Method for removing acidic isomer in monoclonal antibody

Abstract

The invention provides a method for removing an acidic isomer in a monoclonal antibody, and belongs to the technical field of antibody purification. According to the present invention, the monoclonal antibody solution is purified by using the affinity chromatography and mixed mode chromatography combined method, such that the acid isomer in the monoclonal antibody can be efficiently and stably removed, the loss of the target monoclonal antibody is reduced, and the product yield and the product purity are improved. Through detection, the content of the acid isomer in the monoclonal antibody can be reduced by more than 6% by adopting the method provided by the invention. In addition, the method is relatively simple and convenient to operate, is easy for large-scale production, can meet the requirements of industrial production, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of antibody purification technology, and more particularly to a method for removing acidic isomers from monoclonal antibodies. Background Technology

[0002] Therapeutic proteins, such as recombinant monoclonal antibodies, bispecific antibodies, or antibody fragments, exhibit heterogeneity due to post-translational modifications (PTMs) that occur during their manufacturing process. These PTMs alter the overall (or surface) charge distribution of the protein, thus creating charge variants. Based on their net charge, proteins can be broadly classified into acidic charge isomers, neutral principal components, and basic charge isomers. Acidic isomers are more negatively charged, specifically due to aberrant glycosylation modifications (including sialylation and galactosylation) or chemical modifications of amino acid residues (including deamidation, glutamate / aspartate isomerization, and amino acid residue oxidation), ultimately leading to changes in protein structure and indirectly affecting charge distribution.

[0003] Acidic and basic isoforms of therapeutic proteins can exhibit different in vivo clearance rates than the neutral principal component. Numerous reports have observed decreased tissue retention and systemic clearance in acidic variants, while increasing tissue retention and blood clearance in basic variants. Because many acidic variants produce greater negative therapeutic effects compared to basic variants, particularly regarding modifications to complementarity-determining regions (CDRs), reducing the charge of acidic variants is preferred over reducing the charge of basic variants. Therefore, charge heterogeneity is generally considered a critical quality attribute in manufacturing and requires strict control.

[0004] Cation exchange chromatography (CEX) is the preferred technique for separating charged heteromorphs in process development and production. However, the similarity in molecular structure and surface charge properties between the main component and the charged variant makes it difficult for CEX to achieve good separation performance, with significant overlap between the main peak and the charged variant peak. Therefore, many researchers have investigated continuous chromatography to improve separation performance, thereby simultaneously increasing the purity and recovery of the main peak. However, multi-column processes require specialized equipment systems, and optimizing operating parameters to achieve an ideal balance is often difficult. Therefore, it remains necessary to systematically study the impact of separation conditions on the purification of monoclonal antibody charged variants to improve the separation efficiency of charged variants. Summary of the Invention

[0005] The purpose of this invention is to provide a method for removing acidic isomers from monoclonal antibodies. This method can efficiently and stably remove acidic isomers from monoclonal antibodies while reducing the loss of the target monoclonal antibody and improving product yield and purity.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for removing acidic isomers from monoclonal antibodies, comprising the following steps:

[0008] (1) Centrifuge the sample containing monoclonal antibody and acidic isomer, collect the supernatant, filter it, and obtain the pretreated sample;

[0009] (2) Load the pretreated sample described in step (1) onto the affinity chromatography column at a flow rate of 2-5 ml / min, add buffer to equilibrate the chromatography column, elute, collect the eluent, and obtain a preliminarily purified monoclonal antibody solution.

[0010] (3) Equilibrate the multi-mode chromatography column, load the preliminarily purified monoclonal antibody solution described in step (2) onto the multi-mode chromatography column, equilibrate the multi-mode chromatography column again after loading, collect the sample flow-through, and obtain a monoclonal antibody solution free of acidic isomers.

[0011] Preferably, in step (1), the centrifugation method is a two-stage centrifugation method. The rotation speed of the first centrifugation in the two-stage centrifugation method is 3800-4200 r / min, and the time of the first centrifugation is 10-20 min. The rotation speed of the second centrifugation in the two-stage centrifugation method is 7800-8200 r / min, and the time of the second centrifugation is 8-12 min.

[0012] Preferably, in step (1), the pore size of the filter is 0.20 to 0.25 μm.

[0013] Preferably, in step (2), the affinity chromatography column is filled with Protein A affinity packing material, and the volume of the affinity chromatography column is 10-20 ml.

[0014] Preferably, in step (2), the buffer solution is a phosphate buffer with a molar concentration of 0.04 to 0.06 M and a pH of 7.0 to 7.5; the equilibration volume is 4 to 6 times the volume of the affinity chromatography column.

[0015] Preferably, in step (2), the elution is performed using an acetate-sodium acetate buffer solution; the molar concentration of the acetate-sodium acetate buffer solution is 0.04-0.06M, the pH value of the acetate-sodium acetate buffer solution is 3.4-3.6, and the elution flow rate is 2-5 ml / min.

[0016] Preferably, in step (3), the equilibration is performed using Tris-HCl buffer, the molar concentration of which is 0.04 to 0.06 M and the pH of which is 6.0 to 7.5; the equilibration volume is 4 to 6 times the volume of the composite mode chromatography column.

[0017] Preferably, in step (3), the composite mode chromatography column is filled with composite packing material with ion exchange and hydrophobic effects, and the volume of the composite mode chromatography column is 4-6 ml.

[0018] Preferably, in step (3), the pH of the preliminarily purified monoclonal antibody solution is 7.4 to 7.6.

[0019] Preferably, in step (3), the flow rate of the sample loading is 0.9 to 1.0 ml / min, the conductivity of the sample loading is 4 to 6 mS / cm, and the sample loading capacity is 55 to 65 g / L; the conditions for re-equilibration are the same as the conditions for equilibration.

[0020] The beneficial effects of this invention compared to the prior art are as follows:

[0021] This invention employs a combination of affinity chromatography and mixed-mode chromatography to purify monoclonal antibody solutions. Affinity chromatography specifically binds to the monoclonal antibody, achieving preliminary purification and removing most impurities. Mixed-mode chromatography utilizes the ion exchange and hydrophobic properties of the composite packing material to more efficiently separate acidic isomers, improving the purity of the monoclonal antibody. Testing shows that the method provided by this invention can reduce the content of acidic isomers in monoclonal antibodies by more than 6%. Furthermore, the buffer conditions used in mixed-mode chromatography are mild, having minimal impact on the structure and activity of the monoclonal antibody, thus ensuring the effectiveness of the monoclonal antibody drug.

[0022] The method provided by this invention can efficiently and stably remove acidic isomers from monoclonal antibodies while reducing the loss of the target monoclonal antibody, thereby improving product yield and purity. Furthermore, the method is relatively simple to operate, easy to scale up for production, and can meet the needs of industrial production, demonstrating promising application prospects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1The graph shows the trend of the removal rate and experimental yield of acidic isomers under different experimental conditions (sample loading, pH, Cond);

[0025] Figure 2 The contour plot fitted based on the experimental results shows the removal rate of acidic isomers at different loading pH and Cond when the loading is 80 g / L.

[0026] Figure 3 The contour plot fitted based on the experimental results shows the removal rate of acidic isomers at different loading pH and loading when Cond 10mS / cm.

[0027] Figure 4 To present the trend of experimental yield at different loading pH and Cond when the loading is 80 g / L, a contour plot fitted based on the experimental results is used.

[0028] Figure 5 To present the experimental yield trend at pH 6.75 with different loading amounts and Cond, a contour plot fitted based on the experimental results is provided. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] This invention provides a method for removing acidic isomers from monoclonal antibodies, comprising the following steps:

[0035] (1) Centrifuge the sample containing monoclonal antibody and acidic isomer, collect the supernatant, filter it, and obtain the pretreated sample;

[0036] (2) Load the pretreated sample described in step (1) onto the affinity chromatography column at a flow rate of 2-5 ml / min, add buffer to equilibrate the chromatography column, elute, collect the eluent, and obtain a preliminarily purified monoclonal antibody solution.

[0037] (3) Equilibrate the multi-mode chromatography column, load the preliminarily purified monoclonal antibody solution described in step (2) onto the multi-mode chromatography column, equilibrate the multi-mode chromatography column again after loading, collect the sample flow-through, and obtain a monoclonal antibody solution free of acidic isomers.

[0038] In this invention, in step (1), the centrifugation method is preferably a two-stage centrifugation method. The rotation speed of the first centrifugation in the two-stage centrifugation method is preferably 3800-4200 r / min, more preferably 4000 r / min; the time of the first centrifugation is preferably 10-20 min, more preferably 14-18 min, and even more preferably 15 min; the rotation speed of the second centrifugation in the two-stage centrifugation method is preferably 7800-8200 r / min, more preferably 8000 r / min; the time of the second centrifugation is preferably 8-12 min, more preferably 10 min; and the pore size of the filter is preferably 0.20-0.25 μm, more preferably 0.22-0.24 μm, and even more preferably 0.23 μm.

[0039] In this invention, in step (2), the affinity chromatography column is preferably packed with Protein. A. Affinity-modifying packing material, wherein the volume of the affinity chromatography column is preferably 10-20 ml, more preferably 14-18 ml, and even more preferably 15 ml; the buffer solution is preferably phosphate buffer, wherein the molar concentration of the phosphate buffer is preferably 0.04-0.06 M, more preferably 0.05 M; the pH value of the phosphate buffer is preferably 7.0-7.5, more preferably 7.2-7.4, and even more preferably 7.3; the equilibration volume is preferably 4-6 times the volume of the affinity chromatography column, more preferably 5 times; the elution is preferably performed using an acetate-sodium acetate buffer, wherein the molar concentration of the acetate-sodium acetate buffer is preferably 0.04-0.06 M, more preferably 0.05 M; the pH value of the acetate-sodium acetate buffer is preferably 3.4-3.6, more preferably 3.5; the elution flow rate is preferably 2-5 ml / min, more preferably 3-4 ml / min, and even more preferably 3.5 ml / min.

[0040] In this invention, in step (3), the Tris-HCl buffer is preferably used for equilibration, and the molar concentration of the Tris-HCl buffer is preferably 0.04-0.06M, more preferably 0.05M; the pH value of the Tris-HCl buffer is preferably 6.0-7.5, more preferably 6.5-7.0, and even more preferably 6.8; the equilibration volume is preferably 4-6 times the volume of the composite mode chromatography column, more preferably 5 times; the composite mode chromatography column is preferably packed with composite packing material with ion exchange and hydrophobic effects; the volume of the composite mode chromatography column is preferably... The sample loading rate is preferably 0.9-1.0 ml / min, more preferably 0.94-0.98 ml / min, and even more preferably 0.95 ml / min; the pH of the preliminarily purified monoclonal antibody solution is preferably 7.4-7.6, more preferably 7.5; the conductivity of the sample loading rate is preferably 4-6 mS / cm, more preferably 5 mS / cm; the sample loading capacity is preferably 55-65 g / L, more preferably 58-62 g / L, and even more preferably 60 g / L; the conditions for re-equilibration are the same as the equilibration conditions. Steps (1) to (3) are all performed at room temperature.

[0041] All raw materials and consumables used in the examples and experimental cases were commercially or otherwise readily available. The chromatography system used in the examples and experimental cases was the Cytiva AKTA avant25, and the affinity chromatography column was the NMab Protein A affinity packing material from Nanomicro. The MaxTar MMA composite chromatography column used in the examples and experimental cases is a composite packing material from Bailinco, possessing both hydrophobic and ion exchange mechanisms. The cells used in the experimental cases were CHO cells purchased from GenScript.

[0042] Example 1

[0043] A method for removing acidic isomers from monoclonal antibodies, comprising the following steps:

[0044] (1) Centrifuge the sample containing monoclonal antibody and acidic isomer at 4000 r / min for 15 min to remove CHO cells and large particles, collect supernatant 1, then centrifuge the collected supernatant at 8000 r / min for 10 min to remove cell debris and fine particles, collect supernatant 2, filter with a 0.22 μm filter membrane to obtain the pretreated sample;

[0045] (2) Load the pretreated sample described in step (1) into a 15 ml affinity chromatography column (NMab Protein A affinity packing material purchased from Nanomicro) at a flow rate of 3 ml / min. Equilibrate the column with pH 7.2, 0.05 M phosphate buffer at a flow rate of 5 times the volume of the affinity chromatography column until the baseline of the UV detector (280 nm) is stable. Elute with pH 3.5, 0.05 M acetate-sodium acetate buffer at a flow rate of 3 ml / min. Collect the eluent (elution peak component (target antibody acidic isomer content is 26.97%)) to obtain a preliminarily purified monoclonal antibody solution.

[0046] (3) Equilibrate the composite mode chromatography column (5 ml column volume, purchased from Bailinke Pharmaceutical Technology (Shanghai) Co., Ltd.) with pH 7.0, 0.05 M Tris-HCl buffer. The equilibration volume is 5 times the volume of the composite mode chromatography column. Load the preliminarily purified monoclonal antibody solution (pH 7.5) described in step (2) into a 5 ml composite mode chromatography column packed with composite packing material with ion exchange and hydrophobic effects at a flow rate of 0.94 ml / min. The conductivity of the sample loading is 5 mS / cm, and the loading capacity is 60 g / L. After the sample loading starts, collect the sample flow-through. After the sample loading is completed, equilibrate the composite mode chromatography column again (under the same conditions as the equilibration) until the baseline of the UV detector is stable. Stop collecting the sample flow-through and combine all the flow-through to obtain a monoclonal antibody solution with the acid isomer removed.

[0047] Example 2

[0048] A method for removing acidic isomers from monoclonal antibodies, comprising the following steps:

[0049] (1) Centrifuge the sample containing monoclonal antibody and acidic isomer at 3800 r / min for 20 min to remove CHO cells and large particles, collect supernatant 1, then centrifuge the collected supernatant at 7800 r / min for 12 min to remove cell debris and fine particles, collect supernatant 2, filter with a 0.20 μm filter membrane to obtain the pretreated sample;

[0050] (2) Load the pretreated sample described in step (1) into a 10 ml affinity chromatography column packed with Protein A affinity packing material at a flow rate of 2 ml / min. Equilibrate the column with pH 7.0, 0.04 M phosphate buffer at a flow rate of 4 times the volume of the affinity chromatography column until the baseline of the UV detector (280 nm) is stable. Elute with pH 3.4, 0.04 M acetate-sodium acetate buffer at a flow rate of 2 ml / min. Collect the eluent (elution peak component (target antibody acidic isomer content is 26.45%)) to obtain a preliminarily purified monoclonal antibody solution.

[0051] (3) Equilibrate the multi-mode chromatography column using a pH 6.0, 0.04M Tris-HCl buffer. The equilibration volume is 4 times the volume of the multi-mode chromatography column. Load the preliminarily purified monoclonal antibody solution (pH 7.4) described in step (2) into a multi-mode chromatography column with a volume of 4 ml and filled with composite packing material with ion exchange and hydrophobic effects at a flow rate of 0.9 ml / min. The conductivity of the sample is 4 mS / cm and the sample loading capacity is 55 g / L. After the sample loading starts, collect the sample flow-through. After the sample loading is completed, equilibrate the multi-mode chromatography column again (under the same conditions as the equilibration) until the baseline of the UV detector is stable. Stop collecting the sample flow-through and combine all the flow-through to obtain a monoclonal antibody solution with the acid isomer removed.

[0052] Example 3

[0053] A method for removing acidic isomers from monoclonal antibodies, comprising the following steps:

[0054] (1) Centrifuge the sample containing monoclonal antibody and acidic isomer at 4200 r / min for 10 min to remove CHO cells and large particles, collect supernatant 1, then centrifuge the collected supernatant at 8200 r / min for 8 min to remove cell debris and fine particles, collect supernatant 2, filter with a 0.25 μm filter membrane to obtain the pretreated sample;

[0055] (2) Load the pretreated sample described in step (1) into a 20 ml affinity chromatography column packed with Protein A affinity packing material at a flow rate of 5 ml / min. Equilibrate the column with pH 7.5, 0.06 M phosphate buffer at a flow rate of 6 times the volume of the affinity chromatography column until the baseline of the UV detector (280 nm) is stable. Elute with pH 3.6, 0.06 M acetate-sodium acetate buffer at a flow rate of 5 ml / min. Collect the eluent (elution peak component (target antibody acid isomer content is 24.81%)) to obtain a preliminarily purified monoclonal antibody solution.

[0056] (3) Equilibrate the multi-mode chromatography column using a pH 7.5, 0.06M Tris-HCl buffer. The equilibration volume is 6 times the volume of the multi-mode chromatography column. Load the preliminarily purified monoclonal antibody solution (pH 7.6) described in step (2) into a 6ml multi-mode chromatography column packed with composite packing material that has ion exchange and hydrophobic effects at a flow rate of 1.0ml / min. The conductivity of the sample is 6mS / cm and the sample loading capacity is 65g / L. After the sample loading starts, collect the sample flow-through. After the sample loading is completed, equilibrate the multi-mode chromatography column again (under the same conditions as the equilibration) until the baseline of the UV detector is stable. Stop collecting the sample flow-through and combine all the flow-through to obtain a monoclonal antibody solution with the acid isomer removed.

[0057] Experimental Example 1

[0058] Based on the "sample pretreatment-affinity chromatography-complex mode chromatography" basic process established in Example 1, this study investigated the effects of three key parameters—sample loading capacity, conductivity, and pH of the monoclonal antibody solution—on the removal rate of acidic isomers and the yield of the target antibody during the complex mode chromatography stage. The optimal combination of process parameters was determined to improve process stability and efficiency. The specific operating methods are as follows:

[0059] The fermentation broth of ustekinumab expressed in CHO cells (purchased from Genscript) was used as a sample containing monoclonal antibody and acidic isomer. It was processed according to the method in Example 1, except that the loading amount, conductivity and pH of the monoclonal antibody solution in step (3) were changed to the values ​​in Table 1 for the experiment.

[0060] A three-factor, two-level factorial experimental design was adopted, with sample loading, conductivity, and pH of the monoclonal antibody solution selected as variables. Each factor was set with two levels, and a total of nine parallel experiments were designed (numbered 1-9). The specific parameter levels are shown in Table 1.

[0061] Table 1. Three-factor, two-level factorial experimental design

[0062] Variable factors Level 1 Level 2 Sample loading capacity (mg / mL) 60 100 Conductivity mS / cm 5 15 pH 6.0 7.5

[0063] The content of acidic isomers in monoclonal antibody solutions with different acidic isomer removal methods was determined by high performance liquid chromatography (HPLC) using an Agilent 1260 HPLC system. The yield of the target antibody was calculated by ultraviolet spectrophotometry (280 nm) using a NanodropOne micro spectrophotometer. The experimental results are shown in Table 2.

[0064] Table 2 Removal rate and yield at different loading rates, conductivity, and pH values.

[0065] Run sequence number pH Cond / mS / cm Loading capacity / mg / ml Yield / % Acid peak removal percentage point 01 6.75 10 80 94 2.40 02 6.75 10 80 95 2.13 03 7.50 5 60 82 7.76 04 6.00 5 100 96 0.62 05 6.00 15 100 99 0.68 06 6.75 10 80 93 2.06 07 7.50 5 100 87 5.43 08 6.00 5 60 97 0.52 09 6.00 15 60 98 0.49 10 7.50 15 100 97 3.44 11 7.50 15 60 83 4.09

[0066] The results showed that pH significantly affected the removal efficiency of acidic isomers under the same loading and conductivity. When pH = 7.5, the removal percentage of acid peaks was ≥2.8% (up to 7.7%); when pH = 6.0, the removal percentage of acid peaks was ≤0.7%. This indicates that a high pH environment (7.5) is more conducive to the specific binding of acidic isomers with the composite packing material, thus improving the removal rate, while a low pH (6.0) inhibits this binding effect.

[0067] Effect of conductivity: At a loading of 60 mg / ml and pH 7.5, the lower the conductivity, the higher the percentage of acid peak removal. The highest removal rate (7.7%) was achieved at a conductivity of 5 mS / cm, which decreased to 3.1% when the conductivity increased to 15 mS / cm. This indicates that a low conductivity environment (5 mS / cm) can reduce the interference of ions in the buffer solution on the binding of acidic isomers to the packing material.

[0068] Effect of loading capacity: At conductivity = 5 mS / cm and pH = 7.5, the lower the loading capacity, the higher the percentage of acid peak removal. The highest removal rate (7.7%) was achieved at a loading capacity of 60 mg / mL. As the loading capacity increased to 100 mg / mL, the removal rate decreased to 2.8%, but the target antibody yield decreased with decreasing loading capacity. This indicates that a low loading capacity (60 mg / mL) allows the acidic isomer to fully bind to the packing material, avoiding competition for binding sites with the target antibody.

[0069] The above results indicate that, based on the basic process of Example 1, the optimal parameter combination for composite mode chromatography was determined to be: loading capacity of 60 mg / mL, conductivity of 5 mS / cm, and pH of 7.5. Under these conditions, the acid peak of the acid isomer was removed by 7.7%, and the target antibody yield was ≥82%.

[0070] Experimental Example 2

[0071] Based on the "sample pretreatment-affinity chromatography-complex mode chromatography" process established in Example 2, and with the parameters of the affinity chromatography stage and key conditions such as the loading conductivity and pH of the monoclonal antibody solution in the complex mode chromatography stage fixed, only the loading amount in the complex mode chromatography was changed to investigate the effect of different loading amounts on the removal rate of acidic isomers in monoclonal antibodies and the yield of target antibodies. This provides data support for optimizing process parameters and further improving the removal efficiency of acidic isomers. The specific operation method is as follows:

[0072] The fermentation broth of ustekinumab expressed in CHO cells (purchased from Genscript) was used as a sample containing monoclonal antibody and acidic isomer. It was processed according to the method in Example 2, except that the loading amount in step (3) was replaced with 30, 40, 50, 60, 70, and 80 mg / ml, and the conductivity was replaced with 4.37 mS / cm.

[0073] The content of acidic isomers in monoclonal antibody solutions with different acidic isomer removal methods was determined by high performance liquid chromatography (HPLC) using an Agilent 1260 HPLC system. The experimental results are shown in Table 3.

[0074] Table 3 Removal rates at different loading rates when conductivity is 4.37 mS / cm

[0075]

[0076] The results showed that the removal percentage of the acid isomer increased significantly with decreasing loading. The highest removal percentage (15.07%) was observed at a loading of 30 mg / ml; however, as the loading increased to 80 mg / ml, the removal percentage decreased to 2.89%. This is because under low loading conditions, the composite packing material in the composite mode chromatography column has sufficient binding sites to fully bind with the acid isomer, thus retaining it more efficiently. At high loading, the target antibody competes with the acid isomer for binding sites, leading to a decrease in the amount of acid isomer bound and a decline in removal efficiency.

[0077] Experimental Example 3

[0078] Based on the complete "sample pretreatment-affinity chromatography-complex mode chromatography" process established in Example 3, all operating parameters in the affinity chromatography stage and key conditions such as loading conductivity and pH of the monoclonal antibody solution in the complex mode chromatography stage were kept constant. Only the loading amount in the complex mode chromatography was changed. By setting gradient loading levels, the influence of loading amount on the removal rate of acidic isomers in monoclonal antibodies and the yield of target antibodies was systematically investigated. This provides experimental basis for further optimization of process parameters and balancing removal efficiency and product yield. The specific operating method is as follows:

[0079] The fermentation broth of ustekinumab expressed in CHO cells (purchased from Genscript) was used as a sample containing monoclonal antibody and acidic isomer. It was processed according to the method in Example 3, except that the loading amount in step (3) was replaced with 30, 35, and 40 mg / ml, and the loading conductivity was replaced with 4.64 mS / cm.

[0080] The content of acidic isomers in the prepared monoclonal antibody solution after removing the acidic isomers was determined by high performance liquid chromatography (HPLC) using an Agilent 1260 HPLC system. The yield of the target antibody was calculated by ultraviolet spectrophotometry (280 nm) using a NanodropOne micro spectrophotometer. The results are shown in Table 4.

[0081] Table 4. Yield and removal rates at different loading rates when conductivity is 4.64 mS / cm

[0082]

[0083] The results showed that the removal efficiency of acid isomers was related to the loading capacity: under fixed loading conductivity, loading pH, and other process parameters, the percentage of acid isomer removal gradually increased with decreasing loading capacity. Under low loading conditions, the composite packing surface in the composite mode chromatography column had sufficient binding sites, allowing acid isomers to bind effectively to the packing surface through ion exchange and hydrophobic interactions, resulting in efficient retention. However, as the loading capacity increased, the target antibody competed with the acid isomers for the limited binding sites on the packing, leading to a decrease in the amount of acid isomers bound and consequently a decrease in removal efficiency.

[0084] Relationship between target antibody yield and loading capacity: Experimental results show that the yield changes in the opposite direction to the removal rate with increasing loading capacity. At low loading capacities, the packing material not only binds acidic isomers but may also bind a small amount of target antibody due to non-specific adsorption, leading to increased target antibody loss and decreased yield. Conversely, as the loading capacity increases, the proportion of target antibody occupying the packing material binding sites increases, reducing losses caused by non-specific adsorption, thus improving the yield. However, this also sacrifices some removal efficiency of acidic isomers.

[0085] As can be seen from the above embodiments, the present invention provides a method for removing acidic isomers from monoclonal antibodies. This method can reduce the content of acidic isomers in monoclonal antibodies by more than 6%, and can efficiently and stably remove acidic isomers from monoclonal antibodies while reducing the loss of target monoclonal antibodies and improving product yield and purity.

[0086] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for removing acidic isoforms in a monoclonal antibody, characterized by, It comprises the following steps: (1) centrifuging the sample containing monoclonal antibody and acidic isomer, collecting supernatant, filtering, and obtaining pretreated sample; (2) loading the pretreated sample in step (1) to affinity chromatography column at a flow rate of 2-5 ml / min, equilibrating the column with buffer, eluting, collecting eluate, and obtaining preliminary purified monoclonal antibody solution; (3) equilibrating complex mode chromatography column, loading the preliminary purified monoclonal antibody solution in step (2) to the complex mode chromatography column, equilibrating the complex mode chromatography column again after loading, collecting sample flow-through, and obtaining monoclonal antibody solution from which acidic isomer is removed.

2. The method of claim 1, wherein, In step (1), the centrifugation method is twice centrifugation, the first centrifugation is at a speed of 3800-4200 r / min for 10-20 min, and the second centrifugation is at a speed of 7800-8200 r / min for 8-12 min.

3. The method of claim 1, wherein, In step (1), the filter pore size is 0.20-0.25 μm.

4. The method of claim 1, wherein, In step (2), the affinity chromatography column is loaded with Protein A affinity packing, and the volume of the affinity chromatography column is 10-20 ml.

5. The method of claim 1, wherein, In step (2), the buffer is phosphate buffer, the molar concentration of the phosphate buffer is 0.04-0.06 M, the pH value of the phosphate buffer is 7.0-7.5, and the equilibration volume is 4-6 times the volume of the affinity chromatography column.

6. The method of claim 1, wherein, In step (2), acetic acid-sodium acetate buffer is used for elution, the molar concentration of the acetic acid-sodium acetate buffer is 0.04-0.06 M, the pH value of the acetic acid-sodium acetate buffer is 3.4-3.6, and the flow rate of elution is 2-5 ml / min.

7. The method of claim 1, wherein, In step (3), Tris-HCl buffer is used for equilibration, the molar concentration of the Tris-HCl buffer is 0.04-0.06 M, the pH value of the Tris-HCl buffer is 6.0-7.5, and the equilibration volume is 4-6 times the volume of the complex mode chromatography column.

8. The method of claim 1, wherein, In step (3), the complex mode chromatography column is loaded with complex packing with ion exchange and hydrophobic interaction, and the volume of the complex mode chromatography column is 4-6 ml.

9. The method of claim 1, wherein, In step (3), the pH value of the preliminary purified monoclonal antibody solution is 7.4-7.

6.

10. The method of claim 1, wherein, In step (3), the flow rate of loading is 0.9-1.0 ml / min, the conductivity of loading is 4-6 mS / cm, and the loading capacity is 55-65 g / L; the re-equilibration conditions are the same as the equilibration conditions.

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