阅读说明：本技术 聚乙二醇修饰的rhBNP及其制备方法 (Polyethylene glycol modified rhBNP and preparation method thereof ) 是由 彭红卫 韩为跃 张磊 俞亚波 王冲 万云雷 何小林 于 2020-08-31 设计创作，主要内容包括：本发明提供了一种聚乙二醇修饰的rhBNP及其制备方法。具体地,本发明提供了一种新的聚乙二醇修饰的rhBNP制备方法,并基于该方法制备了相应的聚乙二醇修饰的rhBNP的药物组合物。本发明的方法可以提高rhBNP的N-末端单聚乙二醇化的专一性及产品纯度,可以得到基本均一的N-末端单PEG修饰的rhBNP产品。(The invention provides a polyethylene glycol modified rhBNP and a preparation method thereof. Specifically, the invention provides a novel preparation method of polyethylene glycol modified rhBNP, and a corresponding pharmaceutical composition of the polyethylene glycol modified rhBNP is prepared based on the method. The method of the invention can improve the specificity and the product purity of the N-terminal single polyethylene glycol of the rhBNP, and can obtain a basically uniform N-terminal single PEG modified rhBNP product.)

1. A preparation method of modified rhBNP is characterized by comprising the following steps:

(a) carrying out coupling reaction on rhBNP and polyethylene glycol so as to obtain a modified rhBNP crude product; and

(b) purifying the modified rhBNP obtained in the step (a), thereby obtaining the modified rhBNP.

2. The method of claim 1, wherein step (b) comprises:

(b1) subjecting the modified rhBNP obtained in step (a) to a first ion exchange chromatography, performing gradient elution with citrate buffer solution of citric acid and sodium chloride, collecting the elution peak confirmed to contain the modified rhBNP, thereby obtaining an eluate I containing the modified rhBNP, and

(b2) and (b) performing secondary ion exchange chromatography on the eluate I obtained in the step (b1), eluting by using a disodium hydrogen phosphate buffer solution and a sodium dihydrogen phosphate buffer solution, and collecting an elution peak which is proved to contain the modified rhBNP, thereby obtaining the modified rhBNP.

3. The method of claim 1, wherein in step (B1), the gradient elution is performed using buffer A and buffer B, wherein the buffer A is 5-20mM, preferably 8-12mM, citrate buffer, and the buffer B is a mixed solution of buffer A and 1M sodium chloride.

4. The method of claim 1, wherein in step (a), the coupling reaction is carried out in the presence of a reducing agent, preferably selected from the group consisting of: sodium cyanoborohydride, sodium triacetoxyborohydride, sodium acetate borohydride, sodium borohydride, or a combination thereof.

5. The method according to claim 1, wherein the polyethylene glycol is coupled to the serine residue at the N-terminus of rhBNP, and preferably the polyethylene glycol is coupled to one of an amino group and a hydroxyl group of the serine residue.

6. The method of claim 1, wherein the polyethylene glycol is methoxypolyethylene glycol propionaldehyde.

7. The method of claim 1, wherein the polyethylene glycol has a molecular weight of 5KD to 80KD, preferably 10KD to 30KD, and more preferably 15 KD to 25 KD.

8. A composition containing modified rhBNP, which is characterized by comprising the modified rhBNP and polyethylene glycol, wherein the content of the modified rhBNP is more than or equal to 80 percent, preferably more than or equal to 85 percent, and more preferably more than or equal to 92 percent;

in the modified rhBNP, only one of an amino group and a hydroxyl group of an N-terminal serine residue of the rhBNP is coupled with polyethylene glycol.

9. The composition of claim 8, wherein the composition is prepared by the method of claim 1.

10. Use of a composition according to claim 8 for the preparation of a medicament for the treatment of a cardiac disorder.

Technical Field

The invention relates to the field of biomedicine, and more particularly relates to polyethylene glycol modified rhBNP and a preparation method thereof.

Background

B-type natriuretic peptide, also called Brain Natriuretic Peptide (BNP), is a natural hormone with biological activity synthesized by cardiac muscle cells, and is mainly expressed in the ventricle and mainly used to regulate the heart function. BNP secreted by myocardial cells exists in a precursor form consisting of 108 amino acids, and is cleaved into an inactive linear polypeptide amino-terminal fragment (1-76aa) consisting of 76 amino acids and an active cyclic polypeptide BNP-32 consisting of 32 amino acids under the action of an activating enzyme when the myocardial cells are stimulated, wherein the active cyclic polypeptide BNP-32 is released into the blood circulation and is respectively called NT-proBNP and BNP.

The bioavailability of the protein therapeutic drug rhBNP is generally limited by plasma half-life and is sensitive to protease degradation, hindering the greatest clinical potential. The commercially available rhBNP is a short-acting product, has a half-life of 18 minutes, and needs to be slowly infused intravenously for more than 24 hours, which brings great pain to patients.

The PEG-rhBNP is formed by modifying rhBNP with PEG (polyethylene glycol), thereby reducing the immunogenicity and prolonging the half-life of the rhBNP. However, PEG-modified rhBNP has various impurities, such as unreacted raw materials: PEG, rhBNP; such as poly-substituted PEG-rhBNP: di-PEG-rhBNP, tri-PEG-rhBNP; for example, the large amount of impurities such as PEG-rhBNP of other substitution units has certain influence on the quality of products.

Therefore, the development of a new method for preparing rhBNP modified by polyethylene glycol is urgently needed in the field, and the specificity and the product purity of N-terminal monopegylation of the rhBNP are improved.

Disclosure of Invention

The invention aims to provide polyethylene glycol modified rhBNP and a preparation method thereof.

The invention also provides the N-terminal mono-PEG-rhBNP pharmaceutical composition, which comprises the N-terminal mono-PEG-rhBNP pharmaceutical active composition and a pharmaceutically acceptable carrier, and is usually prepared into a freeze-dried product or a liquid product, preferably a freeze-dried powder injection, wherein the preparations can be prepared by adopting corresponding auxiliary materials known by the ordinary skilled person in the art and adopting corresponding known preparation technologies of pharmaceutical preparations.

In another aspect, the present invention also provides the use of the above-mentioned pharmaceutically active composition of N-terminal mono-PEG-rhBNP or a pharmaceutical composition comprising the same for the preparation of a medicament for the treatment of a disease characterized by heart failure.

In a first aspect of the present invention, there is provided a method for preparing modified rhBNP, comprising the steps of:

(a) carrying out coupling reaction on rhBNP and polyethylene glycol so as to obtain a modified rhBNP crude product; and

(b) purifying the modified rhBNP obtained in the step (a), thereby obtaining the modified rhBNP.

In another preferred embodiment, the step (b) includes:

(b1) subjecting the modified rhBNP obtained in step (a) to a first ion exchange chromatography, performing gradient elution with citrate buffer solution of citric acid and sodium chloride, collecting the elution peak confirmed to contain the modified rhBNP, thereby obtaining an eluate I containing the modified rhBNP, and

(b2) and (b) performing secondary ion exchange chromatography on the eluent I obtained in the step (b1), eluting by using a disodium hydrogen phosphate buffer solution and a sodium dihydrogen phosphate buffer solution, and collecting an elution peak to obtain the modified rhBNP.

In another preferred embodiment, in step (b1), the first ion exchange chromatography is performed using a SP Sepharose Fast Flow chromatography column.

In another preferred embodiment, in step (B1), gradient elution is performed by using buffer A and buffer B, wherein buffer A is 5-20mM, preferably 8-12mM, citrate buffer, and buffer B is a mixed solution of buffer A and 1M sodium chloride.

In another preferred example, in the step (b1), the elution peak is an elution peak 2 which is confirmed to contain the modified rhBNP by electrophoresis.

In another preferred embodiment, in step (b1), the modified rhBNP crude product obtained in step (a) is diluted by using buffer A, and the concentration of the diluted modified rhBNP crude product is 350 μ g/mL.

In another preferred embodiment, in step (b1), the diluted modified rhBNP has a loading flow rate of 5 cm/min. + -. 20%, preferably 4.5-5.5 cm/min.

In another preferred embodiment, in step (b1), the amount of the diluted modified rhBNP loaded is 15-25 column volumes.

In another preferred embodiment, in step (b1), before elution, buffer A is used for washing, preferably buffer A is used for washing 2 column volumes at a flow rate of 3cm/min + -10%.

In another preferred embodiment, in step (B1), buffer A and buffer B are eluted at a flow rate gradient of 5 cm/min. + -. 20% for 15-25 column volumes.

In another preferred example, in step (b1), the gradient is 0 to 90% ± 10%.

In another preferred example, in the step (b1), the elution peak is an elution peak 4 which is confirmed to contain the modified rhBNP by electrophoresis.

In another preferred embodiment, in step (b2), a second ion exchange chromatography is performed using a SP Sepharose Fast Flow chromatography column.

In another preferred example, in the step (b2), the eluate I obtained in the step (b1) is diluted by citrate buffer of citric acid-sodium chloride, and the concentration of the diluted modified rhBNP is 350 μ g/mL.

In another preferred embodiment, in step (b2), before elution, the buffer is equilibrated with a buffer solution of disodium hydrogen phosphate and sodium dihydrogen phosphate, wherein the concentration of the buffer solution of disodium hydrogen phosphate and sodium dihydrogen phosphate is 10-30mM, preferably 15-25 mM.

In another preferred embodiment, in step (b2), the rinsing is performed at a flow rate of 5 cm/min. + -. 20%.

In another preferred example, in step (b2), the elution flow rate of the disodium hydrogen phosphate and sodium dihydrogen phosphate buffer is 5cm/min ± 20%.

In another preferred embodiment, the elution in step (b2) is carried out with a buffer solution of disodium hydrogen phosphate and sodium dihydrogen phosphate in a concentration of 5-20mM, preferably 8-12 mM.

In another preferred example, in step (b2), the elution is isocratic.

In another preferred example, the step (b2) further includes a step of removing moisture contained in the elution peak 4.

In another preferred embodiment, in step (a), the coupling reaction is carried out in the presence of a reducing agent, preferably selected from the group consisting of: sodium cyanoborohydride, sodium triacetoxyborohydride, sodium acetate borohydride, sodium borohydride, or a combination thereof.

In another preferred embodiment, the reducing agent is sodium cyanoborohydride.

In another preferred embodiment, the coupling reaction is carried out at a pH of 3-7, preferably at a pH of 4-6.

In another preferred embodiment, the pH is adjusted by using a buffer solution, preferably an acetate buffer or a citrate buffer.

In another preferred embodiment, the reaction time of the coupling reaction is 4 to 20 hours, preferably 8 to 18 hours, and more preferably 10 to 14 hours.

In another preferred embodiment, the reaction temperature of the coupling reaction is 0-28 ℃, preferably 10-25 ℃; more preferably, it is from 15 to 25 ℃.

In another preferred embodiment, the molar ratio of the polyethylene glycol to the rhBNP is 1: (0.2-4), preferably 1: (0.5-3), more preferably 1: (0.8-2).

In another preferred example, the step (a) includes:

(a1) mixing rhBNP and polyethylene glycol, and adjusting the pH to 4-6 to obtain a reaction mixed solution, wherein the molar ratio of the polyethylene glycol to the rhBNP is 1: (0.5-3),

(a2) reacting the reaction mixture in the step (a) for 4 to 20 hours under the condition of stirring at the temperature of between 0 and 28 ℃;

(a3) separating the mixed solution obtained in the step (b) so as to obtain the modified rhBNP.

In another preferred embodiment, in step (a1), the concentration of rhBNP in the reaction mixture is 0.5-8mg/ml, preferably 1-6 mg/ml.

In another preferred example, in the step (a1), the buffer solution is a citrate buffer solution or an acetate buffer solution.

In another preferred embodiment, in step (a2), the reaction mixture is reacted at 10-25 ℃, preferably 15-25 ℃.

In another preferred embodiment, in step (a2), the reaction is carried out in the presence of a reducing agent.

In another preferred embodiment, the reducing agent is selected from the group consisting of: sodium cyanoborohydride, sodium triacetoxyborohydride, sodium acetate borohydride, sodium borohydride, or a combination thereof.

In another preferred embodiment, the molar ratio of the reducing agent to the rhBNP is (3-10): 1, preferably (5-7): 1.

in another preferred embodiment, the polyethylene glycol modification rate of the modified rhBNP is 60-99%.

In another preferred embodiment, the polyethylene glycol is coupled with serine residue at the N-terminal of rhBNP, and preferably, the polyethylene glycol is coupled with one of amino group and hydroxyl group of serine residue.

In another preferred embodiment, one or more amino acid residues of the modified rhBNP are coupled with polyethylene glycol (modified by coupling with polyethylene glycol).

In another preferred embodiment, the sequence of rhBNP is shown in SEQ ID NO. 1 (SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH).

In another preferred embodiment, the polyethylene glycol (i.e., PEG) is coupled with at least one of amino group, thiol group, carboxyl group and hydroxyl group in the amino acid residue.

In another preferred embodiment, the polyethylene glycol is methoxy polyethylene glycol (i.e., mPEG) and its derivatives.

In another preferred embodiment, the polyethylene glycol is alkylated polyethylene glycol or aldehydized polyethylene glycol.

In another preferred embodiment, the methoxypolyethylene glycol derivative has the following general formula:

mPEG-X,

wherein X is a functional group.

In another preferred embodiment, the functional group is an alkylated functional group or an acylated functional group.

In another preferred embodiment, the functional group is an alkylating functional group, and the polyethylene glycol is mPEG-aldehyde or mPEG-trifluoroethane sulfonic acid.

In another preferred embodiment, the polyethylene glycol is selected from the group consisting of: methoxy polyethylene glycol formaldehyde, methoxy polyethylene glycol acetaldehyde, methoxy polyethylene glycol propionaldehyde, methoxy polyethylene glycol butyraldehyde, methoxy polyethylene glycol valeraldehyde, or a combination thereof.

In another preferred embodiment, the polyethylene glycol is methoxypolyethylene glycol propionaldehyde.

In another preferred embodiment, the functional group is an acylated functional group and the polyethylene glycol is mPEG-p-nitrophenyl carbonate.

In another preferred embodiment, the polyethylene glycol has a molecular weight of 5KD-80KD, preferably 10-30KD, more preferably 15-25 KD.

In another preferred embodiment, the molecular weight of the polyethylene glycol is selected from the group consisting of: 5KD, 10KD, 20KD, 30KD, 40KD, 50KD or their combination.

In another preferred embodiment, the polyethylene glycol has a molecular weight of 10kD, 20kD or 30kD, preferably 20 kD.

In another preferred embodiment, the polyethylene glycol is modified on the rhBNP through the N-terminal amino group of the rhBNP.

In another preferred embodiment, the N-terminal amino group of the modified rhBNP is coupled to polyethylene glycol.

In another preferred embodiment, the N-terminal amino group of the modified rhBNP is coupled with a polyethylene glycol aldehyde derivative.

In another preferred embodiment, the modified rhBNP is N-terminal mono-PEG-rhBNP.

In another preferred embodiment, the composition comprises modified rhBNP and polyethylene glycol, wherein the content of the modified rhBNP is more than or equal to 80%, preferably more than or equal to 85%, and more preferably more than or equal to 92%;

in the modified rhBNP, only one of an amino group and a hydroxyl group of an N-terminal serine residue of the rhBNP is coupled with polyethylene glycol.

In another preferred embodiment, the modified rhBNP comprises only one polyethylene glycol coupled to the N-terminal serine residue.

In another preferred embodiment, the composition is prepared by the method of claim 1.

In another preferred embodiment, the content of polyethylene glycol in the composition is less than or equal to 2%, preferably less than or equal to 1.3%, more preferably less than or equal to 0.9%.

In another preferred embodiment, the composition further comprises impurities selected from the group consisting of:

in another preferred embodiment, the composition further comprises impurities selected from the group consisting of:

rhBNP, unmodified rhBNP, non-N-terminal mono-PEG-rhBNP, di-PEG-rhBNP, tri-PEG-rhBNP, or a combination thereof.

In another preferred embodiment, the content of said impurities is less than or equal to 6.5%, preferably less than or equal to 3.8%, more preferably less than or equal to 2%.

In another preferred embodiment, the composition also comprises unmodified rhBNP, and the content of the unmodified rhBNP is less than or equal to 2 percent, and is preferably less than or equal to 1.8 percent.

In another preferred embodiment, the non-N-terminal mono-PEG-rhBNP is formed by coupling any one of the three lysine residues of rhBNP with polyethylene glycol.

In another preferred embodiment, the di-PEG-rhBNP is formed by coupling any two of the three lysine residues of rhBNP with polyethylene glycol.

In another preferred embodiment, the tri-PEG-rhBNP is formed by simultaneously coupling three lysine residues of rhBNP with polyethylene glycol.

In another preferred embodiment, in the non-N-terminal mono-PEG-rhBNP, di-PEG-rhBNP and tri-PEG-rhBNP, the N-terminal serine residue of the rhBNP can be coupled with polyethylene glycol or not coupled with the polyethylene glycol.

In another preferred embodiment, the composition is prepared using the method of claim 1.

In another preferred embodiment, the composition is a pharmaceutical composition.

In another preferred embodiment, the composition further comprises a pharmaceutically acceptable carrier.

In another preferred embodiment, the compound is used for preparing medicaments for treating heart diseases.

In another preferred embodiment, the heart disease comprises heart failure.

In another preferred embodiment, the heart disease is human heart disease.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a schematic diagram of PEG10KD-rhBNP (prepared in example 2) elution by cation exchange chromatography (from left to right, the peaks are respectively a multi-modified peak, a single-modified peak and an unmodified peak, and are respectively an elution peak 1, an elution peak 2 and an elution peak 3)

Fig. 2 shows the PEG20KD-rhBNP/BNP drug-time curve, where the upper curve is the drug-time curve for PEG20KD-rhBNP and the lower curve is the drug-time curve for BNP.

Detailed Description

The inventor of the invention has studied extensively and deeply, discovered a new method for preparing rhBNP modified by polyethylene glycol for the first time unexpectedly, and prepared the corresponding pharmaceutical composition of rhBNP modified by polyethylene glycol based on the method. The method of the invention can improve the specificity and the product purity of the N-terminal single polyethylene glycol of the rhBNP, and can obtain a basically uniform N-terminal single PEG modified rhBNP product.

The inventor of the present application has long dedicated research work on pegylated rhBNP, and through research on PEG-rhBNP products prepared by the prior art method, the following impurities are mainly present in the products:

(1) unreacted starting materials, such as: PEG, rhBNP;

(2) mono (mono) substituted for PEG-rhBNP at other substitution positions;

(3) polysubstituted PEG-rhBNP, such as di-PEG-rhBNP, tri-PEG-rhBNP;

(4) other impurities.

The inventor discovers through a large amount of researches that when the purity of N-terminal mono-PEG-rhBNP in the product reaches more than 92 percent, the total content of the impurities (2) - (3) is less than or equal to 7.8 percent, the content of BNP in the impurity (1) is less than or equal to 4 percent, and the content of the impurity (4) is less than 0.2 percent, the product quality is basically uniform, and animal experiments show that the product is safe and effective. Therefore, the inventor researches the purification method of the crude product of the PEG-rhBNP prepared by the prior art method, and finally finds a method which can lead the purity of the N-terminal mono-PEG-rhBNP to reach more than 96 percent and lead various impurities to be reduced to the indexes or even lower, thereby improving the quality stability of the PEG-rhBNP and ensuring the clinical medication safety of the PEG-rhBNP.

On the basis, the invention provides an N-terminal mono-PEG-rhBNP pharmaceutical active composition, which is characterized by comprising the following components:

a component I: n-terminal mono-PEG-rhBNP with the purity more than or equal to 92.0 percent;

and (2) component II: is selected from one or more of rhBNP, non-N-terminal mono-PEG-rhBNP, di-PEG-rhBNP and tri-PEG-rhBNP, the content is not less than 0 and not more than 6.5 percent, and the content is not less than 0 and not more than 2.0 percent of the rhBNP;

the component III is PEG, the content of 0 is less than or equal to 1.3 percent; and residual impurities, the content of which is less than 0.2 percent.

In another preferred embodiment, the pharmaceutically active composition comprises the following components:

a component I: n-terminal mono-PEG-rhBNP with the purity more than or equal to 95.0 percent;

and (2) component II: is selected from one or more of rhBNP, non-N-terminal mono-PEG-rhBNP, di-PEG-rhBNP and tri-PEG-rhBNP, the content is not less than 0 and not more than 3.8 percent, and the content is not less than 0 and not more than 1.8 percent of the rhBNP;

the component III is PEG, the content of 0 is less than or equal to 1.0 percent; and residual impurities, the content of which is less than 0.2 percent.

In another preferred embodiment, the pharmaceutically active composition comprises the following components:

preferably, component I: the purity is more than or equal to 97.0 percent;

and (2) component II: selected from one or two of rhBNP and di-PEG-rhBNP, the content is not less than 0 and not more than 2.0 percent; and (3) component III: the PEG content is more than or equal to 0 percent and less than or equal to 0.9 percent. And residual impurities, the content of which is less than 0.1 percent.

In any one of the above pharmaceutically active compositions, the PEG is preferably one of PEG10kDa or PEG 5kDa having a molecular weight of 20 kDa.

3 lysines in the sequence of rhBNP are binding sites of PEG, so in the above-mentioned pharmaceutical active composition, the mono-PEG-rhBNP refers to PEG-rhBNP in which any site of the 3 binding sites is combined with PEG;

non-N-terminal mono-PEG-rhBNP refers to mono-PEG-rhBNP formed by any other binding site except the N-terminal; the di-PEG-rhBNP refers to PEG-rhBNP formed by combining any two sites of the 3 combining sites with PEG; the tri-PEG-rhBNP refers to PEG-rhBNP with 3 binding sites combined with PEG.

In the invention, the purity of the component I is the result of SEC-HPLC method determination (the specific method refers to Chinese pharmacopoeia 2015)

The invention also provides a preparation method of the N-terminal mono-PEG-rhBNP pharmaceutical active composition, which comprises the following steps:

(1) ion exchange chromatography a, performing ion exchange chromatography on the crude product of the N-terminal mono-PEG-rhBNP by using SP Sepharose Fastflow, wherein the ion exchange chromatography comprises the steps of loading, washing and gradient elution;

(2) ion exchange chromatography b: and (3) analyzing a collected peak rich in N-terminal mono-PEG-rhBNP by ion exchange chromatography by using a SP Sepharose Fast Flow column, and obtaining the N-terminal mono-PEG-rhBNP by the steps of sampling, post-balancing and eluting.

In the above preparation method, the buffer solution used for loading the ion exchange chromatography a and the ion exchange chromatography b is the same, and is 10mM citric acid buffer solution, optionally, a small amount of surfactant can be added, the surfactant is preferably 0.004% Tween-80 solution, and the 10mM citric acid buffer solution is referred to as buffer solution A hereinafter; the buffer solution used in the gradient elution step in the ion exchange chromatography is 10mM citric acid buffer solution and sodium chloride solution, the concentration of the sodium chloride solution is preferably 1M, the surfactant is preferably 0.004% Tween-80 solution, and the mixed solution of the buffer solution A and the 1M sodium chloride is hereinafter referred to as buffer solution B.

Further, the preparation method of the N-terminal mono-PEG-rhBNP pharmaceutical active composition comprises the following steps:

(1) ion exchange chromatography a: the crude N-terminal PEG-rhBNP was chromatographed by SP Sepharose Fast Flow ion exchange chromatography, comprising:

sampling: diluting and loading the N-terminal PEG-rhBNP crude product by using buffer solution A;

flushing: washing 15-25 column volumes with buffer a;

③ gradient elution: gradient elution is carried out by using buffer solution A and buffer solution B, and a second elution peak which is confirmed by electrophoresis and contains modified rhBNP, namely an elution peak 2, is collected;

(2) ion exchange chromatography b: ion exchange chromatography a peak 2 was subjected to ion exchange chromatography exchange using SP Sepharose Fast Flow, comprising:

sampling: loading the ion exchange chromatography (1) collection peak 2 with a buffer solution A;

secondly, balancing: equilibrating with buffer C;

③ elution: eluting with buffer solution 50% C, collecting elution peak 4, wherein elution peak 4 is N-terminal PEG-rhBNP;

the buffer solution A is a 10mM citric acid buffer solution;

the buffer solution B is a mixed solution of the buffer solution A and 1M sodium chloride;

the buffer C is 20mM disodium hydrogen phosphate and sodium dihydrogen phosphate monohydrate buffer.

Further, the preparation method of the N-terminal mono-PEG-rhBNP pharmaceutical active composition comprises the following steps:

(1) ion exchange chromatography: the crude N-terminal PEG-rhBNP was chromatographed by SP Sepharose Fast Flow ion exchange chromatography, comprising:

sampling: diluting the N-terminal PEG-rhBNP crude product to 350 mu g/mL by using buffer solution A, and loading the crude product at the flow rate of 5cm/min +/-20%;

flushing: washing 2 column volumes with buffer A at a flow rate of 3cm/min + -10%;

③ gradient elution: eluting 20 column volumes by using a buffer solution A and a buffer solution B at a flow rate gradient of 5cm/min +/-20%, wherein the gradient is 0-90% +/-10%, and collecting an elution peak 2 which is proved to contain modified rhBNP;

(2) ion exchange chromatography: subjecting the collected peak 2 of ion exchange chromatography (1) to ion exchange chromatography using SPSepharose Fast Flow, comprising:

sampling: diluting the ion exchange chromatography a to 350 mu g/mL by using the buffer solution A, and loading the sample at the flow rate of 5cm/min +/-20%;

secondly, balancing: equilibrating with buffer C at a flow rate of 5cm/min + -20%;

③ elution: eluting with buffer 50% C at flow rate of 5cm/min + -20%, collecting elution peak 4, and eluting peak 4 to obtain N-terminal PEG-rhBNP.

The elution peaks 1-3 of the PEG10KD-rhBNP are shown in figure 1.

The main advantages of the invention include:

(a) the modified rhBNP has long half-life and low administration frequency.

(b) The sequence used by the modified rhBNP has small heterogeneity with human body, and is not easy to generate immunoreaction.

(c) The modified rhBNP is simple and convenient to operate.

(d) The biological activity of the modified rhBNP is higher than that of other long-acting modification modes.

(e) The modified rhBNP has no toxic or side effect.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.