GB1574508A

GB1574508A - Waterdispersible proteins with polyurethanes

Info

Publication number: GB1574508A
Application number: GB51208/77A
Authority: GB
Original assignee: WR Grace and Co
Current assignee: WR Grace and Co
Priority date: 1976-12-10
Filing date: 1977-12-08
Publication date: 1980-09-10
Also published as: NL7713584A; BE861602A; NL187585B; FR2393005A1; ATA883577A; NL187585C; FR2393005B1; JPH0233356B2; JPS5379094A; MX4534E; DE2755053C2; CH634589A5; AT356620B; DE2755053A1

Abstract

To prepare the novel aqueous dispersion or solution of protein bonded to a urethane polymer, water-soluble or -dispersible bioactive proteins and water-soluble or -dispersible liquid polyurethane prepolymers containing terminal isocyanate groups are mixed under essentially anhydrous conditions, and this mixture is then dissolved or dispersed in water.

Description

(54) WATER-DISPERSIBLE PROTEINS MODIFIED WITH POLYURETHANES (71) We, W. R. GRACE & CO., a Corporation organized and existing under the laws of the State of Connecticut, United States of America, of Grace Plaza, 1114 Avenue of the Americas, New York, New York 10036, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention relates to proteins modified by attachment to organic polymers.

The term "soluble bound enzymes" is employed in the art to designate the reaction product of a protein with a polymeric material (natural or synthetic); the reaction product is soluble or dispersible in liquid media, such as water. The protein/polymer bond is generally covalent although adsorption techniques have also been employed. Such soluble bound proteins frequently exhibit biological reactivity similar in nature to that of the free protein; some have exhibited increased solubility, and/or decreased antigenicity and other improvements in comparison with the free unmodified proteins.

Enzymes have been bound to various polymers in a number of ways. In particular they have been bound to polyurethanes, for example polyurethane foams. Thus United States Patent No. 3,929,574 discloses the preparation of a bound (immobilized) protein, an enzyme, by contacting an isocyanate-capped liquid polyurethane prepolymer with an aqueous dispersion of the enzyme under foam-forming conditions, whereby the polyurethane foams and the enzymes become integrally bound to the resulting polyurethane foam.

This invention provides a process for preparing an aqueous dispersion or solution of protein bound to a urethane polymer which comprises mixing an at least water-dispersible, biologically-active protein and an at least water-dispersible isocyanate-capped liquid polyurethane prepolymer under essentially anhydrous conditions to form a solution, and dispersing the solution in water such that an aqueous dispersion or solution of the protein-bound polymer is obtained and the products obtained thereby. After dispersion in water any water-insoluble material can be separated (e.g. by filtration).

The process is conducted at a temperature at which the isocyanate-capped polyurethane prepolymer exists in the liquid state. Naturally the temperature should be below the denaturation temperature of the protein which is being bound.

The thermal denaturation temperature of proteins is generally above about 35"C.

However, some proteins are stable for relatively short periods (e.g., 5-30 minutes or longer) at higher temperatures (e.g. at temperatures up to about 70"C or somewhat higher).

In the resulting compounds it is believed, although we are not bound by this theory, that the protein is bound to the polyurethane polymer through at least one ureido linkage; such compounds form another aspect of the present invention. It is apparent from the Examples below that a protein/polyurethane product is formed which has different characteristics, e.g. migration through a chromatographic column, migration in an electric field, and solubility properties, than are possessed by either the free protein or the free polymer.

It is, of course, known that the reaction rates of amines with isocyanate groups are much faster than reaction rates between amines and water or hydroxyl groups.

It is also known that proteins e.g. enzymes, antigens, antibodies and simple proteins (e.g. serum albumins) are essentially polymers of amino acids acids having available -NH2 groups. Therefore, it is likely that in the protein/prepolymer solution ureido linkages form between the protein and the prepolymer.

It is accordingly believed that protein/polymer reaction products likely to be present in the protein/prepolymer solution include the following idealized species (where P=protein and Pol=polymer):

It is not unlikely, especially where excess polymer is present, that the protein may serve as a centre for branching resulting in the following type of idealized structure:

The above structures can be extended to form an extensive network which is difficult to disperse in water. Further the presence of large numbers of polymer chains, for example in an enzyme, may create steric interference. This could be reduced where the substrate is small as in the case of peroxidase enzymes; such enzymes are also known to be relatively unstable. It is believed that reaction of peroxidase enzymes with the polyurethane prepolymer is beneficial.

Upon being dispersed in water any unreactedNCO groups are converted to -NH2 by reaction with water by hydrolysis, to give carbon dioxide and form amine groups on the polyurethane molecule. These latter amine groups may react with free isocyanate groups on neighbouring polyurethane molecules, and this reaction (forming a urea linkage) can cause formation and growth (including cross-linking) of a poly(urea-urethane) polymer, which may result in non-dispersible molecules if the reactions proceed far enough.

In the first stage of mixing the protein and prepolymer the relative amount of NCO groups in relation to NH2 groups of the protein is not critical. An excess of NCO groups can be employed, i.e. sufficient isocyanate is employed so that after dissolving the protein in the prepolymer there is unreacted free -NCO more than one hour after the solution has been formed assuming the components were mixed and allowed to dissolve at ambient temperature, e.g. 700F (210C). If the excess of NCO groups is too large, the likelihood of chain extension on dispersing in water is increased and also the likelihood of forming insoluble reaction products is also increased. Preferably the protein amine groups will be in excess so as to reduce the number of polymer chains attached to each protein molecule. However, it is believed that the advantages (e.g. stabilization, reduced antigenicity) of the polymer-modified proteins can be accomplished whether one or a large number of polymer chains are bound to the protein.

In a preferred embodiment ("Embodiment A") of this invention the process comprises: (a) forming a first product by mixing, in the absence of water (i.e. under essentially anhydrous conditions), the protein and a liquid polyisocyanate; and (b) forming a second product comprising an isocyanate-capped liquid polyurethane prepolymer with the protein dissolved therein by mixing and reacting, in the absence of water, the first product and an appropriate amount of a polyol.

Alternatively, the process comprises: (a) forming a first product by mixing, in the absence of water, the protein and a liquid polyol; and (b) forming a second product comprising an isocyanate-capped liquid polyurethane prepolymer with the protein dissolved therein by reacting, in the absence of water, the first product and an appropriate amount of a polyisocyanate.

In these embodiments it is generally preferred to use 2 to 500, especially 50 to 100, mg of protein per gram of polyol. The amount of protein employed in relation to the isocyanate is not critical.

In another preferred embodiment the process comprises: (a) mixing, in the absence of water, the liquid polyurethane prepolymer and a substrate reactable with the enzyme; and (b) mixing, in the absence of water, the resulting mixture and the enzyme.

Here it is generally preferred to use 5 to 100 moles, especially 8 to 12 moles, of substrate per mole of enzyme.

In the process of this invention the isocyanate-capped liquid polyurethane prepolymer acts as: (a) a solvent to dissolve the protein which is to be bound; and (b) a reactant to react with the protein to bind it (the protein) to itself.

The solidification temperature of the isocyanate-capped liquid polyurethane prepolymer used depends on the molecular weight of the prepolymer and on the structure of the backbone of the prepolymer.

The formation of the protein/prepolymer solution is conducted in the absence of water and in the presence or absence of a diluent or mixture of diluents. It will readily be apparent to those skilled in the art that a diluent which would.denature the protein or prevent or substantially reduce dispersibility cannot be employed.

Diluents which are operable include those disclosed in United States Patent No.

3,672,955. The diluents can be very soluble in water, e.g. acetone; moderately soluble in water, e.g., methyl acetate or methyl ethyl ketone; or insoluble in water, e.g. benzene and the other such diluents listed in United States Patent No.

3,672,955.

Diluents serve to reduce the viscosity of: (a) the isocyanate-capped liquid polyurethane prepolymer; and (b) the resulting solution.

Where the diluent is insoluble or substantially insoluble in water, an emulsifying agent can be used to assist dispersion of the protein/methane polymer reaction product in water.

Binding of the protein by reaction with the prepolymer is believed to be a general reaction applicable to all proteins including, but not limited to, enzymes, antibodies, and antigens.

Such enzymes include, oxido reductases, lyases, transferases, isomerases, hydrolases and ligases.

Specific Enzymes Include urease cellulase trypsin ficin lactase bromelain glucose oxidase pancreatin chymotrypsin isoamylase ribonuclease lipase peroxidase malic dehydrogenase pepsin hexokinase rennin lactate dehydrogenase invertase adenosine deaminase papain uricase asparaginase galactose oxidase pectinase diaphorase pectin esterase cholinesterase penicillin amidase aldolase glucose isomerase pyruvate carboxylase lysozyme phosphorylase amine acid acylase cephalosporin amidase pronase isocitric dehydrogenase alcohol dehydrogenase a-glycerolphosphate dehydrogenase a-amylase A-amylase glyceraldehyde-3-phosphate dehydrogenase subtilisin amino acid oxidase malic enzyme catalase glucose-6-phosphate dehydrogenase tannase phenol oxidase 5-dehydroshikimic reductase glucoamylase glutathione reductase pullulanase glycolic acid oxidase yeast cytochrome c reductase nitrate reductase luciferase xanthine oxidase nitrite reductase lipoyl dehydrogenase glutamyl transferase flavin peroxidase glutathione synthetase glycine oxidase glycocyamine phosphokinase carboxylase hippuric acid synthetase a-keto acid dehydrogenase aldehyde oxidase transketolase succinic dehydrogenase as well as human immunoglobulin G. The purity of the protein is not believed to be critical; whether pure or not it would be expected to have the requisite amine groups. Binding can thus be accomplished using: (a) pure crystalline protein; (b) partially purified non-crystalline protein; (c) impure dried extracts containing enzyme, antibody, or antigen activity; or (d) unpurified dried extract from a fermentation broth (e.g. an acetone precipitation product obtained from the broth).

Following formation of the protein/prepolymer solution certain proteins will cause the prepolymer to solidify if the protein is present in sufficiently large amounts. An example of this phenomenon is penicillin amidase. Where the amidase level exceeds about 10 weight percent based on the weight of the prepolymer, the prepolymer solution exhibits increased viscosity and cannot be stirred after about 60 minutes. At concentrations below about 5 weight percent, the solution can still be stirred and dispersed into water. Clearly the onset of solid formation can be determined simply by dissolving the protein at successively larger levels in the different batches of prepolymer.

Isocyanate-capped polyurethane prepolymers are well known to those skilled in the art (see, for example, Kirk-Othmer "Encyclopedia of Chemical Technology", John Wiley and Sons, Inc., New York, 2nd ed. vol 9, and "The Encyclopedia of Chemistry", George L. Clark, Editor, Reinhold Publishing Corporation, New York, 2nd ed.). Those used in the process of this invention are water-dispersible or water-soluble.

Subject to this proviso any liquid polyurethane prepolymer, including those mentioned in United States Patents Nos. 3,672,955 and 3,929,574, which contains at least one free isocyanate group per prepolymer molecule can be used. Preferably the prepolymer contains an average of one to two isocyanate groups per molecule although those with, for example, 2 to 8 or more isocyanate groups per polyurethane molecule can also be used.

The isocyanate-capped (isocyanate-terminated) liquid polyurethane prepolymers used in this invention preferably contain at least two isocyanate groups (reactive isocyanate groups) per molecule of prepolymer. An isocyanatecapped polyurethane prepolymer is a "liquid polyurethane prepolymer" which, as used herein, satisfies the following conditions: it is a free flowine liquid at 40 to 70cC; or it can be dissolved in an inert solvent, such as those listed above to form a solution containing, say, 1 to 50% more particularly 10 to 25 gn by weight of isocyanate-capped polyurethane prepolymer.

As used herein, the term "liquid isocyanate-capped polyurethane prepolymer" preferably means a liquid polyurethane or polyurea molecule containing at least two free isocyanate groups per molecule.

Representative examples of polyisocyanates which can be reacted with an active hydrogen-containing compound (e.g. a glycol. polyol, polyglycol, polyester polyol or polyether polyol) to make an isocyanate-capped polyurethane are as follows: toluene-2,4-diisocyanate, toluene-2,6-diisocyanate, commercial mixtures of toluene-2,4- and 2,6-diisocyanates, ethylene diisocyanate, ethylidene diisocyanate, propylene- 1 ,2-diisocyanate, cyclohexylene-1,2-diisocyanate, cyclohexylene-1,4diisocyanate, m-phenylene diisocyanate, 3,3'-diphenyl-4,4'-biphenylene diisocyanate. 4,4'-biphenylene diisocyanate, 3,3'-dichloro-4,4'-biphenylene diisocyanate, 1,6-hexamethylene diisocyanate, 1 ,4-tetramethylene diisocyanate, 1,10decamethylene diisocyanate, 1,5-naphthalene, diisocyanate, cumene 2,4diisocyanate, 4-methoxy- 1,3-phenylenediisocyanate, 4-chloro- 1 ,3-phenylene diisocyanate, 4-bromo- 1 ,3-phenylenediisocyanate, 4-ethoxy- 1 ,3-phenylenediisocyanate, 2,4'-diisocyanatodiphenylether, 5,6-dimethyl- 1 ,3-phenylene diisocyanate, 2,4 dimethyl- 1 ,3-phenylene diisocyanate, 4,4'-diisocyanatodiphenylether, benzidine, diisocyanate, 4,6-dimethyl-1,3-phenylene diisocyanate, 9,1 0-anthracene diisocyanate, 4,4'-diisocyanatodibenzyl, 3,3'-dimethyl-4,4'-diisocyanatodiphenylmethane, 2,6-dimethyl-4,4'-diisocyanatodiphenyl, 2,4diisocyanatostilbene, 3, 3'-dimethyl-4,4'-diisocyanatodiphenyl, 3, 3'-dimethoxy-4,4'diisocyanatodiphenyl, 1,4 anthracene diisocyanate, 2,5-fluorene diisocyanate, 1,8naphthalene diisocyanate, 2,6-diisocyanatobenzfuran, 2,4,6-toluenetriisocyanate and p,p',p"-triphenylmethane triisocyanate.

A useful class of liquid isocyanate-capped polyurethane prepolymers are those derived from polyether polyols and polyester polyols. These compounds may be prepared, as is well known in the art, by reacting a polyether (or polyester) polyol with a polyisocyanate, using an excess of the latter to ensure provision of free isocyanate groups in the product. A typical example is illustrated in idealized equation form below:

HO ( CH2CH2 3m H Polyether polyol CH3 1 3 NCO \ NCO Polyisocyanate C{3 NH 0 } 2 2 m 011 CH3 OCN CO C-O + CH2CH2 ' m CF ECO Isocyanate-capped liquid polyurethane prepolymer m represents the number of oxyethylene repeating units, this may range, for example, from, say, 5 to 50.

The prepolymers may be prepared from a wide variety of polyols such as are mentioned in United States Patent No. 3,672,955 including simple polyols and polyether polyols and polyester polyols.

Typical simple polyols include: propylene glycol, trimethylene glycol, 1 ,2-butylene glycol, 1,3-butanediol, 1,4butanediol, 1 5-pentanediol, 1 .2-hexylene glycol, 1,1 0-decanediol, 1,2cyclohexanediol, 2-butene- 1 ,4-diol, 3-cyclohexene- 1,1 -dimethanol, 4-methyl3-cyclohexane- 1, 1-dimethanol, 3-methylene- 1,5-pentanediol, diethylene glycol, (2hydroxyethoxy)-l-propanol, 4-(2-hydroxyethoxy) - 1 - butanol, 5 - (2 hydroxypropoxy) - I - 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)methyli - 1,3 propanediol, 3 - (o - propenylphenoxy) - 1,2 - propanediol, thiodiglycol, 2,2' [thiobis(ethyleneoxy)1 - diethanol, polyethylene ether glycol (molecular weight about 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 - trisl(2 hydroxyethoxy)methyl] ethane, 1,1,1 - tris[(2 - hydroxypropoxy)methyl]propane, triethanolamine, triisopropanolamine, resorcinol, pyrogallol, phloroglucinol, hydroquinone, 4,6 - di - tertiarybutyl catechol, catechol, orcinol, methylphloroglucinol, hexylresorcinol, 3 - hydroxy - 2 - naphthol, 2 - hydroxy - 1 - naphthol, 2,5 - dihydroxy - 1 - naphthol, bis - phenols such as 2,2 - bis(p - hydroxyphenyl)propane and bis - (p - hydroxyphenyl)methane, 1,1,2-tris - (hydroxyphenyl)ethane and 1,1,3 - tris - (hydroxyphenyl)propane.

Typical polyether polyols are those prepared by reaction of an alkylene oxide with an initiator containing active hydrogen groups, for example, a polyhydric alcohol such as ethylene glycol; a polyamine such as ethylene diamine; or phosphoric acid. The reaction is usually carried out in the presence of either an acidic or basic catalyst. Examples of suitable alkylene oxides include ethylene oxide, propylene oxide, any of the isomeric butylene oxides, and mixtures of two or more alkylene oxides such as mixtures of ethylene and propylene oxides. The resulting polyether polyols contain a polyether backbone and are terminated by hydroxyl groups. The number of hydroxyl groups per polymer molecule is determined by the functionality of the active hydrogen initiator. For example, a difunctional alcohol such as ethylene glycol leads to polyether chains in which there are two hydroxyl groups per polymer molecule. When polymerization of the oxide is carried out in the presence of glycerol, a trifunctional alcohol, the resulting polyether molecules contain an average of three hydroxyl groups per molecule.

Even higher functionality is obtained when the oxide is polymerized in the presence of such polyols as pentaerythritol, sorbitol, sucrose and dipentaerythritol. Other polyhydric alcohols which may be reacted with alkylene oxides to produce useful polyether polyols are included in the list of simple polyols already given.

An especially useful class of polyether polyols are the polyoxyethylene polyols HOCH2CH2-0)xH in which x is an average number such that the polyol has a number average molecular weight of up to about 1000, or about 2000 or somewhat higher.

The polyester polyols which may be employed are most readily prepared by condensation polymerization of a polyol with a polybasic acid. The polyol and acid reactants are used in such proportions that essentially all the acid groups are esterified and the resulting chain of ester units is terminated by hydroxyl groups.

Representative examples of such polybasic acids are oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, brassylic acid, thapsic acid, maleic acid, fumaric acid, glutaconic acid, a-hydromuconic acid, P-hydromuconic acid, a-butyl-a-ethylglutaric acid, a,-di-ethylsuccinic acid, o-phthalic acid, isophthalic acid, terephthalic acid, hemimellitic acid, trimellitic acid, trimesic acid, mellophanic acid, prehnitic acid, pyromellitic acid, citric acid, benzenepentacarboxylic acid, 1,4 - cyclohexane dicarboxylic acid, diglycollic acid, thiodiglycollic acid, dimerized oleic acid and dimerized linoleic acid. Representative examples of polyols for forming these polymers include ethylene glycol, 1,3-propylene glycol, 1,2-propylene glycol, 1,4butylene glycol, 1,3-butylene glycol, 1,2-butylene glycol, butene-1,4-diol, 1,6hexane diol, hexene - 1,6 - diol, 1,7 - heptane diol, diethylene glycol, glycerine, trimethylol propane 1,3,6 - hexanetriol, triethanolamine, pentaerythritol, sorbitol, and any of the other polyols given above.

Typical preferred isocyanate-capped prepolymers useful in the process of this invention include: (a) the prepolymer prepared by reacting toluene diisocyanate and a polyethylene glycol.

(b) the prepolymer prepared by reacting toluene diisocyanate and a polyethylene glycol having a molecular weight (number average molecular weight) of 80W1,200, preferably about 1,000.

(c) the prepolymer prepared by reacting toluene diisocyanate with ethylene glycol, diethylene glycol, a polyoxyethylene polyol polymer, pentaerythritol, glycerol, trimethylol propane, and a polyoxypropylene polyol polymer-provided at least 50 mole percent of the backbone is polyoxyethylene.

(d) the prepolymer prepared by reacting toluene diisocyanate with a mixture of a polyethylene glycol having a number average molecular weight of about 80 > 1,200, preferably about 1,000, and trimethylol propane, the trimethylol propane and the polyethylene glycol being provided in a mole ratio of about 1:1 to 4 and the toluene diisocyanate being provided in an amount of about 0.85-1.25, preferably 0.951.1, mole of toluene diisocyanate per equivalent (17 g) of-OH provided by the polyethylene glycol plus the trimethylol propane.

(e) the prepolymer prepared from toluene diisocyanate and ethylene glycol according to the method given in Example 1 of United States Patent No. 3,929,574.

The protein/prepolymer solution is then dispersed in water. It is desirable that it is dispersed into enough water and at a rate sufficiently slow so that chain extension is minimized. Otherwise the method of dispersion is not critical except that to minimize chain extension dispersion it should be accompanied by agitation.

Further it will be appreciated that if there is insufficient agitation foaming will take place. It has been found that some chain extension usually occurs to give waterinsoluble material which can be separated by filtration. To increase yields of soluble compounds, adequate dispersion conditions should be maintained. To avoid this situation it is preferred to use water-soluble hydrophilic polyurethanes having at least 50 mole percent of ethylene oxide units in the backbone. The ratio of water to protein plus isocyanate-capped liquid polyurethane prepolymer is not critical.

Additionally to facilitate dispersion a surfactant may be employed. The use of surfactants represents conventional technology, the only caveat being that the surfactant should not of course, interact with the protein to decrease biological activity. Suitable surfactants include nonionic materials such as those represented by the complex mixture of polyoxyethylene derivatives sold under the Registered Trade Mark "Tween". Also useful are the block copolymers of oxyethylene/oxypropylene sold under the Registered Trade Mark "Pluronic". The surfactant can be added either to the water, or the protein/prepolymer solution.

In dispersing the protein/polymer solution in water, it frequently happens that the protein/polymer reaction product is water soluble. This is especially the case where the backbone of the polyurethane contains an appreciable number of oxyethylene units, e.g. 50% or more on a weight basis. The term "dispersions" as used herein includes aqueous "solutions" of the protein/polymer reaction product.

In a preferred embodiment the protein-containing aqueous dispersion comprises about 0.1 to 50 /O by weight of an active protein preparation (anhydrous basis) covalently bound by ureido linkages to a water-dispersible hydrophilic poly(urea-urethane) polymer having an oxyalkylene backbone containing at least 50 mole percent oxyethylene, said protein being an enzyme, an antibody, or an antigen.

Numerous uses for aqueous dispersions or solutions of protein compositions of this invention will be readily apparent to those skilled in the art in view of the increased stability and reduced antigenicity of said materials. They may, inter alia, be employed as standards or controls in test procedures, e.g. stabilized glucose oxidase for use in an assay of serum glucose, or stabilized creatine phosphokinase (CPK) for use as a standard in assaying for serum CPK.

The following Examples (which were actually run) further illustrate the present invention. In these Examples the urethane prepolymer (Prepolymer) was prepared by mixing polyethylene glycol (PEG-M.W. about 1,000) with trimethylol propane (TMOP) and capping the mixture with toluene diisocyanate (TDI). The molar ratio of PEG/TMOP/TDI was about 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 the Prepolymer. These materials were alloWed to react-for one hour in a desiccator at ambient temperature (approximately 700 F).

The enzyme/polyurethane composition was slowly added to 500 ml deionized water containing 3 drops of a poly(oxyethylene/oxypropylene) surfactant ("Pluronic" (Registered Trade Mark) L-61). During addition the water was agitated rapidly. After all the composition had been added it was stirred for an additional 35 minutes. The stirred material separated into two phases.

The soluble phase was separated by filtration through a Whatman &num;40 filter, then through a Millipore 0.22 ,um filter. ("Whatman" and "Millipore" are Registered Trade Marks). From activity against Micrococcus lysodeikticus, it was determined that 50 /" of the enzyme activity was present in the soluble phase.

A control sample was prepared by curing and filtering 1.0 g of Prepolymer (without enzyme) as above.

"Sephadex" (Registered Trade Mark) G-50 (fine) was swollen in 0.05 M ammonium acetate buffer (pH 7.0) and used to fill a 2.5x45 cm glass chromatography column for a finished column of 2.5x41 cm. Samples were eluted with 0.05 M ammonium acetate buffer (pH 7.0) at 1.35 ml minor at ambient temperature and collected in 4.5 ml fractions. Absorbence at A=280 nm was read for each fraction. For each sample the absorbence was measured serially for 60 fractions eluted from the column. The samples were applied to the column as follows.

A 2.0 ml sample of soluble polymer-bound lysozyme was applied to the column and eluted (as described). The soluble polymer-bound lysozyme peaked at fraction 37 with an absorbence of 0.1. The free polyurethane used as a control peaked at 41 and an absorbence of 0.12.

The shapes of the absorbence curves for the polymer-bound enzyme and free polymer were similar and did not give sharp peaks as did the dextran and free enzyme. The relatively broad distribution and lack of a sharp peak is believed to reflect the presence of the broad distribution of polymer chain lengths in the bound enzyme and free polymer samples.

A 2.0 ml sample containing 1.5 mg free dextran (M.W. about 2000) and 4 mg free lysozyme was applied to the column and eluted with buffer. The dextran peaked at fraction 13 (absorbence 0.71) and free lysozyme at fraction 30 (absorbence 0.32).

EXAMPLE 2 Trypsin Bound to Polyurethane Trypsin was bound to polyurethane to yield a water-dispersible product by mixing 0.1 g of trypsin and 0.9 g of the Prepolymer and slowly adding the mixture to 500 ml of deionized water as in the preceding Example.

After filtration, and drying the insoluble material in a 45"C drying oven, 32 X"</

EXAMPLE 4 Invertase Bound to Polyurethane Invertase (250 mg) was dissolved in 4 g of Prepolymer. The solution was slowly added to 300 ml of H2O stirred at 1000 RPM. The resulting dispersion was filtered.

Both the aqueous phase and the solid residual phase were found to possess enzymatic activity.

Free invertase was dispersed in water to provide the same level of enzyme activity as the aqueous dispersion. The two aqueous dispersions (free-enzyme and bound-enzyme) were stored at 100C for 14 days with the activity levels of both dispersions decreasing by about 10%. Simultaneously a similar comparison was conducted only the dispersions were stored at 400 C. After 14 days the polymerbound enzyme retained about 45% of its original activity whereas the free enzyme exhibited only trace amounts of activity amounting to less than 5% of the initial activity.

The aqueous dispersion of polymer-bound enzyme was more thermally stable at 40"C than the free enzyme.

EXAMPLE 5 Trypsin Bound to Polyurethane Precipitation with Trichloroacetic Acid Preparation of Soluble Bound Protein Trypsin (400 mg) was dissolved in the Prepolymer (3600 mg) and was allowed to react at room temperature in a desiccator (dry) for one hour.

The reaction product was dispersed in water by adding to 200 ml water in small increments with rapid stirring for 30 minutes.

Insoluble product was removed by filtering through Whatman &num;40 filter paper, followed by Millipore filtration using a 0.22 ym filter.

The combined precipitates from both filtrations were dried and weighed. The difference (reactant weights less precipitates) was the amount of soluble material in the resultant 200 ml of clear solution.

The above was repeated except no enzyme was used (i.e. 3600 mg Prepolymer reacted with 200 ml water). The resultant polymer solution after filtration served as the control.

Two other controls were made as follows: Control-l contained 6 mg trypsin dissolved in 10 ml water; Control-2 contained 6 mg trypsin dissolved in 10 ml of the blank solution.

To solution (10 ml) of each of the above, 30 ml of a 5% trichloroacetic acid solution was added. After 30 minutes at room temperature, the resultant products were centrifuged (30 minutes; 17,000 rpm). Supernatants were decanted and the resultant precipitates were dried in a vacuum oven.

The weights of the precipitates obtained, as well as the amount of material in each 10 ml solution prior to precipitate were: Amount of Material in Protein in Polymer in Sample Solution Solution Solution Precipitate Blank 72 0 72 2 Control-l 6 6 0 8 Control-2 78 6 72 9 Soluble Bound 59 (6)* (53)* 25 * Calculated from the known amount in solution (obtained as reactants less initial filtration) using the ratio of Prepolymer/enzyme in the reaction mixture.

TCA is a known reagent for precipitating proteins, whereas it is herein shown it does not precipitate soluble polyurethane. When TCA is added to soluble bound proteins, the protein precipitates out, bringing along with it that portion of the soluble polyurethane which was bound thereto.

Similar runs were carried out with BSA and penicillin amidase. The results are set forth below.

BSA/Trichloroacetic Acid Precipitation Total Protein Polymer Precipitate Blank 62 0 62 2 Control 1 8 8 0 11 Control-2 70 8 62 16 Soluble Bound 80 (8)" (72)a 23 Penicillin Amidase/Trichloroacetic Acid Precipitation Total Protein Polymer Precipitate Blank 62 0 62 0 Control-l 8 8 0 8 Control-2 70 8 62 12 Soluble Bound 77 (7)" (70)8 20 a) Calculated as described above for trypsin.

WHAT WE CLAIM IS: 1. A process for preparing an aqueous dispersion or solution of protein bound to a urethane polymer which comprises mixing an at least water-dispersible, biologically-active protein and an at least water-dispersible isocyanate-capped liquid polyurethane prepolymer under essentially anhydrous conditions to form a solution, and dispersing the solution in water such that an aqueous dispersion or solution of the protein-bound polymer is obtained.

2. A process according to Claim 1 wherein the aqueous dispersion is filtered to remove water-insoluble protein/polymer material.

3. A process according to Claim 1 wherein the protein/polymer material is water soluble.

4. A process according to any one of Claims 1 to 3 wherein the prepolymer comprises an oxyalkylene backbone.

5. A process according to Claim 4 wherein the polymer backbone consists of oxyethylene, oxypropylene or oxybutylene units.

6. A process according to Claim 5 wherein the polymer backbone consists of oxyethylene units.

7. A process according to Claim 6 wherein the prepolymer is derived from a polyethylene glycol having a number average molecular weight from 800 to 1200.

8. A process according to Claim 4 wherein the prepolymer contains at least 50 mole % of ethylene oxide units in the backbone.

9. A process according to any one of Claims 1 to 3 wherein the prepolymer comprises a linear polyester backbone.

10. A process according to any one of the preceding claims wherein the prepolymer is formed in the presence of the protein.

11. A process according to Claim 10 wherein the protein is either mixed with a liquid polyol or liquid isocyanate and the resulting composition is mixed with a liquid isocyanate or liquid polyol, respectively, all under essentially anhydrous conditions.

12. A process according to Claim 11 wherein 50 to 100 mg of protein are used per gram of polyol.

13. A process according to any one of the preceding claims wherein a surfactant is added to facilitate the dispersion.

14. A process according to any one of the preceding claims where the protein is an enzyme.

15. A process according to Claim 14 wherein the enzyme is one specifically identified herein.

16. A process according to Claim 1 substantially as hereinbefore described.

17. A process according to Claim 1 substantially as described in any one of the Examples.

18. An aqueous dispersion or solution of protein bound to a urethane polymer whenever prepared by a process as claimed in any one of the preceding claims.

19. A solution or dispersion in water of a water-dispersible, biologically-active protein and a polyurethane having an essentially linear polyoxyalkylene backbone, said protein being bound to said polyurethane through ureido linkages.

20. A solution or dispersion according to Claim 19 which has one or more of the features of Claims 3, 5, 6 and 14.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. Penicillin Amidase/Trichloroacetic Acid Precipitation Total Protein Polymer Precipitate Blank 62 0 62 0 Control-l 8 8 0 8 Control-2 70 8 62 12 Soluble Bound 77 (7)" (70)8 20 a) Calculated as described above for trypsin. WHAT WE CLAIM IS:

1. A process for preparing an aqueous dispersion or solution of protein bound to a urethane polymer which comprises mixing an at least water-dispersible, biologically-active protein and an at least water-dispersible isocyanate-capped liquid polyurethane prepolymer under essentially anhydrous conditions to form a solution, and dispersing the solution in water such that an aqueous dispersion or solution of the protein-bound polymer is obtained.

16. A process according to Claim 1 substantially as hereinbefore described.