KR20180125899A - A method for purifying an antibody fragment or antibody using affinity chromatography - Google Patents

Abstract

The present application relates to a method for isolating and purifying high purity antibodies or antibody fragments using affinity chromatography, and specifically, to a method for isolating high purity antibodies or antibody fragments and isolating and purifying the same using an elution buffer that can increase stability to increase stability.

Description

[0001] The present invention relates to a method for purifying an antibody or an antibody fragment using affinity chromatography,

The present application relates to a method for separating and purifying a high purity antibody or antibody fragment using affinity chromatography, and specifically, a method for separating and purifying a high purity antibody and antibody fragment using an elution solution capable of enhancing stability .

The recombinant protein is expressed and produced by inserting a target protein gene into a bacterium or a cell. A variety of impurities such as protein subtype, dimer, multimer, single antibody fragment, host-derived DNA, host-derived protein and endotoxin exist in the culture stock solution obtained by the expression of the target protein, and the impurity is removed In this protein therapeutic, antibody fragments are used specifically to combine with the target to show its efficacy, and F _ab without F _c does not have a sugar or saccharide, It has no effect and is a very attractive treatment. However, unlike an antibody, there is not a large difference in molecular weight from that of impurities, so separation using chromatography in a purification process is very difficult.

As a method of separating the antibody fragments at present, a method of refining using an affinity resin is widely used. For example, it is known that a desired product can be purified from impurities using a protein A affinity chromatography method as a method of purifying an antibody (International Application WO2011073954). However, in the case of the above method, an acidic buffer solution having a pH of about 2 to about pH 3 should be used to obtain a target protein from the resin. In this case, the pH of the elution solution is low and the target protein becomes unstable. In order to compensate for this problem, the buffer solution for increasing the pH of the eluting solution is directly mixed to secure the stability of the target protein. This method is inconvenient because it is necessary to further concentrate and dialyze to remove the buffer solution, And the yield is reduced.

In addition, as a method for purifying antibody fragments, a method of specifically recovering and purifying only a desired protein using a chromatographic method using a resin that is specifically compatible with a light chain kappa light chain is known Patent No. 2016-0127317). However, there is a disadvantage in the above method. Specifically, in the case of an antibody fragment, a system that expresses a heavy chain and light chain in a vector is used, and a protein in which only two light chains except for two chains are linked can be expressed. That is, when a resin having affinity for the kappa light chain is used, there is a problem that a light chain single slice, which is an impurity, must be further purified and removed. In addition, as the protein A affinity resin needs to elute into a buffer solution having a low pH (approximately 2 to 3), the stability of the obtained antibody fragment is low, and if it is necessary to perform additional concentration and dialysis process, And the purity is also not high.

Under these circumstances, the present inventors have made efforts to find a method of purifying a target protein having high purity and yield even at a pH higher than the pH condition conventionally used in affinity chromatography, and as a result, And a method for purifying antibodies and antibody fragments having high purity and stability when an optimized elution solution was used.

One object of the present invention is to provide a method for detecting an antibody or an antibody fragment comprising the steps of (a) loading an antibody or antibody fragment-containing sample into affinity chromatography comprising a double-sided affinity resin; (b) washing with a washing solution; And (c) recovering the target antibody or antibody fragment from the affinity chromatography column using an elution solution having a pH in the range of pH 3.8 to less than pH 4.7. .

In the following, the present application will be described in more detail.

On the other hand, each description and embodiment disclosed in the present application can be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of the present application. In addition, the scope of the present application is not limited by the specific description to be described below.

In addition, those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described in this application. Such equivalents are also intended to be included in the present application.

In order to achieve the above object, one aspect of the present invention is a method for analyzing a sample comprising (a) loading an antibody or antibody fragment-containing sample into affinity chromatography including a double-sided affinity resin; (b) washing with a washing solution; And (c) recovering the target antibody or antibody fragment from the affinity chromatography column using an elution solution having a pH in the range of pH 3.8 to less than pH 4.7. will be.

Generally, when proteins are purified using affinity chromatography involving an affinity resin, an elution solution in a range of pH 2 to pH 3 is mostly used, resulting in problems in terms of stability of a target protein (Pim Hermans et al., Monoclonal Antibodies, vol 1131, 297-314p; Abhinav A. Shukla et al., BioProcess International, (2005), 36-44p). In order to solve this problem, it is necessary to perform a calibration work which raises the pH to a high pH (4-7) after purification at a low pH (pH 2-3), and the yield of the target protein is reduced . However, the mid-affinity resin of the present application is significant in that it produces a target protein having high purity and stability in the range of pH 3.8 to pH 4.7, which is a mild condition than a general affinity resin.

Each step of the method for purifying the above-mentioned target antibody or antibody fragment will be described in detail as follows. First, step (a) is a step of loading a sample containing an antibody or antibody fragment into an affinity chromatography containing a double-sided affinity resin.

As used herein, " affinity chromatography " refers to a chromatographic method that utilizes a substance that is compatible with and binds to a specific protein. A substance that is compatible with a specific protein and binds to a polymer substance is a functional group that binds to an affinity substance that is dissolved in a polar or nonpolar solution. For purposes of this application, affinity chromatography can be divided into, but not limited to, affinity chromatography and light chain affinity chromatography. Specifically, " heavy chain affinity chromatography " means a chromatography method using a resin capable of specifically binding to a heavy chain, that is, a heavy chain affinity resin, which is a part of an antibody, quot; light chain affinity chromatography " refers to a chromatographic method using a resin capable of specifically binding light chains, that is, a light chain affinity resin, which is a part of an antibody.

For purposes of this application, affinity chromatography may be intermediate affinity chromatography. Specifically, the chromatography can be carried out using a resin capable of specifically binding to the heavy chain, which is a part of the antibody. For example, the mid-affinity resin may be CaptureSelect ^TM CH-1 XL Affinity Matrix (Thermo Fisher), but is not limited thereto. Any resin capable of specifically binding to the core can be used.

In one embodiment, the column may be equilibrated with a buffer solution having a pH of at least 5.5 but not more than pH 7.5 before loading the antibody or antibody fragment-containing sample of step (a). Specifically, the buffer solution may include sodium phosphate, sodium chloride, tris, MES (2- (morpholino) ethanesulfonic acid), MOPS (3-morpholinopropane-1-sulfonic acid), PIPES, potassium phosphate, [4- (2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid], but is not limited thereto.

In one embodiment, the method may further comprise an ion exchange chromatography, concentration and / or dialysis step prior to step (a). This is to remove the primary impurities of the antibody or antibody fragment and to increase the purity of the sample. Specifically, the step of concentrating and dialyzing the antibody fragment-containing sample and purifying it by anion chromatography before the step (a) is performed in advance, and then the antibody fragment is loaded on the affinity chromatography using the affinity- can do. Any work to remove primary impurities that do not bind to the affinity resin and to increase the purity of the sample can be applied without limitation.

In the method for purifying the above-mentioned target antibody or antibody fragment, step (b) is a step of washing with a washing solution, wherein the washing solution is applied to chromatography on which the sample is loaded.

The washing solution may have a pH ranging from pH 5.5 to less than 7.8, and the salt concentration may range from 400 mM or more to 1 M or less, but the present invention is not limited thereto.

In addition, the cleaning solution may be selected from the group consisting of sodium phosphate, potassium chloride, potassium phosphate, sodium chloride, tris, 2- (N-morpholino) ethanesulfonic acid, MOPS (3-morpholinopropane-1-sulfonic acid), PIPES, [4- (2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid], but the present invention is not limited thereto.

For the purposes of the present application, in step (b), impurities that are non-specifically bound to the double bond affinity resin by the wash solution may be removed.

In one embodiment, the purification method may further include the step of discharging an impurity which is not compatible with the resin with the equilibrium solution after the step (a) or (b). The above step may be performed at least once, but generally, it may be performed without limitation until the equilibrium is established.

In one embodiment, the purification method may further include re-equilibrating the re-equilibrium solution after (a) or (b). The rebalancing solution is allowed to flow in the same condition as that of the equilibrium buffer of step (a) in which the desired antibody or antibody fragment is not eluted without any reaction between the washing step and the elution step, and then the solution is flowed It plays a role of bridging between the main washing solution and the eluting solution.

Specifically, the rebalancing solution may have a pH ranging from pH 4.5 to 6.5, and the salt concentration may range from 10 mM or more to 30 mM or less, but the present invention is not limited thereto.

The re-equilibrium solution may also contain sodium phosphate, sodium chloride, tris, MES (2- (morpholino) ethanesulfonic acid), MOPS (3-morpholinopropane-1-sulfonic acid), PIPES, potassium phosphate, - [4- (2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid], but the present invention is not limited thereto.

In the method for purifying the above-mentioned target antibody or antibody fragment, step (c) is a step of recovering the target antibody or antibody fragment from the affinity chromatography column using an elution solution having a pH of not less than 3.8 and not more than pH 4.7.

The elution solution may have a pH ranging from pH 3.8 to 4.7, and the salt concentration may range from 10 mM or more to 100 mM or less, but the present invention is not limited thereto.

Even when the pH of the elution solution is less than 3.8, the target protein is eluted, but the same problem as that in the case of using the affinity chromatography of the prior art, that is, after purification at a low pH (pH 2-3) ) And a concentration and dialysis process are required. When the pH is higher than 4.7, it is not preferable because the ligand which catches the heavy chain in the column does not elute due to hydrogen bonding with the target protein.

In addition, the elution solution may contain at least one salt selected from the group consisting of sodium acetate, sodium citrate, and glycine within a range of salt concentration of 10 mM or more and 100 mM or less. However, in the affinity chromatography Salts which are capable of separating the antibody fragments or antibody fragments under mild conditions can be used without limitation.

Particularly, the purification method of the present application is characterized in that the concentration and dialysis step are not performed after step (C). 'Concentration and dialysis process' is a process generally used for removing substances below a target size on a membrane or for buffer change, and is a process used for further purification and removal of a single slice, which is an impurity when a general affinity resin is used . When the concentrating and dialysis processes are additionally used, the process yield is not high. However, the purification method of the present application is economical in that it can purify antibodies or antibody fragments with high purity and yield without further concentration and dialysis steps through optimization of the elution solution using a mid-affinity resin .

The term " target antibody or antibody fragment " used in the present application is a kind of protein to be separated from the affinity chromatography of the present application, and can be used in combination with a 'target protein'.

The antibody may specifically be a monoclonal antibody, and the term " monoclonal antibody " refers to an antibody that a single antibody-forming cell can produce, and recognizes one antigenic determinant. The antibody of the present application is not limited thereto, and may include all therapeutic antibodies conventionally used in the art.

In addition, the above-mentioned antibody is a concept including both a full-length antibody and an antibody fragment,

The antibody fragment includes all of Fv, Fab, Fab ', F (ab') ₂ , Fd and the like. The Fv comprises both double disulfide Fv (dsFv) and short chain Fv (scFv) forms. Fd refers to the heavy chain portion contained in the Fab fragment. Such as Fv, scFv, and Fab, which is composed of only some fragments essential for the binding of the antibody to the antigen, and has been widely used as a protein therapeutic agent because it exhibits a specific effect by binding to a target. In addition, F _ab without F _c is known to have no effect on the therapeutic effect depending on the type of sugar because there is no sugar or saccharide. However, unlike an antibody, there is not a large difference in molecular weight from that of impurities, so separation using chromatography in a purification process is difficult.

Specifically, the purity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more, but is not limited thereto. The term " purity " means a pure antibody or antibody fragment from which impurities have been removed. For example, when the purity is 92%, it means that the remaining 8% is impurities. The purity may be simply a measure of the purity of the separated material in the eluting solution, but the percent of final purity may also vary depending on how many percent of the loaded sample is purity.

The term " impurity " includes any substance other than the desired antibody or antibody fragment, including but not limited to subtypes, dimers, multimers, host-derived DNA, host-derived proteins, endotoxins, It is not.

In addition, the purity of the target antibody or antibody fragment can be measured by HPLC analysis after purification from the elution solution, and can be specifically analyzed by CEX-HPLC, but the present invention is not limited thereto.

In addition, the antibody or antibody fragment purified by the purification method of the present application can be used as a therapeutic protein. The term " therapeutic protein " as used in the present application is generally used to refer to proteins used in biomedicine, and refers to having various physiological activities. The physiological activity regulates genetic expression and physiological function and corrects abnormal conditions caused by deficiency or excessive secretion of substances involved in function regulation in vivo, and may include a general protein therapeutic agent .

In the present application, the therapeutic protein includes, but is not limited to, an antibody, an antibody fragment, scFv, Fab, Fab 'or F (ab') 2 as long as it has physiological activity in vivo .

According to the affinity chromatography according to the present application, most of the impurities other than the desired protein can be removed, and the antibody or antibody fragment can be purified with high purity and yield without additional steps such as concentration and dialysis.

FIG. 1 shows the procedure for carrying out the experiment of the present application.
FIG. 2A shows the results of elution with a pH linear gradient of pH 3.8 to pH 5.6.
FIG. 2B is a result of analysis by CEX-HPLC of the loading sample before column loading, confirming that the impurities are separated.
FIG. 2C shows the result of analysis by CEX-HPLC of a substance which has no affinity with the heavy-chain so that the impurities are separated from the column.
Figure 2d shows the results of analysis by CEX-HPLC with a washing solution, confirming that some impurities with nonspecific binding to the column are separated.
FIG. 2E shows that the separated material was the target antibody or antibody fragment and the purity was 92% or more as a result of the CEX-HPLC analysis of the separated material using the elution solution.
FIG. 3A shows that the elution solution had an optimum separation ability at pH 4.7, and confirmed that the target protein was separated.
FIG. 3B shows the results of the CEX-HPLC analysis of the separated material using the eluting solution, showing that the separated material in the eluting solution had a purity of 88% or more.
4A shows that the separation ability is maintained under the same pH condition even if the type of the eluting solution is changed.
FIG. 4B shows the results of the CEX-HPLC analysis of the separated material using the eluting solution, showing that the separated material in the eluting solution has a purity of 90% or more.
FIG. 5A shows that the elution solution had an optimal separation ability at pH 4.7, and confirmed that the antibody was separated.
FIG. 5B is a result of analysis by CEX-HPLC of the separated material using the eluting solution, confirming that the material separated from the eluting solution has a purity of 99% or more.
FIG. 6 shows that when the light chain affinity resin was used, the target protein was not separated when the elution solution had a pH of 4.7.
FIG. 7A shows that when the light chain affinity resin was used, the elution solution had an optimum separation ability at pH 2.7, and that the target protein was separated.
7b, when the light chain affinity resin was used, it was confirmed by CEX-HPLC analysis of the separated material using the elution solution that the separated material in the eluting solution had a purity of 53% or more.
Fig. 7C shows the result of comparing the separated material from Experimental Example 2 with the separated materials from Comparative Example 2. Fig.

Hereinafter, preferred embodiments are presented to facilitate understanding of the present application. However, the following embodiments are provided for easier understanding of the present application, and the contents of the present application are not limited by the embodiments.

In order to confirm whether the purification method of the present invention can have a practical effect, a representative antibody fragment drug Ranibizumab was selected for purification.

Experimental Example 1 Experimental Example 2 Experimental Example 3 Experimental Example 4 Comparative Example 1 Comparative Example 2 Condition Suzy CaptureSelect CH1-XL CaptureSelect CH1-XL CaptureSelect CH1-XL CaptureSelect CH1-XL Kappa Select Kappa Select Equilibrium buffer
Salt type and concentration 20 mM sodium phosphate 20 mM sodium phosphate 20 mM sodium phosphate 20 mM sodium phosphate 20 mM sodium phosphate 20 mM sodium phosphate Equilibrium buffer pH pH 6.2 pH 6.2 pH 6.2 pH 6.2 pH 6.2 pH 6.2 Wash buffer salt type and concentration 20 mM sodium phosphate,
400 mM sodium chloride 20 mM sodium phosphate,
400 mM sodium chloride 20 mM sodium phosphate,
400 mM sodium chloride 20 mM sodium phosphate,
400 mM sodium chloride 20 mM sodium phosphate,
40 mM sodium chloride 1X phosphate buffered saline Wash buffer pH pH 5.8 pH 5.8 pH 5.8 pH 5.8 pH 5.8 pH 7.2 Elution buffer salt type and concentration 50 mM sodium acetate 50 mM sodium acetate 50 mM sodium citrate 50 mM sodium acetate 50 mM sodium acetate 0.1 M glycine Elution buffer pH pH 5.6 to 3.8
(0% to 100% linear pH gradient) pH 4.7 pH 4.7 pH 4.7 pH 4.7 pH 2.7 result The target substance could be separated from the eluting solution with high purity.
(Fig. 2) The target substance could be separated from the eluting solution with high purity.
(Fig. 3) The target substance could be separated from the eluting solution with high purity.
(Figure 4) The target substance could be separated from the eluting solution with high purity.
(Fig. 5) No target substance was eluted from the elution solution. (Fig. 7) The target substance could be separated from the eluting solution, but the purity was low and the subsequent purification process could be carried out by concentration and dialysis.
(Fig. 6)

Example 1 Purification of Antibody and Antibody Sections Using Affinity Chromatography

Experimental Example 1: Preparation of antibody fragment purification conditions (conditions of elution solution)

Antibody fragments were purified by loading a sample containing antibody fragments into a chromatography resin (CaptureSelect CH1-XL, Thermo Fisher) that is compatible with the heavy chain. The chromatographic conditions are as follows.

* Chromatographic conditions:

- Resin: CaptureSelect CH1-XL

- Flow rate: 90 cm / h

- equilibrium: 20 mM sodium phosphate, pH 6.2 buffer

- loading: up to 3 g protein / L resin volume

- Wash: 20 mM sodium phosphate, 400 mM sodium chloride, pH 5.8 buffer

Elution: 50 mM sodium acetate, pH 5.6 to 3.8 buffer pH linear concentration gradient

The purification procedure proceeded similarly to the procedure of FIG. 1, and elution solutions of 50 mM sodium acetate, pH 3.8 to pH 5.6 elution solutions were eluted with a pH linear gradient to establish elution solution conditions.

Specifically, the column was equilibrated with an equilibrium solution of 20 mM sodium phosphate, pH 6.2, and the sample containing the antibody fragment was loaded on the affinity chromatography column. The antibody fragment bound to the column was re-equilibrated and then washed. After elution with re-equilibrium, ultraviolet light was collected from 1 mAU to 1 mAU and stored at 5 ± 3 ℃ after filtering.

As a result of elution with a pH linear gradient of pH 3.8 to pH 5.6, it was confirmed that the separation efficiency of the target antibody or antibody fragment was the best when the elution solution had a pH of 4.7 (FIG.

The target antibody or antibody fragment obtained through the purification method was analyzed by CEX-HPLC. As a result, there was no affinity with the loading sample before the column loading (FIG. 2B) and the analysis result of the material which emerged outside the column (FIG. 2C) showed that most of the impurities were not compatible with the double- As a result of using a washing solution of 20 mM sodium phosphate, 400 mM sodium chloride and pH 5.8, it was confirmed that some impurities having nonspecific binding with the column were separated by the washing solution (FIG. 2d). As a result, it was found that only the impurities can be removed from the pre-loading loading sample or washing step, and the target protein is not separated.

Thereafter, the separated material was confirmed to be the target antibody or antibody fragment using the elution solution, and it was confirmed that the purity thereof was 92% or more (FIG. 2E). This means that most of the impurities are removed through the purification process, considering that the product purity of the loading sample is 14%. Also, the process yield was 95% as determined by CEX-HPLC.

Experimental Example 2: Preparation of antibody fragment purification conditions (Determination of proper pH of elution solution)

Antibody fragments were purified by loading the antibody fragment-containing samples onto a mid-affinity chromatography resin (CaptureSelect CH1-XL, Thermo Fisher). The chromatographic conditions are as follows.

* Chromatographic conditions:

- Resin: CaptureSelect CH1-XL

- Flow rate: 90 cm / h

- equilibrium: 20 mM sodium phosphate, pH 6.2 buffer

- loading: up to 3 g protein / L resin volume

- Wash: 20 mM sodium phosphate, 400 mM sodium chloride, pH 5.8 buffer

Elution: 50 mM sodium acetate, pH 4.7 buffer

To confirm whether the elution solution of pH 4.7 set in Experimental Example 1 is suitable for purification of the target antibody or antibody fragment.

The purification procedure proceeded similarly to the procedure of FIG. 1 and eluted with 50 mM sodium acetate, pH 4.7 elution solution to set the elution solution conditions.

Specifically, the column was equilibrated with an equilibrium solution of 20 mM sodium phosphate, pH 6.2, and the sample containing the antibody fragment was loaded on the affinity chromatography column. The antibody fragment bound to the column was re-equilibrated and then washed. After elution with re-equilibrium, ultraviolet light was collected from 1 mAU to 1 mAU and stored at 5 ± 3 ℃ after filtering. A buffer solution (20 mM sodium phosphate, 400 mM sodium chloride, pH 5.8) was used as the washing solution in the same manner as in Experimental Example 1.

As a result, as shown in FIG. 3A, it was confirmed that the elution solution had an optimum resolution when the pH was 4.7. That is, it was confirmed by CEX-HPLC that the impurities and the target protein were separated in stages during the purification process of the present application (FIG. 3A), confirming that the separated material in the eluting solution had a purity of 88% or more (FIG. . In addition, the process yield was 92% as determined by CEX-HPLC.

Experimental Example 3: Detection of Antibody Slice Condition (Determination of whether pH is a key condition)

Antibody fragments were purified by loading the antibody fragment-containing samples onto a mid-affinity chromatography resin (CaptureSelect CH1-XL, Thermo Fisher). The chromatographic conditions are as follows.

* Chromatographic conditions:

- Resin: CaptureSelect CH1-XL

- Flow rate: 90 cm / h

- equilibrium: 20 mM sodium phosphate, pH 6.2 buffer

- loading: up to 3 g protein / L resin volume

- Wash: 20 mM sodium phosphate, 400 mM sodium chloride, pH 5.8 buffer

Elution: 50 mM sodium citrate, pH 4.7 buffer

In order to confirm whether the pH of the elution solution is a key condition in the antibody fragment purification method using affinity chromatography, the elution solution was changed to sodium citrate (NaCitrate) instead of sodium acetate (NaAcetate).

The purification procedure proceeded similarly to the procedure of Figure 1 and was eluted with 50 mM sodium citrate, pH 4.7 eluting solution to set the elution solution conditions.

Specifically, the column was equilibrated with an equilibrium solution of 20 mM sodium phosphate, pH 6.2, and the sample containing the antibody fragment was loaded on the affinity chromatography column. The antibody fragment bound to the column was re-equilibrated and then washed. After elution with re-equilibrium, ultraviolet light was collected from 1 mAU to 1 mAU and stored at 5 ± 3 ℃ after filtering. A buffer solution (20 mM sodium phosphate, 400 mM sodium chloride, pH 5.8) was used as the washing solution in the same manner as in Experimental Example 1.

As a result, as shown in FIG. 4A, it was confirmed that the elution solution had an optimum resolution when the pH was 4.7. As a result, it was confirmed that even if the kind of the eluting solution was changed, the separation ability was maintained at the same pH condition.

Further, the result of the elution was analyzed by CEX-HPLC. As a result, it was confirmed that the product had a purity of 90% or more (FIG. 4B) and the process yield was 92%.

Experimental Example 4: Antibody purification

In order to confirm that not only the antibody fragment but also the antibody can be purified by the purification method according to the present application, chromatography was performed using Erbitux, a representative antibody drug, under the following conditions.

* Chromatographic conditions:

- Resin: CaptureSelect CH1-XL

- Flow rate: 90 cm / h

- equilibrium: 20 mM sodium phosphate, pH 6.2 buffer

- loading: up to 3 g protein / L resin volume

- Wash: 20 mM sodium phosphate, 400 mM sodium chloride, pH 5.8 buffer

Elution: 50 mM sodium acetate, pH 4.7 buffer

The purification procedure proceeded similarly to the procedure of FIG. 1 and eluted with 50 mM sodium acetate, pH 4.7 elution solution to set the elution solution conditions.

Specifically, the column was equilibrated with an equilibrium solution of 20 mM sodium phosphate, pH 6.2, and then ranibizumab was loaded onto a column of the column. The antibody bound to the column was re-equilibrated and then washed. After elution with re-equilibrium, ultraviolet light was collected from 1 mAU to 1 mAU and stored at 5 ± 3 ℃ after filtering. A buffer solution (20 mM sodium phosphate, 400 mM sodium chloride, pH 5.8) was used as the washing solution in the same manner as in Experimental Example 1.

As a result, as shown in FIG. 5A, it was confirmed that the elution solution had an optimum resolution when the pH was 4.7. Further, it was confirmed that the product obtained by elution had a purity of 99% or more (FIG. 5B), and the process yield was found to be 91%.

Comparative Example 1: Condition of antibody fragment purification (using light chain affinity resin)

In order to confirm the excellent effect of the purification method of the present application using the intermediate affinity resin, only the resin (light chain affinity resin) was changed under the same conditions and chromatography was carried out under the same conditions.

* Chromatographic conditions:

- Resin: KappaSelect

- Flow rate: 90 cm / h

- equilibrium: 20 mM sodium phosphate, pH 6.2 buffer

- loading: up to 3 g protein / L resin volume

- Wash: 20 mM sodium phosphate, 400 mM sodium chloride, pH 5.8 buffer

- Sterilization: 10 mM sodium hydroxide solution

Elution: 50 mM sodium acetate, pH 4.7 buffer

The purification procedure was similar to the procedure of FIG. 1 except that the conditions of the resin were the same as those of Experimental Example 2 except that KappaSelect, which is a light chain affinity resin, was used.

Specifically, the column was equilibrated with an equilibrium solution of 20 mM sodium phosphate, pH 6.2, and the antibody fragment-containing sample was loaded on a light chain affinity chromatography column. The antibody fragment bound to the column was re-equilibrated And then washed. After elution with re-equilibrium, ultraviolet light was collected from 1 mAU to 1 mAU and stored at 5 ± 3 ℃ after filtering. A buffer solution (20 mM sodium phosphate, 400 mM sodium chloride, pH 5.8) was used as the washing solution in the same manner as in Experimental Example 1. The re-equilibrium was eluted again. The elution solution was the same as the affinity chromatographic conditions.

As a result, as shown in FIG. 6, it was confirmed that the target protein was separated in the sterilization step, not in the elution step, when the eluting solution had a pH of 4.7.

This confirms that the light chain affinity chromatography does not elute at a high pH range beyond pH 2 to pH 3, as is known in the art.

Comparative Example  2: Antibody fragment  Refining condition setting ( Light chain  Confirm proper pH condition of affinity resin)

In Comparative Example 1, it was confirmed that the target protein was not separated in the resultant elution step using the light chain affinity resin, and when the elution solution suitable for the light chain affinity resin was used, Chromatography was carried out under the conditions.

* Chromatographic conditions:

- Resin: KappaSelect

- Flow rate: 90 cm / h

- equilibrium: 20 mM sodium phosphate, pH 6.2 buffer

- loading: up to 3 g protein / L resin volume

- Washing: 1X Phosphate Buffered Saline, pH 7.2 Buffer

- Sterilization: 10 mM sodium hydroxide solution

Elution: 0.1 M glycine, pH 2.7 buffer

The purification process proceeded similarly to the procedure of Fig.

Specifically, the column was equilibrated with an equilibrium solution of 20 mM sodium phosphate, pH 6.2, and the antibody fragment-containing sample was loaded on a light chain affinity chromatography column. The antibody fragment bound to the column was re-equilibrated And then washed. The eluate was eluted with a re-equilibrium and 0.1 M glycine, pH 2.7 buffer solution was used. The eluate was collected from the time point of 1 mAU increase from the equilibrium ultraviolet ray value until the equilibrium ultraviolet ray value was returned. Due to the stability problems of the antibody fragments, pH was increased using 200 mM sodium phosphate, pH 7.0 to correct for the lower pH of pH 2.7 used in the elution solution. The eluted solution was stored at 5 ± 3 ° C after sterilization.

As a result, as shown in FIG. 7A, it was confirmed that KappaSelect, which is a light chain affinity resin, has an optimum resolution when the elution solution has a pH of 2.7. However, as a result of CEX-HPLC analysis, it was confirmed that a single antibody fragment was separated in addition to the target protein in the elution solution. As a result, it was confirmed by the chromatography of Comparative Example 2 that the single antibody fragment was not properly separated.

The purity of the separated material in the eluting solution was found to be 53%, which is much lower than that of using affinity chromatography (Fig. 7b), and the separated material from Experimental Example 2 and the separated material from Comparative Example 2 In comparison, the purity was again high when using affinity chromatography (Fig. 7c).

When light chain affinity chromatography was used, the yield of the process was also 87%, which was lower than when using the affinity chromatography according to the present application. In addition, in the case of the comparative example, concentration and dialysis are further carried out because the concentration and dialysis process can proceed to the next step. Therefore, when the chromatography of the comparative example is actually used, the target substance is actually obtained at a lower yield .

As described above, in the present application, it was confirmed that, when using affinity chromatography (Experimental Examples 1 to 4), the elution solution of pH 3.8 to pH 4.7, especially the desired antibody or antibody fragment, was separated at pH 4.7 .

However, in Comparative Example 1, when the eluting solution having the same conditions as those in the affinity chromatography was used, the target protein was not separated. In Comparative Example 2, when the eluting solution having pH lower than that of the present experimental example was used, It was confirmed that the target protein was separated.

In the case of using an elution solution having a low pH of pH 2.7 as used in Comparative Example 2, there is a problem in terms of the structural and stability of the protein. Therefore, a correction operation for increasing the pH is necessary. However, in the case of using the purification method of the present application, it is possible not only to omit the above-mentioned correction operation for raising the pH but also to omit the concentration and dialysis process, Is superior in terms of economically obtaining high purity proteins.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present application is to be interpreted as being within the scope of the present application, all changes or modifications derived from the meaning and scope of the appended claims and from their equivalents rather than the detailed description.

Claims (14)

(a) loading an antibody or antibody fragment-containing sample into an affinity chromatography medium containing an affinity resin;
(b) washing with a washing solution; And
(c) recovering the target antibody or antibody fragment from the affinity chromatography column using an elution solution having a pH in the range of pH 3.8 or more to a pH of 4.7 or less.
The method according to claim 1,
Wherein the intermediate affinity resin in step (a) is a resin capable of specifically binding to the midsole.
The method according to claim 1,
Characterized in that the method does not carry out a concentration and dialysis step after step (c).
The method according to claim 1,
Wherein the elution solution in step (c) has a salt concentration ranging from 10 mM or more to 100 mM or less.
The method according to claim 1,
Wherein the elution solution in step (c) comprises at least one salt selected from the group consisting of sodium acetate, sodium citrate, and glycine.
The method according to claim 1,
Wherein the column is equilibrated with a buffer solution having a pH ranging from pH 5.5 to less than pH 7.5 before loading the antibody or antibody fragment containing sample of step (a).
The method according to claim 6,
The buffer solution may be selected from MES (2- (N-morpholino) ethanesulfonic acid), PIPES, potassium phosphate, potassium chloride, 3-morpholinopropane-1-sulfonic acid, sodium phosphate, sodium chloride, tris and HEPES - (2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid).
The method according to claim 1,
Further comprising the step of affinity chromatography, ion exchange chromatography, concentration or dialysis prior to said step (a).
The method according to claim 1,
Wherein the method further comprises the step of, after step (a) or (b), discharging an impurity that is not compatible with the resin into the equilibrium solution.
The method according to claim 1,
The method further comprises the step of re-equilibrating the buffer solution after step (a) or (b).
The method according to claim 1,
Wherein the cleaning solution in step (b) has a pH ranging from pH 4.5 to less than pH 6.5.
The method according to claim 1,
Wherein the washing solution in step (b) has a salt concentration ranging from 400 mM to 1 M or less.
The method according to claim 1,
The cleaning solution in step (b) may be selected from the group consisting of sodium phosphate, potassium chloride, potassium phosphate, sodium chloride, tris, 2- (N-morpholino) ethanesulfonic acid, MOPS (3-morpholinopropane- Wherein the at least one salt is selected from the group consisting of HEPES (2- [4- (2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid).
The method according to claim 1,
Wherein the purified target antibody or antibody fragment is a therapeutic protein.

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