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Process for separating and purifying recombinant human serum albumin and fusion protein thereof

A human albumin and chelation technology, applied in the field of human albumin purification, can solve the problems of long cycle, high cost, complicated purification process, etc.

Active Publication Date: 2014-12-17
ZHEJIANG HISUN PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to similar problems with EP0612761, the purification process is too complex, long cycle, and costly to be an effective method for large-scale production

Method used

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  • Process for separating and purifying recombinant human serum albumin and fusion protein thereof
  • Process for separating and purifying recombinant human serum albumin and fusion protein thereof
  • Process for separating and purifying recombinant human serum albumin and fusion protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Step (1) captures the sample using immobilized metal chelate affinity chromatography (IMAC)

[0066] Packing material IMAC Sepharose 6 Fast Flow 1 liter was purchased from GE Company, self-packed chromatography column, XK50 / 30 column, column diameter 5cm, packing height 15cm, and the preservation solution (20% ethanol) was washed away with purified water.

[0067] Use 0.5 times the column volume of 0.2M copper sulfate solution to pass through the column, and wash away unadsorbed copper ions with purified water.

[0068] Then use buffer A: 20mmol / L phosphate, 600mmol / L sodium chloride, pH7.5, to equilibrate the chromatography column.

[0069] Add phosphate and sodium chloride to the fermentation supernatant containing human serum albumin to make it reach: 20mmol / L phosphate, 600mmol / L sodium chloride, pH7.5.

[0070] Then use AKTA TM The Purify chromatography system was loaded with a flow rate of 50ml / min. After loading, wash with buffer A until the absorbance reaches...

Embodiment 2

[0083] Step (1) Use a chromatographic column of a metal chelate chromatographic medium to capture the sample

[0084] The filler Chelating was purchased from GE Company, and the chromatographic column was filled by itself, with a diameter of 5 cm and a packing height of 15 cm. The preservation solution (20% ethanol) was washed away with purified water

[0085] Then pass through the column with 0.5 times the column volume of 0.2M copper sulfate solution, and then wash away the unadsorbed copper ions with purified water.

[0086] Use equilibrium solution A: 20mmol / L phosphate, 600mmol / L sodium chloride, pH7.5 to balance the chromatographic column, and then adjust the sample containing the target protein to the same condition as the buffer solution A, that is, 20mmol / L phosphate, 600mmol / L sodium chloride, pH7.5. Then use AKTA TM The Purify chromatography system was loaded with a flow rate of 50ml / min.

[0087] After sample loading, wash with equilibrium solution A until the a...

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Abstract

Different from the conventional method for purifying human serum albumin from human plasma, the invention provides a simple method for separating and purifying the human serum albumin from fermentation liquor or a mammal cell culture solution. By an immobilized metal affinity chromatography (IMAC) step, the method is suitable for purifying recombinant proteins at various salt concentrations, fermentation supernate at high salt concentration is allowed to be directly loaded, the total volume of loading buffer is effectively reduced and the cost is reduced. The method is stable and high-efficiency, and is suitable for laboratories, pilot scale tests and industrial production scale-up.

Description

technical field [0001] The invention belongs to the technical field of protein separation and purification, in particular to the purification of recombinant human serum albumin. Background technique [0002] Human serum albumin (HSA), is the protein with the most content in plasma, accounting for 40%-60% of total plasma protein, it is a very important carrier in plasma, in the environment of body fluid pH7.4, albumin is a negative ion, Each molecule can carry more than 200 negative charges. Many poorly soluble substances can be transported by binding to albumin. These substances include bilirubin, long-chain fatty acids (4-6 molecules per molecule can be combined), bile salts, prostaglandins, steroid hormones, metal ions (such as Cu 2+ 、Ni 2+ , Ca 2+ ) drugs (such as aspirin, penicillin, etc.). Another role is to maintain osmotic pressure. [0003] The molecular structure of albumin was elucidated in 1975. It is a globular protein with 585 amino acid residues and a mol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/20C07K1/18C07K14/765C07K1/22
Inventor 聂磊王欢郭婷婷蔡秀云白骅
Owner ZHEJIANG HISUN PHARMA CO LTD
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