JPH0233356B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for preparing aqueous solutions or dispersions of binding proteins. More specifically, the present invention relates to a method for producing an aqueous solution or dispersion of a protein denatured by bonding it to an organic polymer. Even more particularly, the present invention relates to a method of contacting a protein with a liquid isocyanate-terminated polyurethane prepolymer to form a reaction product that is soluble or dispersible in water. Still more particularly, the present invention relates to the above method, wherein said protein is an enzyme, antibody or antigen and the polymer is poly(urea-urethane). Aqueous dispersions of protein/polymer reaction products are formed by dissolving the protein in a liquid isocyanate-terminated polyurethane prepolymer and dispersing the resulting solution in water. First, let's talk about the prior art. For a review of enzyme technology, see “Chemical &
Engineering News” August 18, 1975 issue (22-
Published on page 41). The term "soluble conjugated protein" is used in the prior art to refer to the reaction product of a protein and a polymeric substance (natural or synthetic). The reaction products are soluble or dispersible in the liquid medium, usually water.
Although protein/polymer binding is generally covalent, adsorption techniques have also been used. Soluble, bound proteins often exhibit biological reactivities similar in nature to free proteins. Some of the soluble conjugated proteins exhibited greater solubility and/or reduced antigenicity and other improvements compared to free native protein. Published paper, MG Brattain, “Rutgers
University”, 1974, “L-
"Modification of Asparaginase" describes the binding of asparaginase to polyethylene glycol and other polymers via cyanuric chloride. The conjugated product is dispersible in water and exhibits slightly improved stability and reduced antigenicity. Although the present invention similarly relates to the conjugation of proteins such as enzymes to urethane prepolymers having an oxyalkylene backbone such as polyethylene glycol, the method of conjugation and preparation is very different from that set out in the Brattain article. Additionally, U.S. Patent No. 3574062 (165/63, Sato)
The specification describes a method for making conjugated proteins in which a polyester polyurethane is diazotized with a diazonium salt of an amino acid and then coupled with a non-enzymatic animal protein to form a diazotized polyurethane, which is then combined with an enzyme. The enzyme is reacted to form an immobilized enzyme. U.S. Patent No. 3705084 (195/63, Reynolds)
The specification includes (a) a macroporous reactor core, (b) a polymer surface on the reactor core (which can be a polyurethane resin), and (c) a polymer surface on the polymer surface. Flow-through enzyme reactors have been taught that include an enzyme adsorbed and crosslinked in place on its surface with a bifunctional agent (eg, polyisocyanate). Reynolds was immobilized for reactors by adsorbing the active enzyme onto a polymer surface and further immobilizing the enzyme by crosslinking it in place with a crosslinking agent such as a monomeric polyisocyanate. manufactures enzymes. German Public Bulletin No. 15 November 1973
No. 2319706 describes enzymes bound to polyurethane foams and methods for making enzymes so bound. U.S. Patent No. 3791927 [195/68, Forgione et al.]
The specification shows a water-insoluble bound protein (enzyme) incorporated within the cells of a free-standing reticular cellular material (which can be a polyurethane foam), which protein (enzyme) is incorporated into the cellular material. are combined. U.S. Pat. No. 3,672,955 (195/68, Stanley) discloses that (a) an aqueous dispersion of an enzyme is emulsified with a solution of a polyisocyanate in a volatile water-immiscible solvent (e.g., methyl chloroform); A method for making a conjugated protein (enzyme) is presented that includes b) mixing the resulting emulsion with a solid particulate carrier, and (c) then evaporating the solvent. The Stanley polyisocyanate may be an isocyanate-terminated liquid polyurethane prepolymer. No. 3,672,955 is incorporated herein by reference in its entirety. Stanley states in its Example 3 that it is reported that an enzyme component (peroxidase) of the fermentation broth is bound by mixing a portion of the fermentation broth with polyisocyanate dissolved in methylchloroform. has been done. Under Stanley's reaction conditions, any other enzymes present in the fermentation broth are also immobilized (become insoluble in water,
It seems possible that they are connected (i.e., combined). Silman et al. Annual Review of Biochemistry
1966, Vol. 35 (Part 2), pp. 873-908, describes a review of methods for producing water-insoluble derivatives of enzymes, antigens, and antibodies. Singer, Nature 1959, Vol. 183, pp. 1523-1524, describes a method for reacting proteins with diisocyanates (m-xylene diisocyanates). The specification of U.S. Patent Application No. 250012 (filed May 3, 1972) by inventor Wood et al., assigned to W.R. Grace & Co. and now abandoned, includes
21 discloses a foamed urethane containing an immobilized enzyme (urease), a method for producing the immobilized enzyme, and a method for using the same. The patent application No. 250012 also states in claim 8 that (a) an isocyanate-terminated polyurethane prepolymer, (b) water, and
(c) Biostats Effervescent compositions consisting of fungicides or enzymes are indicated. A similar teaching is found in the above-mentioned DE 2319706 patent claim 7. U.S. Pat. No. 3,929,574 (Wood et al.) describes contacting an isocyanate-terminated liquid polyurethane prepolymer with an aqueous dispersion of an enzyme under foam-forming conditions, whereby a polyurethane foam forms and the enzyme forms a protein bound (immobilized) by a method including resulting in complete binding to a polyurethane foam;
It is taught that enzymes are produced. In the aforementioned U.S. Pat. No. 3,929,574, Wood et al. in Example 1 showed that the enzymes present in the fermentation broth ( It is noted that cellulase) is shown to be immobilized (bound or insolubilized). It seems likely that any of the other enzymes present in the fermentation broth could also be immobilized under the conditions of Example 1 above. U.S. Patent No. 3905923 (260/2.5AD, Klug)
The specification describes an immobilized enzyme system formed from an enzyme and a hydrophilic poly(urea-urethane) foam. In this way, the foam enzyme is surrounded and supported in an uptake-active configuration. Hydrophilic foams are formed by reacting hydrophilic isocyanate-terminated polyoxyalkylene prepolymers with water. Isocyanate-terminated polyurethane prepolymers are well known to those skilled in the art. For example (a)
“Encyclopedia of Chemical” edited by Kirk-Othmer
"Technology", U.S., New York, Willi & Sons, 2nd edition, volume 9, page 854, bottom 2.
or (b) George L. Clark, ed., “The
Encyclopedia of Chemistry, New York Kleinhold Publishing Corporation, 2nd edition, page 872, left (first) column, Section 3 in its entirety. T.Richard and NFOlson, “Immobilized Enzymes in Food and Microbial Processes”
Microbial Processes)” New York, USA
Enzymes, water,
and a method for producing bound (immobilized) enzymes by reacting polyisocyanate polymers is taught. Weetall's "Journal of Bacteriology" No. 93
Vol. 1876-1880 (1967) teaches the isolation and purification of large amounts of bacterial specific antibodies by using polymerized microorganisms as specific immunoadsorbents. The microorganism was polymerized by reaction with tetrazotized benzidine. Next, an outline of the present invention will be described. The present invention relates to a method for preparing aqueous dispersions of water-dispersible biologically active proteins bound to polyurethanes having a substantially linear polyoxyalkylene backbone. The dispersion is prepared by mixing a water-dispersible biologically active protein and an isocyanate-terminated liquid polyurethane prepolymer under substantially anhydrous conditions to form a solution. . This solution can be dispersed in water and any insoluble material separated (eg by filtration). The present invention relates to the production of water-dispersible protein/polymer compounds by preforming a protein/prepolymer solution. Formation of the solution is an integral part of the invention. Solution formation is also disclosed in US Patent Application No. 743,035 (F. Hatdegen and W. Swann, "Protein Immobilization Methods") of Nov. 18, 1976, and solution formation is also an integral part of that invention. The invention of this co-pending application involves the formation of foams to which proteins are attached. The present invention is an improvement on the invention of said co-pending application in the sense that water-dispersible or water-soluble compositions are disclosed. The preparation of the water-dispersible compound is carried out at a temperature at which the isocyanate-terminated liquid polyurethane prepolymer is in liquid form and below the denaturation temperature of the protein to be bound. As shown in the Examples, properties that differ from those possessed by either the free protein or the free polymer, e.g. It is clear that a protein/polyurethane product is formed with migration properties, migration properties in an electric field, and solubility properties. (Saunders and Frisch, "Polyurethanes: Chemistry and Technology")
(compare Interscience Publishers, 1962, Vol. 1, 140, p. 177184) Amino acids with an NH ₂ group that are substantially available to proteins such as enzymes, antigens, antibodies and simple proteins (e.g. serum albumin) It is also known to be a polymer of It therefore seems likely that in the protein/prepolymer solution a ureido bond is formed between the protein and the prepolymer. When dispersed in water, unreacted NCO groups react with water and are converted to _NH2 . Thus, the protein/prepolymer solution is dispersed in a sufficient amount of water at a sufficiently slow rate that chain extension is minimized. The method of dispersion is that dispersion should be done by stirring to minimize main chain extension;
The method of dispersion is not critical, except that the addition of the prepolymer solution to the water should be done at a practical rate slow enough to permit dispersion and minimize chain extension. It has been observed that main chain elongation causes the formation of insoluble material which is separated by filtration. Proper dispersion conditions must be maintained to increase the yield of soluble compounds. Furthermore, a surfactant may be used to promote dispersion. The use of surfactants as used in the present invention corresponds to conventional techniques, but the only thing that must be avoided is that the surfactant interacts with the protein and reduces its biological activity. Suitable surfactants include nonionic materials such as those represented by the complex mixture of polyoxyethylene derivatives sold under the trademark "Tween." Also useful are oxyethylene/oxypropylene block copolymers sold under the trademark Pluronic. This surfactant can be added to either the water or the protein/prepolymer solution. Often, upon dispersing a protein/polymer solution in water, the protein/polymer reaction product becomes soluble in water. This is especially the case if the main chain of the polyurethane contains some amount of oxyethylene units, for example 50% or more on a weight basis. As used herein, the term "dispersion" includes an aqueous "solution" of the protein/polymer reaction product. On the other hand, if the reaction product is, on the contrary, at least partially insoluble or difficult to disperse, surfactants can be used as described above. When mixing proteins with prepolymers, the relative amount of NCO groups with respect to NH ₂ groups on the protein is not important. An excess of NCO groups can be used. The term excess assumes that a sufficient amount of isocyanate is used to dissolve the protein into the prepolymer and then the components are mixed and allowed to dissolve at ambient temperature, e.g., about 21°C (70°C) to form a solution. This shall indicate the presence of unreacted free NCO even after 1 hour or more. excessive
If the number of NCO groups is too large, the possibility of chain extension during dispersion in water increases. The potential for formation of insoluble reaction product powders is also increased. Rather, it is preferable that the protein has an excessive amount of amine groups. This reduces the number of polymer chains attached to each protein molecule. It is believed that the benefits of polymer-denatured proteins (eg, stabilization, reduced antigenicity) are equally well achieved whether a single or multiple polymer chain is attached to the protein. The risk of forming substances that are difficult to disperse when added to water is also reduced. It is believed that the protein/polymer reaction products likely to be present in the protein/polymer solution include the following ideal species, where P=protein and Pol=polymer. That the protein may act as a branching center that leads to the next type of ideal structure is
This is not unlikely, especially if excess polymer is present. The structure can stretch to form an extended network that is difficult to disperse in water. Moreover, the presence of multiple polymer chains may create steric hindrances, such as when the protein is an enzyme and the polymer chains block access between the substrate and the enzyme.
The problem would be reduced if the substrate was small, as is the case with peroxidase enzymes - such enzymes are also known to be relatively unstable. According to the present invention, it is believed that it would be beneficial to modify the peroxidase enzyme with polyurethane. Next, preferred specific examples will be described. In a preferred embodiment of the invention described in the above summary: 1. After dispersing the protein/prepolymer solution, the aqueous dispersion is passed through to remove undispersed materials. It is believed that the non-dispersed material is primarily protein bound to the polyurethane which has become difficult to disperse due to chain extension. The use of a different type of surfactant may be sufficient to correct this condition, or it may be preferable to use a dispersion thereof. It is also preferred to use water-soluble hydrophilic polyurethanes having at least 50 mol % of ethylene oxide units in the main chain. 2 Isocyanate-terminated liquid polyurethane prepolymers are produced by the reaction of toluene diisocyanate and polyethylene glycol. 3. Polyisocyanate-terminated liquid polyurethane prepolymers are prepared by the reaction of toluene diisocyanate with polyethylene glycol having a molecular weight (average molecular weight) of about 800 to 1200 (preferably about 1000). 4 Liquid polyurethane prepolymer having isocyanate at the end is toluene diisocyanate, ethylene glycol, diethylene glycol, polyoxyethylene polyol polymer,
It is prepared by reaction with a member selected from the group consisting of pentaerythritol, glycerin, trimethylolpropane and polyoxypropylene polyol polymers of which at least 50% of the main chain is polyoxyethylene. 5 Proteins are enzymes, antibodies, or antigens. 6 Isocyanate-terminated liquid polyurethane prepolymers are prepared by reacting toluene diisocyanate with a mixture of polyethylene glycol and trimethylolpropane having an average molecular weight of about 800 to 1200 (preferably about 1000). be done. In this case, trimethylolpropane and polyethylene glycol are approximately 1:
Toluene diisocyanate is used in a molar ratio of 1 to 4, and toluene diisocyanate is used in a molar ratio of -OH (17
g) about 0.85 to 1.25 (preferably 0.95 to 1.1)
molar of toluene diisocyanate. 7 Isocyanate-terminated liquid polyurethane prepolymers can be prepared from toluene diisocyanate and ethylene glycol by the method described in Example 1 of the aforementioned U.S. Pat. No. 3,929,574 (Wood et al.). The aforementioned patents are incorporated herein in their entirety. In another preferred embodiment (embodiment A), the invention relates to a process for preparing an aqueous protein/polyurethane dispersion of a protein, which can be an enzyme, an antibody or an antigen, which process comprises a) in the absence of water ( forming a first product by mixing the protein and a liquid polyisocyanate (such as at least one of the liquid polyisocyanates shown in Table 1 below) (i.e. under substantially anhydrous conditions); b) Includes isocyanate-terminated liquid polyurethane prepolymers that dissolve proteins by mixing and reacting a first product with an amount of polyol effective to form a second product in the absence of water. forming a second product; However, the polyol may be at least one of those shown in Table 2 below, and may include at least one of those shown in the section immediately preceding Table 2. and c) dispersing the second product in water. In another preferred embodiment (Embodiment B), the present invention is directed to a process for making an aqueous dispersion of an enzyme, antibody or antigen, comprising: a) mixing the protein and a liquid polyol in the absence of water; 1. Step of forming the product. However, the polyol can be at least one of those shown in Table 2 below,
It is also assumed that at least one of the items shown in the section immediately preceding Table 2 above may be included. b) a first product comprising an isocyanate-terminated liquid polyurethane prepolymer having the protein dissolved therein by reacting a first product with an amount of polyisocyanate effective to form a second product in the absence of water; Step 2 of forming the product. However, the polyisocyanate may be at least one of those shown in Table 1 below. and c) dispersing the second product in water. In the methods of Embodiments A and B, it is generally preferred to use about 2 to 500 mg or 50 to 100 mg of protein per gram of polyol. The amount of protein used with respect to isocyanate is not critical. In another preferred embodiment (Embodiment C), the present invention relates to a method for preparing an aqueous dispersion of enzymes bound to polyurethane, which method comprises: a) reacting a liquid polyurethane prepolymer with an enzyme in the absence of water; b) mixing the first composition and the enzyme in the absence of water to form a second composition; and c) the second composition. The process consists of dispersing the water in water. In the method of Embodiment C, it is generally preferred to use about 5 to 100 moles or 8 to 12 moles of substrate per mole of enzyme. In another preferred embodiment (Embodiment D), the present invention provides a water-dispersible hydrophilic poly(urea-urethane) polymer having an oxyalkylene backbone containing at least 50 mole percent oxyethylene by ureido linkages. The invention is directed to a protein-containing aqueous dispersion comprising about 0.1 to 50% by weight, on an anhydrous basis, of a covalently bound active protein formulation, said aqueous dispersion in an isocyanate-terminated liquid prepolymer. The protein may be selected from the group consisting of enzymes, antibodies and antigens. Next, the present invention will be described in detail. In the method of the present invention, the isocyanate-terminated liquid polyurethane prepolymer is used as (a) a solvent that dissolves the protein to be bound, and (b) a reactant that reacts with the protein and binds it thereto. act. The freezing temperature of the isocyanate-terminated liquid polyurethane prepolymers used in the process of the present invention will vary depending on the molecular weight of the prepolymer and the structure of the prepolymer's backbone. The thermal denaturation temperature of proteins is generally greater than about 35°C. However, some proteins are stable at higher temperatures (eg, up to about 70<0>C or somewhat higher) for relatively short periods of time (eg, 5-30 minutes or longer). Formation of the protein/prepolymer solution takes place in the absence of water. Solutions can be formed in the presence or absence of diluents, or in the presence of mixtures of diluents. Given our disclosure, it will be readily apparent to those skilled in the art that diluents that denature the protein or interfere with or substantially reduce dispersibility cannot be used. Diluents that can be used include, but are not limited to, those shown in Stanley US Pat. No. 3,672,955. The diluent may be very soluble in water, such as acetone; moderately soluble in water, such as methyl acetate, methyl ethyl ketone; or insoluble in water, such as benzene and Stanley, supra, U.S. Pat. No. 3,672,955, column 11, column 45. Other diluents indicated in the section starting with the line. Said Patent No. 3672955
No. 5,007,300 is incorporated herein by reference in its entirety. The diluent serves to reduce the viscosity of (a) the isocyanate-terminated liquid polyurethane prepolymer and (b) the resulting solution. Emulsifiers can be used during the foaming process if the diluent is insoluble or substantially insoluble in water.
Stanley (U.S. Patent No. 3672955 Specification)
The use of such emulsifiers is illustrated. It is believed that conjugation of proteins by reaction with prepolymers is a general reaction applicable to all proteins, including, but not limited to, enzymes, antibodies, and antigens. For example: urease, cellulase, pectinase, papain, bromelain, chymotrypsin,
trypsin, fuicin, lysozyme, glucose isomerase, lactase, human immunoglobulin G, invertase, asparginase, etc.
It is believed that they can be combined. Although our research to form aqueous dispersions has not been extensive to date, we have not found any enzymes, antibodies or antigens or other proteins that cannot be bound. I do not believe that the purity of a protein is important; it is the amine groups that are required for it to be pure or not. Conjugation may be performed on (a) pure crystalline protein, (b) partially purified amorphous protein, (c) impure dry extract containing enzyme, antibody or antigenic activity, or (d) unpurified from fermentation broth. This can be carried out using a dry extract of (eg an acetone precipitated product obtained from the broth). We believe that the methods of the invention can be used to bind proteins of virtually any purity, including enzymes, antibodies, and antigens. After the protein/prepolymer solution is formed, if the protein is present in sufficient quantity,
Certain proteins will cause the prepolymer to coagulate. An example of this phenomenon is penicillin amidase. When the level of amidase exceeds about 10% by weight based on the weight of the prepolymer, the viscosity of the prepolymer solution exhibits an increase and stirring is no longer possible after about 60 minutes. At concentrations less than about 5% by weight, the solution is still stirrable and dispersible in water. It has been found that the coagulated penicillin amidase polyurethane is biologically active, ie the enzyme is bound to the active form. For example, 100 mg of Prepolymer 3 (see Example 2 below), 100 mg of penicillin amidase, and 10 mg of penicillin G (Na salt) were mixed to form a solution. During mixing, the viscosity of the solution increased until approximately 10 minutes after contact with the drug, and the solution could no longer be stirred by hand. After 1 hour the solution was a hard solid material which was crushed and sieved.
Particles with a size of less than 840 micrometers were obtained. Particle activity is 1020 units/g on a dry weight basis
It was hot. Activity is expressed as micromoles of penicillin G cleaved per minute at 30°C and PH8. We believe that perhaps other proteins, if present in sufficient quantities in the prepolymer solution, will also react with the prepolymer to form a solid biologically active polymer. There is currently no way to predetermine which proteins will react with the prepolymer to form a solid or at what level the proteins should be used in solution. However, based on the basic discovery of the present inventors, namely that it is possible to form a protein/prepolymer solution without destroying the biological activity of the protein, it is possible to form a protein/prepolymer solution at a sequentially high level for any protein. Solid formation can be easily determined by dissolving in different batches of prepolymer. In accordance with the teachings of the present invention, the above tests can be easily performed by one of the conventional techniques in enzyme chemistry. Once it is determined that a particular protein is immobilized in its active form by binding it to a polyurethane matrix, successively higher levels of the protein are used as solutes to form solutions; Determining whether solid formation occurs is straightforward. vice versa,
If the formation of solids is to be avoided, the above test can also be performed to determine the maximum binding level of the protein. The aforementioned US Pat. No. 3,672,955 shows that proteins (enzymes) can be attached to isocyanate-terminated polyurethanes. In the method of said patent, an isocyanate-terminated polyurethane is dissolved in a water-immiscible solvent.
This solution is emulsified using an emulsifier in the presence of active enzymes dispersed in water. The method of the present invention is described in Stanley's U.S. Pat.
It is similar in some respects to the method of No. 3672955. We can use the same isocyanate-terminated polyurethane prepolymers (referred to as polyisocyanates in the aforementioned U.S. patents); we can use the same polyols (of the present invention) to We can also use the same enzymes. As per that patent (although we do not wish to be bound by any particular theory), the reaction mechanism is that one or more amine and/or hydroxyl groups on a protein and a polyurethane prepolymer molecule appears to be a reaction with one or more isocyanate groups. As in the Stanley patent, the isocyanate-terminated liquid polyurethane prepolymers of the present invention combine a polyol and a polyisocyanate with excess isocyanate to remove free (unreacted) isocyanate on polyurethane prepolymer molecules. It can be produced by conducting a reaction while ensuring the presence of a nato group. The method of the present invention is also similar in certain respects to the method of the aforementioned US Pat. No. 3,929,574 (Utsudo et al.). We can use the same isocyanate-terminated polyurethane prepolymer (which is shown in the patent application as isocyanate-terminated polyurethane), and we The same protein (enzyme) can be used. However, unlike the method of Patent No. 3,929,574, we mix the prepolymer of the present invention and the enzyme of the present invention in substantially water-free conditions. In said patent application, the prepolymer is mixed with an aqueous dispersion of the enzyme. Any liquid polyurethane prepolymer, including those shown in the aforementioned US Pat. No. 3,929,574, containing at least one free isocyanate group per prepolymer molecule is suitable for protein attachment according to the present invention. We recommend that the prepolymer contain an average of 1 to 2 isocyanate groups per molecule. Higher proportions can also be used, for example from 2 to 8 isocyanate groups per polyurethane molecule. Higher rates can be used but will not provide any advantage. Any excess isocyanate groups left in the protein/prepolymer solution (after protein attachment) are destroyed by hydrolysis when the solution is dispersed in water. The isocyanate-terminated (isocyanate-terminated) liquid polyurethane prepolymer used in the present invention contains at least 1 to 2 isocyanate groups (reactive isocyanate groups) per molecule of the prepolymer. . Isocyanate-terminated polyurethane purpolymer is (a)40
or (b) isocyanate-terminated polyurethane prepolymer if it is a free-flowing liquid at ~70°C; A "liquid polyurethane prepolymer" is defined as a "liquid polyurethane prepolymer" if a solution is formed containing about 1 to 50% by weight (or 10 to 25%) of As used herein, the term "liquid isocyanate-terminated polyurethane prepolymer" refers to liquid polyurethane or polyurea molecules containing at least about 2 free isocyanate groups per molecule. do. Typical examples of polyisocyanates that can be made into isocyanate-terminated polyurethanes by reacting with active hydrogen-containing compounds (e.g., glycols, polyols, polyglycols, polyester polyols, polyether polyols, etc.) according to the present invention. includes those shown in Table 1. Table 1 Toluene-2,4-diisocyanateToluene-2,6-diisocyanateCommercial mixture of toluene-2,4-diisocyanate and toluene 2,6-diisocyanateEthylene diisocyanateEthylidene diisocyanatePropylene -1,2-diisocyanatocyclohexylene-1,2-diisocyanatocyclohexylene-1,4-diisocyanate m-phenylene diisocyanate 3,3'-diphenyl-4,4'-biphenylene di Isocyanate 4,4'-biphenylene diisocyanate 3,3'-dichloro-4,4'-biphenylene diisocyanate 1,6-hexamethylene diisocyanate 1,4-tetramethylene diisocyanate 1,10 -decamethylene diisocyanate 1,5-naphthalene diisocyanate cumene-2,4-diisocyanate 4-methoxy-1,3-phenylene diisocyanate 4-chloro-1,3-phenylene diisocyanate 4 -Bromo-1,3-phenylene diisocyanate 4-ethoxy-1,3-phenylene diisocyanate 2,4'-diisocyanat diphenyl ether 5,6-dimethyl-1,3-phenylene diisocyanate 2,4-dimethyl-1,3-phenylene diisocyanate 4,4'-diisocyanat diphenyl ether benzidine diisocyanate 4,6-dimethyl-1,3-phenylene diisocyanate 9,10- anthracene diisocyanate 4,4'-diisocyanatodibenzyl 3,3'-dimethyl-4,4'-diisocyanatodiphenylmethane 2,6-dimethyl-4,4'-diisocyanatodiphenyl 2, 4-diisocyanatostilbene 3,3'-dimethyl-4,4'-diisocyanatodiphenyl 3,3'-dimethoxy-4,4'-diisocyanatodiphenyl 1,4-anthracene diisocyanate 2, 5-fluorene diisocyanate 1,8-naphthalene diisocyanate 2,6-diisocyanatobenzfuran 2,4,6-toluene diisocyanate p,p′,p″-triphenylmethane triisocyanate Useful types The isocyanate-terminated polyurethane prepolymers are derived from polyether polyols and polyester polyols.These compounds are prepared by reacting polyether (or polyester) polyols with polyisocyanates, as is well known to those skilled in the art. The latter may be reacted in excess to ensure that free isocyanate groups are present in the product. Typical, but by no means limiting, examples include, for example: (where m indicates the number of oxyethylene repeating units, which may range from about 5 to 50, for example). Compounds useful for the purposes of this invention include any of the polyisocyanates exemplified above and (a) simple polyols as shown in Table 2 below, and (b) various polyols, including polyether polyols and polyester polyols. It may also be produced by reacting with any one of the following. Representative examples of those polyols are shown below. Some polyether polyols that may be so used are those prepared by reacting alkylene oxides with initiators containing active hydrogen groups, typical examples of which are polyether polyols such as ethylene glycol. alcohols; polyamines such as ethylenediamine; phosphoric acid, etc. The reaction is usually carried out in the presence of an acid or base catalyst.
Examples of alkylene oxides that may be used in this preparation include ethylene oxide, propylene oxide, any isomer of butylene oxide, and mixtures of two or more different alkylene oxides, such as mixtures of ethylene oxide and propylene oxide. The resulting polyether polyol contains a polyether backbone with terminal hydroxyl groups. The number of hydroxyl groups per polymer molecule is determined by the functionality of the active hydrogen initiator. For example, 2 such as ethylene glycol
The functional alcohol (as active hydrogen initiator) is 1
There are two hydroxyl groups per polymer molecule resulting in a polyether chain. When the oxide is polymerized in the presence of glycerin, which is a trifunctional alcohol, the resulting polyether molecule contains an average of three hydroxyl groups per molecule. Higher functionality (more hydroxyl groups) is obtained when the oxide is polymerized in the presence of polyols such as pentaerythritol, sorbitol, sucrose, dipentaerythritol, etc. In addition to those listed above, other examples of polyhydric alcohols that may be reacted with alkylene oxides to produce useful polyether polyols include those shown in Table 2. Table 2 Propylene glycol trimethylene glycol 1,2-butylene glycol 1,3-butanediol 1,4-butanediol 1,5-pentanediol 1,2-hexylene glycol 1,10-decanediol 1,2-cyclohexanediol 2-Butene-1,4-diol 3-cyclohexene-1,1-dimethanol 4-methyl-3-cyclohexene-1,1-dimethanol 3-methylene-1,5-pentanediol diethylene glycol (2-hydroxyethoxy) -1-propanol 4-(2-hydroxyethoxy)-1-butanol 5-(2-hydroxypropoxy)-1-pentanol 1-(2-hydroxymethoxy)-2-hexanol 1-(2-hydroxypropoxy) -2-octanol 3-allyloxy-1,5-pentanediol 2-allyloxymethyl-2-methyl-1,3
-Propanediol [(4-pentyloxy)methyl]-1,3-
-Propanediol 3-(o-propenylphenoxy-1,2-propanediolthiodiglycol 2,2'-[thiobis(ethyleneoxy)]diethanolpolyethylene ether glycol (molecular weight approx.
200) 2,2'-isopropylidenebis(p-phenyleneoxy)diethanol 1,2,6-hexanetriol 1,1,1-trimethylolpropane 3-(2-hydroxyethoxy)-1,2-propanediol ethylene glycol 3-(2-hydroxypropoxy)-1,2-
Propanediol 2,4-dimethyl-2-(2-hydroxyethoxy)methylpentanediol-1,5 1,1,1-tris[(2-hydroxyethoxy)methyl]ethane 1,1,1-tris[(2 -hydroxypropoxy)methyl]propanetriisopropanolamineresorcinolpyrogallolphloroglucinolhydroquinone4,6-di-t-butylcatecholcatecholorcinolmethylphloroglucinolhexylresorcinol3-hydroxy-2-naphthol2-hydroxy-1-naphthol2 , 5-dihydroxy-1-naphthol 2,2-bis(p-hydroxyphenyl)propane and bisphenol triethanolamine 1,1,2-tris-(hydroxy phenyl)
Ethane 1,1,3-tris-(hydroxyphenyl)
Propane A particularly useful class of polyether polyols is polyoxyethylene polyol, HO-( _CH2 - _CH2 -O-) _x H, where x represents a polyol of up to about 1000 (or about
2000 or somewhat higher) average molecular weight. Isocyanate-terminated polyurethane prepolymers become chemically very active when in close contact with proteins such as enzymes, antibodies, antigens, etc. It is believed that some of the free isocyanate groups of the prepolymer react with the amine groups of the protein, and when subsequently dispersed in water, any remaining isocyanate groups react with water to generate carbon dioxide and are deposited on the polyurethane molecule. Forms an amine group.
This latter amine group reacts with free isocyanate groups on nearby polyurethane molecules, and this reaction (formation of urea bonds) results in the formation and growth (including crosslinking) of poly(urea-urethane) polymers, and the reaction If the reaction proceeds too well, non-dispersible molecules result. The proportion of water to the total amount of isocyanate-terminated liquid polyurethane prepolymer and protein is not critical. The many uses of aqueous dispersions of proteins produced by the method of the invention will be readily apparent to those skilled in the art in view of the greater stability and reduced antigenicity of the materials. Aqueous dispersions can also be used as standards or controls in test procedures, such as stabilized glucose oxidase used in serum glucose assays.
Alternatively, it may be used as a stabilized creatine phosphokinase (CPK) for use as a standard in serum creatine phosphokinase (CPK) assays. All types of enzymes can be coupled by the method of the invention. Such enzymes include: oxidoreductase lyase transferase isomerase hydrolase ligase immobilized (i.e. linked) by the method of the invention
Typical specific enzymes that can be used are shown in Table 3. Table 3 Urease Cellulase Trypsin Huicin lactase Bromelain Glucose Oxidase Pancreatin Chymotrypsin Isoamylase Ribonuclease Lipase Peroxidase Malate Dehydrogenase Pepsin Hexokinase Rennin Lactate Dehydrogenase Invertase Adenosine Deaminase Papain Uricase Asparaginase Galactose Oxidase Pectinase Diaphorase Pectinesterase Cholinesterase Penicillin Amidase Aldolase Glucose Isomerase
Pyruvate carboxylase lysozyme phospharylase amino acid acylase
Cephalosporin amidase pronase isocitrate dehydrogenase alcohol dehydrogenase α-amylase α-glycerol phosphate dehydrogenase β-amylase glyceraldehyde-3-phosphate dehydrogenase subtilisin malic enzyme amino acid oxidase glucose-6-phosphate dehydrogenase catalase tannase 5- Dehydroshikimate reductase phenol oxidase
glutathione reductase glucoamylase glycolate oxidase pullulanase nitrate reductase yeast cytochrome c reductase
xanthine oxidase riboyl dehydrogenase cyfrase flavin peroxidase nitrite reductase
glycine oxidase glutamyl transferase
carboxylase glutathione synthetase
α-keto acid dehydrogenase glycocyamine phosphokinase
transketolase hippurate synthetase aldehyde oxidase
Succinate Dehydrogenase The present invention will be better understood by reference to the following specific but non-limiting examples and procedures. It is understood that the invention is not limited by these examples and procedures, which are provided by way of illustration only, and that they may be modified without departing from the spirit and scope of the invention. . In the following examples, the urethane prepolymer (prepolymer) is prepared by mixing polyethylene glycol (PEG, molecular weight 1000) with trimethylolpropane (TMOP), and ending the mixture with toluene diisocyanate (TDI). Manufactured by. The molar ratio of PEG/TMOP/TDI was approximately 2/0.66/6.3. Example 1 Lysozyme bound to polyurethane A water-dispersible lysozyme/polyurethane reaction product was prepared by mixing 0.1 g of lysozyme and 1.0 g of prepolymer. Store these materials at ambient temperature (approximately
The reaction mixture was allowed to react for 1 hour in a desiccator at 21.1°C (about 70°C). 500 ml of deionized water containing 3 drops of poly(oxyethylene/oxypropylene) surfactant (Pluronik L-61) was slowly added to the enzyme/polyurethane composition. The water was stirred rapidly during the addition.
After adding all the composition, it was stirred for another 35 minutes. The stirred material separated into two phases. The soluble phase was separated by passing through a Watzmann #40 filter and then through a Millipore 0.22 μm filter. Activity against Micrococcus lysodeikticus determined that 50% of the enzyme activity was in the soluble phase. A control sample was prepared by curing 1.0 g of prepolymer (no enzyme) as described above. Sephadex G-50 (thin)
It was swollen in 0.05M ammonium acetate buffer (PH7.0) and used to pack a 2.5 x 45 cm glass chromatography column to form a 2.5 x 41 cm finished column. The sample was eluted with 0.05 M ammonium acetate buffer (PH 7.0) at ambient temperature at 1.35 ml min ^-1 and collected in 4.5 ml fractions. Absorbance was measured at λ=280 nm for each fraction. For each sample, absorbance was measured sequentially for 60 fractions eluted from the column. The sample was applied to the following column. Sample of lysozyme bound to soluble polymer
2.0 ml was applied to the column and eluted (as above). Lysozyme bound to soluble polymers is a fraction
There was a peak at absorbance of 0.1 at 37. The free polyurethane used as a control peaked at 41 with an absorbance of 0.12. The shapes of the absorbance curves for the polymer-bound enzyme and free polymer were similar and did not show sharp peaks like dextran and free enzyme. It is believed that the relatively broad distribution and lack of sharp peaks indicates the presence of a broad distribution of polymer chain lengths in the bound enzyme and free polymer samples. A 2.0 ml sample containing 1.5 mg of free dextran (molecular weight approximately 2000) and 4 mg of free lysozyme was applied to the column and eluted with buffer. Dextran peaked at fraction 13 (absorbance 0.71), and free lysozyme peaked at fraction 30 (absorbance 0.32). Example 2 Trypsin bound to polyurethane Trypsin was prepared by mixing 0.1 g of trypsin and 0.9 g of prepolymer and slowly adding this mixture into 500 ml of deionized water as in the previous example with lysozyme. A product was obtained which was bound to polyurethane and was dispersible in water. After filtration and drying the insoluble matter in a dryer at 45°C, 32% of the initial material was recovered as insoluble matter. A control was prepared as in the previous example but without enzyme. 2. Swelling Cephadex G-50 (thin) in deionized water and connecting short tubules in series.
It was used to fill a 0.9 x 60 cm chromatography column with a final column height of 0.9 x 120 cm. The sample was eluted with deionized water at 0.3 ml min ^-1 at ambient temperature and collected in 2.7 ml fractions. λ= for each fraction
Absorbance was measured at 280 nm. A 1.0 ml sample of solublely bound trypsin was applied to the column and eluted with deionized water. it is
It showed two peaks absorbing at 280 nm, a small peak in fraction 20 and a main peak in fraction 23. 1.0 ml of the control sample was applied to the column and eluted with deionized water, showing a small peak in fraction 18 and a major peak in fraction 21. A 1.0 ml sample containing 2 mg of free trypsin was applied to the column and one peak was shown in fraction 20. For soluble trypsin, the absorbance is approximately
They were 190 nm (main peak) and 173 nm (minor peak). Absorbance is approximately 213 nm for free trypsin
It was hot. The absorbance relative to the polyurethane control is approximately
They were 90 nm (minor peak) and about 205 nm (main peak). Example 3 Bovine serum albumin/polyurethane soluble reaction product 0.1 g of bovine serum albumin (BSA) and 0.9 g of prepolymer were mixed and allowed to react for 1 hour in a desiccator (to keep it dry). Thus, BSA bonded to polyurethane was produced. The material was slowly added to 200 ml of deionized water.
The water was stirred rapidly during the addition. After the addition was complete, the aqueous dispersion was stirred for an additional 35 minutes. The soluble fraction was separated by passing through a Watzmann #40 filter followed by a Millipore 0.22 μm filter. The amount of insoluble material (40%, based on the combined amount of enzyme and prepolymer) was determined by drying the residue present after evaporation. Samples of solubles and equal levels of free BSA in deionized water were collected using LKB's Multiphor isoelectric focusing instrument.
Ampholine on focusing unit
Applied to PAG plates (PH range 3.5-9.5). Electrofocusing until the PH gradient ends
It was carried out at 10°C. Next, the gel plate containing the sample was colored to visualize the proteins. Free BSA (shown in the literature to have pI = 4.9) showed four detectable protein bands in the PH range 4.8-5.3. The sample containing the BSA/prepolymer reaction product showed one uniform band in the PH range 4.8-5.0. Example 4 Invertase conjugated to polyurethane Invertase (250 mg) was added to 4 g of prepolymer.
dissolved in Stir this solution at 1000 RPM
Add slowly to 300ml _H2O . The generated dispersion system was passed. Both the aqueous phase and the solid residual phase were found to have enzymatic activity. Free invertase was dispersed in water to obtain the same level of enzyme activity as an aqueous dispersion. Two aqueous dispersions (free enzyme and bound enzyme) at 10
When stored for 14 days at °C, the activity level of both dispersions was approximately 10
% decreased. At the same time, similar comparisons can be made to simply 40 distributed systems
It was stored at ℃. After 14 days, the enzyme bound to the polymer retains approximately 45% of its original activity;
On the other hand, the free enzyme showed only a trace amount of activity, less than 5% of the initial activity. Aqueous dispersions of polymer-bound enzyme were more thermally stable than free enzyme at 40°C. Example 5 Trypsin conjugated to polyurethane Precipitation with trichloroacetic acid Preparation of soluble conjugated proteins Trypsin (400 mg) was mixed with prepolymer (3600 g)
and allowed to react for 1 hour at room temperature in a desiccator (dry). Add the reaction product to 200 ml of water with rapid stirring.
Dispersed in water by portionwise addition over 30 minutes. After passing through Watzmann #40 paper, undissolved products were removed by Millipore filtration using a 0.22 μm filter. The precipitates obtained from both of the above filtrations were combined, dried and weighed. The weight of the reaction minus that of the precipitate was the weight of soluble material in the resulting 200 ml of clear solution. The above procedure was repeated except that no enzyme was used (ie 3600 mg of prepolymer was reacted with 200 ml of water). The resulting polymer solution was used as a post-purification blank, ie, a polymer control solution. Two other controls were prepared as follows: 10 ml of water.
Control-1 containing 6 mg of trypsin dissolved in;
Control-2 containing 6 mg of trypsin dissolved in 10 ml of blank solution. 30 ml of 5% trichloroacetic acid (TCA) solution was added to each of the above solutions (10 ml). After 30 minutes at room temperature, the resulting product was centrifuged (30 minutes,
17000rpm). The supernatant liquid was decanted and the resulting precipitate was dried in a vacuum oven. The weight of the obtained precipitate and each before precipitation
The amount of material in 10 ml of solution was as follows:

Table: Although TCA is a known chemical that precipitates proteins, it is shown here that it does not precipitate soluble polyurethanes. When TCA is added to soluble bound proteins, the proteins co-precipitate with the soluble polyurethane moieties bound to them. Similar tests were performed with BSA and penicillin amidase. The results are shown below.

【table】

[Table] Calculated.

Claims (1)

[Claims] 1 a) A water-dispersible biologically active protein and a liquid urethane prepolymer having an isocyanate-terminated oxyalkylene main chain are mixed together under substantially anhydrous conditions. forming a solution containing the reaction product of the protein and the prepolymer; and b) adding said solution to water under stirring to obtain an aqueous solution or dispersion of the protein bound to the urethane polymer. A method for producing an aqueous solution or dispersion of a biologically active protein bound to a urethane polymer. 2. The method according to claim 1, wherein the protein is an enzyme.