CN114805473A

CN114805473A - Method for purifying asymmetric fusion protein

Info

Publication number: CN114805473A
Application number: CN202210424493.6A
Authority: CN
Inventors: 吴崇兵; 顾海涛; 姜晓玲; 殷刘松
Original assignee: Sunho China Biopharmaceutical Co Ltd
Current assignee: Sunho China Biopharmaceutical Co Ltd
Priority date: 2022-04-21
Filing date: 2022-04-21
Publication date: 2022-07-29

Abstract

The invention provides a method for purifying asymmetric fusion protein, which comprises the steps of balancing, loading, rebalancing, leaching, eluting and the like. According to the invention, by improving the purification conditions and considering and screening the pH value of the sample, the sample conductance, the eluent and the like, the purification process suitable for the asymmetric fusion protein is obtained, the separation degree of each component in the process is good, the effect of removing impurities is better, the asymmetric fusion protein with high purity, high recovery rate and good stability is finally obtained, and the quality control level and the drug stability of the product are favorably improved.

Description

Method for purifying asymmetric fusion protein

Technical Field

The invention relates to the technical field of biology, in particular to a purification process for effectively purifying asymmetric fusion protein and reducing impurities.

Background

The cell factor is a small molecule protein or glycoprotein, and has the capacity of recognizing and killing tumor cells under specific conditions to prepare the medicine. However, the efficacy of cytokines is often proportional to their dosage, and they have a small molecular weight and a short half-life, and require frequent administration of high doses to maintain a certain blood level in order to ensure the efficacy of antitumor agents. Patients are prone to dose-limiting toxicity and systemic toxicity. To reduce toxicity, antibody-cytokine fusion proteins have been developed.

The antibody-cytokine fusion protein is a fusion protein composed of a targeting recombinant antibody or antibody fragment and a cytokine. Theoretically, antibody-cytokine fusion proteins compare to cytokines: a. the antibody-cytokine fusion protein carrying the same amount of cytokines has larger molecular weight, can improve the stability in vivo, prolong the half-life period and reduce the clearance rate of serum; b. reducing the frequency of administration, reducing DLT and systemic toxicity; c. the cytokine is enriched at the pathological change part, so that the medicine concentration of the pathological change part is improved, and the immunoregulation effect of the cytokine is more effectively exerted; d. increasing the immunogenicity of the lesion, and increasing the reaction of the immunosuppressant by combining with the immunosuppressant.

For antibody-cytokine fusion protein, because the cytokine is fused into the monoclonal antibody structure, the spatial structure of the monoclonal antibody can be changed to a certain extent, and meanwhile, part of the fusion protein such as IL-10 has the characteristic of tending to aggregate, the purification of the antibody-cytokine fusion protein by adopting the traditional cation chromatography condition has great difficulty. More particularly, for asymmetric fusion proteins, impurities such as a dimer, a homodimer, a half antibody, and fragmentation are easily generated, and compared with a double antibody with a symmetric structure, the asymmetric fusion proteins have two special impurities, namely the homodimer and the half antibody, which results in higher purification difficulty, and research and development of a purification scheme applicable to different asymmetric fusion proteins are urgently needed.

Disclosure of Invention

In order to overcome the technical problem, the invention obtains the purification process suitable for the asymmetric fusion protein by improving the purification conditions of the asymmetric fusion protein and screening and optimizing the pH value of a sample, the conductance of the sample, leacheate, eluent and the like. The method has good separation degree of each component of the asymmetric fusion protein and good impurity removal effect, finally obtains the asymmetric fusion protein with high purity, high recovery rate and good stability, and is beneficial to improving the quality control level and the drug stability of the product.

The technical scheme adopted by the invention is as follows:

a method of purifying an asymmetric fusion protein, comprising the steps of:

(1) balancing: adding an equilibration buffer solution with the volume not less than 1 column into a chromatographic column, wherein the equilibration buffer solution contains 40-60mM of sodium salt solution;

(2) sampling;

(3) rebalancing: adding an equilibration buffer solution with the volume not less than 1 column into a chromatographic column, wherein the equilibration buffer solution contains 40-60mM of sodium salt solution;

(4) leaching: adding 2-12 column volumes of elution buffer into the chromatographic column, wherein the elution buffer contains 170-300mM sodium salt solution;

(5) and (3) elution: adding 2-12 column volumes of elution buffer to the chromatography column, the elution buffer containing 310-400mM sodium salt solution;

wherein the pH values of the equilibration buffer, the elution buffer and the elution buffer are 4.5-5.5.

In one embodiment, in the (4) elution step, 2-10 column volumes of elution buffer containing 175. sup. 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 mM 370mM sodium salt are added to the chromatography column.

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

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

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 leaching buffer is selected from sodium acetate-acetic acid, the concentration of sodium chloride is 175-250mM, and the pH is 4.5-5.5 or phosphate buffer, the concentration of sodium chloride is 175-250mM, and the pH is 5.5-6.5; and/or, the elution buffer is selected from sodium acetate-acetic acid, sodium chloride concentration of 325-400mM, pH 4.5-5.5 or phosphate buffer, sodium chloride concentration of 325-400mM, pH 4.5-5.5.

In one embodiment, the pH of the sample in the (2) sample loading step is 4.5-6.5, and the conductance is 3-17 mS/cm; preferably, the loading pH is from 4.5 to 6 and the conductance is from 4 to 15 mS/cm.

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

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

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

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

In one embodiment, the second moiety is a moiety comprising an immunomodulatory agent.

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

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

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

In one embodiment, the second portion comprises an immunomodulator, an antibody Fab region fused to the immunomodulator that specifically binds a tumor antigen, and an Fc region, 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 portion is complexed with the heavy chain of the first portion to form the asymmetric fusion protein.

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, 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, IT, TIGE-3, MYTL 1A, MART-1/MelanA, gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, MAGE-1, MAGE-463, LIP-5, LIPT-2, MAGE-1, MAGE-P-463, MAGE-E-2, MAGE-11, MAGE-E-2, MAGE-E-2, MAGE-E-2, MAGE-E-2, MAGE-E3, MAGE-2, MAGE-E-2, MAGE-E2, MAGE-2, MAGE 3, MAGE-E3, MAGE 2, MAGE 3, 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, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, P15, P16, 43-9F, 5T4, 791Tgp72, alpha fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3/CA 27.29/BCAA, CA 195, CA 242, CA-50, CAM43, CD68/P1, CO-029, FGF-5, G250, Ga/Ep733 CAM, HTgp 175-175, M344, MA-50, MG7-Ag, MOV 23, MOV 24/Ag 4670, CARCA 24/P1, CARCA-462, CARCG-5, CARCG-associated protein, and CARCG 16/C2, TAAL6, TAG 6, TLP, MUC 6, IL13 6 alpha 2, FR alpha, VEGFR 6, Lewis Y, FAP, EphA 6, CEACAM 6, EGFR, CA6, GPNMB, EGP 6, FOLR 6, endothelial receptor, STEAP 6, SLC44A 6, bindin-4, AGS-16, guanidino cyclase 6-1, CFC 16, integrin alpha 3 chain, TPS, CD6, CD 6856854, CD6, CD 6856856854, CD79 6, CD166, CD6, CLL-1/CLEC12, ROR 6, EGFRUCN 3, MP 6, MUL 6/6, IL 6, PSM 6, PSMA-6, or MY-6.

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

In one embodiment, the immunomodulator is selected from the group consisting of IL-1, IL-2 Ra, IL-2 Rbeta, IL-3 Ra, IL-4 Ralpha, IL-5 Ralpha, IL-6 Ralpha, IL-7 Ralpha, IL-8, IL-9 Ralpha, IL-10R1, IL-10R2, IL-11 Ralpha, IL-12 Ralpha, IL-12 Rbeta 2, IL-12 Rbeta 1, IL-13 Ralpha 2, IL-14, IL-15 Ralpha sushi, IL-16, IL-17, IL-13, IL-6 Ralpha, IL-7 Ralpha, IL-8 Ralpha, IL-9 Ralpha, IL-10 Ralpha, IL-11 Ralpha, IL-12 Rbeta 2, IL-12 Rbeta 1, IL-13 Ralpha, IL-14, IL-15 Ralpha sushi, IL-16, IL-17, IL-2, and IL-2, One or more of IL-18, IL-19, IL-20R1, IL-20R2, IL-21 Ra, IL-22, IL-23R, IL-27R, IL-31R, G-CSF-R, LIF-R, OSM-R, GM-CSF-R, R Bc, Ryc, TSL-P-R, EB13, CLF-1, CNTF-Ra, gp130, Leptin-R, PRL-R, GH-R, Epo-R, Tpo-R, IFN-Lambda R1, IFN-Lambda R2, IFNR1 and IFNR 2.

Term(s) for

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 artificially combined to form one protein. Including but not limited to the exemplified form 1, a fusion protein consisting of one polypeptide chain, fused to each other by the same or different polypeptides, to form one polypeptide chain comprising said different polypeptides; 2. for fusion proteins consisting of two or more polypeptide chains, wherein optionally one or more polypeptide chains are fused to each other by the same or different polypeptides to form polypeptide chains comprising said same or different polypeptides, these polypeptide chains are combined with each other in a covalent or non-covalent fashion to form the protein.

As used herein, the term "heterodimer" generally refers to a molecule, e.g., a protein molecule, that is composed of two distinct members. The two members of the heterodimer may differ in structure, function, activity, and/or composition. For example, two different members may comprise polypeptides that differ in the order, number, or type of amino acid residues that form the polypeptides. Each of the two different members of the heterodimer can independently comprise one, two, or more units, polypeptide chains, or portions.

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

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 molecule or compound. Other examples of targeting moieties may include, but are not limited to, aptamers, high affinity multimers, receptor binding ligands, nucleic acids, biotin-avidin binding pairs, binding peptides or proteins, and the like.

As used herein, the term "tumor antigen" generally refers to an antigenic substance produced in or by a tumor cell that may have the ability to trigger an immune response in a host. For example, the tumor antigen may be a protein, polypeptide, peptide or fragment thereof that forms 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 biomolecule (e.g., protein, carbohydrate, glycoprotein, etc.) that is uniquely or preferentially or differentially expressed on and/or found associated with cancer cells to provide a target that is preferential or specific for cancer. For example, preferential expression may be preferential expression compared to any other cell in the organism, or preferential expression within a particular region of the organism (e.g., within a particular organ or tissue).

As used herein, the term "immune checkpoint" generally refers to some inhibitory and activating molecules present in the immune system that modulate the body's anti-tumor immune system by modulating T cell activity. For example, inhibitory molecules include PD1, PDL1, B7H3, CTLA4, and the like, and activating molecules include OX40, 4-1BB, CD40, and the like.

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

As used herein, the term "linear elution" refers to a method, for example, in certain embodiments, where the total volume of elution buffer a and elution buffer B is constant and the volume fraction of elution buffer B is linear with time throughout the elution process, i.e., the volume of elution buffer B increases linearly with time.

Drawings

FIG. 1 is a schematic structural diagram of an asymmetric fusion protein of the present invention.

FIG. 2 is a load test chromatogram of the present invention.

Detailed Description

The present invention will be described in detail with reference to specific examples, which are not intended to limit the scope of the present invention. The experimental method of the present invention, in which the specific conditions are not specified, is generally carried out under the conventional conditions, and the test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

Example 1 chromatography packing screening

Sample purification was investigated using a Capto S inpact cationic filler from GE, a Fractogel COO (M) cationic filler from merck, a Nuvia HR-S cationic filler from burle, and a Diamond SP Mustang cationic filler from bougon, and the purification effects of different chromatographic fillers were investigated under sample loading conditions of retention time 5min, 20mg/mL loading, ph5.5, and conductance 3-6 mS/cm.

Chromatography conditions are as follows:

sample source: antibody sample 2 (prepared with reference to CN202110497420.5 as a heterodimer of an anti-EGFR antibody and interleukin-10);

chromatographic column and packing: omnifit 6.6mm/330mm chromatographic column, filler: GE Healthcare Capto S ImpAct, Merck Fractogel COO (M), Biorad Nuvia HR-S, Booglong Diamond SP Mustang, column volume: 6 mL;

and (3) an equilibrium buffer: 50mM NaAc-HAc, pH 5.5;

elution buffer: 50mM NaAc-HAc +1M NaCl, pH 5.5.

The pH of the antibody sample 2 is adjusted to 5.5, the antibody sample is diluted to the conductance of 3-6mS/cm by water injection, the pipeline is rinsed by an equilibrium buffer solution in advance, then the chromatographic column is washed by a 4-time column volume equilibrium buffer solution at the flow rate of 2mL/min, and the prepared antibody sample 2 is loaded at the flow rate of 1.2 mL/min. Washing with 3 times column volume balance buffer solution at flow rate of 1.2mL/min, washing with 20 times column volume elution buffer solution at flow rate of 1.2mL/min at gradient of 0-30%, and collecting elution peaks in different stages. The purification results of the different chromatography packings are shown in table 1.

TABLE 1

Type of packing	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

From the results, it is clear that the yields after purification of the 4 affinity packings are all higher than 60%, and among them, the best results are obtained by using the Capto S ImpAct cationic packing produced by GE company and the Nuvia HR-S cationic packing produced by Berle company, which are all higher than 70%. The yield ratio of Fractogel COO (M) cationic filler produced by Merck company to Diamond SP Mustang cationic filler produced by Bogelong company is slightly poor, and the yield is also higher than 60%.

Example 2pH and conductivity conditions

And (3) detecting the effect of removing impurities by using a GE Capto S ImpAct filler and fixing a linear elution gradient through different pH values and sample loading conductance conditions when the retention time is 5min and the loading capacity is 15 mg/mL.

Chromatography conditions are as follows:

sample source: antibody sample 1 (prepared with reference to CN202111135075.7, a heterodimer of an anti-B7H 3 antibody and interleukin-10);

equilibration buffer 1: 50mM NaAc-HAc, pH 5.0; elution buffer 1: 50mM NaAc-HAc, 1M NaCl, pH 5.0;

equilibration buffer 2: 50mM NaAc-HAc, pH 5.5; elution buffer 2: 50mM NaAc-HAc, 1M NaCl, pH 5.5;

equilibration buffer 3: 20mM PB, pH 6.0; elution buffer 3: 20mM PB, 1M NaCl, pH 6.0.

Antibody sample 1 was first prepared, pre-formulated to the pH and conductance as shown in the table below. The pipeline is pre-rinsed by an equilibrium buffer solution, then the chromatographic column is flushed by the equilibrium buffer solution with the column volume of 3 times at the flow rate of 1mL/min, the pre-prepared antibody sample 1 is used for sampling at the flow rate of 1mL/min, the sampling loading capacity of each group of samples is 15mg/mL, and the flow rate is 1 mL/min. Then washing with 2 times column volume balance buffer solution at flow rate of 1mL/min, then washing with 20 times column volume elution buffer solution at flow rate of 1mL/min according to 0-50% gradient, and collecting elution peaks in sections. The purification results of the samples under different conditions are shown in table 2.

TABLE 2

From the results, it can be seen that different pH and conductance resulted in higher purity samples under the same linear gradient elution, but the degree of separation was greatly different, with pH having a very large effect on the degree of separation, and the selection of a large degree of separation facilitated the performance of isocratic gradient elution. The degree of separation is optimal when the pH is 5.0; when the pH is 5.5, the separation degree is inferior; at a pH of 6.0, the effect of the degree of separation is not satisfactory. Therefore, when the pH value is 5.0, the separation effect is better when the conductance is selected to be 5-15; when the pH value is 5.5, the separation effect is better when the conductance is 5-10; when the pH is 6.0, the separation effect is better when the conductivity is selected to be around 10.

In one embodiment, the effect of impurity removal was examined using a GE Capto S inpact packing under conditions of a retention time of 5min, a loading of 20mg/mL packing, and a fixed linear elution gradient with eluents having pH of 5.0 and 5.5, respectively.

Chromatography conditions are as follows:

sample source: antibody sample 2;

equilibration buffer 1: 50mM NaAc-HAc, pH 5.0; elution buffer 1: 50mM NaAc-HAc +1M NaCl, pH 5.0;

antibody sample 2 was first prepared, pH adjusted to 5.0, and conductance adjusted to 3-6 mS/cm. The tube was previously rinsed with equilibration buffer, the column was then rinsed with 4 column volumes equilibration buffer at a flow rate of 0.5mL/min, and the pre-prepared antibody sample 1 was loaded at a flow rate of 0.5 mL/min. Then washing with 2 times column volume balance buffer solution at flow rate of 0.5mL/min, then washing with 60 times column volume elution buffer solution at flow rate of 0.5mL/min according to 0-40% gradient, and collecting elution peak by stages. The purification results of the samples under different conditions are shown in table 3.

TABLE 3

The results show that the pH is 5.0, the conductivity is 3-6mS/cm, the separation effect is good, a qualified sample with the polymer less than 2% can be obtained, and the yield is high.

Example 3 elution conditions optimization

The antibody sample 2 contains the polymer and degradation fragments, and a part of the polymer fragments need to be removed by optimizing elution conditions. In this embodiment, 32% and 36% of eluents are respectively used for eluting the chromatography, and 60% and 64% of eluents are correspondingly used for eluting the target protein in the chromatography, so as to detect the removal effect of the polymer, so as to select a relatively suitable elution condition for cation exchange chromatography.

Chromatography conditions are as follows:

sample source: antibody sample 2, 112mg per set of experiments;

chromatographic column and packing: omnifit 6.6mm/330mm chromatographic column, filler: GE Healthcare Capto S ImpAct, column volume: 6 mL;

equilibration buffer: 50mM NaAc-HAc, pH 5.0;

elution buffer: 50mM NaAc-HAc +500mM NaCl, pH 5.0.

Antibody sample 2 was first prepared, adjusted to pH5.0, and diluted with water to a conductance of 3-6 mS/cm. The tubing was pre-rinsed with equilibration buffer, the column was then rinsed with 3 column volumes equilibration buffer at a flow rate of 2mL/min, and the pre-prepared antibody sample 2 was loaded at a flow rate of 1.2mL/min, completing each set of samples. The column was washed with 3 column volumes of equilibration buffer at a flow rate of 1.2mL/min, and eluted with 8 column volumes of elution buffer at a flow rate of 1.2mL/min, with elution gradients of 32% and 36% for the 2 sets of experiments in the order (NaCl concentrations 160mM and 180nM, respectively, in the same manner as calculated below). Eluting with 8 times column volume flow rate 1.2mL/min elution buffer solution, sequentially eluting with 60% and 64% gradient in 2 groups of experiments, and collecting the elution peaks respectively. The results of the purification under different elution conditions are shown in table 4.

TABLE 4

From the results, it can be seen that eluting the chromatography with 36% eluent and 64% eluent effectively removed the polymer, and from the data results, the sample yield was the highest under this elution condition.

Similarly, the antibody sample 1 also contains a polymer and a degradation fragment, in order to verify whether the asymmetric fusion protein with similar structure can remove a part of the polymer fragment by using the same elution conditions, the chromatography is eluted by using 1mL of Captos ImpAct with the loading of 20mg/mL and 20%, 18% and 21% of eluents respectively, and the removal effect of the polymer is detected by eluting the chromatography by using 35%, 33% and 36% of eluents respectively.

Chromatography conditions are as follows:

sample source: antibody sample 1;

and (3) an equilibrium buffer: 50mM NaAc-HAc, pH 5.0;

elution buffer: 50mM NaAc-HAc, 1M NaCl, pH 5.0.

Antibody sample 1 was first prepared, pH adjusted to 5.0, conductance 5.44mS/cm, and assay concentration 5.57 mg/mL. The tube was pre-rinsed with equilibration buffer, the column was then rinsed with 3 column volumes of equilibration buffer at a flow rate of 1mL/min, and the pre-prepared antibody sample 1 was loaded at a flow rate of 1mL/min, 3.59mL per group. Then washing with 2 times column volume balance buffer solution at flow rate of 1mL/min, and finally eluting with 5 times column volume elution buffer solution at flow rate of 1mL/min, wherein the elution gradients in the three groups are 20%, 18% and 21% in sequence (NaCl concentrations are 200mM, 180nM and 210nM, respectively). Then eluting with 5 times column volume flow rate 1mL/min elution buffer solution, wherein the three groups of elution gradients are 35%, 33% and 36% (NaCl concentration is 350mM, 330mM and 360mM respectively), and collecting elution peaks respectively. The results of the purification under different elution conditions are shown in table 5.

TABLE 5

Serial number	Elution conditions	Purity%	The recovery rate is high
				1	20％+35％	99.11％	64.66％
2	18％+33％	99.05％	67.16％
				3	21％+36％	99.09％	69.37％

According to the results, when the elution condition is 18% -21% of the eluent and the elution condition is 33% -35% of the eluent, the purity of the eluted sample is more than 99%, and the recovery rate is high and stable. By combining the two groups of data, it can be clearly seen that the yield is sharply reduced when the leaching salt is as low as 160mM NaCl, and the yield is sharply reduced when the leaching salt is 180mM NaCl, and the yield is sharply increased by about 3 times when the leaching salt is about 320mM NaCl or more.

Example 4 load determination

Experiments were carried out using a chromatography column with an internal diameter of 0.66cm, a column height of 19cm and a column volume of 6.5mL to verify the effect of different loadings.

Sample source: antibody sample 1;

chromatographic column and packing: omnifit 6.6mm/330mm chromatographic column, packing: GE Healthcare Capto S ImpAct, column volume: 6.5 mL;

and (3) an equilibrium buffer: 50mM NaAc-HAc, pH 5.0;

elution buffer: 50mM NaAc-HAc +500mM NaCl, pH 5.0.

Antibody sample 1 was first prepared, pH adjusted to 5.0, and diluted with water to a conductivity of 5.8mS/cm at a concentration of 5.22 mg/mL. The tubing was pre-rinsed with equilibration buffer, the column was then rinsed with 3 column volumes of equilibration buffer at a flow rate of 1.5mL/min, and the pre-prepared antibody sample 1 was loaded at a flow rate of 1.2mL/min, completing each set of samples. Then washing with 2 times column volume balance buffer solution at the flow rate of 1.5mL/min, finally washing with 5 times column volume elution buffer solution at the flow rate of 1.2mL/min, then eluting with 5 times column volume elution buffer solution at the flow rate of 1.2mL/min, and respectively collecting elution peaks. The results of the purification under different elution conditions are shown in table 6.

TABLE 6

Serial number	Sample loading amount	Conditions of elution	Elution conditions	Purity (%)	Recovery (%)
						1	24.9mL(20.0mg/mL)	20％	35％	98.09％	71.76％
2	38.73mL(31.1mg/mL)	19％	34％	98.75％	76.13％

The experimental result shows that the loading capacity of the sample is 20-32mg/mL, the purity of the eluted sample is high, the quality requirement is met, and the recovery rate is high and stable.

In one embodiment, the Capto S inpact packing was tested for sample load flow-through at retention time 5min, sample ph5.0, to determine the load capacity for the most suitable process.

Chromatography conditions are as follows:

sample source: antibody sample 2;

chromatographic column and packing: omnifit 6.6mm/330mm chromatographic column, packing: GE Healthcare Capto S ImpAct, column volume: 6 mL;

and (3) an equilibrium buffer: 50mM NaAc-HAc, pH 5.0;

elution buffer: 50mM NaAc-HAc +500mM NaCl, pH 5.0;

antibody sample 2 was first prepared, adjusted to pH5.0, and diluted with water to a conductance of 3-6 mS/cm. The tubing was pre-rinsed with equilibration buffer, the column was rinsed with 3 column volumes equilibration buffer at a flow rate of 1.2mL/min, and the pre-prepared antibody sample 2 was loaded at a flow rate of 1.2mL/min until flow-through began.

As can be seen from the chromatogram in FIG. 2, when the sample volume reaches 20mL (193.2mg), the UV absorption of the sample tends to increase linearly, so that the overload phenomenon is considered to occur after the sample is loaded by 20mL, and the corresponding loading amount is about 32mg/mL or less.

It should be understood that the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method of purifying an asymmetric fusion protein, comprising the steps of:

(2) sampling;

2. The method as claimed in claim 1, wherein in the (4) elution step, 2-10 column volumes of elution buffer containing 175-250mM 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 mM 370mM sodium salt are added to the chromatography column.

3. The method of any one of claims 1-2, wherein the packing material of the chromatography column is selected from one or more of Capto S inpact, Fractogel COO (M), Nuvia HR-S, Diamond SP Mustang.

4. The method according to claim 1, wherein the sodium salt solution in the buffer system of equilibration, elution and elution buffers is selected from one or more of sodium acetate-acetic acid, sodium chloride, sodium citrate, disodium hydrogen phosphate, sodium dihydrogen phosphate.

5. The method of claim 4, wherein the equilibration buffer is selected from sodium acetate-acetic acid at a pH of 4.5-5.5 or phosphate buffer at a pH of 5.5-6.5; and/or, the leaching buffer is selected from sodium acetate-acetic acid, the concentration of sodium chloride is 175-250mM, and the pH is 4.5-5.5 or phosphate buffer, the concentration of sodium chloride is 175-250mM, and the pH is 5.5-6.5; and/or, the elution buffer is selected from sodium acetate-acetic acid, sodium chloride concentration of 325-400mM, pH 4.5-5.5 or phosphate buffer, sodium chloride concentration of 325-400mM, pH 4.5-5.5.

6. The method according to claim 1, wherein in the (2) loading step, the loading pH is 4.5-6.5, and the conductance is 3-17 mS/cm; preferably, the pH of the sample is 4.5-6 and the conductance is 4-15 mS/cm.

7. The method according to claim 1, wherein the loading in the (2) loading step is less than 100mg/mL, preferably less than 80mg/mL, less than 60mg/mL or less than 40 mg/mL.

8. The method of claim 1, wherein the asymmetric fusion protein is a heterodimer comprising a first portion and a second portion.

9. The method of claim 8, wherein the first moiety is a targeting moiety that specifically binds to a tumor antigen or an immune checkpoint.

10. The method of any one of claims 8-9, wherein the second moiety is a moiety comprising an immunomodulatory agent.