Method for detecting residual protein a in an antibody drug

Abstract

The application provides a method for detecting residual recombinant protein A in an antibody drug, comprising: (a) purifying residual protein A in the antibody drug by affinity chromatography using an affinity filler, to obtain residual protein A; the affinity filler is selected from one of MabSelect Prism A, Nmab Pro, AT Protein A Diamond Plus, MaXtar ARPA, MabPurix A65 Excel, and Praesto Jetted A50 HipH; (b) detecting the content X of the residual protein A by enzyme-linked immunoassay (ELISA) with a Cygnus F610 kit; and (c) bringing the content X of the residual protein A detected in step (b) into a formula corresponding to the affinity filler used in step (a), to calculate the corrected content Y of the residual protein A. The method has universality and is convenient for monitoring the content of residual protein A in multiple batches of antibody drugs.

Description

Technical Field

[0001] This invention relates to a method for detecting residual protein A in antibody drugs, belonging to the field of drug impurity detection; specifically, this invention belongs to the field of detecting impurity proteins after antibody proteins have been purified and separated by affinity chromatography. Background Technology

[0002] Protein A is a protein extracted from Staphylococcus aureus. The molecular weight of natural protein A is approximately 43 kDa, and it contains five homologous domains: EDABC. [1] It can specifically bind to the Fc terminus of the antibody. [2] Currently, protein A affinity chromatography is widely used in the industry as one of the most classic antibody purification steps.

[0003] To improve the purification performance of protein A affinity packing materials, the industry is continuously modifying the gene sequence and structure of natural protein A. [4] Currently, commonly used recombinant protein A affinity fillers on the market include Cytiva's MabSelect Sure / MabSelect SureLX, Cytiva's MabSelect PrismA, Purolite's Jetted A50 HipH, Nanomicro's NmabPro, Borglon's AT ProteinA Diamond Plus, Bailinco's MaXtar ARPA, and Saifen's MabPurix A65Excel.

[0004] During the purification of antibody drugs using protein A affinity chromatography, protein A ligands inevitably detach from the packing material due to factors such as process, column lifespan, and proteases. The protein A ligands remaining in the product, as a process-related impurity, can affect product quality and performance, and pose potential safety risks to human health. Therefore, both domestic and international regulations require the control and detection of residual protein A content during drug production.

[0005] Enzyme-linked immunosorbent assay (ELISA) is currently the main method for detecting residual protein A. The detection steps are generally as follows: (1) (1) On an ELISA plate pre-coated with anti-protein A antibody, protein A sample and protein A standard are added separately and incubated for a certain time to form an anti-protein A antibody-protein A complex; (2) Unbound material is washed away, and horseradish peroxidase (HRP)-labeled protein A detection antibody is added and incubated for a certain time to form an anti-protein A antibody-protein A-HRP-labeled anti-protein A detection antibody complex; (3) Unbound material is washed away, and tetramethylbenzidine solution (TMB) is added and incubated for a certain time. TMB is catalyzed by HRP to form a blue complex; (4) Sulfuric acid stop solution is added, and the blue solution turns into a yellow solution. The intensity of the solution color is positively correlated with the protein A content; (5) A certain wavelength is set, and the ELISA plate is placed in an ELISA reader for reading; (6) The OD value of the sample is substituted into the protein A standard curve to calculate the residual protein A content in the sample.

[0006] The structures and sequences of different recombinant proteins A vary considerably. To ensure data accuracy, affinity reagent manufacturers generally recommend using their respective specific ELISA kits for detection. For example, the corresponding detection kits for MabSelect Sure / MabSelect SureLX are Cygnus F610, for MabSelect PrismA is Cytiva29707299, for Jetted A50HipH is Repligen 9777-1, for Nmab Pro is Primet PA517 with standard 5-Nmab Pro, for AT ProteinA Diamond Plus is Cygnus F610 with AT ProteinA Diamond Plus protein A, for MaXtar ARPA is Bexsys RES-A024, and for MabPurix A65 Excel is Primet PA517 with standard 7-MabPurix.

[0007] Existing methods suffer from drawbacks such as inconsistent detection methods, difficulty in parallel comparison of residual data for different affinity fillers, and the scarcity of specific kits for certain affinity fillers. Therefore, a unified, rapid, and simple detection method is needed. Summary of the Invention

[0008] This invention provides a method for detecting residual protein A in antibody drugs, which has strong versatility and facilitates a unified quality assessment of the residual protein A content in antibody drugs purified by different affinity packing materials.

[0009] A first aspect of the present invention provides a method for detecting residual recombinant protein A in an antibody drug, comprising: (a) The antibody drug was purified by affinity chromatography using an affinity packing material, and the resulting antibody drug still contained residual protein A; the affinity packing material was selected from one of MabSelect PrismA, Nmab Pro, AT ProteinADiamond Plus, MaXtarARPA, MabPurix A65 Excel, and Praesto Jetted A50 HipH; (b) The content X of the residual protein A was detected by enzyme-linked immunosorbent assay (ELISA) with Cygnus F610 kit; (c) Substitute the residual protein A content X obtained in step (b) into the formula corresponding to the affinity filler actually used in step (a) to calculate the corrected residual protein A content Y. MabSelect PrismA <![CDATA[Y=10 ((0.1205±5％)+(1.022±5％)logX) ]]> Nmab Pro <![CDATA[Y=10 ((0.011±5％)+(1.011±5％)logX) ]]> AT ProteinA Diamond Plus <![CDATA[Y=10 ((-0.191±5％)+(1.034±5％)logX) ]]> MaXtar ARPA <![CDATA[Y=10 ((-0.122±5％)+(0.983±5％)logX) ]]> MabPurix A65 Excel <![CDATA[Y=10 ((-0.017±5％)+(0.98±5％)logX)±5％ ]]> Praesto Jetted A50 HipH <![CDATA[Y=10 ((0.57±5％)+(1.134±5％)logX) ]]> .

[0010] In some embodiments, the formula is selected from the following list: MabSelect PrismA <![CDATA[Y=10 (0.1205+1.022logX) ]]> Nmab Pro <![CDATA[Y=10 (0.011+1.011logX) ]]> AT ProteinA Diamond Plus <![CDATA[Y=10 (-0.191+1.034logX) ]]> MaXtar ARPA <![CDATA[Y=10 (-0.122+0.983logX) ]]> MabPurix A65 Excel <![CDATA[Y=10 (-0.017+0.98logX) ]]> Praesto Jetted A50 HipH <![CDATA[Y=10 (0.57+1.134logX) ]]> .

[0011] The method according to a first aspect of the present invention, wherein, The formula Y = 10 (0.1205+1.022logX) The method for obtaining it includes the following steps: (d11) Using the protein A standard solution 1 with a concentration of 0-10 ng / mL in the Cygnus F610 kit, and the anti-protein A antibody in the Cygnus F610 kit, a standard curve 1 was established between the protein A concentration Y and the absorbance value at 450 nm. (d12) Using the ligand protein of the affinity filler MabSelect PrismA, a series of standard solutions with concentrations of 0–10 ng / mL were prepared 2. The anti-protein A antibody in the Cygnus F610 kit was used to establish a standard curve 2 of protein A concentration X and absorbance at 450 nm. (d13) Establish the concentration relationship between standard curve 1 and standard curve 2 under the same absorbance value, i.e., Y = 10 (0.1205 +1.0221ogX) .

[0012] The method according to a first aspect of the present invention, wherein, The formula Y = 10 (0.011+1.0111ogX) The method for obtaining it includes the following steps: (d21) Using the protein A standard solution 1 with a concentration of 0-10 ng / mL in the Cygnus F610 kit, and the anti-protein A antibody in the Cygnus F610 kit, a standard curve 1 of protein A concentration Y and absorbance at 450 nm was established. (d22) Using the ligand protein of the affinity filler Nmab Pro, a series of standard solutions with concentrations of 0-10 ng / mL were prepared. Using the anti-protein A antibody in the Cygnus F610 kit, a standard curve of protein A concentration X and absorbance at 450 nm was established. (d23) Establish the concentration relationship between standard curve 1 and standard curve 2 under the same absorbance value, i.e., Y = 10 (0.011 +1.011logX) .

[0013] The method according to a first aspect of the present invention, wherein, The formula Y = 10 (-0.191+1.034logX) The method of obtaining it includes the following steps: (d31) Using the protein A standard solution 1 with a concentration of 0-10 ng / mL in the Cygnus F610 kit, and the anti-protein A antibody in the Cygnus F610 kit, a standard curve 1 of protein A concentration Y and absorbance at 450 nm was established. (d32) Using the ligand protein of the affinity filler AT ProteinADiamond Plus, a series of standard solutions with concentrations of 0-10 ng / mL were prepared 2. The anti-protein A antibody in the Cygnus F610 kit was used to establish a standard curve 2 of protein A concentration X and absorbance at 450 nm. (d33) Establish the concentration relationship between standard curve 1 and standard curve 2 under the same absorbance value, i.e., Y = 10 (-0.191+1.0341ogX) .

[0014] The method according to a first aspect of the present invention, wherein, The formula Y = 10 (-0.122+0.983logX) The method of obtaining it includes the following steps: (d41) Using the protein A standard solution 1 with a concentration of 0-10 ng / mL in the Cygnus F610 kit, and in combination with the anti-protein A antibody in the Cygnus F610 kit, a standard curve 1 of protein A concentration Y and absorbance at 450 nm was established. (d42) Using the ligand protein corresponding to the affinity filler MaXtar ARPA, a series of standard solutions with concentrations of 0-10 ng / mL were prepared 2. The anti-protein A antibody in the Cygnus F610 kit was used to establish a standard curve 2 of protein A concentration X and absorbance at 450 nm. (d43) Establish the concentration relationship between standard curve 1 and standard curve 2 under the same absorbance value, i.e., Y = 10 (-0.122+0.983logX) .

[0015] The method according to a first aspect of the present invention, wherein, The formula Y = 10 (-0.017+0.981ogX) The method of obtaining it includes the following steps: (d51) Using the protein A standard solution 1 with a concentration of 0-10 ng / mL in the Cygnus F610 kit, and in combination with the anti-protein A antibody in the Cygnus F610 kit, a standard curve 1 of protein A concentration Y and absorbance at 450 nm was established. (d52) Using the ligand protein corresponding to the affinity filler MabPurix A65 Excel, a series of standard solutions with concentrations of 0–10 ng / mL were prepared. Using the anti-protein A antibody in the Cygnus F610 kit, a standard curve of protein A concentration X and absorbance at 450 nm was established. (d53) Establish the concentration relationship between standard curve 1 and standard curve 2 under the same absorbance value, i.e., Y = 10 (-0.017+0.98logX) .

[0016] The method according to a first aspect of the present invention, wherein, The formula Y = 10 (0.57+1.134logX) The method of obtaining it includes the following steps: (d61) Using the protein A standard in the Cygnus F610 kit, a series of standard solutions with concentrations of 0 to 10 ng / mL were prepared. The anti-protein A antibody in the Cygnus F610 kit was used to establish a standard curve of protein A concentration Y and absorbance at 450 nm. (d62) Using the protein A standard in the Repligen 9777-1 kit corresponding to the affinity filler Praesto Jetted A50 HipH, a series of standard solutions with concentrations of 0 to 10 ng / mL were prepared 2. In combination with the anti-protein A antibody in the Cygnus F610 kit, a standard curve of protein A concentration X and absorbance at 450 nm was established 2. (d63) Establish the concentration relationship between standard curve 1 and standard curve 2 under the same absorbance value, i.e., Y = 10 (0.57+1.134logX) .

[0017] According to the method of the first aspect of the present invention, the content Y of the corrected residual protein A is statistically analyzed with the detection values ​​of the fixedly matched affinity filler and detection kit for no less than four samples.

[0018] According to the method of the first aspect of the present invention, the recovery rate calculated by the following formula is between 50% and 150%:

[0019] According to a first aspect of the present invention, the antibody drug comprises a monoclonal antibody, a bispecific antibody, or a trispecific antibody.

[0020] According to a first aspect of the present invention, the method wherein the antibody drug is a PD-1 antibody drug.

[0021] In some embodiments, the antibody drug is a PD-1 antibody.

[0022] In some embodiments, the antibody drug is a CLDN6×CD3 bispecific antibody. Detailed Implementation

[0023] The affinity fillers and kits involved in this invention are all commercially available products.

[0024] This invention combines the Cygnus F610 residual protein A detection kit with different mathematical formulas Y=10. (A +BlogX) This method is highly versatile and can meet the detection requirements of residual protein A in most affinity fillers on the market, improving detection efficiency and ensuring data accuracy and product safety. In the formula, Y is the final calculated concentration of the sample, and X is the concentration of the sample measured using F610.

[0025] Cygnus F610 is a universal reagent kit that can be used with different protein A standards for data calibration when detecting different affinity fillers. Therefore, this invention uses both F610 standards and specific protein A standards for sample testing. After the sample is diluted a certain factor, it produces an absorbance value on the microplate (this value is a relatively fixed value, only related to the sample). Substituting this absorbance value into the different standard curves of the two standards, log Value = A1 + B1log Con.1 and log Value = A2 + B2log Con.2, i.e., A1 + B1log Con.1 = A2 + B2log Con.2, the formula Con2 = 10 can be derived. ((B1÷B2)logCon.1+(A1-A2)÷B2)Based on the relationship between the standard curves of different affinity fillers and the F610 standard curve, this invention derives the following corresponding formula after conducting extensive data statistics.

[0026] The recovery rate (REC%) of the parameters involved in the examples means: the ratio of the measured value using the Cygnus F610 kit to the theoretical value obtained by the specific detection kit corresponding to the packing material. It reflects the accuracy of the data. The closer it is to 100%, the smaller the difference in the data. Right now Example 1: Detection of Protein A in PD-1 Antibody Drugs using the MabSelect PrismA Combination Assay Kit Cygnus F610 Step A: Create the formula (1) The standards in Cygnus F610 are 10, 5, 2.5, 1.25, 0.625, 0.31, 0.16, and 0 ng / mL, respectively. The sample used in curve 1; (2) Dilute the MabSelect PrismA ligand protein with dilution buffer to concentrations of 10, 5, 2.5, and 1.25, respectively. 0.625, 0.31, 0.16, 0 ng / mL, which are the samples used in standard curve 2; (3) Take 60 μL of the above standard solution and add 30 μL of denaturing solution, then mix by shaking / piping. (5) Centrifuge the prepared solution at 13000 rpm / min for 5 min and let it stand at room temperature for 10 min; (5) Add 100 μL of anti-protein A antibody to the pre-coated ELISA plate: HRP detection antibody; ⑹Add 25 μL of the supernatant of the above standard to each well plate and incubate at 25°C for 60 min; (7) Wash the plate, add 100 μL of TMB solution, and incubate in the dark for 30 min; (8) Add 100 μL of stop solution; (9) Place the sample in an ELISA reader and take readings at a detection wavelength of 450 nm and a reference wavelength of 650 nm; (10) Select Log-Log in SoftMax Pro 7.2 to establish standard curves. The formula for standard curve 1 is logValue=(-0.629)+0.851logX, and the formula for standard curve 2 is logValue=(-0.722)+0.819log Y. (11) The OD value of the same sample in a single test is constant, meaning the Value in both formulas is the same. Therefore, subtracting Standard Curve 2 from Standard Curve 1 yields (-0.629) + 0.851logX = (-0.722) + 0.819logY. After conversion, we get Y = 10. ((0.851÷0.819)logX+(0.722-0.629)÷0.819) That is, Y = 10 (1.0391ogX+0.114) . Repeat experiments (1) to (11) multiple times, calculate the average value, and obtain the final formula Y = 10. (1.022logX+0.1205) . Step B: According to the formula, correct the MabSelect PrismA formula-matched detection kit (Cygnus F610) to detect the content of residual protein A in antibody drugs, and statistically analyze the correction results with the detection results of its specificity test kit (Cytiva 29707299). The content of residual protein A in the MabSelect PrismA purified sample was detected using its specificity Cytiva 29707299: (1) Dilute the sample and protein A standard with dissociation buffer, respectively. The standard curve range is 2.5 to 0.078 ng / mL. The protein A concentration of the diluted sample must be within the standard curve range. (2) Add 100 μL of analytical solution to 200 μL of the above sample and standard solutions and mix well; (3) Add 100 μL of the above solution to the pre-coated enzyme-linked plate and incubate for 30 min; (4) Wash the plate, add 100 μL of detection antibody, and incubate for 30 min; (5) Wash the plate, add 100 μL of TMB, and incubate for 15 min; (6) Add sulfuric acid as a stop solution; (7) Place the plate into the microplate and take readings at the detection wavelength of 450 nm and the reference wavelength of 650 nm. Use the 4-parameter logic in SoftMaxPro 7.2 to build the standard curve. (8) Substitute the sample OD value into the protein A standard curve to calculate the actual residual protein A content (C) in the sample. T ). The residual protein A content in the MabSelect PrismA purified sample was detected using a Cygnus F610: (1) Dilute the sample with dilution buffer. The concentration of protein A in the diluted sample should be within the standard curve range (10-0.0 ng / mL). (2) Add 30 μL of denaturing solution to 60 μL of the above sample and standard solutions respectively and mix by shaking / piping. (3) Centrifuge the above sample at 13000 rpm / min for 5 min and let it stand at room temperature for 10 min; (4) Add 100 μL of anti-protein A antibody to the pre-coated ELISA plate: HRP detection antibody; (5) Add 25 μL of the supernatant of the above sample and standard to the well plate respectively, and incubate at 25°C for 60 min; (6) Wash the plate, add 100 μL of TMB solution, and incubate in the dark for 30 min; (7) Add 100 μL of stop solution; (8) Place the plate into the microplate and take readings at the detection wavelength of 450nm and the reference wavelength of 650nm. Use the 4-parameter logic in SoftMaxPro7.2 to build the standard curve. (9) (8) Substitute the OD value of the sample into the protein A standard curve to obtain the residual protein A content in the sample. (10) (9) Substitute the above residual protein A content into the formula Y = 10 (1.022logX+0.1205) From X, we can calculate the residual protein A content Y.

[0027] The calculated residual protein A content Y in the above samples was compared with the actual residual protein A content (C). T The recovery rate is obtained, and the difference between two sets of data is compared based on the magnitude of the recovery rate. In this invention, the acceptable recovery rate is 50%–150%. As shown in the table below, using Cygnus F610 combined with Y=10… (0.1205+1.022log0) The calculated recoveries for the detection method were all in the range of 80% to 100%, indicating that this method can accurately detect the content of residual protein A in samples purified by Prism A affinity chromatography column. Table 1A. Residual protein A content of samples after purification by Prism A affinity chromatography column, as detected by Cytiva 29707299. Sample 1-1 407.9 Sample 1-2 39.1 Samples 1-3 34.8 Samples 1-4 10.2 Samples 1-5 11.9 Samples 1-6 7.2 Samples 1-7 30.3 Table 1B. The content of residual protein A in the samples after purification by Prism A affinity chromatography was determined using a Cygnus F610. Sample 1-1 328.9 Sample 1-2 29.1 Samples 1-3 27.4 Samples 1-4 7.5 Samples 1-5 9.4 Samples 1-6 9.6 Samples 1-7 22.5 Table 1C. Substitute the data from Table 1B into the formula Y = 10 (0.1205+1.022logX) Then, calculate REC% using the data from Table 1A. Sample 1-1 493.1 82.7 Sample 1-2 41.3 94.5 Samples 1-3 38.9 89.3 Samples 1-4 10.4 98.3 Samples 1-5 13.1 90.9 Samples 1-6 13.4 94.1 Samples 1-7 31.8 95.3 Example 2: Detection of Protein A in PD-1 Antibody Drugs Using Nmab Pro Combined with Cygnus F610 Detection Kit

[0028] Similar to the steps in Implementation 1, the difference lies in the use of different affinity packing materials to purify the samples in steps A and B, the use of different protein A standards to establish the formula, and the use of different specific residual protein A kits to detect the samples.

[0029] Table 2A shows the residual protein A content in samples purified by Nmab Pro affinity chromatography and detected using a Primite PA517 analyzer. Table 2B shows the residual protein A content in samples purified by Nmab Pro affinity chromatography and detected using a Cygnus F610 analyzer. Table 2C shows the residual protein A content in samples obtained by substituting the data from Table 2B into the formula Y = 10. (0.011+1.0logX) After calculating the Y value from X, the REC value is then calculated using the data in Table 2A. Table 2A. Residual protein A content of samples purified by Nmab Pro affinity chromatography column, as determined by Primate PA517. Sample 2-1 529.5 Sample 2-2 289.7 Sample 2-3 113.1 Samples 2-4 92.8 Table 2B. The content of residual protein A in the samples after purification by Prism A affinity chromatography was determined by Cygnus F610. Sample 2-1 419.2 Sample 2-2 267.1 Sample 2-3 101.0 Samples 2-4 102.1 Table 2C. Substitute the data from Table 2B into the formula Y = 10 (0.011+1.011logX) Then, calculate REC% using the data from Table 2A. Sample 2-1 459.4 115.2 Sample 2-2 291.4 99.4 Sample 2-3 109.0 103.8 Samples 2-4 110.1 84.2 Example 3: Detection of Protein A in CLDN6×CD3 Bispecific Antibody Drug using Jetted A50 HipH Assay Kit and Cygnus F610

[0030] Similar to the steps in Implementation 1, the difference lies in the use of different affinity packing materials to purify the samples in steps A and B, the use of different formulas for establishing protein A standards, and the use of different specific residual protein A detection kits to detect the samples.

[0031] Table 3A shows the residual protein A content in samples purified using a Jetted A50 HipH affinity chromatography column, as detected by a Repligen 9777-1. Table 3B shows the residual protein A content in samples purified using a Jetted A50 HipH affinity chromatography column, as detected by a Cygnus F610. Table 3C shows the residual protein A content in samples purified using a Jetted A50 HipH affinity chromatography column, as detected by a Cygnus F610. (0.57+1.134logX) Then, calculate REC% using the data from Table 3A. Table 3A. Residual protein A content of samples after purification using a Jetted A50 HipH affinity chromatography column, as determined by a Repligen 9777-1. Sample 3-1 36.2 Sample 3-2 23.9 Sample 3-3 103.7 Sample 3-4 14.9 Samples 3-5 14.6 Table 3B. The content of residual protein A in the samples after purification by Jetted A50 HipH affinity chromatography was determined by Cygnus F610. Sample 3-1 8.5 Sample 3-2 6.8 Sample 3-3 26.1 Sample 3-4 3.3 Samples 3-5 2.5 Table 3C. Substitute the data from Table 3B into the formula Y = 10 (0.57+1.134logX) Then, calculate REC% using the data from Table 3A. Sample 3-1 42.3 85.7 Sample 3-2 32.7 73.1 Sample 3-3 150.2 69.1 Sample 3-4 14.2 105.0 Samples 3-5 10.3 142.3 Example 4: Detection of Protein A in PD-1 Antibody Drugs Using the AT Protein A Diamond Plus Combined Detection Kit with Cygnus F610

[0032] Similar to the steps in Implementation 1, the difference lies in the use of different affinity packing materials to purify the samples in steps A and B, the establishment of different formulas for protein A standards, and the detection of the samples using different specific residual protein A detection kits.

[0033] Table 4A shows the residual protein A content in samples purified by AT ProteinA Diamond Plus affinity chromatography column and detected using Cygnus F610 with AT ProteinA Diamond Plus protein A. Table 4B shows the residual protein A content in samples purified by AT ProteinA Diamond Plus affinity chromatography column and detected using Cygnus F610. Table 4C shows the residual protein A content in samples purified by AT ProteinA Diamond Plus affinity chromatography column and detected using Cygnus F610. (-0.191+1.034logX) Then, calculate REC% using the data from Table 4A. Table 4A. Residual protein A content detected by Cygnus F610 with ATProteinA Diamond Plus affinity chromatography after purification of sample AT ProteinA Diamond Plus. Sample 4-1 48.8565 Sample 4-2 183.7145 Sample 4-3 42.974 Sample 4-4 31.196 Table 4B. The content of residual protein A in the samples after purification by AT ProteinA Diamond Plus affinity chromatography was determined by Cygnus F610. Sample 4-1 57.6 Sample 4-2 234.0 Sample 4-3 51.0 Sample 4-4 39.2 Table 4C. Substitute the data from Table 4B into the formula Y = 10 (-0.191+1.034logX) Then, calculate REC% using the data from Table 4A. Example 5: Detection of Protein A in PD-1 Antibody Drugs using the AT MaXtar ARPA Combination Assay Kit Cygnus F610

[0034] Similar to the steps in Implementation 1, the difference lies in the use of different affinity packing materials to purify the samples in steps A and B, the use of different formulas for establishing protein A standards, and the use of different specific residual protein A detection kits to detect the samples.

[0035] Table 5A shows the residual protein A content in samples purified by MaXtar ARPA affinity chromatography column, detected using a Bipsys RES-A024 analyzer; Table 5B shows the residual protein A content in samples purified by AT ProteinAD Diamond Plus affinity chromatography column, detected using a Cygnus F610 analyzer; Table 5C shows the residual protein A content in samples obtained by substituting the data from Table 5B into the formula Y = 10. (-0.122+0.983logX) Then, calculate REC% using the data from Table 5A. Table 5A. Residual protein A content of samples after purification by MaXtar ARPA affinity chromatography column, as determined by Bipsys RES-A024. Sample 5-1 528.7 Sample 5-2 224.9 Sample 5-3 626.0 Sample 5-4 224.4 Sample 5-5 64.0 Samples 5-6 36.4 Table 5B. The content of residual protein A in the samples after purification by MaXtar ARPA affinity chromatography was determined by Cygnus F610. Sample 5-1 856.3 Sample 5-2 303.0 Sample 5-3 910.0 Sample 5-4 338.7 Sample 5-5 129.7 Samples 5-6 48.6 Table 5C. Substitute the data from Table 5B into the formula Y = 10 (-0.122+0.983logX) Then, calculate the RSD% using the data from Table 5A. Example 6: Detection of Protein A in PD-1 Antibody Drugs using MabPurix A65 Excel Combined with Cygnus F610 Detection Kit

[0036] Similar to the steps in Implementation 1, the difference lies in the use of different affinity packing materials to purify the samples in steps A and B, the use of different formulas for establishing protein A standards, and the use of different specific residual protein A detection kits to detect the samples.

[0037] Table 6A shows the samples purified using a MabPurix A65 Excel affinity chromatography column, with the residual protein A content detected using a Primit PA517 analyzer. Table 6B shows the samples purified using a MabPurix A65 Excel affinity chromatography column, with the residual protein A content detected using a Cygnus F610 analyzer. Table 6C shows the results of substituting the data from Table 6B into the formula Y = 10. (-0.017+0.98logX) Then, calculate the RSD% using the data from Table 6A. Table 6A. Residual protein A content of samples purified by MabPurix A65 Excel affinity chromatography column, as determined by Primit PA517. Sample 6-1 126.1 Sample 6-2 96.1 Sample 6-3 107.3 Sample 6-4 102.6 Sample 6-5 29.7 Table 6B. The content of residual protein A in the samples after purification by MabPurix A65 Excel affinity chromatography was determined by Cygnus F610. Table 6C. Substitute the data from Table 6B into the formula Y = 10 (-0.017+0.98logX) Then, calculate REC% using the data from Table 4A. Sample 6-1 130.4 96.7 Sample 6-2 90.6 106.1 Sample 6-3 87.9 122.1 Sample 6-4 98.8 103.8 Sample 6-5 28.9 102.9 REFERENCES 1.M.Zarrineh,et al,Mechanism of antibodies purification by proteinA.Analytical Biochemistry 609(2020)113909 2.H.Hjelm, et al, Protein A from Staphylococcus aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins, FEBS Lett. 28 (1972) 73–76. 3.A.Ramos,et al,Protein A chromatography:Challenges and progress in the purification of monoclonal antibodies.J Sep Sci(42)2019 1816–1827. 4. V. Amritkara, et al, Engineering Staphylococcal Protein A for high-throughput affinity purification of monoclonal antibodies. Biotechnology Advances 44 (2020) 107632.

[0038] It should be understood that if any prior art publications are cited in this document, such citation does not imply an admission that such publications constitute part of the common knowledge in the field in any country.

[0039] All publications, patents, patent applications, and published patent applications mentioned herein by way of reference are incorporated herein by reference in their entirety.

[0040] Although the foregoing invention has been described in detail by way of illustration and examples for purposes of clarity, it will be apparent to those skilled in the art that some minor changes and modifications will be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention.

Claims

1. A method for detecting residual protein A in an antibody drug, comprising: (a) The antibody drug is purified by affinity chromatography using an affinity packing material to obtain the purified antibody drug; wherein the affinity packing material is selected from one of MabSelect PrismA, Nmab Pro, AT ProteinA Diamond Plus, MaXtar ARPA, MabPurix A65 Excel, and Praesto Jetted A50 HipH; (b) The content X of residual protein A in the purified antibody drug was detected by enzyme-linked immunosorbent assay (ELISA) with Cygnus F610 kit; (c) Substitute the residual protein A content X obtained in step (b) into the formula corresponding to the affinity filler actually used in step (a) to calculate the corrected residual protein A content Y; the formula is selected from the following list: 。 2. The method of claim 1, wherein the formula is selected from the following list:

3. The method according to claim 2, wherein, The formula Y = 10 (0.1205+1.022logX) The method for obtaining it includes the following steps: (d11) Using the protein A standard solution 1 with a concentration of 0-10 ng / mL in the Cygnus F610 kit, and the anti-protein A antibody in the Cygnus F610 kit, a standard curve 1 was established between the protein A concentration Y and the absorbance value at 450 nm. (d12) Using the ligand protein of the affinity filler MabSelect PrismA, a series of standard solutions with concentrations of 0–10 ng / mL were prepared 2. The anti-protein A antibody in the Cygnus F610 kit was used to establish a standard curve 2 of protein A concentration X and absorbance at 450 nm. (d13) Establish the concentration relationship between standard curve 1 and standard curve 2 under the same absorbance value, i.e., Y = 10 (0.1205 +1.022logX) ; The formula Y = 10 (0.011+1.011logX) The method for obtaining it includes the following steps: (d21) Using the protein A standard solution 1 with a concentration of 0-10 ng / mL in the Cygnus F610 kit, and the anti-protein A antibody in the Cygnus F610 kit, a standard curve 1 of protein A concentration Y and absorbance at 450 nm was established. (d22) Using the ligand protein of the affinity filler Nmab Pro, a series of standard solutions with concentrations of 0-10 ng / mL were prepared. Using the anti-protein A antibody in the Cygnus F610 kit, a standard curve of protein A concentration X and absorbance at 450 nm was established. (d23) Establish the concentration relationship between standard curve 1 and standard curve 2 under the same absorbance value, i.e., Y = 10 (0.011 +1.011logX) ; The formula Y = 10 (-0.191+1.034logX) The method of obtaining it includes the following steps: (d31) Using the protein A standard solution 1 with a concentration of 0-10 ng / mL in the Cygnus F610 kit, and the anti-protein A antibody in the Cygnus F610 kit, a standard curve 1 of protein A concentration Y and absorbance at 450 nm was established. (d32) Using the ligand protein of the affinity filler AT ProteinA Diamond Plus, a series of standard solutions with concentrations of 0-10 ng / mL were prepared 2. The anti-protein A antibody in the Cygnus F610 kit was used to establish a standard curve 2 of protein A concentration X and absorbance at 450 nm. (d33) Establish the concentration relationship between standard curve 1 and standard curve 2 under the same absorbance value, i.e., Y = 10 (-0.191+1.034logX) ; The formula Y = 10 (-0.122+0.983logX) The method of obtaining it includes the following steps: (d41) Using the protein A standard solution 1 with a concentration of 0-10 ng / mL in the Cygnus F610 kit, and in combination with the anti-protein A antibody in the Cygnus F610 kit, a standard curve 1 of protein A concentration Y and absorbance at 450 nm was established. (d42) Using the ligand protein corresponding to the affinity filler MaXtar ARPA, a series of standard solutions with concentrations of 0-10 ng / mL were prepared 2. The anti-protein A antibody in the Cygnus F610 kit was used to establish a standard curve 2 of protein A concentration X and absorbance at 450 nm. (d43) Establish the concentration relationship between standard curve 1 and standard curve 2 under the same absorbance value, i.e., Y = 10 (-0.122+0.983logX) ; The formula Y = 10 (-0.017+0.981ogX) The method of obtaining it includes the following steps: (d51) Using the protein A standard solution 1 with a concentration of 0-10 ng / mL in the Cygnus F610 kit, and in combination with the anti-protein A antibody in the Cygnus F610 kit, a standard curve 1 of protein A concentration Y and absorbance at 450 nm was established. (d52) Using the ligand protein corresponding to the affinity filler MabPurix A65 Excel, a series of standard solutions with concentrations of 0–10 ng / mL were prepared. Using the anti-protein A antibody in the Cygnus F610 kit, a standard curve of protein A concentration X and absorbance at 450 nm was established. (d53) Establish the concentration relationship between standard curve 1 and standard curve 2 under the same absorbance value, i.e., Y = 10 (-0.017+0.98logX) ; The formula Y = 10 (0.57+1.134logX) The method of obtaining it includes the following steps: (d61) Using the protein A standard solution 1 with a concentration of 0-10 ng / mL in the Cygnus F610 kit, and the anti-protein A antibody in the Cygnus F610 kit, a standard curve 1 was established between the protein A concentration Y and the absorbance value at 450 nm. (d62) Using the ligand protein corresponding to the affinity filler Praesto Jetted A50 HipH, a series of standard solutions with concentrations of 0-10 ng / mL were prepared 2. The anti-protein A antibody in the Cygnus F610 kit was used to establish a standard curve 2 of protein A concentration X and absorbance at 450 nm. (d63) Establish the concentration relationship between standard curve 1 and standard curve 2 under the same absorbance value, i.e., Y = 10 (0.57 +1.134logX) .

4. The method according to claim 1, wherein, The content Y of the corrected residual protein A was statistically analyzed against the detection values ​​of the fixed-pair affinity filler and detection kit for no less than four samples.

5. The method according to claim 1, wherein the recovery rate calculated by the following formula is in the range of 50% to 150%:

6. The method according to claim 1, wherein, The antibody drugs include monoclonal antibody drugs, bispecific antibodies, and trispecific antibodies.

7. The method according to claim 6, wherein, The antibody drug is a PD-1 antibody.

8. The method according to claim 6, wherein, The antibody drug is a CLDN6×CD3 bispecific antibody.

Images