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Compound containing immunoglobulin fc fragment and granulocyte-macrophage colony-stimulating factor and pharmaceutical composition thereof

A colony stimulating factor and immunoglobulin technology, which can be used in drug combinations, medical preparations containing active ingredients, extracellular fluid diseases, etc. and the effect of swallowing

Active Publication Date: 2016-08-03
BEIJING HANMI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although articles and patents on genetic modification have been reported from time to time, such as patent CN95101619, it only modifies the N-terminal gene and stop codon, and this optimization is far from enough to obtain high expression of the product

Method used

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  • Compound containing immunoglobulin fc fragment and granulocyte-macrophage colony-stimulating factor and pharmaceutical composition thereof
  • Compound containing immunoglobulin fc fragment and granulocyte-macrophage colony-stimulating factor and pharmaceutical composition thereof
  • Compound containing immunoglobulin fc fragment and granulocyte-macrophage colony-stimulating factor and pharmaceutical composition thereof

Examples

Experimental program
Comparison scheme
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example

[0050] The present invention will be described in further detail below in conjunction with examples, but they are not intended to limit the present invention.

example 1

[0051] Example 1 Construction of expression vector and screening of high-expressing strains

[0052]In order to make GM-CSF highly expressed in E. coli, the GM-CSF gene (SEQ ID No: 4) was redesigned and synthesized, including: reducing the GC content of the upstream gene and reducing the possibility of secondary structure formation; and for the whole GM-CSF The gene was optimized, and the rare codon was replaced with the preferred codon of E. coli, and the stop codon was also designed as TAA preferred by E. coli; at the same time, an NdeI restriction site was placed in front of the start codon of the gene, and the stop codon was A BamHI restriction site is added after the sub.

[0053] Then, the synthesized and sequenced gene of SEQ ID No: 4 was double digested with NdeI (New England Biolabs, Cat. R0111S) and BamHI (New England Biolabs, Cat. R0136S) restriction enzymes, and recovered. At the same time, the vector pET22b(+) (Novagen, Cat: 69744-3) was also double digested with...

example 2

[0056] Example 2 Fermentation tank production

[0057] One of the glycerol bacteria obtained in Example 1 was inoculated into 50ml of LB / Amp+ basal medium, and after culturing for 12 hours at 37°C 200rpm, 1.8ml of the culture solution was inoculated into 180ml of LB / Amp+ basal medium, and under the condition of 37°C and 200rpm Incubate for 16h. 180ml of seed bacteria were all inoculated into a 5L fermenter containing sterilized 1.7L basal medium LB. During the fermentation process, the culture temperature was 37° C., pH 6.8, the initial aeration rate was 2 L / min, and the initial stirring speed was 200 rpm. The stirring speed was adjusted according to the dissolved oxygen. When the dissolved oxygen dropped to 0, the stirring speed was increased by 50 rpm each time. After incubation for 3 hours, the stirring speed was not changed after the rotation speed was increased to 800 rpm. The culture was continued for 1 hour when the pH began to rise, and the feed rate was adjusted by...

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Abstract

The invention discloses a compound containing an immunoglobulin Fc segment and a granulocyte macrophage-colony stimulating factor and a pharmaceutical composition of the compound. The immunoglobulin Fc segment is connected with the granulocyte macrophage-colony stimulating factor through a chemical molecule, wherein the chemical molecule can be a polyethylene glycol polymer. According to the compound and the pharmaceutical compositions of the compound, the serum half-life of the granulocyte macrophage-colony stimulating factor can be remarkably prolonged while the higher level biological activity of the granulocyte macrophage-colony stimulating factor is maintained. Compared with a traditional PEG (Polyethylene Glycol)-granulocyte macrophage-colony stimulating factor, the compound has better uniformity.

Description

technical field [0001] The present invention relates to a complex containing immunoglobulin Fc fragment and granulocyte-macrophage colony stimulating factor and its pharmaceutical composition. Background technique [0002] Granulocyte-macrophage colony-stimulating factor (GranulocyteMacrophage-Colony Stimulating Factor, referred to as GM-CSF) belongs to the cytokine class recombinant protein, since Wong et al. cloned the cDNA sequence of GM-CSF in 1985 (WongG Getal., Science, 1985, 228:810-815), successively different kinds of GM-CSF analogs were cloned (US 5,391,485, US 5,942,253 and US 6,120,807). The development of genetic engineering technology makes it possible to express GM-CSF on a large scale. Clinically, GM-CSF mainly acts on hematopoietic progenitor cells to promote their proliferation and differentiation. Its important role is to stimulate the maturation of granulocytes and monocyte-macrophages, to promote the release of mature cells into peripheral blood, and to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K17/08A61K38/19A61K47/48A61P7/00A61P7/06A61K47/60
Inventor 连忠辉杜伯雨周小平张世奇阚洪囡周筠李钟守文圣焕
Owner BEIJING HANMI PHARMA CO LTD
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