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Method for purifying recombinant human serum albumin protein and application thereof

A technology of supernatant and application, applied in the purification of recombinant human serum albumin and its application field, can solve the problems of reduction and difficulty in controlling endotoxin levels, etc.

Active Publication Date: 2010-07-07
NCPC NEW DRUG RES & DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with other methods, the level of impurities such as chromogenic substances and polysaccharides in the purified recombinant HSA purified by this method is greatly reduced, but the level of endotoxin is difficult to control, and further optimization treatment is required to control endotoxin and increase the yield to obtain a reliable product. Industrial purification process

Method used

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  • Method for purifying recombinant human serum albumin protein and application thereof
  • Method for purifying recombinant human serum albumin protein and application thereof
  • Method for purifying recombinant human serum albumin protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The purification of embodiment 1 recombinant human serum albumin (rHSA)

[0025] The various buffers used in the purification process are shown in the table below:

[0026] name

Buffer formulation

Solution A

25mM Disodium Hydrogen Phosphate - Sodium Dihydrogen Phosphate at pH 4.5

Solution B

50mM Disodium Hydrogen Phosphate - Sodium Dihydrogen Phosphate at pH 7.0,

0.1M NaCl, 10mM Sodium Octanoate

Solution C

50mM Disodium Hydrogen Phosphate - Sodium Dihydrogen Phosphate, 0.1M Sodium Chloride, pH 6.0

Solution D

50mM Disodium Hydrogen Phosphate - Sodium Dihydrogen Phosphate, 0.2M Sodium Chloride, pH 6.0

[0027] a) Clarification and heat treatment of fermentation broth

[0028] The fermentation broth was clarified using a ceramic membrane with a pore size of 0.5um, and 80L of the fermentation supernatant was taken, added with 5mM sodium octanoate and 5mM EDTA, heated at 70°C for 20 minutes, and ...

Embodiment 2

[0046] Example 2 The application of rHSA in the rabies vaccine rotary bottle culture process

[0047] a) Cell preparation:

[0048] Vero cells (derived from ATCC CRL-1586 in the United States) were revived, expanded in spinner bottles, and passaged into 3.5L double-layer spinner bottles. Culture conditions 37°C, CO 2 5% (v / v).

[0049] b) Virus inoculation:

[0050] When the Vero cells were cultured into a monolayer, the virus seed of CTN strain (National Institute for the Control of Pharmaceutical and Biological Products) was inoculated at an MOI of 0.025-0.125. After inoculation, the DMEM medium maintenance solution supplemented with 0.35% rHSA for vaccine production was replaced. The culture temperature was 34°C.

[0051] c) Virus collection liquid:

[0052] After changing to the maintenance solution, the solution was changed every three days, and after the solution was collected, the fresh maintenance solution was continued, and the solution was collected three times ...

Embodiment 3

[0060] Example 3 Application of rHSA in Rabies Vaccine NBS Reactor Cultivation Process

[0061] a) Cell preparation:

[0062] The Vero cells were revived and expanded in spinner bottles at 4×10 5 The density of cells / ml was inoculated in NBS5L bioreactor, with 160g polyester sheet in the reactor ester sheet basket, working volume 2.5L, culture condition 37°C, dissolved oxygen 45%, stirring speed 70rpm, pH value 7.4.

[0063] b) Virus inoculation:

[0064] When the Vero cells were cultured for 4-5 days, the DMEM medium supplemented with 0.35% rHSA for vaccine production was replaced, and the CTN strain virus seed was inoculated at an MOI of 0.025-0.125. The culture temperature is 34° C., the dissolved oxygen is 45%, the stirring speed is 70 rpm, and the pH value is 7.4.

[0065] c) Virus collection liquid:

[0066] After continuous perfusion for 20 days, the collection volume was 24L, and the virus titer of the mixed virus collection reached 6.51gLD50.

[0067] d) Purifica...

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Abstract

The present invention relates to a method for purifying recombinant human serum albumin (rHSA) protein. The method comprises the following steps: fermented liquid containing rHSA is processed by a ceramic membrane, supernatant liquid is orderly purified by high salt cation exchange chromatography, hydrophobic layer exchange chromatography and weak anion exchange chromatography, and purified rHSA is obtained. The present invention is characterized in that solution processed by high salt cation exchange chromatography is processed by borate and then filtered by hollow fibers. The rHSA obtained can be used for producing vaccines for humans against viruses with a cell culture method, particularly rabies vaccines.

Description

technical field [0001] The invention describes a purification method of recombinant human serum albumin (rHSA) expressed by Pichia pastoris and its application in the production of virus vaccine for human by cell culture method. Background technique [0002] Bovine serum albumin or human serum albumin is an indispensable stabilizer in the production of virus vaccines by cell culture, but bovine serum albumin is gradually replaced by human blood-derived human serum albumin due to its origin from animals and potential contamination such as mad cow disease. replaced. However, blood-derived human serum albumin inevitably contains the risk of potential pathogen infection, and at the same time, blood-derived human serum albumin is derived from human plasma, which limits its yield and batch-to-batch stability, and its quality is unstable. [0003] WO98 / 06822 on February 19, 1998 disclosed the use of recombinant human serum albumin (rHSA) in animal cell culture, wherein the recombi...

Claims

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Application Information

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IPC IPC(8): C07K1/20C07K1/18C12N5/06C12N7/00
Inventor 贾茜刘文献赵伟王志明魏敬双杜力黄顺军李梅彦高健任乐民贺建功
Owner NCPC NEW DRUG RES & DEV
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