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Preparation method of human serum albumin standard substance raw material as well as product and application thereof

A technology of human serum albumin and standard substances, which is applied in the preparation method of peptides, serum albumin, animal/human protein, etc., can solve the problems of low purity and co-precipitation of albumin products, and achieve high purity and low production cost. Low, cost-saving effect

Inactive Publication Date: 2019-12-13
NAT INST OF METROLOGY CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the possibility of co-precipitation during the cold ethanol precipitation process, the purity of the albumin product is not high

Method used

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  • Preparation method of human serum albumin standard substance raw material as well as product and application thereof
  • Preparation method of human serum albumin standard substance raw material as well as product and application thereof

Examples

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Effect test

Embodiment 1

[0033] Purification of albumin in plasma: Take 50 mL of fresh frozen plasma at -20°C, freeze and thaw slowly in a refrigerator at 4°C, and then centrifuge (4°C, 10,000 r / min, 30 min), and dilute the supernatant with normal saline (0.9% NaCl) to The protein concentration was 45g / L, the pH value was adjusted to 5.8 with hydrochloric acid, and pre-cooled in a low-temperature circulating water bath at -5°C for 30 minutes. Take 95% ethanol at -20°C, add it to the pre-cooled sample, stir until the final volume ratio of ethanol is 19%, stir and react at -5°C for more than 1 hour, centrifuge and discard the supernatant to obtain the pretreatment solution .

[0034] The pretreatment solution is a membrane bag with a molecular weight cut-off of 30KDa (Pellicon XL ultrafiltration membrane bag Ultracel 30kDa0.005m 2 , the U.S. Millipore Company) replacement buffer is 20mmol / L Na 2 HPO 4 and citric acid buffer system (pH6.0) and diluted to a protein concentration of about 10mg / mL.

[003...

Embodiment 2

[0040] Crude albumin was obtained according to the cold ethanol precipitation and ultrafiltration steps in Example 1, and the HPLC spectra of the CM Sepharose eluted fractions were obtained under different pH citrate equilibrium buffer conditions. The crude product obtained under three kinds of conditions compares the following table:

[0041] Summary of HPLC Analysis of CM Sepharose Breakthrough Peak Components under Three pH Conditions

[0042]

[0043] Under three different pH conditions, considering the factors of purity and yield, the preferred pH is 6.0.

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Abstract

The invention relates to a preparation method of a human serum albumin standard substance raw material as well as a product and an application thereof, which relate to the field of protein purification. The preparation method of the human serum albumin standard substance raw material comprises the following steps: (1) treating human serum by adopting a cold ethanol precipitation method, discardingprecipitation components I, II and III, and reserving a supernate; (2) carrying out ultrafiltration on the supernate to remove ethanol, and replacing a buffer solution; (3) performing cation exchangechromatography; (4) performing anion exchange chromatography; (5) replacing with water through ultrafiltration; and (6) performing freeze drying. The albumin product prepared by the process is high in purity which can reach 99% or above, low in monomer content which is superior to that of a sample prepared by a Sigma company, and worthy of popularization and application.

Description

technical field [0001] The invention relates to the field of protein purification, and more specifically, to a preparation method of a human serum albumin standard substance raw material and its product and application. Background technique [0002] Albumin is the most abundant protein in plasma. Each 100mL plasma contains 3500mg~5500mg, which accounts for more than 50% of the total protein content in plasma, and is easy to extract in large quantities and with high purity. The molecular weight of albumin is 66kDa, the isoelectric point is 4.7, and the molecule is elliptical. The albumin molecule is a spherical molecule formed by coiling a single peptide chain composed of 585 amino acids. The structure of albumin contains 3 functional regions and 9 subfunctional regions, and there are 17 disulfide bond cross-links between cysteine ​​residues in the chain, maintaining the natural quaternary structure and good stability. [0003] Albumin is currently the most widely used medi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K1/30C07K1/34C07K1/18C07K1/36
CPCC07K14/765
Inventor 全灿孔金隆孙永跃李红梅罗坚何雅娟
Owner NAT INST OF METROLOGY CHINA
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