WO2018190677A2

WO2018190677A2 - Method for purifying analogous antibody using cation-exchange chromatography

Info

Publication number: WO2018190677A2
Application number: PCT/KR2018/004345
Authority: WO
Inventors: Sung-Muk KANG; Hong Jai Lee; Kyunghwa Kim; Jung Min Yoo; Chung Min Lee; Gyung-Hwa LEE; Dong Eok Lee; Sung In Lim; Gyong-sik HA
Original assignee: CJ Healthcare Corp
Current assignee: HK Inno N Corp
Priority date: 2017-04-14
Filing date: 2018-04-13
Publication date: 2018-10-18
Anticipated expiration: 2019-10-14
Also published as: WO2018190677A3; KR20180116159A; KR102140693B1; AR111465A1; TW201841933A; TWI679209B

Abstract

The present disclosure relates to a method for separating impurities of analogous antibodies using cation-exchange chromatography, and more specifically, to a method for separating the single fragments and isoforms of analogous antibodies, which are produced during the process of producing analogous antibodies, and purifying only target antibody fragments in high purity and yield using an elution buffer for the cation-exchange chromatography.

Description

METHOD FOR PURIFYING ANALOGOUS ANTIBODY USING CATION-EXCHANGE CHROMATOGRAPHY

The present disclosure relates to a method for separating impurities of an analogous antibody using cation-exchange chromatography, and more specifically, to a method for purifying target fragments of an analogous antibody in high purity and yield by using an elution buffer for cation-exchange chromatography and by removing single fragments of an analogous antibody and isoforms of an analogous antibody, which are produced during the process of producing an analogous antibody.

During the process of producing analogous antibodies using bacteria, various types of isoforms and single antibody fragments of an analogous antibody are produced. Therefore, it is necessary to develop a method for maximally reducing the non-product impurities through studies to improve a fermentation process along with construction of optimal clones. However, for the production of a desired optimal product, it is necessary to conduct studies for removing most of the non-product impurities during the purification process and find suitable conditions for preventing a further increase of the non-product impurities during storage.

Monoclonal antibodies, which are representative materials of protein therapeutics, have become a very attractive tool for the development of therapeutics because these have the ability to specifically bind to targets, and thereby most of these can be used in the body.

Monoclonal antibodies also produce various types of isoforms during the intracellular expression process. In order to maximize the efficacy without producing product-related impurities among these isoforms, it should be solved by the development of clone selection and cultivation processes. However, if the isoforms of the product-related impurities still remain even after the processes above, the purification and storage processes are carried out to prevent further increase of the isoforms.

Product-related impurities have properties very similar to those of products, and thus they are very difficult to separate through chromatography used in a purification process. However, with the development of technology, attempts have recently been made to remove such product-related impurities through the development of a purification process (Korean Patent Publication No.　10-2013-0064803).

Under the circumstances, the present inventors have confirmed that most non-product impurities can be removed by cation-exchange chromatography using a specific elution buffer described in the present disclosure, through a method for separating impurities of analogous antibodies, thereby completing the present disclosure.

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

The present disclosure relates to a method for separating impurities from an analogous antibody by cation-exchange chromatography, and more specifically, to a method for purifying single fragments and isoforms of the analogous antibody, which are produced during the production of the analogous antibody, using a buffer for cation-exchange chromatography, thereby obtaining the analogous antibody in high purity and yield. Therefore, the method of the present disclosure can be applied to the purification of analogous antibodies produced by genetic recombinant technology from bacterial fermentation.

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

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

Fig.　2b shows the analysis results of the isoforms of the analogous antibody and the single fragments of the analogous antibody using CEX-HPLC, confirming that the purity is 98% or higher.

Fig.　2c shows the results of the elution buffer in a range of 5　mM to 300　mM eluted in a concentration gradient. Specifically, No.　1 indicates the single fragments of the analogous antibody (i.e., single chain), and No.　2 indicates the antibody fragment of the acidic isoform (i.e., acidic variants).

Fig.　2d shows the results of Fig.　2c through SDS-PAGE.

Fig.　3a shows that the elution buffer has the optimum separation ability at a concentration of 110　mM, and that the target fragments of the analogous antibody are separated.

Fig.　3b shows the results of CEX-HPLC analysis of the separated substances using the elution buffer, confirming that the substances separated from the elution buffer have a purity of 98% or higher.

Fig.　4a shows that the separation ability is maintained under the pH and salt concentration of the same elution buffer even when the type of an affinity resin is changed.

Fig.　4b shows the results of CEX-HPLC analysis of the substances separated using the elution buffer, confirming that the substances separated from the elution buffer have a purity of 98% or higher.

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

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

Fig.　5c shows the results of CEX-HPLC analysis of the substances separated using the 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, confirming that the acidic isoform fragment and the basic isoform fragment are not properly separated.

Figs.　6c, 6d, and 6e show the results of CE-SDS analysis of the substances separated using the elution buffer; each of Figs.　6c, 6d, and 6e show the results of the control group, Comparative Example 1, and Experimental Example 2, respectively.

Figs.　6f and 6g show the results of SE-SDS analysis of the substances separated using the elution buffer; each of Figs.　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.

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 method for purifying a target fragment of an analogous antibody, comprising:

(a) loading a sample comprising a mixture of analogous antibodies into a cation-exchange chromatography column equilibrated with an equilibration buffer;

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

(c) removing a target fragment of an analogous antibody from the cation-exchange chromatography column using an elution buffer of sodium acetate, which has a pH ranging from pH　4.9 to pH　5.2.

Previously, cation-exchange chromatography was used to separate Ranibizumab in high purity, which is a representative antibody fragment medicine; however, it has been reported that due to the presence of a large amount of impurities, inflammation was induced in clinical trials, and thus it failed to perform clinical replacement. This teaches that even when the antibody fragments with high purity are separated using cation-exchange chromatography, problems may occur at the actual clinical stage, thereby suggesting that optimized purification conditions must be found. However, the present inventors have discovered a method for effectively separating unnecessary single fragments of an analogous antibody and isoforms of an analogous antibody through optimization of an elution buffer, and for purifying a target antibody fragment with high yield.

In particular, when the method of the present disclosure is applied to the preparation of a biosimilar of the analogous antibody, the fragments of the analogous antibody, which contain the fragment of the main active antibody and the isoforms of the analogous antibody as compositions identical or corresponding to a control drug, can be prepared.

Each step in the method for purifying the target antibody or the antibody fragment is described in detail as follows. First, Step (a) is a step of loading a sample comprising a mixture of analogous antibodies into a cation-exchange chromatography column equilibrated with an equilibration buffer.

As used herein, the term "sample comprising a mixture of analogous antibodies" is a sample which is partially purified from a culture supernatant of cells producing analogous antibodies or a lysate of the cells, and thus refers to a partially purified sample containing a mixture of analogous antibodies including both the main active antibody fragment and the isoforms of the antibody fragment. The partial purification refers to a state in which proteins other than target fragments of an analogous antibody, i.e., single fragments of an analogous antibody, isoforms of an analogous antibody, etc., are present even if a filtration process has been performed.

As used herein, the term "main active antibody fragment" is a main component included in the antibody group of the present disclosure; that is, it refers to an antibody fragment in which some amino acids in the antibody fragment are modified by deamination or oxidation such that the biological activity is not lowered, i.e., an antibody fragment that is not an acidic or basic isoform of the antibody fragment. The main active antibody fragment is the most important component for controlling the quality of a desired antibody fragment, and thus is the antibody fragment having the highest biological activity among the components of the antibody.

As used herein, the term "isoform of antibody fragment" refers to an antibody fragment in which some of the amino acids in the main active antibody fragment are modified by deamination or oxidation, and includes antibody fragments of an acidic isoform and antibody fragments of a basic isoform. Examples thereof include antibody fragments of an isoform in which asparagine among the amino acids is modified into aspartate through deamination, antibody fragments of an isoform in which methionine among the amino acids is modified into methionine sulfate through oxidization, etc. Additionally, when glutamate is present at the N-terminus of the heavy chain, the glutamate forms a pentagonal ring structure, and thus includes antibody fragments of an isoform, in which the glutamate is modified into pyroglutamate. When the antibody fragments are produced from host cells such as bacterial cells, the antibody fragments of the isoform are contained in the culture medium of the host cells at a high rate, and therefore, the antibody fragments of the isoform should be removed by chromatography in order to be included in the antibody fragments at a desired rate.

As used herein, the term "ion-exchange chromatography" refers to a method of performing chromatography which utilizes a chromatography material that exchanges ions. Specifically, the term "a chromatography material that exchanges ions", in which a functional group is bound to a polymer material, refers to a material which purifies ionic materials dissolved in polar and non-polar solutions by mutual exchange. In addition, the ion-exchange efficiency and purpose of ion-exchange chromatography vary depending 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 purpose of the present disclosure, a cation-exchange chromatography column may be used as the ion-exchange chromatography. Specifically, the term "cation-exchange chromatography column" refers to a column filled with cation-exchange resins. In the present disclosure, cation-exchange chromatography is performed to remove isoforms of analogous antibodies, impurities, etc. Cation-exchange resins can be classified into sulfonic acid (S) and carboxymethyl (CM) groups; and effective pH, exchange efficiency, regeneration efficiency, ion adsorption, etc. differ depending on the properties of each group. In addition, the cation-exchange resins are synthetic resins that exchange cations in the aqueous solution with cations in the resins. In this regard, since the isoelectric point of the antibody is high, these become cations in the buffer with a pH below the isoelectric point value. Therefore, the quality of the antibody group can be improved by using cation-exchange resins capable of adsorbing antibodies exhibiting the cations. The cation-exchange resins may be those conventionally used in the art. Specifically, for the cation-exchange resins, the column having a functional group of COO^- or SO₃ may be used, and more specifically carboxymethyl (CM), fractogel, sulfoethyl (SE), sulfopropyl (SP), phosphate (P), or sulfonate (S), etc. may be used, and most specifically carboxymethyl sepharose (CM sepharose) or fractogel COO^- may be used, but the functional group is not limited thereto.

Additionally, before loading the sample of Step (a), which contains the mixture of antibodies, the column may be equilibrated with an equilibration buffer having a pH ranging from pH　4.5 to pH　5.5. The equilibration buffer of Step (a) may have a salt concentration ranging from 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 it is not limited thereto.

Additionally, the method may further include carrying out ion-exchange chromatography, concentration, and dialysis before Step (a). The purpose thereof is to remove the primary impurities of the antibody or antibody fragment, and to increase the concentration of the sample. Specifically, a step of concentrating and dialyzing the sample containing the mixture of antibodies may be performed in advance before carrying out Step (a), and then the sample may be loaded into a cation-exchange affinity chromatography column. However, any work to remove the primary impurities and increase the concentration of the sample may be applied without limitation. For example, a step of purifying the sample containing the mixture of analogous antibodies by using CH-1 affinity chromatography may be carried out in advance before Step (a), but is not limited thereto.

In the method for purifying the target fragments of an analogous antibody, Step (b) is a step of washing the column with a washing buffer; that is, it is a step of applying a washing buffer to the chromatography column into which the sample is loaded.

The pH values of the washing buffer may range from a value exceeding the pI value of the single fragments of the analogous antibody to a value below the pH value of the target fragments of the analogous antibody. 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 fragments of analogous antibody, more specifically, the washing buffer may have a pH ranging from pH　6.7 to pH　7.3, but the pH values are not limited thereto. In addition, a salt concentration of the washing buffer may range from 5mM to 25mM, but the salt concentration values are not limited thereto.

In the case of the fragments of the analogous antibody, the isoelectric point is high such that coagulation may not occur under acidic conditions with a lower pH, whereas in the case of the sample containing the mixture of analogous antibodies, in which the isoelectric point is lower than that of the fragments of the analogous antibody, the coagulation may not generally occur by van der Waals forces because charges are removed under acidic conditions with a lower pH. Therefore, a pH value may be used which is lower than the isoelectric point of the fragments of the analogous antibody to be purified in the present disclosure and which is concurrently higher than the isoelectric point of the single fragments of the analogous antibody. That is, the pH may be a value lower than the isoelectric point 1.0 of the fragments of the analogous antibody to be purified, but is not limited thereto.

Additionally, the washing buffer may include at least one salt selected from the group consisting of sodium phosphate, sodium chloride, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (Bis-Tris), and 3-morpholinopropane-1-sulfonic acid (MOPS), but is not limited thereto.

For the purpose of the present disclosure, the substances separated by the washing buffer in Step (b) may be single fragments of an analogous fragment or isoforms of the analogous fragment.

As used herein, the term "isoforms of analogous antibody" may be acidic and/or basic isoforms. Since several isoforms of analogous antibodies are present in analogous antibody products, it is important that the quality of a biosimilar to be produced should be mostly similar to its comparator in order to demonstrate equivalence. The isoforms of the analogous fragments are in the form in which several amino acids of the main active antibody fragment are modified, and thus there is a slight difference in charge between the main active antibody fragment, the antibody fragment of the acidic isoform, and the antibody fragment of the basic isoform. Accordingly, the antibody fragments of the isoform can be separated using this difference in charge. However, since this difference in charge is a minute difference due to only a few amino acids, it is necessary to set very precise conditions for separation. In this regard, the cation-exchange column of the present disclosure is capable of effectively removing the antibody fragments of the acidic isoform and the antibody fragments of the basic isoform.

In the present disclosure, 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 ranging from pH　4.9 to pH　5.2 and have a salt concentration ranging from 1　mM to 5　mM, but these are not limited thereto.

In the method for purifying a target antibody fragment, Step (c) may be a step of recovering the target fragments of the analogous antibody from the cation-exchange chromatography column using an elution buffer of sodium acetate having a pH ranging from pH　4.9 to pH　5.2.

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

When the pH of the elution buffer is less than pH　4.9, there are problems in that the target fragments of the analogous antibody are not separated and that impurities such as the single fragment of the analogous fragment, the isoform of the analogous fragment, etc. are not separated. In addition, when the pH of the elution buffer is higher than pH　5.2, the target fragments of the analogous antibody are not in any way separated. Accordingly, such pH ranges of the elution buffer are preferably not to be used.

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

As used herein, the term "target fragment of analogous antibody" is a kind of protein to be separated, and thus may interchangeably be used with the term "target protein"

For the purpose of the present disclosure, the target fragments of the analogous antibody are those in which the single fragments of the analogous antibody or the isoforms of the analogous fragment are removed by the purification method, and thus CEX-HPLC, SDS-PAGE, and CE-SDS may be used to confirm whether the impurities are separated from the target fragments of the analogous antibody; however, these are not limited as long as they can identify the acidic and basic isoforms of the analogous antibodies and the single antibody fragments. Further, SE-HPLC analysis may be conducted by setting the optimal concentration of the elution buffer so as to confirm that dimers and multimers are mostly separated, but is not limited thereto.

The single fragments of the analogous antibody may include an scFv form, a single heavy chain, a single light chain, etc., but are not limited thereto.

The types of the antibody fragments all include Fv, Fab, Fab′, F(ab′)₂, Fd, etc. The Fv include both forms of double disulfide Fv(dsFv) and single chain Fv(scFv). Fd refers to a heavy chain component included in the Fab fragment.

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 F_ab without F_c 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. For the purpose of the present disclosure, the purified antibody fragment may be a Ranibizumab antibody fragment in the F_ab form or various F_ab(s), but is not limited thereto.

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

Additionally, the purity of the protein 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 target fragments of the analogous antibody, which were purified by the purification method of the present disclosure, may be used as therapeutic proteins. 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 Fab, Fab′, or F(ab′')₂, but are not limited thereto.

For the purpose of the present disclosure, when the purification method is used, the target fragments of the analogous antibody, which have a high purity, can be separated. For the purpose of the present disclosure, even when a sample containing the mixture of target analogous antibodies, which had been separated using any kind of chromatography, is purified by the purification method, the separated target fragments of the analogous antibody, which have high purity, can be obtained. For example, when the target fragments of the analogous antibody having 92% purity, which were separated using the affinity chromatography, are re-purified using the cation-exchange chromatography of the present disclosure, the target fragments of the analogous antibody, which have 99% purity, can be separated.

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.

	Condition	Experimental Example 1	Experimental Example 2	Experimental Example 3	Experimental Example 4	Comparative Example 1	Comparative Example 2
Affinity Chromatography (Example 1)	Resin	CH-1	CH-1	Kappa	CH-1	CH-1	CH-1
Cation-exchange Chromatography	Resin	Capto SP ImpRes	Capto SP ImpRes	Capto SP ImpRes	Capto SP ImpRes	Capto SP ImpRes	Capto SP ImpRes
	Equilibration Buffer, Type and Conc. of Salts	50　mM sodium acetate	50　mM sodium acetate	50　mM sodium acetate	50　mM sodium acetate	20　mM histidine, 30　mM sodium chloride	50　mM sodium acetate
	Equilibration Buffer (pH)	pH　4.5	pH　4.5	pH　4.5	pH　4.5	pH　5.2	pH　4.5
	Re-equilibration Buffer, Type and Conc. of Salts	5　mM sodium acetate	5　mM sodium acetate	5　mM sodium acetate	-	20　mM histidine, 30　mM sodium chloride	5　mM sodium acetate
	Re-equilibration Buffer (pH)	pH　4.9	pH　4.9	pH　4.9	-	pH　5.2	-
	Washing Buffer, Type and Conc. of Salts	25　mM MOPS	25　mM MOPS	25　mM MOPS	44　mM sodium acetate	-	25　mM MOPS
	Washing Buffer (pH)	pH　7.0	pH　7.0	pH　7.0	pH　5.2	-	pH　7.0
	Elution Buffer, Type and Conc. of Salts	5　mM - 300　mM sodium acetate(0% - 100% liner conc. gradient)	110　mM sodium acetate	110　mM sodium acetate	85　mM sodium acetate	20　mM histidine, 30　mM sodium chloride	5　mM - 300　mM sodium acetate(0% - 100% liner conc. gradient)
	Elution Buffer(pH)	pH　4.9	pH　4.9	pH　4.9	pH　5.2	pH　5.7	pH　4.5
Results		Separation of single fragments and acidic isoforms in the washing buffer; Obtained antibody fragments in which most of the impurities were removed in the elution process (Fig.　2)	Separation of single fragments and acidic isoforms in the washing buffer; Obtained antibody fragments in which most of the impurities were removed in the elution process (Fig.　3)	Separation of single fragments and acidic isoforms in the washing buffer; Obtained antibody fragments in which most of the impurities were removed in the elution process (Fig.　4)	Separation of only single fragments in the washing buffer; Obtained antibody fragments in which most of the impurities were removed in the elution process (Fig.　5)	No separation of single fragments and antibody isoforms (Fig.　6)	Separation of single fragments and acidic isoforms in the washing buffer; impurities were separated in the elution process, but antibody fragments were not separated (Fig.　7)

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

Example 1: Purification of analogous antibody by CH-1 affinity chromatography

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

Heavy chain affinity chromatography refers to a chromatographic method using a heavy chain affinity resin that is capable of specifically binding to a heavy chain, a part of an antibody. In Example 1, the case of using CaptureSelect^TM CH-1 XL Affinity Matrix (Thermo Fisher; hereinafter referred to as CH-1) was given as an example of the heavy chain affinity resin, but the heavy chain resin is not limited thereto. Any resin capable of specifically binding to the heavy chain can be used. In addition, in the present disclosure, the affinity chromatography using CH-1 as the heavy chain affinity resin can be referred to as CH-1 affinity chromatography.

The elution from the affinity chromatography column was performed in an acidic condition (50　mM sodium acetate, pH　4.5±0.1). The elution buffer having a conductivity of 3.5±0.3　mS/cm was used and the elution buffer was filtered with a sterilizing filter, and the filtered elution buffer was loaded into a cation chromatography column. The fragments purified using the CH-1 affinity chromatography had a purity of about 92% to 93%. Hereinafter, the present disclosure disclosed on the premise thereof will be described.

Additionally, in order to confirm whether the purification method of the present disclosure has a substantial effect, Ranibizumab, a representative antibody fragment medicine, was selected for purification.

Example 2: Purification of fragment of analogous antibody using cation -exchange chromatography

Experimental Example 1: Setting purification condition for fragments of analogous antibody using cation -exchange chromatography (condition setting of elution buffer)

The elution buffer obtained by purification using the CH-1 affinity chromatography in Example 1 was loaded into a cation-exchange chromatography resin (Capto SP ImpRes, GE Healthcare) to separate the fragments of the analogous antibody. The chromatography conditions were as follows:

* Chromatography conditions:

- Resin: Capto SP ImpRes

- Flow Rate: 90　cm/h

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

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

- Re-equilibration: 5　mM sodium acetate buffer (pH　4.9)

- Washing: 25　mM MOPS buffer (pH　7.0)

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

The purification process using the cation-exchange chromatography was performed similarly to the procedure of Fig.　1. The elution buffer obtained by purification using the CH-1 affinity chromatography was loaded into the cation-exchange chromatography column, and a sample containing the mixture of analogous antibodies bound to the column was washed after re-equilibration.

For a washing buffer, a method of using a buffer (25　mM MOPS, pH　7.0) was introduced. The substances separated from the washing buffer were analyzed by CEX-HPLC, and the results thereof are shown in Fig.　2c. Specifically, the substance which appeared on No.　1 of Fig.　2c was single fragments of an antibody (i.e., a single chain), while the substance which appeared on No.　2 of Fig.　2c was acidic isoforms of an antibody (i.e., acidic variants). These substances were analyzed by SDS-PAGE and also confirmed in the same manner as the CEX-HPLC results as shown in Fig.　2d.

In order to set elution conditions after re-equilibration, sodium acetate (5　mM to 300　mM) was used for elution with a linear concentration gradient. Thereafter, all of the products ranging from the highest point of UV rays to the top 30% point of UV rays were collected. The remaining parts were collected in divisions, analyzed by CEX-HPLC, and the results were confirmed, but they were not additionally included in the products.

It was confirmed through the experiment above that the elution buffer had the optimum separation ability at a concentration of 110　mM (Fig.　2a). The isoforms and single fragments of analogous antibodies, which were 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 the acidic and basic isoforms and the single fragments of the analogous antibody were separated. As a result of the CEX-HPLC analysis, the final process yield was confirmed to be 61%.

Experimental Example 2: Setting purification conditions for isoforms and single fragments of analogous antibody using cation -exchange chromatography (appropriate salt concentration of elution buffer)

* Chromatography conditions:

- Resin: Capto SP ImpRes

- Flow Rate: 90　cm/h

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

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

- Re-equilibration: 5　mM sodium acetate buffer (pH　4.9)

- Washing: 25　mM MOPS buffer (pH　7.0)

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

Experiments were performed to confirm whether the elution buffer having a concentration of 110　mM set in Experimental Example 1 was suitable for purification of the target fragments of the analogous antibody.

For a washing buffer, a buffer (25　mM MOPS, pH　7.0) was used as in Experimental Example 1. In addition, the resultant was subjected to re-equilibration and then eluted using a sodium acetate elution buffer (110　mM). Thereafter, all of the products ranging from the highest point of UV rays to the top 30% point of UV rays were collected. The remaining parts were collected in divisions, analyzed by CEX-HPLC, and the results were confirmed, but they were not additionally included in the products.

It was confirmed through the experiment above that the elution buffer had the optimum separation ability at a concentration of 110　mM (Fig.　3a). The fragments of the analogous antibody, which were 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 most of the acidic and basic isoforms and the single fragments of the analogous antibodies were separated.

Experimental Example 3: Purification of fragments of analogous antibody using cation -exchange chromatography according to change in affinity resin

Unlike Experimental Example 2, experiments were performed to confirm whether the purification method using the cation-exchange chromatography was affected when the kinds of the affinity chromatography column changed. Specifically, experiments were performed to confirm whether the fragments of the analogous antibody were separated 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).

* Chromatography conditions:

- Resin: Capto SP ImpRes

- Flow Rate: 90　cm/h

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

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

- Re-equilibration: 5　mM sodium acetate buffer (pH　4.9)

- Washing: 25　mM MOPS buffer (pH　7.0)

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

The elution buffer purified using the Kappa affinity resin in the affinity chromatography was loaded into a cation-exchange column, and the analogous antibody bound to the column was washed after re-equilibration.

For a washing buffer, the elution buffer (25　mM MOPS, pH　7.0) was used as in Experimental Example 2. Then, the resultant was subjected to re-equilibration and then eluted. For elution, all of the products ranging from the highest point of UV rays to the top 30% point of UV rays were collected. The remaining parts were collected in divisions, analyzed by CEX-HPLC, and the results were confirmed, but they were not additionally included in the products.

As the elution buffer, sodium acetate having a concentration of 110　mM was used based on Experimental Example 2, and it was confirmed that the elution buffer had the optimum separation ability at the concentration above (Fig.　4a). The fragments of the analogous antibody 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 most of the acidic and basic isoforms and the single fragments of the analogous antibodies were separated.

As a result of the experimentations, it was confirmed that even when the amounts of the single fragments of the analogous antibody and the acidic isoforms, which are impurities, were relatively increased by using a Kappa affinity resin instead of a CH-1 affinity resin in the affinity chromatography column, the separation of the target antibody fragments was still possible by performing the cation-exchange chromatography.

In other words, it was confirmed that the target antibody fragments can be purified in high purity under the elution buffer conditions optimized for the cation-exchange chromatography irrespective of the kinds of affinity chromatography columns.

Experimental Example 4: Setting purification conditions for isoforms and single fragments of analogous antibodies using cation -exchange chromatography (setting concentration range of elution buffer)

The elution buffer obtained by purification using the CH-1 affinity chromatography in Experimental Example 1 was loaded into a cation-exchange chromatography resin (Capto SP ImpRes, GE Healthcare) to separate the fragments of the analogous antibody. The chromatography conditions were as follows:

* Chromatography conditions:

- Resin: Capto SP ImpRes

- Flow Rate: 90　cm/h

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

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

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

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

Depending on the pH, the charge of a protein generally changes, and thus the salt concentration also varies. In the present disclosure, experiments were performed by varying the pH and the salt concentration of sodium acetate in order to determine the upper limit of the elution buffer.

The elution buffer obtained by purification using the CH-1 affinity chromatography was loaded into a cation-exchange chromatography column, and a sample containing the mixture of analogous antibodies bound to the column was eluted after the washing process. For elution, all of the products ranging from the highest point of UV rays to the top 50% point of UV rays were collected. The remaining parts were collected in divisions, analyzed by CEX-HPLC, and the results were confirmed, but they were not additionally included in the products.

For the washing buffer, an elution buffer (44　mM sodium acetate, pH　5.2) was used. As a result of analyzing the substances separated from the washing buffer, it was confirmed that the single fragments of the analogous antibody were removed during the washing process (Fig.　5c). The sodium acetate (44　mM) used as the washing buffer was the same in type and concentration as the elution buffer used in the next elution process. In the washing process, only the single fragments of the analogous antibody were removed, and the target fragments of the analogous antibody were not eluted. Therefore, it could be derived that it is preferable to use sodium acetate having a concentration greater 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 fragments of the analogous antibody were eluted at this concentration. (Fig.　5a). As a result of analyzing the fragments of the analogous antibody, which were obtained by the purification method according to Experimental Example 4, by CEX-HPLC, it was confirmed that they had a product purity of 98% or higher (Fig.　5b). In addition, as a result of CEX-HPLC analysis, it was confirmed that the yield of the final process was 39%. Comparing with the yield of 61% in Experimental Example 2, it was confirmed that the antibody fragments of the acidic isoform were not removed during the washing process, but only the single fragments of the analogous antibody were removed (Fig.　5c). As a result, it was found that the antibody fragments of the acidic isoform were contained in the elution buffer and the amount that could have been contained as a product was reduced from the aspect of product yield, although there was no problem from the aspect of purity.

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

The elution buffer obtained by purification using the CH-1 affinity chromatography in Example 1 was loaded into a cation-exchange chromatography resin (Capto SP ImpRes, GE Healthcare) to proceed with the purification process. The chromatography conditions were as follows:

* Chromatography conditions:

- Resin: Capto SP ImpRes

- Flow Rate: 90　cm/h

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

- Loading: max. 30　g 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 proteins to be achieved in the present disclosure were separated even when the kinds of the buffer solution, pH, and salt concentration used in Experimental Examples 1 to 4 changed.

Additionally, in order to confirm whether the purification method of the present disclosure has a substantial effect, Lucentis^® (Ranibizumab), an antibody fragment medicine, was selected as a control group.

The elution buffer obtained by purification using the CH-1 affinity chromatography was loaded into a cation-exchange chromatography column, and the antibody bound to the column was eluted after re-equilibration. For elution, all of the products ranging from the highest point of UV rays to the top 30% point of UV rays were collected. The remaining parts were collected in divisions, analyzed by CEX-HPLC, and the results were confirmed, but they were not additionally included in the products.

A buffer of 20　mM histidine-hydrochloric acid and 30　mM sodium chloride at pH　5.7 was used as the elution buffer, and it was confirmed that the optimum separation ability was shown in this condition (Fig.　6a). The fragments of the analogous antibody obtained by the purification method were analyzed by CEX-HPLC. As a result, it was confirmed that the antibody fragments of the acidic and basic isoforms, which are impurities, were not separated (Fig.　6b).

Additionally, as a result of CE-SDS, it was confirmed that most of the single fragments of the analogous antibody were removed in Experimental Example 2. However, it was also confirmed from Figs.　6c (control group), 6d (Comparative Example 1), and 6e (Experimental Example 2) that about 2% of the single fragments of the analogous antibody still remained in Comparative Example 1.

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

Therefore, it was confirmed that when the conditions of the elution buffer for the cation-exchange chromatography set in the present disclosure were changed, separation of the impurities (i.e., the single fragments and isoforms of the analogous antibodies) was not possible.

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

The elution buffer obtained by purification using the CH-1 affinity chromatography in Example 1 was loaded into a cation-exchange chromatography resin (Capto SP ImpRes, GE Healthcare). The chromatography conditions were as follows:

* Chromatography conditions:

- Resin: Capto SP ImpRes

- Flow Rate: 90　cm/h

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

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

- Re-equilibration: 5　mM sodium acetate buffer (pH　4.5)

- Washing: 25　mM MOPS buffer (pH 7.0)

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

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

The purification process using the cation-exchange chromatography was performed similarly to the procedure of Fig.　1. The elution buffer obtained by purification using the CH-1 affinity chromatography was loaded into a cation-exchange chromatography column, and a sample containing the mixture of the analogous antibodies bound to the column was washed after re-equilibration. For a washing buffer, a method of using a buffer (25　mM MOPS, pH　7.0) was introduced. The substances separated from the washing buffer were analyzed by CEX-HPLC. As a result, it was confirmed that the single fragments of the analogous antibody and the antibody fragments of the acidic isoform are separated from the washing buffer.

In addition, in order to set elution conditions after re-equilibration, the resultant was eluted in a linear concentration gradient using sodium acetate (5　mM to 300　mM; pH　4.5) as the elution buffer. Thereafter, all of the products ranging from the highest point of UV rays to the top 30% point of UV rays were collected. The remaining parts were collected in divisions, analyzed by CEX-HPLC, and the results were confirmed, but they were not additionally included in the products.

Based on the results that the isoforms and single fragments of the analogous antibodies, which are impurities, were separated by the purification method and that the target fragments of the analogous antibody were not eluted, the lowest limit of the pH was able to be set (Fig.　7).

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 fragment of an analogous antibody, comprising:

(a) loading a sample comprising a mixture of analogous antibodies into a cation-exchange chromatography column equilibrated with an equilibration buffer;

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

(c) recovering a target fragment of an analogous antibody from the cation-exchange chromatography column using an elution buffer of sodium acetate, which has a pH ranging from pH　4.9 to pH　5.2.
The method of claim 1, wherein the elution buffer of Step (c) has a salt concentration ranging from 85　mM to 110　mM.
The method of claim 1, which further comprises removing the single fragment of the analogous antibody and an isoform of the analogous antibody during Step (b) or Step (c).
The method of claim 1, wherein the column is equilibrated with an equilibration buffer having a pH ranging from pH　4.5 to pH　5.5 before loading the sample comprising the mixture of analogous antibodies of Step (a).
The method of claim 4, wherein the equilibration 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 ranging from 5　mM to 50　mM.
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 re-equilibrating the column using a re-equilibration buffer after Step (a) or Step (b).
The method of claim 1, wherein the washing buffer in Step (b) has a pH in a range which is greater than the pI value of the fragment of an analogous antibody and smaller than the pH value of the target fragment of the analogous antibody.
The method of claim 1, wherein the washing buffer in Step (b) has a salt concentration ranging from 5　mM to 25　mM.
The method of claim 1, wherein the washing buffer in Step (b) comprises at least one salt selected from the group consisting of sodium phosphate, sodium chloride, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (Bis-Tris), and 3-morpholinopropane-1-sulfonic acid (MOPS).
The method of claim 1, wherein the purified target fragment of the analogous antibody is a therapeutic protein.