Method for quality detection of anti-interleukin 17A antibodies

Abstract

The present application relates to a method for detecting the quality of anti-interleukin 17A antibodies. Specifically, the present application provides a method for detecting anti-interleukin 17A (IL-17A) antibodies based on cation exchange high performance liquid chromatography (CEX-HPLC), and a method for further detecting anti-interleukin 17A (IL-17A) antibodies based on radioactive markers.

Description

Technical Field

[0001] The present application belongs to the field of biochemistry and analytical chemistry, and mainly relates to a method for detecting the quality of anti-interleukin 17A (IL-17A) antibodies based on cation exchange high performance liquid chromatography (CEX-HPLC) and radioactive markers. Background Art

[0002] The interleukin 17 (IL-17) family currently includes six cytokines: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. The members of the IL-17 family have no sequence similarity to any other known cytokines, and the sequence similarity between family members is also relatively low (Gaffen SL, Nat. Rev. Immunol. 2009 Aug; 9(8): 556-67). The function of IL-17 family members is mainly to participate in the regulation of immune response. IL-17A (hereinafter referred to as IL-17) is a 20-30kD homodimeric glycoprotein, which is mainly produced by activated CD4 + T cells produce and act as proinflammatory cytokines. IL-17 is secreted by activated T cells at inflammatory sites, but not in the systemic circulation. IL-17 has a variety of biological properties, including upregulating adhesion molecules and inducing a large number of inflammatory cytokines and chemokines in a variety of cell types including synovial cells, chondrocytes, fibroblasts, endothelial cells, epithelial cells, keratinocytes and macrophages. Similarly, IL-17 induces neutrophils to gather at inflammatory sites by inducing the release of chemokines, stimulates the production of prostaglandins and metalloproteinases, and inhibits the synthesis of proteoglycans. In addition, IL-17 plays an important role in the maturation of hematopoietic progenitor cells. IL-17 has signal transduction functions in different organs and tissues including lungs, articular cartilage, bones, brain, hematopoietic cells, kidneys, skin and intestines. Therefore, the immune response mediated by IL-17A / Th17 is systemic, and the inflammatory response caused thereby is mainly manifested as neutrophil infiltration.

[0003] Due to their unique biological properties and functions, antibodies are increasingly being used in disease treatment. Currently, there are a variety of antibodies targeting IL-17A, which are expected to prevent or treat diseases caused by IL-17A by binding to IL-17A.

[0004] As a drug, the quality control and detection of antibodies are crucial. Therefore, those skilled in the art need to develop new and effective detection methods for anti-IL-17A antibodies to ensure the safe application of antibodies. Summary of the invention

[0005] In a first aspect, the present application provides a method for detecting the quality of an anti-interleukin 17A (IL-17A) antibody based on cation exchange high performance liquid chromatography (CEX-HPLC), comprising:

[0006] Adding the sample containing the anti-IL-17A antibody to a CEX-HPLC column, wherein the column temperature of the cation exchange high performance liquid chromatography column is 30° C. or 40° C.;

[0007] The quality of the anti-IL-17A antibody is judged according to the separation degree between the main peak of the anti-IL-17A antibody and the adjacent acidic peaks and basic peaks in the chromatogram, wherein when the separation degree between the main peak of the anti-IL-17A antibody and the adjacent acidic peaks and basic peaks is greater than 1.2 or 1.5, the quality of the anti-IL-17A antibody meets the requirements;

[0008] in:

[0009] The buffer salt used in the mobile phase of the CEX-HPLC is MES, glycine, disodium hydrogen phosphate or sodium dihydrogen phosphate;

[0010] The pH value of the mobile phase of the CEX-HPLC is lower than the isoelectric point (PI) value of the anti-IL-17A antibody; and

[0011] The mobile phase of the CEX-HPLC includes mobile phase A and mobile phase B, and the elution gradients of the mobile phase A and the mobile phase B are as follows:

[0012]

[0013] In some embodiments of the first aspect, the pH of the mobile phase is about 6.4.

[0014] In some embodiments of the first aspect, the pH value of the mobile phase of the CEX-HPLC is adjusted by directly weighing sodium hydroxide.

[0015] In some embodiments of the first aspect, the mobile phase of the CEX-HPLC comprises mobile phase A and mobile phase B; in mobile phase A, the amount of sodium hydroxide added is 0.5225-0.5775 g / L; and / or in mobile phase B, the amount of sodium hydroxide added is 0.573 g / L; and / or in mobile phase B, the amount of sodium chloride added is 26.298-32.142 g / L.

[0016] In some embodiments of the first aspect, the column temperature of the chromatographic column of the CEX-HPLC is about 30°C or about 40°C.

[0017] In some embodiments of the first aspect, the sample comprising the anti-IL-17A antibody has a concentration of 4-15 mg / mL.

[0018] In some embodiments of the first aspect, the sample loading volume comprising the anti-IL-17A antibody is 10 μL.

[0019] According to the above method, the quality of the anti-IL-17A antibody is judged based on the separation degree between the main peak of the anti-IL-17A antibody and the adjacent acidic peaks and basic peaks in the chromatogram, wherein when the separation degree between the main peak of the anti-IL-17A antibody and the adjacent basic peaks is greater than 1.17 or 1.45, the quality of the anti-IL-17A antibody meets the requirements.

[0020] In a second aspect, the present application provides a method for detecting anti-interleukin 17A (IL-17A) antibodies based on radioactive markers, which can be used in combination with the method for detecting anti-interleukin 17A (IL-17A) antibodies based on cation exchange high performance liquid chromatography (CEX-HPLC) described in the first aspect, wherein the method for detecting anti-interleukin 17A (IL-17A) antibodies based on radioactive markers comprises:

[0021] Labeling the anti-IL-17A antibody in the sample with a radioactive label;

[0022] The labeled samples are separated by gel column;

[0023] Determine the purity of the anti-IL-17A antibody in the sample by measuring the radioactivity intensity of the corresponding peak;

[0024] Wherein, the radioactive marker is 125 I. 124 I. 131 I. 89 Zr or 68 Ga.

[0025] In some embodiments of the second aspect, the tyrosine, histidine and / or arginine residues in the anti-IL-17A antibody in the sample are labeled with a radioactive label.

[0026] In some embodiments of the second aspect, the radioactivity intensity is gamma radioactivity intensity.

[0027] In some embodiments of the second aspect, a method for detecting the quality of an anti-interleukin 17A (IL-17A) antibody based on a radioactive marker is performed using a kit for detecting the quality of an anti-interleukin 17A (IL-17A) antibody, wherein the kit for detecting the quality of an anti-interleukin 17A (IL-17A) antibody comprises:

[0028] Radioactive label and instructions for use; and / or

[0029] Amphoteric electrolytes, sodium hydroxide, sodium chloride, and instructions for use;

[0030] Wherein, the radioactive marker is 125 I. 124 I. 131 I. 89 Zr or 68 Ga; and / or

[0031] The ampholytes are MES, glycine, disodium hydrogen phosphate or sodium dihydrogen phosphate.

[0032] By controlling and checking the quality of the anti-IL-17A antibody molecules by the method described in the first aspect and / or the second aspect, the quality of the antibodies can be ensured so as to be better applied in the treatment of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of the comparison of CEX-HPLC chromatograms of GR1501 samples under different mobile phase pH conditions are shown;

[0034] Figure 2 The results of the comparison of CEX-HPLC chromatograms of GR1501 samples under different mobile phase gradient conditions are shown;

[0035] Figure 3 The results of the comparison of CEX-HPLC chromatograms of GR1501 samples under different column temperature conditions are shown;

[0036] Figure 4 The linear relationship between the acidic peak area and the injection amount in the range of 40-150 μg is shown;

[0037] Figure 5 The linear relationship between the main peak area and the injection amount in the range of 40-150 μg is shown;

[0038] Figure 6 The linear relationship between the basic peak area and the injection amount in the range of 40-150 μg is shown;

[0039] Figure 7 Shows the adoption 125 I is the linear fitting diagram of the protein concentration of the eluate after labeling GR1501;

[0040] Figure 8 Shows the adoption 125 SHPLC purity spectrum of GR1501 before and after I labeling. 125 I represents the result of using UV detector at 280nm before labeling GR1501, B represents the result of using 125The results of the FSA detector after I labeled GR1501;

[0041] Fig. 9 Shows the adoption 125 Comparison of immunoreactivity before and after I-labeling of GR1501. DETAILED DESCRIPTION

[0042] The inventors of the present application have established a quality detection method for anti-IL-17A antibody molecules (such as GR1501) based on CEX-HPLC by optimizing the parameters of CEX-HPLC according to the characteristics of anti-IL-17A antibody molecules (such as GR1501). The inventors of the present application have also used radioactive markers (such as 125 I) labeling the anti-IL-17A antibody molecule (e.g., GR1501), and separating it using a gel column (e.g., a dextran gel column), and determining the quality (e.g., purity) of the anti-IL-17A antibody molecule (e.g., GR1501) in the sample by measuring the radioactivity intensity of the corresponding peak. The quality of the anti-IL-17A antibody molecule (e.g., GR1501) is controlled and checked by the above method to ensure the quality of the antibody and better apply it to the treatment of diseases.

[0043] The practice of this application employs, unless otherwise indicated, conventional biochemistry and analytical chemistry techniques within the skill of the art.

[0044] To facilitate understanding of this application, certain terms used herein are first defined.

[0045] As used herein, "ion exchange high performance liquid chromatography (IEX-HPLC)" is based on the reversible exchange between ions on the stationary phase of ion exchange chromatography and solute ions with the same charge in the mobile phase, and its separation mechanism is mainly ion exchange.

[0046] As used herein, "cation exchange high performance liquid chromatography (CEX-HPLC)" is a method of using a cation exchange chromatography column to make the mobile phase pH lower than the isoelectric point of the protein to be separated so that each isoform has a different positive charge. In some embodiments of the present application, CEX-HPLC uses a gradient elution, and before the start of the gradient elution, the mobile phase is balanced with a low-salt mobile phase to adsorb each isoform with a positive charge onto the stationary phase of the cation exchange chromatography column. During elution, the salt concentration is gradually increased, and the Na + It will compete with the negative ion groups of the stationary phase, thereby desorbing each heterogeneous substance from the stationary phase. Since the pI values ​​of each heterogeneous substance are different, the number of positive charges carried in the same mobile phase is different, and the strength of interaction with the stationary phase of the chromatographic column is also different, then the salt concentration required for desorption is also different, so the charged heterogeneous substances can be separated.

[0047] As used herein, "radioactive marker" refers to a compound that replaces one or several atoms of a compound molecule with a radioactive nuclide so that it can be identified and used as a tracer. It has the same chemical and biological properties as the corresponding unlabeled compound, except that it is radioactive and can be tracked using radioactive detection technology. Common labeling methods for radioactive markers include chemical synthesis, isotope exchange, and biochemical methods. Usually, the appropriate radionuclide is selected based on the composition, structure, and application requirements of the required labeled substance, and then the labeling route is designed based on the available raw materials containing the required radionuclide and the application requirements. The labeling routes used are different for different raw materials with different physicochemical properties. The method of "marking" a substance with one or two easily detectable radionuclides is generally called isotope labeling if the radionuclide used as the "marker" is an isotope of an inherent element in the radioactive marker, otherwise it is called non-isotope labeling. In addition to having a specific radioactivity and sufficient specific activity and radiochemical purity, the chemical and biological properties of the labeled substance are the same as those of the unlabeled corresponding substance, or are very similar in practical terms.

[0048] As used herein, the "main peak of a CEX-HPLC chromatogram" refers to the chromatographic peak produced by the main component in the effluent of the chromatographic column. The main peak is not a complete antibody molecule without modification or degradation, but is actually composed of several major modifications. For the charge isomer analysis of antibodies, common components of the main peak include some specific modification forms. These modifications may cause changes in the surface charge of the antibody, thereby generating charge heterogeneity.

[0049] As used herein, "acidic peaks in CEX-HPLC chromatograms" refer to chromatographic peaks corresponding to substances with acidic properties or more negative charges in the chromatogram. These substances are usually formed after certain modifications or changes of antibodies or proteins. For example, during the glycosylation process, the negatively charged sialic acid at the end of the sugar chain will cause the charge to change. The antibody component with sialic acid will be eluted before the main peak in the cation exchange chromatography, forming an acidic variant, which appears as an acidic peak; asparagine loses its amino group to form succinimide, and then forms an acidic variant. This process is also irreversible and will also produce acidic peaks. The charge variants of antibodies have a key influence on the functional effects of proteins and the nature, location and degree of their post-translational modifications. The analysis and study of these acidic peaks will help to gain a deeper understanding of the properties, structure and changes that occur during the production, storage, transportation and other processes of antibodies or proteins, and are of great significance for the development, quality control and stability studies of antibody drugs.

[0050] As used herein, "basic peaks in a CEX-HPLC chromatogram" refer to chromatographic peaks corresponding to substances with alkaline properties or more positive charges. These substances may be formed after certain modifications or changes to antibodies or proteins. For example, alkaline peaks may appear when certain amino acid residues of a protein are deprotonated, resulting in an increase in the overall positive charge. Analysis of alkaline peaks helps to gain a deeper understanding of the properties, structure, and possible changes of these components with alkaline characteristics in the sample, which is of great significance for the development, quality control, and stability studies of antibody drugs.

[0051] As used in this article, "separation" is also called resolution, which is a measure of the degree of separation between two adjacent peaks in a chromatogram. The degree of separation between the two peaks is restricted by the distance between the two peak tips and the peak widths of the two peaks. If the peak width remains unchanged, increasing the distance between the peaks will increase the degree of separation.

[0052] As used herein, "about" defines a range including plus or minus 10% of a reference value. For example, "about 40" defines a range of 40+ / -4, i.e., 36-44.

[0053] As used herein, "instructions for use" is a written material that uses description as the main expression method to summarize the knowledge of the use, performance, characteristics, use and storage methods of the product. In some embodiments of the present application, the user can use the radioactive marker in the kit for quality detection of anti-interleukin 17A (IL-17A) antibodies according to the instructions for use to detect the quality of anti-interleukin 17A (IL-17A) antibodies. In some embodiments of the present application, the user can use the ampholytes, sodium hydroxide and sodium chloride in the kit for quality detection of anti-interleukin 17A (IL-17A) antibodies based on cation exchange high performance liquid chromatography (CEX-HPLC) according to the instructions for use to detect the quality of anti-interleukin 17A (IL-17A) antibodies. In some embodiments of the present application, the user can use the radioactive markers, ampholytes, sodium hydroxide and sodium chloride in the kit for quality detection of anti-interleukin 17A (IL-17A) antibodies according to the instructions for use to detect the quality of anti-interleukin 17A (IL-17A) antibodies.

[0054] In some embodiments of the present application, the anti-IL-17A antibody is a monoclonal antibody, which is the antibody 9GC5 in Chinese patent application No. CN201510097117.0, referred to as GR1501 in the present application, and the above patent application is incorporated herein by reference in its entirety.

[0055] In the present application, the amino acid sequence of HCDR1 of the anti-IL-17A antibody GR1501 is GMSMSDY (SEQ ID NO: 1), the amino acid sequence of HCDR2 is NQDGDE (SEQ ID NO: 2), the amino acid sequence of HCDR3 is DYYDLISDYYIHYWYFDL (SEQ ID NO: 3), the amino acid sequence of LCDR1 is RASQNVHNRLT (SEQ ID NO: 4), the amino acid sequence of LCDR2 is GASNLES (SEQ ID NO: 5), and the amino acid sequence of LCDR3 is QQYNGSPTT (SEQID NO: 6).

[0056] In the present application, the amino acid sequence of the heavy chain of the anti-IL-17A antibody GR1501 is as follows:

[0057] EVQLVESGGGLVQPGGSLRLSCAASGMSMSDYWMNWVRQAPGKGLEWVAAINQDGDEKYYVGSVKGRFTISRDNAKNSLYLQMNSLRVEDTAVYYCVRDYYDLISDYYIHYWY FDLWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:7);

[0058] The amino acid sequence of the light chain of the anti-IL-17A antibody GR1501 is shown below:

[0059] DIQMTQSPSSSLSASVGDRVTITCRASQNVHNRLTWYQQKPGKAPKLLIYGASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNGSPTTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:8).

[0060] In a first aspect, the present application provides a method for detecting anti-interleukin 17A (IL-17A) antibodies based on cation exchange high performance liquid chromatography (CEX-HPLC), comprising:

[0061] Adding the sample containing the anti-IL-17A antibody to a CEX-HPLC column, wherein the column temperature of the cation exchange high performance liquid chromatography column is 30° C. or 40° C.;

[0062] in:

[0063] The buffer salt used in the mobile phase of the CEX-HPLC is MES, glycine, disodium hydrogen phosphate or sodium dihydrogen phosphate;

[0064] The pH value of the mobile phase of the CEX-HPLC is lower than the isoelectric point (PI) value of the anti-IL-17A antibody; and

[0065] The mobile phase of the CEX-HPLC includes mobile phase A and mobile phase B, and the elution gradients of the mobile phase A and the mobile phase B are as follows:

[0066]

[0067] In some embodiments of the first aspect, the buffer salt used in the mobile phase of the CEX-HPLC is MES.

[0068] In some embodiments of the first aspect, the buffer salt used in the mobile phase of the CEX-HPLC is an ampholyte. In some embodiments, the ampholyte is MES, glycine, disodium hydrogen phosphate or sodium dihydrogen phosphate. In some embodiments, the ampholyte is MES.

[0069] In some embodiments of the first aspect, the pH value of the mobile phase of the CEX-HPLC is lower than the isoelectric point (PI) value of the anti-IL-17A antibody. In some embodiments, the pH value of the mobile phase is lower than the PI value of the anti-IL-17A antibody, so that the charge heterogeneous bodies carry positive charges, and each heterogeneous body carries a different amount of charge, so that it can be separated in CEX-HPLC.

[0070] In some embodiments of the first aspect, the pH value of the mobile phase is about 6.30-about 6.60, for example, it can be about 6.30, about 6.31, about 6.32, about 6.33, about 6.34, about 6.35, about 6.36, about 6.37, about 6.38, about 6.39, about 6.40, about 6.41, about 6.42, about 6.43, about 6.44, about 6.45, about 6.46, about 6.47, about 6.48, about 6.49, about 6.50, about 6.51, about 6.52, about 6.53, about 6.54, about 6.55, about 6.56, about 6.57, about 6.58, about 6.59, about 6.60, or a value between any two of the above values.

[0071] In some embodiments of the first aspect, the pH value of the mobile phase is about 6.40, such as in the range of 6.40+ / -0.64, i.e., 5.76-7.04. In some specific embodiments, the pH value of the mobile phase is 6.40. In some embodiments, when the pH value of the mobile phase is 6.40, the CEX-HPLC separation of the anti-IL-17A antibody can achieve the best separation effect between the main peak and the acidic peak and the basic peak.

[0072] In some embodiments of the first aspect, the pH value of the mobile phase of the CEX-HPLC is adjusted by directly weighing sodium hydroxide. In some embodiments, the CEX-HPLC salt gradient method is very sensitive to the pH of the mobile phase. However, the method of using sodium hydroxide solution to adjust the pH when preparing the mobile phase will be affected by various aspects such as pH meter calibration and ambient temperature, thereby making the pH of the mobile phase inaccurate, thereby affecting the separation effect. The precision of the electronic balance is high and it is less affected by the outside world, so the pH value can be adjusted more accurately by directly weighing a certain amount of sodium hydroxide to prepare a mobile phase of a specific pH.

[0073] In some embodiments of the first aspect, the mobile phase of the CEX-HPLC comprises mobile phase A and mobile phase B.

[0074] In some embodiments of the first aspect, the amount of sodium hydroxide added to the mobile phase A is 0.550 g / L, which can ensure that the pH value of the mobile phase A is about 6.40 (preferably 6.40).

[0075] In some embodiments of the first aspect, the amount of sodium hydroxide added to mobile phase A is 0.550 g / L, and an increase or decrease of 5% in the amount of sodium hydroxide added can ensure that the pH value of mobile phase A is about 6.40 (preferably 6.40), without affecting the pH value of mobile phase A or the quality detection of anti-IL-17A.

[0076] In some embodiments of the first aspect, in mobile phase A, the amount of sodium hydroxide added is 0.5225-0.5775 g / L, for example 0.5225, 0.53625, 0.550, 0.56375, 0.5775 g / L, or a value between any two of the above values.

[0077] In some specific embodiments of the first aspect, in mobile phase A, the amount of sodium hydroxide added is 0.5225, 0.550 or 0.5775 g / L.

[0078] In some embodiments of the first aspect, in mobile phase B, the amount of sodium hydroxide added is 0.573 g / L. In some embodiments, the amount of sodium hydroxide added to mobile phase B is 0.573 g / L, which can ensure that the pH value of mobile phase B is about 6.40 (preferably 6.40).

[0079] In some embodiments of the first aspect, the amount of sodium chloride added to 300 mL of mobile phase B is 8.766 g, which is converted to an amount of sodium chloride added to 1 L of mobile phase B of 29.22 g / L.

[0080] In some embodiments of the first aspect, the amount of sodium chloride added to mobile phase B is 29.22 g / L.

[0081] In some embodiments of the first aspect, the amount of sodium chloride added to mobile phase B is 29.22 g / L, wherein an increase or decrease of 10% in the amount of sodium chloride added will not affect the pH value of mobile phase B or the quality detection of anti-IL-17A.

[0082] In some embodiments of the first aspect, in mobile phase B, the amount of sodium chloride added is 26.298-32.142 g / L, for example 26.298 g / L, 27.759, 29.22, 30.681, 32.142 g / L, or a value between any two of the above values.

[0083] In some specific embodiments of the first aspect, in mobile phase B, the amount of sodium chloride added is 26.298, 29.22 or 32.142 g / L.

[0084] In some embodiments of the first aspect, the column temperature of the chromatographic column of the CEX-HPLC is about 25-45°C, for example, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45°C, or a value between any two of the above values.

[0085] In some embodiments of the first aspect, the column temperature of the chromatographic column of the CEX-HPLC is about 30°C or about 40°C.

[0086] In some embodiments of the first aspect, the column temperature of the chromatographic column of the CEX-HPLC is about 30°C, such as 30+ / -3°C, ie 27-33°C.

[0087] In some embodiments of the first aspect, the column temperature of the chromatographic column of the CEX-HPLC is about 40°C, such as 40+ / -4°C, ie 36-44°C.

[0088] In some specific embodiments of the first aspect, the column temperature of the CEX-HPLC chromatographic column is 40°C.

[0089] In some embodiments of the first aspect, the sample comprising the anti-IL-17A antibody has a concentration of 4-15 mg / mL, such as 4, 5, 6, 8, 10, 15 mg / mL, or a value between any two of the foregoing values.

[0090] In some embodiments of the first aspect, the sample containing the anti-IL-17A antibody is loaded in an amount of 10 μL.

[0091] According to the above method, the quality of the anti-IL-17A antibody is judged based on the separation degree between the main peak of the anti-IL-17A antibody and the adjacent acidic peaks and basic peaks in the chromatogram, wherein when the separation degree between the main peak of the anti-IL-17A antibody and the adjacent basic peaks is greater than 1.17 or 1.45, the quality of the anti-IL-17A antibody meets the requirements.

[0092] In some embodiments of the first aspect, when the concentration of the sample containing the anti-IL-17A antibody is 4-15 mg / mL, for example 4, 5, 6, 8, 10, 15 mg / mL, or a value between any two of the above values; and the sample loading volume containing the anti-IL-17A antibody is 10 μL, the loading amount of the CEX-HPLC column is 40-150 μg, for example 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 μg, or a value between any two of the above values.

[0093] In some embodiments of the first aspect, the chromatographic column of the CEX-HPLC can be Thermo ProPacWCX 4.6*250 mm, 10 μm, product number: 028000.

[0094] In some embodiments of the first aspect, the CEX-HPLC chromatographic conditions of the anti-IL-17A antibody (eg, GR1501) are a method using a Thermo ProPac WCX chromatographic column and a salt gradient, and the specific chromatographic condition parameters are as follows:

[0095] Chromatographic column: Thermo ProPac WCX 4.6*250 mm, 10 μm

[0096] Flow rate: 0.8 mL / min; column temperature: 40°C; detection wavelength: 280 nm

[0097] Injection volume: 100 μg

[0098] Mobile phase A: 20 mM MES, pH 6.4

[0099] Preparation example: Weigh MES•H 2 O 4.266 g, NaOH 0.550 g, dissolve in appropriate amount of water and dilute to 1000 mL with water, filter and obtain.

[0100] Mobile phase B: 20 ​​mM MES + 0.5 M NaCl, pH 6.4

[0101] Preparation example: Weigh MES•H 2 O 1.280 g, NaCl 8.766 g, NaOH 0.172 g, dissolve in appropriate amount of water and dilute to 300 mL with water, filter and obtain.

[0102] The gradient elution program is as follows:

[0103]

[0104] After investigation, it was found that when the injection volume was in the range of 40-150 μg, the sodium chloride concentration in mobile phase B was ±10%, and the amount of sodium hydroxide added in mobile phase A was ±5%, there was no effect on the CEX purity determination of GR1501.

[0105] In some embodiments of the first aspect, the method of detecting anti-IL-17A antibodies using CEX-HPLC has good repeatability, and the relative standard deviations (RSD) of the main peak area percentage, acidic peak area percentage, and basic peak area percentage are all below 2%.

[0106] In some embodiments of the first aspect, the method of detecting anti-IL-17A antibodies using CEX-HPLC is not affected by the batch of chromatography columns.

[0107] In a second aspect, the present application provides a method for detecting anti-interleukin 17A (IL-17A) antibodies based on radioactive markers, which can be used in combination with the method for detecting anti-interleukin 17A (IL-17A) antibodies based on cation exchange high performance liquid chromatography (CEX-HPLC) described in the first aspect, wherein the method for detecting anti-interleukin 17A (IL-17A) antibodies based on radioactive markers comprises:

[0108] Labeling the anti-IL-17A antibody in the sample with a radioactive label;

[0109] The labeled samples are separated by gel column;

[0110] Determine the purity of the anti-IL-17A antibody in the sample by measuring the radioactivity intensity of the corresponding peak;

[0111] Wherein, the radioactive marker is 125 I. 124 I. 131 I. 89 Zr or 68 Ga.

[0112] In some embodiments of the second aspect, the tyrosine, histidine and / or arginine residues in the anti-IL-17A antibody in the sample are labeled with a radioactive label.

[0113] In some specific embodiments of the second aspect, the tyrosine residues in the anti-IL-17A antibody in the sample are labeled with a radioactive label.

[0114] In some specific embodiments of the second aspect, the radioactive marker is 125 I, such as Na 125 I.

[0115] ICH-S6 Guidelines for Preclinical Safety Evaluation of Biotechnology-derived Drugs suggest that when using radiolabeled proteins, it is important to show that the radiolabeled test substance still retains the same activity and biological properties as the unlabeled substance. The inventors of this application have repeatedly explored and determined that Na 125 I is suitable for radiolabeling of anti-IL-17A antibodies.

[0116] In some embodiments of the second aspect, for using 125 The anti-IL-17A antibody labeled with I is subsequently purified using a gel purification column (e.g., a dextran gel column).

[0117] In some embodiments of the second aspect, for the separation by gel purification column 125 The main peak of the anti-IL-17A antibody was determined by measuring the γ radioactivity intensity, and the purity of the antibody was determined by measuring the peak area.

[0118] In some embodiments of the second aspect, the bicinchoninic acid method can be used to determine 125 I is the concentration of the anti-IL-17A antibody in the samples before and after labeling.

[0119] In some embodiments of the second aspect, ELISA can be used to detect 125 I-1 immunoreactivity of the anti-IL-17A antibody before and after labeling.

[0120] In some embodiments of the second aspect, using 125 Labeling the anti-IL-17A antibody with I does not affect the immunological activity of the anti-IL-17A antibody.

[0121] In some embodiments of the second aspect, a method for detecting the quality of an anti-interleukin 17A (IL-17A) antibody based on a radioactive marker is performed using a kit for detecting the quality of an anti-interleukin 17A (IL-17A) antibody, wherein the kit for detecting the quality of an anti-interleukin 17A (IL-17A) antibody comprises:

[0122] Radioactive label and instructions for use; and / or

[0123] Amphoteric electrolytes, sodium hydroxide, sodium chloride, and instructions for use;

[0124] Wherein, the radioactive marker is 125 I. 124 I. 131 I. 89 Zr or 68 Ga; and / or

[0125] The ampholytes are MES, glycine, disodium hydrogen phosphate or sodium dihydrogen phosphate.

[0126] In some specific embodiments of the second aspect, the radioactive marker is 125 I.

[0127] In some specific embodiments of the second aspect, the ampholyte is MES.

[0128] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application, and is not intended to be limiting in any aspect. Those skilled in the art can make various modifications and changes to the embodiments.

[0129] The following examples are for illustration purposes only and are not intended to limit the scope of this application. Example

[0130] Example 1. Detection of the quality of GR1501 using CEX-HPLC

[0131] 1 Development of CEX-HPLC method

[0132] GR1501 is a monoclonal antibody, so the CEX-HPLC method was first developed using a salt gradient method, and the cation exchange column used was Thermo ProPac WCX 4.6*250 mm, 10 μm.

[0133] 1.1 Optimizing the pH value of the mobile phase

[0134] Since the pI value of GR1501 is around 6.7, the pH of the mobile phase of the CEX-HPLC method should be below 6.7 so that the charge heterogeneous species carry a positive charge. Because the pH of the mobile phase is different, the number of charges carried by each charge heterogeneous species is different, resulting in different separation effects in the cation exchange chromatography column elution, so it is necessary to find the mobile phase pH that can best separate the main peak from the acidic peak and the alkaline peak.

[0135] First, fixed chromatographic conditions were used:

[0136] Chromatographic column: Thermo ProPac WCX 4.6*250 mm, 10 μm, product number: 028000

[0137] Flow rate: 1.0 mL / min; column temperature: 30 °C; detection wavelength: 280 nm

[0138] Sample: GR1501-E3, Batch No.: GR1501-20151103 CPX

[0139] Amphoteric electrolyte MES was used as the buffer salt of the mobile phase, the initial mobile phase A was 20 mM MES, pH 5.5, and the mobile phase B was 20 mM MES + 0.5 M NaCl, pH 5.5. The pH of mobile phases A and B was adjusted, and the elution gradient of mobile phases A and B was adjusted to an appropriate start-end ratio under each pH value condition, and then the sample was injected for analysis.

[0140] 1.2 Optimize the elution gradient of the mobile phase:

[0141] Sample: GR1501-E3, Batch No.: GR1501-20151103 CPX

[0142] According to the optimal mobile phase pH value, the ratio of mobile phase gradient elution was adjusted, and the peak retention time and peak shape were compared and analyzed.

[0143] 1.3 Optimize column temperature:

[0144] Sample: GR1501-E3, Batch No.: GR1501-20151103 CPX

[0145] After determining the best mobile phase pH and the best mobile phase elution gradient, and after numerous column temperature tests, column temperatures of 30°C and 40°C were selected for injection to observe the column efficiency and separation.

[0146] 1.4 Optimize the preparation method of mobile phase:

[0147] The CEX-HPLC salt gradient method is very sensitive to the pH of the mobile phase. However, the method of using sodium hydroxide solution to adjust the pH when preparing the mobile phase will be affected by various factors such as the calibration of the pH meter and the ambient temperature, which will make the pH of the mobile phase inaccurate and thus affect the separation effect.

[0148] Because electronic balances have high precision and are less affected by external factors, the method of directly weighing a certain amount of sodium hydroxide to prepare a mobile phase with a specific pH can be used as an alternative method. The specific method for determining the mass of sodium hydroxide to be added is as follows:

[0149] 20% sodium hydroxide solution: Accurately weigh 2.0 g of sodium hydroxide, dilute with appropriate amount of water and make up to 10 mL.

[0150] Optimization of mobile phase A preparation method: weigh MES•H 2 O 4.266 g, add 1000 mL of water to dissolve and mix, gradually add 20% sodium hydroxide solution, when the pH value is 6.40, the amount of 20% sodium hydroxide solution added is 2.56 mL, and the mass of sodium hydroxide added is calculated to be 0.512 g.

[0151] Weigh MES•H 2 O 4.266 g, sodium hydroxide 0.510 g, add 1000 mL of water to dissolve and mix, the pH value is 6.32 at 25°C. Add 0.040 g of sodium hydroxide, mix, and the pH value is 6.39 at 25°C. Therefore, the amount of sodium hydroxide required to prepare 1000 mL of mobile phase A is 0.550 g.

[0152] Optimization of mobile phase B preparation method: weigh MES•H 2O 1.280 g, sodium chloride 8.766 g, add appropriate amount of water to dissolve and add water to 300 mL, gradually add 20% sodium hydroxide solution, when the pH value is 6.39, the amount of 20% sodium hydroxide solution added is 0.83 mL, and the mass of sodium hydroxide added is calculated to be 0.166 g.

[0153] Weigh MES•H 2 O 1.280 g, sodium chloride 8.766 g, sodium hydroxide 0.166 g, add appropriate amount of water to dissolve and add water to 300 mL, the pH value is 6.34 at 25℃. Add 0.006 g of sodium hydroxide, mix well, and the pH value is 6.41 at 25℃. Therefore, the amount of sodium hydroxide required to prepare 300 mL of mobile phase B is 0.172 g.

[0154] 2 Verification of CEX-HPLC method

[0155] After determining the optimal chromatographic conditions of CEX-HPLC for GR1501, the stability and durability of the method were tested.

[0156] 2.1 Investigating the influence of injection volume

[0157] Take GR1501-A1 sample, dilute it with water to 20 mg / mL, 15 mg / mL, 10 mg / mL, 8 mg / mL, 6 mg / mL, 5 mg / mL, 4 mg / mL, 2.5 mg / mL, and 1.0 mg / mL, respectively, and inject 10 μL of each sample. The injection volumes are 200 μg, 150μg, 100μg, 80μg, 60μg, 50μg, 40μg, 25μg and 10μg, respectively.

[0158] Compare the purity analysis results of GR1501 at different injection volumes. Verify the linearity of injection volume and peak area for GR1501 purity results within the acceptable range.

[0159] 2.2 Repeatability

[0160] Take the GR1501-B1 sample, prepare 6 portions of GR1501 test solution (10.0 mg / mL), and inject each portion once. Calculate the RSD of the main peak area percentage, acidic peak area percentage, and basic peak area percentage of the 6 samples.

[0161] 2.3 Column batch-to-batch consistency

[0162] Take GR1501-B1 sample, prepare GR1501 test solution (10.0 mg / mL), use different batches of chromatographic columns, and investigate the ratio of the main peak area percentage, acidic peak area percentage and basic peak area percentage.

[0163] 2.4 Effect of salt concentration in mobile phase B

[0164] Take GR1501-B1 sample and prepare GR1501 test solution (10.0 mg / mL). When preparing mobile phase B, the amount of sodium chloride added was increased by 10% and decreased by 10%, and the ratios of the main peak area percentage, acidic peak area percentage, and basic peak area percentage to those under normal conditions were examined.

[0165] 2.5 Effect of the amount of sodium hydroxide added to mobile phase A

[0166] Take GR1501-B1 sample and prepare GR1501 test solution (10.0 mg / mL). Prepare mobile phase A with the amount of sodium hydroxide added increased by 10% and decreased by 10%, and examine the ratio of the main peak area percentage, acidic peak area percentage, and alkaline peak area percentage to those under normal conditions.

[0167] 3. Results and Discussion

[0168] 3.1 Optimizing the pH value of the mobile phase

[0169] Compare the CEX-HPLC chromatograms of GR1501 samples under different mobile phase pH conditions. Figure 1 As shown: It was unexpectedly found that the separation effect of the main peak from the acidic peak and the basic peak was best when the mobile phase pH value was 6.4. Therefore, pH 6.4 was selected as the mobile phase pH value for the CEX-HPLC detection method of GR1501.

[0170] 3.2 Optimizing the elution gradient of the mobile phase

[0171]

[0172] Comparing the chromatograms under three gradients, the results are as follows Figure 2 As shown: Gradient 1: Mobile phase B: 2%→12% has the best effect when the main peak emerges in the middle of the elution time and the acid and base peaks are not affected by the solvent peak.

[0173] 3.3 Optimizing column temperature

[0174] The CEX-HPLC chromatograms of the GR1501 sample at column temperatures of 30°C and 40°C are shown in Figure 2. Figure 3 The separation is shown in Table 1. The results show that when the column temperature is 40°C, the separation effect of the GR1501 main peak and the alkaline peak is better, and the column efficiency of the main peak is higher.

[0175]

[0176] 3.4 Optimize the mobile phase preparation method:

[0177] The CEX-HPLC mobile phase of GR1501 was optimized and prepared as follows:

[0178] Mobile phase A: 20 mM MES, pH 6.4

[0179] Preparation example: Weigh MES•H 2 O 4.266 g, NaOH 0.550 g, dissolve in appropriate amount of water and dilute to 1000 mL with water, filter and obtain.

[0180] Mobile phase B: 20 ​​mM MES + 0.5 M NaCl, pH 6.4

[0181] Preparation example: Weigh MES•H 2 O 1.280 g, NaCl 8.766 g, NaOH 0.172 g, dissolve in appropriate amount of water and dilute to 300 mL with water, filter and obtain.

[0182] 3.5 Investigate the influence of injection volume:

[0183] The effects of different injection volumes on the purity of GR1501 were compared. The results are shown in Table 2. In the range of injection volume of 40 μg-150 μg (equivalent to 40-150% of the injection volume of 100 μg), the ratio of the main peak area percentage to the injection volume of 100 μg was between 98-102%, the ratio of the acidic peak area percentage to the injection volume of 100 μg was between 95-105%, and the ratio of the basic peak area percentage to the injection volume of 100 μg was between 95-105%.

[0184] When the injection volume of GR1501 is in the range of 40 μg-150 μg, the linear relationship between the injection volume and the peak area is as follows: Figure 4-Figure 6 shown.

[0185]

[0186] 3.6 Repeatability

[0187] The RSDs of the main peak area percentage, acidic peak area percentage, and alkaline peak area percentage of the six samples were calculated, and the results are shown in Table 3. The RSDs of the main peak area percentage, acidic peak area percentage, and alkaline peak area percentage were all below 2.0%, indicating that the repeatability of the method met the requirements.

[0188]

[0189] 3.7 Column Batch-to-Batch Consistency

[0190] Different batches of chromatographic columns were used to investigate the ratio of the main peak area percentage, the acidic peak area percentage, and the basic peak area percentage. The results are shown in Table 4.

[0191]

[0192] 3.8 Effect of salt concentration in mobile phase B

[0193] When the amount of sodium chloride added in mobile phase B increased by 10% or decreased by 10%, the ratios of the main peak area percentage, acidic peak area percentage and basic peak area percentage to those under normal conditions are shown in Table 5.

[0194]

[0195] 3.9 Effect of the amount of sodium hydroxide added to mobile phase A

[0196] When the amount of sodium hydroxide added in mobile phase A increased by 5% or decreased by 5%, the ratios of the main peak area percentage, acidic peak area percentage and basic peak area percentage to those under normal conditions are shown in Table 6.

[0197]

[0198] 4. Conclusion

[0199] The optimal CEX-HPLC chromatographic conditions for GR1501 are to use a Thermo ProPac WCX column and a salt gradient method. The specific chromatographic condition parameters are as follows:

[0200] Chromatographic column: Thermo ProPac WCX 4.6*250 mm, 10 μm

[0201] Flow rate: 0.8 mL / min; column temperature: 40 °C; detection wavelength: 280 nm

[0202] Injection volume: 100 μg

[0203] Mobile phase A: 20 mM MES, pH 6.4

[0204] Preparation example: Weigh MES•H 2 O 4.266 g, NaOH 0.550 g, dissolve in appropriate amount of water and dilute to 1000 mL with water, filter and obtain.

[0205] Mobile phase B: 20 ​​mM MES + 0.5 M NaCl, pH 6.4

[0206] Preparation example: Weigh MES•H 2O 1.280 g, NaCl 8.766 g, NaOH 0.172 g, dissolve in appropriate amount of water and dilute to 300 mL with water, filter and obtain.

[0207] Gradient elution:

[0208]

[0209] After investigation, it was found that when the injection volume was in the range of 40-150 μg, the salt concentration in mobile phase B was ±10%, and the amount of sodium hydroxide added in mobile phase A was ±5%, there was no effect on the CEX-HPLC purity determination of GR1501.

[0210] Example 2. Use 125 I marker to detect the quality of GR1501

[0211] 1. 125 Preparation of I-GR1501

[0212] 1.1 Experimental Materials

[0213] Test product 1: GR1501 injection, batch number: 20160601; clear solution, concentration 100 mg / mL, provided by Zhixiang (Shanghai) Pharmaceutical Technology Co., Ltd. The sample was stored at 2-8°C. Used for tissue distribution and excretion experiments.

[0214] Test product 2: GR1501 injection replacement formulation, batch number: GR1501-20161221WF; clear solution, concentration 11.6 mg / mL, provided by Zhixiang (Shanghai) Pharmaceutical Technology Co., Ltd. The sample was stored at 2-8°C until use. For labeling.

[0215] Iodination reagent: Iodogen, batch number: 80K4088, Sigma.

[0216] Labeled isotope: Radioactive Na 125 I, batch number: I121416L, PerkinElmer.

[0217] Purification column filler: Sephadex TM G-10, lot number: 10222168, GE Healthcare.

[0218] Cinchona acid protein quantification kit, batch number: 10121, Kangwei Century Company.

[0219] ELISA plate: 96-well, Costar 2592, Corning, USA.

[0220] Coating reagent: recombinant human IL-17A, batch number: 0330312, 0.5 mg / mL, provided by Zhixiang (Shanghai) Pharmaceutical Technology Co., Ltd.

[0221] Detection reagent: Horseradish peroxidase-labeled goat anti-human IgG, batch number: A80-319P-21, Bethyl, USA.

[0222] Enzyme substrate: TMB, batch number: 10130, Kangwei Century Company.

[0223] Chemical reagents such as disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, chloroform, and trichloroacetic acid are produced by Sinopharm Chemical Reagent Co., Ltd.

[0224] 1.2 Experimental methods and steps

[0225] 1.2.1 Preparation of atmospheric pressure gel purification column

[0226] Soak the Sephadex G-10 gel in distilled water overnight (more than 16 h), take a clean glass column with a diameter of 1 cm and a height of 35 cm, install it vertically on a test tube rack, slowly infuse the fully expanded Sephadex gel into the glass column, connect a peristaltic pump, and use PBS buffer (pH7.2) to balance the purification column at a flow rate of 1 mL / min for 1 h. Take 1 mL of the test product GR1501 (11.6 mg / mL) and perform normal pressure separation on the purification column to saturate the purification column. After preparation, store it in a refrigerator at 2-8°C.

[0227] 1.2.2 Radiolabeling

[0228] Weigh 1 mg of chlorouril and dissolve it in 0.5 mL of chloroform. Take 50 µL and add it to the bottom of the test tube. Spread it evenly and blow it dry with nitrogen. Add 1 mL of GR1501 (11.6 mg / mL) at the mark, and then add about 15 mCi of Na 125 I (50 μL), react at room temperature for 10 min, and shake continuously during the reaction to ensure complete reaction.

[0229] 1.2.3 Separation and purification of labeling reactants

[0230] The labeled reaction mixture was added to the pre-infused gel purification column for separation and purification. The eluent was PBS buffer at a flow rate of 1 mL / min. The eluent was collected with a fraction collector at 1 mL / tube, and the gamma radioactivity of each fraction was determined. The protein concentration was determined at the peak of the highest radioactivity count and the vicinity of the peak.

[0231] 1.2.4 Radiochemical purity identification

[0232] The purity of the marker was identified by SHPLC. The samples collected after separation and purification were diluted appropriately and injected to measure the γ-radioactive chromatogram. Eluent: 0.01 mol / L phosphate buffer, containing 0.1 mol / L NaCL, pH 7.2; flow rate 1.0 mL / min. The radioactive peak area was directly analyzed and processed using the workstation software Chromera v 3.4.1 provided by the Flexar HPLC system (Perkin Elmer). The area percentage of the radioactive peak with the same elution behavior as the standard non-labeled GR1501 was calculated.

[0233] 1.2.5 Protein content determination

[0234] Diquinone acid method: add a series of concentration levels (0, 0.2, 0.4, 0.8, 1.2, 1.6 and 2 mg / mL) of standard bovine serum albumin (BSA) or 10 mL of unknown sample to a 96-well plate, duplicate wells, and then add 200 mL of protein determination solution (dichononone acid: 4% copper sulfate pentahydrate solution = 1:50) to each well, shake and mix, incubate in a 37 ℃ biochemical incubator for 30 minutes, cool to room temperature, and use a plate reader to measure the light absorption value at 570 nm. Draw a standard curve with the OD value of the BSA series concentration, and calculate the protein content of the unknown sample by linear regression. The standard curve is as follows Figure 7 shown.

[0235] 1.2.6. Identification of GR1501 immunoreactivity after labeling

[0236] The ELISA method was used to detect the immune activity of GR1501 before and after labeling. The steps are as follows:

[0237] (1) Dilute the coating protein concentrate (recombinant human IL-17A) 667 times with coating solution (50 mM sodium carbonate buffer, pH 9.6) to make coating protein working solution (0.75 μg / mL). Add 100 mL to each well and place in a refrigerator at 2-8°C overnight (16-18 hours).

[0238] (2) After washing, add 200 mL / well of I-block blocking solution and incubate at room temperature (25±3℃) for 2 h.

[0239] (3) After washing the plate, add 100 μL of GR1501 (pre- and post-labeling samples) at different concentrations (400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 ng / mL) to two more wells. Incubate at room temperature (25 ± 3°C) for 2 h, then pour off the solution in the wells and wash the plate and pat dry.

[0240] (4) Dilute the detection reagent horseradish peroxidase-labeled goat anti-human IgG at a ratio of 1:100,000 in PBS buffer containing 0.5% BSA and 0.05% Tween-20, and add 100 mL / well. Incubate at room temperature (25±3℃) for 1 h, then pour off the solution in the wells and wash the plate and pat dry.

[0241] (5) Add 100 mL of TMB working solution into each well. Incubate at room temperature in the dark for 15 min, and then add 50 mL of 1 M dilute sulfuric acid to terminate the reaction.

[0242] (6) After a brief shake, the optical absorption value (OD) was measured on a plate reader. The detection wavelength was 450 nm and the reference wavelength was 630 nm. The GR1501 drug concentration and OD value were fitted using a four-parameter equation, and the curve equations before and after labeling were compared.

[0243] 2. Results

[0244] 2.1 Isolation, purification and protein quantification of labeled drugs

[0245] After the labeled drug was purified, the radioactivity of the collected sample was measured to be 72.61 MBq / mL, and the protein quantification results showed that the protein concentration was 1.69 mg / mL, the volume was about 5 mL, and the specific activity of the labeled drug was: 42.89 MBq / mg. The results are shown in Table 7-8.

[0246]

[0247]

[0248] 2.2 125 Radiochemical Purity Identification of I-GR1501

[0249] Figure 8 Figure A in FIG is the HPLC purity chromatogram of GR1501 before labeling, and the elution volume is 7.762 mL. Figure 8 Figure B is the product after labeling, and the elution volume is about 7.72 mL. Since the liquid phase system is connected in series with the flow scintillation, the elution time of the liquid scintillation peak is later, but the elution volume of the chromatograms before and after labeling is basically the same, and the radiochemical purity after labeling is close to 100%. The results show that the labeled product is the target product ( 125 I-GR1501), with a radiochemical purity greater than 95%, which meets the requirements for tissue distribution research.

[0250] 2.3 Comparison of activity before and after labeling

[0251] The drugs before and after labeling were diluted into a series of solutions with the same concentration and then subjected to ELISA assay. Table 9 shows the concentrations and corresponding OD values ​​and fitting equations of the two curves before and after labeling. The results show that the two curves have a good fit, and the coefficient of variation between duplicate wells CV% <14.256%. The parameters of the equation of the immunoactivity curve before and after drug labeling are similar, and the trends of the two curves are consistent and basically overlap ( Fig. 9 ). After labeling, the curves move slightly downward in parallel, indicating that the affinity of the two to the antigen is similar.

[0252]

[0253] The above results show that after labeling 125 The specific activity, radiochemical purity and immunological activity of I-GR1501 all meet the requirements of pharmacokinetic studies.

[0254] All patents, patent application disclosures, and non-patent documents mentioned and / or listed in this application are incorporated herein by reference in their entirety. The exemplary embodiments of the inventions of this application are described above, but without departing from the essence and scope of this application, those skilled in the art can modify or improve the exemplary embodiments described in this application, and the resulting variants or equivalents also belong to the scope of this application.

Claims

1. A method for detecting anti-IL-17A antibodies based on cation exchange high performance liquid chromatography, comprising: Adding the sample containing the anti-IL-17A antibody to a cation exchange high performance liquid chromatography column, wherein the column temperature of the cation exchange high performance liquid chromatography column is 40° C.; in: The cation exchange high performance liquid chromatography column is a Thermo ProPac WCX chromatography column; The mobile phase of the cation exchange high performance liquid chromatography includes mobile phase A and mobile phase B, and the elution gradients of the mobile phase A and the mobile phase B are as follows: At 0 min, mobile phase A was 98% and mobile phase B was 2%; At 5 min, mobile phase A was 98% and mobile phase B was 2%; At 30 min, mobile phase A was 88% and mobile phase B was 12%; At 31 min, mobile phase A was 0% and mobile phase B was 100%; At 33 min, mobile phase A was 0% and mobile phase B was 100%; At 34 min, mobile phase A was 98% and mobile phase B was 2%; At 40 min, mobile phase A was 98% and mobile phase B was 2%; The mobile phase A is 20 mM MES, and the pH value of the mobile phase A is 6.4; The mobile phase B is 20 mM MES and 0.5 M NaCl, and the pH value of the mobile phase B is 6.4; The anti-IL-17A antibody comprises a heavy chain as shown in SEQ ID NO:7 and a light chain as shown in SEQ ID NO:

8.

2. The method according to claim 1, wherein the pH value of the mobile phase of the cation exchange high performance liquid chromatography is adjusted by directly weighing and preparing sodium hydroxide.

3. The method according to claim 2, wherein the mobile phase of the cation exchange high performance liquid chromatography comprises mobile phase A and mobile phase B; In mobile phase A, the amount of sodium hydroxide added is 0.5225-0.5775 g / L; and / or In mobile phase B, the amount of sodium hydroxide added was 0.573 g / L.

4. The method of claim 1, wherein the sample containing the anti-IL-17A antibody has a concentration of 4-15 mg / mL.

5. The method of claim 1, wherein the sample loading volume of the anti-IL-17A antibody is 10 μL.

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