Method of preparing physiologically active polypeptide conjugate

A technology for physiologically active and conjugated compounds, applied in the field of preparing physiologically active polypeptide conjugates, can solve the problems of reducing peptide activity, reducing preparation yield, inefficiently preparing physiologically active polypeptides, etc., and achieving the effect of increasing in vivo activity

Active Publication Date: 2018-05-11
HANMI PHARMA
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  • Description
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AI Technical Summary

Problems solved by technology

[0003] However, the problem with the preparation of fusion proteins is that the non-specific binding of the polymer to the physiologically active peptide blocks the active domain of the peptide, thereby reducing the activity of the peptide, and thus preventing the site-specific binding of non-specific binding reduces the preparation yield Or manual manipulation of physiologically active peptides is required
[0004] In order to solve the problem of inefficiently preparing a conjugate of a physiologically active polypeptide and a non-peptidyl polymer, attempts have been made to modify a specific site of the physiologically active polypeptide in a physiologically active polypeptide conjugate using a non-peptidyl polymer (Korean Patent No. 10-1164453)

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  • Method of preparing physiologically active polypeptide conjugate
  • Method of preparing physiologically active polypeptide conjugate
  • Method of preparing physiologically active polypeptide conjugate

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preparation example Construction

[0111] The preparation method of the present invention may further include isolating the physiologically active polypeptide conjugate after step 2). This procedure can be carried out using various separation methods such as chromatography known in the art. Specifically, ion exchange chromatography, and more specifically, high pressure ion exchange chromatography may be used, but not limited thereto.

[0112] In the present invention, the physiologically active polypeptide conjugate can be separated from the first reaction mixture to prepare an aqueous solution by applying selective precipitation to the first reaction mixture, the second reaction mixture, or a combination thereof.

[0113] In particular, when separating a physiologically active polypeptide conjugate from a mixture of an aqueous solution prepared by applying selective precipitation to the first reaction mixture and a secondary reaction mixture prepared by using the precipitated physiologically active polypeptide...

Embodiment 1

[0128] Example 1. Pegylation of imidazo-acetylexendin-4 and isolation of positional isomers

[0129]Using 3.4K ALD(2) PEG (3.4 kDa PEG with two aldehyde groups) at the Lys of imidazo-acetyl-exendin-4 (CA-exendin-4, Bachem, USA) , NOF, Japan) for PEGylation.

[0130] To pegylate the Lys position of imidazo-acetyl-exendin-4 using 3.4K ALD(2)PEG, the peptide and PEG were separated at about 1: Molar ratios of 3 to 1:30 and peptide concentrations of about 5 mg / mL to 18 mg / mL were reacted for 0.5 hours to 4.0 hours. At this point, the reaction was allowed to react at a pH of 6.5 to 7.5 in 100 mM HEPES buffer (supplemented with reducing agent 20 to 30 mM SCB (NaCNBH 3 ))conduct. Specific reaction conditions and results isolated by the following methods are shown in Tables 1 and 2 below. In detail, they were reacted at a peptide concentration of about 15 mg / mL at 4°C to 8°C for 3 hours, and the results of separation by the following method are shown in figure 2 .

[0131] In a ...

Embodiment 2

[0138] Example 2. Pegylated insulin analogs and separation of positional isomers

[0139] PEGylation was performed at the N-terminus of the β-chain of the insulin analog using 3.4K ALD(2)PEG (WO2014-133324, Hanmi Pharmaceutical Co., Ltd, Korea).

[0140] To pegylate the N-terminal position of an insulin analogue using 3.4K ALD(2)PEG, peptide and PEG were prepared at a molar ratio of about 1:4 and about 5 mg / mL to A peptide concentration of 18 mg / mL was reacted for 0.5 hours to 4.0 hours. At this point, the reaction was allowed to proceed in 50 mM sodium citrate buffer solution at a pH of 4.0 to 6.0, and the reducing agent 3-5 mM SCB (NaCNBH 3 ). In detail, they were reacted at a peptide concentration of about 5 mg / mL at 4°C to 8°C for 2 hours, and the results of separation by the following method are shown in image 3 .

[0141] Insulin analogue-PEG was separated from each reaction solution using a SP-HP column with a linear concentration gradient of KCl in a citrate buffe...

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Abstract

Provided is a method of preparing a physiologically active polypeptide conjugate, in which a physiologically active polypeptide and a non-peptidyl polymer are linked to each other via a covalent bond.The method is to improve an overall yield of the physiologically active polypeptide conjugate by improving a reaction of the non-peptidyl polymer and the physiologically active polypeptide, and particularly, the method is to prepare a physiologically active polypeptide conjugate in a high yield by performing a two-step reaction through selective precipitation. The preparation method of the present invention is used to produce a non-peptidyl polymer-physiologically active polypeptide conjugate and a physiologically active polypeptide-physiologically active carrier conjugate in a high yield, and therefore, the method may be used in the development of long-acting formulations of various peptide drugs which maintain in vivo activity at a relatively high level and have remarkably increased blood half-life.

Description

technical field [0001] The present invention relates to a method for producing a physiologically active polypeptide conjugate, wherein a physiologically active polypeptide and a non-peptidyl polymer are linked to each other via a covalent bond. The method improves the overall yield of a physiologically active polypeptide conjugate by improving the reaction of the non-peptidyl polymer with the physiologically active polypeptide; and particularly, the method performs a two-step reaction by selective precipitation to obtain Preparation of physiologically active polypeptide conjugates with high yield. Background technique [0002] Peptides tend to be easily denatured due to their low stability and degraded by proteolytic enzymes in vivo, thus losing activity. Peptides have a relatively small size and thus pass easily through the kidneys. Therefore, in order to maintain the blood level and efficacy of a drug including a peptide as a pharmaceutically active ingredient, it is nec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/107C07K1/30C07K19/00
CPCC07K14/605C07K2319/30A61K47/60A61K47/68C07K1/1077C07K1/30C07K19/00C07K14/52C07K14/59C07K14/62
Inventor 文瑄真申清星洪性熙金大振权世昌
Owner HANMI PHARMA
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