Purification method of monoclonal antibody

Abstract

The invention relates to a purification method of a monoclonal antibody. According to the purification method disclosed by the invention, the anti-human CCR8 monoclonal antibody is purified by adopting two-step chromatography, the purification steps are few, the sample recovery rate is high, the process is stable, linear amplification can be realized, the high-purity monoclonal antibody can be obtained, residues of host cell protein, DNA and the like in a purified product are few, and the purified monoclonal antibody has relatively high biological activity and biological safety.

Description

Technical Field

[0001] The present invention belongs to the field of protein purification, and specifically relates to a method for purifying a monoclonal antibody. Background Art

[0002] CC chemokine receptor 8 protein (CCR8) is specifically and highly expressed on the surface of Treg cells in human tumor tissues. Anti-CCR8 antibodies can eliminate Treg cells in tumor tissues but not in normal tissues, and have good safety. At the same time, they can block the CCL1 / CCR8 pathway, and combined with PD-1 / PD-L1, it is expected to increase the drug response rate of tumor patients and enhance tumor immunity.

[0003] The monoclonal antibody J06 against human CCR8 is an innovative target monoclonal antibody drug expressed by CHO cells independently developed by the applicant. The complete molecule of this monoclonal antibody consists of 2 heavy chains (HC) and 2 light chains (LC), each heavy chain consists of 453 amino acid residues; each light chain consists of 219 amino acid residues.

[0004] The above-mentioned anti-human CCR8 monoclonal antibody J06 can block the CCL1 / CCR8 pathway, and is expected to improve the drug response rate of tumor patients in combination with PD-1 / PD-L1 to enhance tumor immunity. Since CCR8 is specifically and highly expressed on regulatory T cells (Treg) infiltrating tumors, it can bind to CCR8 on the surface of Treg cells in the tumor site and initiate ADCC to specifically eliminate Treg cells in tumor tissues, so it has good safety.

[0005] The anti-human CCR8 monoclonal antibody J06 produces a large amount of process-related impurities (such as host cell protein residues, exogenous DNA residues, etc.) and product-related impurities (high molecular weight substances (HMWS), low molecular weight substances (LMWS), charge isomers, etc.) during the expression process. These impurities pose a huge challenge to downstream purification. The traditional three-step chromatography, affinity capture + preliminary purification + fine purification, can achieve the purpose of removing impurities and related residual standards. However, due to the high impurity content and many chromatography steps, the recovery rate of the entire process will be low. Summary of the invention

[0006] The present disclosure provides a method for purifying the anti-human CCR8 monoclonal antibody J06 by two-step chromatography. DETAILED DESCRIPTION OF THE INVENTION

[0008] 1. Terminology

[0009] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0010] Before describing the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodology, scheme and reagent described herein, because these can change. It should also be understood that the terms used in this article are only for describing specific embodiments, and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used in this article have the same meaning as those of ordinary skill in the art to which the present disclosure belongs.

[0011] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure may be described in a range manner. Unless otherwise stated, it should be understood that numerical ranges or the manner of description in a range are only for the purpose of brevity and convenience, and should not be considered as a strict limitation of the scope of the present disclosure. Therefore, the description using a range manner should be considered to specifically disclose all possible sub-ranges and all possible specific numerical points within the range, as these sub-ranges and numerical points have been clearly written in this article. Regardless of the width of the numerical value, the above principles are equally applicable. When a range description is adopted, the range includes the endpoints of the range.

[0012] When referring to a measurable value such as an amount, a temporal duration, etc., the term "about" is meant to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0013] The term "antibody" as used herein typically refers to a Y-shaped tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies have such a structure. Each light chain comprises a light chain variable domain (VL) and a light chain constant domain (CL). Each heavy chain comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH) (or referred to as a heavy chain constant region (CH)).

[0014] The term "monoclonal antibody" (or "MAB") refers to an antibody that is essentially homogeneous and directed only against a specific antigenic epitope and is produced by a single cell clone. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or a combination of the above techniques.

[0015] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or antibody binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including a single epitope, multiple epitopes, a single domain, multiple domains, a complete extracellular domain (ECD) or a protein. Peptides, proteins, glycoproteins, polysaccharides and lipids, parts thereof and combinations thereof can all constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, etc. "Antigen" can also refer to a molecule that triggers an immune response. Any form of antigen or a cell or preparation containing the antigen can be used to generate antibodies specific to the antigenic determinant. The antigen can be an isolated full-length protein, a cell surface protein (e.g., immunized with a cell expressing at least a portion of the antigen on its surface), or a soluble protein (e.g., immunized with only the ECD portion of the protein) or a protein construct (e.g., Fc antigen). The antigen can be produced in genetically modified cells. Any of the aforementioned antigens can be used alone or in combination with one or more immunogenicity enhancing adjuvants known in the art. The DNA encoding the antigen may be genomic or non-genomic (e.g., cDNA) and may encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector may be used to transform cells in which the antigen is expressed, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.

[0016] The term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). D " refers to the dissociation constant for a specific antibody-antigen interaction. Binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance, biolayer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0017] The term "biological activity" refers to the ability of an antibody to bind an antigen and result in a measurable biological response, which can be measured in vitro or in vivo.

[0018] The term "stable" formulation is a formulation in which the protein substantially maintains its physical stability and / or chemical stability and / or biological activity after storage. Preferably, the formulation substantially maintains its physical and chemical stability, as well as its biological activity after storage. The storage period is generally selected based on the shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art. Stability can be measured at a selected temperature for a selected time. Stability can be assessed qualitatively and / or quantitatively in many different ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and / or by visual observation); assessing charge heterogeneity by using cation exchange chromatography or capillary partition electrophoresis; amino-terminal or carboxyl-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis to compare reduced and intact antibodies; peptide mapping; assessing biological activity or antigen binding function of antibodies; and the like. Instability can include any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), cleavage / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extension, C-terminal processing, differences in glycosylation, and the like.

[0019] The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during manufacturing, storage and application. Stabilizers include, but are not limited to, sugars, amino acids, polyols, cyclodextrins, etc.

[0020] Methods for producing and purifying antibodies and antigen-binding fragments are well known and available in the art, such as Cold Spring Harbor Laboratory Manual of Antibody Laboratory Techniques, Chapters 5-8 and 15.

[0021] The engineered antibodies disclosed herein can be prepared by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in a bioreactor to produce antibodies. 2. Summary of the invention

[0023] The purpose of the present disclosure is to provide a two-step chromatography method for purifying monoclonal antibodies containing anti-human CCR8.

[0024] A technical solution provided by the present disclosure is: a method for purifying a monoclonal antibody, comprising the following steps:

[0025] The cell culture harvest fluid containing the anti-human CCR8 monoclonal antibody expressed by CHO cells and filtered was subjected to two-step column chromatography, wherein the first step of column chromatography used a MabSelect SuRe LX affinity chromatography column, and the second step of column chromatography used a Toyopearl NH2-750F chromatography column. The three CDR sequences in the heavy chain variable region of the monoclonal antibody were: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the three CDR sequences in the light chain variable region were: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0026] Preferably, the purification method comprises the following steps:

[0027] (1) passing the clarified cell culture harvested fluid containing the anti-human CCR8 monoclonal antibody through a MabSelect SuReLX affinity chromatography column, eluting, and collecting the elution peak containing the target antibody protein;

[0028] (2) incubating the affinity elution peak sample in step (1) under low pH conditions;

[0029] (3) deep filtration of the sample in step (2);

[0030] (4) The sample in step (3) was passed through a Toyopearl NH2-750F chromatography column in flow-through mode, and the flow-through sample was collected;

[0031] (5) the sample in step (4) is passed through a nanofiltration membrane;

[0032] (6) The sample in step (5) is concentrated, the buffer is replaced, and the sample is diluted.

[0033] Preferably, in step (1), the impurities are washed with eluent 1 and eluent 2 respectively, and then the eluent isocratic elution is performed to collect the elution peak; the eluent 1 comprises 50 mmol / L Tris, 0.2 mol / L to 0.6 mol / L Arg-HCl, pH 8.1±0.5; the eluent 2 comprises 25 mmol / L Tris-HCl, pH 8.3±0.5; the eluent comprises 10 mmol / L to 50 mmol / L NaAc-HAc, pH 3.4±0.4.

[0034] Preferably, in step (2), the pH of the affinity elution peak sample is adjusted to 3.5±0.3 with 2 mol / L HAc, and after incubation at room temperature for 1 to 4 hours, the pH is adjusted to 5.3±0.5 with 1 mol / L Tris.

[0035] Preferably, in step (3), a Merck A1HC deep filter is used to deep filter the sample, and the loading capacity is

[0036] ≤5000g / m 2 .

[0037] Preferably, in step (4), the chromatography column is equilibrated with 25 mmol / L NaAc-HAc, pH 5.3±0.5 solution before loading the sample.

[0038] Preferably, in step (5), the sample is first Pro Shield pre-filter; Pro Device nanofiltration membrane; nanofiltration membrane Pro Device load is less than 5000g / m 2 .

[0039] Preferably, in the step (6), the membrane package selected for sample concentration and replacement buffer is a Merck ultrafiltration membrane package; the replacement buffer is 8-12 mmol / L HAc-NaOH, 7%-11% sucrose, pH 5.0±0.5; the concentration of the sample stock solution is finally controlled at 20 g / L±2 g / L; the auxiliary material added to the sample stock solution is a final concentration of 0.03%-0.07% polysorbate 80.

[0040] Preferably, the sequence of the heavy chain variable region of the anti-human CCR8 monoclonal antibody is shown in SEQ ID NO:7, and the sequence of the light chain variable region is shown in SEQ ID NO:8.

[0041] Preferably, the sequence of the heavy chain of the anti-human CCR8 monoclonal antibody is shown in SEQ ID NO:9, and the sequence of the light chain is shown in SEQ ID NO:10.

[0042] A technical solution provided by the present disclosure is: the monoclonal antibody containing anti-human CCR8 obtained by the above purification method.

[0043] A technical solution provided by the present disclosure is: use of the above-mentioned anti-human CCR8 monoclonal antibody in the preparation of a drug for treating advanced solid tumors.

[0044] The purification method disclosed in the present disclosure has the following beneficial effects:

[0045] Compared with the existing purification technology, the advantages of the present disclosure are: the present disclosure has fewer purification steps, high sample recovery rate, stable process, linear scalability, and can obtain relatively high-purity target monoclonal antibodies with less residual host cell proteins, DNA, etc., and has relatively high biological activity and biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the structure of the monoclonal antibody J06 against human CCR8.

[0047] Figure 2 This is the flowthrough mode chromatogram of Toyopearl NH2-750F filler.

[0048] Figure 3 This is the chromatogram of Capto adhere mixed medium in flow-through mode. DETAILED DESCRIPTION

[0049] The present disclosure is further illustrated by the following examples.

[0050] Specific embodiments are listed below to illustrate the present disclosure. However, it should be understood that these embodiments are listed only to illustrate the present disclosure rather than to limit the scope of the present disclosure.

[0051] Example 1 Acquisition and sequence information of anti-human CCR8 monoclonal antibody J06

[0052] According to the contents described in PCT / US2023 / 060686, the anti-human CCR8 monoclonal antibody J06 (corresponding to anti-CCR8Mab 1187 in the patent) was obtained. The three CDR sequences in the heavy chain variable region of the anti-human CCR8 monoclonal antibody J06 are: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; the three CDR sequences in the light chain variable region are: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and the sequence of the light chain variable region is shown in SEQ ID NO: 8. The sequence of the heavy chain is shown in SEQ ID NO: 9, and the sequence of the light chain is shown in SEQ ID NO: 10.

[0053] Example 2 Preparation of anti-human CCR8 monoclonal antibody J06

[0054] The monoclonal antibody J06 against human CCR8 is a monoclonal antibody expressed by CHO cells. The complete molecule of this monoclonal antibody consists of 2 heavy chains (HC) and 2 light chains (LC), each heavy chain consists of 453 amino acid residues; each light chain consists of 219 amino acid residues. Its structure is shown in the attached figure. Figure 1 The above anti-human CCR8 monoclonal antibody J06 was expressed in CHO cells and purified by the method of Example 3 for further experiments.

[0055] Example 3 Purification of anti-human CCR8 monoclonal antibody J06 using affinity chromatography and Toyopearl NH2-750F column chromatography

[0056] The monoclonal antibody J06 against human CCR8 was purified by two-step chromatography using affinity chromatography and Toyopearl NH2-750F column chromatography, comprising the following steps:

[0057] (1) Affinity chromatography: The clarified culture harvest fluid expressing the monoclonal antibody J06 against human CCR8 was loaded onto a MabSelect SuRe LX affinity chromatography column (Cytiva, batch number: 10287596) equilibrated with a buffer (25mmol / L Tris-HCl, pH8.28), and the impurities were washed with eluent 1 (50mmol / L Tris, 0.5mol / L Arg-HCl, pH8.27) and eluent 2 (25mmol / LTris-HCl, pH8.28), respectively, and then the elution was eluted isocratically with an eluent (20mmol / L NaAc-HAc, pH 3.20), and the elution peak was collected. The peak range was 100mAU-100mAU. The chromatography column was regenerated with a regeneration solution (0.1mol / L NaOH);

[0058] (2) Low pH incubation: The affinity eluted sample in step (1) was adjusted to pH 3.65 with 2 mol / L HAc and incubated at room temperature for 2 hours. After the incubation, the pH was adjusted to 5.22 with 1 mol / L Tris.

[0059] (3) Deep filtration: A1HC deep filter (Merck, batch number: CP0DB11443) was equilibrated with a balancing solution (25 mmol / L NaAc-HAc, pH 5.23), and then the sample neutralized in step (2) was subjected to A1HC deep filtration. After the sample was filtered, the deep filter was top-washed with a balancing solution (25 mmol / L NaAc-HAc, pH 5.23), with a loading of 2060.63 g / m 2 .

[0060] (4) Toyopearl NH2-750F chromatography column (TOSOH, batch number: 75NH2FD003GA): Equilibrate the Toyopearl NH2-750F chromatography column with 3 column volumes of buffer (25 mmol / L NaAc-HAc, pH 5.28). Load the sample obtained in step (3) onto the equilibrated Toyopearl NH2-750F chromatography column. During the loading process, UV 280When the flow-through reaches 100 mAU, collect the liquid. After the sample is loaded, use buffer (25 mmol / L NaAc-HAc, pH 5.28) to top-wash the column. The top-wash process continues to collect peaks until UV 280 Stop collecting peaks when the peak drops to 200 mAU. Regenerate the column with regeneration solution (0.5 mol / L NaOH). Figure 2 shown.

[0061] (5) Nanofiltration: Rinse with water for injection pro shield membrane (Merck, batch number: C9SA80093V) and pro Device membrane (Merck, batch number: C1BB23982). Toyopearl NH2-750F flowthrough sample first passes through pro shield pre-filter membrane; Pro Device nanofiltration membrane. The nanofiltration membrane load is 4999.93g / m 2 .

[0062] (6) Concentration and replacement buffer: The membrane package selected for concentration and replacement buffer is Merck ultrafiltration membrane package (batch number: C0NB31760, pore size 30KD, C flow channel, regenerated cellulose material). Use replacement buffer (10mmol / L HAc-NaOH, 9% sucrose, pH5.0) to balance the ultrafiltration membrane package; load the sample after nanofiltration and concentrate it to 30mg / ml to start replacement buffer (replacement buffer: 10mmol / L HAc-NaOH, 9% sucrose, pH 5.0); the volume of replacement buffer is greater than or equal to 10 times the volume of the sample loaded; after the replacement buffer is completed, the top wash is emptied and polysorbate 80 is added to a final concentration of polysorbate 80 of 0.05%. The concentration of the prepared stock solution is 20.0±2mg / ml.

[0063] When scaled up to 500L, the two-step chromatography purification process achieved a total yield of 91.55%, which is at the industry-leading level.

[0064] Step 4 Toyopearl NH2-750F chromatography results are shown in Table 1:

[0065] Table 1 Toyopearl NH2-750F chromatography results

[0066]

[0067] Note: N / A means not tested or not applicable

[0068] Comparative Example: Purification of anti-human CCR8 monoclonal antibody J06 by two-step chromatography using affinity chromatography and Capto adhere column chromatography

[0069] The monoclonal antibody J06 against human CCR8 was purified by two-step chromatography using affinity chromatography and Capto adhere column chromatography, comprising the following steps:

[0070] (1) Affinity chromatography: The clarified culture harvest fluid expressing the anti-human CCR8 monoclonal antibody J06 was loaded onto a MabSelect SuRe LX affinity chromatography column (Cytiva, batch number: 10287596) equilibrated with a buffer (25mmol / L Tris-HCl, 200mmol / L NaCl, pH 7.15), and the impurities were washed with eluent 1 (25mmol / L Tris, 0.5mol / L Arg-HCl, pH7.43) and eluent 2 (20mmol / L NaAc, pH5.65), respectively, and then the eluent (30mmol / L NaAc-HAc, pH3.36) was used for isocratic elution, and the elution peak was collected. The peak range was 100mAU-100mAU. The chromatography column was regenerated with a regeneration solution (0.1mol / LNaOH).

[0071] (2) Capto adhere column (Cytiva, batch number: 10292076): Equilibrate the Capto adhere column with 8 column volumes of buffer (50 mmol / L NaAc-HAc, 0.4 mol / L NaCl, pH 5.99). Use sample treatment solution (4 mol / L NaCl) to adjust the sample conductivity to 37.30 mS / cm and the sample pH to 6.03. Load the treated sample onto the equilibrated Capto adhere column. During the loading process, UV 280 When the flow-through reaches 100mAU, collect the liquid. After the loading is completed, use buffer (50mmol / L NaAc-HAc, 0.4mol / L NaCl, pH 5.99) to top-wash the column. The peak is collected during the top-wash process until UV 280 Stop collecting peaks when the peak drops to 200 mAU. Regenerate the column with 3 column volumes of regeneration solution (50 mmol / L HAc, pH 3.0). Figure 3 shown.

[0072] The results of step (2) Capto adhere chromatography are shown in Table 2:

[0073] Table 2 Capto adhere chromatography results

[0074]

[0075] Note: N / A means not tested or not applicable

[0076] From the comparison of the yield results in Table 1 and Table 2, it can be seen that the two chromatography methods in Example 3 and the comparative example have little difference in the effect on the SEC purity of the sample. The yield of Toyopearl NH2-750F chromatography is 98.95%, which is higher than the 95.00% yield of Capto adhere chromatography, and Toyopearl NH2-750F chromatography is better than Capto adhere chromatography in terms of residual removal effect.

[0077] Example 4 Binding activity detection of anti-human CCR8 monoclonal antibody J06

[0078] The anti-human CCR8 monoclonal antibody J06 purified by the method of Example 3 was tested for protein binding ability. The test method and results are as follows:

[0079] The ELISA method was used to determine the binding ability of the anti-human CCR8 monoclonal antibody J06 to the CCR8 protein. First, the human CCR8 protein was coated on a 96-well plate, and then the test sample was added in gradient dilutions. The anti-human CCR8 monoclonal antibody J06 binds to the human CCR8 protein coated on the well plate, and then a peroxidase-labeled sheep anti-human IgG secondary antibody is added to the well plate. These detection antibodies can bind to the anti-human CCR8 monoclonal antibody J06 captured in the previous step. Finally, TMB substrate solution was added to the well plate for color development and the absorbance at 450nm was read. Using the SoftMax Pro computer software, a four-parameter fitting regression model was used to draw the curve and calculate the EC 50 The results are shown in Table 3. The results show that the anti-human CCR8 monoclonal antibody J06 has a good binding activity with the CCR8 protein.

[0080] Table 3 Binding activity results of monoclonal antibody J06 against human CCR8

[0081]

Claims

1. A method for purifying a monoclonal antibody, characterized in that: The following steps are involved: The cell culture harvest fluid containing the anti-human CCR8 monoclonal antibody expressed by CHO cells and filtered was subjected to two-step column chromatography, wherein the first step of column chromatography used a MabSelect SuRe LX affinity chromatography column, and the second step of column chromatography used a Toyopearl NH2-750F chromatography column. The three CDR sequences in the heavy chain variable region of the monoclonal antibody were: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the three CDR sequences in the light chain variable region were: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

2. The purification method according to claim 1, characterized in that The following steps are involved: (1) passing the clarified cell culture harvest containing the anti-human CCR8 monoclonal antibody through a MabSelect SuRe LX affinity chromatography column, eluting, and collecting the elution peak containing the target antibody protein; (2) incubating the affinity elution peak sample in step (1) under low pH conditions; (3) deep filtering the sample in step (2); (4) passing the sample in step (3) through a Toyopearl NH2-750F chromatography column in flow-through mode, and collecting the flow-through sample; (5) passing the sample in step (4) through a nanofiltration membrane; (6) Concentrate the sample in step (5), replace the buffer, and dilute.

3. The purification method according to claim 2, characterized in that: In the step (1), the impurities are washed with eluent 1 and eluent 2 respectively, and then the eluent isocratic elution is performed to collect the elution peak; the eluent 1 comprises: 50mmol / L Tris, 0.2mol / L-0.6mol / L Arg-HCl, pH 8.1±0.5; the eluent 2 comprises: 25mmol / L Tris-HCl, pH 8.3±0.5; the eluent comprises: 10mmol / L-50mmol / L NaAc-HAc, pH 3.4±0.

4.

4. The purification method according to any one of claims 2 to 3, characterized in that: In the step (2), the pH of the affinity elution peak sample is adjusted to 3.5±0.3 with 2 mol / L HAc, and after incubation at room temperature for 1 to 4 hours, the pH is adjusted to 5.3±0.5 with 1 mol / L Tris.

5. The purification method according to any one of claims 2 to 4, characterized in that: In step (3), a Merck A1HC deep filter is used to deep filter the sample, with a loading of ≤5000 g / m 2 In step (4), the chromatography column is equilibrated with 25 mmol / L NaAc-HAc, pH 5.3 ± 0.5 solution and then the sample is loaded.

6. The purification method according to any one of claims 2 to 5, characterized in that: In step (5), the sample is first passed through Pro Shield pre-filter; Pro Device nanofiltration membrane; nanofiltration membrane ProDevice load is less than 5000g / m 2 .

7. The purification method according to any one of claims 2 to 6, characterized in that: The membrane package selected for sample concentration and replacement buffer in step (6) is a Merck ultrafiltration membrane package; the replacement buffer is 8-12 mmol / L HAc-NaOH, 7%-11% sucrose, pH 5.0±0.5; the concentration of the sample stock solution is finally controlled at 20 g / L±2 g / L; the auxiliary material added to the sample stock solution is a final concentration of 0.03%-0.07% polysorbate 80.

8. The purification method according to any one of claims 1 to 7, characterized in that: The sequence of the heavy chain variable region of the anti-human CCR8 monoclonal antibody is shown in SEQ ID NO:7, and the sequence of the light chain variable region is shown in SEQ ID NO:8; preferably, the sequence of the heavy chain of the anti-human CCR8 monoclonal antibody is shown in SEQ ID NO:9, and the sequence of the light chain is shown in SEQ ID NO:

10.

9. The anti-human CCR8 monoclonal antibody obtained by the purification method according to any one of claims 1 to 8.

10. Use of the anti-human CCR8 monoclonal antibody according to claim 9 in the preparation of a medicament for treating advanced solid tumors.

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