Physiologically active polypeptide conjugate having prolonged in vivo half-life

a polypeptide and in vivo half-life technology, applied in the field of long-acting proteins having a prolonged in vivo half-life, can solve the problems of reducing the activity and production yield of an active substance, affecting the activity of the active substance, so as to prolong the circulating half-life of a physiologically active polypeptide, and enhance the in vivo stability

Inactive Publication Date: 2005-08-11
HANMI PHARMA
View PDF0 Cites 40 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009] Accordingly, a primary object of the present invention is to provide a protein conjugate having a prolonged in vivo half-life of a physiologically active polypeptide without inducing an immune response in a subject, while minimizing the reduction in the polypeptide's activity.
[0012] A still further object of the present invention is to provide a method for enhancing the in vivo stability and prolonging the circulating half-life of a physiologically active polypeptide, without sacrificing the activity thereof.

Problems solved by technology

Polypeptides are susceptible to denaturation or enzymatic degradation in the blood, liver or kidney.
Moreover, since peptide drugs are usually administrated by infusion, frequent injection of peptide drugs causes considerable discomfort to a subject.
However, such pegylated polypeptides have the disadvantages of lowering both the activity and production yield of an active substance as the molecular weight of PEG increases.
However, the stability of the modified G-CSF-PEG-albumin is merely 4 times higher than that of authentic G-CSF and, thus, it has not yet been put to practical use.
However, a fusion protein coupled with albumin still has the problem of reduced activity.
In addition, the use of chemical compounds such as carbodiimide, glutaraldehyde, acid chloride, etc. as a cross-linking agent is disadvantageous due to their potent toxicity and non-specificity of reaction.
Although there have been many attempts to combine a physiologically active polypeptide with various macromolecules, all have failed to simultaneously increase the stability and the activity.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Physiologically active polypeptide conjugate having prolonged in vivo half-life
  • Physiologically active polypeptide conjugate having prolonged in vivo half-life
  • Physiologically active polypeptide conjugate having prolonged in vivo half-life

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of hGH-PEG-IgG conjugate I

(Step 1) Preparation of hGH-PEG Complex

[0061] Human growth hormone (hGH, M.W. 22 kDa) was dissolved in 100 mM phosphate buffer solution to a concentration of 5 mg / ml, and polyethylene glycol containing aldehyde groups at both ends (ALD-PEG-ALD, Shearwater Inc, USA) which has a molecular weight of 3.4 kDa was added to the resulting buffer solution in an amount corresponding to an hGH:PEG molar ratio of 1:1, 1:2.5, 1:5, 1:10 or 1:20. Sodium cyanoborohydride (NaCNBH3, Sigma) was added thereto to a final concentration of 20 mM as a reducing agent, and the reaction mixture was stirred at 4° C. for 3 hours. To separate an hGH-PEG complex in which PEG is selectively linked to the terminal amino residue of hGH in a molar ratio of 1:1, the reaction mixture was subjected to Superdex® (Pharmacia, USA) size exclusion chromatography. The hGH-PEG complex was eluted and purified from the column with 10 mM potassium phosphate buffer (pH 6.0) to remove contam...

example 2

Preparation of hGH-PEG-IgG Conjugate II

(Step 1) Preparation of I2G-PEG Complex

[0064] IgG (Green Cross, Korea) was dissolved in 100 mM phosphate buffer to a concentration of 15 mg / ml, and 3.4 kDa of ALD-PEG-ALD (Shearwater Inc, USA) was added to the resulting buffer solution in an amount corresponding to an IgG:PEG molar ratio of 1:1, 1:2.5, 1:5, 1:10 or 1:20. NaCNBH3 was added thereto to a final concentration of 20 mM as a reducing agent, and the reaction mixture was stirred at 4° C. for 3 hours. To separate IgG-PEG complex in which PEG is selectively linked to the terminal amino residue of IgG in a molar ratio of 1:1, the reaction mixture was subjected to Superdex® (Pharmacia, USA) size exclusion chromatography. The IgG-PEG complex was eluted and purified from the column with 10 mM potassium phosphate buffer (pH 6.0) to remove contaminants such as unmodified IgG, unreacted PEG and dimmeric by-products having two molecules of IgG linked at both ends of PEG. The purified IgG-PEG c...

example 3

Preparation of IFN α-PEG-IgG Conjugate

[0066] An IFN α-PEG-IgG conjugate was prepared and purified according to the same method described in Example 1, except that interferon alpha 2b (IFN α 2b, M. W. 20 kDa) was employed instead of hGH and the IFN α 2b:ALD-PEG-ALD (M.W. 3.4 kDa) molar ratio was 1:5.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
concentrationaaaaaaaaaa
pHaaaaaaaaaa
Login to view more

Abstract

A protein conjugate having a prolonged in vivo half-life of a physiological activity, comprising i) a physiologically active polypeptide, ii) a biocompatible non-peptidic polymer, and iii) an immunoglobulin, is useful for the development of a peptide drug due to the enhanced in vivo stability and prolonged half-life in blood, while reducing the possibility of inducing an immune response.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a long acting protein having a prolonged in vivo half-life and a preparation method thereof. BACKGROUND OF THE INVENTION [0002] Polypeptides are susceptible to denaturation or enzymatic degradation in the blood, liver or kidney. Because of the low stability of polypeptides, it has been required to administer a peptide drug in a sustained frequency to a subject in order to maintain an effective plasma concentration of the active substance. Moreover, since peptide drugs are usually administrated by infusion, frequent injection of peptide drugs causes considerable discomfort to a subject. Thus, there have been many studies to develop a peptide drug which has an increased circulating half-life in the blood, while maintaining a high pharmacological efficacy thereof. Such desirous peptide drugs should also meet the requirements of enhanced serum stability, high activity, applicability to various polypeptides and a low probabil...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(United States)
IPC IPC(8): A61K47/48C07K16/00C07K19/00
CPCA61K47/48215A61K47/48284C07K16/00A61K47/48692A61K47/48415A61K47/60A61K47/643A61K47/6811A61K47/6883C07K19/00
Inventor KIM, YOUNG-MINKIM, DAE-JINBAE, SUNG-MINLIM, CHANG-KIKWON, SE-CHANGLEE, GWAN-SUN
Owner HANMI PHARMA
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap