CN118240917A - A method for detecting the biological activity of GLP-1, GLP-1 analogs or GLP-1 fusion proteins - Google Patents

Abstract

The application relates to the field of biological medicine, in particular to a method for detecting the biological activity of GLP-1, GLP-1 analogues or GLP-1 fusion proteins, which is a luciferase reporter gene assay method based on HEK293/CRE-Luc/GLP1R cells, and comprises the steps of diluting the cells with 2% FBS analysis medium, plating, incubating for 1 hour, and adding a sample to be detected for activity detection.

Description

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

Technical Field

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 technique

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.

At present, the luciferase reporter gene method is widely used to determine 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, the fluorescence signal can be detected. The RLU value (relative light unit) is measured by an ELISA instrument, and the experimental data is analyzed by software to draw a dose-response curve. The biological activity of the corresponding GLP-1, GLP-1 analog or GLP-1 fusion protein can be calculated based on the EC ₅₀ value.

However, the existing luciferase reporter gene assay has a long test cycle, high material consumption, and large sample usage, which makes the experiment time-consuming and material-intensive. For example, patent document CN115703766A Example 13 discloses a typical GenScript luciferase reporter gene assay method, in which HEK293/CRE-Luc/GLP1R cells need to be incubated overnight after being plated, using 10% FBS complete medium. In addition, according to the fitting curve provided in the corresponding product manual of GenScript (Cat. No. M00562), it is speculated that the effective concentration of the GLP-1(1-37) sample is 1.0E-12 ~ 1.0E-6 M.

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

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.

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.

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, seeding the plates, incubating for 1 hour, and then adding the sample to be tested for activity detection.

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.

In one aspect, the method of the present invention uses a luciferase detection reagent, such as a commercially available luciferase reporter gene detection reagent such as the Bio-Lite ^™ Luciferase Assay System, Fire-Lumi™ Assay System or Bright-Glo ^™ Luciferase Assay System reagent, to detect the RLU value of the chemiluminescent signal after stimulation with GLP-1, a GLP-1 analog or a GLP-1 fusion protein using an ELISA reader.

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

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

In one aspect, the cells in the method of the present invention are diluted to a concentration of 8×10 ⁵ cells/ml.

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.

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.

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

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

FIG. 1a is a fitting curve of liraglutide measured when the cells were seeded and incubated overnight; and FIG. 1b is a fitting curve of liraglutide measured when the cells were seeded and incubated for 1 hour.

FIG. 2 is a fitting curve of semaglutide measured after the cells were incubated for 1 hour after seeding.

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

Figure 4a is a fitting curve of the relative accuracy in the verification of the method of the present invention when liraglutide reference is used as the reference and 100% S of the verification sample is added on Day 1 for detection; Figure 4b is a fitting curve of the relative accuracy in the verification of the method of the present invention when liraglutide reference is used as the reference and 100% S of the verification sample is added on Day 2 for detection; Figure 4c is a fitting curve of the relative accuracy in the verification of the method of the present invention when liraglutide reference is used as the reference and 100% S of the verification sample is added on Day 3 for detection; and Figure 4d is a fitting curve of the relative accuracy in the verification of the method of the present invention when liraglutide reference is used as the reference and 100% S of the verification sample is added on Day 4 for detection.

Figure 5a is a durability fitting curve in the verification of the method of the present invention when liraglutide reference is used as a reference and HEK293/CRE-Luc/GLP1R cells of the F5 generation are used; Figure 5b is a durability fitting curve in the verification of the method of the present invention when liraglutide reference is used as a reference and HEK293/CRE-Luc/GLP1R cells of the F7 generation are used; and Figure 5c is a durability fitting curve in the verification of the method of the present invention when liraglutide reference is used as a reference and HEK293/CRE-Luc/GLP1R cells of the F31 generation are used.

Detailed ways

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

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, and Bio-Lite ^™ Luciferase Assay System detection kit purchased from Vazyme (DD1201-03). Liraglutide and semaglutide reference products were homemade by Chia Tai Tianqing Pharmaceutical Group Co., Ltd.

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.

Solution preparation instructions:

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

Preparation of Bio-Lite ^TM Luciferase reagent: Take out the Bio-Lite ^TM Luciferase Assay System kit and equilibrate it to room temperature. After the Bio-Lite ^TM Luciferase Assay Buffer is completely dissolved, add it to the Bio-Lite ^TM Luciferase Assay Substrate, mix well, and store it at -20℃ in the dark after aliquoting.

Biological activity calculation formula:

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:

Where, Y is the RLU value;

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

Bottom is the asymptote estimate under the curve;

Top is the asymptote valuation on the curve;

HillSlope is the slope of the curve;

_EC50 is the half-maximal effect concentration, mol/L.

Example 1: Comparative Example of Overnight Incubation after Cell Seeding

1. Cell dilution, plating and incubation

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 live cell density to 8×10 ⁵ cells/ml with 2% FBS analysis medium.

Cell seeding and incubation: After mixing the above cell suspension, add 50 μl/well to a 96-well all-white plate and incubate in a 37°C, 5% CO ₂ incubator overnight.

2. Preparation of sample solution

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.

3. Compound stimulation

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 2 parallel wells for each dilution and incubate in a 37°C, 5% CO ₂ incubator for 6 hours.

4. Detection

Take out the mixed and packaged Bio-Lite ^TM Luciferase reagent in advance, melt it at room temperature, add 100μl/well to the above experimental wells, incubate at room temperature in the dark for 2-3 minutes. Read the RLU value on the chemiluminescence detection module of the microplate reader.

5. Data processing

GraphPad Prism software was used to analyze the experimental data. 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 _EC50 value of the sample.

The results are shown in Figure 1a. The R ² of the fitting curve of this method is 0.9208, which is poorly fitted and the signal window is only 1.2 times. The EC ₅₀ of the liraglutide sample was measured to be 1.0E-9 M.

Example 2 Improved method for determining the activity of liraglutide samples

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.

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.

The results are shown in Figure 1b. Compared with before adjustment, the fitting curve is approximately "S" shaped, ^R2 is 0.9989, the fitting degree is good, and the signal window is increased to 336.4 times. The _EC50 of liraglutide measured by this method is 1.4E-11 M. This shows that the use of this method is not only fast, saves materials and samples, but also has a good dose-effect curve for the detection of liraglutide.

Example 3 Improved method for determining the activity of semaglutide samples

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 after addition of sample was 1.9E-8 M) with 2% FBS analysis medium as the first compound concentration point, and then a 4-fold gradient dilution was performed 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.

The results are shown in Figure 2. The fitting curve is approximately "S" shaped, ^R2 is 0.9992, and the fitting degree is good. The _EC50 of semaglutide measured by this method 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.

Example 4 Method Verification Example

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.

(1) Exclusivity

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.

The results are shown in Figure 3a. When only the blank control or negative control is added, the measured RLU value is equivalent to the platform area under the dose-effect curve of liraglutide, and there is no dose dependence, indicating that the blank buffer does not interfere with the determination of the biological activity of the sample by this method, and the method has no response to degludec insulin. In contrast, as shown in Figure 3b, when Novo Nordisk's liraglutide preparation is added, a dose-response curve can be fitted. The above results show that the method has good specificity.

(2) Intermediate precision

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.

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.

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

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.

The theoretical value is the ideal value of biological activity obtained by theoretical calculation under ideal conditions at different potency levels. Since the validation sample and the reference substance are derived from the same substance, i.e., the homemade liraglutide reference substance, their structure and properties are the same, and 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 prepared and 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 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, i.e., EC ₅₀ of the reference substance / EC ₅₀ of the validation sample × 100%. The average value is the average of the 4 measured values, and the RSD is the standard deviation of the 4 measured values/average value × 100%.

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

Table 2 Intermediate precision verification results of the method of the present invention

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.

(3) Relative accuracy

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.

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%.

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.

Table 3 Relative accuracy verification results of the method of the present invention

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.

Among them, the four test results of the representative verification sample 100%S Day1, Day2, Day3, and Day4 are shown in Figure 4a (Day1), Figure 4b (Day2), Figure 4c (Day3), and Figure 4d (Day4), respectively. The dose-effect curves of the reference and the verification samples are parallel, and the recovery rate of the 100%S verification sample is good.

(4) Durability

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.

Table 4 Durability verification results of the method of the present invention

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

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 (experimental results omitted). All technical features disclosed in this specification can be combined in any combination.

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

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 (10)

1. A method for detecting the biological activity of GLP-1, a GLP-1 analogue or a GLP-1 fusion protein, characterized in that the method is a luciferase reporter assay based on HEK293/CRE-Luc/GLP1R cells, comprising diluting the cells with 2% FBS assay medium, plating, incubating for 1 hour, and adding a sample to be tested for activity detection.

2. The method of claim 1, further comprising formulating the sample solution of GLP-1, GLP-1 analogue or GLP-1 fusion protein with 2% FBS assay medium.

3. The method of claim 1 or 2, further comprising detecting the post-stimulation chemiluminescent signal RLU value of GLP-1, a GLP-1 analog, or a GLP-1 fusion protein by a microplate reader using a luciferase detection reagent.

4. The method of claim 1 or 2, wherein the 2% FBS assay medium is DMEM medium supplemented with 2% FBS.

5. The method of claim 1 or2, wherein the cell dilution concentration is 8 x 10 ⁵ cells/ml.

6. The method of claim 1 or 2, wherein the GLP-1 analogue or GLP-1 fusion protein is liraglutide, semraglutide, dolraglutide, risraglutide, apride, benraglutide, tasraglutide, su Palu peptide, exenatide, or telipopeptide.

7. The method of claim 1 or 2, wherein the GLP-1 analogue is liraglutide.

8. The method of claim 7, wherein the range of action concentration of the liraglutide sample is 2.7E-14 to 1.6E-9M.

9. The method of claim 1 or 2, wherein the GLP-1 analogue is semaglutinin.

10. The method of claim 9, wherein the concentration range of the semaglutin sample is 1.9E-14 to 1.9E-8M.