Purification method and application of target protein with Fc structure

Abstract

The invention relates to a purification method and application of a target protein with an Fc structure, and belongs to the technical field of biology. The invention provides a purification method of a target protein with an Fc structure. The purification method comprises two steps of antibody capture and antibody refined purification, the antibody capture comprises the following steps: purifying the feed liquid through an affinity chromatography system to obtain an affinity collection liquid; the antibody refined purification comprises the following steps: purifying the affinity collection liquid through an improved cation membrane chromatography system to obtain cation collection liquid; the step of purifying the antibody is carried out on an improved cation membrane chromatography system; the improved cation membrane chromatography system is a cation membrane chromatography system which is connected with an anion chromatography membrane in front of a sample loading pipeline. The improved cation membrane chromatography system used in the purification method can efficiently realize removal of host impurities, and has higher aggregate control capability.

Description

Technical Field

[0001] The invention relates to a method for purifying a target protein with an Fc structure and application thereof, belonging to the field of biotechnology. Background Art

[0002] Monoclonal antibodies are highly uniform antibodies produced by a single B cell clone that target only a specific antigen epitope. At present, monoclonal antibodies are usually produced by cell culture processes. The cell culture process requires the construction of an antibody expression sequence by genetic engineering, and then the antibody expression sequence is recombined into an expression vector. The recombinant expression vector is then transfected into a host cell, and finally the transfected host cells are cultured in vitro to achieve the expression of the recombinant monoclonal antibody. After the host cells are cultured in vitro to obtain a feed solution containing monoclonal antibodies, the monoclonal antibodies need to be purified from the feed solution for subsequent use.

[0003] With the continuous improvement of cell culture technology, the output of antibodies has been continuously improved. The technical bottleneck and production cost of large-scale antibody production have gradually shifted from antibody expression to antibody purification. Therefore, improving the flux and efficiency of antibody purification technology and reducing the cost of antibody purification technology have become the top priority of antibody production process research. At present, the traditional three-step chromatography method (affinity chromatography + anion column chromatography + cation column chromatography) or two-step chromatography method (affinity chromatography + anion column chromatography / cation column chromatography) is mainly used to purify monoclonal antibodies. Among them, the traditional three-step chromatography method can provide good impurity removal and aggregate removal (anion chromatography is mainly used for impurity removal, and cation chromatography is mainly used for aggregate control), but it requires two sets of chromatography equipment and a matching anion / cation buffer system, the steps are cumbersome and time-consuming, and the purification cost is high; and the traditional two-step chromatography method simplifies the equipment and steps and reduces the purification cost, but it cannot take into account the removal of impurities and aggregates at the same time, and there is a defect of a long purification cycle. Therefore, there is an urgent need to find a protein purification method that can take into account both the removal of impurities and the removal of aggregates, and is fast (short purification cycle) and efficient (good quality and high yield) to achieve the purification of monoclonal antibodies. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for purifying a target protein having an Fc structure, the purification method comprising the following steps:

[0005] Antibody capture: The feed solution is purified by an affinity chromatography system to obtain an affinity collection solution;

[0006] Antibody purification: the affinity collection solution is purified by an improved cationic membrane chromatography system to obtain a cationic collection solution; the antibody purification step is performed on the improved cationic membrane chromatography system; the improved cationic membrane chromatography system is a cationic membrane chromatography system connected to an anionic chromatography membrane before the sample loading pipeline;

[0007] Alternatively, the purification method comprises the following steps:

[0008] Antibody capture: The feed solution is purified by an affinity chromatography system to obtain an affinity collection solution;

[0009] Deep filtration: The affinity collection solution is purified by deep filtration to obtain a deep collection solution;

[0010] Antibody purification: The deep collection liquid is purified by an improved cationic membrane chromatography system to obtain a cationic collection liquid; the antibody purification step is performed on the improved cationic membrane chromatography system; the improved cationic membrane chromatography system is a cationic membrane chromatography system connected to an anionic chromatography membrane before the sample loading pipeline.

[0011] In one embodiment of the present invention, the improved cationic membrane chromatography system purification includes a loading step; the loading step includes: using affinity collection fluid or deep collection fluid for loading; when loading, the loading capacity of the cationic chromatography membrane is 18 to 35 g / L, and the loading capacity of the anion membrane is 1 to 1200 g / L; the loading conditions of the affinity collection fluid or the deep collection fluid are: pH 5.25 to 6.25.

[0012] In one embodiment of the present invention, in the loading step, when loading, the loading capacity of the cationic chromatography membrane is 18-32 g / L, and the loading capacity of the anionic membrane is 10-1000 g / L; the loading conditions of the affinity collection liquid or the deep collection liquid are: pH 5.5-6.25.

[0013] In one embodiment of the present invention, in the loading step, when loading, the loading capacity of the cationic chromatography membrane is 20-30 g / L, and the loading capacity of the anionic membrane is 100-1000 g / L; the loading conditions of the affinity collection liquid or the deep collection liquid are: pH 5.5-6.0.

[0014] In one embodiment of the present invention, the improved cationic membrane chromatography system purification comprises the following steps:

[0015] Cleaning: Use cleaning solution to clean at least 10 membrane volumes with a retention time of at least 0.1 min;

[0016] Pre-equilibrium: flush at least 2 membrane volumes with pre-equilibrium solution, with a retention time of at least 0.1 min;

[0017] Balance: flush with balance solution until the pH and conductivity of the effluent are consistent with those of the solution, and the retention time is at least 0.1 min;

[0018] Sample loading: Use affinity collection solution or deep collection solution for sample loading, with a retention time of at least 0.1 min; when loading, the loading capacity of the cation chromatography membrane is 18-35 g / L, and the loading capacity of the anion membrane is 1-1200 g / L; the loading conditions of the affinity collection solution or deep collection solution are: pH 5.25-6.25, conductivity <5 mS / cm;

[0019] Balance: Use balance solution to rinse until the pH and conductivity of the effluent are consistent with the solution, and the retention time is at least 0.1 min;

[0020] Elution: first use eluent A for elution, then use eluent B for gradient elution, eluting a total of 20 to 25 membrane volumes, the collection conditions of the cation collection solution are 100 to 250 mAU / mm, and the retention time is at least 0.1 min.

[0021] In one embodiment of the present invention, the improved cationic membrane chromatography system purification comprises the following steps:

[0022] Cleaning: Use cleaning solution to clean 10 to 15 membrane volumes, retention time 0.1 to 0.2 min;

[0023] Pre-equilibrium: Use pre-equilibrium solution to flush 2 to 3 membrane volumes, retention time 0.1 to 0.2 min;

[0024] Balance: flush with balance solution until the pH and conductivity of the effluent are consistent with those of the solution, with a retention time of 0.1 to 0.2 min;

[0025] Sample loading: Use affinity collection solution or deep collection solution for sample loading, retention time 0.1-0.2min; when loading, the loading capacity of cation chromatography membrane is 20-30g / L, and the loading capacity of anion membrane is 100-1000g / L; the loading conditions of affinity collection solution or deep collection solution are: pH 5.5-6.0, conductivity <5mS / cm;

[0026] Balance: Use balance solution to rinse until the pH and conductivity of the effluent are consistent with those of the solution, with a retention time of 0.1 to 0.2 min;

[0027] Elution: first use eluent A for elution, then use eluent B for gradient elution, eluting a total of 20 to 25 membrane volumes, the collection conditions of the cation collection solution are 100 to 250 mAU / mm, and the retention time is 0.1 to 0.2 min.

[0028] In one embodiment of the present invention, in the cleaning step of the improved cationic membrane chromatography system purification, the cleaning solution is a NaOH solution with a concentration of 0.1 to 0.5 M;

[0029] In the pre-equilibrium step of the purification of the improved cationic membrane chromatography system, the pre-equilibrium solution is a NaAc-HAc buffer solution with a concentration of 10 to 100 mM and a pH of 5.25 to 6.25 containing 0.1 to 0.5 M NaCl;

[0030] In the equilibration step of the improved cationic membrane chromatography system purification, the equilibration solution is a NaAc-HAc buffer solution with a concentration of 10 to 100 mM and a pH of 5.25 to 6.25;

[0031] In the elution step of the improved cationic membrane chromatography system purification, the eluent A is a NaAc-HAc buffer with a concentration of 10-100 mM and a pH of 5.25-6.25, and the eluent B is a NaAc-HAc buffer with a concentration of 10-100 mM and a pH of 5.25-6.25 containing 0.7-1.2 M NaCl.

[0032] In one embodiment of the present invention, in the cleaning step of the improved cationic membrane chromatography system purification, the cleaning solution is a NaOH solution with a concentration of 0.1 to 0.5 M;

[0033] In the pre-equilibrium step of the purification of the improved cationic membrane chromatography system, the pre-equilibrium solution is a NaAc-HAc buffer solution with a concentration of 20 to 50 mM and a pH of 5.5 to 6.0 containing 0.1 to 0.5 M NaCl;

[0034] In the equilibration step of the improved cationic membrane chromatography system purification, the equilibration solution is a NaAc-HAc buffer solution with a concentration of 20 to 50 mM and a pH of 5.5 to 6.0;

[0035] In the elution step of the improved cationic membrane chromatography system purification, the eluent A is a NaAc-HAc buffer with a concentration of 20-50 mM and a pH of 5.5-6.0, and the eluent B is a NaAc-HAc buffer with a concentration of 20-50 mM and a pH of 5.5-6.0 containing 0.8-1.0 M NaCl.

[0036] In one embodiment of the present invention, the gradient elution is performed using eluent B with a gradient elution rate from 0% to 50%.

[0037] In one embodiment of the present invention, the affinity chromatography system purification comprises the following steps:

[0038] Rinse: Rinse the column with purified water for at least 1 column volume, with a retention time of at least 5 minutes;

[0039] Cleaning: Use at least 3 column volumes of cleaning solution and the retention time should be at least 5 minutes;

[0040] Rinse: Rinse the column with purified water for at least 1 column volume, with a retention time of at least 5 minutes;

[0041] Balance: flush the column with the balance solution until the pH and conductivity of the effluent are consistent with those of the solution, and the retention time is at least 5 minutes;

[0042] Sample loading: Sample loading with a retention time of at least 5 min;

[0043] Equilibration: flush the column with equilibration solution for at least 3 column volumes, with a retention time of at least 5 min;

[0044] Elution 1: Elute with eluent A for at least 3 column volumes with a retention time of at least 5 min.

[0045] Wash 2: Wash with eluent B for at least 3 column volumes with a retention time of at least 5 min.

[0046] Elution: elute with eluent for at least 3 column volumes, with a retention time of at least 5 minutes, and the collection conditions of the affinity collection solution are 100-100 mAU / 2 mm. The pH of the collected affinity collection solution is adjusted to 5.25-6.25 using a pH adjusting solution.

[0047] In one embodiment of the present invention, the affinity chromatography system purification comprises the following steps:

[0048] Rinse: rinse the column with purified water for 1 to 2 column volumes, retention time 5 to 7 minutes;

[0049] Cleaning: Use 3 to 5 column volumes of cleaning solution for 5 to 7 minutes of retention time;

[0050] Rinse: rinse the column with purified water for 1 to 2 column volumes, retention time 5 to 7 minutes;

[0051] Balance: flush the column with the balance solution until the pH and conductivity of the effluent are consistent with those of the solution, and the retention time is 5 to 7 minutes;

[0052] Sample loading: Sample loading at a retention time of 5 to 7 minutes;

[0053] Balance: flush the column with balance solution for 3 to 5 column volumes, retention time 5 to 7 minutes;

[0054] Elution 1: Use eluent A to wash 3 to 5 column volumes, retention time 5 to 7 minutes;

[0055] Elution 2: Elute 3 to 5 column volumes with eluent B, retention time 5 to 7 minutes;

[0056] Elution: use eluent to elute for 3 to 5 column volumes, retention time 5 to 7 min, collection conditions of affinity collection solution 100 to 100 mAU / 2 mm, collect the affinity collection solution and adjust the pH to 5.5 to 6.0 using pH adjusting solution.

[0057] In one embodiment of the present invention, in the cleaning step of the affinity chromatography system purification, the cleaning solution is a NaOH solution with a concentration of 0.1 to 0.2 M;

[0058] In the equilibration step of the affinity chromatography system purification, the equilibration solution is a Tris-HAc buffer with a concentration of 10 to 100 mM and a pH of 7.2 to 7.6, containing 80 to 170 mM NaCl;

[0059] In the elution step 1 of the affinity chromatography system purification, the eluent A is a NaAc-HAc buffer solution with a concentration of 10 to 100 mM and a pH of 5.2 to 5.8 containing 0.5 to 1.0 M NaCl;

[0060] In the elution step 2 of the affinity chromatography system purification, the eluent B is a NaAc-HAc buffer with a concentration of 10 to 100 mM and a pH of 5.2 to 5.8;

[0061] In the elution step of the affinity chromatography system purification, the eluent is a NaAc-HAc buffer with a concentration of 10-100 mM and a pH of 3.4-3.8, and the pH adjustment solution is a Tris buffer with a concentration of 1.0-2.0 M.

[0062] In one embodiment of the present invention, in the cleaning step of the affinity chromatography system purification, the cleaning solution is a NaOH solution with a concentration of 0.1 to 0.2 M;

[0063] In the equilibration step of the affinity chromatography system purification, the equilibration solution is a Tris-HAc buffer with a concentration of 20 to 50 mM and a pH of 7.2 to 7.6, containing 100 to 150 mM NaCl;

[0064] In the elution step 1 of the affinity chromatography system purification, the eluent A is a NaAc-HAc buffer solution with a concentration of 20 to 50 mM and a pH of 5.4 to 5.6 containing 0.5 to 1.0 M NaCl;

[0065] In the elution step 2 of the affinity chromatography system purification, the eluent B is a NaAc-HAc buffer with a concentration of 20 to 50 mM and a pH of 5.4 to 5.6;

[0066] In the elution step of the affinity chromatography system purification, the eluent is a NaAc-HAc buffer with a concentration of 20-50 mM and a pH of 3.5-3.7, and the pH adjustment solution is a Tris buffer with a concentration of 1.0-2.0 M.

[0067] In one embodiment of the present invention, the deep filtration purification comprises the following steps:

[0068] Flushing: flush the membrane package with purified water, volume ≥ 100L / m 2 , pump speed 200~600LMH;

[0069] Balance: flush the membrane package with the balance solution until the pH and conductivity of the effluent are consistent with those of the solution, and the pump speed is ≤100LMH;

[0070] Loading: Adjust the pH of affinity collection solution to 5.25-6.25 with pH adjustment solution and load the sample. The pump speed is ≤100LMH.

[0071] Post-balance: flush the membrane package with balance solution, volume ≥ 30L / m 2 , pump speed ≤100LMH.

[0072] In one embodiment of the present invention, the deep filtration purification comprises the following steps:

[0073] Flushing: flush the membrane package with purified water at a volume of 100-120 L / m 2 , pump speed 200~600LMH;

[0074] Balance: flush the membrane package with the balance solution until the pH and conductivity of the effluent are consistent with those of the solution, and the pump speed is 80-100LMH;

[0075] Loading: Adjust the pH of affinity collection solution to 5.5-6.0 with pH adjustment solution and load the sample at a pump speed of 80-100 LMH;

[0076] Post-balance: flush the membrane package with balance solution at a volume of 30-50 L / m 2 , pump speed 80~100LMH.

[0077] In one embodiment of the present invention, in the equilibration step of deep filtration purification, the equilibration solution is a NaAc-HAc buffer with a concentration of 10 to 100 mM and a pH of 5.25 to 6.25;

[0078] In the loading step of deep filtration purification, the pH adjustment solution is a Tris buffer with a concentration of 1.0 to 2.0 M.

[0079] In one embodiment of the present invention, in the equilibration step of deep filtration purification, the equilibration solution is a NaAc-HAc buffer with a concentration of 20 to 50 mM and a pH of 5.5 to 6.0;

[0080] In the loading step of deep filtration purification, the pH adjustment solution is a Tris buffer with a concentration of 1.0 to 2.0 M.

[0081] In one embodiment of the present invention, the purification method uses a membrane chromatography device to purify the monoclonal antibody in the feed solution; the membrane chromatography device comprises an affinity chromatography system and an improved cationic membrane chromatography system connected in series; the improved cationic membrane chromatography system is a cationic membrane chromatography system connected to an anionic chromatography membrane before the sample loading pipeline;

[0082] Alternatively, the purification method uses a membrane chromatography device to purify the monoclonal antibody in the feed solution; the membrane chromatography device includes an affinity chromatography system, a deep filtration device and an improved cationic membrane chromatography system connected in series in sequence; the improved cationic membrane chromatography system is a cationic membrane chromatography system connected to an anionic chromatography membrane before the sample loading pipeline.

[0083] In one embodiment of the present invention, the membrane chromatography device further comprises a feed liquid pool, an affinity collection pool and a cation collection liquid; the membrane chromatography device comprises a cell feed liquid pool, an affinity chromatography system, an affinity collection pool, an improved cationic membrane chromatography system and a cation collection liquid connected in series in sequence;

[0084] Alternatively, the membrane chromatography equipment further comprises a feed liquid pool, an affinity collection pool, a deep collection pool and a cation collection liquid; the membrane chromatography equipment comprises a cell feed liquid pool, an affinity chromatography system, an affinity collection pool, a deep filtration device, a deep collection pool, an improved cation membrane chromatography system and a cation collection liquid which are connected in series in sequence.

[0085] In one embodiment of the present invention, the feed liquid pool, affinity chromatography system, affinity collection pool, improved cationic membrane chromatography system and cationic collection liquid are connected by pipelines;

[0086] Alternatively, the feed liquid pool, affinity chromatography system, affinity collection pool, deep filtration device, deep collection pool, improved cationic membrane chromatography system and cationic collection liquid are connected through pipelines.

[0087] In one embodiment of the present invention, the affinity chromatography filler used in the affinity chromatography system includes Cityva-MabSelect SuRe LX and / or Nano-Nmab Pro; the anion chromatography membrane used in the improved cationic membrane chromatography system includes Sartorius-Sartobind Q, 3M-Polisher ST and / or Cobetter-Purcise Q; the cationic chromatography membrane used in the improved cationic membrane chromatography system includes Sartorius-Sartobind S.

[0088] In one embodiment of the present invention, the preparation method of the affinity chromatography system is: loading affinity chromatography filler on an AKTA avant150 chromatography system, an AKTApure 150 chromatography system, an SCG-P chromatography system or an SCG-100 chromatography system.

[0089] In one embodiment of the present invention, the preparation method of the improved cationic membrane chromatography system is: loading a cationic chromatography membrane on an AKTAavant 150 chromatography system, an AKTApure 150 chromatography system, an SCG-P chromatography system or an SCG-100 chromatography system, and connecting an anionic chromatography membrane in front of the sample loading pipeline of the chromatography system.

[0090] In one embodiment of the present invention, the target protein having an Fc structure comprises a monoclonal antibody.

[0091] The present invention also provides application of the purification method in preparing a target protein with an Fc structure.

[0092] In one embodiment of the present invention, the target protein having an Fc structure comprises a monoclonal antibody.

[0093] The present invention also provides a target protein with an Fc structure, and the target protein with an Fc structure is prepared by the above purification method.

[0094] In one embodiment of the present invention, the target protein having an Fc structure comprises a monoclonal antibody.

[0095] The technical solution of the present invention has the following advantages:

[0096] The present invention provides a method for purifying a target protein with an Fc structure, the purification method comprising two steps of antibody capture and antibody purification; the antibody capture is: purifying the feed liquid through an affinity chromatography system to obtain an affinity collection liquid; the antibody purification is: purifying the affinity collection liquid through an improved cationic membrane chromatography system to obtain a cationic collection liquid; the antibody purification step is performed on an improved cationic membrane chromatography system; the improved cationic membrane chromatography system is a cationic membrane chromatography system connected to an anionic chromatography membrane before a sample loading pipeline. The improved cationic membrane chromatography system used in the purification method can efficiently remove host impurities and has a higher ability to control aggregates. In the purification method of the present invention, the affinity chromatography step is responsible for rapidly capturing proteins, and the improved cationic membrane chromatography step is responsible for providing host impurity removal and aggregate control.

[0097] Experiments have shown that, compared with the traditional two-step chromatography method (affinity chromatography + anion chromatography / cation chromatography), the purification method of the present invention (affinity chromatography + improved cationic membrane chromatography) requires a greatly reduced process time while providing effective impurity removal capabilities. In the purification method of the present invention, the speed of the entire chromatography step (5 membrane volumes per minute) is 50 times that of the traditional two-step chromatography method (5 minutes per column volume), which can not only provide qualified products, but also has a higher sample recovery rate than the traditional chromatography process; and, in the traditional two-step chromatography method (sample → affinity chromatography → deep filtration → anion column chromatography pH 6.2 → cation column chromatography pH 5.3), due to the different types and pH of buffers for anion chromatography and cation chromatography, the sample needs to adjust the pH and replace the buffer after the anion chromatography is completed, while the purification method of the present invention (sample → affinity chromatography → deep filtration → anion membrane pH 5.5 + cation membrane pH 5.5) does not need to adjust the sample pH and replace the buffer; in addition, the purification method of the present invention also has the advantage of a large sample loading capacity of the anion chromatography membrane.

[0098] Furthermore, the improved cationic membrane chromatography system purification includes a loading step; the loading step includes: using affinity collection solution or deep collection solution for loading; when loading, the loading capacity of the cationic chromatography membrane is 18-35 g / L, and the loading capacity of the anionic membrane is 1-1200 g / L; the loading conditions of the affinity collection solution or deep collection solution are: pH 5.25-6.25. Under these parameters, the purification effect of the purification method of the present invention is better.

[0099] In one embodiment of the present invention, in the loading step, when loading, the loading capacity of the cationic chromatographic membrane is 18-32 g / L, and the loading capacity of the anionic membrane is 10-1000 g / L; the loading conditions of the affinity collection solution or the deep collection solution are: pH 5.5-6.25. Under this parameter, the purification effect of the purification method of the present invention is better.

[0100] Furthermore, in the loading step, when loading, the loading capacity of the cationic chromatographic membrane is 20-30 g / L, and the loading capacity of the anionic membrane is 100-1000 g / L; the loading conditions of the affinity collection solution or the deep collection solution are: pH 5.5-6.0. Under these parameters, the purification effect of the purification method of the present invention is the best. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 : Diagram of the structure of membrane chromatography equipment for the traditional two-step chromatography method.

[0102] Figure 2 : Improved membrane chromatography equipment structure diagram.

[0103] Figure 3 : Chromatographic spectra of cationic membrane loading 40.3 g / L and loading 35.3 g / L in Experiment 2 (Experiment 1 and Experiment 2). Figure 3 In the figure, the sample flows through at the red mark.

[0104] Figure 4 : Chromatographic spectrum of cationic membrane loading 28.6 g / L in Experiment 2 (Experiment 3).

[0105] Figure 5 : Chromatographic spectrum of cationic membrane loading 20.2 g / L in Experiment 2 (Experiment 4).

[0106] Figure 6 : Chromatographic spectrum of cationic membrane loading 15.1 g / L in Experimental Example 2 (Experiment Five).

[0107] Figure 7 : Chromatographic spectrum of Experiment 1 in Experimental Example 3 (loading pH 5.01 / conductivity 2.774 mS / cm). Figure 7 In the figure, the red mark is the UV280 absorption peak of the sample flow-through.

[0108] Figure 8 : Chromatographic spectrum of Experiment 2 in Experimental Example 3 (loading pH 5.52 / conductivity 3.566 mS / cm).

[0109] Fig. 9 : Chromatographic spectrum of Experiment 3 (loading pH 6.03 / conductivity 3.255 mS / cm) in Experimental Example 3. DETAILED DESCRIPTION

[0110] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0111] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0112] Example 1: A method for purifying a monoclonal antibody

[0113] This example provides a method for purifying a monoclonal antibody. Figure 2 (The improved membrane chromatography equipment used in the purification method is composed of a cell feed liquid pool, an affinity chromatography system, an affinity collection pool, an improved cationic membrane chromatography system and a cationic collection liquid which are sequentially connected in series through a pipeline, wherein the improved cationic membrane chromatography system is a cationic membrane chromatography system connected to an anionic chromatography membrane before the sample loading pipeline), and the purification method comprises the following steps:

[0114] 1. Antibody capture (completed in affinity chromatography system)

[0115] Rinse: rinse the column with purified water for 2 column volumes, retention time 5 min;

[0116] Cleaning: Wash the column with 0.1 M NaOH for 3 column volumes, retention time 10 min;

[0117] Rinse: rinse the column with purified water for 2 column volumes, retention time 5 min;

[0118] Balance: flush the column with 50 mM Tris-HAc buffer containing 150 mM NaCl, pH 7.4 until the pH and conductivity of the effluent are consistent with those of the solution, and the retention time is 5 min;

[0119] Sample loading: Sample loading under the condition of retention time 5min;

[0120] Equilibration: Wash the column with 3 column volumes of 50 mM Tris-HAc buffer containing 150 mM NaCl, pH 7.4, retention time 5 min;

[0121] Wash 1: Wash with 3 column volumes of NaAc-HAc buffer containing 1 M NaCl at a concentration of 50 mM and pH 5.5, with a retention time of 5 min;

[0122] Wash 2: Wash with 50 mM NaAc-HAc buffer at pH 5.5 for 3 column volumes, retention time 5 min;

[0123] Elution: Use 50mM NaAc-HAc buffer with pH3.6 for 5 column volumes, retention time 5min, collection conditions 100-100mAU / 2mm, after sample elution, adjust pH to 5.2±0.1 with 1M Tris buffer, and name the collected sample as affinity collection solution.

[0124] 2. Antibody purification (completed in improved cationic membrane chromatography system)

[0125] Rinse: rinse the column with purified water for 2 column volumes, retention time 0.1 min;

[0126] Cleaning: Wash the column with 0.5 M NaOH for 10 column volumes, retention time 0.1 min;

[0127] Pre-equilibration: 3 column volumes of NaAc-HAc buffer containing 1 M NaCl at a concentration of 50 mM and pH 5.5, with a retention time of 0.1 min;

[0128] Balance: flush with 50mM NaAc-HAc buffer at pH 5.5 until the pH and conductivity of the effluent are consistent with those of the solution, with a retention time of 0.1min;

[0129] Loading: The affinity collection solution was adjusted to pH 5.5 with 1M Tris buffer and then loaded (the loading sample pH was 5.5, the conductivity was <5mS / cm), and the retention time was 0.2min; when loading, the loading capacity of the cation chromatography membrane was 28.8g / L, and the loading capacity of the anion membrane was 301.2g / L;

[0130] Balance: flush with 50 mM NaAc-HAc buffer at pH 5.5 until the pH and conductivity of the effluent are consistent with those of the solution, with a retention time of 0.2 min;

[0131] Elution: Use eluent A (50 mM NaAc-HAc buffer with pH 5.5) and eluent B (50 mM NaAc-HAc buffer containing 1 M NaCl with pH 5.5) for elution, wherein the elution volume of eluent B is increased from 0% to 50% for gradient elution, and a total of 20 membrane volumes are eluted (the gradient elution setting can improve the resolution of protein impurity removal), the starting collection condition is 100 mAU / mm—MAX—the collection end condition is 250 mAU / mm, and the retention time is 0.2 min.

[0132] Comparative Example 1: A method for purifying a monoclonal antibody

[0133] This comparative example provides a method for purifying a monoclonal antibody. Figure 1 , the purification method comprises the following steps:

[0134] 1. Antibody capture (completed in affinity chromatography system)

[0135] Rinse: rinse the column with purified water for 2 column volumes, retention time 5 min;

[0136] Cleaning: Wash the column with 0.1 M NaOH for 3 column volumes, retention time 10 min;

[0137] Rinse: rinse the column with purified water for 2 column volumes, retention time 5 min;

[0138] Balance: flush the column with 50 mM Tris-HAc buffer containing 150 mM NaCl, pH 7.4 until the pH and conductivity of the effluent are consistent with those of the solution, and the retention time is 5 min;

[0139] Sample loading: Sample loading under the condition of retention time 5min;

[0140] Equilibration: Wash the column with 3 column volumes of 50 mM Tris-HAc buffer containing 150 mM NaCl, pH 7.4, retention time 5 min;

[0141] Wash 1: Wash with 3 column volumes of NaAc-HAc buffer containing 1 M NaCl at a concentration of 50 mM and pH 5.5, with a retention time of 5 min;

[0142] Wash 2: Wash with 50 mM NaAc-HAc buffer at pH 5.5 for 3 column volumes, retention time 5 min;

[0143] Elution: Use 50mM NaAc-HAc buffer with pH3.6 for elution for 5 column volumes, retention time 5min, collection conditions 100-100mAU / 2mm, after sample elution, adjust pH to 5.2±0.1 with 1M Tris buffer, and name the collected sample as affinity collection solution.

[0144] 2. Antibody Purification

[0145] 2.1. Anion chromatography (completed in anion chromatography system, column chromatography)

[0146] Cleaning: Use 0.5M NaOH solution to clean 3 column volumes, linear flow rate 100cm / h, cleaning for 40min;

[0147] Pre-equilibrium: flush 3 column volumes with 200 mM PB solution, pH 5.9, linear flow rate 100 cm / h;

[0148] Balance: flush the column with 20 mM, pH 6.2 PB solution until the pH and conductivity of the effluent are consistent with those of the solution, with a linear flow rate of 100 cm / h;

[0149] Sample loading: After adjusting the pH of the affinity collection solution to 6.2 with 1M Tris buffer, the sample was loaded at a linear flow rate of 100 cm / h with a loading capacity of 38 g / L. When the UV detection reached 50 mAU / mm, the flow-through sample was collected.

[0150] Balance: Wash the column with 20mM PB solution at pH 6.2 for 3 column volumes, collect when UV reaches 50mAU / mm, adjust pH to 5.3 with 1M citric acid solution, and name the collected sample as anion flow-through;

[0151] 2.2. Cationic chromatography (performed in a cationic chromatography system, column chromatography)

[0152] Cleaning: Use 0.5M NaOH solution to clean 3 column volumes, linear flow rate 100cm / h, cleaning for 40min;

[0153] Pre-equilibrium: flush 3 column volumes with 200 mM PB solution, pH 5.9, linear flow rate 100 cm / h;

[0154] Balance: flush the column with 20 mM NaAc-HAc buffer at pH 5.3, with a linear flow rate of 120 cm / h;

[0155] Sample loading: Anion flow-through was used for sample loading, with a linear flow rate of 120 cm / h and a loading capacity of 39 g / L;

[0156] Balance: flush the column with 20 mM NaAc-HAc buffer at pH 5.3 for 3 column volumes at a linear flow rate of 120 cm / h;

[0157] Elution: flush the column volume with eluent (PB solution with a concentration of 100 mM and pH 6.2 containing 250 mM NaCl) and collect samples. The starting collection condition is 1450 mAU / mm—MAX—the ending collection condition is 600 mAU / mm. The pH of the sample is adjusted to 6.2±0.1 with 1 M Tris buffer, and the linear flow rate is ≤150 cm / h.

[0158] Experimental Example 1: Performance Verification of Purification Method

[0159] This experimental example verifies the performance of the purification method in Example 1 and the comparative example, and the specific process is as follows:

[0160] The fermentation liquid of the anti-VEGF monoclonal antibody (purchased from Dongyao Pharmaceutical, obtained by shaking flask culture and fed batch culture of CHO cells containing VEGF antibody bevacizumab) was used as a sample, and the purification methods in Example 1 and Comparative Example 1 were used for purification, and the purified products were detected with reference to the methods recorded in the pharmacopoeia (aggregate detection was detected by HPLC-SEC method, HCP host residue detection was detected by Elisa method, and the Elisa method was performed using the F550-1 kit purchased from Cygnus Company). The test results are shown in Table 1;

[0161] Among them, the purification method of comparative example 1 uses an AKTA chromatography system, and in the purification method of comparative example 1, the chromatography column of the affinity chromatography system uses Cityva-MabSelect SuRe LX filler, the chromatography column of the anion chromatography system uses Cityva-QFF filler, and the chromatography column of the cationic chromatography system uses Merck-Fractogel EMD COO filler;

[0162] The purification method of Example 1 uses an AKTA chromatography system, and in the purification method of Example 1, the chromatography column of the affinity chromatography system uses Cityva-MabSelect SuRe LX filler, the cationic membrane chromatography system is loaded with Sartorius-Sartobind S cationic chromatography membrane, and the sample loading pipeline is connected to a Sartorius-Sartobind Q anionic chromatography membrane (during purification, in the loading step of the antibody purification process, the pH of the loaded sample is 5.5, and the conductivity is 1.526 mS / cm, which meets the loading requirements).

[0163] From the purification process, it can be seen that in the antibody purification step of Comparative Example 1, the anion chromatography takes 4.8 hours, the cation chromatography takes 4 hours, and the antibody purification step of Example 1 takes a total of 40 minutes. The process time required for the purification method of Example 1 is greatly reduced. As can be seen from Table 1, under the premise that the product quality meets the standard, the conventional process of Comparative Example 1 requires two-step chromatography, the buffer solution during the anion column chromatography is 6.2, and the buffer solution after the cationic membrane is 5.3. Due to the different pH of the buffer solution, the cationic chromatography cannot be directly performed after the anion column chromatography is completed, and both the anion column chromatography and the cationic column chromatography require a complete set of chromatography systems, which is inefficient and will cause feed liquid loss. There is no way to proceed in one step, and the time consumption is 13.2 times that of the improved process; while the improved process of Example 1 only requires a one-step chromatography process, does not require the replacement of the buffer solution, and only requires a set of chromatography system connection, that is, the anion chromatography membrane is connected to the sample loading pipeline through the chromatography column connector, and the cationic chromatography membrane is connected to the column position of the chromatography system, and the SEC of the cationic collection liquid is 98.8%, the HCP is 91ppm, and the yield is 89.3%, which meets the standard. In addition, the loading capacity of the anion membrane of Example 1 reaches 301.2g / L, which is nearly 8 times that of Comparative Example 1, and the purification flux and efficiency are significantly improved. In summary, compared with the traditional process of Comparative Example 1, the improved process of Example 1 requires a simpler solution and does not require sample adjustment due to changes in the chromatography steps, thereby reducing the risks caused by sample adjustment.

[0164] Table 1 Purification results

[0165]

[0166] The experimental results need to meet the following standards: quality standard: SEC ≥ 95%, HCP < 100ppm; process standard: Yield ≥ 85%.

[0167] Example 2: A method for purifying a monoclonal antibody

[0168] This example provides a method for purifying a monoclonal antibody. Figure 2 (The improved membrane chromatography equipment used in the purification method is composed of a cell feed liquid pool, an affinity chromatography system, an affinity collection pool, a deep filtration device, a deep collection pool, an improved cationic membrane chromatography system and a cationic collection liquid which are sequentially connected in series through a pipeline, wherein the improved cationic membrane chromatography system is a cationic membrane chromatography system connected to an anion chromatography membrane before the sample loading pipeline), and the purification method comprises the following steps:

[0169] 1. Antibody capture (completed in affinity chromatography system)

[0170] Rinse: rinse the column with purified water for 2 column volumes, retention time 5 min;

[0171] Cleaning: Wash the column with 0.1 M NaOH for 3 column volumes, retention time 10 min;

[0172] Rinse: rinse the column with purified water for 2 column volumes, retention time 5 min;

[0173] Balance: flush the column with 50 mM Tris-HAc buffer containing 150 mM NaCl, pH 7.4 until the pH and conductivity of the effluent are consistent with those of the solution, and the retention time is 5 min;

[0174] Sample loading: Sample loading under the condition of retention time 5min;

[0175] Equilibration: Wash the column with 3 column volumes of 50 mM Tris-HAc buffer containing 150 mM NaCl, pH 7.4, retention time 5 min;

[0176] Wash 1: Wash with 3 column volumes of NaAc-HAc buffer containing 1 M NaCl at a concentration of 50 mM and pH 5.5, with a retention time of 5 min;

[0177] Wash 2: Wash with 50 mM NaAc-HAc buffer at pH 5.5 for 3 column volumes, retention time 5 min;

[0178] Elution: Use 50mM NaAc-HAc buffer with pH3.6 for 5 column volumes, retention time 5min, collection conditions 100-100mAU / 2mm, after sample elution, adjust pH to 5.2±0.1 with 1M Tris buffer, and name the collected sample as affinity collection solution.

[0179] 2. Deep filtration (completed on the filter membrane package)

[0180] Rinse: Rinse the membrane package with purified water at a volume of 100L / m 2 , pump speed 200LMH;

[0181] Balance: flush the membrane package with 50mM NaAc-HAc buffer at pH 5.5 until the pH and conductivity of the effluent are consistent with those of the solution, and the pump speed is 100LMH;

[0182] Sample loading: adjust the pH of affinity collection solution to 5.5 with 1M Tris buffer and load the sample at a pump speed of 100LMH;

[0183] Post-equilibrium: flush the membrane package with 50 mM NaAc-HAc buffer at a volume of 30 L / m 2 , pump speed 100LMH, the collected sample was named deep collection liquid.

[0184] 3. Antibody purification (completed in improved cationic membrane chromatography system)

[0185] Cleaning: 40 membrane volumes were cleaned with 0.5 M NaOH solution, retention time 0.2 min;

[0186] Pre-equilibration: flush 20 membrane volumes with 50 mM NaAc-HAc buffer, pH 5.5, containing 1 M NaCl, retention time 0.1 min;

[0187] Balance: flush with 50mM NaAc-HAc buffer at pH 5.5 until the pH and conductivity of the effluent are consistent with those of the solution, with a retention time of 0.1min;

[0188] Loading: Use deep collection solution for loading (sample pH 5.5, conductivity <5mS / cm), retention time 0.2min; when loading, the loading capacity of the cation chromatography membrane is 35g / mL, and the loading capacity of the anion membrane is 550g / L;

[0189] Balance: flush with 50 mM NaAc-HAc buffer at pH 5.5 until the pH and conductivity of the effluent are consistent with those of the solution, with a retention time of 0.2 min;

[0190] Elution: Use eluent A (50 mM NaAc-HAc buffer with pH 5.5) and eluent B (50 mM NaAc-HAc buffer containing 1 M NaCl with pH 5.5) for elution, wherein the elution volume of eluent B is increased from 0% to 50% for gradient elution, and a total of 20 membrane volumes are eluted (the gradient elution setting can improve the resolution of protein impurity removal), the starting collection condition is 100 mAU / mm—MAX—the collection end condition is 250 mAU / mm, and the retention time is 0.2 min.

[0191] Experimental Example 2: Effect of loading capacity on purification effect

[0192] This experimental example explores the effect of loading capacity on purification effect. The specific process is as follows:

[0193] The fermentation liquid of anti-VEGF monoclonal antibody (purchased from Dongyao Pharmaceutical, obtained by shaking flask culture and fed batch culture of CHO cells containing VEGF antibody bevacizumab) was used as a sample, and the purification method in Example 2 was used for purification, and the purified product was detected with reference to the method described in the pharmacopoeia (aggregate detection was detected by HPLC-SEC method, HCP host residue detection was detected by Elisa method, and Elisa method was performed using F550-1 kit purchased from Cygnus Company). The test results are shown in Tables 2 and Figure 3 to Figure 6; The loading capacity for each purification was set as follows:

[0194] Experiment 1: Cationic membrane loading 40.3 g / L, anionic membrane loading 1006.4 g / L;

[0195] Experiment 2: Cationic membrane loading 35.3 g / L, anionic membrane loading 1006.4 g / L;

[0196] Experiment 3: Cationic membrane loading 28.6 g / L, anionic membrane loading 301.2 g / L;

[0197] Experiment 4: Cationic membrane loading 20.2 g / L, anionic membrane loading 1006.4 g / L;

[0198] Experiment 5: Cationic membrane loading 15.1 g / L, anionic membrane loading 100.4 g / L;

[0199] Among them, the purification method of Example 2 uses an AKTA chromatography system, and in the purification method of Example 2, the chromatography column of the affinity chromatography system uses Nano-Nmab Pro filler, the deep filtration uses Cobetter Cat:CDFCDCSDH2HPPCP membrane package, the cationic membrane chromatography system is loaded with Sartorius-Sartobind S cationic chromatography membrane, and the Sartorius-Sartobind Q anionic chromatography membrane is connected to the sample loading pipeline, and the experimental loading amount is controlled by controlling the loading volume.

[0200] From Table 2 and Figure 3 to Figure 6 It can be seen that Figure 3 Corresponding to Experiment 1 and Experiment 2, Figures 4 to 6 Corresponding to Experiments 3 to 5, respectively, in Experiment 5, when the cation loading was 15.1 g / L, the sample yield was <85%, and in Experiment 1, when the cation loading was 40.3 g / L, the sample had flowed through (see Figure 3 The above two loading conditions do not meet the experimental expectations. According to Experiments 2, 3, and 4, the cation loading range is determined to be 20-35 g / L, the safety factor is 0.85, and the final cation loading range is 20-30 g / L. In addition, the recovery rate of anion membrane chromatography from Experiments 1 to 5 is between 100% and 102.4%, and the recovery rate is high and stable. The anion yields of Experiments 2 and 5 are 100.9% and 101.4%, respectively, indicating that the change in anion membrane loading has almost no effect on the overall yield, so the anion membrane loading range is set to 100-1000 g / L. In addition, the cation loading is independent of the anion loading, and the cation loading depends on the sample flow-through and yield.

[0201] Table 2 Purification results

[0202]

[0203] NA: stands for not tested; the sample in experiment one has flowed through, and the yield in experiment five is low. Both groups did not meet expectations and were not tested.

[0204] The experimental results need to meet the following standards: quality standard: SEC ≥ 95%, HCP < 100ppm; process standard: Yield ≥ 85%.

[0205] Experimental Example 3: Effects of equilibrium and loading conditions on purification results

[0206] This experimental example explores the effects of equilibrium and loading conditions on purification effects. The specific process is as follows:

[0207] The fermentation liquid of anti-VEGF monoclonal antibody (purchased from Dongyao Pharmaceutical, obtained by shaking flask culture and fed batch culture of CHO cells containing VEGF antibody bevacizumab) was used as a sample, and the purification method in Example 2 was used for purification, and the purified product was detected with reference to the method described in the pharmacopoeia (aggregate detection was detected by HPLC-SEC method, HCP host residue detection was detected by Elisa method, and Elisa method was performed using F550-1 kit purchased from Cygnus Company). The test results are shown in Tables 3 and Figure 7 to Figure 9 ; During each purification, the equilibration solution, pre-equilibration solution and loading conditions in the antibody purification step were set as follows:

[0208] Experiment 1:

[0209] Balance solution: NaAc-HAc buffer with a concentration of 50 mM and pH 5.0;

[0210] Pre-equilibrium solution (regeneration solution): NaAc-HAc buffer with a concentration of 50 mM and pH 5.0 containing 1 M NaCl;

[0211] Loading conditions: pH 5.01 / conductivity 2.774 mS / cm;

[0212] Experiment 2:

[0213] Equilibrium solution: NaAc-HAc buffer with a concentration of 50 mM and pH 5.5;

[0214] Pre-equilibrium solution (regeneration solution): NaAc-HAc buffer with a concentration of 50 mM and pH 5.5 containing 1 M NaCl;

[0215] Loading conditions: pH 5.52 / conductivity 3.566 mS / cm;

[0216] Experiment 3:

[0217] Equilibrium solution: NaAc-HAc buffer with a concentration of 50 mM and pH 6.0;

[0218] Pre-equilibrium solution (regeneration solution): NaAc-HAc buffer with a concentration of 50 mM and pH 6.0 containing 1 M NaCl;

[0219] Loading conditions: pH 6.03 / conductivity 3.255 mS / cm;

[0220] Among them, the purification method of Example 2 uses an AKTA chromatography system, and in the purification method of Example 2, the chromatography column of the affinity chromatography system uses Nano-Nmab Pro filler, the deep filtration uses Cobetter Cat:CDFCDCSDH2HPPCP membrane package, the cationic membrane chromatography system is loaded with Sartorius-Sartobind S cationic chromatography membrane, and the Sartorius-Sartobind Q anionic chromatography membrane is connected to the sample loading pipeline. The pH value of the sample is adjusted using a 1M HAc solution and a 1M Tris solution.

[0221] From Table 3 and Figure 7 to Figure 9 It can be seen that the sample has flow-through under the loading conditions of pH 5.0 and conductivity <5mS / cm (see Figure 7 The red mark indicates that the sample did not flow through when the pH value was 6.0 and the conductivity was less than 5 mS / cm. Therefore, the final sample loading conditions were pH 5.5-6.0 and the conductivity was less than 5 mS / cm.

[0222] Table 3 Purification results

[0223]

[0224] Experimental Example 4: Verification of the universality of the purification method

[0225] This experimental example verifies the universality of the purification method. The specific process is as follows:

[0226] Experiment 1: The fermentation liquid of anti-HER2 monoclonal antibody (purchased from Dongyao Pharmaceutical, obtained by shaking flask culture and fed batch culture of CHO cells containing HER2 trastuzumab) was used as a sample, and purified using the purification method in Example 2, and the purified product was detected with reference to the method recorded in the pharmacopoeia (aggregate detection was detected by HPLC-SEC method, HCP host residue detection was detected by Elisa method, and the Elisa method was performed using F550-1 kit purchased from Cygnus Company). The test results are shown in Table 4;

[0227] Among them, the purification method of Example 2 uses an AKTA chromatography system, and in the purification method of Example 2, the chromatography column of the affinity chromatography system uses Nano-Nmab Pro filler, the deep filtration uses Cobetter Cat:CDFCDCSDH2HPPCP membrane package, the cationic membrane chromatography system is loaded with Sartorius-Sartobind S cationic chromatography membrane, and the Sartorius-Sartobind Q anionic chromatography membrane is connected before the sample loading pipeline.

[0228] Experiment 2: Using the fermentation broth of anti-ILT2 / 4 monoclonal antibody (purchased from Dongyao Pharmaceutical, obtained by shaking flask culture and fed batch culture of CHO cells containing HER2 trastuzumab, the monoclonal antibody sequence is shown in PR302264-2 of the patent application text with publication number WO2024120526A1) as a sample, the purification method in Example 2 was used for purification, and the purified product was detected with reference to the method recorded in the pharmacopoeia (aggregate detection was detected by HPLC-SEC method, HCP host residue detection was detected by Elisa method, and the Elisa method was performed using the F550-1 kit purchased from Cygnus). The test results are shown in Table 4;

[0229] Among them, the purification method of Example 2 uses an AKTA chromatography system, and in the purification method of Example 2, the chromatography column of the affinity chromatography system uses Nano-Nmab Pro filler, the deep filtration uses Cobetter Cat:CDFCDCSDH2HPPCP membrane package, the cationic membrane chromatography system is loaded with Sartorius-Sartobind S cationic chromatography membrane, and the Sartorius-Sartobind Q anionic chromatography membrane is connected before the sample loading pipeline.

[0230] It can be seen from Table 4 that, in addition to anti-VEGF monoclonal antibody, the purification method based on the improved cationic membrane chromatography system is also applicable to anti-HER2 monoclonal antibody and anti-ILT2 / 4 monoclonal antibody. It can be seen that the purification method based on the improved cationic membrane chromatography system has a certain universality and can be used for the purification of a variety of monoclonal antibodies.

[0231] Table 4 Purification results

[0232]

[0233] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for purifying a target protein having an Fc structure, characterized in that: The purification method comprises the following steps: Antibody capture: The feed solution is purified by an affinity chromatography system to obtain an affinity collection solution; Antibody purification: The affinity collection solution is purified by an improved cationic membrane chromatography system to obtain a cationic collection solution; The antibody purification step is carried out on an improved cationic membrane chromatography system; the improved cationic membrane chromatography system is a cationic membrane chromatography system connected to an anionic chromatography membrane before the sample loading pipeline; Alternatively, the purification method comprises the steps of: Antibody capture: The feed solution is purified by an affinity chromatography system to obtain an affinity collection solution; Deep filtration: The affinity collection solution is purified by deep filtration to obtain a deep collection solution; Antibody purification: The deep collection solution is purified by an improved cationic membrane chromatography system to obtain a cationic collection solution; The antibody purification step is carried out on an improved cationic membrane chromatography system; the improved cationic membrane chromatography system is a cationic membrane chromatography system in which an anionic chromatography membrane is connected before the sample loading pipeline.

2. The purification method according to claim 1, characterized in that The improved cationic membrane chromatography system purification includes a sample loading step; The loading step comprises: using affinity collection solution or deep collection solution for loading; when loading, the loading capacity of the cation chromatography membrane is 18-35 g / L, and the loading capacity of the anion membrane is 1-1200 g / L; the loading conditions of the affinity collection solution or deep collection solution are: pH 5.25-6.

25.

3. The purification method according to claim 1 or 2, characterized in that The improved cationic membrane chromatography system purification comprises the following steps: Cleaning: Use cleaning solution to clean at least 10 membrane volumes with a retention time of at least 0.1 min; Pre-equilibrium: flush at least 2 membrane volumes with pre-equilibrium solution, with a retention time of at least 0.1 min; Balance: flush with balance solution until the pH and conductivity of the effluent are consistent with those of the solution, and the retention time is at least 0.1 min; Sample loading: Use affinity collection solution or deep collection solution for sample loading, with a retention time of at least 0.1 min; when loading, the loading capacity of the cation chromatography membrane is 18-35 g / L, and the loading capacity of the anion membrane is 1-1200 g / L; the loading conditions of the affinity collection solution or deep collection solution are: pH 5.25-6.25, conductivity <5 mS / cm; Balance: Use balance solution to rinse until the pH and conductivity of the effluent are consistent with the solution, and the retention time is at least 0.1 min; Elution: first use eluent A for elution, then use eluent B for gradient elution, eluting a total of 20 to 25 membrane volumes, the collection conditions of the cation collection solution are 100 to 250 mAU / mm, and the retention time is at least 0.1 min.

4. The purification method according to claim 3, characterized in that In the cleaning step of the improved cationic membrane chromatography system purification, the cleaning solution is a NaOH solution with a concentration of 0.1 to 0.5 M; In the pre-equilibrium step of the purification of the improved cationic membrane chromatography system, the pre-equilibrium solution is a NaAc-HAc buffer solution with a concentration of 10 to 100 mM and a pH of 5.25 to 6.25 containing 0.1 to 0.5 M NaCl; In the equilibration step of the improved cationic membrane chromatography system purification, the equilibration solution is a NaAc-HAc buffer solution with a concentration of 10 to 100 mM and a pH of 5.25 to 6.25; In the elution step of the improved cationic membrane chromatography system purification, the eluent A is a NaAc-HAc buffer with a concentration of 10-100 mM and a pH of 5.25-6.25, and the eluent B is a NaAc-HAc buffer with a concentration of 10-100 mM and a pH of 5.25-6.25 containing 0.7-1.2 M NaCl.

5. The purification method according to any one of claims 1 to 4, characterized in that: The affinity chromatography system purification comprises the following steps: Rinse: Rinse the column with purified water for at least 1 column volume, with a retention time of at least 5 minutes; Cleaning: Use at least 3 column volumes of cleaning solution and the retention time should be at least 5 minutes; Rinse: Rinse the column with purified water for at least 1 column volume, with a retention time of at least 5 minutes; Balance: flush the column with the balance solution until the pH and conductivity of the effluent are consistent with those of the solution, and the retention time is at least 5 minutes; Sample loading: Sample loading with a retention time of at least 5 min; Equilibration: flush the column with equilibration solution for at least 3 column volumes, with a retention time of at least 5 min; Elution 1: Elute with eluent A for at least 3 column volumes with a retention time of at least 5 min. Wash 2: Wash with eluent B for at least 3 column volumes with a retention time of at least 5 min. Elution: elute with eluent for at least 3 column volumes, with a retention time of at least 5 minutes, and the collection conditions of the affinity collection solution are 100-100 mAU / 2 mm. The pH of the collected affinity collection solution is adjusted to 5.25-6.25 using a pH adjusting solution.

6. The purification method according to claim 5, characterized in that In the cleaning step of the affinity chromatography system purification, the cleaning solution is a NaOH solution with a concentration of 0.1 to 0.2 M; In the equilibration step of the affinity chromatography system purification, the equilibration solution is a Tris-HAc buffer with a concentration of 10 to 100 mM and a pH of 7.2 to 7.6, containing 80 to 170 mM NaCl; In the elution step 1 of the affinity chromatography system purification, the eluent A is a NaAc-HAc buffer solution with a concentration of 10 to 100 mM and a pH of 5.2 to 5.8 containing 0.5 to 1.0 M NaCl; In the elution step 2 of the affinity chromatography system purification, the eluent B is a NaAc-HAc buffer with a concentration of 10 to 100 mM and a pH of 5.2 to 5.8; In the elution step of the affinity chromatography system purification, the eluent is a NaAc-HAc buffer with a concentration of 10-100 mM and a pH of 3.4-3.8, and the pH adjustment solution is a Tris buffer with a concentration of 1.0-2.0 M.

7. The purification method according to any one of claims 1 to 6, characterized in that: The deep filtration purification comprises the following steps: Flushing: flush the membrane package with purified water, volume ≥ 100L / m 2 , pump speed 200~600LMH; Balance: flush the membrane package with the balance solution until the pH and conductivity of the effluent are consistent with those of the solution, and the pump speed is ≤100LMH; Loading: Adjust the pH of affinity collection solution to 5.25-6.25 with pH adjustment solution and load the sample. The pump speed is ≤100LMH. Post-balance: flush the membrane package with balance solution, volume ≥ 30L / m 2 , pump speed ≤100LMH.

8. The purification method according to claim 7, characterized in that In the equilibration step of deep filtration purification, the equilibration solution is a NaAc-HAc buffer solution with a concentration of 10 to 100 mM and a pH of 5.25 to 6.25; In the loading step of deep filtration purification, the pH adjustment solution is a Tris buffer with a concentration of 1.0 to 2.0 M.

9. Use of the purification method according to any one of claims 1 to 8 in preparing a target protein having an Fc structure.

10. A target protein having an Fc structure, characterized in that: The target protein having an Fc structure is prepared by the purification method according to any one of claims 1 to 8.

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