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Preparation method and purification method of human serum albumin

A technology of human serum albumin and purification method, which is applied in the preparation methods of serum albumin and peptides, albumin peptides, etc., can solve the problem that the proportion recovery rate of the final product is not very ideal, the culture medium is expensive, and it is easy to introduce animal origin. virus, etc.

Pending Publication Date: 2020-09-15
SHANGHAI INST OF PHARMA IND CO LTD +1
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0008] In order to overcome the Pichia pastoris engineering bacterium (Pichia pastoris) expressing human serum albumin in the prior art, in the process of producing human serum albumin, while ensuring production efficiency, the output of human serum albumin, human serum albumin The proportion of the total protein, the recovery rate of downstream separation and purification and the purity of the final product are not very ideal, and the culture medium used in the production process is relatively expensive, and it is easy to introduce animal-derived viruses. A human serum albumin Preparation method and purification method of human serum albumin

Method used

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  • Preparation method and purification method of human serum albumin
  • Preparation method and purification method of human serum albumin
  • Preparation method and purification method of human serum albumin

Examples

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Effect test

Embodiment 1

[0085] Embodiment 1: four kinds of culture medium yield comparison experiment on 5L fermentor tank level

[0086] Comparing the human serum albumin production of BMGY medium, BSM medium, 1 / 2BSM medium, 1 / 4BSM medium on the level of 5L fermenter, the components of the medium are as follows:

[0087] BMGY (glycerol buffer compound medium): 20g / L tryptone, 10g / L yeast extract, 40g / L glycerol, 10g / L ammonium sulfate, 3.4g / L yeast basic nitrogen base, 0.1M potassium phosphate buffer ( pH6.0), 0.0004g / L biotin. The price is about 50 yuan / liter.

[0088] BSM (basic salt medium): 40g / L glycerol, 26.7mL / L phosphoric acid (85%), 0.93g / L calcium sulfate dihydrate, 18.2g / L potassium sulfate, 14.9g / L magnesium sulfate heptahydrate, 4.13g / L potassium hydroxide, 4.0mL / L PTM1 (Pichia pastoris trace element 1, the specific components are: 6g / L copper sulfate pentahydrate, 0.08g / L sodium iodide, 3g / L manganese sulfate monohydrate, 0.2g / L dihydrate and sodium molybdate, 0.02g / L boric acid, ...

Embodiment 2

[0108] Embodiment 2: Separation and purification of human serum albumin

[0109] In this example, the supernatant of the fermentation broth induced in 1 / 2 BSM medium for 192 hours was filtered through a 0.45 μm microporous membrane, and the filtrate was used as the raw material solution for separation and purification. When performing Blue Sepharose 6FF affinity chromatography, the pH of the loading buffer was selected as 5.

[0110] Prepare affinity chromatography solution A [25mM KPB (potassium phosphate buffer) + 0.1M NaCl] with different pH (5.0, 6.0, 7.0, 8.0 and 9.0), mix it with the fermentation supernatant at 9:1 and load it And use pH 7.0 affinity chromatography B liquid (25mM KPB+2.0M NaCl) to carry out elution, collect flow-through fluid and eluate and carry out protein gel electrophoresis, the result is as follows Figure 14 shown. It can be seen from the electrophoresis results that with the increase of the loading pH (the pH value of the loading solution when l...

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Abstract

The invention provides a preparation method of human serum albumin. The preparation method comprises the following steps: inoculating pichia pastoris for producing human serum albumin into a culture medium, performing fermenting, and obtaining the human serum albumin from fermentation liquor. The culture medium is a BSM culture medium in which the concentrations of other components are reduced to1 / 4 at most except glycerol and pichia pastoris microelements 1, and preferably, the concentrations of other components are reduced to 1 / 4-1 / 2. The invention also provides a purification method of thehuman serum albumin. The yield of the human serum albumin prepared by the preparation method and the proportion of the human serum albumin in total protein are remarkably higher than those in the prior art, cost of a culture medium used in the preparation method is remarkably reduced, and the risk of animal-derived virus pollution is avoided, so that cost of the preparation method is remarkably reduced. According to the purification method, the recovery rate of purification is remarkably increased, and the purity of a final product is remarkably and further improved.

Description

technical field [0001] The invention relates to a preparation method of human serum albumin and a purification method thereof. Background technique [0002] Human serum albumin (HSA) is the most abundant protein in human plasma, accounting for 60% of total serum protein, about 35-40g / L, and its main function is to maintain plasma osmotic pressure and transport various substances. HSA is widely used in shock and burn treatment, postoperative recovery, drug carrier, cell culture medium, etc. Its global demand has reached about 600 tons per year, and my country's demand accounts for about one-third of it. At present, human serum albumin is mainly extracted from human blood, and the corresponding purification process is low-temperature ethanol precipitation. The requirements also lead to its inefficiency. [0003] The use of transgenic technology to construct engineering bacteria and produce foreign proteins first originated in the 1970s. Since then, a series of foreign protei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12N15/81C07K14/765C07K1/22C12R1/84
CPCC12P21/02C07K14/765C12N15/815
Inventor 谢丽萍胡又佳朱文吴珺艺阮江雄韩姝徐磊
Owner SHANGHAI INST OF PHARMA IND CO LTD
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