A method for purifying a monoclonal antibody

Abstract

The present disclosure relates to a purification method of a monoclonal antibody. The purification method of the present disclosure adopts two-step chromatography to purify a monoclonal antibody against human CCR8, has fewer purification steps, high sample recovery rate, stable process, linear amplification, can obtain a monoclonal antibody with high purity, has less residual host cell protein, DNA, etc. in the purified product, and the purified monoclonal antibody has relatively high biological activity and biosafety.

Description

TECHNICAL FIELD

[0001] The present disclosure belongs to the field of protein purification, and in particular to a method for purifying a monoclonal antibody. BACKGROUND

[0002] C-C chemokine receptor 8 protein (CCR8) is specifically highly expressed on the surface of Treg cells in human tumor tissues, and an anti-CCR8 antibody can remove Treg cells in tumor tissues without removing Treg cells in normal tissues, and has good safety; meanwhile, it can block the CCL1 / CCR8 pathway, and the combination with PD-1 / PD-L1 is expected to improve the drug response rate of tumor patients and enhance tumor immunity.

[0003] The anti-human CCR8 monoclonal antibody J06 is an innovative target monoclonal antibody drug expressed by CHO cells independently developed by the applicant. The complete molecule of the monoclonal antibody is composed of two heavy chains (Heavy Chain, HC) and two light chains (Light Chain, LC), each heavy chain is composed of 453 amino acid residues; and each light chain is composed of 219 amino acid residues.

[0004] The above-mentioned anti-human CCR8 monoclonal antibody J06 can block the CCL1 / CCR8 pathway, and the combination with PD-1 / PD-L1 is expected to improve the drug response rate of tumor patients and enhance tumor immunity. Since CCR8 is specifically highly expressed on the tumor-infiltrating regulatory T cells (Treg), by binding to CCR8 on the surface of Treg cells in the tumor site, ADCC-specific removal of Treg cells in the tumor tissue is initiated, and therefore 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 expression. These impurities bring great challenges to downstream purification work. Although the traditional three-step chromatography, affinity capture + preliminary purification + fine purification, can achieve the purpose of removing impurities and related residues standards. However, due to the high content of impurities and multiple chromatography steps, it will lead to a low recovery rate of the whole process. SUMMARY

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

[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 the present disclosure is described in detail below, it is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described herein as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0011] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure can be described in terms of ranges of values. Unless otherwise stated, it is to be understood that numerical ranges or ranges described in terms of ranges are for the purpose of describing the embodiments only, and are not to be considered as limiting the scope of the present disclosure. Thus, descriptions in ranges should be considered as specifically disclosing all possible subranges and all possible individual numerical values within that range, as if each and every such subrange and numerical value were specifically written herein. The same applies regardless of whether the range is expressed in lower or higher limit form. When ranges are used to describe elements, unless otherwise stated the ranges are "open" ranges, i.e., the range excludes the range endpoints.

[0012] The term "about" when used in relation to a measurable value such as an amount, temporal duration, and the like, means ±20% of the specified value, or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±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 noncovalent interactions. Native IgG antibodies have this 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 heavy chain constant region (CH)).

[0014] The term "monoclonal antibody" (or "mAb") refers to an antibody produced by a single clone of cells, which is essentially homogenous and directed to only one specific epitope of an antigen. Monoclonal antibodies can be produced using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic techniques, or a combination of the foregoing.

[0015] The term“antigen” refers to a substance that is recognized and specifically bound by an antibody or antibody binding fragment. Broadly, an antigen can include any immunogenic fragment or determinant of a selected target, including a single epitope, a polyepitope, a single domain, a multi-domain, an entire extracellular domain (ECD) or protein. Peptides, proteins, glycoproteins, polysaccharides, and lipids, portions thereof, and combinations thereof can all constitute an antigen. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, among others. An“antigen” can also refer to a molecule that elicits an immune response. Any form of an antigen or a cell or preparation containing the antigen can be used to generate antibodies specific for an antigenic determinant. An 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 a genetically modified cell. Any of the foregoing antigens can be used alone or in combination with one or more immunogenicity-enhancing adjuvants known in the art. DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA), and can encode at least a portion of an ECD sufficient to elicit an immunogenic response. Any vector can be used to transform the cell 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 total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The term“K D ” refers to the dissociation constant of a particular antibody-antigen interaction. Binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance, bio-layer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry, among others.

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

[0018] The term "stable" formulation is one in which the protein substantially retains its physical stability and / or chemical stability and / or biological activity upon storage. Preferably, the formulation substantially retains its physical and chemical stability, as well as its biological activity upon 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 period. Stability can be qualitatively and / or quantitatively assessed in a number of different ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and / or by visual inspection); assessing charge heterogeneity by using cation exchange chromatography or capillary zone electrophoresis; amino terminal or carboxy terminal sequence analysis; mass spectrometry; SDS-PAGE analysis to compare reduced and intact antibody; peptide mapping; assessing biological activity or antigen binding function of the antibody; 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), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extensions, C-terminal processing, glycosylation differences, and the like.

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

[0020] Methods for producing and purifying antibodies and antigen binding fragments are well known and available in the art, such as in Antibody Engineering Protocols, 5-8 and 15 chapters of the Methods in Molecular Biology series, Cold Spring Harbor Laboratory Press.

[0021] The engineered antibodies of the present disclosure can be produced using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vectors can be stably transfected into CHO cells. As a more preferred prior art, mammalian expression systems result in glycosylation of the antibodies, particularly in the highly conserved N-terminal of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free media in bioreactors to produce antibodies. 2. SUMMARY

[0023] It is an object of the present disclosure to provide a two-step chromatography method for purifying a monoclonal antibody comprising anti-human CCR8.

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

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

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

[0027] (1) passing the clarified cell culture harvest containing anti-human CCR8 monoclonal antibody through a MabSelect SuRe LX 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 layer filtering the sample in step (2);

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

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

[0032] (6) concentrating, replacing the buffer, and diluting the sample in step (5).

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

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

[0035] Preferably, in step (3), the sample is subjected to depth filtration using a Merck A1 HC depth filter, with a loading capacity of

[0036] ≤5000g / m 2 .

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

[0038] Preferably, in step (5), the sample is first subjected to pre-filtration using a Pro Shield pre-filtration membrane, and then subjected to nanofiltration using a Pro Device nanofiltration membrane. Preferably, in step (5), the sample is first subjected to pre-filtration using a Pro Shield pre-filtration membrane, and then subjected to nanofiltration using a Pro Device nanofiltration membrane. Preferably, in step (5), the sample is first subjected to pre-filtration using a Pro Shield pre-filtration membrane, and then subjected to nanofiltration using a Pro Device nanofiltration membrane. Preferably, in step (5), the sample is first subjected to pre-filtration using a Pro Shield pre-filtration membrane, and then subjected to nanofiltration using a Pro Device nanofiltration membrane. 2 .

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

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

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

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

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

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

[0045] Compared with existing purification techniques, the present disclosure has the advantages of fewer purification steps, high sample recovery rate, stable process, linear scalability, high purity of the obtained target monoclonal antibody, low residual host cell protein and DNA, and high biological activity and biological safety. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 Structure of monoclonal antibody J06 against human CCR8.

[0047] Figure 2 Toyopearl NH2-750F packing flow-through mode chromatogram.

[0048] Figure 3 Capto adhere mixed packing flow-through mode chromatogram. DETAILED DESCRIPTION

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

[0050] The following specific examples are set forth to illustrate the present disclosure, however, it is understood that these examples are not meant to limit the scope of the present disclosure.

[0051] Example 1 Obtaining of monoclonal antibody J06 against human CCR8 and sequence information

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

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

[0054] Monoclonal antibody J06 against human CCR8 is a monoclonal antibody expressed by CHO cells. The complete molecule of the monoclonal antibody is composed of 2 heavy chains (Heavy Chain, HC) and 2 light chains (Light Chain, LC), each heavy chain is composed of 453 amino acid residues; each light chain is composed of 219 amino acid residues. Its structure is shown in the attached Figure 1 The above-mentioned monoclonal antibody J06 against human CCR8 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 two-step chromatography of affinity chromatography and Toyopearl NH2-750F column chromatography

[0056] Purification of anti-human CCR8 monoclonal antibody J06 using two-step chromatography of affinity chromatography and Toyopearl NH2-750F column chromatography, comprising the following steps:

[0057] (1) Affinity chromatography: The clarified culture harvest of anti-human CCR8 monoclonal antibody J06 was loaded onto a MabSelect SuRe LX affinity chromatography column (Cytiva, batch number: 10287596) equilibrated with buffer (25 mmol / L Tris-HCl, pH 8.28), and the impurities were washed with eluent 1 (50 mmol / L Tris, 0.5 mol / L Arg-HCl, pH 8.27) and eluent 2 (25 mmol / L Tris-HCl, pH 8.28), respectively, and then eluted with eluent (20 mmol / L NaAc-HAc, pH 3.20) at a gradient, and the elution peak was collected. The peak range was 100 mAU-100 mAU. The chromatography column was regenerated with regeneration solution (0.1 mol / L NaOH);

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

[0059] (3) Depth filtration: The A1HC depth filter (Merck, batch number: CP0DB11443) was equilibrated with equilibration buffer (25 mmol / L NaAc-HAc, pH 5.23), and then the sample after neutralization in step (2) was subjected to A1HC depth filtration. After the sample was filtered, the depth filter was washed with equilibration buffer (25 mmol / L NaAc-HAc, pH 5.23) at a load of 2060.63 g / m 2 .

[0060] (4) Toyopearl NH2-750F column chromatography (TOSOH, batch number: 75NH2FD003GA): The Toyopearl NH2-750F column was equilibrated with 3 column volumes of buffer (25 mmol / L NaAc-HAc, pH 5.28). The sample obtained in step (3) was loaded onto the equilibrated Toyopearl NH2-750F column. During loading, the UV 280Collect the flow-through when the volume reaches 100 mAU. After sample loading, wash the column with buffer (25 mmol / L NaAc-HAc, pH 5.28), continuing peak collection during the wash process until UV concentration is reached. 280 Peak collection ceased when the column reached 200 mAU. The column was regenerated with regeneration buffer (0.5 mol / L NaOH). The chromatogram is shown below. Figure 2 As shown.

[0061] (5) Nanofiltration: Rinse with water for injection separately. Pro Shield membrane (Merck, batch number: C9SA80093V) and Pro Device membrane (Merck, batch number: C1BB23982). Toyopearl NH2-750F flow-through sample was first passed through... Pro Shield pre-filter membrane; then... Pro Device nanofiltration membrane. The nanofiltration membrane loading is 4999.93 g / m³. 2 .

[0062] (6) Concentration and Replacement Buffers: The membrane packs selected for concentration and replacement buffers were Merck ultrafiltration membrane packs (batch number: C0NB31760, pore size 30KD, C-channel, regenerated cellulose material). The ultrafiltration membrane packs were equilibrated with replacement buffer (10 mmol / L HAc-NaOH, 9% sucrose, pH 5.0); the nanofiltration sample was loaded and concentrated to 30 mg / ml before starting the replacement buffer (replacement buffer: 10 mmol / L HAc-NaOH, 9% sucrose, pH 5.0); the volume of the replacement buffer was greater than or equal to 10 times the volume of the loaded sample; after the replacement buffer was completed, the sample was flushed and drained, and polysorbate 80 was added until the final concentration of polysorbate 80 was 0.05%. The resulting stock solution concentration was 20.0 ± 2 mg / ml.

[0063] When scaled up to 500L production, the overall yield of this two-step chromatography purification process was 91.55%, which is at the leading level in the industry.

[0064] The results of the Toyopearl NH2-750F chromatography in step 4 are shown in Table 1:

[0065] Table 1. Chromatographic results of Toyopearl NH2-750F

[0066]

[0067] Note: N / A indicates not detected or not applicable.

[0068] The comparative example used a two-step chromatography method, affinity chromatography and Capto adhere column chromatography, to purify the anti-human CCR8 monoclonal antibody J06.

[0069] The two-step purification of anti-human CCR8 monoclonal antibody J06 by affinity chromatography and Capto adhere column chromatography includes the following steps:

[0070] (1) Affinity chromatography: The clarified culture harvest of anti-human CCR8 monoclonal antibody J06 was loaded into a MabSelect SuRe LX affinity chromatography column (Cytiva, batch number: 10287596) equilibrated with buffer (25 mmol / L Tris-HCl, 200 mmol / L NaCl, pH 7.15). The impurities were washed with eluent 1 (25 mmol / L Tris, 0.5 mol / L Arg-HCl, pH 7.43) and eluent 2 (20 mmol / L NaAc, pH 5.65), respectively, and then eluted with eluent (30 mmol / L NaAc-HAc, pH 3.36) at a gradient, and the elution peak was collected. The peak range was 100 mAU-100 mAU. The chromatography column was regenerated with regeneration solution (0.1 mol / L NaOH).

[0071] (2) Capto adhere chromatography column (Cytiva, batch number: 10292076): The Capto adhere chromatography column was equilibrated with 8 column volumes of buffer (50 mmol / L NaAc-HAc, 0.4 mol / L NaCl, pH 5.99). The sample was adjusted to a conductivity of 37.30 mS / cm with sample treatment solution (4 mol / L NaCl), and the sample pH was adjusted to 6.03. The treated sample was loaded into the equilibrated Capto adhere chromatography column. During the loading process, the UV 280 flow-through was collected when the UV reached 100 mAU. After the loading was completed, the chromatography column was top-washed with buffer (50 mmol / L NaAc-HAc, 0.4 mol / L NaCl, pH 5.99), and the peak was continued to be collected until the UV 280 dropped to 200 mAU, and the collection of the peak was stopped. The chromatography column was regenerated with 3 column volumes of regeneration solution (50 mmol / L HAc, pH 3.0). The chromatography profile is shown in Figure 3 .

[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 detected or not applicable

[0076] From the comparison of the yield results of Table 1 and Table 2, the effect of the two chromatography methods of Example 3 and Comparative Example on the SEC purity of the sample is not much different. The Toyopearl NH2-750F chromatography yield is 98.95%, higher than the Capto adhere chromatography yield of 95.00%, and the Toyopearl NH2-750F chromatography is better than the Capto adhere chromatography in the effect of removing residual.

[0077] Example 4 Anti-human CCR8 monoclonal antibody J06 binding activity detection

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

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

[0080] Table 3 Anti-human CCR8 monoclonal antibody J06 binding activity results

[0081]

Claims

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

2. The purification method of claim 1, wherein, The method comprises the following steps: (1) The clarified cell culture harvest containing the anti-human CCR8 monoclonal antibody is subjected to a MabSelect SuRe LX affinity chromatography column, elution is performed, and the elution peak containing the target antibody protein is collected; (2) The affinity elution peak sample in step (1) is incubated under a low pH condition; (3) The sample in step (2) is subjected to deep filtration; (4) The sample in step (3) is subjected to a Toyopearl NH2-750F chromatography column in a flow-through mode, and the flow-through sample is collected; (5) The sample in step (4) is subjected to a nanofiltration membrane; (6) The sample in step (5) is concentrated, the buffer is replaced, and dilution is performed.

3. The purification method of claim 2, wherein: In step (1), the impure proteins are washed with elution liquid 1 and elution liquid 2 respectively, and then gradient elution is performed with an elution liquid, and the elution peak is collected; the composition of elution liquid 1 is 50 mmol / L Tris, 0.2 mol / L-0.6 mol / L Arg-HCl, pH 8.1±0.5; the composition of elution liquid 2 is 25 mmol / L Tris-HCl, pH 8.3±0.5; and the composition of the elution liquid is 10 mmol / L-50 mmol / L NaAc-HAc, pH 3.4±0.

4.

4. The purification method of claim 2, wherein: In step (2), the pH of the affinity elution peak sample is adjusted to 3.5±0.3 with 2 mol / L HAc, and the incubation is performed at room temperature for 1-4 hours, and then the pH is adjusted to 5.3±0.5 with 1 mol / L Tris.

5. The purification method of claim 2, wherein: The step (3) adopts Merck A1 HC depth filter to perform depth filtration on the sample, and the load is ≤5000 g / m 2 ; in the step (4), the chromatography column is equilibrated with a 25 mmol / L NaAc-HAc solution with pH 5.3±0.5, and then the sample is loaded.

6. The purification method of claim 2, wherein: The sample in step (5) is first passed through Viresolve ® Pro Shield prefilter membrane; Viresolve ® Pro Device nanofiltration membrane; nanofiltration membrane Viresolve ® Pro Device loadings of less than 5000 g / m 2 .

7. The purification method of claim 2, wherein: In step (6), the membrane package selected for sample concentration and buffer replacement 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 final concentration of the sample stock solution is controlled to be 20 g / L±2 g / L; and the auxiliary material added to the sample stock solution is polysorbate 80 with a final concentration of 0.03%-0.07%.

8. The purification method of any one of claims 1-7, wherein: 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.

9. The purification method of any one of claims 1-7, wherein: 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.

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