Preparation method and application of universal detection kit for residual protein of E. coli host

Abstract

This invention relates to the field of biopharmaceutical quality control technology, and particularly to a method for preparing and applying a universal detection kit for residual E. coli host proteins. The method provided in this application offers a method for preparing a universal detection kit for residual E. coli host proteins, which boasts high detection accuracy, a limit of quantitation sensitivity of 1.56 ng / mL, high antibody coverage, and no cross-reactivity with the closely related Pichia yeast. Furthermore, this method uses a one-step incubation process involving the mixed incubation of antigen and detection antibody, resulting in fewer steps, shorter processing time, and a low error rate due to calculations based on standard concentrations. The method of this invention allows for rapid, efficient, and accurate calculation of residual E. coli host protein content, making it applicable to biopharmaceutical quality control.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical quality control technology, and in particular to a method for preparing and applying a universal detection kit for residual proteins in E. coli hosts. Background Technology

[0002] Host cell protein (HCP) residues are a major impurity associated with the production of biopharmaceuticals. The main risk they pose is their immunogenicity. HCPs, complex mixtures with various physiological, chemical, and immunological properties, may trigger an immune response in the human body, inducing the production of corresponding HCP antibodies. Some HCPs also have adjuvant effects, which can cause inflammatory responses in the human body, or combine with antibody drugs and other components to induce immunogenicity against drugs, producing antibodies against the drugs and reducing their efficacy. Therefore, almost all HCPs pose clinical safety risks as foreign protein drugs.

[0003] Existing HCP detection technologies suffer from long detection cycles, lengthy preparation times, and complex procedures, and are significantly affected by environmental factors, thus placing certain demands on the detection environment. Current ELISA kits, some of which provide pre-coated plates and prepared reagents to mitigate the complexity and high error rates of existing experimental procedures, still fail to address the issue of linear range accuracy. Other kits offer high specificity, sensitivity, and accuracy, but the cumbersome pre-preparation process remains unresolved.

[0004] Therefore, in order to solve the above problems, this application provides a method for preparing a universal detection kit for residual proteins in E. coli hosts and its application. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing and applying a universal detection kit for residual proteins in E. coli hosts, in order to solve the problems mentioned in the background art.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] A method for preparing a universal detection kit for residual proteins in E. coli hosts includes the following steps:

[0008] S1. Preparation of Escherichia coli antigen;

[0009] S11. Culture Escherichia coli and centrifuge to obtain bacterial cells;

[0010] S12. Homogenize the bacterial cells from step S11 under high pressure to obtain E. coli antigen;

[0011] S2. Prepare antibodies against sheep anti-Escherichia coli antigen;

[0012] S21. Select goats of appropriate age for immunization;

[0013] S22. After immunization, antiserum was collected and purified to obtain Escherichia coli host residual protein antibody;

[0014] S3. Labeling of Escherichia coli host residual protein antibodies: Horseradish peroxidase was used to label the Escherichia coli host residual protein antibodies prepared in step S22.

[0015] S4. Establishment of an ELISA method for detecting residual proteins in Escherichia coli hosts;

[0016] S41. Coating: Dilute the purified antibody in step S22 with carbonate buffer or phosphate buffer to 5 μg / mL to 25 μg / mL, and coat it at 4°C overnight;

[0017] S42. Blocking: Remove the coating solution by spin-drying, wash 3 times with a washing solution consisting of 0.01% to 0.1% Tween 20 phosphate buffer, and block at 25℃ to 37℃ for 0.5h to 2h with a blocking solution consisting of 1 to 10 wt% sucrose, 1 to 8 wt% trehalose and 0.1 to 6 wt% fish collagen. After blocking, wash 3 times with the washing solution.

[0018] S43. Conjugate the antigen and detection antibody mixture: Use the E. coli antigen prepared in step S12 as a calibrator or the sample to be tested and mix it with the host residual protein labeled antibody diluted at a ratio of 1:1 to 1:3 using a dilution solution consisting of 2% to 8 wt% sucrose, 3% to 8 wt% trehalose and 0.5% to 3 wt% fish collagen at a ratio of 1:50 to 1:400. Add the mixture to the corresponding wells and incubate for 1 to 2 hours.

[0019] S44. After removing the plate solution from step S43 by spin-drying, wash with washing solution, add TMB chromogenic substrate to each well, and incubate at room temperature in the dark for 15-35 min.

[0020] S45. Add 30μL to 150μL of stop solution to each well to stop the reaction, and measure the OD450 value using an enzyme-linked immunosorbent assay reader.

[0021] S46. Use a four-parameter fitting equation to plot a calibration curve, and calculate the concentration of the sample to be tested based on the calibration curve and the OD450 value of the sample to be tested.

[0022] Furthermore, in step S41, the host residual protein antibody purified in step S22 is coated with 0.02-0.2M carbonate buffer.

[0023] Furthermore, the concentration of the calibrator selected in step S43 is 0–400 ng / mL.

[0024] Furthermore, in step S43, the E. coli host residual protein labeled antibody is diluted with a diluent consisting of 3%–6 wt% sucrose, 3.5%–7 wt% trehalose, and 1%–2 wt% fish collagen.

[0025] Furthermore, the sealing solution used in step S42 contains 3-8 wt% sucrose, 2-6 wt% trehalose, and 0.5-5 wt% fish collagen.

[0026] Moreover, the one-step method using antigen and detection antibody mixed incubation can complete the test in just 2 hours.

[0027] Moreover, the antibody coverage rate is shown to be over 80%.

[0028] Moreover, this method showed no cross-reaction with the closely related Pichia yeast.

[0029] Moreover, it has high detection accuracy, with a quantitation limit sensitivity of 1.56 ng / mL, high antibody coverage, and no cross-reactivity with closely related Pichia yeast. In addition, this method uses a one-step method of mixing and incubating antigen and detection antibody, which is simple, quick, and calculates sample concentration using a fixed standard concentration, resulting in a low error rate.

[0030] The second aspect of this invention provides an application of a method for preparing a universal E. coli HCP detection kit, including the application of this E. coli HCP ELISA detection kit in rapid and simple methods for detecting E. coli HCP content.

[0031] The advantages and positive effects of this invention are:

[0032] 1. The E. coli host residual protein universal detection kit prepared by the method of the present invention has high detection accuracy. The detection uses a one-step method of mixing and incubating antigen and detection antibody. The limit of quantitation can reach 1.56 ng / mL. It also has a fixed standard concentration for calculating sample concentration, resulting in a low error rate, which facilitates the smooth conduct of experiments and obtains more reliable and accurate data.

[0033] 2. The preparation method of the universal detection kit for E. coli host residual proteins described in this invention can achieve an antibody coverage rate of over 80% and has no cross-reactivity with the closely related Pichia yeast.

[0034] 3. This invention can omit the step of coating the plate the day before, and the reagent kit contains pre-prepared reagents that can be used directly in the experiment, saving the entire operation time. Furthermore, the one-step method of mixing and incubating the antigen and detection antibody can complete the test in just 2 hours. Attached Figure Description

[0035] Figure 1 This is a graph showing the antibody coverage results of the E. coli HCP ELISA detection kit of the present invention.

[0036] Figure 2 This is a graph showing the four-parameter equations for the E. coli HCP ELISA detection kit of the present invention.

[0037] Figure 3 This is a diagram showing the specificity verification results of the E. coli HCP ELISA detection kit of the present invention.

[0038] Figure 4 This is a flowchart comparing the preparation steps of the conventional kit of this invention and the E. coli HCP ELISA detection kit. Detailed Implementation

[0039] The technical embodiments of the present invention will be clearly and comprehensively described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] It should be noted that the relational terms used in this article, such as first and second, are only used to distinguish between operations and do not require that operations be performed in this relationship or order.

[0041] Example 1

[0042] Sheep anti-E. coli HCP antibody coverage assay

[0043] Using E. coli HCP antigen and goat anti-E. coli HCP antibody as materials, the coverage of goat anti-E. coli HCP antibody was verified by immunoaffinity chromatography-liquid chromatography-mass spectrometry (AAE-MS).

[0044] Based on the final LC-MS analysis data, the protein count was retrieved, and the coverage of the goat anti-E. coli HCP polyclonal antibody was found to be 82%. The results are as follows... Figure 1 As shown.

[0045] Example 2

[0046] Establishment and application of E. coli HCP ELISA detection method

[0047] 1. Establishment of E. coli HCP ELISA Detection Method: To quantify the prepared E. coli HCP, standards were prepared for use in the E. coli HCP residue detection kit. The preparation method is as follows: The prepared E. coli HCP antigen was diluted with diluent to ST1-ST7 and NTC to prepare E. coli HCP standards. The OD450 value was detected using a sandwich ELISA method, and a four-parameter fitting curve was plotted.

[0048] The setup process is as follows:

[0049] (1) Coating: The purified goat anti-E. coli HCP antibody was diluted with coating buffer to 12 μg / mL and 24 μg / mL for coating, and then coated overnight at 4℃.

[0050] (2) Blocking: Spin dry to remove coating liquid, wash 3 times with washing liquid, add blocking solution, and incubate at 37°C for 1 hour;

[0051] (3) Combining antigen and detection antibody: Dilute E. coli HCP standard to 100 ng / mL and 0 ng / mL with dilution buffer for later use. Dilute the freshly prepared HCP-labeled antibody in Example 3 at a ratio of 1:100 with dilution buffer for later use. Mix the two at a volume ratio of 1:1 for later use. Pour off the blocking solution, wash 3 times with washing solution, spin dry, and pat dry upside down on filter paper.

[0052] (4) Discard the liquid, wash 6 times with washing solution, spin dry, and pat dry upside down on filter paper. Add TMB substrate and incubate for 30 minutes;

[0053] (5) Add 50 μL of stop solution to each well to terminate the reaction, and measure the OD450 value using an enzyme-linked immunosorbent assay (ELISA) reader.

[0054] Based on the detection data, the concentration of the coating antibody was determined to be 12 μg / mL, and the optimal dilution ratio of the HCP-labeled antibody was 1:100.

[0055] 2. Correlation of the E. coli HCP ELISA kit calibration curve: Based on the optimal working concentrations of the coating antibody and detection antibody determined above, and while keeping the concentrations of the coating antibody and detection antibody constant, the E. coli HCP calibrator was diluted 2-fold to seven different concentration gradients, with each concentration used in replicates to minimize interference from experimental errors. ELISA detection was performed on the same strip using the antibody pairing method described above to obtain the detection reaction values, and a calibration curve was plotted based on these values. The calibrator concentrations and corresponding reaction values ​​are shown in Table 1.

[0056] Table 1

[0057]

[0058] Using the calibration value and measured OD value of the calibrator as the X and Y axes, a four-parameter fit is performed to plot the calibration curve, as shown below. Figure 2 As shown. The calibrator was diluted to 100 ng / mL, and then serially diluted two-fold until it reached 1.56 ng / mL, with no antigen added at the last point. The antigen concentration was plotted on the x-axis, and the OD450 value obtained from the detection was plotted on the y-axis. The above data were then fitted using a four-parameter algorithm, and the goodness of fit obtained met R0. 2 The requirement of greater than 0.99 indicates that this method has good linearity in the E. coli HCP concentration range of 0-100 ng / mL.

[0059] Example 3

[0060] Performance validation of the E. coli HCP ELISA kit

[0061] 1. Pichia yeast is the organism most likely to cross-contaminate with E. coli in nature. Therefore, in the specificity validation stage, the currently established double-antibody sandwich ELISA method was used to detect Pichia yeast at three concentrations: 400 ng / mL, 100 ng / mL, and 25 ng / mL. A blank value was used as a control reference. Each concentration was tested in two groups. The experimental results are as follows: Figure 3 As shown, the detection values ​​of the three concentrations of Pichia yeast were all lower than the blank value, which confirms that the double-antibody sandwich ELISA method established in this experiment does not have cross-reactivity with Pichia yeast.

[0062] 2. For the limit of quantitation analysis of the E. coli HCP ELISA kit, the lowest concentration of the calibration curve (1.5625 ng / mL) was selected as the limit of quantitation. The calibrator was mixed with an equal volume of the test sample so that the concentration of the calibrator was at the lowest detection point of 1.5625 ng / mL. The recovery rate was determined and the RSD was calculated. The results are shown in Table 2.

[0063] Table 2

[0064]

[0065] According to the experimental results, the recovery rate of the lowest detection limit ranged from 99.84% to 117.76%, which met the standard of 70%-130%, and the recovery rate was qualified. The calculated RSD was 8.41%, which met the requirement of RSD less than 15%. Therefore, the limit of quantitation of this method is 1.5625 ng / mL.

[0066] 3. Repeatability analysis of E. coli HCP ELISA kit

[0067] Using the ELISA method and kit established in Example 1, calibrators of high, medium, and low concentrations were selected. Six independent aliquots of each calibrator were prepared and mixed with the same concentration of the test sample for spike recovery verification. RSD was calculated. The results are shown in Table 3.

[0068] Table 3

[0069]

[0070] The test results showed that, at a high concentration (50 ng / mL), the RDS (n=6) of the calibrator HCP detection in the six samples was 13.10%; at a medium concentration (6.25 ng / mL), the RDS (n=6) was 9.03%; and at a low concentration (1.56 ng / mL), the RDS (n=6) was 13.40%. The RSD values ​​at all three concentrations were less than 15%, therefore the repeatability was satisfactory.

[0071] 4. Precision analysis of E. coli HCP ELISA kit

[0072] Using the ELISA method and kit established in Example 1, another experimenter selected three concentrations of calibrators (high, medium, and low), prepared six independent samples for each concentration, mixed them with the test samples of the same concentration, performed spike recovery verification, and calculated the CV. The results are shown in Table 4.

[0073] Table 4

[0074]

[0075] The test results showed that the RDS (n=6) of the intermediate precision for six samples was 11.62% at high concentration (50 ng / mL), 10.13% at medium concentration (6.25 ng / mL), and 13.85% at low concentration (1.56 ng / mL). The RDS (n=12) of the intermediate precision and repeatability for 12 samples at high concentration was 11.62%, for medium concentration it was 10.13%, and for low concentration it was 13.85%, all meeting the standard of RSD less than 15%, indicating that the intermediate precision was qualified.

[0076] 5. Accuracy analysis of E. coli HCP ELISA kit

[0077] Using the ELISA method and kit established in Example 1, the HCP calibrator was mixed into the test sample in equal volumes at high, medium and low concentrations to determine the recovery rate. The concentration of the test sample was calculated based on the calibration curve and the OD of the test sample obtained in the experiment, and compared with the theoretical concentration. At the same time, a spike recovery experiment was performed using quality control samples to calculate the back-calculated concentration value and spike recovery rate of each sample. The spike recovery rate results are shown in Table 5.

[0078] Table 5

[0079]

[0080] The results show that the recovery rate ranges from 84.12% to 108.8% at high concentrations, from 91.92% to 97.12% at medium concentrations, and from 96% to 104% at low concentrations, all meeting the requirement of 70% to 130%. Furthermore, the RSD (n=3) of the recovery rate is 14.51% at high concentrations, 2.81% at medium concentrations, and 4.15% at low concentrations, all meeting the standard of RSD less than 15%. Therefore, the accuracy of this method is satisfactory.

[0081] In summary, methodological validation has demonstrated that this method exhibits good linearity when fitting the standard curve using a four-parameter equation within a calibrator concentration range of 0-100 ng / ml, with R0. 2 >0.99; the sensitivity detection limit of quantitation is 1.56 ng / ml; the spiked recoveries at high, medium and low calibrator concentrations are all between 80% and 120%, and the RSDs are all less than 15%; the repeatability and intermediate precision RSDs are all less than 15%; and the method does not have cross-reactivity with Pichi yeast.

[0082] Figure 4 This is a flowchart comparing the preparation steps of the traditional kit of this invention and the E. coli HCP ELISA detection kit. The method of this kit takes approximately 2 hours and involves 7 experimental steps; the method of the traditional kit takes approximately 3 hours and involves 9 experimental steps.

[0083] Finally, it should be noted that the above descriptions are only some preferred embodiments of the present invention, and the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made, as long as they are within the spirit and principles of the present invention, should be included within the protection scope of the present invention.

Claims

1. A method for preparing a universal detection kit for residual proteins in E. coli hosts, characterized in that: Includes the following steps: S1. Preparation of Escherichia coli antigen; S11. Culture Escherichia coli and centrifuge to obtain bacterial cells; S12. Homogenize the bacterial cells from step S11 under high pressure to obtain E. coli antigen; S2. Prepare antibodies against sheep anti-Escherichia coli antigen; S21. Select goats of appropriate age for immunization; S22. After immunization, antiserum was collected and purified to obtain Escherichia coli host residual protein antibody; S3. Labeling of Escherichia coli host residual protein antibodies: Horseradish peroxidase was used to label the Escherichia coli host residual protein antibodies prepared in step S22. S4. Establishment of an ELISA method for detecting residual proteins in Escherichia coli hosts; S41. Coating: Dilute the purified antibody in step S22 with carbonate buffer or phosphate buffer to 5 μg / mL to 25 μg / mL, and coat it at 4°C overnight; S42. Blocking: Remove the coating solution by spin-drying, wash 3 times with a washing solution consisting of 0.01% to 0.1% Tween 20 phosphate buffer, and block at 25℃ to 37℃ for 0.5h to 2h with a blocking solution consisting of 1 to 10 wt% sucrose, 1 to 8 wt% trehalose and 0.1 to 6 wt% fish collagen. After blocking, wash 3 times with the washing solution. S43. Conjugate the antigen and detection antibody mixture: Use the E. coli antigen prepared in step S12 as a calibrator or the sample to be tested and mix it with the host residual protein labeled antibody diluted at a ratio of 1:1 to 1:3 using a dilution solution consisting of 2% to 8 wt% sucrose, 3% to 8 wt% trehalose and 0.5% to 3 wt% fish collagen at a ratio of 1:50 to 1:

400. Add the mixture to the corresponding wells and incubate for 1 to 2 hours. S44. After removing the plate solution from step S43 by spin-drying, wash with washing solution, add TMB chromogenic substrate to each well, and incubate at room temperature in the dark for 15-35 min. S45. Add 30μL to 150μL of stop solution to each well to stop the reaction, and measure the OD450 value using an enzyme-linked immunosorbent assay reader. S46. Use a four-parameter fitting equation to plot a calibration curve, and calculate the concentration of the sample to be tested based on the calibration curve and the OD450 value of the sample to be tested.

2. The method for preparing a general detection kit for residual protein of E. coli host according to claim 1, characterized in that: In step S41, the host residual protein antibody purified in step S22 is coated with 0.02-0.2M carbonate buffer.

3. The method for preparing a general detection kit for residual protein of E. coli host according to claim 2, characterized in that: The concentration of the calibrator selected in step S43 is 0–400 ng / mL.

4. The method for preparing a general detection kit for residual protein of E. coli host according to claim 3, characterized in that: In step S43, the E. coli host residual protein labeled antibody is diluted with a diluent consisting of 3%–6 wt% sucrose, 3.5%–7 wt% trehalose, and 1%–2 wt% fish collagen.

5. The method for preparing a general detection kit for residual protein of E. coli host according to claim 4, characterized in that: The blocking solution used in step S42 contains 3-8 wt% sucrose, 2-6 wt% trehalose, and 0.5-5 wt% fish collagen.

6. The method for preparing a general detection kit for residual protein of E. coli host according to claim 5, characterized in that: Using a one-step method that involves mixing and incubating the antigen and detection antibody, the test can be completed in just 2 hours.

7. The method for preparing a universal detection kit for residual E. coli host proteins according to claim 6, characterized in that: The antibody coverage rate is shown to be over 80%.

8. The method for preparing a general detection kit for residual protein of E. coli host according to claim 7, characterized in that: This method showed no cross-reactivity with the closely related Pichia yeast.

9. The method for preparing a universal detection kit for residual E. coli host proteins according to claim 8, characterized in that: It has high detection accuracy, with a quantitation limit sensitivity of 1.56 ng / mL, high antibody coverage, and no cross-reactivity with closely related Pichia yeast. In addition, this method uses a one-step method of mixing and incubating antigen and detection antibody, which is simple, quick, and calculates sample concentration using a fixed standard concentration, resulting in a low error rate.

10. The application of an E. coli host residual protein ELISA detection kit prepared by the method of any one of claims 1-9 in a rapid and simple method for detecting the content of E. coli host residual protein.

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