KR100353392B1 - Method of Preparing the PEG-Protein Composition Having Enhanced Biological Activity - Google Patents

Abstract

The present invention relates to a method for preparing a conjugate of a bioactive protein and PEG having a high bioactivity, the composition comprises the steps of blocking the active site of the bioactive protein with a hydrolyzable blocking material; Coupling a PEG derivative resistant to hydrolysis to a site other than the active site of the bioactive protein; Hydrolyzing and removing the blocking material bound to the active site of the bioactive protein; and having the effect of increasing the retention time in the body and reducing antigenicity, which are advantages of PEG binding, It is possible to maintain high levels of bioactivity.

Description

Method of preparing the PEG-Protein Composition Having Enhanced Biological Activity}

The present invention relates to a manufacturing method having a high bioactivity as a combination of a polyethylene glycol (hereinafter referred to as "PEG") and a bioactive protein such as interferon, the increase in the residence time and antigenic properties of the advantages of PEG binding The present invention relates to a method for preparing a conjugate of a bioactive protein and PEG, which has a reduction effect and can maintain a high level of bioactivity of a bioactive protein such as interferon.

As a representative example of a bioactive protein, interferon (hereinafter referred to as 'IFN') is a substance that modulates the action of various types of white blood cells that make up the immune system, and its properties inhibit antiviral properties and cell division. There is a property to let. Human IFN can be classified into three types according to its source and antigenicity. There are α-IFN filed in white blood cells, β-IFN filed in fibroblast, and γ-IFN filed in B cell. Recombinent forms of IFN produced by genetic engineering have already been developed and are commercially available. These three IFNs can be subdivided again due to their antigenic and structural properties, and there are currently more than 24 α-IFNs known. α-IFN is currently approved by the FDA and is used in patients, including hairy cell leukemia, genital edema, Kaposi's Sarcoma, non-A, and non-B types. Chronic hepatitis and hepatitis C. Other diseases in which α-IFN can be applied include myelogenous leukemia, multiple myeloma, superficial bladder cancer, skin cancer, and linalcell carsi. Renal cell carcinoma, ovarian cancer, T-cell lymphoma, and glioma are also effective when used in combination with drug therapy for lung cancer, rectal cancer, and breast cancer. Has been reported.

α-IFN is a substance involved in the function of the cell and affects the replication of DNA and RNA, protein synthesis, and the like. Α-IFN, which has cell killing ability, therefore acts not only on diseased cells but also on healthy cells, resulting in side effects. Common side effects include flu-like symptoms such as fever, tiredness, headache, chills, dizziness, cough, abnormal bowel function, liver damage, decreased red blood cell count, and reduced white blood cell and platelet count.

Many kinds of natural or genetically produced proteins have medical and pharmacological effects. However, when injected as a therapeutic agent, it may express antigenicity, have low water solubility, may have difficulty as an injection, and the pharmacological effect may not be good due to a short period of stay in the body. To overcome this difficulty, a method of combining protein and PEG has been used, which was first disclosed in US Pat. No. 4,179,337. The patent, which was published in 1979 and lost its effectiveness in 1996, shows that the use of PEG-associated proteins and enzymes as therapeutic agents has the advantages of reduced antigenicity, increased water solubility, and increased retention in the body. It was. The current trend of expiration of this patent, the first patent on the use of PEG, is gradually being subdivided into material patents for specific proteins bound to PEG, and material patents for the corresponding PEG-proteins.

It is true that the side effects are greatly reduced due to the above advantages when the protein and PEG are combined, but at the same time, the drawback is that the biological activity of the protein is greatly reduced after binding to PEG. When PEG binds to a protein, amino acids on the surface of the protein and PEG form a covalent bond, so that PEG is attached to the surface of the protein. When bound, the site is no longer capable of biological activity, thereby reducing its activity. Therefore, how PEG proceeds while maintaining protein activity has been the focus of recent research.

U. S. Patent 5,382, 657 (1995.1.17) acknowledged that the activity was significantly reduced when PEG bound IFN. In addition, it was recognized that the number of PEG attached to the surface of the IFN should be minimized in order to maintain the maximum activity of the IFN. This is because the greater the number of PEG attached, the greater the probability of touching the active site of IFN. For this reason, in order to minimize the number of PEG to be bound and at the same time show the advantages of PEG, it is inevitable to select the largest molecular weight of the PEG used. If only one or two PEGs are bound to each IFN molecule, the molecular weight of the PEG should be chosen to be at least 5,000, even 10,000 to 20,000. According to the patent, when PEG of about 5,000 molecular weight was used, IFN activity was reduced by 60% when one PEG was bound, and IFN activity was reduced by 96% when two PEGs were bound. When PEG with molecular weight of about 10,000 was used, IFN activity was reduced by 75% when one PEG was bound, and IFN activity was reduced by 98% when two PEGs were bound. As a result, IFN activity decreased by 96-98% when two PEGs were combined, and this condition was not selectable. When one PEG was bound, IFN activity decreased by 60-75% Resulted in a loss.

According to US Pat. No. 5,951,974 (1999.9.14), PEG is intended to selectively react with His34 of IFN to avoid PEG reacting with the active site of IFN in binding PEG with IFN-α 2b. IFN-α 2b has eight amino acids reactive to the surface, and they are Cys1, Lys31, His34, Lys49, Lys83, Lys121, Lys131, Lys134. When 34 was reacted with PEG by targeting His34 of IFN, His34, Cys1, and Lys121 were reacted. His34 reacted 55%, Cys1 reacted 15%, Lys121 reacted 15% appear. This is the result of trying to attach only one PEG per molecule of IFN. In addition, as a result of investigating the bioactivity through the cytopathic assay, 1.0-1.3 PEG (molecular weight 12,000) was combined for each IFN molecule, resulting in a 74-76% decrease in bioactivity.

According to U.S. Patent 5,981,709 (November 9, 1999), it was recognized that it was disadvantageous to bind a large number of PEGs per molecule of IFN, and thus, one PEG per molecule of IFN was intended. The PEG derivatives used were PEG-succinimidyl carbonate (SC-PEG) having a molecular weight of 12,000. As a method of reaction, IFN: SC-PEG was mixed at a molar ratio of 1: 2 and reacted at pH 6.5 to react with one PEG-linked IFN (1-PEG-IFN) and two PEG-linked IFNs (2- A mixture of PEG-IFN) was obtained. The mixture was acid treated with trifluoroacetic acid to give 1-PEG-IFN. There is no explanation as to whether one of the two PEGs of 2-PEG-IFN is separated by acid treatment or whether only 2-PEC-IFN molecules are selectively degraded or treated. The bioactivity of 1-PEG-IFN thus obtained was examined by CPE (Cytophathic Effect) assay. The CPE assay used was a model in which A549 human lung cancer cells were infected with EMC virus. As a result, the bioactivity of 1-PEG-IFN was reduced by 71%.

According to US Pat. No. 5,985,265 (Nov. 16, 1999), PEG-binding reaction was attempted by targeting the N-terminal amino acid of IFN. The PEG derivative used at this time was PEG-aldehyde having a molecular weight of 12,000. This reaction produced a mixture, but PEG-IFN was isolated from the N-terminus by PEG. The bioactivity of PEG-IFN thus obtained was measured in a model testing the antiviral properties of human HeLa cells, where the bioactivity was found to be 80% reduced.

As shown in the above example, it was found that it is not easy to PEG bond while maintaining the bioactivity of IFN to the maximum. In all cases, the number of PEGs that could be bound per molecule of IFN was one. When two PEGs were combined, the activity was reduced so much that the number of PEGs was limited to one. Despite only one PEG bound, bioactivity was reduced by 60-80%.

In general, it is known that it is more advantageous to combine several PEGs than one PEG per molecule of a target protein to increase the effects of PEG, such as reduction of antigenicity and reduction of allergic reaction. However, when more than one PEG binds, the decrease in bioactivity was so severe that it could not be adopted.

The present invention is to solve the above problems, the advantage of the present invention over the known technology is that PEG can be attached to one or more PEG in bioactive proteins such as IFN so that the decrease in bioactivity is not significant It is to maximize the advantages that can be given.

When a large number of PEGs are combined, the site of antigenic expression on the protein surface can be covered so broadly that the likelihood of side effects related to the immune response is reduced. In addition, if a large number of PEG can be combined, the total molecular weight of the PEG is also increased by that amount, so the effect of pharmacological effects such as increase in the half-life of the body is improved. For example, if only one PEG is capable of binding, the total molecular weight increases by 10,000 if the molecular weight of the used PEG is 10,000, whereas if the molecular weight of the PEG used is 10,000, the total molecular weight is 10,000. That's an increase of 30,000.

As described above, if one or more PEGs can be combined, the pharmacological effect can be expected to be significantly improved than the conventional technology.

That is, an object of the present invention is to provide a method capable of binding a large number of PEGs to one molecule of bioactive protein while maintaining most of the bioactivity.

1 is a graph of HPLC analysis results according to Example 1,

2 is a graph of HPLC analysis results according to Example 2.

In order to achieve the above object, the present invention provides a method for preparing a conjugate of a bioactive protein and PEG having a high bioactivity, comprising the steps of: blocking the active site of the bioactive protein with a hydrolyzable blocking material; Binding a PEG derivative resistant to hydrolysis to a site other than an active site of the bioactive protein; It provides a method for producing a conjugate of a bioactive protein and PEG consisting of; hydrolyzing and removing the blocking material bound to the bioactive protein active site.

In the preparation method, the bioactive protein is interferon, G-CSF, interleukin, insulin, TNF, nub growth factor, growth factor, EGF, EFO, GM- It may be selected from the group consisting of CSF and the like.

In the above production method, the blocking substance is preferably a PEG derivative or a hydrolyzable sugar that is sensitive to hydrolysis.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The purpose of the present invention can be achieved through the configuration of the present invention is that the present invention induces PEG to be bound to a site other than the active site of the bioactive protein.

As mentioned above, the process of the present invention can be largely divided into three stages, and in the first stage, the amino acid forming the active site of IFN is bound to PEG or sugar by reacting a weakly reactive PEG derivative or sugar with IFN as the blocking substance. Induce. In the second step, the reactive PEG species are reacted again with the intermediate obtained in the first step to obtain a bioactive protein and PEG conjugate in the form of both weak PEG or sugar and strong PEG. In the third step, it is possible to obtain the desired bioactive protein and PEG conjugate, under hydrolysis conditions, inducing the weak PEG or sugar to hydrolyze off and consequently leaving only the strong PEG bound.

First of all, the use of the PEG reductant with weak reaction force as a blocking material will be described. First, the PEG reductant with low reaction force is first bound to the active site of the bioactive protein, and then the strong PEG derivative is bound to the weak PEG through hydrolysis. Deriving the derivative, the reason for this process was confirmed in the present invention that the active site of the protein in the reaction of the PEG derivative and the protein preferentially binds to the PEG, and thus to apply such fact usefully In order to react the PEG derivative with a weak reaction force first to preemptively occupy the active site.

As a next step, the PEG derivatives that are resistant to hydrolysis are almost permanently bound to the protein, and the sites where they react are out of the active site of the protein, so that at the last step, the weak PEG derivatives drop off the active site. Is again exposed to its original state to obtain a biologically active protein and a PEG conjugate, which are the final target substances of the present invention, which retain most of the biological activity.

The PEG derivative used in the first step is PEG-succinimidyl succinate (SS-PEG), which has a property of separating PEG through hydrolysis due to the presence of ester groups in the backbone. It is used in the invention.

The PEG derivative used in the second step is a kind of PEG derivative having no ester group in the backbone, and there is no fear of PEG separation. This property is used in the present invention. There are several types of PEG derivatives that can be used, for example PEG-succinamide (PEG-succinamide, SSA-PEG), PEG-succinimidyl propionate (SP-PEG), PEG- Succinimidyl carboxymethyl (PEG-succinimidyl carbonate, SC-PEG), and the like.

In another method of the present invention, a blocking material is used to block the active site using sugar. Maltose, trehalose, sialic acid, hyaluronic acid, and the like may be used as the sugar to be used. An appropriate amount of sugar is first reacted with a bioactive protein to block the active site, and the second step is to react with PEG derivatives that are resistant to hydrolysis, ie, SSA-PEG, SP-PEG, SCM-PEG, SC-PEG, etc. . And the third step is to separate the sugar blocking the active site of the biologically active protein through a hydrolysis process.

As described above, the present invention is unique in that the binding to PEG is performed while maintaining the bioactivity of the biopharmaceutical, which enables the first step of blocking the active site of the biopharmaceutical as a PEG derivative or a sugar. Such a concept would be universally used not only for bioactive proteins but also for many other types of biopharmaceuticals.

Other biologics include, for example, granulocyte-colony stimulating factor (G-CSF), erythropoietin (EFO), interleukin (IL), growth factor, insulin, and nerve growth factor. , IFN beta, IFN gamma, IFN omega, IFN tau, tumor necrosis factor (TNF), epidermal growth factor (EGF), and granulocyte macrophage-colony stimulating factor (GM-CSF).

Example 1.

Dissolve IFN alpha (40 mg) in 10 ml of sodium-phosphate (0.15 M, pH 7.8). The mixture is stirred at room temperature, and then, as the first step, the ratio of SSN-PEG (molecular weight 5,000) is added to the IFNalpha solution by molar ratio of IFN alpha: SS-PEG of 1: 3, and the stirring is continued. . The reaction proceeds for about two hours, then excess glycine is added and the reaction is quenched. A small amount of sample is taken here and this is called Sample A. Sample A performs a CPE assay for analysis via HPLC and measurement of bioactivity along with other samples. The solution is dialed out to separate the reacted SS-PEG and glycine.

As a next step, the amount of SSA-PEG (molecular weight 5,000) is added to the PEG-IFN solution already obtained by quantifying the amount of PEG-IFN: SSA-PEG by about 1: 5 and stirred. The reaction proceeds for about two hours, then excess glycine is added and the reaction is quenched. A small sample is taken here and this is called Sample B. The resulting solution is buffer exchanged through dialisis so that the pH of the final buffer is 5.5-6.5 and the concentration of sodium-phosphate is 0.5M. The PEG-IFN solution thus obtained is incubated at a temperature of 28-30 ° C. for 24 hours and stirred slowly. Afterwards, the buffer of PEG-IFN solution was adjusted to pH 7.0-7.5 and sodium-phosphate concentration of 0.15M by buffer exchange again. The sample is taken again and named sample C. For the purpose of this experiment, Sample A is a PEG-IFN solution with SS-PEG bound to hydrolysis, Sample B is a PEG-IFN solution with SSA-PEG bound to PEG-IFN with SS-PEG bound, Sample C is PEG-IFN in which SS-PEG is separated and separated through hydrolysis, and only SSA-PEG is bound to IFN. HPLC analysis of samples A, B and C is to estimate the number of bound PEG by measuring the approximate molecular weight using a size exclusion column.

HPLC analysis results are shown in the accompanying drawings, FIG. 1.

HPLC analysis showed that Sample A was a mixture of 1-PEG-IFN with one PEG bound and 2-PEG-IFN with two PEG bound, with the ratio 1-PEG-IFN: 2-PEG- IFN = 25%: 75%.

Sample B showed a mixture of 1-PEG-IFN, 2-PEG-IFN, 3-PEG-IFN, 4-PEG-IFN, with a ratio of 1-PEG-IFN: 2-PEG-IFN: 3 -PEG-IFN: 4-PEG-IFN = 2%: 15%: 48%: 35% was analyzed.

Sample C showed a mixture of 1-PEG-IFN, 2-PEG-IFN, 3-PEG-IFN, and the ratio was 1-PEG-IFN: 2-PEG-IFN: 3-PEG-IFN = It was analyzed as 32%: 47%: 21%.

This suggests that the reaction of SS-PEG and IFN proceeded in the first stage, and the reaction of SSA-PEG and PEG-IFN proceeded in the second stage, and in the third hydrolysis stage, the PEG was partially hydrolyzed. It was confirmed to fall off. The three samples were subjected to a Cytopathic Effect Assay to investigate bioactivity. The CPE assay used a standard method for measuring CPE assays using A549 human lung cancer cells infected with the EMC virus (Human Cytokines, Handbook for Basic Clinical Research, B. Aggarwal S. Gutterman, Blackwell Scientific Publications, 1991)

The CPE assay results of samples A, B and C were analyzed in comparison with the CPE assay results of IFN itself (original native IFN without PEG reaction). When the bioactivity of native IFN was 100%, the activity of sample A was 6%, the activity of sample B was 5%, and the activity of sample C was 46%. The above assay was repeated three times and then averaged. As a result, Sample A exhibited a 94% decrease in activity, which indirectly confirmed that the active site of IFN was blocked. Sample B showed a 95% decrease in activity, indicating that no further decrease in activity occurred. Sample C showed 46% activity, indicating that activity was significantly restored.

CPE Assay Results from PEG-IFN Samples division CPE Assay (% of control) Assay # 1 Assay # 2 Assay # 3 Average Sample A 5 8 4 6 Sample B 4 4 6 5 Sample C 45 54 39 46

Example 2

Dissolve IFN alpha (40 mg) in 10 ml of sodium-phosphate (0.15 M, pH 7.8). After stirring at room temperature, as the first step, the sialic acid (or N-acetylneuraminic acid) was quantified by the molar ratio of IFN: sialic acid in an amount of about 1:10 and then the solution described above. Mix and stir. The sample is taken here and is called sample D. After the reaction was performed for about 4 hours, as a second step, SSA-PEG (molecular weight 5,000) was quantified by the molar ratio of IFN: SSA-PEG in the amount of about 1: 5, mixed in the solution and stirred. The reaction is allowed to proceed for about 2 hours, then excess glycine is added and the reaction is quenched. Dialisis is then performed to isolate and purify the unreacted materials. The sample is taken again and this is called sample E. The PEG-IFN solution obtained here is buffer exchanged, and the final buffer has a pH of 4.5-6.5 and a concentration of sodium-phosphate of 0.5M. This buffer-exchanged PEG-IFN solution was incubated at a temperature of 25-30 ° C. for 24 hours and stirred slowly. Afterwards, the buffer of PEG-IFN solution was adjusted to pH 7.0-7.5 and sodium-phosphate concentration of 0.15M by buffer exchange again. The sample is taken again and named sample F. Samples D, E and F were subjected to HPLC analysis and CPE assay as in Example 1.

HPLC analysis results are shown in FIG. 2.

HPLC analysis showed that sample D showed an increase in molecular weight as much as the amount of sialic acid bound to IFN. Sample E showed a mixture of three substances with PEG bound to 1-PEG-IFN: 2-PEG-IFN: 3-PEG-IFN = 26%: 63%: 11%. It is assumed that sample F had separated sialic acid and PEG binding state showed no change with sample E.

According to the CPE assay, Sample D showed 21% activity, Sample E 17%, and Sample F 38%, indicating that activity was restored at the last step.

CPE Assay Results from PEG-IFN Samples division CPE Assay (% of control) Assay # 1 Assay # 2 Assay # 3 Average Sample D 18 25 21 21 Sample E 17 14 19 17 Sample F 35 42 41 39

As shown in Examples 1 and 2 above, the concept of protecting the active site in the next step of the PEG reaction by appropriately blocking the active site of the target protein as a previous step when PEG-reacting a bioactive protein such as IFN is effective. Proved that there is. At this time, it can be seen that it is effective to use a PEG derivative or a sugar sensitive to hydrolysis as a method that can be used as the active site blocking. When the above method was used, the bioactivity was maintained at a high level of 39-46%. As a feature of the present invention, the existing method maintains about 20 to 30% of activity even when only one PEG is bound, whereas the present invention is one-peg-IFN, 2-PEG by binding a large number of PEGs. Despite being present as a mixture of -IFN and 3-PEG-IFN, it was able to maintain a high activity of 39-46%.

In the method for preparing a conjugate of the bioactive protein and PEG of the present invention, the active site is blocked with a hydrolyzable PEG derivative or a sugar, and then the PEG derivative, which is resistant to hydrolysis, is bound to a non-active site. By hydrolysing and removing PEG derivatives or sugars, the bioactivity of the bioactive protein can be maintained high, and thus the pharmacological effect can be enhanced when applied to biopharmaceuticals.

Claims (7)

In the method for producing a conjugate of a bioactive protein and PEG having a high bioactivity, Blocking the active site of the bioactive protein with a hydrolyzable blocking material; Binding a PEG derivative resistant to hydrolysis to a site other than an active site of the bioactive protein; Hydrolyzing and removing the blocking material bound to the bioactive protein active site; a method for preparing a conjugate of a bioactive protein and PEG consisting of: The bioactive protein of claim 1, wherein the bioactive protein is interferon, G-CSF, interleukin, insulin, TNF, nucleus growth factor, growth factor, EGF, EFO, GM-. Method for producing a conjugate of a bioactive protein and PEG, characterized in that selected from the group consisting of CSF and the like. The method of claim 1, wherein the blocking material is a PEG-derived bioactive protein, characterized in that the PEG derivative is sensitive to hydrolysis. The method of claim 3, wherein the PEG derivative is PEG-succinimidyl succinate. The method of claim 1, wherein the blocking substance is a hydrolyzable sugar. The bioactive protein and PEG according to claim 5, wherein the sugar is selected from the group consisting of maltose, trehalose, sialic acid, hyaluronic acid, and the like. Method of preparation of the conjugate. The method of claim 1, wherein the PEG derivative is PEG-succinamide (PEG-succinimidyl propionate), PEG-succinimidyl carboxymethyl (PEG-succinimidyl carboxymethyl), PEG-succinimidyl carbonate (PEG-succinimidyl carbonate), etc. A method for preparing a conjugate of a bioactive protein and PEG, characterized in that selected from the group consisting of.