CN117069792A

CN117069792A - Method for eliminating cation exchange chromatography eluting acromion

Info

Publication number: CN117069792A
Application number: CN202311270033.3A
Authority: CN
Inventors: 黄飞; 刘娜; 徐天丹
Original assignee: Wuxi Biologics Shanghai Co Ltd
Current assignee: Wuxi Biologics Shanghai Co Ltd
Priority date: 2023-09-27
Filing date: 2023-09-27
Publication date: 2023-11-17

Abstract

A method for eliminating shoulder during purification of proteins by cation exchange chromatography is disclosed, characterized in that it comprises the steps of: a) Treating the cation exchange chromatography packing with an equilibration buffer selected from the group consisting of Tris-HCl buffer system and NaAc-HAc buffer system, at a pH ranging from 5.5 to 7.0, at a concentration of about 20mM to 1.0M, adding a protein-containing solution to the packing; b) Eluting the cation exchange chromatography packing with a first eluting buffer, the first eluting buffer being the same as the equilibration buffer, followed by eluting the cation exchange packing with a second eluting buffer, the second eluting buffer having the same buffer system as the equilibration buffer, the buffer system having a concentration of about 20mM to 1.0M, optionally wherein the concentration of the buffer system is higher than the concentration in the equilibration buffer; c) Eluting the protein with an elution buffer having the same buffer system as the equilibration buffer at a concentration of about 50mM to about 80mM, optionally having a higher concentration of counter ions relative to the equilibration buffer or wash buffer; and d) collecting the product.

Description

Method for eliminating cation exchange chromatography eluting acromion

(1) Technical field

The invention belongs to the field of biological pharmacy, in particular to the field of protein separation and purification, and particularly relates to a method for eliminating the occurrence of shoulder peaks of elution peaks when utilizing cation exchange chromatography to purify proteins.

(2) Background art

Ion exchange chromatography is a widely used technique for purifying biological macromolecules, and the principle is that under a certain pH condition, proteins have different charges, and when passing through an ion exchange matrix, the proteins are separated under different binding or eluting conditions. Among them, cation exchange chromatography (Cation Exchange Chromatography, CEX) refers to chromatography using negatively charged ion exchange matrices to bind positively charged proteins. Cation exchange chromatography is widely used for purification and industrial production of protein molecules (e.g., antibodies).

In the purification of antibody proteins, cation exchange chromatography generally employs a bind-elute mode. In this mode, the loading pH is below the isoelectric point (pI) of the antibody, the antibody molecule is positively charged, and the impurity molecule is negatively or uncharged. In the loading step, the antibody protein is bound to the cation exchange matrix, and the impurity molecules are not bound to the matrix, so that the separation of the antibody molecules and the impurity molecules is realized. In the elution step, the antibody molecules are eluted using an elution buffer containing a high concentration of salt ions.

In some research and production practices, the elution step of cation exchange chromatography is prone to the phenomenon of shoulder, which is often more pronounced during the amplification process, due to the variation in the concentration of the salt ions used or the pH. Theoretically, for a protein of high purity (SEC purity>90%) of the target protein is usually a single peak form when subjected to cation exchange chromatography, because the loaded protein has a very high purity and no obvious multiple elution peaks occur. However, in some production and development cases, high purity protein cation exchange elution peaks also appear as shoulder peaks. This means in particular the phenomenon that the A280 absorption curve of the elution step is inflection or bimodal during the chromatography process. The reason for the generation of shoulder during ion exchange chromatography is quite complex. Studies have shown that local ion exchange and dissociation between charged ion exchangers and buffer substances in cation exchanged solutions can lead to transient jumps in pH. The Ghose et al study (Biotechnol. Prog.2002,18, 530-537) showed that in the cation exchange chromatography process, na was present when the system NaCl concentration was shifted from low to high concentrations ⁺ Competing protons bound to the filler ligand causes a short time pH abnormality drop in the downward pH jump, and then the pH returns to the pH of the system due to the buffer system, although there is no direct evidence of protein washingDeacromion is associated with such pH jumps, which can be one of the reasons for the elution of proteins due to frequent pH jumps in the cation exchange elution step. In addition, cation exchange chromatography may also exhibit a transient increase in the pH of the elution step when a different buffer system is selected (e.g., citrate).

This transient change in pH can significantly affect the quality of the target protein molecule. Studies have shown that in chromatographic purification steps, the biological activity of recombinant proteins is significantly reduced when the pH deviates from the desired value by only 0.5 pH units (biotechnol. Prog.2002,18, 530-537). Therefore, when the elution shoulder occurs in cation exchange chromatography, sufficient attention needs to be paid and corresponding measures should be taken. Some theoretical studies suggest that mathematical models can effectively predict pH jumps in chromatography, indicating that pH jumps are widely present in ion exchange chromatography using different buffer systems and ion concentrations. The extent to which different buffer systems can undergo pH jumps is different, for example MES buffer systems are less prone to pH jumps (Biotechnol. Prog.2005,21, 902-910). It has been reported that increasing the buffering capacity of the buffer system of the leaching step or changing the pH of the leaching step can reduce the pH jump in ion exchange. However, there are problems that the stability of the protein is affected and the impurity removal ability is affected by changing the buffer system in production. In the production of protein products, the problem of elution acromion caused by pH jump is not yet effectively overcome. Accordingly, there remains a need in the art to find a solution that can effectively overcome the pH jump and eliminate the shoulder.

(3) Summary of the invention

The present invention is based on the finding by the inventors that: the pH jump in the cation exchange elution step is one of the causes of protein shoulder. Thus, the present inventors have avoided such pH jumps by seeking an effective buffer system that reduces the pH jumps, thereby avoiding the occurrence of shoulder peaks during elution.

It is therefore an object of the present invention to provide a method for eliminating shoulder during purification of proteins by cation exchange chromatography. In particular, the method comprises the steps of:

a) Treating the cation exchange chromatography packing with an equilibration buffer selected from the group consisting of Tris-HCl buffer system and NaAc-HAc buffer system, at a pH ranging from 5.5 to 7.0, at a concentration of about 20mM to 200mM, preferably 50mM, and adding a protein-containing solution to the packing;

b) Eluting the cation exchange chromatography packing with a first eluting buffer, the first eluting buffer being the same as the equilibration buffer, followed by eluting the cation exchange packing with a second eluting buffer, the second eluting buffer having the same buffer system as the equilibration buffer, at a concentration of about 20mM-1.0M, preferably at a pH of equal to or greater than the pH of the equilibration buffer, more preferably 50mM Tris-HCl,100mM NaCl,pH 7.5, or increasing the buffer system concentration relative to the equilibration buffer, more preferably 50-200mM NaAc-HAc pH 5.5, more preferably 100mM NaAc-HAc pH 5.5 or 162mM NaAc-HAc pH 5.5;

c) The protein is eluted with an elution buffer having the same buffer system as the equilibration buffer, preferably having the same pH as the second elution buffer, at a concentration of about 50mM-80mM, preferably selected from the group consisting of 80mM Tris-HCl,220mM NaCl,pH 7.5, 80mM NaAc-HAc,110mM NaCl, pH 5.5, 50mM NaAc-HAc140mM NaCl pH 5.5.

In a specific embodiment of the method, the equilibration buffer and the first and second elution buffers are used for equilibration and elution in a volume of 2-7 times, preferably 3-5 times the volume of the chromatography column.

In a preferred embodiment, the cationic chromatographic filler is a sulfotype cationic filler, preferably a sulfopropyl type cationic filler, more preferably a POROS XS cation exchange chromatographic filler.

In a preferred embodiment, the protein is selected from antibodies or antibody derivatives, preferably antibody-like proteins (including monoclonal antibodies, FC fusion proteins, diabody proteins, and the like), more preferably monoclonal antibodies.

In a preferred embodiment, wherein the elution buffer and the elution buffer further comprise salt ions, preferably selected from NaCl, in a concentration range of 0-250mM.

In another embodiment of the present invention, detecting the presence or absence of an absorption peak at UV280nm is also included. Preferably the assay is selected from GE AKTA PureM.

In another embodiment of the invention, the method further comprises step d) collecting the product.

In another embodiment of the invention, the method further comprises detecting purity with SEC-HPLC and detecting residual levels of host proteins with ELISA.

The invention improves the problem of elution acromion in the cation exchange chromatography process by selecting a proper buffer system and increasing the concentration of the buffer system in the cation exchange chromatography, has simple and convenient operation, does not influence the purity and quality of the eluted product, and does not negatively influence the stability of protein and the capability of removing impurities by chromatography.

Additional features and advantages of various embodiments will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the various embodiments. The objectives and other advantages of the various embodiments will be realized and attained by means of the elements and combinations particularly pointed out in the description and appended claims.

Unless otherwise indicated, reagents and instrumentation employed in the present invention are all commonly commercially available and publicly available.

(4) Description of the drawings

FIG. 1 depicts the experimental effect chromatography (local, elution peak) of the Tris-HCl system of example 1 before use of the present invention.

FIG. 2 depicts the experimental effect chromatography (local, elution peak) of the Tris-HCl system of example 1 after use of the present invention.

FIG. 3 depicts a chromatographic profile (local, elution peak) of the NaAc-HAc system of example 2 using the pre-experimental effect of the invention.

FIG. 4 depicts an experimental effect chromatographic profile (local, elution peak) of the NaAc-HAc system of example 2 after use of the invention.

FIG. 5 depicts a chromatographic profile (local, elution peak) of the NaAc-HAc system of example 3 using the pre-experimental effect of the invention.

FIG. 6 depicts a chromatographic profile (local, elution peak) of the experimental effect of the NaAc-HAc system of example 3 after use of the invention.

(5) Detailed description of the preferred embodiments

Reference will now be made in detail to some embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents as may be included within the invention as defined by the appended claims.

Definition of the definition

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Also, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

As used herein, the term "about," "approximately" or "approximately," when preceded by a numerical value, refers to a range defined by an approximation of 1%, 2%, 3%, 4%, 5%, 10% or more around the indicated value.

In this disclosure, one or more features of one embodiment may be combined with any one or more features of another implementation without departing from the spirit and concepts of the present invention.

In this disclosure, unless otherwise specified, all ranges, including ranges defined between the two specified endpoints, are inclusive of the specified endpoints. For example, a range between 1 and 10 means a range between 1 and 10 (including 1 and 10).

Detailed description of the invention

The "target protein" or "target molecule" to be cationically purified, or "protein to be purified", "molecule to be purified", as described herein, may be used interchangeably and may include antibody derivatives, such as Fc fusion proteins, monoclonal antibodies (e.g. IgG1, igG2, igG4, igGA, etc.), bispecific antibodies, which may be derived from, for example, mammalian cell fermentation processes or blood products, which are comprised in, for example, supernatants, or serum, etc., obtained from fermentation processes. The person skilled in the art can perform conventional separation and extraction according to the steps of protein A affinity chromatography, anion exchange and the like, and load the protein A affinity chromatography and the anion exchange chromatography on a cation exchange chromatography column.

Reference herein to "shoulder" refers to the phenomenon of inflection or bimodality of the absorption curve at a280nm of the elution step during cationic chromatography.

"cation exchange chromatography" as referred to herein is chromatography that utilizes a negatively charged ion exchange matrix to bind positively charged proteins. The medium used for cation exchange chromatography may be a sulfo-type cationic filler, for example a strong cationic filler with a ligand type of sulfopropyl, and in the present invention POROS XS (Life Technology, USA) is used.

Depending on the nature of the purified protein, neutral salt ions may be added to the elution buffer and elution buffer to maintain a certain ion concentration, e.g., 0-250mM NaCl, which acts to maintain ion concentration, stabilize the protein and separate impurities, which is a means of formulating chromatography buffers well known to those skilled in the art. Other neutral salt ions, such as potassium chloride (KCl), may also be used.

Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention unless explicitly stated.

Examples

EXAMPLE 1 chromatographic Effect test Using Tris-HCl System

1. Materials and equipment:

the target protein molecule adopted in the test is monoclonal antibody IgG1 molecule with the molecular weight of 143kDa. The instrument was used as AKTA PureM (GE, USA). The cation exchange chromatography packing material is POROS XS (Life Technology, USA). The diameter of the chromatographic column is more than or equal to 0.66cm, and the height of the column is 20-24cm. The sample is a purified target monoclonal antibody protein solution subjected to affinity chromatography and anion exchange chromatography. Host cell protein residues were detected by an enzyme-linked immunosorbent assay kit (CHO HCP ELISA Kit, cygnus, usa).

2. The experimental steps are as follows:

the cation exchange chromatography step comprises: equilibration, loading, elution 1, elution 2, elution:

in the equilibration step, the column was equilibrated with 3-5 column volumes of 50mM Tris-HCl, pH 7.0 solution, with a retention time of 5 minutes.

And in the loading step, the solution containing the target protein to be purified is loaded on a chromatographic column, the retention time is 5 minutes, and the loading capacity is 20-25g/L of packing.

In the step 1, the column was rinsed with 3-5 column volumes of 50mM Tris-HCl, pH 7.0 solution for a retention time of 5 minutes.

In the step 2, the column was rinsed with 3-5 column volumes of 50mM Tris-HCl,100mM NaCl,pH 7.5 solution for a retention time of 5 minutes.

In the elution step, the target protein in the column was eluted with 3-5 column volumes of 50mM Tris-HCl,250mM NaCl,pH 7.5 (control) (FIG. 1) or 80mM Tris-HCl,220mM NaCl,pH 7.5 (experimental) (FIG. 2) solution for a retention time of 5 minutes.

Table 1 shows the specific chromatographic operating conditions in this example:

TABLE 1 cation exchange chromatography conditions (filler POROS XS, buffer system Tris-HCl)

NA: not Applicable.

3. Analysis test:

the elution step shoulder condition was determined by looking at the AKTA instrument UV280 graph (fig. 1, fig. 2). The eluted samples were tested for host cell protein (Host Cell Protein, HCP) residue for analysis of the product quality of the experimental and control groups. Host cell protein residues were detected by enzyme-linked immunosorbent assay. The results are shown in Table 2, with no significant difference in HCP content between the eluted samples of the experimental and control groups.

TABLE 2 Tris-HCl elution buffer to be tested and key element and eluent HCP residue thereof

ppm: concentration in parts per million.

As shown in the results of FIGS. 1 and 2, the concentration of Tris-HCl in the elution buffer was increased from 50mM to 80mM, effectively eliminating the shoulder of the 280nm absorption peak during the monoclonal antibody IgG1 chromatography. Increasing the concentration of the elution buffer system as described herein resulted in an increase in the ionic concentration of the solution, which was manifested as an increase in conductivity, and in order to maintain the ionic strength and conductivity of the experimental and control groups consistent, the concentration of neutral salt ions (NaCl) in the buffer of the experimental group was reduced by an amount that was dependent on the actual measured conductivity data. As shown in table 2, the elution buffer of experimental group 1 contained 250mM NaCl and the elution buffer of experimental group 2 contained 220mM NaCl, such that the pH and conductivity of the elution buffers used in experimental groups 1 and 2 were the same. Conductivity measurements used a pH meter/conductivity meter (Mettler Toledo, usa). It was demonstrated that increasing the buffer system concentration of the elution buffer had a significant elimination of pH transitions and elution shoulders in ion exchange chromatography without changing pH and conductivity.

Example 2: chromatographic effect test Using NaAc-HAc System

1. Materials and instrumentation:

the target protein molecule used in the assay was an Fc fusion protein, 22kDa. The instrument was used as AKTA PureM (GE, USA). The cation exchange chromatography packing material is POROS XS (Life Technology, USA). The diameter of the chromatographic column is more than or equal to 0.66cm, and the height of the column is 20-24cm. The sample is purified target protein solution through affinity chromatography and anion exchange chromatography.

2. The experimental steps are as follows:

in the equilibration step, the column was equilibrated with 3-5 column volumes of 50mM NaAc-HAc, pH 5.5 solution, with a retention time of 5 minutes.

In the step 1 elution, the column was eluted with 3-5 column volumes of 50mM NaAc-HAc pH 5.5 for a retention time of 5 minutes.

In the elution 2 step, the column was eluted with 3-5 column volumes of 50mM NaAc-Hac,60mM NaCl pH 5.5 (control) (FIG. 3) or 162mM NaAc-HAc pH 5.5 (experimental) (FIG. 4) for a retention time of 5 minutes.

In the elution step, the target protein in the column was eluted with 3-5 column volumes of 80mM NaAc-HAc,110mM NaCl,pH 5.5 solution with a retention time of 5 minutes.

TABLE 3 cation exchange chromatography conditions (filler POROS XS, buffer system NaAc-HAc)

NA: not Applicable.

3. Analysis test:

the elution step shoulder condition was determined by looking at the AKTA instrument UV280 graph (fig. 3, fig. 4). The eluted samples were subjected to SEC-HPLC purity detection for analysis of product quality in the experimental and control groups. The results are shown in table 4, with no significant difference in SEC-HPLC purity of the eluted samples for the experimental and control groups.

TABLE 4 Leaching buffer to be tested and key element and eluent SEC-HPLC purity

HMW: a high polymer; LMW: an oligomer.

As shown in the results of FIGS. 3 and 4 of example 2, increasing the NaAc-HAc concentration of the wash buffer from 50mM to 162mM effectively eliminates the shoulder of the 280nm absorption peak during the ion exchange chromatography of the Fc-fusion protein, and has no effect on the purity of the product. To maintain the ionic strength and conductivity of the experimental and control groups consistent, the buffer neutral salt ion (NaCl) concentration of the experimental group was reduced by an amount that depends on the actual measured conductivity data. As shown in table 2, the elution buffer of experimental group 1 contained 60mM NaCl and the elution buffer of experimental group 2 contained no NaCl, so that the pH and conductivity of the elution buffers used in experimental groups 1 and 2 were the same. Conductivity measurements used a pH meter/conductivity meter (Mettler Toledo, usa). It is proved that the increase of the buffer system concentration of the eluting buffer has a remarkable elimination effect on the pH transition and the eluting shoulder in the ion exchange chromatography without changing the pH and the conductivity.

Example 3: chromatographic effect test Using NaAc-HAc System

1. Materials and instrumentation:

the target molecule adopted in the test is a monoclonal antibody IgG1 molecule with the molecular weight of 145kDa. The instrument was used as AKTA PureM (GE, USA). The cation exchange chromatography packing material is POROS XS (Life Technology, USA). The diameter of the chromatographic column is more than or equal to 0.66cm, and the height of the column is 20-24cm. The sample is a target monoclonal antibody protein solution purified by affinity chromatography and anion exchange chromatography.

2. The experimental steps are as follows:

in the equilibration step, the column was equilibrated with 3-5 column volumes of 50mM NaAc-HAc pH 5.5 for a retention time of 5 minutes.

In the elution 2 step, the column was eluted with 3-5 column volumes of 50mM NaAc-HAc 60mM NaCl pH 5.5 (control) (FIG. 5) or 100mM NaAc-HAc pH 5.5 (experimental) (FIG. 6) for a retention time of 5 minutes.

In the elution step, the target protein in the column was eluted with 3-5 column volumes of 50mM NaAc-HAc140mM NaCl pH 5.5 solution with a retention time of 5 minutes.

TABLE 5 cation exchange chromatography conditions (filler POROS XS, buffer system NaAc-HAc)

NA: not Applicable.

3. Analysis test:

the elution step shoulder condition was determined by looking at the AKTA instrument UV280 graph (fig. 3, fig. 4). The eluted samples were subjected to SEC-HPLC purity detection for analysis of the product quality of the experimental group pre-control group. The results are shown in table 6, with no significant difference in SEC-HPLC purity of the eluted samples for the experimental and control groups. The specific chromatographic operating conditions of examples 1, 2 and 3 are shown in Table 1, table 2 and Table 3, respectively.

TABLE 6 Leaching buffer to be tested and key element and eluent SEC-HPLC purity thereof

HMW: a high polymer; LMW: an oligomer.

As shown in the results of FIGS. 5 and 6 of example 3, increasing the NaAc-HAc concentration of the wash buffer from 50mM to 100mM effectively eliminates the shoulder of the 280nm absorbance peak during IgG1 protein ion exchange chromatography with little effect on HMW/LMW removal. To maintain the ionic strength and conductivity of the experimental and control groups consistent, the buffer neutral salt ion (NaCl) concentration of the experimental group was reduced by an amount that depends on the actual measured conductivity data. As shown in table 2, the elution buffer of experimental group 1 contained 60mM NaCl and the elution buffer of experimental group 2 contained no NaCl, so that the pH and conductivity of the elution buffers used in experimental groups 1 and 2 were the same. Conductivity measurements used a pH meter/conductivity meter (Mettler Toledo, usa). It is proved that the increase of the buffer system concentration of the eluting buffer has a remarkable elimination effect on the pH transition and the eluting shoulder in the ion exchange chromatography without changing the pH and the conductivity.

The comparison of elution peak-to-peak patterns for the different experimental groups in examples 1-3 is shown in FIGS. 1-6. As can be seen from the graph comparison results, increasing the concentration of the elution buffer (fig. 1 and 2) or increasing the concentration of the elution buffer (fig. 3 and 4, fig. 5 and 6) can effectively eliminate the shoulder of the CEX elution peak. The HCP residue in example 1 is an important indicator of the purification of the target protein, and it can be seen from table 2 that increasing the elution buffer concentration does not affect the HCP removal capacity of the process. SEC-HPLC purity in examples 2 and 3 is an important indicator of purification of the target protein, and it can be seen from tables 4 and 6 that increasing the concentration of the elution buffer does not affect the process product quality.

It will be apparent to those of ordinary skill in the art that various modifications and variations can be made to the various embodiments described herein without departing from the spirit or scope of the teachings herein. Thus, it is intended that the various embodiments cover other modifications and variations of the various embodiments within the scope of the present teachings.

Claims

1. A method for eliminating shoulder peaks in purifying proteins by cation exchange chromatography, comprising the steps of:

a) Treating the cation exchange chromatography packing with an equilibration buffer selected from the group consisting of Tris-HCl buffer system and NaAc-HAc buffer system, pH in the range of 5.0-7.5, at a concentration of about 20mM-200mM, preferably 50mM, and adding a protein-containing solution to the packing;

b) Eluting the cation exchange chromatography packing with a first eluting buffer, the first eluting buffer being the same as the equilibration buffer, followed by eluting the cation exchange packing with a second eluting buffer, the second eluting buffer having the same buffer system as the equilibration buffer, at a concentration of about 20mM to 1.0M, optionally wherein the concentration of the buffer system is higher than the concentration in the equilibration buffer;

c) Eluting the protein with an elution buffer having the same buffer system as the equilibration buffer, preferably having the same pH as the second elution buffer, at a concentration of about 50mM to 80mM, preferably having a higher buffer system concentration relative to the equilibration buffer; and

d) And collecting the product.

2. The method of claim 1, wherein the second elution buffer is selected from 50mM Tris-HCl,100mM NaCl,pH 7.5, or 50-100mM NaAc-HAc pH 5.5.

3. The method of claim 1, wherein the second elution buffer is selected from 100mM NaAc-HAc pH 5.5 or 162mM NaAc-HAc pH 5.5.

4. The method of claim 1, wherein the elution buffer is selected from 80mM Tris-HCl,220mM NaCl,pH 7.5;80mM NaAc-HAc,110mM NaCl,pH 5.5; or 50mM NaAc-HAc140mM NaCl pH 5.5.

5. The method of any one of claims 1-4, wherein the buffer system is Tris-HCl.

6. The method of any one of claims 1-4, wherein the buffer system is NaAc-HCl.

7. The method of claim 1, wherein the equilibration buffer and the first and second elution buffers are 2-7 times, preferably 3-5 times the volume of the chromatography column.

8. The process according to claim 1, wherein the cationic chromatographic filler is a sulfotype cationic filler, preferably a sulfopropyl type cationic filler, preferably a POROS XS cation exchange chromatographic filler.

9. The method of claim 1, wherein the protein is selected from antibodies or antibody derivatives, preferably antibody-like proteins (including monoclonal antibodies, FC fusion proteins, diabody proteins, etc.), more preferably monoclonal antibodies.

10. The method of claim 1, wherein the elution buffer and elution buffer further contain salt ions, preferably NaCl, at a concentration in the range of 0-250mM.