WO2018212556A1

WO2018212556A1 - A method for purifying an antibody or an antibody fragment thereof using affinity chromatography

Info

Publication number: WO2018212556A1
Application number: PCT/KR2018/005552
Authority: WO
Inventors: Kyunghwa Kim; Sung-Muk KANG; Jung Min Yoo; Hong Jai Lee; Se Jun Kim; Chung Min Lee; Yoo Hee Yang; Dong Eok Lee; Gyong-sik HA
Original assignee: CJ Healthcare Corp
Current assignee: HK Inno N Corp
Priority date: 2017-05-16
Filing date: 2018-05-15
Publication date: 2018-11-22
Anticipated expiration: 2019-11-16

Abstract

The present disclosure relates to a method for separating and purifying an antibody or an antibody fragment thereof in high purity using affinity chromatography, and specifically, to a method for separating the antibody and antibody fragment thereof in high purity and purifying the same using an elution buffer capable of increasing stability for the enhancement of stability.

Description

A METHOD FOR PURIFYING AN ANTIBODY OR AN ANTIBODY FRAGMENT THEREOF USING AFFINITY CHROMATOGRAPHY

The present disclosure relates to a method for separating and purifying a high-purity antibody or antibody fragment thereof using affinity chromatography, and specifically to a method for separating a high-purity antibody and antibody fragment thereof and separating the same using an elution buffer capable of enhancing stability.

Recombinant proteins are expressed and produced by inserting a target protein gene into bacteria or cells. In addition to target proteins, a variety of impurities, such as protein isoforms, dimers, multimers, single antibody fragments, host-derived DNAs, host-derived proteins, endotoxins, etc., are present in the stock culture obtained upon expression, and a purification process for removing the impurities is required to commercialize the target proteins. An antibody fragment of such protein therapeutic agents is used to show drug efficacy by binding specifically to the target. In addition, since Fab without Fc has no glycone or glycosylation, it is a very attractive therapeutic agent as the therapeutic effect is not affected by the type of sugars. However, unlike antibodies, because the difference between the molecular weights of impurities and antibody fragments is not large, it is difficult to separate the same in the entire purification process using chromatography.

A purification method using an affinity resin is widely used for the method for separating antibody fragments. For example, it is known that for the method of purifying antibodies, impurities in a target product can be purified by using protein A affinity chromatography (International Patent Application No.　WO2011073954). However, in this method, a target protein can be obtained from a resin as long as an acidic buffer solution having a pH of about 2 to 3 is used; that is, the method has a problem in that a target protein becomes unstable as a result of the low pH of an elution solution. In order to overcome such problem, a buffer solution that increases the pH of the elution buffer is immediately added and mixed to the resultant so that the stability of a target protein is secured. Because a buffer solution is mixed to ensure the stability of a target protein, inconvenience and yield reduction are accompanied as the method requires an additional concentration/dialysis process in order to remove the mixed buffer solution.

In addition, for the method for purifying an antibody fragment, a method for specifically recovering and purifying a target protein using a chromatographic method utilizing a resin that has a specific affinity for kappa located in a light chain is known (Korean Patent Application No.　2016-0127317). However, this method also has drawbacks. Specifically, an antibody fragment uses a system expressing each of a heavy chain and a light chain in a vector, wherein a protein in which a light chain is solely present, in addition to the type to which the two chains are linked, may be expressed. That is, there is a problem in that when a resin having an affinity for a kappa located in a light chain is used, impurities such as single fragments of light chains should be further purified and removed. Additionally, similar to a protein A affinity resin, elution with a buffer solution having a low pH (approximately pH　2 to pH　3) is required, and thus the stability of an antibody fragment to be obtained is low. That is, it has a drawback in that an additional concentration/dialysis process must be carried out in order to compensate for the defect. Additionally, the purity and yield of the process are not high.

With this background, the present inventors have made efforts to find a method for purifying a target protein having high purity and yield even under pH conditions which are higher than conventionally used in affinity chromatography. As a result, they have discovered a method for purifying an antibody and antibody fragment with high purity and stability when using heavy chain affinity chromatography and utilizing an optimized elution buffer.

An object of the present disclosure is to provide a target antibody or an antibody fragment thereof, comprising: (a) loading a sample containing an antibody or an antibody fragment thereof into an affinity chromatography column containing a heavy chain affinity resin; (b) washing the column with a washing buffer; and (c) recovering a target antibody or an antibody fragment thereof from the affinity chromatography column using an elution buffer which has a pH ranging from pH　3.8 to pH　4.7.

Hereinbelow, the present disclosure will be described in detail.

Meanwhile, each of the explanations and exemplary embodiments disclosed herein can be applied to other explanations and exemplary embodiments. That is, all combinations of various factors disclosed herein belong to the scope of the present disclosure. Furthermore, the scope of the present disclosure should not be limited by the specific disclosure provided hereinbelow.

Additionally, those skilled in the art will be able to recognize or confirm, based on routine experimentation, many equivalents to the specific embodiments of the present disclosure described in this application, and such equivalents are intended to be included in the present disclosure.

An aspect of the present disclosure is to provide a target antibody or an antibody fragment thereof, comprising: (a) loading a sample containing an antibody or an antibody fragment thereof into an affinity chromatography column containing a heavy chain affinity resin; (b) washing the column with a washing buffer; and (c) recovering a target antibody or an antibody fragment thereof from the affinity chromatography column using an elution buffer which has a pH ranging from pH　3.8 to pH　4.7.

In general, when a protein is purified using affinity chromatography containing an affinity resin, an elution buffer in the range of pH　2 to pH　3 is mostly used, resulting in problems in terms of the 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 to increase the pH to pH　4 to pH　7 after purification at a low pH (pH　2 to pH　3), and it has a problem in that the yield of a target protein is reduced in the step of performing such an additional process. However, the heavy chain affinity resin of the present disclosure is significant in that a target protein having high purity and stability is produced in the pH range of pH　3.8 to pH　4.7, which is a condition milder than that required for a general affinity resin.

Each step of the method for purifying a target antibody or an antibody fragment thereof will be described in detail as follows. First, Step (a) is a step of loading a sample containing an antibody or an antibody fragment thereof into an affinity chromatography column containing a heavy chain affinity resin.

As used herein, the term "affinity chromatography" refers to a chromatographic method using a binding substance having an affinity for a specific protein. A binding substance having an affinity for a specific protein is a polymeric substance binding to a functional group; that is, it binds to a substance having an affinity, which is dissolved in polar and non-polar solutions. For the purpose of the present disclosure, the affinity chromatography can be classified into heavy chain affinity chromatography and light chain affinity chromatography, but is not limited thereto. Specifically, heavy chain affinity chromatography refers to a chromatographic method using a resin, e.g., a heavy chain affinity resin, capable of specifically binding to a heavy chain which is a part of an antibody. In addition, light chain affinity chromatography refers to a chromatographic method using a resin, e.g., a light chain affinity resin, capable of specifically binding to a light chain which is a part of an antibody.

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

In an embodiment of the present disclosure, the column may be equilibrated using a buffer having a pH ranging from pH　5.5 to pH　7.5 before loading the sample of Step (a), which contains the antibody or antibody fragment thereof. The buffer may specifically comprise one or more salts selected from the group consisting of sodium phosphate, sodium chloride, Tris, 2-(N-morpholino)ethanesulfonic acid (MES), 3-morpholinopropane-1-sulfonic acid (MOPS), PIPES, potassium phosphate, potassium chloride, and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), but is not limited thereto.

In an embodiment of the present disclosure, the method may further comprise carrying out ion-exchange chromatography, concentration, and/or dialysis before Step (a). The purpose thereof is to remove primary impurities of the antibody or antibody fragment thereof and to increase the purity of the sample. Specifically, the antibody fragment-containing sample is subjected to concentration and dialysis before Step (a), the step of purifying the sample using anion-exchange chromatography is performed in advance, and then the sample can be loaded into affinity chromatography utilizing a heavy chain affinity resin. Any work can be applied without limitation as long as it removes the primary impurities that do not conjugate with an affinity resin and can increase the purity of the sample.

In the method for purifying a target antibody or an antibody fragment thereof, Step (b) is a step of washing the column with a washing buffer, wherein the washing buffer is applied to the chromatography into which the sample is loaded.

The washing buffer may have a pH ranging from pH　5.5 to pH　7.8, and have a salt concentration ranging from 400　mM to 1　M, but the ranges of the pH and salt concentration are not limited thereto.

Additionally, the washing buffer may comprise one or more salts selected from the group consisting of sodium phosphate, potassium chloride, potassium phosphate, sodium chloride, Tris, 2-(N-morpholino)ethanesulfonic acid (MES), 3-morpholinopropane-1-sulfonic acid (MOPS), PIPES, and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), but is not limited thereto.

For the purpose of the present disclosure, in Step (b), the impurities that are non-specifically bound to the heavy chain affinity resin may be removed by the washing buffer.

In an embodiment of the present disclosure, the purification method may further comprise discharging impurities with no affinity for the resin using an equilibration buffer after Step (a) or Step (b). Specifically, this step may be performed at least one time, but generally, the step may be performed without limitation until the equilibration is established.

In an embodiment of the present disclosure, the purification method may further comprise re-equilibrating the column using a re-equilibration buffer after Step (a) or Step (b). The re-equilibration buffer is flowed under the same condition as the equilibration buffer of Step (a) in which nothing is reacted and a target antibody or an antibody fragment thereof is not eluted between the washing and elution processes, and then is flowed once again before the elution buffer is flowed. Therefore, the re-equilibration buffer serves as a bridge between the washing buffer and the elution buffer.

Specifically, the re-equilibration buffer may have a pH ranging from pH　4.5 to pH　6.5, and have a salt concentration ranging from 10　mM to 30　mM, but the ranges of the pH and salt concentration are not limited thereto.

Additionally, the re-equilibration buffer may comprise one or more salts selected from the group consisting of sodium phosphate, sodium chloride, Tris, 2-(N-morpholino)ethanesulfonic acid (MES), 3-morpholinopropane-1-sulfonic acid (MOPS), PIPES, potassium phosphate, potassium chloride, and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), but is not limited thereto.

In the method for purifying a target antibody or an antibody fragment thereof, Step (c) is a step of recovering a target antibody or an antibody fragment thereof from the affinity chromatography column using an elution buffer which has a pH ranging from pH　3.8 to pH　4.7.

The elution buffer may have a pH ranging from pH　3.8 to pH　4.7, and have a salt concentration ranging from 10　mM to 100　mM, but the ranges of the pH and salt concentration are not limited thereto.

Even when the pH of the elution buffer is less than pH　3.8, a target protein is still eluted. However, there is a problem in that it requires a concentration/dialysis process and calibration work of increasing the pH (pH　4 to pH　7) after purification at a low pH (pH　2 to pH　3); and this problem also occurs when using conventional affinity chromatography. In addition, a pH higher than pH　4.7 is not preferable because the ligand which holds the heavy chain in the column forms a hydrogen bond with a target protein, and is thereby not eluted.

Additionally, the elution buffer may comprise one or more salts selected from the group consisting of sodium acetate, sodium citrate, and glycine in a salt concentration ranging from 10　mM to 100　mM, but any salts may be used without limitation as long as they can separate a target antibody or an antibody fragment thereof under the mild conditions of the heavy chain affinity chromatography.

In particular, the purification method of the present disclosure is characterized in that a concentration/dialysis process is not carried out after Step (c). The "concentration/dialysis process" is a process generally used for removing substances having a size smaller than that of a target on a membrane or for changing a buffer, and thus is a process used to further purify and remove single fragments, i.e., impurities, when a general affinity resin is used. When the concentration/dialysis process is additionally used, the process yield is not high. However, the purification method of the present disclosure has economic superiority in that even if the additional concentration/dialysis process is not carried out, the antibody or antibody fragment thereof can be purified in a high purity and yield by using the heavy chain affinity resin and optimization of the elution buffer.

As used herein, the term "target antibody or antibody fragment thereof" refers to a kind of protein to be separated from the affinity chromatography of the present disclosure, and thus can interchangeably be used with "target protein".

The antibody may specifically be a monoclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody which can be formed by a cell with an antibody-encoding sequence and which recognizes a specific antigen. The antibody of the present disclosure, although not limited thereto, may preferably include all therapeutic antibodies conventionally used in the art.

Additionally, the antibody is a concept encompassing both full length antibodies and antibody fragments, and examples of the antibody fragments include all of Fv, Fab, Fab′, F(ab′')₂, Fd, etc. The Fv includes both forms of disulfide-stabilized Fv (dsFv) and single chain Fv (scFv). Fd refers to the heavy chain component included in Fab. The antibody fragment refers to one that is constituted using only some fragments essential for binding to an antigen of antibodies such as Fv, scFv, and Fab. Further, the antibody fragment is widely used as a protein therapeutic agent because it specifically binds to a target to exhibit a medicinal effect. In addition, it is known that Fab without Fc has no influence on the therapeutic effect depending on the type of sugars because no glycone or saccharification exists therein. However, unlike an antibody, since the antibody fragment does not exhibit a large difference in molecular weight compared to that of impurities, separation of the antibody fragment using the chromatography in the purification method is difficult.

The target antibody or antibody fragment thereof separated using the purification method of the present disclosure refers to one having a purity of 88% or higher, and specifically, the purity may be 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, or 98% or higher, but is not limited thereto. The term "purity" refers to a pure antibody or antibody fragment thereof from which impurities have been removed. For example, if the purity is 92%, it means that the remaining 8% is impurities. In addition, the purity may simply indicate the purity of the material separated from the elution buffer, but the final purity percentage may vary depending on the purity percentage of the loaded sample.

As used herein, the term "impurities" includes any material other than the target antibody or antibody fragment thereof. Examples of the impurities include isoforms, dimers, multimers, host-derived DNAs, host-derived proteins, endotoxins, etc., but are not limited thereto.

Additionally, the purity of the target antibody or antibody fragment thereof can be measured by HPLC analysis after purification from the elution buffer, and specifically can be analyzed by CEX-HPLC, but is not limited thereto.

Additionally, the antibody or antibody fragment thereof purified by the purification method of the present disclosure can be used as a therapeutic protein. As used herein, the term "therapeutic protein" is a concept collectively referring to a protein conventionally used in biomedicine, and thus refers to a protein having various physiological activities. The physiological activities regulate genetic expressions and physiological functions to rectify abnormal conditions caused by deficiency or excessive secretion of substances involved in functional regulations in vivo, and thus may be included in general protein therapeutic agents.

In the present disclosure, the therapeutic protein can be included without limitation as long as the protein has the physiological activities in vivo. Examples of the therapeutic protein may be scFv, Fab, Fab′', or F(ab′)₂, which is the antibody or antibody fragment thereof, but are not limited thereto.

According to the affinity chromatography of the present disclosure, most impurities in addition to the target protein can not only be removed, but also an antibody or an antibody fragment thereof can be purified in high purity and yield without an additional process, such as a concentration/dialysis process.

Fig.　1 shows the procedure for carrying out the experiments of the present disclosure.

Fig.　2a shows the results of the elution buffer (pH　3.8 to pH　5.6) eluted in a pH linear concentration gradient.

Fig.　2b shows the results of CEX-HPLC analysis of the sample before loading into the column, confirming that impurities are separated.

Fig.　2c shows the results of CEX-HPLC analysis of the substance which exits the column due to no affinity for a heavy chain, confirming that impurities are separated.

Fig.　2d shows the results of CEX-HPLC analysis of the substance separated using the washing buffer, confirming that some impurities which are non-specifically binding to the column are separated.

Fig.　2e shows the results of CEX-HPLC analysis of the substance separated using the elution buffer, confirming that the separated substance is the target antibody or antibody fragment thereof, and that the purity thereof is 92% or higher.

Fig.　3a shows that the elution buffer had optimum separation ability at pH　4.7, and that the target protein was separated.

Fig.　3b shows the results of CEX-HPLC analysis of the substance separated using the elution buffer, confirming that the substance separated from the elution buffer had a purity of 88% or higher.

Fig.　4a shows that the separation ability was maintained under the same pH condition even when the type of the elution buffer changed.

Fig.　4b shows the results of CEX-HPLC analysis of the substance separated using the elution buffer, confirming that the substance separated from the elution buffer had a purity of 90% or higher.

Fig.　5a shows that the elution buffer had the optimum separation ability at pH　4.7, and that the antibody was separated.

Fig.　5b shows the results of CEX-HPLC analysis of the substance separated using the elution buffer, confirming that the substance separated from the elution buffer had a purity of 99% or higher.

Fig.　6 shows that when the light chain affinity resin was used, the target protein was not separated when the elution buffer was at pH　4.7.

Fig.　7a shows that when the light chain affinity resin was used, the elution buffer had the optimum separation ability at pH　2.7, confirming that the target protein was separated.

Fig.　7b shows the results of CEX-HPLC analysis of the substance separated using the elution buffer when the light chain affinity resin was used, confirming that the substance separated from the elution buffer had a purity of 53% or higher.

Fig.　7c shows the results of comparing the substance separated in Experimental Example 2 and that separated in Comparative Example 2.

Hereinafter, the present disclosure will be described in more detail with reference to the following Examples. However, these Examples are provided to assist in further understanding of the present disclosure and the present disclosure is not intended to be limited by these Examples.

In order to confirm whether the purification method of the present disclosure can exhibit a substantial effect, ranibizumab, a representative antibody fragment medicine, was selected for purification.

		Experimental Example 1	Experimental Example 2	Experimental Example 3	Experimental Example 4	Comparative Example 1	Comparative Example 2
Condition	Resin	CaptureSelect CH1-XL	CaptureSelect CH1-XL	CaptureSelect CH1-XL	CaptureSelect CH1-XL	Kappa Select	Kappa Select
	Type and concentration of equilibration buffer	Sodium phosphate (20　mM)	Sodium phosphate (20　mM)	Sodium phosphate (20　mM)	Sodium phosphate (20　mM)	Sodium phosphate (20　mM)	Sodium phosphate (20　mM)
	pH of equilibration buffer	pH　6.2	pH　6.2	pH　6.2	pH　6.2	pH　6.2	pH　6.2
	Type and concentration of washing buffer	Sodium phosphate (20　mM) and sodium chloride (400　mM)	Sodium phosphate (20　mM) and sodium chloride (400　mM)	Sodium phosphate (20　mM) and sodium chloride (400　mM)	Sodium phosphate (20　mM) and sodium chloride (400　mM)	Sodium phosphate (20　mM) and sodium chloride (400　mM)	1X Phosphate - buffer saline
	pH of washing buffer	pH　5.8	pH　5.8	pH　5.8	pH　5.8	pH　5.8	pH　7.2
	Type and concentration of elution solution	Sodium acetate (50　mM)	Sodium acetate (50　mM)	Sodium citrate (50　mM)	Sodium acetate (50　mM)	Sodium acetate (50　mM)	Glycine (0.1　M)
	pH of elution buffer	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 with high purity could be separated from the elution buffer (Fig.　2).	The target substance with high purity could be separated from the elution buffer (Fig.　3).	The target substance with high purity could be separated from the elution buffer (Fig.　4).	The target substance with high purity could be separated from the elution buffer (Fig.　5).	The target substance was not eluted from the elution buffer(Fig.　7).	Although the target substance could be separated from the elution buffer, the purity thereof was low. In addition, concentration/dialysis processes were required in order to proceed with the next purification process(Fig.　6).

Example 1: Purification of antibody and antibody fragment using affinity chromatography

Experimental Example 1: Setting purification condition for antibody fragment (condition setting of elution buffer)

The antibody fragment-containing sample was loaded into a chromatographic resin (CaptureSelect^TM CH1-XL, Thermo Fisher) having an affinity for a heavy chain, and then purified. The chromatography conditions are as follows:

* Chromatography conditions:

- Resin: CaptureSelect CH1-XL

- Flow Rate: 90　cm/h

- Equilibration: 20　mM sodium phosphate buffer (pH　6.2)

- Loading: max. 3　g protein/L (resin volume)

- Washing: Bbuffer (20　mM sodium phosphate, 400　mM sodium chloride; pH　5.8)

- Elution: linear pH concentration gradient sodium acetate (50　mM) buffer (pH　3.8 to pH　5.6)

The purification process was performed similarly to the procedure of Fig.　1. In order to set the conditions of the elution buffer, a sodium acetate (50　mM) elution buffer (pH　3.8 to pH　5.6) was eluted with a linear pH concentration gradient.

Specifically, the column was equilibrated with an equilibration buffer (sodium phosphate (20　mM); pH　6.2), and the antibody fragment-containing sample was loaded into an affinity chromatography column. Thereafter, the antibody fragment bound to the column was washed after re-equilibration. The resultants were once more eluted through re-equilibration and collected from the point where ultraviolet rays at the time of elution were higher than 1　mAU until the point where the ultraviolet rays reached 1　mAU again, and then stored at 5±3°C upon antibacterial filtration.

As a result of eluting the elution buffer (pH　3.8 to pH　5.6) with a linear pH concentration gradient, it was confirmed that the elution buffer had optimum separation ability of the target antibody or antibody fragment thereof at pH　4.7 (Fig.　2a).

The target antibody or antibody fragment thereof obtained through the purification method was analyzed with CEX-HPLC. Considering the analysis results (Fig.　2c) that a substance was released out of the column because it had no affinity with the heavy chain and the loading sample before loading into the column (Fig. 2b), it was confirmed that most impurities had no affinity for the heavy chain affinity resin, and were thus separated and purified. In addition, as a result of using the washing buffer (sodium phosphate (20　mM) and sodium chloride (400　mM); pH　5.8), it was confirmed that some impurities having non-specific binding with the column were separated by the washing buffer (Fig.　2d). Accordingly, it was confirmed from the results above that only the impurities could be removed from the sample before loading or by the washing step, but the target protein was not separated.

Thereafter, it was confirmed that the substance separated using the elution buffer was the target antibody or antibody fragment thereof, and that the purity thereof was 92% or higher (Fig.　2e). Considering that the product purity of the loading sample was 14%, it was confirmed that most impurities were removed through this purification process. Additionally, it was confirmed that, as a result of analyzing with CEX-HPLC, the process yield was 95%.

Experimental Example 2: Setting purification condition for antibody fragment (Determination of proper pH of elution buffer)

The antibody fragment-containing sample was loaded into a heavy chain affinity chromatography resin (CaptureSelect CH1-XL, Thermo Fisher), and then the antibody fragment was purified. The chromatography conditions are as follows:

* Chromatography conditions:

- Resin: CaptureSelect CH1-XL

- Flow Rate: 90　cm/h

- Equilibration: 20　mM sodium phosphate buffer (pH　6.2)

- Loading: max. 3　g protein/L (resin volume)

- Washing: buffer (20　mM sodium phosphate, 400　mM sodium chloride; pH　5.8)

- Elution: buffer (50　mM sodium acetate; pH　4.7)

Experiments were performed in order to determine whether the elution buffer (pH　4.7) set in Experimental Example 1 was suitable for purification of the target antibody or antibody fragment thereof.

The purification process was performed similarly to the procedure of Fig.　1. In order to set the conditions of the elution buffer, a sodium acetate (50　mM) buffer (pH　4.7) was eluted.

Specifically, the column was equilibrated with an equilibration buffer (sodium phosphate (20　mM); pH　6.2), and the antibody fragment-containing sample was loaded into an affinity chromatography column. Thereafter, the antibody fragment bound to the column was washed after re-equilibration. The resultants were once more eluted through re-equilibration and collected from the point where ultraviolet rays at the time of elution were higher than 1　mAU until the point where the ultraviolet rays reached 1　mAU again, and then stored at 5±3°C upon antibacterial filtration. For the washing buffer, a buffer (sodium phosphate (20　mM) and sodium chloride (400　mM); pH　5.8) was used as in Experimental Example 1.

As a result, as shown in Fig.　3a, it was confirmed that the elution buffer had optimum separation ability at pH　4.7. That is, it was confirmed by the CEX-HPLC analysis that impurities and the target protein were separated in each step of the purification process of the present disclosure (Fig.　3a), and that the substance separated from the elution buffer had a purity of 88% or higher (Fig.　3b). Additionally, as a result analyzing with CEX-HPLC, it was confirmed that the process yield was 92%.

Experimental Example 3: Setting purification condition for antibody fragment (Determination on whether pH is essential condition)

The antibody fragment-containing sample was loaded into an affinity chromatography resin (CaptureSelect CH1-XL, Thermo Fisher), and then the antibody fragment was purified. The chromatography conditions are as follows:

* Chromatography conditions:

- Resin: CaptureSelect CH1-XL

- Flow Rate: 90　cm/h

- Equilibration: 20　mM sodium phosphate buffer (pH　6.2)

- Loading: max. 3　g protein/L (resin volume)

- Washing: buffer (20　mM sodium phosphate, 400　mM sodium chloride; pH　5.8)

- Elution: 50　mM sodium citrate buffer (pH　4.7)

In order to determine whether the pH of the elution buffer is an essential condition in the purification method of the antibody fragment using affinity chromatography, the experiments were performed by changing the type of the elution buffer to sodium citrate (NaCitrate) instead of sodium acetate (NaAcetate).

The purification process was performed similarly to the procedure of Fig.　1. In order to set the condition of the elution buffer, a sodium citrate (50　mM) buffer (pH　4.7) was eluted as the elution buffer.

Specifically, the column was equilibrated with an equilibration buffer (sodium phosphate (20　mM); pH　6.2), and then the antibody fragment-containing sample was loaded into an affinity chromatography column. Thereafter, the antibody fragment bound to the column was washed after re-equilibration. The resultants were once more eluted through re-equilibration and collected from the point where ultraviolet rays at the time of elution were higher than 1　mAU until the point where the ultraviolet rays reached 1　mAU again, and then stored at 5±3°C upon antibacterial filtration. For the washing buffer, a buffer (sodium phosphate (20　mM) and sodium chloride (400　mM); pH　5.8) was used as in Experimental Example 1.

As a result, as shown in Fig.　4a, it was confirmed that the elution buffer had optimum separation ability at pH　4.7. Accordingly, it was confirmed that even if the type of the elution buffer changed, the separation ability was still maintained at the same pH condition.

Additionally, as a result of analyzing the resultants obtained by the elution with CEX-HPLC, it was confirmed that the resultants have a purity of 90% or higher (Fig.　4b), and that the process yield thereof was 92%.

Experimental Example 4: Purification of antibody

In order to determine whether not only the antibody fragment but also the antibody can be purified by the purification method according to the present disclosure, chromatography was performed using Erbitux, a representative antibody medicine, under the following conditions:

* Chromatography conditions:

- Resin: CaptureSelect CH1-XL

- Flow Rate: 90　cm/h

- Equilibration: 20　mM sodium phosphate buffer (pH　6.2)

- Loading: max. 3　g protein/L (resin volume)

- Washing: buffer (20　mM sodium phosphate, 400　mM sodium chloride; pH　5.8)

- Elution: 50　mM sodium acetate buffer (pH　4.7)

The purification process was performed similarly to the procedure of Fig.　1. In order to set the conditions of the elution buffer, a sodium acetate (50　mM) elution buffer (pH　4.7) was eluted.

Specifically, the column was equilibrated with an equilibration buffer (sodium phosphate (20　mM); pH　6.2), and ranibizumab was loaded into an affinity chromatography resin containing heavy chain domain. Thereafter, the antibody bound to the column was washed after re-equilibration. The resultants were once more eluted through re-equilibration and collected from the point where ultraviolet rays at the time of elution were higher than 1　mAU until the point where the ultraviolet rays reached 1　mAU again, and then stored at 5±3°C upon antibacterial filtration. For the washing buffer, a buffer (sodium phosphate (20　mM) and sodium chloride (400　mM); pH　5.8) was used as in Experimental Example 1.

As a result, as shown in Fig.　5a, it was confirmed that the elution buffer had optimum separation ability at pH　4.7. Additionally, it was confirmed that the resultants obtained by the elution had a purity of 99% or higher (Fig.　5b), and that the process yield thereof was 91%.

Comparative Example 1: Setting purification condition for antibody fragment (using light chain affinity resin)

In order to once again confirm the excellent effect of the purification method of the present disclosure using the heavy chain affinity resin, chromatography was performed under the same conditions except that the resin was changed to a light chain affinity resin. Chromatography conditions are as follows:

* Chromatography conditions:

- Resin: KappaSelect

- Flow Rate: 90　cm/h

- Equilibration: 20　mM sodium phosphate buffer (pH　6.2)

- Loading: max. 3　g protein/L (resin volume)

- Washing: buffer (20　mM sodium phosphate, 400　mM sodium chloride; pH　5.8)

- Sterilization: 10　mM sodium hydroxide solution

- Elution: 50　mM sodium acetate buffer (pH　4.7)

The purification process was performed similarly to the procedure of Fig.　1. Experiments were carried out under the same conditions as in Experimental Example 2, except that KappaSelect, a light chain affinity resin, was used as the resin.

Specifically, the column was equilibrated with an equilibration buffer (sodium phosphate (20　mM); pH　6.2). Thereafter, the antibody fragment-containing sample was loaded into a light chain affinity chromatography column, and then the antibody fragment bound to the column was washed after re-equilibration. The resultants were once more eluted through re-equilibration and collected from the point where ultraviolet rays at the time of elution were higher than 1　mAU until the point where the ultraviolet rays reached 1　mAU again, and then stored at 5±3°C upon antibacterial filtration. For the washing buffer, a buffer (sodium phosphate (20　mM) and sodium chloride (400　mM); pH　5.8) was used as in Experimental Example 1. The resultants were once more eluted through re-equilibration; at the time of the elution, the same elution buffer as that for the heavy chain affinity chromatography was used.

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

This result confirms that the elution does not occur when the elution buffer in light chain affinity chromatography has a high pH beyond pH　2 to pH　3, as is already known in the art.

Comparative Example 2: Setting purification condition for antibody fragment (Determination of suitable pH condition of light chain affinity resin)

It was confirmed in Comparative Example 1 that the target protein was not separated in the elution step when using the light chain affinity resin. In addition, in order to confirm whether the target protein is separated when using an elution buffer suitable for the light chain affinity resin, chromatography was performed under the following conditions:

* Chromatography conditions:

- Resin: KappaSelect

- Flow Rate: 90　cm/h

- Equilibration: 20　mM sodium phosphate buffer (pH　6.2)

- Loading: max. 3　g protein/L (resin volume)

- Washing: 1X phosphate-buffer saline (pH　7.2)

- Sterilization: 10　mM sodium hydroxide solution

- Elution: glycine (0.1　M) buffer (pH　2.7)

The purification process was performed similarly to the procedure of Fig.　1.

Specifically, the column was equilibrated with an equilibration buffer (sodium phosphate (20　mM); pH　6.2). The antibody fragment-containing sample was loaded into a light chain affinity chromatography column, and the antibody fragment bound to the column was washed after re-equilibration. The resultants were once more eluted through re-equilibration. For the elution buffer, a buffer (glycine (0.1　M); pH　2.7) was used. The resultants were collected from the point where ultraviolet ray value at the equilibration state during elution was increased by 1　mAU until the point where the value was returned to the UV value at the equilibration state. In order to resolve the stability problem of the antibody fragment, the low pH (pH　2.7) of the elution buffer was increased using sodium phosphate (200　mM; pH　7.0). The corrected elution buffer was stored at 5±3°C upon antibacterial filtration.

As a result, as shown in Fig.　7a, it was confirmed that in the KappaSelect, which is a light chain affinity resin, the elution buffer had optimum separation ability at pH　2.7, and the obtained resultants were analyzed using CEX-HPLC. As a result, it was confirmed that in the elution buffer, a single antibody fragment was also separated in addition to the target protein. Consequently, it was confirmed that a single antibody fragment was not properly separated using the chromatography of Comparative Example 2.

The purity of the substance separated from the elution buffer was 53%, confirming that the purity was much lower than that using the heavy chain affinity chromatography (Fig.　7b). In addition, as a result of comparing the substance separated in Experimental Example 2 with that separated in Comparative Example 2, it was once again confirmed that the purity was higher when using the heavy chain affinity chromatography (Fig.　7c).

When the light chain affinity chromatography was used, the process yield was 87%, which was also lower than in the case of using the heavy chain affinity chromatography according to the present disclosure. In addition, in the Comparative Examples, a concentration/dialysis process must be carried out in order to proceed with the next process. Therefore, in consideration of the fact that the concentration/dialysis process is required to be additionally carried out, it was confirmed that a target substance was in fact secured in a lower yield by using the chromatography of the Comparative Examples.

As described above, in the present disclosure, it was confirmed that when the heavy chain affinity chromatography was used (Experimental Examples 1 to 4), a target antibody or an antibody fragment thereof was properly separated when the elution buffer was at pH　3.8 to pH　4.7, particularly at pH　4.7.

However, it was confirmed in Comparative Example 1 that when the elution buffer having the same conditions as that for the heavy chain affinity chromatography was used, a target protein was not separated. In addition, it was confirmed in Comparative Example 2 that when using the elution buffer having pH　2.7, which is lower than that in the Experimental Examples of the present disclosure, a target protein with a lower yield was separated.

In the case of using the elution buffer having a low pH of pH 2.7 used in Comparative Example 2, there is a problem in terms of the structural and stability of the protein. Therefore, a calibration process for increasing the pH is required. However, in the case of using the purification method of the present disclosure, such calibration process for increasing the pH can be omitted, as well as a concentration/dialysis process; therefore, the purification method of the present disclosure is superior in terms of ensuring the stability of the separated protein and economically obtaining the protein with a high purity, from which impurities have been removed.

While the present disclosure has been described with reference to the particular illustrative embodiments, it will be understood by those skilled in the art to which the present disclosure pertains that the present disclosure may be embodied in other specific forms without departing from the technical spirit or essential characteristics of the present disclosure. Therefore, the embodiments described above are considered to be illustrative in all respects and not restrictive. Furthermore, the scope of the present disclosure is defined by the appended claims rather than the detailed description, and it should be understood that all modifications or variations derived from the meanings and scope of the present disclosure and equivalents thereof are included in the scope of the appended claims.

Claims

A method for purifying a target antibody or an antibody fragment thereof, comprising:

(a) loading a sample comprising an antibody or an antibody fragment thereof into an affinity chromatography column comprising a heavy chain affinity resin;

(b) washing the column with a washing buffer; and

(c) recovering the target antibody or the antibody fragment thereof from the affinity chromatography column using an elution buffer, which has a pH ranging from pH　3.8 to pH　4.7.
The method of claim 1, wherein the heavy chain affinity resin is a resin capable of specifically binding to a heavy chain.
The method of claim 1, which does not carry out a concentration/dialysis process after Step (c).
The method of claim 1, wherein the elution buffer in Step (c) has a salt concentration ranging from 10　mM to 100　mM.
The method of claim 1, wherein the elution buffer in Step (c) comprises one or more salts selected from the group consisting of sodium acetate, sodium citrate, and glycine.
The method of claim 1, wherein the column is equilibrated with a buffer having a pH ranging from pH　5.5 to pH　7.5 before loading the sample of Step (a), which comprises the antibody or antibody fragment thereof.
The method of claim 6, wherein the buffer comprises one or more salts selected from the group consisting of 2-(N-morpholino)ethanesulfonic acid (MES), PIPES, potassium phosphate, potassium chloride, 3-morpholinopropane-1-sulfonic acid (MOPS), sodium phosphate, sodium chloride, Tris, and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES).
The method of claim 1, which further comprises carrying out affinity chromatography, ion-exchange chromatography, concentration, or dialysis before Step (a).
The method of claim 1, which further comprises discharging impurities with no affinity for the resin using an equilibrium buffer after Step (a) or Step (b).
The method of claim 1, which further comprises re-equilibrating the column using a re-equilibrium buffer after Step (a) or Step (b).
The method of claim 1, wherein the washing buffer in Step (b) has a pH ranging from pH　4.5 to pH　6.5.
The method of claim 1, wherein the washing buffer in Step (b) has a salt concentration ranging from 400　mM to 1　M.
The method of claim 1, wherein the washing buffer of Step (b) comprises one or more salts selected from the group consisting of sodium phosphate, potassium chloride, potassium phosphate, sodium chloride, Tris, 2-(N-morpholino)ethanesulfonic acid (MES), 3-morpholinopropane-1-sulfonic acid (MOPS), PIPES, and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES).
The target antibody or antibody fragment thereof of Claim 1, wherein the purified target antibody or antibody fragment thereof is a therapeutic protein.