Monoclonal antibody purification device and concentration measurement method

Abstract

The invention belongs to the technical field of bioassay, and discloses a monoclonal antibody purification device and a concentration determination method. The purification device can be used for separating and purifying the monoclonal antibody in the monoclonal antibody cell fermentation liquid supernatant, and comprises an injector, an affinity chromatography medium and an injection pump. The concentration determination method is carried out by using the purification device, and comprises the following steps: carrying out affinity chromatography on the fermentation supernatant of the monoclonal antibody, collecting an eluent containing the target monoclonal antibody, determining the concentration of the target monoclonal antibody in the eluent, and calculating the concentration of the target monoclonal antibody in the fermentation supernatant. The determination method is at least suitable for purification and content determination of fermentation supernatant of existing monoclonal antibodies QX004N, QX005N, QX006N, QX007N, QX008N, QX002N, QX013N and QX001S, and has the advantages that expensive equipment is not needed, the cost is low, the operation is simple, the universality is high, and the result is basically consistent with that of a high performance liquid chromatography.

Description

Technical Field

[0001] The present application belongs to the field of bioassay technology, and specifically relates to a simple device for separating and purifying monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth and a concentration determination method. Background Art

[0002] Antibody drugs are a type of protein drugs produced after cell culture. They need to go through a series of purification processes to obtain high-purity antibody drug molecules for preclinical evaluation and drugability assessment. Affinity chromatography purification of antibody drugs is a very critical process step. This process uses the reversible specific binding between Protein A and antibody molecules to capture and concentrate antibodies in the fermentation broth. This is the first step to achieve crude purification of antibodies, and it can also quantitatively detect the production of monoclonal antibodies in the fermentation broth. In the fermentation process of genetically engineered cell lines such as Chinese hamster ovary cells (CHO), the production determination of monoclonal antibodies is of great significance for cell line screening in the early stage of the process and for scale-up production after the process is determined.

[0003] The current existing technology uses a high performance liquid chromatograph combined with a Protein A affinity chromatography column to crudely purify the antibody fermentation broth and quantitatively detect the antibody content in the fermentation broth. This technology has the following disadvantages:

[0004] 1. High performance liquid chromatography and chromatography columns are expensive;

[0005] 2. The operation is complicated and requires high experimental skills of the operator;

[0006] 3. The testing conditions are not uniform. For each monoclonal antibody, different buffers are prepared, different affinity chromatography columns are used, different testing conditions are set, and even different groups of operators are arranged to avoid confusion. For CDMO companies, this leads to a significant increase in production burden;

[0007] 4. High performance liquid chromatography has low throughput and is not suitable for sample preparation. Summary of the invention

[0008] In view of the above technical problems existing in the prior art, the purpose of the present application is to provide a simple device that can be used to replace high performance liquid chromatography for separation and purification of monoclonal antibodies, and a universal monoclonal antibody concentration determination method using the simple device.

[0009] 1. A simple device for separating and purifying monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth, comprising:

[0010] syringe,

[0011] Affinity chromatography media, and

[0012] Syringe pump;

[0013] Wherein, the syringe is fixed on the syringe pump, and the sample outlet of the syringe is connected to the sample inlet of the affinity chromatography medium. When the monoclonal antibody cell fermentation liquid supernatant is loaded in the syringe, the syringe pump can push the injection rod of the syringe, so that the monoclonal antibody cell fermentation liquid supernatant enters the affinity chromatography medium from the syringe at a set flow rate, and flows out from the sample outlet of the affinity chromatography medium after being separated and purified by the affinity chromatography medium.

[0014] The simple device can be, for example, Figure 1 shown.

[0015] The syringe can be a conventional medical syringe as required.

[0016] The affinity chromatography medium is preferably a Protein A affinity chromatography medium, and more preferably an AT Protein ADiamond Plus affinity chromatography column. The AT Protein A Diamond Plus affinity chromatography column can be purchased from Boglong Boglong (Shanghai) Biotechnology Co., Ltd. It is an affinity medium synthesized by epoxidation chemistry using an alkali-resistant Protein A and a high-rigidity agarose frame. The advantage is that its ligand is a genetically engineered protein A fragment, and alkali-resistant amino acids are used to replace alkali-intolerant amino acids during upstream construction, and protease-sensitive amino acids are replaced, so that the ligand has good alkali stability, and the ligand fermentation and subsequent purification processes are all free of any animal-derived raw materials. The medium can be cleaned in place with 0.1~0.5M NaOH, avoiding the use of expensive and corrosive in-place cleaning reagents, which can effectively save costs. At the same time, compared with AT Protein A Diamond, it has a higher loading capacity, and a smaller column volume can be selected during the process amplification to reduce production costs.

[0017] The affinity chromatography medium can be provided in the form of a prepacked column. There is no restriction on the number of affinity chromatography media, which can be adjusted according to actual needs, and can generally be 1 to 30, such as 3 to 20, or 5 to 10.

[0018] The syringe pump can be a conventional laboratory syringe pump, which can have multiple channels (e.g., 1 to 20 channels, or 3 to 12 channels), and can simultaneously mount multiple syringes of the same or different specifications. The syringe pump can be a push-pull micro-injection pump, which generally has five working modes: perfusion, extraction, perfusion first and then extraction, extraction first and then perfusion, and continuous mode. The syringe pump can be, for example, a LSP10-1B 10-channel syringe pump produced by Baoding Lange Constant Flow Pump Co., Ltd.

[0019] 2. A method for determining the concentration of monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth, which is carried out using the simple device described in item 1, and comprises the following steps:

[0020] Using the first equilibration buffer to equilibrate the affinity chromatography medium to obtain an equilibrated affinity chromatography medium;

[0021] Loading the monoclonal antibody cell fermentation broth supernatant onto the equilibrated affinity chromatography medium;

[0022] re-equilibrate the affinity chromatography medium using a second equilibration buffer;

[0023] The affinity chromatography medium is eluted with an elution buffer, and the eluate containing the target monoclonal antibody is collected;

[0024] Determine the concentration of the monoclonal antibody in the collected eluate, and calculate the concentration of the monoclonal antibody in the supernatant of the monoclonal antibody cell fermentation broth:

[0025] Add pH adjustment buffer to the collected eluate to make the pH of the eluate 6.5-8.0, mix well, adjust the dilution multiple to dilute, and measure the A280nm absorbance.

[0026] The concentration of monoclonal antibody in the eluent = A280nm absorbance ÷ monoclonal antibody extinction coefficient × dilution multiple;

[0027] The concentration of monoclonal antibody in the supernatant of monoclonal antibody cell fermentation broth = the concentration of monoclonal antibody in the elution buffer × the volume of elution buffer ÷ the loading volume of the supernatant of monoclonal antibody cell fermentation broth;

[0028] The first balancing buffer is an aqueous solution of 20 mM Na2HPO4-NaH2PO4 and 150 mM NaCl, with a pH of 6.8, and the conductivity of the first balancing buffer is 18 mS / cm;

[0029] The second balancing buffer is a 10 mM Na2HPO4-NaH2PO4 aqueous solution with a pH of 7.2, and the conductivity of the second balancing buffer is 1.3 mS / cm;

[0030] The elution buffer is an aqueous solution of 15 mM citric acid and 7 mM Na2HPO4, with a pH of 3.1, and the conductivity of the elution buffer is 1.3 mS / cm.

[0031] 3. The method according to claim 2, wherein:

[0032] The monoclonal antibody is an IgG1 monoclonal antibody and / or an IgG4 monoclonal antibody.

[0033] 4. The method according to claim 2, wherein:

[0034] The affinity chromatography medium is an AT Protein A Diamond Plus affinity chromatography column.

[0035] 5. The method according to claim 2, wherein:

[0036] The monoclonal antibody cell fermentation supernatant is a eukaryotic cell fermentation supernatant.

[0037] 6. The method according to claim 2, wherein:

[0038] The volume of the first equilibration buffer is 3 to 6 times the volume of the affinity chromatography medium;

[0039] The volume of the second equilibration buffer is 3 to 6 times the volume of the affinity chromatography medium; and / or

[0040] The volume of the elution buffer is 3 to 6 times the volume of the affinity chromatography medium.

[0041] 7. The method according to item 2, wherein the monoclonal antibody is one selected from the following a to j, or a combination of any two, three, four, five, six, seven or all of them:

[0042] a. Anti-human interleukin-23 monoclonal antibody QX004N;

[0043] b. Anti-human interleukin-4 receptor α monoclonal antibody QX005N;

[0044] c. Anti-human interferon α receptor 1 monoclonal antibody QX006N;

[0045] d. Human interleukin-33 monoclonal antibody QX007N;

[0046] e. Anti-human TSLP monoclonal antibody QX008N;

[0047] f. Anti-human interleukin 17A monoclonal antibody QX002N;

[0048] g. anti-human CD117 monoclonal antibody QX013N; and

[0049] j. Anti-human IL-12 / IL-23p40 subunit fully human monoclonal antibody QX001S.

[0050] 8. The method according to item 2, wherein the monoclonal antibody is an anti-human interleukin 17A monoclonal antibody, which comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein:

[0051] The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1 SYYVG;

[0052] The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2 VIRIGGGTAYASWAKG;

[0053] The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3 SSINGGDI;

[0054] The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4 QASQDIRYDLS;

[0055] The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5 AASKLAS;

[0056] The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6 LGIFSFTDDDGLA.

[0057] 9. The method according to item 8, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0058] Wherein, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7 EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWVGVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSSINGGDIWGQGTLVTVSS;

[0059] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8 AIQMTQSPSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDDGLAFGGGTKVEIK.

[0060] 10. The method according to claim 8, wherein the monoclonal antibody comprises a heavy chain and a light chain; wherein,

[0061] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 9;

[0062] The amino acid sequence of the light chain is shown in SEQ ID NO:10.

[0063] 11. The method according to claim 2, wherein:

[0064] The pH adjustment buffer was a 2 M Tris-HCl aqueous solution, pH 9.5.

[0065] 12. The method according to claim 2, wherein the dilution factor is 5 to 20.

[0066] Compared with the prior art, the beneficial effects of this application are:

[0067] The simple device of the present application can be applied to multiple samples that are the same or different from each other at the same time, and each sample does not affect each other. When using this simple device, it is easy to operate, low cost, and high accuracy. The assay method described in the present application can be used for the purification and content determination of monoclonal antibodies, with no need for expensive equipment, low cost, simple operation, good versatility (the same purification and assay conditions are applicable to both IgG1 type monoclonal antibodies and IgG4 type monoclonal antibodies, which greatly reduces the production burden of CDMO companies), and the results are basically consistent with the advantages of high performance liquid chromatography. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 The schematic diagram of the simple device of the present invention is shown in FIG. 1- affinity chromatography medium; 2- syringe; 3- syringe pump.

[0069] Figure 2 This is the SDS-PAGE electrophoresis diagram of the anti-human interleukin 17A monoclonal antibody HZD-19 prepared in Example 2. DETAILED DESCRIPTION

[0070] The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

[0071] Unless otherwise defined, the technical and scientific terms in this specification have the same meaning as those generally understood by those skilled in the art. Although similar or identical methods and materials as described herein may be used in experiments or practical applications, materials and methods are described herein below. In the event of a conflict, the present specification including the definitions therein shall prevail. In addition, materials, methods and examples are provided for illustration only and are not restrictive. The present application is further described below in conjunction with specific embodiments, but is not intended to limit the scope of the present application.

[0072] The monoclonal antibody represents an antibody derived from a colony of substantially homologous antibodies, that is, each antibody constituting the colony is identical and / or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody products), such variants are usually present in trace amounts. Different from the polyclonal antibody products that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of the monoclonal antibody product is directed to a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the antibody is derived from the characteristics of a substantially homologous antibody colony, and should not be interpreted as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present application can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals comprising all or part of human immunoglobulin loci.

[0073] like Figure 1 As shown, the present application provides a simple device for separating and purifying monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth, which comprises: a syringe, an affinity chromatography medium and a syringe pump, wherein the syringe is arranged on the syringe pump, and the sample outlet of the syringe is connected to the sample inlet of the affinity chromatography medium, the monoclonal antibody cell fermentation broth supernatant is injected into the syringe, enters the affinity chromatography medium after being acted upon by the syringe pump, and flows out from the sample outlet of the affinity chromatography medium after being separated and purified by the affinity chromatography medium; when the device is used for separating and purifying monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth, it comprises the following steps:

[0074] Using the first equilibration buffer to equilibrate the affinity chromatography medium to obtain an equilibrated affinity chromatography medium;

[0075] Loading the monoclonal antibody cell fermentation broth supernatant onto the equilibrated affinity chromatography medium;

[0076] re-equilibrate the affinity chromatography medium using a second equilibration buffer;

[0077] The affinity chromatography medium is eluted with an elution buffer, and the eluate containing the target monoclonal antibody is collected;

[0078] Determine the concentration of the monoclonal antibody in the collected eluate, and calculate the concentration of the monoclonal antibody in the supernatant of the monoclonal antibody cell fermentation broth:

[0079] Add pH adjustment buffer to the collected eluate to make the pH of the eluate 6.5-8.0, mix well, adjust the dilution multiple to dilute, and measure the A280nm absorbance.

[0080] The concentration of monoclonal antibody in the eluent = A280nm absorbance ÷ monoclonal antibody extinction coefficient × dilution multiple;

[0081] The concentration of monoclonal antibody in the supernatant of monoclonal antibody cell fermentation broth = the concentration of monoclonal antibody in the elution buffer × the volume of elution buffer ÷ the loading volume of the supernatant of monoclonal antibody cell fermentation broth;

[0082] The first balancing buffer is an aqueous solution of 20 mM Na2HPO4-NaH2PO4 and 150 mM NaCl, with a pH of 6.8, and the conductivity of the first balancing buffer is 18 mS / cm;

[0083] The second balancing buffer is a 10 mM Na2HPO4-NaH2PO4 aqueous solution with a pH of 7.2, and the conductivity of the second balancing buffer is 1.3 mS / cm;

[0084] The elution buffer is an aqueous solution of 15 mM citric acid and 7 mM Na2HPO4, with a pH of 3.1, and the conductivity of the elution buffer is 1.3 mS / cm.

[0085] In some embodiments of the present application, the syringe is fixed on a syringe pump, and the sample outlet of the syringe is connected to the sample inlet of the affinity chromatography medium, so that when the monoclonal antibody cell fermentation broth supernatant is loaded into the syringe, the syringe pump can push the injection rod of the syringe, so that the monoclonal antibody cell fermentation broth supernatant enters the affinity chromatography medium from the syringe at a set flow rate, and flows out from the sample outlet of the affinity chromatography medium after separation and purification by the affinity chromatography medium.

[0086] The present application has no restrictions on the syringe, which can be purchased commercially or homemade, as long as it meets its purpose.

[0087] In one embodiment of the present application, the syringe is purchased from a commercial source.

[0088] The present application has no restrictions on the affinity chromatography medium, which can be purchased commercially or homemade, as long as it meets its purpose.

[0089] In one embodiment of the present application, the affinity chromatography medium is purchased from a commercial source.

[0090] The present application has no restrictions on the syringe pump, which can be purchased commercially or homemade, as long as it meets its purpose.

[0091] In one embodiment of the present application, a conventional laboratory syringe pump can be selected, which may have multiple channels (e.g., 1 to 20 channels, or 3 to 12 channels), and multiple syringes of the same or different specifications can be simultaneously mounted. The syringe pump can be a push-pull micro-injection pump, which generally has five working modes: perfusion, extraction, perfusion first and then extraction, extraction first and then perfusion, and continuous mode. The syringe pump can be, for example, a LSP10-1B 10-channel syringe pump produced by Baoding Lange Constant Flow Pump Co., Ltd.

[0092] In one embodiment of the present application, the syringe pump is commercially available.

[0093] In some embodiments of the present application, the syringe pump has a plurality of channels for placing syringes.

[0094] This application provides a method of using Figure 1 The simple device shown in the figure is used to determine the concentration of monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth, which includes the following steps: using a first equilibration buffer to equilibrate an affinity chromatography medium to obtain an equilibrated affinity chromatography medium; loading the monoclonal antibody cell fermentation broth supernatant onto the equilibrated affinity chromatography medium; using a second equilibration buffer to re-equilibrate the affinity chromatography medium; using an elution buffer to elute the affinity chromatography medium, and collecting the eluate containing the target monoclonal antibody; determining the concentration of the monoclonal antibody in the collected eluate, and calculating the concentration of the monoclonal antibody in the supernatant of the monoclonal antibody cell fermentation broth: adding a pH adjustment buffer to the collected eluate so that the pH of the eluate is 6.5-8.0, mixing, adjusting the dilution multiple for dilution, and determining the A280nm absorbance value, The concentration of monoclonal antibody in the eluent = A280nm absorbance ÷ monoclonal antibody extinction coefficient × dilution multiple; the concentration of monoclonal antibody in the supernatant of monoclonal antibody cell fermentation broth = the concentration of monoclonal antibody in the eluent × the volume of elution buffer ÷ the loading volume of the supernatant of monoclonal antibody cell fermentation broth.

[0095] In the present application, the Protein A affinity chromatography medium is a new type of affinity medium synthesized by epoxidation chemistry using an alkali-resistant Protein A and a high-rigidity agarose frame, and is purchased from Boglon. The advantage of the AT Protein A Diamond Plus affinity chromatography column is that its ligand is a genetically engineered recombinant protein A fragment, and alkali-resistant amino acids are used to replace alkali-intolerant amino acids during upstream construction, and protease-sensitive amino acids are replaced, so that the ligand has good alkali stability, and the ligand fermentation and subsequent purification processes are all free of any animal-derived raw materials. The medium can be cleaned in place with 0.1~0.5M NaOH, avoiding the use of expensive and corrosive in-place cleaning reagents, which can effectively save costs. At the same time, compared with AT Protein A Diamond, it has a higher loading capacity, and a smaller column volume can be selected during the process scale-up to reduce production costs.

[0096] In some embodiments of the present application, the affinity chromatography medium can be an AT Protein A Diamond Plus affinity chromatography column.

[0097] In the present application, there is no restriction on the number of affinity chromatography media, and it can be adjusted according to actual needs. For example, if you want to purify 10 fermentation supernatants, the number of affinity chromatography media is 10.

[0098] In some embodiments of the present application, a pH adjusting buffer is added to the collected eluate so that the pH of the eluate is 6.5-8.0; for example, the sample pH adjusting buffer is added so that the pH of the eluate of the monoclonal antibody can be 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0 or any range therebetween.

[0099] In some embodiments of the present application, the affinity chromatography medium may be washed with a washing buffer before being equilibrated with the first equilibration buffer.

[0100] In some embodiments of the present application, after the monoclonal antibody elution buffer is collected, the affinity chromatography medium is washed with a washing buffer, equilibrated with a first equilibration buffer, and then stored with a storage solution.

[0101] In some embodiments of the present application, the first equilibration buffer is an aqueous solution of 20 mM Na2HPO4-NaH2PO4 and 150 mM NaCl, the pH of the first equilibration buffer is 6.8, and the conductivity of the first equilibration buffer is 18 mS / cm.

[0102] In some embodiments of the present application, the second equilibration buffer is a 10 mM Na2HPO4-NaH2PO4 aqueous solution, the pH of the second equilibration buffer is 7.2, and the conductivity of the second equilibration buffer is 1.3 mS / cm.

[0103] In some embodiments of the present application, the elution buffer is an aqueous solution of 15 mM citric acid and 7 mM Na2HPO4, the pH of the elution buffer is 3.1, and the conductivity of the elution buffer is 1.3 mS / cm.

[0104] In some embodiments of the present application, the pH adjusting buffer is a 2M Tris-HCl aqueous solution, and the pH of the pH adjusting buffer is 9.5.

[0105] In some embodiments of the present application, the dilution factor is 5 to 20; for example, the dilution factor can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any range therebetween.

[0106] In some embodiments of the present application, before the affinity chromatography medium is equilibrated with the first equilibration buffer, it is first washed with a washing buffer.

[0107] In some embodiments of the present application, the washing buffer is an aqueous solution of 0.1 M NaOH and 1.0 M NaCl.

[0108] In some embodiments of the present application, the volume of the washing buffer is 3 to 6 times the volume of the affinity chromatography medium, for example, it can be 3 times, 4 times, 5 times, 6 times or any range therebetween.

[0109] In some embodiments of the present application, after the monoclonal antibody eluate is collected, the affinity chromatography medium is washed with a washing buffer, equilibrated with a first equilibration buffer, and then stored with a storage solution.

[0110] In some embodiments of the present application, the preservation solution is a 20% ethanol aqueous solution.

[0111] In some embodiments of the present application, the volume of the washing buffer is 3 to 6 times the volume of the affinity chromatography medium, for example, it can be 3 times, 4 times, 5 times, 6 times or any range therebetween.

[0112] In some embodiments of the present application, the volume of the first equilibration buffer is 3 to 6 times the volume of the affinity chromatography medium, for example, it can be 3 times, 4 times, 5 times, 6 times or any range therebetween.

[0113] In some embodiments of the present application, the monoclonal antibody is an IgG1 monoclonal antibody and / or an IgG4 monoclonal antibody.

[0114] In some embodiments of the present application, the monoclonal antibody cell fermentation supernatant is a eukaryotic cell fermentation supernatant.

[0115] In some embodiments of the present application, the volume of the first equilibration buffer is 3 to 6 times the volume of the affinity chromatography medium; for example, it can be 3 times, 4 times, 5 times, 6 times or any range therebetween.

[0116] In some embodiments of the present application, the volume of the second equilibration buffer is 3 to 6 times the volume of the affinity chromatography medium; for example, it can be 3 times, 4 times, 5 times, 6 times or any range therebetween.

[0117] In some embodiments of the present application, the volume of the elution buffer is 3 to 6 times the volume of the affinity chromatography medium; for example, it can be 3 times, 4 times, 5 times, 6 times or any range therebetween.

[0118] In some embodiments of the present application, the monoclonal antibody is selected from the following a~j, and specifically can be one selected from the following a~j, or a combination of any two, three, four, five, six, seven, or all of them:

[0119] a. Anti-human interleukin-23 monoclonal antibody;

[0120] b. Anti-human interleukin-4 receptor α monoclonal antibody;

[0121] c. Anti-human interferon α receptor 1 monoclonal antibody;

[0122] d. Anti-human interleukin-33 monoclonal antibody;

[0123] e. anti-human TSLP monoclonal antibody; and

[0124] f. Anti-human interleukin-17A monoclonal antibody;

[0125] g. Anti-human CD117 monoclonal antibody;

[0126] j. Fully human monoclonal antibody against human IL-12 / IL-23p40 subunit; and

[0127] i. anti-human interleukin 17A monoclonal antibody as described below;

[0128] Among them, they are: anti-human interleukin 23 monoclonal antibody QX004N described in patent document CN111718414B, anti-human interleukin 4 receptor α monoclonal antibody QX005N described in patent document CN110746507B, anti-human interferon α receptor 1 monoclonal antibody QX006N described in patent document CN113278071B, human interleukin-33 monoclonal antibody QX007N described in patent document CN113603775B, anti-human TSLP monoclonal antibody QX008N described in patent document CN113683694B, anti-human interleukin 17A monoclonal antibody QX002N described in patent document CN108640991B, and anti-human CD117 monoclonal antibody QX005N described in patent document CN202410059200. Monoclonal antibody QX013N, anti-human IL-12 / IL-23p40 subunit fully human monoclonal antibody QX001S described in patent document CN112852743B.

[0129] In some embodiments of the present application, the monoclonal antibody is an anti-human interleukin 17A monoclonal antibody, which comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein:

[0130] The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1 SYYVG;

[0131] The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2 VIRIGGGTAYASWAKG;

[0132] The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3 SSINGGDI;

[0133] The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4 QASQDIRYDLS;

[0134] The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5 AASKLAS;

[0135] The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6 LGIFSFTDDDGLA.

[0136] Preferably, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0137] Wherein, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7 EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWVGVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSSINGGDIWGQGTLVTVSS;

[0138] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8 AIQMTQSPSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDDGLAFGGGTKVEIK.

[0139] Preferably, the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 9, SEQ ID NO: 9

[0140] EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWVGVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSSINGGDIWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0141] The amino acid sequence of the light chain is shown in SEQ ID NO: 10.

[0142] AIQMTQSPSSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDDGLAFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0143] In some embodiments of the present application, the affinity chromatography medium is first washed with a washing buffer, and the affinity chromatography medium is balanced with a first equilibration buffer to obtain a balanced affinity chromatography medium; the monoclonal antibody cell fermentation broth supernatant is loaded onto the balanced affinity chromatography medium; the affinity chromatography medium is re-equilibrated with a second equilibration buffer; the affinity chromatography medium is eluted with an elution buffer, and the eluate containing the target monoclonal antibody is collected; the concentration of the monoclonal antibody in the collected eluate is determined, and the concentration of the monoclonal antibody in the monoclonal antibody cell fermentation broth supernatant is calculated: a pH adjustment buffer is added to the collected eluate so that the pH of the eluate is 6.5 to 8.0, the mixture is mixed, the dilution multiple is adjusted for dilution, and the A280nm absorbance is determined. The concentration of monoclonal antibody in the eluate = A280nm absorbance ÷ monoclonal antibody extinction coefficient × dilution multiple; the concentration of monoclonal antibody in the supernatant of monoclonal antibody cell fermentation broth = the concentration of monoclonal antibody in the eluate × the volume of elution buffer ÷ the loading volume of the supernatant of monoclonal antibody cell fermentation broth; after collecting the monoclonal antibody eluate, wash the affinity chromatography medium with the washing buffer, balance it with the first equilibration buffer, and then store it with the preservation solution.

[0144] Example 1 Simple method for determining the production of existing monoclonal antibodies

[0145] 1.1 Simple method for determining the yield of target protein of IgG1 and IgG4 monoclonal antibodies

[0146] Take 50 mL of the cell fermentation broth supernatant containing IgG1 monoclonal antibody or IgG4 monoclonal antibody respectively, IgG1 monoclonal antibody or IgG4 monoclonal antibody is shown in Table 2, remove cells and debris particles by centrifugation, centrifuge at 8000 rpm for 30 minutes, and collect the fermentation broth supernatant for subsequent steps;

[0147] The separation and purification of monoclonal antibodies in various fermentation broth supernatants was carried out using Figure 1The simple device shown in the figure is carried out. The simple device includes: a syringe (10mL disposable sterile syringe, Shandong Ande Medical Products Co., Ltd.), an affinity chromatography medium (AT Protein A Diamond Plus affinity chromatography column) and a syringe pump (LSP10-1B type 10-channel syringe pump). Among them, the syringe is fixed on the syringe pump, and the sample outlet of the syringe is connected to the sample inlet of the affinity chromatography medium, so that when the supernatant of the monoclonal antibody cell fermentation liquid is loaded in the syringe, the syringe pump can push the injection rod of the syringe, so that the supernatant of the monoclonal antibody cell fermentation liquid enters the affinity chromatography medium from the syringe at a set flow rate, and flows out from the sample outlet of the affinity chromatography medium after separation and purification by the affinity chromatography medium.

[0148] AT Protein A Diamond Plus affinity chromatography columns (column volume 1 mL) were used, and each affinity chromatography column was washed with 5 column volumes of washing buffer at a flow rate of 1 mL / min. Each affinity chromatography column was then balanced with 5 column volumes of the first equilibration buffer at a flow rate of 1 mL / min. Samples containing IgG1 monoclonal antibodies or IgG4 monoclonal antibodies were loaded onto two affinity chromatography columns, respectively, at a flow rate of 0.2 mL / min. Each affinity chromatography column was re-equilibrated with 5 column volumes of the second equilibration buffer, wherein the first column volume was washed with a flow rate of 0.2 mL / min, and then 4 column volumes were washed with a flow rate of 1 mL / min. Each affinity chromatography column was eluted with 4 column volumes of elution buffer at a flow rate of 1 mL / min. 15 mL centrifuge tubes were used to collect the target IgG1 monoclonal antibody elution buffer or IgG4 monoclonal antibody elution buffer, and pH adjustment buffer was added immediately after collection to make the pH of the IgG1 monoclonal antibody solution or IgG4 monoclonal antibody solution 6.5-8.0. Each affinity chromatography column was cleaned with 5 column volumes of cleaning buffer at a flow rate of 1 mL / min, then equilibrated with 5 column volumes of first equilibration buffer at a flow rate of 1 mL / min, and finally preserved with 5 column volumes of preservation solution at a flow rate of 1 mL / min.

[0149] in:

[0150] The first equilibrium buffer: 20 mM Na2HPO4-NaH2PO4 and 150 mM NaCl, pH 6.8, conductivity 18 mS / cm;

[0151] Second equilibrium buffer: 10 mM Na2HPO4-NaH2PO4, pH 7.2, conductivity 1.3 mS / cm;

[0152] Elution buffer: 15 mM citric acid and 7 mM Na2HPO4, pH 3.1, conductivity 1.3 mS / cm;

[0153] Sample pH adjustment buffer: 2 M Tris-HCl, pH 9.5;

[0154] Wash buffer: 0.1 M NaOH and 1.0 M NaCl;

[0155] Preservative solution: 20% ethanol.

[0156] For the collected IgG1 monoclonal antibody or IgG4 monoclonal antibody (IgG1 monoclonal antibody or IgG4 monoclonal antibody is recorded as the target protein) eluate, the absorbance value at 280 nm is measured by ultraviolet spectrophotometer, so as to determine the concentration of IgG1 monoclonal antibody or IgG4 monoclonal antibody therein: the concentration of monoclonal antibody in the eluate = A280nm absorbance value ÷ monoclonal antibody extinction coefficient × dilution multiple.

[0157] Then, the concentration of IgG1 monoclonal antibody or IgG4 monoclonal antibody in the cell fermentation broth supernatant (i.e., the target protein yield) is calculated:

[0158] Target protein yield = protein concentration of target protein elution buffer × volume of target protein elution buffer ÷ volume of injected fermentation broth supernatant.

[0159] 1.2 Liquid phase determination of target protein yield of IgG1 or IgG4 monoclonal antibodies

[0160] High performance liquid chromatograph: brand: thermo, model: UltiMate3000;

[0161] Chromatographic column: Sepax, ProAqa Excel, specification 20μm, 2.1×30mm,

[0162] Mobile phase A (20mmol / L PB+0.15mol / L NaCl): weigh 4.30g Na2HPO4.12H2O, 1.25g NaH2PO4.2H2O, and 8.76g NaCl, add 1L ultrapure water to dissolve them, filter, sonicate for 15min, set aside, and store at room temperature. The shelf life is 3 days. The flow rate is 2.0mL / min.

[0163] Mobile phase B (7mmol / L Na2HPO4.12H2O + 27mmol / L citric acid): weigh 1.25g Na2HPO4.12H2O and 2.83g citric acid, add 500mL ultrapure water to dissolve, filter, ultrasonicate for 15min, set aside, store at room temperature, valid for 3 days, flow rate is 2.0mL / min.

[0164] 20% ethanol solution: Measure 80 mL of ultrapure water, add 20 mL of ethanol, and store at room temperature. The shelf life is 1 month.

[0165] The method comprises the following steps: (1) taking a reference substance, diluting the reference substance with mobile phase A to 10 mg / mL and marking it as ST1, and then diluting it in a gradient manner to 9 mg / mL, 8 mg / mL, 6 mg / mL, 4 mg / mL, and 2 mg / mL and marking them as ST2, ST3, ST4, ST5, and ST6, respectively.

[0166] (2) Take 100 μl of the fermentation broth supernatant containing IgG1 monoclonal antibody or IgG4 monoclonal antibody in Example 1 above, carefully pipette it into a sample injection bottle equipped with an inner insert.

[0167] (3) Load the standard sample with a sample volume of 5ul and obtain the linear equation (R 2 ≥0.99);

[0168] (4) Load IgG1 monoclonal antibody sample or IgG4 monoclonal antibody sample. If the sample peak area exceeds the peak area of ​​the standard curve ST1 or ST6, adjust the injection volume and re-inject and analyze so that the sample peak area is within the range of the standard curve ST1-ST6 peak area. The flow rate is 2.0 mL / min; the detection wavelength is 280 nm.

[0169] (5) Elution: Perform the experiment according to the elution gradient described in Table 1

[0170] Table 1

[0171]

[0172] (5) Establish a standard curve and calculate the sample content.

[0173] The target protein yields of the following monoclonal antibodies were determined using the simple determination method in 1.1 and the liquid phase determination method in 1.2, respectively. The determination results are shown in Table 2.

[0174] The anti-human interleukin 23 monoclonal antibody QX004N described in the patent document CN111718414B, the anti-human interleukin 4 receptor α monoclonal antibody QX001S described in the patent document CN112852743B, the anti-human interleukin 4 receptor α monoclonal antibody QX005N described in the patent document CN110746507B, the anti-human interferon α receptor 1 monoclonal antibody QX006N described in the patent document CN113278071B, the human interleukin-33 monoclonal antibody QX007N described in the patent document CN113603775B, the anti-human TSLP monoclonal antibody QX008N described in the patent document CN113683694B, the anti-human interleukin 17A monoclonal antibody QX002N described in the patent document CN108640991B, and the anti-human CD117 monoclonal antibody QX013N described in the patent document CN202410059200.8.

[0175] Table 2 Results of simple method and liquid phase method for different monoclonal antibodies

[0176]

[0177] Wherein, deviation = (target protein yield determined by the simple method - target protein yield determined by the liquid phase method) ÷ target protein yield determined by the liquid phase method × 100%.

[0178] The measurement results in Table 2 show that the measurement results of the simplified measurement method of the present application have a small deviation from the measurement results of the liquid phase method, and are applicable to various IgG1 monoclonal antibodies or IgG4 monoclonal antibodies.

[0179] Comparative Example 1

[0180] The first equilibrium buffer used in Comparative Example 1 has a pH of 6.5 and a conductivity of 18 mS / cm. Other measurement conditions and the monoclonal antibody measured are the same as those in Example 1.

[0181] The measurement results showed that, at least for the above-mentioned monoclonal antibody QX001S, the target protein yield measured by the simple method was 4.88 mg / mL, with a deviation of -4% from the result measured by the liquid phase method.

[0182] Comparative Example 2

[0183] The first equilibrium buffer used in Comparative Example 2 has a pH of 6.8 and a conductivity of 16 mS / cm, and other measurement conditions and the measured monoclonal antibody are the same as those in Example 1. It should be noted that in the examples and comparative examples of the present application, the conductivity is adjusted by adding 4 M NaCl or purified water.

[0184] The measurement results showed that, at least for the above-mentioned monoclonal antibody QX005N, the target protein yield measured by the simple method was 5.23 mg / mL, with a deviation of -4% from the result measured by the liquid phase method.

[0185] Comparative Example 3

[0186] The first equilibrium buffer used in Comparative Example 3 has a pH of 7.2 and a conductivity of 20 mS / cm. Other measurement conditions and the monoclonal antibody measured are the same as those in Example 1.

[0187] The measurement results showed that, at least for the above-mentioned monoclonal antibody QX002N, the target protein yield measured by the simple method was 7.78 mg / mL, which deviated from the result measured by the liquid phase method by 4%.

[0188] Comparative Example 4

[0189] The first equilibrium buffer used in Comparative Example 4 has a pH of 7.2 and a conductivity of 16 mS / cm. Other measurement conditions and the monoclonal antibody measured are the same as those in Example 1.

[0190] The measurement results showed that, at least for the above-mentioned monoclonal antibody QX006N, the target protein yield measured by the simple method was 5.58 mg / mL, with a deviation of -3% from the result measured by the liquid phase method.

[0191] Comparative Example 5

[0192] The pH of the elution buffer used in Comparative Example 5 was 3.1, the conductivity was 1.8 mS / cm, and the other measurement conditions and the monoclonal antibody measured were the same as those in Example 1.

[0193] The results showed that, at least for the monoclonal antibody QX013N, the target protein yield determined by the simplified method was 5.22 mg / mL, which deviated from the result determined by the liquid phase method by 4%.

[0194] Comparative Example 6

[0195] The pH of the elution buffer used in Comparative Example 6 was 2.8, the conductivity was 1.3 mS / cm, and the other measurement conditions and the monoclonal antibody measured were the same as those in Example 1.

[0196] The measurement results showed that, at least for the above-mentioned monoclonal antibody QX008N, the target protein yield measured by the simple method was 6.01 mg / mL, which deviated from the result measured by the liquid phase method by 6%.

[0197] Comparative Example 7

[0198] The pH of the elution buffer used in Comparative Example 7 was 3.4, the conductivity was 1.8 mS / cm, and the other measurement conditions and the monoclonal antibody measured were the same as those in Example 1.

[0199] The results showed that, at least for the monoclonal antibody QX007N, the target protein yield determined by the simplified method was 8.05 mg / mL, with a deviation of -7% from the result determined by the liquid phase method.

[0200] Comparative Example 8

[0201] The pH of the elution buffer used in Comparative Example 8 was 3.4, the conductivity was 0.8 mS / cm, and the other measurement conditions and the monoclonal antibody measured were the same as those in Example 1.

[0202] The measurement results showed that, at least for the above-mentioned monoclonal antibody QX006N, the target protein yield measured by the simple method was 5.27 mg / mL, with a deviation of -9% from the result measured by the liquid phase method.

[0203] Example 2 A simple method for determining the yield of a novel anti-human interleukin 17A monoclonal antibody

[0204] 2.1 Preparation of new anti-human interleukin-17A monoclonal antibody

[0205] Human interleukin 17A (hIL-17A) was purchased from Shanghai Jinan Technology Co., Ltd. for immunization of rabbits, and monoclonal antibodies with inhibitory activity against human interleukin 17A were screened using B cell cloning technology. First, the cell supernatant was detected by binding ELISA to select clones that bind to hIL-17A; then, blocking ELISA was used to detect and select clones that can inhibit the binding of hIL-17A to its receptor. Eight positive clones were selected for recombinant expression and sequenced, including clone 19#, and the anti-human IL-17A monoclonal antibody produced by it was named 19# monoclonal antibody. Monoclonal antibody 19# was humanized to obtain a humanized anti-human IL-17A monoclonal antibody, which was named HZD-19. The SDS-PAGE electrophoresis diagram of the purified HZD-19 is shown in the figure. Figure 2 ; HZD-19 (Reducing) indicates HZD-19 under reducing conditions, and HZD-19 (non-reducing) indicates HZD-19 under non-reducing conditions.

[0206] HZD-19 comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein:

[0207] The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1 SYYVG;

[0208] The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2 VIRIGGGTAYASWAKG;

[0209] The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3 SSINGGDI;

[0210] The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4 QASQDIRYDLS;

[0211] The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5 AASKLAS;

[0212] The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6 LGIFSFTDDDGLA.

[0213] HZD19 contains a heavy chain variable region and a light chain variable region.

[0214] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7

[0215] EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWVGVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSSINGGDIWGQGTLVTVSS is shown;

[0216] The amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8

[0217] AIQMTQSPSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDDGLAFGGGTKVEIK.

[0218] The amino acid sequence of the heavy chain of HZD19 is shown in SEQ ID NO:9; the amino acid sequence of the light chain is shown in SEQ ID NO:10.

[0219] SEQ ID NO: 9

[0220] EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWVGVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSSINGGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0221] SEQ ID NO: 10

[0222] AIQMTQSPSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDDGLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0223] Biacore T200 was used to detect the affinity of anti-human IL-17A monoclonal antibody (HZD-19) to human IL-17A, and all processes were carried out at 25°C. The appropriate amount of antibody was captured using a Protein A chip so that Rmax was less than 50 RU, and the capture flow rate was 10μL / min. The antigen was gradient diluted, and the instrument flow rate was switched to 30μL / min. The reference channel and the capture antibody channel were flowed in order from low to high concentration, and the buffer was flowed as a negative control. After each binding and dissociation, the chip was regenerated with pH1.5 glycine. The 1:1 binding model in the Kinetics option was selected using the instrument's own analysis software for fitting, and the antibody's binding rate constant ka, dissociation rate constant kd, and dissociation equilibrium constant KD values ​​were calculated. The results showed that the affinity of HZD-19 for binding to human IL-17A was in the nM~pM order, and KD was 8.31×10 -10 M.

[0224] 2.2 Determination of anti-human interleukin-17A monoclonal antibody (HZD-19) by simple method and liquid phase method

[0225] The target protein content in the supernatant of HZD-19 cell fermentation broth was determined by the simple method and the liquid phase method as in Example 1. The target protein yield determined by the simple method was 5.40 mg / mL, and the target protein yield determined by the liquid phase method was 5.33 mg / mL, with a deviation of 1%.

Claims

1. A method for determining the concentration of monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth, which is carried out using a simple device and comprises the following steps: Using the first equilibration buffer to equilibrate the affinity chromatography medium to obtain an equilibrated affinity chromatography medium; Loading the monoclonal antibody cell fermentation broth supernatant onto the equilibrated affinity chromatography medium; re-equilibrate the affinity chromatography medium using a second equilibration buffer; The affinity chromatography medium is eluted with an elution buffer, and the eluate containing the target monoclonal antibody is collected; Determine the concentration of the monoclonal antibody in the collected eluate, and calculate the concentration of the monoclonal antibody in the supernatant of the monoclonal antibody cell fermentation broth: Add pH adjustment buffer to the collected eluate to make the pH of the eluate 6.5-8.0, mix well, adjust the dilution multiple to dilute, and measure the A280nm absorbance. The concentration of monoclonal antibody in the eluent = A280nm absorbance ÷ monoclonal antibody extinction coefficient × dilution multiple; The concentration of monoclonal antibody in the supernatant of monoclonal antibody cell fermentation broth = the concentration of monoclonal antibody in the elution buffer × the volume of elution buffer ÷ the loading volume of the supernatant of monoclonal antibody cell fermentation broth; The first balancing buffer is an aqueous solution of 20 mM Na2HPO4-NaH2PO4 and 150 mM NaCl, with a pH of 6.8, and the conductivity of the first balancing buffer is 18 mS / cm; The second balancing buffer is a 10 mM Na2HPO4-NaH2PO4 aqueous solution with a pH of 7.2, and the conductivity of the second balancing buffer is 1.3 mS / cm; The elution buffer is an aqueous solution of 15 mM citric acid and 7 mM Na2HPO4, with a pH of 3.1, and the conductivity of the elution buffer is 1.3 mS / cm; The simple device is used for separation and purification of monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth, and comprises: syringe, Affinity chromatography media, and Syringe pump; Wherein, the syringe is fixed on the syringe pump, and the sample outlet of the syringe is connected to the sample inlet of the affinity chromatography medium. When the monoclonal antibody cell fermentation liquid supernatant is loaded in the syringe, the syringe pump can push the injection rod of the syringe, so that the monoclonal antibody cell fermentation liquid supernatant enters the affinity chromatography medium from the syringe at a set flow rate, and flows out from the sample outlet of the affinity chromatography medium after being separated and purified by the affinity chromatography medium.

2. The method according to claim 1, wherein: The monoclonal antibody is an IgG1 monoclonal antibody and / or an IgG4 monoclonal antibody.

3. The method according to claim 1, wherein: The affinity chromatography medium is an AT Protein A Diamond Plus affinity chromatography column.

4. The method according to claim 1, wherein: The monoclonal antibody cell fermentation supernatant is a eukaryotic cell fermentation supernatant.

5. The method according to claim 1, wherein: The volume of the first equilibration buffer is 3 to 6 times the volume of the affinity chromatography medium; The volume of the second equilibration buffer is 3 to 6 times the volume of the affinity chromatography medium; and / or The volume of the elution buffer is 3 to 6 times the volume of the affinity chromatography medium.

6. The method according to claim 1, wherein: The monoclonal antibody is one selected from the following a~j, or a combination of any two, three, four, five, six, seven or all of them: a. Anti-human interleukin-23 monoclonal antibody QX004N; b. Anti-human interleukin-4 receptor α monoclonal antibody QX005N; c. Anti-human interferon α receptor 1 monoclonal antibody QX006N; d. Human interleukin-33 monoclonal antibody QX007N; e. Anti-human TSLP monoclonal antibody QX008N; f. Anti-human interleukin 17A monoclonal antibody QX002N; g. anti-human CD117 monoclonal antibody QX013N; and j. Anti-human IL-12 / IL-23p40 subunit fully human monoclonal antibody QX001S.

7. The method according to claim 1, wherein: The monoclonal antibody is an anti-human interleukin 17A monoclonal antibody, which comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein: The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1 SYYVG; The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2 VIRIGGGTAYASWAKG; The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3 SSINGGDI; The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4 QASQDIRYDLS; The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5 AASKLAS; The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6 LGIFSFTDDDGLA.

8. The method according to claim 7, wherein: The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; Wherein, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7 EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWVGVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSSINGGDIWGQGTLVTVSS; The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8 AIQMTQSPSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDDGLAFGGGTKVEIK.

9. The method according to claim 7, wherein: The monoclonal antibody comprises a heavy chain and a light chain, wherein The amino acid sequence of the heavy chain is shown in SEQ ID NO: 9; The amino acid sequence of the light chain is shown in SEQ ID NO:10.

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