Preparation method and pharmaceutical composition of PEGylated recombinant human granulocyte colony-stimulating factor

A technology of PEGylation and stimulatory factors, which is applied in the field of preparation of recombinant human granulocyte stimulatory factors, can solve the problems of unfavorable industrial production, complicated operation steps, increased costs, etc., and achieve fewer types of reagents, simple process, and improved safety Effect

Inactive Publication Date: 2016-01-27
JIANGSU AOSAIKANG PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The method for preparing PEG-rhG-CSF known in the prior art has complex operation steps and high cost, which is not conducive to industrial production. For example, the method for preparing PEG-rhG-CSF disclosed in CN1824324A needs to replace the buffer at the beginning of the preparation , an additional reagent DMMAn (dimethylmaleic anhydride) needs to be added during the preparation process, and the reaction temperature reaches 37°C; the method for preparing PEG-rhG-CSF disclosed in CN101711876A needs to add a large amount of Glycine terminates the reaction, and three kinds of chromatographic columns such as S-200, DEAE and CM column need to be used in the purification process, which increases the cost of industrialization; the method for preparing PEG-rhG-CSF disclosed in CN102485742A uses Phosphate-buffered saline, terminated by the addition of cystine at the end of the reaction, and requires gradient elution during purification

Method used

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  • Preparation method and pharmaceutical composition of PEGylated recombinant human granulocyte colony-stimulating factor
  • Preparation method and pharmaceutical composition of PEGylated recombinant human granulocyte colony-stimulating factor
  • Preparation method and pharmaceutical composition of PEGylated recombinant human granulocyte colony-stimulating factor

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Experimental program
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Effect test

Embodiment 1

[0040] Take 1 L of mother solution of recombinant human granulocyte-stimulating factor (containing 1 grhG-CSF, 20 mmol NaAc and 50 mmol NaCl), cool down to 4 °C, and add 0.5 mol / L NaOH solution to adjust the pH to 6. Add methoxy-polyethylene glycol-propionaldehyde (mPEG-PAL, molecular weight about 20 kilodaltons), stir to dissolve, add sodium nitrile borohydride (NaBH3CN), and make the final concentration of sodium nitrile borohydride 15mmol / L, stirring at low speed for 15 hours, adding 0.5 mol / L hydrochloric acid solution to adjust the pH of the reaction solution to 3.

[0041] According to the different amounts of mPEG-PAL added, the ratio of rhG-CSF monomer, PEG single modification of rhG-CSF (PEG-rhG-CSF), PEG double modification of rhG-CSF, and PEG multi-modification of rhG-CSF in the reaction solution The ratio is shown in Table 1.

[0042] The reaction solution was purified by a chromatographic column, and the filler was SPSepharoseFF, which was first equilibrated in ...

Embodiment 2

[0046] Take 1 L of mother solution of recombinant human granulocyte-stimulating factor (containing 1 grhG-CSF, 20 mmol NaAc and 50 mmol NaCl), lower the temperature to 10° C., and add 1 mol / L NaOH solution to adjust the pH to 5.5. Add 7 g of methoxy-polyethylene glycol-propionaldehyde (mPEG-PAL, molecular weight about 20 kilodaltons), stir to dissolve, add sodium nitrile borohydride (NaBH3CN), stir at low speed for 18 hours, add 0.5 mol / L Hydrochloric acid solution adjusted the pH of the reaction solution to 4.

[0047] According to the final concentration of sodium nitrile borohydride, the rhG-CSF monomer, the PEG single modification of rhG-CSF (PEG-rhG-CSF), the PEG double modification of rhG-CSF, and the PEG multi-modification of rhG-CSF in the reaction liquid were different. The proportions of the substances are shown in Table 2.

[0048] The reaction solution was purified by a chromatographic column, and the filler was SPSepharoseFF, which was first equilibrated in the e...

Embodiment 3

[0052] Take 1 L of mother solution of recombinant human granulocyte-stimulating factor (containing 1 grhG-CSF, 20 mmol NaAc and 50 mmol NaCl), lower the temperature to 8°C, and add 1 mol / L NaOH solution to adjust the pH to 5-7. Add 6 g of methoxy-polyethylene glycol-propionaldehyde (mPEG-PAL, molecular weight about 20 kilodaltons), stir to dissolve, add sodium nitrile borohydride (NaBH CN), and make the final concentration of sodium nitrile borohydride 35 mmol / L, stirred at low speed for 16 hours, and added 1mol / L hydrochloric acid solution to adjust the pH of the reaction solution to 3.5.

[0053] According to the pH adjusted by the sodium hydroxide solution, the rhG-CSF monomer, the PEG single modification of rhG-CSF (PEG-rhG-CSF), the PEG double modification of rhG-CSF, and the PEG multi-modification of rhG-CSF in the reaction liquid The proportions of the substances are shown in Table 3.

[0054] The reaction solution was purified by a chromatographic column, and the fil...

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Abstract

The invention provides a preparation method of PEGylated recombinant human granulocyte colony-stimulating factor. The preparation method comprises following steps: (a), an alkali conditioning agent is used for adjusting pH value of a mother liquor containing recombinant human granulocyte colony-stimulating factor to a first pH value; (b), methoxy-polyethylene glycol-propionaldehyde is added into the mother liquor, and an obtained solution is stirred for dissolving; (c), NaBH3CN is added, and an obtained mixture is stirred for reaction; and (d) an acid conditioning agent is used for adjusting pH value of an obtained reaction liquid to a second pH value so as to stop reaction and obtain the PEGylated recombinant human granulocyte colony-stimulating factor. The preparation method is simple; buffer system of a reaction liquid is the same to buffer system of the mother liquor of the recombinant human granulocyte colony-stimulating factor, so that buffer solution replacing is avoided; reaction is stopped by adjusting pH value with hydrochloric acid; product purifying difficulty is reduced; and the preparation method is suitable for industrialized production. The kinds of added reagent are few; and safety of the finished products in clinical application is improved.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a preparation method of polyethylene glycol derivatized recombinant human granulocyte stimulating factor and a pharmaceutical composition containing the stimulating factor. Background technique [0002] Granulocyte stimulating factor (G-CSF) binds to surface receptors on hematopoietic cells to stimulate proliferation and prevent proliferation of functionally activated cells. The U.S. FDA approved recombinant human granulocyte-stimulating factor in 1991. The trade name is The generic name is Filgrastim. Filgrastim is a water-soluble protein containing 175 amino acids and a molecular weight of about 19 kilodaltons, which is fermented by E.coli. The amino acid sequence of Filgrastim is shown in SEQ ID No.1. [0003] The polyethylene glycol derivatization of G-CSF can reduce the plasma clearance rate and prolong the half-life. In 2002, the U.S. FDA approved the marketing of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/535C07K1/107C07K1/18A61K38/19A61K47/48A61P35/00
Inventor 陈庆财文勇鲍曼张超李文
Owner JIANGSU AOSAIKANG PHARMA CO LTD
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