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Novel stable preparation of recombinant human pancreatic glucagon-like peptide-1 analogue fusion protein

A technology of glucagon and fusion protein, applied in the field of new pharmaceutical preparations, can solve the problems of decreased protein activity, decreased therapeutic effect, toxic and side effects, etc.

Pending Publication Date: 2018-06-29
JIANGSU T MAB BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These changes can reduce the activity of the protein, reduce the therapeutic effect and even cause serious side effects

Method used

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  • Novel stable preparation of recombinant human pancreatic glucagon-like peptide-1 analogue fusion protein
  • Novel stable preparation of recombinant human pancreatic glucagon-like peptide-1 analogue fusion protein
  • Novel stable preparation of recombinant human pancreatic glucagon-like peptide-1 analogue fusion protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Preparation of recombinant human glucagon-like peptide-1 analogue fusion protein: the strain is inoculated in YPD medium, shaken at 220-280rpm at 30°C until the wet weight of the bacteria is about 50g / L, put into the tank according to 10% inoculation amount, and culture After 20 hours, the induction was started by continuous feeding of methanol. After 4 hours of induction, the temperature was lowered to 22°C. After 50 hours, the induction was terminated, and the fermentation supernatant was collected by centrifugation at 10000 g for 15 minutes. Purification adopts four-step chromatography of BLUE affinity, PHE hydrophobicity, DEAE ion exchange and gel exclusion. Firstly, the fermentation supernatant was diluted 3 times with 20mmol / L pH7.0 sodium phosphate solution, passed through the Blue Sepharose Fast Flow affinity chromatography column with 2M NaCl, 20mmol / L pH6.5 sodium phosphate solution to elute the target protein. Add (NH4) to the collected protein solution 2 SO...

Embodiment 2

[0031] The effect of pH on the stability of recombinant human glucagon-like peptide-1 analogue fusion protein composition was investigated.

[0032] The purity of recombinant human glucagon-like peptide-1 analogue fusion protein was evaluated by size exclusion HPLC. Samples were analyzed using a size exclusion column (7.8mm*30cm) with an isocratic mobile phase consisting of phosphate and sulfate. Purity was determined by calculating the ratio of the main peak area to the total peak area at a detection wavelength of 214 nm. Each group contained recombinant human glucagon-like peptide-1 analogue fusion protein 1.0mg / ml, mannitol 30mg / ml, histidine 10mg / ml, polysorbate 80 1mg / ml, 20mmol / L of different pH Citric acid / sodium citrate buffer or disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, pH 4.0 to pH 8.0, with intervals of 0.5 between groups. The samples of each group were sterile filtered and distributed into sterile pyrogen-free vials. Accelerated investigati...

Embodiment 3

[0037] The selection of embodiment 3 excipients

[0038] Some excipients and auxiliary materials suitable for human use were screened, and their effects on the formation of recombinant human glucagon-like peptide-1 analogue fusion protein preparations during freeze-drying were studied. In each group, the recombinant human glucagon-like peptide-1 analogue fusion protein solution was ultrafiltered into 20mmol / L pH 6.5 phosphate buffer, the protein concentration was 50.0mg / ml, and various excipients, types and The concentration is detailed in Table 2. After the sample solution was filtered and sterilized, it was divided into sterile vials with a volume of 1.0ml, half stoppered, and freeze-dried. The lyophilized products were visually inspected, and protein aggregates were detected by SEC-HPLC.

[0039] Table 2 Excipient results and SEC-HPLC results of several excipients after freeze-drying

[0040]

[0041] The results show that, from the excipient effect, mannitol is used ...

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Abstract

The invention describes a novel stable preparation of a recombinant human pancreatic glucagon-like peptide-1 analogue fusion protein. The medicinal preparation can be used for treating diabetes and related diseases.

Description

technical field [0001] The invention relates to a novel pharmaceutical preparation of recombinant human glucagon-like peptide-1 analogue fusion protein. Background technique [0002] Glucagon-like peptide-1 (GLP-1) analogs and derivatives thereof are useful in the treatment of type 2 diabetes. GLP-1 can induce a variety of biological effects, such as acting on pancreatic beta cells in a glucose-dependent manner, promoting the transcription of insulin genes, increasing the biosynthesis and secretion of insulin; stimulating the proliferation and differentiation of beta cells, inhibiting beta cell apoptosis, Thereby increasing the number of islet β cells, inhibiting the secretion of glucagon, suppressing appetite and feeding, and delaying the emptying of gastric contents. Therefore, GLP-1 analogues and derivatives thereof can maintain blood sugar at a constant level by improving metabolic disorders in patients with type 2 diabetes. [0003] GLP-1(1-37) activity is very low, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/38A61K38/26A61K9/19A61P3/10
CPCA61K38/26A61K38/385A61K47/26A61K9/19
Inventor 严守升顾迎迎陆游于东安郭斌任杰李雪松范敏
Owner JIANGSU T MAB BIOPHARMA
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