CN116769049A - Purification method of bifunctional fusion protein - Google Patents

Abstract

The invention relates to a purification method of a bifunctional fusion protein. The purification method adopts two-step chromatography to purify the IL15/PD-L1 double-function fusion protein, has few purification steps, low sample loss rate, stable process and linear amplification, can obtain the high-purity double-function fusion protein, has little residue of host cell proteins, DNA and the like in the purified product, and has higher bioactivity and biosafety after purification.

Description

Purification method of bifunctional fusion protein

Technical Field

The invention belongs to the field of protein purification, and particularly relates to a purification method of a bifunctional fusion protein.

Background

Grabstein et al found a cytokine with a molecular weight of 12-14KD, interleukin 15 in 1994. Interleukin 15, as a T cell and NK cell growth factor, plays an important role in the production, replication and activation of immune cells. Interleukin 15 is listed by the national cancer institute as one of the most potential targets in cancer immunotherapy. Another related cytokine, interleukin 2, has been approved by the FDA in the united states for the treatment of renal cell cancer and malignant melanoma.

PD-L1, apoptosis ligand 1, is a protein in humans encoded by the CD274 gene and has a molecular weight of 40KD. PD-L1 is typically expressed on the surface of tumor cells. PD-1, the apoptosis receptor-1 (PD-1), is a CD28 superfamily member and is expressed on the surface of T cells. When PD-L1 is linked to PD-1, T cells are unable to discover the tumor and signal an attack on the immune system. By designing PD-L1 antibodies, the connection between PD-L1 and PD-1 can be cut off, so that tumor cells are recognized by immune cells and killed.

The IL15/PD-L1 double-function fusion protein is an innovative target double-function fusion protein drug expressed by CHO cells independently developed by the applicant. The bifunctional fusion protein is formed by connecting an anti-PD-L1 monoclonal antibody of an IgG1 type with IL15RαSu and IL-15 through the C-terminal of an Fc segment, wherein the C-terminal of each heavy chain of the anti-PD-L1 monoclonal antibody is connected with 1 IL15RαSu and 1 IL-15 through a G4S Linker (G4S Linker), and the IL15RαSu and the IL-15 are also connected through the G4S Linker.

The IL15/PD-L1 bifunctional fusion protein is combined with PD-L1 on the surface of a tumor cell through an anti-PD-L1 antibody, so that the PD-L1/PD-1 mediated immune checkpoint inhibition effect can be relieved; meanwhile, the functional activities of T lymphocytes and NK cells can be promoted by activating the downstream STAT5 signal pathway of the IL-15 receptor on the surface of the primary lymphocyte.

IL15/PD-L1 bifunctional fusion proteins produce a large number of aggregates during expression. These polymers present a significant challenge for downstream purification work. Conventional three-step chromatography, affinity capture+anion flow through+cation binding elution, has failed to achieve the objective of impurity removal and related residual criteria for this antibody. The high impurity content can result in lower recovery of the whole process.

Disclosure of Invention

In order to overcome the defects, the invention provides a purification method for purifying IL15/PD-L1 bifunctional fusion protein by a two-step chromatography method.

Detailed Description

1. Terminology

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.

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may 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 invention. 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 invention belongs.

Certain embodiments disclosed herein encompass a range of values, and certain aspects of the invention may be described by way of the range. Unless otherwise indicated, it should be understood that the numerical ranges or the manner in which the ranges are described are for the purpose of brevity and convenience only and should not be construed as a strict limitation on the scope of the invention. Accordingly, the description of a range format should be considered to specifically disclose all possible sub-ranges and all possible specific numerical points within the range as if such sub-ranges and numerical points had been explicitly written herein. The above principle applies equally regardless of the breadth of the values. When a range description is employed, the range includes the endpoints of the range.

The term "about" when referring to a measurable value such as an amount, temporal duration, or the like, is meant to include a change 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.

The term "antibody" as used herein, typically refers to a Y-type 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 consists of one variable domain (VL) and one constant domain (CL). Each heavy chain comprises a variable domain (VH) and a constant region.

As used herein, the broad class of "antibodies" can include, for example, polyclonal antibodies (polyclonal antibodies), monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies (CDR-grafted antibodies), human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bifunctional fusion proteins, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies (including muteins and variants thereof), and the like.

The term "monoclonal antibody" (or "mab") refers to an antibody that is produced by a single cell clone that is substantially homogeneous and directed against only a particular epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma techniques, recombinant techniques, phage display techniques, transgenic animals, synthetic techniques, combinations thereof, or the like.

The term "antibody fragment" encompasses at least a portion of an intact antibody. As used herein, a "fragment" of an antibody molecule includes an "antigen-binding fragment" of an antibody, and the term "antigen-binding fragment" refers to an immunoglobulin or antibody that specifically binds or reacts with a selected antigen or immunogenic determining portion thereofPolypeptide fragments, or fusion protein products further derived from such fragments, e.g., single chain antibodies, extracellular binding regions in chimeric antigen receptors, and the like. Exemplary antibody fragments or antigen-binding fragments thereof include, but are not limited to: variable light chain fragments, variable heavy chain fragments, fab fragments, F (ab') ₂ Fragments, fd fragments, fv fragments, single domain antibodies, linear antibodies, single chain antibodies (scFv), bifunctional fusion proteins formed from antibody fragments, or multispecific antibodies, and the like.

The term "antigen" refers to a substance recognized and specifically bound by an antibody or antibody binding fragment, and in a broad sense, an antigen may include any immunogenic fragment or determinant of a selected target, including a single epitope, multiple epitopes, a single domain, multiple domains, an intact extracellular domain (ECD), or a protein. Peptides, proteins, glycoproteins, polysaccharides and lipids, portions thereof and combinations thereof may all constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, and the like. An "antigen" may also refer to a molecule that initiates an immune response. Any form of antigen or cell or preparation containing the antigen can be used to generate antibodies specific for an antigenic determinant. The antigen may 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 may be produced in a genetically modified cell. Any of the foregoing antigens may 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.

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).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, biolayer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, flow cytometry, and the like.

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

The term "stable" formulation is a formulation in which the protein substantially retains its physical and/or chemical stability and/or biological activity after storage. Preferably, the formulation substantially retains its physical and chemical stability after storage, as well as its biological activity. The shelf life is generally selected based on the shelf life of the formulation. Various analytical techniques for measuring protein stability are known in the art. Stability may 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 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 antibodies; peptide profile analysis; assessing biological activity or antigen binding function of the antibody; etc. Instability may 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, glycosylation differences, and the like.

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 manufacture, storage and use. Stabilizers include, but are not limited to, sugars, amino acids, polyols, cyclodextrins, and the like.

Methods for producing and purifying antibodies and antigen binding fragments are well known and available in the art, such as the guidelines for antibody experimentation in Cold spring harbor, chapters 5-8 and 15.

The engineered antibodies or antigen-binding fragments thereof of the invention can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc region. Stable clones were obtained by expressing antibodies that specifically bound to human antigens. Positive clones were expanded in serum-free medium of the bioreactor to produce antibodies. The antibody-secreting culture may be purified and collected using conventional techniques. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange.

2. Summary of the invention

The invention aims to provide a purification method for purifying IL15/PD-L1 bifunctional fusion protein by a two-step chromatography.

The technical scheme provided by the invention is as follows: a method for purifying a bifunctional fusion protein comprising the steps of:

the filtered cell fermentation broth containing the IL15/PD-L1 double-function fusion protein expressed by CHO cells is subjected to twice chromatographic column chromatography, wherein MabSelect SuRe LX affinity chromatographic column is adopted in the first chromatography, and Capto sphere chromatographic column is adopted in the second chromatography.

Preferably, the purification method comprises the steps of:

(1) Passing the clarified cell fermentation broth containing the IL15/PD-L1 bifunctional fusion protein through a MabSelect SuRe LX affinity chromatography column, eluting, and collecting an eluting peak containing the target antibody protein;

(2) Incubating the affinity elution peak sample in step (1) under low pH conditions;

(3) Deep filtration of the sample in step (2);

(4) The sample in the step (3) is passed through a Capto sphere chromatographic column, a flow-through mode is adopted, and the flow-through sample is collected;

(5) Passing the sample in the step (4) through a nanofiltration membrane;

(6) Concentrating the sample in the step (5) to replace the buffer solution and diluting the buffer solution.

Preferably, in the step (1), eluting the hybrid protein with eluent 1 and eluent 2 respectively, then isocratically eluting with eluent, and collecting elution peaks; the leaching solution 1 is: 50mM Tris-HAC,0.5M Arginine,pH5.0 + -0.5; the leaching 2 solution is as follows: 50mM NaAC-HAC, pH 5.0.+ -. 0.5; the elution solution was 50mM NaAC-HAC, pH 3.9.+ -. 0.3.

Preferably, the pH of the affinity eluted sample in the step (2) is adjusted to 3.6+ -0.2 by using 1M HAC, and the pH is adjusted to 5.0-7.0 by using 2M Tris after the incubation at room temperature is completed for 1-4 hours.

Preferably, in the step (3), the sample is subjected to deep filtration by using a Millipore XOHC membrane package, and the loading is less than or equal to 240L/m ² 。

Preferably, the chromatography column in step (4) is equilibrated with a 50mM NaAC-HAC,0.05-0.6M NaCl, pH 4.0-7.0 solution; samples were loaded after pH adjustment to 4.0-7.0 with 2M Tris and conductivity adjustment to 5-60ms/cm with 4M NaCl.

Preferably, the sample in the step (5) is first filtered by a Nano pre-filter A1HC prefilter; then passing through Viresolve pro shield nanofiltration membrane; nanofiltration membrane Viresolve pro shield loading is less than 956.6L/m ² 。

Preferably, the membrane package used for replacing the sample buffer solution in the step (6) is a membrane package selected for concentrating and replacing the sample buffer solution and is a Merck ultrafiltration replacement membrane package; the displacement buffer was 5mM succinic acid, pH 5.5.+ -. 0.3; finally diluting the concentration of the sample stock solution to 2.0mg/mL; the auxiliary material is 9% sucrose and 0.05% tween 80.

Preferably, the IL15/PD-L1 bifunctional fusion protein is formed by connecting an IgG1 type anti-PD-L1 monoclonal antibody with IL15RαSu and IL-15 through the C-terminal of an Fc segment, and the fusion heavy chain sequence of the bifunctional fusion protein is shown as SEQ ID NO:1, wherein the light chain sequence of the bifunctional fusion protein is shown as SEQ ID NO: 2.

The technical scheme provided by the invention is as follows: IL15/PD-L1 double-function fusion protein obtained by the purification method.

The purification method for purifying the IL15/PD-L1 bifunctional fusion protein by using the two-step chromatography has the following beneficial effects:

compared with the prior purifying technology, the invention has the advantages that: the invention has the advantages of few purification steps, low sample loss rate, stable process, linear amplification, capability of obtaining target bifunctional fusion protein with relatively high purity, less residue of host cell protein, DNA and the like, and relatively high bioactivity and biosafety.

Drawings

FIG. 1 is a schematic diagram of the structure of an IL15/PD-L1 bifunctional fusion protein.

FIG. 2 is a Capto sphere mixed packing flow-through mode chromatographic profile.

FIG. 3 is a chromatographic chart of Eshmuno CPX cation binding elution pattern.

Detailed Description

The invention is further illustrated by the following examples.

The invention is illustrated by the following specific examples, however, it should be understood that these examples are presented by way of illustration only and are not intended to limit the scope of the invention.

EXAMPLE 1 preparation of IL15/PD-L1 bifunctional fusion protein

The IL15/PD-L1 bifunctional fusion protein is a CHO cell expressed bifunctional fusion protein. The bifunctional fusion protein is formed by connecting an anti-PD-L1 monoclonal antibody of an IgG1 type with IL15RαSu and IL-15 through the C-terminal of an Fc segment, wherein the C-terminal of each heavy chain of the anti-PD-L1 monoclonal antibody is connected with 1 IL15RαSu and 1 IL-15 through a G4S Linker (G4S Linker), and the IL15RαSu and the IL-15 are also connected through a G4 SLlinker. The structure is shown in figure 1. The protein complete molecule contains 2 fusion heavy chains (comprising PD-L1 heavy chain, IL15RαSu/IL-15 and G4 SLinker) composed of 658 amino acid residues and 2 light chains composed of 215 amino acid residues, and the chains are connected through disulfide bonds. The sequence of the fusion heavy chain is shown as SEQ ID NO:1, wherein the sequence of the light chain is shown as SEQ ID NO: 2.

Expression of the above IL15/PD-L1 bifunctional fusion protein in CHO cells was purified by the method of example 2 for further experiments.

EXAMPLE 2 purification of IL15/PD-L1 bifunctional fusion proteins Using two-step chromatography

The IL15/PD-L1 bifunctional fusion protein is purified by a two-step chromatography method, which comprises the following steps:

(1) Affinity chromatography: the clarified fermentation broth expressing IL15/PDL1 bifunctional fusion protein was loaded onto MabSelect SuRe LX affinity column (Cytiva, lot number: 10287596) equilibrated with buffer (50 mM Tris-HAC,150mM NaCl,pH7.41), and the hetero-proteins were washed with eluent 1 (50 mM Tris-HAC,500mM Arginine,pH 5.02) and eluent 2 (50 mM NaAC-HAC, pH 4.97), respectively, and eluted isocratically with eluent (50 mM NaAC-HAC, pH 3.94), and the elution peaks were collected. The peak-collecting range is 100mAu-100mAu. Regenerating the column with a regeneration solution (50 mM HAC, pH 3.0);

(2) Low pH incubation: the affinity eluted sample in step (1) was pH-adjusted to 3.60 with 1M HAC and incubated at room temperature for 1 hour. After incubation was completed, the pH was adjusted to 5.99 with 2M Tris.

(3) Deep filtration: equilibrating the X0HC membrane package (Merck, cat# MX0HC23CL 3) with equilibration solution (50 mM NaAC-HAC, pH 5.99), then subjecting the neutralized sample in step (2) to X0HC membrane package filtration, top washing the membrane package with equilibration solution (50 mM NaAC-HAC, pH 5.99) of 3 times dead volume, loading 158.47L/m ² 。

(4) Capto sphere column (cytova, lot number: 10292076): the Capto sphere column was equilibrated with 5 column volumes of buffer (50 mM NaAC-HAC,0.4M NaCl,pH 5.93). The deeply filtered sample was adjusted to a conductivity of 37.90ms/cm with sample treatment fluid (4M NaCl) and the pH of the sample was adjusted to 5.93,0.22um membrane filtration sample. And loading the treated sample to a well-balanced Capto sphere chromatographic column, and collecting the flow-through liquid when the UV280 reaches 100mAU in the loading process. After loading, the chromatographic column was top-washed with 13 column volumes of buffer (50 mM NaAC-HAC,0.4M NaCl,pH 5.93), and peak collection was continued during top-washing until UV280 was reduced to 100mAU. The column was regenerated with 3 column volumes of regeneration solution (50 mM HAC, pH 3.0). The chromatographic pattern is shown in figure 2.

(5) Nanofiltration: nano pre-filter A1HC (Merck, cat# SSPVA40NB 9) and Viresolve pro shield membranes (Merck, cat# VPMSKITNB 9) were rinsed with water for injection, respectively. The Capto sphere flow-through sample is firstly filtered by a Nano pre-filter A1HC prefilter; then passing through Viresolve pro shield nanofiltration membrane. Nanofiltration membrane loading is 798.57L/m ² 。

(6) Concentrating and changing liquid: the membrane package selected for the concentrated liquid exchange is Merck ultrafiltration displacement membrane package (batch number: C0BB36207-0020, pore diameter 30KD, A flow channel, polyether sulfone material). Equilibrate the ultrafiltration displacement membrane pack with displacement fluid (5 mM succinic acid, pH 5.59); the nanofiltration sample is loaded and concentrated to 1.412mg/mL to begin the liquid change (liquid change solution: 5mM succinic acid, pH 5.59); the volume of the exchanged liquid is more than 10 times of the volume of the sample to be sampled; after the completion of the liquid exchange, the concentration of the sample was concentrated to 5.571mg/mL, and sucrose and Tween 80 were added to a final concentration of 9% sucrose and 0.05% Tween 80. Diluting the stock solution with sample diluent (5 mM succinic acid, 9% sucrose, 0.05% Tween 80, pH 5.59) to 2.0 mg/mL.

Step 4Capto sphere chromatography results are shown in Table 1:

TABLE 1Capto sphere chromatography results

Comparative example IL15/PD-L1 bifunctional fusion protein was purified by two-step chromatography using affinity chromatography and cationic chromatography

The IL15/PD-L1 bifunctional fusion protein is purified by adopting an affinity chromatography and cation chromatography two-step chromatography mode, and the method comprises the following steps of:

(1) Affinity chromatography: the clarified fermentation broth expressing IL15/PDL1 bifunctional fusion protein was loaded onto MabSelect SuRe LX affinity column (Cytiva, lot number: 10287596) equilibrated with buffer (50 mM Tris-HAC,150mM NaCl,pH 7.41), and the hetero-proteins were washed with eluent 1 (50 mM Tris-HAC,500mM Arginine,pH 5.02) and eluent 2 (50 mM NaAC-HAC, pH 4.97), respectively, and eluted isocratically with eluent (50 mM NaAC-HAC, pH 3.94), and the elution peaks were collected. The peak-collecting range is 100mAu-100mAu. The column was regenerated with regeneration solution (50 mM HAC, pH 3.0).

(2) Cationic chromatography column: the Eshmuno CPX (Merck, lot number TA 2215983930) column was equilibrated with 3 column volumes of buffer (50 mM NaAC-HAC, pH 5.03). The pH of the 2M Tris-adjusted sample was 5.00,0.22 μm membrane filtered. The column was top washed with 3 column volumes of buffer (50 mM NaAC-HAC, pH 5.03) after loading to equilibrated Eshmuno CPX column. Samples were eluted with 5 column volumes of buffer (50 mM NAAC-HAC,0.4MNaCl,pH 5.02) and collection was started when UV280 reached 100mAU and stopped when UV280 was reduced to 100mAU. The column was regenerated with 3 column volumes of regeneration solution (50 mM NaAC-HAC,1M NaCl,pH 5.03). The chromatographic pattern is shown in figure 3.

The results of step (2) Eshmuno CPX chromatography are shown in Table 2:

TABLE 2Eshmuno CPX chromatography results

From comparison of the yield results in tables 1 and 2, the two chromatographic modes do not differ much from the sample yields. From the SEC purity results in Table 1, the sample SEC purity increased from 66% to 98.5% after Capto sphere mixed mode chromatography, allowing removal of the majority of the polymer. From the SEC purity results in table 2, after Eshmuno CPX cationic analysis, the SEC purity of the sample was reduced from 69.9% to 60.28%, without increasing the sample purity. Therefore, SEC purity results show that Capto sphere mixed mode chromatography shows excellent separation effect.

EXAMPLE 3 detection of the biological Activity of IL15/PD-L1 bifunctional fusion proteins

The IL15/PD-L1 bifunctional fusion protein obtained by purifying the method of example 2 was subjected to detection of the protein binding ability. The detection method and the detection result are as follows:

the binding capacity of the IL-15/PD-L1 bifunctional fusion protein to IL-15 Rbeta and PD-L1 proteins was determined by ELISA. Human IL-15Rβ protein was first coated onto 96-well plates, then the test samples were added in a gradient dilution, and then biotin-labeled PD-L1 protein was added at a fixed concentration. The Fc end of the IL15/PD-L1 bifunctional fusion protein is combined with IL-15 Rbeta protein, and the Fab end is combined with biotin-labeled PD-L1 protein. And adding horseradish peroxidase-labeled streptavidin into the 96-well plate, and finally adding TMB substrate solution for color development, and reading the absorbance at 450 nm. EC50 values were obtained by calculation using SoftMax Pro computer software, using a four parameter fitting regression model to draw a curve, and the results are shown in table 3. The results show that the IL-15/PD-L1 bifunctional fusion protein has good binding activity to both IL-15R mu and PD-L1 proteins.

TABLE 3IL15/PD-L1 bifunctional fusion protein binding Activity results

Sample name	EC50 value (ng/mL)
		IL15/PD-L1 bifunctional fusion proteins	0.0134

Claims (10)

1. A method for purifying a bifunctional fusion protein, comprising the steps of:

the filtered cell fermentation broth containing the IL15/PD-L1 double-function fusion protein expressed by CHO cells is subjected to twice chromatographic column chromatography, wherein MabSelect SuRe LX affinity chromatographic column is adopted in the first chromatography, and Capto sphere chromatographic column is adopted in the second chromatography.

2. The purification method of claim 1, comprising the steps of:

(1) Passing the clarified cell fermentation broth containing the IL15/PD-L1 bifunctional fusion protein through a MabSelect SuRe LX affinity chromatography column, eluting, and collecting an eluting peak containing the target antibody protein;

(2) Incubating the affinity elution peak sample in step (1) under low pH conditions;

(3) Deep filtration of the sample in step (2);

(4) The sample in the step (3) is passed through a Capto sphere chromatographic column, a flow-through mode is adopted, and the flow-through sample is collected;

(5) Passing the sample in the step (4) through a nanofiltration membrane;

(6) Concentrating the sample in the step (5) to replace the buffer solution and diluting the buffer solution.

3. The purification method of claim 2, wherein: washing the hybrid protein with eluent 1 and eluent 2 in the step (1), then carrying out isocratic elution with eluent, and collecting elution peaks; the leaching solution 1 is: 50mM Tris-HAC,0.5M Arginine,pH5.0 + -0.5; the leaching 2 solution is as follows: 50mM NaAC-HAC, pH 5.0.+ -. 0.5; the elution solution was 50mM NaAC-HAC, pH 3.9.+ -. 0.3.

4. The purification method of claim 2, wherein: and (3) adjusting the pH of the affinity elution sample in the step (2) to 3.6+/-0.2 by using 1M HAC, and adjusting the pH to 5.0-7.0 by using 2M Tris after the incubation at room temperature is completed for 1-4 hours.

5. The purification method of claim 2, wherein: in the step (3), a Millipore XOHC membrane package is adopted to carry out deep filtration on the sample, and the loading capacity is less than or equal to 240L/m ² 。

6. The purification method of claim 2, wherein: the chromatography column in the step (4) is balanced by 50mM NaAC-HAC,0.05-0.6M NaCl and pH 4.0-7.0; samples were loaded after pH adjustment to 4.0-7.0 with 2M Tris and conductivity adjustment to 5-60ms/cm with 4M NaCl.

7. The purification method of claim 2, wherein: the sample in the step (5) is firstly filtered by a Nano pre-filter A1HC prefilter; then passing through Viresolve pro shield nanofiltration membrane; nanofiltration membrane ViresolveThe pro shield loading is less than 956.6L/m ² 。

8. The purification method of claim 2, wherein: the membrane package used for replacing the sample buffer solution in the step (6) is a membrane package selected for concentrating and replacing the sample buffer solution and is a Merck ultrafiltration replacement membrane package; the displacement buffer was 5mM succinic acid, pH 5.5.+ -. 0.3; finally diluting the concentration of the sample stock solution to 2.0mg/mL; the auxiliary material is 9% sucrose and 0.05% tween 80.

9. The purification method of claims 1-8: the method is characterized in that: the IL15/PD-L1 bifunctional fusion protein is formed by connecting an anti-PD-L1 monoclonal antibody of an IgG1 type with IL15RαSu and IL-15 through the C-terminal of an Fc segment, and the fusion heavy chain sequence of the bifunctional fusion protein is shown as SEQ ID NO:1, wherein the light chain sequence of the bifunctional fusion protein is shown as SEQ ID NO: 2.

10. The IL15/PD-L1 bifunctional fusion protein obtained by the purification method of any one of claims 1-9.