Method for purifying asymmetric fusion protein

Abstract

The invention provides a method for purifying asymmetric fusion protein. The method comprises the following steps: balancing, loading a sample, rebalancing, leaching, eluting and the like. By improving purification conditions and considering and screening sample pH, sample conductivity, leacheate, eluent and the like, a purification process suitable for the asymmetric fusion protein is obtained, the separation degree of each component in the process is good, the impurity removal effect is better, and finally the asymmetric fusion protein with high purity, high recovery rate and good stability is obtained. The quality control level and the medicine stability of the product are favorably improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a purification process for effectively purifying asymmetric fusion proteins and reducing impurities. Background Art

[0002] Cytokines are small molecule proteins or glycoproteins that have the ability to recognize and kill tumor cells under certain conditions and are prepared as drugs. However, the efficacy of cytokines is often proportional to their dose, and they have a small molecular weight and a short half-life. High doses and frequent administration are required to maintain a certain blood concentration to ensure anti-tumor efficacy. However, patients are prone to dose-limiting toxicity and systemic toxicity. In order to reduce toxicity, people have developed antibody-cytokine fusion proteins.

[0003] Antibody-cytokine fusion protein refers to a fusion protein composed of targeted recombinant antibodies or antibody fragments and cytokines. In theory, compared with cytokines, antibody-cytokine fusion proteins have the following advantages: a. Antibody-cytokine fusion proteins carrying the same amount of cytokines have a larger molecular weight, which can improve in vivo stability, prolong half-life, and reduce serum clearance; b. Reduce the number of dosing, reduce DLT and systemic toxicity; c. Cytokines are enriched in the lesion site, which increases the drug concentration in the lesion site and more effectively exerts the immunoregulatory effect of cytokines; d. Increase the immunogenicity of the lesion site, and increase the response of immunosuppressants when used in combination with immunosuppressants.

[0004] For antibody-cytokine fusion proteins, since cytokines are fused to the monoclonal antibody structure, the spatial structure of the monoclonal antibody will change to a certain extent. At the same time, some fusion proteins such as IL-10 have the characteristic of tending to aggregate. Therefore, it is very difficult to purify antibody-cytokine fusion proteins using traditional cationic chromatography conditions. More specifically, for asymmetric fusion proteins, impurities such as aggregates, homodimers, half antibodies, and fragments are easily generated. Compared with bispecific antibodies with symmetrical structures, asymmetric fusion proteins have two special impurities, homodimers and half antibodies, which make purification more difficult. It is urgent to research and develop a purification scheme that can be applied to different asymmetric fusion proteins. Summary of the invention

[0005] In order to overcome this technical difficulty, the present invention improves the purification conditions of asymmetric fusion proteins, screens and optimizes sample pH, sample conductivity, eluent, eluent, etc., and obtains a purification process suitable for asymmetric fusion proteins. The method has good separation of the components of the asymmetric fusion protein and good effect of removing impurities, and finally obtains an asymmetric fusion protein with high purity, high recovery rate and good stability, which is conducive to improving the quality control level of the product and drug stability.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A method for purifying an asymmetric fusion protein, characterized in that the method comprises the following steps:

[0008] (1) Equilibration: adding no less than 1 column volume of equilibration buffer to the chromatography column, wherein the equilibration buffer contains 40-60 mM sodium salt solution;

[0009] (2) Loading the sample;

[0010] (3) Re-equilibration: adding no less than 1 column volume of equilibration buffer to the chromatography column, wherein the equilibration buffer contains 40-60 mM sodium salt solution;

[0011] (4) Elution: Add 2-12 column volumes of elution buffer to the chromatography column, wherein the elution buffer contains 170-300 mM sodium salt solution;

[0012] (5) Elution: adding 2-12 column volumes of elution buffer to the chromatography column, wherein the elution buffer contains 310-400 mM sodium salt solution;

[0013] The pH values ​​of the equilibration buffer, the washing buffer and the elution buffer are 4.5-5.5.

[0014] In one embodiment, in the (4) elution step, 2-10 column volumes of an elution buffer containing 175-250 mM sodium salt are added to the chromatography column; and / or, in the (5) elution step, 2-10 column volumes of an elution buffer containing 310-370 mM sodium salt are added to the chromatography column.

[0015] In one embodiment, the filler of the chromatography column is selected from one or more of Capto S ImpAct, Fractogel COO(M), Nuvia HR-S, and Diamond SP Mustang.

[0016] In one embodiment, the sodium salt solution in the buffer system of the equilibration buffer, the washing buffer and the elution buffer is selected from one or more of sodium acetate-acetic acid, sodium chloride, sodium citrate, disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0017] In one embodiment, the equilibration buffer is selected from sodium acetate-acetic acid, pH 4.5-5.5 or phosphate buffer, pH 5.5-6.5; and / or, the elution buffer is selected from sodium acetate-acetic acid, sodium chloride concentration is 175-250mM, pH 4.5-5.5 or phosphate buffer, sodium chloride concentration is 175-250mM, pH 5.5-6.5; and / or, the elution buffer is selected from sodium acetate-acetic acid, sodium chloride concentration is 325-400mM, pH 4.5-5.5 or phosphate buffer, sodium chloride concentration is 325-400mM, pH 4.5-5.5.

[0018] In one embodiment, in the (2) loading step, the loading pH is 4.5-6.5, and the conductivity is 3-17 mS / cm; preferably, the loading pH is 4.5-6, and the conductivity is 4-15 mS / cm.

[0019] In one embodiment, the loading capacity in the (2) loading step is less than 100 mg / mL, preferably less than 80 mg / mL, less than 60 mg / mL or less than 40 mg / mL.

[0020] In one embodiment, the asymmetric fusion protein is a heterodimer comprising a first portion and a second portion.

[0021] In one embodiment, the first moiety is a targeting moiety that specifically binds to a tumor antigen or an immune checkpoint.

[0022] In one embodiment, the first portion comprises the light chain and the heavy chain of an antibody that specifically binds to a tumor antigen.

[0023] In one embodiment, the second part is a part comprising an immunomodulator.

[0024] In one embodiment, the second portion comprises, from N-terminus to C-terminus, an immunomodulator and an antibody Fc region fused to the immunomodulator.

[0025] In one embodiment, the second part comprises an immunomodulator, an antibody Fab region that specifically binds to a tumor antigen and an Fc region fused to the immunomodulator.

[0026] In one embodiment, the second part comprises, from N-terminus to C-terminus, an immunomodulator, an antibody Fab region that specifically binds to a tumor antigen and an Fc region fused to the immunomodulator; preferably, the immunomodulator is fused to the N-terminus of the heavy chain or light chain of the antibody Fab region.

[0027] In one embodiment, the second part comprises an immunomodulator, an antibody Fab region that specifically binds to a tumor antigen and an Fc region fused to the immunomodulator, and the immunomodulator is fused to the C-terminus of the light chain of the antibody Fab region.

[0028] In one embodiment, the antibody Fc region of the second part is complexed with the heavy chain of the first part to form the asymmetric fusion protein.

[0029] In one embodiment, the tumor antigen or immune checkpoint is B7H3, B7H4, B7H5, BTLA, CD27, CD28, CD153, CD40, CD40L, CD70, CD80, CD86, CD96, CD112, CD134, CD137, CD137L, CD152 / CTLA-4, CD155, CD223, CD226, CD252 / OX40L, CD258, CD273 / PD-L2, CD274 / PD-L1, CD278, CD279, CD357, DR3, Galectin-9, GITRL, HVEM, ICOSL / B7RP1 / B7H2, IDO, TIGIT, TIM-3, TL1A, MART-1 / MelanA, gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, CEA, p53, Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen EBVA, human papillomavirus antigen E6 or E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, erbB, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA15-3 / CA27.29 / BCAA, CA195, CA242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophilin C-related protein, TAAL6, TAG72, TLP, MUC16, IL13Rα2, FRα, VEGFR2, Lewis Y, FAP, EphA2, CEACAM5, EGFR, CA6, CA9, GPNMB, EGP1, FOLR1, endothelial receptor, STEAP1, SLC44A4, integrin-4, AGS-16, guanidinyl cyclase C, MUC-1, CFC1B, integrin α3 chain, TPS, CD19, CD20, CD22, CD30, CD72, CD180, CD171, CD123, CD133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166, CD71, CLL-1 / CLEC12A, ROR1, glypican 3, mesothelin, CD33 / IL3Ra, c-Met, PSCA, PSMA, glycolipid F77, EGFRvIII, BCMA, GD-2, MY-ESO-1, or MAGE One or more of A3.

[0030] In one embodiment, the immunomodulator is a cytokine, cytokine receptor, growth factor, hormone, or extracellular matrix molecule.

[0031] In one embodiment, the immunomodulatory agent is selected from the group consisting of IL-1, IL-2, IL-2Rα, IL-2Rβ, IL-3, IL-3Rα, IL-4, IL-4Rα, IL-5, IL-5Rα, IL-6, IL-6Rα, IL-7, IL-7Rα, IL-8, IL-9, IL-9 Rα, IL-10, IL-10R1, IL-10R2, IL-11, IL-11Rα, IL-12, IL-12Rα, IL-12Rβ2, IL-12Rβ1, IL-13, IL-13Rα, IL-13Rα2, IL-14, IL-15, IL-15Rαsus hi, IL-16, IL-17, IL-18, IL-19, IL-20, IL-20R1, IL-20R2, IL-21, IL-21Rα, IL-22, IL-23, IL-23R, IL-27R, IL-31R, G-CSF-R, LIF-R, OSM-R, GM-CSF-R, Rβc, Rγc, TSL-PR, EB13, CLF-1, CNTF-Rα, gp130, Leptin-R, PRL-R, GH-R, Epo-R, Tpo-R, IFN-λR1, IFN-λR2, IFNR1, and IFNR2.

[0032] the term

[0033] As used herein, the term "fusion protein" refers to a protein that includes one, two or more polypeptides derived from different naturally occurring proteins or engineered proteins that are artificially combined to form a protein. Including but not limited to the following forms: 1. For a fusion protein composed of one polypeptide chain, it is composed of the same or different polypeptides fused to each other to form a polypeptide chain containing the different polypeptides; 2. For a fusion protein composed of two or more polypeptide chains, wherein one or more optional polypeptide chains are composed of the same or different polypeptides fused to each other to form a polypeptide chain containing the same or different polypeptides, and these polypeptide chains are combined with each other in a covalent or non-covalent form to form a protein.

[0034] As used herein, the term "heterodimer" generally refers to a molecule, such as a protein molecule, composed of two different members. The two members of a heterodimer may differ in structure, function, activity and / or composition. For example, the two different members may contain polypeptides that differ in the order, number or kind of amino acid residues that form these polypeptides. Each of the two different members of a heterodimer may independently comprise one, two or more units, polypeptide chains or parts.

[0035] As used herein, the term "asymmetric fusion protein" refers to a protein molecule composed of two different members, wherein the two members differ in structure, function, activity and / or composition. For example, two different members may include polypeptides that are different in the order, number or type of amino acid residues that form these polypeptides. Each of the two different members of the asymmetric fusion protein may independently include one, two or more units, polypeptide chains or parts. In one embodiment, the asymmetric fusion protein is a heterodimer including a first part and a second part. In one embodiment, the first part is a targeting part that specifically binds to a tumor antigen or an immune checkpoint. In one embodiment, the first part comprises a light chain and a heavy chain of an antibody that specifically binds to a tumor antigen. In one embodiment, the second part is a part comprising an immunomodulator. In one embodiment, the second part comprises an immunomodulator and an antibody Fc region fused to the immunomodulator from the N-terminus to the C-terminus. In one embodiment, the second part comprises an immunomodulator, an antibody Fab region and an Fc region that specifically binds to a tumor antigen fused to an immunomodulator. In one embodiment, the second part comprises an immunomodulator, an antibody Fab region and an Fc region that specifically binds to a tumor antigen fused to the immunomodulator from the N-terminus to the C-terminus; preferably, the immunomodulator is fused to the N-terminus of the heavy chain or light chain of the antibody Fab region. In one embodiment, the second part comprises an immunomodulator, an antibody Fab region and an Fc region that specifically binds to a tumor antigen fused to the immunomodulator, and the immunomodulator is fused to the C-terminus of the light chain of the antibody Fab region. In one embodiment, the antibody Fc region of the second part is complexed with the heavy chain of the first part to form the asymmetric fusion protein.

[0036] As used herein, the term "targeting moiety" generally refers to a molecule, complex or aggregate that specifically, selectively or preferentially binds to a target molecule, cell, particle, tissue or aggregate. For example, the targeting moiety can be an antibody, an antigen-binding antibody fragment, a bispecific antibody or other antibody-based molecules or compounds. Other examples of targeting moieties can include, but are not limited to, aptamers, high-affinity polymers, receptor binding ligands, nucleic acids, biotin-avidin binding pairs, binding peptides or proteins, etc.

[0037] As used herein, the term "tumor antigen" generally refers to an antigenic substance produced in or by a tumor cell, which may have the ability to trigger an immune response in a host. For example, a tumor antigen may be a protein, polypeptide, peptide, or fragment thereof that constitutes a part of a tumor cell and is capable of inducing tumor-specific cytotoxic T lymphocytes. In some embodiments, the term "tumor antigen" may also refer to a biological molecule (e.g., protein, carbohydrate, glycoprotein, etc.) that is uniquely or preferentially or differentially expressed on a cancer cell and / or is found to be associated with a cancer cell, thereby providing a preferential or specific target for cancer. For example, preferential expression may be preferential expression compared to any other cell in an organism, or preferential expression in a specific region of an organism (e.g., in a specific organ or tissue).

[0038] As used herein, the term "immune checkpoint" generally refers to some inhibitory molecules and activating molecules present in the immune system, which can regulate the body's anti-tumor immune system by regulating T cell activity. For example, inhibitory molecules include PD1, PDL1, B7H3, CTLA4, etc., and activating molecules include OX40, 4-1BB, CD40, etc.

[0039] As used herein, the term "immunomodulator" generally refers to a substance that affects the function of the immune system. An immunomodulator can enhance or reduce an immune response. For example, an immunomodulator can be an active agent of immunotherapy, including but not limited to, for example, cytokines, granulocyte colony stimulating factor (G-CSF), interferon, imiquimod, cell membrane fragments from bacteria, chemokines, interleukins, cytosine phosphate-guanosine (CpG) oligodeoxynucleotides and recombinant, synthetic and / natural preparations of dextran. In some embodiments, the immunomodulator is a cytokine.

[0040] As used herein, the term "linear elution" refers to a method in which, for example, in certain embodiments, the total volume of elution buffer A and elution buffer B remains unchanged throughout the elution process, and the volume fraction of elution buffer B is linearly related to time, that is, the volume of elution buffer B increases linearly with time. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a schematic diagram of the structure of the asymmetric fusion protein of the present invention.

[0042] Figure 2 It is the chromatogram of the loading test of the present invention. DETAILED DESCRIPTION

[0043] The present invention is described in detail below in conjunction with specific examples, but these examples are not intended to limit the scope of the present invention. The experimental methods of the present invention that do not specify specific conditions are usually carried out under conventional conditions, and the test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores.

[0044] Example 1 Screening of Chromatographic Fillers

[0045] The sample purification was explored using Capto S ImpAct cationic filler produced by GE, Fractogel COO(M) cationic filler produced by Merck, Nuvia HR-S cationic filler produced by Bio-Rad, and Diamond SP Mustang cationic filler produced by Borgron. The purification effects of different chromatographic fillers were studied under the retention time of 5 min, loading capacity of 20 mg / mL, pH 5.5, and conductivity 3-6 mS / cm.

[0046] Chromatography conditions:

[0047] Sample source: Antibody sample 2 (prepared with reference to CN202110497420.5, a heterodimer of anti-EGFR antibody and interleukin-10);

[0048] Chromatography column and filler: Omnifit 6.6mm / 330mm chromatography column, filler: GE Healthcare Capto SImpAct, Merck Fractogel COO(M), Biorad Nuvia HR-S, Bogelon Diamond SP Mustang, column volume: 6mL;

[0049] Equilibration buffer: 50 mM NaAc-HAc, pH 5.5;

[0050] Elution buffer: 50 mM NaAc-HAc + 1 M NaCl, pH 5.5.

[0051] The pH of antibody sample 2 was adjusted to 5.5, diluted with injection water to a conductivity of 3-6 mS / cm, the pipeline was pre-rinsed with equilibrium buffer, and then the chromatography column was rinsed with 4 column volumes of equilibrium buffer at a flow rate of 2 mL / min, and the pre-prepared antibody sample 2 was loaded at a flow rate of 1.2 mL / min. First, rinse with 3 column volumes of equilibrium buffer at a flow rate of 1.2 mL / min, then rinse with 20 column volumes of elution buffer at a gradient of 0-30%, at a flow rate of 1.2 mL / min, and collect the elution peak in sections. The purification results of different chromatography fillers are shown in Table 1.

[0052] Table 1

[0053] Filling Type Concentration (mg / mL) Volume (mL) Protein content (mg) Yield (%) Capto S ImpAct 2.48 36 89.28 74.4 Fractogel COO(M) 3.22 25.5 82.11 68.4 Nuvia HR-S 2.94 30 88.20 73.5 Diamond SP Mustang 3.16 24 75.84 63.2

[0054] The results show that the yields of the four affinity fillers after purification are all higher than 60%, among which the yields of Capto S ImpAct cationic filler produced by GE and Nuvia HR-S cationic filler produced by Bio-Rad are both higher than 70%, which is the best effect. The yields of Fractogel COO(M) cationic filler produced by Merck and Diamond SPMustang cationic filler produced by Bogelon are slightly lower, but the yields are also higher than 60%.

[0055] Example 2 pH and conductivity conditions

[0056] Using GE Capto S ImpAct filler, at a retention time of 5 min, a loading capacity of 15 mg / mL filler, and a fixed linear elution gradient, the effect of removing impurities was tested by varying pH and sample conductivity conditions.

[0057] Chromatography conditions:

[0058] Sample source: Antibody sample 1 (prepared with reference to CN202111135075.7, a heterodimer of anti-B7H3 antibody and interleukin-10);

[0059] Equilibration buffer 1: 50 mM NaAc-HAc, pH 5.0; Elution buffer 1: 50 mM NaAc-HAc, 1 M NaCl, pH 5.0;

[0060] Equilibration buffer 2: 50 mM NaAc-HAc, pH 5.5; Elution buffer 2: 50 mM NaAc-HAc, 1 M NaCl, pH 5.5;

[0061] Equilibration buffer 3: 20 mM PB, pH 6.0; Elution buffer 3: 20 mM PB, 1 M NaCl, pH 6.0.

[0062] First, prepare antibody sample 1, and preform the pH and conductivity as shown in the table below. The pipeline is pre-rinsed with equilibrium buffer, and then the chromatography column is rinsed with 3 column volumes of equilibrium buffer at a flow rate of 1 mL / min, and the pre-prepared antibody sample 1 is loaded at a flow rate of 1 mL / min. The sample loading capacity of each group of samples is 15 mg / mL, and the flow rate is 1 mL / min. Then rinse with 2 column volumes of equilibrium buffer at a flow rate of 1 mL / min, and then rinse with 20 column volumes of elution buffer at a gradient of 0-50%, at a flow rate of 1 mL / min, and collect the elution peak in sections. The purification results of samples under different conditions are shown in Table 2.

[0063] Table 2

[0064] sample Loading pH Loading conductivity purity Separation 1 5 5 98.99 1.57 2 5 10 99.03 1.68 3 5 15 99.02 1.67 4 5.5 5 97.98 1.39 5 5.5 10 98.82 1.45 6 5.5 15 98.27 1.16 7 6 5 97.99 1.08 8 6 10 95.87 1.33 9 6 15 97.99 1.18

[0065] From the results, we can see that different pH and conductivity can obtain samples with higher purity under the same linear gradient elution, but the separation degree is quite different. Among them, pH has a great influence on the separation degree. Choosing a large separation degree is helpful for isocratic gradient elution. When the pH is 5.0, the separation degree is the best; when the pH is 5.5, the separation degree is second; when the pH is 6.0, the separation effect is not ideal. Therefore, when the pH is 5.0, the separation effect is better when the conductivity is 5-15; when the pH is 5.5, the separation effect is better when the conductivity is 5-10; when the pH is 6.0, the separation effect is better when the conductivity is around 10.

[0066] In one embodiment, GE Capto S ImpAct filler is used to detect the effect of removing impurities under the conditions of 5 min retention time, 20 mg / mL filler loading, fixed linear elution gradient, and eluent pH of 5.0 and 5.5, respectively.

[0067] Chromatography conditions:

[0068] Sample source: Antibody sample 2;

[0069] Equilibration buffer 1: 50 mM NaAc-HAc, pH 5.0; Elution buffer 1: 50 mM NaAc-HAc + 1 M NaCl, pH 5.0;

[0070] First, prepare antibody sample 2, adjust pH to 5.0, and conductance to 3-6mS / cm. Rinse the pipeline with equilibrium buffer in advance, then rinse the chromatography column with 4 column volumes of equilibrium buffer at a flow rate of 0.5mL / min, and load the pre-prepared antibody sample 1 at a flow rate of 0.5mL / min. Rinse with 2 column volumes of equilibrium buffer at a flow rate of 0.5mL / min, then rinse with 60 column volumes of elution buffer at a gradient of 0-40%, at a flow rate of 0.5mL / min, and collect the elution peak in sections. The purification results of samples under different conditions are shown in Table 3.

[0071] Table 3

[0072] Sample No. SEC-HPLC HMW(%) SEC-HPLC purity (%) 5.0-3B10 Not detected 7.06 5.0-3C2 Not detected 100 5.0-3C4 Not detected 100 5.0-3C6 Not detected 100 5.0-3C8 1.29 98.71 5.0-3C10 3.34 96.66 5.0-3C12 4.77 95.23 5.0-3D2 20.10 79.90 5.0-3D4 76.06 23.94

[0073] From the results, we can see that when the pH is 5.0 and the conductivity is 3-6 mS / cm, the separation effect is good, and qualified samples with less than 2% aggregates can be obtained, and the yield is also high.

[0074] Example 3 Optimization of elution conditions

[0075] Antibody sample 2 contains aggregates and degradation fragments, and some of the aggregate fragments need to be removed by optimizing the elution conditions. In this example, 32% and 36% eluents are used to elute the chromatography, and 60% and 64% eluents are used to elute the target protein, respectively, to detect the removal effect of aggregates, so as to select a more suitable elution condition for cation exchange chromatography.

[0076] Chromatography conditions:

[0077] Sample source: Antibody sample 2, 112 mg per experiment;

[0078] Chromatography column and filler: Omnifit 6.6mm / 330mm chromatography column, filler: GE Healthcare CaptoSImpAct, column volume: 6mL;

[0079] Equilibration buffer: 50 mM NaAc-HAc, pH 5.0;

[0080] Elution buffer: 50 mM NaAc-HAc + 500 mM NaCl, pH 5.0.

[0081] First, prepare antibody sample 2, adjust pH to 5.0, and dilute with water injection to a conductivity of 3-6mS / cm. The pipeline is pre-rinsed with equilibrium buffer, and then the chromatography column is rinsed with 3 times the column volume equilibrium buffer at a flow rate of 2mL / min, and the pre-prepared antibody sample 2 is loaded at a flow rate of 1.2mL / min, and each group of samples is loaded. Then rinse with 3 times the column volume equilibrium buffer at a flow rate of 1.2mL / min, and finally rinse with 8 times the column volume, 1.2mL / min elution buffer for elution, and the elution gradients of the two groups of experiments are 32% and 36% (NaCl concentrations are 160mM and 180nM, respectively, and the calculation method is the same below). Then elute with 8 times the column volume flow rate 1.2mL / min elution buffer, and the elution gradients of the two groups of experiments are 60% and 64%, respectively, and the elution peaks are collected. The purification results of different elution conditions are shown in Table 4.

[0082] Table 4

[0083]

[0084]

[0085] The results show that the aggregates can be effectively removed by washing the chromatogram with 36% of the eluent and eluting the chromatogram with 64% of the eluent. The data also show that the sample yield under the elution conditions is the highest.

[0086] Similarly, antibody sample 1 also contains aggregates and degradation fragments. In order to verify whether the asymmetric fusion protein of the present invention with a similar structure can use the same elution conditions to remove a part of the aggregate fragments, 1 mL of CaptoS ImpAct, a loading capacity of 20 mg / mL, and the chromatogram was eluted with 20%, 18%, and 21% eluents, respectively, and 35%, 33%, and 36% eluents, respectively, to detect the removal effect of the aggregates.

[0087] Chromatography conditions:

[0088] Sample source: Antibody sample 1;

[0089] Equilibration buffer: 50 mM NaAc-HAc, pH 5.0;

[0090] Elution buffer: 50 mM NaAc-HAc, 1 M NaCl, pH 5.0.

[0091] First, prepare antibody sample 1, adjust pH to 5.0, conductivity 5.44mS / cm, and detect concentration 5.57mg / mL. Rinse the pipeline with equilibrium buffer in advance, then rinse the chromatography column with 3 column volumes of equilibrium buffer at a flow rate of 1mL / min, and load the pre-prepared antibody sample 1 at a flow rate of 1mL / min, 3.59mL per group. Rinse with 2 column volumes of equilibrium buffer at a flow rate of 1mL / min, and finally elute with 5 column volumes and 1mL / min elution buffer. The three groups of elution gradients are 20%, 18%, and 21% (NaCl concentrations are 200mM, 180nM, and 210nM, respectively). Elute with 5 column volumes of elution buffer at a flow rate of 1mL / min, and the three groups of elution gradients are 35%, 33%, and 36% (NaCl concentrations are 350mM, 330mM, and 360mM, respectively), and collect the elution peaks respectively. The purification results under different elution conditions are shown in Table 5.

[0092] Table 5

[0093]

[0094]

[0095] The results show that when the elution conditions are 18%-21% of the eluent and 33%-35% of the eluent, the purity of the eluted sample is greater than 99%, and the recovery rate is high and stable. Combining the two sets of data, it can be clearly seen that when the elution salt is as low as 160mM NaCl and the elution salt is as low as 300mM NaCl, the yield drops sharply, and when the elution salt is 180mM NaCl and the elution salt is around 320mM NaCl and above, the yield rises sharply, about 3 times.

[0096] Example 4 Loading capacity determination

[0097] The experiment was conducted using a chromatography column with an inner diameter of 0.66 cm, a packing height of 19 cm, and a column volume of 6.5 mL to verify the effects of different loading capacities.

[0098] Sample source: Antibody sample 1;

[0099] Chromatography column and filler: Omnifit 6.6mm / 330mm chromatography column, filler: GE Healthcare Capto SImpAct, column volume: 6.5mL;

[0100] Equilibration buffer: 50 mM NaAc-HAc, pH 5.0;

[0101] Elution buffer: 50 mM NaAc-HAc + 500 mM NaCl, pH 5.0.

[0102] First, prepare antibody sample 1, adjust the pH to 5.0, and dilute with water to a conductivity of 5.8mS / cm and a concentration of 5.22mg / mL. The pipeline is pre-rinsed with equilibrium buffer, and then the chromatography column is rinsed with 3 column volumes of equilibrium buffer at a flow rate of 1.5mL / min, and the pre-prepared antibody sample 1 is loaded at a flow rate of 1.2mL / min. Each group of samples is loaded. Then rinse with 2 column volumes of equilibrium buffer at a flow rate of 1.5mL / min, and finally rinse with 5 column volumes of elution buffer at a flow rate of 1.2mL / min, and then elute with 5 column volumes of elution buffer at a flow rate of 1.2mL / min, and collect the elution peaks respectively. The purification results of different elution conditions are shown in Table 6.

[0103] Table 6

[0104] Serial number Sample volume Elution conditions Elution conditions purity(%) Recovery rate (%) 1 24.9mL (20.0mg / mL) 20％ 35％ 98.09％ 71.76％ 2 38.73mL (31.1mg / mL) 19％ 34％ 98.75％ 76.13％

[0105] From the experimental results, it can be concluded that the sample loading capacity is 20-32 mg / mL, the eluted sample has a high purity, meets the quality requirements, and the recovery rate is high and stable.

[0106] In one embodiment, the Capto S ImpAct medium is tested for sample flowthrough at a retention time of 5 min and a sample pH of 5.0 to determine the most appropriate sample loading capacity for the process.

[0107] Chromatographic conditions:

[0108] Sample source: Antibody sample 2;

[0109] Chromatography column and filler: Omnifit 6.6mm / 330mm chromatography column, filler: GE Healthcare Capto SImpAct, column volume: 6mL;

[0110] Equilibration buffer: 50 mM NaAc-HAc, pH 5.0;

[0111] Elution buffer: 50 mM NaAc-HAc + 500 mM NaCl, pH 5.0;

[0112] First, prepare antibody sample 2, adjust pH to 5.0, and dilute with injection water to a conductivity of 3-6 mS / cm. Rinse the pipeline with equilibrium buffer in advance, then flush the chromatography column with 3 column volumes of equilibrium buffer at a flow rate of 1.2 mL / min, and load the pre-prepared antibody sample 2 at a flow rate of 1.2 mL / min until it starts to flow through.

[0113] from Figure 2 It can be seen from the chromatogram that when the sample volume reaches 20 mL (193.2 mg), the sample UV absorption shows a linear upward trend. Therefore, it is considered that overloading occurs after 20 mL of sample is loaded, and the corresponding loading capacity is about 32 mg / mL or less.

[0114] It should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for purifying an asymmetric fusion protein, It is characterized in that The method comprises the following steps: (1) Equilibration: adding no less than 1 column volume of equilibration buffer to the chromatography column, wherein the equilibration buffer contains 40-60 mM sodium salt solution; (2) Loading the sample; (3) Re-equilibration: adding no less than 1 column volume of equilibration buffer to the chromatography column, wherein the equilibration buffer contains 40-60 mM sodium salt solution; (4) Elution: Add 2-12 column volumes of elution buffer to the chromatography column, wherein the elution buffer contains 40-60 mM sodium acetate and 170-300 mM sodium chloride solution; (5) Elution: adding 2-12 column volumes of elution buffer to the chromatography column, wherein the elution buffer contains 40-60 mM sodium acetate and 310-400 mM sodium chloride solution; Wherein, the pH values ​​of the equilibration buffer, the washing buffer and the elution buffer are 4.5-5.5; The filler of the chromatography column is selected from one or more of Capto S ImpAct, Fractogel COO(M), Nuvia HR-S, and Diamond SP Mustang; The equilibration buffer is selected from sodium acetate-acetic acid; In the (2) loading step, the loading pH is 4.5-6.5 and the conductivity is 3-17 mS / cm; The loading amount in the (2) loading step is less than 60 mg / mL; The asymmetric fusion protein is a heterodimer including a first part and a second part, wherein the first part comprises a light chain and a heavy chain of an antibody that specifically binds to a tumor antigen, and the second part comprises an immunomodulator and an antibody Fc region fused to the immunomodulator from the N-terminus to the C-terminus.

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