CN103113466B - Recombinant human interferon beta-1b modified by polyethylene glycol and preparation method of recombinant human interferon beta-1b - Google Patents

Abstract

The invention relates to polyethylene glycol-modified recombinant human interferon beta-1b (IFN-beta-1b) and a preparation method thereof. By oxidizing the nitrogen-terminal serine (Ser) of recombinant human interferon β-1b into an aldehyde group, and then reacting with polyethylene glycol derivatives (PEG-NH ₂ , PEG-O-NH ₂ , PEG-HZ) was covalently coupled to prepare the nitrogen-terminal polyethylene glycol site-specific modification product of recombinant human interferon β-1b. The polyethylene glycol modified recombinant human interferon beta-1b of the present invention still has the physiological activity of recombinant human interferon beta-1b, but is superior to the original protein in terms of immunogenicity, pharmacology, pharmacokinetics and drug efficacy.

Description

Recombinant human interferon beta-1b modified with polyethylene glycol and its preparation method

technical field

The invention belongs to the field of protein chemistry, and relates to a polyethylene glycol-modified protein and a preparation method, in particular to a polyethylene glycol-point-modified product of the nitrogen terminal of recombinant human interferon beta-1b and a preparation method.

Background technique

With the development of biotechnology, biomacromolecular therapeutic drugs such as peptides and proteins are emerging, and have been more and more widely used in the treatment of diseases. Protein and peptide drugs have the advantages of high activity, low toxicity, clear biological function and strong specificity. However, there are also disadvantages such as easy hydrolysis by enzymes, short circulation half-life, high immunogenicity and low solubility. Therefore, the development of safe, effective, and highly bioavailable drug delivery systems is of great significance for improving the clinical effectiveness of protein drugs. After more than 40 years of development, the polyethylene glycol modification technology of proteins has become a mature technology for improving the physiological properties of proteins, which can be used to solve or alleviate many problems in the process of pharmaceutical use of proteins and peptides. Compared with the original protein, the protein covalently coupled with polyethylene glycol has weaker immunogenicity and longer half-life in vivo, which can significantly improve the pharmacological and pharmacokinetic properties of the drug. So far, a variety of polyethylene glycol-modified drugs have passed FDA certification [Alconcel, S.N.S., Baas, A.S., Maynard H.D., et al. FDA-approved poly(ethylene glycol) protein conjugate drugs.Polymer Chemistry, 2011,2: 1442–1448.].

For polyethylene glycol modification of proteins, the usual modification sites are the epsilon amino group of lysine residues in the protein sequence, the amino group at the N-terminal, and the sulfhydryl group of cysteine. Since there are relatively many free amino groups in the protein structure, a variety of modified products will appear in the modification of the amino group, even if the single modified product is obtained through separation and purification, there may be isomers. The sulfhydryl modification has higher requirements on the protein itself, which requires the protein itself to have free sulfhydryl groups or be introduced by genetic engineering methods. For the PEG modification of proteins, site-directed single modification is a development trend, because such a modification strategy produces a uniform modified product, the separation and purification of the product is greatly simplified, and it is beneficial to the activity of protein drugs reserve. Specifically for proteins whose nitrogen terminal is serine, a highly specific oxidation method can be used to derivatize the protein N-terminal serine into an aldehyde group, and covalently couple with an activated polyethylene glycol whose terminal group can react with the aldehyde group to achieve this The purpose of site-directed modification of protein-like N-terminus.

Human interferon (IFN) is a cytokine protein drug produced by activated fibroblasts and certain epithelial cells, which has antiviral infection, antitumor and strong immune regulation effects. Most of the interferons in the application have short half-lives and strong immunogenicity. In response to these problems, Pegintron (PEG-IFN-α-2b) and Pegasys (PEG-IFN-α-2a), the polyethylene glycol-modified products of interferon α, were approved by the FDA and launched in 2000 and 2002, respectively. Compared with the original protein, PEG-modified interferon α has a longer half-life and better pharmacology and pharmacokinetic properties, showing great medicinal advantages in clinical practice [Yagyu, H., Okada, K., Sato, S., et al. Pegylated interferon-α2b and ribavirin combination therapy induces Hashitoxicosis followed by type 1 diabetes mellitus. Diabetes research and clinical practice, 2012, 95:52-54.]. It has been proven that interferon beta can reduce the number of relapses of multiple sclerosis, prolong the interval of relapses, reduce the degree of pain and disability, delay the occurrence of disability, and improve the histopathological changes of the central nervous system brain and spinal cord, although it also exists in the body Short half-life, immunogenicity, certain side effects, etc., but because it is a strongly hydrophobic protein, it needs to add a solubilizer as an auxiliary agent for dissolution in water, and it is easy to form aggregates in the solution state, which leads to its analysis and quantification. There are difficulties, and the separation and purification of products after PEG modification is also difficult to carry out effectively due to the existence of aggregates. Over the years, scientists have explored the polyethylene glycol modification of interferon β. For example, Katre et al. modified the amino group of interferon β-1a with polyethylene glycol (see US Patent Nos. 4766106 and 4917888 and International Publication No. WO87/00056), and Alessandra modified the sulfhydryl group (Cys ¹⁷ ) of interferon β-1a. Modifications (see US Patent No. 0239529). For recombinant human interferon β-1b, there is no free sulfhydryl group in its sequence, and its modification is mainly concentrated on the amino group. For example, Basu et al. used a variety of PEG modifiers to chemically modify recombinant human interferon β-1b, and compared the advantages and disadvantages of each other [Basu, A., Yang, K., Wang, ML, et al. Structure-function engineering of interferon-β-1b for improving stability, solubility, potency, immunogenicity, and pharmacokinetic properties by site-selective mono-PEGylation. Bioconjugate chemistry, 2006,17(3):618-630.]. Recently, there has been a new exploration of chemical modification of recombinant human interferon β-1b in organic phase solution, using polyethylene glycol nitrogen hydroxysuccinimide carbonate (mPEG-SC) to modify recombinant human interferon β-1b , the modification in the organic phase can effectively increase the modification rate compared with the water phase modification [Su, ZG, Peng, F., Wang, YJ, et al. PEGylation of Proteins in Organic Solution: a Case Study for Interferon beta-1b. Bioconjugate chemistry, 2012, 23:1812-1820.]. However, these explorations are all aimed at modifying the amino groups of recombinant human interferon β-1b, and there are problems such as multiple modifications and separation difficulties, and finally no uniform site-specific single-modification products have been obtained. As for the widely used method of N-terminal amino modification of protein by controlling the pH value of the solution (see Japanese Patent Publication No. 9-25298A), it has also been reported recently that this method can also produce multiple modified products [Yu, DQ, Ghosh R., Purification of pegylated protein using membrane chromatography. Journal of Pharmaceutical Sciences, 2010, 99(8): 3326-3333]. The stability and physiological activity of the products obtained by traditional amino modification cannot be guaranteed, which makes it difficult for these products to apply for new drugs. At present, there are no polyethylene glycol-modified interferon-β drugs on the market at home and abroad.

Therefore, there is a need in the art for a polyethylene glycol-modified recombinant human interferon β-1b that is stable and can improve the pharmacological and pharmacokinetic properties of recombinant human interferon β-1b.

Contents of the invention

The present invention is based on the structure that the N-terminal of recombinant human interferon β-1b is serine, oxidizes the protein N-terminal serine to aldehyde group, and carries out covalent coupling with activated polyethylene glycol whose end group can react with aldehyde group to obtain N Recombinant human interferon beta-1b with single-terminal modification.

Concrete technical scheme of the present invention is as follows:

In the first aspect, the present invention provides a polyethylene glycol (PEG) modified recombinant human interferon β-1b, characterized in that the structure of the modified product is shown in Formula I:

PEG-IFN-β-1b (I)

Wherein, the N-terminal of the recombinant human interferon β-1b is serine, and the polyethylene glycol is covalently coupled to the N-terminal of the recombinant human interferon β-1b;

Preferably, the end group structure of the polyethylene glycol is as shown in formula II:

PEG-A (II)

Wherein, A is amino group (-NH ₂ ), oxyamino group (-O-NH ₂ ) or hydrazide group (-CO-NH-NH ₂ );

Preferably, the polyethylene glycol is monomethoxypolyethylene glycol (mPEG).

In the polyethylene glycol-modified recombinant human interferon β-1b of the present invention, the link between the polyethylene glycol and the recombinant human interferon β-1b can be an imine Oxime and acylhydrazone one of, or an amine Oxyamine and amides

One of.

In the polyethylene glycol-modified recombinant human interferon β-1b of the present invention, the polyethylene glycol may be linear, branched or multi-arm polyethylene glycol, preferably linear polyethylene glycol.

In the polyethylene glycol-modified recombinant human interferon β-1b of the present invention, the molecular weight of polyethylene glycol may range from 2000Da to 80000Da, preferably from 5000Da to 50000Da, more preferably from 10000Da to 30000Da.

In the second aspect, the present invention provides the preparation method of recombinant human interferon beta-1b modified with polyethylene glycol as described in the first aspect, characterized in that, the method comprises the preparation of recombinant human interferon beta-1b The N-terminal serine is oxidized to an aldehyde group, followed by covalent coupling with polyethylene glycol terminated with ammonia, oxammonium, or hydrazide.

The preparation method of the recombinant human interferon beta-1b modified by polyethylene glycol of the present invention may comprise the following two steps:

(A) Oxidize the N-terminal serine of recombinant human interferon β-1b to an aldehyde group, as shown in formula (1):

and

(B1) Reaction of polyethylene glycol whose end group is ammonia, oxammonium or hydrazide with the product obtained in step (A), and covalently coupling to obtain recombinant human interferon β- 1b, the reaction is shown in formula (2)-(4):

or

(A) Oxidize the N-terminal serine of recombinant human interferon β-1b to an aldehyde group, as shown in formula (1):

and

(B2) Reaction of activated polyethylene glycol whose end group is ammonia, oxammonium or hydrazide with the product obtained in step (A), adding a reducing agent during the reaction, to obtain recombinant human interference with the N-terminal single-modification of polyethylene glycol The reduction product of prime β-1b, the reaction formula is shown in (5)-(7):

In the preparation method of polyethylene glycol-modified recombinant human interferon β-1b of the present invention, the oxidant used in step (A) can be ammonium chloride, chromium trioxide, manganese dioxide, periodic acid and periodate , preferably periodate, more preferably sodium periodate.

In the preparation method of polyethylene glycol-modified recombinant human interferon β-1b of the present invention, the reducing agent used in step (B) can be sodium borohydride, sodium acetate borohydride and sodium cyanoborohydride, preferably cyanide sodium borohydride.

In the third aspect, the present invention provides a pharmaceutical composition comprising the polyethylene glycol-modified recombinant human interferon β-1b as described in the first aspect.

In the fourth aspect, the present invention provides the use of polyethylene glycol-modified recombinant human interferon beta-1b as described in the first aspect in the preparation of medicines for the prevention and treatment of acute, chronic and recurrent Viral infectious diseases and inflammatory immune diseases of the nervous system, remission and treatment of relapsing multiple sclerosis diseases, and treatment of malignant tumors such as cervical intraepithelial tumors, neoplastic pleural effusions, hairy cell leukemia, breast tumors or endometrial tumors .

Compared with the prior art in this field, the advantage of the present invention is:

(1) The only aldehyde group at the N-terminal of recombinant human interferon β1b is obtained by a highly specific oxidation method, and then covalently coupled with polyethylene glycol to obtain a recombinant human interferon β-1b with N-terminal single modification , single product, easy to separate;

(2) The polyethylene glycol modifier and aldehyde group used have high reactivity, and the modification reaction yield is higher than that in the organic phase, which greatly improves the use efficiency of protein and polyethylene glycol modifiers. The obtained modified product can largely retain the antiviral and anti-tumor activity of the original recombinant human interferon beta-1b, and can also achieve the purpose of improving the pharmacological and pharmacokinetic properties of the recombinant human interferon beta-1b.

Description of drawings

Fig. 1 is the electrophoresis image of mPEG _5k -HZ modified recombinant human interferon β-1b detected by SDS-PAGE. Use 12% separating gel and 4.5% stacking gel.

in:

1. Standard protein samples, the molecular weights are 14.4KDa, 20.1KDa, 29.0KDa, 43.1KDa, 66.4KDa, 97.2KDa.

2. The reaction mixture of mPEG _5k -HZ modified recombinant human interferon β-1b.

3. Recombinant human interferon beta-1b proprotein.

4. Recombinant human interferon β-1b modified by mPEG _5k -HZ (Mono HZ-5k-PEG-IFN-β-1b, product before reduction).

Fig. 2 is the electrophoresis image of mPEG _20k -HZ modified recombinant human interferon β-1b detected by SDS-PAGE. Use 15% separating gel and 4.5% stacking gel.

in:

1. Standard protein samples, the molecular weights are 14.4KDa, 20.1KDa, 29.0KDa, 43.1KDa, 66.4KDa, 97.2KDa.

2. Recombinant human interferon beta-1b proprotein.

3. The reaction mixture of mPEG _20k -HZ modified recombinant human interferon β-1b.

4. Recombinant human interferon β-1b modified with mPEG _20k -HZ (Mono HZ-20k-PEG-IFN-β-1b, product before reduction).

Fig. 3 is the electrophoresis diagram of mPEG _20k -NH ₂ and mPEG _20k -O-NH ₂ modified recombinant human interferon β-1b detected by SDS-PAGE. Use 15% separating gel and 4.5% stacking gel.

in:

1. Standard protein samples, the molecular weights are 14.4KDa, 20.1KDa, 29.0KDa, 43.1KDa, 66.4KDa, 97.2KDa.

2. Recombinant human interferon β-1b modified with mPEG _20k -NH ₂ (Mono NH ₂ -20k-PEG-IFN-β-1b, product before reduction).

3. Recombinant human interferon β-1b modified by mPEG _20k -O-NH ₂

(Mono ONH ₂ -20k-PEG-IFN-β-1b, product before reduction).

Detailed ways

The present invention will be described in detail below in conjunction with the examples, but the examples are only for the purpose of illustration rather than limitation of the present invention.

Nitrogen terminal oxidation of embodiment 1 recombinant human interferon beta-1b

Weigh 2 mg of IFN-β-1b lyophilized powder and dissolve it in 2 mL of 50 mM PB, pH 4.0, 0.1% SDS buffer solution. Add 200 μL of 1 mg/mL NaIO ₄ aqueous solution, the molar ratio of NaIO ₄ to IFN-β-1b is 10:1. React for 1 h at 25°C. Then, 100 μL of ethylene glycol was added to the reaction system to terminate the reaction, and the reaction was carried out at room temperature for 10 min. The protein peak was collected by gel column chromatography, and the sample was added with achromatic magenta color development reagent for color development. The color development result shows that the oxidized IFN-β-1b sample is bright red, which is the characteristic color of achromatic magenta to detect the aldehyde group. The color development result of the unoxidized sample is colorless, indicating that NaIO ₄ oxidizes IFN-β-1b to form aldehyde base product.

Example 2 Preparation of polyethylene glycol hydrazide 5000 (mPEG _5k -HZ) site-directed single-modification recombinant human interferon β-1b

Take the oxidized recombinant human interferon β-1b solution, add polyethylene glycol hydrazide powder, the molecular weight is 5000Da, and the molar feed ratio of polyethylene glycol hydrazide and recombinant human interferon β-1b is 10:1. The reaction was shaken at pH 4.5 and 25° C. for 24 h.

After the reaction was completed, the reaction solution was chromatographically separated through a Sephacryl S-200HR column, and the mobile phase was a buffer system (pH7.4) containing 20mM PB and 0.1M Na ₂ SO ₄ . The flow rate is 0.8mL/min, and the single loading volume is 500μL. The detection wavelength was 280nm, and the peak was collected. The collected sample was dialyzed overnight in 20mM PB, 5% mannitol system, and concentrated by ultrafiltration.

The results of the reaction and separation were detected by SDS-PAGE, and the results showed that the monomodified recombinant human interferon beta-1b was obtained after modification with polyethylene glycol hydrazide 5000 and separation and purification. see picture 1.

Example 3 Preparation of polyethylene glycol hydrazide 20000 (mPEG _20k -HZ) site-directed single-modification recombinant human interferon β-1b

Take the oxidized recombinant human interferon β-1b solution, add polyethylene glycol hydrazide powder, the molecular weight is 20000Da, and the molar feed ratio of polyethylene glycol hydrazide and recombinant human interferon β-1b is 10:1. The reaction was shaken at pH 4.5 and 25° C. for 24 h.

After the reaction was completed, the reaction solution was chromatographically separated through a Sephacryl S-200HR column, and the mobile phase was a buffer system (pH7.4) containing 20mM PB and 0.1M Na ₂ SO ₄ . The flow rate is 0.8mL/min, and the single loading volume is 500μL. The detection wavelength was 280nm, and the peak was collected. The collected sample was dialyzed overnight in 20mM PB, 5% mannitol system, and concentrated by ultrafiltration.

The results of the reaction and separation were detected by SDS-PAGE, and the results showed that polyethylene glycol hydrazide 20000 was modified, separated and purified to obtain a single-modified recombinant human interferon β-1b. See Figure 2.

Example 4 Preparation of recombinant human interferon β-1b reduction product of polyethylene glycol hydrazide 20000 (mPEG _20k -HZ) site-directed single modification

Take the oxidized recombinant human interferon β-1b solution, add polyethylene glycol hydrazide powder, molecular weight 20000Da, the molar ratio of polyethylene glycol hydrazide and recombinant human interferon β-1b is 10:1, add reducing Sodium cyanoborohydride was added to give a final concentration of 10 mM. The reaction was shaken at pH 4.5 and 25° C. for 24 h.

After the reaction was completed, the reaction solution was chromatographically separated through a Sephacryl S-200HR column, and the mobile phase was a buffer system (pH7.4) containing 20mM PB and 0.1M Na ₂ SO ₄ . The flow rate is 0.8mL/min, and the single loading volume is 500μL. The detection wavelength was 280nm, and the peak was collected. The collected sample was dialyzed overnight in 20mM PB, 5% mannitol system, and concentrated by ultrafiltration.

The results of the reaction and separation were detected by SDS-PAGE, and the results showed that polyethylene glycol hydrazide 20000 was modified, separated and purified to obtain a single-modified recombinant human interferon β-1b reduction product.

Example 5 Preparation of polyethylene glycol hydrazide 40000 (mPEG _40k -HZ) site-directed single-modification recombinant human interferon β-1b

Take the oxidized recombinant human interferon beta-1b solution, add polyethylene glycol hydrazide powder, molecular weight 40000Da, the molar feed ratio of polyethylene glycol hydrazide and recombinant human interferon beta-1b is 10:1, at pH The value was 4.5, and the reaction was shaken at 25°C for 24h.

After the reaction was completed, the reaction solution was chromatographically separated through a Sephacryl S-200HR column, and the mobile phase was a buffer system (pH7.4) containing 20mM PB and 0.1M Na ₂ SO ₄ . The flow rate is 0.8mL/min, and the single loading volume is 500μL. The detection wavelength was 280nm, and the peak was collected. The collected sample was dialyzed overnight in 20mM PB, 5% mannitol system, and concentrated by ultrafiltration.

The results of the reaction and separation were detected by SDS-PAGE, and the results showed that the monomodified recombinant human interferon beta-1b was obtained after modification with polyethylene glycol hydrazide 40000 and separation and purification.

Example 6 Preparation of polyethylene glycol ammonium 20000 (mPEG _20k -NH ₂ ) site-directed single-modification recombinant human interferon β-1b

Take the oxidized recombinant human interferon beta-1b solution, add polyethylene glycol ammonia powder, molecular weight 20000Da, the molar feed ratio of polyethylene glycol ammonia and recombinant human interferon beta-1b is 10:1, at pH 4.5 , Shaking reaction at 25°C for 24h.

After the reaction was completed, the reaction liquid was separated by Sephacryl S-200HR column chromatography, and the collected samples were tested by SDS-PAGE. The results showed that polyethylene glycol ammonia 20000 was modified and separated and purified to obtain mono-modified recombinant human interferon β-1b . See Figure 3.

Example 7 Preparation of polyethylene glycol oxamine 20000 (mPEG _20k -O-NH ₂ ) site-directed single-modification recombinant human interferon β-1b

Get the oxidized recombinant human interferon beta-1b solution, add polyethylene glycol ammonium oxide powder, molecular weight 20000Da, the molar feed ratio of polyethylene glycol ammonium oxide and recombinant human interferon beta-1b is 10:1, at pH The value was 4.5, and the reaction was shaken at 25°C for 24h.

After the reaction was completed, the reaction liquid was separated by chromatography on a Sephacryl S-200HR column, and the collected samples were tested by SDS-PAGE. The results showed that the monomodified recombinant human interferon β- 1b. See Figure 3.

Example 8 Preparation of polyethylene glycol oxamine 20000 (PEG _20k -O-NH ₂ ) site-directed single-modification recombinant human interferon β-1b

Get the oxidized recombinant human interferon beta-1b solution, add polyethylene glycol ammonium oxide powder, molecular weight 20000Da, the molar feed ratio of polyethylene glycol ammonium oxide and recombinant human interferon beta-1b is 10:1, at pH The value was 4.5, and the reaction was shaken at 25°C for 24h.

After the reaction was completed, the reaction liquid was separated by Sephacryl S-200HR column chromatography, and the collected samples were tested by SDS-PAGE. The results showed that polyethylene glycol ammonia 20000 was modified and separated and purified to obtain mono-modified recombinant human interferon β-1b .

Example 9 Preparation of polyethylene glycol hydrazide 20000 (PEG _20k -HZ) site-directed single-modification recombinant human interferon β-1b reduction product

Take the oxidized recombinant human interferon β-1b solution, add polyethylene glycol hydrazide powder, molecular weight 20000Da, the molar ratio of polyethylene glycol hydrazide and recombinant human interferon β-1b is 10:1, add reducing Sodium cyanoborohydride was added to give a final concentration of 10 mM. The reaction was shaken at pH 4.5 and 25° C. for 24 h.

After the reaction was completed, the reaction solution was chromatographically separated through a Sephacryl S-200HR column, and the mobile phase was a buffer system (pH7.4) containing 20mM PB and 0.1M Na ₂ SO ₄ . The flow rate is 0.8mL/min, and the single loading volume is 500μL. The detection wavelength was 280nm, and the peak was collected. The collected sample was dialyzed overnight in 20mM PB, 5% mannitol system, and concentrated by ultrafiltration.

The results of the reaction and separation were detected by SDS-PAGE, and the results showed that polyethylene glycol hydrazide 20000 was modified, separated and purified to obtain a single-modified recombinant human interferon β-1b reduction product.

Example 10 Preparation of polyethylene glycol ammonia 20000 (PEG _20k -NH ₂ ) site-directed single-modification recombinant human interferon β-1b

Take the oxidized recombinant human interferon beta-1b solution, add polyethylene glycol ammonia powder, molecular weight 20000Da, the molar feed ratio of polyethylene glycol ammonia and recombinant human interferon beta-1b is 10:1, at pH 4.5 , Shaking reaction at 25°C for 24h.

After the reaction was completed, the reaction liquid was separated by Sephacryl S-200HR column chromatography, and the collected samples were tested by SDS-PAGE. The results showed that polyethylene glycol ammonia 20000 was modified and separated and purified to obtain mono-modified recombinant human interferon β-1b .

In vitro antiviral activity detection of recombinant human interferon β-1b before and after modification in Example 11

The WISH-VSV system was selected for the in vitro antiviral activity assay.

An inhibition microassay based on the inhibition of cytopathic effects was used. The challenge dose of VSV virus is 100 times of the dose to make half of the cells lesion (TCID ₅₀ ). Take the VSV virus preserved in the laboratory and dilute it to the working concentration with the challenge culture medium. Cell counting was selected as the CCK-8 kit, and the absorbance value was detected at 450nm. The results of the activity assay are shown in Table 1.

Table 1. Antiviral activity of recombinant human β-1b interferon before and after modification

Note: *1 is the non-reduced product, *2 is the reduced product.

The applicant declares that the present invention illustrates the detailed features and methods of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed features and methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed features and methods to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of selected materials and steps in the present invention, addition of auxiliary materials and steps, selection of specific methods, etc., all fall within the protection scope and disclosure of the present invention. within range.

Claims (13)

1. recombinant human interferon beta-the 1b that modifies of a polyoxyethylene glycol (PEG), it is characterized in that, described modified outcome structure is such as formula shown in I:

PEG-IFN-β-1b (I)

Wherein, the N-terminal of described recombinant human interferon beta-1b is Serine, the N-terminal covalent coupling of described polyoxyethylene glycol and described recombinant human interferon beta-1b;

The end-group structure of described polyoxyethylene glycol is such as formula shown in II:

PEG-A (II)

Wherein, A is hydrazide group (-CO-NH-NH ₂);

Described polyoxyethylene glycol is mono methoxy polyethylene glycol (mPEG), and described polyoxyethylene glycol is straight chain or multi-arm polyethylene glycol, and the molecular weight ranges of described polyoxyethylene glycol is 5000Da-30000Da;

The connecting key of described polyoxyethylene glycol and described recombinant human interferon beta-1b is acylhydrazone or acid amides

2. polyethyleneglycol modified recombinant human interferon beta-1b according to claim 1, is characterized in that, described polyoxyethylene glycol is straight chain polyoxyethylene glycol.

3. recombinant human interferon beta-1b polyethyleneglycol modified as claimed in claim 1, is characterized in that, the molecular weight ranges of described polyoxyethylene glycol is 10000Da-30000Da.

4. the preparation method of the polyethyleneglycol modified recombinant human interferon beta-1b according to any one of claim 1-3, it is characterized in that, it is aldehyde radical that described method comprises the serine oxidation of the N-terminal of recombinant human interferon beta-1b, is the polyoxyethylene glycol covalent coupling of hydrazides afterwards with end group.

5. preparation method according to claim 4, is characterized in that, said method comprising the steps of:

(A) be aldehyde radical by the serine oxidation of the N-terminal of recombinant human interferon beta-1b, shown in (1):

with

(B1) be that the product that the polyoxyethylene glycol of hydrazides and step (A) obtain reacts by end group, carry out covalent coupling and obtain polyoxyethylene glycol and to fix a point the recombinant human interferon beta-1b that mono-modified N holds, react such as formula shown in (4):

or

(A) be aldehyde radical by the serine oxidation of the N-terminal of recombinant human interferon beta-1b, shown in (1):

with

(B2) be that the product that the activated polyethylene glycol of hydrazides and step (A) obtain reacts by end group, reductive agent is added in reaction process, obtain polyoxyethylene glycol fix a point mono-modified N hold recombinant human interferon beta-1b reduzate, reaction formula is as shown in (7):

6. preparation method according to claim 5, is characterized in that, the oxygenant used in step (A) is ammonium chlorochromate, Manganse Dioxide, Periodic acid or periodate.

7. preparation method according to claim 6, is characterized in that, the oxygenant used in step (A) is periodate.

8. preparation method according to claim 7, is characterized in that, the oxygenant used in step (A) is sodium periodate.

9. the preparation method according to any one of claim 5-8, is characterized in that, the reductive agent used in step (B2) is sodium borohydride, acetic acid sodium borohydride or sodium cyanoborohydride.

10. preparation method according to claim 9, is characterized in that, the reductive agent used in step (B2) is sodium cyanoborohydride.

11. 1 kinds of pharmaceutical compositions, it comprises recombinant human interferon beta-1b polyethyleneglycol modified as claimed any one in claims 1 to 3.

12. polyethyleneglycol modified as claimed any one in claims 1 to 3 recombinant human interferon beta-1b are preparing the application in medicine, described medicine is used for acute, the chronic and recurrent viral infection disease of prevention and therapy and neural system inflammatory immunological disease, alleviate and treat relapsive sclerosis disease, and treating malignant tumour and neoplastic pleural effusion.

13. application according to claim 12, is characterized in that, described malignant tumour is cervical intraepithelial neoplasia, hairy cell, breast tumor or endometrial tumors.