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METHOD FOR PREPARING GLY-T[beta]4

A technology of thymosin and buffer solution, applied in the field of preparation of Gly-Tβ4, can solve the problems of formation of culture medium precipitate and acetate accumulation, low proliferation rate, high unit cost, etc., and achieve the effect of excellent industrial practical value.

Pending Publication Date: 2021-05-14
HUONS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has been pointed out that a large amount of protein is lost in the purification and the Gly-Tβ4 obtained by the above process ends up having a low productivity
In addition, when fermenting GST-Tβ4 was recorded according to a conventional 30 L culture batch manufactured by Northland Biotech in Beijing, China (hereinafter referred to as "NR culture method"), it was also pointed out that there were conditions such as Disadvantages of formation of media precipitates and acetate accumulation and low productivity
In addition, when purified according to the purification method of Northland Biotechnology Co., Ltd., there are many problems for use as a drug due to low proliferation rate, high unit cost and low purity of the final product

Method used

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  • METHOD FOR PREPARING GLY-T[beta]4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment (1

[0180] Embodiment (1) bacterial strain information

[0181] Escherichia coli BL21 strain was used to prepare a recombinant strain producing GST-fused human thymosin β4 (GST-Tβ4), and RCB was prepared for use in the present invention (Table 1)

[0182] Table 1

[0183]

[0184] Example (2) confirms the repeatability of China's Northland process

[0185] (2-1) 5L scale culture

[0186] Referring to the 30L culture batch record of Beijing Northland Biotechnology Co., Ltd. (hereinafter referred to as "NR process"), the conditions for the 5L scale-down culture process (Tables 2, 3 and 4) were derived and the first to 4th cultivation.

[0187] Table 2

[0188]

[0189]

[0190] Culture Conditions for 5L Validation Run

[0191] table 3

[0192]

[0193]

[0194] Media Composition for 5L Validation Run

[0195] Table 4

[0196] composition Preparation standard (L -1 )

ferric chloride 16.0g Cobalt dichloride 2.0g Copper chloride ...

Embodiment (3

[0208] Example (3): Development of a fed-batch fermentation process for increasing GST-Tβ4 productivity

[0209] It was confirmed that the above-mentioned NR process has several problems, such as low productivity, formation of medium precipitates and accumulation of acetate. Therefore, it is aimed to develop basic medium composition and process conditions that can solve the above-mentioned problems.

[0210] (3-1) Flask culture (batch culture)

[0211] Flask cultivation experiments (flasks 1, 2) were performed according to the presence or absence of yeast extract (hereinafter referred to as "YE") in the medium composition of the BNS platform (Tables 7 and 8). Strain-specific growth parameters for fed-batch process development were determined, and seed culture conditions for primary culture in fermentors were determined.

[0212] Table 7

[0213]

[0214] Flask Test Medium Composition

[0215] Table 8

[0216]

[0217] Flask test culture conditions

[0218] (3-2) 5L...

Embodiment

[0257] The optimal condition scale-up of embodiment (4) 50L scale process (sample production)

[0258] (4-1) Conditions of culture process

[0259] By scaling up the optimal conditions for developing a 5L scale process, a 50L culture process for sample production was performed (Tables 18 and 19).

[0260] Table 18

[0261]

[0262]

[0263] Conditions for optimized 50L scale-up culture

[0264] *Incubation temperature and pH are the same as BNS-platform conditions.

[0265] **Reflects the maximum stirring speed of 75L equipment (700rpm)

[0266] Table 19

[0267]

[0268]

[0269] The medium composition of 50L optimal condition scale-up culture

[0270] (4-2) Recycling conditions

[0271] (4-2-1) Cytoplasm recovery

[0272] (A) Recovery of culture medium

[0273] - Recovery time: Overnight after cooling the culture solution (≤15.0°C)

[0274] - Recovery volume: 40 to 42L

[0275] (B) Recovery of cytoplasm (continuous centrifugation)

[0276] ①The first r...

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Abstract

The present invention relates to a method for preparing glycine-thymosin [beta]4 (Gly-T[beta]4). The present invention has an excellent effect in that, according to the present invention, a large amount of high-purity Gly-T[beta]4 can be obtained with high productivity through chromatography, enzyme treatment, and filtration of a sample containing GST fusion thymosin [beta]4 (GST-T[beta]4).

Description

technical field [0001] The present invention relates to a method for preparing glycine-thymosin β4 (Gly-Tβ4), more specifically, relates to a method for preparing Gly-Tβ4, which comprises expressing GST fusion recombinant human thymus as the precursor of Gly-Tβ4 Gly-Tβ4 (hereinafter referred to as "GST-Tβ4") expression process and Gly-Tβ4 purification process. Background technique [0002] Large-scale protein purification is a problem that is becoming increasingly important in the biotechnology industry. Typically, proteins are produced by cell culture using mammalian or bacterial cell lines engineered to produce the target protein by insertion of a recombinant plasmid containing the gene for the target protein. Since the cell lines used herein are living organisms, the cells will be provided with a complex medium comprising carbohydrates, amino acids and growth factors, usually provided by animal serum preparations. It is very difficult to isolate a desired protein in suf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/575C07K1/12C07K1/18C07K1/22C07K1/34C07K1/36C12P21/00
CPCC07K14/57581C07K2319/23C07K1/12C07K1/18C07K1/22C07K1/34C07K1/36C12P21/00C12P21/02
Inventor 金渶睦张道水姜锡瓒金完燮严基安
Owner HUONS
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