Method for removing trispecific antibody polymer

Abstract

The invention discloses a method for removing a trispecific antibody polymer, which comprises the following steps: firstly, filling a chromatographic column by using Monomix Mab60 SP; secondly, balancing the chromatographic column by using a citric acid buffer solution; thirdly, adding the loading solution containing the three specific antibodies into a chromatographic column; washing impurities by using a phosphate buffer solution and balancing the chromatographic column again by using a balancing solution; and finally, carrying out gradient elution by using the eluent, collecting a chromatographic signal, and collecting effluent by using a branch pipe. Based on a chromatographic filler Monomix Mab60 SP, the content of polymer impurities in the three-specific antibody can be reduced to 1.042% from 42.397% through one-step chromatography, and the method is efficient, simple and convenient and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to a method for removing specific antibody aggregates, in particular to a method for removing trispecific antibody aggregates, and belongs to the field of protein purification. Background Art

[0002] The proportion of macromolecular biological drugs in anti-tumor drugs has increased year by year, especially antibody-based drugs have developed rapidly and have become one of the most important types of drugs. Monoclonal antibody drugs have shown excellent therapeutic effects, and bispecific antibodies can improve efficacy and safety by simultaneously recognizing and binding to two different antigens or antigenic epitopes. Trispecific antibodies, referred to as tri-antibodies, add a specific antigen binding site on the basis of bi-antibodies, which is beneficial for redirecting drugs to the local tumor, increasing binding specificity, improving the accuracy of drug targeting tumor cells, enhancing the killing of target cells, and reducing off-target toxicity. Preclinical data suggest that tri-antibodies have the potential to be superior to bi-antibodies or bi-antibody combination therapy.

[0003] The inherent heterogeneity of antibody drugs (such as charge heterogeneity and size heterogeneity), various post-translational modifications, and quality changes that may occur during the production process, purification and storage (such as the increase of impurities or other contaminants) may have a significant impact on product safety and effectiveness. Invention patent application CN118459603A discloses a purification method for removing homodimers in bispecific antibodies. The supernatant culture fluid expressing the bispecific antibody is subjected to mixed mode chromatography and anion exchange chromatography in sequence to obtain a purified bispecific antibody, removing mismatch products in the assembly process of the bispecific antibody molecule and homodimers that are highly similar to the bispecific antibody molecule. The preparation of tertiary antibodies also faces the problem of removing polymer impurities. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for effectively removing trispecific antibody aggregates.

[0005] Technical solution: The present invention provides a method for removing trispecific antibody aggregates, comprising the following steps: Step 1: Column filling: Take the chromatographic filler Monomix Mab60 SP and fill the chromatographic column; Step 2: Equilibration; 20-30 mM Na 3 Cit-H 3 Cit, 40-60 mM NaCl, pH 4.5-5.5 as a balancing solution to balance the chromatographic column; Step 3: Loading: Adding the loading solution containing the trispecific antibodies to the chromatographic column; Step 4: Washing: Use 20-30 mM PB, 70-90 mM NaCl, pH 6.0-7.0 as the washing solution for washing; Step 5: Post-equilibrium: equilibrate the chromatographic column again with the equilibration solution described in step 2; Step 6: Elution; 20-30 mM Na 3 Cit-H 3 Cit, 500-550 mM NaCl, pH 4.5-5.5 as the eluent, use the balance solution in step 2 and the eluent for gradient elution, the gradient is set to 20%-55% of the eluent, collect the chromatographic signal, and collect the effluent in separate tubes.

[0006] The chromatographic filler Monomix Mab60 SP is modified with sulfonic acid groups and is a strong cationic medium with good hydrophilicity. It is suitable for the separation and purification of biological samples such as antibodies, vaccines, insulin, proteins, nucleic acids, and heparin. After loading, the target three antibodies, fragments, and polymers are all bound to the filler. Due to their different charge distribution and larger molecular size, polymers may form a stronger bond with the resin. Therefore, polymers require a stronger ion concentration to be washed out. During elution, increasing the pH will reduce the binding force between the fragment and the resin, so the fragment is washed out; the role of post-equilibrium is to lower the pH environment of the chromatography column to facilitate subsequent elution; during elution, the target is washed out; finally, the filler is cleaned by high salt and strong alkali washing. Based on Monomix Mab60 SP, the present invention effectively removes polymer impurities generated during the preparation of trispecific antibodies through one-step chromatography.

[0007] Furthermore, the sample solution was filtered through a 0.22 μm filter membrane before being added to the chromatographic column.

[0008] Preferably, in step 1, the plate number of the chromatographic column is not less than 2000 N / m, and the symmetry is 0.8-1.5.

[0009] After the chromatographic filler is loaded into the chromatographic column, the column efficiency measurement is helpful to judge the performance of the chromatographic column and ensure the effect of separation and purification. The number of plates and symmetry are commonly used indicators to measure column efficiency.

[0010] Preferably, in step 3, the protein concentration in the loading solution is 3-6 mg / mL, pH 4.5-5.5, and conductivity 0-5 mS / cm.

[0011] Detecting the protein concentration, pH and conductivity in the sample to be tested is beneficial to adjusting the sample loading amount and ensuring the effect of separation and purification.

[0012] Preferably, in step 6, the chromatographic signal is UV280, and the peak cutoff range is 50mAU-50mAU.

[0013] Preferably, in step 6, the branch tubes are used to collect the effluent of every 0.2-3 column volumes into one tube.

[0014] Preferably, in step 6, the gradient elution volume is 25-30 column volumes.

[0015] Preferably, in step 2, the amount of the equilibration solution is 5-8 column volumes.

[0016] Preferably, in step 4, the amount of the eluent used is 5-8 column volumes.

[0017] Preferably, in step 5, the amount of the equilibration solution is 5-8 column volumes.

[0018] Preferably, after the elution is completed, the chromatographic column is cleaned with the eluent or alkaline solution.

[0019] The alkaline solution may be 0.5-1 M NaOH. The cleaning includes cleaning in place (CIP).

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: Based on the chromatographic filler Monomix Mab60 SP, the present invention reduces the polymer impurity content in the trispecific antibody from 42.397% to 1.042% through one-step chromatography. The method is efficient and simple and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a chromatogram of the purified trispecific antibody in Example 1 of the present invention; Figure 2 This is a chromatogram of the purified trispecific antibody in Comparative Example 1 of the present invention; Figure 3 This is the SEC graph of the original effluent of Example 1 of the present invention; Figure 4 SEC diagram of the eluted effluent of Example 1 of the present invention; Figure 5 This is the SEC graph of the original effluent of Comparative Example 1 of the present invention; Figure 6 This is the SEC graph of the effluent of Comparative Example 1 of the present invention when the elution gradient is 37%. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0023] The fillers used in the examples and comparative examples are all from Suzhou Saifen Technology Co., Ltd.

[0024] Example 1 A method for removing trispecific antibody aggregates This embodiment provides a method for removing trispecific antibody aggregates, comprising the following steps: Step 1: Column loading; Take 5 mL of filler (Monomix Mab60 SP) and wash with purified water, drain the water to obtain sedimentation gel, weigh 3.7 g of sedimentation gel and add an equal amount of 0.1 M sodium chloride solution, resuspend it and add it to a column tube with specifications of 6.6*100 mm with a disposable dropper, let it stand for 1 hour and press into the upper plug. The column volume is 3.421 mL. After the column is loaded, measure the column efficiency. The column efficiency requires the number of plates to be above 2000 N / m and the symmetry to be between 0.8-1.5.

[0025] Step 2: Equilibration; use equilibration solution (20 mM Na 3 Cit-H 3 Cit, 50 mM NaCl, pH 5.0) for 5 CV at a flow rate of 0.684 mL / min.

[0026] Step 3: Sample loading; the sample is a human GPRCSD-BCMA-CD3 trispecific antibody sample (eukaryotic expression, isoelectric point 8.4, the main impurities are mismatches, aggregates and fragments, the impurity content is relatively high, and the SEC purity is only 55-57%). The sample concentration is 4.07 mg / mL, pH 5.0, and conductivity 2.27 mS / cm. The sample solution needs to be filtered with a 0.22 μm filter membrane before loading. The loading volume is 25 mL, the loading capacity is 29.74 g / L, and the flow rate is 0.684 mL / min.

[0027] Step 4: Elution: Use eluent (20 mM PB, 80 mM NaCl, pH 6.5) to elute for 5CV at a flow rate of 0.684 mL / min. The collected signal is UV280 with a cutoff range of 50 mAU-50 mAU. Collect in separate tubes, 1 mL per tube.

[0028] Step 5: Post-equilibrium; use the equilibration solution to equilibrate again for 5CV at a flow rate of 0.684 mL / min; collect chromatographic signals using the same method as step 4, and collect the effluent in separate tubes.

[0029] Step 6: elution; use the equilibration solution and elution solution (20 mM Na 3 Cit-H 3 Cit, 500 mM NaCl, pH 5.0), the linearity was 20%-55% eluent, the linearity was 25CV, and the flow rate was 0.684 mL / min; the chromatographic signal was collected by the same method as step 4, and the effluent was collected in separate tubes.

[0030] Step 7: Cleaning; Use the eluent to clean the chromatography column for 5CV at a flow rate of 0.684 mL / min; Use the same method as step 4 to collect chromatographic signals, and collect the effluent in separate tubes.

[0031] Step 8: Cleaning in place: The column was cleaned in place (CIP) using a cleaning solution (0.5 M NaOH) for 5 CV at a flow rate of 0.684 mL / min.

[0032] The chromatograms collected in steps 4-7 of this embodiment are Figure 1 .

[0033] Example 2 A method for removing trispecific antibody aggregates Based on the chromatographic column and sample of Example 1, this example provides a method for removing trispecific antibody aggregates, comprising the following steps: Step 1: Equilibration; use equilibration solution (30 mM Na 3 Cit-H 3 Cit, 60 mM NaCl, pH 5.5) for 8 CV at a flow rate of 0.6 mL / min.

[0034] Step 2: Sample loading: Before loading, the sample solution needs to be filtered with a 0.22 μm filter membrane. The loading volume is 25 mL and the flow rate is 0.6 mL / min.

[0035] Step 3: Elution: Use eluent (30 mM PB, 90 mM NaCl, pH 6.5) to elute for 8CV at a flow rate of 0.6 mL / min; collect chromatographic signals using the method of Example 1, and collect the effluent in separate tubes.

[0036] Step 4: Post-equilibrium; use the equilibration solution to equilibrate again for 8CV at a flow rate of 0.6 mL / min; collect chromatographic signals using the method of Example 1, and collect the effluent in separate tubes.

[0037] Step 5: elution; use the equilibration solution and elution solution (30 mM Na 3 Cit-H 3 Cit, 550 mM NaCl, pH 5.5), the linearity was 20%-55% eluent, the linearity was 25CV, and the flow rate was 0.6 mL / min; the chromatographic signal was collected by the method of Example 1, and the effluent was collected in separate tubes.

[0038] Step 6: Cleaning; use the eluent to clean the chromatography column for 8CV at a flow rate of 0.6 mL / min; collect chromatographic signals using the method of Example 1, and collect the effluent in separate tubes.

[0039] Step 7: Cleaning in place: CIP the column using a cleaning in place solution (1 M NaOH) for 8 CV at a flow rate of 0.6 mL / min.

[0040] Comparative Example 1 A method for removing trispecific antibody aggregates This comparative example provides a method for removing trispecific antibody aggregates, comprising the following steps: Step 1: Column loading; Take 2 mL of filler (Monomix HC45 SP) and wash it with purified water, drain the water to obtain sedimentation gel, weigh 1.1 g of sedimentation gel and add an equal amount of 0.1 M sodium chloride solution, resuspend it and add it to the 1 mL pre-loaded column tube with a disposable dropper, let it stand for 1 hour and then press the upper plug to make the column volume 1 mL. After the column is loaded, measure the column efficiency, which requires the number of plates to be above 2000 N / m and the symmetry to be between 0.8-1.5.

[0041] Step 2: Equilibration; use equilibration solution (20 mM Na 3 Cit-H 3 Cit, 50 mM NaCl, pH 5.0) for 5 CV at a flow rate of 0.5 mL / min.

[0042] Step 3: Sample loading; the sample is a human GPRCSD-BCMA-CD3 trispecific antibody sample with a sample concentration of 6.1 mg / mL, pH 5.04, and a conductivity of 3.13 mS / cm. The sample solution needs to be filtered with a 0.22 μm filter membrane before loading. The loading volume is 2 mL, the loading capacity is 12.2 g / L, and the flow rate is 0.5 mL / min.

[0043] Step 4: Post-equilibrium: Use the equilibration solution to equilibrate again for 5CV at a flow rate of 0.5 mL / min.

[0044] Step 5: elution; use the equilibration solution and elution solution (20 mM Na 3 Cit-H 3 Cit, 500 mM NaCl, pH5.0), linearity was 20%-37% eluent (8CV), 37% eluent (16CV), linearity was 37%-45% eluent (6CV), 45% eluent (10CV), linearity was 45%-55% eluent (8CV), flow rate was 0.5 mL / min, collection signal was UV280, peak cutoff range was 50mAU-50mAU, collection was in separate tubes, 3 mL was collected in one tube.

[0045] Step 6: Cleaning; Use the eluent to clean the chromatography column for 5CV at a flow rate of 0.5 mL / min; Use the same method as step 5 to collect chromatographic signals, and collect the effluent in separate tubes.

[0046] Step 7: Cleaning in place: CIP the column with a cleaning solution (0.5 M NaOH) for 5 CV at a flow rate of 0.5 mL / min.

[0047] The chromatogram collected in steps 5-6 of this comparative example is Figure 2 .

[0048] Purity testing The effluent collected in Example 1 and Comparative Example 1 was subjected to purity detection to determine the removal rate of polymer impurities in the trispecific antibody sample. The purity detection method was size exclusion chromatography (SEC), using a TSKgel G3000SWXL analytical column provided by Tosoh (Shanghai) Biotechnology Co., Ltd., with a mobile phase of 150 mM PB, pH 7.0, a flow rate of 1 mL / min, a detector of UV 280 nm, and a column temperature of room temperature.

[0049] The SEC test results of Example 1 are shown in Table 1. Figure 1 As shown, three peaks appeared when Example 1 was eluted. The SEC test results showed that the SEC purity of Example 1 was 57.603% (see Figure 3 ), the proportion of macromolecules was 42.397%; peak two combined sample (corresponding to the sample name "24-38" in Table 1, i.e., elution effluent, see Figure 4 ) has an SEC purity of 98.958%, with macromolecules accounting for 1.042%. The macromolecule removal rate is as high as 97.542%, and the yield is 17.56%. The macromolecule content of peak 2 also shows a certain trend, with a gradual increase from the front of the peak to the top of the peak and then to the end of the peak. The reason for this phenomenon may be that the isoelectric point of the target and the aggregate is different, and the isoelectric point of the aggregate is lower than that of the target protein.

[0050] The SEC test results of Comparative Example 1 are shown in Table 2. Figure 2 As shown, two peaks appeared during the elution of Comparative Example 1. The SEC test results showed that the SEC purity of Comparative Example 1 was 55.121% (see Figure 5 ), the proportion of macromolecules was 37.207%; the SEC purity of the sample eluted with 37% eluent was 79.473% (see Figure 6 ), the proportion of large molecules was 10.453%, the removal rate of aggregates was 71.91%, and the recovery rate was 70.15%.

[0051] In summary, the aggregate removal rate of Example 1 is significantly better than that of Comparative Example 1, that is, the method for removing trispecific antibody aggregates provided by the present invention can effectively remove aggregate impurities in trispecific antibodies.

[0052] Table 1 Effect of removing aggregates in Example 1 Sample name Peak area Volume (mL) Peak area × volume Recovery rate (%) SEC purity (%) Polymer ratio (%) Small molecule ratio (%) As is 18269.6 25 456740 N / A 57.603 42.397 0 24 1010.7 1 1010.7 0.22 98.25 0.251 1.499 27 3886.2 1 3886.2 0.85 99.624 0.376 0 29 7015.3 1 7015.3 1.54 99.427 0.573 0 31 9356.9 1 9356.9 2.05 99.256 0.744 0 33 10811 1 10811 2.37 99.039 0.961 0 35 11000.1 1 11000.1 2.41 98.760 1.240 0 38 10094.7 1 10094.7 2.21 98.083 1.917 0 40 9000.3 1 9000.3 1.97 97.351 2.649 0 24-38 5348.3 15 80224.5 17.56 98.958 1.042 0 Table 2 Effect of removing aggregates in Comparative Example 1 Sample name Peak area Volume (mL) Peak area × volume Recovery rate (%) SEC purity (%) Polymer ratio (%) As is 15374.4 2 30748.80 N / A 55.121 37.207 37% elution 1859.5 11.6 21570.20 70.15 79.473 10.453 45% elution 16 3.2 51.20 0.17 1.9 94.094 Cleaning 22.6 0.8 18.08 0.06 3.447 92.275

Claims

1. A method for removing trispecific antibody aggregates, characterized in that: The following steps are involved: Step 1: Column filling: Take the chromatographic filler Monomix Mab60 SP and fill the chromatographic column; Step 2: equilibration; using 20-30 mM Na3Cit-H3Cit, 40-60 mM NaCl, pH 4.5-5.5 as an equilibration solution to equilibrate the chromatographic column; Step 3: Sample loading; Add the loading solution containing the trispecific antibodies to the chromatography column; Step 4: Washing: Use 20-30 mM PB, 70-90 mM NaCl, pH 6.0-7.0 as the washing solution for washing; Step 5: Post-equilibrium: equilibrate the chromatographic column again with the equilibration solution described in step 2; Step 6: elution; using 20-30 mM Na3Cit-H3Cit, 500-550 mM NaCl, pH 4.5-5.5 as the eluent, using the balance solution and the eluent in step 2 for gradient elution, with the gradient set to 20%-55% of the eluent, collecting the chromatographic signal, and collecting the effluent in separate tubes.

2. The method for removing trispecific antibody aggregates according to claim 1, characterized in that: In step 1, the plate number of the chromatographic column is not less than 2000 N / m, and the symmetry is 0.8-1.

5.

3. The method for removing trispecific antibody aggregates according to claim 1, characterized in that: In step 3, the protein concentration in the loading solution is 3-6 mg / mL, pH 4.5-5.5, and conductivity 0-5 mS / cm.

4. The method for removing trispecific antibody aggregates according to claim 1, characterized in that: In step 6, the chromatographic signal is UV280, and the peak cutoff range is 50mAU-50mAU.

5. The method for removing trispecific antibody aggregates according to claim 1, characterized in that: In step 6, the branch tubes are collected so that each 0.2-3 column volumes of the effluent are collected into one tube.

6. The method for removing trispecific antibody aggregates according to claim 1, characterized in that: In step 6, the gradient elution volume is 25-30 column volumes.

7. The method for removing trispecific antibody aggregates according to claim 1, characterized in that: In step 2, the amount of the equilibration solution is 5-8 column volumes.

8. The method for removing trispecific antibody aggregates according to claim 1, characterized in that: In step 4, the amount of the eluent used is 5-8 column volumes.

9. The method for removing trispecific antibody aggregates according to claim 1, characterized in that: In step 5, the amount of the equilibration solution is 5-8 column volumes.

10. The method for removing trispecific antibody aggregates according to claim 1, characterized in that: After the elution is completed, the chromatographic column is cleaned with the eluent or alkaline solution.

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