Method for detecting concentration of host residual protein and application thereof

Abstract

The invention relates to a host residual protein detection method and application thereof. According to the method disclosed by the invention, the acid-soluble I-type collagen with the triple-helix structure is hydrolyzed by adopting the I-type collagenase through sample pretreatment, and the triple-helix structure is decomposed, so that the host residual protein is fully exposed, the host residual protein and the enzyme-labeled antibody are fully combined, and the detection accuracy of the host residual protein is improved; in addition, no cross reaction exists between the type I collagenase and the enzyme-labeled antibody, the existence of the type I collagenase does not interfere with the detection result, and the specific detection of the host residual protein can be realized. Therefore, the method for detecting the host residual protein is simple, effective and high in accuracy, and has a wide application prospect in detection of the host residual protein in collagen related biological products with triple helix structures.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to a method for detecting residual host protein and application thereof. Background Art

[0002] Host Cell Protein (HCP) refers to the proteins remaining in the host cells during the production of protein drugs, including intracellular proteins, cell wall proteins and extracellular proteins. The presence of HCP may have a negative impact on the purity and efficacy of protein drugs, such as immunogenicity and contamination of degraded proteins. Therefore, accurate and comprehensive HCP detection is a crucial link in the development and production of biologics.

[0003] Up to now, the detection methods of host cell protein residues (hereinafter referred to as HCP) mainly include capillary electrophoresis, high performance liquid chromatography, mass spectrometry, and enzyme-linked immunosorbent assay (hereinafter referred to as ELISA). Each of the above methods has its own advantages and disadvantages: although high performance liquid chromatography has high resolution, its specificity is poor, it cannot accurately identify host proteins, and the data analysis is subjective; capillary electrophoresis and mass spectrometry instruments are expensive, and it is necessary to establish a professional analysis map database, which is difficult and complicated; ELISA, as a mature analytical method, has strong specificity, high sensitivity, and short detection time, and is widely used in the field of antibody and protein quantification.

[0004] Due to its high cost-effectiveness, ELISA has become the most commonly used method for HCP detection in recent years. The main principle of ELISA for HCP detection is to capture HCPs in the sample by coating with polyclonal antibodies against HCPs, and adding HRP markers at the same time so that all HCPs in the sample are marked with this marker, thereby catalyzing the color development of TMB substrate to detect HCP concentration. The optimal pH range for the reaction of enzyme-labeled antibodies and antigens is 6.0-8.0. Overly acidic or alkaline environments will cause significant interference to the test results.

[0005] In the process of preparing collagen products, it is necessary to introduce gene fragments capable of expressing collagen into host cells for expression, and then produce collagen through host cells. In this process, whether it is type I collagen, type II collagen or type III collagen, it has a triple helix structure, which is easy to wrap host residual protein during the production process, and then causes difficulties in detecting host residual protein. In particular, for acid-soluble recombinant type I collagen with a triple helix structure, the optimal pH range for its hydrolysis is between 2.5-3.5. At this time, if you want to hydrolyze type I collagen to expose host cells, the ELISA detection environment will be acidic, which may cause problems such as a decrease in the efficiency of polyclonal antibodies to capture HCPs, resulting in a large deviation from the true value of the test results. For host residual proteins wrapped by acid-soluble recombinant type I collagen with a triple helix structure, ELISA cannot be directly applied for detection. Therefore, it is urgent to establish a method for detecting HCPs wrapped by collagen with a triple helix structure, especially acid-soluble recombinant type I collagen. Summary of the invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art that the residual host protein is wrapped by collagen and cannot be accurately detected.

[0007] To solve the above technical problems, the present invention provides a method for detecting residual host protein and its application. The acid-soluble collagen with a triple helix structure produced by the host cell in the process of producing type I collagen will wrap the residual host protein in its triple helix structure to form an inclusion. Since the triple helix structure wraps the hydrophilic bond and exposes the hydrophobic bond, the inclusion has poor solubility in a neutral pH environment, resulting in the inability of the residual host protein to fully bind to the antibody. The hydrolysis of type I collagen under acidic conditions to expose the residual host protein will affect the ELISA reaction performance. Therefore, it is necessary to obtain a suitable sample pretreatment method so that the antibody and HCP can be normally combined, and the pretreatment of the sample will not affect the concentration detection of the residual host protein. The present invention uses type I collagenase to hydrolyze type I collagen by sample pretreatment, and the triple helix structure is untied, so that the residual host protein is fully exposed, which is convenient for the full combination of the residual host protein and the enzyme-labeled antibody. And after detection, the products of type I collagenase and type I collagen hydrolysis will not affect the concentration detection of the residual host protein, so the concentration of the residual host protein can be accurately detected.

[0008] The first object of the present invention is to provide a method for detecting residual host proteins, comprising the following steps:

[0009] S1. Co-incubate different concentrations of host residual protein standards with enzyme-labeled antibodies, add a colorimetric solution, and detect the optical density value to establish a standard curve between the host residual protein concentration and the optical density value, wherein the pH of the co-incubation is 6.0-8.0;

[0010] S2, incubating the sample to be tested with the type I collagenase solution to obtain the sample to be tested after enzymatic hydrolysis;

[0011] S3, performing the operation of the sample to be tested after enzymatic hydrolysis according to S1, detecting the optical density value, and substituting it into the standard curve of the host residual protein concentration and the optical density value, and calculating the concentration of the host residual protein in the sample to be tested;

[0012] Wherein, the sample to be tested is a host residual protein wrapped by type I collagen, the enzyme-labeled antibody is a Chinese hamster ovary cell K1 host residual protease-labeled antibody, and the enzyme-labeled antibody specifically binds to the host residual protein.

[0013] Furthermore, the Chinese hamster ovary cell K1 host residual protease labeled antibody is provided by Huzhou Shenke Biotechnology Co., Ltd., with the item number: 1301305. The CHO-K1 HCP enzyme labeled antibody is a broad-spectrum antibody with a coverage rate of about 80% for the host protein of CHO cells.

[0014] Furthermore, the type I collagen is acid-soluble and has a triple helical structure, and the optimal pH for dissolution is 2.5-4.0.

[0015] Further, the pH of the co-incubation in step S1 is 6.0-8.0. An overly acidic or alkaline environment may change the structure of the antigen or antibody, affecting its immunological activity, thereby affecting the specificity and sensitivity of the ELISA test. In some cases, an overly acidic or alkaline environment may cause cross-reactions because extreme pH values ​​may expose new antigenic determinants, resulting in nonspecific antibody binding.

[0016] Furthermore, the type I collagenase solution in step S2 is prepared by dissolving type I collagenase in a buffer solution, wherein the ratio of the mass of type I collagenase to the volume of the buffer solution is (5-9) mg: (1-4) mL.

[0017] Furthermore, the buffer is a phosphate buffer.

[0018] Furthermore, the color developing solution in step S1 is 3,3',5,5'-tetramethylbenzidine color developing solution.

[0019] Furthermore, after adding the color developing solution in step S1, incubation is performed in the dark, the incubation temperature is 20-28° C., and the incubation time is 10-20 minutes.

[0020] Furthermore, the co-incubation temperature in step S2 is 30-40° C., and the co-incubation time is 0.5-2 hours.

[0021] Furthermore, in step S2, the ratio of the mass of the sample to be tested to the volume of the type I collagenase solution is (0.1-0.5) g: (0.5-0.7) mL.

[0022] Furthermore, the sample to be tested in step S2 is diluted, and the detection effect is best when the sample to be tested is 5-10 ng / mL, and the diluent is a phosphate buffer containing bovine serum albumin. Due to the specificity of the ELISA reaction, there is a problem of excessive or insufficient binding of antigen-antibody. If excessive binding occurs, non-specific products will increase, and the increase in non-specific reactions will lead to the appearance of false positive results, affecting the accuracy of the experiment; if the binding is insufficient or there is too much remaining sample to be tested, it will lead to a "hook effect", and the excess sample (antigen) may form an antigen-antibody complex, which can no longer bind to the antibody on the solid phase, resulting in a low measurement result or even a false negative.

[0023] Beneficial effects of the present invention:

[0024] The present invention uses type I collagenase to hydrolyze acid-soluble type I collagen with a triple helical structure by sample pretreatment, and the triple helical structure is untied, so that the host residual protein is fully exposed, which is convenient for the full combination of the host residual protein and the antibody, and improves the accuracy of the host residual protein detection; in addition, type I collagenase will not hydrolyze the host residual protein, and there is no cross reaction with the enzyme-labeled antibody. The presence of type I collagenase will not interfere with the detection result of the host residual protein concentration, so it is possible to achieve specific detection of the host residual protein. Therefore, the host residual protein detection method of the present invention is simple, effective and highly accurate, and has broad application prospects for the detection of host residual proteins in collagen-related biological products with a triple helical structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0026] Figure 1 is a standard curve diagram of the host residual protein concentration and optical density value of the present invention;

[0027] Figure 2 This is an experimental diagram of comparative example 1 after hydrolysis by type II collagenase;

[0028] Figure 3 This is the repeatability test result of Example 1;

[0029] Figure 4 It is the test result of the spiked recovery rate of comparative example 1;

[0030] Figure 5 This is an experimental diagram of comparative example 2 after hydrolysis by type III collagenase;

[0031] Figure 6 This is the repeatability test result of Example 2;

[0032] Figure 7 It is the test result of the recovery rate of spiked sample 2. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0034] Example 1

[0035] (1) Preparation of enzymatic solution

[0036] Weigh 7 mg of type I collagenase and dissolve it in 2 mL of sterile PBS buffer.

[0037] (2) Enzymatic hydrolysis

[0038] Obtaining the sample to be tested: introducing a plasmid containing type I collagen gene into CHO-K1 cells for fermentation culture to obtain the sample to be tested, wherein the sample to be tested is a host residual protein wrapped by type I collagen;

[0039] Weigh about 0.3 g of the sample to be tested, add 0.6 mL of enzymatic hydrolysis solution and mix well, incubate at 37° C. for 1 h, and then dilute the sample with 1% BSA diluent to obtain the sample to be tested after enzymatic hydrolysis.

[0040] (3) Preparation of CHO-K1 HCP Standard

[0041] CHO-K1 HCP standard was purchased from Huzhou Shenke Biotechnology Co., Ltd.

[0042] Prepare HCP standards of various concentrations according to Table 1, where the concentration of the standard stock solution is 0.5 mg / mL.

[0043] Table 1 Configuration of CHO-K1 HCP standard in Example 1

[0044]

[0045]

[0046] (4) Antigen-enzyme-labeled antibody incubation reaction

[0047] Add calibrators and samples to be tested: Accurately pipette 50μL of series calibrator solution, diluent (NCS), and the sample to be tested after enzymatic hydrolysis into the corresponding microplate, and make 2 duplicate wells for each; take 1×CHO-K1 HCP enzyme-labeled antibody solution into the sample loading slot, and use a multi-channel pipette to quickly add 100μL / well of CHO-K1 HCP enzyme-labeled antibody solution to the bottom of the microplate well, without introducing bubbles; after the sample is added, seal the microplate with a sealing film, place it on a microplate constant temperature oscillator, and incubate at 500rpm at 25±3℃, away from light, and shake for 2h. Among them, the incubation pH is 6.0-8.0, and the CHO-K1 HCP enzyme-labeled antibody is provided by Huzhou Shenke Biotechnology Co., Ltd. The CHO-K1 HCP enzyme-labeled antibody is a broad-spectrum antibody with a coverage rate of about 80% for the host protein of CHO cells.

[0048] (5) Washing and color development

[0049] After the incubation, wash the microplate with 1×PBST buffer, 300μL / well, and repeat the washing 3 times. The microplate should be immediately operated after washing and should not be left standing; take a suitable volume of TMB color development solution into the sample loading slot, use a multi-channel pipette to quickly add 100μL / well of TMB color development solution to the above microplate, and incubate at room temperature in the dark for 15 minutes. Do not seal with a sealing film in this step.

[0050] (6) Termination and reading

[0051] Set the wavelength of the microplate reader to 450nm and 650nm, and measure the OD value of each well. Do not cover the sealing film or lid during the measurement; use low-concentration hydrochloric acid as the stop solution, take an appropriate volume of the stop solution into the sample loading groove, and use a multi-channel pipette to quickly add 100μL / well of the stop solution to the above microplate, and read the value immediately.

[0052] (7) Data processing

[0053] OD of each well 450 The nm value needs to subtract the OD of each well. 650 nm value;

[0054] After subtracting the OD value of the negative control (NCS) from the OD values ​​of each calibration point and sample, the average of the duplicate wells was taken;

[0055] The standard curve point concentration value and the OD value after the previous step were used as the X-axis and Y-axis parameters for curve fitting (a total of 6 points), and the standard curve and standard curve equation were obtained by four-parameter fitting (such as Figure 1 The standard curve equation is y=8.93068+((0.000235328-8.93068) / (1+(x / 436.78)^1.03527)).

[0056] Substitute the sample's OD value as the Y value into the equation obtained in the previous step, and calculate the sample's HCP concentration (ng / mL) by back-calculating the X value. If the sample is tested after dilution, the sample's HCP concentration (ng / mL) = the measured value after dilution × the dilution factor.

[0057] (8) Test results

[0058] A. According to the experimental steps of (2), 6 samples to be tested after enzymatic hydrolysis were obtained, and the test results are shown in Table 2. The test results show that the RSD of Example 1 is only 2.3%, and the repeatability and consistency of the test results are good. The actual concentration is the measured concentration × the dilution factor.

[0059] Table 2 RSD test results of Example 1

[0060]

[0061] B. The cross reaction between the enzymatic hydrolysis buffer and the CHO-K1 enzyme-labeled antibody was detected. The test results are shown in Table 3. The results show that type I collagenase, diluent, hydrochloric acid and CHO-K1 enzyme-labeled antibody have no cross reaction, and the blank OD values ​​are all lower than the lowest point ST6 of the calibration curve. Therefore, the detection method of this embodiment has good specificity.

[0062] Table 3 Cross-reaction results of Example 1

[0063] name <![CDATA[OD 450 -OF 650 ]]> Diluent (NCS) Blank 0.034640 Enzyme blank 0.034155 ST6 0.086990

[0064] C. Accuracy verification was performed according to the guidelines for analytical method verification in Part IV, 9101 of the 2020 edition of the Chinese Pharmacopoeia. The unspiked group was only used to test the sample obtained after enzymatic hydrolysis in step (2); the low-concentration spiked group was used to test the sample obtained after enzymatic hydrolysis in step (2) after adding 10 ng / mL of CHO-K1 HCP standard; the medium-concentration spiked group was used to test the sample obtained after enzymatic hydrolysis in step (2) after adding 20 ng / mL of CHO-K1 HCP standard; and the high-concentration spiked group was used to test the sample obtained after enzymatic hydrolysis in step (2) after adding 50 ng / mL of CHO-K1 HCP standard. The sample to be tested after enzymatic hydrolysis obtained in step (2) contains both type I collagenase and a mixture after type I collagen hydrolysis, and also contains residual host proteins released after hydrolysis of the triple helix structure of type I collagen. Regardless of whether a low-concentration standard, a medium-concentration standard or a high-concentration standard is added, the recoveries of the three concentrations of high, medium and low are all between 80-120%, which meets the requirements, indicating that type I collagenase or the mixture after type I collagen hydrolysis will not affect the concentration detection of residual host proteins.

[0065] Table 4: Test results of spiked recovery of Example 1

[0066] name Measurement concentration (ng / mL) Theoretical spike concentration (ng / mL) Recovery rate (%) Unspecified 4.821 N / A N / A Low concentration spike 14.250 10 94.29 Medium concentration spike 22.940 20 90.60 High concentration spike 53.260 50 96.88

[0067] D. The optimal concentration range of the sample to be tested and the enzyme-labeled antibody was explored, and the dilution linear recovery rate was between 80-120%, indicating that the accuracy of the results was further guaranteed.

[0068] Table 5 Dilution linear recovery test results of Example 1

[0069] Dilution multiple Measurement concentration (ng / mL) Dilution linearity (%) 25000 6.902 N / A 50000 3.428 99.33 15000 8.608 N / A 30000 4.821 112.01

[0070] Comparative Example 1

[0071] The steps of this comparative example are the same as those of Example 1, except that the enzyme used in this comparative example is type II collagenase. The specific steps are as follows:

[0072] (1) Preparation of enzymatic solution

[0073] Weigh 7 mg of type II collagenase and dissolve it in 2 mL of sterile PBS buffer;

[0074] (2) Enzymatic hydrolysis

[0075] Obtaining the sample to be tested: introducing a plasmid containing the type I collagen gene into CHO-K1 cells for fermentation culture to obtain the sample to be tested, wherein the sample to be tested is a host residual protein encapsulated with type I collagen;

[0076] Weigh about 0.3 g of the sample to be tested, add 0.6 mL of enzymatic hydrolysis solution and mix well, incubate at 37° C. for 1 h, and then use 1% BSA diluent to dilute the sample to a suitable multiple to obtain the enzymatically hydrolyzed sample to be tested.

[0077] (3) Preparation of CHO-K1 HCP Standard

[0078] Prepare HCP standards of various concentrations according to the table below.

[0079] Table 6 Configuration of CHO-K1 HCP standard in Comparative Example 1

[0080] Calibration curve samples Dilution method Concentration (ng / mL) A 10μL standard stock solution + 490μL diluent 10000 ST1 10μL A solution + 990μL diluent 100 ST2 300μL ST1 solution + 100μL diluent 75 ST3 200μL ST2 solution + 175μL diluent 40 ST4 150μL ST3 solution + 350μL diluent 12 ST5 200μL ST4 solution + 200μL diluent 6 ST6 200μL ST5 solution + 200μL diluent 3

[0081] (4) Antigen-enzyme-labeled antibody incubation reaction

[0082] Add calibrators and samples to be tested: Accurately pipette 50 μL of series calibrator solutions, diluent (NCS), and test samples into the corresponding microplates, and make 2 duplicate wells for each well; take 1×CHO-K1 HCP enzyme-labeled antibody solution into the sample loading slot, and use a multichannel pipette to quickly add 100 μL / well of the antibody solution to the bottom of the microplate wells without introducing bubbles; after the sample loading is completed, seal the microplate with a sealing film, place it on a microplate constant temperature oscillator, and incubate at 500 rpm at 25±3℃ in the dark for 2 hours.

[0083] (5) Washing and color development

[0084] After the incubation, wash the microplate with 1×PBST buffer, 300μL / well, and repeat the washing 3 times. The microplate should be immediately operated after washing and should not be left standing; take a suitable volume of TMB color development solution into the sample loading slot, use a multi-channel pipette to quickly add 100μL / well of TMB color development solution to the above microplate, and incubate at room temperature in the dark for 15 minutes. Do not seal with a sealing film in this step.

[0085] (6) Termination and reading

[0086] Set the wavelength of the microplate reader to 450nm and 650nm, and measure the OD value of each well. Do not cover the sealing film or lid during the measurement; use low-concentration hydrochloric acid as the stop solution, take an appropriate volume of the stop solution into the sample loading groove, and use a multi-channel pipette to quickly add 100μL / well of the stop solution to the above microplate, and read the value immediately.

[0087] (7) Data processing

[0088] OD of each well 450 The nm value needs to subtract the OD of each well. 650 nm value;

[0089] After subtracting the OD value of the negative control (NCS) from the OD values ​​of each calibration point and sample, the average of the duplicate wells was taken;

[0090] The standard curve point concentration value and the OD value after the previous step were used as the X-axis and Y-axis parameters for curve fitting (a total of 6 points), and the standard curve was obtained by four-parameter fitting (such as Figure 1 shown).

[0091] Substitute the sample's OD value as the Y value into the equation obtained in the previous step, and calculate the sample's HCP concentration (ng / mL) by back-calculating the X value. If the sample is tested after dilution, the sample's HCP concentration (ng / mL) = the measured value after dilution × the dilution factor.

[0092] (8) Test results

[0093] A. Figure 2As shown, type I collagen after enzymatic hydrolysis by type II collagenase dissolves well under neutral conditions, indicating that type II collagenase can also unravel the triple helix structure of type I collagen and increase the solubility of type I collagen under neutral conditions, thereby fully exposing the residual host protein and fully combining with the enzyme-labeled antibody.

[0094] B. Specificity verification found that the blank OD value of type II collagenase far exceeded the lowest point ST6 of the calibration curve, indicating that although type II collagenase can hydrolyze type I collagen, it will cross-react with the CHO-K1 enzyme-labeled antibody, thus affecting the test results. The method specificity does not meet the requirements of the pharmacopoeia. It is speculated that type II collagenase has a capture site for the enzyme-labeled antibody, competing with the HCP in the sample to be tested for the antibody binding site, or enhancing interference with the TMB color development reaction.

[0095] Table 7 Cross-reaction results of Comparative Example 1

[0096] name <![CDATA[OD 450 -OF 650 ]]> Diluent Blank 0.034256 Collagenase type II blank 0.414109 ST6 0.089314

[0097] C. Perform HCP testing according to steps (1)-(7) and test 6 parallel samples. The test results are as follows: Figure 3 The test results showed that although the average values ​​of the repeatability test results of type I collagenase and type II collagenase were not much different, the test results of type II collagenase were unstable and had large errors. The repeatability of the test results after treatment with type II collagenase was extremely poor, with an RSD of about 57.3%.

[0098] D.Accuracy verification is performed according to the guidelines for analytical method verification in Part IV 9101 of the 2020 edition of the Chinese Pharmacopoeia. Figure 4 As shown, the sample to be tested after enzymatic hydrolysis in this comparative example contains type II collagenase, a mixture of type I collagen hydrolyzed by type II collagenase, and residual host protein, and the recoveries of the three concentrations of high, medium, and low are 160.2%, 200.6%, and 188.2%, respectively, indicating that the sample containing type II collagenase and a mixture of type I collagen hydrolyzed by type II collagenase will affect the concentration detection of residual host protein, which does not meet the requirements of pharmacopoeia methodology validation. If type II collagenase is used for treatment, the test results will be seriously biased high.

[0099] Comparative Example 2

[0100] The steps of this comparative example are the same as those of Example 1, except that the enzyme used in this comparative example is type III collagenase. The specific steps are as follows:

[0101] (1) Preparation of enzymatic solution

[0102] Weigh 7 mg of type III collagenase and dissolve it in 2 mL of sterile PBS buffer;

[0103] (2) Enzymatic hydrolysis

[0104] Obtaining the sample to be tested: introducing a plasmid containing the type I collagen gene into CHO-K1 cells for fermentation culture to obtain the sample to be tested, wherein the sample to be tested is a host residual protein encapsulated with type I collagen;

[0105] Weigh about 0.3 g of the sample to be tested, add 0.6 mL of enzymatic hydrolysis solution and mix well, incubate at 37°C for 1 h, and then use 1% BSA diluent to dilute the sample to a suitable multiple to obtain the enzymatically hydrolyzed sample to be tested.

[0106] (3) Preparation of CHO-K1 HCP Standard

[0107] Prepare HCP standards of various concentrations according to the table below.

[0108] Table 8 Configuration of CHO-K1 HCP standard in Comparative Example 2

[0109]

[0110]

[0111] (4) Antigen-enzyme-labeled antibody incubation reaction

[0112] Add calibrators and samples to be tested: Accurately pipette 50 μL of series calibrator solutions, diluent (NCS), and test samples into the corresponding microplates, and make 2 duplicate wells for each well; take 1×CHO-K1 HCP enzyme-labeled antibody solution into the sample loading slot, and use a multichannel pipette to quickly add 100 μL / well of the antibody solution to the bottom of the microplate wells without introducing bubbles; after the sample loading is completed, seal the microplate with a sealing film, place it on a microplate constant temperature oscillator, and incubate at 500 rpm at 25±3℃ in the dark for 2 hours.

[0113] (5) Washing and color development

[0114] After the incubation, wash the microplate with 1×PBST buffer, 300μL / well, and repeat the washing 3 times. After washing, the microplate should be operated immediately and should not be left standing; take a suitable volume of TMB color development solution into the sample loading slot, use a multi-channel pipette to quickly add 100μL / well of TMB color development solution to the above microplate, and incubate at room temperature in the dark for 15 minutes. Do not seal with a sealing film in this step.

[0115] (6) Termination and reading:

[0116] Set the wavelength of the microplate reader to 450nm and 650nm, and measure the OD value of each well. Do not cover the sealing film or lid during the measurement; use low-concentration hydrochloric acid as the stop solution, take an appropriate volume of the stop solution into the sample loading groove, and use a multi-channel pipette to quickly add 100μL / well of the stop solution to the above microplate, and read the value immediately.

[0117] (7) Data processing:

[0118] OD of each well 450 The nm value needs to subtract the OD of each well. 650 nm value;

[0119] After subtracting the OD value of the negative control (NCS) from the OD values ​​of each calibration point and sample, the average of the duplicate wells was taken;

[0120] The standard curve concentration value and the OD value after the previous step were used as the X-axis and Y-axis parameters for curve fitting (a total of 6 points), and the standard curve and equation (such as Figure 1 shown).

[0121] Substitute the sample's OD value as the Y value into the equation obtained in the previous step, and calculate the sample's HCP concentration (ng / mL) by back-calculating the X value. If the sample is tested after dilution, the sample's HCP concentration (ng / mL) = the measured value after dilution × the dilution factor.

[0122] (8) Test results

[0123] A. Figure 5 As shown, after treatment with type III collagenase, milky white precipitate was found, and type I collagen still could not be dissolved in a neutral pH solution, indicating that type III collagenase could not effectively increase the solubility of type I collagen in a neutral buffer.

[0124] B. Perform HCP testing according to steps (1)-(7) and test 6 parallel samples. The test results are as follows: Figure 6 As shown, the results showed that compared with the test results after type I collagenase treatment, the repeatability of the results after type III collagenase treatment was extremely poor, with an RSD of approximately 157.6%, and the average test result was low, indicating that the performance of the detection method was poor.

[0125] C.According to the guidelines for validation of analytical methods in Part IV 9101 of the 2020 edition of the Chinese Pharmacopoeia, the accuracy is verified. The test results are as follows: Figure 7 As shown, the recoveries of high, medium and low concentrations were 34.1%, 15.3% and 46.0%, respectively, which did not meet the requirements for pharmacopoeia methodology validation. If type III collagenase was used for treatment, the test results would be seriously low.

[0126] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for detecting residual host protein, characterized in that: The following steps are involved: S1. Co-incubate different concentrations of host residual protein standards with enzyme-labeled antibodies, add a colorimetric solution, and detect the optical density value to establish a standard curve between the host residual protein concentration and the optical density value, wherein the pH of the co-incubation is 6.0-8.0; S2, incubating the sample to be tested with the type I collagenase solution to obtain the sample to be tested after enzymatic hydrolysis; S3, performing the operation of the sample to be tested after enzymatic hydrolysis according to S1, detecting the optical density value, and substituting it into the standard curve of the host residual protein concentration and the optical density value, and calculating the concentration of the host residual protein in the sample to be tested; Wherein, the sample to be tested is a host residual protein wrapped by type I collagen, the enzyme-labeled antibody is a Chinese hamster ovary cell K1 host residual protease-labeled antibody, and the enzyme-labeled antibody specifically binds to the host residual protein.

2. The detection method according to claim 1, characterized in that: The co-incubation temperature in step S1 is 22-28°C.

3. The detection method according to claim 1, characterized in that: The co-incubation time in step S1 is 1.5-3 hours.

4. The detection method according to claim 1, characterized in that: The color developing solution in step S1 includes 3,3',5,5'-tetramethylbenzidine color developing solution.

5. The detection method according to claim 4, characterized in that: After adding the color developing solution in step S1, incubation is carried out in the dark, the incubation temperature is 20-28° C., and the incubation time is 10-20 minutes.

6. The detection method according to claim 1, characterized in that: The type I collagenase solution in step S2 is prepared by dissolving type I collagenase in a buffer solution, wherein the ratio of the mass of type I collagenase to the volume of the buffer solution is (5-9) mg: (1-4) mL.

7. The detection method according to claim 6, characterized in that: The buffer is phosphate buffer.

8. The detection method according to claim 1, characterized in that: The co-incubation temperature in step S2 is 30-40°C.

9. The detection method according to claim 1, characterized in that: The co-incubation time in step S2 is 0.5-2 hours.

10. The detection method according to claim 1, characterized in that: In step S2, the ratio of the mass of the sample to be tested to the volume of the type I collagenase solution is (0.1-0.5) g: (0.5-0.7) mL.

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