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A mixed-mode chromatographic method for separating human albumin from yeast fermentation broth

A human serum albumin and yeast fermentation technology, which is applied in the direction of serum albumin, albumin peptide, peptide preparation methods, etc., to achieve the effects of simplified pretreatment steps, good salt-resistant adsorption characteristics, and easy scale-up

Active Publication Date: 2021-03-23
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Checking domestic and foreign patents and literature, no MX-Trp-650M and similar media were found to be used to separate human serum albumin from yeast fermentation broth

Method used

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  • A mixed-mode chromatographic method for separating human albumin from yeast fermentation broth
  • A mixed-mode chromatographic method for separating human albumin from yeast fermentation broth
  • A mixed-mode chromatographic method for separating human albumin from yeast fermentation broth

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Take Pichia fermented liquid containing recombinant human serum albumin, the conductivity is about 24mS / cm, and the concentration of human serum albumin is 10mg / ml. After being clarified by centrifugation, adjust the pH to 6.0, add sodium octanoate to 5 mM, heat at 68°C for 30 min, precipitate impurity proteins and inactivate protease, and centrifuge at 8000 rpm for 20 min to obtain a supernatant. Adjust the pH value of the supernatant to 4.0, filter through a 0.45 μm filter membrane, and take 10 ml as an injection sample. The chromatographic column (0.5cm inner diameter) is filled with 2ml of MX-Trp-650M medium, the equilibration buffer is 20mM acetic acid-sodium acetate buffer (pH 4.0), and the eluent is 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.0), the eluted fractions were collected, desalted, and freeze-dried to obtain human serum albumin. The HPLC analysis showed a purity of 96.6%, a yield of 96.0%, and a pigment removal ra...

Embodiment 2

[0034]Take Pichia fermented liquid containing recombinant human serum albumin, the conductivity is about 24mS / cm, and the concentration of human serum albumin is 10mg / ml. After clarification by centrifugation, adjust the pH to 6.0, add sodium octanoate to 20 mM, heat at 68°C for 30 min, precipitate impurity proteins and inactivate protease, and centrifuge at 10,000 rpm for 10 min to obtain a supernatant. Adjust the pH value of the supernatant to 4.5, filter through a 0.45 μm filter membrane, and take 10 ml as an injection sample. The chromatographic column (0.5cm inner diameter) is filled with 2ml of MX-Trp-650M medium, the equilibration buffer is 20mM acetic acid-sodium acetate buffer (pH 4.5), and the eluent is 20mM sodium dihydrogen phosphate- Disodium hydrogen phosphate buffer solution (pH 7.0), collected eluted fractions, desalted, freeze-dried to obtain human serum albumin, the HPLC analysis purity was 96.7%, the yield was 97.8%, and the pigment removal rate was 91.4%. ...

Embodiment 3

[0036] Take Pichia fermented liquid containing recombinant human serum albumin, the conductivity is about 24mS / cm, and the concentration of human serum albumin is 10mg / ml. After clarification by centrifugation, adjust the pH to 6.0, add sodium octanoate to 15 mM, heat at 68°C for 30 min, precipitate impurity proteins and inactivate protease, and centrifuge at 9000 rpm for 15 min to obtain a supernatant. Adjust the pH value of the supernatant to 4.2, filter through a 0.45 μm filter membrane, and take 10 ml as an injection sample. The chromatographic column (0.5cm inner diameter) is filled with 2ml of MX-Trp-650M medium, the equilibrium buffer is 20mM acetic acid-sodium acetate buffer (pH 4.2), and the eluent is 20mM sodium dihydrogen phosphate with 0.4M NaCl added - Disodium hydrogen phosphate buffer solution (pH 7.0), collect the elution fraction, desalt, and freeze-dry to obtain human serum albumin, and the chromatographic separation spectrum is as follows figure 2 Shown, H...

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Abstract

The invention belongs to the protein chromatographic separation technology in the field of biochemical industry and discloses a mixed model chromatographic method for separating human serum albumin from yeast fermentation liquid. The method includes steps: 1) fermentation liquid pretreatment, to be more specific, removing yeast cells by centrifuging the yeast fermentation liquid which contains the human serum albumin, adding sodium caprylate, heating to remove impurity proteins and inactivating protease, performing centrifugal separation, and taking supernatant; 2) column chromatography, to be more specific, adopting a mixed mode medium with tryptophan as ligand, performing fixed bed chromatographic separation of the supernatant, and collecting elution peaks, wherein a sample loading pH value is 4.0-4.5, and an elution pH value is 7.0-9.0; 3) desalting and drying, desalting collected liquid, performing freeze drying to obtain the human serum albumin with purity larger than 95%. The mixed model chromatographic method is characterized in that by development of a novel separation process, the high-purity human serum albumin can be directly separated from the yeast fermentation liquid. By adoption of the mixed mode medium with tryptophan as the ligand, ion strength adjustment of the yeast fermentation liquid is avoided, adsorption under acid conditions and elution under neutral and weak alkali conditions are realized, and the method has advantages of mild elution conditions, simple process, high separation efficiency and high yield.

Description

technical field [0001] The invention belongs to the technical field of protein separation, and relates to a mixed-mode chromatography method for separating human serum albumin from yeast fermentation liquid. Background technique [0002] Human albumin is the most abundant protein in human plasma, accounting for about 60% of plasma protein. Its main physiological function is to maintain plasma osmotic pressure, combine and transport nutrients. It is clinically used in the treatment of hemorrhagic shock, traumatic shock, acute hypovolemia and hypoalbuminemia. It can also be used as an additive for cell culture, drug adjuvant, excipient, etc., and has a wide range of application values. [0003] Clinically used human serum albumin products are mainly extracted and purified from human plasma. The preparation methods include cold ethanol precipitation, ammonium sulfate precipitation, rivanol precipitation, octanoate precipitation, chromatography, etc. The raw materials are tight...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/765C07K1/16
CPCC07K14/765
Inventor 林东强褚文宁吴启赐姚善泾
Owner ZHEJIANG UNIV
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