, phytase and pectinmethyl esterase.
- the linker group may be a saccharide which may have six or five membered rings as shown in formulas I or II below.
- the six-membered ring is selected from the group consisting of Formulas Ia, Ib, Ic, and Id each of which is shown below.
- the five-membered ring saccharide is selected from the group consisting of Formulas IIa, IIb, IIc and lid each of which are shown below.
- a and B are shown in the formulas below to show how the saccharide ring(s) are bonded to the polymer through A and the protein through B.
- n 0 or 1;
- R 1 through R 9 selected from the group consisting of H, OH, alkoxy, acetoxy, all of the
- R 1 through R 9 are H, OH, or an alkyl having 1 to 4 carbon atoms.
- the polymer can be bound to the second sugar in the disaccharide or the third saccharide ring from the ring bonded to the protein. Further, although not shown in the above formulas, five and six membered rings may be bonded together to form one
- the polymer protein compound of the invention can be prepared in several ways.
- the polymer protein compound of the invention may be made by bonding a polymer or monomer without linker to a separate linker with the linker being bonded to the protein.
- the polymer monomer or the polymer portion of the compounds of the invention is the residue of a monomer or polymer having a pendant group with a functional group reactive with a functional group on the linker.
- the polymer portion of the compound of the invention is the residue of a polymer selected from the group consisting of a polyvinyl polymer, an acrylic polymer, a polyester polymer and a polyamide polymer.
- the linker also includes a second functional group which is reactive with the protein.
- the functional group on the protein which is reactive with the linker preferably is an amine group.
- monomer units which incorporate the linker are made. Thereafter the polymer which includes the linker is made by polymerizing the monomer units which include the linker making the linker available for bonding to the protein. After that polymerization, the polymer with the linker extending therefrom is reacted with a protein or mixture of proteins to link the protein to the polymer through the linker to form the polymer protein compound of the invention.
- the monomer-linker units contain linkers that are derivatives of glucose and galactose.
- the monomer-linker units can be, but are not limited to 2-N-methacrylamido-2-deoxy-D-glucose, 3-N-methacrylamido-3-deoxy-1,2;5,6-diisopropylidine-D-glucose, 3-N-methacrylamido-3-deoxy-D-glucose, 6-N-methacrylamido-6-deoxy-D-glucose, 6-O-methacryloyl-D-glucose, 6-O-vinyl-1,2;5,6-diisopropylidene-D-galactose and 2-N-methacrylamido-2-deoxy-D-glucose-6-phosphate.
- the individual monomer-linker units are 2-N-methacrylamido-2-deoxy-D-glucose, 3-N-methacrylamido-3-deoxy-3-D-glucose or methacrylamido-6-deoxy-D-glucose.
- a monomer-linker unit suitable for the purposes of the invention described herein can be prepared by treatment of 2 -amino-2 -deoxy-D-glucose hydrochloride, which is commercially available from chemical suppliers such as Aldrich Chemical Co., with one mole equivalent of methacryloyl chloride and two equivalents of sodium methoxide in methanol at about 25oC to give
- a monomer-linker unit can be prepared by treatment of a glucose derivative such as 3-amino-3- deoxy-D-glucose with 1 mole equivalent of methacryloyl chloride and 1 mole equivalent of a base such as sodium methoxide at about OoC to about 60oC in a solvent such as methanol, to give 3-N-methacrylamido-3-deoxy-D-glucose where A is equal to -CONH-.
- a glucose derivative such as 3-amino-3- deoxy-D-glucose
- methacryloyl chloride 1 mole equivalent of a base such as sodium methoxide at about OoC to about 60oC
- a solvent such as methanol
- the 3-amino-3-deoxy-D- glucose can be prepared by treatment of 3-amino-3-deoxy- 1,2:5,6-di-O-isopropylidene-D-glucofuranose with about 1N acid, such as hydrochloric acid, in water for about 4 hours at about 25oC.
- the 3-amino-1,2:5,6-di-O- isopropylidene-D-glucofuranose can be prepared by the reduction of 3-azido-3-deoxy-1,2:5,6-di-O-isopropylidene- D-glucofuranose (prepared by the method described by Richardson. 6 Methods in Carbohydrate Chemistry at 218- 224 (1972) with ammonium carbonate and platinum oxide.
- a monomer-linker unit can be prepared by treatment of 3-amino-3-deoxy-1,2:5,6-di-O-isopropylidene-D-glucofuranose with 1 mole equivalent of methacryloyl chloride and 1 mole equivalent of a base such as sodium methoxide at about 0oC to about 60oC in a solvent such as methanol, to give 3-N-methacrylamido-3-deoxy-1,2:5,6-di-O-isopropylidene-D-glucofuranose where A is equal to -CONH-.
- a monomer can be attached to the linker at the 6-position by treatment of a glucose derivative such as 6-amino-6-deoxy-D-glucose with 1 mole equivalent of methacryloyl chloride and 1 mole equivalent of a base such as sodium methoxide at about 0oC to about 60 °C in a solvent such as methanol, to give 6-N-methacrylamido-6-deoxy-D-glucose where A is equal to -CONH-.
- a glucose derivative such as 6-amino-6-deoxy-D-glucose
- methacryloyl chloride 1 mole equivalent of a base such as sodium methoxide at about 0oC to about 60 °C in a solvent such as methanol
- the 6-amino-6-deoxy-D-glucose can be prepared by treatment of 6-amino-1,2-O-isopropylidene-D-glucofuranose with about 1N acid, such as hydrochloric acid, in water for about 4 hours at about 25oC.
- the 6-amino-1,2-O-isopropylidene-D-glucofuranose can be prepared by the method described by Cramer. 1 Methods in Carbohydrate Chemistry at 242-246 (1962).
- a monomer-linker unit can be formed by treating 2-amino-2-deoxy-D-glucose with hexokinase and then treating with an equivalent of methacryloyl chloride and two equivalents of sodium methoxide in methanol at about 25oC gives 2-N-methacrylamido-2-deoxy-D-glucose 6-phosphate.
- Adenosine triphosphate formed by pyruvate kinase phosphorylation of adenosine diphosphate with phosphoenol pyruvate serves as the source of phosphate for hexokinase.
- the individual linker unit is prepared as
- the maltose or maltotriose linker is joined to the monomer with methacryloyl anhydride in a solvent such as pyridine or triethylamine to form the monomer-linker unit.
- a solvent such as pyridine or triethylamine
- anhydride used is about 1 mole equivalent to about 2 mole equivalents based on the linker.
- polymerization is carried out using a free-radical initiator such as ammonium persulfate or 2,2'-azobis(2-amidinopropane) hydrochloride in water.
- a free-radical initiator such as ammonium persulfate or 2,2'-azobis(2-amidinopropane) hydrochloride in water.
- the polymerization is conducted at 0oC to about 100oC, preferably at 0o to about 70oC.
- polymerization may be carried out in water using a free-radical initiator, but the reaction could also contain an emulsifier.
- the free- radical initiator could include azobis(isobutyronitrile) or benzoyl peroxide and the like.
- polymerization may be carried out using free-radical initiator such as azobis(isobutyronitrile) or benzoyl peroxide in solvents such as benzene, acetonitrile, hexane, diethyl ether and the like.
- the amount of initiator is used in an amount of about 0.5 to about 20 mole % based on the linker. Preferably, the initiator is used in an amount of about 1.0 to about 5.0 mole %.
- the emulsifying agent used herein could be Triton X-405 or Triton X-100, which can be purchased from Rohm and Haas.
- the emulsifier is used in an amount of about 5 to about 20 weight percent based on the weight of monomer-linker unit.
- the polymerization is conducted at about 0° to about 100°C, preferably at about 25° to about 70oC.
- Polymerization of monomer-linker units may be carried out under acid-catalyzed conditions.
- Catalysts which may be used include titanium trichloride, titanium tetrachloride, aluminum trichloride, boron trifluoride etherate and the like in hexane, tetrahydrofuran, or ether solution and the like, and used in an amount of about 1 mole percent to about 20 mole percent based on the monomer, preferably about 1 mole percent to about 10 mole percent.
- the temperature of the polymerization under acid-catalyst conditions is about
- Another way of making the polymer protein compound of the invention is to react the protein with the linker to form a derivatized protein; and thereafter react the derivatized protein with polymerizable group to form a protein-linker-monomer which is polymerized in excess of monomer containing linkers to form the polymer protein compound of the invention.
- Yet another way of making the polymer protein compound of the invention is to react the protein with the monomer containing a linker to form a protein-linker-monomer and thereafter polymerize the protein-linker-monomer in an excess of monomer containing linker to form the polymer protein compound of the invention, see below.
- Each of these synthetic routes are shown below.
- the individual linker also may be reacted or grafted onto a polymer chain with the protein thereafter being reacted or grafted onto the linker on the polymer chain.
- the polymer chain has been formed from an acrylic monomer the chain can have the following formula:
- repeating units are derived from the monomers which provide the acrylic polymer as defined above.
- the linker units can be attached or grafted onto the polymer chain with lipase at about 4oC to about 60°C, preferably about 37oC, in borate, acetate, bicarbonate or phosphate buffer as the solvent.
- the reaction is carried out in phosphate buffer.
- glucose can be attached to poly(methacrylic acid) or poly(acrylic acid) with lipase in phosphate buffer.
- the monomers when making random, regular, or block copolymers, the monomers can be different from each other and the linkers can be
- the comonomers as described in connection with Formula III can be independently selected from methacrylamide, acrylamide, methacrylic acid, acrylic acid, hydroxyethyl methacrylate, N-(2-hydroxypropyl)methacrylamide, and other monomers which include the reaction of a linker as described herein.
- linkers including saccharide linkers such as mono-, di-, and trisaccharides as described herein.
- Polymers from the polymerization of the monomer linker or the polymer with the linker bonded thereto may be water soluble and have an absolute molecular weight of over 4 ⁇ 10 6 daltons.
- the resulting polymers which include the linker exhibit solubility in distilled water and in buffered water solutions. Preferably the polymer exhibits a solubility of greater than 1 mg/ml.
- the molecular weight of the polymer can vary from low molecular weight
- the polymers also exhibit solubility in various organic solvents including acetonitrile,
- the polymer can also form gels in water, buffered water, and organic solvent solutions.
- Cross-linked gels can be formed by reaction of the polymer with a suitable diamine under reductive amination conditions.
- the saccharide linker is in equilibrium between the linear and cyclic forms. In the cyclic form it is protected. In the aldehyde linear form, it is not protected and could react with an amino functional group.
- linker After a linker is coupled with a protein to form a linker group, the availability of an abundant number of hydroxyl groups on the linker group such as on a saccharide linker group provides enhancement of
- the coupling reaction with an aldehyde group on the saccharide linker group is accomplished by treatment of a protein such as an enzyme or antibody or the like with the polymer which includes the linker, whether the polymer is a homopolymer, random or block copolymer in a buffer solution.
- a protein such as an enzyme or antibody or the like
- the buffer solution is sodium borate solution, but the buffer solution can also be HEPES or phosphate buffer. Even though sodium
- cyanoborohydride is preferred, sodium borohydride, lithium cyanoborohydride or potassium cyanoborohydride can be used, this mild reaction is preferably carried out at a pH of about 6 to about 10, preferably about 8 to about 9.5.
- the reaction temperature is between about 4oC to about 50oC, at about 2 hours to about 48 hours.
- the reaction temperature is between about 20°C and about 37oC, for about 24 hours to about 48 hours.
- the coupling reaction with a lactone group on the saccharide linker group is accomplished by treatment of a protein such as an enzyme or antibody or the like with the polymer which includes the linker, whether the polymer is a homopolymer, random or block copolymer in a buffer solution.
- the buffer solution is sodium borate solution, but the buffer solution can also be HEPES or phosphate buffer.
- This mild reaction is preferably carried out at a pH of about 6 to about 10, preferably about 7 to about 9.
- the reaction temperature is between about 4oC to about 50oC, at about 2 hours to about 48 hours.
- the reaction temperature is between about 20oC and about 37oC, for about 24 hours to about 48 hours.
- the weight equivalents of the ingredients used in the reductive reaction coupling the protein to linker is about 1 to about 100 weight equivalents of polymer and about 0.1 to about 10 weight equivalents of sodium cyanoborohydride.
- the amount of the ingredients used is about 50 weight equivalents of polymer and about 2 weight equivalents of sodium cyanoborohydride.
- the progress of the reaction can be monitored by gel filtration chromatography and the yield of the reaction can be determined, for example, by measurement of the relative activity of the native enzyme and the protein polymer conjugate. Generally the yield of the coupling reaction is greater than about 25%.
- the solubility of the compounds of the invention and their compatibility with a particular solvent system is at least in part a function of the alkyl groups on the linker group. While these alkyls may have up to 30 or more carbon atoms, organic solvents will be compatible with polymer protein
- the polymer protein compounds of the invention may be water soluble.
- An alternative way of preparing the compounds of this is to attach the polymerizable saccharides, especially monosaccharides to the protein first.
- the smaller size of the functional groups substantially reduces the amount of steric hinderance which may occur about densely packed lysine residues in a protein.
- lysine residues located inside a cleft which are not accessible by bulky polymer-saccharide chains, become accessible and may be coupled by small monomeric units. As a result, a greater number of lysine residues in a protein are bonded to saccharides.
- the monomeric units can be polymerized (as described in C above) to produce a carbohydrate polymer bonded to an enzyme with a higher degree of substitution between polymer and protein.
- This polymerization step also provides a great deal of control in the nature of the coupled product. By introducing a varied amount of uncoupled monomer to the polymerization step, the length of the polymer chain between lysine residues of the protein can be controlled. Furthermore, the
- concentration of the enzyme can be altered in the
- enzymes were coupled to 2-N-methacrylamido-D-glucose monomer and were polymerized by free radical polymerization conducted in an aqueous solution using ammonium persulfate as the initiator.
- the copolymerized enzymes are separated by HPLC chromatography. High molecular weight fractions of the enzyme bonded to the saccharide linker polymer were collected and desiccated by lyophilization, at which point the polymer protein product could be stored indefinitely.
- Compound 1 can be prepared by acid catalyzed methods to give 2 using, by way of example, by using boron trifluoride etherate or titanium chloride as catalysts in solvents such a diethyl ether or
- Compound 3 is prepared by standard reductive amination.
- Compound 5 may be made by acid catalyzed polymerization of 4. Compound 5 when treated with the desired protein and sodium cyanoborohydride in aqueous solution gives 6.
- a protein conjugate with an ester linkage between the polymer backbone and the linker can be prepared by free-radical polymerization of the monomer 7 to give 8 which, when treated with the desired protein and sodium cyanoborohydride in aqueous solution gives the conjugate 9.
- a protein conjugate with an amide linkage between the polymer backbone and a saccharide linker which contains a ring in addition to the open form of the sugar (via a disaccharide) can be prepared by attachment of a polymerizable side-group to an amino-substituted disaccharide followed by its polymerization and reductive amination with a protein.
- a polymerizable amino-substituted disaccharide (12) can be prepared by the silver oxide mediated coupling of 10 with 11 followed by removal of the acetate groups with ammonia and
- poly(2-methacrylamido-2-deoxy-D-glucose) with bromine in water to give poly(2-methacrylamido-2-deoxy-D-gluconolactone) which can be used directly to couple with a desired protein in
- Controlling the solubility of the polymer protein compounds of the invention is another important aspect of this invention.
- this solubility may be controlled, for example, by cross-linking, by formation of hydrogels or by attaching the polymers to a glass as shown below.
- Gels of the polymer can be made by using high concentrations of monomer with the least amount of solvent effective for solubilizing the monomer or monomer linker for polymerizing the monomer or monomer linker at a given pH. Using low amounts of water as a solvent for polymerization is particularly effective for producing hydrogels of the compounds of the invention.
- control of the reaction conditions for the polymerization of saccharides substituted with polymerizable side-groups can effect the formation of such a hydrogel rather than a water-soluble polymer.
- Cross-linking the compounds of the invention is another way to control their solubility and to make them insoluble.
- the compounds of the invention are cross-linking the compounds of the invention.
- the polymer and linker may be cross-linked to form an insoluble gel before protein is bonded to other unreacted linkers on the polymer or after the protein is bonded to the linker on the polymer.
- a cross-linked polymer or a polymeric matrix may be formed through bonding a protein to multiple polymers or through cross-linking the
- the soluble form of the compounds can be made for ease of preparation and then be made insoluble.
- an insoluble form of the conjugate is the reaction of the polyer conjugate (15) with a diamine linker, such as 1,6-hexanediamine, with sodium cyanoborohydride to give the cross-linked material shown as 32.
- a diamine linker such as 1,6-hexanediamine
- sodium cyanoborohydride to give the cross-linked material shown as 32.
- the level of cross-linking preferably about 1 percent, affects the rigidity of the gel formed.
- An alternative method for formation of an insoluble form of the conjugate is the reaction of the polymer protein conjugate (15) with alkylamine glass using EDC to affix the conjugate to the solid support giving 31.
- Hostile environments affect the activity of proteins including enzymes.
- an intended functional property is either maintained to be at least equal to the native protein or it is enhanced over a reduced activity that the protein would have in a hostile environment.
- the method of the invention particularly can be seen in connection with maintaining or enhancing the half life of an enzyme or antibody.
- half life means the time it takes for an enzyme to lose one half of a given activity.
- the half life of the enzyme in a hostile environment will be at least greater and preferably at least about 110% or more of the half life of the native enzyme in the same harsh environment.
- most preferably the half life of the enzyme when a part of the polymer protein compound of the invention will be enhanced from about 110% to about 1000% or more over that of the native enzyme in a hostile environment which would reduce the half life of the native enzyme.
- the stability of antibodies also is improved.
- the "half life of an antibody” means the time it takes for an antibody to lose one half of its ligand binding ability.
- the half life of the antibody in a hostile environment will be at least about 110% or more of the half life of the antibody in the same hostile environment.
- the half life of the antibody when made into a polymer protein compound of the invention will be enhanced from about 110% to about 1000% or more over that of the native antibody in a hostile environment which would reduce the activity of the native antibody.
- ⁇ -chymotrypsin is deactivated immediately when exposed to about 60oC borate buffer solution.
- the ⁇ -chymotrypsin-poly(6-N- methacrylamido-6-deoxy- D-glucose) conjugate made herein maintains greater than 30 percent activity after exposure to about 60oC borate buffer solution for about 1 hour, greater than about 20 percent activity after about 6 hours, and greater than 15 percent activity after about 15 hours, see Figure 2.
- Native trypsin enzyme loses greater than 90 percent of its activity within 1 hour when exposed to a temperature of 55oC.
- the lack of buffer is another factor which can result in denaturation or inactivation of enzymes.
- the trypsin enzyme in a phosphate buffer solution does not lose its activity quickly. The enzyme loses
- solution at about 45oC falls to about 75% of its initial activity within about 10 minutes, and less than about 10 percent after about 9 hours, and to about 0 percent after about 18 hours.
- the activity of ⁇ -chymotrypsin in distilled water solution falls to approximately 30% activity within about 10 min and falls to about 0 percent after about 9 hours.
- the activity at about 45oC is maintained at greater than 80% over about 18 hours in both buffer and distilled water solutions, see Figure 1.
- Enzymes which can be part of the polymer protein compounds of the invention include enzymes related to the production of fructose, such as glucose isomerase, which operates at 60" to 65" C, hydrolysis of starch by ⁇ -amylase, which occurs at 85' to 110oC and resolution of D,L-amino acids by amino acetolase at 50oC.
- Other enzymes which can be a part of the polymer protein compounds of the invention are nucleases, including endonucleases.
- Enzymes which can be stabilized as described here include oxidoreductases, transferases, dehydrogenases, transaminases, peptidases, hydrolases, isomerases, mutases and the like.
- other enzymes which could be stabilized herein include but are not limited to malate dehydrogenase;
- CK creatine kinase
- AP alkaline phosphatase
- ALP alkaline phosphatase
- AST aspartate aminotransferase
- alanine aminotransferase ALT, PT, SGPT
- gammaGT gammaGTP
- glutamic oxalacetic transaminase SGOT
- glutamic pyruvic transaminase GTP
- alpha amylase beta amylase
- lactate dehydrogenase LD, LDH, lactic dehydrogenase
- glucose-6-phosphate alanine aminotransferase
- G6PDH dehydrogenase
- HK hexokinase
- dehydrogenase glucose oxidase; peroxidase (HRP, HPO, HRPO, PO); glycerol dehydrogenase; glutamate
- dehydrogenase cholesterol oxidase
- cholesterol esterase cholesterol esterase
- lipase lipase
- uricase urease
- glycerol kinase aldolases
- the compounds of the invention could be used in applications of enzymatic analysis represented by the diagnostic reagents used to determine and quantitate the following constituents in fluids include but are not limited to glutamic-oxalacetic transaminase (SGOT); glutamic-pyruvic transaminase
- SGPR lactic dehydrogenase
- LDH lactic dehydrogenase
- CPK phosphokinase
- alpha-hydroxybuteric dehydrogenase alpha-HBD
- glucose via hexokinase-glucose-6-phosphate dehydrogenase, glucose oxidase, glucose dehydrogenase
- BUN blood urea nitrogen
- Antibodies or antigens may be coupled to the polymer with linkers by treating, based on the weight of the antibody or antigen, about 1 to about 100 weight equivalents of polymer and about 0.1 to about 10 weight equivalents of sodium cyanoborohydride in 0.05 M sodium borate buffer.
- the reaction temperature is between about 25oC to about 50oC, for about 2 to about 48 hours.
- the coupling reaction is carried out, based on the weight of the antibody or antigen, with about 50 weight equivalents of polymer and about 2 weight
- the antibody-polymer and antigen-polymer complex can be isolated using gel filtration chromatography. The concentration of antibody-polymer and antigen-polymer complex is determined by measurement of optical density.
- antibodies and antigens maintained their competency when incorporated into normal enzyme-linked immunosorbent assay methodology (Tables 3 and 4 in
- Example 11 Furthermore, conjugated antibody and antigen performed similarly to nonconjugated antibody and antigen in competition assays (Table 5).
- antigens incorporated into the compounds of the invention allows the detection of antigens in non-optimal aqueous solutions and organic solvents. Potentially,
- the polymer herein can also be used to synthesize peptide bonds.
- equimolar solutions containing modified amino acid(s) are dissolved in a solvent system containing an organic solvent such as dioxane or THF (tetrahydrofuran), or a tertiary amine such as (iPr) 2 NEt, to form an amino acid mixture.
- the organic solvent is acetonitrile.
- the solutions dissolved in the solvent system are defined herein as amino acid acceptor and amino acid donor solutions. Examples of such solutions can be found in Table 2 of Example 10.
- a conjugated enzyme such as protease may be added to the amino acid mixture in an amount of about 10 -6 to about 10 -1 mole fraction relative to the amount of amino acid.
- the conjugated enzyme can be prepared as in Example 8. The reaction is performed at from about 0o to 70oC, at about 1 to about 24 hours. The enzyme polymer conjugate is removed from the formed peptide by filtering and the solvent is evaporated to obtain the resulting peptide.
- a water-soluble monosaccharide suitable for this application was prepared by treatment of 3-amino-3- deoxy-1,2:5,6-diisopropylidine-D-glucose (1.78 mmol, 0.46 g) in 150 mL 1N HCl (prepared with triply glass distilled water) with stirring under argon at room temperature for 4 days. The resulting pale yellow solution was filtered and evaporated to a light brown brittle froth. The froth was dissolved in water, stirred with activated charcoal, and filtered through Celite 545 yielding a colorless filtrate. Removal of the water in vacuo gave 0.41 g (100%) of a pale yellow glassy solid of 3-amino-3-deoxy- D-glucose hydrochloride.
- a water-soluble monomer suitable for this application was prepared by treatment of 3-amino-3-deoxy-D-glucose hydrochloride with sodium methoxide (prepared by the addition of sodium (3.35 mmol, 0.077 g) to 7 mL methanol) added dropwise to a solution of 3-amino-3-deoxy-D-glucose hydrochloride (1.84 mmol, 0.397 g) in 6 mL of methanol. The mixture was stirred at room
- a water-soluble monosaccharide suitable for this application was prepared by treatment of 6-amino-6-deoxy-1,2-isopropylidine-D-glucose (1.98 mmol, 0.51 g) in 150 mL 1 N HCl (prepared with triply glass distilled water) with stirring under argon at room temperature for 2 days. The resulting pale yellow solution was filtered and evaporated to a light brown brittle froth. The froth was dissolved in water, stirred with activated charcoal, and filtered through Celite 545 yielding a colorless filtrate. Removal of the water in vacuo gave 0.49 g (100%) of a pale yellow glassy solid of 6-amino-6-deoxy-D-glucose hydrochloride.
- a water-soluble monomer suitable for this application was prepared by treatment of 6-amino-6-deoxy-D-glucose hydrochloride (0.49 g, 2.29 mmol) with sodium methoxide (prepared by the addition of sodium (3.70 mmol, 0.085 g) to 7 mL methanol) added dropwise to a solution of 6-amino-6-deoxy-D-glucosamine hydrochloride (1.84 mmol, 0.397 g) in 16 mL of methanol at 0oC. The mixture was stirred at room temperature for 30 min.
- a water-soluble individual backbone-linker unit was prepared by the addition of 47.97 g (0.22 mole) of glucosamine hydrochloride to 12.16 g (0.23 mole) of sodium methoxide in 500 mL of methanol.
- Free-radical polymerization of 2-N-methacrylamido-2-deoxy-D-glucose was carried out under an inert atmosphere in quadruply distilled and freeze-thawdegassed water solution.
- a solution of 5.00 g (0.0202 mole) of 2-N-methacrylamido-2-deoxy-D-glucose was
- a protein polymer conjugate of poly(2-N-methacrylamido-2-deoxy-D-glucose) and trypsin was prepared by the reductive amination procedure as shown below: To 20 mL of a 0.05 M sodium borate solution at pH 8 was added 148 mg (3 ⁇ 10 -8 mole) of poly(2-N-methacrylamido-2-deoxy-D-glucose) with 30 mg (1.2 ⁇ 10 -6 mole) of trypsin and 10 mg (9.55 x 10 -4 mole) of purified sodium cyanoborohydride. The solution was placed in a shaking water bath at 60 oscillations/min at 35oC for 48 hours. The progress of the reaction was followed by gel
- the yield of the active enzyme was determined by comparison of the relative activity of the native trypsin and the trypsin-polymer conjugate for N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide as the substrate in 0.05 M sodium phosphate buffer at pH 8 containing 10% methanol.
- Kinetic analyses of the trypsin-polymer conjugate and native trypsin for k cat and K m are listed in Table 1.
- EXAMPLE 8 (a) (i) was repeated in every essential detail except that poly(6-N-methacrylamido-6-deoxy-D-glucose) was used instead of poly(2-N-methacrylamido-2-deoxy-D-glucose) and BPN'-subtilisin was used instead of trypsin.
- EXAMPLE 8 (a) (i) was repeated in every essential detail except that poly(3-N-methacrylamido-3-deoxy-D-glucose) was used instead of poly(2-N-methacrylamido-2-deoxy-D-glucose) and Carlsbergsubtilisin was used instead of trypsin.
- CPC is the carbohydrate protein conjugate for poly-(2-N-methacrylamido-2-deoxy-D-glucose)-enzyme conjugate.
- a series of stabilized soluble proteases were prepared having general utility for the synthesis of peptide bonds denoted by the carbohydrate protein
- CPC-proteases conjugates of proteases
- protease-polymer conjugates for the formation of peptide bonds are contained in Table 2. Note that despite the presence of organic solvent, peptide bond formation can be accomplished at elevated temperatures.
- the use of protease-polymer conjugates for the synthesis of peptide bonds is important, not only for the preparation of peptides and proteins, but also because the chemistry demonstrates the stability of protein-polymer conjugates in organic solvents. Under the same conditions and at the same activity levels used for the preparation of peptide bonds using protease-polymer conjugates, the native enzymes gave less than 5% of the desired product.
- the stabilization of antibodies is highly desirable for their use in diagnostic applications.
- the stabilization of antibodies for use in an enzyme-linked immunosorbent assay (ELISA) detection of avermectins has been accomplished.
- the avermectins which are fermentation products of the soil microorganism Streptomyces avermitilis. have potent antiparasitic, insecticidal, and antihelmintic properties. As a result of these properties, the avermectins are finding
- Ivermectin an approximately 80:20 mixture of 22,23-dihydroavermectins B1a and B1b
- the antibody Mab B11C2.1 (0.374 mg) was treated with 2.5 mg of poly(2-N-methacrylamido-2-deoxy-D-glucose and 0.40 mg of sodium cyanoborohydride at 25°C in 0.05 M sodium borate buffer for 12 hours.
- the antibody-polymer complex (CPC-M ab B11C2.1) was isolated by gel filtration chromatography using 0.05 M sodium borate as the eluant on a Superose 12 column.
- the concentration of M ab B11C2.1 in CPC-M ab B11C2.1 was determined by measurement of its optical density at 280 nm.
- CPC-Ive-Con immunogen-polymer complex
- ivermectin-conalbumin immunogen was used in place of M ab B11C2.1.
- concentration of Ive-Con in CPC-Ive-Con was determined by measurement of its optical density at 280 nm.
- the immunogen-polymer complex is also competent for the detection of the antibody. Plates were coated with CPC-Ive-Con with concentrations from 1 to 100 ng/well. The presence of the protein-polymer conjugate was then determined using M ab B11C2.1 (Table 4). The data clearly demonstrate that the CPC-Ive-Con protein-polymer conjugate gives significant and easily detectable EIA rates. Table 4
- the competency of the protein-polymer conjugates was determined by comparison of the response of the control experiment of Ive-Con exposure to M ab
- CPC Poly(2-N-methacrylamido-2-deoxy-D-glucose) .
- antibody-polymer conjugates can be used for the
- antibody-polymer conjugates in the ELISA assay allows measurements in high concentrations of organic solvent. Therefore, detection of water insoluble chemicals is possible.
- Aldrin is a pesticide used in the
- Aldrin contains 95 percent or more of 1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro-1,4,5,8,-endo, exo-dimethanonaphthalene.
- Aldrin was succinylated and coupled to a carrier protein using 1-ethyl-3-(3'-N,N,dimethylaminopropyl)carbodiimide to give Con-Aldrin conjugate. This macromolecule was then used to raise a monoclonal antibody, M ab 8H11.
- the M ab 8H11 antibody was conjugated to poly
- the column was eluted with 20 mM sodium phosphate and 50 mM sodium chloride at a pH of 7.0 at a rate of 0.5 mL/min.
- the conjugated antibody concentration was determined by measuring absorption of the CPC-M ab 8H11 solution at 280 mM and subtracting the contribution of the polymer.
- the overall yield of the conjugation reaction and purification was about 20%.
- the purified CPC-M ab 8H11 was concentrated using a membrane filtration apparatus.
- the purified CPC-M ab 8H11 was
- the stability of the CPC-M ab 8H11 was studied in a non-ideal environment using a general enzymelinked immunoassay (EIA).
- EIA enzymelinked immunoassay
- the CPC-M ab 8H11 and M ab 8H11 were exposed to a non-ideal environment and then assayed for their remaining activity in an enzyme immunoassay.
- the plates were treated with CPC-M ab 8H11 and M ab 8H11, and followed by washing with buffer solution to remove unbound antibodies.
- the native M ab 8H11 (1.6 mg/ml) and 220 ⁇ L of the CPC-M ab 8H11 (protein concentration 1.6 ⁇ g/mL) were placed in separate micro test tubes with 20 mM sodium phosphate and 50 mM sodium chloride, pH 8.0. The tubes were immersed in a water bath at an elevated temperature.
- the CPC-M ab 8H11 is more stable than the native M ab 8H11 over time.
- the denatuation of both the CPC-M ab 8H11 and the M ab 8H11 was found to be a first order kinetic process, as can be further seen in FIG. 10.
- Lyophilized powders of Mab 8H11 and CPC-M ab 8H11 were treated with a number of anhydrous organic solvents, as can be seen in Table 7 below.
- Hexane 65 69 As shown in Table 7 above, the native antibody powder is totally denatured in acetonitrile in about five hours, while the CPC-M ab 8H11 is intact. In methanol, isopropanol and ethyl acetate, CPC-M ab 8H11 also showed higher stability than M ab 8H11.
- a series of stabilized enzymes were prepared, where the enzymes were modified using the poly (2-N-methacrylamido-2-deoxy-D-glucose) of Example 5. The treatment for all of the enzymes was equivalent, except where noted.
- the enzymes were conjugated to the polymer.
- the enzyme of interest was dissolved at 1 mg/mL in 50 mM borate buffer, having a pH of 9.0. Two samples were made, one sample where the enzyme was conjugated to the polymer, and a second sample containing native enzyme.
- the polymer was added to the first sample in an amount o 10 mg/mL to the conjugation buffer. Both samples, control and conjugated enzyme polymer, were assayed at 25o C.
- NaCNBH 3 was added at 10 mg/m and the reaction mixture was incubated at 37oC. for 48 hours. The enzyme activity was monitored periodically. After 48 hours, the activity was measured and the sample were dialyzed against 100 mM phosphate buffer having a p of 6. The retention of the initial activity was
- the assay temperature was maintained at 25oC.
- Phosphate buffer was initially tried as a conjugation buffer (100 mM at pH 8.5). Glucose oxidase was added at 1 mg/mL to phosphate buffer and NaCNBH 3 was added. To the native enzyme in 1 mg/mL amount,
- cellobiose was added at 2 mg/mL along with NaCNBH 3 at 10 mg/mL.
- NaCNBH 3 was added at 10 mg/mL to 1 mg/mL of native enzyme.
- Enzyme was conjugated to the polymer as stated above.
- the conjugated enzyme was dialysized against phosphate buffer using 300 K Da dialysis tubing. After dialysis, 69% of the native enzyme's activity and 78% of the conjugated enzyme's activity was retained (on a volume basis).
- the tl/2 of the conjugated enzyme was less than the native enzyme.
- the t1/2 of the native enzyme was 12.1 minutes, but 4.9 minutes for the conjugated enzyme; and at 60oC the tl/2 of the native enzyme was 47.7, but 24.9 minutes for the conjugated enzyme.
- the activity of lactic dehydrogenase was measured.
- the enzyme was conjugated as described above in Example 11 and compared to the native enzyme. As can be seen in FIG. 13, the conjugated enzyme was more active than the native enzyme at temperatures between about 35o to about 75°C.
- the thermal stability of pyruvate kinase at different temperatures was measured. Mean half-life, measured in minutes, along with the standard deviation (in parentheses) are listed in Trial 1.
- the enzyme was conjugated using 100 mM borate buffer (pH 8.5).
- the EcoR1 enzyme conjugate with poly(2-N-methacryloyl-2-deoxy-D-aminoglucose) (300,000 daltons) was prepared by adding purified sodium cyanoborohydride (10 mg, 1.6 ⁇ 10 -4 mol) and (4 mg, 7 ⁇ 10 -7 mol) of poly(2-N-methacryloyl-2-deoxy-D-aminoglucose) in 200 ⁇ L of 0.2 M sodium borate to a 200 ⁇ L solution of EcoRI (1250 units of activity per ⁇ L).
- the reaction solution was prepared by adding purified sodium cyanoborohydride (10 mg, 1.6 ⁇ 10 -4 mol) and (4 mg, 7 ⁇ 10 -7 mol) of poly(2-N-methacryloyl-2-deoxy-D-aminoglucose) in 200 ⁇ L of 0.2 M sodium borate to a 200 ⁇ L solution of EcoRI (1250 units of activity per ⁇ L).
- the reaction solution was
- the digested lambda DNA was placed on a 0.8%
- BIO RAD ultra pure DNA agarose gel and developed with a Fotodyne minigel apparatus at 80 volts for one hour. Gel electrophoresis was done to determine the cleavage patterns of the lambda DNA treated by the native enzyme and the EcoRl-conjugate.
- the digested PBR322 plasmid was placed on a 0.8% BIO RAD ultra pure DNA agarose gel and developed with a Fotodyne minigel apparatus at 80 volts for one hour. Gel electrophoresis was done to determine the cleavage patterns of the PBR322 plasmid treated by C-2 and the EcoR1-conjugate.
- the samples were loaded also with a buffer comprising 0.25% bromophenol blue, 0.25% xylene cyanol FF, and 15% Ficol in water.
- Gels were developed by staining with ethidium bromide (20 ⁇ L of a 5 mg/mL solution was diluted to 100 ml). After staining the gels were photographed.
- a protease, trypsin was conjugated to poly(2-N-methacrylamido-2-deoxy-D-glucose).
- the polymer was prepared as in Example 5.
- the conjugation was done under the conditions as described in Example 8.
- the conjugated trypsin was separated from the trypsin that was not conjugated to the polymer using gel filtration
- the conjugated trypsin in an amount of about 2.7 mg/mL, 795 units/mL was mixed with bovine serum albumin (BSA) in an amount of about 50 mg/mL at 30°C for 48 hours.
- BSA bovine serum albumin
- Control-1 containing BSA in an amount of about 50 mg/mL in 100 mM Tris-HCL buffer pH 8.6 was prepared and combined with native trypsin in an amount of 0.05 mg/mL, 795 units/mL. Control-1 mixture was incubated at 30 oC for 48 hours. After digestion, a SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was
- PhastGelhomgeneous 20 SDS-PAGE was used.
- the gel was stained with ethidium bromide.
- lanes 3 through 5 are molecular weight markers (66000, 45000, 36000, 29000, 24000, 20100 daltons).
- the conjugate was isolated by HPLC (Sephacryl S-200) using 0.05 M sodium borate buffer (pH 8). The solution was then dialyzed against distilled water using a Spectra Por 7 10K MWCO membrane. The solution was lyophilized to give a fluffy white powder with 28% of the original activity.
- Example 19 Preparation of Disaccharide Polymer System Part a. Preparation of 1,2,3,4-tetra-O-acetyl-6-0- (2,3,4-tri-O-acetyl-6-deoxy-6-trifluoroacetamido-ß-D- glucopyranosyl)-ß-D-glucopyranose.
- 1,2,2,3-tetra-O- acetyl-ß-D-glucopyranose 1.1 g, 3.2 mmol
- silver oxide 0.9 g, 3.9 mmol
- drierite 2.5 g
- Chloroform 5 mL
- Iodine 0.2 g was then added as a catalyst.
Abstract
Description
Claims
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US5789219A (en) * | 1994-12-02 | 1998-08-04 | Abbott Laboratories | Phosphatase activated crosslinking conjugating and reducing agents; methods of using such agents; and reagents comprising phosphatase activated crosslinking and conjugating agents |
US6160153A (en) * | 1994-12-02 | 2000-12-12 | Abbott Laboratories | Phosphatase activated crosslinking conjugating and reducing agents; methods of using such agents; and reagents comprising phosphatase activated crosslinking and conjugating |
US8048659B1 (en) * | 1999-10-08 | 2011-11-01 | Leif Robert C | Conjugated polymer tag complexes |
WO2010039384A2 (en) * | 2008-09-30 | 2010-04-08 | Fresenius Medical Care Holdings, Inc. | Covalently immobilized enzyme and method to make the same |
WO2010039384A3 (en) * | 2008-09-30 | 2010-06-10 | Fresenius Medical Care Holdings, Inc. | Covalently immobilized enzyme and method to make the same |
US8497107B2 (en) | 2008-09-30 | 2013-07-30 | Fresenius Medical Care Holdings, Inc. | Covalently immobilized enzyme and method to make the same |
US9187744B2 (en) | 2008-09-30 | 2015-11-17 | Fresenius Medical Care Holdings, Inc. | Covalently immobilized enzyme and method to make the same |
Also Published As
Publication number | Publication date |
---|---|
US5736625A (en) | 1998-04-07 |
US5492821A (en) | 1996-02-20 |
CA2073511A1 (en) | 1992-05-29 |
US5639633A (en) | 1997-06-17 |
EP0513332A1 (en) | 1992-11-19 |
EP0513332A4 (en) | 1993-03-17 |
US5691154A (en) | 1997-11-25 |
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