Method for purifying antibody by using cation exchange chromatography and application thereof

Abstract

The invention provides a method for purifying an antibody by using cation exchange chromatography and application of the method, the method comprises the following steps: carrying out cation exchange chromatography treatment on an antibody solution, the eluent adopted by the cation exchange chromatography treatment comprises 80-120mM of NaAc-HAc and 140-170mM of NaCl, and the pH value is 5.0-5.5; or, 250-370 mM of NaAc-HAc, and the pH value of the NaAc-HAc is 5.0-5.5. The antibody purification method provided by the invention can effectively solve the double-peak phenomenon in elution on the premise of ensuring the polymer removal capacity, and the method is stable and controllable in process flow, is suitable for industrial production, and lays a solid foundation for later large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of protein separation and purification, and specifically relates to a method for purifying antibodies by using cation exchange chromatography and an application thereof. Background Art

[0002] At present, in the field of antibody purification, cation exchange chromatography is widely used in antibody purification production as a method with low cost and strong ability to remove polymer impurities. Sodium chloride is widely used in cation exchange chromatography as a cheap and non-toxic material. After years of development, the basic development of cation exchange chromatography has tended to be standardized, but in the process of pursuing the improvement of polymer removal ability, double peaks often appear during the elution step due to the properties of the protein itself, changes in the loading capacity of the filler, or different resolutions of the cationic filler.

[0003] As an effective means of removing aggregates in antibody purification, the cation exchange chromatography binding mode often strictly controls the loading capacity of the filler, the pH value of the chromatography, and the molar concentration range of salt ions such as sodium chloride in the eluent. The working principle of the cation exchange chromatography binding mode is to adsorb the target protein and some impurities under pH conditions lower than the isoelectric point (PI) of the target protein and under conditions of lower ion concentration, and then elute the target protein by increasing the ionic strength in a specific eluent, while impurities such as aggregates are kept in a bound state as much as possible during the elution process. Because the protein itself is charged, it will be enriched to a certain extent during the binding process with the chromatography column. In the process of increasing the ionic strength in the buffer and leaving the chromatography column, the overall pH may fluctuate to a certain extent due to the insufficient buffering capacity of the buffer. The commonly used sodium chloride itself has no ability to buffer pH and cannot suppress the change of pH. This kind of fluctuation often causes the phenomenon of double peaks in the cation exchange chromatography during the elution process. During drug registration and production, the collection parameters of the entire production steps are strictly controlled. Automated program settings are generally used in the actual production process. Therefore, the double peak phenomenon in the elution process may cause deviations from the preset program, resulting in variables in the production process and huge economic losses.

[0004] Therefore, developing an antibody purification method that can solve the double peak phenomenon occurring during the elution process has important application value. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a method for purifying antibodies using cation exchange chromatography and its application. The present invention uses sodium acetate-acetic acid buffer to partially and completely replace sodium chloride in the elution buffer, thereby reducing the cation elution double peak phenomenon caused by insufficient buffering capacity.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for purifying an antibody using cation exchange chromatography, the method comprising: subjecting an antibody solution to cation exchange chromatography treatment, wherein the eluent used in the cation exchange chromatography treatment comprises: 80-120 mM NaAc-HAc, 140-170 mM NaCl, and a pH of 5.0-5.5; or,

[0008] 250-370 mM NaAc-HAc, pH 5.0-5.5.

[0009] Preferably, before loading the sample, the cation exchange chromatography medium is subjected to equilibration treatment with an equilibration solution, wherein the equilibration solution comprises: 20-80 mM NaAc-HAc, and a pH of 5.0-5.5.

[0010] Preferably, after loading the sample, the cation exchange chromatography medium is eluted with a elution buffer 1, wherein the elution buffer 1 comprises: 20-80 mM NaAc-HAc, and a pH of 5.0-5.5.

[0011] Preferably, after eluting with the elution buffer 1, the method further comprises eluting with the elution buffer 2, wherein the elution buffer 2 comprises: 50-120 mM NaAc-HAc, and pH is 5.0-5.5.

[0012] Preferably, the pH of the antibody solution is 50-5.5.

[0013] Preferably, the loading capacity of the antibody solution is 40%-80% of the actual dynamic loading capacity of the chromatography medium.

[0014] Preferably, the cation exchange chromatography medium is selected from any one of NanoGel 50SP HP, Monomix Mab60-SP or Diamond SP Mustang.

[0015] Preferably, when the eluent used in the cation exchange chromatography treatment does not contain sodium chloride, the method comprises:

[0016] (1) Before loading, the cation exchange chromatography medium is equilibrated with an equilibrium solution, wherein the equilibrium solution comprises: 20-80 mM NaAc-HAc, pH 5.0-5.5;

[0017] (2) After loading, the cation exchange chromatography medium is eluted with a elution buffer 1, wherein the elution buffer 1 comprises: 20-80 mM NaAc-HAc, pH 5.0-5.5;

[0018] (3) Using an eluent for elution treatment, the eluent includes: 250-370 mM NaAc-HAc, pH 5.0-5.5.

[0019] Preferably, when the eluent used in the cation exchange chromatography treatment contains sodium chloride, the method comprises:

[0020] (1) Before loading, the cation exchange chromatography medium is equilibrated with an equilibrium solution, wherein the equilibrium solution comprises: 20-80 mM NaAc-HAc, pH 5.0-5.5;

[0021] (2) After loading, the cation exchange chromatography medium is sequentially eluted with elution buffer 1 and elution buffer 2, wherein the elution buffer 1 comprises: 20-80 mM NaAc-HAc, pH 5.0-5.5; the elution buffer 2 comprises: 110-280 mM NaAc-HAc, pH 5.2-5.7;

[0022] (3) Using an eluent for elution treatment, the eluent includes: 80-120 mM NaAc-HAc, 140-170 mM NaCl, pH 5.0-5.5.

[0023] In a second aspect, the present invention provides use of the method for purifying antibodies using cation exchange chromatography as described in the first aspect in the preparation of antibody drugs.

[0024] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides a new idea for developing a cation exchange chromatography process using a sodium acetate-acetic acid buffer pair. The method for purifying antibodies using cation exchange chromatography can effectively solve the double peak phenomenon in elution while ensuring the ability to remove aggregates. Under the condition that the production cost is not significantly increased, the method can effectively improve the robustness of the process and lay a solid foundation for later large-scale production. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0028] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0029] In the following specific embodiments, the antibody protein in Example 14 is the sequence of the hCQ137 clone shown in Table 6 of patent CN112552406B; the antibody protein used in the remaining embodiments and comparative examples is the BC-1C3 antibody sequence disclosed in patent CN116333123A.

[0030] Example 1

[0031] This embodiment provides a method for purifying antibodies using cation exchange chromatography, the purification method comprising the following steps:

[0032] Loading capacity: 28 g / L (the loading capacity refers to the amount of antibody adsorbed per liter of filler); filler: NanoGel 50SP HP equilibrium solution: 50 mM NaAc-HAc, pH 5.0; elution buffer 1: 50 mM NaAc-HAc, pH 5.0; elution buffer 2: 80 mM NaAc-HAc, pH 5.0; eluent: 80 mM NaAc-HAc, 170 mM NaCl, pH 5.0.

[0033] (1) Before loading the sample, the cation exchange chromatography medium is equilibrated with an equilibrium solution.

[0034] (2) Loading the sample. The pH of the antibody solution is 5.0, and the sample loading capacity is 28 g / L. After loading the sample, the cation exchange chromatography medium is eluted with elution buffer 1, and then the cation exchange chromatography medium is eluted with elution buffer 2.

[0035] (3) The antibody was eluted in a good state with no impurities in the front part of the main peak by using the above-mentioned elution solution.

[0036] Example 2

[0037] This embodiment provides a method for purifying antibodies using cation exchange chromatography, the purification method comprising the following steps:

[0038] Loading capacity: 56 g / L; Filler: NanoGel 50SP HP; Equilibration solution: 50 mM NaAc-HAc, pH 5.0; Elution buffer 1: 50 mM NaAc-HAc, pH 5.0; Elution buffer 2: 100 mM NaAc-HAc, pH 5.0; Eluent: 100 mM NaAc-HAc, 148 mM NaCl, pH 5.0.

[0039] (1) Before loading the sample, the cation exchange chromatography medium is equilibrated with an equilibrium solution;

[0040] (2) Loading the sample. The pH of the antibody solution is 5.0, and the sample loading capacity is 56 g / L. After loading the sample, the cation exchange chromatography medium is eluted with elution buffer 1, and then the cation exchange chromatography medium is eluted with elution buffer 2.

[0041] (3) The antibody was eluted in a good state with no impurities in the front part of the main peak by using the above-mentioned elution solution.

[0042] Example 3

[0043] This embodiment provides a method for purifying antibodies using cation exchange chromatography, the purification method comprising the following steps:

[0044] Loading capacity: 28 g / L; Filler: NanoGel 50SP HP; Equilibration solution: 50 mM NaAc-HAc, pH 5.0; Elution buffer 1: 50 mM NaAc-HAc, pH 5.0; Elution buffer 2: 100 mM NaAc-HAc, pH 5.0; Eluent: 100 mM NaAc-HAc, 148 mM NaCl, pH 5.0.

[0045] (1) Before loading the sample, the cation exchange chromatography medium is equilibrated with an equilibrium solution;

[0046] (2) Loading the sample. The pH of the antibody solution is 5.0, and the sample loading capacity is 28 g / L. After loading the sample, the cation exchange chromatography medium is eluted with elution buffer 1, and then the cation exchange chromatography medium is eluted with elution buffer 2.

[0047] (3) The antibody was eluted in a good state with no impurities in the front part of the main peak by using the above-mentioned elution solution.

[0048] Example 4

[0049] This example provides a method for purifying antibodies using cation exchange chromatography. The purification method is different from that of Example 1 in that:

[0050] Loading capacity: 56 g / L; Filler: NanoGel 50SP HP; Equilibration solution: 50 mM NaAc-HAc, pH 5.0; Elution buffer 1: 50 mM NaAc-HAc, pH 5.0; Elution buffer 2: 100 mM NaAc-HAc, pH 5.0; Eluent: 100 mM NaAc-HAc, 158 mM NaCl, pH 5.0.

[0051] The antibody was eluted in a good state with no impurities in the front section of the main peak by using the above-mentioned elution solution.

[0052] Example 5

[0053] This example provides a method for purifying antibodies using cation exchange chromatography. The purification method is different from that of Example 1 in that:

[0054] Loading capacity: 56 g / L; Filler: NanoGel 50SP HP; Equilibration solution: 50 mM NaAc-HAc, pH 5.0; Elution buffer 1: 50 mM NaAc-HAc, pH 5.0; Elution buffer 2: 100 mM NaAc-HAc, pH 5.0; Eluent: 100 mM NaAc-HAc, 168 mM NaCl, pH 5.0.

[0055] The antibody was eluted in a good state with no impurities in the front section of the main peak by using the above-mentioned elution solution.

[0056] Example 6

[0057] This example provides a method for purifying antibodies using cation exchange chromatography. The purification method is different from that of Example 1 in that:

[0058] Loading capacity: 64g / L; Filler: Monomix Mab60-SP; Equilibration solution: 50mM NaAc-HAc, pH 5.0; Elution buffer 1: 50mM NaAc-HAc, pH 5.0; Elution buffer 2: 120mM NaAc-HAc, pH 5.0; Eluent: 120mMNaAc-HAc, 160mM NaCl, pH 5.0.

[0059] The antibody was eluted in a good state with no impurities in the front section of the main peak by using the above-mentioned elution solution.

[0060] Example 7

[0061] This example provides a method for purifying antibodies using cation exchange chromatography. The purification method is different from that of Example 1 in that:

[0062] Loading capacity: 32g / L; Filler: Monomix Mab60-SP; Equilibration solution: 50mM NaAc-HAc, pH 5.0; Elution buffer 1: 50mM NaAc-HAc, pH 5.0; Elution buffer 2: 120mM NaAc-HAc, pH 5.0; Eluent: 120mMNaAc-HAc, 160mM NaCl, pH 5.0.

[0063] The antibody was eluted in a good state with no impurities in the front section of the main peak by using the above-mentioned elution solution.

[0064] Example 8

[0065] This example provides a method for purifying antibodies using cation exchange chromatography. The purification method is different from that of Example 1 in that:

[0066] Loading capacity: 28g / L; Filler: Diamond SP Mustang; Equilibration solution: 50mM NaAc-HAc, pH 5.0; Elution buffer 1: 50mM NaAc-HAc, pH 5.0; Elution buffer 2: 100mM NaAc-HAc, pH 5.0; Eluent: 100mM NaAc-HAc, 150mM NaCl, pH 5.0.

[0067] The antibody was eluted in a good state with no impurities in the front section of the main peak by using the above-mentioned elution solution.

[0068] Example 9

[0069] This example provides a method for purifying antibodies using cation exchange chromatography. The purification method is different from that of Example 1 in that:

[0070] Loading capacity: 56 g / L; Filler: Diamond SP Mustang; Equilibration solution: 50 mM NaAc-HAc, pH 5.0; Elution buffer 1: 50 mM NaAc-HAc, pH 5.0; Elution buffer 2: 100 mM NaAc-HAc, pH 5.0; Eluent: 100 mM NaAc-HAc, 150 mM NaCl, pH 5.0.

[0071] The antibody was eluted in a good state with no impurities in the front section of the main peak by using the above-mentioned elution solution.

[0072] Example 10

[0073] This example provides a method for purifying antibodies using cation exchange chromatography. The purification method is different from that of Example 1 in that:

[0074] Loading capacity: 28 g / L; Filler: NanoGel 50SP HP; Equilibration solution: 50 mM NaAc-HAc, pH 5.5; Elution buffer 1: 50 mM NaAc-HAc, pH 5.5; Eluent: 250 mM NaAc-HAc, pH 5.5.

[0075] The antibody was eluted in a good state with no impurities in the front section of the main peak by using the above-mentioned elution solution.

[0076] Embodiment 11

[0077] This example provides a method for purifying antibodies using cation exchange chromatography. The purification method is different from that of Example 1 in that:

[0078] Loading capacity: 56 g / L; Filler: NanoGel 50SP HP; Equilibration solution: 50 mM NaAc-HAc, pH 5.5; Elution buffer 1: 50 mM NaAc-HAc, pH 5.5; Eluent: 250 mM NaAc-HAc, pH 5.5.

[0079] The antibody was eluted in a good state with no impurities in the front section of the main peak by using the above-mentioned elution solution.

[0080] Example 12

[0081] This example provides a method for purifying antibodies using cation exchange chromatography. The purification method is different from that of Example 1 in that:

[0082] Loading capacity: 56 g / L; Filler: NanoGel 50SP HP; Equilibration solution: 50 mM NaAc-HAc, pH 5.5; Elution buffer 1: 50 mM NaAc-HAc, pH 5.5; Eluent: 280 mM NaAc-HAc, pH 5.5.

[0083] The antibody was eluted in a good state with no impurities in the front section of the main peak by using the above-mentioned elution solution.

[0084] Example 13

[0085] This example provides a method for purifying antibodies using cation exchange chromatography. The purification method is different from that of Example 1 in that:

[0086] Loading capacity: 32 g / L; Filler: Monomix Mab60-SP; Equilibration solution: 50 mM NaAc-HAc, pH 5.5; Elution buffer 1: 50 mM NaAc-HAc, pH 5.5; Eluent: 370 mM NaAc-HAc, pH 5.5.

[0087] The antibody was eluted in a good state with no impurities in the front section of the main peak by using the above-mentioned elution solution.

[0088] Embodiment 14

[0089] This embodiment provides a method for purifying antibodies using cation exchange chromatography. The difference between this embodiment and embodiment 1 is that the type of purified antibodies is different. The purification method comprises the following steps:

[0090] Loading capacity: 28g / L; Filler: NanoGel 50SP HP; Equilibration solution: 50mM NaAc-HAc, pH 5.0; Elution buffer 1: 50mM NaAc-HAc, pH 5.0; Elution buffer 2: 80mM NaAc-HAc, pH 5.0; Eluent: 80mM NaAc-HAc, 170mM NaCl, pH 5.0.

[0091] (1) Before loading the sample, the cation exchange chromatography medium is equilibrated with an equilibrium solution.

[0092] (2) Loading the sample. The pH of the antibody solution is 5.0, and the sample loading capacity is 28 g / L. After loading the sample, the cation exchange chromatography medium is eluted with elution buffer 1, and then the cation exchange chromatography medium is eluted with elution buffer 2.

[0093] (3) The antibody was eluted in a good state with no impurities in the front part of the main peak by using the above-mentioned elution solution.

[0094] Comparative Example 1

[0095] This comparative example provides a method for purifying antibodies using cation exchange chromatography, and the purification method is different from that of Example 1 in that:

[0096] Loading capacity: 25 g / L; Filler: NanoGel 50SP HP; Equilibration solution: 50 mM NaAc-HAc, pH 5.0; Elution buffer 1: 50 mM NaAc-HAc, pH 5.0; Eluent: 50 mM NaAc-HAc, 180 mM NaCl, pH 5.0.

[0097] When the above-mentioned eluent was used for elution, the elution state of the antibody was poor, and there were miscellaneous peaks in the front part of the main peak.

[0098] Comparative Example 2

[0099] This comparative example provides a method for purifying antibodies using cation exchange chromatography, and the purification method is different from that of Example 1 in that:

[0100] Loading capacity: 56 g / L; Filler: NanoGel 50SP HP; Equilibration solution: 50 mM NaAc-HAc, pH 5.5; Elution buffer 1: 50 mM NaAc-HAc, pH 5.5; Elution buffer 2: 50 mM NaAc-HAc, pH 5.2; Eluent: 50 mM NaAc-HAc, 110 mM NaCl, pH 5.5.

[0101] When the above-mentioned eluent was used for elution, the elution state of the antibody was poor, and there were miscellaneous peaks in the front part of the main peak.

[0102] Comparative Example 3

[0103] This comparative example provides a method for purifying antibodies using cation exchange chromatography, and the purification method is different from that of Example 1 in that:

[0104] Loading capacity: 28 g / L; Filler: NanoGel 50SP HP; Equilibration solution: 50 mM NaAc-HAc, pH 5.5; Elution buffer 1: 50 mM NaAc-HAc, pH 5.5; Elution buffer 2: 50 mM NaAc-HAc, pH 5.7; Eluent: 50 mM NaAc-HAc, 110 mM NaCl, pH 5.5.

[0105] When the above-mentioned eluent was used for elution, the elution state of the antibody was poor, and there were miscellaneous peaks in the front part of the main peak.

[0106] Comparative Example 4

[0107] This comparative example provides a method for purifying antibodies using cation exchange chromatography, and the purification method is different from that of Example 1 in that:

[0108] Loading capacity: 28g / L; Filler: NanoGel 50SP HP; Equilibration solution: 50mM NaAc-HAc, pH 5.0; Elution buffer 1: 50mM NaAc-HAc, pH 5.0; Elution buffer 2: 80mM NaAc-HAc, pH 5.0; Eluent: 250mM NaAc-HAc, 150mM NaCl, pH 5.0.

[0109] In this comparative example, since the ion concentration in the eluent is too high, impurities are also eluted, and the elution effect is poor.

[0110] Comparative Example 5

[0111] This comparative example provides a method for purifying antibodies using cation exchange chromatography, and the purification method is different from that of Example 1 in that:

[0112] Loading capacity: 28g / L; Filler: NanoGel 50SP HP; Equilibration solution: 50mM NaAc-HAc, pH 5.0; Elution buffer 1: 50mM NaAc-HAc, pH 5.0; Elution buffer 2: 80mM NaAc-HAc, pH 5.0; Eluent: 180mM NaAc-HAc, 150mM NaCl, pH 5.0.

[0113] In this comparative example, since the ion concentration in the eluent is too high, impurities are also eluted, and the elution effect is poor.

[0114] Comparative Example 6

[0115] This comparative example provides a method for purifying antibodies using cation exchange chromatography, and the purification method is different from that of Example 1 in that:

[0116] Loading capacity: 28g / L; Filler: NanoGel 50SP HP; Equilibration solution: 50mM NaAc-HAc, pH 5.0; Elution buffer 1: 50mM NaAc-HAc, pH 5.0; Elution buffer 2: 80mM NaAc-HAc, pH 5.0; Eluent: 180mM NaAc-HAc, pH 5.0.

[0117] In this comparative example, due to the low concentration of NaAc-HAc in the eluent, it is difficult to completely elute the target protein, and the elution effect is poor.

[0118] Comparative Example 7

[0119] This comparative example provides a method for purifying antibodies using cation exchange chromatography, and the purification method is different from that of Example 1 in that:

[0120] Loading capacity: 28g / L; Filler: NanoGel 50SP HP; Equilibration solution: 50mM NaAc-HAc, pH 5.0; Elution buffer 1: 50mM NaAc-HAc, pH 5.0; Elution buffer 2: 80mM NaAc-HAc, pH 5.0; Eluent: 400mM NaAc-HAc, pH 5.0.

[0121] In this comparative example, since the concentration of NaAc-HAc in the eluent is too high, a good elution effect cannot be achieved, the target protein and impurities cannot be separated, and the elution effect is poor.

[0122] The test data results of the above embodiments and comparative examples are summarized in Table 1.

[0123] Table 1

[0124]

[0125] Note: The sum of the percentages of polymers, monomers and fragments in Table 1 is in the range of 99.9-100.1%.

[0126] According to the results of Comparative Examples 1-3, when using the acetic acid-sodium acetate buffer and sodium chloride combined elution method, under different chromatography pH and loading conditions, when the sodium acetate-acetic acid buffer concentration in the eluent is lower than 80mM, the polymer removal capacity of the three experiments is about 0.6% to 0.8%. However, there are two pH fluctuations in the elution process, and the pH fluctuations under low loading conditions are more obvious. A double peak phenomenon also occurs in the low loading elution process. This shows that when the molar concentration of NaAc-HAc is too low, simply changing the chromatography pH value cannot effectively increase the buffering capacity of the buffer in the eluent process, and a double peak elution phenomenon occurs.

[0127] According to the results of Example 1, Comparative Example 4 and Comparative Example 5, when the elution method using the acetic acid-sodium acetate buffer pair and sodium chloride is combined, under the same loading amount conditions, when the molar concentration of the NaAc-HAc buffer pair in the eluent is higher than 120 mM, the elution force is too strong and impurities such as polymers cannot be effectively separated. This shows that although a high molar concentration of NaAc-HAc can avoid the elution double peak phenomenon, it also loses the ability to separate impurities such as polymers.

[0128] According to the results of Example 10, Example 11, Example 12 and Example 13, when the concentration of the sodium acetate-acetic acid buffer in the elution solution is continued to increase to 250mM and 280mM under different loading conditions, the aggregate removal capacity is about 0.4% to 0.6%, and the aggregate removal capacity does not decrease significantly. During the elution process, the pH value fluctuates once, and there is no elution peak phenomenon.

[0129] According to the results of Example 2, Example 3, Example 3, Example 4 and Example 5, when the sodium acetate concentration in the eluent is increased to 100mM under different loading conditions, the polymer removal capacity is about 0.4% to 0.6% after the sodium chloride concentration in the eluent is appropriately changed from 148mM to 168mM, which will not cause a significant impact on the polymer removal capacity. During the elution process, the pH value fluctuates once, and there is no elution peak phenomenon. The results of the two groups of experiments show that the process development method is robust and controllable, and is suitable for industrial production.

[0130] According to the results of Example 2, Example 3, Example 6, Example 7, Example 8 and Example 9, under the conditions of different loadings and different filler types, the polymer removal capacity is about 0.4% to 0.6%, which will not cause a significant impact on the polymer removal capacity. During the elution process, the pH value fluctuates once, and there is no elution peak phenomenon. The results of the six groups of experiments show that the process development method can be applied to most cationic fillers on the market, and the process has a wide range of applicability.

[0131] According to the results of Example 1 and Example 14, the aggregate removal capacity of two different antibody proteins at the same loading and process conditions can reach about 0.8% and 1.2%, which will not have a significant impact on the aggregate removal capacity. The pH value fluctuation is maintained once during the elution process, and there is no elution peak phenomenon. The results of the two groups of experiments show that the process development method can be applied to a variety of monoclonal antibody proteins, and the process has a wide range of applicability.

[0132] In summary, the present invention uses an eluent to effectively solve the double peak phenomenon in elution while ensuring the ability to remove aggregates. Under the condition that the production cost is not significantly increased, this method can effectively improve the robustness of the process and lay a solid foundation for later large-scale production.

[0133] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for purifying antibodies using cation exchange chromatography, characterized in that: The method comprises: loading the antibody solution onto a cation exchange chromatography medium, and sequentially performing elution and elution, wherein the eluent comprises: 80-120 mM NaAc-HAc, 140-170 mM NaCl, and a pH of 5.0-5.5; or, 250-370 mM NaAc-HAc, pH 5.0-5.

5.

2. The method for purifying antibodies using cation exchange chromatography according to claim 1, characterized in that: Before loading, the cation exchange chromatography medium is equilibrated with an equilibrium solution, wherein the equilibrium solution comprises: 20-80 mM NaAc-HAc, and the pH is 5.0-5.

5.

3. The method for purifying antibodies using cation exchange chromatography according to claim 1 or 2, characterized in that: After loading, the cation exchange chromatography medium is eluted with elution buffer 1, wherein the elution buffer 1 comprises: 20-80 mM NaAc-HAc, pH 5.0-5.

5.

4. The method for purifying antibodies using cation exchange chromatography according to claim 3, characterized in that: After the elution treatment with the elution buffer 1, the elution treatment is further performed with the elution buffer 2, wherein the elution buffer 2 comprises: 50-120 mM NaAc-HAc, and the pH is 5.2-5.

7.

5. The method for purifying antibodies using cation exchange chromatography according to any one of claims 1 to 4, characterized in that: The pH of the antibody solution is 5.0-5.

5.

6. The method for purifying antibodies using cation exchange chromatography according to any one of claims 1 to 5, characterized in that: The loading capacity of the antibody solution is 40-80% of the actual dynamic loading capacity of the chromatography medium.

7. The method for purifying antibodies using cation exchange chromatography according to any one of claims 1 to 6, characterized in that: The cation exchange chromatography medium is selected from any one of NanoGel 50SP HP, Monomix Mab60-SP or Diamond SP Mustang.

8. The method for purifying antibodies using cation exchange chromatography according to any one of claims 1 to 7, characterized in that: When the eluent used in the cation exchange chromatography treatment does not contain sodium chloride, the method comprises: (1) Before loading, the cation exchange chromatography medium is equilibrated with an equilibrium solution, wherein the equilibrium solution comprises: 20-80 mM NaAc-HAc, pH 5.0-5.5; (2) After loading, the cation exchange chromatography medium is eluted with a elution buffer 1, wherein the elution buffer 1 comprises: 20-80 mM NaAc-HAc, pH 5.0-5.5; (3) Using an eluent for elution treatment, the eluent includes: 250-370 mM NaAc-HAc, pH 5.0-5.

5.

9. The method for purifying antibodies using cation exchange chromatography according to any one of claims 1 to 8, characterized in that: When the eluent used in the cation exchange chromatography treatment contains sodium chloride, the method comprises: (1) Before loading, the cation exchange chromatography medium is equilibrated with an equilibrium solution, wherein the equilibrium solution comprises: 20-80 mM NaAc-HAc, pH 5.0-5.5; (2) After loading, the cation exchange chromatography medium is sequentially eluted with elution buffer 1 and elution buffer 2, wherein the elution buffer 1 comprises: 20-80 mM NaAc-HAc, pH 5.0-5.5; the elution buffer 2 comprises: 50-120 mM NaAc-HAc, pH 5.2-5.7; (3) Using an eluent for elution treatment, the eluent comprises: 80-120 mM NaAc-HAc, 140-170 mM NaCl, and pH 5.0-5.

5.

10. Use of the method for purifying antibodies using cation exchange chromatography according to any one of claims 1 to 9 in the preparation of antibody drugs.

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