CH639673A5 - Process for the preparation of polyurethane foam containing bonded protein, and the use thereof - Google Patents

Description

The invention relates to a process for the production of bound protein-containing foam, containing 0.1 to 50% by weight of an active protein preparation on an anhydrous basis and a hydrophilic poly (urea urethane) foam matrix made of a polyurethane prepolymer with terminal isocyanate groups, protein and water, the foam produced in this way and its use for contacting with substances which react biologically on the protein of the foam.

Immobilized proteins are already known. For example, U.S. Patent 3,557,4062 describes a method for making bound protein (enzyme) in which a polyester polyurethane diazotizes with a diazonium salt of an amino acid and then with a non-enzymatic animal protein to form a diazotized Coupled polyurethane and then reacted with an enzyme to form the immobilized enzyme.

U.S. Patent 3,705,084 describes a flow-through reactor composed of (a) a macroporous reactor core, (b) a polymeric surface (which may be made of a polyurethane resin) on the reactor core, and (c) an enzyme absorbed on the polymeric surface by a cross-linking, bifunctional agent (eg a polyisocyanate) is held in its place.

US Pat. No. 3,791,927 describes a water-insoluble bound protein (enzyme) which is introduced into the cells of a self-supporting cellular material (which can be a polyurethane foam), the protein being bound to the cell-forming material.

U.S. Patent 3,672,955 also describes a method; for the production of bound protein (enzyme) by (a) emulsifying an aqueous dispersion of the enzyme with a solution of a polyisocyanate in a volatile, water-immiscible solvent (for example methyl chloroform), (b) mixing the emulsion obtained with a solid, finely divided carrier and (c) evaporating the solvent. The polyisocyanate used according to this patent can consist of a liquid polyurethane prepolymer with terminal isocyanate groups.

Example 3 of the above patent describes the binding of an enzyme component (a peroxidase) of a fermentation broth by mixing part of the broth with a polyisocyanate dissolved in methyl chloroform. It seems likely that under the reaction conditions given there, other enzymes contained in the broth will also be immobilized (insoluble in water, i.e. bound).

US Pat. No. 3,929,574 describes the preparation of a bound (immobilized) enzyme by contacting a liquid polyurethane prepolymer having terminal isocyanate groups with an aqueous dispersion of the enzyme under foam-forming conditions, as a result of which the polyurethane and the enzyme are firmly bonded to one another in the polyurethane foam obtained.

According to US Pat. No. 3,905,923, an immobilized enzyme system is formed from an enzyme and a 5 hydrophilic poly (urea-urethane) foam, the foam surrounding and enclosing the enzyme and keeping it in active form. The hydrophilic foam is produced by reacting water with a hydrophilic polyoxyalkylene prepolymer with terminal isocyanate groups.

T. Richard and NF Olson, "Immobilized Enzymes in Food and Microbial Processes", Plenum Press, New York, NY, 1974, pages 35 to 36 explain the formation of a bound (immobilized) enzyme by reacting the enzyme with water and a polyisocyanate polymer .

In its own DT-OS 2 319 706 the production of an enzyme bound in foam is described, in which the enzyme is dispersed in water and the aqueous dispersion of the enzyme is mixed with a polyurethane prepolymer, so that the prepolymer hardens and foams.

The process according to the invention for the production of bound protein-containing foam, containing 0.1 to 50% by weight of an active protein preparation on an anhydrous basis, and a hydrophilic poly- (urea-urethane) -25 foam matrix from a polyurethane prepolymer with terminal isocyanate groups, protein and water is in the previous claim 1, the use of the foam thus obtained is characterized in the preceding claim 11.

The process is carried out at a temperature at which the liquid polyurethane prepolymer with terminal isocyanate groups is in liquid form and below the denaturation temperature of the protein to be immobilized.

The process according to the invention preferably comprises the preliminary stage of the formation of the prepolymer by reacting a polyhydroxy compound with a polyisocyanate and is characterized in that the protein is mixed with the polyhydroxy compound or with the polyisocyanate before these are reacted with their reactant.

It has been found that it is advantageous to wash the bound (immobilized) protein-containing foam (preferably with water) in order to remove unbound protein and to hydrolyze unreacted isocyanate groups.

The liquid polyurethane prepolymer with terminal isocyanate groups can be prepared by reacting toluenediiso cyanate with a polyethylene glycol, advantageously a Poso ethylene glycol with a molecular weight (average molecular weight) of about 800 to 1200 and preferably of about 1000.

It is also possible to prepare the liquid polyurethane prepolymer with terminal isocyanate groups by reacting 55 toluene diisocyanate with a polyhydric alcohol, namely a polyoxybutylene polyol polymer, ethylene glycol, di-ethylene glycol, a polyoxyethylene polyol polymer, pentaerythritol, glycerol, polyoxyol propylene or a trimethylol propylene.

6o The liquid polyurethane prepolymer with terminal isocyanate groups can also be prepared by reacting toluene diisocyanate with a mixture of a polyethylene glycol, advantageously one with an average molecular weight of about 800 to 1200 and preferably 65 to about 1000 and trimethylolpropane, the trimethylolpropane and the polyethylene glycol being in one Molar ratio of about 1: 1 to 4 and the toluene diisocyanate in an amount of about 0.85 to 1.25 (preferably 0.95

639673

4th

to 1.1) mol of toluenediisocyanate per equivalent (17 g) of OH groups from the polyethylene glycol and the trimethylolpropane can be used.

The liquid polyurethane prepolymer with terminal isocyanate groups can be prepared from toluene diisocyanate and ethylene glycol in accordance with Example 1 of US Pat. No. 3,929,574.

According to preferred embodiments of the process according to the invention, the protein is first mixed with a polyisocyanate or a polyol, whereupon, in the absence of water, the mixture is reacted with a polyol or a polyisocyanate to give the polyurethane prepolymer which foams and cures in the presence of water becomes.

In these preferred embodiments of the invention, it is generally preferred to use about 2 to 500 or 50 to 100 mg of protein per gram of polyol. The ratio of polyisocyanate to polyol plus enzyme must be such that there are sufficient isocyanate groups for reaction with the water with foaming in the polyurethane prepolymer. In general, about 1.25 to 4.5 and in particular 1.8 to 2.2 milliequivalent NCO groups per gram of polyurethane prepolymer are preferred. According to a further embodiment of the invention, liquid polyurethane prepolymer and a substrate which can react with the enzyme are first mixed, and this first mixture is then mixed with the enzyme in the absence of water to form a second mixture which is added by adding water a poly (urea urethane) foam is foamed.

When a substrate is used, it is generally preferred to use about 5 to 100 moles, in particular 8 to 12 moles, of substrate per mole of enzyme. E.g. can be used as the enzyme penicillin amidase and as the substrate benzylpenicillin, or as the enzyme glucoamylase and as the substrate starch or as the enzyme amino acid acylase and as the substrate an acylated amino acid.

The invention also includes the protein-containing foam produced according to the invention, which contains 0.1 to 50% by weight of an active protein preparation on an anhydrous basis and a hydrophilic poly (urea urethane) foam matrix with an oxyalkylene skeleton which contains at least 50 mol. % Has oxyethylene. The hydrophilic foam encloses and carries the protein in an active form for biological effectiveness. The protein can be an enzyme, an antibody or an antigen.

In the process according to the invention, the liquid polyurethane prepolymer with terminal isocyanate groups acts as (a) solvent for the protein to be bound and (b) as reaction partner for the reaction of the protein in order to bind it in the poly (urea-urethane) foam which arises when the aforementioned solution is mixed with water.

The solidification temperature of the liquid polyurethane prepolymer with terminal isocyanate groups depends on the molecular weight of the prepolymer and the structure of the skeleton of the prepolymer.

The thermal denaturation temperature of protein is generally above about 35 ° C. However, some proteins are stable for a relatively short time (e.g. 5 to 30 minutes or longer) at higher temperatures (e.g. at temperatures up to about 70 ° C or slightly higher).

In general, it is preferred to use about 2 to 10 times or more (e.g. up to 100 to 100 times or more) stoichiometric amount of the liquid polyurethane prepolymer having terminal isocyanate groups to prepare the product of embodiment A (which is identical to the intermediate of the above composition is). This intermediate product can then be foamed in a subsequent step by mixing with water, as indicated above, in order to obtain a protein intimately connected to the polyurethane foam.

The invention thus provides a method for binding 5 (immobilization) of proteins in an active and usable form to a polyurethane foam. First, by reacting an excess of di- and triisocyanate and other polyisocyanates (including mixtures of polyisocyanates) with active hydrogen-containing compounds, in particular glycols, polyglycols, polyester polyols, polyether polyols, other polyols and mixtures of two or more such polyols in a known manner a polyurethane prepolymer. This reaction leads to a liquid polyurethane prepolymer 15 with terminal isocyanate groups. The protein and the prepolymer are mixed in the absence of water to form a solution which then forms a poly (urea urethane) foam which contains the immobilized or bound protein in a chemically and / or biologically active form Water mixed and reacted.

The solution (step «(a)» of the above compilation) is imaged in the absence of water. A diluent or a mixture of diluents may or may not be used for step "(a)". It is clear that a. Diluent that would denature the protein or significantly reduce foaming cannot be used. Useful diluents are e.g. in U.S. Patent 3,672,955. The diluents can be highly soluble in water, e.g. Acetone and the like, moderately soluble, e.g. Methyl acetate, methyl ethyl ketone and the like or insoluble, such as benzene and the other diluents listed in column 1, line 45, following U.S. Patent 3,672,955.

35 The diluents serve to reduce the viscosity of the (a) liquid polyurethane prepolymer with terminal isocyanate groups and (b) the mixture formed.

The foaming step (step “(b)” of the above composition) can also be carried out in the absence of a diluent or in the presence of one or more of the diluents mentioned above. However, if the foaming step is carried out in the presence of a diluent, care must be taken to ensure that there is not too much diluent, so that the viscosity of the mixture containing the diluent and the solution formed plus the water added for the foaming is not reduced as much that the carbon dioxide formed by the reaction of the water with the isocyanate groups in this way does not result in foaming or inadequate foaming with the formation of a self-supporting poly (urea urethane) foam which contains the immobilized protein.

If the diluent is insoluble or essentially insoluble in water, an emulsifier can be used for the foaming step, cf. U.S. Patent 3,672,955.

The protein binding (immobilization) reaction can be applied to all proteins, including enzymes, antibodies and antigens. For example, the following can be bound: urease, cellulase, pectinase, papain, bromelain, chy-motrypsin, trypsin, ficin, lysozyme, glucose isomerase, lac-tase, penicillin amidase, human immunoglobulin G, invertase, asparginase and the like. No En-65 enzyme, antibody, or antigen or other protein was found that could not be bound. The purity of the protein is not critical. The binding (immobilization) of the protein can be effected with: (a) pure, crystalline

5

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Protein; (b) partially purified, non-crystalline protein; (c) impure, dried extracts containing enzyme, antibody or antigen, or (d) unpurified, dried extract from a fermentation broth (e.g., a product obtained from the broth by precipitation with acetone). According to the invention, it was found that proteins, including enzymes, antibodies and antigens, of essentially any degree of purity can be bound.

According to the aforementioned US Pat. No. 3,672,955, proteins (enzymes) can be bound to polyurethanes with terminal isocyanate groups. According to the process described there, the polyurethane having terminal isocyanate groups is dissolved in a water-immiscible solvent. This solution is emulsified using an emulsifier in the presence of an active enzyme dispersed in water.

The method according to the invention is similar in several respects to that of US Pat. No. 3,672,955. The same polyurethane prepolymer with terminal isocyanate groups (which is referred to there as polyisocyanate) can be used, the same polyols can also be used for the preparation of the prepolymers and the same enzymes can be used. Without being bound by any particular theory, as with the patent mentioned, the proteins are probably bound by reacting one or more amino and / or

or hydroxyl groups of the protein with one or more isocyanate groups of the polyurethane prepolymer.

As with the process of U.S. Patent 3,672,955, the liquid polyurethane prepolymer having terminal isocyanate groups can be made by reacting a polyol with an excess of polyisocyanate that ensures the presence of free (unreacted) isocyanate groups in the polyurethane prepolymer.

The method according to the invention is also similar in several respects to that of US Pat. No. 3,929,574. The same polyurethane prepolymer with terminal isocyanate groups (which is referred to there as polyurethane with terminal isocyanate groups) can be used, and the same proteins (enzymes) can also be used. Like the method of US Pat. No. 3,929,574, the method according to the invention is directed towards the production of a protein bound to polyurethane foam.

In contrast to the method of the last-mentioned US patent, the prepolymer and the enzyme are mixed with one another under essentially water-free conditions. In the process of the US patent, the prepolymer is mixed with an aqueous dispersion of an enzyme.

Accordingly, the mixing and foaming takes place in one step in the process of the * US patent, while the process according to the invention takes place in two steps. These consist of:

1. a mixing step in which the protein (which may be an enzyme) and the prepolymer are mixed together in the absence of water to form a solution, and

2. a foaming step in which water is mixed into the previously prepared solution to form a foam which contains the protein bound.

In the process according to the invention, compare the examples below, a much larger amount of protein is bound to the foamed polyurethane (ie it is present in the poly (urea-urethane) foam in an active but water-insoluble form) than in the process of the attracted Patent specification. In other words, the method according to the invention is much more effective in binding the protein than the known method.

Any liquid polyurethane prepolymer, including 5 mentioned in US Pat. No. 3,929,574, which contains at least two free isocyanate groups per molecule of prepolymer, is suitable for binding the proteins according to the invention. The prepolymer preferably contains an average of two isocyanate groups per molecule. A higher ratio can be used, e.g. 2 to 8 isolo cyanate groups per molecule of polyurethane. Even higher ratios can be used, but they have no advantage. Any excess of isocyanate groups remaining in the polyurethane foam (after the enzyme has bound) is hydrolyzed the first time the foam contacts water, e.g. destroyed during a washing step prior to application of the bound enzyme.

The liquid polyurethane prepolymers with terminal isocyanate groups used according to the invention contain at least two isocyanate groups (reactive 20 isocyanate groups) per molecule of prepolymer. A polyurethane prepolymer with terminal isocyanate groups is a "liquid polyurethane prepolymer" if

(a) it flows freely at 40 to 70 ° C, or if it dissolves in an inert solvent (e.g. an inert solvent such as those listed in U.S. Patent 3,672,955) to form a solution which is about Contains 1 to 50 (or 10 to 25) wt .-% of a polyurethane prepolymer with terminal isocyanate groups.

The expression “liquid poly-30 urethane prepolymer with terminal isocyanate groups” used above denotes a liquid polyurethane or a polyurea molecule which contains at least about two free isocyanate groups per molecule.

Examples of polyisocyanates which can be reacted with an active hydrogen-containing compound (for example a glycol, polyol, polyglycol, polyester polyol, polyether polyol and the like) in order to produce a polyurethane having terminal isocyanate groups in the manner according to the invention are in the table 1 listed.

40

TABLE 1

Toluene-2,4-diisocyanate Toluene-2,6-diisocyanate Commercially available mixtures of toluene-2,4- and 2,6-diisocyanate 45 ethylene diisocyanate Ethylene diisocyanate propylene 1,2-diisocyanate cyclohexylene 1,2-diisocyanate cyclohexylene -1,4-diisocyanate so 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 55 1, 10-decamethylene diisocyanate

1,5-naphthalene diisocyanate, cumene-2,4-diisocyanate, 4-methoxy-1,3-phenylene diisocyanate, 4-chloro-1,3-phenylene diisocyanate

60 4-bromo-1,3-phenylene diisocyanate 4-ethoxy-l, 3-phenylidene diisocyanate 2 4'-diisocyanatodiphenyl ether

5.6-dimethyl-1,3-phenylene diisocyanate 2,4-dimethyl-1,3-phenylene diisocyanate

65 4,4'-diisocyanatodiphenyl ether benzidine diisocyanate 4,6-dimethyl-1,3-phenylene diisocyanate 9,10-anthracene diisocyanate

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4,4'-diisocyanatodibenzyl 3,3'-dimethyl-4,4'-diisocyanatodiphenylmethane 2,6-dimethyl-4,4'-diisocyanatodiphenyl 2,4-diisocyanatostilbene

3,3'-dimethyl-4,4'-diisocyanatodiphenyl 5

3,3'-dimethoxy-4,4'-diisocyanatodiphenyl

1,4-anthracene diisocyanate

2.5-fluoro diisocyanate

1,8-naphthalene diisocyanate

2,6-Diisocyanatobenzfuran io 2,4,6-toluene triisocyanate p, p ', p "-triphenylmethane triisocyanate

1,4-tetramethylene diisocyanate

A useful class of liquid polyurethane prepolymers with terminal isocyanate groups is derived from polyether polyols and polyester polyols. These compounds can be prepared in a known manner by reacting a polyether (or polyester) polyol with an excess of polyisocyanate, which ensures that

that the end product contains two isocyanate groups. 20 Typically, such an implementation follows, for example, the following equation:

HO— (—CH2 CH2 CH2 CH2 0—) —H polyether polyol m

+

CH.

-NCO

Polyisocyanate

OCN

0

) -c-

• NCO

liquid polyurethane prepolymer with terminal isocyanate groups

In the above formulas, m means the number of repeating tetramethylene ether units. These can e.g. be about 5 to 50.

Compounds useful in the present invention can be made by reacting one of the polyisocyanates described above with a variety of polyols, e.g. (a) with simple polyols as listed in Table 2 below and (b) polyether polyols and polyester polyols. Examples of these polyols are described below.

Polyether polyols which can be used are those which are obtained by reacting an alkylene oxide with an initiator which contains active hydrogen. Typical examples of such initiators are polyhydric alcohols, such as ethylene glycol; Polyamines such as ethylenediamine; Phosphoric acid, etc. The reaction is usually carried out in the presence of an acidic or basic catalyst. Examples of alkylene oxides which can be used are ethylene oxide, propylene oxide, the isomeric butylene oxides and mixtures of two or more different alkylene oxides, e.g. Mixtures of ethylene and propylene oxide. The polyether polyols 60 obtained contain a polyether backbone and terminal hydroxyl groups. The number of hydroxyl groups per polymer molecule is determined by the functionality of the initiator with active hydrogen. E.g. a dihydric alcohol, such as ethylene glycol, as an initiator with active hydrogen-65 leads to polyether chains in which two hydroxyl groups are contained per polymer molecule. If the polymerization of the oxide is carried out in the presence of glycerol, a trihydric alcohol, the polymers formed contain

7

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ether molecules average three hydroxyl groups per molecule. An even higher functionality, i.e. more hydroxyl groups are obtained when the oxide is polymerized in the presence of polyols such as pentaerythritol, sorbitol, sucrose, dipentaerythritol and the like. Examples of polyhydric alcohols which can be reacted with the alkylene oxides to give usable polyether polyols are listed in Table 2 in addition to those given above.

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 -Cy clohexene-1,1-dimethanol 4-methyl ~ 3 -cyclohexene-1,1-dimethanol

3-methylene-1,5-pentanediol diethylene glycol (2-hydroxyethoxy) -l-propanol

4- (2-hydroxyethoxy) - 1-butanol

5- (2-hydroxypropoxy) -l-pentanol 1 - (2-hydroxymethoxy) -2-hexanol

1 - (2-hydroxypropoxy) -2-octanol 3-allyloxy-1,5-pentanediol

2-AHyloxymethyl-2-methyl-1,3-propanediol [(4-pentyloxy) methyl] -1,3-propanediol

3 - (o-prop enylphenoxy) -1,2-propanediol thiodiglycol

2,2 '- [Thiobis- (äthyIenoxy) J-diethanol Polyethylene ether glycol (molecular weight about 200) 2,2'-IsopropyIiden-bis- (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] propane triethanolamine

Triisopropanolamine

Resorcinol

Pyrogallol

Phloroglucin

Hydroquinone

4,6-di-tert.butylcatechin catechin

Orcin

Methylphloroglucin 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-hydroxyphenylimethane)

1.1.2-T ris- (hydroxyphenyl) -ethane

1.1.3-Tris - ((hydroxyphenyl) propane

A particularly useful class of polyether polyols are the polytetramethylene glycols. They are obtained by polymerizing tetrahydrofuran with ring opening and contain the repeating unit in the polymer structure

-ch2-ch2-ch2-ch2-o-

5

The end positions of the polymer chains are thus occupied by hydroxyl groups.

Also particularly useful are the polyoxyethylene polyols H0- (CH2CH2-0) x-H, in which x represents an average number such that the polyol has an average molecular weight of up to about 1000 (or about 2000 or slightly higher).

The polyester polyols which can be used as precursors can be easily prepared by condensation polypolymerization of a polyol with a polybasic acid. The polyol and the acids are used in such proportions that essentially all acid groups are esterified and hydroxyl groups are in the end positions of the chains formed from ester units. Representative examples of polybasic acids for the preparation of these polymers 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, 25ß-hydromonic acid, 25β-hydromonic acid a-Butyl-a-ethyl glutaric acid, a, ß-di-ethyl succinic acid, o-phthalic acid, isophthalic acid, terephthalic acid, hemimellitic acid, trimellitic acid, trimesic acid, citric acid, benzene pentacarbonic acid, 1,4-benzoic acid , Diglycolic acid, thiodiglycolic acid, dimerized oleic acid, dimerized linoleic acid and the like. Representative examples of polyols for the preparation of these polymers are ethylene glycol, 1,3-propylene glycol, 1,2-propylene glycol, 1,4-butylene glycol, 1,3-butylene glycol, 1,2-butylene glycol, 35 butene-1,4-diol , 1,5-pentanediol, 1,4-pentanediol, 1,3-pentanediol, 1,6-hexanediol, hexen-1,6-diol, 1,7-heptanediol, diethylene glycol, glycerol, trimethylolpropane, 1,3 , 6-hexanetriol, trimethanolamine, pentaerythritol, sorbitol and the other polyols which are given in connection with the production of polyether polyols.

When a protein, such as an enzyme, antibody, antigen or the like, comes into contact with a polyurethane prepolymer, the latter becomes chemically active. It is believed that some of the free isocyanate groups of prepolymer 45 react with the amino groups of the protein and, if water is subsequently added, some isocyanate groups with water to form carbon dioxide and amino groups on the polyurethane molecule. These last-mentioned amino groups react with free isocyanate groups on neighboring polyurethane molecules, and this reaction leading to the formation of a urea bond causes the formation and growth of a poly (urea-ure-than) polymer with cross-linking of the polymer molecules. This further growth and crosslinking are essential for the formation of a good foam. Without being bound by any particular theory, it is believed that the superior results obtained by the present invention, i.e. the binding of a much larger amount of enzyme than is possible with the known methods is based on the reaction between the protein and the isocyanate groups of the liquid polyurethane prepolymer in the absence of a competing reaction between water and the isocyanate groups of the corpolymer. In the known methods, water and unbound protein, i.e. Protein that has not yet been able to react with the prepolymer is present at the same time and the competing reactions, i.e. the reaction between the protein and the prepolymer

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on one side and water and the prepolymer on the other side run simultaneously. As stated above, in the method according to the invention, the protein is added before the water is added.

Other additives such as crosslinking agents (polyamines, polythiols, polyacids), surface-active agents, wetting agents, anti-foaming agents, dyes, antioxidants, fillers, etc. may also be present during foaming.

The foaming is of course caused by the release of carbon dioxide.

The ratio of protein to liquid polyurethane prepolymers with terminal isocyanate groups is not critical. It is important, however, that this ratio is such that not all of the prepolymer's isocyanate groups are consumed by reaction with the protein, but rather unreacted isocyanate groups remain which can react with the water to form carbon dioxide.

The ratio of water to protein plus liquid polyurethane prepolymer is not critical. In general, however, the use of about 0.5 to 3 or 0.9 to 2 parts by weight of water per part by weight of prepolymer plus protein is preferred. Bound enzymes produced according to the invention are useful for analytical chemistry. For example, urea can be determined by passing a urea solution through a column containing urease bound to polyurethane foam and quantitatively converting the urea to ammonia, which can be determined by titration or colorimetric.

Invertase bound to polyurethane foam can be used to convert sucrose to invert sugar.

Lipase bound to polyurethane foam can be used for the hydrolysis of organic esters by passing a mixture of water and ester through a column packed with lipase bound to polyurethane foam. The acid and alcohol can then be separated and recovered using conventional methods. This method can also be used for the analysis of ester mixtures.

Numerous other uses for the bound enzymes produced according to the invention are known to the person skilled in the art.

The bound protein-containing polyurethane foam produced according to the invention can be used for any reaction in which contact between the protein and another substance is to be effected. If the protein is an enzyme, the enzyme-containing foam can be used for a catalytic reaction for which the enzyme is the suitable catalyst. If the protein is an antibody, the foam containing the antibody can be used to remove an antigen from a biological sample, such as human blood, and vice versa, an antigen-containing foam can be used to remove an antibody from a biological sample. The invention also includes the use of the proteinaceous foam to effect contact between the protein and another substance.

Antigens bound according to the invention can be used for the removal of antibodies from biological samples. For example, as stated later, bound human immunoglobulin G (IgG), an antigen, is useful for removing rheumatoid arthritis factor (an antibody) from human blood.

Antibodies bound according to the invention are suitable for removing antigens from biological samples. For example, the antibody d'er hepatitis can be bound to polyurethane in the manner according to the invention and the bound antibody obtained for removing the hepatitis antigen from blood, e.g. the blood in blood banks are removed.

The protein bound according to the invention can be used in a batch or in a continuous system. An example of a batch process is the hydrolysis of urea present in an aqueous system. In this process, one or more pieces of foamed polyurethane, i.e. self-supporting poly (urea-urethane) foam with the enzyme bound to it (urease) added to a batch with aqueous urea, which is to be hydrolyzed to ammonia. When the hydrolysis is complete, the pieces with the bound enzyme are removed from the hydrolyzed sample, if desired, washed and placed in another batch with aqueous urea to be hydrolyzed.

However, it may also be desirable to use the bound protein in a manner in which the foam with the bound protein is packed into a column and the solution to be treated is passed through the column. This can e.g. with bound invertase can be done in a completely continuous manner for the hydrolysis of a sucrose solution. A packed column can also be used for batch determination, e.g. for the determination of urea in a series of urea solutions by hydrolysis with bound urease and analysis of the hydrolyzed samples, i.e. the solutions leaving the packed column on ammonia. Using this method, after each individual sample has left the packed column, the column is washed with water and the washing water is combined with the hydrolyzed urea solution = ammonia solution, after which the ammonia content of the combined liquids is determined.

The protein bound according to the invention has a long service life, e.g.

1. if the bound protein is an enzyme, it does not lose its effectiveness even after 100 hours of use;

2. If the bound protein is an antigen that can be used to remove an antibody from an aqueous system, it is consumed ("saturated") if it has taken up an equivalent amount of antibody. The bound protein must then be regenerated, i.e. be freed from the antibody. This can be done

that an aqueous regeneration solution is passed through the packed column and the regeneration solution is washed out of the regenerated column. An excellent regeneration solution consists of an aqueous glycine hydrochloride solution, e.g. Is 0.15 to 3 molar and preferably about 0.5 molar. Such a glycine hydrochloride solution has a pH of about 2.5.

3. If the bound protein is an antibody that can be used to remove an antigen from an aqueous system, it is consumed (saturated) when it has taken up an equivalent amount of antibody. The bound protein must then be regenerated, i.e. to be freed from the antigen. Again, this can be done by using an aqueous regeneration solution, e.g. passes the glycine hydrochloride solution described above through the packed column and washes the regenerated column as above.

With the method according to the invention, enzymes of any kind can be bound. These include enzymes

8th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

9

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Oxidoreductases transferases hydrolases lyases

Isomerases and ligases

Examples of specific enzymes that are immobilized according to the invention, i.e. can be bound are listed in Table 3 below.

TABLE 3

Urease trypsin lactase

Glucose oxidase

Chymotrypsin

Ribonuclease

Peroxidase

pepsin

Rennin

Invertase

Papain

Asparaginase

Pectinase

Pectin esterase

Pencillin amidase

Glucose isomerase

Lysozyme

Amino acid acylase pronase

Alcohol dehydrogenase a-amylase ß-amylase subtilisin

Amino acid oxidase

Catalase

Tannase

Phenol oxidase

Glucoamylase

PuIIulanase

Cellulase

Ficin

Bromelain pancreatin isoamylase lipase

Malic acid dehydrogenase

Hexokinase

Lactate dehydrogenase

Adenosine deaminase

Uricase

Galactose oxidase diaphorase cholinesterase aldolase

Pyruvate carboxylase

Phospharylase

Cephalosporin amidase

Isocitric acid dehydrogenase a-glycerol phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase

Malic acid enzyme

Glucose-6-phosphate dehydrogenase

5-dehydroshikimine reductase

Glutathione reductase

Glycolic acid oxidase

Yeast cytochrome reductase

Luciferase nitrite reductase Glutamyltransferase Glutathione synthetase 5 Glycocyamine phosphokinase Hippuric acid synthase Aldehyde oxidase Succinic acid dehydrogenase Nitrate reductase io Xanthine oxidase Lipoyl dehydrogenase Flavin peroxidase Glycine oxidase Carboxylase 15 a-Keto acid acid dehydrogenase

The following examples illustrate the invention.

20 Example 1

A liquid polyurethane prepolymer with terminal isocyanate groups was prepared by reacting 1 g (2 milliequivalents) of polyethylene glycol with an average molecular weight of 1000 with 0.229 g (2.63 25 milliequivalents) of toluene diisocyanate. The prepolymer obtained was referred to as “prepolymer 1”.

This procedure was repeated using 20 milliequivalents of polyethylene glycol and 26.3 milliequivalents of toluene diisocyanate. The resulting liquid polyurethane prepolymer with terminal isocyanate groups was referred to as “prepolymer 1-A”.

Example 2

A mixture was prepared from 1 g of prepolymer 2 described in Example 9 and 100 mg of human immunoglobulin G (IgG). The IgG solution obtained was stirred for 15 minutes in a dry atmosphere at about 25 ° C. Then 2 g of water was added to the solution at about 25 ° C with stirring. The resulting 40 water system started to foam and within 10 minutes the foaming was complete. The foam obtained was washed carefully with water. The wash water was collected and analyzed for IgG by UV spectrophotometry. It was found that 45 less than 1% of the initially added IgG was washed out of the foam by washing with water. In other words, 99% of the IgG used was bound to the polyurethane foam. The washed foam with the bound IgG was designated "Foam A" 50.

Example 3

Foam A was packed into a column. Human blood containing 100 mg rheumatoid arthritis factor 55 was slowly passed through the column. The red blood cells were not destroyed. Analysis of the blood exiting the column showed that essentially all of the rheumatic arthritis factor had been removed from it.

A second human blood, containing approximately 100 mg 60 rheumatic arthritis factor, was slowly passed through the same column. Only a small portion of the rheumatoid arthritis factor was removed from the blood sample, indicating that the activity of the bound protein was exhausted.

65 The bound protein was reactivated by passing about 100 ml of glycine hydrochloride solution (about 0.5 molar with a pH of 2.5) through the packed column. The column was then washed with water from the glycine hydro-

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washed chloride free. A third portion of human blood containing approximately 100 mg rheumatoid arthritis factor was slowly passed through the column. Analysis of the blood exiting the column indicated that all of the rheumatic arthritis factor was removed from it. This confirms that the bound IgG has been reactivated by treatment with the glycine hydrochloride.

The bound IgG can be reactivated for use in a very large number of experiments - 15 to 20 or more.

Example 4 Comparative Experiment

This experiment illustrates the superiority of the method according to the invention for binding protein to polyurethane over the known methods.

1 g of liquid polyurethane prepolymer (prepolymer 2) according to Example 9 was mixed with a mixture of 100 mg of human IgG and 2 g of water. The resulting mixture of prepolymer, IgG and 1 water was stirred for about 10 minutes. The foaming was complete in about a further 5 minutes. The foam was washed carefully with water and the washings were analyzed as above. It was found that 35% of the IgG supplied was present in the wash water. The IgG bound in this way was found to effectively remove the rheumatoid arthritis factor from human blood without destroying the red blood cells. However, it only removed about 65% of the amount removed from the IgG bound according to Example 2 before regeneration by treatment with glycine hydrochloride solution was required.

Example 5

A protein bound to polyurethane was prepared according to the general procedure of example 2, but invertase was used instead of the IgG and prepolymer 3 according to example 12 instead of prepolymer 2.

It was found that 91% of the enzyme (invertase) was bound to the polyurethane foam [poly (urea urethane) foam].

The enzyme bound in this way proved to be highly effective in the inversion of sucrose solutions. Enzyme activity was maintained for many hours (1440) during which sucrose solution was continuously passed through a column packed with the bound invertase. Even after this time, the bound enzyme was still fully effective.

Example 6

The procedure of Example 5 was modified by mixing the enzyme (invertase) with the water and then adding the enzyme-water mixture to the prepolymer. Only 68% of the enzyme supplied was bound to the polyurethane. The rest of the enzyme (42%) was found in the wash water obtained by washing the foamed polyurethane with the bound enzyme.

Example 7

Asparaginase was bound to polyurethane foam using the general procedure of Example 5. 75% of the asparaginase was bound. The bound enzyme was found to be highly effective and maintained its potency in 12 runs, washing between 1 hour to convert the asparagine, an amide, to aspartic acid.

Example 8

If the procedure of example 7 was repeated, but modified in such a way that the asparaginase was mixed with the em water before the enzyme was brought into contact with the polyurethane prepolymer, no enzyme was bound to the foam formed. The entire enzyme was washed out of the foam with water. The washed foam had no enzymatic activity.

Example 9

2 moles of polyethylene glycol with an average molecular weight of 1000 and 1 mole of trimethylolpropane were mixed and dried at 100 to 110 ° C under a pressure of 5 to 15 torr to remove the water. The resulting dried mixture, containing a total of 7 moles (119 g) of reactive terminal hydroxyl groups, was slowly added with stirring to a reaction vessel containing 6.65 moles of toluene diisocyanate over a period of about 1 hour. The toluene diisocyanate and the mixture formed in the reaction vessel were kept at 60 ° C. The mixture was then stirred for 3 hours while again being kept at about 60 ° C. A further 1.05 mol of toluene diisocyanate was then added, and the mixture was stirred for a further 1 hour, while maintaining the temperature at about 60 ° C. In this way, an excess of 10 mol% of toluene diisocyanate was added to the mixture of polyethylene glycol with a molecular weight of 1000 and trimethylolpropane. This ensured that all the hydroxyl groups of the polyol were reacted with isocyanate and that a certain chain extension occurred as a result of crosslinking of the polyols with the excess toluenediisocyanate.

The resulting liquid polyurethane prepolymer with terminal isocyanate groups was referred to as “prepolymer 2”. It can be used in the manner according to the invention for binding proteins.

The physical properties of the foams containing bound proteins can be varied by varying the proportions of polyols used to prepare the liquid polyurethane prepolymers. The physical properties can also be improved by using other polyols, e.g. of the polyols listed above can be varied.

Example 10

A mixture was prepared from 1 g prepolymer 2 and 5 mg fungal lactase at about 25 ° C. This mixture was stirred at about 25 ° C in a dry atmosphere for 15 minutes. Then 2 g of water were added, the mixture formed being stirred at about 25 ° C. This mixture started to foam and within 10 minutes the foaming was complete. The foam obtained was washed carefully with water at about 25 ° C. The wash water was collected and examined for lactase by UV spectrophotometry. It was found that less than 1% of the lactase originally supplied was washed out of the foam by washing with water. In other words, 99% of the lactase originally used was bound to the polyurethane foam.

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639 673

Example 11

The lactase bound and washed according to Example 10 was examined with a buffered lactose solution. The following procedure was used:

Trial no. 1: All of the bound enzyme was placed in a flask containing a certain amount of buffered lactose solution and the rate of glucose formation as a function of time was determined.

Experiment no. 2: At essentially the same time, a similar experiment with 5 mg free, i.e. unbound lactase, as used for the production of the bound lactase.

The hydrolysis rate in experiment no. 1 and trial no. 2 was the same, indicating that the bound lactase had retained its full potency, i.e. no activity has been lost due to the enzyme binding.

The bound enzyme was removed from the flask and washed with water. The trial no. 1 and 2 were repeated, whereby for the repetition of experiment no. 1 the washed foam and for repeating test no. 2 5 mg free enzyme of the same origin as for the first experiment no. 2 used.

The results obtained in the repetitions of experiments No. 1 and 2 were identical to those of the first experiments No. 1 and 2.

Example 12

A liquid polyurethane prepolymer with terminal isocyanate groups was prepared by reacting 31 g of glycerol with sufficient ethylene oxide to produce 500 g of polyether with terminal hydroxyl groups and an equivalent weight of about 500 (ie a content of about 17 g of OH groups per 500 g of the intermediate product ) were obtained. This intermediate was reacted with commercially available toluene diisocyanate using 1.05 mol toluene diisocyanate per 500 g of intermediate. The resulting liquid polyurethane prepolymer with terminal isocyanate groups was referred to as “prepolymer 3”.

Example 13

3 g of the prepolymer 2 prepared according to Example 9 were mixed with 2 ml of benzene and stirred for 5 minutes. 10 mg of Jackbean urease were added to this first mixture, and the mixture thus formed was stirred at about 25 ° C. for 15 minutes and water was added to cause foaming. The mixture was then stirred for about 5 minutes.

After the foam formation was complete, the self-supporting foam was cut into small pieces (about 25 mm 3). These small pieces of immobilized urease were washed for 18 hours.

A sample of the washed small pieces of foam with immobilized urease was examined for their urease effectiveness. To this end, the rate of ammonia formation in a standard urea solution by the immobilized urease was compared with the rate of ammonia formation in another part of the standard urea solution by a predetermined amount of free, i.e. non-immobilized jackbean urease.

The results of this experiment showed that 11% of the urease originally used was contained as active immobilized urease in the foam.

Example 14

The general procedure of Example 13 was repeated, but the benzene was replaced by ethylene glycol. In this case, the examination of the foam according to the general procedure of Example 13 showed that 3.5% of the urease originally used was bound in the foam in active form.

Example 15

s The general procedure of Example 2 was repeated except that (a) the IgG was replaced with 10 mg urease and (b) after the mixture of prepolymer 2 and urease was stirred for 15 minutes, 2 ml of ethylene glycol was added and the resulting mixture was stirred for about 5 minutes before adding 2 g of water to form the self-supporting foam.

Examination of this immobilized urease in bound form-containing foam according to the general procedure of Example 13 showed that 21% of the ur-15 originally used were present in the form of immobilized, active urease.

Example 16

The general procedure of Example 15 was repeated. However, it was modified in such a way that (a) 10 mg of urease was mixed with 2 ml of ethylene glycol and (b) 3 g of prepolymer 2 was added to the urease / ethylene glycol mixture. The mixture of urease, ethylene glycol and prepolymer was then mixed with water as in Example 15 to give a foam with bound urease.

Examination of this foam using the general procedure of Example 13 showed that 3.7% of the urease originally used was present as active, immobilized urease 30 in the foam.

The diluent used in Examples 13 to 16 reduced the viscosity of the liquid polyurethane prepolymer with terminal isocyanate groups. The amount of diluent used is not critical. 35 It often reduces the viscosity of the prepolymer in the sense of easier handling, so that the diluted and therefore less viscous prepolymer is easier to pour and stir. However, the viscosity must not be reduced to such an extent that the prepolymer plus diluent plus protein no longer forms a self-supporting foam when mixed with water.

Example 17

It has been found that certain enzymes, e.g. Pe-45 nicininamidase, glucoamylase, glucose isomerase and amino acid acylase, which can be bound according to the invention, have a lower enzymatic activity than the free form, based on the amount of enzyme actually present in the immobilized form. 50 Without being bound by any particular theory,

it is believed that this phenomenon is due to primary or secondary amino groups at the active sites of the enzymes, and that these amino groups on some of the enzyme molecules bound to the polyurethane foam react with the isocyanate groups of the liquid polyurethane prepolymer to form urea units and the like Active sites of the enzymes having amino groups are inactivated.

It has now been found that this reduction in the enzymatic activity by admixing a so-called “substrate” (a material on which the specific enzyme acts, for example benzylpenicillin in the case of penicillin amidase, starch in the case of glucoamylase, glucose in the case of glucose isomerase and acylated Amino-65 acid, such as acetylated glycine in the case of amino acid acylase) to the liquid polyurethane prepolymer before the prepolymer is mixed with the enzyme can be greatly reduced.

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Experiment 1 below compared to Experiment 2 shows the advantage of this procedure.

Trial 1

The general procedure of Example 2 was repeated. However, in this case (a) 50 mg of benzylpenicillin were mixed with the prepolymer (prepolymer 2 according to Example 9) before adding the protein and (b) the protein consisted of 50 mg penicillin amidase instead of the IgG used in Example 2.

It was found that 65% of the penicillin amidase was bound to the poly (urea urethane) foam and 42% of the bound penicillin amidase retained its enzymatic activity.

Trial 2

The general procedure of Experiment 1 was repeated. However, no benzylpenicillin was added to the prepolymer.

It was found that 66% of the penicillin amidase used was bound, but only 14% of the bound penicillin amidase retained its enzymatic activity.

In experiment 1, the amount of enzyme washed out of the foam was determined by spectrophotometry. The difference between this value and the enzyme used represents the amount of enzyme bound in the foam.

The amount of bound enzyme with enzymatic activity was determined according to the general method of Example 13 to determine the enzymatic activity of bound urease. In this case, however, the standard urea solution was replaced by a standard benzylpenicillin solution and the free urease was replaced by free penicillin amidase. The same general procedures were used to determine (a) the amount of bound penicillin amidase and (b) the enzymatic activity of the bound penicillin amidase in Experiment 2.

Procedure 1

A liquid polyurethane prepolymer, which is referred to as “prepolymer P-1”, can be prepared by mixing 100 g of ethylene glycol and 561 g of toluene diisocyanate and holding the mixture obtained at about 65 ° C. for about half an hour.

Procedure 2

The general procedure of Example 2 is repeated, but the prepolymer 2 used there is replaced by the prepolymer P-1 prepared above.

Essentially the same results are obtained as in Example 2. The washed-out, bound IgG-containing foam is referred to as "foam A-P".

Procedure 3

The general procedure of Example 3 is repeated using Foam A-P instead of Foam A.

The results obtained essentially correspond to those of Example 3.

Procedure 4

The general procedure of Example 5 is repeated using prepolymer P-1 instead of prepolymer 2.

Essentially the same results are obtained as in Example 5.

Procedure 5

In the absence of water, 6 g of toluene diisocyanate and 100 mg of urease are mixed. This mixture is mixed with 1 g of ethylene glycol in the absence of water. By adding a sufficient amount of water, e.g. From 1 g water per g mixture, a poly (urea urethane) foam is formed which contains bound (immobilized) form enzymatically active urease, i.e. by adding the foam to an aqueous urea solution, e.g. Containing 0.03 mol of urea per liter, the urea is hydrolyzed to form ammonia and carbon dioxide.

Procedure 6

The ethylene glycol used in process 5 is replaced by the equivalent amount (based on the OH groups) of polyethylene glycol with an average molecular weight of 1000. Essentially the same results are achieved as in Method 5, i.e. when the foam is added to an aqueous urea solution containing 0.03 mol of urea per liter, the urea is hydrolyzed.

Procedure 7

In the absence of water, 1 g of ethylene glycol and 100 mg of urease are mixed. In the absence of water, 6 g of toluene diisocyanate are added. By adding a sufficient amount of water, e.g. 1 g of water per g of mixture forms a poly (urea urethane) foam which contains enzymatically active urease in bound (immobilized) form. If you add the foam to an aqueous urea solution, e.g. Containing 0.03 mol of urea per liter, the urea is hydrolyzed to form ammonia and carbon dioxide.

Procedure 8

The ethylene glycol used in process 7 is replaced by the equivalent amount (based on the OH groups) of polyethylene glycol with an average molecular weight of 1000. Essentially the same results are obtained as in Method 7, i.e. the addition of the foam, which contains urease in bound form, to an aqueous urea solution with 0.03 mol of urea per liter leads to hydrolysis of the urea.

The term “polyisocyanate” used here also includes diisocyanates.

The liquid polyurethane prepolymers with terminal isocyanate groups used according to the invention contain at least two reactive isocyanate groups per molecule of prepolymer.

Unless otherwise stated, the term “polyurethane foam” and “foamed polyurethane” denotes a self-supporting poly (urea urethane) foam. If the foaming in the manner according to the invention is carried out in the presence of a protein, the foam formed contains the active protein in bound (immobilized) form.

Unless otherwise specified, the temperature is based on ° C and the percentages are based on weight.

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Claims (17)

639 673 1. Process for the production of bound protein-containing foam, containing 0.1 to 50% by weight of an active protein preparation on an anhydrous basis, and a hydrophilic poly (urea-urethane) foam matrix, from a polyurethane prepolymer with terminal isocyanate groups, Protein and water, characterized in that one (a) preparing a solution of the protein in the liquid polyurethane prepolymer in the absence of water; and (b) the solution obtained is foamed by admixing water. 2. The method according to claim 1, characterized in that the foam matrix is one with polyoxyalkylene chain-forming oxyalkylene units, of which at least 50 mol% are oxyethylene units. 2nd PATENT CLAIMS 3rd 639673 removal of an antibody from a biological sample, characterized in that the biological sample is passed through a column which is loaded with the protein immobilized in the foam. 3. The method according to claim 1, characterized in that the foam formed is washed to remove unbound protein and for the hydrolysis of unreacted isocyanate groups. 4. The method according to claim 1, characterized in that an enzyme, an antibody or an antigen is used as the protein. 5. The method according to claim 4, characterized in that human immunoglobulin G is used as the protein. 6. The method according to claim 4, characterized in that one of the following enzymes is used as protein: Urease Trypsin Lactase Glucose oxide loop Chymotrypsin Ribonuclease Peroxidase pepsin Rennin Invertase Papain Asparaginase Pectinase Pectin esterase Penicillin amidase Glucose isomerase Lysozyme Amino acid acylase pronase Alcohol dehydrogenase a-amylase ß-amylase subtilisin Amino acid oxidase Catalase Tannase Phenol oxidase Glucoamylase Pullulanase Cellulase Ficin Bromelain pancreatin isoamylase lipase Malic acid dehydrogenase hexokinase lactate dehydrogenase adenosine deaminase Uricase Galactose oxidase diaphorase cholinesterase 5 aldolase Pyruvate carboxylase phospharylase cephalosporin amidase isocitric acid dehydrogenase io a-glycerol phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase Malic acid enzyme Glucose-6-phosphate dehydrogenase 5-Dehydroshikimin reductase 15 glutathione reductase Glykolsäureoxidase yeast cytochrome c reductase lucif erase nitrite 20 glutamyltransferase glutathione glycocyamine-phosphokinase Hippursäuresynthetase aldehyde 25 Bernsteinsäuredehydrogenase nitrate xanthine oxidase Lipoyldehydrogenase Flavinperoxidase 30 Glycinoxidase carboxylase a-Ketosäuredehyd ' rogenase transketolase 35 7. The method according to claim 1, characterized in that one adds a substrate to the mixture of polyurethane and protein before adding the water, which is able to react with the protein1. 8. The method according to claim 7, characterized in that the protein is an enzyme. 9. The method according to claim 8, characterized in that the enzyme penicillin amidase and the substrate benzylpenicillin, the enzyme glucoamylase and the substrate starch or the enzyme amino acid acylase and that 45 substrate are an acylated amino acid. 10. Bound protein-containing foam, produced by the method according to claim 1, characterized in that the foam contains the protein in a biologically active form. so 11. Use of the bound protein-containing foam according to claim 10 for contacting substances which react biologically on the protein of the foam. 12. Use according to claim 11, of bound-55 nes protein-containing foam, which is produced by the method according to one of claims 3 to 9. 13. Use according to claim 11, wherein the protein in the protein-containing foam is an antibody for removing an antigen from a biological sample, 60 characterized in that the biological sample is passed through a column which is loaded with the protein immobilized in the foam. 14. Use according to claim 13, characterized in that the antibody hepatitis antibody, the 65 antigen hepatitis antigen and the biological sample is human blood. 15. Use according to claim 11, wherein the protein in the protein-containing foam is an antigen for ent 16. Use according to claim 15, characterized in that the antigen is human immunoglobulin G, the antibody is rheumatic arthritis factor and the biological sample is human blood. 17. Use according to claim 11, characterized in that the protein is an enzyme and that the substance contacted with it is one which undergoes a reaction catalyzed by the enzyme.