Kit and method for detecting residual recombinant carboxypeptidase b in insulin aspart

Abstract

The present application relates to a kind of chemiluminescence immunoassay kit for detecting the residual amount of recombinant carboxypeptidase B in aspart insulin, it is characterized in that, it includes: enzyme-labeled plate, recombinant carboxypeptidase B antibody, recombinant carboxypeptidase B standard solution, diluent and biotin-labeled recombinant carboxypeptidase B antibody, and streptavidin-biotin-enzyme combination or the reagent combination for generating streptavidin-biotin-enzyme combination, and the developing substrate of enzyme in streptavidin-biotin-enzyme combination;Wherein, the diluent is 0.1-10mg / mL recombinant human insulin buffer solution.The present application also relates to a kind of method for detecting the residual amount of recombinant carboxypeptidase B in aspart insulin.The method of the present application eliminates the interference of aspart insulin to detection system, so that the detection result is more accurate and reliable, the residual amount of recombinant carboxypeptidase B in aspart insulin can be accurately detected.The method is good in repeatability, high in accuracy, and can be used for the product quality control of aspart insulin.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a chemiluminescent immunoassay kit for detecting the residual amount of recombinant carboxypeptidase B in aspart insulin, and also to a method for detecting the residual amount of recombinant carboxypeptidase B in aspart insulin. Background Technology

[0002] In the production of aspart insulin, recombinant carboxypeptidase B (CPB) is used to enzymatically cleave the arginine and lysine residues at the C-terminus of the aspart insulin precursor peptide chain. This process introduces new impurities, posing a potential risk of CPB contamination and affecting the quality of aspart insulin. These impurities may be toxic or trigger immune responses. To ensure the quality of aspart insulin, the residual amount of CPB must be kept to a minimum. Currently, there are no national, pharmacopoeia, or industry standards for detecting residual CPB in biopharmaceuticals. Existing detection methods have low accuracy and reliability. Therefore, there is an urgent need to develop a method that can accurately and reliably detect the residual amount of CPB in aspart insulin. Summary of the Invention

[0003] The purpose of this invention is to provide a chemiluminescent immunoassay kit for detecting the residual amount of recombinant carboxypeptidase B in aspart insulin. This kit can be used to accurately detect the residual amount of recombinant carboxypeptidase B in aspart insulin.

[0004] Another objective of this invention is to provide a method for detecting the residual amount of recombinant carboxypeptidase B in insulin aspart. This method effectively eliminates the interference of insulin aspart on the detection system, making the detection results more accurate and reliable. Furthermore, this method has good repeatability and high accuracy, and can be used for product quality control of insulin aspart.

[0005] To achieve the above objectives, the present invention provides the following technical solutions.

[0006] A chemiluminescent immunoassay kit for detecting recombinant carboxypeptidase B residues in aspart insulin, characterized in that it comprises: an enzyme-labeled plate, recombinant carboxypeptidase B antibody, recombinant carboxypeptidase B standard solution, diluent, and biotin-labeled recombinant carboxypeptidase B antibody, as well as a streptavidin-biotin-enzyme conjugate or a reagent combination for generating the streptavidin-biotin-enzyme conjugate, and a chromogenic substrate for the enzyme in the streptavidin-biotin-enzyme conjugate;

[0007] The diluent is a buffer solution containing 0.1-10 mg / mL recombinant human insulin.

[0008] Preferably, the diluent is obtained by diluting recombinant human insulin with HEPES buffer, carbonate buffer, phosphate buffer, or acetate-sodium acetate buffer.

[0009] Preferably, the concentration of recombinant human insulin in the diluent is 0.625-5 mg / mL, and more preferably 0.625-3.0 mg / mL.

[0010] Preferably, the kit further includes one or more of coating buffer, blocking solution, washing solution and stop solution.

[0011] Preferably, the reagent combination includes streptavidin, biotin, and an enzyme, or includes streptavidin and a biotin-enzyme conjugate; preferably, the enzyme is horseradish peroxidase or alkaline phosphatase.

[0012] Preferably, the chromogenic substrate is o-phenylenediamine OPD, 3,3',5,5'-tetramethylbenzidine TMB, 3,3'-diaminobenzidine DAB, benzidine, tetramethylbenzidine sulfate TMB-PS, 5-aminosalicylic acid, 2,2'-diazo-bis(3-ethylbenzothiazole-6-sulfonic acid) ammonium salt, 3-amino-9-ethylcarbazole AEC, 4-chloro-1-naphthol, 4-nitrophenyl phosphate disodium salt PNPP, 5-bromo-4-chloro-3-indole phosphate p-toluidine salt BCIP, or nitrotetrazolium chloride NBT.

[0013] Preferably, the recombinant carboxypeptidase B standard solution is either undiluted or diluted with the diluent. Preferably, the concentration of recombinant carboxypeptidase B in the recombinant carboxypeptidase B standard solution diluted with the diluent is 0.5-1000 ng / mL, more preferably 0.7-500 ng / mL, and even more preferably 0.781-400 ng / mL.

[0014] A method for detecting the residual amount of recombinant carboxypeptidase B in insulin aspart, characterized by comprising the following steps:

[0015] The ELISA plate was coated with recombinant carboxypeptidase B antibody;

[0016] Dilute the recombinant carboxypeptidase B standard solution with diluent to obtain working standard solutions of different concentrations;

[0017] Dilute the sample to be tested with a diluent to obtain the test solution;

[0018] Then, add the working standard solution or the test solution to the ELISA plate coated with the recombinant carboxypeptidase B antibody, and incubate the reaction.

[0019] Then, biotin-labeled recombinant carboxypeptidase B antibody was added, and the reaction was incubated.

[0020] Then, streptavidin-biotin-enzyme conjugate was added, and the reaction was incubated.

[0021] Then, the chromogenic substrate of the enzyme in the streptavidin-biotin-enzyme conjugate is added to carry out the chromogenic reaction;

[0022] The absorbance of the solution after the colorimetric reaction is measured;

[0023] A standard curve was established by performing correlation analysis between the concentration of the working standard solution and its corresponding absorbance.

[0024] The residual amount of recombinant carboxypeptidase B in the sample is calculated using the working standard curve and the absorbance of the sample solution to be tested.

[0025] The diluent is a buffer solution containing 0.1-10 mg / mL recombinant human insulin.

[0026] Preferably, the correlation analysis method is four-parameter fitting.

[0027] Preferably, the diluent is obtained by diluting recombinant human insulin with HEPES buffer, carbonate buffer, phosphate buffer, or acetate-sodium acetate buffer.

[0028] Preferably, the enzyme in the streptavidin-biotin-enzyme conjugate is horseradish peroxidase or alkaline phosphatase.

[0029] Preferably, the chromogenic substrate is o-phenylenediamine OPD, 3,3',5,5'-tetramethylbenzidine TMB, 3,3'-diaminobenzidine DAB, benzidine, tetramethylbenzidine sulfate TMB-PS, 5-aminosalicylic acid, 2,2'-diazo-bis(3-ethylbenzothiazole-6-sulfonic acid) ammonium salt, 3-amino-9-ethylcarbazole AEC, 4-chloro-1-naphthol, 4-nitrophenyl phosphate disodium salt PNPP, 5-bromo-4-chloro-3-indole phosphate p-toluidine salt BCIP, or nitrotetrazolium chloride NBT.

[0030] Preferably, the method further includes diluting the recombinant carboxypeptidase B antibody with a coating buffer, wherein the concentration after dilution is preferably 20-100 ng / μl, for example 30-70 ng / μl.

[0031] Preferably, the method further includes diluting the biotin-labeled recombinant carboxypeptidase B antibody with a sample dilution buffer, wherein the concentration after dilution is preferably 2.0-5.0 μg / ml, and more preferably, the concentration of the added biotin-labeled recombinant carboxypeptidase B antibody is 2.2-3.5 μg / ml.

[0032] Preferably, the concentration of recombinant human insulin in the diluent is 0.625-5 mg / mL, and more preferably 0.625-3.0 mg / mL.

[0033] Preferably, the concentration of recombinant carboxypeptidase B in the working standard solution is 0.5-1000 ng / mL, more preferably 0.7-500 ng / mL, and even more preferably 0.781-400 ng / mL.

[0034] Preferably, the concentration of insulin aspart in the test solution is 0.1-10 mg / mL, for example 1.25-5 mg / mL.

[0035] Preferably, the four-parameter fitting equation is as follows:

[0036]

[0037] in:

[0038] Y represents the absorbance value;

[0039] X represents the concentration of recombinant carboxypeptidase B, in ng / ml;

[0040] B is the slope of the curve;

[0041] A is the estimate of the asymptote below the curve;

[0042] D is the asymptote estimate on the curve;

[0043] C represents the concentration at which the maximum binding is halved, in ng / ml.

[0044] The beneficial effects achieved by this invention are:

[0045] To accurately detect the residual amount of recombinant carboxypeptidase B in insulin aspart, this invention uses a buffer solution containing recombinant human insulin without carboxypeptidase B as a diluent for preparing the working standard solution and the test solution. This eliminates the interference of insulin aspart on the detection system, making the detection results more accurate and reliable. The method was validated with reference to the Technical Guideline 9101 of Part III of the 2020 edition of the Chinese Pharmacopoeia. The method has good repeatability and high accuracy and can be used for the product quality control of insulin aspart. Attached Figure Description

[0046] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention.

[0047] Figure 1This is a four-parameter equation fitting plot for the absorbance values ​​at each concentration point of the calibration standard curve (with 2.5 mg / ml recombinant human insulin as an additive) and the uncalibrated standard curve. Detailed Implementation

[0048] To facilitate understanding of the present invention, the technical solutions described below are further illustrated with specific embodiments, but the present invention is not limited thereto. Reagents, instruments, or operating procedures not described herein are all matters that can be conventionally determined by those skilled in the art.

[0049] Example

[0050] Example 1

[0051] Solution Preparation

[0052] 1) Prepare coating buffer

[0053] The coating buffer is a carbonate buffer, and its preparation steps include: weighing about 1.5g of anhydrous sodium carbonate powder and about 2.9g of sodium bicarbonate, dissolving them in water and making up to 1000ml, and measuring the pH value to be about 9.6.

[0054] 2) Prepare a 20mM sample dilution buffer.

[0055] Take one packet of PBS powder, dissolve it completely in ultrapure water and bring the volume to 2L, then mix well.

[0056] 3) Prepare a 40mM washing solution (also known as PBST solution).

[0057] Take one packet of PBS powder, dissolve it in 2L of ultrapure water, add 1ml of Tween 20, mix well, and store at 25℃ for later use.

[0058] 4) Prepare the blocking solution (3% commercial serum)

[0059] Take 1.5 ml of rabbit serum, add 48.5 ml of PBST solution, and mix well.

[0060] 5) Prepare the stop solution

[0061] Measure 54.3 ml of concentrated sulfuric acid and slowly add it along the wall of a beaker containing 400 ml of ultrapure water. After it cools, add more ultrapure water to make the final volume 500 ml, shake well, and store at 25°C for later use.

[0062] 6) Prepare dilution buffer / blank solution (sample dilution buffer containing 2.5 mg / ml recombinant human insulin)

[0063] Weigh 125.0 mg of recombinant human insulin lyophilized powder, add 50 ml of sample dilution buffer, shake for 10 s, then place at 37 ℃ until completely dissolved, shake for another 20 s to mix thoroughly.

[0064] 7) Prepare SABC working solution

[0065] Measure 10 ml of the secondary antibody dilution buffer and add 10 μl of reagent A (streptavidin) and reagent B (biotin-labeled horseradish peroxidase (Biotin-HRP)) from the SABC-HRP kit (manufacturer: Beyotime, catalog number: P0603). Mix well and incubate at 25°C in the dark for at least 30 min. (The SABC working solution should be prepared immediately after adding the biotin-labeled recombinant carboxypeptidase B antibody to the ELISA plate).

[0066] 8) Preparation of test solution

[0067] ① Test sample stock solution (5mg / ml): Weigh approximately 25mg of insulin aspart accurately, add the prepared diluent to dissolve and dilute to 5mg / ml, mix well, and the solution is ready.

[0068] ② Test solution (2.5 mg / ml): Take 200 μl of the test sample stock solution, add 200 μl of the prepared diluent, and vortex for 20 seconds to mix thoroughly. Prepare two parallel solutions.

[0069] 9) Prepare working standard solution

[0070] One vial of recombinant carboxypeptidase B standard solution was thawed at 25°C, and the concentration of recombinant carboxypeptidase B was calibrated to 1543.5 μg / ml.

[0071] The preparation and dilution process of working standard solutions of various concentrations are shown in Table 1 below.

[0072] Table 1 Working standard solutions of different concentrations

[0073]

[0074] <Determination Method>

[0075] (1) Dilute the recombinant carboxypeptidase B antibody with a concentration of 2.97 mg / ml to 50 ng / μl using the coating buffer prepared above, add 100 μl / well to a 96-well microplate, and incubate at 37°C for 2 h.

[0076] (2) Pour out the liquid in the well, pat dry the remaining liquid on absorbent paper, add the sealing solution prepared above at 200 μl / well, incubate at 37°C for 2 hours, pour out the liquid in the well, and pat dry the remaining liquid on absorbent paper.

[0077] (3) Add the working standard solution (tube number 3-12), test solution and blank solution prepared above to the microplate at 100 μl / well, three wells for each concentration, and incubate at 37℃ for 1.5 h; pour out the liquid in the well and pat dry the remaining liquid on absorbent paper; add 300 μl of the washing solution prepared above to each well and wash, pour out the liquid in the well and pat dry on absorbent paper, repeat this operation four times.

[0078] (4) Add 100 μl of biotin-labeled recombinant carboxypeptidase B antibody (3 μg / ml) diluted with the sample dilution buffer prepared above to each well, and incubate at 37°C for 1 h; discard the liquid in the well and pat dry the remaining liquid on absorbent paper; add 300 μl of washing buffer to each well and wash, discard the liquid in the well and pat dry on absorbent paper, and repeat this operation four times.

[0079] (5) Add the prepared SABC working solution to the microplate at 100 μl / well, and then place it in a dark place at 25°C for 30 min; pour out the liquid in the well and pat dry the remaining liquid on absorbent paper; add 300 μl of washing buffer to each well and wash, pour out the liquid in the well and pat dry on absorbent paper, and repeat this operation four times.

[0080] (6) Add 100 μl of the high-sensitivity TMB substrate solution (manufacturer: Array Bridge, catalog number: AB000802) (used directly in commercial form, undiluted) to the microplate and incubate at 37°C in the dark for 10 min without shaking.

[0081] (7) Add the stop solution prepared above to the microplate at a rate of 50 μl / well, and measure the absorbance using a microplate reader at a wavelength of 450 nm.

[0082] <Calculation>

[0083] Based on the concentrations and corresponding absorbance values ​​of the working standard solutions, a standard curve was plotted using four-parameter fitting. The absorbance values ​​of the two prepared test solutions were substituted into the following standard curve equation to calculate the concentration (average value) of recombinant carboxypeptidase B in the test solutions:

[0084]

[0085] in:

[0086] Y represents the absorbance value;

[0087] X represents the concentration of recombinant carboxypeptidase B, in ng / ml;

[0088] B is the slope of the curve;

[0089] A is the estimate of the asymptote below the curve;

[0090] D is the asymptote estimate on the curve;

[0091] C represents the concentration at which half of the maximum binding is achieved, in ng / ml;

[0092] Example 2

[0093] The accuracy of standard curve calibration for recombinant human insulin and other additives was verified and compared. The experimental steps are as follows:

[0094] Working standard solutions and test solutions of various concentrations (5 mg / ml, 2.5 mg / ml, and 1.25 mg / ml) were prepared using sample dilution buffers containing 2.5 mg / ml recombinant human insulin, 2.5 mg / ml degludec insulin, 2.5 mg / ml M18K, and sample dilution buffers, respectively. Spiked solutions for the test solutions were also prepared (the 5 mg / ml test solution contained recombinant carboxypeptidase B spikes of 100 ng / ml, 50 ng / ml, 25 ng / ml, 12.5 ng / ml, and 6.25 ng / ml; the 2.5 mg / ml test solution contained...). The recombinant carboxypeptidase B was spiked at concentrations of 50 ng / ml, 25 ng / ml, 12.5 ng / ml, and 6.25 ng / ml; the corresponding spiked solutions for the 1.25 mg / ml test sample solution had recombinant carboxypeptidase B spikes of 25 ng / ml, 12.5 ng / ml, 6.25 ng / ml, 3.125 ng / ml, and 1.563 ng / ml. The determination was performed according to the method described in Example 1. The differences in accuracy verification results under the four dilution conditions were compared, and the results are shown in Table 2 below.

[0095] Table 2. Comparison of the accuracy of standard curve calibration using recombinant human insulin and other additives.

[0096]

[0097]

[0098] As can be seen from Table 2, using recombinant human insulin as an additive for curve calibration is the best choice for accurately detecting the residual amount of recombinant carboxypeptidase B in insulin aspart.

[0099] Example 3

[0100] The accuracy of standard curve calibration was verified and compared using different concentrations of recombinant human insulin as additives. The experimental steps are as follows:

[0101] Working standard solutions and test solutions (2.5 mg / ml) of various concentrations were prepared using sample dilution buffers containing 2.5 mg / ml, 1.25 mg / ml, and 0.625 mg / ml recombinant human insulin, respectively. Spiked solutions of the test samples were also prepared (with recombinant carboxypeptidase B spikes of 37.5 ng / ml, 25 ng / ml, 12.5 ng / ml, 6.25 ng / ml, 3.125 ng / ml, and 1.563 ng / ml). The assays were performed according to the method described in Example 1. The differences in accuracy verification results under different concentrations of recombinant human insulin dilution buffers were compared, and the results are shown in Table 3 below.

[0102] Table 3. Comparison of the accuracy of standard curve calibration using different concentrations of recombinant human insulin as additives.

[0103]

[0104]

[0105] As shown in Table 3, after standard curve correction with different concentrations of recombinant human insulin as additives, there was no significant difference in the spiking recovery rate at different spiking amounts of recombinant carboxypeptidase B.

[0106] Example 4

[0107] The experimental procedure for plotting the absorbance values ​​at various concentration points of the calibration standard curve (with 2.5 mg / ml recombinant human insulin as an additive) and the uncalibrated standard curve is as follows:

[0108] Working standard solutions of various concentrations were prepared using sample dilution buffer containing 2.5 mg / ml recombinant human insulin and sample dilution buffer as diluents. The assays were performed according to the method described in the example. The absorbance values ​​and the differences in the four-parameter equation fitting curves for each concentration of standard solution were compared. The results are shown in Table 4 below, and the four-parameter equation fitting graph is shown in the figure below. Figure 1 As shown.

[0109] Table 4 compares the absorbance values ​​at various concentration points used to plot the calibration standard curve (with 2.5 mg / ml recombinant human insulin as an additive) and the uncalibrated standard curve.

[0110]

[0111]

[0112] The accuracy of the calibrated standard curve (with 2.5 mg / ml recombinant human insulin as an additive) and the uncalibrated standard curve were verified and compared. The experimental steps are as follows:

[0113] Working standard solutions and test solutions of various concentrations (5 mg / ml, 2.5 mg / ml, and 1.25 mg / ml) were prepared using sample dilution buffer and sample dilution buffer containing 2.5 mg / ml recombinant human insulin, respectively. Spiked solutions of the test solutions were also prepared (the 5 mg / ml test solution contained recombinant carboxypeptidase B spikes of 100 ng / ml, 50 ng / ml, 25 ng / ml, 12.5 ng / ml, and 6.25 ng / ml; the 2. ...). The corresponding spiking amounts of recombinant carboxypeptidase B in the spiking solutions were 50 ng / ml, 25 ng / ml, 12.5 ng / ml, and 6.25 ng / ml; the corresponding spiking amounts of recombinant carboxypeptidase B in the spiking solutions for the 1.25 mg / ml test sample solution were 25 ng / ml, 12.5 ng / ml, 6.25 ng / ml, 3.125 ng / ml, and 1.563 ng / ml. The determination was performed according to the determination method in Example 1, and the accuracy verification results of the calibration curve and the non-calibration curve were compared. The results are shown in Table 5 below.

[0114] Table 5. Accuracy verification comparison between the calibrated standard curve (with 2.5 mg / ml recombinant human insulin as an additive) and the uncalibrated standard curve.

[0115]

[0116]

[0117] The results in Tables 4 and 5 show that the test solution (insulin aspart) significantly interfered with the detection system of recombinant carboxypeptidase B. When recombinant carboxypeptidase B and insulin aspart were present simultaneously, the absorbance of the enzyme-linked immunosorbent assay (ELISA) system increased significantly, resulting in a significantly higher spiked recovery rate. Therefore, under these conditions, recombinant human insulin, which is produced without recombinant carboxypeptidase B and has high amino acid sequence similarity, was used as an additive in the standard curve (sample) diluent to correct the standard curve before sample detection. This effectively eliminated the interference of insulin aspart in the detection system, ensuring that the spiked recovery rate met the accuracy verification requirements and guaranteeing the accuracy and reliability of the detection results.

[0118] Methodological Validation

[0119] Example 5

[0120] The specificity of the detection method of the present invention was verified by the following experimental steps:

[0121] Prepare appropriate amounts of the following spiked solutions: one aliquot of the test sample spiking solution (the spiking amount of recombinant carboxypeptidase B in the 2.5 mg / ml test sample solution is 1.563 ng / ml), one aliquot of the standard solution (0.781 ng / ml), one blank solution (sample dilution buffer containing 2.5 mg / ml recombinant human insulin), and one test sample solution (2.5 mg / ml). Perform the assay according to the method described in Example 1 to examine the specificity of the method. The specificity verification results are shown in Table 6 below.

[0122] Table 6 Specificity Validation Results

[0123]

[0124]

[0125] As shown in Table 6, the average absorbance of the spiked test solution (0.311) increased compared to the average absorbance of the test solution (0.157). The ratio of the average absorbance of the working standard solution (0.244) at the linear minimum (0.781 ng / ml) to the average absorbance of the blank solution (0.169) was 1.44, which is greater than 1.0. The specificity verification meets the requirements.

[0126] Example 6

[0127] The accuracy of the detection method of the present invention was verified by the following experimental steps:

[0128] Prepare three aliquots (numbered 1-9) each of the following accuracy solutions (37.5 ng / ml (150%), 25 ng / ml (100%), and 12.5 ng / ml (50%): the spiked solutions of the test sample (the spiked amounts of recombinant carboxypeptidase B in the corresponding 2.5 mg / ml test sample solutions are 37.5 ng / ml, 25 ng / ml, and 12.5 ng / ml, respectively). Also prepare two aliquots of the test sample solution (2.5 mg / ml) and standard solutions of each concentration. Add each solution in triplicate to three wells and determine the recombinant carboxypeptidase B concentration according to the determination method described in section 4.2. Report the measured value of recombinant carboxypeptidase B in the test sample solution, the actual measured value of recombinant carboxypeptidase B in each accuracy solution, the inter-well CV value, the single recovery value, the average recovery rate (n=9), and the RSD value of the recovery rate (n=9). The accuracy verification results are shown in Table 7 below.

[0129] Table 7 Accuracy Verification Results

[0130]

[0131]

[0132] As shown in Table 7, the interwell CV values ​​of each accuracy solution were 1.4%, 2.7%, 2.4%, 4.4%, 1.9%, 0.6%, 1.7%, 0.9%, and 2.0%, respectively, all less than 20.0%. The single and average recoveries at each concentration point were between 70.0% and 130.0%, and the RSD value (n=9) of the recoveries at each concentration point was 5.32%, less than 20.0%. The accuracy verification results meet the requirements.

[0133] Example 7

[0134] The precision of the detection method of the present invention was verified by the following experimental steps:

[0135] Prepare three aliquots each of the following accuracy solutions (numbered 1-9): 37.5 ng / ml (150%), 25 ng / ml (100%), and 12.5 ng / ml (50%) (i.e., the spiked solutions of the test sample (the spiked amounts of recombinant carboxypeptidase B in the spiking solutions corresponding to the 2.5 mg / ml test sample solution are 37.5 ng / ml, 25 ng / ml, and 12.5 ng / ml, respectively)); two aliquots of the test sample solution (2.5 mg / ml); working standard solutions of each concentration; and blank solution (sample dilution buffer containing 2.5 mg / ml recombinant human insulin). Each solution is added to three wells in parallel. The determination is performed according to the method described in Example 1. The measured absorbance values ​​are substituted into the standard curve. Report the sample weight, the actual measured value of recombinant carboxypeptidase B in each accuracy solution, the interwell CV value, the content of recombinant carboxypeptidase B per milligram of lyophilized aspart insulin powder, and the RSD value of recombinant carboxypeptidase B measured in each accuracy solution (n=3). The precision verification results are shown in Table 8 below.

[0136] Table 8 Precision Verification Results

[0137]

[0138] As shown in Table 8, the inter-well CV values ​​for parallel addition of the accuracy solutions at each level were 1.4%, 2.7%, 2.4%, 4.4%, 1.9%, 0.6%, 1.7%, 0.9%, and 2.0%, respectively, all not exceeding 20.0%. The RSD values ​​(n=3) for the recombinant carboxypeptidase B content measured in the precision solutions at each level were 7.70%, 3.22%, and 3.86%, respectively, all not exceeding 15.0%. The method precision verification results meet the requirements.

[0139] Example 8

[0140] The daytime precision of the detection method of the present invention was verified, and the experimental steps are as follows:

[0141] Accuracy solutions (i.e., the spiked solutions of the test sample (2.5 mg / ml test sample corresponding to 37.5 ng / ml (150%), 25 ng / ml (100%), and 12.5 ng / ml (50%)) were prepared by the same analyst at different times in the same laboratory. Three aliquots of each solution were prepared (numbered 1-9), along with two aliquots of the test sample solution (2.5 mg / ml). Also prepared were working standard solutions of various concentrations. The absorbance of the sample and blank solution (sample dilution buffer containing 2.5 mg / ml recombinant human insulin) was measured according to the method in Example 1. The measured absorbance values ​​were substituted into the standard curve. The sample weight, the actual measured value of recombinant carboxypeptidase B in each level of accuracy solution operated by the individual, the inter-well CV value, the content of recombinant carboxypeptidase B in each level of accuracy solution, the mean content and RSD value (n=3), and the RSD value of the content of recombinant carboxypeptidase B in each level of accuracy solution operated by the individual on two different days (n=6) were reported. The results of the daytime precision verification are shown in Table 9 below.

[0142] Table 9. Results of Daytime Precision Verification

[0143]

[0144] As shown in Table 9, the interwell CV values ​​of the precision solutions at each level were 1.4%, 2.7%, 2.4%, 4.4%, 1.9%, 0.6%, 1.7%, 0.9%, 2.0%, 6.5%, 10.7%, 6.2%, 4.4%, 5.3%, 6.0%, 7.5%, 6.9%, and 8.1%, respectively, all not exceeding 20.0%. The recombinant carboxypeptides measured in the precision solutions at each level used by the individual on the day of operation... The RSD values ​​(n=3) of enzyme B content were 7.70%, 3.22%, 3.86%, 2.78%, 5.61%, and 1.53%, respectively, all not exceeding 15.0%. The RSD values ​​(n=6) of recombinant carboxypeptidase B content measured by an individual on two different dates at various precision levels were 5.22%, 4.19%, and 4.49%, respectively, all not exceeding 20.0%. The results of the daytime precision verification met the requirements.

[0145] Example 9

[0146] The intermediate precision verification of the detection method of the present invention was carried out through the following experimental steps:

[0147] Three aliquots (numbered 1-9) of accuracy solutions (i.e., test sample spiking solutions with recombinant carboxypeptidase B spikes of 37.5 ng / ml (150%), 25 ng / ml (100%), and 12.5 ng / ml (50%), respectively, were prepared at different times in the same laboratory. These included two aliquots of the test sample solution (2.5 mg / ml test sample solution corresponding to spiking solutions with recombinant carboxypeptidase B spikes of 37.5 ng / ml, 25 ng / ml, and 12.5 ng / ml, respectively). Two aliquots of the test sample solution (2.5 mg / ml), standard solutions of various concentrations, and a blank solution (containing 2...) were also prepared. The sample dilution buffer (5 mg / ml recombinant human insulin) was used to determine the recombinant carboxypeptidase B content according to the assay method in Example 1. The report includes the sample weight, the actual measured value of recombinant carboxypeptidase B in each level of accuracy solution performed by the individual on two different dates, the inter-well CV value, the content of recombinant carboxypeptidase B in each level of accuracy solution, the mean content, and the RSD value (n=3); the RSD value of recombinant carboxypeptidase B content measured by the individual in each level of accuracy solution performed on two different dates (n=6); and the RSD value of recombinant carboxypeptidase B content measured by two experimenters in each level of accuracy solution performed on different dates (n=12). Intermediate precision verification results are shown in Table 10 below.

[0148] Table 10 Intermediate Precision Validation Results

[0149]

[0150]

[0151] As shown in Table 10, the RSD values ​​(n=3) of the recombinant carboxypeptidase B content measured in the precision solutions at each level operated by an individual on the same day were 0.76%, 2.30%, 2.87%, 7.22%, 0.87%, and 2.26%, respectively, none exceeding 15.0%. The RSD values ​​(n=6) of the recombinant carboxypeptidase B content measured in the accuracy solutions at each level operated by an individual on two different days were 10.50%, 8.34%, and 6.05%, respectively, none exceeding 20.0%. The RSD values ​​(n=12) of the recombinant carboxypeptidase B content measured in the accuracy solutions at each level operated by two researchers on different days were 9.00%, 8.23%, and 5.33%, respectively, none exceeding 20.0%. These meet the requirements, and the intermediate precision is qualified.

[0152] Example 10

[0153] The inter-well precision of the detection method of the present invention was verified by the following experimental steps:

[0154] Prepare a 25 ng / ml (100%) accuracy solution (i.e., the spiked solution of the test sample (the spiking amount of recombinant carboxypeptidase B in the spiked solution corresponding to the 2.5 mg / ml test sample solution is 25 ng / ml)), add the sample to 6 wells in parallel, and determine the result according to the method in Example 1. Report the actual measured value of recombinant carboxypeptidase B and its RSD value for each well (n=6). The results of the inter-well precision verification are shown in Table 11 below.

[0155] Table 11 Results of inter-well precision verification

[0156]

[0157] As shown in Table 11, the actual measured RSD value of the recombinant carboxypeptidase B concentration in the 6-well interwell precision solution was 10.10%, which is less than 15.0%, and the interwell precision meets the requirements.

[0158] Example 11

[0159] The detection limit of the detection method of the present invention was verified by the following experimental steps:

[0160] A blank solution (containing 2.5 mg / ml sample dilution buffer for recombinant human insulin) and a 0.781 ng / ml recombinant carboxypeptidase B standard solution were prepared. The absorbance of three replicates was measured according to the method in Example 1, and the average absorbance was calculated. The average absorbance of the 0.781 ng / ml recombinant carboxypeptidase B standard solution was taken as the absorbance value of the detection limit, and the ratio of the average absorbance of the detection limit to the average absorbance of the blank solution was calculated. The detection limit verification results are shown in Table 12 below.

[0161] Table 12 Detection Limit Validation Results

[0162]

[0163] As shown in Table 12, the average absorbance of the 0.781 ng / ml recombinant carboxypeptidase B standard solution was taken as the absorbance value of the detection limit. The ratio of the average absorbance of the detection limit to the average absorbance of the blank solution was 1.44, which is greater than 1.0. The detection limit verification result meets the requirements. The linear minimum point of the standard curve, 0.781 ng / ml, was taken as the LOD value of this detection method, which is equivalent to a residual level of 0.31 ppm of recombinant carboxypeptidase B in the test solution.

[0164] Example 12

[0165] The detection method of the present invention was validated for its limit of quantitation. The experimental steps are as follows:

[0166] Prepare three portions of the following: 6.25 ng / ml (25%) accuracy solution (i.e., the spiking solution of the test sample (the spiking solution corresponding to the 2.5 mg / ml test sample solution contains 6.25 ng / ml of recombinant carboxypeptidase B)); three portions of the following: 3.125 ng / ml (12.5%) accuracy solution (i.e., the spiking solution of the test sample (the spiking solution corresponding to the 2.5 mg / ml test sample solution contains 3.125 ng / ml of recombinant carboxypeptidase B)); and one portion of the following: 1.563 ng / ml (6.25%) accuracy solution. Three aliquots of the test sample spiking solution (i.e., the spiking amount of recombinant carboxypeptidase B in the spiking solution corresponding to the 2.5 mg / ml test sample solution was 1.563 ng / ml) (numbered 10-18), two aliquots of the test sample solution (2.5 mg / ml), and working standard solutions of various concentrations were prepared. The absorbance values ​​were measured according to the method described in Example 1. The measured absorbance values ​​were substituted into the standard curve, and the actual measured value of recombinant carboxypeptidase B for each solution, the inter-well CV value, the single recovery value, the average recovery rate, the RSD value of the recovery rate (n=3), and the LOQ value were reported. The results of the limit of quantitation validation are shown in Table 13 below.

[0167] Table 13 Results of Limit of Quantitation Validation

[0168]

[0169]

[0170] As shown in Table 13, the inter-well CV values ​​for parallel loading of the 12.5% ​​level accuracy solution group were 3.3%, 5.5%, and 1.6%, respectively, all not exceeding 20.0%; the individual recovery rates (55.3%-61.2%) and the average recovery rate (59.0%) were both between 50.0% and 200.0%; the RSD value of the recovery rate (n=3) was 5.48%, less than 20.0%. Therefore, 3.125 ng / ml was selected as the LOQ value for this method, which is equivalent to a residual level of 1.25 ppm of recombinant carboxypeptidase B in the test sample.

[0171] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An immunoassay kit for detecting residual recombinant carboxypeptidase B in insulin aspart, characterized in that, It includes: The enzyme-labeled plate, recombinant carboxypeptidase B antibody, recombinant carboxypeptidase B standard solution, diluent and biotin-labeled recombinant carboxypeptidase B antibody, as well as streptavidin-biotin-enzyme conjugate or reagent combination for generating streptavidin-biotin-enzyme conjugate, and chromogenic substrate of enzyme in streptavidin-biotin-enzyme conjugate; The diluent is a buffer solution containing 0.625-3.0 mg / mL recombinant human insulin.

2. The kit according to claim 1, wherein, The diluent was obtained by diluting recombinant human insulin with HEPES buffer, carbonate buffer, phosphate buffer, or acetate-sodium acetate buffer.

3. The kit according to claim 1, wherein, The kit also includes one or more of coating buffer, blocking solution, washing solution and stop solution.

4. The reagent kit according to claim 1, characterized in that, The reagent combination includes streptavidin, biotin, and an enzyme, or includes streptavidin and a biotin-enzyme conjugate.

5. The reagent kit according to claim 4, characterized in that, The enzyme is horseradish peroxidase or alkaline phosphatase.

6. The reagent kit according to claim 1, characterized in that, The chromogenic substrate is o-phenylenediamine, 3,3',5,5'-tetramethylbenzidine, 3,3'-diaminobenzidine, benzidine, tetramethylbenzidine sulfate, 5-aminosalicylic acid, 2,2'-diazo-bis(3-ethylbenzothiazole-6-sulfonic acid)ammonium salt, 3-amino-9-ethylcarbazole, 4-chloro-1-naphthol, disodium 4-nitrophenyl phosphate, 5-bromo-4-chloro-3-indole phosphate p-toluidine salt, or nitrotetrazazole blue chloride.

7. The kit according to claim 1, characterized in that, The recombinant carboxypeptidase B standard solution is either undiluted or diluted with the diluent.

8. The reagent kit according to claim 7, characterized in that, The concentration of recombinant carboxypeptidase B in the recombinant carboxypeptidase B standard solution diluted with the diluent is 0.5-1000 ng / mL.

9. The reagent kit according to claim 7, characterized in that, The concentration of recombinant carboxypeptidase B in the recombinant carboxypeptidase B standard solution diluted with the diluent is 0.7-500 ng / mL.

10. The kit according to claim 7, characterized in that, The concentration of recombinant carboxypeptidase B in the recombinant carboxypeptidase B standard solution diluted with the diluent is 0.781-400 ng / mL.

11. A method for detecting the residual amount of recombinant carboxypeptidase B in insulin aspart, characterized in that, Includes the following steps: The ELISA plate was coated with recombinant carboxypeptidase B antibody; Dilute the recombinant carboxypeptidase B standard solution with diluent to obtain working standard solutions of different concentrations; Dilute the sample to be tested with a diluent to obtain the test solution; Then, add the working standard solution or the test solution to the ELISA plate coated with the recombinant carboxypeptidase B antibody, and incubate the reaction. Then, biotin-labeled recombinant carboxypeptidase B antibody was added, and the reaction was incubated. Then, streptavidin-biotin-enzyme conjugate was added, and the reaction was incubated. Then, the chromogenic substrate of the enzyme in the streptavidin-biotin-enzyme conjugate is added to carry out the chromogenic reaction; The absorbance of the solution after the colorimetric reaction is measured; A working standard curve was established by performing correlation analysis between the concentration of the working standard solution and its corresponding absorbance. The residual amount of recombinant carboxypeptidase B in the sample was calculated using the working standard curve and the absorbance of the test solution. The diluent is a buffer solution containing 0.625-3.0 mg / mL recombinant human insulin.

12. The method according to claim 11, characterized in that, The correlation analysis method is: four-parameter fitting.

13. The method according to claim 11, characterized in that, The diluent was obtained by diluting recombinant human insulin with HEPES buffer, carbonate buffer, phosphate buffer, or acetate-sodium acetate buffer.

14. The method according to claim 11, characterized in that, The enzyme in the streptavidin-biotin-enzyme conjugate is horseradish peroxidase or alkaline phosphatase.

15. The method according to claim 11, characterized in that, The chromogenic substrate is o-phenylenediamine, 3,3',5,5'-tetramethylbenzidine, 3,3'-diaminobenzidine, benzidine, tetramethylbenzidine sulfate, 5-aminosalicylic acid, 2,2'-diazo-bis(3-ethylbenzothiazole-6-sulfonic acid)ammonium salt, 3-amino-9-ethylcarbazole, 4-chloro-1-naphthol, disodium 4-nitrophenyl phosphate, 5-bromo-4-chloro-3-indole phosphate p-toluidine salt, or nitrotetrazazole blue chloride.

16. The method according to any one of claims 11-15, characterized in that, The concentration of recombinant carboxypeptidase B antibody during coating is 20-100 ng / μl.

17. The method according to any one of claims 11-15, characterized in that, The concentration of recombinant carboxypeptidase B antibody during coating is 30-70 ng / μl.

18. The method according to any one of claims 11-15, characterized in that, The concentration of the added biotin-labeled recombinant carboxypeptidase B antibody was 2.0-5.0 ug / ml.

19. The method according to any one of claims 11-15, characterized in that, The concentration of the added biotin-labeled recombinant carboxypeptidase B antibody was 2.2-3.5 ug / ml.

20. The method according to any one of claims 11-15, characterized in that, The concentration of recombinant carboxypeptidase B in the working standard solution is 0.5-1000 ng / mL.

21. The method according to any one of claims 11-15, characterized in that, The concentration of recombinant carboxypeptidase B in the working standard solution is 0.7-500 ng / mL.

22. The method according to any one of claims 11-15, characterized in that, The concentration of recombinant carboxypeptidase B in the working standard solution is 0.781-400 ng / mL.

23. The method according to any one of claims 11-15, characterized in that, The concentration of insulin aspart in the test solution is 0.1-10 mg / mL.

24. The method according to any one of claims 11-15, characterized in that, The concentration of insulin aspart in the test solution was 1.25-5 mg / mL.

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