Preparation method and separation and purification method of polyethylene glycol single modified recombinant human granulocyte-colony stimulating factor

A solution and buffer technology, applied in the field of biomedicine, can solve the problems of low product purity and specific activity, and it is difficult to be completely removed by purification, so as to achieve the effects of cost saving, purity improvement and simplification of steps.

Inactive Publication Date: 2012-06-06
SHANDONG NEWTIME PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the production process reported in the literature, a common problem is that the purity and specific activity of the product obtained by separation and purification after the PEGylation reaction are very low. Existence, making it difficult to completely remove in later purification

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation of embodiment 1.PEG-rhG-CSF

[0028] Take 500 mL of recombinant human granulocyte colony-stimulating factor solution, adjust the protein concentration to 5 mg / mL, pH 4.0 with pH 4.0, 10 mmol / L phosphate buffer;

[0029] Add the catalyst sodium triacetoxyborohydride with a final concentration of 20mmol / L into the adjusted rhG-CSF solution, and stir to dissolve it rapidly;

[0030] Calculated according to the molar mass ratio of PEG:rhG-CSF of 3:1, weigh monomethoxypolyethylene glycol butyraldehyde (20KDa), add the above solution and stir to make it dissolve rapidly, and stir and react at 4°C in the dark; After 4h, 8h, 12h, 16h, and 20h of reaction, samples were taken for HPLC to detect the PEG modification efficiency and double-modified product content. After 24h, cystine with a final concentration of 40mmol / L was added to terminate the reaction to obtain PEG-rhG-CSF.

[0031] Table 1 The detection results of different reaction times

[0032] g...

Embodiment 2

[0034] Embodiment 2. Preparation of PEG-rhG-CSF

[0035]Take recombinant human granulocyte colony-stimulating factor solution 1000mL, use pH3.8, 20mmol / L NaAc-HAc buffer to adjust the protein concentration to 2mg / mL, and adjust the pH to 3.8;

[0036] Add the catalyst sodium triacetoxyborohydride with a final concentration of 10mmol / L into the adjusted rhG-CSF solution, and stir to dissolve it rapidly;

[0037] Calculated according to the molar mass ratio of PEG:rhG-CSF of 8:1, weigh PEG (5KDa), add the above solution and stir to make it dissolve rapidly, stir and react at 25°C in the dark, and add PEG with a final concentration of 60mmol / L after 4h Cystine terminates the reaction to yield PEG-rhG-CSF.

[0038] Under the condition of PEG:rhG-CSF modification ratio of 8:1, the reaction time was 4 hours, the single modification efficiency reached 71.50%, and the double modification product was only 2.21%.

Embodiment 3

[0039] The preparation of embodiment 3.PEG-rhG-CSF

[0040] Take 500 mL of recombinant human granulocyte colony-stimulating factor solution, adjust the protein concentration to 10 mg / mL, and pH to 5.0 with NaAc-HAc buffer solution of pH 5.0 and 15 mmol / L;

[0041] Add the catalyst sodium triacetoxyborohydride with a final concentration of 40mmol / L into the adjusted rhG-CSF solution, and stir to dissolve it rapidly;

[0042] Calculated according to the molar mass ratio of PEG:rhG-CSF of 2:1, weigh PEG (10KDa), add the above solution and stir to make it dissolve rapidly, stir and react at 15°C in the dark, and add PEG with a final concentration of 20mmol / L after 22h Cystine terminates the reaction, resulting in a PEG-rhG-CSF mixture.

[0043] Under the condition of PEG:rhG-CSF modification ratio of 2:1, the reaction time was 22 hours, the single modification efficiency reached 71.90%, and the double modification product was only 2.26%.

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Abstract

The invention belongs to the field of biomedicine, specifically relates to polyethylene glycol modification and separation and purification of protein and especially relates to preparation and purification of a polyethylene glycol single modified recombinant human granulocyte-colony stimulating factor (PEG-rhG-CSF). According to optimized modification on a reaction system and reaction conditions, single modified PEG-rhG-CSF larger than 70% and multiple modified PEG-rhG-CSF less than 2.3% are obtained; according to control on technological parameters in the separation and purification process, single modified PEG-rhG-CSF with high purity and high activity are obtained, and a yield of the PEG-rhG-CSF reaches higher than 85%. The purification method of the invention employs a one-step cation exchange chromatography to simplify steps and save cost, and is suitable for industrialized large-scale production.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to protein pegylation modification and separation and purification, and more specifically, relates to a preparation and separation and purification method of recombinant human granulocyte colony-stimulating factor modified with polyethylene glycol. Background technique [0002] Recombinant Human Granulocyte-Colony Stimulating Factor (rhG-CSF) is a hematopoietic growth factor that promotes the formation of granulocyte colonies. proliferation, differentiation. For mature neutrophils, it can promote migration, phagocytosis, enzyme production, release of active oxygen, bactericidal ability and adhesion to foreign bodies. It also mobilizes mature neutrophils from the bone marrow to the periphery. The earliest indication of rhG-CSF is to promote the recovery of neutrophils after cancer chemotherapy and bone marrow transplantation, and then gradually expand to neutropenia caused by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/113C07K14/535
Inventor 赵志全刘忠张培彪
Owner SHANDONG NEWTIME PHARMA
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