CN105017418B

CN105017418B - Monoclonal antibody purification process

Info

Publication number: CN105017418B
Application number: CN201410120032.5A
Authority: CN
Inventors: 朱健; 万艳; 孙旭东; 陈广勇; 鲁钱达; 瞿丽莉; 谢晋; 胡可可; 高金湖; 沈克强; 周伟昌; 陈智胜
Original assignee: Wuxi Yaoming Biotechnology Co ltd; Wuxi Apptec Co Ltd; Wuxi Biologics Shanghai Co Ltd
Current assignee: Shanghai Yaoming Biomedical Co.,Ltd.
Priority date: 2014-03-27
Filing date: 2014-03-27
Publication date: 2021-02-23
Anticipated expiration: 2034-03-27
Also published as: CN105017418A

Abstract

The invention discloses a monoclonal antibody purification process method, which comprises the following steps: 1) affinity chromatography; 2) adjusting the pH value of the affinity chromatography eluent to 3.3-3.8, and inactivating viruses; 3) adjusting pH to neutral, and deep filtering; 4) anion exchange chromatography; 5) cation exchange chromatography. According to the invention, a washing step of low pH and high salt is added in the affinity chromatography to remove host protein and DNA, and meanwhile, deep filtration is adopted for clarification treatment after low pH virus inactivation, so that the separation effect of the affinity chromatography on antibody monomers and high polymers is improved, and the purity of the monoclonal antibody product is improved.

Description

Monoclonal antibody purification process

Technical Field

The invention relates to the field of biology, in particular to a purification process method of a monoclonal antibody.

Background

In 1997, the first humanized monoclonal antibody drug was successfully developed by the gene tack in the United states, which led to the first step of the industrialization of monoclonal antibody drugs. The antibody drug presents an inconsistent development situation in the history of more than 10 years, and several figures can indicate the status and development prospect of the antibody drug: the total sales of globally used monoclonal antibody drugs in 2010 reaches $ 440 billion, and if 100 billion of monoclonal antibody diagnostic and research reagents are added, the total market amount of monoclonal antibody drugs reaches $ 550 billion, and the total market amount of monoclonal antibody drugs reaches $ 628 billion in 2011. And in the last 2009 and 2008 the numbers were 400 and 370 billion dollars, respectively (data from the national bureau of statistics). Currently, nearly all large pharmaceutical companies have monoclonal antibody development programs.

Although the increasing of the world economic glide risk trend influences the growth of the monoclonal antibody medicines, the rise of the monoclonal antibody medicines is not blocked, and with the continuous deepening of the research of the monoclonal antibody medicines and the continuous release of new medicines, the global monoclonal antibody medicines still keep a higher growth rate in the future. Analysis predicts that by 2015, global monoclonal antibody drug sales are expected to reach around 980 billions of dollars. However, the single batch yield of the antibody drug reaches the kilogram level at most, and the single dose of the antibody drug is microgram and milligram. The value of antibody drugs is far superior to that of gold. Such expensive products, the manufacturing process is of great importance and any imperfections in the process can cause significant losses.

The manufacture of antibody drugs is most complicated and tedious in the downstream process. The downstream process comprises a plurality of steps, and liquid chromatography separation methods of affinity chromatography, cation exchange chromatography and anion exchange chromatography are currently commonly used. Although these several chromatographic methods are used, the specific parameters and the choice of the filler will produce completely different results.

Affinity chromatography is the first step of chromatographic separation and generally functions to capture the product in the fermentation supernatant. Based on the principle of specific washing-free adsorption, the product after affinity chromatography can generally reach the monomer purity of more than 80 percent, and simultaneously can also ensure higher recovery rate. However, antibody drugs need not only ensure monomer purity, but also minimize the residues of host proteins and DNA. Most antibody product purification processes rely on the removal of host proteins (HCP) and DNA in subsequent steps, but from an absolute removal perspective, affinity chromatography is the most efficient step for removing host proteins and DNA, so removing as much host proteins and DNA as possible in this step greatly reduces the stress on subsequent steps.

The elution pool from affinity chromatography is typically turbid when neutralized with alkaline buffer after viral inactivation at low pH, which is typically caused by co-precipitation of small amounts of residual HCP and DNA at pH 4-6. If the impurity is removed, the recovery rate can be slightly reduced, but more HCPs and DNAs can be removed, and the load of a subsequent virus removal filter membrane can be increased, so that the cost is reduced.

Cation exchange chromatography is mainly used for removing high polymers, and can further remove host proteins and DNA. The conventional method is to use high-salt elution after low-salt sample loading, and select proper salt concentration to elute the target protein, thereby achieving the separation effect. However, the conventional elution mode has lower resolution, can only slightly improve the purity of the target protein, and has poor separation effect on the acid-base analogues with smaller charge difference with the target protein.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a monoclonal antibody purification process method, which can improve the purity of a monoclonal antibody product.

In order to solve the technical problems, the monoclonal antibody purification process method provided by the invention comprises the following steps:

1) affinity chromatography;

2) adjusting the pH value of the affinity chromatography eluent to 3.3-3.8, and inactivating viruses;

3) adjusting pH to neutral, and deep filtering;

4) anion exchange chromatography;

5) cation exchange chromatography.

Preferably, after the sample is loaded and before elution, the affinity chromatography in the step 1) is carried out, and a chromatographic column is washed by a high-concentration salt solution with the pH value of 5.0-5.5 and the concentration of 1-3M. For example, the column may be washed with a mixed solution of 20mM citric acid-sodium citrate (or acetic acid-sodium acetate) and 1M sodium chloride, and then washed with 20mM citric acid-sodium citrate (or acetic acid-sodium acetate). Wherein, the sodium chloride can also be replaced by potassium chloride or potassium thiocyanate.

In the step 3), a secondary deep filter with the pore diameter of 0.1-0.5 mu m can be adopted for deep filtration.

Preferably, the cation exchange chromatography in step 5) may be performed by washing the cation exchange chromatography column with a buffer solution with a high pH value (pH value 6.0-9.0) before elution, and then changing to a balance solution with a low pH value before the peak of the target protein comes out.

Compared with the existing purification method, the monoclonal antibody purification process method has the following advantages and beneficial effects:

1. by adding a washing step in affinity chromatography, impurities such as high polymers, fragments, HCP, DNA and the like are effectively removed, the purity of the antibody is improved, and multiple dilutions and pH adjustment are avoided.

2. After the elution liquid of the affinity chromatography is inactivated and neutralized at low pH, a deep filtration step is added, so that host protein, DNA and high polymer are further removed, and the purity of the antibody is improved. The product after the filtration and clarification treatment has higher carrying capacity during the later nanofiltration virus removal and is not easy to block. And the product which is not subjected to deep filtration is easy to block the membrane by late nanofiltration, and is difficult to pass a virus removal verification experiment.

3. The acidic and alkaline impurities are removed by adding a high pH salt washing step in cation exchange chromatography, so that the purity of the CEX main peak of the product is improved by at least 5 percent.

Detailed Description

For a more detailed understanding of the technical content, the characteristics and the functions of the present invention, the technical solutions of the present invention will now be described in detail with reference to specific embodiments. The following detection methods are adopted for various indexes involved in the embodiment: SEC (monomer purity) is detected by using a molecular exclusion high performance liquid chromatography, CEX (charge purity) is detected by using a cation exchange high performance liquid chromatography, residual HCP is detected by using an enzyme-linked immunosorbent assay, residual DNA is detected by using a quantitative polymerase chain reaction, and residual ProA (protein A) is detected by using an enzyme-linked immunosorbent assay.

Example 1 purification of Herceptin monoclonal antibody

1. Protein A affinity chromatography

Step 1, eluting the chromatographic column with 3 times of column volume of water for injection, then disinfecting the chromatographic column with 3 times of column volume of mixed solution of NaOH (50 mM) and NaCl (1M), and eluting the chromatographic column with 3 times of column volume of water for injection.

Step 2, the column was equilibrated with 5 column volumes of buffer (20 mM disodium hydrogenphosphate-sodium dihydrogenphosphate +150mM NaCl, pH 7.4).

Step 3, load and allow the sample to remain on the column for 4 minutes.

Step 4, the column was rinsed to a280 base line with 20mM disodium hydrogen phosphate-sodium dihydrogen phosphate +150mM NaCl, ph7.4, then washed with 5 column volumes of buffer (20 mM citric acid-sodium citrate +1M NaCl, ph 5.5), and then washed with 3 column volumes of buffer (20 mM citric acid-sodium citrate, ph 5.5).

By adding two buffer wash steps in step 4, HCP residue in the affinity chromatography eluate is greatly reduced. The residual amount of HCP in the eluate from affinity chromatography is generally 10 before the washing step is not added³ppm level, HCP residual reduction by 60% to 80% with additional washing steps, specific data as shown in table 1:

TABLE 1 residual amount of HCP after addition of washing step in affinity chromatography

As can be seen from Table 1, the residual amount of HCP significantly decreased from 8374ppm to 2520ppm after addition of appropriate washing steps to affinity chromatography.

Step 5, the target protein was completely eluted with a buffer (20 mM citric acid-sodium citrate, pH 3.6).

And 6, regenerating the chromatographic column by using 1M acetic acid with 3 times of column volume.

2. Low pH inactivation followed by depth filtration

The affinity chromatography eluent is adjusted to pH 3.3-3.8 by 1M acetic acid for virus inactivation, then adjusted to neutrality by 1M Tris, and then subjected to deep filtration by a Millipore Millistat Filter A1HC (Millipore Millistat + pod Filter A1 HC) to remove a large amount of HCP. HCP and DNA residual amounts and SEC purity are shown in table 2:

TABLE 2 HCP and DNA residual amount and SEC purity of affinity chromatography eluate after depth filtration

As can be seen from Table 2, after the affinity chromatography eluent is subjected to one-step deep filtration, the residual amount of HCP is obviously reduced from 737ppm to 303ppm or less, and the DNA residue also completely meets the FDA requirement.

3. Anion exchange chromatography

The filtrate A1HC after depth filtration was passed directly through an anion exchange chromatography column with primary amino functions and the flow-through was collected.

4. Cation exchange chromatography

Step 1, eluting the chromatographic column with the sulfonic functional group by using 3 times of column volume of water for injection, then disinfecting the chromatographic column by using 3 times of column volume of 1M NaOH, and eluting the chromatographic column by using 3 times of column volume of water for injection.

Step 2, the column was equilibrated with 5 column volumes of buffer (20 mM citric acid-sodium citrate, pH 5.5).

And 3, adjusting the pH value of the anion flow-through solution containing the target protein to 5.5, loading the sample, and allowing the sample to remain on the chromatographic column for 6 minutes.

Step 4, the column was rinsed to a280 baseline with 20mM citric acid-sodium citrate solution ph5.5, then washed with 1 column volume of buffer (20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, ph 7.3), and immediately washed with 3 column volumes of 20mM citric acid-sodium citrate solution ph 5.5.

Through adding the buffer solution washing step, wash the acidic isomer with lower pI with the short-lived high pH buffer solution first, change into the equilibrium liquid of low pH value immediately before the protein peak of interest comes out to under the prerequisite of guaranteeing the rate of recovery the purpose of separating acidic impurity has been reached, specific result is seen in table 3:

TABLE 3 purity of monomers after high pH wash before cation exchange chromatography elution

As can be seen from Table 3, the addition of a high pH wash step prior to cation exchange chromatography elution removed most of the acidic impurities, increased CEX, and also increased monomer purity.

Step 5, eluting the target protein by using a mixed solution of 20mM citric acid-sodium citrate and 130mM sodium chloride as an eluent (pH 5.5) until a protein peak is completely discharged;

and 6, regenerating the chromatographic column by using 1M sodium chloride.

Example 2 purification of a Xiametalle monoclonal antibody

The purification process was the same as in example 1, and the HCP and DNA residual amounts and SEC purity of the affinity chromatography eluate after one-step depth filtration are shown in table 4:

TABLE 4 HCP and DNA residual amount after deep filtration of affinity chromatography eluent and SEC purity

As can be seen from Table 4, after the affinity chromatography eluent is subjected to one-step deep filtration, the residual amount of HCP is obviously reduced from 226ppm to 86ppm, and the DNA residue also completely meets the FDA requirement.

See table 5 for purity and residue index of final product:

TABLE 5 purity and residue index of the product prepared by purification according to the invention

As can be seen from Table 5, the purity and residue index of the Xumeele monoclonal antibody product purified and prepared by the process of the invention all meet the requirements of sFDA.

Example 3 rituximab purification

The purification process was the same as in example 1, and the HCP and DNA residual amounts and SEC purity of the affinity chromatography eluate after one-step depth filtration are shown in table 6:

TABLE 6 HCP and DNA residual amount after deep filtration of affinity chromatography eluent and SEC purity

As can be seen from Table 6, after the affinity chromatography eluate was further subjected to one-step depth filtration, the residual amount of HCP was significantly reduced from 794ppm to 127ppm, and the DNA residue also completely met the FDA requirement.

See table 7 for purity and residue index of final product:

TABLE 7 purity and residue index of the product prepared by purification according to the invention

As can be seen from Table 7, the rituximab products purified and prepared by the process of the present invention all have purity and residue indexes meeting the requirements of sFDA.

Example 4 purification of rituximab

The purification process was the same as in example 1, and the purity and residue index of the final product are shown in Table 8:

TABLE 8 purity and residue index of the product prepared by purification according to the invention

As can be seen from Table 8, the purity and residue index of the rituximab product purified and prepared by the process of the present invention also meet the requirements of sFDA.

Claims

1. The monoclonal antibody purification process is characterized by comprising the following steps:

1) affinity chromatography; after sample loading and before elution, affinity chromatography firstly uses a salt solution with the pH value of 5.0-5.5 and the concentration of 1-3M to wash a chromatographic column once, wherein the salt is selected from sodium chloride, potassium chloride and potassium thiocyanate, and then the chromatographic column is washed once by using a buffer solution which is different from the salt solution and only does not contain the salt;

3) adjusting the pH value to be neutral, and performing deep filtration by using a secondary deep filter with the pore diameter of 0.1-0.5 mu m;

4) anion exchange chromatography;

5) cation exchange chromatography; before elution, cation exchange chromatography is carried out, a buffer solution with the pH value of 6.0-9.0 is used for washing a cation exchange chromatography column, and a balance solution is replaced before a target protein peak appears;

the monoclonal antibody is a herceptin monoclonal antibody, a trumetile monoclonal antibody, a rituximab monoclonal antibody or a rituximab monoclonal antibody.

2. The method of claim 1, wherein in step 1), the affinity chromatography is performed by washing the column with a mixed solution of 20mM citric acid-sodium citrate and 1M sodium chloride, and then washing the column with 20mM citric acid-sodium citrate after loading and before elution.

3. The method according to claim 1, wherein the buffer of step 5) is a 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate solution at pH 7.3.

4. The method according to claim 1, wherein in step 1), affinity chromatography is performed by washing the column with a mixed solution of 20mM acetic acid-sodium acetate and 1M sodium chloride and then washing the column with 20mM acetic acid-sodium acetate after loading and before elution.