Method for purifying antibody by using Protein A affinity chromatography and application thereof

Abstract

The invention provides a method for purifying an antibody by using Protein A affinity chromatography and application of the method, and the method comprises the following steps: loading a supernatant solution to be purified into a Protein A affinity chromatography medium, and sequentially carrying out leaching and elution for three times to obtain the purified antibody. The method comprises the following steps: adding sodium caprylate into a supernatant solution; or sodium caprylate, a combination of sodium caprylate and NaCl and a combination of sodium caprylate and Arg-HCl are added into the leacheate, so that the host protein and esterase removal capability of Protein A affinity chromatography is improved. According to the antibody purification method, the host protein and esterase removal capacity of Protein A affinity chromatography can be effectively enhanced on the premise that the recovery rate is guaranteed and the polymer removal capacity is not reduced, the technological process of the method is stable and controllable, the method can effectively reduce impurity residues in drugs, and the method is suitable for industrial production. The effective guarantee is provided for the safety and the stability of the medicine.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for purifying antibodies using Protein A affinity chromatography and an application thereof. Background Art

[0002] At present, in the field of antibody purification, Protein A affinity chromatography is widely used in antibody purification production as an effective and simple method for antibody capture. Sodium octanoate, sodium chloride and Arg-HCl are widely used in Protein A affinity chromatography as cheap and less toxic materials. After years of development, the basic development of Protein A affinity chromatography has tended to be standardized, but the large amount of host protein and esterase residues produced during cell expression is still a problem that plagues many companies. Under certain conditions, esterases can cause degradation of common excipients in biological drugs, such as Tween 80. Excessive residues of host proteins and esterases pose a major hidden danger to the safety and stability of the final drug.

[0003] As an effective means of capturing antibodies in antibody purification, the protein A affinity chromatography binding mode often strictly controls the sample handling and harvesting methods, the loading capacity of the filler, the type of reagents in the elution step, the pH selection, and the pH value of the eluent. The working principle of the protein A affinity chromatography binding mode is that it can bind to the protein containing the Fc tag in the supernatant containing a large number of impurities in a specific binding manner under neutral conditions, elute a part of the non-specifically bound host protein and other impurities by selecting a specific eluent, and then elute the target protein by lowering the pH value in the specific eluent, while impurities such as polymers are kept in a bound state as much as possible during the elution process. Because the supernatant contains a large number of impurity products expressed by cells, including host proteins, some of them will flow through and not bind to the column during the binding process with the chromatography column, but some impurities will still bind to the chromatography column or the target protein non-specifically. Adding a high concentration of sodium chloride to the eluent can remove a small amount of this part of the host protein, but the effect is often not very obvious. There are strict regulations on host protein residues during drug registration and production. Excessive host protein and esterase residues will pose a major threat to the stability and safety of drugs.

[0004] Therefore, developing an antibody purification method that can increase the ability of Protein A affinity chromatography to remove host proteins and esterases has important application value. Summary of the invention

[0005] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a method for purifying antibodies using Protein A affinity chromatography and its application. In the supernatant to be purified, sodium caprylate is added; or sodium caprylate, a combination of sodium caprylate and NaCl, or a combination of sodium caprylate and Arg-HCl is added to the eluent for the second elution, so as to increase the ability of Protein A affinity chromatography to remove host proteins and esterases.

[0006] To achieve the object of the present invention, the following technical solutions are adopted:

[0007] In the first aspect, the present invention provides a method for purifying antibodies using Protein A affinity chromatography, and the method includes:

[0008] Loading the supernatant solution to be purified onto a Protein A affinity chromatography medium, and sequentially performing the first elution, the second elution, the third elution and elution to obtain purified antibodies;

[0009] The elution buffer 2 used for the second elution is selected from any one or a combination of at least two of eluent A, eluent B or eluent C;

[0010] The eluent A includes, in terms of molar concentration: 20 - 70 mM Tris-HAc and 0 - 300 mM sodium caprylate, and the pH is 7.3 - 7.7;

[0011] Among them, "20 - 70 mM Tris-HAc" can be, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM or 70 mM, etc.; "0 - 300 mM sodium caprylate" can be, for example, 0 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM or 300 mM, etc.; "the pH is 7.3 - 7.7" can be, for example, 7.3, 7.4, 7.5, 7.6 or 7.7, etc.;

[0012] The eluent B includes, in terms of molar concentration: 20 - 70 mM Tris-HAc, 0 - 200 mM sodium caprylate and 0.8 - 1.5 M NaCl, and the pH is 7.3 - 7.7;

[0013] Among them, "20 - 70 mM Tris-HAc" can be, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM or 70 mM, etc.; "0 - 200 mM sodium caprylate" can be, for example, 0 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM or 200 mM, etc.; "0.8 - 1.5 M NaCl" can be, for example, 0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M or 1.5 M, etc.; "pH is 7.3 - 7.7" can be, for example, 7.3, 7.4, 7.5, 7.6 or 7.7, etc.

[0014] The eluent C, in terms of molar concentration, comprises: 20 - 80 mM Tris-HAc, 0 - 100 mM sodium caprylate and 0.25 - 0.5 M Arg-HCl, with a pH of 7.3 - 7.7;

[0015] Among them, "20 - 80 mM Tris-HAc" can be, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM or 80 mM, etc.; "0 - 100 mM sodium caprylate" can be, for example, 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, etc.; "0.25 - 0.5 M Arg-HCl" can be, for example, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M or 0.5 M, etc.; "pH is 7.3 - 7.7" can be, for example, 7.3, 7.4, 7.5, 7.6 or 7.7, etc.

[0016] In the present invention, the Tris-HAc buffer pair has a strong pH buffering capacity within the range of pH 7.3 - 7.3, which can reduce the uncontrollable influence on the experiment caused by the pH fluctuation.

[0017] Preferably, the eluent A, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 100 - 300 mM sodium caprylate, with a pH of 7.3 - 7.7.

[0018] Among them, "20 - 70 mM Tris-HAc" can be, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM or 70 mM, etc. "100 - 300 mM sodium caprylate" can be, for example, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM or 300 mM, etc. "pH is 7.3 - 7.7" can be, for example, 7.3, 7.4, 7.5, 7.6 or 7.7, etc.

[0019] Preferably, the eluent B contains, in terms of molar concentration: 20 - 70 mM Tris-HAc, 50 - 200 mM sodium caprylate, and 0.8 - 1.5 M NaCl, with a pH of 7.3 - 7.7.

[0020] Among them, for "20 - 70 mM Tris-HAc", it can be, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, or 70 mM, etc. For "50 - 200 mM sodium caprylate", it can be, for example, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, or 200 mM, etc. For "0.8 - 1.5 M NaCl", it can be, for example, 0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, or 1.5 M, etc. For "pH of 7.3 - 7.7", it can be, for example, 7.3, 7.4, 7.5, 7.6, or 7.7, etc.

[0021] Preferably, the eluent C contains, in terms of molar concentration: 20 - 80 mM Tris-HAc, 50 - 100 mM sodium caprylate, and 0.25 - 0.5 M Arg-HCl, with a pH of 7.3 - 7.7.

[0022] Among them, for "20 - 80 mM Tris-HAc", it can be, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, or 80 mM, etc. For "50 - 100 mM sodium caprylate", it can be, for example, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM, etc. For "0.25 - 0.5 M Arg-HCl", it can be, for example, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M, or 0.5 M, etc. For "pH of 7.3 - 7.7", it can be, for example, 7.3, 7.4, 7.5, 7.6, or 7.7, etc.

[0023] Preferably, before loading, sodium caprylate is added to the supernatant solution to be purified, and the final concentration of sodium caprylate in the supernatant solution to be purified is 10 - 25 mM, for example, it can be 10 mM, 12 mM, 14 mM, 15 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM, or 25 mM, etc.

[0024] Preferably, the eluent A contains, in terms of molar concentration: 20 - 70 mM Tris-HAc, with a pH of 7.3 - 7.7.

[0025] Among them, for "20 - 70 mM Tris-HAc", it can be, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, or 70 mM, etc. For "pH of 7.3 - 7.7", it can be, for example, 7.3, 7.4, 7.5, 7.6, or 7.7, etc.

[0026] Preferably, the eluent B contains, in terms of molar concentration: 20 - 70 mM Tris-HAc and 0.8 - 1.5 M NaCl, with a pH of 7.3 - 7.7.

[0027] Among them, "20 - 70 mM Tris-HAc" can be, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, or 70 mM, etc. "0.8 - 1.5 M NaCl" can be, for example, 0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, or 1.5 M, etc. "pH of 7.3 - 7.7" can be, for example, 7.3, 7.4, 7.5, 7.6, or 7.7, etc.

[0028] Preferably, the eluent C contains, in terms of molar concentration: 20 - 80 mM Tris-HAc and 0.25 - 0.5 M Arg-HCl, with a pH of 7.3 - 7.7.

[0029] Among them, "20 - 80 mM Tris-HAc" can be, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, or 80 mM, etc. "0.25 - 0.5 M Arg-HCl" can be, for example, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M, or 0.5 M, etc. "pH of 7.3 - 7.7" can be, for example, 7.3, 7.4, 7.5, 7.6, or 7.7, etc.

[0030] In the present invention, sodium caprylate is added to the supernatant solution to be purified; or sodium caprylate, a combination of sodium caprylate and NaCl, or a combination of sodium caprylate and Arg-HCl is added to the eluent for the second elution, so as to enhance the ability of Protein A affinity chromatography to remove host proteins and esterases.

[0031] In the present invention, when using the method of adding sodium caprylate to the supernatant, a final concentration of sodium caprylate of 10 - 25 mM has a better elution effect; if no sodium caprylate is added at all or an excessive amount of sodium caprylate is added, a better removal effect on host proteins and esterases cannot be achieved.

[0032] Preferably, the elution buffer 1 used for the first elution contains, in terms of molar concentration: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0.

[0033] Among them, "20 - 70 mM Tris-HAc" can be, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, or 70 mM, etc. "0 - 200 mM NaCl" can be, for example, 0 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, or 200 mM, etc. "pH is 7.0 - 8.0" can be, for example, 7, 7.5, or 8, etc.

[0034] Preferably, the elution buffer 3 used for the third elution comprises, in terms of molar concentration: 20 - 80 mM Tris-HAc, with a pH of 7.3 - 7.7.

[0035] Among them, "20 - 80 mM Tris-HAc" can be, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, or 80 mM, etc. "pH is 7.3 - 7.7" can be, for example, 7.3, 7.4, 7.5, 7.6, or 7.7, etc.

[0036] Preferably, before loading the sample, the Protein A affinity chromatography medium is also equilibrated with an equilibration solution, which, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0.

[0037] Among them, "20 - 70 mM Tris-HAc" can be, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, or 70 mM, etc. "0 - 200 mM NaCl" can be, for example, 0 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, or 200 mM, etc. "pH is 7.0 - 8.0" can be, for example, 7, 7.5, or 8, etc.

[0038] In the present invention, before loading the sample, the Protein A affinity chromatography medium needs to be equilibrated with an equilibration solution. The equilibration solution and the elution buffer 1 are the same buffer solution, and its use is to equilibrate the chromatography column before and after loading the sample to make it enter the equilibrium state.

[0039] In the present invention, the conventional binding mode of the affinity chromatography packing material is that after the chromatography column is equilibrated with a neutral buffer solution, the affinity packing material can bind to the protein with an Fc tag by affinity. The equilibration solution has relatively high requirements for the pH value range and no strict regulations for the conductivity. The addition of NaCl can slightly reduce some non-specific adsorption effects, but the overall impact is not significant. The buffer components are selected as the Tris-HAc combination, and this buffer solution can play a strong pH buffering role in the pH range of 7.0 - 8.0, reducing the uncontrollable effects caused by pH fluctuations. Compared with the PB buffer solution, Tris-HAc has no problem of phosphate discharge, and has obvious advantages in cost control for large-scale production.

[0040] Preferably, the elution buffer solution used for elution comprises, in terms of molar concentration: 20 - 80 mM NaAc-HAc, with a pH of 3.8 - 4.0.

[0041] Among them, "20 - 80 mM NaAc-HAc" can be, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM or 80 mM, etc. "With a pH of 3.8 - 4.0" can be, for example, 3.8, 3.9 or 4.0, etc.

[0042] Preferably, the Protein A affinity chromatography medium binds to the Fc-tagged protein by affinity.

[0043] In the present invention, the Protein A affinity chromatography medium can be selected from any one of AT Diamond Plus or Nmab Pro.

[0044] In the present invention, two commercially available Protein A affinity packing materials have been tested, and the results are all in line with expectations. Given that the binding and elution principles of the currently commercially available Protein A affinity packing materials are the same, only there may be slight differences in the loading capacity, so this method is applicable to most of the commercially available Protein A affinity packing materials.

[0045] In the present invention, the loading capacity of the antibody for sample loading depends on the dynamic loading capacity of the Protein A affinity packing material; for example, when the dynamic loading capacity is 55 g / L, the safe loading capacity is 80% of the dynamic loading capacity, that is, 44 g / L; the sample loading amount can be 50 - 100% of the safe loading capacity, or can be 32 - 80% of the dynamic loading capacity. The method for measuring the loading capacity is the 10% breakthrough dynamic loading capacity measurement method.

[0046] Preferably, the method for purifying an antibody using Protein A affinity chromatography comprises:

[0047] (1) Before loading, equilibrate the Protein A affinity chromatography medium with an equilibration solution, which, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0;

[0048] (2) Add sodium caprylate to the supernatant solution to be purified, with the final concentration of sodium caprylate in the supernatant solution to be purified being 10 - 25 mM; load the supernatant solution to be purified onto the Protein A affinity chromatography medium;

[0049] (3) After loading, perform the first wash, and the wash buffer 1 used for the first wash, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0;

[0050] (4) Perform the second wash with eluent A, and eluent A, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc, with a pH of 7.3 - 7.7;

[0051] (5) Perform the third wash, and the wash buffer 3 used for the third wash, in terms of molar concentration, comprises: 20 - 80 mM Tris-HAc, with a pH of 7.3 - 7.7;

[0052] (6) Perform elution with an elution buffer, and the elution buffer, in terms of molar concentration, comprises: 20 - 80 mM NaAc-HAc, with a pH of 3.8 - 4.0.

[0053] Preferably, the method for purifying an antibody using Protein A affinity chromatography comprises:

[0054] (1) Before loading, equilibrate the Protein A affinity chromatography medium with an equilibration solution, which, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0;

[0055] (2) Add sodium caprylate to the supernatant solution to be purified, with the final concentration of sodium caprylate in the supernatant solution to be purified being 10 - 25 mM; load the supernatant solution to be purified onto the Protein A affinity chromatography medium;

[0056] (3) After loading, perform the first wash, and the wash buffer 1 used for the first wash, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0;

[0057] (4) Perform a second elution with eluent B, which, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0.8 - 1.5 M NaCl, with a pH of 7.3 - 7.7;

[0058] (5) Perform a third elution. The elution buffer 3 used for the third elution, in terms of molar concentration, comprises: 20 - 80 mM Tris-HAc, with a pH of 7.3 - 7.7;

[0059] (6) Perform elution with an elution buffer, which, in terms of molar concentration, comprises: 20 - 80 mM NaAc-HAc, with a pH of 3.8 - 4.0.

[0060] Preferably, the method for purifying an antibody using Protein A affinity chromatography comprises:

[0061] (1) Before loading, equilibrate the Protein A affinity chromatography medium with an equilibration solution, which, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0;

[0062] (2) Add sodium caprylate to the supernatant solution to be purified, with the final concentration of sodium caprylate in the supernatant solution to be purified being 10 - 25 mM; load the supernatant solution to be purified onto the Protein A affinity chromatography medium;

[0063] (3) After loading, perform a first elution. The elution buffer 1 used for the first elution, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0;

[0064] (4) Perform a second elution with eluent C, which, in terms of molar concentration, comprises: 20 - 80 mM Tris-HAc and 0.25 - 0.5 M Arg-HCl, with a pH of 7.3 - 7.7;

[0065] (5) Perform a third elution. The elution buffer 3 used for the third elution, in terms of molar concentration, comprises: 20 - 80 mM Tris-HAc, with a pH of 7.3 - 7.7;

[0066] (6) Perform elution with an elution buffer, which, in terms of molar concentration, comprises: 20 - 80 mM NaAc-HAc, with a pH of 3.8 - 4.0.

[0067] Preferably, the method for purifying an antibody using Protein A affinity chromatography comprises:

[0068] (1) Before sample loading, the Protein A affinity chromatography medium is equilibrated with an equilibration solution which, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0;

[0069] (2) Load the supernatant solution to be purified onto the Protein A affinity chromatography medium;

[0070] (3) After sample loading, perform the first elution. The elution buffer 1 used for the first elution, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0;

[0071] (4) Perform the second elution with eluent A. The eluent A, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 100 - 300 mM sodium caprylate, with a pH of 7.3 - 7.7;

[0072] (5) Perform the third elution. The elution buffer 3 used for the third elution, in terms of molar concentration, comprises: 20 - 80 mM Tris-HAc, with a pH of 7.3 - 7.7;

[0073] (6) Perform elution with an elution buffer. The elution buffer, in terms of molar concentration, comprises: 20 - 80 mM NaAc-HAc, with a pH of 3.8 - 4.0.

[0074] Preferably, the method for purifying an antibody using Protein A affinity chromatography comprises:

[0075] (1) Before sample loading, the Protein A affinity chromatography medium is equilibrated with an equilibration solution which, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0;

[0076] (2) Load the supernatant solution to be purified onto the Protein A affinity chromatography medium;

[0077] (3) After sample loading, perform the first elution. The elution buffer 1 used for the first elution, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0;

[0078] (4) Perform the second elution with eluent B. The eluent B, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc, 50 - 200 mM sodium caprylate and 0.8 - 1.5 M NaCl, with a pH of 7.3 - 7.7;

[0079] (5) Perform the third elution. The elution buffer 3 used for the third elution comprises, in terms of molar concentration: 20 - 80 mM Tris-HAc, with a pH of 7.3 - 7.7;

[0080] (6) Perform elution using an elution buffer. The elution buffer comprises, in terms of molar concentration: 20 - 80 mM NaAc-HAc, with a pH of 3.8 - 4.0.

[0081] Preferably, the method for purifying an antibody using Protein A affinity chromatography comprises:

[0082] (1) Before loading, equilibrate the Protein A affinity chromatography medium with an equilibration solution. The equilibration solution comprises, in terms of molar concentration: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0;

[0083] (2) Load the supernatant solution to be purified onto the Protein A affinity chromatography medium;

[0084] (3) After loading, perform the first elution. The elution buffer 1 used for the first elution comprises, in terms of molar concentration: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0;

[0085] (4) Perform the second elution using elution solution C. Elution solution C comprises, in terms of molar concentration: 20 - 80 mM Tris-HAc, 50 - 100 mM sodium caprylate, 0.25 - 0.5 M Arg-HCl, with a pH of 7.3 - 7.7;

[0086] (5) Perform the third elution. The elution buffer 3 used for the third elution comprises, in terms of molar concentration: 20 - 70 mM Tris-HAc, with a pH of 7.3 - 7.7;

[0087] (6) Perform elution using an elution buffer. The elution buffer comprises, in terms of molar concentration: 20 - 80 mM NaAc-HAc, with a pH of 3.8 - 4.0.

[0088] In a second aspect, the present invention provides the application of the method for purifying an antibody using Protein A affinity chromatography according to the first aspect in the preparation of antibody drugs.

[0089] The antibody is selected from any one of the following or a combination thereof: anti-CD73 antibody, anti-TNFα antibody, anti-CLDN18.2 antibody, anti-CD47 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-EGFR antibody, anti-HER2 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF antibody, or anti-CLDN18.2 / CD47 antibody, etc., including monoclonal antibodies and polyclonal antibodies.

[0090] The numerical ranges described in the present invention not only include the point values listed above, but also any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of brevity, the specific point values included in the ranges of the present invention are not exhaustively listed herein.

[0091] Compared with the prior art, the present invention has the following beneficial effects:

[0092] The present invention provides a new idea for the process development of removing host proteins and esterases with Protein A affinity chromatography. The method for purifying antibodies using Protein A affinity chromatography according to the present invention can effectively improve the removal ability of host proteins and esterases on the premise of ensuring the ability to remove aggregates. Under the condition of no major change in the production process, this method can effectively increase the removal amount of impurities such as host proteins, making a significant contribution to the safety and stability of drugs in the later stage. Detailed implementation manners

[0093] The technical solutions of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0094] For those not specifying specific techniques or conditions in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular commercial channels.

[0095] In the following specific implementation manners, the antibody protein used in Example 18 is the BC-1C3 antibody sequence disclosed in Patent CN116333123A; the antibody proteins in the remaining examples and comparative examples are the sequences of the hCQ137 clone shown in Table 6 of Patent CN112552406B.

[0096] Tris, product number: 288737, manufacturer: Angus.

[0097] HAC, product number: N / A, manufacturer: Hunan Erkang.

[0098] Sodium caprylate, product number: 1984-06-1, manufacturer: Hunan Jiudian Hongyang Pharmaceutical.

[0099] Arg-HCl, Catalog Number: N / A, Source: Ajinomoto Amino Acid Co., Ltd., Shanghai.

[0100] AT Diamond Plus, Catalog Number: AA402307, Source: BioGram (Shanghai) Biotechnology Co., Ltd.

[0101] Nmab Pro, Catalog Number: 17013 - 070100, Source: Suzhou NanoMicro Tech Co., Ltd.

[0102] MaXtar ARPA, Catalog Number: 1024 - 1821, Source: BioLinc Pharmaceutical Technology (Shanghai) Co., Ltd.

[0103] Example 1

[0104] This example provides a method for purifying antibodies using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows, and the purification method includes the following steps:

[0105] Loading capacity: 36 g / L (the loading capacity refers to the amount of antibody adsorbed per liter of packing material); Packing material: AT Diamond Plus, the dynamic loading capacity of the packing material is 55 g / L, and the safety loading capacity is 80% of the dynamic loading capacity, that is, 44 g / L. In this example, the sample is loaded within 50 - 100% of the safety loading capacity.

[0106] Equilibration buffer and washing buffer 1: 50 mM Tris - HAc, 150 mM NaCl, pH 7.5.

[0107] Washing buffer 2: 50 mM Tris - HAc, 1 M NaCl, pH 9.0.

[0108] Washing buffer 3: Tris - HAc, pH 7.5.

[0109] Elution buffer: 30 mM NaAc - HAc, pH 3.9.

[0110] (1) Before sample loading, equilibrate the Protein A affinity chromatography medium with the equilibration buffer.

[0111] (2) Add 300 mM sodium caprylate stock solution to the supernatant to be purified so that the final concentration of sodium caprylate in the supernatant reaches 10 mM; Load the supernatant to be purified onto the Protein A affinity chromatography medium; The loading capacity is 36 g / L.

[0112] (3) After sample loading, perform the first wash with washing buffer 1.

[0113] (4) Perform the second wash with washing buffer 2.

[0114] (5) The third elution is performed using elution buffer 3.

[0115] (6) Elution is performed using the elution buffer.

[0116] In this example, sodium caprylate is added to the supernatant and then elution treatment is carried out, and the removal effects of host proteins and esterase are good.

[0117] Example 2

[0118] This example provides a method for purifying an antibody using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows, and the purification method includes the following steps:

[0119] Loading capacity: 36 g / L; packing material: AT Diamond Plus.

[0120] Equilibration buffer and elution buffer 1: 30 mM Tris-HAc, 170 mM NaCl, pH 7.

[0121] Elution buffer 2: 30 mM Tris-HAc, 1.5 M NaCl, pH 9.0.

[0122] Elution buffer 3: 20 mM Tris-HAc, pH 7.3.

[0123] Eluent: 30 mM NaAc-HAc, pH 3.9.

[0124] (1) Before loading, the Protein A affinity chromatography medium is equilibrated using the equilibration buffer.

[0125] (2) A 300 mM sodium caprylate stock solution is added to the supernatant to be purified so that the final concentration of sodium caprylate in the supernatant reaches 25 mM; the supernatant solution to be purified is loaded onto the Protein A affinity chromatography medium; the loading capacity is 36 g / L.

[0126] (3) After loading, the first elution is performed using elution buffer 1.

[0127] (4) The second elution is performed using elution buffer 2.

[0128] (5) The third elution is performed using elution buffer 3.

[0129] (6) Elution is performed using the eluent.

[0130] In this example, sodium caprylate is added to the supernatant and then elution treatment is carried out, and the removal effects of host proteins and esterase are good.

[0131] Example 3

[0132] This example provides a method for purifying antibodies using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows, and the purification method includes the following steps:

[0133] Loading capacity: 36 g / L; Packing material: AT Diamond Plus.

[0134] Equilibration buffer and washing buffer 1: 70 mM Tris-HAc, 120 mM NaCl, pH 8.

[0135] Washing buffer 2: 30 mM Tris-HAc, 100 mM sodium caprylate, pH 7.5.

[0136] Washing buffer 3: 70 mM Tris-HAc, pH 7.7.

[0137] Elution buffer: 30 mM NaAc-HAc, pH 3.9.

[0138] (1) Before loading, equilibrate the Protein A affinity chromatography medium with the equilibration buffer.

[0139] (2) Load the supernatant solution to be purified (without sodium caprylate) onto the Protein A affinity chromatography medium; the loading capacity is 36 g / L.

[0140] (3) After loading, perform the first wash with washing buffer 1.

[0141] (4) Perform the second wash with washing buffer A.

[0142] (5) Perform the third wash with washing buffer 3.

[0143] (6) Perform elution with the elution buffer.

[0144] After the above washing steps are performed in this example and then elution is carried out, the removal effects of host proteins and esterases are good.

[0145] Example 4

[0146] This example provides a method for purifying antibodies using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows:

[0147] Loading capacity: 36 g / L; Packing material: AT Diamond Plus.

[0148] Equilibration buffer and washing buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0149] Elution buffer 2: 50 mM Tris-HAc, 150 mM sodium caprylate, pH 7.5.

[0150] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0151] Eluent: 30 mM NaAc-HAc, pH 3.9.

[0152] The purification steps refer to Example 3. Before loading, no sodium caprylate needs to be added to the supernatant.

[0153] In this example, after the above elution steps are carried out, the host protein and esterase removal effects are good.

[0154] Example 5

[0155] This example provides a method for purifying an antibody using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows, and the purification method includes the following steps:

[0156] Loading capacity: 36 g / L; packing material: AT Diamond Plus.

[0157] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0158] Elution buffer 2: 50 mM Tris-HAc, 300 mM sodium caprylate, pH 7.5.

[0159] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0160] Eluent: 30 mM NaAc-HAc, pH 3.9.

[0161] The purification steps refer to Example 3. Before loading, no sodium caprylate needs to be added to the supernatant.

[0162] In this example, after the above elution steps are carried out, the host protein and esterase removal effects are good.

[0163] Example 6

[0164] This example provides a method for purifying an antibody using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows, and the purification method includes the following steps:

[0165] Loading capacity: 44 g / L; packing material: AT Diamond Plus.

[0166] Equilibration buffer and washing buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0167] Washing buffer 2: 50 mM Tris-HAc, 50 mM sodium caprylate, 1.5 M NaCl, pH 7.5.

[0168] Washing buffer 3: 50 mM Tris-HAc, pH 7.5.

[0169] Elution buffer: 30 mM NaAc-HAc, pH 3.9.

[0170] The purification steps refer to Example 3. Before loading, no sodium caprylate needs to be added to the supernatant.

[0171] In this example, after the above washing steps are performed, elution is carried out, and the removal effects of host proteins and esterases are good.

[0172] Example 7

[0173] This example provides a method for purifying an antibody using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows, and the purification method includes the following steps:

[0174] Loading capacity: 44 g / L; packing material: AT Diamond Plus.

[0175] Equilibration buffer and washing buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0176] Washing buffer 2: 50 mM Tris-HAc, 100 mM sodium caprylate, 1 M NaCl, pH 7.5.

[0177] Washing buffer 3: 50 mM Tris-HAc, pH 7.5.

[0178] Elution buffer: 30 mM NaAc-HAc, pH 3.9.

[0179] The purification steps refer to Example 3. Before loading, no sodium caprylate needs to be added to the supernatant.

[0180] In this example, after the above washing steps are performed, elution is carried out, and the removal effects of host proteins and esterases are good.

[0181] Example 8

[0182] This example provides a method for purifying an antibody using Protein A affinity chromatography. The purification method includes the following steps:

[0183] Loading capacity: 22 g / L (the loading capacity refers to the amount of antibody adsorbed by the packing per liter, and 22 g / L corresponds to 50% of the safe loading capacity); Packing: AT Diamond Plus.

[0184] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0185] Elution buffer 2: 50 mM Tris-HAc, 100 mM sodium caprylate, 1 M NaCl, pH 7.5.

[0186] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0187] Eluent: 30 mM NaAc-HAc, pH 3.9.

[0188] The purification steps refer to Example 3. Before loading, no sodium caprylate needs to be added to the supernatant.

[0189] In this example, after the above elution steps are used for treatment, the removal effects of host proteins and esterases are good.

[0190] Example 9

[0191] This example provides a method for purifying an antibody using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows, and the purification method includes the following steps:

[0192] Loading capacity: 36 g / L; Packing: AT Diamond Plus.

[0193] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0194] Elution buffer 2: 50 mM Tris-HAc, 150 mM sodium caprylate, 1 M NaCl, pH 7.5.

[0195] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0196] Eluent: 30 mM NaAc-HAc, pH 3.9.

[0197] The purification steps refer to Example 3. Before loading, no sodium caprylate needs to be added to the supernatant.

[0198] In this example, after the above elution steps are used for treatment, the removal effects of host proteins and esterases are good.

[0199] Example 10

[0200] This example provides a method for purifying antibodies using Protein A affinity chromatography. The purification method includes the following steps:

[0201] Loading capacity: 36 g / L; packing material: AT Diamond Plus.

[0202] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0203] Elution buffer 2: 50 mM Tris-HAc, 200 mM sodium caprylate, 0.8 M NaCl, pH 7.5.

[0204] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0205] Elution solution: 30 mM NaAc-HAc, pH 3.9.

[0206] The purification steps refer to Example 3. Before loading, no sodium caprylate needs to be added to the supernatant.

[0207] After the above elution steps are used in this example for elution treatment, the removal effects of host proteins and esterases are good.

[0208] Example 11

[0209] This example provides a method for purifying antibodies using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows, and the purification method includes the following steps:

[0210] Loading capacity: 36 g / L; packing material: AT Diamond Plus.

[0211] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0212] Elution buffer 2: 50 mM Tris-HAc, 150 mM sodium caprylate, 1 M NaCl, pH 7.7.

[0213] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0214] Elution solution: 30 mM NaAc-HAc, pH 3.9.

[0215] The purification steps refer to Example 3. Before loading, no sodium caprylate needs to be added to the supernatant.

[0216] After the above elution step treatment in this example, the elution treatment is carried out, and the removal effects of host proteins and esterases are good.

[0217] Example 12

[0218] This example provides a method for purifying an antibody using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows, and the purification method includes the following steps:

[0219] Loading capacity: 36 g / L; packing material: AT Diamond Plus.

[0220] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0221] Elution buffer 2: 50 mM Tris-HAc, 150 mM sodium caprylate, 1 M NaCl, pH 7.3.

[0222] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0223] Elution solution: 30 mM NaAc-HAc, pH 3.9.

[0224] The purification steps refer to Example 3. Before loading, no sodium caprylate needs to be added to the supernatant.

[0225] After the above elution step treatment in this example, the elution treatment is carried out, and the removal effects of host proteins and esterases are good.

[0226] Example 13

[0227] This example provides a method for purifying an antibody using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows, and the purification method includes the following steps:

[0228] Loading capacity: 36 g / L; packing material: AT Diamond Plus.

[0229] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0230] Elution buffer 2: 50 mM Tris-HAc, 50 mM sodium caprylate, 0.25 M Arg-HCl, pH 7.5.

[0231] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0232] Elution solution: 30 mM NaAc-HAc, pH 3.9.

[0233] The purification steps refer to Example 3. Before loading, no sodium caprylate needs to be added to the supernatant.

[0234] In this example, after the above elution steps are used for treatment, elution treatment is carried out, and the removal effects of host proteins and esterases are good.

[0235] Example 14

[0236] This example provides a method for purifying an antibody using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows, and the purification method includes the following steps:

[0237] Loading capacity: 22 g / L; packing material: AT Diamond Plus.

[0238] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0239] Elution buffer 2: 50 mM Tris-HAc, 50 mM sodium caprylate, 0.5 M Arg-HCl, pH 7.5.

[0240] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0241] Eluent: 30 mM NaAc-HAc, pH 3.9.

[0242] The purification steps refer to Example 3. Before loading, no sodium caprylate needs to be added to the supernatant.

[0243] In this example, after the above elution steps are used for treatment, elution treatment is carried out, and the removal effects of host proteins and esterases are good.

[0244] Example 15

[0245] This example provides a method for purifying an antibody using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows, and the purification method includes the following steps:

[0246] Loading capacity: 44 g / L (the loading capacity refers to the amount of antibody adsorbed by each liter of packing material, and 44 g / L corresponds to 100% of the safe loading capacity); packing material: AT Diamond Plus.

[0247] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0248] Washing buffer 2: 50 mM Tris-HAc, 50 mM sodium caprylate, 0.5 M Arg-HCl, pH 7.5.

[0249] Washing buffer 3: 50 mM Tris-HAc, pH 7.5.

[0250] Elution buffer: 30 mM NaAc-HAc, pH 3.9.

[0251] The purification steps refer to Example 3. Before loading, no sodium caprylate needs to be added to the supernatant.

[0252] In this example, after the above washing steps are performed, elution is carried out, and the removal effects of host proteins and esterases are good.

[0253] Example 16

[0254] This example provides a method for purifying antibodies using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows, and the purification method includes the following steps:

[0255] Loading capacity: 44 g / L; packing material: AT Diamond Plus.

[0256] Equilibration buffer and washing buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0257] Washing buffer 2: 50 mM Tris-HAc, 100 mM sodium caprylate, 0.5 M Arg-HCl, pH 7.5.

[0258] Washing buffer 3: 50 mM Tris-HAc, pH 7.5.

[0259] Elution buffer: 30 mM NaAc-HAc, pH 3.9.

[0260] The purification steps refer to Example 3. Before loading, no sodium caprylate needs to be added to the supernatant.

[0261] In this example, after the above washing steps are performed, elution is carried out, and the removal effects of host proteins and esterases are good.

[0262] Example 17

[0263] This example provides a method for purifying antibodies using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows, and the purification method includes the following steps:

[0264] Loading capacity: 44 g / L; packing material: Nmab Pro. The dynamic loading capacity of the packing material is 55 g / L, and the safety loading capacity is 80% of the dynamic loading capacity, that is, 44 g / L.

[0265] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0266] Elution buffer 2: 50 mM Tris-HAc, 100 mM sodium caprylate, 1 M NaCl, pH 7.5.

[0267] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0268] Eluent: 30 mM NaAc-HAc, pH 3.9.

[0269] The purification steps refer to Example 3. Before loading, no sodium caprylate needs to be added to the supernatant.

[0270] In this example, after the above elution steps are performed, elution treatment is carried out, and the removal effects of host proteins and esterases are good.

[0271] Example 18

[0272] This example provides a method for purifying an antibody using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows, and the purification method includes the following steps:

[0273] Loading capacity: 44 g / L; packing material: MaXtar ARPA. The dynamic loading capacity of the packing material is 55 g / L, and the safety loading capacity is 80% of the dynamic loading capacity, that is, 44 g / L.

[0274] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0275] Elution buffer 2: 50 mM Tris-HAc, 100 mM sodium caprylate, 1 M NaCl, pH 7.5.

[0276] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0277] Eluent: 30 mM NaAc-HAc,, pH 3.9.

[0278] The purification steps refer to Example 3. Before loading, no sodium caprylate needs to be added to the supernatant.

[0279] In this example, after the above elution steps are performed, elution treatment is carried out, and the removal effects of host proteins and esterases are good.

[0280] Comparative Example 1

[0281] This comparative example provides a method for purifying antibodies using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows. The difference between the purification method and Example 1 is that:

[0282] Treatment method for supernatant: no sodium caprylate is added; loading capacity: 36 g / L; packing material: AT Diamond Plus.

[0283] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0284] Elution buffer 2: 50 mM Tris-HAc, 1 M NaCl, pH 9.0.

[0285] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0286] Eluent: 30 mM NaAc-HAc, pH 3.9.

[0287] In this comparative example, after no sodium caprylate is added to the supernatant and then elution treatment is carried out, the removal effects of host proteins and esterases are poor.

[0288] Comparative Example 2

[0289] This comparative example provides a method for purifying antibodies using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows. The difference between the purification method and Example 1 is that:

[0290] Treatment method for supernatant: 300 mM sodium caprylate stock solution is added to make the final concentration of sodium caprylate in the supernatant reach 50 mM; loading capacity: 36 g / L; packing material: AT Diamond Plus.

[0291] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0292] Elution buffer 2: 50 mM Tris-HAc, 1 M NaCl, pH 9.0.

[0293] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0294] Eluent: 30 mM NaAc-HAc,, pH 3.9.

[0295] In this comparative example, after excessive sodium caprylate is added to the supernatant and then elution treatment is carried out, the removal effects of host proteins and esterases are poor.

[0296] Comparative Example 3

[0297] This comparative example provides a method for purifying an antibody using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows. The difference between the purification method and Example 3 is that:

[0298] Loading capacity: 36 g / L; packing material: AT Diamond Plus.

[0299] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0300] Elution buffer 2: 50 mM Tris-HAc, 1 M NaCl, pH 7.5.

[0301] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0302] Eluent: 30 mM NaAc-HAc, pH 3.9.

[0303] Under the condition that no appropriate amount of sodium caprylate was added to the supernatant in this comparative example, when elution buffer 2 contained only NaCl and no sodium caprylate, the removal effects of host proteins and esterase were poor.

[0304] Comparative Example 4

[0305] This comparative example provides a method for purifying an antibody using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows. The difference between the purification method and Example 3 is that:

[0306] Loading capacity: 36 g / L; packing material: AT Diamond Plus.

[0307] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0308] Elution buffer 2: 50 mM Tris-HAc, 50 mM sodium caprylate, pH 7.5.

[0309] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0310] Eluent: 30 mM NaAc-HAc, pH 3.9.

[0311] Under the condition that no appropriate amount of sodium caprylate was added to the supernatant in this comparative example, when elution buffer 2 contained only a small amount of sodium caprylate and no NaCl, the removal effects of host proteins and esterase were poor.

[0312] Comparative Example 5

[0313] This comparative example provides a method for purifying antibodies using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows. The difference between this purification method and Example 6 is that:

[0314] Loading capacity: 36 g / L; packing material: AT Diamond Plus.

[0315] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0316] Elution buffer 2: 50 mM Tris-HAc, 250 mM sodium caprylate, 1 M NaCl, pH 7.5.

[0317] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0318] Eluent: 30 mM NaAc-HAc, pH 3.9.

[0319] In this comparative example, when an appropriate amount of sodium caprylate was not added to the supernatant and an excessive amount of sodium caprylate was added in the presence of NaCl in elution buffer 2, although the removal effects of host proteins and esterases were good, the recovery rate was significantly reduced.

[0320] Comparative Example 6

[0321] This comparative example provides a method for purifying antibodies using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows. The difference between this purification method and Example 13 is that:

[0322] Loading capacity: 36 g / L; packing material: AT Diamond Plus.

[0323] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0324] Elution buffer 2: 50 mM Tris-HAc, 0.25 M Arg-HCl, pH 7.5.

[0325] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0326] Eluent: 30 mM NaAc-HAc, pH 3.9.

[0327] Under the condition that an appropriate amount of sodium caprylate was not added to the supernatant in this comparative example, when elution buffer 2 contained only Arg-HCl, the removal effects of host proteins and esterase were poor.

[0328] Comparative Example 7

[0329] This comparative example provides a method for purifying antibodies using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows. The difference between the purification method and Example 13 is that:

[0330] Loading capacity: 36 g / L; packing material: AT Diamond Plus.

[0331] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0332] Elution buffer 2: 50 mM Tris-HAc, 0.5 M Arg-HCl, pH 7.5.

[0333] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0334] Elution solution: 30 mM NaAc-HAc, pH 3.9.

[0335] Under the condition that an appropriate amount of sodium caprylate was not added to the supernatant in this comparative example, when elution buffer 2 contained only Arg-HCl, the removal effects of host proteins and esterase were poor.

[0336] Comparative Example 8

[0337] This comparative example provides a method for purifying antibodies using Protein A affinity chromatography. The buffers and materials used in the purification method are as follows. The difference between the purification method and Example 13 is that:

[0338] Loading capacity: 36 g / L; packing material: AT Diamond Plus.

[0339] Equilibration buffer and elution buffer 1: 50 mM Tris-HAc, 150 mM NaCl, pH 7.5.

[0340] Elution buffer 2: 50 mM Tris-HAc, 150 mM sodium caprylate, 0.5 M Arg-HCl, pH 7.5.

[0341] Elution buffer 3: 50 mM Tris-HAc, pH 7.5.

[0342] Elution solution: 30 mM NaAc-HAc, pH 3.9.

[0343] When an excessive amount of sodium caprylate is added under the condition that an appropriate amount of sodium caprylate is not added to the supernatant and the eluent 2 contains Arg-HCl, although the removal effects of host protein and esterase are good, the recovery rate is significantly reduced.

[0344] The detection data results in the above-mentioned examples and comparative examples are summarized in Table 1 as follows.

[0345] The SEC-HPLC detection method uses high performance liquid chromatography for detection. The chromatographic conditions include: chromatographic column model: TSKgel G3000SWxl (7.8×300mm, 5μm), or other equivalent products can be used for substitution. Mobile phase: 100mM KH2PO4, 100mM Na2SO4, pH6.7.

[0346] The detection method for HCP residue is: enzyme-linked immunosorbent assay (ELISA). Using a CHO cell (Host Cell Protein) host cell residue detection kit (product number F550-1) for detection, and the specific steps of the detection method refer to the instructions of the kit.

[0347] The detection method for HCP residue esterase activity is: fluorescence color development with an enzyme-labeled instrument. The materials used include: DMSO, product number D2438-5X10ML; MU-C8 (substrate stock solution), product number Y19151-250 mg; MU (product standard stock solution), product number M1381-25G.

[0348] Table 1

[0349]

[0350]

[0351] Note: The sum of the percentages of polymer, monomer and fragment in Table 1 is in the range of 99.9 - 100.1%. "ND" in the table means not detected.

[0352] According to the results of Example 1, Example 2, Comparative Example 1 and Comparative Example 2, when using the method of adding sodium caprylate to the supernatant, neither adding no sodium caprylate nor adding an excessive amount of sodium caprylate can achieve better removal effects on host protein and esterase.

[0353] According to the results of Example 3 - 5, Comparative Example 3 and Comparative Example 4, when using the method of elution 2 with only NaCl or with a concentration of sodium caprylate less than 50mM alone under the condition that no sodium caprylate is added to the supernatant, under the same sample loading amount condition, better removal effects on host protein and esterase cannot be achieved.

[0354] From the results of Examples 3 - 5, Comparative Example 3 and Comparative Example 4, it can be seen that under the condition of not adding sodium caprylate to the supernatant, when using the method of elution 2 with only 0.8 - 1.5 M NaCl or the concentration of sodium caprylate less than 50 mM alone, under the condition of the same sample loading amount, better removal effects on both host protein and esterase cannot be achieved.

[0355] From the results of Example 6, Example 7, Example 9, Example 10, Comparative Example 3 and Comparative Example 5, it can be seen that under the condition of not adding sodium caprylate to the supernatant, when using the method of elution 2 with the combination of 0.8 - 1.5 M NaCl and sodium caprylate, under the condition of similar sample loading amounts, when the concentration of sodium caprylate is less than 50 mM, better removal effects on both host protein and esterase cannot be achieved; when the concentration of sodium caprylate exceeds 200 mM, the recovery rate decreases significantly.

[0356] From the results of Example 13, Example 15, Example 16 and Comparative Examples 6 - 8, it can be seen that under the condition of not adding sodium caprylate to the supernatant, when using Arg - HCl alone for elution 2, better removal effects on both host protein and esterase cannot be achieved; when using the combination of Arg - HCl and sodium caprylate for elution 2, under the condition of similar sample loading amounts, when the concentration of Arg - HCl is less than 0.25 M and the concentration of sodium caprylate is less than 50 mM, better removal effects on both host protein and esterase cannot be achieved, and when the concentration of Arg - HCl is greater than 0.5 M and the concentration of sodium caprylate is greater than 100 mM, the recovery rate decreases significantly.

[0357] According to the result analysis of Example 7, Example 11 and Example 12, under different loading conditions, after appropriately changing the pH value in eluent 2 from 7.3 to 7.7, good removal effects on both host protein and esterase are shown. The results of the three - group experiments show that this process development method is robust and controllable, and suitable for industrial production.

[0358] According to the result analysis of Example 7, Example 8, Example 14, Example 15, Example 17 and Example 18, under the conditions of different loadings and different types of packing materials, good removal effects on both host protein and esterase are shown. The results of the six - group experiments show that this process development method can be applied to most of the affinity packing materials on the market, and the process has a wide application range.

[0359] According to the result analysis of Example 7 and Example 18, two different antibody proteins can effectively remove host protein and esterase under the same loading and process conditions. The results of the two - group experiments show that this process development method can be applied to a variety of monoclonal antibody proteins, and the process has a wide application range.

[0360] In summary, the present invention can effectively remove the ability of host proteins and esterases by affinity chromatography using an eluent on the premise of ensuring the recovery rate and not reducing the ability to remove aggregates. Under the condition that the production cost is not significantly increased, this method can effectively reduce the residue of impurities such as host proteins in the drug, providing an effective guarantee for the safety and stability of the drug.

[0361] The applicant declares that the above description is only a specific embodiment 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 conceived by any person skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for purifying antibodies using Protein A affinity chromatography, characterized in that, The method includes: Loading the supernatant solution to be purified onto a Protein A affinity chromatography medium, and successively performing a first elution, a second elution, a third elution, and an elution to obtain a purified antibody; The elution buffer 2 used for the second elution is selected from any one or a combination of at least two of elution solution A, elution solution B, or elution solution C; The elution solution A includes, in terms of molar concentration: 20 - 70 mM Tris-HAc and 0 - 300 mM sodium caprylate, with a pH of 7.3 - 7.7; The elution solution B includes, in terms of molar concentration: 20 - 70 mM Tris-HAc, 0 - 200 mM sodium caprylate, and 0.8 - 1.5 M NaCl, with a pH of 7.3 - 7.7; The elution solution C includes, in terms of molar concentration: 20 - 80 mM Tris-HAc, 0 - 100 mM sodium caprylate, and 0.25 - 0.5 M Arg-HCl, with a pH of 7.3 - 7.

7.

2. The method for purifying an antibody using Protein A affinity chromatography according to claim 1, wherein The elution solution A includes, in terms of molar concentration: 20 - 70 mM Tris-HAc and 100 - 300 mM sodium caprylate, with a pH of 7.3 - 7.7; Preferably, the elution solution B includes, in terms of molar concentration: 20 - 70 mM Tris-HAc, 50 - 200 mM sodium caprylate, and 0.8 - 1.5 M NaCl, with a pH of 7.3 - 7.7; Preferably, the elution solution C includes, in terms of molar concentration: 20 - 80 mM Tris-HAc, 50 - 100 mM sodium caprylate, and 0.25 - 0.5 M Arg-HCl, with a pH of 7.3 - 7.

7.

3. The method for purifying an antibody using Protein A affinity chromatography according to claim 1, wherein Before the loading, sodium caprylate is further added to the supernatant solution to be purified, and the final concentration of sodium caprylate in the supernatant solution to be purified is 10 - 25 mM; Preferably, the elution solution A includes, in terms of molar concentration: 20 - 70 mM Tris-HAc, with a pH of 7.3 - 7.7; Preferably, the elution solution B includes, in terms of molar concentration: 20 - 70 mM Tris-HAc and 0.8 - 1.5 M NaCl, with a pH of 7.3 - 7.7; Preferably, the elution solution C includes, in terms of molar concentration: 20 - 80 mM Tris-HAc and 0.25 - 0.5 M Arg-HCl, with a pH of 7.3 - 7.

7.

4. The method for purifying an antibody using Protein A affinity chromatography according to any one of claims 1-3, characterized in that, The elution buffer 1 used for the first elution includes, in terms of molar concentration: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0; Preferably, the elution buffer 3 used for the third elution includes, in terms of molar concentration: 20 - 80 mM Tris-HAc, with a pH of 7.3 - 7.7; Preferably, before the loading, the Protein A affinity chromatography medium is further balanced with a balancing solution, and the balancing solution includes, in terms of molar concentration: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0; Preferably, the elution buffer used for elution comprises, in terms of molar concentration: 20 - 80 mM NaAc - HAc, with a pH of 3.8 - 4.0; Preferably, the Protein A affinity chromatography medium binds to the Fc - tagged protein in an affinity manner.

5. The method for purifying an antibody using Protein A affinity chromatography according to claim 3 or 4, characterized in that, The method comprises: (1) Before loading, the Protein A affinity chromatography medium is equilibrated with an equilibration solution which comprises, in terms of molar concentration: 20 - 70 mM Tris - HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0; (2) Sodium caprylate is added to the supernatant solution to be purified, and the final concentration of sodium caprylate in the supernatant solution to be purified is 10 - 25 mM; the supernatant solution to be purified is loaded onto the Protein A affinity chromatography medium; (3) After loading, the first washing is carried out, and the washing buffer 1 used for the first washing comprises, in terms of molar concentration: 20 - 70 mM Tris - HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0; (4) The second washing is carried out with washing solution A, and the washing solution A comprises, in terms of molar concentration: 20 - 70 mM Tris - HAc, with a pH of 7.3 - 7.7; (5) The third washing is carried out, and the washing buffer 3 used for the third washing comprises, in terms of molar concentration: 20 - 80 mM Tris - HAc, with a pH of 7.3 - 7.7; (6) Elution is carried out with an elution buffer which comprises, in terms of molar concentration: 20 - 80 mM NaAc - HAc, with a pH of 3.8 - 4.

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6. The method for purifying an antibody using Protein A affinity chromatography according to claim 3 or 4, characterized in that, The method comprises: (1) Before loading, the Protein A affinity chromatography medium is equilibrated with an equilibration solution which comprises, in terms of molar concentration: 20 - 70 mM Tris - HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0; (2) Sodium caprylate is added to the supernatant solution to be purified, and the final concentration of sodium caprylate in the supernatant solution to be purified is 10 - 25 mM; the supernatant solution to be purified is loaded onto the Protein A affinity chromatography medium; (3) After loading, the first washing is carried out, and the washing buffer 1 used for the first washing comprises, in terms of molar concentration: 20 - 70 mM Tris - HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0; (4) The second washing is carried out with washing solution B, and the washing solution B comprises, in terms of molar concentration: 20 - 70 mM Tris - HAc and 0.8 - 1.5 M NaCl, with a pH of 7.3 - 7.7; (5) The third washing is carried out, and the washing buffer 3 used for the third washing comprises, in terms of molar concentration: 20 - 80 mM Tris - HAc, with a pH of 7.3 - 7.7; (6) Elution is carried out with an elution buffer which comprises, in terms of molar concentration: 20 - 80 mM NaAc - HAc, with a pH of 3.8 - 4.

0.

7. The method for purifying an antibody using Protein A affinity chromatography according to claim 3 or 4, characterized in that, The method comprises: (1) Before loading the sample, equilibrate the Protein A affinity chromatography medium with an equilibration solution, which, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0; (2) Add sodium caprylate to the supernatant solution to be purified, with the final concentration of sodium caprylate in the supernatant solution to be purified being 10 - 25 mM; load the supernatant solution to be purified onto the Protein A affinity chromatography medium; (3) After loading the sample, perform the first washing, and the washing buffer 1 used for the first washing, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0; (4) Perform the second washing with washing solution C, which, in terms of molar concentration, comprises: 20 - 80 mM Tris-HAc and 0.25 - 0.5 M Arg-HCl, with a pH of 7.3 - 7.7; (5) Perform the third washing, and the washing buffer 3 used for the third washing, in terms of molar concentration, comprises: 20 - 80 mM Tris-HAc, with a pH of 7.3 - 7.7; (6) Elute with an elution buffer, which, in terms of molar concentration, comprises: 20 - 80 mM NaAc-HAc, with a pH of 3.8 - 4.

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8. The method for purifying an antibody using Protein A affinity chromatography according to claim 2 or 4, characterized in that, The method comprises: (1) Before loading the sample, equilibrate the Protein A affinity chromatography medium with an equilibration solution, which, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0; (2) Load the supernatant solution to be purified onto the Protein A affinity chromatography medium; (3) After loading the sample, perform the first washing, and the washing buffer 1 used for the first washing, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0; (4) Perform the second washing with washing solution A, which, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 100 - 300 mM sodium caprylate, with a pH of 7.3 - 7.7; (5) Perform the third washing, and the washing buffer 3 used for the third washing, in terms of molar concentration, comprises: 20 - 80 mM Tris-HAc, with a pH of 7.3 - 7.7; (6) Elute with an elution buffer, which, in terms of molar concentration, comprises: 20 - 80 mM NaAc-HAc, with a pH of 3.8 - 4.

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9. The method for purifying an antibody using Protein A affinity chromatography according to claim 2 or 4, characterized in that, The method comprises: (1) Before loading the sample, equilibrate the Protein A affinity chromatography medium with an equilibration solution, which, in terms of molar concentration, comprises: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0; (2) Load the supernatant solution to be purified onto the Protein A affinity chromatography medium; (3) After sample loading, the first elution is performed. The elution buffer 1 used for the first elution comprises, in terms of molar concentration: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0; (4) The second elution is performed using eluent B. The eluent B comprises, in terms of molar concentration: 20 - 70 mM Tris-HAc, 50 - 200 mM sodium caprylate, and 0.8 - 1.5 M NaCl, with a pH of 7.3 - 7.7; (5) The third elution is performed. The elution buffer 3 used for the third elution comprises, in terms of molar concentration: 20 - 80 mM Tris-HAc, with a pH of 7.3 - 7.7; (6) Elution is performed using the elution buffer. The elution buffer comprises, in terms of molar concentration: 20 - 80 mM NaAc-HAc, with a pH of 3.8 - 4.

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10. The method for purifying an antibody using Protein A affinity chromatography according to claim 2 or 4, characterized in that, The method comprises: (1) Before sample loading, the Protein A affinity chromatography medium is equilibrated with the equilibration solution. The equilibration solution comprises, in terms of molar concentration: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0; (2) The supernatant solution to be purified is loaded onto the Protein A affinity chromatography medium; (3) After sample loading, the first elution is performed. The elution buffer 1 used for the first elution comprises, in terms of molar concentration: 20 - 70 mM Tris-HAc and 0 - 200 mM NaCl, with a pH of 7.0 - 8.0; (4) The second elution is performed using eluent C. The eluent C comprises, in terms of molar concentration: 20 - 80 mM Tris-HAc, 50 - 100 mM sodium caprylate, 0.25 - 0.5 M Arg-HCl, with a pH of 7.3 - 7.7; (5) The third elution is performed. The elution buffer 3 used for the third elution comprises, in terms of molar concentration: 20 - 80 mM Tris-HAc, with a pH of 7.3 - 7.7; (6) Elution is performed using the elution buffer. The elution buffer comprises, in terms of molar concentration: 20 - 80 mM NaAc-HAc, with a pH of 3.8 - 4.

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11. Use of the method for purifying an antibody using Protein A affinity chromatography according to any one of claims 1 - 10 in the preparation of an antibody drug.

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