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Preparation method of polyethylene glycol modified rhG-CSF

A polyethylene glycol and modifier technology, applied in the field of biomedicine, can solve the problems of increasing the cost of modification, the cost is not advantageous, and increasing the pressure of subsequent purification of process-related impurities, so as to achieve the effect of reducing production costs

Pending Publication Date: 2022-08-05
SHANDONG NEWTIME PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method will firstly increase the cost of modification, and secondly, it will increase the pressure of process-related impurities and subsequent purification. After commercial scale-up production, the cost is not advantageous, and there is still the possibility that the sulfhydryl group of the 18-position cysteine ​​will be modified.

Method used

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  • Preparation method of polyethylene glycol modified rhG-CSF
  • Preparation method of polyethylene glycol modified rhG-CSF
  • Preparation method of polyethylene glycol modified rhG-CSF

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] a. Sample loading: take an appropriate amount of chromatography filler Capto MMC, load it into an XK26 chromatography column, the column height is 20cm, and connect it to the protein purification system; use buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate 25mM) , sodium chloride 400mM, adjust pH to 4.5) to equilibrate the chromatography column, 3 to 5 column volumes, flow rate 10mL / min; use the above buffer A to prepare rhG-CSF into a solution with a concentration of 2mg / mL, and load the sample In the chromatography column, the loading capacity is 20 mg / mL. After the sample loading is completed, rinse the above buffer A for 3 to 5 column volumes until no protein penetrates.

[0049] b. Modification reaction: weigh mPEG-butyraldehyde (20kDa) according to the molar ratio of rhG-CSF:mPEG modifier=1:4, prepare it into a solution with a concentration of 2 mg / mL with the above buffer A, and add The final concentration is 20mM sodium cyanobor...

Embodiment 2

[0052] a. Loading: take an appropriate amount of the chromatography filler Capto MMC, load it in the XK26 chromatography column, the column height is 20cm, and connect it to the protein purification system; use buffer A (sodium acetate buffer containing sodium chloride, in which sodium acetate is used) 20mM, 400mM sodium chloride, adjusted to pH 4) to equilibrate the chromatography column, 3 to 5 column volumes, flow rate 10mL / min; rhG-CSF was prepared into a solution with a concentration of 3mg / mL with the above buffer A, and the sample was loaded on Chromatography column with a loading capacity of 20 mg / mL, and then rinsed with the above buffer A for 3 to 5 column volumes until no protein penetrated.

[0053] b. Modification reaction: weigh mPEG-propionaldehyde (20kDa) according to the molar ratio of rhG-CSF:mPEG modifier=1:2, prepare it into a solution with a concentration of 3 mg / mL with the above-mentioned buffer A, and add The final concentration is 10mM sodium cyanoboro...

Embodiment 3

[0056] a. Loading: take an appropriate amount of chromatography filler Capto MMC, load it into an XK26 chromatography column, the column height is 20cm, and connect it to the protein purification system; use buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate 30mM) , NaCl 400mM, adjust pH to 5) to equilibrate the chromatography column, 3 to 5 column volumes, flow rate 10mL / min; rhG-CSF was prepared into a solution with a concentration of 2mg / mL with the above buffer A, and the sample was applied to the layer The column was separated, with a loading capacity of 20 mg / mL, and then washed with the above buffer A for 3 to 5 column volumes until no protein penetrated.

[0057] b. Modification reaction: weigh mPEG-succinate (20kDa) according to the molar ratio rhG-CSF:mPEG modifier=1:10, prepare it into a solution with a concentration of 1 mg / mL with the above-mentioned buffer A, and Add sodium cyanoborohydride with a final concentration of 10 mM, and ...

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Abstract

The invention belongs to the field of biological medicine, and particularly relates to a preparation method of polyethylene glycol modified rhG-CSF. The method specifically comprises the following steps: by taking a composite chromatography medium as a solid-phase reaction carrier, loading an rhG-CSF protein sample and adsorbing the rhG-CSF protein sample on a chromatography filler, by taking a polyethylene glycol modifier as a mobile phase, completing a modification reaction under the condition of catalysis of a catalyst, and after the reaction is completed, carrying out gradient elution to obtain the polyethylene glycol modified protein mPEG-rhG-CSF. By adopting the preparation method, the mPEG-rhG-CSF modified protein with higher single modification rate and purity can be obtained, the single modification rate of the obtained mPEG modified protein reaches 91% or above, and the purity reaches 99.3% or above; and meanwhile, the preparation method simplifies modification and purification reaction operations, greatly reduces the production cost, provides a new process for realizing mPEG modification of the protein, and has a very good application prospect.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a preparation method of polyethylene glycol modified rhG-CSF. Background technique [0002] In the late 1950s, using chemical modification methods to study the relationship between the structure and function of protein molecules became a hot spot in the fields of biochemistry and molecular biology. The purpose of protein modification is for biomedical and biotechnological applications. In biomedicine, chemical modification can reduce immunogenicity or immunoreactivity, inhibit the production of immunoglobulin E, etc. In the field of biotechnology, after chemical modification, enzymes can efficiently catalyze in organic solvents, and show Novel catalytic properties. [0003] The nature of the reaction between protein modifiers and proteins can be mainly divided into four types: (1) acylation and related reactions, such modifiers can undergo acylation reactions with the sid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/535C07K1/107C07K1/22C07K1/04C07K1/14
CPCC07K14/535
Inventor 刘忠王磊刘文财
Owner SHANDONG NEWTIME PHARMA