Detection method and detection kit for biological activity of degludec insulin

Abstract

The present invention relates to the field of biological detection, and more particularly to a detection method and a detection kit for the biological activity of degludec insulin. The present invention tests the biological activity of degludec insulin by culturing cells containing insulin receptors in vitro. After the cells are cultured, prepared samples to be tested and standard products of different concentrations are added to interact with the cells, and a lysis solution containing an inhibitor is added to lyse the cells. After lysis, the cell lysate is transferred to a coated detection plate coated with an antibody, combined, a PY-20 reporter antibody is added, and a color developing solution is added for color development. Finally, a stop solution is used to terminate the reaction, and a multifunctional microplate reader is used for reading. The method provided by the present invention is experimented by an ELISA kit, which is not easily affected by exogenous factors compared with the in vitro cell and mouse blood glucose tests of the prior art, shortens the detection time, and improves the accuracy of the experimental results.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a detection method and a detection kit for the biological activity of degludec insulin. Background Art

[0002] According to statistics from the International Diabetes Federation (IDF), approximately 463 million adults aged 20-79 worldwide suffered from diabetes in 2019. Insulin has a long history as the main drug for the treatment of diabetes. Based on the research and development concept of reducing drug immunogenicity and better simulating the physiological secretion of insulin, the insulin series of products has undergone three generations of product changes: animal insulin, human insulin, and insulin analogs. Many diabetic patients have benefited from this and their quality of life has been significantly improved.

[0003] Degludec insulin is a new generation of insulin analogs: the threonine at position B30 is removed from the human insulin molecule structure, and a 16-carbon fatty diacid is linked to the lysine at position B29 through an L-γ-glutamic acid linker, thereby obtaining an ultra-long-acting insulin analog. This structure allows it to exist in the preparation (containing substances such as zinc, phenol and meta-cresol) in a stable, soluble, double hexamer form. After subcutaneous injection, phenol diffuses rapidly, and degludec insulin forms multiple hexamers and deposits at the injection site, forming a reservoir, which becomes the main delayed action mechanism. As time goes on, the hexamer gradually dissociates into monomers, which diffuse into the capillaries and bind to albumin to be buffered in the blood, becoming a secondary delayed action mechanism. After reaching the target organ, degludec insulin dissociates from the protein and binds to the insulin receptor with higher affinity, activating the insulin receptor to exert its efficacy.

[0004] In the process of drug development, biological activity detection methods are important evaluation indicators for drug screening and quality control. This method must pass method validation and meet the requirements of method validation such as specificity, precision, and accuracy. Only in this way can the reliability of biological activity detection results be guaranteed and can be used to guide drug screening and quality control. Therefore, establishing a practical and reliable biological activity detection method is of great significance for the research and development, quality control and even clinical application of degludec insulin injection. Summary of the invention

[0005] In view of this, the present invention provides a method for detecting the biological activity of insulin degludec and a detection kit. The detection method provided by the present invention detects the biological activity of insulin degludec by an in vitro cell assay, which is less susceptible to the influence of exogenous factors compared with the in vitro cell and mouse blood glucose tests in the prior art, shortens the detection time, and improves the accuracy of the experimental results.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for detecting the biological activity of insulin degludec, comprising the following steps:

[0008] Step A: Resuscitate, subculture, plate, and culture cells containing insulin receptors overnight;

[0009] Step B: Dilute the capture antibody with coating buffer and spread on the plate. Seal the plate and incubate overnight in the dark.

[0010] Step C: The standard sample and the test sample are serially diluted and transferred to the cell plate obtained in step A after washing, respectively, and the plate is sealed and incubated, and then lysed with a lysis buffer to obtain a cell lysate;

[0011] Step D: The detection plate obtained in step B is washed, blocked with a blocking solution, cultured, the blocking solution is removed, washed, and transferred to the cell lysate obtained in step C for culture;

[0012] Step E: After washing, adding a reporter antibody for incubation; removing the reporter antibody, washing, developing with a color developing solution, incubating in the dark, then adding a stop solution, detecting, and obtaining an EC50 value;

[0013] The biological activity of the insulin degludec is obtained based on the EC50 value of the standard, the EC50 value of the test sample, the labeled specific activity of the standard, the starting concentration of the standard and the starting concentration of the test sample.

[0014] In some specific embodiments of the present invention, the cell density of the plating in step A comprises 5×10 4 / ml~1×10 5 / ml, and plated at 100 μl / well.

[0015] In some specific embodiments of the present invention, the cell density of the plate in step A is 7.5×10 4 Pieces / ml.

[0016] In some specific embodiments of the present invention, the serial gradient dilution in step C is a doubling dilution, and the initial concentration of the standard or the test sample is 3000-6000 pmol / ml.

[0017] In some specific embodiments of the present invention, the multiple of the dilution ratio is 2 times.

[0018] In some specific embodiments of the present invention, the initial concentration of the standard or the test product is 3000 pmol / ml.

[0019] In some specific embodiments of the present invention, the incubation in step C is specifically: incubating at 37° C., 5% CO 2 for 30 min±10 min or 2 h±10 min; preferably, the incubation time in step C is 30 min±10 min.

[0020] In some specific embodiments of the present invention, step E obtains the biological activity of insulin degludec by:

[0021] Biological activity of degludec (nmol / mg) = EC50 of standard substance / EC50 of test substance × labeled specific activity of standard substance × correction factor

[0022] The labeled specific activity of the standard is 163.9 nmol / mg, and the correction factor = the initial concentration of the standard / the initial concentration of the test sample.

[0023] In some specific embodiments of the present invention, the cell containing insulin receptor is a CHO-K1-INSR cell, and the preparation method comprises:

[0024] Cell recovery and passaging

[0025] Cell recovery: CHO-K1-INSR cells (purchased from WuXi AppTec) were used to recover one tube of CHO-K1-INSR cells. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and the cells were resuspended in 1 ml of 10% DMEM / F12 (1:1) medium and transferred to a culture dish containing 15 ml of 10% DMEM / F12 (1:1) medium, and placed in a culture dish at 37°C and 5% CO2.

[0026] Cell passaging: After culturing for 2-3 days, take a bottle of CHO-K1-INSR cells in the logarithmic growth phase (i.e., the cells cover 70%-90% of the culture bottle), remove the culture medium in the culture bottle, add 2 ml of 1×PBS, shake the culture bottle to cover the entire culture bottle, then aspirate the 1×PBS, add 1 ml of 0.25% Trypsin EDTA (trypsin) to the culture bottle and digest at room temperature for 2-3 minutes until the cells can be observed to fall off the culture bottle by gently shaking the culture bottle, add 6 ml of 10% DMEM / F12 (1:1) culture medium to wash the cells from the culture bottle, transfer the cell fluid to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes, remove the supernatant, and resuspend the cells in 1 ml of 10% DMEM / F12 (1:1) culture medium to make a single cell suspension. Take 10 μl of single cell suspension and add it to 10 μl of trypan blue staining solution. Count the cells using a cell counter. The cell density is 1×10 6 Pieces / ml.

[0027] In some specific embodiments of the present invention, the capture antibody is MAb IR83-14.

[0028] In some specific embodiments of the present invention, step B is specifically as follows: prepare coating buffer, dilute MAb IR83-14 capture antibody to 3 μg / ml with 1×PBS buffer. Use a multichannel pipette to add the diluted MAb IR83-14, 100 μl / well, to a black detection plate. Seal the plate and incubate overnight at 4°C in the dark.

[0029] In some specific embodiments of the present invention, the step C is specifically:

[0030] The standard and test solutions were pre-diluted to a certain concentration using DMEM / F12 (1:1) culture medium, and then serially diluted.

[0031] Take the cell culture plate that has been cultured overnight, gently pour off the culture medium, add 300 μl of washing buffer to each well for washing, tap gently by hand, and invert and tap on an adsorbent surface to remove the buffer.

[0032] Immediately use the cell culture plate, transfer the standard solution and the test solution to the cell plate, 100 μl / well, parallel 2-3 replicates, seal the plate and incubate at 37°C with shaking for an appropriate time.

[0033] 120 μl / well lysis buffer was added to the cell culture plate, the plate was sealed, and lysis was performed at 350 rpm at room temperature for 2 h.

[0034] In some specific embodiments of the present invention, the step D is specifically:

[0035] Take out the test plate incubated overnight at 4°C, remove the sealing film, discard the coating buffer, and wash the test plate 3 times with 300μl / well washing buffer. Use a multichannel pipette to add 200μl of blocking solution. Seal the plate and incubate it on a shaker at room temperature and about 300rpm for at least 2h.

[0036] Remove the blocking solution, wash the test plate three times with 300 μl / well washing buffer, transfer 100 μl / well lysate from the drug-added lysate plate to the test plate, and incubate on a shaker at room temperature for at least 1 h at 300 rpm.

[0037] In some specific embodiments of the present invention, the step E is specifically:

[0038] The detection plate was washed three times with 300 μl / well washing buffer, and 100 μl / well of PY-20 reporter antibody was added and incubated at room temperature in the dark for 1 hour.

[0039] Remove the PY-20 reporter antibody, wash the detection plate three times with 300 μl / well washing buffer, add 100 μl / well color development solution, and incubate at room temperature in the dark for 20-30 minutes.

[0040] Add 50 μl / well of stop solution and detect at 450 nm.

[0041] The plate was read on a multifunctional microplate reader to obtain the EC50 value.

[0042] Calculation: Biological activity of degludec insulin (nmol / mg) = EC50 of standard substance / EC50 of test substance*labeled specific activity of standard substance*correction factor (labeled specific activity of standard substance is 163.9nmol / mg, correction factor = initial concentration of standard substance / initial concentration of test substance)

[0043] Based on the above, the present invention also provides a detection kit for the biological activity of degludec insulin, comprising: cells containing insulin receptors; standard substances; lysate; a detection carrier coated with a capture antibody; a blocking solution; a reporter antibody; a color developing solution; and a stop solution.

[0044] In some specific embodiments of the present invention, the initial concentration of the standard substance includes 3000-6000 pmol / ml; preferably, the initial concentration of the standard substance is 3000 pmol / ml.

[0045] In some specific embodiments of the present invention, the multiple of the dilution of the standard is 2-fold.

[0046] In some specific embodiments of the present invention, the plating density of the cells containing insulin receptors comprises 5×10 4 / ml~1×10 5 / ml, and plated at 100 μl / well.

[0047] In some specific embodiments of the present invention, the plating density of the cells containing insulin receptor is 7.5×10 4 Pieces / ml.

[0048] The present invention also provides a method for using the detection kit, comprising the following steps:

[0049] Step A: Resuscitate, subculture, plate, and culture cells containing insulin receptors overnight;

[0050] Step B: Dilute the capture antibody with coating buffer and spread on the plate. Seal the plate and incubate overnight in the dark.

[0051] Step C: The standard sample and the test sample are serially diluted and transferred to the cell plate obtained in step A after washing, respectively, and the plate is sealed and incubated, and then lysed with a lysis buffer to obtain a cell lysate;

[0052] Step D: The detection plate obtained in step B is washed, blocked with a blocking solution, cultured, the blocking solution is removed, washed, and transferred to the cell lysate obtained in step C for culture;

[0053] Step E: After washing, adding a reporter antibody for incubation; removing the reporter antibody, washing, developing with a color developing solution, incubating in the dark, then adding a stop solution, detecting, and obtaining an EC50 value;

[0054] The biological activity of the insulin degludec is obtained based on the EC50 value of the standard, the EC50 value of the test sample, the labeled specific activity of the standard, the starting concentration of the standard and the starting concentration of the test sample.

[0055] In some specific embodiments of the present invention, the incubation in step C is specifically: incubating at 37° C., 5% CO 2 for 30 min±10 min or 2 h±10 min; preferably, the incubation time in step C is 30 min±10 min.

[0056] In some specific embodiments of the present invention, step E obtains the biological activity of insulin degludec by:

[0057] Biological activity of degludec (nmol / mg) = EC50 of standard substance / EC50 of test substance × labeled specific activity of standard substance × correction factor

[0058] The labeled specific activity of the standard is 163.9 nmol / mg, and the correction factor = the initial concentration of the standard / the initial concentration of the test sample.

[0059] The present invention tests the biological activity of degludec insulin by culturing cells containing insulin receptors in vitro. After the cells are cultured, prepared samples to be tested and standards of different concentrations are added to interact with the cells, and a lysis solution containing an inhibitor is added to lyse the cells. After lysis, the cell lysate is transferred to a coated detection plate coated with an antibody, combined, a PY-20 reporter antibody is added, and then a color developing solution is added for color development. Finally, a stop solution is used to terminate the reaction, and the multifunctional microplate reader is used for reading.

[0060] The method provided by the present invention uses an ELISA kit to conduct experiments. Compared with the in vitro cell and mouse blood glucose tests in the prior art, it is not easily affected by exogenous factors, shortens the detection time, and improves the accuracy of the experimental results. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0062] Figure 1The four-parameter fitting curve of degludec insulin API is shown; among them, DY2021050002 is the API of degludec insulin;

[0063] Figure 2 The four-parameter fitting curve of degludec insulin API is shown; among them, DY202105003 is degludec insulin API;

[0064] Figure 3 The four-parameter fitting curve of degludec insulin API is shown; among them, DY202106004 is degludec insulin API. DETAILED DESCRIPTION

[0065] The invention discloses a detection method and a detection kit for the biological activity of insulin degludec, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0066] 1.1 Cell recovery: CHO-K1-INSR cells (purchased from WuXi AppTec) were recovered. One tube of CHO-K1-INSR cells was centrifuged at 1000 rpm for 5 min, the supernatant was removed, and the supernatant was resuspended in 1 ml of 10% DMEM / F12 (1:1) medium and transferred to 15 ml of 10% DMEM.

[0067] / F12 (1:1) culture medium in a culture dish and placed in an incubator at 37°C and 5% CO2.

[0068] 1.2 Cell passaging: After culturing for 2-3 days, take a bottle of CHO-K1-INSR cells in the logarithmic growth phase (i.e., the cells cover 70%-90% of the culture bottle), remove the culture medium in the culture bottle, add 2 ml of 1×PBS, shake the culture bottle to cover the entire culture bottle, then aspirate the 1×PBS, add 1 ml of 0.25% Trypsin EDTA (trypsin) to the culture bottle and digest at room temperature for 2-3 minutes until the cells can be observed to fall off the culture bottle by gently shaking the culture bottle, add 6 ml of 10% DMEM / F12 (1:1) culture medium to wash the cells from the culture bottle, transfer the cell fluid to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes, remove the supernatant, and resuspend the cells in 1 ml of 10% DMEM / F12 (1:1) culture medium to make a single cell suspension. Take 10 μl of single cell suspension and add it to 10 μl of trypan blue staining solution. Count the cells using a cell counter. The cell density is 1×106 Pieces / ml.

[0069] 1.3. Day 1

[0070] 1.3.1 Cell plating: Take CHO-K1-INSR cells in the logarithmic growth phase, prepare a cell suspension, plate it in a 96-well plate, and then place the 96-well plate in an incubator for culture.

[0071] 1.3.2 Prepare coating buffer and dilute MAb IR83-14 capture antibody to 3 μg / ml with 1× PBS buffer. Use a multichannel pipette to add 100 μl / well of the diluted MAb IR83-14 to the black detection plate. Seal the plate and incubate overnight at 4°C in the dark.

[0072] 1.4. The next day

[0073] 1.4.1 Pre-dilute the standard and test solutions to 3000 pmol / ml with DMEM / F12 (1:1) medium, and then perform serial gradient dilutions.

[0074] 1.4.2 Take the cell culture plate that has been cultured overnight, gently pour off the culture medium, add 300 μl of washing buffer to each well for washing, tap it gently with your hands, and invert and tap it on an adsorption surface to remove the buffer.

[0075] 1.4.3 Adding drugs: Immediately use the cell culture plate, transfer the standard solution and the test solution to the cell plate, 100 μl / well, parallel 2-3 replicates, seal the plate and incubate at 37°C with shaking for an appropriate time.

[0076] 1.4.4 Add 120 μl / well lysis buffer to the cell culture plate, seal the plate, and lyse the cells at room temperature at 350 rpm for 2 hours.

[0077] 1.4.5 Take out the test plate incubated overnight at 4°C, remove the sealing film, discard the coating buffer, and wash the test plate 3 times with 300μl / well washing buffer. Use a multichannel pipette to add 200μl of blocking solution. Seal the plate and incubate it on a shaker at room temperature and about 300rpm for at least 2h.

[0078] 1.4.6 Remove the blocking solution, wash the test plate three times with 300 μl / well washing buffer, transfer 100 μl / well lysis buffer from the drug-added lysis plate to the test plate, and incubate on a shaker at room temperature for at least 1 hour at 300 rpm.

[0079] 1.4.7 Remove the blocking solution, wash the test plate three times with 300 μl / well washing buffer, transfer 100 μl / well lysis buffer from the drug-added lysis plate to the test plate, and incubate on a shaker at room temperature for at least 1 hour at 300 rpm.

[0080] 1.4.8 Wash the detection plate three times with 300 μl / well washing buffer. Add 100 μl / well of PY-20 reporter antibody and incubate at room temperature in the dark for 1 hour.

[0081] 1.4.9 Remove the PY-20 reporter antibody, wash the detection plate three times with 300 μl / well washing buffer, add 100 μl / well color development solution, and incubate at room temperature in the dark for 20-30 minutes.

[0082] 1.4.10 Add 50 μl / well of stop solution and detect at 450 nm.

[0083] 1.4.11 Read the plate on a multifunctional microplate reader and obtain the EC50 value.

[0084] 1.4.12 Calculation: Biological activity of degludec (nmol / mg) = EC50 of standard substance / EC50 of test substance*labeled specific activity of standard substance*correction factor (labeled specific activity of standard substance is 163.9 nmol / mg, correction factor = initial concentration of standard substance / initial concentration of test substance)

[0085] In the examples of the present invention, the cells used in experimental step 1.1 are in vitro constructed cells, CHO-K1 cells into which the human insulin receptor gene (INSR) has been inserted and can be efficiently expressed, so the cells used in the present invention are called CHO-K1-INSR cells.

[0086] In the embodiment of the present invention, the experimental step 1.3.1 is to take CHO-K1-INSR cells in the logarithmic growth phase, digest them with trypsin, prepare a cell suspension, count and adjust the cell density to a suitable cell density, which is generally 5×10 4 / ml-1×10 5 100 μl / well was added into a 96-well cell culture plate.

[0087] In the embodiment of the present invention, the serial gradient dilution described in step 1.4.1 is a doubling dilution, and more preferably, the concentrations of the reference solution and the test solution are pre-diluted to 3000-6000 pmol / ml.

[0088] In the embodiment of the present invention, after sealing in step 1.4.3, the plate is placed in a 37° C., 5% CO 2 incubator and incubated for 30 min ± 10 min or 2 h ± 10 min.

[0089] The method provided by the present invention uses an ELISA kit to conduct experiments. Compared with the in vitro cell and mouse blood glucose tests in the prior art, it is not easily affected by exogenous factors, shortens the detection time, and improves the accuracy of the experimental results.

[0090] In the detection method and detection kit for the biological activity of degludec insulin provided by the present invention, the raw materials and reagents used can be purchased from the market. The cells used in experimental step 1 are in vitro constructed cells, CHO-K1 cells into which the human insulin receptor gene (INSR) has been inserted and can be efficiently expressed, so the cells used in the present invention are called CHO-K1-INSR cells.

[0091] The present invention will be further described below in conjunction with embodiments:

[0092] Preliminary Experiment Key Parameter Screening Experiment

[0093] 1. Cell plating density screening

[0094] In order to screen out the optimal cell plating density, experiments were conducted on different plating densities (according to the above experimental scheme), and the statistical data are as follows:

[0095] Table 1 ELISA data statistics of different cell plating densities

[0096]

[0097] Table 2 Statistical analysis of experimental data

[0098]

[0099] According to the experimental results in Table 1 and Table 2, when the cell plating density is 7.5-10.0×10 4 When the cell density was 2.5-5.0×10 / ml, the R2 value of the four-parameter S-shaped curve was higher, and the fitting effect was better than that when the cell density was 2.5-5.0×10 4 cells / mL, so the cell plating density is 7.5×10 4 Pieces / ml.

[0100] 2. Screening of incubation time after drug addition

[0101] In order to select the optimal incubation time, experiments were conducted on different incubation times (according to the above experimental scheme), and the statistical data are as follows:

[0102] Table 3 ELISA data statistics at different incubation times

[0103]

[0104] Table 4 Statistical analysis of experimental data

[0105]

[0106] According to the experimental results in Tables 3 and 4, when the incubation time is 30 min ± 10 min, the R2 value of the four-parameter fitting S-type curve is higher, and the fitting effect is better than the incubation time of 2 h ± 10 min, so 30 min ± 10 min should be selected as the optimal incubation time.

[0107] 3. Screening of initial dilution concentration and dilution ratio of reference and test products

[0108] In order to select the optimal dilution factor, experiments were conducted on different dilution factors (according to the above experimental scheme), and the statistical data are as follows:

[0109] Table 5 Statistics of experimental data of ELISA reader with different dilution ratios

[0110]

[0111] Table 6 Statistical analysis of experimental data

[0112]

[0113] Table 7 ELISA data statistics of different dilution starting concentrations

[0114]

[0115] Table 8 Statistical analysis of experimental data

[0116]

[0117] According to the results in Tables 5 and 6, the four-parameter fitting S-shaped curve fitting effect of 2-fold dilution multiple is better than 3-fold and 5-fold dilution multiples, and the biological activity is 17%-60% higher than 3-fold and 5-fold, so the dilution multiple is selected as 2-fold.

[0118] According to the results in Tables 7 and 8, the four-parameter fitting S-curve fitting effect of 3000 pmol / ml starting concentration is better than 4000 pmol / ml, and the biological activity is 9.5% higher, so 3000 pmol / ml is selected as the starting concentration of the reference and test products.

[0119] Example 1

[0120] 1.1 Cell recovery: CHO-K1-INSR cells (purchased from WuXi AppTec) were used to recover one tube of CHO-K1-INSR cells. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 1 ml of 10% DMEM / F12 (1:1) medium and transferred to a culture dish containing 15 ml of 10% DMEM / F12 (1:1) medium, and placed in a culture dish at 37°C and 5% CO2.

[0121] The cells used in experimental step 1.1 are in vitro constructed cells, CHO-K1 cells into which the human insulin receptor gene (INSR) has been inserted and can be efficiently expressed, so the cells used in the present invention are called CHO-K1-INSR cells.

[0122] 1.2 Cell passaging: After culturing for 2-3 days, take a bottle of CHO-K1-INSR cells in the logarithmic growth phase (i.e., the cells cover 70%-90% of the culture bottle), remove the culture medium in the culture bottle, add 2 ml of 1×PBS, shake the culture bottle to cover the entire culture bottle, then aspirate the 1×PBS, add 1 ml of 0.25% Trypsin EDTA (trypsin) to the culture bottle and digest at room temperature for 2-3 minutes until the cells can be observed to fall off the culture bottle by gently shaking the culture bottle, add 6 ml of 10% DMEM / F12 (1:1) culture medium to wash the cells from the culture bottle, transfer the cell fluid to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes, remove the supernatant, and resuspend the cells in 1 ml of 10% DMEM / F12 (1:1) culture medium to make a single cell suspension. Take 10 μl of single cell suspension and add it to 10 μl of trypan blue staining solution. Count the cells using a cell counter. The cell density is 1×10 6 Pieces / ml.

[0123] 1.3. Day 1

[0124] 1.3.1 Cell plating: Take CHO-K1-INSR cells in the logarithmic growth phase, prepare a cell suspension, plate it in a 96-well plate, and then place the 96-well plate in an incubator for culture.

[0125] Experimental step 1.3.1 is to take CHO-K1-INSR cells in the logarithmic growth phase, digest them with trypsin, make a cell suspension, count and adjust to the appropriate cell density, which is generally 5.0×10 4 -1.0×10 5 100 μl / well was added into a 96-well cell culture plate.

[0126] 1.3.2 Prepare coating buffer and dilute MAb IR83-14 capture antibody to 3 μg / ml with 1× PBS buffer. Use a multichannel pipette to add 100 μl / well of the diluted MAb IR83-14 to the black detection plate. Seal the plate and incubate overnight at 4°C in the dark.

[0127] 1.4. The next day

[0128] 1.4.1 Pre-dilute the standard and test solutions to a certain concentration using DMEM / F12 (1:1) medium, and then perform a series of gradient dilutions.

[0129] The serial gradient dilutions described in step 1.4.1 are doubling dilutions, and more preferably the concentrations of the standard solution and the test solution are pre-diluted to 3000 pmol / ml.

[0130] 1.4.2 Take the cell culture plate that has been cultured overnight, gently pour off the culture medium, add 300 μl of washing buffer to each well for washing, tap it gently with your hands, and invert and tap it on an adsorption surface to remove the buffer.

[0131] 1.4.3 Dosing: Immediately use the cell culture plate, transfer the reference solution and the test solution to the cell plate, 100 μl / well, parallel 2-3 replicates, seal the plate and incubate at 37°C with shaking for an appropriate time.

[0132] After sealing the plate in step 1.4.3, incubate it in a 37°C, 5% CO2 incubator for 30 min ± 10 min.

[0133] 1.4.4 Add 120 μl / well lysis buffer to the cell culture plate, seal the plate, and lyse the cells at room temperature at 350 rpm for 2 hours.

[0134] 1.4.5 Take out the test plate incubated overnight at 4°C, remove the sealing film, discard the coating buffer, and wash the test plate 3 times with 300μl / well washing buffer. Use a multichannel pipette to add 200μl of blocking solution. Seal the plate and incubate it on a shaker at room temperature and about 300rpm for at least 2h.

[0135] 1.4.6 Remove the blocking solution, wash the test plate three times with 300 μl / well washing buffer, transfer 100 μl / well lysis buffer from the drug-added lysis plate to the test plate, and incubate on a shaker at room temperature for at least 1 hour at 300 rpm.

[0136] 1.4.7 Wash the test plate three times with 300 μl / well washing buffer. Add 100 μl / well of PY-20 reporter antibody and incubate at room temperature in the dark for 1 hour.

[0137] 1.4.8 Remove the PY-20 reporter antibody, wash the detection plate three times with 300 μl / well washing buffer, add 100 μl / well color development solution, and incubate at room temperature in the dark for 20 min to 30 min.

[0138] 1.4.9 Add 50 μl / well of stop solution and detect at 450 nm.

[0139] 1.4.10 Read the plate on a multifunctional microplate reader and obtain the EC50 value.

[0140] 1.4.11 Calculation: Biological activity of degludec insulin (nmol / mg) = EC50 of standard substance / EC50 of test substance*labeled specific activity of standard substance*correction factor (where the labeled specific activity of standard substance is 163.9 nmol / mg, correction factor = initial concentration of standard substance / initial concentration of test substance).

[0141] Table 9 ELISA data statistics for pre-dilution concentration of 3000pmol / ml

[0142]

[0143]

[0144] Table 10 Statistical analysis of experimental data

[0145] Sample name <![CDATA[R 2 ]]> EC50 Biological activity (nmol / mg) Novo Nordisk Degludec Insulin 1.000 292.0 163.8 Insulin degludec API 0.994 343.8 139.1

[0146] Example 2

[0147] The specific operation steps refer to Example 1, wherein the pre-dilution concentration of the standard and test samples in 1.4.1 is adjusted to 4000 pmol / ml.

[0148] Table 11 ELISA data statistics for pre-dilution concentration of 4000pmol / ml

[0149]

[0150] Table 12 Statistical analysis of experimental data

[0151] Sample name <![CDATA[R 2 ]]> EC50 Biological activity (nmol / mg) Novo Nordisk Degludec Insulin 0.966 161.9 163.8 Insulin degludec API 0.950 251.7 105.4

[0152] Example 3

[0153] The specific operation steps refer to Example 1, wherein the pre-dilution concentration of the standard and test samples in 1.4.1 is adjusted to 6000 pmol / ml.

[0154] Table 13 ELISA data statistics for pre-dilution concentration of 6000pmol / ml

[0155]

[0156] Table 14 Statistical analysis of experimental data

[0157] Sample name <![CDATA[R 2 ]]> EC50 Biological activity (nmol / mg) Novo Nordisk Degludec Insulin 0.963 276.2 163.8 Insulin degludec API 0.966 330.4 136.9

[0158] Embodiment 4:

[0159] 1.1 Cell recovery: CHO-K1-INSR cells (purchased from WuXi AppTec) were used to recover one tube of CHO-K1-INSR cells. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 1 ml of 10% DMEM / F12 (1:1) medium and transferred to a culture dish containing 15 ml of 10% DMEM / F12 (1:1) medium, and placed in a culture dish at 37°C and 5% CO2.

[0160] The cells used in experimental step 1.1 are in vitro constructed cells, CHO-K1 cells into which the human insulin receptor gene (INSR) has been inserted and can be efficiently expressed, so the cells used in the present invention are called CHO-K1-INSR cells.

[0161] 1.2 Cell passaging: After culturing for 2-3 days, take a bottle of CHO-K1-INSR cells in the logarithmic growth phase (i.e., the cells cover 70%-90% of the culture bottle), remove the culture medium in the culture bottle, add 2 ml of 1×PBS, shake the culture bottle to cover the entire culture bottle, then aspirate the 1×PBS, add 1 ml of 0.25% Trypsin EDTA (trypsin) to the culture bottle and digest at room temperature for 2-3 minutes until the cells can be observed to fall off the culture bottle by gently shaking the culture bottle, add 6 ml of 10% DMEM / F12 (1:1) culture medium to wash the cells from the culture bottle, transfer the cell fluid to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes, remove the supernatant, and resuspend the cells in 1 ml of 10% DMEM / F12 (1:1) culture medium to make a single cell suspension. Take 10 μl of single cell suspension and add it to 10 μl of trypan blue staining solution. Count the cells using a cell counter. The cell density is 1×10 6 Pieces / ml.

[0162] 1.3. Day 1

[0163] 1.3.1 Cell plating: Take CHO-K1-INSR cells in the logarithmic growth phase and make 5.0×10 4 After the cell suspension was plated in a 96-well plate, the 96-well plate was placed in an incubator for culture.

[0164] 1.3.2 Prepare coating buffer and dilute MAb IR83-14 capture antibody to 3 μg / ml with 1× PBS buffer. Use a multichannel pipette to add 100 μl / well of the diluted MAb IR83-14 to the black detection plate. Seal the plate and incubate overnight at 4°C in the dark.

[0165] 1.4. The next day

[0166] 1.4.1 Pre-dilute the standard and test solutions to 3000 pOmL / mL with DMEM / F12 (1:1) medium, and then perform serial gradient dilutions.

[0167] 1.4.2 Take the cell culture plate that has been cultured overnight, gently pour off the culture medium, add 300 μl of washing buffer to each well for washing, tap it gently with your hands, and invert and tap it on an adsorption surface to remove the buffer.

[0168] 1.4.3 Dosing: Immediately use the cell culture plate, transfer the reference solution and the test solution to the cell plate, 100 μl / well, parallel 2-3 replicates, seal the plate and incubate at 37°C with shaking for an appropriate time.

[0169] After sealing the plate in step 1.4.3, incubate it in a 37°C, 5% CO2 incubator for 30 min ± 10 min.

[0170] 1.4.4 Add 120 μl / well lysis buffer to the cell culture plate, seal the plate, and lyse the cells at room temperature at 350 rpm for 2 hours.

[0171] 1.4.5 Take out the test plate incubated overnight at 4°C, remove the sealing film, discard the coating buffer, and wash the test plate 3 times with 300μl / well washing buffer. Use a multichannel pipette to add 200μl of blocking solution. Seal the plate and incubate it on a shaker at room temperature and about 300rpm for at least 2h.

[0172] 1.4.6 Remove the blocking solution, wash the test plate three times with 300 μl / well washing buffer, transfer 100 μl / well lysis buffer from the drug-added lysis plate to the test plate, and incubate on a shaker at room temperature for at least 1 hour at 300 rpm.

[0173] 1.4.7 Wash the test plate three times with 300 μl / well washing buffer. Add 100 μl / well of PY-20 reporter antibody and incubate at room temperature in the dark for 1 hour.

[0174] 1.4.8 Remove the PY-20 reporter antibody, wash the detection plate three times with 300 μl / well washing buffer, add 100 μl / well color development solution, and incubate at room temperature in the dark for 20 min to 30 min.

[0175] 1.4.9 Add 50 μl / well of stop solution and detect at 450 nm.

[0176] 1.4.10 Read the plate on a multifunctional microplate reader and obtain the EC50 value.

[0177] 1.4.11 Calculation: Biological activity of degludec insulin (nmol / mg) = EC50 of standard substance / EC50 of test substance*labeled specific activity of standard substance*correction factor (where the labeled specific activity of standard substance is 163.9 nmol / mg, correction factor = initial concentration of standard substance / initial concentration of test substance).

[0178] Table 15 Cell plating density is 5.0×10 4 Microplate reader data statistics

[0179]

[0180]

[0181] Table 16 Statistical analysis of experimental data

[0182] Sample name <![CDATA[R 2 ]]> EC50 Biological activity (nmol / mg) Novo Nordisk Degludec Insulin 0.999 247.2 163.8 Insulin degludec API 0.996 361.8 111.9

[0183] Embodiment 5:

[0184] The specific operation steps refer to Example 1, wherein the density of the cell suspension in 1.3.1 is adjusted to 7.5×10 4 Pieces / ml.

[0185] Table 17 Cell plating density is 7.5×10 4 Microplate reader data statistics

[0186]

[0187] Table 18 Statistical analysis of experimental data

[0188] Sample name <![CDATA[R 2 ]]> EC50 Biological activity (nmol / mg) Novo Nordisk Degludec Insulin 0.997 326.4 163.8 Insulin degludec API 0.993 355.0 150.6

[0189] Example 6

[0190] The specific operation steps refer to Example 1, wherein the density of the cell suspension in 1.3.1 is adjusted to 1.0×10 5 Pieces / ml.

[0191] Table 19 Cell plating density is 1.0×10 5 Microplate reader data statistics

[0192]

[0193] Table 20 Statistical analysis of experimental data

[0194] Sample name <![CDATA[R 2 ]]> EC50 Biological activity (nmol / mg) Novo Nordisk Degludec Insulin 0.992 234.0 163.8 Insulin degludec API 0.992 279.2 137.3

[0195] Effect Example 1

[0196] 1. Cell recovery: CHO-K1-INSR cells (purchased from WuXi AppTec) were used to recover one tube of CHO-K1-INSR cells. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 1 ml of 10% DMEM / F12 (1:1) medium and transferred to a culture dish containing 15 ml of 10% DMEM / F12 (1:1) medium, and placed in a culture dish at 37°C and 5% CO2.

[0197] 2. Cell passaging: After culturing for 2-3 days, take a bottle of CHO-K1-INSR cells in the logarithmic growth phase (i.e., the cells cover 70%-90% of the culture bottle), remove the culture medium in the culture bottle, add 2 ml of 1×PBS, shake the culture bottle to cover the entire culture bottle, then aspirate the 1×PBS, add 1 ml of 0.25% Trypsin EDTA (trypsin) to the culture bottle and digest at room temperature for 2-3 minutes until the cells can be observed to fall off the culture bottle by gently shaking the culture bottle, add 6 ml of 10% DMEM / F12 (1:1) culture medium to wash the cells from the culture bottle, transfer the cell fluid to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes, remove the supernatant, and resuspend the cells in 1 ml of 10% DMEM / F12 (1:1) culture medium to make a single cell suspension. Take 10 μl of single cell suspension and add it to 10 μl of trypan blue staining solution. Count the cells using a cell counter. The cell density is 1×10 6 Pieces / ml.

[0198] 3. Day 1

[0199] 3.1 Cell plating: Take CHO-K1-INSR cells in the logarithmic growth phase, prepare a cell suspension, count and adjust to 7.5×10 4 100 μl / well was added into a 96-well cell culture plate.

[0200] 3.2 Prepare coating buffer and dilute MAb IR83-14 capture antibody to 3 μg / ml with 1× PBS buffer. Use a multichannel pipette to add 100 μl / well of the diluted MAb IR83-14 to the black detection plate. Seal the plate and incubate overnight at 4°C in the dark.

[0201] 4. Day 2

[0202] 4.1 Pre-dilute the standard and test solution to 3000 using DMEM / F12 (1:1) medium.

[0203] pmol / ml, and then perform 2-fold serial dilutions.

[0204] 4.2 Take the cell culture plate that has been cultured overnight, gently pour off the culture medium, add 300 μl of washing buffer to each well for washing, tap it gently with your hands, and invert and tap it on an adsorption surface to remove the buffer.

[0205] 4.3 Dosing: Immediately use the cell culture plate, transfer the reference solution and the test solution to the cell plate, 100 μl / well, parallel 2-3 replicates, seal the plate and incubate at 37°C with shaking for an appropriate time.

[0206] After sealing the plate in step 1.4.3, incubate it in a 37°C, 5% CO2 incubator for 30 min ± 10 min.

[0207] 4.4 Add 120 μl / well lysis buffer to the cell culture plate, seal the plate, and lyse at room temperature at 350 rpm for 2 hours.

[0208] 4.5 Take out the test plate incubated overnight at 4°C, remove the sealing film, discard the coating buffer, and wash the test plate 3 times with 300μl / well washing buffer. Use a multichannel pipette to add 200μl of blocking solution. Seal the plate and incubate it on a shaker at room temperature and about 300rpm for at least 2h.

[0209] 4.6 Remove the blocking solution, wash the test plate three times with 300 μl / well washing buffer, transfer 100 μl / well lysis buffer from the drug-added lysis plate to the test plate, and incubate on a shaker at room temperature for at least 1 hour at 300 rpm.

[0210] 4.7 Wash the detection plate 3 times with 300 μl / well washing buffer. Add 100 μl / well of PY-20 reporter antibody and incubate at room temperature in the dark for 1 hour.

[0211] 4.8 Remove the PY-20 reporter antibody, wash the detection plate three times with 300 μl / well washing buffer, add 100 μl / well color development solution, and incubate at room temperature in the dark for 30 min ± 10 min.

[0212] 4.9 Add 50 μl / well of stop solution and detect at 450 nm.

[0213] 4.10 Read the plate on a multifunctional microplate reader and obtain the EC50 value.

[0214] 4.11 Calculation: Biological activity of degludec (nmol / mg) = EC50 of standard substance / EC50 of test substance*labeled specific activity of standard substance*correction factor (labeled specific activity of standard substance is 163.9 nmol / mg, correction factor = initial concentration of standard substance / initial concentration of test substance).

[0215] After reading using an ELISA reader, the data results are shown in Table 21.

[0216] Table 21 ELISA data statistics

[0217]

[0218] Experimental data analysis

[0219] Table 22 Experimental data analysis statistics

[0220]

[0221] According to Table 21, the CV values ​​of this experiment are all ≤20%, which is in compliance with the regulations. The CV value requirement is ≤20%.

[0222] According to Table 22, the R 2 All ≥0.950, in compliance with regulations, R 2 The value is specified as ≥0.950.

[0223] Experimental map: Figure 1 Note: DY2021050002 is the API of degludec insulin

[0224] Effect Example 2

[0225] The specific operation steps are the same as those in Example 1.

[0226] After reading using an ELISA reader, the data results are shown in Table 23.

[0227] Table 23 ELISA data statistics

[0228]

[0229] Experimental data analysis:

[0230] Table 24 Experimental data analysis statistics

[0231]

[0232] According to Table 23, the CV values ​​of this experiment are all ≤20%, which is in compliance with the regulations. The CV value regulation is ≤20%.

[0233] According to Table 24, the R 2 All ≥0.950, in compliance with regulations, R 2 The value is specified as ≥0.950.

[0234] Experimental map: Figure 2 Note: DY202105003 is the raw material of degludec insulin.

[0235] Effect Example 3

[0236] The specific operation steps are the same as those in Example 1.

[0237] After reading using an ELISA reader, the data results are shown in Table 25.

[0238] Table 25 ELISA data statistics

[0239]

[0240] Experimental data analysis:

[0241] Table 26 Experimental data analysis statistics

[0242]

[0243] According to Table 25, the CV values ​​of this experiment are all ≤20%, which is in compliance with the regulations. The CV value regulation is ≤20%.

[0244] According to Table 26, the R 2 All ≥0.950, in compliance with regulations, R 2 The value is specified as ≥0.950.

[0245] Experimental map: Figure 3 Note: DY202106004 is the raw material of degludec insulin.

[0246] In summary: the ELISA data results of the three experiments were all within the specified range, the biological activity results of degludec insulin raw materials were not much different from the biological activity of degludec insulin purchased from Novo Nordisk, and the three experiments all proved the feasibility of the experimental method.

[0247] Effect Example 4 Cell Growth Data Analysis

[0248] 1. Cell recovery: CHO-K1-INSR cells (purchased from WuXi AppTec) were used to recover one tube of CHO-K1-INSR cells. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 1 ml of 10% DMEM / F12 (1:1) medium and transferred to a culture dish containing 15 ml of 10% DMEM / F12 (1:1) medium, and placed in a culture dish at 37°C and 5% CO2.

[0249] 2. Cell passaging: After culturing for 2-3 days, take a bottle of CHO-K1-INSR cells in the logarithmic growth phase (i.e., the cells cover 70%-90% of the culture bottle), remove the culture medium in the culture bottle, add 2 ml of 1×PBS, shake the culture bottle to cover the entire culture bottle, then aspirate the 1×PBS, add 1 ml of 0.25% Trypsin EDTA (trypsin) to the culture bottle and digest at room temperature for 2-3 minutes until the cells can be observed to fall off the culture bottle by gently shaking the culture bottle, add 6 ml of 10% DMEM / F12 (1:1) culture medium to wash the cells from the culture bottle, transfer the cell fluid to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes, remove the supernatant, and resuspend the cells in 1 ml of 10% DMEM / F12 (1:1) culture medium to make a single cell suspension. Take 10 μl of single cell suspension and add it to 10 μl of trypan blue staining solution. Count the cells using a cell counter. The cell density is 1×10 6 Pieces / ml.

[0250] 3. Perform cell counting and subculture operations on the cells one day after cell culture according to the above operation. Perform the same operation after two days of cell culture and three days of culture, and record the experimental data (cell density, cell viability).

[0251] Table 27 Cell growth (cell density, cell viability) data

[0252]

[0253] It can be seen from the data in Table 27 that the cells can maintain stable growth regardless of whether they are cultured for 1 day, 2 days, or 3 days, and the viability is maintained above 90%.

[0254] In summary, the accuracy of the experimental test results of the present invention is better than that of the conventional mouse blood glucose method, the test results are more accurate, and the cost of experimental investment and the influence of exogenous factors are reduced.

[0255] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting the biological activity of insulin degludec, characterized in that: The steps include: Step A: Resuscitate, subculture, plate, and culture cells containing insulin receptors overnight; Step B: Dilute the capture antibody with coating buffer and spread on the plate. Seal the plate and incubate overnight in the dark. Step C: The standard sample and the test sample are serially diluted and transferred to the cell plate obtained in step A after washing, respectively, and the plate is sealed and incubated, and then lysed with a lysis buffer to obtain a cell lysate; Step D: The detection plate obtained in step B is washed, blocked with a blocking solution, cultured, the blocking solution is removed, washed, and transferred to the cell lysate obtained in step C for culture; Step E: After washing, adding a reporter antibody for incubation; removing the reporter antibody, washing, developing with a color developing solution, incubating in the dark, then adding a stop solution, detecting, and obtaining an EC50 value; Obtaining the biological activity of insulin degludec according to the EC50 value of the standard, the EC50 value of the test sample, the labeled specific activity of the standard, the starting concentration of the standard and the starting concentration of the test sample; The cell density of the plate in step A was 7.5×10 4 Pieces / ml; The serial gradient dilution in step C is a multiple dilution, and the multiple of the multiple dilution is 2 times; The initial concentration of the standard or the test product is 3000 pmol / ml The incubation in step C is specifically: incubating at 37°C, 5% CO2 for 30min±10min; Step E: obtaining the biological activity of insulin degludec is specifically as follows: Biological activity of degludec insulin (nmol / mg) = EC50 of standard substance / EC50 of test substance × labeled specific activity of standard substance × correction factor; The labeled specific activity of the standard is 163.9 nmol / mg, and the correction factor = the initial concentration of the standard / the initial concentration of the test sample.

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