Improved monoclonal antibody purification device and concentration determination method

Abstract

The invention discloses an improved 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 simple device disclosed by the invention can be suitable for purifying a plurality of samples at the same time, and the samples do not influence each other. The simple device is at least suitable for the purification and concentration determination of the fermentation supernatant of the existing monoclonal antibodies QX004N, QX005N, QX006N, QX007N, QX008N, QX002N, QX013N and QX001S. By using the simple device, the operation is simple and convenient, and complex training on an operator is not needed; the cost of equipment and consumables is low, and an expensive high performance liquid chromatograph is not occupied.

Description

Technical Field

[0001] The present patent application belongs to the field of bioassay, and specifically relates to an improved 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] Monoclonal antibody drugs are widely used in the treatment of autoimmune diseases (such as asthma, rheumatoid arthritis, multiple sclerosis, lupus erythematosus, etc.) and tumors. For monoclonal antibody drugs, in addition to paying attention to the effectiveness of the product, we should also focus on safety and molecular stability, especially residual HCP and molecular aggregation in the production process of monoclonal antibodies. Therefore, it is necessary to purify and measure the concentration of monoclonal antibodies in each link of monoclonal antibody drug research and development and production.

[0003] In the prior art, a high performance liquid chromatograph is used in combination with a Protein A affinity chromatography column to crudely purify the supernatant of a monoclonal antibody cell fermentation broth and quantitatively detect the content of the monoclonal antibody in the supernatant of the cell fermentation broth. The prior art has the following disadvantages in terms of the device used:

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

[0005] 2. The instruments and equipment are complex and require high experimental skills of operators;

[0006] 3. High performance liquid chromatography has low throughput and is not suitable for sample preparation.

[0007] Moreover, this prior art has a big problem in terms of experimental operation - the experimental operation is not uniform. That is, for each monoclonal antibody, it is necessary to prepare various buffers, use various affinity chromatography columns, set different detection conditions, and even arrange different groups of operators to avoid confusion. This undoubtedly leads to a significant increase in production burden for CDMO companies that need to develop and produce multiple monoclonal antibody drug products in parallel during the same period. 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 to separate and purify monoclonal antibodies in the supernatant of cell fermentation broth, and a universal method for determining the concentration of monoclonal antibodies using the simple device.

[0009] The technical solution of this patent application includes:

[0010] The technical solution of this patent application includes:

[0011] 1. A simple device for separating and purifying monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth, comprising:

[0012] syringe;

[0013] Affinity chromatography media, and

[0014] Syringe pump;

[0015] 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.

[0016] The syringe is for example Figure 1 As shown, a conventional medical syringe can be selected as needed.

[0017] Affinity chromatography media such as Figure 2 As shown, it is preferably a Protein A affinity chromatography medium, and more preferably an ATProtein A Diamond 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 ADiamond, the loading capacity is higher, and a smaller column volume can be selected during the process amplification to reduce production costs.

[0018] 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.

[0019] The syringe pump is for example Figure 3As shown, 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, the LSP10-1B 10-channel syringe pump produced by Baoding Lange Constant Flow Pump Co., Ltd.

[0020] The simple device of this patent application can be used to purify multiple samples at the same time, and each sample does not affect each other. These samples can be samples of the same monoclonal antibody or samples of different monoclonal antibodies, and their volumes can be the same or different. The simple device is easy to operate and does not require complex training for operators; the equipment and consumables cost is low and does not occupy expensive high-performance liquid chromatography. However, the inventors also found that there are two shortcomings in using this simple device - one is that there is a lack of universal detection conditions for different monoclonal antibodies; the other is that after long-term and multiple uses, the detection results of the same sample will be biased.

[0021] In response to the first shortcoming mentioned above, the inventors explored and studied the detection conditions for determining the concentration of monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth using the simple device, and developed detection conditions applicable to various IgG1 and IgG4 monoclonal antibodies. The detection results were basically consistent with those of high-performance liquid chromatography, thereby making the method for determining the concentration of monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth using the simple device universal, greatly reducing the production burden of CDMO companies.

[0022] Therefore, the technical solution of this patent application also includes:

[0023] 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:

[0024] Using the first equilibration buffer to equilibrate the affinity chromatography medium to obtain an equilibrated affinity chromatography medium;

[0025] Loading the monoclonal antibody cell fermentation broth supernatant onto the equilibrated affinity chromatography medium;

[0026] re-equilibrate the affinity chromatography medium using a second equilibration buffer;

[0027] The affinity chromatography medium is eluted with an elution buffer, and the eluate containing the target monoclonal antibody is collected;

[0028] 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:

[0029] 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.

[0030] The concentration of monoclonal antibody in the eluent = A280nm absorbance ÷ monoclonal antibody extinction coefficient × dilution multiple;

[0031] 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;

[0032] 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;

[0033] 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;

[0034] 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.

[0035] 3. The method according to claim 2, wherein:

[0036] The monoclonal antibody is an IgG1 monoclonal antibody and / or an IgG4 monoclonal antibody.

[0037] 4. The method according to claim 2, wherein:

[0038] The affinity chromatography medium is an AT Protein A Diamond Plus affinity chromatography column.

[0039] 5. The method according to claim 2, wherein:

[0040] The fermentation supernatant containing the monoclonal antibody is a eukaryotic cell fermentation supernatant.

[0041] 6. The method according to claim 2, wherein:

[0042] The volume of the first equilibration buffer is 3 to 6 times the volume of the affinity chromatography medium;

[0043] The volume of the second equilibration buffer is 3 to 6 times the volume of the affinity chromatography medium; and / or

[0044] The volume of the elution buffer is 3 to 6 times the volume of the affinity chromatography medium.

[0045] 7. The method according to item 2, 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:

[0046] a. Anti-human interleukin-23 monoclonal antibody QX004N;

[0047] b. Anti-human interleukin-4 receptor α monoclonal antibody QX005N;

[0048] c. Anti-human interferon α receptor 1 monoclonal antibody QX006N;

[0049] d. Human interleukin-33 monoclonal antibody QX007N;

[0050] e. Anti-human TSLP monoclonal antibody QX008N;

[0051] f. Anti-human interleukin 17A monoclonal antibody QX002N;

[0052] g. anti-human CD117 monoclonal antibody QX013N; and

[0053] j. Anti-human IL-12 / IL-23p40 subunit fully human monoclonal antibody QX001S.

[0054] 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:

[0055] The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1SYYVG;

[0056] The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2VIRIGGGTAYASWAKG;

[0057] The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3SSINGGDI;

[0058] The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4QASQDIRYDLS;

[0059] The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5AASKLAS;

[0060] The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6LGIFSFTDDDGLA.

[0061] 9. The method according to item 8, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0062] Wherein, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWV GVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSSINGGDIWGQGTLVTVSS;

[0063] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8AIQMTQSPSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKLLIYA ASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDDGLAFGGGTKVEIK.

[0064] 10. The method according to item 8, wherein the monoclonal antibody comprises a heavy chain and a light chain; wherein,

[0065] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 9;

[0066] The amino acid sequence of the light chain is shown in SEQ ID NO:10.

[0067] 11. The method according to claim 2, wherein:

[0068] The pH adjustment buffer was a 2 M Tris-HCl aqueous solution, pH 9.5.

[0069] 12. The method according to item 2, wherein the dilution factor is 5 to 20.

[0070] In response to the second shortcoming mentioned above, the inventors found in long-term performance tests that the reason for the above deviation is that the syringe pump is usually placed horizontally on the desktop. When the flow rate is set to a slow rate, it is easy for the droplets to fail to drip into the sink, resulting in the phenomenon of sample liquid hanging on the wall, flowing along the device to the syringe pump body, causing serious corrosion of the contact surface and damage to the electrical equipment, seriously affecting the performance and service life of the equipment. In addition, alkaline samples may also cause harm to users during cleaning.

[0071] In response to the above technical problems, the inventors designed a syringe pump bracket, which is used to place the syringe pump so that the syringe pump is placed at a certain height from the horizontal plane and at a certain angle to the horizontal plane, so that the sample liquid can flow out smoothly and effectively prevent the sample liquid from sticking to the wall.

[0072] That is, the technical solution of this patent application also includes:

[0073] 13. The simple device according to item 1, further comprising:

[0074] A syringe pump bracket for placing the syringe pump;

[0075] The injection pump bracket has a bottom surface that can be placed on a horizontal tabletop, and a top inclined surface that is located above the bottom surface and forms a certain inclination angle with the bottom surface, and the injection pump can be placed on the top inclined surface;

[0076] The lower edge of the top inclined surface is at a certain height from the bottom surface.

[0077] 14. The simple device according to item 13, wherein the inclination angle is 45° to 75°.

[0078] 15. The simple device according to item 13, wherein the lower edge of the top slope is 5 to 15 cm away from the bottom surface. Preferably, the lower edge of the top slope is 20 to 50 cm away from the bottom surface.

[0079] 16. The simple device according to item 13, wherein a blocking component is provided at the lower edge of the top slope to prevent the injection pump from sliding off the top slope.

[0080] 17. The simple device according to item 13, wherein the injection pump bracket is made of stainless steel.

[0081] Beneficial Effects

[0082] This application develops detection conditions that are applicable to various IgG1 and IgG4 monoclonal antibodies, and the detection results are basically consistent with the detection results of high-performance liquid chromatography, thereby making the method for determining the concentration of monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth using this simple device universal, greatly reducing the production burden of CDMO companies.

[0083] The present application aims to address the problem that after long-term and multiple uses, the test results of the same sample may deviate. The inventors have found in long-term performance tests that the reason for the above-mentioned deviation is that the syringe pump is usually placed horizontally on a desktop. When the flow rate is set to a slow rate, it is easy for droplets to fail to drip into the sink, resulting in the phenomenon of sample liquid hanging on the wall, flowing along the device to the syringe pump body, causing serious corrosion of the contact surface and damage to electrical equipment, seriously affecting the performance and service life of the equipment. In addition, alkaline samples may also cause harm to users during cleaning.

[0084] The inventor of the present application has designed a syringe pump bracket for placing the syringe pump so that the syringe pump is placed at a certain height from the horizontal plane and at a certain angle to the horizontal plane, thereby allowing the sample liquid to flow out smoothly and effectively preventing the sample liquid from adhering to the wall. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 is a picture of an example of a syringe that can be used in this application.

[0086] Figure 2 is a picture of an example of an affinity chromatography medium that can be used in the present application.

[0087] Figure 3 is a picture of an example of a syringe pump (multi-channel) that can be used in this application.

[0088] Figure 4 This is a picture of an example of a simple device of the present application (without the syringe pump holder).

[0089] Figure 5 This is a picture of another example of the simple device of the present application (with a syringe pump bracket).

[0090] Figure 6 This is the SDS-PAGE electrophoresis diagram of the anti-human interleukin 17A monoclonal antibody HZD-19 prepared in Example 2. DETAILED DESCRIPTION

[0091] 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.

[0092] 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.

[0093] 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.

[0094] In response to the lack of universal detection conditions for different monoclonal antibodies, the inventors of this application explored and studied the detection conditions for determining the concentration of monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth using the simple device, and developed detection conditions applicable to various IgG1 and IgG4 monoclonal antibodies. The detection results are basically consistent with those of high-performance liquid chromatography, thereby making the method for determining the concentration of monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth using the simple device universal, greatly reducing the production burden of CDMO companies.

[0095] The present application aims to address the problem that after long-term and multiple uses, the test results of the same sample may deviate. The inventors have found in long-term performance tests that the reason for the above-mentioned deviation is that the syringe pump is usually placed horizontally on a desktop. When the flow rate is set to a slow rate, it is easy for droplets to fail to drip into the sink, resulting in the phenomenon of sample liquid hanging on the wall, flowing along the device to the syringe pump body, causing serious corrosion of the contact surface and damage to electrical equipment, seriously affecting the performance and service life of the equipment. In addition, alkaline samples may also cause harm to users during cleaning.

[0096] The inventor has designed a syringe pump bracket for placing the syringe pump so that the syringe pump is placed at a certain height from the horizontal plane and at a certain angle to the horizontal plane, thereby allowing the sample liquid to flow out smoothly and effectively preventing the sample liquid from adhering to the wall.

[0097] like Figure 5As 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, a syringe pump; and a syringe pump bracket for placing the 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;

[0098] When the device is used for separation and purification of monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth, it comprises the following steps:

[0099] Using the first equilibration buffer to equilibrate the affinity chromatography medium to obtain an equilibrated affinity chromatography medium;

[0100] Loading the monoclonal antibody cell fermentation broth supernatant onto the equilibrated affinity chromatography medium;

[0101] re-equilibrate the affinity chromatography medium using a second equilibration buffer;

[0102] The affinity chromatography medium is eluted with an elution buffer, and the eluate containing the target monoclonal antibody is collected;

[0103] 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:

[0104] 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.

[0105] The concentration of monoclonal antibody in the eluent = A280nm absorbance ÷ monoclonal antibody extinction coefficient × dilution multiple;

[0106] 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;

[0107] 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;

[0108] 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;

[0109] 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.

[0110] 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.

[0111] The present application has no restrictions on the syringe, which can be purchased commercially or homemade, as long as it meets its purpose.

[0112] In one embodiment of the present application, a syringe such as Figure 1 shown.

[0113] In one embodiment of the present application, the syringe is purchased from a commercial source.

[0114] 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.

[0115] In one embodiment of the present application, an affinity chromatography medium such as Figure 2 shown.

[0116] In one embodiment of the present application, the affinity chromatography medium is purchased from a commercial source.

[0117] The present application has no restrictions on the syringe pump, which can be purchased commercially or homemade, as long as it meets its purpose.

[0118] In one embodiment of the present application, a syringe pump such as Figure 3 As shown. The injection pump is for example Figure 3 As shown, 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, the LSP10-1B 10-channel syringe pump produced by Baoding Lange Constant Flow Pump Co., Ltd.

[0119] In one embodiment of the present application, the syringe pump is commercially available.

[0120] In some embodiments of the present application, Figure 3 As shown, the syringe pump has a plurality of channels for placing syringes.

[0121] like Figure 5 As shown, the injection pump bracket includes a top slope and a bottom surface from top to bottom, and the top slope includes a top upper part (upper edge) and a top lower part (lower edge); the bottom surface is parallel to the horizontal plane, and the top slope is inclined at an angle to the bottom surface; the top slope is in contact with the injection pump.

[0122] In some embodiments of the present application, the inclination angle is 45° to 75°; for example, the inclination angle may be 45°, 50°, 55°, 60°, 65°, 70°, 75° or any range therebetween.

[0123] In some embodiments of the present application, the straight-line distance between the top portion and the bottom surface is 20 to 50 cm, for example, 25 cm, 30 cm, 35 cm, 40 cm, or 45 cm.

[0124] In some embodiments of the present application, the straight-line distance between the top lower portion and the bottom surface is 5 to 15 cm; for example, the straight-line distance between the top lower portion and the bottom surface can be 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, 15 cm or any range therebetween.

[0125] In some embodiments of the present application, the top lower portion is provided with a blocking component for preventing the injection pump from sliding off the top lower portion. In some embodiments of the present application, the injection pump bracket is made of stainless steel.

[0126] This application provides a method of using Figure 5The method for determining the concentration of monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth is carried out using the simple device shown in the figure, wherein the method comprises the following steps: using a first equilibration buffer to equilibrate an affinity chromatography medium to obtain an equilibrated affinity chromatography medium; loading the supernatant of the monoclonal antibody cell fermentation broth 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 to collect the eluate containing the target monoclonal antibody; determining the concentration of the monoclonal antibody in the collected eluate, and calculating 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.

[0127] In this application,

[0128] Affinity chromatography media such as Figure 2 As shown, it is preferably a Protein A affinity chromatography medium, and more preferably an ATProtein A Diamond Plus affinity chromatography column. The AT Protein ADiamond Plus affinity chromatography column can be purchased from Boglong Boglong (Shanghai) Biotechnology Co., Ltd. It is an affinity medium synthesized by epoxidation chemistry with 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 ADiamond, the loading capacity is higher, and a smaller column volume can be selected during the process amplification to reduce production costs.

[0129] 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.

[0130] In some embodiments of the present application, the affinity chromatography medium may be an AT Protein ADiamond Plus affinity chromatography column.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] In some embodiments of the present application, the washing buffer is an aqueous solution of 0.1 M NaOH and 1.0 M NaCl.

[0142] 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.

[0143] 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.

[0144] In some embodiments of the present application, the preservation solution is a 20% ethanol aqueous solution.

[0145] 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.

[0146] 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.

[0147] In some embodiments of the present application, the monoclonal antibody is an IgG1 monoclonal antibody and / or an IgG4 monoclonal antibody.

[0148] In some embodiments of the present application, the fermentation supernatant containing the monoclonal antibody is a eukaryotic cell fermentation supernatant.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] In some embodiments of the present patent 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 adjusting buffer is added to the eluate so that the pH of the eluate is 6.5-8.0, the mixture is mixed, the dilution multiple is adjusted for dilution, 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 balancing buffer, and then stored with the preservation solution.

[0153] The monoclonal antibodies applicable to the present application include 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:

[0154] a. Anti-human interleukin-23 monoclonal antibody;

[0155] b. Anti-human interleukin-4 receptor α monoclonal antibody;

[0156] c. Anti-human interferon α receptor 1 monoclonal antibody;

[0157] d. Anti-human interleukin-33 monoclonal antibody;

[0158] e. anti-human TSLP monoclonal antibody; and

[0159] f. Anti-human interleukin 17A monoclonal antibody;

[0160] g. Anti-human CD117 monoclonal antibody;

[0161] j. fully human monoclonal antibody against human IL-12 / IL-23p40 subunit; and

[0162] i. anti-human interleukin-17A monoclonal antibody as described below;

[0163] Specifically, the anti-human interleukin-23 monoclonal antibody QX004N described in the patent document CN111718414B, 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, and the anti-human interleukin-4 receptor α monoclonal antibody QX008N described in the 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.

[0164] In some embodiments of the present application, the monoclonal antibody may be 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:

[0165] 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] Preferably, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0172] Wherein, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWVGVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSSINGGDIWGQGTLVTVSS;

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

[0174] Preferably, the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 9, SEQ ID NO: 9

[0175] EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWVGVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSSINGGDIWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0176] The amino acid sequence of the light chain is shown in SEQ ID NO: 10.

[0177] AIQMTQSPSSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDDGLAFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0178] Example 1 Simple method for determining the production of existing monoclonal antibodies

[0179] 1.1 Simple method for determining the yield of target protein of IgG1 monoclonal antibody and IgG4 monoclonal antibody

[0180] 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;

[0181] The separation and purification of monoclonal antibodies in various fermentation broth supernatants was carried out using Figure 5 The simple device shown in the figure is used. The simple device comprises: Figure 1 The syringe shown (10mL disposable sterile syringe, Shandong Ande Medical Supplies Co., Ltd.), Figure 2 Affinity chromatography medium (AT Protein ADiamond Plus affinity chromatography column) shown Figure 3The syringe pump (LSP10-1B type 10-channel syringe pump) shown and the syringe pump bracket for placing the syringe pump, wherein the syringe pump has multiple channels; the syringe pump bracket includes a top slope and a bottom surface from top to bottom, the top slope includes an upper top portion (upper edge) and a lower top portion (lower edge), the bottom surface is parallel to the horizontal plane, the top slope is inclined at an angle to the bottom surface, the inclination angle is 60°, the top slope is in contact with the syringe pump, the straight-line distance between the top upper portion and the bottom surface is 28 cm, the straight-line distance between the top lower portion and the bottom surface is 7 cm, and a blocking component is provided at the top lower portion to prevent the syringe pump from sliding off the top lower portion. The syringe is mounted 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 liquid supernatant is loaded into 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.

[0182] Using AT Protein ADiamond Plus affinity chromatography column (column volume 1 mL), each affinity chromatography column is washed with 5 column volumes of washing buffer at a flow rate of 1 mL / min, each affinity chromatography is then balanced with 5 column volumes of the first equilibration buffer at a flow rate of 1 mL / min, samples containing IgG1 monoclonal antibody or IgG4 monoclonal antibody are loaded onto two affinity chromatography columns respectively at a flow rate of 0.2 mL / min, each affinity chromatography column is re-equilibrated with 5 column volumes of the second equilibration buffer, wherein the first column volume adopts a flow rate of 0.2 mL / min, and then 4 column volumes adopt a flow rate of 1 mL / min, each affinity chromatography column is eluted with 4 column volumes of elution buffer at a flow rate of 1 mL / min, and 15 mL centrifuge tubes are used to collect the target IgG1 monoclonal antibody elution buffer or IgG4 monoclonal antibody elution buffer, and pH adjustment buffer is 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.

[0183] in:

[0184] The first equilibrium buffer: 20 mM Na2HPO4-NaH2PO4 and 150 mM NaCl, pH 6.8, conductivity 18 mS / cm;

[0185] Second equilibrium buffer: 10 mM Na2HPO4-NaH2PO4, pH 7.2, conductivity 1.3 mS / cm;

[0186] Elution buffer: 15 mM citric acid and 7 mM Na2HPO4, pH 3.1, conductivity 1.3 mS / cm;

[0187] Sample pH adjustment buffer: 2 M Tris-HCl, pH 9.5;

[0188] Wash buffer: 0.1 M NaOH and 1.0 M NaCl;

[0189] Preservative solution: 20% ethanol.

[0190] 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.

[0191] 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:

[0192] Target protein yield = protein concentration of target protein elution buffer × volume of target protein elution buffer ÷ volume of fermentation broth supernatant injected.

[0193] It should also be noted that the above-mentioned simple purification device was used for one year and carried out at least 1,000 sample purifications. There was no obvious corrosion on the contact surface, no damage to the electrical equipment, and the equipment performance remained good.

[0194] 1.2 Liquid phase determination of target protein yield of IgG1 monoclonal antibody or IgG4 monoclonal antibody High performance liquid chromatography: brand thermo, model UltiMate3000;

[0195] Chromatographic column: Sepax, ProAqa Excel, specification 20μm, 2.1×30mm,

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] (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.

[0201] (3) Load the standard sample with a sample volume of 5 μl and obtain the linear equation (R 2 ≥0.99);

[0202] (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.

[0203] (4) Elution: Perform the experiment according to the following elution gradient

[0204]

[0205]

[0206] (5) Establish a standard curve and calculate the sample content.

[0207] 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 1.

[0208] 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.

[0209] Table 1 Results of simple method and liquid phase method for different monoclonal antibodies

[0210]

[0211]

[0212] 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%.

[0213] The measurement results in Table 1 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.

[0214] Comparative Example 1

[0215] 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.

[0216] 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 result determined by the liquid phase method.

[0217] Comparative Example 2

[0218] 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 patent application, the conductivity is adjusted by adding 4 M NaCl or purified water.

[0219] The results showed that, at least for the monoclonal antibody QX005N, the target protein yield determined by the simplified method was 5.23 mg / mL, with a deviation of -4% from the result determined by the liquid phase method.

[0220] Comparative Example 3

[0221] 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.

[0222] 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 result determined by the liquid phase method.

[0223] Comparative Example 4

[0224] 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.

[0225] The results showed that, at least for the monoclonal antibody QX006N, the target protein yield determined by the simplified method was 5.58 mg / mL, with a deviation of -3% from the result determined by the liquid phase method.

[0226] Comparative Example 5

[0227] 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.

[0228] 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%.

[0229] Comparative Example 6

[0230] 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.

[0231] The results showed that, at least for the monoclonal antibody QX008N, the target protein yield determined by the simplified method was 6.01 mg / mL, which deviated from the result determined by the liquid phase method by 6%.

[0232] Comparative Example 7

[0233] 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.

[0234] 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.

[0235] Comparative Example 8

[0236] 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.

[0237] The results showed that, at least for the monoclonal antibody QX006N, the target protein yield determined by the simplified method was 5.27 mg / mL, with a deviation of -9% from the result determined by the liquid phase method.

[0238] Example 2 A simple method for determining the yield of a novel anti-human interleukin 17A monoclonal antibody

[0239] 2.1 Preparation of new anti-human interleukin-17A monoclonal antibody

[0240] 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 5 .

[0241] 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:

[0242] The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1SYYVG;

[0243] The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2VIRIGGGTAYASWAKG;

[0244] The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3SSINGGDI;

[0245] The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4QASQDIRYDLS;

[0246] The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5AASKLAS;

[0247] The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6LGIFSFTDDDGLA.

[0248] HZD19 contains a heavy chain variable region and a light chain variable region.

[0249] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7

[0250] EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGL EWVGVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY CSRSSINGGDIWGQGTLVTVSS;

[0251] The amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8

[0252] AIQMTQSPSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKL LIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDD GLAFGGGTKVEIK.

[0253] 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.

[0254] SEQ ID NO: 9

[0255] EVQLVESGGGLVQPGGSLRLSCAASGFSLNSYYVGWVRQAPGKGLEWVGVIRIGGGTAYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRSSINGGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0256] SEQ ID NO: 10

[0257] AIQMTQSPSSLSASVGDRVTITCQASQDIRYDLSWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGIFSFTDDDGLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0258] 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 in 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 measurement results showed that the affinity of HZD-19 for binding to human IL-17A was in the order of nM to pM, and KD was 8.31×10 -10 M.

[0259] 2.2 Determination of anti-human interleukin-17A monoclonal antibody (HZD-19) by simple method and liquid phase method

[0260] 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 simple device for separating and purifying monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth, comprising: syringe; Affinity chromatography media, Syringe pump; and A syringe pump bracket for placing the 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 broth supernatant is injected 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 being separated and purified by the affinity chromatography medium; 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 being separated and purified by the affinity chromatography medium.

2. The simple device according to claim 1, wherein: The affinity chromatography medium is an AT Protein ADiamondPlus affinity chromatography column.

3. The simple device according to claim 1, wherein: The syringe pump has a plurality of channels for placing syringes.

4. The simple device according to claim 1, wherein: The injection pump bracket has a bottom surface that can be placed on a horizontal tabletop, and a top inclined surface that is located above the bottom surface and forms a certain inclination angle with the bottom surface, and the injection pump can be placed on the top inclined surface; The lower edge of the top inclined surface is at a certain height from the bottom surface.

5. The simple device according to claim 4, wherein: The inclination angle is 45° to 75°.

6. The simple device according to claim 4, wherein: The lower edge of the top slope is 5 to 15 cm away from the bottom surface; preferably, the upper edge of the top slope is 20 to 50 cm away from the bottom surface.

7. The simple device according to item 4, wherein: A blocking component is provided at the lower edge of the top inclined surface to prevent the injection pump from sliding off the top inclined surface.

8. A method for determining the concentration of monoclonal antibodies in the supernatant of monoclonal antibody cell fermentation broth, which is performed using the simple device according to claim 1, comprising 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 monoclonal antibody is one selected from the following a to k, 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; j. QX001S, a fully human monoclonal antibody against human IL-12 / IL-23p40 subunit; and k. An anti-human interleukin 17A monoclonal antibody comprising a heavy chain with an amino acid sequence as shown in SEQ ID NO: 9 and a light chain with an amino acid sequence as shown in SEQ ID NO:

10.

9. The method according to claim 8, wherein: The fermentation supernatant containing the monoclonal antibody is a eukaryotic cell fermentation supernatant.

10. The method according to claim 8, 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.

Images