TWI679209B - Method for purifying analogous antibody using cation-exchange chromatography - Google Patents

Abstract

The present invention relates to a method for separating impurities of isokinetic antibodies using cation exchange chromatography, and more specifically, to separate single fragments and isoforms of isokinetic antibodies generated during the process of isokinetic antibody production and use A method in which cation exchange chromatography elutes a buffer and purifies only a target antibody fragment with high purity and high yield.

Description

   Method for purifying isoantibodies using cation exchange chromatography   

The present invention relates to a method for separating impurities of isoantibodies using cation exchange chromatography. More specifically, the present invention relates to a method for purifying target fragments of isokinetic antibodies with high purity and high yield. The method uses a cation exchange layer. The assay uses an elution buffer and removes single fragments of isoforms and isoforms of isoforms produced during the process of making isoforms.

During the process of producing isoantibodies using bacteria, various types of isoforms and single antibody fragments of isokinetic antibodies are produced. Therefore, research and development of a method to minimize non-product impurities is needed to improve the fermentation process and the construction of the best clones.

However, in order to produce the ideal optimal product, research is needed to remove most of the non-product impurities during the purification process and find suitable conditions to prevent further increase of non-product impurities during storage.

Monoclonal antibodies are representative materials for protein therapeutics, and because of their ability to bind to specific targets, they have become a very attractive tool for developing therapeutics, and most of them can be used on the body.

Monoclonal antibodies also produce various types of isoforms during the process of intracellular expression. In order to achieve the maximum efficacy without producing product-related impurities between these isoforms, it should be solved by selecting plantlets and developing culture methods. However, if isoforms of product-related impurities remain after the above process, purification and storage treatments are required to prevent further increase of isoforms.

Product-related impurities have properties very similar to those of these products, making it difficult to separate them by chromatography used in purification processes. However, with the development of technology, attempts have recently been made to remove these product-related impurities through the development of purification processes (Korean Patent Publication No. 10-2013-0064803).

In this case, the inventors have confirmed that by removing the impurities of the isoantibodies, most of the non-product impurities can be removed by cation exchange chromatography using the specific elution buffer described in the present invention, thereby The present invention has been completed.

An object of the present invention is to provide a method for purifying a target antibody fragment using cation exchange chromatography and an optimized elution buffer.

Hereinafter, the present invention will be explained in detail.

At the same time, various explanations and exemplary specific examples disclosed herein can be applied to other explanations and exemplary specific examples. That is, all combinations of the various factors disclosed herein are within the scope of the present invention. Furthermore, the scope of the invention should not be limited by the specific disclosure provided below.

In addition, based on conventional experimental methods, those skilled in the art will be able to understand or confirm many equivalents of the specific examples of the invention described in this application, and these equivalents are determined to be included in the invention.

One aspect of the present invention is to provide a method for purifying a target fragment of a homogeneous antibody, comprising: (a) loading a sample containing a homogeneous antibody mixture into a cation exchange chromatography column equilibrated with an equilibration buffer; (b) The washing buffer rinses the column; and (c) removes the target fragment of the isoantibody from the cation exchange chromatography column using a sodium acetate elution buffer having a pH of pH 4.9 to pH 5.2.

Previously, ranibizumab was isolated with high purity using cation exchange chromatography. Ranibizumab is a representative antibody fragment drug; however, it has been reported that inflammation is induced in clinical trials due to the presence of a large amount of impurities , And therefore cannot be clinically replaced. This shows that even if antibody fragments are separated with high purity using cation exchange chromatography, problems may occur in the actual clinical stage, and it is recommended that optimized purification conditions must be found. However, the present inventors have found a method for efficiently separating a single fragment of an isotropic antibody and an isoform of an isotropic antibody by optimizing an elution buffer, and purifying a target antibody fragment with high yield.

In particular, when the method of the present invention is used to prepare a biosimilar of an isokinetic antibody, a fragment of the isokinetic antibody can be prepared, and the fragment of the isokinetic antibody contains the same or corresponding main active antibody fragment as the control drug And isoforms of isoantibodies are used as components.

The steps of a method for purifying a target antibody or antibody fragment in detail are as follows. First, step (a) is a step of loading a sample containing a homogeneous antibody mixture into a cation exchange chromatography column equilibrated with an equilibration buffer.

As used herein, the term "sample containing a homogeneous antibody mixture" refers to a partially purified sample from a cell culture clarified solution or cell decomposed solution that produces isotropic antibodies, and thus means a partially purified sample that contains Isotropic antibody mixture of active antibody fragments and isoforms of antibody fragments. Partial purification means a state in which a protein other than the target fragment of the isokinal antibody, that is, a single fragment of the isokinetic antibody, a isoform of the isokinetic antibody, or the like is present even after the filtering treatment is performed.

As used herein, the term "mainly active antibody fragment" is a major component in the antibody group of the invention; that is, means an antibody fragment in which some amino acids in the antibody fragment are modified via deamination or oxidation There is no reduction in biological activity, that is, antibody fragments that are not acidic or basic isoforms of antibody fragments. The main antibody active fragment is the most important component that controls the properties of the desired antibody fragment, and is therefore the antibody fragment with the highest biological activity among the antibody components.

As used herein, the term "isoforms of antibody fragments" means that some of the amino acids in the main active antibody fragments in the antibody fragments are modified by deamination or oxidation, and include acidic isoform antibody fragments and basic isoform antibodies Fragment. Examples thereof include asparagine in amino acids in isoform antibody fragments modified to aspartate by deamination, and methionine in amino acids in isoform antibody fragments to be modified by oxidation Methionine sulfate, etc. In addition, when glutamate is present at the N-terminus of the heavy chain, the glutamate forms a pentagonal ring structure and thus contains isoform antibody fragments, where the glutamate is modified to pyroglutamate. When an antibody fragment is produced from a host cell such as a bacterial cell, a high ratio of the isotype antibody fragment is contained in the culture medium of the host cell, so in order to include the desired ratio in the antibody fragment, the isotype antibody fragment should be removed by chromatography.

As used herein, the term "ion exchange chromatography" means a method of performing chromatography using a chromatography material that exchanges ions. Specifically, the term "chromatographic material that exchanges ions," in which a functional group is combined with a polymer material, means a material that purifies ionic materials in polar and non-polar solutions via mutual exchange. In addition, the efficiency and purpose of ion exchange in ion exchange chromatography depends on the exchanger. When cations are exchanged, the material is called a cation exchange resin, and when anions are exchanged, the material is called an anion exchange resin.

For the purposes of the present invention, cation exchange chromatography columns can be used in ion exchange chromatography. Specifically, the term "cation exchange chromatography column" means a column packed with a cation exchange resin. In the present invention, cation exchange chromatography is performed to remove isoforms, impurities, and the like of isoantibodies. Cation exchange resins can be classified into sulfonic acid (S) and carboxymethyl (CM) groups; effective pH, exchange efficiency, regeneration efficiency, ion adsorption, etc. vary depending on the properties of each group. The cation exchange resin is a synthetic resin that exchanges cations in an aqueous solution with cations in the resin. In this regard, because the isoelectric point of the antibody is high, it becomes a cation in a buffer whose pH is lower than the isoelectric point value. Therefore, a cation exchange resin capable of adsorbing an antibody exhibiting a cation can be used to improve the properties of the antibody group. The cation exchange resin may be a cation exchange resin commonly used in the art. Specifically, for the cation exchange resin, a column having a COO ^- or SO ₃ functional group can be used, and more specifically, carboxymethyl (CM), fractogel, sulfoethyl (SE), and sulfopropyl (SP ), A phosphate group (P) or a sulfonic acid group (S), and the like, and most specifically, CM sepharose or fractogel COO ⁻ may be used, but the functional group is not limited thereto.

In addition, before loading the sample containing the antibody mixture in step (a), the column can be equilibrated with an equilibration buffer having a pH of pH 4.5 to pH 5.5. The equilibrium buffer of step (a) may have a salt concentration of 5 mM to 50 mM, and may be any one or more salts selected from the group consisting of sodium acetate, sodium chloride, sodium phosphate, and sodium citrate, but not Limited to this.

In addition, before step (a), the method may further include performing ion exchange chromatography, concentration, and dialysis. The purpose is to remove the main impurities of antibodies and antibody fragments and increase the concentration of the sample. Specifically, before performing step (a), the sample containing the antibody mixture can be concentrated and dialyzed, and then the sample can be loaded into a cation exchange affinity chromatography column. However, any work that removes the major impurities of the sample and increases the concentration can be applied without limitation. For example, the step of purifying a sample containing a homogeneous antibody mixture using CH-1 affinity chromatography may be performed before step (a), but is not limited thereto.

In the method of purifying the target fragment of the isoantibody, step (b) is a step of washing the column with a washing buffer; that is, this step is a step of applying the washing buffer to a column of a chromatography column loaded with a sample.

The pH of the wash buffer can range from exceeding the pi value of a single fragment of the isoantibody to below the pH of the target fragment of the isoantibody. Specifically, the pH value of the washing buffer may be 1.0 lower than the value measured by pI, and may be higher than the pI value of a single fragment of the same antibody. More specifically, the washing buffer may have a pH of 6.7 to pH 7. 3, but the pH is not limited to this. In addition, the salt concentration of the washing buffer may be 5 mM to 25 mM, but the value of the salt concentration is not limited thereto.

In the case of isotopic antibody fragments, the isoelectric point is high so that aggregation does not occur under acidic conditions at low pH, while in the case of samples containing isotopic antibody mixtures, where the isoelectric point is lower than the isofunctional antibody fragment Since the charge is removed under acidic conditions at low pH, the isoelectric point is generally not condensed by Van der Waals force. Therefore, a pH value lower than the isoelectric point of a homologous antibody fragment to be purified according to the present invention and higher than the isoelectric point of a single fragment of the isofunctional antibody can be used. That is, the pH value may be a value lower than the isoelectric point 1.0 of the isofunctional antibody fragment to be purified, but is not limited thereto.

-1-基]乙磺酸(HEPES)、2-[雙(2-羥乙基)胺基]-2-(羥甲基)丙烷-1,3-二醇(Bis-Tris)及3-嗎啉基 丙烷-1-磺酸(MOPS)，但不限於此。 In addition, the washing buffer may contain at least one salt selected from the group consisting of sodium phosphate, sodium chloride, 2- [4- (2-hydroxyethyl) piperate

-1-yl] ethanesulfonic acid (HEPES), 2- [bis (2-hydroxyethyl) amino] -2- (hydroxymethyl) propane-1,3-diol (Bis-Tris), and 3- Morpholine propane-1-sulfonic acid (MOPS), but is not limited thereto.

For the purpose of the present invention, the substance separated by the washing buffer in step (b) may be a single segment of the same work fragment or an isoform of the same work fragment.

As used herein, the term "isoforms of isoantibodies" may be acidic and / or basic isoforms. There are various isoforms of isokinetic antibodies in isokinetic antibody products. In order to prove their equivalence, it is important that the properties of the biosimilars to be produced should be mostly the same as those of their comparators. The isoform of the isoform is a modified form of some amino acids in the main active antibody fragment, so the charge between the main active antibody fragment, the acid isoform antibody fragment and the basic isoform antibody fragment will be a little difference. Based on this, the difference in charge can be used to isolate isoform antibody fragments. However, because this difference in charge comes from slight differences in only a few amino acids, it is necessary to establish very precise separation conditions. In this regard, the cation exchange column of the present invention can effectively remove acid isoform antibody fragments and basic isoform antibody fragments.

In the present invention, the purification method may further include a step of re-equilibrating the column using a re-equilibration buffer.

Specifically, the re-equilibration buffer may have a pH of pH 4.9 to pH 5.2 and a salt concentration of 1 mM to 5 mM, but these are not limited thereto.

In the method for purifying a target antibody fragment, step (c) may be to recover the target fragment of a homogeneous antibody from a cation exchange chromatography column using a sodium acetate elution buffer having a pH of 4.9 to pH 5.2. step.

The elution buffer may have a pH of pH 4.9 to pH 5.2 and a salt concentration of 85 mM to 110 mM, but these are not limited thereto.

When the pH of the elution buffer is lower than pH 4.9, there is a problem that the target fragment of the isoform antibody cannot be separated, and impurities such as a single fragment of the isoform fragment, an isoform of the isoform fragment, and the like cannot be separated. In addition, when the pH of the elution buffer is higher than pH 5.2, the target fragment of the isofunctional antibody cannot be separated in any way. Accordingly, an elution buffer of this pH range is preferably not used.

In addition, the elution buffer may include, but is not limited to, one or more salts selected from the group consisting of sodium acetate, sodium citrate, and glycine.

As used herein, the term "target fragment of an isotropic antibody" is a protein to be separated, and therefore can be used interchangeably with the term "target protein".

For the purpose of the present invention, the target fragment of the isokinetic antibody is a single fragment of the isokinetic antibody and the isoform system of the isokinetic fragment is removed by a purification method. Therefore, it is possible to determine whether the Isolate impurities from the target fragment of the isoantibodies; however, as long as the acidic and basic isoforms of the isobodies and single antibody fragments can be confirmed, there is no limit. Furthermore, SE-HPLC analysis can be performed by establishing the optimal concentration of the elution buffer to determine that the dimers and multimers have been largely separated, but not limited thereto.

A single fragment of an isotropic antibody may include, but is not limited to, a scFv format, a single heavy chain, a single light chain, and the like.

All types of antibody fragments include Fv, Fab, Fab ', F (ab') ₂ , Fd, and the like. Fv includes two forms of double disulfide Fv (dsFv) and single chain Fv (scFv). Fd means the heavy chain component contained in the Fab fragment.

The antibody fragment means that it is constituted by using only some fragments necessary for binding an antibody such as Fv, scFv, and Fab. Furthermore, antibody fragments are widely used as protein therapeutics because they specifically bind to a target and exhibit a drug effect. In addition, known not to have the type F _c when the F _ab sugar treatment has no effect, because the absence of glycosylation or glycation. However, unlike antibodies, because antibody fragments do not show large molecular weight differences compared to impurities, it is difficult to isolate antibody fragments using chromatography in a purification method. For the purpose of the present invention, the purified antibody fragment may be, but is not limited to, a ranibizumab antibody fragment in the form of F _ab or various F _ab (s).

As used herein, the term "impurity" includes any substance other than the protein of interest. Examples of impurities include, but are not limited to, isoforms, dimers, multimers, single antibody fragments, host-derived DNAs, host-derived proteins, endotoxins, and the like.

In addition, after purification from the elution buffer, the purity of the protein can be determined via HPLC analysis, and specifically, can be analyzed via CEX-HPLC, but is not limited thereto.

In addition, the target fragment of the isofunctional antibody purified by the purification method of the present invention can be used as a therapeutic protein. As used herein, the term "therapeutic protein" collectively means the concept of proteins commonly used in biomedicine, and thus means proteins with various physiological activities. The physiological activity regulates gene expression and physiological function and corrects abnormal conditions caused by insufficient secretion or excessive secretion of substances related to the regulation of in vivo functions, and therefore can be included in general protein therapeutics.

The present invention may include a therapeutic protein without limitation as long as the protein has physiological activity in vivo. An example of a therapeutic protein may be, but is not limited to, Fab, Fab 'or F (ab') ₂ .

For the purpose of the present invention, when a purification method is used, a target fragment of a homogeneous antibody with high purity can be isolated. For the purpose of the present invention, even if a sample containing a homogeneous antibody mixture that has been separated by any kind of chromatography is purified by a purification method, a target fragment of the homogeneous antibody having high purity can be obtained after isolation. For example, when the target fragment of the isofunctional antibody separated by affinity chromatography has a purity of 92%, the target fragment of the isofunctional antibody having a purity of 99% can be separated by further purification using the cation exchange chromatography method of the present invention.

The present invention relates to a method for separating impurities from isokinetic antibodies via cation exchange chromatography, and more specifically, a method for purifying single fragments and isoforms of isokinetic antibodies produced during the production of isokinetic antibodies, which method uses a cation The chromatography buffer was exchanged to obtain high-purity and high-efficiency isoantibodies. Therefore, the method of the present invention can be applied to the purification of isoantibodies produced from bacterial fermentation by genetic recombination technology.

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

Figure 2a shows that the elution buffer has the best separation ability at a concentration of 110 mM, and the target protein is separated.

Figure 2b shows the results of analysis of isoforms and single fragments of isoforms using CEX-HPLC, confirming a purity of 98% or higher.

Figure 2c shows the elution results of the elution buffer at a concentration gradient from 5 mM to 300 mM. Specifically, No. 1 indicates a single fragment (i.e., single chain) of an isotropic antibody, and No. 2 indicates an acid isoform antibody fragment (i.e., acidic variant).

Figure 2d shows the results of Figure 2c by SDS-PAGE.

Figure 3a shows that the elution buffer has the best separation ability at a concentration of 110 mM, and can isolate the target fragments of isoantibodies.

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 98% or higher.

Figure 4a shows that at the same pH and salt concentration of the elution buffer, the separation ability is maintained even if the type of affinity resin is changed.

Figure 4b 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 98% or higher.

Figure 5a shows whether the separation ability is exhibited when the conditions of the elution buffer are changed.

Figure 5b shows the results of CEX-HPLC analysis of the material separated using the elution buffer of Figure 5a, confirming that the material separated from the elution buffer has a purity of 98% or higher.

Figure 5c shows the results of CEX-HPLC analysis of the material isolated using an elution buffer (44 mM sodium acetate, pH 5).

Fig. 6a shows whether the separation ability is exhibited when the conditions of the elution buffer (20 mM histidine-hydrochloric acid, 30 mM sodium chloride, pH 5.7 buffer) are changed.

Fig. 6b shows the results of CEX-HPLC analysis of the substances separated using the elution buffer of Fig. 6a, and it was confirmed that the acid isoform fragment and the basic isoform fragment were not properly separated.

Figures 6c, 6d, and 6e show the results of CE-SDS analysis of the substances separated using the elution buffer; Figures 6c, 6d, and 6e show the results of the control group, comparative example 1, and experimental example 2, respectively.

Figures 6f and 6g show the results of SE-SDS analysis of the substances separated using the elution buffer; Figures 6f and 6g show the results of the control group, comparative example 1 and experimental example 2, respectively.

Fig. 7 shows whether the separation ability is exhibited when the conditions of the elution buffer are changed.

Hereinafter, the present invention 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 invention, rather than to limit the present invention to these examples.

Unlike antibodies, it is difficult to purify antibody fragments with high purity. In the present invention, in order to purify the antibody fragment with high purity, a purification process using affinity chromatography (Example 1) is performed, followed by a purification process using cation exchange chromatography (Example 2).

Example 1: Purification of isoantibodies via CH-1 affinity chromatography

For affinity chromatography, except for Experimental Example 3, heavy chain affinity chromatography was applied.

Heavy chain affinity chromatography means chromatography using a heavy chain affinity resin that is capable of specifically binding a portion of the heavy chain of an antibody. Example 1 provides a case where a CaptureSelect ^™ CH-1 XL affinity matrix (Thermo Fisher; hereinafter referred to as CH-1) is used as an example of a heavy chain affinity resin, but the heavy chain resin is not limited thereto. Any resin capable of specifically binding a heavy chain may be used. In addition, in the present invention, affinity chromatography using CH-1 as a heavy-chain affinity resin may be referred to as CH-1 affinity chromatography.

Elution from an affinity chromatography column was performed under acidic conditions (50 mM sodium acetate, pH 4.5 ± 0.1). An elution buffer having a conductivity of 3.5 ± 0.3 mS / cm was used and the elution buffer was filtered with a sterile filter, and the filtered elution buffer was packed into a cation chromatography column. The fragments purified using CH-1 affinity chromatography have a purity of about 92% to 93%. Hereinafter, the present invention disclosed on the premise thereof will be explained.

In addition, in order to confirm whether the purification method of the present invention has a substantial effect, ranibizumab, which is a representative antibody fragment drug, was selected for purification.

Example 2: Purification of homologous antibody fragments using cation exchange chromatography

Experimental Example 1: Establishing Purification Conditions of Identical Antibody Fragments Using Cation Exchange Chromatography (Conditions of Establishing Elution Buffer)

The elution buffer obtained by purification using CH-1 affinity chromatography in Example 1 was charged into a cation exchange chromatography resin (Capto SP ImpRes, GE Healthcare) to isolate isoantibody fragments. The chromatographic conditions are as follows:

* Chromatographic conditions:

-Resin: Capto SP ImpRes

-Flow rate: 90cm / h

-Equilibrium: 50mM sodium acetate buffer (pH 4.5)

-Loading capacity: max.30g protein / L (resin volume)

-Rebalance: 5mM sodium acetate buffer (pH 4.9)

-Rinse: 25mM MOPS buffer (pH 7.0)

-Elution: linear concentration gradient sodium acetate (5mM to 300mM) buffer (pH 4.9)

A purification process using cation exchange chromatography was performed in a process similar to that shown in FIG. 1. The elution buffer obtained by purification by CH-1 affinity chromatography was loaded into a cation exchange chromatography column, and after reequilibration, the sample containing the homogeneous antibody mixture bound to the column was washed.

For the washing buffer, a method using a buffer (25 mM MOPS, pH 7.0) was introduced. The material separated from the washing buffer was analyzed by CEX-HPLC, and the results are shown in Figure 2c. Specifically, the substance shown in No. 1 in FIG. 2c is a single fragment (i.e., single chain) of the antibody, and the substance shown in No. 2 in FIG. 2c is an acid isoform (i.e., acidic variant) of the antibody. body). These materials were analyzed by SDS-PAGE and confirmed in the same manner as the CEX-HPLC results shown in Figure 2d.

To establish the elution conditions after re-equilibration, a linear concentration gradient of sodium acetate (5 mM to 300 mM) was used for elution. After that, all products were collected from the highest UV point to 30% before the UV high point. The remainder was collected in sections and analyzed by CEX-HPLC to confirm the results, but they were not additionally included in the product.

The above experiments confirmed that the elution buffer had the best separation ability at a concentration of 110 mM (Figure 2a). The isoforms and single fragments of the isoantibodies obtained by the purification method were analyzed by CEX-HPLC. As a result, it was confirmed that they had a product purity of 98% or higher (FIG. 2b). It was also confirmed that most of the antibody fragments of acidic and basic isoforms and the single fragments of isoform antibodies were isolated. As a result of CEX-HPLC analysis, the final process yield was confirmed to be 61%.

Experimental Example 2: Establishment of purification conditions of isoforms and single fragments using cation exchange chromatography (appropriate salt concentration of elution buffer)

The elution buffer obtained by purification using CH-1 affinity chromatography in Example 1 was charged into a cation exchange chromatography resin (Capto SP ImpRes, GE Healthcare) to isolate isoantibody fragments. The chromatographic conditions are as follows:

* Chromatographic conditions:

-Resin: Capto SP ImpRes

-Flow rate: 90cm / h

-Equilibrium: 50mM sodium acetate buffer (pH 4.5)

-Loading capacity: max.30g protein / L (resin volume)

-Rebalance: 5mM sodium acetate buffer (pH 4.9)

-Rinse: 25mM MOPS buffer (pH 7.0)

-Elution: 110mM sodium acetate buffer (pH 4.9)

Experiments were performed to confirm whether the elution buffer having a concentration of 110 mM established in Experimental Example 1 is suitable for purification of the target fragment of isoantibodies.

A purification process using cation exchange chromatography was performed in a process similar to that shown in FIG. 1. The elution buffer obtained by CH-1 affinity chromatography was loaded into a cation exchange chromatography column, and after re-equilibration, the sample containing the homogeneous antibody mixture bound to the column was washed.

Regarding the washing buffer, Experimental Example 1 used a buffer (25 mM MOPS, pH 7.0). In addition, the resultant was re-equilibrated and eluted using a sodium acetate elution buffer (110 mM). After that, all products from the highest UV point to 30% before the UV high point were collected. The remainder was collected in sections and analyzed by CEX-HPLC to confirm the results, but they were not additionally included in the product.

The above experiments confirmed that the elution buffer had the best separation ability at a concentration of 110 mM (Figure 3a). The isofunctional antibody fragments obtained by the purification method were analyzed by CEX-HPLC. As a result, it was confirmed that they had a product purity of 98% or higher (FIG. 3b). It was also confirmed that the acidic and basic isoforms and single fragments of most isoantibodies were isolated.

Experimental Example 3: Purification of homologous antibody fragments using cation exchange chromatography based on changes in affinity resin

Unlike Experimental Example 2, experiments were performed to confirm whether the purification method using cation exchange chromatography was affected when the type of the affinity chromatography column was changed. Specifically, experiments were performed to confirm whether isotopic antibody fragments were isolated when a sample purified using a Kappa affinity resin instead of a CH-1 affinity resin was loaded into a cation exchange chromatography resin (Capto SP ImpRes, GE Healthcare).

* Chromatographic conditions:

-Resin: Capto SP ImpRes

-Flow rate: 90cm / h

-Equilibrium: 50mM sodium acetate buffer (pH 4.5)

-Loading capacity: max.30g protein / L (resin volume)

-Rebalance: 5mM sodium acetate buffer (pH 4.9)

-Rinse: 25mM MOPS buffer (pH 7.0)

-Elution: 110mM sodium acetate buffer (pH 4.9)

The elution buffer obtained using Kappa affinity resin purification in affinity chromatography was loaded into a cation exchange column, and after re-equilibration, isotropic antibodies bound to the column were washed.

For the washing buffer, a buffer solution (25 mM MOPS, pH 7.0) was used in Experimental Example 2. The resultant was then re-equilibrated and then eluted. For elution, collect all products from the highest UV point to the 30% point before UV. The remainder was collected in sections and analyzed by CEX-HPLC to confirm the results, but they were not additionally included in the product.

As for the elution buffer, sodium acetate having a concentration of 110 mM was used according to Experimental Example 2, and it was confirmed that the elution buffer has the best separation ability at the above concentration (FIG. 4 a). The isofunctional antibody fragments obtained by the purification method were analyzed by CEX-HPLC. As a result, it was confirmed that they had a product purity of 98% or higher (FIG. 4b). It was also confirmed that acid and basic isoforms and single fragments of most isoantibodies were isolated.

The results of the experiment confirmed that even if Kappa affinity resin was used instead of CH-1 affinity resin in the affinity chromatography column to increase the amount of impurity isoantibodies single fragments and acid isoforms, they could still be separated by cation exchange chromatography. Target antibody fragment.

In other words, it was confirmed that the target antibody fragment can be purified with high purity under the optimized elution buffer conditions of the cation exchange chromatography regardless of the type of the affinity chromatography column.

Experimental Example 4: Establishment of purification conditions of isoforms and single fragments using cation exchange chromatography (concentration range of elution buffer)

The elution buffer obtained by purification using CH-1 affinity chromatography in Example 1 was charged into a cation exchange chromatography resin (Capto SP ImpRes, GE Healthcare) to isolate isoantibodies. The chromatographic conditions are as follows:

* Chromatographic conditions:

-Resin: Capto SP ImpRes

-Flow rate: 90cm / h

-Equilibrium: 50mM sodium acetate buffer (pH 4.5)

-Loading capacity: max 30g protein / L (resin volume)

-Rinse: 44mM sodium acetate buffer (pH 5.2)

-Elution: 85mM sodium acetate buffer (pH 5.2)

Depending on the pH, the charge of the protein usually changes, so the salt concentration also changes. In the present invention, in order to measure the upper limit of the elution buffer, the experiment was performed by changing the pH and salt concentration of sodium acetate.

The elution buffer obtained by purification using CH-1 affinity chromatography was loaded into a cation exchange chromatography column, and after the washing process, the sample containing the isotropic antibody mixture bound to the column was eluted. For elution, collect all products from the UV peak to 50% before the UV peak. The remainder was collected in sections and analyzed by CEX-HPLC to confirm the results, but they were not additionally included in the product.

For washing buffer, use elution buffer (44 mM sodium acetate, pH 5.2). Analysis of the material separated from the washing buffer confirmed the removal of a single fragment of isoantibodies during the washing process (Figure 5c). The use of sodium acetate (44 mM) as a wash buffer is the same type and concentration as the elution buffer used in the next elution procedure. During the washing process, only a single fragment of the isoantibody was removed, but the target fragment of the isoantibody was not eluted. Therefore, it can be seen that it is preferable to use sodium acetate having a concentration of more than 44 mM as the elution buffer.

Therefore, sodium acetate (85 mM) was used as the elution buffer, and it was confirmed that the target fragment of the isozyme was eluted at this concentration (Fig. 5a). As a result of analyzing the isofunctional antibody fragments obtained according to the purification method of Experimental Example 4 by CEX-HPLC, it was confirmed that they had a product purity of 98% or higher (FIG. 5b). In addition, CEX-HPLC analysis results confirmed that the final process yield was 39%. Compared with the 61% yield in Experimental Example 2, it was confirmed that acidic isoform antibody fragments were not removed during the washing process, and only a single fragment of the isoform antibody was removed (Figure 5c). As a result, it was found that the acidic isoform antibody fragment was contained in the elution buffer, and the content of the product may be reduced from the viewpoint of product yield, although there was no problem in terms of purity.

Comparative Example 1: Different purification conditions of isoforms and single fragments using cation exchange chromatography

The elution buffer obtained by CH-1 affinity chromatography purification in Example 1 was charged into a cation exchange chromatography resin (Capto SP ImpRes, GE Healthcare) to perform a purification process. The chromatographic conditions are as follows:

* Chromatographic conditions:

-Resin: Capto SP ImpRes

-Flow rate: 90cm / h

-Equilibrium: 50mM sodium acetate buffer (pH 4.5)

-Loading capacity: max.30g protein / L (resin volume)

-Re-equilibration: buffer (20 mM histidine-hydrochloric acid, 30 mM sodium chloride; pH 5.2)

-Elution: buffer (20 mM histidine-hydrochloric acid, 30 mM sodium chloride; pH 5.7)

Experiments were performed to confirm whether the target protein to be obtained in the present invention can be isolated even if the kinds of buffer solution, pH, and salt concentration in Experimental Examples 1 to 4 are changed.

In addition, in order to confirm whether the purification method of the present invention has a substantial effect, the antibody fragment drug Lucentis ^® (ranibizumab) was selected as a control group.

An elution buffer obtained by purification using CH-1 affinity chromatography was loaded into a cation exchange chromatography column, and after reequilibration, the antibody bound to the column was eluted. During elution, collect all products from the highest UV point to 30% before the UV high point. The remainder was collected in sections and analyzed by CEX-HPLC to confirm the results, but they were not additionally included in the product.

A buffer of 20 mM histidine-hydrochloric acid and 30 mM sodium chloride at pH 5.7 was used as an elution buffer, and it was confirmed that this condition showed the best separation ability (Figure 6a). The isofunctional antibody fragments obtained by the purification method were analyzed by CEX-HPLC. As a result, it was confirmed that the impurity acidic and basic isoform antibody fragments were separated (Fig. 6b).

In addition, the results of CE-SDS confirmed that most of the single fragments of the same antibody were removed in Experimental Example 2. However, it is confirmed from FIG. 6c (control group), FIG. 6d (comparative example 1), and FIG. 6e (experimental example 2) that about 2% of a single fragment of the isofunctional antibody still remains in the comparative example 1.

In addition, as a result of SE-HPLC analysis, it was confirmed that most dimers and multimers were removed in Experimental Example 2. However, it is confirmed from the graphs 6f (Comparative Example 1 and the control group) and 6g (Experimental Example 2 and the control group) that dimers and multimers are still present in Comparative Example 1.

Therefore, it was confirmed that when the conditions of the elution buffer of the cation exchange chromatography established by the present invention are changed, it is impossible to separate impurities (ie, single fragments and isoforms of isoantibodies).

Comparative Example 2: Purification method using cation exchange chromatography according to pH gradient (confirmation of establishment of elution buffer concentration range)

The elution buffer purified using CH-1 affinity chromatography in Example 1 was charged into a cation exchange chromatography resin (Capto SP ImpRes, GE Healthcare). The chromatographic conditions are as follows:

* Chromatographic conditions:

-Resin: Capto SP ImpRes

-Flow rate: 90cm / h

-Equilibrium: 50mM sodium acetate buffer (pH 4.5)

-Loading capacity: max.30g protein / L (resin volume)

-Rebalance: 5mM sodium acetate buffer (pH 4.5)

-Rinse: 25mM MOPS buffer (pH 7.0)

-Elution: 5mM to 300mM sodium acetate buffer (pH 4.5)

In Comparative Example 2, in order to determine the lower limit of the pH value of the elution buffer used in the purification process of the present invention, experiments were performed using a salt concentration gradient (pH 4.5) of sodium acetate.

A purification process using cation exchange chromatography was performed in a process similar to that shown in FIG. 1. The elution buffer obtained by CH-1 affinity chromatography was loaded into a cation exchange chromatography column, and after re-equilibration, the sample containing the homogeneous antibody mixture bound to the column was washed. For the washing buffer, a method using a buffer (25 mM MOPS, pH 7.0) was introduced. The material separated from the washing buffer was analyzed by CEX-HPLC. As a result, it was confirmed that a single fragment of the isotropic antibody and an acid isoform antibody fragment were separated from the washing buffer.

In addition, in order to establish elution conditions after re-equilibration, a linear concentration gradient of sodium acetate (5 mM to 300 mM; pH 4.5) was used as an elution buffer to elute the resultant. After that, all products were collected from the highest UV point to 30% before the UV high point. The remainder was collected in portions and analyzed by CEX-HPLC to confirm the results, but they were not additionally included in the product.

Based on the results of isolating the isoforms and single fragments of the impurity isoantibodies by the purification method without eluting the target fragments of the isoantibodies, the lower limit of the pH can be established (FIG. 7).

Although the invention has been described with reference to specific illustrative specific examples, those skilled in the art will understand that the invention may be embodied in other specific forms without departing from the technical spirit or essential characteristics of the invention. Therefore, the above specific examples are to be considered in all respects as illustrative and not restrictive. Furthermore, the scope of the present invention is defined by the appended claims rather than the detailed description, and it should be understood that all modifications or variations derived from the meaning and scope of the present invention and its equivalents are included in the appended rights In the required range.

Claims (11)

A method for purifying a target fragment of isokinetic antibodies, comprising: (a) loading a sample containing a homogeneous antibody mixture into a cation exchange chromatography column equilibrated with an equilibration buffer; (b) rinsing the column with a washing buffer; And (c) recovering a target fragment of isotropic antibody from the cation exchange chromatography column using a sodium acetate elution buffer, the sodium acetate elution buffer having a pH of pH 4.9 to pH 5.2 and a salt concentration of 85 mM to 110 mM. The method according to item 1 of the scope of patent application, further comprising removing a single fragment of the isotropic antibody and an isoform of the isotropic antibody during step (b) or step (c). The method according to item 1 of the scope of patent application, wherein the column is equilibrated with an equilibration buffer having a pH of pH 4.9 to pH 5.2 before loading the sample containing the homogeneous antibody mixture in step (a). The method of claim 3, wherein the equilibrium buffer comprises at least one salt selected from the group consisting of sodium acetate, sodium chloride, sodium phosphate, and sodium citrate. The method of claim 1, wherein the equilibration buffer of step (a) has a salt concentration of 5 mM to 50 mM. The method according to item 1 of the patent application scope, further comprising performing affinity chromatography, ion exchange chromatography, and concentration or dialysis before step (a). The method according to item 1 of the patent application scope, further comprising re-equilibrating the column using a re-equilibration buffer after step (a) or step (b). The method according to item 1 of the scope of patent application, wherein the washing buffer in step (b) has a pH range greater than the pI value of the fragment of the isotropic antibody and a pH range less than the pH value of the target fragment of the isofunctional antibody. The method of claim 1, wherein the washing buffer in step (b) has a concentration of 5 mM to 25 mM. The method of claim 1, wherein the washing buffer in step (b) comprises at least one selected from the group consisting of sodium phosphate, sodium chloride, and 2- [4- (2-hydroxyethyl) piperazine -1-yl] ethanesulfonic acid (HEPES), 2- [bis (2-hydroxyethyl) amino] -2- (hydroxymethyl) propane-1,3-diol (Bis-Tris), and 3- Salts of the group consisting of morpholinylpropane-1-sulfonic acid (MOPS). The method according to item 1 of the scope of patent application, wherein the purified target fragment of the isotropic antibody is a therapeutic protein.