Method for purifying target protein

文档序号：445340 发布日期：2021-12-28 浏览：20次中文

阅读说明：本技术 目的蛋白纯化方法 (Method for purifying target protein ) 是由赵雪冰王妮于 2020-06-28 设计创作，主要内容包括：本发明提供了一种目的蛋白纯化方法。该方法包括：利用洗脱缓冲液对亲和层析柱进行洗脱处理,以便获得含有目的蛋白的洗脱液,所述亲和层析柱加载有待测样品,所述待测样品中含有待纯化目的蛋白,其中,所述洗脱缓冲液中含有糖类物质。利用本发明提供的方法进行目的蛋白亲和层析,可以显著提高洗脱蛋白质的纯度,抑制洗脱过程中多聚体的产生,提高蛋白回收率。(The invention provides a target protein purification method. The method comprises the following steps: eluting the affinity chromatography column by using an elution buffer solution so as to obtain an eluent containing the target protein, wherein a sample to be detected is loaded on the affinity chromatography column, the sample to be detected contains the target protein to be purified, and the elution buffer solution contains saccharides. The method provided by the invention is used for carrying out target protein affinity chromatography, can obviously improve the purity of eluted protein, inhibits the generation of polymers in the elution process, and improves the recovery rate of the protein.)

1. A method for purifying a target protein, comprising the steps of:

eluting the affinity chromatography column by using an elution buffer solution so as to obtain an eluent containing the target protein, loading a sample to be detected on the affinity chromatography column, wherein the sample to be detected contains the target protein to be purified,

wherein the elution buffer contains a saccharide.

2. The method of claim 1, wherein the saccharide comprises at least one selected from the group consisting of sucrose, glucose, and lactose.

3. The method of claim 2, wherein the saccharide is present in the elution buffer at a concentration of 0.1M to 1M.

4. The method according to claim 1, wherein the elution buffer comprises at least one selected from the group consisting of a citric acid buffer, an acetate buffer, and a glycine buffer.

5. The method according to any one of claims 2 to 5, wherein the elution buffer has a pH of 3 to 4.

6. The method of claim 6, wherein the elution buffer has a pH of 3.4 to 3.6.

7. The method of claim 1, wherein the eluate is collected using a container pre-filled with a neutralizing solution.

8. The method of claim 1, wherein the test sample comprises at least one selected from the group consisting of a fermentation broth and a cell culture.

9. The method of claim 1, wherein the affinity chromatography column is a Protein A affinity chromatography column, a Protein G affinity chromatography column, or a Protein L affinity chromatography column.

10. A method for purifying a Protein of interest by a Protein a affinity chromatography column, comprising the steps of:

wherein the elution buffer solution is 100mM citric acid buffer solution, the elution buffer solution comprises 0.1M-1M carbohydrate, the pH value of the elution buffer solution is 3.4-3.6, the sample to be detected is cell culture,

optionally, the saccharide substance comprises at least one selected from sucrose, glucose and lactose.

Technical Field

The invention relates to the technical field of biology, in particular to a target protein purification method.

Background

The protein affinity chromatography technology is suitable for proteins which are difficult to separate due to small difference of physicochemical properties, the affinity chromatography utilizes the difference of affinity between the ligand and mixed biomolecules in a solid phase medium to separate, when protein mixed liquor passes through a chromatography column, the protein which can be specifically combined with the ligand can be adsorbed and fixed in the chromatography column, the combination is reversible under certain conditions, after the affinity chromatography is finished, the protein can be eluted by an elution buffer solution, and other proteins which do not have specific combination ability to the ligand flow through the column in the processes of sample loading and washing and can not be combined on the column. The protein affinity chromatography achieves the purpose of separation and purification through specific affinity binding between target protein and ligand, and has the characteristic of biological specificity.

However, some types of proteins, particularly Fc fusion proteins, are easily aggregated and even visibly precipitated when purified by protein affinity chromatography in an acidic solution, resulting in low purity and recovery of the protein obtained after purification.

Therefore, methods for protein purification using protein affinity chromatography techniques still need to be further developed and improved.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, an object of the present invention is to provide a method capable of suppressing the production of multimers during elution, increasing the purity of a target protein, and increasing the recovery rate of the target protein.

In a first aspect of the invention, a method of purifying a protein of interest is provided. According to an embodiment of the invention, the method comprises: eluting the affinity chromatography column by using an elution buffer solution so as to obtain an eluent containing the target protein, wherein a sample to be detected is loaded on the affinity chromatography column, the sample to be detected contains the target protein to be purified, and the elution buffer solution contains saccharides. The inventors surprisingly found that by adding a saccharide substance into the elution buffer solution, a protein polymer is not easy to generate in the elution process, and the purity of the target protein can be greatly improved.

According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:

according to an embodiment of the invention, the saccharide substance comprises at least one selected from sucrose, glucose and lactose. The inventor finds that the generation of polymer of the elution protein can be obviously inhibited by independently adding sucrose, glucose or lactose into the elution buffer solution, and the purity of the target protein can be greatly improved.

According to the embodiment of the present invention, the concentration of the saccharide in the elution buffer is 0.1M-1M, and according to the method of the embodiment of the present invention, the saccharide has a good inhibitory effect on the production of protein multimers within the concentration range.

According to an embodiment of the present invention, the elution buffer comprises at least one of a citrate buffer, an acetate buffer and a glycine buffer.

According to an embodiment of the invention, the elution buffer has a pH of 3-4.

According to an embodiment of the invention, the elution buffer has a pH of 3.4-3.6. The inventors have found that the addition of an elution buffer containing a saccharide ensures a higher recovery at a pH of 3.4 to 3.6.

According to an embodiment of the invention, the eluate is collected using a container pre-filled with a neutralising solution. In the prior art, the target protein-containing eluent is collected and then neutralized by adding the neutralizing solution, and the inventor finds that the purity and recovery rate of the target protein can be further improved by collecting the eluent by using a container pre-filled with the neutralizing solution compared with the prior art.

According to an embodiment of the present invention, the sample to be tested includes at least one selected from a fermentation broth and a cell culture.

According to an embodiment of the invention, the affinity chromatography column is a Protein a affinity chromatography column, a Protein G affinity chromatography column or a Protein L affinity chromatography column.

In a second aspect of the invention, the invention provides a method for purifying a Protein of interest by means of a Protein A affinity chromatography column. According to an embodiment of the invention, the method comprises: eluting an affinity chromatography column by using an elution buffer solution so as to obtain an eluent containing target protein, wherein a sample to be detected is loaded on the affinity chromatography column, the sample to be detected contains the target protein to be purified, the elution buffer solution is 100mM citric acid buffer solution, the elution buffer solution contains 0.1M-1M of carbohydrate, the pH value of the elution buffer solution is 3.4-3.6, and the sample to be detected is a cell culture.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative and are intended to be illustrative of the invention and are not to be construed as limiting the invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

In a first aspect of the invention, a method of purifying a protein of interest is provided. According to an embodiment of the invention, the method comprises the steps of: eluting the affinity chromatography column by using an elution buffer solution so as to obtain an eluent containing the target protein, wherein a sample to be detected is loaded on the affinity chromatography column, the sample to be detected contains the target protein to be purified, and the elution buffer solution contains saccharides.

According to the method provided by the embodiment of the invention, the carbohydrate is added into the elution buffer solution, so that a protein polymer is not easy to generate in the elution process, and the purity of the target protein can be greatly improved.

According to an embodiment of the present invention, before loading the sample containing the target protein onto the stationary phase of the affinity chromatography column, the affinity chromatography column is further equilibrated to allow the target protein to be better bound to the stationary phase.

According to the specific embodiment of the invention, after a sample containing a target protein is loaded on the stationary phase of the affinity chromatography column and before the target protein is eluted by using an elution buffer, the affinity chromatography column needs to be leached to elute the foreign protein; the collected target protein is further subjected to protein neutralization so as to finally obtain the purified target protein.

According to an embodiment of the invention, the carbohydrate comprises at least one selected from sucrose, glucose and lactose. According to the method provided by the embodiment of the invention, the generation of the polymer of the eluted protein can be obviously inhibited by independently adding sucrose, glucose and lactose into the elution buffer, and the purity of the target protein can be greatly improved.

According to an embodiment of the invention, the concentration of the saccharide in the elution buffer is between 0.1M and 1M.

According to an embodiment of the present invention, the elution buffer comprises at least one of a citrate buffer, an acetate buffer and a glycine buffer.

According to an embodiment of the invention, the elution buffer has a pH of 3-4.

According to an embodiment of the invention, the elution buffer has a pH of 3.4-3.6. According to the method provided by the embodiment of the invention, the elution buffer solution added with the saccharide can ensure higher recovery rate when the pH value is 3.4-3.6.

According to an embodiment of the invention, the eluate is collected using a container pre-filled with a neutralising solution. In the art, the target protein is neutralized by collecting the eluate containing the target protein and adding a neutralizing solution to the eluate. According to the method provided by the embodiment of the invention, the eluent is collected by using the container pre-filled with the neutralizing solution, so that the purity and the recovery rate of the target protein can be further improved compared with the conventional method in the field.

According to an embodiment of the invention, the sample comprises at least one selected from the group consisting of a fermentation broth, a cell culture and a cell line culture. According to the specific embodiment of the invention, the sample to be detected needs to be subjected to preliminary impurity removal before sample loading so as to remove cells and debris particles therein, the preliminary impurity removal method is not limited, and the cells and debris particles in the sample can be removed, for example, a two-step centrifugation method can be adopted.

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.

Example 1

Experimental groups: the concentration of sucrose was 0.1M

The target protein purification method comprises the following steps:

1. primary purification: taking a cell culture containing target protein, removing cells and debris particles by adopting a two-step centrifugation method, firstly centrifuging for 10min by using 1000g, transferring and taking supernate, centrifuging for 30min by using 6000g, and collecting the supernate for the subsequent steps;

2. and (3) capture chromatography: by using Mabselect^TMSuRe^TMAnd (2) an affinity chromatography column, wherein 5 column volume balance buffer solutions are used for balancing, the sample primarily purified in the step (1) is loaded on the affinity chromatography column, 5 column volume elution buffer solutions are used for elution, a container with a neutralization solution is used for collecting the obtained target protein eluate, and the pH value of the neutralized target protein solution is 6.5-8.0. Regenerating with 5 column volumes of regenerating solution, cleaning the chromatographic column with 3 column volumes of cleaning solution, balancing the chromatographic column with 5 column volumes of balance buffer solution, and storing the chromatographic column with a storage solution.

The buffer solution used in each step was as follows:

and (3) an equilibrium buffer: 20mM Tris-HCl, 150mM NaCl, pH 7.2;

and (3) eluting a buffer solution: 20mM Tris-HCl, 150mM NaCl, pH 7.2;

elution buffer: 100mM citrate buffer, 0.1M sucrose, pH 3.4;

neutralizing liquid: 1M Tris-HCl, pH 8.5;

regeneration liquid: 100mM HAc;

cleaning solution: 0.1M NaOH;

preservation solution: 20% by volume of ethanol.

Control group: the elution buffers used for the control group were: 100mM citrate buffer, pH3.4, and other elution conditions were the same as those in the experimental group for the purification method of the objective protein.

After neutralization, the purity (by SEC-HPLC) and concentration of the target protein were measured, and the purity of the target protein purified in example 1 was 89.58%, and the recovery rate was 88.94%. The purity of the target protein of the control group was 49.55%, and the recovery rate was 71.97%.

Example 2: the concentration of sucrose was 0.25M

The purification method of the target protein was carried out under the same conditions as those in the experimental group of example 1 except that the concentration of sucrose was different from that in the experimental group of example 1, and the concentration of sucrose in example 2 was 0.25M.

The purity of the target protein was measured after neutralization, and the purity of the target protein purified in example 2 was 92.09%. .

Example 3: the concentration of sucrose was 0.5M

After neutralization, the purity and concentration of the target protein were measured, and the purity of the target protein purified in example 2 was 93.40%, and the recovery rate was 85.36%.

Example 4: the concentration of sucrose is 1M

A target protein was purified under the same conditions as those in the experimental group of example 1 except that the concentration of sucrose was different from that in the experimental group of example 1, and the concentration of sucrose was 1M in example 4.

The purity of the target protein was measured after neutralization, and the purity of the target protein purified in example 4 was 93.62%.

From embodiments 1-4, it can be known that, after sucrose is added into an elution buffer solution, the monomer ratio can be effectively improved, the generation of polymers is reduced, the protein purity is remarkably improved, and the recovery rate is effectively improved. When the concentration of sucrose in the elution buffer solution is respectively 0.1M, 0.25M, 0.5M and 1M, the purity of the target protein after elution and neutralization can reach 89.58%, 92.09%, 93.40% and 93.62%, and is respectively improved by 81%, 86%, 88% and 89% compared with the protein elution purity of 49.55% when sucrose is not added; when the volume fractions of sucrose in the elution buffer solution are 0.1M and 0.5M respectively, the recovery rates of the target protein after elution and neutralization are 88.94 percent and 85.36 percent respectively, and are improved by 24 percent and 19 percent respectively compared with the recovery rate of the protein when sucrose is not added, which is 71.97 percent.

Example 5: the pH of the elution buffer was 3.5

The target protein was purified under the same conditions as those in the experimental group of example 1 except that the pH of the elution buffer was different from that in the experimental group of example 1, and the pH of the elution buffer in example 5 was 3.5.

After neutralization, the purity and concentration of the target protein were measured, and the purity of the target protein purified in example 5 was 94.20%, and the recovery rate was 53.13%. Therefore, compared with the prior art, the purity of the target protein can be improved by using the elution buffer solution containing sucrose and having the pH of 3.5, and the protein recovery rate can reach more than 50%.

Example 6: the saccharide in the elution buffer is glucose with a concentration of 1M

The target protein was purified under the same conditions as those in the experimental group of example 1 except that the saccharide added to the elution buffer was different in concentration from those in the experimental group of example 1, and the saccharide in example 6 was glucose in a concentration of 1M.

After neutralization, the purity and concentration of the target protein are detected, and the purity of the target protein purified in example 6 is 93.35%, and the recovery rate is 82.89%. From this, it was found that the purity and recovery rate of the target protein can be greatly improved by adding glucose to the elution buffer compared to the elution buffer to which no saccharide was added to the control group in example 1.

Example 7: adjusting the pH of the elution buffer

The target protein was purified under the same conditions as in example 6 except that the pH of the elution buffer was different from that in example 6, and the pH of the elution buffer in example 7 was 3.5.

The purity of the target protein was measured after neutralization, and the purity of the target protein purified in example 7 was 95.82%.

Example 8: adjusting the pH of the elution buffer

The purity of the target protein was measured after neutralization, and the purity of the target protein purified in example 8 was 90.02%.

According to examples 7 to 8, the purity of the target protein can be increased to 90% or more by using an elution buffer containing glucose and having a pH of 3.5 or 3.6.

Example 9: collecting the eluate containing target protein without using a container containing a neutralizing solution

The target protein purification method is the same as example 3, except that the target protein-containing eluate collected in a pre-placed neutralization solution container is not used in the present example, and the specific steps are as follows:

2. and (3) capture chromatography: by using Mabselect^TMSuRe^TMAnd (2) an affinity chromatography column, wherein 5 column volume balance buffer solutions are used for balancing, the sample primarily purified in the step (1) is loaded on the affinity chromatography column, 5 column volume elution buffer solutions are used for elution, the obtained target protein eluate is collected, then a neutralizing solution is added into the target protein eluate for neutralization, and the pH value of the neutralized target protein solution is 6.5-8.0. Regenerating with 5 column volumes of regenerating solution, cleaning the chromatographic column with 3 column volumes of cleaning solution, balancing the chromatographic column with 5 column volumes of balance buffer solution, and storing the chromatographic column with a storage solution.

The buffer solution used in each step was as follows:

and (3) an equilibrium buffer: 20mM Tris-HCl, 150mM NaCl, pH 7.2;

and (3) eluting a buffer solution: 20mM Tris-HCl, 150mM NaCl, pH 7.2;

elution buffer: 100mM citrate buffer, 0.5M sucrose, pH 3.4;

neutralizing liquid: 1M Tris-HCl, pH 8.5;

regeneration liquid: 100mM HAc;

cleaning solution: 0.1M NaOH;

preservation solution: 20% by volume of ethanol.

After neutralization, the purity and concentration of the target protein were determined, and the purity of the target protein purified in example 9 was 85.33%, and the recovery rate was 82.36%. Compared with the purification effect of example 3, the purity and recovery rate of the target protein were reduced, indicating that the purity and recovery rate of the target protein could be further improved by performing neutralization using the target protein eluate collected in a container in which a neutralization solution was preliminarily placed.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

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