Detection method for insulin analogue stimulated cell glycogen synthesis as well as product and application thereof

Abstract

The invention belongs to the field of biological activity detection, and particularly relates to a detection method for insulin analogue stimulated cell glycogen synthesis as well as a product and application thereof. The cells are 3T3L1 precursor cells subjected to induced differentiation, and the induced differentiation comprises the steps of inducing for 9 days by using a cell differentiation culture medium (d), and continuing to differentiate for 3 days by using an adipose cell maintenance culture medium. The insulin analogue is diluted through a diluent, the diluent is a GM culture medium added with dexamethasone, and the final concentration of the dexamethasone is 0.5-2 [mu] M. The method provided by the invention can quantify the insulin analogue, and is simple and safe to operate, good in stability and reproducibility and high in sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of biological activity detection, and specifically relates to a detection method for insulin analogs to stimulate cell glycogen synthesis, and a product and application thereof. Background Art

[0002] Biological activity is an important indicator of product quality and clinical effectiveness. During the research and development stage of new drugs, it is necessary to establish a biological activity analysis method that is the same as or related to the product's in vivo mechanism of action for the functional activity research of the product. Biological activity detection has a large number of applications throughout the life cycle of new drug products. The methods used for biological activity determination mainly include animal-based biological activity detection methods and cell culture-based biological activity detection methods. Cell biological activity determination uses the reactivity of cells cultured in vitro to samples to evaluate the biological activity of the product.

[0003] With the establishment of high-throughput drug screening platforms and the expansion of production scale, as well as the deepening understanding of the 3R (Reduction, Refinement, Replacement) principle, people are increasingly seeking alternatives to animal experiments. In vitro bioactivity analysis methods based on cell lines are increasingly favored by researchers and manufacturers due to their advantages such as high throughput, high efficiency, and high precision.

[0004] In general, biological activity is expressed in potency, which is an activity unit obtained by comparison with a reference substance. Quantitative biological activity assays should select reaction systems with low background and high signal-to-noise ratio to obtain better repeatable dose-response curves.

[0005] Glycogen is a large intracellular glucose polymer synthesized by cells. As a way to store glucose, it can meet the energy needs at the cellular and organismal levels. Insulin and insulin analogs are a class of protein hormones that can promote the synthesis and storage of glycogen by activating glycogen synthase. Therefore, the biological activity level of insulin can be reflected by detecting the glycogen content in tissues and cells. Specifically, insulin enhances phosphodiesterase activity, reduces cAMP levels, and increases cGMP concentrations through covalent modification, thereby increasing glycogen synthase activity and reducing phosphorylase activity, accelerating glycogen synthesis, and inhibiting glycogenolysis.

[0006] Fully differentiated 3T3-L1 adipocytes are currently considered to have good stability and high sensitivity to insulin, and are suitable as a cell model for insulin-stimulated glycogen synthesis. Existing reports on the differentiation of 3T3-L1 into adipocytes generally use IBMX, dexamethasone and insulin. Generally, the three factors are used for stimulation for 2 days, and then the adipocyte maintenance medium is used for 2 days, and then the growth medium is used for culture for a certain period of time. However, the inventor tried to conduct experiments using the methods reported in the literature and found that the effect was not good, which was manifested in the repeatability of the results, resulting in a low proportion of adipocytes in the cells used for the detection reaction, and thus unable to effectively accept insulin stimulation to produce glycogen.

[0007] In addition, there are currently no reports on the effects of insulin stimulation on glycogen synthesis under different concentration gradients, that is, it is impossible to quantify the test index in a repeatable dose-response curve manner. Moreover, the existing common technologies for detecting glycogen, the ethanol precipitation method and the radioactive element method, both have great limitations. The ethanol precipitation method is complicated and can easily cause glycogen loss during operation, resulting in inaccurate test results, poor repeatability, and the method cannot be further optimized to generate a dose-response curve. The radioactive element method is inconvenient to implement due to the incorporation of radioactive elements, and has high requirements for the experimental environment and experimental personnel, and cannot meet the needs of general companies for biological activity detection.

[0008] Therefore, it is urgent to develop a detection method that can stably induce 3T3L1 precursor cells to differentiate into adipocytes and quantify insulin-stimulated glycogen synthesis. Summary of the invention

[0009] In order to solve the above problems, the present invention provides an improved method for detecting the biological activity of insulin, which comprises stably inducing 3T3L1 precursor cells to differentiate into adipocytes, stimulating adipocyte glycogen synthesis through insulin, and indirectly reflecting the biological activity of insulin.

[0010] In one aspect, the present invention provides a method for detecting insulin analogs stimulating cell glycogen synthesis, the method comprising the following steps:

[0011] S1. Cultivating preadipocytes using cell culture medium (CM culture medium) to obtain cell A;

[0012] S2, using a cell differentiation medium (DM medium) to induce differentiation of the above-mentioned cell A, and then using an adipocyte maintenance medium (IM medium) to continue differentiation to obtain cell B;

[0013] S3, adding insulin analogs to the above-mentioned cell B to stimulate cell B to synthesize glycogen, thereby obtaining cell C;

[0014] S4, lysing the cell C with acid to obtain a lysate;

[0015] S5, detecting the glycogen content in the lysate;

[0016] The induction differentiation described in step S2 is: using a cell differentiation medium to induce for 9 days, and then using an adipocyte maintenance medium to continue differentiation for 3 days;

[0017] In step S3, the insulin analog is diluted with a diluent; the diluent contains dexamethasone.

[0018] Specifically, the above cell culture operation was carried out at 37°C and 5% CO2.

[0019] Specifically, the cell differentiation medium in step S2 includes DMEM, fetal bovine serum, penicillin-streptomycin, dexamethasone, IBMX and human insulin.

[0020] Preferably, the cell differentiation medium comprises 83.5-91.5% DMEM, 8-15% fetal bovine serum, 0.5-1.5% penicillin-streptomycin, 0.5-1.5 μM dexamethasone, 0.2-0.5 mM IBMX and 0.5-1.5 μg / mL human insulin.

[0021] Further preferably, the cell differentiation medium comprises 89% DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin, 1 μM dexamethasone, 0.5 mM IBMX and 1 μg / mL human insulin.

[0022] Specifically, the adipocyte maintenance medium in step S2 includes DMEM, fetal bovine serum, penicillin-streptomycin and human insulin.

[0023] Preferably, the adipocyte maintenance medium comprises 83.5-91.5% DMEM, 8-15% fetal bovine serum, 0.5-1.5 penicillin-streptomycin and 0.5-1.5 μg / mL human insulin.

[0024] Further preferably, the adipocyte maintenance medium comprises 89% DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin and 1 μg / mL human insulin.

[0025] Specifically, the diluent in step S3 also includes growth medium (GM medium).

[0026] More specifically, the final concentration of dexamethasone is 0.5-2 μM; the GM culture medium includes DMEM, bovine serum and penicillin-streptomycin.

[0027] Preferably, the GM culture medium comprises 83.5-91.5% DMEM, 8-15% fetal bovine serum and 0.5-1.5% penicillin-streptomycin.

[0028] Further preferably, the GM culture medium comprises 89% DMEM, 10% fetal bovine serum and 1% penicillin-streptomycin.

[0029] Specifically, the insulin analogue in step S3 is insulin glargine, insulin degludec, insulin degludec, insulin lispro, insulin aspart or insulin detemir.

[0030] Preferably, the insulin analog in step S3 is insulin glargine or insulin degludec.

[0031] Specifically, the precursor adipocytes in step S1 are derived from embryonic development, adipose tissue, stem cell differentiation, cell lines, and in vitro culture or induced pluripotent stem cells.

[0032] Preferably, the preadipocytes are derived from a cell line and cultured in vitro.

[0033] More preferably, the preadipocytes are 3T3-L1 preadipocytes.

[0034] Specifically, the CM culture medium in step S1 includes DMEM, calf serum and penicillin-streptomycin.

[0035] Preferably, the CM culture medium comprises 83.5-91.5% DMEM, 8-15% calf serum and 0.5-1.5% penicillin-streptomycin.

[0036] More preferably, the CM culture medium comprises 89% DMEM, 10% calf serum and 1% penicillin-streptomycin.

[0037] Specifically, the method for detecting the glycogen content in step S5 is enzyme-linked detection, acid hydrolysis, colorimetry, fluorescence detection, high performance liquid chromatography, mass spectrometry or immunology.

[0038] More specifically, the method for detecting glycogen content comprises the following steps:

[0039] (1) Lysing cells;

[0040] (2) terminating lysis;

[0041] (3) diluting different concentrations of insulin analog standards and the lysate produced by cell lysis after stimulation with the test sample;

[0042] (4) taking the lysate, adding glucoamylase solution and incubating;

[0043] (5) adding glucose detection reagent and incubating;

[0044] (6) measuring the luminescent signal on an ELISA reader;

[0045] (7) Analyze the luminescence signal, use the four-parameter regression of the constraint model to fit the standard curve and the sample curve, and calculate the relative potency of the sample.

[0046] More specifically, analyzing the luminescent signal in (7) of the detection method comprises the following steps:

[0047] 1) Use the four-parameter regression of the unconstrained model to fit each curve and evaluate the system suitability:

[0048]

[0049] In the formula, A represents the lower limit of absorbance, which represents the lower asymptote of the S-shaped curve; D represents the upper limit of absorbance, which represents the upper asymptote of the S-shaped curve; B is the absorbance increase rate parameter, which is equivalent to the slope of the curve; subscript TS represents the sample; subscript RS represents the standard.

[0050] 2) The four-parameter regression of the constraint model was used to fit the standard curve and the sample, and the relative potency of the sample was calculated as follows:

[0051]

[0052] In the formula, C is the concentration value at which the absorbance growth rate begins to change, that is, the half-effective reaction concentration (EC50); the subscript TS represents the sample; and the subscript RS represents the standard.

[0053] In yet another aspect, the present invention provides a kit, comprising the reagents or culture medium in the aforementioned detection method.

[0054] In yet another aspect, the present invention provides the use of the aforementioned detection method or kit in detecting the stimulation of glycogen synthesis by insulin analogs.

[0055] The technical effects achieved by the present invention are:

[0056] (1) The method provided by the present invention can be quantified and compared.

[0057] (2) The method provided by the present invention is stable and has good reproducibility.

[0058] (3) The method provided by the present invention is easy to operate and will not cause glycogen loss and affect the results.

[0059] (4) The method provided by the present invention has good sensitivity and specificity and has few restrictions on samples, that is, it can detect low-concentration samples and is not affected by other components in the sample.

[0060] (5) The method provided by the present invention avoids the use of radioactive elements, and the entire experiment is non-toxic and harmless and will not cause harm to the body of the experimenter. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Schematic diagram of the glycogen detection principle in the basic example.

[0062] Figure 2 It is a curve chart of the detection results of Example 1, A is the experimental results of plate 1, circles represent standards, squares represent samples with a theoretical potency of 1, triangles represent samples with a theoretical potency of 0.75, and diamonds represent samples with a theoretical potency of 1.25; B is the experimental results of plate 2, circles represent standards, squares represent samples with a theoretical potency of 1, triangles represent samples with a theoretical potency of 0.5, and diamonds represent samples with a theoretical potency of 1.5.

[0063] Figure 3 This is the linear graph of Example 4.

[0064] Figure 4 It is a curve chart of the detection results of Example 5, A is the experimental results of plate 1, circles represent standard products, squares represent samples with a theoretical titer of 1, and triangles represent samples with batch number DBJL083; B is the experimental results of plate 2, circles represent standard products, squares represent samples with a theoretical titer of 1, and diamonds represent samples with batch number B02F20240303.

[0065] Figure 5 8 is a curve chart of the test results of Example 8, A is the experimental results of plate 1, circles represent standards, squares represent samples with a theoretical potency of 1, triangles represent samples with a theoretical potency of 0.75, and diamonds represent samples with a theoretical potency of 1.25; B is the experimental results of plate 2, circles represent standards, squares represent samples with a theoretical potency of 1, triangles represent samples with a theoretical potency of 0.5, and diamonds represent samples with a theoretical potency of 1.5.

[0066] Figure 6 It is a curve chart of the detection results of Comparative Example 1, A is the experimental result of Comparative Example 1-1, the circle represents the standard, the square represents the sample with a theoretical potency of 1, and the triangle represents the sample of glargine insulin injection excipient (B02F20240302); B is the experimental result of Comparative Example 1-2, the circle represents the standard, the square represents the sample with a theoretical potency of 1, and the diamond represents the BSA sample. DETAILED DESCRIPTION

[0067] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0068] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is only an exemplary explanation of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0069] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0070] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Products from different manufacturers have no significant effect on the effects.

[0071] The term "theoretical potency level (relative potency theoretical value)" refers to the potency value obtained under specific conditions based on theoretical calculations or standard regulations, which is used to compare with the actual measured potency value to evaluate the accuracy and reliability of the measurement.

[0072] The term "relative potency measurement value" refers to the comparison of the biological response of the test article with the response produced by a known standard in the biological activity / potency assay of biological products, thereby quantitatively determining the potency of the test article relative to the standard. The relative potency theoretical value is dimensionless.

[0073] Basic Example

[0074] 1.1 Glycogen detection process

[0075] The cells were cultured normally in a T75 culture flask with a cell culture medium (CM) until contact inhibition. The culture medium was replaced with a cell differentiation medium (DM) to induce differentiation for 9 days, and then the cells were seeded into a 96-well plate at a certain density. The adipocyte maintenance medium (IM) was used to continue differentiation for 3 days. Two days later, a certain concentration of synergistic factors (2.5 mM dexamethasone solution, final concentration of 1 μM) were added to the growth medium (GM) to inject the insulin analog injection sample (composition: taking glargine insulin injection as an example, each milliliter of glargine insulin injection contains 3.64 mg of the main component of insulin, Zinc ion 30μg, m-cresol 2.7mg and glycerol 17mg) were diluted into 10 concentration gradients, with the specific concentrations being 3000nM, 1500nM, 750nM, 250nM, 83.33nM, 27.78nM, 9.26nM, 1.85nM, 0.37nM and 0.074nM, respectively, and then added into 96-well plates to stimulate the induced adipocytes to synthesize glycogen. After 3-5 days of stimulation, the cells were lysed and the intracellular glycogen content was detected using a glycogen detection kit (manufacturer: Promega, catalog number: J5051). The principle of glycogen detection is that glucoamylase decomposes glycogen into glucose, followed by glucose dehydrogenase catalyzing the oxidation of glucose and reducing NAD to NADH. Then, NADH reacts with the reductase substrate luciferin (Proluciferin) under the action of reductase to generate luciferin. Luciferin is the substrate of luciferase and can generate light signals under the action of luciferase. The light signals generated are proportional to the starting amounts of glucose and glycogen, see Figure 1 .

[0076] 1.2 Preparation of main solution

[0077] (1) 0.012 M HCl solution: Add 10 μL of 12 M HCl to 10 mL of water and mix thoroughly to obtain 0.012 M HCl.

[0078] (2) 2.5 mM dexamethasone solution: Weigh 10 mg of dexamethasone, add anhydrous ethanol filtered through a 0.22 μm syringe filter to dissolve, and dilute to 10 mL. Store at 2-8°C, with a shelf life of one month.

[0079] (3) 250 mM IBMX solution: Weigh 555 mg of 3-isobutyl-1-methylxanthine, add dimethyl sulfoxide to dissolve, dilute to 10 mL, dispense 0.15 mL per tube, store at -20°C, valid for one year.

[0080] (4) 10 mg / mL insulin (human) solution: Add 1 mL of 0.012 M HCl solution to 10 mg / vial of insulin (human) to dissolve it.

[0081] (5) Glucoamylase solution: Glucose starch buffer and glucoamylase were mixed in a volume ratio of 200:1.

[0082] (6) Glucose detection reagent: according to the volume ratio of fluorescein detection solution: reductase substrate: reductase: NAD: glucose dehydrogenase = 200:1:1:1:8, pipette corresponding amounts and mix them.

[0083] 1.3 Culture medium composition (percentages are by volume)

[0084] (a) Cell culture medium (CM): 89% DMEM (ATCC), 10% calf serum and 1% penicillin-streptomycin.

[0085] (b) Cell differentiation medium (DM): 89% DMEM (Gibco), 10% fetal bovine serum, 1% penicillin-streptomycin, 1 μM dexamethasone, 0.5 mM IBMX and 1 μg / mL human insulin.

[0086] (c) Adipocyte maintenance medium (IM): 89% DMEM (Gibco), 10% fetal bovine serum, 1% penicillin-streptomycin and 1 μg / mL human insulin.

[0087] (d) Growth medium (GM): 89% DMEM (Gibco), 10% fetal bovine serum and 1% penicillin-streptomycin.

[0088] (e) Sample dilution medium (SDM): 10 mL GM, 4 μL 2.5 mM dexamethasone solution, prepared in equal proportions.

[0089] 1.3 3T3-L1 precursor cell culture and differentiation induction

[0090] (1) Count the cells and add cell culture medium (CM) to adjust the cell density. 6 The cells were inoculated into culture flasks at a density of 10 cells / T75 flask and cultured in a 37°C, 5% CO2 incubator for 3 days.

[0091] (2) Aspirate the cell culture medium (CM) from the T75 flask and add 14 mL of cell differentiation medium (DM) for 9 days.

[0092] 1.4 Preparation of cells and preparation of insulin analog injection samples

[0093] (1) Discard cell differentiation medium (DM).

[0094] (2) Cells were cultured at 5×10 4The cells were seeded at a density of 10 cells / mL in a 96-well cell culture plate, 200 μL / well, and cultured in a 37° C., 5% CO 2 incubator for 3 days.

[0095] (3) Dilution of insulin analog standard (Reference Sample, RS, the same below) and test sample (Test Sample, TS, the same below): The standard and test sample were diluted from the stock solution to 3000 nM. The samples were diluted in a gradient manner using sample dilution medium (SDM) to make 10 gradient dilution concentration points.

[0096] Note: In general, biological activity is expressed in potency, which is an activity unit obtained by comparison with a reference substance. Therefore, in order to obtain a quantifiable potency value, both the standard and the sample need to be prepared simultaneously in each experiment.

[0097] (4) Aspirate the cell supernatant and try to aspirate all the liquid. Add 200 μL of the insulin analog standard and the sample to be tested at different concentrations prepared by gradient dilution to each well and culture in a 37°C, 5% CO2 incubator for 3-5 days.

[0098] 1.5 Glycogen determination

[0099] (1) Cell lysis: Aspirate the cell supernatant, add 200 μL PBS to each well, wash twice, then add 30 μL PBS and 15 μL 0.3N HCl, and incubate at 300 rpm and room temperature for 5 minutes.

[0100] (2) Termination of lysis: Add 15 μL Tris buffer to each well, mix gently, and then blow the cells with a pipette to blow off all the cells and mix well.

[0101] (3) Diluting the lysate produced by cell lysis after stimulation with different concentrations of insulin analog standards and test samples: Take 10 μL of the cell lysate in the cell culture plate and dilute it 5 times with ultrapure water.

[0102] (4) Take 25 μL of the diluted lysate and add it to a 96-well white photometer plate. Add 25 μL of glucoamylase solution to the wells and incubate at 300 rpm and room temperature for 60 minutes.

[0103] (5) Add 50 μL of glucose detection reagent to each well, mix gently, and incubate at room temperature for 60-90 minutes.

[0104] (6) Measure the luminescent signal on an ELISA reader.

[0105] 1.6 Analysis

[0106] (1) The four-parameter regression of the unconstrained model was used to fit each curve and the applicability of the system was evaluated as follows:

[0107]

[0108] In the formula, A represents the lower limit of absorbance, which represents the lower asymptote of the S-shaped curve; D represents the upper limit of absorbance, which represents the upper asymptote of the S-shaped curve; B is the absorbance increase rate parameter, which is equivalent to the slope of the curve; subscript TS represents the sample; subscript RS represents the standard;

[0109] (2) Applicability of curve system:

[0110] R of standards and samples 2 Not less than 0.95;

[0111] The analysis window is not less than 2;

[0112] The maximum signal ratio is between 0.6 and 1.4;

[0113] The dynamic range ratio is between 0.6-1.4;

[0114] The effective slope ratio is between 0.6 and 1.4.

[0115] (4) The four-parameter regression of the constraint model was used to fit the standard curve and the sample, and the relative potency and potency recovery rate of the sample were calculated as follows:

[0116]

[0117] In the formula, C is the concentration value at which the absorbance growth rate begins to change, that is, the half-effective reaction concentration (EC50).

[0118] Example 1 Establishment of a method for detecting glycogen synthesis stimulated by glargine insulin

[0119] According to the record in the validation of the ICHQ2 (R2) analytical method, five relative potency levels (0.5, 0.75, 1, 1.25, 1.5; that is, the theoretical potency is 0.5 times, 0.75 times, 1 times, 1.25 times and 1.5 times of RS, respectively, and the specific operation is to dilute the standard to 1500nM (0.5), 2250nM (0.75), 3000nM (1), 3750nM (1.25), 4500nM (1.5) as the starting concentration, and then perform gradient dilution simultaneously, the same below) were determined by the method of the basic embodiment. The test result curve is shown in FIG. Figure 2 The curve system suitability results are shown in Table 1, and the relative potency determination values ​​and recovery results of five relative potency levels (0.5, 0.75, 1, 1.25, 1.5) are shown in Table 2.

[0120] Table 1 Curve system applicability results

[0121]

[0122] Table 2 Relative potency determination values ​​and potency recovery results

[0123]

[0124]

[0125] Result analysis:

[0126] The results show that R 2 All of them can reach above 0.99 and close to 1, and the concentration points of the upper platform, lower platform and intermediate transition period of the curve are evenly distributed, indicating that the four-parameter curve fit is excellent. The analysis window, maximum signal ratio, dynamic range ratio, and effective slope ratio all meet the system applicability requirements, indicating that the method can be used for comparative research and quantitative indicators.

[0127] Example 2 Confirmation of the accuracy of the detection method for glargine insulin stimulating glycogen synthesis

[0128] In order to further confirm the accuracy of the glycogen synthesis detection method, according to the records in the validation of the ICHQ2 (R2) analytical method, two experimenters performed relative titer determinations on five relative titer levels (0.5, 0.75, 1, 1.25, 1.5) according to the method of the basic embodiment at different time points.

[0129] The relative bias (RB), geometric mean (GM) of titer recoveries for each titer level and across all titer levels were calculated.

[0130] Relative bias calculation formula:

[0131]

[0132] The accuracy results are shown in Table 3:

[0133] Table 3 Accuracy results analysis

[0134]

[0135]

[0136] Result analysis:

[0137] The results showed that the relative bias (RB) of the titer measurement value at each titer level was between -40% and 40%; the geometric mean (GM) of the relative titer at each titer level and the overall level was between 0.60 and 1.40, indicating that the detection method had good accuracy.

[0138] Example 3 Precision Confirmation of the Detection Method for Glycogen Synthesis Stimulation by Glargine Insulin

[0139] In order to further confirm the glycogen synthesis detection method, based on the experiment in Example 2, the precision of the glycogen synthesis detection method was analyzed according to the precision requirements in the bioanalysis example in the ICHQ2 (R2) analysis method, mainly by calculating the geometric standard deviation (GSD) and geometric coefficient of variation (GCV) based on the relative activity. The calculation method is as follows:

[0140] GSD=antilog(SD)

[0141] GCV=(GSD-1)×100%

[0142] Where: SD is the logarithmic standard deviation of the relative potency of each potency level and the overall level. The precision results are shown in Table 4:

[0143] Table 4 Precision results analysis

[0144]

[0145]

[0146] Result analysis:

[0147] The results showed that the geometric standard deviation (GSD) of each titer level and the overall relative titer was between 0.6 and 1.4, and the geometric coefficient of variation (GCV) of each titer level and the overall relative titer was no more than 30%, indicating that the detection method had good precision.

[0148] Example 4: Linearity and range confirmation of the detection method for glargine insulin-stimulated glycogen synthesis

[0149] In order to further confirm the glycogen synthesis detection method, based on the experiment in Example 2, according to the requirements for linearity and range in the bioanalysis example in the ICH Q2 (R2) analysis method, the linearity and range of the glycogen synthesis detection method were evaluated using the data of the five titer levels of all test samples. The correlation coefficient R was obtained by linear fitting with the theoretical titer value as the X-axis and the measured titer value as the Y-axis. 2 , and report the equation and graph at the same time. Linearity and range results are shown in Tables 5 and Figure 3 .

[0150] Table 5 Linearity and range results analysis

[0151]

[0152] Result analysis:

[0153] The results show that the linear regression equation correlation coefficient R 2 ≥0.95. This indicates that the linearity of the glycogen synthesis detection method is good, and the detection range is between 50% and 150%.

[0154] Example 5 Confirmation of the detection method of glargine insulin stimulating glycogen synthesis from different batches from different sources

[0155] To further confirm the applicability of the method, different batches of glargine insulin from different sources were used to confirm the glycogen method, and the operation method was the same as in Example 1. The test result curve is shown in FIG. Figure 4 The curve system suitability results are shown in Table 6, and the relative potency determination values ​​and recovery results are shown in Table 7.

[0156] Table 6 Curve system applicability results

[0157]

[0158] Table 7 Relative potency determination values ​​and potency recovery results

[0159]

[0160] Note: The manufacturer of glargine insulin injection with batch number DBJL083 is Sanofi of France; the manufacturer of glargine insulin injection with batch number B02F20240303 is Yifan Pharmaceutical.

[0161] Result analysis:

[0162] The test results of glycogen synthesis of insulin glargine from different batches and different sources showed good curves, and the results were consistent with the system applicability, indicating that the glycogen synthesis detection method was suitable for insulin glargine and the method was stable and reproducible.

[0163] Example 6

[0164] The difference between Example 6 and Example 1 is that the final concentration of dexamethasone in GM is 0.5 μM, and the rest is the same as Example 1. The curve system applicability results are shown in Table 8, and the relative potency measurement values ​​and recovery results of five relative potency levels (0.5, 0.75, 1, 1.25, 1.5) are shown in Table 9.

[0165] Table 8 Curve system applicability results

[0166]

[0167] Table 9 Relative potency determination values ​​and potency recovery results

[0168]

[0169] The results showed that when the final concentration of dexamethasone in GM was adjusted to 0.5 μM, the glargine insulin glycogen synthesis detection result curve was still good, and the results all met the system suitability requirements, indicating that the glycogen synthesis detection method has a certain durability, and the fluctuation of dexamethasone concentration in the range of 0.5-1 μM will not affect the detection results.

[0170] Example 7

[0171] The difference between Example 7 and Example 1 is that the final concentration of dexamethasone in GM is 2 μM, and the rest is the same as Example 1. The curve system applicability results are shown in Table 10, and the relative potency measurement values ​​and recovery results of five relative potency levels (0.5, 0.75, 1, 1.25, 1.5) are shown in Table 11.

[0172] Table 10 Curve system applicability results

[0173]

[0174] Table 11 Relative potency determination values ​​and potency recovery results

[0175]

[0176]

[0177] The results showed that when the final concentration of dexamethasone in GM was adjusted to 2 μM, the glargine insulin glycogen synthesis detection result curve was still good, and the results were consistent with the system applicability, indicating that the glycogen detection method has a certain durability, and the fluctuation of dexamethasone concentration in the range of 1-2 μM will not affect the detection results.

[0178] Example 8 Establishment of a method for detecting glycogen synthesis stimulated by degludec and insulin aspart

[0179] In order to further determine the applicable scope of the glycogen synthesis method, the detection method of Example 1 was used to detect insulin degludec and insulin aspart. The detection result curve is as follows: Figure 5 As shown, the curve system suitability results are shown in Table 12, and the relative potency measurement values ​​and recovery results of five relative potency levels (0.5, 0.75, 1, 1.25, 1.5) are shown in Table 13.

[0180] Table 12 Curve system applicability results

[0181]

[0182] Table 13 Relative potency determination values ​​and potency recovery results

[0183]

[0184]

[0185] Result analysis:

[0186] The results show that R 2All of them can reach above 0.99 and close to 1, and the concentration points of the upper platform, lower platform and intermediate transition period of the curve are evenly distributed, indicating that the four-parameter curve fitting is excellent. The analysis window, maximum signal ratio, dynamic range ratio, effective slope ratio and relative activity all meet the system adaptability requirements, indicating that the method can also be applied to degludec dual insulin aspart.

[0187] Comparative Example 1

[0188] The comparative example is set with reference to Example 1. The difference between the comparative example and Example 1 is shown in Table 14, and the test results are shown in Tables 15 and 16. Figure 6 .

[0189] Table 14

[0190]

[0191] Table 15

[0192]

[0193] Result analysis:

[0194] The results of different concentrations of BSA were comparable to the lowest concentration of RS (0.074nM), indicating that the non-related protein BSA had no dose response. The results of different dilution multiples of the excipients of insulin glargine injection were comparable to the lowest concentration of RS (0.074nM), indicating that it also had no dose response. Overall, the method has good specificity and is specifically suitable for the detection of insulin.

[0195] Comparative Example 2

[0196] A comparative example was set with reference to Example 1. The difference between the comparative example and Example 1 and the test results are shown in Table 16:

[0197] Table 16

[0198]

[0199] Result analysis:

[0200] The duration of DM treatment has a great impact on cell differentiation, and the proportion of semi-induced adipocytes increases with the increase of DM treatment duration. Glycogen analysis requires a high proportion of semi-induced adipocytes. If it is too low, it will affect the response to insulin, and if it is too high, adipocytes will aggregate and fall off in the late stage of the reaction, resulting in unstable result curves and cannot be used for sample testing.

[0201] Comparative Example 3

[0202] A comparative example was set with reference to Example 1. The difference between the comparative example and Example 1 and the test results are shown in Table 17:

[0203] Table 17

[0204]

[0205] Result analysis:

[0206] Since DM is carried out in a culture flask, and IM is treated after cell plating, too short an IM treatment time will result in poor cell adhesion and easy cell detachment during the loading stage, leading to the termination of the experiment. IM contains insulin, and too long an IM treatment time will cause the differentiated adipocytes to become desensitized to insulin stimulation and lose their ability to respond to insulin stimulation.

[0207] Comparative Example 4

[0208] A comparative example was set with reference to Example 1. The difference between the comparative example and Example 1 and the test results are shown in Table 18:

[0209] Table 18

[0210]

[0211]

[0212] 10mg / mL rosiglitazone solution: Add 1mL of dimethyl sulfoxide to 10mg / bottle of rosiglitazone to dissolve. Dispense into 10μL tubes and store at -20℃. The shelf life is one year.

[0213] Result analysis:

[0214] Compared with Example 1, which uses GM+dexamethasone as a sample diluent to well reflect the stimulation of insulin, this comparative example shows that not adding factors, replacing other factors, and superimposing other factors cannot well reflect the stimulation of insulin, indicating that only by specifically using GM and dexamethasone as sample diluents can glycogen synthesis detection be effectively carried out.

[0215] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting insulin analogs stimulating cell glycogen synthesis, characterized in that: The detection method comprises the following steps: S1. Cultivating preadipocytes using cell culture medium to obtain cell A; S2, inducing differentiation of the above-mentioned cell A using a cell differentiation medium, and then continuing differentiation using an adipocyte maintenance medium to obtain cell B; S3, adding insulin analogs to the above-mentioned cell B to stimulate cell B to synthesize glycogen, thereby obtaining cell C; S4, lysing the cell C with acid to obtain a lysate; S5, detecting the glycogen content in the lysate; The induction differentiation in step S2 is: using the cell differentiation medium to induce for 9 days, and then using the adipocyte maintenance medium to continue differentiation for 3 days; In step S3, the insulin analog is diluted with a diluent; the diluent contains dexamethasone.

2. The detection method according to claim 1, characterized in that: The cell differentiation medium in step S2 includes, by volume, 83.5-91.5% DMEM, 8-15% fetal bovine serum, 0.5-1.5% penicillin-streptomycin, 0.5-1.5 μM dexamethasone, 0.2-0.5 mM IBMX and 0.5-1.5 μg / mL human insulin.

3. The detection method according to claim 1, characterized in that: The adipocyte maintenance medium in step S2 includes, by volume, 83.5-91.5% DMEM, 8-15% fetal bovine serum, 0.5-1.5% penicillin-streptomycin, and 0.5-1.5 μg / mL human insulin.

4. The detection method according to claim 1, characterized in that: The final concentration of dexamethasone in step S3 is 0.5-2.0 μM; the diluent also includes growth medium.

5. The detection method according to claim 4, characterized in that: The growth medium comprises, by volume, 83.5-91.5% DMEM, 8-15% fetal bovine serum and 0.5-1.5% penicillin-streptomycin.

6. The detection method according to claim 1, characterized in that: In step S3, the insulin analogue is selected from at least one of insulin glargine, insulin degludec, insulin degludec, insulin lispro, insulin aspart or insulin detemir.

7. The detection method according to claim 1, characterized in that: The precursor adipocytes described in step S1 are derived from embryonic development, adipose tissue, stem cell differentiation, cell lines, and in vitro culture or induced pluripotent stem cells.

8. The detection method according to claim 7, characterized in that: The preadipocytes are 3T3-L1 preadipocytes.

9. The detection method according to claim 1, characterized in that: The cell culture medium in step S1 includes 83.5-91.5% DMEM, 8-15% calf serum and 0.5-1.5% penicillin-streptomycin.

10. The detection method according to claim 1, characterized in that: The method for detecting the glycogen content in step S5 is enzyme-linked detection, acid hydrolysis, colorimetry, fluorescence detection, high performance liquid chromatography, mass spectrometry or immunology.

11. The detection method according to claim 10, characterized in that: The method for detecting the glycogen content in step S5 comprises the following steps: (1) Lysing cells; (2) terminating lysis; (3) diluting different concentrations of insulin analog standards and the lysate produced by cell lysis after stimulation with the test sample; (4) taking the lysate, adding glucoamylase solution and incubating; (5) adding glucose detection reagent and incubating; (6) measuring the luminescent signal on an ELISA reader; (7) Analyze the luminescent signal, use the four-parameter regression of the constraint model to fit the standard curve and the sample curve, and calculate the relative potency of the sample.

12. A kit, characterized in that: The kit comprises the reagent or culture medium in the detection method according to any one of claims 1 to 11.

13. Use of the detection method according to any one of claims 1 to 11 or the kit according to claim 12 in detecting glycogen synthesis stimulated by insulin analogs.

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