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Conjugates of human insulin and analogues with sustained hypoglycemia and high receptor binding

A technology of recombinant human insulin and conjugates, applied in the direction of insulin, animal/human proteins, hormone peptides, etc.

Active Publication Date: 2015-09-16
CHONGQING FAGEN BIOMEDICAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Novo Nordisk's long-acting insulin detemir is coupled to the 29th lysine in the B chain of insulin analogs by fatty acid myristic fatty acid (CN101784563A, CN101389650A and EP2008 / 060734), which has obvious hypoglycemic effect in vivo, and can achieve hypoglycemic effect in the human body 24 hours, but the hypoglycemic effect is only maintained within 12 hours in animals

Method used

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  • Conjugates of human insulin and analogues with sustained hypoglycemia and high receptor binding
  • Conjugates of human insulin and analogues with sustained hypoglycemia and high receptor binding
  • Conjugates of human insulin and analogues with sustained hypoglycemia and high receptor binding

Examples

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Effect test

example 1

[0030] Example 1. Recombinant expression and purification of recombinant human insulin analog (DesB30)

[0031] In this example, according to the amino acid sequence of the human insulin analog DesB30 (Seq ID NO.2), the corresponding cDNA sequence of the yeast preferred codon is designed, which contains the leader peptide sequence EVFK and the middle short C peptide AAK corresponding to The cDNA sequence contains the restriction endonuclease Xho I and Not I sites at the 5' end and the 3' end respectively. The entire cDNA sequence is as follows (Seq ID NO.3):

[0032]CTCGAGAAGAGAGAAGTCTTCAAGTTTGTTAACCAACATTTGTGTGGTTCCCACTTGGTTGAAGCTTTGTACTTGGTTTGTGGTGAAAGAGGTTTCTTCTACACTCCAAAGGCTGCTAAGGGTATCGTTGAACAATGTTGTACTTCCATCTGTTCCTTGTACCAATTGGAAAACTACTGTAACTGATAAGCGGCCGC,委托宝生物(大连)有限公司人工合成Seq ID NO.3的cDNA基因片段,并嵌入pUC18载体且命名为pUC18-DesB30。 Plasmid pUC18-DesB30 and expression vector plasmid pICZαA were double-digested with restriction endonucleases Xho I and Not I, recovered by gel, ligated ...

example 2

[0037] Example 2. Preparation of recombinant human insulin analogue conjugate (DesB30-5K)

[0038] Take 100ml of recombinant human insulin analog (DesB30) solution (containing 20mmol / L Tris-HCl) with a concentration of 2mg / ml, adjust the pH to 10.5, stir at 100rpm at room temperature, first add mPEG-SPA ( The molecular weight is 5K, purchased from Beijing Jiankai Technology Co., Ltd.), after reacting for 30 minutes, add mPEG-SPA at a molar mass ratio of 1:1.5, continue to react for 30 minutes, and adjust the pH to 3.0 with 1% trifluoroacetic acid to terminate the reaction , The modified sample was subjected to reverse-phase preparative C18 column chromatography to separate and remove unmodified DesB30, free PEG molecules, and other conjugates not modified at the B29 position. The separation conditions are: liquid A is 0.1% trifluoroacetic acid and 5% acetonitrile, liquid B is 0.1% trifluoroacetic acid and 95% acetonitrile, the DesB30-5K conjugate is collected by linear gradien...

example 3

[0039] Example 3. Preparation of recombinant human insulin analogue conjugate (DesB30-10K)

[0040] Take 100ml of recombinant human insulin analogue (DesB30) solution (containing 20mmol / L Tris-HCl) with a concentration of 2mg / ml, adjust the pH to 10.5, stir at 100rpm at room temperature, first add mPEG-SPA ( The molecular weight is 10K, purchased from Beijing Jiankai Technology Co., Ltd.), after reacting for 30 minutes, add mPEG-SPA at a molar mass ratio of 1:2, continue to react for 30 minutes, adjust the pH to 3.0 with 1% TFA to terminate the reaction, modify Afterwards, the sample was subjected to reverse-phase preparative C18 column chromatography to separate and remove unmodified DesB30, free PEG molecules, and other conjugates not modified at the B29 position. The separation conditions are: liquid A is 0.1% trifluoroacetic acid and 5% acetonitrile, liquid B is 0.1% trifluoroacetic acid and 95% acetonitrile, the DesB30-10K conjugate is collected by linear gradient elution...

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Abstract

Disclosed is a recombinant human insulin or analog conjugate thereof. Said conjugate is formed by connecting the µ-amino group of lysine 29 of the B chain to activated polyethylene glycol by means of an amide bond. Further disclosed is a method of preparing said conjugate, a pharmaceutical compound, and a use for said conjugate in preparing medication for treating Type 1 and Type 2 diabetes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to the recombinant preparation, modification, purification and application of human insulin and analogues. Background technique [0002] Insulin is a protein secreted by pancreatic beta cells stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc. It contains 51 amino acids and has a molecular weight of about 5800. Insulin The molecule has an A chain (21 amino acids) and a B chain (30 amino acids) bound by two disulfide bonds. If the disulfide bond is opened, it will lose its activity. B cells first synthesize a macromolecule of pre-proinsulin, which is then processed into proinsulin of octahexapeptide, which is then hydrolyzed into insulin and connecting peptide. The main physiological function of insulin is to regulate the metabolic process: 1. Promote the uptake and utilization of glucose by tissue ce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/62A61K47/48A61K38/28A61P3/10
CPCC07K14/62A61K38/28A61K47/48215A61K47/60A61P3/10
Inventor 范开曹宇陈勇李红亮陈海容万颖
Owner CHONGQING FAGEN BIOMEDICAL
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