A method for detecting the biological activity of GLP-1, GLP-1 analogs or GLP-1 fusion proteins

Abstract

The present application relates to the field of biomedicine, and specifically, to a method for detecting the biological activity of GLP-1, GLP-1 analogs or GLP-1 fusion proteins, the method being based on a luciferase reporter gene assay of HEK293 / CRE‑Luc / GLP1R cells, comprising diluting the cells with 2% FBS analysis medium, seeding the plates, incubating for 1 hour, and then adding a sample to be tested for activity detection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for detecting the biological activity of GLP-1, GLP-1 analogs or GLP-1 fusion protein. Background Art

[0002] Glucagon-like peptide-1 (GLP-1) is an intestinal polypeptide hormone in the human body. It specifically acts on the GLP-1 receptor of pancreatic islet cells, triggering a GLP-1R-mediated signaling pathway cascade reaction, thereby stimulating insulin secretion in a glucose concentration-dependent manner and exerting a hypoglycemic function. In order to overcome the short half-life of natural GLP-1 in the body, many GLP-1 analogs or GLP-1 fusion protein hypoglycemic drugs have been launched to meet the clinical application of the increasing number of diabetic patients around the world, especially in China.

[0003] At present, the luciferase reporter gene method is widely used to measure the biological activity of GLP-1, GLP-1 analogs or GLP-1 fusion proteins. The principle of the determination is that GLP-1, GLP-1 analogs or GLP-1 fusion proteins interact with GLP-1 receptors on the cell membrane, activate intracellular adenylate cyclase, lead to increased intracellular cAMP levels, and then activate the cell's luciferase signaling pathway. When the luciferase detection substrate is added, a fluorescent signal can be detected. The RLU value (relative light unit) is measured by an enzyme reader, and the experimental data is analyzed using software to draw a dose-response curve. According to the EC 50 The biological activity of the corresponding GLP-1, GLP-1 analog or GLP-1 fusion protein can be calculated from the value.

[0004] However, the existing luciferase reporter gene assay method has a long experimental cycle, high material consumption, and large sample volume, which makes the experiment time-consuming and material-intensive. For example, Example 13 of patent document CN115703766A discloses a typical GenScript luciferase reporter gene assay method. After HEK293 / CRE-Luc / GLP1R cells are plated, Overnight incubation , using 10% FBS In addition, according to the fitting curve provided in the corresponding product manual of GenScript (Cat. No. M00562), the effective concentration of GLP-1(1-37) sample is estimated to be 1.0E-12 ~ 1.0E-6 M .

[0005] Therefore, there is an urgent need in the art to develop more economical and efficient methods to save time costs, save materials, and / or reduce sample usage. Summary of the invention

[0006] In order to solve the above problems, the present invention provides an improved method for detecting the biological activity of GLP-1, GLP-1 analogs or GLP-1 fusion proteins. The 10% FBS complete medium is changed to a 2% FBS analysis medium, and the overnight incubation after cell plating is shortened to 1 hour.

[0007] Through a large number of experiments, it was found that the improved method can stably and accurately obtain the dose-effect curve of GLP-1, GLP-1 analogs or GLP-1 fusion protein, and the method has good specificity, relative accuracy, intermediate precision and durability.

[0008] Therefore, the present invention provides a method for detecting the biological activity of GLP-1, GLP-1 analogs or GLP-1 fusion proteins, which is based on the luciferase reporter gene assay of HEK293 / CRE-Luc / GLP1R cells, comprising diluting the cells with 2% FBS analysis medium, plating, incubating for 1 hour, and then adding the sample to be tested for activity detection.

[0009] In one aspect, the method of the present invention further comprises preparing the sample solution of the GLP-1, GLP-1 analog or GLP-1 fusion protein using 2% FBS analysis medium.

[0010] In one aspect, the methods of the invention use a luciferase detection reagent, such as a commercially available luciferase reporter gene detection reagent such as Bio-Lite TM Luciferase Assay System, Fire-Lumi™ Assay System or Bright-Glo TM Luciferase Assay System reagent, use an ELISA instrument to detect the RLU value of the chemiluminescent signal after stimulation with GLP-1, GLP-1 analogs or GLP-1 fusion protein.

[0011] In one aspect, the data analysis software of the method of the present invention can utilize GraphPad Prism or other suitable data analysis software.

[0012] In one aspect, the 2% FBS assay medium of the methods of the present invention is DMEM medium supplemented with 2% FBS.

[0013] In one aspect, the cell dilution concentration in the method of the present invention is 8×10 5 cells / ml.

[0014] The method of the present invention can be used to analyze the activity of GLP-1, GLP-1 analogs or GLP-1 fusion proteins (such as GLP-1 / antibody fusion proteins, GLP-1 / Fc fusion proteins). Preferably, the GLP-1 analogs or GLP-1 fusion proteins include liraglutide, semaglutide, dulaglutide, lixisenatide, albiglutide, benaglutide, tasiroglutide, supaglutide, exenatide, telportide and CPX101 products.

[0015] Preferably, the GLP-1 analog is liraglutide. Preferably, the effective concentration range of the liraglutide sample is 2.7E-14 to 1.6E-9 M.

[0016] Preferably, the GLP-1 analog is semaglutide. Preferably, the effective concentration range of the semaglutide sample is 1.9E-14 ~ 1.9E-8 M.

[0017] The beneficial effect of the present invention is that compared with the existing technology such as the general GenScript detection method, it saves FBS material and greatly shortens the test cycle, and the required sample concentration is lower, ranging from about 1.0E-14 to 1.0E-8 M. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1a is the fitting curve of liraglutide measured after cells were seeded and incubated overnight; Figure 1b Fitting curve of liraglutide measured after 1 hour of incubation after cell seeding.

[0019] Figure 2 Fitting curve of semaglutide measured after 1 hour of incubation after cell seeding.

[0020] Figure 3a When liraglutide reference product is used as a control product and a buffer blank control or degludec insulin negative control is added, the fitting curve of the property investigation test in the verification of the method of the present invention is obtained; and Figure 3b When the liraglutide reference product is used as the control product and the reference drug Novo Nordisk's liraglutide preparation is added, the fitting curve of the property investigation test in the verification of the method of the present invention is obtained.

[0021] Figure 4a The relative accuracy fitting curve in the verification of the method of the present invention is obtained when 100% S of the verification sample is added to Day 1 for detection with liraglutide reference as the reference; Figure 4b The relative accuracy fitting curve in the verification of the method of the present invention is obtained when 100% S of the verification sample is added to Day 2 for detection with liraglutide reference as the reference; Figure 4c The relative accuracy fitting curve in the verification of the method of the present invention is obtained by taking liraglutide as the reference substance and adding 100%S of the verification sample on Day 3 for detection; Figure 4dThe relative accuracy fitting curve in the verification of the method of the present invention was obtained by taking liraglutide as the reference substance and adding 100%S of the verification sample on Day 4 for detection.

[0022] Figure 5a The durability fitting curve in the validation of the method of the present invention is shown when liraglutide reference is used as the control and HEK293 / CRE-Luc / GLP1R cells of the F5 generation are used; Figure 5b The durability fitting curve in the validation of the method of the present invention is obtained by using liraglutide as a reference substance and HEK293 / CRE-Luc / GLP1R cells of the F7 generation; and Figure 5c The durability fitting curve in the validation of the method of the present invention is shown in Figure 1, when liraglutide reference is used as a control substance and HEK293 / CRE-Luc / GLP1R cells of F31 generation are used. DETAILED DESCRIPTION

[0023] The present invention is further described below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

[0024] Note: The cells in the examples are HEK293 / CRE-Luc / GLP1R cells purchased from Nanjing GenScript Biotechnology Co., Ltd. (M00562), 96-well all-white plates purchased from Corning, Bio-Lite TM Luciferase Assay System test kit was purchased from Vazyme (DD1201-03). Liraglutide and semaglutide reference products were made by Chia Tai Tianqing Pharmaceutical Group Co., Ltd.

[0025] Unless otherwise specified, the reagents are all commercially available products. Unless otherwise specified, the methods used in the examples are all conventional methods in the art.

[0026] Solution preparation instructions:

[0027] 2% FBS analysis medium: Under sterile conditions, pipette 2 ml of FBS and add 98 ml of DMEM GlutaMax basal medium, mix well, and store at 2-8°C.

[0028] Bio-Lite TM Luciferase reagent preparation: Bio-Lite TM Take out the Luciferase Assay System kit, equilibrate to room temperature, and wait for Bio-Lite TM After the Luciferase Assay Buffer is completely dissolved, add Bio-Lite TMLuciferase Assay Substrate, mix well, and store at -20℃ in aliquots away from light.

[0029] Biological activity calculation formula:

[0030] The biological activity calculation uses log (actual concentration of the compound) as the horizontal axis and chemiluminescence value RLU as the vertical axis to perform four-parameter fitting. The specific formula is as follows:

[0031]

[0032] Where, Y is the RLU value;

[0033] X is the log(compound concentration) value, mol / L;

[0034] Bottom is the asymptote estimate under the curve;

[0035] Top is the asymptote estimate on the curve;

[0036] HillSlope is the slope of the curve;

[0037] EC 50 is the half-maximal effect concentration, mol / L.

[0038] Example 1: Comparative Example of Overnight Incubation after Cell Seeding

[0039] 1. Cell dilution, plating and incubation

[0040] Cell collection and counting: After taking out the cells (HEK293 / CRE-Luc / GLP1R), discard the culture supernatant, aspirate trypsin to wash the residual culture medium, discard the trypsin, add 5ml trypsin to each T75 cell culture bottle, digest at 37℃ for 2~3 minutes, wait for the cells to be completely digested, add 5ml / bottle analysis medium to elute, transfer to a sterile centrifuge tube, centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cells with 2% FBS analysis medium, count, and adjust the viable cell density to 8×10 with 2% FBS analysis medium. 5 cells / ml.

[0041] Cell seeding and incubation: Mix the above cell suspension and add 50 μl / well to a 96-well white plate and place at 37°C and 5% CO 2 Incubate overnight in an incubator.

[0042] 2. Preparation of sample solution

[0043] The liraglutide sample was diluted to 1.1E-5 M (effective concentration after addition 5.3E-6 M) with 2% FBS analysis medium as the first compound concentration point, followed by 5-fold gradient dilutions to obtain a total of 12 compound solutions of different concentrations.

[0044] 3. Compound stimulation

[0045] Add 50 μl / well of the compound solution prepared in step 2 to the 96-well white plate incubated overnight in step 1. Set up two parallel wells for each dilution and place at 37°C, 5% CO 2 Incubate in the incubator for 6 hours.

[0046] 4. Detection

[0047] Take out the mixed and packaged Bio-Lite in advance TM Luciferase reagent was melted at room temperature and added to the above experimental wells at 100 μl / well. Incubated at room temperature in the dark for 2-3 minutes. The RLU value was read on the chemiluminescence detection module of the microplate reader.

[0048] 5. Data Processing

[0049] The experimental data were analyzed using GraphPad Prism software. The logarithm of the sample molar concentration was used as the X-axis and the corresponding RLU value was used as the Y-axis. A four-parameter equation was used for fitting and the sample dose-response curve was drawn to obtain the EC of the sample. 50 value.

[0050] The results are as follows Figure 1a As shown, this method fits the curve R 2 The EC value of the liraglutide sample was 0.9208, which was poorly fitted and the signal window was only 1.2 times. 50 is 1.0E-9 M.

[0051] Example 2 Improved method for determining the activity of liraglutide samples

[0052] In view of the poor signal window of the overnight incubation method explored in the comparative example of Example 1, in this example, the incubation time after cell seeding was adjusted to 1 hour, and the concentration was adjusted at the same time to obtain a more complete result fitting curve of the upper and lower platforms.

[0053] The experimental method of Example 1 was used with reference to the difference that the incubation time after cell plating was adjusted to 1 hour, and the liraglutide sample concentration was adjusted to 3.2E-9 M (effective concentration 1.6E-9 M after sample addition) with 2% FBS analysis medium as the first compound concentration point, followed by 3-fold gradient dilution to obtain 11 compound solutions of different concentrations. That is, the effective concentration range of the compound reference / test product was 2.7E-14 ~ 1.6E-9 M.

[0054] The results are as follows Figure 1b As shown in the figure, compared with before adjustment, the fitting curve is approximately "S" shaped, R 2 The EC value of liraglutide measured by this method is 0.9989, with good fitting degree and the signal window increased to 336.4 times. 50 It is 1.4E-11 M. This shows that the method is not only fast, saves materials and samples, but also has a good dose-effect curve for the detection of liraglutide.

[0055] Example 3 Improved method for determining the activity of semaglutide samples

[0056] The experimental method of Example 2 was referred to, except that the effective concentration of the semaglutide sample was adjusted. The semaglutide sample was diluted to 3.8E-8 M (effective concentration 1.9E-8 M after addition) with 2% FBS analysis medium as the first compound concentration point, followed by a 4-fold gradient dilution to obtain a total of 11 compound solutions of different concentrations. That is, the effective concentration range of the compound reference / test sample was 1.9E-14 ~ 1.9E-8 M.

[0057] The results are as follows Figure 2 As shown, the fitting curve is approximately "S" shaped, R 2 The fitting degree is 0.9992, and the EC value of semaglutide measured by this method is good. 50 It is 3.7E-11 M. This shows that the method is not only fast, saves materials and samples, but also has a good dose-effect curve for the detection of semaglutide.

[0058] Example 4 Method Verification Example

[0059] This example uses liraglutide as a test sample, and from the perspective of professional analysis, verifies the scientific nature of the determination method of the present invention from four aspects: specificity, intermediate precision, relative accuracy, and durability.

[0060] (1) Exclusivity

[0061] Using the self-made liraglutide reference product as the control product, the following three situations were investigated for their effects on the biological activity detection. a) Blank control test: Using the buffer solution without liraglutide as the blank control, whether it interferes with the determination of this method. b) Negative control test: Using degludec insulin as the negative control, whether this method responds to it. c) Reference drug comparison test: Using Novo Nordisk's liraglutide preparation as the control, whether this method can specifically fit the dose-response curve.

[0062] The results are as follows Figure 3a As shown in the figure, when only blank control or negative control was added, the measured RLU value was equivalent to the platform area under the dose-effect curve of liraglutide and had no dose dependence, indicating that the blank buffer did not interfere with the determination of the biological activity of the sample by this method, and that this method had no response to degludec insulin. Figure 3b As shown in the figure, when Novo Nordisk's liraglutide preparation was added, a dose-response curve could be fitted. The above results show that the method has good specificity.

[0063] (2) Intermediate precision

[0064] The homemade liraglutide reference product was used as the control product (STD) and pre-diluted to 1.0E-8 M with 2% FBS analysis medium. Then, it was diluted according to Table 1 to obtain the first concentration of the verification samples 64%S, 80%S, 100%S, 125%S, and 156%S. Subsequently, 3-fold gradient dilutions were performed to obtain a total of 11 verification sample solutions of different concentrations.

[0065] The different potency levels of the aforementioned validation samples are set according to the guidelines for validation of biological activity / potency determination methods for biological products specified in Part IV of the Pharmacopoeia of the People's Republic of China (2020 Edition) (11th Edition, China Medical Science and Technology Press, Beijing) of the National Pharmacopoeia Commission.

[0066] Table 1 Dilution scheme for the first concentration point of reference and verification samples

[0067]

[0068] Two different testers (Analyst1 and Analyst2) used two cell generations on different dates to perform relative potency tests on the control substance STD and verification samples 64%S, 80%S, 100%S, 125%S, and 156%S for four times each, and calculated the biological activity and RSD value of each sample.

[0069] The theoretical value is the ideal value of biological activity obtained by theoretical calculation under ideal conditions at different potency levels, because the validation sample and the reference substance are derived from the same substance, that is, the homemade liraglutide reference substance, and their structure and properties are the same, while the validation samples at different potency levels are prepared from the reference substance at different concentration ratios. For example, the first concentration point of the validation sample 64%S prepared in Table 1 above is calculated as STD concentration × 64%. Correspondingly, its biological activity should be the biological activity of the reference substance (assigned 100%) × 64%. Therefore, the theoretical value of the biological activity of the validation sample 64%S is 64%, and so on. The measured value is the biological activity value actually measured in the test, that is, the EC value of the reference substance. 50 / Verification Sample EC 50 ×100%, the average is the mean of 4 measured values, and the RSD is the standard deviation of the 4 measured values / average × 100%.

[0070] RSD≤20% meets the intermediate precision verification requirements. Within this range, the smaller the RSD, the higher the intermediate precision of the method.

[0071] Table 2 Intermediate precision verification results of the method of the present invention

[0072]

[0073] The results are shown in Table 2. The RSD of the intermediate precision test results of the validation samples 64%S, 80%S, 100%S, 125%S, and 156%S was between 3.6% and 11.9%, indicating that the intermediate precision of this method was good.

[0074] (3) Relative accuracy

[0075] Under this method, the test results of the intermediate precision experiment were shared, and the recovery of the average of the four test results of 64%S, 80%S, 100%S, 125%S, and 156%S of the validation sample was investigated. The dose-effect curves of the homemade liraglutide reference substance and the validation sample were compared.

[0076] The theoretical value, measured value and average value have the same meanings as described above. The recovery rate is the average value of 4 measured values / theoretical value × 100%.

[0077] The recovery rate was between 70% and 130%, which met the acceptable standard for relative accuracy, and the closer it was to 100%, the higher the relative accuracy of the method.

[0078] Table 3 Relative accuracy verification results of the method of the present invention

[0079]

[0080] The results are shown in Table 3. The recovery rates of the relative accuracy test results of the validation samples were between 96.2% and 105.1%, indicating that the relative accuracy of this method was good.

[0081] Among them, the four test results of the representative verification sample 100%S Day1, Day2, Day3, and Day4 are as follows: Figure 4a (Day 1), Figure 4b (Day 2), Figure 4c (Day3), Figure 4d As shown in (Day4), the dose-effect curves of the control and verification samples were parallel, and the recovery rate of the 100%S verification sample was good.

[0082] (4) Durability

[0083] Cells of F5, F7 and F31 generations were used to investigate the effects of different cell generations on the 100%S determination results of the validation samples.

[0084] Table 4 Durability verification results of the method of the present invention

[0085]

[0086] The results are shown in Table 4 and Figure 5a (F5 generation), Figure 5b (F7 generation), Figure 5c As shown in Figure 3 (F31 generation), the liraglutide dose-effect curves obtained using HEK293 / CRE-Luc / GLP1R cells of F5, F7, and F31 generations were well parallelized, with an RSD of 11.5%, indicating that this method has good durability.

[0087] In summary, the specificity, intermediate precision, relative accuracy and durability of this method are good, meeting the acceptable standards for biological activity method validation. This method was also successfully used to complete the method validation of semaglutide (test results omitted). All technical features disclosed in this specification can be combined in any combination.

[0088] Each feature disclosed in this specification can also be replaced by other features with the same, equivalent or similar functions. Therefore, unless otherwise specified, each feature disclosed is only an example of a series of equal or similar features.

[0089] From the above description, those skilled in the art can easily understand the key features of the present invention. Without departing from the spirit and scope of the present invention, many modifications can be made to the invention to adapt to various different purposes and conditions of use. Therefore, such modifications are also intended to fall within the scope of the appended claims.

Claims

1. A method for detecting the biological activity of liraglutide or semaglutide, characterized in that: The method is based on a luciferase reporter gene assay of HEK293 / CRE-Luc / GLP1R cells, comprising diluting the cells with 2% FBS assay medium, seeding the plates, incubating for 1 hour, and then adding the sample to be tested for activity detection; The sample to be tested is prepared with 2% FBS analysis medium; the effective concentration range of the liraglutide sample is 2.7E-14 to 1.6E-9M; or the effective concentration range of the semaglutide sample is 1.9E-14 to 1.9E-8; and the activity detection includes using a luciferase detection reagent to detect the RLU value of the chemiluminescent signal after stimulation with liraglutide or semaglutide by an enzyme marker.

2. The method according to claim 1, characterized in that The 2% FBS assay medium is DMEM medium supplemented with 2% FBS.

3. The method according to claim 1, characterized in that The cell dilution concentration is 8×10 5 cells / ml.

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