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Rhbnp modified with polyethylene glycol and preparation method thereof

A technology of polyethylene glycol and disodium hydrogen phosphate, which is applied in the field of biomedicine and can solve problems such as product quality impact

Active Publication Date: 2022-03-18
JINGZE PHARMA (HEFEI) CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many impurities in PEG-modified rhBNP, such as unreacted raw materials: PEG, rhBNP; such as multi-substituted PEG-rhBNP: di-PEG-rhBNP, tri-PEG-rhBNP; such as other substituted units of PEG-rhBNP, and other impurities A large number of existence, have a certain impact on product quality

Method used

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  • Rhbnp modified with polyethylene glycol and preparation method thereof
  • Rhbnp modified with polyethylene glycol and preparation method thereof
  • Rhbnp modified with polyethylene glycol and preparation method thereof

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preparation example Construction

[0117] The present invention also provides a preparation method of the above-mentioned N-terminal mono-PEG-rhBNP pharmaceutically active composition, comprising the following steps:

[0118] (1) Ion-exchange chromatography a: The N-terminal mono-PEG-rhBNP crude product is subjected to SP Sepharose FastFlow ion-exchange chromatography, including loading, washing, and gradient elution steps;

[0119] (2) Ion-exchange chromatography chromatography b: The collected peak of ion-exchange chromatography rich in N-terminal mono-PEG-rhBNP is analyzed with SP Sepharose Fast Flow column, including loading, post-equilibrium, and elution steps to obtain N-terminal mono-PEG-rhBNP.

[0120] In the above-mentioned preparation method, the buffer used in the ion exchange chromatography chromatography a and the ion exchange chromatography chromatography b loading is the same, which is 10mM citric acid buffer, optionally, a small amount of surfactant can be added, and the surfactant Preferably 0...

Embodiment 15

[0150] Embodiment 1 The reaction of 5KD mPEG propionaldehyde and rhBNP

[0151] Measure 16ml of biosynthesized rhBNP with a concentration of 4mg / ml, weigh 92.38mg of 5KD mPEG propionaldehyde (methoxy polyethylene glycol propionaldehyde) and dissolve it in the measured rhBNP, adjust pH5 with citrate buffer, 25°C Mix for 5 minutes under the same conditions (the molar ratio of rhBNP to 5KD mPEG propionaldehyde is 1:1). Sodium triacetoxyborohydride (the molar ratio of rhBNP to sodium triacetoxyborohydride is 1:5) was added as a reducing agent, and reacted for 10 hours.

[0152] The PEGylated compound was separated from free PEG and free polypeptide by a cation exchange chromatography column using an acidic pH NaCl gradient on a SP Sepharose Fast Flow column (GE) to obtain PEG 5KD-rhBNP.

Embodiment 2

[0153] Embodiment 2 The reaction of 10KD mPEG propionaldehyde and rhBNP

[0154] Measure 16ml of biosynthesized rhBNP with a concentration of 4mg / ml, weigh 184.76mg of 10KD mPEG propionaldehyde (methoxypolyethylene glycol propionaldehyde) and dissolve it in the measured rhBNP, adjust the pH to 4.8 with citric acid buffer, Mix for 5 minutes at 25°C (the molar ratio of rhBNP to 10KD mPEG propionaldehyde is 1:1). The reducing agent sodium cyanoborohydride (the molar ratio of rhBNP to sodium cyanoborohydride is 1:7) was added and reacted for 14 hours.

[0155] The PEGylated compound was separated from free PEG and free polypeptide by a cation exchange chromatography column using an acidic pH NaCl gradient on a SP Sepharose Fast Flow column (GE) to obtain PEG10KD-rhBNP.

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Abstract

The invention provides a rhBNP modified with polyethylene glycol and a preparation method thereof. Specifically, the present invention provides a new preparation method of polyethylene glycol-modified rhBNP, and a corresponding pharmaceutical composition of polyethylene glycol-modified rhBNP is prepared based on the method. The method of the present invention can improve the specificity and product purity of N-terminal mono-PEGylation of rhBNP, and can obtain a substantially uniform N-terminal mono-PEG-modified rhBNP product.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a rhBNP modified with polyethylene glycol and a preparation method thereof. Background technique [0002] B-type natriuretic peptide, also known as brain natriuretic peptide (BNP), is a biologically active natural hormone synthesized by cardiomyocytes, mainly expressed in the ventricle, and mainly used to regulate heart function. The BNP secreted by cardiomyocytes first exists in the form of a precursor consisting of 108 amino acids. When the cardiomyocytes are stimulated, they are cleaved into an inactive linear polypeptide amino-terminal fragment (1-76aa) composed of 76 amino acids under the action of activating enzymes. ) and BNP-32, an active cyclic polypeptide composed of 32 amino acids, are released into the blood circulation and are called NT-proBNP and BNP, respectively. [0003] The bioavailability of protein therapeutic rhBNP is often limited by plasma half-life and is susce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/58C07K1/18C07K1/107A61K38/22A61K47/60A61P9/00A61P9/04
CPCC07K14/58A61K47/60A61P9/00A61P9/04A61K38/00
Inventor 彭红卫韩为跃张磊俞亚波王冲万云雷何小林
Owner JINGZE PHARMA (HEFEI) CO LTD