CN117603359A - Purification method for purifying bispecific antibody - Google Patents

Abstract

The invention provides a purification method for purifying bispecific antibody, which comprises the following steps: s1, clarifying fermentation liquor; s2, performing affinity chromatography, and capturing a bispecific antibody by using capto L; s3, carrying out bispecific antibody fine purification by anions: removing HCP, fragments and aggregates by using anionic fillers; s4, carrying out bispecific antibody fine purification by using cations: the HCP, charge isomer, a plurality of fragments and aggregates are further removed by the cationic filler, so that the purity of the HCP, charge isomer, a plurality of fragments and aggregates is more than 95%, and a plurality of HCPs are removed. The method for purifying the bispecific antibody provided by the invention adopts light chain select chromatography to purify the bispecific antibody and combines with anion and cation purification, has simple operation, strong specificity and high load, and has better effect of removing impurity fragments, polymers, charge isomers and HCP by affinity chromatography and anion and cation exchange compared with the conventional purification method of Protein L after SEC or ion exchange or hydrophobic chromatography.

Description

Purification method for purifying bispecific antibody

Technical Field

The invention relates to the technical field of bispecific antibodies, in particular to a purification method for purifying bispecific antibodies.

Background

Immunotherapy is a new direction of current tumor treatment, and by mobilizing the immune system of the organism, the anti-tumor immunity of the tumor microenvironment is enhanced, so that tumor cells are controlled and killed, and the common tumor therapeutic antibody can only bind to a single antigen and cannot be effectively accumulated at tumor tissues, which is probably a big reason for low curative effect or low response rate.

Bispecific antibodies (bsAbs) are a class of bifunctional artificial antibody molecules capable of simultaneously specifically binding two different antigens, which can bind immune cells, viral molecules, etc. to tumor cells, enhancing the killing effect on target cells; meanwhile, different antigens can be combined on the same tumor cell to enhance the combination specificity and reduce side effects such as off-target toxicity and the like.

The bispecific antibody can erect a bridge between target cells and functional molecules (cells), excite immune response with guidance, is one of genetic engineering antibodies, becomes a hotspot in the field of antibody engineering, has wide application prospect in the immunotherapy of tumors, and has application in the fields of autoimmune diseases, infectious diseases, tissue regeneration and clinical diagnosis.

Bispecific antibodies are artificial antibodies containing 2 specific antigen binding sites, generally divided into two broad classes, one class being bispecific antibodies containing an Fc region and one class being bispecific antibodies not containing an Fc region. However, most of them have Fc segment structure, so that Protein a affinity chromatography or Protein a combined SEC or ion exchange or hydrophobic chromatography can be used for separation and purification, but for bispecific antibodies having a common IgG heavy chain and two different light chains, that is, kappa/Lambda integrated bispecific antibodies, various forms of mismatching or chain dropping can occur during expression, and then the above methods are generally used in combination, so that it is difficult to find conditions, time and effort are wasted, and it is difficult to achieve high-efficiency separation effect, and fusion proteins assembled from antigen binding regions (Fab) have poor stability and easy aggregation in cell expression, and degradation and multimerization are easy to occur.

Disclosure of Invention

The present invention is directed to a method for purifying bispecific antibodies, which solves the above-mentioned problems of the prior art.

In order to achieve the above purpose, the present invention provides the following technical solutions: a purification method for purifying bispecific antibodies, comprising the steps of:

s1, clarifying fermentation liquor;

s2, performing affinity chromatography, and capturing a bispecific antibody by using capto L;

s3, carrying out bispecific antibody fine purification by anions:

removing HCP, fragments and aggregates by using anionic fillers;

s4, carrying out bispecific antibody fine purification by using cations:

the HCP, charge isomer, a plurality of fragments and aggregates are further removed by the cationic filler, so that the purity of the HCP, charge isomer, a plurality of fragments and aggregates is more than 95%, and a plurality of HCPs are removed.

Preferably, the fermentation broth clarification may take any of three forms:

(1) balancing and centrifuging the fermentation liquor, and clarifying;

(2) adding diatomite into the fermentation liquor, filtering by a coarse filtration membrane, and clarifying;

(3) clarifying and filtering the fermentation liquor by membrane package treatment.

Preferably, the fermentation broth is clarified in the following manner:

the fermentation broth is clarified after being filtered by a coarse filtration membrane with diatomite.

Preferably, the affinity chromatography adopts a protein L filler capto L, and the specific steps of the affinity chromatography are as follows:

s2.1, balance:

balancing by using a balancing buffer solution, wherein the balancing solution is as follows: 50mM PB, pH6.5;

s2.2, loading:

loading is less than or equal to 5mg/ml, the retention time is 5min, and after loading is finished, unbound materials are removed by flushing with balance liquid;

s2.3, balance:

balancing by using a balancing buffer solution, wherein the balancing solution is as follows: 50mM PB, pH6.5;

s2.4, flushing:

washing impurities with 100mM Gly, pH 4.5;

s2.5, eluting:

eluting with eluent 1:100mM HAc-Na, pH3.0 eluent 2:100mM Gly-HCl, pH3.0, optionally one of the two eluents;

sample elution treatment: diluting with eluent to 0.5-1.5mg/ml, and filtering with 0.22 μm needle filter after adjusting pH to 6.0-8.0.

Preferably, the anion filler for performing bispecific antibody fine purification is selected from one of capto Q and capto sphere; the specific steps of the anion for carrying out the fine purification of the bispecific antibody are as follows:

s3.1, balance:

balancing by using a balancing buffer solution, wherein the balancing solution is as follows: 20mM Tris, pH8.0;

s3.2, loading:

the PH of the sample is regulated to 8.0, the sample is loaded, the loading capacity is less than or equal to 5mg/ml, the retention time is 5min, the sample is not combined with the filler after passing through a chromatographic column at the beginning of loading, and the flow through sample is collected;

s3.3, balance:

balancing by using a balancing buffer solution, wherein the balancing solution is as follows: 20mM Tris, pH8.0.

Preferably, the filler for performing the fine purification of the bispecific antibody by the cation is selected from one of POROS 50HS, nanoGel-50SP and capto S im pact, and the specific steps for performing the fine purification of the bispecific antibody by the cation are as follows:

s4.1, balance:

balancing by using a balancing buffer solution, wherein the balancing solution is as follows: balancing solution: 20mM Citric-Na, pH6.0-6.5;

s4.2, loading:

loading the sample with the loading capacity of less than or equal to 5mg/ml, adjusting the pH value of the sample to 6.5, loading the sample, keeping the sample for 5min, and flushing the sample with a balancing solution to remove unbound materials after loading the sample;

s4.3, eluting:

eluent: 20mM Citric-Na, 0.5M NaCl, pH6.0-6.5;

the current elution mode is linear elution range: 0-250mmol NaCl,PH6.0-6.5;

the sample has a majority of polymer removed and fragment SEC purity > 99%, charge main peak purity > 90% and HCP < 5ppm.

The invention has at least the following beneficial effects:

the method for purifying the bispecific antibody provided by the invention adopts light chain select chromatography to purify the bispecific antibody and combines with anion and cation purification, has simple operation, strong specificity and high load, and has better effect of removing impurity fragments, polymers, charge isomers and HCP by affinity chromatography and anion and cation exchange compared with the conventional purification method of Protein L after SEC or ion exchange or hydrophobic chromatography.

Drawings

FIG. 1 is a chromatographic chart of eluent 1 elution;

FIG. 2 is a chromatographic chart of eluent 2 elution;

FIG. 3 is a chromatogram of filler POROS 50 HS;

FIG. 4 is a chromatogram of the packing NanoGel-50 SP;

FIG. 5 is a chromatogram of a filler capto S im;

FIG. 6 is a chromatographic chart of column 1 for the fine purification of bispecific antibodies by cations;

FIG. 7 is a chromatographic chart of column 2 for the fine purification of bispecific antibodies by cations.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Examples

A purification method for purifying bispecific antibodies, comprising the steps of:

s1, clarifying fermentation liquor:

the fermentation broth may be clarified in any of three ways:

(1) clarifying the fermentation liquor after balancing and centrifuging:

balancing the fermentation liquor (7245 g), centrifuging (0.5 h, room temperature), and filtering with 0.22um membrane after centrifuging;

(2) and adding diatomite into the fermentation liquor, filtering by a coarse filtration membrane, and clarifying:

adding diatomite (the diatomite content is 2-4%, more preferably 3%) into the fermentation broth, filtering with a coarse filtration membrane, and filtering with a 0.22um membrane;

(3) clarifying and filtering the fermentation liquor through membrane package treatment:

treating the fermentation liquor with a first membrane bag (4.0-18 um, material: cellulose, diatomite and resin), treating with a second membrane bag (0.02-0.2 um, material: cellulose, diatomite and resin), and clarifying and filtering;

the three better choices are clarification by adding diatomite;

s2, affinity chromatography, capturing a bispecific antibody by capto L:

the affinity chromatography adopts protein L filler capto L, and the specific steps of the affinity chromatography are as follows:

s2.1, balance:

balancing by using a balancing buffer solution, wherein the balancing solution is as follows: 50mM PB, pH6.5;

s2.2, loading:

loading is less than or equal to 5mg/ml, the retention time is 5min, and after loading is finished, unbound materials are removed by flushing with balance liquid;

s2.3, balance:

balancing by using a balancing buffer solution, wherein the balancing solution is as follows: 50mM PB, pH6.5;

s2.4, flushing:

washing impurities with 100mM Gly, pH 4.5;

s2.5, eluting:

eluting with eluent 1:100mM HAc-Na, pH3.0 eluent 2:100mM Gly-HCl, pH3.0, optionally one of the two eluents;

sample elution treatment: diluting to 0.5-1.5mg/ml with eluent, and filtering with a 0.22 μm needle filter after adjusting pH to 6.0-8.0 to obtain a filtered sample, detecting SEC and electric charge, and obtaining the result:

the chromatographic chart eluted with eluent 1 is shown in figure 1;

the chromatographic chart eluted with eluent 2 is shown in figure 2;

eluting the obtained filtered sample with eluent 1, and performing SEC > 60%; the purity of the charge is more than 60 percent;

eluting the obtained filtered sample with eluent 2, wherein SEC is more than 70%; the purity of the charge is more than 65%;

both eluents are possible, and eluent 2 is more preferred;

s3, carrying out bispecific antibody fine purification by anions:

removing HCP, fragments and aggregates by using an anionic filler, wherein the filler for performing bispecific antibody fine purification by using anions is selected from one of capto Q and capto sphere; the specific steps of anion for the fine purification of bispecific antibodies are as follows:

s3.1, balance:

balancing by using a balancing buffer solution, wherein the balancing solution is as follows: 20mM Tris, pH8.0;

s3.2, loading:

the PH of the sample is regulated to 8.0, the sample is loaded, the loading capacity is less than or equal to 5mg/ml, the retention time is 5min, the sample is not combined with the filler after passing through a chromatographic column at the beginning of loading, and the flow through sample is collected;

s3.3, balance:

balancing by using a balancing buffer solution, wherein the balancing solution is as follows: 20mM Tris, pH8.0;

s4, carrying out bispecific antibody fine purification by using cations:

the HCP, the charge isomer, a large number of fragments and aggregates are further removed by using a cationic filler, so that the purity of the HCP is more than 95%, a large number of HCP is removed, the filler for performing the fine purification of the bispecific antibody by the cation is selected from one of POROS 50HS, nanogel-50SP and capto S image, and the specific steps for performing the fine purification of the bispecific antibody by the cation are as follows:

s4.1, balance:

balancing by using a balancing buffer solution, wherein the balancing solution is as follows: balancing solution: 20mM Citric-Na, pH6.0-6.5;

s4.2, loading:

loading the sample with the loading capacity of less than or equal to 5mg/ml, adjusting the pH value of the sample to 6.5, loading the sample, keeping the sample for 5min, and flushing the sample with a balancing solution to remove unbound materials after loading the sample;

s4.3, eluting:

eluent: 20mM Citric-Na, 0.5M NaCl, pH6.0-6.5;

the current elution mode is linear elution range: 0-250mmol NaCl,PH6.0-6.5; preferred elution ranges are:

20mM Citric-Na+67.5mMol-85mMol NaCl，PH6.0；

the sample has a majority of polymer removed and fragment SEC purity > 99%, charge main peak purity > 90% and HCP < 5ppm.

Results:

filler POROS 50HS chromatography: FIG. 3;

packing NanoGel-50SP chromatography: FIG. 4;

packing capto S image chromatograph: FIG. 5;

the result is that the yield of the filler capto S imact is maximum, and the result of SEC and charge is basically consistent with the three types of fillers, preferably the filler capto S imact.

The results of column 1 are shown in the figure: FIG. 6;

the results of column 2 are shown in the figure: FIG. 7;

as a result, the yield, SEC, charge and HCP were all within acceptable ranges, and the results of column 2 were slightly better.

While the fundamental and principal features of the invention and advantages of the invention have been shown and described, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (6)

1. A purification method for purifying a bispecific antibody, comprising the steps of:

s1, clarifying fermentation liquor;

s2, performing affinity chromatography, and capturing a bispecific antibody by using capto L;

s3, carrying out bispecific antibody fine purification by anions:

removing HCP, fragments and aggregates by using anionic fillers;

s4, carrying out bispecific antibody fine purification by using cations:

the HCP, charge isomer, a plurality of fragments and aggregates are further removed by the cationic filler, so that the purity of the HCP, charge isomer, a plurality of fragments and aggregates is more than 95%, and a plurality of HCPs are removed.

2. The method of claim 1, wherein the fermentation broth is clarified in any of three ways:

(1) balancing and centrifuging the fermentation liquor, and clarifying;

(2) adding diatomite into the fermentation liquor, filtering by a coarse filtration membrane, and clarifying;

(3) clarifying and filtering the fermentation liquor by membrane package treatment.

3. The method of claim 2, wherein the fermentation broth is clarified in the following manner:

the fermentation broth is clarified after being filtered by a coarse filtration membrane with diatomite.

4. The method for purifying bispecific antibody according to claim 1, wherein the affinity chromatography uses protein L as filler capto L, and the specific steps of the affinity chromatography are as follows:

s2.1, balance:

balancing by using a balancing buffer solution, wherein the balancing solution is as follows: 50mM PB, pH6.5;

s2.2, loading:

loading is less than or equal to 5mg/ml, the retention time is 5min, and after loading is finished, unbound materials are removed by flushing with balance liquid;

s2.3, balance:

balancing by using a balancing buffer solution, wherein the balancing solution is as follows: 50mM PB, pH6.5;

s2.4, flushing:

washing impurities with 100mM Gly, pH 4.5;

s2.5, eluting:

eluting with eluent 1:100mM HAc-Na, pH3.0 eluent 2:100mM Gly-HCl, pH3.0, optionally one of the two eluents;

sample elution treatment: diluting with eluent to 0.5-1.5mg/ml, and filtering with 0.22 μm needle filter after adjusting pH to 6.0-8.0.

5. The method for purifying a bispecific antibody according to claim 1, wherein the filler for fine purification of the bispecific antibody by the anion is selected from one of capto Q, capto sphere; the specific steps of the anion for carrying out the fine purification of the bispecific antibody are as follows:

s3.1, balance:

balancing by using a balancing buffer solution, wherein the balancing solution is as follows: 20mM Tris, pH8.0;

s3.2, loading:

the PH of the sample is regulated to 8.0, the sample is loaded, the loading capacity is less than or equal to 5mg/ml, the retention time is 5min, the sample is not combined with the filler after passing through a chromatographic column at the beginning of loading, and the flow through sample is collected;

s3.3, balance:

balancing by using a balancing buffer solution, wherein the balancing solution is as follows: 20mM Tris, pH8.0.

6. The method for purifying a bispecific antibody according to claim 1, wherein the filler for performing the fine purification of the bispecific antibody by the cation is selected from one of POROS 50HS, nanoGel-50SP, capto S image, and the specific steps for performing the fine purification of the bispecific antibody by the cation are as follows:

s4.1, balance:

balancing by using a balancing buffer solution, wherein the balancing solution is as follows: balancing solution: 20mM Citric-Na, pH6.0-6.5;

s4.2, loading:

loading the sample with the loading capacity of less than or equal to 5mg/ml, adjusting the pH value of the sample to 6.5, loading the sample, keeping the sample for 5min, and flushing the sample with a balancing solution to remove unbound materials after loading the sample;

s4.3, eluting:

eluent: 20mM Citric-Na, 0.5M NaCl, pH6.0-6.5;

the current elution mode is linear elution range: 0-250mmol NaCl,PH6.0-6.5;

the sample has a majority of polymer removed and fragment SEC purity > 99%, charge main peak purity > 90% and HCP < 5ppm.