Pichia pastoris fermenting method for improving precursor expression of insulin and analogue thereof

A technology of Pichia pastoris and a fermentation method, applied in the field of Pichia pastoris fermentation, can solve the problems of increasing the risk of contamination, supplementation, and major risks, and achieve the effects of reducing the degradation of target proteins, prolonging the fermentation period, and increasing the utilization rate

Active Publication Date: 2017-08-08
YICHANG HEC CHANGJIANG PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because ammonia water is highly corrosive, irritating and explosive, there are greater risks during use and storage
In addition, since ammonia water cannot be sterilized, it can only be sterilized through a filter, which increases the cost of the filter and its verification
[0006] Determination of bacterial cell concentration in Pichia pastoris fermentation process usually adopts wet weight method (WCW) or absorbance method (OD 600 ), both of these two measurement methods have large errors, and particles and dead cells in the medium can be detected, so the measured value cannot reflect the amount of real living cells
At the same time, the operation of these two methods is cumbersome and involves sampling operations, which increases the risk of bacterial contamination
In addition, this method is usually measured every few hours, which cannot reflect the real-time status of the bacteria, and many critical stages and abnormal situations may be missed
[0007] The carbon source (glycerol, methanol, etc.) in the fermentation process of Pichia pastoris is usually supplemented by constant speed, increasing or decreasing, or constant specific growth rate. These methods cannot be carried out according to the real-time growth characteristics of Pichia pastoris. carbon supplementation

Method used

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  • Pichia pastoris fermenting method for improving precursor expression of insulin and analogue thereof

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Effect test

Embodiment 1

[0042] A Pichia fermentation method for improving the expression of recombinant human insulin precursor, said fermentation method comprising the steps of:

[0043] 1) Initial growth stage: the inoculum concentration is 2.0×10 5 cfu / ml, the temperature is controlled at 32°C, the air intake ratio is 1.5vvm, and the dissolved oxygen value is controlled to be ≥50% by stirring;

[0044] 2) Glycerin supplementation stage: When the dissolved oxygen value rebounds to 80%, start to supplement glycerin solution, and set the initial glycerin supplementation rate F 0 =12g / l / h, then adjust the glycerol supplementation rate in real time based on the microbial growth kinetic model, so that the living cells of the bacteria are exponential (X=1.25*1.1 t , X represents the concentration of living cells, that is, the permittivity of living cells, t represents the time) to grow, so as to obtain a large number of bacteria with high activity, the control temperature is 32 ° C, the air ratio is 1.0...

Embodiment 2

[0059] A Pichia fermentation method for improving the expression of recombinant human insulin precursor, said fermentation method comprising the steps of:

[0060] 1) Initial growth stage: the inoculum concentration is 6.0×10 5 cfu / ml, the temperature is controlled at 28°C, the air intake ratio is 1.0vvm, and the dissolved oxygen value is controlled to be ≥50% by stirring;

[0061] 2) Glycerin supplementation stage: When the dissolved oxygen value rebounds to 80%, start to supplement glycerin solution, and set the initial glycerin supplementation rate F 0 =8g / l / h, then adjust the glycerol supplementation rate in real time based on the microbial growth kinetic model, so that the living cells of the bacteria are exponential (X=1.5*1.2 t , X represents the concentration of living cells, that is, the permittivity of living cells, t represents the time) to grow, so as to obtain a large number of bacteria with high activity, the control temperature is 28 ° C, the air intake ratio i...

Embodiment 3

[0076] A Pichia fermentation method for improving the expression of recombinant human insulin precursor, said fermentation method comprising the steps of:

[0077] 1) Initial growth stage: the inoculum concentration is 2.0×10 5 cfu / ml, the temperature is controlled at 32°C, the air intake ratio is 1.5vvm, and the dissolved oxygen value is controlled to be ≥50% by stirring;

[0078] 2) Glycerin supplementation stage: When the dissolved oxygen value rebounds to 80%, start to supplement glycerin solution, and set the initial glycerin supplementation rate F 0 =12g / l / h, then adjust the glycerol supplementation rate in real time based on the microbial growth kinetic model, so that the living cells of the bacteria are exponential (X=1.15*1.3 t , X represents the concentration of living cells, that is, the permittivity of living cells, t represents the time) to grow, so as to obtain a large number of bacteria with high activity, the control temperature is 32 ° C, the air intake ratio...

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Abstract

The invention relates to the field of biological pharmacy, in particular to a pichia pastoris fermenting method for improving precursor expression of insulin and analogue thereof. The fermenting culture comprises the following stages of an initial growth phase, a glycerin supplementing phase and a methanol inducing phase. The method has the advantages that by controlling a formula of an initial culture medium, and the nitrogen source type and carbon source supplementing type, and continuously or semi-continuously supplementing a small amount of microorganisms or plant-source organic nitrogen sources in the middle and later period of fermenting, the fermenting cycle is extended; while the quality of the target product is guaranteed, the output is increased by not less than 30%.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a Pichia yeast fermentation method for improving the expression of insulin and its analog precursors. Background technique [0002] Diabetes is a common endocrine and metabolic disease. In recent years, the prevalence of diabetes has been increasing rapidly all over the world. In 2015, the number of adult diabetic patients in the world reached 415 million, among which the number of diabetic patients in China exceeded 100 million. Diabetes has become another important chronic non-communicable disease that seriously endangers people's health after cardiovascular and cerebrovascular diseases and tumors. The acute and chronic complications of diabetes, especially the complications of chronic diseases, involve multiple organs, causing disability and high mortality, seriously affecting the physical and mental health of patients, and bringing heavy burdens to individuals, families an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12R1/84
CPCC07K14/62C12P21/02
Inventor 李爽曾进戴祖冰赵慧郑强刘爱丽贾永峰
Owner YICHANG HEC CHANGJIANG PHARMA CO LTD
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