KR100473659B1 - Process for preparing hybrid protein replaced by available polymer at the saccharide chain by site-specific modification - Google Patents

Abstract

The present invention relates to glycosylation of existing cells through regioselective protein modifications performed to confer or enhance the in vivo stability and biological activity of glycoproteins produced for therapeutic, research and industrial purposes. By artificially manipulating the gene, a part of the glycoprotein is removed, and the glycoprotein is expressed using a glycoprotein prepared using a transformed yeast system in a form in which only glycoprotein is attached, or by expressing the protein in a conventional manner. By using a mutated glycoprotein that has been cleaved with a sugar chain cleavage enzyme, and leaving only acetyl glucosamine, a modified protein useful for the human body instead of the existing oligosaccharide is prepared in a hybrid glycoprotein. It is about a method.

Description

Process for preparing hybrid protein replaced by available polymer at the saccharide chain by site-specific modification}

The present invention relates to glycosylation of existing cells through regioselective protein modifications performed to confer or enhance the in vivo stability and biological activity of glycoproteins produced for therapeutic, research and industrial purposes. By artificially manipulating the gene, a part of the glycoprotein is removed, and the glycoprotein is expressed using a glycoprotein prepared using a transformed yeast system in a form in which only glycoprotein is attached, or by expressing the protein in a conventional manner. By using a mutated glycoprotein that has been cleaved with a sugar chain cleavage enzyme, and leaving only acetyl glucosamine, a modified protein useful for the human body instead of the existing oligosaccharide is prepared in a hybrid glycoprotein. It is about a method.

More specifically, sugars containing only en-acetylglucosamine produced in a glycoprotein or transgenic yeast system in which existing glycoproteins produced by a recombinant protein production method for the production of hybrid proteins are treated with glycoproteinases. By producing a hybrid protein by regioselectively modifying a polymer useful for the human body at an additional position of the sugar chain of the protein, the sugar chain (oligosaccharide chain) of the existing glycoprotein is replaced or supplemented with a polymer useful for the human body, and blood Etc. A method for producing hybrid proteins for increasing the half-life in vivo and conferring the provision of medical efficacy and activity, biological efficacy and activity, and the number of natural or synthetic polymers useful for the human body. To provide a plant.

To date, various attempts have been made to enhance the in vivo bioactivity of therapeutic proteins. The main purpose of these methods was to increase the size of the therapeutic protein. That is, a method of increasing the size of protein molecules by adding a polymer material such as PEG to the protein chain.

The best known of these methods is known as "PEGylation", which is a chemical addition of PEG to proteins. This method was initially used to reduce antigenicity, but is now widely used to enhance biological activity by increasing the duration of in vivo protein.

However, these conventional methods bind regioselectively to proteins because they bind all amino acids with similar reactivity at positions accessible to functional groups of modifying materials due to the nature of chemical bonding. There is a restriction that can not be. That is, the positional specificity of the bond is either missing or very limited. In order to solve this problem of positional nonspecificity, a method of using the active structure of the protein itself has been attempted (Korean Patent Registration No. 353,392), but it is not yet generalized and cannot be applied to a formal method for all proteins.

Polypeptides of erythropoietin (EPO) produced by microorganisms such as Escherichia coli cannot be used for therapeutic purposes because of the lack of glycans. Administration of EPO derived from a microorganism, which consists solely of polypeptides without added sugars, exhibits inferior biological activity due to lack of sugar chains. EPO produced from microorganisms lacking such glycosylation can be easily purified by an in vivo purification system, thereby reducing the therapeutic effect significantly.

In addition, eukaryotic cells such as yeast are capable of glycosylation, but the production of EPO using these cells is different from that of EPO produced in Chinese hamster ovary (CHO) cells, which are commonly used as therapeutic agents. Despite its high productivity, it could not be actually used for commercial production due to the induction of antigen-antibody reaction due to the difference in sugar chain structure.

The problem of reducing the therapeutic effect by the antigen-antibody reaction when produced in eukaryotic cells other than CHO cells can be improved by preparing a hybrid protein in which the polymer material according to the method of the present invention is modified.

In other words, the sugar chain of EPO produced in these cells was left with only N-acetylglucosamine using sugar chain cleavage enzyme, or the inventors developed a hybrid protein therapeutic agent by the position-selective modification method filed by Korean Patent Application No. 2002-65470. A polymer useful for a human body that does not induce an antigen-antibody reaction after producing a transformed yeast strain disclosed in 'transforming yeast system for the production of E-glycoprotein in the form of attached only N-acetylglucosamine instead of the existing sugar chain. The substance is solved by regioselective modification using en-acetylglucosamine or a derivative thereof added to the glycoprotein.

In other words, modifying the polymer material useful for the human body to the existing sugar chain position of glycoproteins, these proteins produced in ordinary Escherichia coli are not modified properly, so the in vivo stay is too short to lose efficacy as a therapeutic agent. In addition, even when produced using eukaryotic cells, the sugar chains added to proteins vary depending on the host cell, and as a result, the drug may be toxic through antigen-antibody reaction or may be treated due to low body titer. This is because their use is often limited to their purpose.

Regioselective protein modification requires the presence of functional groups specific to the protein to be modified. In general, the 20 amino acids present in the protein theoretically contains a reaction reagent, that is, 13 functional groups that can act chemically with those corresponding to the modified substance in the present invention.

There are 14 different groups that differ according to chemical reactivity, including disulfide bonds of cystine and secondary amide bonds in the protein chain. These groups are: thio- (Cys, or rarely seleno-Cys), methylthio- (Met), disulfide bonds, primary and secondary hydroxyls (Ser, Thr), phenolic hydroxyls (Tyr) ), Indole (Trp), primary amino (Lys, N-terminus of all amino acids), carboxyl (Asp, Glu, C-terminus of all amino acids), guanidine (Arg), primary amide (Asn, Gln), Secondary and tertiary amides (Pro) and imidazole (His). It may also include functional groups introduced by modification after protein expression, including amidation, phosphorylation, and the like. Although these various reaction groups exist, the functional groups in each group have similar reactivity.

Therefore, in the case of using the conventional chemical modification method, all functional groups in each group are modified by reacting all functional groups having similar reactivity in each group with respect to a certain modifier. As a result, when it is necessary to regioselectively modify some amino acids at a specific position instead of all amino acids having functional groups belonging to the same group, conventional chemical modification methods cannot be used.

In order to prepare the best protein hybrids, the polymer should be regioselectively modified at a desired position in the protein, and the present invention is to solve various problems according to the modification of the polymer.

The technical problem to be achieved by the present invention is to prepare a hybrid protein that is regioselectively modified with a suitable polymer modifier that can replace the sugar chain added to the protein. In other words, the best protein modification method should be position selectivity and a new modification method that can selectively modify the protein only at the desired position. Therefore, the present invention is to prepare a hybrid protein by modifying a polymer material useful for the human body only at a desired position, unlike the conventional modification method having no position selectivity at all.

The present invention provides a method for producing a hybrid protein by removing oligosaccharides added to glycoproteins and regioselectively modifying the oligosaccharides with polymer modified substances useful for humans at the oligosaccharide position, i) transgenic yeast strains (Accession Number: KCCM). -10441) to produce glycoproteins with added N-acetylglucosamine alone, or the sugar chain structure of glycoproteins produced in normal production cells is removed with sugar chain cleavage enzyme, and only N-acetylglucosamine is used. Preparing a glycoprotein modified into an added form; Ii) a method of producing a hybrid protein comprising regioselectively modifying a polymeric modifier useful for the human body by using en-acetylglucosamine or a derivative thereof present in the glycoprotein as a modified position.

The polymer material useful for the human body is a biocompatible polymer having a number average molecular weight of about 300 to 100,000 Daltons, polyethylene glycol, polypropylene glycol, polyoxyethylene, polytrimethylene glycol, polylactic acid and its derivatives, polyacrylic acid and its derivatives, poly At least one polymeric modifier selected from amino acids, polyurethanes, polyphosphazines, polyalkylene oxides, polysaccharides, dextran, polyvinylpyrrolidone, polyvinyl alcohols, polyacrylamides or other similar non-immune polymeric modifiers It is characterized by that.

In addition, the polymer modifier more preferable in the present invention is characterized in that the PEG derivative, that is, PEG-amine, PEG-isocyanate, PEG-hydrazide.

The present invention also provides a hybrid protein produced by the above method.

Hereinafter, the present invention will be described in more detail.

In order to perform regioselective modification only at the desired position of a protein, amino acids with functional groups that have specific reactivity with the chemicals to be used in the modification must be present at the position of the protein to be modified. Proteins in which amino acids with functional groups with such specific reactivity exist only at the modified sites are naturally less likely to be used for regioselective protein modification.

Therefore, the present invention is to propose a method for modifying the protein regioselectively without affecting the amino acid of the protein chain, preferably by using the N-acetylglucosamine, which is a sugar whose reactivity is distinct from the functional group of the amino acid in the protein modification. .

In the case of using a sugar chain, due to the many functional groups derived from the various sugars present in the sugar chain, it is not easy to control the modification reaction such as the number of modified substances modified in one molecule of the protein and to ensure uniformity of the modified product. In the case of long sugar chains having a structure, the possibility of inducing an antigen-antibody reaction by sugar chains is higher than that of using a monosaccharide.

In addition, if the sugar chain contains mannose or galactose, even after modifying the sugar chains containing the modified sugars as a therapeutic agent, the protein is removed from the body such as endocytosis, which is recognized by mannose or galactose. Clearance is likely to cause a drastic shortening of the stay in the body.

It is most preferable that the sugar is en-acetylglucosamine when the single sugar is used as a modification site for the modification reaction.

In the sugar chains of most glycoproteins in nature, the first sugar of the sugar chain that binds to the protein is en-acetylglucosamine, so it is possible to produce en-acetylglucosamine-attached proteins or by using sugar chain structural modification enzymes. It is easier to produce proteins with derivatives of en-acetylglucosamine formed through acetylglucosamine or derivatization reactions than to produce or prepare sugar adducts with only other monosaccharides.

In the present invention, the amino acid corresponding to the site to be modified is an amino acid corresponding to the glycosylation site in the producing cell itself, and the sugar must be attached before the modification by glycosylation of the producing cell.

In addition, in the present invention, the term "formula" refers to all substances useful to the human body that can be added to proteins, such as natural or artificially synthesized polymer materials. As a modifier, PEG has a low risk of inducing an antigen-antibody reaction and many related technologies have been developed.

Modification of therapeutic proteins with modified substances such as PEG provides several advantages. Most importantly, increasing the length of stay in vivo, such as plasma, increases the bioavailability of blood and tissues in vivo. Second, the modifier may mask the antigenic determinants of non-human proteins and may also alter the immunogenicity of the protein. Modifications of these proteins can often change their interaction with receptors, including receptors involved in plasma clearance or immune system recognition, by forming new structures on the protein surface.

Many therapeutic proteins can be modified by modifiers such as synthetic polymers. The polymers applied to the formulas are biocompatible polymer chains that are easily soluble in various solvents and exhibit substantially non-antigenic properties, and their number average molecular weight is preferably about 300 daltons to about 100,000 daltons, more preferably about 2,000 Daltons to about 40,000 daltons.

Representative biocompatible polymers include, but are not limited to, polypropylene glycol (PPG), polyoxyethylene (POE), polytrimethylene glycol, polylactic acid in addition to PEG. ) And derivatives thereof, polyacrylic acid and derivatives thereof, poly (amino acid), polyurethane, polyphosphazene, polyalkylene oxide (PAO) , Polysaccharides, dextran, polyvinyl pyrrolidone, polyvinyl alcohol (PVA), polyacryl amide or other similar non-immune polymers. .

Of these modified substances, PEG is the most preferred because of the wide range of commercially available molecular weights, the oxyethylene backbone has hydrophilic properties that allow 2-3 water molecules per unit, and from methoxypolyethylene glycol It is easy to synthesize derivatives having a single functional group.

Various types of chemical bonds have been studied to bind functional groups of protein such as PEG and protein. U.S. Patent No. 4,179,337 refers to protein-polymer conjugates having reduced antigenicity and anti-immunity while maintaining biological activity by binding proteins or polypeptides to PEG or water-soluble polymers with molecular weights of 500 to 20,000.

U.S. Patent No. 4,301,144 also describes increased oxygen transport capacity when hemoglobin is combined with PEG or a water soluble polymer. Abuchowski et al ., Cancer Biochem. Biophys . 7: 175-186 (1984) demonstrated that PEG-associated proteins increase the body's half-life and decrease immunogenicity in plasma, and Gilbert et al. (Gilbert et al. , US Pat . No. 5,951,974 (1999); Algranati et al. , Hepatology 40 (suppl): 190A (1999)) reported the rate of loss in the body by binding PEG 12,000 and branched PEG 40,000 to alpha interferon. Interferon, which was administered three times a week, was reduced to make it sufficient once.

Davies, etc. (Davis et al, Lancet 2: . 281-283 (1981)) is, when combined with the Eureka claim (uricase) PEG increased the half-life of the body and the side effects upon the metabolism of uric acid prove reduced, such niben (Niven et al. , J. of Contr., Rel. 32: 177-189 (1994)) show long-term leukocyte growth effect by incorporating recombinant human granulocyte-colony stimulating factor (rhG-CSF) with PEG. Reported.

The most commonly used method for binding PEG to proteins or polypeptides is to react the activated PEG to amino residues of the protein or polypeptide. The amino residues of the polypeptide include lysine and en-terminal amino groups, and the method of activating PEG is as follows.

One hydroxyl group of the PEG is substituted with a methyl ether group, and the other hydroxyl group is bonded to a functional group containing an electrophilic substance.

Examples of activated PEG include PEG-ene-hydroxysuccinimide-activated esters with amide bonds, PEG-epoxides and PEG-tresylate with alkyl bonds, and urethanes. PEG-carbonyl imidazole with a bond, PEG-nitrophenyl carbonates, PEG-aldehyde with a Schiff's base at the N-terminus, and the like. have.

However, since lysine residues are present in proteins and polypeptides randomly, using PEG reacting with amino groups causes PEG to bind nonspecifically, resulting in a heterogeneous mixture. Therefore, in recent years, methods for binding PEG to specific sites using cysteine, hydroxy, and arginine groups of proteins or polypeptides have been attempted to make uniform PEG conjugates.

However, although PEG modifications to specific sites, such as cysteine, are quantitative and site-specific, there are complexities in which cysteine must be artificially introduced into proteins or modified proteins. In addition, the PEG formula in a position such as a hydroxyl group or arginine is difficult to determine the exact position of the modification.

However, the present invention has the characteristic of specifically binding the PEG derivative to the sugar site after producing or modifying the protein so that en-acetylglucosamine is added to specific three positions of the EPO. To prepare a hybrid type EPO (hybrid EPO) by modifying a specific site with a PEG derivative commonly used, that is, PEG-amine, PEG-isocyanate, PEG-hydrazide do. The PEG used at this time is not only linear, but also multi-armed PEG.

Some polymers, such as ethylene / maleic anhydride copolymers, are inherently reactive, but most require initially to be specifically activated by reagents with polyfunctional groups. Partial substitution of halogens in tri- or di-halotriazines was a common early method of activating amino- and hydroxyl-containing polymers.

However, because of concerns about side effects and triazine reactivity, this method is not currently commonly used. Carbonyl diimidazole is a very effective reagent that activates the hydroxyl group of PEG to acylimidazoles having good reactivity to protein amino groups. Some binding methods produce an active ester of a reversible polymer-protein conjugate such as succinyl-PEG, where the polymer can be removed by hydrolysis.

Methods based on disulfide bond exchanges are not widely used for the connection of synthetic polymers other than the preparation of protein-protein conjugates. For PEG-modified therapeutic proteins, increasing the molecular weight, usually above about 60 kilodaltons, has a significant effect on plasma purification. The magnitude of the effect here depends largely on the purification mechanism of the unmodified protein.

In the present invention, a strain capable of producing a glycoprotein to which en-acetylglucosamine is attached or using a conventional sugar chain addition using a transformed yeast strain having a sugar chain addition reaction in which an existing sugar chain portion is added with only en-acetylglucosamine at a position. First, the target protein is produced in the form of glycoprotein to which sugar chains are added, and then, secondly, N-acetylglucosamine alone or an enzyme formed by derivatization reaction using a sugar chain structural modification enzyme such as glycoclease is appropriate. Only derivatives of acetylglucosamine are attached to form.

The present invention is a variant produced by replacing the sugar chain of the conventional pharmaceutical, industrial, research glycoproteins with a modified substance or by changing the sugar chain portion of the existing glycoprotein or introducing the sugar chain portion into the ordinary protein (mutein) It can be applied to the formula of) and this means the development of a new drug.

The protein or peptide used in the present invention is not limited to a specific therapeutic drug, any biologically active substance can be used. Specifically, alpha, beta, gamma interferon (α-, β-, γ-interferon), asparaginase, arginase, adenosine deaminase, superoxide dismutase (superoxide dismutase), catalase, lipase, free case, adenosine diphosphatase, tyrosinase, glucose oxidase, glucosidase, blood Blood factor VII, VIII and IX, immunoglobulins, cytokines, ie interleukins, granulocyte colony stimulating factor (G-CSF) Granulocyte macrophage colony stimulating factor (GM-CSF), platelet derived growth factor (PDGF), lectins, lysine (tumins necros) is factor (TNF), transforming growth factors (TGFs), thrombopoietin (TPO) and tissue plasminogen activator (tPA), and the like. In addition, the present invention can be applied to variants of existing therapeutic proteins such as ARANESP of Amgen, USA.

Hereinafter, specific examples of therapeutic proteins will be described in detail for modifying EPO using the method of the present invention. However, EPO is merely presented as an example of a therapeutic protein, and the present invention is not limited to EPO.

EPO can be produced by removing or removing all parts except en-acetylglucosamine from the existing sugar chains and modifying them with modified substances such as PEG to reduce antigen-antibody response when administered as a therapeutic agent. Increasing the length of stay can increase biological activity. In particular, the method of removing the rest of sugar chains except specific sugars is not influenced by the glycosylation pattern inherent in the production cell, so the production strain may be freely selected without any restriction regardless of the difference in the glycosylation reaction of the production strain.

The present inventors used EPO produced in specially engineered yeast and EPO produced in general yeast and then treated with sugar chain structural modification enzymes for modification. Modification of EPO was selectively modified by PEGylation methods designed for the purposes of the present invention.

Of course, the conventional modification method, but modified the protein at random, in the present invention was modified to the modified material by using the en-acetylglucosamine or its derivatives are bound only at the amino acid site of the desired position.

The final product, hybrid EPO, was chemically modified by covalent bonds with one to three PEG molecules per molecule. PEG was bound to the hydroxyl group of en-acetylglucosamine or derivatives thereof added to EPO. The PEG polymer derivatives used in the present invention are not only linear but also branched PEG derivatives.

Hereinafter, an Example demonstrates this invention more concretely. These examples are merely illustrative of the present invention and the scope of the present invention is not limited to these examples.

Example 1 Expression and Production of EPO with Only N-acetylglucosamine Added

EPO added only en-acetylglucosamine using MPY (Accession No .: KCCM-10441) to the transformed yeast strain Saccharomyces cerevisiae disclosed in detail in Korean Patent Application No. 2002-65470 of the present inventors. Produced. In order to produce EPO with added N-acetylglucosamine alone, mutant yeast strains with the EPO gene were inoculated in YPD medium (1% yeast extract, 1% peptone, 2% glucose) and then incubated for 16 hours at 28 ° C. Was done.

Subsequently, the cell concentration was 0.4-0.6 OD ₆₀₀ / ml (wherein OD ₆₀₀ was absorbed at a wavelength of 600 nm in a spectrophotometer) in YPD0.5 (1% yeast extract, 1% peptone, 0.5% glucose) medium used as the production medium. After the inoculation, the shaking culture was performed at 30 ° C. for 5 hours. After incubation, the culture solution was centrifuged (12,000 rpm, 4 ° C, 10 minutes) to recover the supernatant, and the antigen-antibody reaction was performed without refolding the EPO containing only N-acetylglucosamine instead of the sugar chain at the sugar chain portion. Separated using. En-acetylglucosamine attached to EPO was used for subsequent PEGylation reactions.

Example 2 Production of EPO Using Yeast and Modification of Sugar Chain Using Glucose Modifiers

In order to obtain EPO having only the same form of the product of Example 1 as that of en-acetylglucosamine, EPO genes were introduced into conventional yeast cells to transform yeast cells, and sugar chains were added to the sugar chain producing strains using the cells. The added EPO was secreted and produced according to the reaction form (in this case, the yeast cells and therefore the high-mannose type). The yeast strain used at this time is Saccharomyces cerevisiae. The vector used for the transformation of the yeast is pEY-EPO disclosed in detail in Korean Patent Application No. 2002-65470 of the present inventors.

EPO secreted into the culture of transformed yeast is isolated by antigen-antibody reaction without refolding process, and the sugar chain attached to EPO is used by the endoglycosidase H, a sugar chain cleavage enzyme, As a result, the remaining sugar was removed from the sugar chain of EPO, leaving only en-acetylglucosamine. EPO from which all other sugars were removed except endo-acetylglucosamine added to EPO was separated from the endoglycosidase H reaction solution by size exclusion chromatography. This was used for subsequent PEGylation reaction.

Example 3 Preparation of PEG 5,000 Amine-EPO Using En-acetylglucosamine

The reaction with 2 mg of EPO with N-acetylglucosamine, PEG 5,000-amine (PEG 5,000-amine) with a molecular weight of 5,000, and 7 mg was carried out in 50 ml of 50 mM sodium acetate buffer at pH 6.0 in 1 mM cyanoborohydride ( Cyanoborohydride) was carried out by stirring at 4 ℃ for 24 hours so that the molecular ratio between EPO and PEG is 1: 10. Modifications of PEGylated EPO were isolated using size exclusion chromatography on unmodified EPO and remaining PEG. The biological activity of the modified bodies of isolated PEGylated EPO was measured.

Example 4 Preparation of PEG 20,000 Amine-EPO Using En-acetylglucosamine

The same procedure as in Example 3 was performed but PEG 20,000-amine was used instead of PEG 5,000-amine.

Example 5 Preparation of PEG 5,000 Hydrazide-EPO Using En-acetylglucosamine

Reaction with 2 mg of EPO with N-acetylglucosamine, PEG 5,000-hydrazide with molecular weight of 5,000, and 7 mg of 1 mM cyanoborohydride in 50 ml of 50 mM sodium acetate buffer at pH 6.0 It was carried out by stirring for 24 hours at 4 ℃ to a molecular ratio of 1: 10 EPO and PEG in the presence of a ride. Modifications of PEGylated EPO were isolated using size exclusion chromatography on unmodified EPO and remaining PEG. The biological activity of the modified bodies of isolated PEGylated EPO was measured.

Example 6 Preparation of PEG 20,000 Hydrazide-EPO Using En-acetylglucosamine

The same procedure as in Example 5 was carried out but PEG 20,000-hydrazide was used instead of PEG 5000-hydrazide.

Example 7 Determination of Biological Activity of Modified Body of PEGylated EPO

In vitro bioactivity of PEGylated EPO strains was determined in EPO cultured in RPMI 1640 medium (Kitamura, T. et al. , Blood 73: 375-380 (1989)) containing 10% embryonic calf serum . The growth of human cell line TF-1 showing dependent growth was measured. In vivo bioactivity quantified ⁵⁹ Fe introduced into rat blood cells according to Goldwizer's method (Goldwasser, E. and Gross, M., Methods in Enzymol. 37: 109-121 (1975)). It was measured by.

Each measurement was determined according to a parallel line assay (Dunn, CDR., Napier, JAP., Exp. Hematol. (N.Y.) 6: 577-584 (1978)). That is, nine measurements were taken per sample, in vitro activity was measured by two assays per measurement, and three measurements per sample and three mice were used for each measurement. Other substances, such as salts in the modified form of PEGylated EPO diluted to 1/500, did not significantly affect biological activity assays.

In biological activity assays, recombinant human EPO (rHuEPO) purified to high purity according to The second International Reference Preparation was used as standard. As a result of the biological activity measurement, the modified EPO PEGylated with PEG having molecular weight of 5000 was 30% higher in vitro activity than rHuEPO, and the in vivo activity was based on the quantitative value of ⁵⁹ Fe introduced into blood cells in vivo. It was higher than 50%.

In addition, the modified body of EPO PEGylated with PEG having a molecular weight of 20,000 was 15% higher in vitro activity than rHuEPO, and the in vivo activity was 70% or more based on the quantitative value of ⁵⁹ Fe introduced into blood cells in vivo. High. In both cases of PEG-amine or PEG-hydrazide used in the modification, no difference in biological activity with different PEG derivatives was observed. Some experimental variation in the in vivo activity of PEGylated EPO's modifications appears to be due to the degree of hydration of PEG molecules added to EPO.

The effect of the present invention is to provide a hybrid protein which is regioselectively modified with an appropriate polymer modifier that can replace the sugar chain added to the protein. In other words, the best protein modification methods should be regioselective. According to the novel modification method that can selectively modify the protein only at the desired position disclosed in the present invention, the polymer material useful for the human body is selectively modified in the protein to increase the system period in the living body such as blood, thereby increasing the medical efficacy and biological It is to provide a hybrid protein that enhances and enhances the activity efficacy.

1 is a diagram showing an electrophoretic band of erythropoietin isolated after producing in a form in which only acetylglucosamine is added from the transformed yeast of the present invention.

FIG. 2 is an electrophoretic image of hybrid erythropoietin obtained by erythropoietin shown in FIG. 1 using en-acetylglucosamine (A) PEG 5,000-amine, (B) PEG as PEG 20,000-amine Electrophoresis picture of hybridized erythropoietin.

Claims (4)

In the method for producing hybrid protein by removing oligosaccharide added to glycoprotein and regioselectively modifying oligosaccharide position with a polymer modified substance useful for human body, i) transgenic yeast strain (Accession No .: KCCM-10441) ) To produce the glycoprotein added with only en-acetylglucosamine or the sugar chain structure of glycoproteins produced in normal producing cells using sugar chain structure modified enzyme, and only en-acetylglucosamine is added. Preparing a modified glycoprotein that has been modified into the present form; And Ii) a method of producing a hybrid protein comprising regioselectively modifying a polymeric modifier useful for the human body by using en-acetylglucosamine or a derivative thereof present in the glycoprotein as a modified position; The method of claim 1, wherein the polymer modifier useful for the human body is a biocompatible polymer having a number average molecular weight of about 300 to 100,000 Daltons, polyethylene glycol, polypropylene glycol, polyoxyethylene, polytrimethylene glycol, polylactic acid and derivatives thereof, Among polyacrylic acid and its derivatives, polyamino acids, polyurethanes, polyphosphazines, polyalkylene oxides, polysaccharides, dextran, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylamide or other similar non-immune polymeric modifiers Method for producing a hybrid protein, characterized in that at least one polymer modified material selected The method of claim 2, wherein the polymer modifier is a linear or multi-stranded form of PEG derivative, that is, PEG-amine, PEG-isocyanate, PEG-hydrazide. delete