WO2005077421A1

WO2005077421A1 - A method of preparing polyethylene glycol modified interferon alpha 1b

Info

Publication number: WO2005077421A1
Application number: PCT/CN2005/000168
Authority: WO
Inventors: Xiantai Meng; Ying Peng; Piaoyang Sun
Original assignee: Jiangsu Hengrui Medicine Co., Ltd; Lianyungang Xinyang Pharmaceutical Co., Ltd.
Priority date: 2004-02-12
Filing date: 2005-02-06
Publication date: 2005-08-25
Also published as: CN100355784C; CN1654478A

Abstract

The invention related to a method of preparing pegylated interferon alpha 1b covalently bounding to actived polyethylene glycol. To achieve the modified interferon alpha 1b protein, where the histidine and lysine on the protein surface were selectively conjugated to PEG the pH value of reaction system was modulated. There were no needs to separate interferon alpha 1b material and intermediate products from reaction system, and the modified interferon alpha 1b still maintained biological activity of original interferon alpha 1b, and excels as pharmacology, immunogenicity, drug metabolism and drug efficiency.

Description

Preparation method of polyethylene glycol modified α-interferon lb Technical field

The invention relates to a novel and simple technology for chemical modification of biological protein macromolecule drugs, and the products obtained by the technology and the use of the products. This method can chemically modify specific amino acids on the surface of the protein by covalent bonding without affecting or reducing the biological activity of the biological protein macromolecule drug. This is a special method for the preparation of polyethylene glycol-biomacromolecules, especially for the preparation of polyethylene glycol modified α-interferon lb. Background technique

Generally speaking, many products (such as interferon, peptide, or protein biotechnology drugs) obtained through the expression of biotechnology (such as genetic recombination), or biomacromolecular pharmaceutical products obtained by natural extraction and purification are all parenteral Through the route of administration into the body to exert its pharmacological and pharmacological effects. Such biological macromolecular drug products for parenteral administration usually have the following problems: (1) Biological macromolecular drugs often have an allergic reaction, and antibodies produced in the body can cause serious damage and affect the organism. The progress of treatment; (2) The biological macromolecular drug itself is greatly shortened in its biological half-life due to the metabolic effects caused by antibodies or proteolytic enzymes; (3) The stability of biological macromolecules is poor and it is difficult to store.

At present, the above-mentioned problems can be solved by using some chemical modification technology of biological macromolecules. In 1980, Davis et al., In U.S. Patent No. 4,179,337, disclosed the chemical modification of protein drugs using a single molecule of polyethylene glycol with different degrees of polymerization. While maintaining the biological activity of the drug, the antigenicity of the drug was reduced. And its water solubility and the biological half-life of the drug are improved. Similar to the Davis patent, Verones et al. Published in Applied Biochem and Biotech 11, 142 (1985) published a polyethylene glycol modified ribonuclease and superoxide dismutase activated by phenyl chloropotassium ester, which increased protein Biological half-life. The above-mentioned prior art literature reports have shown that the polyethylene glycol modification technology can indeed solve some problems existing in parenteral administration of biological macromolecular drugs. However, some technical difficulties have arisen during the application of this technology, such as:

(1) The modification of polyethylene glycol to biological macromolecules is a non-selective chemical reaction: there are multiple reactive amino acids on the surface of the protein, and these reactive amino acids Both may react with polyethylene glycol. In the prior art, the modification of polyethylene glycol often cannot be modified for specific amino acids, which causes some amino acids that affect the activity of the protein to be modified by polyethylene glycol, which reduces the activity of the modified product or changes the activity of the protein.

(2) The chemical reaction of polyethylene glycol can only covalently bind polyethylene glycol with the same chain length in a single phase: Sometimes, a polyethylene glycol with a uniform chain length in a single phase cannot completely increase bioavailability and extend biological activity. The purpose of half-life. In addition, it has been proved that the binding product between a single polyethylene glycol with a molecular weight of 5000 and histidine on the protein surface is unstable, and the already formed polyethylene glycol-amino acid covalent bond will be rapidly broken. Biological macromolecular drugs have completely lost the advantages of polyethylene glycol modification (Wang et al., Advanced Drug Delivery Review 54, 547, 2002). At present, no report has been reported to improve or overcome the above-mentioned problems from the perspective of the modified biomacromolecule preparation process. According to the published technical literature reports, it can be known that the two main factors affecting the biological half-life and bioavailability of polyethylene glycol-protein biomacromolecules are: (1) the surface of the selected protein is modified by polyethylene glycol (2) Select the chain length, chain type, and degree of branching of polyethylene glycol.

Therefore, there is still a need to find a method that can specifically and selectively modify specific amino acids on a protein molecule, and that different types of amino acids on the surface of the protein can be modified using modifiers with different structures, respectively. .

In the present invention, the term "polyethylene glycol-biomacromolecule" means a product formed by the reaction of a specific group in a biological macromolecule (protein, nucleotide, etc.) and activated polyethylene glycol through covalent bonding. , Such as polyethylene glycol-α-interferon. In addition, PEGylated biomacromolecules (proteins), PEG-modified biomacromolecules (proteins), or PEG biomacromolecules (proteins) have the same meaning.

"Polyethylene glycol" is divided into linear and branched chains according to the polymerization method of the polymerization unit; according to the degree of polymerization, polyethylene glycols of different molecular weights can be separated, such as PEG 5000, PEG 8000, and PEG 20000. and many more.

The term "single-phase reaction" refers to the modification of biological macromolecules using polyethylene glycol with the same polymerization method and degree of polymerization (same molecular weight). The modification reaction can be completed at one time or repeatedly. The term "heterogeneous reaction" refers to the modification of biological macromolecules with polyethylene glycols with different polymerization methods or different degrees of polymerization (different molecular weights). The modification reaction requires at least two or more repeated reactions to complete.

"SS-PEG" means Succinimi dyl Succinate Polyethelene Glycol.

"SC-PEG" means Succinimide Carbonate Polyethylene Glycol. Summary of the invention

In order to overcome the shortcomings of the existing interferon-polyethylene glycol modification technology, the purpose of the present invention is to provide a new method for preparing polyethylene glycol-biomacromolecules and to prepare a brand new polyethylene glycol-06- Interferon lb. -In order to achieve the purpose of the present invention, the technical solution of the present invention is as follows-A method for preparing α-interferon lb modified by covalent bonding of polyethylene glycol, by adjusting the pH value in the reaction system, using different polymerization methods Activated linear polyethylene glycol with different polymer chain length performs specific chemical modification of different amino acids on the surface of α-interferon lb protein step by step. The activated polyethylene glycol is a succinic acid group of CI-C6. Polyethylene glycol ester, including the following two steps:

(A) At a pH of 5-7, contacting activated linear polyethylene glycol with α-interferon lb, reacting polyethylene glycol with α-interferon lb, and reacting with α-interferon lb protein Specific chemical modification of histidine on the surface;

(B) at a pH of 7-9, reacting the activated linear polyethylene glycol with ci-interferon lb to specifically chemically modify the lysine on the surface of the α-interferon lb protein;

The order of the above two steps (A) and (B) can be reversed, and the pH of the first reaction system can be adjusted to a pH suitable for the second reaction by using a pH adjuster between the two reactions.

In the above preparation method, the reaction pH in step (A) is preferably controlled between 5.5 to 6.5, preferably 6.0 to 6.5, and most preferably 6.0 ± 0.1; the pH of reaction in step (B) 0。 It is preferably controlled in the range of 7.5 to 8.5, preferably 8.0 to 8.5, and most preferably 8.0 ± 0.1.

Those skilled in the art can select the structure type, molecular weight, and amount required for the reaction of polyethylene glycol based on common sense. Through the preparation method provided by the present application, different kinds of A class of polyethylene glycol specifically modifies different amino acids on the surface of CL-interferon lb protein. When different activated polyethylene glycols used in the present invention react with α-interferon lb, different covalent bonds can be formed. The specific reactions are relatively independent reactions. Those skilled in the art can adjust the content of polyethylene glycol and the structure of polyethylene glycol by selecting the structure type, molecular weight, and amount required for the reaction of polyethylene glycol according to the needs. Other factors are used to control the structure and properties of the modified protein molecule. According to the requirements of the solubility, the biological half-life, and the bioavailability of the drug protein in clinical use, those skilled in the art can select the structure type, molecular weight, and amount required for reaction of polyethylene glycol by the method of the present invention. According to the research of the present invention, it is found that the activated linear polyethylene glycol used for modifying the interferon preferably has a molecular weight of 12,000 to 40,000 Daltons, preferably 12,000 to 25,000 Daltons, and most preferably 12 10,000 to 20,000 Daltons; the ratio of the amount of α-interferon lb to the activated linear polyethylene glycol in the reactions of the above steps can be selected from 1: 10-1: 30, preferably 1: 20-1: 30 , Most preferably 1:20. The application specifically provides the molecular weight of the activated linear polyethylene glycol used in step (A) is 12,000, the molecular weight of the activated linear polyethylene glycol used in step (B) is 20,000, and steps (A) and (B) A method in which the activated linear polyethylene glycols each have a molecular weight of 20,000. Those skilled in the art can understand that the choice of the structure type, molecular weight, and amount required for the reaction of polyethylene glycol is not limited to the above specific range.

The reaction temperature of the present application can be selected according to common knowledge of those skilled in the art. The ambient temperature of a protein modification reaction can be generally selected, preferably 25 to 30 ° C, especially 25 ± 0. Γ C.

The present invention also relates to a novel polyethylene glycol-α-interferon lb prepared by the above method of the present invention. After modification, the histidine and lysine on the surface protein of ct-interferon lb are respectively the same or different. The alkanoic acid polyethylene glycol ester of the imino CI-C6 is modified, and preferably the activated linear polyethylene glycol is SS-activated linear polyethylene glycol or SC-activated linear polyethylene glycol.

The present invention also relates to opportunistic infections caused by the above-mentioned novel polyethylene glycol-α-interferon lb prepared in the present invention for the prevention and treatment of infectious diseases and malignant tumor diseases such as infectious acute and chronic hepatitis and AIDS virus infection. Application of medicines for diseases, and polyethylene glycol modified α-interferon lb in preparation for treating malignant tumors including Hairy's cell leukemia, Kaposi tumor, non-Hawkins nose Application of drugs for pharyngeal malignant T-cell lymphoma or nasopharyngeal virus (Epstein Barr) lymphoma. Gather Glycol-modified - α- interferon lb clinically effective dose of at least 1.0 X 10 ° to 3.0 χΐθ "IU / square meter / 7 days ^{(1.0 X 10 6 to 3.0 X} 10 6 IU / m 2/7 day).

The present invention also provides a pharmaceutical composition containing a polyethylene glycol modified a-interferon lb prepared according to the method of the present invention, and a pharmaceutically acceptable carrier. This application specifically gives the following two preferred technical solutions:

1. Modification reaction based on Succinimidyl Succinate Polyethelene Glycol (SS-PEG), or SS-activated linear polyethylene glycol.

(1) Chemical modification of the covalent bond of histidine on the surface of α-interferon protein: contacting activated linear polyethylene glycol (SS-PEG) with ex-interferon, the length of the linear chain of polyethylene glycol is 12000 To 20,000 Daltons, the dosage ratio (w / w) of the reaction between polyethylene glycol and α-interferon ranges from 1: 30, and the preferred dosage ratio is 1:20. The pH of the reaction is 6.0-6.5.

(2) Chemical modification of covalent bond of lysine on α-interferon protein surface: contacting activated linear polyethylene glycol (SS-PEG) with α-interferon, the reaction uses the length range of the linear chain of polyethylene glycol For 12,000-20,000 Daltons, the ratio of α-interferon to polyethylene glycol (w

/ w) The range is 1: 10-1: 30, the preferred dosage ratio is 1:20, and the pH of the reaction is 8.0

-8.5.

In said step (1), the preferred reaction uses a polyethylene glycol linear chain with a length of 20,000 Daltons. In step (1), the preferred ratio of α-interferon to polyethylene glycol is 1:20. In step (2), the preferred length of the ethylene glycol linear chain is 20,000 Daltons. The preferred ratio of a-interferon to polyethylene glycol is 1:20.

The ρΗ range of the reaction in step (1) should be controlled to 6.0 to 6.5, preferably 6.0 ± 0.1; the ρΗ range of the PEG reaction in step (2) should be controlled to 8.0-8.5, preferably 8.0 ± 0.1. The pH of the reaction solution can be adjusted by adding a 10-fold concentration of sodium phosphate buffer. Γ C。 The temperature range of the reaction of step (1) and (2) should be controlled to 25 to 30 ° C, and the preferred temperature is controlled to 25 ± 0. Γ C. '' Second, based on succinimide polyethylene glycol carbonate (SC-PEG) (Succinimide Carbonate Polyethylene Glycol (SC-PEG), called SC-activated linear polyethylene glycol modification reaction.

The step (1) of SC-activated polyethylene glycol modification of biological macromolecular protein is to control the pH of the reaction to optimize the pH between 6.0 and 1.0 to make histamine on the surface of α-interferon biomacromolecular protein. The acid can fully react with the activated polyethylene glycol to obtain a polyethylene glycol α-interferon modified product with a specific molecular weight (specific chain length). SC-activated linear polyethylene glycol (SC-PEG) with high molecular weight and a narrow unidirectional distribution (molecular weight distribution between 1.0 and 1.05) with an average molecular weight of 20,000 is used for α-interferon polyorganisms. The molecular protein is further modified to obtain a polyethylene glycol-modified α-interferon product. The PEGylated macromolecules formed through amide bonds maintain relative stability and biological activity, and will not break under normal laboratory conditions. After the reaction, the unreacted raw materials or other reaction by-products in the solution can be removed, or the pH of the reaction can be changed for the next chemical reaction.

Step (2) The polyethylene glycol chemical reaction is the most important control step of the present invention. Without the need for purification and separation of proteins and other intermediates in the chemical reaction, the addition of a 10-fold concentration of sodium phosphate buffer solution will rapidly change the pH of the reaction and reach the optimal reaction pH value to 8. 0 ± 0.1. Then add SC-activated linear polyethylene glycol (SC-PEG) to polyethylene glycol α- with high purity, narrow unidirectional distribution (molecular weight distribution between 1.01-1.05) and average molecular weight of 20,000. Interferon is further modified to obtain highly reactive but stable alpha-interferon protein products.

In the step (2) reaction, an activated polyethylene glycol having the same molecular weight as that in the step (1) can be used to continue the reaction with the α-interferon biomacromolecule protein, and finally a single molecular weight (single chain length) polyethylene is obtained. Glycol modified products. In addition, the α-interferon biomacromolecule protein can be further modified by using activated polyethylene glycol with a completely different molecular weight or different polymerization method from the reaction in step (1), and finally a mixed polyethylene glycol modified α can be obtained. -Interferon production

The new α-interferon lb (INFalb) prepared by long-chain PEG modification using the polyethylene glycol covalent binding modification method of the present invention has a molecular weight different from that of a-interferon lb, and exhibits a completely different SDS-PAGE measures behavior and has a specifically extended biological half-life and significantly increased bioavailability.

And currently reported pegylated a-interferon 2a (PEG-INFa2a) and polyethylene Glycolated α-Interferon 2b (PEG-INFa2b) Compared with PEG-INFa2b, the novel PEGylated a-interferon lb (PEG-INFalb) obtained by the present invention is chemically organized in composition and molecular weight with PEG-INFa2a and PEG- The two protein compounds of INFa2b are absolutely different. Therefore, the above three PEG proteins should be regarded as completely different compounds. By using a-interferon lb, a preferred unbranched long-chain polyethylene glycol and the polyethylene glycol a-interferon lb obtained by the preparation process of this patent, the polyethylene glycol chain length is much longer than that of a-interferon 2a ( PEG-INFa2a). Therefore, the biological half-life and bioavailability of the new polyethylene glycol α-interferon lb will be better than similar PEG-INFa2a.

From the biological activity of interferon, the new pegylated α_interferon lb obtained by using this method has a mild effect on different amino acids on the protein surface in the PEG chemical reaction. The activity has not been largely inactivated by chemical action. Compared with other interferon proteins, this method and the novel pegylated α-interferon lb (PEG-INFalb) obtained by the present invention and other interferon pharmaceutical preparations obtained by other methods, including a-interferon 2a (PEG -INFa2a) and PEG-INFa2b (PEG-INFa2b) are far superior to other similar PEG-modified protein products in relative biological half-life and bioavailability.

Polyethylene glycol modified a-interferon lb can be made into corresponding pharmaceutical dosage forms for powders, liquids, suspensions and other medicinal products obtained through various chemically or non-chemically modified reagents, carriers and methods of administration. Dosage forms are for subcutaneous, intradermal, intermembrane, suppository and aerosol administration.

It is found through research that the succinimide-substituted CI-C6 alkanoate polyethylene glycol ester has a very good specific modification function at a specific pH value. The present invention uses a succinimide-substituted CI-C6 alkanoate polyethylene glycol ester as a modifier, and the method of modifying site transfer through a change in pH can be used for a new or improved polyethylene glycol-biological Production of molecular drugs. The traditional preparation method of polyethylene glycol-biomacromolecules is to use a uniform molecular weight of polyethylene glycol and biomacromolecules as a main preparation process through a single chemical reaction. According to the characteristics of the modified biological macromolecule, the present invention can use single molecular weight or different molecular weight polyethylene glycol and polyethylene glycol with different substituent groups to perform different ways and different degrees of surface amino acids of biological macromolecules such as proteins. Modification to extend the biologically active half-life of biological macromolecular drugs, and to obtain the expected therapeutic effect under various administration conditions, especially parenteral administration conditions, so the development of polyethylene glycol modified a-interferon therapeutic drugs, It is of great significance to meet the urgent needs of clinical treatment. The method of the present invention uses a chemical reaction to simplify the chemical reaction process between a biological macromolecular protein and a polyethylene glycol, and does not require intermediate purification and separation for a multi-step reaction, so that the reaction is performed under optimized conditions and effectively controlled to overcome The shortcomings of the current modification of protein molecules are not strong, making polyethylene glycol more targeted modification of specific amino acids on protein molecules. By adjusting the acid-base environment in the reaction kettle, the reaction proceeds directly, avoiding the intermediate steps (such as separation, purification, etc.) usually existing in the reaction, simplifying the synthetic steps, and thus providing a biologically improved product in the preparation process. Loss of activity and ways to increase product yield make the entire process easier to implement on a large scale in industry. The new compound retains the original biological activity of α-interferon lb, but the new compound is superior to α-interferon lb in pharmacology, immunogenicity and pharmacodynamics; and it is also superior in terms of stability and biological activity. Existing single-phase or non-specifically modified (X-interferon lb o

The pH value and reaction temperature of the present invention can be considered based on the stability of the α-interferon lb and the acceptability of the reaction results. The results are measured by the degree of retention of biological activity (see results listed in Tables 1 and 2 below). BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is obtained by SDS-polyacrylamide gel electrophoresis analysis technology using activated polyethylene glycol SS-PEG of the same or different molecular weights, modified in two steps to modify α-interferon lb (INF a lb). Protein molecule products.

among them-

1. Gel band molecular weight (200 KD, 116. 3 KD, 66.3 KD, 55.4 KD, 36. 5 D, 31.0 KD, 21. 5 KD, 14. 4 KD, 6. 0 KD) Labeled protein.

2. Unpegylated α-Interferon Standard (INF ci lb).

3. Alpha-interferon (INF a lb-PEG12pH6) after modification with activated polyethylene glycol 12000 (SS-PEG) under pH 6.0 reaction conditions.

4. A-interferon (INF a lb_PEG20pH6) after modification with activated polyethylene glycol 20000 (SS-PEG) at pH 6.0.

5. A-interferon (INF a lb-PEG12pH8) after modification with activated polyethylene glycol 12000 (SS-PEG) under pH 8. 0 reaction conditions.

6. Modified with activated polyethylene glycol 20000 (SS-PEG) at pH 8. 0 Alpha-interferon (INF a lb-PEG20pH8).

7. Activated polyethylene glycol 12000 (SS-PEG) reacted at pH 6.0 for the first reaction and modified a-interferon (INF a lb-PEG12 pH6, pH8).

8. Activated polyethylene glycol 20000 (SS-PEG) reacts at pH 6.0 for the first reaction and a-interferon (INF a lb-PEG20 pH6, modified at pH 8. 0 for the second reaction). pH8). Figure 2 is a SDS-polyacrylamide gel electrophoresis analysis technique to analyze the protein molecule products obtained by modifying a-interferon lb (INF (i lb) with activated polyethylene glycol SC-PEG).

1. Gel band molecular weight (200 KD, 116. 3 KD, 66. 3 KD, 55.4 KD, 36.5 KD, 31.0 KD, 21. 5 KD, 14. 4 KD, 6. 0 KD) Labeled protein.

2. Unpegylated a-interferon standard (INF a lb).

3. A-interferon (INF a lb-PEG20pH6) after modification with activated polyethylene glycol 20000 (SC-PEG) under pH 6.0 reaction conditions. Figure 3 is a SDS-polyacrylamide gel electrophoresis analysis technology to analyze the protein molecules obtained by using activated polyethylene glycol SC-PEG and modifying a-interferon lb (INF a lb) in one or two steps. product.

among them

1. Gel band molecular weight (200 KD, 116. 3 KD, 66.3 KD, 55.4 KD, 36.5 KD, 31.0 KD, 21. 5 D, 14. 4 KD, 6. 0 KD) Labeled protein.

2. Unpegylated a-interferon lb standard (INF a lb).

3. A-interferon (INF a lb-PEG20pH6) modified by activated polyethylene glycol 20000 (SC-PEG) under pH 6.0 reaction conditions.

4. A-interferon (INF a lb-PEG20pH8) modified by activated polyethylene glycol 20000 (SC-PEG) under pH 8 · 0 reaction conditions.

5. Modified a-interferon (INF a lb-PEG20pH6) modified with activated polyethylene glycol 20000 (SC-PEG) under the first reaction condition at pH 6.0 and the second reaction at pH 8. 0. , pH8). detailed description

Hereinafter, the present invention will be described in detail with reference to the examples, but the examples are only for the purpose of illustration, but not to limit the present invention. Example 1 Preparation of polyethylene glycol 12000/20000 α-interferon lb

Step (1) Reaction

Take 1.0 mg of oc-interferon and dissolve it in 1 ml of 100 mM (pH 6.0) sodium phosphate buffer. Take 20 mg of SS-activated linear polyethylene glycol (SS-PEG12000) with a molecular weight of 12,000, quickly add it to the above solution, and shake it appropriately to completely dissolve the activated linear polyethylene glycol (SS-PEG12000) within 30 seconds. The reaction was carried out at 25 ° C for 60 minutes. The reaction solution can be further purified or subjected to the second step without having to be concentrated and washed.

The pH range of the reaction in step (1) is controlled between 6.0 and 6.5.

Step (2) Reaction

To the solution obtained in the above step 1, 0.1 ml, 500 mM, (pH 8. 0) sodium phosphate buffer was added. Place this solution in a water bath to preheat to 25 ° C, take another 20 mg of molecular weight 20000 activated polyethylene glycol (SS-PEG20000) and quickly add to the above solution to stir appropriately to make molecular weight 20000 activated polyethylene glycol completely within 30 seconds Dissolve. The reaction lasted 30 minutes. The reaction solution was concentrated by centrifugation. After separation, purification and aseptic filtration, the final product of pegylated α-interferon lb (α-interferon-PEG12000 I 20, 000) was obtained and stored at 4 ° C. in a sealed, sterile, dark place .

The pH range of the SS-activated linear polyethylene glycol chemical reaction in step (2) should be controlled between 8. 0 and 8. 5. Example 2 Preparation of polyethylene glycol 20000/20000 alpha-interferon lb

The same procedure as in Example 1 was used to prepare polyethylene glycol 20000/2000 α-interferon lb, except that in the first step, SS-activation with a linear length ranging from 30 mg to 20,000 Daltons The linear polyethylene glycol is contacted with 1 mg of α-interferon lb, and the reaction uses the length of the linear chain of polyethylene glycol, that is, the mass ratio of α-interferon lb to polyethylene glycol ranges from 1:30.

In the second step, SS-activated linear polyethylene glycol with a linear length ranging from 30 mg to 20,000 Daltons was contacted with 1 mg of α-interferon lb. The length, that is, the mass ratio of polyethylene glycol to α-interferon lb ranges from 1 ·· 30. Example 3 Preparation of polyethylene glycol 12000/20000 α-interferon lb

Step (1) Reaction

Take 1.0 mg of interferon-lb in 1 ml of 100 mM (pH 6.0) sodium phosphate buffer. Take 30 milligrams of SC-activated linear polyethylene glycol (SC-PEG12000) with a molecular weight of 12,000, quickly add to the above solution, and shake it appropriately to completely dissolve the activated linear polyethylene glycol (SC-PEG12000) within 30 seconds. The reaction is carried out at 25 ° C for 60 minutes. The reaction solution can be further purified or subjected to a second reaction step without concentrating and washing.

The pH range of the reaction in step (1) is controlled between 6.0 and 6.5.

Step (2) Reaction

To the solution obtained in the above step 1, 0.1 ml, 500 mM, (pH 8. 0) sodium phosphate buffer was added. Preheat this solution to 25 ° C in a water bath, take another 20 mg of molecular weight 20000 activated polyethylene glycol (SC-PEG20000) and quickly add to the above solution to stir appropriately to make molecular weight 20000 activated polyethylene glycol completely within 30 seconds. Dissolve. The reaction lasted 30 minutes. The reaction solution was concentrated by centrifugation, separated, purified, and sterile filtered to obtain the final product of pegylated o-interferon (α-interferon-PEG12000 / 20,000), and stored at 4 ° C. in a sealed, sterile, and protected from light.

The pH range of the SC-activated linear polyethylene glycol chemical reaction in step (2) should be controlled between 8. 0 and 8. 5. The test samples used in the following experimental methods were obtained in Example 1. Experimental example 4

Analysis method: protein concentration analysis

Method 1: The Bradford-Lowry method was used to determine the protein concentration. This method determines the protein concentration based on the amount of Coomassie Brilliant Blue (protein dye) bound to the protein. First, draw a standard curve based on the known amount of dyes bound to the standard protein at different concentrations, and infer the protein sample concentration by measuring the dye's bound amount of the protein.

Method 2: Take the test solution of α-interferon lb activity measurement, according to spectrophotometry (Chinese Pharmacopoeia 1995 edition Appendix II page 20) Measure the absorbance at the wavelengths of 280 ± 1 nm and 260 1 nm, and calculate the milligrams of protein contained in each ml of solution according to the following formula.

Protein content (mg I ml) 1. 55 x D _{280 nm} — 0.76 x D ₂₆ Experimental example 5 Analysis of protein purity

Biological macromolecules, such as proteins, enzymes, peptides, and amino acids, have different moving directions and mobilities under the same electric field condition due to different charging properties. The more charged and the smaller the molecular weight, the faster the electrophoresis speed. Therefore, SDS-polyacrylamide gel electrophoresis can separate biological macromolecules such as different molecular weights and different types of proteins. Therefore, this method can determine the purity of unknown proteins and the size of protein molecules while determining the presence of heteroproteins. Experimental Example 6 Purification of ct-interferon lb modified by polyethylene glycol by high-pressure liquid phase separation method Single or double molecule (^ _interferon lb can be modified by molecular sieve depending on the molecular weight of the protein) Separation and purification by high pressure liquid chromatography. Molecular sieve column-200 can be purchased from Pharmacia Co. The mobile phase consists of 100 mM sodium phosphate buffer (pH 5.0) physiological saline (150 mM sodium chloride). Mobile phase flow rate 0 5 ml / min. The separation process can be monitored by a UV detector at 214 nm. Experimental Example 7 Determination of the biological activity of ot-interferon lb-polyethylene glycol

The biological activity of interferon can be determined by the color response of the host cell pathogenic effect test. Host cell pathogenic effect (CytoPathic Effect, or CPE) test method was proposed by Foti (Methods in Enzymology, 119, 533, 198). This method is designed as a simple and quick color absorption response test method based on the principle that human host cells are treated with interferon to produce anti-virus infection activity and prevent cytopathic death. In simple terms, host cells are resistant to virus invasion after interferon treatment and thus prevent host cell death. The antiviral biological activity of interferon can be directly and quantitatively measured by the dye absorbed by the living cells. In a specific test, first, based on the known antiviral pathogenic effects of host cell dyes produced by standard bioactive interferon proteins of different concentrations, draw a standard titer curve for dyes absorbed by surviving host cells. Α-interferon modified by unknown polyethylene glycol The lb protein titer can be directly and quantitatively determined by the dye absorbed by the antiviral pathogenic effect produced by the host cell. Experimental Example 8

Results of PEGylated ex-interferon protein

A. SDS-polyacrylamide gel electrophoresis protein analysis

Two methods are commonly used to evaluate the effect of polyethylene glycol on chemical modification of amino acids on the surface of proteins. The first method is to use SDS-polyacrylamide gel electrophoresis to determine the degree of modification by measuring the moving rate of the modified protein in an electric field. The second method is to directly determine the difference between the number of amino acids before and after the protein is modified by polyethylene glycol, and directly analyze. Alpha-interferon lb modified by polyethylene glycol chemical reaction is shown in Figure 1. SDS-polyacrylamide gel electrophoresis results show that: Alpha-interferon lb is modified by polyethylene glycol to significantly increase its molecular weight, which results in modification. The subsequent protein moves at a reduced speed in the electrophoretic field. The significant increase in molecular weight is due to the covalent binding of amino acids on the surface of e-interferon lb.

The results of gel electrophoresis in Figure 1 also show another experimental result, that is, the comparison of the movement rate of α-interferon lb protein in the electrophoresis field. Under the same reaction conditions, α-interferon lb has an activated molecular weight of 20,000 polyethylene. After the chemical modification of diol (SS-PEG), compared with the moving speed of the protein after chemical modification using molecular weight 12,000 polyethylene glycol (SS-PEG modification), the electrophoretic rate of the former protein is The increased result is significantly lower than the latter (compare items 4 and 3 in Figure 1). Experimental Example 9

Activity analysis of pegylated α-interferon lb

The activity of pegylated α-interferon lb is indicated by the data listed in Table 1. The experimental data in Table 1 also show that the two-step reaction can be used to finally achieve the purpose of chemically modifying α_interferon lb with activated polyethylene glycol (SS-PEG) with a molecular weight of 12,000 and 20,000 without causing a large amount of α- Interferon lb loses interferon activity during the chemical reaction.

Judging from the antiviral pathogenic effect (CPE) data, a-interferon lb was chemically modified in a weakly acidic (pH 6.0) environment to be far more active than in a slightly alkaline (pH 8.0) environment. Chemistry The product obtained by the modification is highly active (compare Table 1, columns 4, 5; columns 6, 7). At the same time, under the same pH chemical modification environment, the antiviral activity of products with higher molecular weight (SS-PEG 22,000) is higher than that of chemical products with lower molecular weight (SS-PEG12,000) (compared with Table 1, columns 4, 6; columns 5, 7). From the experimental data in Table 1, it is known that during the modification of interferon chemical reaction, the activity of the product obtained by chemical modification is not only affected by the transfer of modification sites caused by changes in pH, but also by the use of activated polyethylene glycols of different molecular weights. influences.

Table 1 Polyethylene glycol α-interferon lb (PEG-INF a _lb ) activity relationship

The experimental data in Table 2 also show that the use of activated SC-PEG polyethylene glycol with a molecular weight of 20,000 Daltons can also achieve the purpose of modifying the surface amino acids of a-interferon protein without causing a large amount of a-interferon lb Loss of interferon biological activity during chemical reactions.

Table 2 Activity relationship of polyethylene glycol a-interferon lb (SC-PEG-INF a lb) Product Viral pathogenic effect (CPE) titer x 10 "item ICso (ng / mL)

1. Standard a-interferon 2b (INF a 2b) 0. 03 219

2. Standard a-interferon lb (INF a lb) 0. 11 65

3. Standard a-interferon 2a (INF a 2a) 0. 04 171

4. c Interferon INF a lb- PEG20 pH6 1. 5 4. 6

5. a-Interferon INF a lb- PEG20 pH8 1. 8 3. 8

6. a-interferon INF a lb- PEG20 pH6, 8 1. 5 4. 6

Claims

Claim

1. A method for preparing co-bonded polyethylene glycol modified α-interferon lb, by adjusting the _PH value in the reaction system of polyethylene glycol and α-interferon lb, using different polymerization methods or different Activated linear polyethylene glycol with length of polymerized chain to specifically chemically modify different amino acids on the surface of α-interferon lb protein. The activated polyethylene glycol is succinimyl CI-C6 alkanoic acid polyethylene Glycol esters, including the following two steps:

(A) contacting the activated linear polyethylene glycol with α-interferon lb at a pH value of 5-7, reacting the linear polyethylene glycol with α-interferon lb, and reacting with α-interferon lb Specific chemical modification of histidine on the protein surface;

(B) contacting the activated linear polyethylene glycol with α-interferon lb at a pH value of 7-9 to specifically chemically modify lysine on the surface of the α-interferon lb protein;

2. The preparation method according to claim 1, characterized in that the pH range of the reaction in step (A) is controlled to 5.5 to 6.5; the pH range of the reaction in step (B) should be controlled to 7.5 to 8. 5.

3. The preparation method according to claim 2, characterized in that the pH range of the reaction in step (A) is controlled at 6.0 ± 0.1, and the pH range of the reaction in step (B) is controlled at 8. 0 ± 0.1.

4. The method according to claim 1, 2 or 3, characterized in that the molecular weight of the activated linear polyethylene glycol linear chain is 12,000 to 40,000 Daltons.

5. The method according to claim 4, wherein the molecular weight of the activated linear polyethylene glycol is 12,000 to 25,000 Daltons. 5. The method according to claim 5, wherein the molecular weight of the activated linear polyethylene glycol is 12,000 to 20,000 Daltons. The preparation method according to claim 1, 2 or 3, wherein the ratio of the amount of ct-interferon lb to the activated linear polyethylene glycol in steps (A) and (B) is 1: 10-1: 30. 7. The method according to claim, wherein the ratio of the amount of α-interferon lb reacted with the activated linear polyethylene glycol in steps (A) and (B) is 1: 20-1: 30. A method according to claim 8, characterized in that in steps (A) and (B)

-The ratio of interferon lb to activated linear polyethylene glycol is 1:20. 0. The method according to claim 1, characterized in that the two-step reaction is a relatively independent and separate reaction, and the molecular weight of the activated linear polyethylene glycol used in step (A) is 12,000, and that used in step (B) The activated linear polyethylene glycol has a molecular weight of 20,000, and the resulting product activates different types of amino acids on the surface of the linear polyethylene glycol modified α-interferon lb protein macromolecules, respectively, having 12,000 and 20,000. Road

'Dalton polyethylene glycol chain-length molecules. 1. The method according to claim 1, wherein the activated linear polyethylene glycols used in steps (A) and (B) each have a molecular weight of 20,000, and the reactions are relatively independent reactions. 2. The method according to claim 1, wherein the activated linear polyethylene glycol is SS-activated linear polyethylene glycol. 3. The method according to claim 1, wherein the activated linear polyethylene glycol is SC-activated linear polyethylene glycol. 4. The method according to claim 1, characterized in that step (A) and step (B) The temperature range of the step reaction should be controlled between 25 and 30 ° (:.

15. The method according to claim 14, characterized in that the reaction temperature range of steps (A) and (B) is controlled to 25 ± 0. Γ Co

16. The method according to claim 1, comprising the following specific steps: Chemically modifying α-interferon lb by using SS-activated polyethylene glycol with different polymerization methods or different polymerization chain lengths, characterized in that: It includes the following steps:

(A1) The SS-activated linear polyethylene glycol is contacted with ct-interferon lb, and the molecular weight of the linear chain of polyethylene glycol is 12,000 to 20,000 Daltons, and α-interferon lb and polyethylene are used. The mass ratio of diol is from 1: 10 to 1: 30;

(B1) After changing the pH of the reaction, contacting SS-activated linear polyethylene glycol with ct-interferon lb for a second reaction contact, the length of the linear chain of SS-activated polyethylene glycol ranges from 12,000 to 20, 000 Daltons, the mass ratio of α-interferon lb to SS-activated polyethylene glycol ranges from 1: 10 to 1: 30.

17. The method according to claim 1, comprising the following specific steps: Chemical modification of α-interferon lb by SC-activated linear polyethylene glycol with different polymerization methods or different polymerization chain lengths:

(A2) The SC-activated linear polyethylene glycol is contacted with α-interferon lb. The reaction uses SC-activated linear polyethylene glycol with a linear chain length of 12,000 to 20,000 Daltons. Ct -interferon The dosage ratio of lb to SC-activated linear polyethylene glycol ranges from 1: 10 to 1: 30;

(B2) After changing the pH of the reaction, the SC-activated linear polyethylene glycol is contacted with ci-interferon lb for a second reaction. The length of the linear chain of SC-activated linear polyethylene glycol is 12,000-20, 000 Daltons, the dosage ratio of the reaction between α-interferon lb and activated polyethylene glycol ranges from 1: 10 to 1: 30. 8. The preparation method according to claim 16 or 17, characterized in that the steps (A1),

(A2) The pH range of the reaction is controlled to be 5.5 to 6.5; the pH range of the steps (Bl) and (B2) should be controlled to be 7.5 to 8.5. The method according to claim 17 or 18, characterized in that the mass ratio of the reaction between α-interferon lb and activated linear polyethylene glycol in each step ranges from 1:20. 2. The polyethylene glycol-modified α-interferon lb prepared by the method of claims 1-19, and the modified histidine and lysine on the surface protein of the α-interferon lb are modified by the same or different activated linear polymers, respectively. Glycol modification, characterized in that the activated linear polyethylene glycol is a succinyl-based CI-C6 alkanoate polyethylene glycol ester. 2. The polyethylene glycol-modified α-interferon lb according to claim 20, wherein the activated linear polyethylene glycol is SS-activated linear polyethylene glycol. 2. The polyethylene glycol-modified α-interferon lb according to claim 20, wherein the activated linear polyethylene glycol is SC-activated linear polyethylene glycol. The use of polyethylene glycol-modified α-interferon lb according to claim 20 in the preparation of a medicament for preventing and treating infectious diseases and malignant tumor diseases such as infectious acute and chronic hepatitis, AIDS virus The opportunity to treat infections infects infectious diseases and treats malignant tumors. The use according to claim 23, characterized in that the malignant tumor is Harley's cell leukemia, Kaposi tumor, non-Hawkins nasopharyngeal malignant T cell lymphoma or nasopharyngeal virus lymphoma. 0. The application according to claim 23 or 24, characterized in that the polyethylene glycol modified α-interferon lb clinically effective drug dose is at least 1.0 X 10 ^fi to 3.0 X 10 ⁶ International Unit / square meter body surface area / 7 days. A pharmaceutical composition comprising the polyethylene glycol-modified α-interferon lb according to claim 20 and a pharmaceutically acceptable carrier.