阅读说明：本技术 一种人血白蛋白标准物质原料的制备方法及其产品和应用 (Preparation method of human serum albumin standard substance raw material, product and application thereof ) 是由 全灿 孔金隆 孙永跃 李红梅 罗坚 何雅娟 于 2018-06-06 设计创作，主要内容包括：本发明涉及蛋白纯化领域的一种人血白蛋白标准物质原料的制备方法及其产品和应用；所述人血白蛋白标准物质原料的制备方法,包括以下步骤：(1)采用冷乙醇沉淀法处理人血清,弃沉淀组分Ⅰ+Ⅱ+Ⅲ,留上清液；(2)上清液超滤脱乙醇,置换缓冲液；(3)阳离子交换层析；(4)阴离子交换层析；(5)超滤置换为水；(6)冷冻干燥。本工艺制备的白蛋白产品纯度较高,可达99％以上,单体含量较低,优于Sigma公司样品,值得推广应用。(the invention relates to a preparation method of a human serum albumin standard substance raw material, a product and application thereof, belonging 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 cold ethanol precipitation, removing precipitation components I + II + III, and collecting supernatant; (2) ultrafiltering the supernatant to remove ethanol, and replacing the buffer solution; (3) cation exchange chromatography; (4) anion exchange chromatography; (5) replacing the ultrafiltration with water; (6) and (5) freeze drying. The albumin product prepared by the process has high purity which can reach more than 99 percent, has low monomer content, is superior to a sample of a Sigma company, and is worthy of popularization and application.)

1. A preparation method of a human serum albumin standard substance raw material comprises the following steps:

(1) Treating human serum by cold ethanol precipitation, removing precipitation components I + II + III, and collecting supernatant;

(2) Ultrafiltering the supernatant to remove ethanol, and replacing the buffer solution;

(3) cation exchange chromatography;

(4) anion exchange chromatography;

(5) Replacing the ultrafiltration with water;

(6) and (5) freeze drying.

2. The method of claim 1, wherein:

And (3) in the step (2), membrane-packed ethanol removal treatment is adopted, wherein the used ultrafiltration diluent is the equilibrium buffer solution used in the cation exchange chromatography in the step (3), and the ultrafiltration diluent is diluted in the post-treatment step until the protein concentration ranges from 5mg/mL to 25 mg/mL.

3. The method of claim 1, wherein:

The pH value of the balance buffer solution used in the cation exchange chromatography in the step (3) is 5.0-7.0, and the balance buffer solution contains a buffer agent with the concentration of 5-100 mmol/L, wherein the buffer agent is selected from phosphate and/or citrate.

4. the method of claim 1, wherein:

The elution buffer solution used in the cation exchange chromatography in the step (3) contains sodium chloride with the concentration of 0.1-1 mol/L and a buffer agent with the concentration of 5-100 mmol/L, and the buffer agent is selected from phosphate and/or citrate.

5. the method of claim 1, wherein:

the cation exchange chromatography in the step (3) uses a weak acid type cation exchange medium, and preferably, the charge group of the weak acid type cation exchange medium is carboxymethyl.

6. the method of claim 1, wherein:

The anion exchange chromatography in the step (4) uses a weak base type anion exchange medium, and the preferred weak base type anion exchange medium has a charge group of diethylaminoethyl.

7. The method of claim 6, wherein:

The matrix of the weak base type anion exchange medium in the step (4) is at least one selected from agarose, cellulose, dextran, polyacrylamide and polystyrene.

8. albumin produced by the production method according to any one of claims 1 to 7.

9. The albumin of claim 8, wherein the albumin has a purity greater than 99%.

10. Use of albumin according to claim 8 or 9 as a standard substance.

Technical Field

The invention relates to the field of protein purification, and in particular relates to a preparation method of a human serum albumin standard substance raw material, and a product and application thereof.

Background

Albumin is the highest protein content in plasma. The content of 3500 mg-5500 mg in each 100mL of plasma is about more than 50% of the total protein content of the plasma, and the extraction with large amount and high purity is easy. The molecular weight of albumin is 66kDa, the isoelectric point is 4.7, the molecule is elliptic, and the albumin molecule is a spherical molecule formed by a single peptide coil consisting of 585 amino acids. The structure of albumin contains 3 functional regions and 9 subfunction regions, and 17 disulfide bonds are crossed and connected among cysteine residues in chains, so that a natural quaternary structure is maintained, and the stability is good.

Albumin is currently the most clinically used pharmaceutical protein. The development of the albumin standard substance can provide quality control samples for various analysis and detection methods, and on the other hand, the standard substance is a traceability basis, is a guarantee of measurement and analysis and can also provide evaluation for the analysis method. Due to the large population and the improvement of medical level in China, the clinical demand of albumin is increasing year by year. This means that, under the condition of the shortage of the existing plasma resources, the improvement of the purity and the yield of the existing albumin purification process in China has important significance. Besides, in scientific research projects and production processes, quantitative and qualitative detection of albumin are indispensable operations. Therefore, the development of albumin standards is not slow. On one hand, the quality control sample can be provided for various analysis and detection methods, and on the other hand, the standard substance is a traceability basis, is a guarantee of test analysis and can provide evaluation. The United States Pharmacopeia (USP) uses recombinant human albumin from Sigma as a control, and has a purity of 97% or more. The national standard substance center queries can obtain the components of frozen human serum albumin, a prealbumin standard substance (GBW (E)090619) and human serum albumin with the purity of more than 85 percent. Therefore, the development of the high-purity albumin standard substance has important significance.

The currently marketed human serum albumin products are all prepared by purifying human serum by a cold ethanol precipitation method, and the ethanol precipitation has the advantages that the used solvent ethanol has a sterilization function, viruses such as HIV can be inhibited in the precipitation process, and the production process is relatively simple. However, due to the phenomenon of co-precipitation in the cold ethanol precipitation process, the purity of the albumin product is not high.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a raw material of a human serum albumin standard substance, and particularly relates to a preparation method, a product and an application of the raw material of the human serum albumin standard substance. The invention considers that the method combining cold ethanol precipitation and chromatography can complement the advantages with the development of the chromatographic technology, and the human blood albumin is better purified.

One of the purposes of the invention is to provide a preparation method of a human serum albumin standard substance raw material, which can comprise the following steps:

(1) treating human serum by cold ethanol precipitation, removing precipitation components I + II + III, and collecting supernatant;

(2) Ultrafiltering supernatant to remove ethanol, and replacing buffer solution;

(3) Cation exchange chromatography;

(4) Anion exchange chromatography;

(5) Replacing the ultrafiltration with water;

(6) And (5) freeze drying.

Wherein the content of the first and second substances,

The step (1) is based on the traditional cold ethanol precipitation process, which is a five-step precipitation method, and in the invention, the precipitation components I + II + III in the process production process are discarded, and the supernatant is left. In the step (1), the serum can be diluted by using physiological saline to ensure that the protein concentration is 45 +/-1 g/L, hydrochloric acid with the concentration of 0.1-0.5 mol/L is used for adjusting the pH value to 5.8 +/-0.2, the serum is placed in a low-temperature circulating water bath for precooling, the temperature of a low-temperature circulating tank is set to be minus 5 +/-1 ℃, 95 percent ethanol is precooled at minus 20 ℃, the mixture is added into a precooled sample and stirred while adding until the final volume ratio of the ethanol is 19 +/-1 percent, and the mixture is stirred at minus 5 +/-1 ℃ for more than 1 hour; centrifuging and removing the precipitate to obtain a supernatant, thereby obtaining a pretreatment solution.

In the step (2), ethanol removal treatment can be performed by using a membrane, specifically, a 30kDa ultrafiltration membrane (e.g. 30kDa 0.005 m)²Specification), wherein the used ultrafiltration diluent is the equilibrium buffer solution used in the cation exchange chromatography in the step (3), and the ultrafiltration diluent is diluted in the post-treatment step of ultrafiltration until the protein concentration range is selected from 5-25 mg/mL, 5-20 mg/mL, 5-15 mg/mL, 5-10 mg/mL, 10-20 mg/mL or 10-15 mg/mL, preferably 5-15 mg/mL, and most preferably 5-10 mg/mL. Adjusting the pH value to 5.5-6.5 by using 0.1-0.3 mol/L sodium hydroxide or hydrochloric acid solution to obtain a proper chromatography sample loading solution.

The flow rate of the cation exchange chromatography and the anion exchange chromatography can be 8-12 mL/min, and the ultraviolet detection wavelength is set to be 280 nm.

The equilibration buffer used in the cation exchange chromatography in the step (3) is pH 5.0-7.0, and contains a buffer with a concentration of 5-100 mmol/L (preferably 10-30 mmol/L), wherein the buffer is selected from phosphate and/or citrate (namely the equilibration buffer is selected from at least one of phosphate buffer and citrate buffer), preferably citrate (namely the equilibration buffer is preferably citrate buffer);

The elution buffer solution used in the cation exchange chromatography in the step (3) contains sodium chloride with the concentration of 0.1-1 mol/L and a buffer agent with the concentration of 5-100 mmol/L (preferably 10-30 mmol/L), and the buffer agent is selected from phosphate and/or citrate, preferably citrate.

the cation exchange chromatography in the step (3) uses a weak acid type cation exchange medium, and preferably, the charge group of the weak acid type cation exchange medium is carboxymethyl.

In some embodiments of the present invention, the pH of the equilibration buffer used in the cation exchange chromatography in step (3) may be selected from 5.0 to 7.0, 5.0 to 6.5, 5.0 to 6.0, 5.0 to 5.5, 5.5 to 6.5, 5.5 to 6.0, or 6 to 6.5, preferably 5.5 to 6.5, and most preferably 6.0 to 6.5.

In some embodiments of the present invention, the pH of the equilibration buffer in step (3) may be any value between 5.0 and 7.0, such as 5.2, 5.8, 6.0, 6.2, etc.

Anion exchange chromatography may comprise the following steps: adjusting the pH value of the penetrating peak component collected by chromatography to 5.2 +/-0.2, loading the penetrating peak component to an anion exchange column well balanced by 10-50 mmol/L (preferably 15-30 mmol/L) of acetic acid and a sodium acetate buffer system, and eluting by using an acetic acid buffer solution with the pH value of 4.6 +/-0.2 to obtain an eluent which is albumin.

and (3) replacing the eluent obtained by the two-step chromatography with a buffer solution by using ultrapure water with the volume of 8-12 times, and freeze-drying and storing.

the anion exchange chromatography in the step (4) uses a weak base type anion exchange medium, and the preferred weak base type anion exchange medium has a charge group of diethylaminoethyl.

The matrix of the weak base type anion exchange medium in the step (4) is selected from at least one of agarose, cellulose, dextran, polyacrylamide and polystyrene; preferably, the matrix of the ion exchange medium is agarose, for example Capto medium, Sepharose FF, Sepharose and the like.

The inventor considers that the main hetero protein in the supernatant of the cold ethanol precipitation is immunoglobulin, the isoelectric point of the immunoglobulin is 6-7, and in the cation exchange chromatography, the pH value is 6.0, part of the immunoglobulin is adsorbed on the column and removed, but part of the immunoglobulin is penetrated out along with the albumin. Thus, in anion exchange chromatography, protein with isoelectric point greater than 5.2 or less than 4.6 is separated from albumin by means of pH elution, pH5.2 loading, pH4.6 elution, and either breakthrough or distributed elution. Finally, the albumin as a final product reaches high purity of more than 99 percent.

The product prepared by the technical scheme of the invention has high purity, low preparation cost (because the adopted conventional media are all), and short period (in the prior art, the cold ethanol precipitation and anion-cation exchange chromatography method adopts a pH elution mode, and the process adopts one-step pH elution and one-step salt elution, and the salt elution mode is faster and more convenient than the pH elution, and is easy to amplify). And the cation exchange chromatography in the step (3) is penetration chromatography, the flow-through chromatography can increase the sample processing amount and save the cost while saving time, and simultaneously, because the protein directly penetrates and does not interact with the medium, the possibility of protein denaturation and inactivation is reduced.

The invention also aims to provide the albumin prepared by the preparation method of the human serum albumin standard substance raw material, wherein the purity of the albumin is more than 99 percent, and preferably more than 99.3 percent. The purity calculation herein is carried out according to methods well known to those skilled in the art, for example, based on the integration of the 280nm UV absorbance in HPLC.

The invention also relates to the application of the albumin prepared by the preparation method of the human serum albumin standard substance raw material as a standard substance, in particular to the application of the albumin as a transferrin standard substance. The albumin standard substance can provide quality control samples for various analysis and detection methods, is a traceability basis, guarantees test and analysis, and can provide evaluation for the analysis method. The product prepared by the technical scheme of the invention fills the blank of China in the field of albumin standard substances.

Drawings

FIG. 1 is a graph showing the comparison between human serum albumin standard material prepared in example 1 and a sample produced by Sigma.

Detailed Description

the present invention will be further described with reference to the following examples. However, the present invention is not limited to these examples.