Medical preparation containing recombinant human granulocytecolony stimulating factor

A technology of colony stimulating factor and pharmaceutical preparation, applied in the field of biopharmaceuticals, can solve problems such as difficulty in excision of methionine

Inactive Publication Date: 2010-07-07
BEIJING SL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The direct purpose of the present invention is to obtain the pharmaceutical preparation of recombinant human G-CSF containing the natural N-terminus, overcome the difficult problem of excision of N-terminal methionine in existing products, and improve product quality

Method used

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  • Medical preparation containing recombinant human granulocytecolony stimulating factor
  • Medical preparation containing recombinant human granulocytecolony stimulating factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1. Cloning of human granulocyte colony-stimulating factor without methionine at the N-terminus

[0019] The expression vector pBV220 was donated by the State Key Laboratory of Viral Genetic Engineering, Institute of Virology, Chinese Academy of Preventive Medicine. The recipient strain DH5α is a strain preserved by our company, and its genotype is SupE44△lac U169(80lacZ△M15)hsdR17recALend Al gyr A96thi-1rel AL. Plasmid DNA extraction and purification: refer to the alkaline denaturation method in "Molecular Cloning" edited by Sambeook J et al. (Chinese version, second edition, translated by Jin Dongyan et al., p16).

[0020] Primer design: through computer-aided PCR primer design, the two primers have no hairpin structure, no complementarity of more than 4 bases, and the upstream primer (G+C) is 47.6%, which is an ideal primer design.

[0021] The upstream primers were: - AAGCTT ATG CCA TTA GGC CCT-3'. The first code was originally CCC, but because of the low f...

Embodiment 2

[0028] Embodiment 2. fermentation purification

[0029] Seed medium formula: 1000mL contains 5g of yeast powder, 10g of peptone, 10g of sodium chloride, 2g of glucose, adjust the pH to 7.2-7.4 with sodium hydroxide, and autoclave at 121°C for 20 minutes. Fermentation medium formula: 5g of peptone per liter, Na 2 HPO 4 2H 2 O 6g, KH 2 PO 4 3g, NH 4 CL 1g, NaCl 0.5g, MgSO 4 ·7H 2 O 0.13g, glucose 4g (the latter two components need to be autoclaved separately). The above medium was autoclaved at 115°C for 30 min.

[0030] Seed culture:

[0031] The primary seeds were shaken and cultivated at 30°C and 140r / min for 10h, then transferred to the second-grade seeds with 0.5% inoculum, 30°C and 140r / min were shaken overnight (15h), and transferred to the fermenter with 5% amount.

[0032] Fermenter culture:

[0033] Incubate at 30°C for 3-4h, OD 600 When >1.5, increase the culture temperature to 42°C, continuously aerate for 3.5 hours, and finally collect the bacteria by c...

Embodiment 3

[0056] Example 3 Activity Determination of Recombinant Human Granulocyte Colony Stimulating Factor

[0057] The biological activity of G-CSF was determined by MTT colorimetry. The specific activity that embodiment 1 sample records is 8 * 10 7 IU / mg. The specific method is as follows:

[0058] Reagent: RPMI1640 culture solution: Add 2.0g sodium bicarbonate, 2.383g HEPES, 0.2923L-glutamine per liter, filter and sterilize through a 0.22μm filter membrane. Store at 4°C; basal culture medium: take 100ml of fetal bovine serum (FBS), add it to 900ml RPMI1640 culture medium, store at 4°C; complete culture medium: add recombinant human granulocyte colony-stimulating factor to the basal culture medium to a final concentration of 20ng / ml or 3000IU / ml; Phosphate buffered saline (PBS): Weigh 8g sodium chloride, 0.2g potassium chloride, 1.44g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, add water to dissolve into 1000ml, after 121℃, 15 Sterilize by autoclaving in m...

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Abstract

The invention discloses a medical preparation containing a recombinant human recombination granulocytecolony stimulating factor (G-CSF) with the same structure as a natural human granulocytecolony stimulating factor and a preparation method thereof. The active ingredient of the preparation is G-CST with proline at the N terminal, is 173-numbered amino acids and is one of the natural structures of G-CST in human body. The preparation overcomes the defect that the N terminal of the existing recombinant human G-CSF product contains methionine. The invention also provides the method for preparing the preparation.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a pharmaceutical preparation of recombinant human granulocyte colony-stimulating factor and a preparation method thereof. Background of the invention [0002] Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that acts on bone marrow neutrophils to promote their proliferation and differentiation, promote the maturation and release of neutrophils, and enhance the function of mature neutrophils. Its early clinical indications are neutropenia after cancer chemotherapy and bone marrow transplantation, and then gradually expanded to neutropenia caused by various etiologies. [0003] With the development of molecular biology and genetic engineering technology, the production of drugs through protein recombination technology has been widely used. Cells for the production of exogenous proteins include eukaryotic cells, yeast and prokaryotic cells. Some prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/19A61P7/00C12N15/70C12P21/02C12R1/19
Inventor 徐明波梁果义杨仲璠连治国刘迎王俊玲赵紫岭倪娜何瑞峰李亚军齐燕明王勇波吴彦卓
Owner BEIJING SL PHARMA
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