Fermentation medium of pichia pastoris for improving expression of insulin precursor and fermentation method

A fermentation medium and Pichia pastoris technology, which is applied in the field of biopharmaceuticals, can solve the problems of difficulty in achieving a balance between product yield and quality, complicated operation process control methods, unstable medium component sources, etc., so as to shorten the pre-induction culture time, The effect of avoiding product quality and accelerating bacterial growth

Active Publication Date: 2020-07-14
BEIJING HUIZHIHENG BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] It can be seen that when the above medium or method is used to produce insulin precursors, it is difficult to achieve a balance between the output and quality of the product, the instability of the source of medium components, the increase of by-products when the cycle is extended, and the operation process control method is too complicated.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The Pichia fermentation medium of the present embodiment is: urea 8.0g / L, potassium dihydrogen phosphate 12.0g / L, dipotassium hydrogen phosphate 4.5g / L, calcium carbonate 0.9g / L, magnesium sulfate heptahydrate 15.0g / L L, ammonium sulfate 6.0g / L, ammonium dihydrogen phosphate 13.0g / L, ferrous sulfate 0.8g / L, glycerol 40.0g / L, PTM1 solution 4.0ml / L, solvent is water, pH 5.5.

[0035] Using the mixed feeding method of glycerol / methanol, follow the steps below to ferment Pichia pastoris to produce insulin precursor:

[0036] (1) Initial culture stage: insert the Pichia pastoris seed liquid into the fermentation medium, the inoculation ratio is 5%, and the inoculation OD 600 = 8, the initial culture conditions are 28°C, pH 5.0, and ventilation rate 0.5vvm. As the culture progresses, increase the rotation speed and ventilation rate accordingly, and control dissolved oxygen ≥ 30%. When the initial glycerol in the fermentation medium is exhausted, it can be judged that the DO ...

Embodiment 2

[0043] The Pichia fermentation medium of the present embodiment is: urea 5.0g / L, potassium dihydrogen phosphate 10.0g / L, dipotassium hydrogen phosphate 4.0g / L, calcium carbonate 0.9g / L, magnesium sulfate heptahydrate 15.0g / L L, ammonium sulfate 5.0g / L, ammonium dihydrogen phosphate 14.0g / L, ferrous sulfate 1.0g / L, glycerol 40.0g / L, PTM1 solution 4.0ml / L, solvent is water, pH 5.5.

[0044] Using the mixed feeding method of glycerol / methanol, follow the steps below to ferment Pichia pastoris to produce insulin precursor:

[0045] (1) Initial culture stage: insert the Pichia pastoris seed liquid into the fermentation medium, the inoculation ratio is 7%, and the inoculation OD 600 = 9, the initial culture conditions are 29°C, pH 5.4, ventilation rate 0.5vvm, as the culture progresses, increase the speed and ventilation rate accordingly, and control the dissolved oxygen ≥ 30%. When the initial glycerol in the fermentation medium is exhausted, it can be judged that the DO value qui...

Embodiment 3

[0052] The Pichia fermentation medium of the present embodiment is: urea 5.0g / L, potassium dihydrogen phosphate 15.0g / L, dipotassium hydrogen phosphate 7.0g / L, calcium carbonate 0.8g / L, magnesium sulfate heptahydrate 16.0g / L L, ammonium sulfate 6.0g / L, ammonium dihydrogen phosphate 13.0g / L, ferrous sulfate 0.8g / L, glycerol 40.0g / L, PTM1 solution 4.0ml / L, solvent is water, pH 5.5.

[0053] Using the mixed feeding method of glycerol / methanol, follow the steps below to ferment Pichia pastoris to produce insulin precursor:

[0054] (1) Initial culture stage: insert the Pichia pastoris seed liquid into the fermentation medium, the inoculation ratio is 7%, and the inoculation OD 600 =9, the initial culture conditions are 29°C, pH 5.4, ventilation rate 0.5vvm, as the culture progresses, increase the rotation speed and ventilation rate accordingly, and control dissolved oxygen ≥ 30%. When the initial glycerol in the fermentation medium is exhausted, it can be judged that the DO value...

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Abstract

The invention belongs to the technical field of biopharmaceuticals, and discloses a fermentation medium of pichia pastoris for improving the expression of insulin precursor and a method for fermentedproduction of the insulin precursor. The fermentation medium of pichia pastoris in the present invention adopts a phosphate buffer system, supplemented by inorganic salts such as ammonium sulfate andferrous sulfate, as well as nitrogen sources such as urea and ammonium dihydrogen phosphate. The fermentation medium has definite compositions with stable sources and does not require fed-batch of nitrogen sources in the induction stage. According to the method for fermented production of the insulin precursor, a glycerol/methanol mixed solution is adopted for induction of culture after initial culture and glycerol fed-batch culture. A certain proportion of glycerol is fed by batches in the induction stage, so as to accelerate the growth of microorganisms. As time increases, the mixing ratio of glycerol gradually decreases while methanol gradually increases, and a large number of microorganisms enter the high-speed expression period in a short time, so that a target product can obtain a higher level of expression, greatly shortening the culture time and avoiding influence on product quality caused by excessive by-products.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a fermentation medium of Pichia pastoris for improving the expression of insulin precursors and a method for fermenting and producing insulin precursors. Background technique [0002] Diabetes is one of the most important chronic non-communicable diseases in the world. With the rising prevalence in recent years, the disease burden brought by diabetes has become more and more serious. Diabetes and its complications have seriously affected the quality of life of human beings. . [0003] Insulin is the most effective therapeutic drug for controlling blood sugar, and its preparation methods include chemical synthesis and gene recombination. Insulin has been used clinically for 90 years and has experienced three generations of milestone progress. The development of genetic recombination technology has made insulin preparations from the first generation of insul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P21/02C12R1/84
CPCC12N1/16C12P21/02C07K14/62Y02A50/30
Inventor 曹海燕王洪宇张世野王宝泉王田园王虹池营吴海洋郭富饶牟禹
Owner BEIJING HUIZHIHENG BIOTECHNOLOGY CO LTD
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