CN113845562A - Method for purifying target protein - Google Patents
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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Abstract
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非离子表面活性剂。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 present invention provides a method for purifying a target protein. The method comprises: using an elution buffer to elute an affinity chromatography column, so as to obtain an eluent containing a target protein, the affinity chromatography column is loaded with a sample to be tested, and the sample to be tested contains The target protein to be purified, wherein the elution buffer contains a nonionic surfactant. Using the method provided by the present invention to carry out the target protein affinity chromatography can significantly improve the purity of the eluted protein, inhibit the generation of multimers during the elution process, and improve the protein recovery rate.
Description
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 inhibiting the generation of multimers during the elution of protein affinity chromatography, 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 a nonionic surfactant. The inventors surprisingly found that by adding a nonionic surfactant into the elution buffer, 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 non-ionic surfactant comprises at least one of tween and triton. The inventor finds that the tween or the triton added into the elution buffer solution can obviously inhibit the generation of polymer of the eluted protein and improve the purity of the target protein.
According to an embodiment of the present invention, the nonionic surfactant includes at least one of tween 80 and tween 20. The inventor finds that the tween 80 or tween 20 is added into the elution buffer solution independently, so that the generation of the polymer of the eluted protein can be obviously inhibited, and the purity of the target protein is improved.
According to the embodiment of the invention, the volume fraction of the nonionic surfactant in the elution buffer is 0.5-2%, and according to the method of the embodiment of the invention, the nonionic surfactant has a better inhibition effect on the generation of protein polymers 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 the embodiment of the invention, the pH value of the elution buffer is 3-4.
According to an embodiment of the invention, the pH of the elution buffer is 3.4-3.6. The inventor finds that the elution buffer solution added with the nonionic surfactant 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 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 invention, the sample to be tested comprises at least one selected from fermentation broth or 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 0.5-2 vol% of nonionic surfactant, the pH 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: 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 a nonionic surfactant.
According to the method provided by the embodiment of the invention, the nonionic surfactant 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 present invention, the nonionic surfactant includes at least one of tween 80 and tween 20. According to the method provided by the embodiment of the invention, the Tween 80 or Tween 20 is independently added into the elution buffer, so that the generation of the polymer of the eluted protein can be remarkably inhibited, and the purity of the target protein is improved.
According to an embodiment of the invention, the volume fraction of the non-ionic surfactant in the elution buffer is between 0.5% and 2%.
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 the embodiment of the invention, the pH value of the elution buffer is 3-4.
According to an embodiment of the invention, the pH of the elution buffer is 3.4-3.6. According to the method provided by the embodiment of the invention, the elution buffer solution added with the nonionic surfactant can ensure a 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, for the neutralization of the target protein, a method of collecting the eluate containing the target protein and then adding a neutralizing solution to the eluate containing the target protein is generally adopted. 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.
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 0.5-2 vol% of nonionic surfactant, the pH of the elution buffer solution is 3.4-3.6, and the sample to be detected is a cell culture.
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 volume fraction of tween 80 is 1 percent
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 MabselectTM SuReTMAnd (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 regeneration solution, cleaning chromatography column with 3 column volumes of cleaning solution, balancing chromatography column with 5 column volumes of balance buffer solution, and maintaining with a certain volumeStoring the chromatographic column in the 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, 1 vol% Tween 80, 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 and concentration of the target protein (detected by SEC-HPLC) are detected, and the purity of the target protein purified by the experimental group in example 1 is 82.23%, and the recovery rate is 85.81%. The purity of the target protein of the control group was 49.55%, and the recovery rate was 71.97%.
Example 2: tween 80 volume fraction of 0.5%
The purification method of the target protein is the same as that of the experimental group of example 1 except that the concentration of tween 80 is different from that of the experimental group of example 1, and the volume fraction of tween 80 in example 2 is 0.5%.
After neutralization, the purity and concentration of the target protein were measured, and the purity of the target protein purified in example 2 was 67.78%, and the recovery rate was 83.27%.
Example 3: the volume fraction of tween 80 is 2 percent
The purification method of the target protein is the same as that of the experimental group of example 1 except that the concentration of tween 80 is different from that of the experimental group of example 1, and the volume fraction of tween 80 in example 3 is 2%.
After neutralization, the purity and concentration of the target protein are detected, and the purity of the target protein purified in example 2 is 54.96%, and the recovery rate is 85.26%.
From the embodiments 1 to 3, referring to table 1, after tween 80 is added to the elution buffer, 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 volume fractions of the Tween 80 in the elution buffer solution are respectively 0.5%, 1% and 2%, the purity of the target protein after elution and neutralization can reach 67.78%, 82.23% and 54.96%, and compared with the protein elution purity 49.55% when the Tween 80 is not added, the protein elution purity is respectively improved by 37%, 66% and 11%, and higher recovery rate can be kept; when the volume fraction of the tween 80 in the elution buffer solution is 0.5%, 1% and 2%, the recovery rates of the target protein after elution and neutralization are 83.27%, 85.81% and 85.26%, respectively, and are improved by 16%, 19% and 18% respectively compared with the recovery rate of the target protein 71.97% when the tween 80 is not added.
Table 1: protein purity corresponding to different concentrations of Tween 80 in elution buffer
| Tween 80 concentration | Purity (SEC-HPLC) |
| 0 | 49.55％ |
| 0.5％ | 67.78％ |
| 1％ | 82.23％ |
| 2％ | 54.96％ |
Example 4: collecting the eluate containing the target protein with a container containing a neutralizing solution
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 MabselectTM SuReTMAnd (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, 1 vol% Tween 80, 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 4 was 88.52%, and the recovery rate was 89.14%. Compared with the purification effect of example 1, the purity and recovery rate of the target protein are further improved, which shows that the purity and recovery rate of the target protein can be further improved by performing neutralization by using the eluent containing the target protein and collected by a container in which the neutralization solution is pre-placed.
Example 5: the pH of the elution buffer was 3.5
In example 5, the purification conditions were the same as in example 4 except that the pH of the elution buffer was different from that in example 4, 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 obtained by purification in example 5 was 90.2%, and the recovery rate was 88.33%. It was found that the purity and recovery of the target protein were also high when the pH of the elution buffer was 3.5, as compared with the pH of the elution buffer in example 4, which was 3.4.
Example 6: adjusting the pH of the elution buffer
In example 6, the experimental conditions were the same as those in the experimental group of example 1 except that there was a difference in elution buffer.
The elution buffer used in example 6 was: 1)100mM citrate buffer, pH 3.6; 2)100mM citrate buffer, pH 3.5; 3)100mM citrate buffer, pH 3.4.
As a result, referring to Table 2, the recovery rate of 50% or more was ensured when the pH of the elution buffer was 3.4 to 3.6 during the affinity elution.
Table 2: protein elution recovery rate corresponding to elution buffers with different pH values
| pH | Elution recovery rate |
| 3.6 | 51.87％ |
| 3.5 | 67.18％ |
| 3.4 | 71.97％ |
Comparative example 1
In comparative example 1, the purification conditions were the same as those in the experimental group of example 1 except that there was a difference in the concentration of tween 80 in the elution buffer, and the volume fraction of tween 80 in comparative example 1 was 0.2%, as shown in table 3.
Table 3: protein purity corresponding to 0.2% Tween 80 in elution buffer
| Tween 80 concentration | Purity (SEC-HPLC) |
| 0.2％ | 49.32％ |
The purity of the target protein obtained after purification in comparative example 1 is 49.32%, and the recovery rate is 67.11%, and compared with the results of adding 0.5%, 1% and 2% tween 80 in examples 1 to 3, the purity and recovery rate of the target protein obtained in the comparative example are both obviously reduced.
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.
Claims (10)
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 nonionic surfactant.
2. The method of claim 1, wherein the non-ionic surfactant comprises at least one of tween and triton.
3. The method of claim 2, wherein the tween comprises at least one of tween 80 and tween 20.
4. The method of claim 3, wherein the volume fraction of the non-ionic surfactant in the elution buffer is between 0.5% and 2%.
5. The method of claim 1, wherein the elution buffer comprises at least one of a citrate buffer, an acetate buffer, and a glycine buffer.
6. The method according to any one of claims 2 to 5, wherein the elution buffer has a pH of 3 to 4, preferably 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:
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 solution is 100mM citric acid buffer solution, the elution buffer solution contains 0.5-2 vol% of nonionic surfactant, the pH of the elution buffer solution is 3.4-3.6, and the sample to be detected is a cell culture;
optionally, the non-ionic surfactant comprises at least one of tween and triton;
optionally, the tween comprises at least one of tween 80 and tween 20.
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Application publication date: 20211228 |