Solid-liquid separation method for Pichia pastoris fermentation broth

A Pichia pastoris, solid-liquid separation technology, applied in the field of separation and purification of Pichia pastoris expression recombinant protein fermentation liquid, can solve the problems of difficult removal of bacteria, high cost of solid-liquid separation, suitable for industrial production, and reduce the purification process Effect of pressure and easy solid-liquid separation

Active Publication Date: 2012-12-26
ZHEJIANG HISUN PHARMA CO LTD
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The invention provides a brand-new method for solid-liquid separation of Pichia pastoris fermentation liquid, which can effectively solve the problems of difficulty in removing bacteria and high cost of solid-liquid separation in high-density fermentation liquid of Pichia pastoris

Method used

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  • Solid-liquid separation method for Pichia pastoris fermentation broth
  • Solid-liquid separation method for Pichia pastoris fermentation broth
  • Solid-liquid separation method for Pichia pastoris fermentation broth

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1 (comparative example)

[0034] Using our own strains and fermentation technology to produce recombinant human serum albumin in a 100L fermenter, the OD600 of the fermentation broth was measured to be 421, and the target protein content in the supernatant was detected after centrifugation. 1 9.5g / L, after dilution, detect C2 is 3.5g / L, and the calculated bacterial cell concentration p is 41.7%.

[0035] After the fermentation, take 400ml of recombinant human albumin fermentation liquid (calculated total protein is 2.2g), put it in a centrifuge cup, centrifuge at 10000rpm in a Beckman centrifuge at 4°C for 5min, obtain 210ml of supernatant, and detect the supernatant Protein content is 8.1g / L (protein total amount is 1.70g) in the liquid, and macromolecule impurity content is 7.5% (attached figure 1 RT=8.853 and 9.451 two impurity peaks), the OD600 of the fermentation broth after centrifugation was 22.3, the calculated bacterium removal rate was 94.7%, and t...

Embodiment 2

[0037] 0.176g chitosan is dissolved in the acetic acid solution of 39.6ml 1%, then adds 0.4ml dehydrated alcohol, stirs, then joins in the 400ml recombinant human albumin fermented liquid described in embodiment 1, controls operating temperature Stir at 15°C for 30 minutes, add sodium bicarbonate, adjust the pH to 9.5, and stir to flocculate chitosan.

[0038] The above mixed solution was placed in a centrifuge cup, centrifuged at 10000rpm in a Beckman centrifuge at 4°C for 5min to obtain 250ml of supernatant, and the protein content in the supernatant was detected to be 7.06g / L (the total amount of protein was 1.765g). The high molecular impurity content was 3.0%, the OD600 of the fermented liquid after centrifugation was 2.8, the calculated cell removal rate was 99.3%, and the protein recovery rate was 80.2%.

Embodiment 3

[0040] 0.44g chitosan was dissolved in 40ml 1% acetic acid solution, then added in the 400ml recombinant human albumin fermentation broth described in Example 1, controlled operating temperature 15°C, stirred for 30min, added sodium carbonate, adjusted pH To 8.5, stir to flocculate the chitosan.

[0041] The above mixed solution was placed in a centrifuge cup, centrifuged at 10000rpm in a Beckman centrifuge at 4°C for 5min to obtain 250ml of supernatant, and the protein content in the supernatant was detected to be 7.2g / L (the total amount of protein was 1.8g). The high molecular impurity content was 3.3%, the OD600 of the fermented liquid after centrifugation was 1.1, the calculated cell removal rate was 99.7%, and the protein recovery rate was 81.8%.

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Abstract

The invention discloses a solid-liquid separation method for Pichia pastoris fermentation broth. The method comprises the following steps: uniformly mixing chitosan solution and Pichia pastoris fermentation broth, adjusting pH and stirring, subjecting chitosan to flocculation in the Pichia pastoris fermentation broth, and finally removing strains and macromolecule impurity through centrifugation or filtering to obtain solution comprising recombinant protein. By adopting the method disclosed by the invention, the removal rate of Pichia pastoris strains and the recovery rate of recombinant protein are both significantly improved, the time for separation is short, the cost for separation is low, the obtained solution has high clarity, and the method is more suitable for industrial production.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for separating and purifying fermented liquid for expressing recombinant protein by Pichia pastoris. Background technique [0002] The Pichia pastoris expression system is a new type of exogenous protein expression system developed in the early 1980s. It has the advantages of simple operation, easy culture, fast growth, high expression, and low cost. At present, this system has become widely used in the production of recombinant proteins, such as recombinant human serum albumin and its fusion proteins, and industrial enzymes. [0003] With the maturity and development of Pichia pastoris high-density fermentation technology, increasing the fermentation density has become an important strategy to increase protein expression. At present, the content of Pichia pastoris fermentation cells can reach 40-50% of the total fermentation broth, or even more High, which put...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/30C07K14/765C07K19/00
Inventor 徐杰徐有富聂磊王海彬杨仲毅李丹张赛平徐期白骅
Owner ZHEJIANG HISUN PHARMA CO LTD
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