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Method for separating human serum albumin by expanded bed adsorption based on mixed mode

A human albumin and mixed-mode technology, which is applied in the field of mixed-mode expansion-bed adsorption and separation of human albumin, can solve the problems of decreasing target concentration, prolonging process time, decreasing adsorption capacity, etc. Simplified preprocessing steps, easy-to-scale effects

Active Publication Date: 2017-05-31
ZHEJIANG UNIV
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Problems solved by technology

However, the cation exchange method is adopted in the protein capture stage, and the feed liquid needs to be adjusted to pH 4.0 and diluted. The acidic environment is easy to activate protease, resulting in the degradation of human serum albumin. Dilution will reduce the concentration of the target substance, reduce the adsorption capacity, and increase the processing capacity. , the process time is prolonged

Method used

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  • Method for separating human serum albumin by expanded bed adsorption based on mixed mode

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Embodiment 1

[0023] Take the Pichia fermented liquid containing recombinant human serum albumin, adjust the pH to 6.0, add sodium octanoate to 5 mM, and heat at 68° C. for 30 minutes to obtain a crude human serum albumin solution. The mixed-mode adsorbent (agarose microspheres with quartz sand as a weighting agent, L-tryptophan as a ligand) is packed into an expanded bed with an inner diameter of 1 cm, a column height of about 20 cm, and pH 5.0 acetic acid-sodium acetate buffer Pre-equilibrate to reach 2.0 times the expansion rate, keep for 20 minutes; adjust the pH of the crude human serum albumin solution to 5.0, and load 10ml of the sample. Wash 5 times column volume with equilibration buffer, wash 10 times column volume with 20mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 7.5), wash 10 times column volume with 20mM acetic acid-sodium acetate buffer (pH 4.0), The eluted II fraction was collected, desalted, and freeze-dried to obtain human serum albumin. The purit...

Embodiment 2

[0025]Take the Pichia fermented liquid containing recombinant human serum albumin, adjust the pH to 6.0, add sodium caprylate to 10 mM, and heat at 68° C. for 30 min to obtain a crude human serum albumin solution. Mixed mode adsorbent (agarose microspheres with titanium dioxide added as a weighting agent, L-tryptophan methyl ester as a ligand) is packed into an expanded bed with an inner diameter of 1 cm, a column height of about 20 cm, pH 5.5 acetic acid-sodium acetate The buffer was pre-equilibrated to achieve a 1.8-fold expansion rate, and kept for 20 minutes; the pH of the crude human serum albumin solution was adjusted to 5.5, and 10ml of the sample was loaded. Wash 5 times the column volume with equilibration buffer, wash 5 times the column volume with 20mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 7.0), wash with 10 times the column volume with 20mM acetic acid-sodium acetate buffer (pH 3.5), The eluted II fraction was collected, desalted, and f...

Embodiment 3

[0027] Take the Hansenula fermented liquid containing recombinant human serum albumin, adjust the pH to 6.0, add sodium octanoate to 15 mM, and heat at 68° C. for 30 min to obtain a crude human serum albumin solution. Mixed mode adsorbent (agarose microspheres added with tungsten carbide as a weighting agent, L-tryptophan ethyl ester as a ligand) is packed into an expanded bed with an inner diameter of 1 cm, the column height is about 20 cm, pH 6.0 disodium hydrogen phosphate - Sodium dihydrogen phosphate buffer pre-equilibrated to achieve 2.2 times the expansion rate, keep for 20 minutes; adjust the pH of the crude human serum albumin solution to 6.0, and load 10ml of the sample. Wash 5 times column volume with equilibration buffer, wash 15 times column volume with 20mM disodium hydrogen phosphate-citrate buffer (pH8.0), and elute with 10 times column volume with 20mM citric acid-sodium citrate buffer (pH 4.5) , collected the eluted II fraction, desalted, and freeze-dried to ...

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Abstract

The invention discloses a method for adsorbing and separating human serum albumin by expanded bed adsorption based on a mixed mode, which can directly separate and recombine human serum albumin from a yeast fermentation solution, and belongs to the protein separation technology in the biochemical field. The method comprises the following steps: (1) preprocessing a fermentation solution; obtaining the yeast fermentation solution containing the human serum albumin, adding sodium caprylate, and heating and deactivating protease; (2) performing expanded bed adsorption, directly separating the yeast fermentation solution by using an expanded bed filled with a mixed-mode adsorption agent, and collecting elution peaks; and (3) desalting and drying: desalting a collected solution, freeze drying to obtain the human serum albumin with the purity greater than 95 percent. The invention is characterized by developing a novel separation process, so that the high-purity recombined human serum albumin can be directly separated from the yeast fermentation solution. A critical point of the invention lies in adopting the expanded bed adsorption agent based on the mixed mode, cells do not need to be removed from the yeast fermentation solution, the ion strength does not need to be adjusted, the elution condition is moderate, and the method has the characteristics of 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-based expanded bed adsorption separation method for human serum albumin. 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, caprylate precipitation, etc. The process is complicated, and viruses or other potential ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K1/14
CPCC07K14/765
Inventor 林东强吴启赐姚善泾
Owner ZHEJIANG UNIV
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