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Purification method for obtaining high-purity recombinant human serum albumin

A technology for human serum albumin and a purification method, which is applied in the field of purification for obtaining high-purity recombinant human serum albumin, can solve the problems of complex composition of fermentation broth, decomposition, low yield and the like, and achieves good application prospects, less impurities, and difficulty in Degradation effect

Pending Publication Date: 2019-08-06
山东健通生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with plasma protein, the separation of recombinant albumin has some challenges: the yield of the target product in the expression system is low; the magnitude of the product is grams or milligrams; additional product proteases will be produced, and proteases may decompose the target protein; the composition of the fermentation broth Complex, including recombinant human albumin, yeast cells, pigments, salt ions, cell excretion, etc.; endotoxin will be produced during the fermentation process, but there is no endomycin in human plasma

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] This example provides a purification method for obtaining high-purity recombinant human serum albumin, and the specific processing method includes the following steps:

[0029] (1) Centrifuge the fermented broth containing recombinant human serum albumin at a speed of 4000r / min for 15min, then add heat stabilizers sodium octanoate and sodium acetyl tryptophan with a mass ratio of 2:1, and then heat at 66°C for 20min , to obtain recombinant human serum albumin supernatant;

[0030] (2) Perform primary ultrafiltration and secondary ultrafiltration on the supernatant of recombinant human serum albumin successively. The primary ultrafiltration adopts an ultrafiltration membrane with a molecular mass of 80kD. is 10kD, the pH value of the two ultrafiltrations is 6.8, and the operating pressure of the ultrafiltration is 0.14MPa to obtain recombinant human serum albumin ultrafiltrate;

[0031] (3) Add the recombinant human serum albumin ultrafiltrate to the pre-balanced Ni-NTA...

Embodiment 2

[0034] This example provides a purification method for obtaining high-purity recombinant human serum albumin, and the specific processing method includes the following steps:

[0035] (1) Centrifuge the fermented broth containing recombinant human serum albumin at a speed of 6000r / min for 35min, then add heat stabilizers sodium octanoate and sodium acetyl tryptophan with a mass ratio of 4:1, and then heat at 70°C for 40min , to obtain recombinant human serum albumin supernatant;

[0036](2) Perform primary ultrafiltration and secondary ultrafiltration on the supernatant of recombinant human serum albumin successively. The primary ultrafiltration adopts an ultrafiltration membrane with a molecular mass of 80kD. is 50kD, the pH value of the two ultrafiltrations is 7.2, and the operating pressure of the ultrafiltration is 0.16MPa to obtain recombinant human serum albumin ultrafiltrate;

[0037] (3) Add the recombinant human serum albumin ultrafiltrate to the pre-balanced Ni-NTA ...

Embodiment 3

[0040] This example provides a purification method for obtaining high-purity recombinant human serum albumin, and the specific processing method includes the following steps:

[0041] (1) Centrifuge the fermented broth containing recombinant human serum albumin at a speed of 8000r / min for 25min, then add heat stabilizers sodium caprylate and sodium acetyl tryptophan with a mass ratio of 3:1, and then heat at 68°C for 30min , to obtain recombinant human serum albumin supernatant;

[0042] (2) Perform primary ultrafiltration and secondary ultrafiltration on the supernatant of recombinant human serum albumin successively. The primary ultrafiltration adopts an ultrafiltration membrane with a molecular mass of 80kD. is 30kD, the pH value of the two ultrafiltrations is 7, and the operating pressure of the ultrafiltration is 0.15MPa to obtain recombinant human serum albumin ultrafiltrate;

[0043] (3) Add the recombinant human serum albumin ultrafiltrate to the pre-balanced Ni-NTA c...

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Abstract

The invention relates to a purification method for obtaining high-purity recombinant human serum albumin. The method comprises the following steps: centrifugal ultrafiltration and secondary ultrafiltration are carried out after centrifuging and heating the fermentation liquid, the impurities encapsulated in the recombinant human serum albumin are then removed using 30% of alcohol and 6 of mol / L urea, the protein is renatured, eluted, and dialyzed to obtain the decontamination liquid, finally, the obtained decontamination liquid is sequentially subjected to cation exchange chromatography, anionexchange chromatography, and hydrophobic chromatography to obtain the high-purity recombinant human serum albumin. The recombinant human serum albumin obtained by the method has the advantages of high purity, less impurities, low degradation, high stability, and the like, and has the good application prospect. The final obtained recombinant human serum albumin reaches the level of pharmaceuticalgrade (the purity is greater than 99.999999%), which fully meets the requirements of the US Pharmacopoeia and the Japanese Pharmacopoeia for the injection-level recombinant human serum albumin.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a purification method for obtaining high-purity recombinant human serum albumin. Background technique [0002] At present, with the development of economic conditions, people pay more and more attention to medical health. Human serum albumin (HSA) has played various roles in clinical treatment in recent decades. The amount of albumin, a blood product, is also in short supply. Due to the improvement of the safety of blood products, the production of albumin from blood is very limited, and the use of biological gene technology to produce a blood substitute has become a hot spot. [0003] After several years, researchers have successfully used various expression systems to express recombinant human serum albumin (rHSA). However, there are many impurities in the fermentation broth, and the main components are miscellaneous proteins, nucleic acids, fatty acids, pigments, pol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K1/36C07K1/34C07K1/22C07K1/20C07K1/18
CPCC07K14/765
Inventor 齐乐齐智史杰
Owner 山东健通生物科技有限公司
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