CH616942A5 - Method for suppressing the immunogenic action of a biocatalyst. - Google Patents

Description

The invention relates to a method for suppressing the immunogen activity of a biocatalyst, i. H. of an enzyme or hormone with a polypeptide structure, without the biological or enzymatic activity of the biocatalyst being abolished.

The medical use of hormonal biocatalysts, which are polypeptides, for the circulation to achieve certain physiological effects is known, e.g. in the case of insulin, which is used for the treatment of diabetes. Another group of biocatalytic polypeptides to which great therapeutic capabilities are attributed is that of various classes of enzymes. The therapeutic use, in particular of the enzymatic biocatalysts, is often restricted by the fact that these compounds trigger immunogenic reactions in the circulation, as a result of the generation of polypeptide antibodies. This leads to the inactivation or destruction of the polypeptides and may trigger dangerous allergic effects.

Antibody polypeptide destruction is probably also the reason for the relatively short duration of action of insulin in the circulatory system, which is why diabetes patients must receive insulin at relatively short intervals. In the case of enzymatic biocatalysts with a polypeptide structure, the allergy reaction is usually added to the inactivation or destruction.

If it were possible to modify enzymatic polypeptides in such a way that their desired physiological activity would be retained in whole or to a considerable extent and at the same time the immunogen reaction would be switched off, these extremely valuable compounds could be increasingly used for human and veterinary medicine without the disadvantages mentioned and in use much smaller amounts than previously possible.

The problems of the therapeutic use of biocatalysts mentioned are known and there has been no lack of attempts to solve these problems. The addition or attachment of enzymes to insoluble substrates is the subject of extensive work, see e.g. Ann. Rev. Biochem. 35 (1966), 873, and "Fermentation Advances", New York (1969), page 391. However, injectable polypeptides with a sustained action were not obtained. Another proposal for the production of enzymatic polypeptides with extended in vivo activity is based on microencapsulation, see e.g. Science, 146 (1964), 524; Trans. On. Soc. 12 (1966), 13; Nature, 218: 243,268; Can. J. Physiol. Phar-macol., 47 (1969), 1043; Canad. J. Physiol. Pharmacol., 45 (1967), 705. Another proposal is based on heat stabilization by adding carboxymethyl cellulose to enzymes such as trypsin (see Nature, 189 [1961], 576) or on the combination of proteases with hydrophilic substrates (see, for example Eur. J. Biochem., 25 [1972], 129); in both methods, however, the polypeptides are not obtained in a soluble form, as is generally desirable for injectable therapeutic preparations and their effectiveness monitoring. Another known method is based on the addition or addition, also called fixation, of synthetic polymers to polypeptide proteins, as described in “Advances in Immunology”, 5 (1966), 30; The work just mentioned shows that although almost all amino acid homopolymers are not immunogenic, the immunogenic activity is not shielded by fixing these polymers to immunogenic proteins, which is shown by the formation of antibodies in the circulatory systems used for the experiments. Polyglycine is e.g. not immunogenic itself; but if it is fixed to a protein, a hapten is formed as the derived protein. Similarly, e.g. dextran itself is also only weakly immunogenic; however, if it is fixed on insulin, the insulin coupled with dextran has a considerable immunogenic effect.

The method according to the invention for suppressing the immunogenic effect of a biocatalyst which has a multiplicity of peptide bonds, while at least partially maintaining its physiological activity by fixing polymers to the biocatalyst, makes it possible to obtain essentially non-immunogenic polypeptide biocatalysts which have a considerable part of the desired physiological Preserve activities of the starting polypeptide, and is characterized in that the biocatalyst is fixed to a practically linear polyalkyl or polyalkoxy polymer via at least one end carbon atom thereof, the polyalkyl or polyalkoxy polymer being unsubstituted or having an alkoxy containing less than 5 carbon atoms. or alkyl group is substituted.

The attached drawing shows test results, which are explained below.

To attach to one or both end carbon atoms of the polymer, its end group can be changed or modified by adding a coupling group that is reactive with the polypeptide. The polypeptides obtainable according to the invention with a polyalkyl or polyalkoxy polymer fixed thereon can introduce the circulation of humans and mammals as aqueous solutions, e.g. are injected intramuscularly and cause practically no immunogenic effects.

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The basic chain of the polyalkyl or polyalkoxy polymers used in the process according to the invention is a practically linear C-C-ICette or an ether chain. It has been found that when using corresponding polymers with highly branched chains, products are formed which lead to immunogenic effects. A relatively low substitution on the basic chain is permissible, however, if this substitution is limited to substituting alkyl or alkoxy groups with less than 5 and preferably 2 or less carbon atoms. Examples of useful polymers are polyethylene glycol, polypropylene glycol; Polyethylene glycol is particularly preferred.

Polymers of this type with chain lengths between 500 and 5000 daltons, in particular from 750 to 2000 daltons, are also preferably used.

Various examples of the type of coupling of polymer with polypeptide are described below. It must be taken into account here that the proportions of a specific polypeptide which has a desired physiological effect can depend on the polypeptide used in each case. With certain enzymes, one or two amino acid residues may be responsible for the desired physiological activity. When using a coupling agent, it is then expedient to choose one which has no affinity for these special active groups. If this cannot be fully achieved, a compromise can be made in the sense that one sacrifices activity in favor of non-immunogenicity. However, the final results that can be achieved mostly depend not only on the coupling agent used, but also on the relative proportions of the components and on the molecular weight of the polymer. For most polypeptides it is expedient to work with 10 to 100 moles and preferably 15 to 50 moles of polymer per mole of polypeptide. These relative proportions generally preserve at least 15% of the physiological activity. However, it is preferred to work in such a way that at least 40% of the physiological activity of the polypeptide is retained.

The method according to the invention is generally applicable to biocatalysts with a large number of peptide bonds - here called polypeptides for short. Of particular interest is the application to enzymatic polypeptides and insulin. Examples of suitable enzyme categories are the following (the system numbers correspond to the EC numbers according to IUP AC):

1. Oxidoreductases, e.g. Urate-Oxygen-Oxidoreductase, System No. 1.7.3.3. “Also called uricase; Hydrogen peroxide: -hydrogen peroxide oxidoreductase, system No. 1.11.1.6., Also called catalase; and cholesterol-red. NADP: Oxygen-oxedoreductase, 20-ß hydroxylating, system No. 1.14.1.9., Also called cholesterol-20-hydroxylase.

2. Transferases, e.g. UDP-glucuronate-glucuronyltransferase with unspecific acceptor, system No. 2.4.1.17., Also called UDP-glucoronyltransferase, and UDP-glucose-a-D-galactose-1-phosphaturidylyltransferase, system No. 2.7.7.12.

3. Hydrolases, e.g. Mucopeptide-N-acetylmuramylhydroIase, system No. 3.2.1.17., Also called lysozyme; Trypsin, System No. 3.4.4.4 .; L-Asparaginaminohydrolase, System No. 3.5.1.1., Also called Asparaginase.

4. Lyases, e.g. Fructose-l, 6-diphosphate-D-glyceraldehyde-3-phosphate lyase, system No. 4.1.2.12., Also called aldolase.

5. isomerases, e.g. D-xylose-keto-isomerase, system No. 5.3.1.5., Also called xylose isomerase.

6. ligases, e.g. L-citrulline: L-aspartate ligase (AMP), system No. 6.3.4.5.

The nonimmunogenicity of the products obtained by the process according to the invention could be confirmed by the usual immunological methods. For example, the free polypeptide can first be injected into a test animal and the antiserum thus formed can be tested for the antigen reaction against the polypeptide which is fixed to the polymer according to the invention - hereinafter also referred to as "protected" - e.g. through gel diffusion, complementary fixation or similar methods. When the protected polypeptide was injected intradermally, there was no immediate or delayed hypersensitivity. On the other hand, when the test animals are injected with protected polypeptide, the antisera obtained from the test animals show no reaction in the gel diffusion test or in the complementary fixation test, and the injection with unprotected polypeptide showed neither an immediate nor a delayed hypersensitivity reaction.

As mentioned above, the polymer can be fixed to the biocatalyst using various methods. In one method, the polymer end group is provided with a coupling group; in another preferred method, a group on the terminal C atom, e.g. the hydroxy group, is modified by conversion and e.g. converted into an amino group which can then be coupled to the polypeptide, for example using coupling agents known per se.

Examples of suitable coupling groups for the first method are cyanuric chloride or cyanuric fluoride. The polymer, preferably polyethylene glycol, hereinafter referred to as PEG, can be taken up in a suitable inert solvent such as hydrocarbon, suitably anhydrous benzene which contains a small amount of a weak base such as sodium carbonate, and the cyanuric chloride can then be added. The reaction mixture can then be poured into excess water, separated from insoluble constituents and finally freed from solvent, preferably under reduced pressure; in this way 2-PEG-4-hydroxy-6-chloro-1,3,5-triazine is obtained. The “activated” polymer thus obtained can then be reacted with a solution of the chosen polypeptide in a suitable buffer solution to achieve the desired fixation. The unreacted polymer can then be removed, preferably by treatment with a gel permeation resin such as "Sephadex" -G-50; the protected polypeptide obtained as the target product can then be purified by conventional methods which avoid denaturation of the polypeptide component. The target product is expediently left in a buffered aqueous solution, but can also be isolated in a solid state using gentle methods, e.g. by freeze drying.

Another suitable end group is the acyl azide group. To form it, terminal hydroxyl of the polymer can be reacted with chloroacetic anhydride and then reacted with diazo-methane to give the methyl ester of the carbomethoxy ether, which, by treatment with hydrazine, gives the corresponding hydrazide, which, when treated with nitrous acid, gives the desired acylazide.

The succinate group can also be used as a coupling group. For this purpose, the polymer glycol, for example PEG or PPG (polypropylene glycol), can be taken up in an inert solvent in the presence of a mild base, such as sodium bicarbonate, and treated with a dihalosuccinic anhydride, such as dibromosuccinic anhydride. The PEG-dihalosuccinate or

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polymer treated analogously can then be reacted with polypeptide.

The anthranilate group is also suitable. In this variant, the polymer glycol, again in a reaction-inert solvent, can be reacted with isatoic anhydride to form the anthranilate ester, which can then be used for the next step, namely the diazotization, without further purification; this can be carried out in a conventional manner, e.g. by taking up the anthranilate ester in water, acidifying the solution, e.g. with glacial acetic acid, cooling and addition of sodium nitrite. The diazonium salt thus obtained can be reacted with the polypeptide.

An interesting further variant using azido groups is based on the photochemical addition of an azide coupling group, for example by treating the glycol in buffer solution with 4-fluoro-3-nitrophenylazide and removing the unreacted azide, preferably by dialysis. To fix the enzyme, for example lysozyme, it can be taken up in water, brought together with the reactant and irradiated again with it, which, for example, gives the PEG-2-nitrophenyllysozyme.

A terminal hydroxyl group of the polymer can be converted to an amino group for fixation to the polypeptide. To this end, the polymer at its terminal hydroxyl group can be treated either with a sulfonating agent such as toluenesulfonyl chloride. or with a halogenating agent, such as triphenylphosphine in carbon tetrachloride, or triphenylphosphine together with a suitable N-halosuccinimide. The halide or tosylate produced in this way can then, after treatment with sodium azide with lithium aluminum hydride, be reduced to the corresponding polymer with an amino group at the end.

Such polymers with a terminal amino group can then be coupled via a carboxyl group of the polypeptide to the latter according to methods known per se. For this, e.g. water-soluble carbodiimides, such as 1-cyclohexyl-3 (2-morpholinethyl) carbodiimide, in particular metho-p-toluene, are used and an amido bond is formed from the nitrogen of the polymer and the carbonyl group of the enzyme.

Instead of directly coupling the amino group to form an amido bond with the polypeptide, the coupling can also take place via a maleimide group; for this e.g. cu-amino-PEG reacted with maleic anhydride and the resulting N-PEG-maleimide can be reacted with the desired polypeptide.

In the following examples, the numerical values after the name of the polymer (e.g. PEG 750) relate to the molecular weight of the polymer in daltons. Degrees ° refer to the temperature in ° C.

Example 1 Fixation of Uricase to PEG

(A) Preparation of the PEG component (stages 1-3) 1. PEG 750 (2.0 g) is dissolved in liquid ammonia (30 ml); the solution is treated with sodium until the blue color persists for 5 minutes. Then the ammonia is evaporated with a dry stream of nitrogen. The residue is treated with methyl chloroacetate (5 ml), the mixture is left to stand at room temperature overnight and finally heated to 100 ° for 1 hour. After removal of excess reagent under reduced pressure, the PEG-methylcarbomethoxy ester remains.

2. The PEG-methyl carbomethoxy ester (2.0 g) is kept at reflux with methanol (300 ml) and hydrazine hydrate (15 ml) overnight and the solution is then evaporated under reduced pressure to give the PEG-methoxycar-bohydrazide.

3. The hydrazide (1.0 g) thus obtained is dissolved in 2% hydrochloric acid (150 ml) and 5% sodium nitrite solution (9 ml) is slowly added while stirring. The solution is then kept at room temperature for 20 minutes. The PEG-carboxymethylazide solution thus formed is used for the coupling described below.

(B) Coupling uricase with the PEG component

The azide solution (16 ml) obtained in stage (A) 3 is adjusted to pH 8.7 by adding sodium phosphate. Uricase (25 mg) is added and the solution is stirred for 2 hours at room temperature. The solution is then dialyzed and the protected enzyme is isolated by chromatography on “Sephadex” G-50. The product can also be obtained in solid form by freeze-drying.

The corresponding PEG-asparaginase is obtained in an analogous manner, but using asparaginase instead of uricase. If PPG is used instead of PEG, PPG uricase or PPG asparaginase is obtained.

Example 2 Fixation of catalase to PEG

(A) Preparation of the PEG component

PEG 750 (30 g, 0.04 mol) or PEG 2000 (80 g, 0.04 mol) is dissolved in 150 ml of anhydrous benzene containing 8 g of NaiCCb. The solution is cooled to 10 ° and cyanuric chloride (7.38 g, 0.04 mol) is added. The solution is stirred at 10 ° overnight. Then water (5 ml) is added, the solution is kept at room temperature for a few hours and finally warmed to 40 ° overnight. The insoluble components are then centrifuged off and the solvent is removed in a rotary evaporator at 40 ° under reduced pressure. Small amounts of precipitate that occur during concentration can be removed by adding a little benzene to reduce the viscosity, then centrifuging off and evaporating again. The product is PEG-4-hydroxy-6-chloro-l, 3,5-triazine, a 40 "viscous liquid that can be stored in the refrigerator until use.

(B) Coupling of catalase to the PEG component

Catalase (60 mg, 8.7xl (H mol) is dissolved in 3 ml of 0.05-m borate buffer solution (pH = 9). Then the PEG component (470 mg) prepared from PEG 750 according to paragraph (A) is added. After 3 hours the pH is brought back to 9.0 with sodium hydroxide, the solution is left to stand at room temperature overnight, then the pH is readjusted to 9.0 and the unreacted PEG component is removed by removing the solution is passed over a column with “Sephadex G-50.” The PEG-HTA catalase obtained is in a rotary evaporator up to a concentration of

1 mg protein / ml concentrated and stored in the refrigerator (HTA = -4-hydroxy-l, 3,5-triazin-6-yI).

A similar product is obtained in an analogous manner, but with “Carbowax” 2000 - a technical PEG. Furthermore, the corresponding PEG-HTA xylose isomerase or the PEG-HTA insulin is obtained in an analogous procedure, but with D-xyloseketoli somerase (xylose isomerase) or insulin instead of catalase.

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Example 3

Fixation of cholesterol-20-hydroxylase to PEG-maleimide

(A) Preparation of N-PEG-maleimide maleic anhydride (1.0 g, 0.001 mol), benzene (50 ml)

and co-amino-PEG (0.005 mol) are refluxed for 2 hours. The solution is evaporated under reduced pressure and heated to 200 ° in a dry nitrogen stream for 2 hours.

(B) Reaction of N-PEG-maleimide with cholesterol-20-hydroxylase

The enzyme (25 mg) is added to a solution of N-PEG-maleimide (70 mg) in 0.1-m phosphate buffer solution (pH 7.0; 10 ml) and the resulting solution is left to stand at room temperature for 1 hour. The solution is then dialyzed and the target product is isolated from the dialysate by chromatography on "Sephadex" G-50 in the form of a solution which can optionally be freeze-dried.

Example 4

Fixation of UDP-glycuronyl transferase to PEG succinate

(A) Preparation of PEG dibromosuccinate (PEG component)

PEG (1.0 g) is dissolved in dry benzene (10 ml) containing sodium bicarbonate (1.0 g). Then dibromosuccinic anhydride (0.5 g) is added, the solution stirred overnight, then filtered and the filtrate concentrated under reduced pressure. This gives PEG dibromosuccinate.

In an analogous manner, the corresponding PEG diiodosuccinate is obtained when diiodosuccinic anhydride is used instead of dibromosuccinic anhydride.

(B) Coupling of UDP-glucuronyltransferase with PEG-succinate

The enzyme (50 mg) in buffer solution (10 ml; pH 7.0) is slowly treated with PEG-dibromosuccinate (100 mg) in water (5 ml) at room temperature. The pH is kept between 7 and 8. The target product is isolated by chromatography on “Sephadex” G-50 and obtained as a solid by freeze-drying.

Example 5

Fixation of UDP-glucose-a-D-galactose-1-phosphate-turidyl-transferase (system No. 2.7.7.12.) On PEG-anthranilate

(A) Preparation of the PEG component

1. PEG (1.0 g) is dissolved in dry benzene (10 ml) containing sodium bicarbonate (1.0 g), isatoic anhydride (0.25 g) added and stirred overnight. The resulting solution is filtered and the filtrate is evaporated at 40 ° under reduced pressure. The PEG-anthranilate ester thus obtained is used in the next step without further purification.

2. The PEG anthranilate ester thus obtained is dissolved in water (5.0 ml) and glacial acetic acid (0.5 ml) is added. This solution is cooled to 0-2 ° and, while stirring, a concentrated sodium nitrite solution is added dropwise until nitrous acid is formed.

(B) Coupling of the transferase (system No. 2.7.7.12.) With PEG-anthranilate ester diazonium salt

The enzyme (25 mg) is dissolved in buffer solution (5 ml; pH 7 to 7.5), the solution is cooled to 0-2 ° and added dropwise to the diazotized solution obtained according to paragraph (A) 2, the pH Holds 7 to 7.5. After 2 hours the solution is brought to room temperature and filtered. The target product is isolated by «Sephadex» chromatography and can be obtained in a solid form by freeze-drying.

Example 6

Fixation of lysozyme to 4-azido-2-nitrophenyl-PEG

(A) Preparation of the PEG component

PEG (1.0 g) is dissolved in borate buffer at pH 9.8 (100 ml) and the solution is treated with 4-fluor-3-nitrophenylazide (1.0 g) in acetone (10 ml). The reaction mixture is stirred at 40 ° overnight and filtered. The filtrate is dialyzed against water, filtered and freeze-dried.

(B) Photochemical coupling of lysozyme to the PEG component

Lysozyme (60 mg) is dissolved in 3 ml of water and 4-azido-2-nitrophenyl-PEG (50 mg) is added in accordance with paragraph (A). The resulting solution is irradiated for 18 hours at 40 ° with two tungsten lamps (125 W each) which were immersed in sodium nitrite solution in order to filter out radiation with wavelengths of less than 400 nm. The product was purified by chromatography on “Sephadex” G-50, obtained as a solution by elution and could be isolated from the solution by freeze-drying.

Example 7 Fixation of trypsin to PEG amide

(A) Preparation of Co-Amino-PEG (PEG Component)

1.a. Obtaining the azide precursor by tosylation PEG 6000 (50 g) is dissolved in toluene (400 ml) and the

Solution for azeotropic removal of traces of water heated with a portion (60 ml) of toluene. Anhydrous triethylamine (5.5 ml) and then p-toluenesulfonyl chloride (3.4 g) are added to the cooled solution. The solution obtained is kept at room temperature overnight, then filtered and the filtrate is cooled to 5 °. The precipitate formed is collected, dissolved in absolute ethanol (200 ml) and sodium azide (1.0 g) is added. The mixture is heated to reflux for 36 hours, which gives the PEG-Cü azide in ethanolic solution.

1 .b. Obtaining the azide precursor by halogenation

As in paragraph l.a. worked, but with thionyl bromide (2.5 ml) instead of p-toluenesulfonyl chloride. The solution is refluxed 15 min after the addition of thionyl bromide and the PEG-co-bromide obtained is converted to the azide as above.

2. Conversion to co-amino PEG

The according to 1 .a. or 1 .b. The ethanolic solution of co-azido-PEG obtained is mixed with Adams catalyst and treated with hydrogen until no more hydrogen is absorbed. The hydrogenation product is filtered and concentrated under reduced pressure. Then dry ether (150 ml) is added and the mixture is cooled to 3 ° overnight. The amino PEG is obtained as a precipitate.

(B) Coupling of trypsin and co-amino PEG

The co-amino-PEG (100 mg) obtained according to paragraph (A) 2 is dissolved in buffer (10 ml; pH 6.0) and subsequently trypsin (50 mg) and ethyldimethylaminopropyl-carbodiimide (200 mg) are added. The pH is kept at 6. After 1 hour, the reaction is stopped by adding excess acetate buffer solution (pH 6). The target product is isolated by chromatography on «Sephadex» G-50.

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In an analogous procedure, the L-citrulline-L: aspartate ligase-PEG-amide is obtained using AMP instead of trypsin.

Example 8 Fixation of aldolase to PEG-amide

Aldolase (30 rag) is dissolved in 2m sodium acetate (3ml), 0.1m sodium glyoxylate (1.5ml) and 10mmol copper sulfate (1ml). Enough 0.4-M acetic acid is added to bring the pH to 5.5. The solution is kept at 20 ° for 1 hour. The protein modified in this way is isolated by gel filtration on "Sephadex" and prepared at 37 ° overnight in a mixture with 2-m sodium acetate (3 ml), 2-m acetic acid (1 ml) and co-amino-PEG (100 mg) - as above - left to incubate. The target product is isolated by chromatography on «Sephadex» G-50.

Example 9 Fixing Insulin to PEG-HTA

(A) Preparation of the PEG component

The PEG component, namely PEG-4-hydroxy-6-chloro-1,3,5-triazine, is produced from PEG 2000 according to Example 2, paragraph (A).

(B) Coupling of insulin and PEG component Insulin (10 mg) is in 1 ml 0.1-m borate buffer solution

(pH 9.2) and the PEG component (179 mg) was added. After 2 hours of reaction, the unreacted PEG component is removed by chromatography on “Sephadex” G-10. The PEG-HTA insulin is concentrated to 1 protein / ml in a rotary evaporator and stored in the freezer (HTA = 4-hydroxy-l, 3,5-triazin-6-yl).

If the procedure is analogous, a corresponding product is obtained starting from PEG 750. Furthermore, similar products are obtained when the reaction is carried out at pH values of 8.5 and 10.

Test methods and results are described below.

I. Enzymatic activity of PEG catalase

Catalase was tested as such and after fixation to PEG according to the method of Beers and Sizer (J. Biol. Chem. 195 [1952] 133). Both PEG 750 catalase and PEG 2000 catalase proved to be somewhat more active than unmodified catalase:

Unmodified catalase: 41,500 units / mg protein PEG 750 catalase: 43,750 units / mg protein

PEG 2000 catalase: 43,500 units / mg protein

The thermal stability values of unmodified catalase and PEG 750 catalase were similar at 37 ° and 60 °.

II. Antigenicity test

(a) Immunization

White adult New Zealand female rabbits were immunized intravenously with a 1.5 mg / ml stock solution of beef liver catalase for 4 weeks; at the end of the immunization, each animal had received a total of 55 mg of catalase; 7 and 14 days after the immunization was completed, blood was drawn from the animals by heart puncturing. About 1 month later, the animals were injected with 5 mg of the corresponding antigen; a week later, blood was again drawn by heart puncture.

(b) In vivo and in vitro tests

After the last blood draw according to II.a, the animals were injected with 0.1 ml of the corresponding antigen in borate-buffered physiological saline in order to determine the occurrence of immediate and / or delayed hypersensitivity. All animals were observed for skin reactions for a period of 96 hours after injection.

The antisera obtained from the animals after immunization according to Il.a were also examined in vitro for precipitation (1) and for complementary fixation activity (2, 3). Each antiserum (undiluted, dilution V10, dilution ■ / see above) was applied to gel diffusion plates (1.0% agar in physiological saline with addition of 1.0% azide as preservative) against both catalase and PEG-HTA catalase solutions tested, namely at concentrations from 1000 Y / ml to 5 y / ml. The plates were incubated for 48 hours at room temperature and for 3-4 days at 4 °. The complementary fixation activity of the heat-inactivated antisera absorbed on sheep rythrocytes was determined according to the microliter complementary fixation test (test set from Cooke Engineering Co., USA). The antisera were tested in 1: 1 and 1:10 dilution against catalase and PEG-HTA catalase solutions in the dilution range from 1000 ylml to 15 y / ml. After 30 min incubation at 37 °, the plates were tested for the presence or absence of hemolysis.

(c) Intravenous immunization with catalase

1. Gel diffusion

The rabbit antiserum, undiluted and tested in '/ m dilution, gave positive results of antibody precipitation against catalase. The examination of the antiserum produced against native enzyme against the enzyme modified by polyoxyethylene residues showed no evidence of “cross reactions”.

2. Complementary fixation

Both the first and second sera showed positive complementary fixation activities when tested against native catalase. In contrast, when the catalase fixed to polymers according to the invention was used as the reactive antigen, the results were negative.

III. PEG immunoassay of PEG insulin

The PEG-HTA insulin produced according to Example 2 was tested for antigen activity in comparison with insulin by the methods mentioned in the table below; the results are given in the table.

test

(1)

(2)

Insulin activity

Insulin antibodies

ji-i; units / ml activity

H-units / ml

material

(Animal experiment)

(Radioimmunoassay)

Unmodified insulin

137

127

PEG-HTA insulin (750)

72 *

0

PEG-HTA insulin (2000)

90 *

0

* These numbers are dilution numbers, not actual test values.

The results summarized in the table above show that the PEG-HTA insulin has no antigenic activity against insulin antiserum.

IV. Blood sugar level

(a) Insulin and PEG-HTA insulin with the same insulin content

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rabbit was injected; the resulting blood sugar values were measured using the glucostat method (test by the Worthington Biochemical Corporation, USA). The results after 3 hours were as follows:

Blood glucose value

PEG-HTA insulin

50% of the normal value

insulin

20% of normal

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These results show that the PEG-HTA insulin has an insulin activity that is about 50% that of insulin when referenced to the weight of the PEG-HTA insulin.

(b) Usual test rats (220/225 g) were diabetic using the method of Goldner (Bull. N. Y. Acad. Med. 21, 44 [1945]) with alloxan injections. The animals were given no food during the test period. The animals were administered insulin or PEG 2000-HTA insulin preparations (produced at pH values of 9.2 and 10.5). The product made at pH 10.5 showed no decrease in blood sugar levels. The results are summarized in the following table and shown in the figure.

Insulin (intramuscular)

Time blood glucose level

Abfali towards the beginning

% Waste

(Hours.)

level compared to mg / 100 ml mg / 100 ml

control

0 410

1 295

135

32.9

2,270

140

34.1

4,290

120

29.2

6,400

10th

2.4

Control (no feed during the test period)

0 520

0

0

2,510

10th

1.9

4,490

30th

5.7

6 470

50

9.6

10 395

125

24.0

PEG-HTA insulin (pH 9.2) (intravenous)

0 305

2 72.5

232

76.2

4 101.5

203.5

66.7

5 *

8 230

75

24.6

* Animals were fed after this point.

In general, the non-immunogenic biocatalysts obtained by the process according to the invention can be processed in the same way as the biocatalysts from which they are derived. Products of this type can be standardized in vivo in terms of their polypeptide content before use. The strength of an administration can thus be assumed to be known. The amounts administered, based on the active material, can be chosen approximately the same as is customary for the biocatalyst. If e.g.

40 the usual insulin dose is 50, the product obtained according to the invention can also be administered with an insulin content of 50. As a result of the long-term effect, the required dose can be administered at frequencies which are approximately half or a quarter of the frequency required for the free material. So if e.g. If insulin was administered at intervals of 6 hours, intervals of 12 to 24 hours are recommended for the fixed material obtained according to the invention.

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1 sheet of drawings

Claims (9)

616 942 1. A method for suppressing the immunogenic effect of a biocatalyst which has a multiplicity of peptide bonds while at least partially maintaining its physiological activity by fixing polymers to the biocatalyst, characterized in that the biocatalyst is attached to a practically linear polyalkyl or polyalkoxy polymer via at least an end carbon atom thereof is fixed, the polyalkyl or polyalkoxy polymer being unsubstituted or substituted by an alkoxy or alkyl group containing less than 5 carbon atoms. 2. The method according to claim 1, characterized in that the polyalkyl or polyalkoxy polymer has a molecular weight of between 500 and 5000 daltons. 2nd PATENT CLAIMS 3. The method according to claim 2, characterized in that 10 to 100 polymer units per molecule of the biocatalyst are fixed thereon. 4. The method according to claim 1 or 3, characterized in that polyethylene glycol is used as the polyalkoxy polymer. 5. The method according to claim 1, characterized in that an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase or the insulin is used as the biocatalyst. 6. The method according to claim 1 or 5, characterized in that an enzyme is used as the biocatalyst, and that the biocatalyst with polymer fixed thereon has at least 40% of the enzyme activity of the biocatalyst without fixed polymer per mole. 7. The method according to claim 1, characterized in that a hydrolase is used as the biocatalyst and polyethylene glycol as the polymer. 8. The method according to claim 1, characterized in that asparaginase is used as the biocatalyst and polyethylene glycol as the polymer. 9. The method according to claim 1 or 6, characterized in that insulin is used as the biocatalyst and polyethylene glycol as the polymer.