EA023323B1 - Branched acyl azide pegylating agent, method for preparing the same and method for preparing pegylated interferon - Google Patents

Abstract

The invention relates to pharmaceutical industry, in particular to branched acyl azide pegylating agent, capable of binding to free amino groups of proteins with formation of amide bond by reaction of azide condensation, as well as to a method for preparing the same and method for pegylation of biologically active proteins, for instance, interferons. The invention provides the method for preparing branched acyl azide agent and respective method for pegylation of interferons (IFN), in particular IFN α-2b. Technical results of the invention include pegylating agent, capable of binding to free amino groups of proteins with formation of amide bond by means of azide condensation without causing racemisation, oxidation and/or aggregation of substrate, and the method for preparing the same.

Description

The invention relates to the pharmaceutical industry, in particular to a pegylating agent capable of attaching to the free amino groups of proteins to form an amide bond through an azide condensation reaction, as well as to a method for its preparation and a method for pegylating biologically active proteins, for example, interferons. Pegylated interferons can be used for the preparation of antiviral drugs.

State of the art

Polyethylene oxide (polyethylene glycol, PEG) is a widely used polymer for covalent modification (pegylation) of biological macromolecules, for example polypeptides, which is of great importance in modern medicine. The purpose of pegylation is to prevent protein degradation by proteolytic enzymes and to protect against antigenic and immunogenic epitopes. In addition, pegylation increases the size of the polypeptides, reducing renal filtration, and also changes the volume of biodistribution. All this helps to lengthen the residence time of modified physiologically active proteins in the patient's body, which increases their value as medicines.

The PEG molecule is not able to directly interact with protein molecules, because it does not contain reactive functional groups that can react with any functional groups of proteins. Therefore, it is required to introduce into the molecule of this polymer such reactive groups or their precursors that are converted to the active form shortly before the reaction with proteins.

Of greatest importance in the pegylation of polypeptides is the choice of the pegylating agent and the conjugation method, which determine the type and structure of the final conjugate, and therefore its biological activity, stability and circulation time in the blood.

So, for example, the antiviral activity of pegylated interferons (INF) depends to a large extent both on the size and geometry of the PEG molecule, and on the location and method of attachment of the polymer to the protein molecule, i.e. from the INF functional group, with which the corresponding pegylating agent interacts directly (Рёёгй M.Е е (а1. SNETMGU GOGREROE aib rgo! es RESU1oyoi. Αάν. Aegid OePuegu Keu. Уо1. 54. (2002), p. 459-476) .

The introduction of various active functional groups capable of interacting with functional groups of proteins under mild conditions to produce the corresponding covalent conjugates is described, in particular, Κοζ1ο \ ν5ΐ <ί Α. e (a1. Oyue1ortep1 oG ReduSeb 1n1egGegop5 Gog (Ne TgeaGteiG oG SNGoshe Nerajk S. BüOgidv.Io. 15 (7) (2001). P. 419-429.

From the chemistry of peptides (Greenstein J., Vinitz M. Chemistry of amino acids and peptides. M .: Mir, 1965) it is known that, when an amide bond is formed with optically active α-amino acids, as a rule, racemization of the substrate is observed to one extent or another, moreover to the least extent, racemization is observed using the azide method, which consists in the interaction of the acylazide of the corresponding protected amino acid or peptide with the free amino group of the corresponding amine substrate. The main disadvantage of acyl azides is their low stability, acyl azides are obtained from the corresponding stable hydrazides by treatment with nitrous acid or its esters immediately before use (A. Gershkovich, V. Kibirev. Chemical synthesis of peptides. Kiev: Nukova Dumka, 1992).

Currently, a fairly wide range of linear PEG hydrazide derivatives is known, which are used as pegylating agents directly in hydrazide form without conversion to acyl azide and the use of azide condensation. Most of these PEG hydrazide derivatives are reacted with proteins having carboxylic moieties activated by carbodiimides. The disadvantage of this conjugation method is a side reaction of the interaction of activated carboxyl groups with free amino groups both between and within protein molecules, which leads either to the fusion of protein molecules into larger covalent aggregates (intermolecular interaction), or to an irreversible change in the protein conformation (intramolecular interaction) . Branched PEG hydrazide derivatives do not find significant use for protein modification.

In the patent EP 0605963 (publ. 13.07.1994) disclosed, in particular, hydrazide derivatives of PEG with a molecular weight of from 2 to 12 kDa, having the structure:

Ρ-Ο-ίΊΕ-ΓΟ-ΝΙΙΝΗ? Ρ ΝΗ-ΟΪ-ΝΗΝΙΗ Ρ ΝΗΝΗΓΌ-ΝΗΝΗ?

P-O-CO-ΝΗΝΙΕ Ρ-ΝΗΟΟ-ΝΗΝΗσΟ-ΝΗΝΗζ Ρ-ΝΗΝΉΓδ-ΝΗΝΗ,

Ρ ΝΗ <.Ο-ΝΗΝΗ ₂ P-O-CO-CHaCH.-CO-NICK; Ρ ΝΗΥΟΑΜΕ-ΝΗΝΗ ;.

where P represents PEG, as well as methods for their preparation and use. These PEG derivatives modify, for example, erythropoietin preoxidized by periodate to form a hydrazone bond between the polymer and protein domains.

The application I8 20050118277 (publ. 02.06.2005) describes hydrazide compounds of structural formula (I)

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Κ _3- ΝΗ-ΝΗ-Ο (= Θ) -Κ _2- Ρ-Κ ₁ , in which P represents a water-soluble biodegradable polymer with a molecular weight of from 250 to 50,000 Da, K ₁ represents hydrogen, lower alkyl, lower alkoxy or -Κ _2- Ο (= Θ) -ΝΗ-ΝΗ-Κ ₃ , K ₃ represents the residue of a natural α-b-amino acid or dipeptide having angiogenic activity, which is obtained by reacting PEG dihydrazide with the corresponding carboxylic acid derivatives activated, for example, 1-ethyl -3- (3-dimethylaminopropyl) carbodiimide. PEG dihydrazide is obtained by treating polyethylene glycol (2 kDa) with an excess of phosgene, and then with hydroxy succinimide in the presence of triethylamine. The obtained PEG disuccinimidyl carbonate is converted to PEG dihydrazide by the action of an excess of hydrazine.

In the application νθ 1992/016555 (publ. 01.10.1992) PEG hydrazide is disclosed, which is obtained by a process comprising the steps of (a) treating monomethoxypolyethylene glycol with phosgene to form PEG chloroformate, (b) treating the resulting product with an excess of β-alanine ethyl ester hydrochloride in the presence of an excess triethylamine to form PEG-β-alanine ethyl ester and (c) treating PEG-β-alanine ethyl ester with hydrazine to form PEG-β-alanine hydrazide:

Protein pegylation (Prgo1c1n) is carried out by the interaction of PEG hydrazide with a carboxyl group of a protein activated by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, with the formation of an acylhydrazine linker:

or with a carbohydrate fragment of a glycosylated protein (O1usorgo1st), an oxidized periodate, followed by fixing the formed hydrazone bond by reducing sodium cyanoborohydride to the corresponding amine:

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The possibility of using the specified PEG-agent in the reaction of azide condensation in the known invention is not assumed.

The main disadvantages of the pegylating agent under consideration are the presence of a relatively easily hydrolyzable carbamate bond, and the use of highly toxic phosgene as one of the intermediate reagents. In addition, the disadvantages also include the use of conjugation of carbodiimide or periodate, which cause a number of side reactions, for example, inter- and intramolecular interaction of activated carboxyl groups with amino groups in proteins, oxidation and racemization.

PEG agents that interact with protein by means of azide condensation include, for example, a crosslinking PEG agent with a molecular weight of about 2.5 kDa, which is a polyethylene glycol diacyl azide described in I8 4101380 (publ. 06/18/1978). The specified agent is obtained immediately before crosslinking of proteins from PEG dihydrazide, which, in turn, is obtained by treating the polyethylene oxide diglycolate with thionyl chloride, followed by hydrazinolysis of the resulting diacyl chloride. This crosslinking agent can be used in the preparation of protein membranes, in particular gelatine and chymotrypsin membranes.

In the patent I8 4179337 (publ. 12/18/1979) describes PEG azide with a molecular weight of about 0.75 kDa, which is obtained from PEG hydrazide by treatment with sodium nitrite in a hydrochloric acid solution at room temperature immediately before conjugation with uricase. In turn, PEG hydrazide is obtained by hydrazinolysis of PEG glycolic acid methyl ester, which is formed by the interaction of polyethylene glycol with chloroacetic acid methyl ester in liquid ammonia in the presence of sodium metal.

In the application I8 20060115450 (publ. 01.06.2006) proposed PEG agent of General formula (1)

CH ₂ —Υ (OA'C — X

CH (OA ² ) _I , OK (1),

CH ₂ (OA ² ) _u OK where K represents a hydrocarbon group having from 1 to 24 carbon atoms;

OA ¹ and OA ² each represents an alkylene oxide group having from 2 to 4 carbon atoms; t denotes the average number of moles of attached alkylene oxide groups and is equal to from 10 to 1000;

Υ means a linker representing an ether, amide, urethane, ester or carbonate bond, a secondary amino group or an alkylene group including these bonds;

X represents a functional group capable of chemically interacting with biological substances, having the structure

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where V ¹ represents a halogen selected from C1, Br or I;

Ζ represents an alkylene group or an alkylene group comprising an ether or ester bond, a urethane, amide or carbonate bond, a secondary amino group.

PEG hydrazide derivatives can be prepared by condensation of PEG carboxyl derivatives with hydrazine tert-butoxycarbonyl (ΝΗ ₂ -ΝΗ-Βοε) in the presence of condensing agents, for example dicyclohexylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or hexafluorophosphate (benzotrietazole) - (dimethylamino) phosphonium, followed by removal of the protective group.

This PEG agent interacts with the aldehyde group of the substrate with the formation of a hydrazone bond:

Ltd

II + II - II - Ζ — C - ΝΗΝΗ ₂ t — C — Η —Ζ —C — ΝΗΝ = ΟΗ-Τ, where T denotes the residual group of the bioactive substance.

As the closest analogue of the present invention, the authors consider the invention disclosed in application I8 2006 0073113 (publ. 06.04.2006) relating to branched derivatives of PEG of structural formula (1)

CH ₂ —X

I ₂

CH (OA ² ;, _t OG (1),

CH ₂ (OA ² ) _t OK where K represents a hydrocarbon group having from 1 to 24 carbon atoms;

OA ² represents an alkylene oxide group having from 2 to 4 carbon atoms; t denotes the average number of moles of attached alkylene oxide groups and is equal to from 10 to 1000;

X represents a functional group capable of chemically interacting with biological substances, having the structure

νη ₂ ? about II - Ζ — ονη ₂ , —Ζ- -from- -POWER ¹ , % about II X —Ο- —CO- —Νο ₂ —ζ — COOH -Ν ] —Ζ-ZN, ABOUT ABOUT about ABOUT II II II with— BUT ^{Ί _ζ} ' -from- ΝΗΝΗ>_; —Ζ — CH, N

where V ¹ represents a halogen selected from C1, Br or I;

Ζ represents an alkylene group or an alkylene group comprising an ether or ester bond, a urethane, amide or carbonate bond, a secondary amino group.

PEG hydrazide derivatives can be prepared by condensation of carboxyl derivatives

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PEG with tert-butoxycarbonyl hydrazine (ΝΗ ₂ -ΝΗ-Βοο) in the presence of condensing agents, for example dicyclohexylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or hexafluorophosphate (benzotriazolyloxy) tris- (dimethylamino) phosphonium, protective group.

There is an indication of the possibility of using the obtained PEG hydrazide to form a hydrazone bond with a protein, for example, with interferon α:

Ltd

II + II - II —Ζ — C — ΝΗΝΗ ₂ T — C — Η —Ζ — C — ΝΗΝ = € Ή-Τ.

where T denotes the residual group of a bioactive substance.

The disadvantage of the above similar PEG agents is the impossibility of their direct conjugation with proteins, such as interferons, due to the absence of aldehyde groups in the proteins. PEG agents of a similar structure can form protein conjugates only after preliminary oxidation of the glycosylated domain, for example, periodate, or targeted introduction of the formyl group into the polypeptide, which leads to unpredictable changes in substrate activity and the formation of by-products, for example, due to inter- and intramolecular formation of disulfide bonds in proteins from thiols of cysteine fragments or inter- and intramolecular interaction of formyl groups with thiols or amino groups of a protein with images Niemi Schiff bases or thioacetals respectively.

Thus, the technical objectives of the present invention are to develop pegylating agents suitable for the preparation of stable derivatives of proteins, and expanding the arsenal of pegylating agents and methods for pegylating biologically active proteins, for example, interferons.

The technical results of the invention are a pegylating agent capable of attaching to the free amino groups of proteins to form an amide bond through azide condensation without causing racemization, oxidation and / or aggregation of the substrate, and a method for producing it.

SUMMARY OF THE INVENTION

As a result of extensive research, the authors found that the identified shortcomings of the prior art can be overcome by the creation and use of a branched acyl azide pegylating agent (PEG agent) of the general formula (I)

where η is an integer in the range from 200 to 950; c] is an integer in the range from 1 to 5; t is an integer in the range from 1 to 5.

The invention also provides a method for producing a branched acyl azide pegylating agent (PEG agent) of the general formula (I)

where η is an integer in the range from 200 to 950; c] is an integer in the range from 1 to 5; t is an integer in the range from 1 to 5, characterized in that the activated ether (III)

where Θ-Κ is the residue of a hydroxyl-containing compound selected from Ν-hydroxysuccinimide, 4-nitrophenolate, 2,4-dinitrophenolate, pentachlorophenolate or pentafluorophenolate, treated with an excess of hydrazine or hydrazine hydrate in an aqueous, aqueous, or organic gas in the atmosphere or argon, at a temperature of from -10 to 30 ° C, where the organic medium is selected from the group consisting of methanol, ethanol, acetonitrile, dioxane, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, and aqueous An anicic medium is a mixture of an organic medium with water in any proportions, to obtain hydrazide (II)

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where η, s | and m have the above meanings;

hydrazide (II) is isolated by extraction with a chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, after which it is precipitated with diethyl ether and filtered off;

hydrazide (II) in a medium of hydrochloric acid at a temperature of 0-5 ° C is treated with an aqueous solution of an excess of nitrite of an alkali or alkaline earth metal, and after completion of the reaction, the excess of nitrous acid is removed by the addition of sodium azide.

In a preferred embodiment of the method for producing a PEG agent, the activated ester (III) is treated with a 1% aqueous hydrazine hydrate solution in a molar ratio of hydrazine hydrate to ether (III) of 6: 1, extracted five times with dichloromethane, precipitated with diethyl ether, filtered off and precipitated an additional three times with diethyl ether. from a solution in isopropanol; hydrazide (II) in a medium of 0.5 M hydrochloric acid is treated with an aqueous solution of sodium nitrite at a molar ratio of sodium nitrite to hydrazide (II) equal to 10: 1.

Further, the invention provides a method for producing pegylated interferon a-2b 2 a-2b), characterized in that the activated ester (III)

where O-K, as described above, is treated with an excess of hydrazine or hydrazine hydrate in an aqueous, aqueous-organic or organic medium in an inert gas atmosphere, which is nitrogen or argon, at a temperature of from -10 to 30 ° C, where the organic medium is selected from a group consisting of methanol, ethanol, acetonitrile, dioxane, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, and the aqueous-organic medium is a mixture of the organic medium with water in any ratio to obtain hydrazide (II)

where η, s | and m have the above meanings;

hydrazide (II) is isolated by extraction with a chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, after which it is precipitated with diethyl ether and filtered off;

hydrazide (II) in a medium of hydrochloric acid at a temperature of 0-5 ° C is treated with an aqueous solution of an excess of nitrite of an alkali or alkaline earth metal, and after completion of the reaction, the excess of nitrous acid is removed by the addition of sodium azide;

to a solution of ΉΝ a-2b in a phosphate buffer with a pH of 8.0-8.7 at 0-5 ° C, a 4-5-fold molar excess of a solution of the PEG agent of general formula (I) is added;

when the degree of conversion ΣΡΝ a-2b reaches 70-85%, the reaction is stopped by adding excess glycine;

the reaction mixture is diluted 5-15 times, the pH value is set to 4.5-5.5 required for the chromatographic separation of products on CM-sepharose, and then gradient elution is carried out from 0.1 to 0.8 M No. C1;

fractions are selected that analyze reversed-phase HPLC for the content of mono-PEG-IΡN a-2b, according to the results of which the target fractions are determined, which are then purified to a degree sufficient to prepare the drug.

An expression selected from the group consisting of, in addition to individual alternatives, also includes possible and rational combinations of two or more such alternatives. For example, the expression chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride includes, in particular, mixtures of dichloroethane and chloroform, chloroform and carbon tetrachloride, irichlorethylene and carbon tetrachloride.

The pegylating agent (PEG agent) in accordance with the invention is obtained from the corresponding activated ester (II) in accordance with the reaction scheme

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where the values of the indices n, s | and t are indicated above;

Κ-ΘΗ is a hydroxyl-containing compound selected from Ν-hydroxysuccinimide, 4 nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol. Activated PEG esters of structure (III) are commercially available agents.

The preparation of compound (II) involves treating the corresponding activated ester (III) with an excess of hydrazine or hydrazine hydrate in an aqueous, aqueous-organic or organic medium. The reaction is carried out in an inert gas atmosphere, which is nitrogen or argon, at a temperature of from -10 to 30 ° C. The organic medium is methanol, ethanol, acetonitrile, dioxane, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, or mixtures thereof. An aqueous-organic medium is a mixture of an organic medium with water in any ratio. The molar ratio of hydrazine or hydrazine hydrate to activated ester (III) is from 5: 1 to 30: 1, preferably it is from 6: 1 to 10: 1.

Hydrazide (II) is isolated by extraction with a chlorinated solvent, which may be dichloromethane, chloroform, trichlorethylene, carbon tetrachloride or mixtures thereof, followed by precipitation with diethyl ether.

The amount of introduced hydrazide groups in (III) is determined spectrophotometrically in the visible region with a reagent 4-dimethylaminobenzaldehyde or nitrite.

PEG agent (I) is obtained immediately before conjugation with protein by treating hydrazide (II) with an excess of nitrous acid at a reduced temperature in the range from -5 to 10 ° C. The source of nitrous acid is a mixture of alkali or alkaline earth metal nitrite and an acid selected from sulfuric, hydrochloric, hydrobromic, phosphoric, citric, acetic or mixtures thereof. The molar ratio of nitrous acid to hydrazide (II) is from 10: 1 to 50: 1, preferably it is from 10: 1 to 20: 1.

The amount of azide groups in the polymer is determined, for example, spectrophotometrically with ReCl3 reagent.

At the end of the reaction, the excess nitrous acid is removed by the addition of an alkali metal azide, after which the resulting compound (I) is used without isolation for conjugation with a protein. Conjugation is carried out by adding a PEG azide (I) solution to a protein solution in a neutral or slightly alkaline buffer selected from phosphate, acetate, citrate, borate, carbonate, or mixtures thereof.

The invention is further illustrated by the preferred examples of its implementation, confirming the achievement of the technical result.

Information confirming the possibility of carrying out the invention

Example 1. Obtaining PEG hydrazide (II).

As hydroxysuccinimide ester (III), di-branched succimidyl glutarates are used (in the formula (I) c = m = 3. K. =) with a molar mass of 40 kDa (δυΝΒΚΙΟΗΤ 0Ь2-400082) or 60 kDa (δυΝΒΚΙΟΗΤ 0Ь2-600 Οδ2), manufactured by the company ΝΘΡ Sogrogabop.

In 30 ml of a 1% aqueous solution of hydrazine hydrate cooled to 0 ° C, 100 μmol of the corresponding hydroxysuccinimide ester (III) is dissolved. The mixture was stirred for 5 hours under argon at room temperature, acidified with 5% sulfuric acid to a pH of 1-2, and extracted with 5 ml of 30 ml portions of dichloromethane. The organic layer was washed with 10% sodium sulfate until neutral and dried over anhydrous sodium sulfate overnight.

The dried dichloromethane solution of PEG hydrazide was filtered, evaporated under reduced pressure, and the product was precipitated by the addition of diethyl ether. The precipitate is filtered off and precipitated three times with diethyl ether from a solution in isopropanol.

The yield for both hydroxysuccinimide esters (III) is 96-98%. The number of hydrazide groups in the polymer according to spectrophotometric analysis is 98-101%.

Example 2. Obtaining PEG-azide (I).

To a solution cooled to 0 ° C, 7 μmol of PEG-phenylalanine hydrazide in 5 ml of 0.5 M HCl is added 70 μmol Ν; · ιΝΘ ₂ in 1 ml of water. The mixture is stirred while cooling for 20 minutes, then the excess nitrous acid is removed by adding 70 μmol Ν; · ιΝ ₃ in 0.5 ml of water and immediately used for conjugation with protein.

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Example 3. Obtaining pegylated interferon a-2b (ΙΡΝ a-2b).

A solution of 7 μmol of PEG azide (I) obtained in Example 2 is added to a solution of 1 μmol of α-2b cooled to 0-5 ° C in a phosphate buffer with a pH of 8.5. The reaction mixture was stirred for 5 hours while cooling. Next, the reaction is stopped by adding excess glycine.

The reaction mixture was diluted 10-fold, pH 5.0 was adjusted by adding CH ₃ COOH and applied to CM-Sepharose, previously equilibrated with 20 mM acetate buffer at pH 5.0. After applying the reaction mixture, the sorbent is washed with a balancing buffer and then with a gradient of 0.1 to 0.8 M No. C1. Selected fractions were analyzed by reverse phase HPLC for mono-PEG-2a-2b content. The desired fractions are combined and reanalyzed to determine the purity and content of monopegylated ΙΡΝ a-2b. The yield (in terms of ΙΡΝ a-2b) of the purified PEG _40- SHL a-2b is 42%, the purity is 98.1%; for purified PEG _60- SHL a-2b, the yield is 37%, the purity is 99.0%.

Claims (4)

1. A branched acyl azide pegylating agent (PEG agent) of the general formula (Ι) where η is an integer in the range from 200 to 950; c] is an integer in the range from 1 to 5; t is an integer in the range from 1 to 5. 2. A method of obtaining a branched acyl azide pegylating agent (PEG agent) of the general formula (Ι) where η is an integer in the range from 200 to 950; c] is an integer in the range from 1 to 5; t is an integer in the range from 1 to 5, characterized in that the activated ester (ΙΙΙ) where Θ-Κ is the residue of a hydroxyl-containing compound selected from Ν hydroxy succinimide, 4-nitrophenolate, 2,4-dinitrophenolate, pentachlorophenolate or pentafluorophenolate, is treated with excess hydrazine or hydrazine hydrate in an aqueous, aqueous-organic or organic medium in an inert gas atmosphere, which is nitrogen or argon, at a temperature of from -10 to 30 ° C, where the organic medium is selected from the group consisting of methanol, ethanol, acetone nitrile, dioxane, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, and aqueous-organic medium is a mixture of the organic medium with water in any ratio, to give a hydrazide (ΙΙ) where η, with | and m have the above meanings; hydrazide (ΙΙ) is isolated by extraction with a chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, after which it is precipitated with diethyl ether and filtered off; hydrazide (ΙΙ) in a medium of hydrochloric acid at a temperature of 0-5 ° C is treated with an aqueous solution of an excess of nitrite of an alkali or alkaline earth metal, and after completion of the reaction, the excess of nitrous acid is removed by the addition of sodium azide. 3. The method of producing the PEG agent according to claim 2, characterized in that the activated ester (ΙΙΙ) is treated with a 1% aqueous hydrazine hydrate solution in a molar ratio of hydrazine hydrate to ether (ΙΙΙ) of 6: 1, extracted five times with dichloromethane, precipitated with diethyl ether, filtered off and additionally three times reprecipitated with diethyl ether from a solution in isopropanol; hydrazide (ΙΙ) in a medium of 0.5 M hydrochloric acid is treated with an aqueous solution of sodium nitrite - 8 023323 with a molar ratio of sodium nitrite to hydrazide (II) equal to 10: 1. 4. A method of producing pegylated interferon a-2b (ΙΡΝ a-2b), characterized in that the activated ester (III) where Θ-Κ is the residue of a hydroxyl-containing compound selected from β-hydroxysuccinimide, 4-nitrophenolate, 2,4-dinitrophenolate, pentachlorophenolate or pentafluorophenolate, treated with an excess of hydrazine or hydrazine hydrate in an aqueous, aqueous-organic or organic medium in an inert gas atmosphere, which is nitrogen or argon, at a temperature of from -10 to 30 ° C, where the organic medium is selected from the group consisting of and h methanol, ethanol, acetonitrile, dioxane, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, and the aqueous-organic medium is a mixture of the organic medium with water in any ratio to obtain hydrazide (II) where p, s | and m have the above meanings; hydrazide (II) is isolated by extraction with a chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, after which it is precipitated with diethyl ether and filtered off; hydrazide (II) in a medium of hydrochloric acid at a temperature of 0-5 ° C is treated with an aqueous solution of an excess of nitrite of an alkali or alkaline earth metal and, after completion of the reaction, an excess of nitrous acid is removed by the addition of sodium azide to obtain the PEG agent of general formula (I) according to paragraph 1; to a solution of IΡN a-2b in a phosphate buffer with a pH of 8.0-8.7 at 0-5 ° C, a 4-5-fold molar excess of a solution of the PEG agent of general formula (I) is added; upon reaching the degree of conversion ΓΡΝ a-2b 70-85%, the reaction is stopped by adding excess glycine; the reaction mixture is diluted 5-15 times, the pH value is set to 4.5-5.5 required for the chromatographic separation of products on CM-sepharose, and then gradient elution is carried out from 0.1 to 0.8 M No. C1; fractions are selected that analyze reverse-phase HPLC for the content of mono-PEG ΡΝ a-2b, according to the results of which the target fractions are determined, which are then purified to a degree sufficient to prepare the drug.