TWI679052B - A method for purifying an antibody or an antibody fragment thereof using affinity chromatography - Google Patents

Abstract

The present disclosure relates to a method for isolating and purifying a high-purity antibody or an antibody fragment thereof using affinity chromatography, and in particular, to a method for isolating and purifying a high-purity antibody and an antibody fragment thereof, and purifying the same using an elution buffer capable of increasing stability Antibodies and antibody fragments to enhance stability.

Description

   Method for purifying antibody or antibody fragment thereof using affinity chromatography   

The present disclosure relates to a method for isolating and purifying a high-purity antibody or an antibody fragment thereof using affinity chromatography, and in particular, to separating a high-purity antibody and an antibody fragment thereof, and using an elution buffer capable of enhancing stability Methods of antibodies and antibody fragments thereof.

Recombinant proteins are expressed and produced by inserting a target protein gene into a bacterium or cell. In addition to the target protein, various impurities are present in the original culture obtained during expression, such as protein isoforms, dimers, multimers, single antibody fragments, host-derived DNA, host-derived proteins, endotoxin Etc., and purification procedures for removing impurities are needed to commercialize the target protein. The antibody fragments of these protein therapeutics are used to show drug efficacy by specifically binding targets. In addition, since Fab without Fc has no glycosyl or glycosylation, it is a very attractive therapeutic agent because the therapeutic effect is not affected by the type of sugar. However, unlike antibodies, because the difference between the molecular weight of the impurity and the antibody fragment is small, it is difficult to separate the impurity and antibody throughout the purification procedure using chromatography.

Purification methods using affinity resins are widely used as methods for isolating antibody fragments. For example, a method for purifying an antibody is known, and impurities in a target product can be purified by using Protein A affinity chromatography (International Patent Application No. WO2011073954). However, in this method, the target protein can be obtained from the resin as long as an acidic buffer solution having a pH of about 2 to 3 is used; that is, the problem with this method is that the target protein is due to the low pH value of the eluate Become restless. To overcome this problem, immediately add a buffer solution that increases the pH of the elution buffer and mix it into the resultant to ensure the stability of the target protein. Because the buffer solution is mixed to ensure the stability of the target protein, this method requires an additional concentration / dialysis process to remove the mixed buffer solution with inconvenience and reduced yield.

In addition, as a method of purifying an antibody fragment, a method of specifically recovering and purifying a target protein using a chromatography method using a resin having a specific affinity for kappa in a light chain is known (Korean Patent Application No. 2016-0127317) ). However, this method also has disadvantages. Specifically, the antibody fragment uses a system that expresses each of a heavy chain and a light chain in a vector, in which, in addition to the type of two chains, a protein in which only a light chain is present can be expressed. That is, there is a problem that when a resin having an affinity for kappa in the light chain is used, impurities such as a single fragment of the light chain should be further purified and removed. In addition, similar to the Protein A affinity resin, it is necessary to elute with a buffer solution having a low pH (about pH 2 to pH 3), so the stability of the obtained antibody fragment is low. That is, the disadvantage of this method is that an additional concentration / dialysis process must be performed to compensate for the defects. In addition, the purity and yield of the method are not high.

In this context, the inventors of the present case have worked hard to find a method that can be used to purify a target protein with high purity and yield even at a pH higher than that conventionally used for affinity chromatography. As a result, they have discovered a method for purifying antibodies and antibody fragments with high purity and stability when using heavy chain affinity chromatography and utilizing optimized elution buffers.

An object of the present disclosure is to provide a target antibody or an antibody fragment thereof, comprising: (a) loading a sample containing the antibody or an antibody fragment thereof onto an affinity chromatography column containing a heavy chain affinity resin; (b) using a wash buffer Wash the analytical column; (c) recover the target antibody or antibody fragment thereof from the affinity chromatography column using an elution buffer having a pH ranging from pH 3.8 to pH 4.7.

Hereinafter, the present disclosure will be described in detail.

At the same time, the descriptions and exemplary embodiments disclosed herein can be applied to other descriptions and exemplary embodiments. That is, all combinations of various factors disclosed herein belong to the scope of this disclosure. In addition, the scope of this disclosure should not be limited to the specific disclosure provided below.

In addition, those skilled in the art to which their inventions belong will be able to identify or confirm many equivalents to the specific embodiments of the present disclosure described in this application based on routine experimentation, and these equivalents are intended to be included in this application. Revealed.

One aspect of the present invention is to provide a target antibody or an antibody fragment thereof, comprising: (a) loading a sample containing the antibody or an antibody fragment thereof onto an affinity chromatography column containing a heavy chain affinity resin; (b) washing and analyzing Buffer washing the column; (c) recovering the target antibody or its antibody fragment from the affinity chromatography column using an elution buffer having a pH ranging from pH 3.8 to pH 4.7.

In general, when using affinity chromatography with affinity resins to purify proteins, mainly the use of elution buffers in the pH 2 to pH 3 range causes problems with the stability of the target protein (Pim Hermans et al., Monoclonal Antibodies, vol. 1131, 297-314p; Abhinav A. Shukla et al., BioProcess International, 2005, 36-44p). To solve this problem, calibration work is needed to increase the pH to pH 4 to pH 7 after purification at low pH (pH 2 to pH 3), and there is a problem of reducing the target protein in the steps of performing this additional process. However, the importance of the heavy-chain affinity resin of the present disclosure is that a target protein having high purity and stability is produced in a pH range of pH 3.8 to pH 4.7, which is milder than the conditions required for general affinity resins. .

Each step of the method for purifying an antibody of interest or an antibody fragment thereof will be described in detail below. 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 chromatography method that uses a binding substance that has an affinity for a particular protein. A binding substance having an affinity for a specific protein is a polymeric substance that binds to a functional group; that is, the binding substance binds to a substance having an affinity, and the substance is dissolved in polar and non-polar solutions. For the purposes of this disclosure, affinity chromatography can be divided into, but not limited to, heavy chain affinity chromatography and light chain affinity chromatography. Specifically, heavy chain affinity chromatography refers to a chromatography method using a resin (for example, a heavy chain affinity resin) capable of specifically binding a heavy chain that is part of an antibody. In addition, light chain affinity chromatography refers to a chromatography method using a resin (for example, a light chain affinity resin) capable of specifically binding a light chain that is part of an antibody.

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

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

In a specific embodiment of the present disclosure, the method may further include performing ion exchange chromatography, concentration, and / or dialysis before step (a). The purpose is to remove the main impurities of the antibody or antibody fragment and improve the purity of the sample. Specifically, before the step (a), the sample containing the antibody fragment is concentrated and dialyzed, and the step of purifying the sample using anion exchange chromatography is performed beforehand, and then the sample can be loaded onto the affinity chromatography using a heavy chain affinity resin. Any operation can be applied without limitation as long as it removes major impurities that do not bind to the affinity resin, and can improve the purity of the sample.

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

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

In addition, the wash buffer may include one or more salts selected from the group consisting of sodium phosphate, potassium chloride, potassium phosphate, sodium chloride, Tris, 2- (N-morpholinyl) ethanesulfonic acid (MES ), 3-morpholinylpropane-1-sulfonic acid (MOPS), PIPES and 2- [4- (2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid (HEPES), but are not limited thereto.

For the purposes of this disclosure, in step (b), impurities that are non-specifically bound to the heavy chain affinity resin can be removed by washing buffer.

In a specific embodiment of the present disclosure, the purification method may further include using an equilibration buffer after step (a) or step (b) to discharge impurities having no affinity for the resin. Specifically, this step can be performed at least once, but generally, this step can be performed without restrictions until equilibrium is established.

In a specific embodiment of the present disclosure, the purification method may further include re-equilibrating the column with a re-equilibration buffer after step (a) or step (b). The re-equilibration buffer flows under the same conditions as the equilibration buffer of step (a), in which there is no reaction, and the target antibody or antibody fragment thereof is not eluted between the washing and elution processes, and then the elution buffer is Flow again before flowing. Therefore, the re-equilibration buffer is used as a bridge between the washing buffer and the elution buffer.

Specifically, the reequilibration buffer may have a pH ranging from pH 4.5 to pH 6.5 and a salt concentration of 10 mM to 30 mM, but the range of pH and salt concentration is not limited thereto.

In addition, the rebalance buffer may include one or more salts selected from the group consisting of: sodium phosphate, sodium chloride, Tris, 2- ( N -morpholinyl) ethanesulfonic acid (MES), 3-morpholine Propane-1-sulfonic acid (MOPS), PIPES, potassium phosphate, potassium chloride, and 2- [4- (2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid (HEPES), but are not limited thereto .

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

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

Even when the pH of the elution buffer is lower than pH 3.8, the target protein is still eluted. However, there are problems that require a concentration / dialysis process and calibration operations that increase the pH (pH 4 to pH 7) after purification at low pH (pH 2 to pH 3); and this problem also occurs when using traditional affinity chromatography . In addition, a pH higher than pH 4.7 is not preferable because the ligand that maintains the heavy chain in the column forms a hydrogen bond with the target protein, and therefore is not eluted.

In addition, the elution buffer may include one or more salts having a salt concentration ranging from 10 mM to 100 mM selected from the group consisting of sodium acetate, sodium citrate, and glycine, but any salt may be used without limitation as long as they are An antibody of interest or an antibody fragment thereof can be isolated under the mild conditions of heavy chain affinity chromatography.

In particular, the purification method of the present disclosure is characterized in that no concentration / dialysis process is performed after step (c). A "concentration / dialysis process" is a process that is commonly used to remove objects smaller than the size of the membrane or to change the buffer, and is therefore used to further purify and remove single fragments (i.e., impurities) when using universal affinity resins. process. When a concentration / dialysis process is additionally used, the process yield is not high. However, the purification method of the present disclosure has economic advantages in that it can be purified to high purity and yield by using optimization of a heavy chain affinity resin and an elution buffer without performing an additional concentration / dialysis process. Antibodies or antibody fragments thereof.

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

The antibody may be specifically a monoclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody that can be formed from cells having an antibody coding sequence and recognizes a specific antigen. The antibodies of the present disclosure may include, but are not limited to, all therapeutic antibodies traditionally used in the technical field to which the invention belongs.

In addition, an antibody is a concept including a full-length antibody and an antibody fragment, and examples of the antibody fragment include all Fv, Fab, Fab ', F (ab') ₂ , Fd, and the like. Fv includes two forms of disulfide stable Fv (dsFv) and single-chain Fv (scFv). Fd refers to the heavy chain component contained in the Fab. An antibody fragment refers to an antibody fragment constructed using only some fragments (eg, Fv, scFv, and Fab) necessary for binding to an antibody antigen. In addition, an antibody fragment is widely used as a protein therapeutic because it specifically binds to a target to show a drug effect. In addition, Fabs without Fc have no effect on the therapeutic effect depending on the type of sugar, because there is no glycosyl or glycosylation therein. However, unlike antibodies, because antibody fragments do not exhibit large molecular weight differences compared to impurities, it is difficult to isolate antibody fragments using chromatography in purification methods.

The target antibody or antibody fragment thereof isolated using the purification method of the present disclosure refers to an antibody or antibody fragment thereof 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 not limited thereto. The term "purity" refers to a pure antibody or antibody fragment from which impurities have been removed. For example, a purity of 92% means that the remaining 8% are impurities. In addition, purity can simply represent the purity of the material isolated from the elution buffer, but the final purity percentage can vary depending on the purity percentage of the loaded sample.

As used herein, the term "impurity" includes any material other than the antibody of interest or an antibody fragment thereof. Examples of impurities include, but are not limited to, isoforms, dimers, multimers, host-derived DNA, host-derived proteins, endotoxins, and the like.

In addition, 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.

In addition, 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 referred to as a protein traditionally used in biomedicine, and thus refers to a protein having various physiological activities. Physiological activity regulates genetic expression and physiological functions to correct abnormal conditions caused by the lack or excessive secretion of substances involved in the regulation of in vivo functions, and can therefore be included in general protein therapeutics.

In the present disclosure, the therapeutic protein that can be included is not limited as long as the protein has physiological activity in the body. An example of a therapeutic protein may be, but is not limited to, scFv, Fab, Fab ', or F (ab') ₂ of its antibody or antibody fragment.

According to affinity chromatography of the present disclosure, not only can most impurities other than the target protein be removed, but the antibody or antibody fragment thereof can be purified to high purity and yield without additional processes (e.g., concentration / dialysis). .

Figure 1 shows the procedure for carrying out the experiments of the present invention.

Figure 2a shows the results of the elution buffer (pH 3.8 to pH 5.6) which was eluted at a linear concentration gradient of pH.

Figure 2b shows the results of CEX-HPLC analysis of the sample before loading on the column, confirming that impurities were separated.

Figure 2c shows the results of CEX-HPLC analysis of the material leaving the column due to its lack of affinity for the heavy chain, confirming the separation of impurities.

Figure 2d shows the results of CEX-HPLC analysis of the material separated using the wash buffer, confirming that some impurities that did not specifically bind to the column were separated.

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

Figure 3a shows that the elution buffer has the best separation ability at pH 4.7, and the target protein is separated.

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

Figure 4a shows that the separation ability is maintained at the same pH conditions even when the type of elution buffer is changed.

Figure 4b shows CEX-HPLC analysis of the material separated using the elution buffer.

As a result, it was confirmed that the substance separated from the elution buffer had a purity of 90% or more.

Figure 5a shows that the elution buffer has the best separation ability at pH 4.7 and the antibodies are separated.

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

Figure 6 shows that when the light chain affinity resin is used, the target protein is not separated when the pH of the elution buffer is 4.7.

Figure 7a shows that when light chain affinity resin is used, the elution buffer has the best separation ability at pH 2.7, confirming that the target protein is separated.

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

Fig. 7c shows the results of the substance isolated in Experimental Example 2 and the substance isolated 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 help further understand the present invention, and the present invention is not limited by these examples.

In order to confirm whether the purification method of the present disclosure can exhibit substantial effects, ranibizumab (which is a representative antibody fragment drug) was selected for purification.

Example 1: Purification of antibodies and antibody fragments using affinity chromatography

Experimental Example 1: Setting the purification conditions of antibody fragments (condition setting of the elution buffer)

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

* Chromatographic conditions:

-Resin: CaptureSelect CH1-XL

-Flow rate: 90 cm / hour

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

-Loading: up to 3 grams protein / liter (resin volume)

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

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

The purification process was performed in a similar procedure to that in FIG. 1. To set the conditions of the elution buffer, 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 a sample containing the antibody fragment was loaded onto an affinity chromatography column. Thereafter, the antibody fragments bound to the column were washed after re-equilibration. The resultant was washed again by re-equilibrium, and collected from the point where the ultraviolet ray was higher than 1 mAU during washing, until the ultraviolet ray reached 1 mAU again, and then stored at 5 ± 3 ° C after antibacterial filtration.

The results of the elution with a linear pH concentration gradient elution buffer (pH 3.8 to pH 5.6) confirmed that the elution buffer had the best separation ability of the target antibody or antibody fragment at pH 4.7 (Figure 2a).

The target antibody or antibody fragment thereof obtained by the purification method was analyzed by CEX-HPLC. Considering that the substance has no affinity for the heavy chain and sample loaded on the column (Figure 2b), the analysis results released from the column (Figure 2c) confirm that most impurities have no affinity for the heavy chain affinity resin, so Isolated and purified. In addition, due to the use of elution buffers (sodium phosphate (20mM) and sodium chloride (400mM); pH 5.8), it was confirmed that some impurities that were not specifically bound to the column were separated by the elution buffer (Figure 2d) ). Therefore, it is confirmed from the above results that impurities can be removed from the sample only before loading or by a washing step, but the target protein is not isolated.

Thereafter, it was confirmed that the substance separated using the elution buffer was the target antibody or an antibody fragment thereof, and its purity was 92% or higher (FIG. 2e). Considering the 14% purity of the loaded sample, it was confirmed that most impurities were removed by this purification process. In addition, it was confirmed that the analysis result by CEX-HPLC was that the process yield was 95%.

Experimental Example 2: Setting the purification conditions of antibody fragments (measurement of the appropriate pH of the elution buffer)

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

* Chromatographic conditions:

-Resin: CaptureSelect CH1-XL

-Flow rate: 90 cm / hour

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

-Loading: up to 3 grams protein / liter (resin volume)

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

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

An experiment was performed to determine whether the eluent buffer (pH 4.7) set in Experimental Example 1 was suitable for purifying the target antibody or its antibody fragment.

The purification process was performed in a manner similar to that of FIG. 1. In order to set the conditions of the elution buffer, the elution was performed with sodium acetate (50 mM) buffer (pH 4.7).

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

As a result, as shown in Fig. 3a, it was confirmed that the elution buffer had the best separation ability at pH 4.7. That is, it was confirmed by CEX-HPLC analysis that impurities and target proteins were separated in each step of the purification process of the present disclosure (Figure 3a), and the material separated from the elution buffer had a purity of 88% or more High (Figure 3b). In addition, as a result of analysis by CEX-HPLC, it was confirmed that the process yield was 92%.

Experimental Example 3: Setting the purification conditions of antibody fragments (determining whether pH is a necessary condition)

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

* Chromatographic conditions:

-Resin: CaptureSelect CH1-XL

-Flow rate: 90 cm / hour

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

-Loading: up to 3 g protein / liter (resin amount)

-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 a necessary condition in the purification method of antibody fragments using affinity chromatography, experiments were performed by replacing the type of the elution buffer with sodium citrate (NaCitrate) instead of sodium acetate ( NaAcetate).

The purification process was performed in a manner similar to that of FIG. 1. In order to set the conditions of the elution buffer, it was washed with a sodium citrate (50 mM) buffer (pH 4.7) as an elution buffer.

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

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

In addition, when analyzing the results obtained by CEX-HPLC elution, it was confirmed that the obtained product had a purity of 90% or higher (Figure 4b), and the process yield was 92%.

Experimental Example 4: Purification of Antibodies

In order to determine whether it is possible to purify not only antibody fragments but also antibodies by the purification method according to the present invention, chromatographic analysis was performed using the representative antibody drug Erbitux under the following conditions:

* Chromatographic conditions:

-Resin: CaptureSelect CH1-XL

-Flow rate: 90 cm / hour

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

-Loading: up to 3 grams protein / liter (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 in a manner similar to that of FIG. 1. In order to set the conditions of the elution buffer, the elution was performed with sodium acetate (50 mM) as an elution buffer (pH 4.7).

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

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

Comparative Example 1: Setting purification conditions of antibody fragments (using light chain affinity resin)

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

* Chromatographic conditions:

-Resin: KappaSelect

-Flow rate: 90 cm / hour

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

-Loading: up to 3 g protein / liter (resin volume)

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

-Sterilization: 10mM sodium hydroxide solution

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

The purification process was performed in a manner similar to that of FIG. 1. The experiment was performed under the same conditions as in Experimental Example 2 except that a light chain affinity resin KappaSelect was used as the resin.

Specifically, the column was equilibrated with an equilibration buffer (sodium phosphate (20 mM); pH 6.2). Thereafter, a sample containing the antibody fragment is loaded onto a light chain affinity chromatography column, and the antibody fragment bound to the column is then washed out after re-equilibration. The resultant was washed again by re-equilibrium, and collected from the point where the ultraviolet ray was higher than 1 mAU during washing, until the ultraviolet ray reached 1 mAU again, and then stored at 5 ± 3 ° C after antibacterial filtration. For the elution buffer, the buffers (sodium phosphate (20 mM) and sodium chloride (400 mM); pH 5.8) as in Experimental Example 1 were used. The resultant was washed again by re-equilibration. For elution, the same elution buffer was used as for heavy chain affinity chromatography.

As a result, as shown in FIG. 6, it was confirmed that when the pH of the elution buffer was 4.7, the target protein was separated in the sterilization step instead of the elution step.

This result confirms that, as is known in the technical field to which the invention belongs, elution does not occur when the elution buffer in light chain affinity chromatography has a high pH exceeding pH 2 to pH 3.

Comparative Example 2: Setting the purification conditions of antibody fragments (measurement of suitable pH conditions for light chain affinity resins)

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

* Chromatographic conditions:

-Resin: KappaSelect

-Flow rate: 90 cm / hour

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

-Loading: up to 3 grams protein / liter (resin volume)

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

-Sterilization: 10mM sodium hydroxide solution

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

The purification process was performed in a manner similar to that of FIG. 1.

Specifically, the column was equilibrated with an equilibration buffer (sodium phosphate (20 mM); pH 6.2). A sample containing the antibody fragment was loaded onto a light chain affinity chromatography column, and the antibody fragment bound to the column was washed after reequilibration. The resultant was washed again by re-equilibration. For the elution buffer, a buffer (glycine (0.1M); pH 2.7) was used. The resultant was collected from the point where the ultraviolet value in the equilibrium state increased by 1 mAU during the elution, until the value returned to the UV value in the equilibrium state. To solve the stability problem of antibody fragments, sodium phosphate (200 mM; pH 7.0) was used to increase the low pH (pH 2.7) of the elution buffer. The calibrated wash buffer was stored at 5 ± 3 ° C after antibacterial filtration.

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

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

When light chain affinity chromatography is used, the process yield is 87%, which is also lower than when heavy chain affinity chromatography according to the present disclosure is used. In addition, in the comparative example, a concentration / dialysis procedure must be performed to proceed to the next procedure. Therefore, in consideration of the fact that an additional concentration / dialysis procedure is required, the use of the chromatography of the comparative example confirms that the target substance is actually ensured at a relatively low yield.

As described above, in the present disclosure, it is confirmed that when heavy chain affinity chromatography is used (Experimental Examples 1 to 4), when the elution buffer is at pH 3.8 to pH 4.7, particularly at pH 4.7, Isolate the antibody of interest or its antibody fragment.

However, it was confirmed in Comparative Example 1 that when an eluent buffer having the same conditions as those of heavy chain affinity chromatography was used, the target protein was not separated. In addition, it was confirmed in Comparative Example 2 that when an eluent buffer having a pH of 2.7, which is lower than that in the experimental example of the present disclosure, was used, the target protein was isolated in a lower yield.

When the eluent buffer having a low pH value of 2.7 was used in Comparative Example 2, there were problems in the structure and stability of the protein. Therefore, a calibration process for increasing pH is needed. However, in the case of using the purification method of the present disclosure, such a calibration process for increasing the pH, and a concentration / dialysis procedure can be omitted; therefore, the purification method of the present disclosure ensures the stability and saving of the isolated protein. Qiandi is superior in obtaining high-purity proteins from which impurities are removed.

Although this disclosure has been described with reference to specific illustrative specific embodiments, those skilled in the art to which this disclosure pertains will appreciate that this disclosure can be implemented in other specific forms without departing from the technical spirit or necessary technical characteristics of this disclosure. . Features of this disclosure. Accordingly, the specific embodiments described above are to be considered in all respects as illustrative and not restrictive. In addition, the scope of the present disclosure is defined by the scope of the attached application patents, rather than the embodiments, and it should be understood that all modifications or changes derived from the meaning and scope of the present disclosure and equivalents thereof are included in the present disclosure. Within the scope of patent application.

Claims (14)

A method for purifying an antibody of interest or an antibody fragment thereof, comprising: (a) loading a sample containing the antibody or an antibody fragment thereof onto an affinity chromatography column including a heavy chain affinity resin; and (b) washing with a washing buffer A column; and (c) recovering a target antibody or an antibody fragment thereof from the affinity chromatography column using an elution buffer having a pH ranging from pH 3.8 to pH 4.7. The method according to item 1 of the scope of patent application, wherein the heavy-chain affinity resin is capable of specifically binding a heavy-chain resin. The method according to item 1 of the scope of patent application does not perform a concentration / dialysis procedure 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 according to item 1 of the aforementioned patent application range, wherein before loading the sample in step (a), equilibrate the column with a buffer having a pH ranging from pH 5.5 to pH 7.5, the sample including the antibody or Antibody fragments. The method of claim 6, wherein the buffer comprises one or more salts selected from the group consisting of 2- ( N -morpholinyl) ethanesulfonic acid (MES), PIPES, phosphoric acid Potassium, potassium chloride, 3-morpholinylpropane-1-sulfonic acid (MOPS), sodium phosphate, sodium chloride, Tris, and 2- [4- (2-hydroxyethyl) piperazin-1-yl] ethyl Sulfonic acid (HEPES). The method according to item 1 of the patent application scope, further comprising performing affinity chromatography, ion exchange chromatography, concentration or dialysis before step (a). The method according to item 1 of the patent application scope, further comprising, after step (a) or step (b), using an equilibrium buffer to discharge impurities having no affinity for the resin. The method according to item 1 of the patent application scope, further comprising, after step (a) or step (b), re-equilibrating the column with a re-equilibration buffer. 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 1M. The method of claim 1, wherein the buffer comprises one or more salts selected from the group consisting of sodium phosphate, potassium chloride, potassium phosphate, sodium chloride, 2- ( N- Morpholinyl) ethanesulfonic acid (MES), PIPES, 3-morpholinylpropane-1-sulfonic acid (MOPS), Tris, and 2- [4- (2-hydroxyethyl) piperazin-1-yl] ethyl Sulfonic acid (HEPES). The method according to item 1 of the patent application scope, wherein the purified target antibody or antibody fragment thereof is a therapeutic protein.