Method for detecting concentration of monoclonal antibody in supernatant of monoclonal antibody cell fermentation liquor

Abstract

The invention provides a method for detecting the concentration of a monoclonal antibody in a monoclonal antibody cell fermentation broth supernatant. The detection method comprises the following steps: carrying out affinity chromatography on a fermentation supernatant of the monoclonal antibody, collecting an eluent containing the target monoclonal antibody, measuring the concentration of the target monoclonal antibody in the eluent, and calculating the concentration of the target monoclonal antibody in the fermentation supernatant. The detection 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] This patent application belongs to the field of bioassay, and specifically relates to a method for detecting the concentration of monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth. 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. During 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 chromatographic 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] 3. High performance liquid chromatography has low throughput and is not suitable for sample preparation. Summary of the invention

[0008] The technical solution of this application is as follows:

[0009] 1. A method for detecting the concentration of monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth, comprising the following steps:

[0010] Using the first equilibration buffer to equilibrate the affinity chromatography medium to obtain an equilibrated affinity chromatography medium;

[0011] Loading the monoclonal antibody cell fermentation broth supernatant onto the equilibrated affinity chromatography medium;

[0012] re-equilibrate the affinity chromatography medium using a second equilibration buffer;

[0013] The affinity chromatography medium is eluted with an elution buffer, and the eluate containing the target monoclonal antibody is collected;

[0014] 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:

[0015] 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.

[0016] The concentration of monoclonal antibody in the eluent = A280nm absorbance ÷ monoclonal antibody extinction coefficient × dilution multiple;

[0017] 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;

[0018] 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;

[0019] 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;

[0020] 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.

[0021] 2. The method according to claim 1, wherein:

[0022] The monoclonal antibody is an IgG1 monoclonal antibody and / or an IgG4 monoclonal antibody.

[0023] 3. The method according to claim 1, wherein:

[0024] The affinity chromatography medium is an AT Protein A Diamond Plus affinity chromatography column.

[0025] 4. The method according to claim 1, wherein:

[0026] The fermentation supernatant containing the monoclonal antibody is a eukaryotic cell fermentation supernatant.

[0027] 5. The method according to claim 1, wherein:

[0028] The volume of the first equilibration buffer is 3 to 6 times the volume of the affinity chromatography medium;

[0029] The volume of the second equilibration buffer is 3 to 6 times the volume of the affinity chromatography medium; and / or

[0030] The volume of the elution buffer is 3 to 6 times the volume of the affinity chromatography medium.

[0031] 6. The method according to item 1, wherein the monoclonal antibody is selected from one of the following a to j, or a combination of any two, three, four, five, six, seven, or eight, or all of them:

[0032] a. Anti-human interleukin-23 monoclonal antibody QX004N;

[0033] b. Anti-human interleukin-4 receptor α monoclonal antibody QX005N;

[0034] c. Anti-human interferon α receptor 1 monoclonal antibody QX006N;

[0035] d. Human interleukin-33 monoclonal antibody QX007N;

[0036] e. Anti-human TSLP monoclonal antibody QX008N;

[0037] f. Anti-human interleukin 17A monoclonal antibody QX002N;

[0038] g. anti-human CD117 monoclonal antibody QX013N; and

[0039] j. Anti-human IL-12 / IL-23p40 subunit fully human monoclonal antibody QX001S.

[0040] 7. The method according to item 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:

[0041] The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1SYYVG;

[0042] The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2VIRIGGGTAYASWAKG;

[0043] The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3SSINGGDI;

[0044] The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4QASQDIRYDLS;

[0045] The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5AASKLAS;

[0046] The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6LGIFSFTDDDGLA.

[0047] 8. The method according to item 7, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0048] Wherein, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWV GVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSSINGGDIWGQGTLVTVSS;

[0049] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8AIQMTQSPSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKLLIYA ASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDDGLAFGGGTKVEIK.

[0050] 9. The method according to item 7, wherein the monoclonal antibody comprises a heavy chain and a light chain; wherein,

[0051] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 9;

[0052] The amino acid sequence of the light chain is shown in SEQ ID NO:10.

[0053] 10. The method according to claim 1, wherein:

[0054] The pH adjustment buffer is a 2M Tris-HCl aqueous solution with a pH of 9.5; and / or the dilution multiple is 5-20.

[0055] Compared with the prior art, the beneficial effects of this patent application are:

[0056] The detection method described in the present application can be used for the purification and content determination of monoclonal antibodies. It has the advantages of not requiring expensive equipment, low cost, simple operation, good versatility (the same purification and detection conditions are applicable to both IgG1 and IgG4 monoclonal antibodies, which greatly reduces the production burden of CDMO companies), and the results are basically consistent with those of high performance liquid chromatography. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the SDS-PAGE electrophoresis diagram of the anti-human interleukin 17A monoclonal antibody HZD-19 prepared in Example 2. DETAILED DESCRIPTION

[0058] 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.

[0059] 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.

[0060] 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.

[0061] The present application provides a method for detecting the concentration of a monoclonal antibody in a supernatant of a monoclonal antibody cell fermentation broth, which comprises the following steps: balancing an affinity chromatography medium with a first balancing buffer to obtain a well-balanced affinity chromatography medium; loading the monoclonal antibody cell fermentation broth supernatant onto the well-balanced affinity chromatography medium; rebalancing the affinity chromatography medium with a second balancing buffer; eluting the affinity chromatography medium with an elution buffer to collect an 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; Calculate the concentration of the monoclonal antibody in the supernatant of the monoclonal antibody cell fermentation broth: add a pH adjustment buffer to the collected eluate so that the pH of the eluate is 6.5-8.0, mix well, adjust the dilution multiple for dilution, and measure the A280nm absorbance. The concentration of the monoclonal antibody in the eluate = A280nm absorbance / monoclonal antibody extinction coefficient × dilution multiple; the concentration of the monoclonal antibody in the supernatant of the monoclonal antibody cell fermentation broth = the concentration of the monoclonal antibody in the eluate × the volume of the elution buffer / the loading volume of the monoclonal antibody cell fermentation broth supernatant.

[0062] 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 Bogelon. 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 process scale-up to reduce production costs.

[0063] In some embodiments of the present application, the affinity chromatography medium can be an AT Protein ADiamond Plus affinity chromatography column.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] In some embodiments of the present application, the washing buffer is an aqueous solution of 0.1 M NaOH and 1.0 M NaCl.

[0075] 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.

[0076] 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.

[0077] In some embodiments of the present application, the preservation solution is a 20% ethanol aqueous solution.

[0078] 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.

[0079] 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, 3 times, 4 times, 5 times, 6 times or any range therebetween.

[0080] In some embodiments of the present application, the monoclonal antibody is an IgG1 monoclonal antibody and / or an IgG4 monoclonal antibody.

[0081] In some embodiments of the present application, the fermentation supernatant containing the monoclonal antibody is a eukaryotic cell fermentation supernatant.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] In some embodiments of the present application, the monoclonal antibody is selected from one of the following a to j, or a combination of any two, three, four, five, six, seven, or eight, or all of them:

[0086] a. Anti-human interleukin-23 monoclonal antibody;

[0087] b. Anti-human interleukin-4 receptor α monoclonal antibody;

[0088] c. Anti-human interferon α receptor 1 monoclonal antibody;

[0089] d. Anti-human interleukin-33 monoclonal antibody;

[0090] e. anti-human TSLP monoclonal antibody; and

[0091] f. Anti-human interleukin 17A monoclonal antibody;

[0092] g. Anti-human CD117 monoclonal antibody;

[0093] j. fully human monoclonal antibody against human IL-12 / IL-23p40 subunit; and

[0094] i. anti-human interleukin-17A monoclonal antibody as described below;

[0095] 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, and anti-human interleukin-4 receptor α monoclonal antibody QX008N described in patent document CN113603775B. The anti-human TSLP monoclonal antibody QX008N described in the patent document 13683694B, the anti-human interleukin 17A monoclonal antibody QX002N described in the patent document CN108640991B, the anti-human CD117 monoclonal antibody QX013N described in the patent document CN202410059200.8, and the anti-human IL-12 / IL-23p40 subunit fully human monoclonal antibody QX001S described in the patent document CN112852743B.

[0096] 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:

[0097] The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1SYYVG;

[0098] The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2VIRIGGGTAYASWAKG; the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3SSINGGDI;

[0099] The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4QASQDIRYDLS;

[0100] The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5AASKLAS;

[0101] The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6LGIFSFTDDDGLA.

[0102] Preferably, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0103] Wherein, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWV GVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSSINGGDIWGQGTLVTVSS;

[0104] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8AIQMTQSPSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKLLIYA ASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDDGLAFGGGTKVEIK.

[0105] Preferably, the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 9. SEQ ID NO: 9 EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWVGVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSSINGGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. The amino acid sequence of the light chain is as shown in SEQ ID NO: 10. SEQ ID NO: 10 AIQMTQSPSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDDGLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0106] 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 well-equilibrated affinity chromatography medium; the monoclonal antibody cell fermentation broth supernatant is loaded onto the well-equilibrated 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 make the pH of the eluate be 6.5-8.0, mixed, diluted by adjusting the dilution multiple, and the A280nm absorbance value is measured. The concentration of the monoclonal antibody in the eluate = A280nm absorbance value ÷ monoclonal antibody extinction coefficient × dilution multiple; the concentration of the monoclonal antibody in the supernatant of the monoclonal antibody cell fermentation broth = the concentration of the monoclonal antibody in the eluate × the volume of the elution buffer ÷ the loading volume of the supernatant of the monoclonal antibody cell fermentation broth; after collecting the monoclonal antibody eluate, the affinity chromatography medium is washed with the washing buffer, balanced with the first equilibration buffer, and then stored with the preservation solution.

[0107] Example 1 Simple method for detecting the production of existing monoclonal antibodies

[0108] 1.1 Simple detection method for the target protein production of IgG1 monoclonal antibody and IgG4 monoclonal antibody

[0109] 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 1, remove cells and debris particles by centrifugation, centrifuge at 8000 rpm for 30 minutes, and collect the fermentation broth supernatant for subsequent steps;

[0110] An AT Protein A Diamond Plus affinity chromatography column (column volume 1 mL) was 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 at a flow rate of 0.2 mL / min, and then 4 column volumes were at 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 immediately added after collection to make the pH of the IgG1 monoclonal antibody solution or IgG4 monoclonal antibody solution be 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.

[0111] in:

[0112] The first equilibrium buffer: 20 mM Na2HPO4-NaH2PO4 and 150 mM NaCl, pH 6.8, conductivity 18 mS / cm;

[0113] Second equilibrium buffer: 10 mM Na2HPO4-NaH2PO4, pH 7.2, conductivity 1.3 mS / cm;

[0114] Elution buffer: 15 mM citric acid and 7 mM Na2HPO4, pH 3.1, conductivity 1.3 mS / cm;

[0115] Sample pH adjustment buffer: 2 M Tris-HCl, pH 9.5;

[0116] Wash buffer: 0.1 M NaOH and 1.0 M NaCl;

[0117] Preservative solution: 20% ethanol.

[0118] 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 factor.

[0119] 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:

[0120] Target protein yield = protein concentration of target protein elution buffer × volume of target protein elution buffer ÷ volume of fermentation broth supernatant injected.

[0121] 1.2 Liquid phase detection of target protein production of IgG1 monoclonal antibody or IgG4 monoclonal antibody High performance liquid chromatography: brand thermo, model UltiMate3000;

[0122] Chromatographic column: Sepax, ProAqa Excel, specification 20μm, 2.1×30mm,

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] (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.

[0128] (3) Load the standard sample with a sample volume of 5 μl and obtain the linear equation (R 2 ≥0.99);

[0129] (4) Load an IgG1 monoclonal antibody sample or an 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 perform another injection analysis 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.

[0130] (4) Elution: Perform the experiment according to the following elution gradient

[0131] Time(min) Flow(mL / min) Mobile phase A (%) Mobile phase B (%) 0-0.100 2.0 100 0 0.100-0.110 2.0 100 0 0.110-0.600 2.0 0 100 0.600-0.610 2.0 0 100 0.610-2.000 2.0 100 0

[0132] (5) Establish a standard curve and calculate the sample content.

[0133] The simple detection method in 1.1 and the liquid phase detection method in 1.2 were used to detect the target protein production of the following monoclonal antibodies, respectively. The detection results are shown in Table 1.

[0134] 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.

[0135] Table 1 Detection results of simple method and liquid phase method for different monoclonal antibodies

[0136]

[0137] 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%.

[0138] The test results in Table 1 show that the test results of the simple detection method of the present patent application have a small deviation from the test results of the liquid phase method, and are applicable to various IgG1 monoclonal antibodies or IgG4 monoclonal antibodies.

[0139] Comparative Example 1

[0140] The first equilibrium buffer used in Comparative Example 1 has a pH of 6.5 and a conductivity of 18 mS / cm. Other detection conditions and the monoclonal antibody detected are the same as those in Example 1.

[0141] The results showed that, at least for the monoclonal antibody QX001S, the target protein yield determined by the simplified method was 4.88 mg / mL, with a deviation of -4% from the liquid phase method result.

[0142] Comparative Example 2

[0143] The first equilibrium buffer used in Comparative Example 2 has a pH of 6.8 and a conductivity of 16 mS / cm, and other detection conditions and the detected monoclonal antibody are the same as those in Example 1. It should be noted that in the examples and comparative examples of the present patent application, the conductivity is adjusted by adding 4M NaCl or purified water.

[0144] The results showed that, at least for the monoclonal antibody QX005N, the target protein yield determined by the simple method was 5.23 mg / mL, with a deviation of -4% from the liquid phase method result.

[0145] Comparative Example 3

[0146] The first equilibrium buffer used in Comparative Example 3 has a pH of 7.2 and a conductivity of 20 mS / cm. Other detection conditions and the monoclonal antibody detected are the same as those in Example 1.

[0147] The results showed that, at least for the monoclonal antibody QX002N, the target protein yield determined by the simplified method was 7.78 mg / mL, with a deviation of 4% from the liquid phase method result.

[0148] Comparative Example 4

[0149] The first equilibrium buffer used in Comparative Example 4 has a pH of 7.2 and a conductivity of 16 mS / cm. Other detection conditions and the monoclonal antibody detected are the same as those in Example 1.

[0150] The results showed that, at least for the monoclonal antibody QX006N, the target protein yield determined by the simple method was 5.58 mg / mL, with a deviation of -3% from the liquid phase method result.

[0151] Comparative Example 5

[0152] The pH of the elution buffer used in Comparative Example 5 was 3.1, the conductivity was 1.8 mS / cm, and the other detection conditions and the monoclonal antibody detected were the same as those in Example 1.

[0153] 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, with a deviation of 4% from the liquid phase method result.

[0154] Comparative Example 6

[0155] The pH of the elution buffer used in Comparative Example 6 was 2.8, the conductivity was 1.3 mS / cm, and the other detection conditions and the monoclonal antibody detected were the same as those in Example 1.

[0156] The results showed that, at least for the monoclonal antibody QX008N, the target protein yield determined by the simple method was 6.01 mg / mL, which was 6% different from the result of the liquid phase method.

[0157] Comparative Example 7

[0158] The pH of the elution buffer used in Comparative Example 7 was 3.4, the conductivity was 1.8 mS / cm, and the other detection conditions and the monoclonal antibody detected were the same as those in Example 1.

[0159] 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 liquid phase method result.

[0160] Comparative Example 8

[0161] The pH of the elution buffer used in Comparative Example 8 was 3.4, the conductivity was 0.8 mS / cm, and the other detection conditions and the monoclonal antibody detected were the same as those in Example 1.

[0162] The results showed that, at least for the monoclonal antibody QX006N, the target protein yield determined by the simple method was 5.27 mg / mL, with a deviation of -9% from the liquid phase method result.

[0163] Example 2 Simple Detection Method for the Production of Novel Anti-Human Interleukin 17A Monoclonal Antibodies 2.1 Preparation of Novel Anti-Human Interleukin 17A Monoclonal Antibodies

[0164] Human interleukin 17A (hIL-17A) was purchased from Shanghai Jinan Technology Co., Ltd. and used to immunize rabbits. B cell cloning technology was used to screen monoclonal antibodies with inhibitory activity against human interleukin 17A. 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#. 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 1 .

[0165] 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: the amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1SYYVG;

[0166] The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2VIRIGGGTAYASWAKG;

[0167] The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3SSINGGDI;

[0168] The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4QASQDIRYDLS;

[0169] The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5AASKLAS;

[0170] The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6LGIFSFTDDDGLA.

[0171] HZD19 contains a heavy chain variable region and a light chain variable region.

[0172] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWV GVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSSINGGDIWGQGTLVTVSS;

[0173] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8AIQMTQSPSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKLLIYA ASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDDGLAFGGGTKVEIK.

[0174] 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.

[0175] SEQ ID NO: 9

[0176] EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWV

[0177] GVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSS

[0178] INGGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP

[0179] EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV

[0180] NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

[0181] SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR

[0182] VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT

[0183] LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

[0184] GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 10

[0185] AIQMTQSPSSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKLLIYA

[0186] ASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDDGLAF

[0187] GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW

[0188] KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT

[0189] HQGLSSPVTKSFNRGEC

[0190] 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 the Protein A chip so that Rmax was less than 50RU, 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 to pM order, and KD was 8.31×10 -10 M.

[0191] 2.2 Detection of anti-human interleukin-17A monoclonal antibody (HZD-19) using a simple method and liquid phase method

[0192] The target protein content in the supernatant of HZD-19 cell fermentation broth was determined by the simple method and 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 detecting the concentration of monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth, wherein: The steps include: 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.

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 fermentation supernatant containing the monoclonal antibody 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 to j, or a combination of any two, three, four, five, six, seven or eight, 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: 1SYYVG; The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2VIRIGGGTAYASWAKG; The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3SSINGGDI; The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4QASQDIRYDLS; The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5AASKLAS; The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6LGIFSFTDDDGLA.

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: 7EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWV GVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSSINGGDIWGQGTLVTVSS; The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8AIQMTQSPSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKLLIYA ASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDDGLAFGGGTKVEIK.

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.

10. The method according to claim 1, wherein: The pH adjustment buffer is a 2M Tris-HCl aqueous solution with a pH of 9.5; and / or the dilution multiple is 5-20.

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