CN118240917A - Method for detecting biological activity of GLP-1, GLP-1 analogue or GLP-1 fusion protein - Google Patents
Method for detecting biological activity of GLP-1, GLP-1 analogue or GLP-1 fusion protein Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 69
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 title claims abstract description 61
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- UKVFVQPAANCXIL-FJVFSOETSA-N glp-1 (1-37) amide Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 UKVFVQPAANCXIL-FJVFSOETSA-N 0.000 description 1
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- GCYXWQUSHADNBF-AAEALURTSA-N preproglucagon 78-108 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 GCYXWQUSHADNBF-AAEALURTSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/605—Glucagons
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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
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a method for detecting the biological activity of GLP-1, GLP-1 analogues or GLP-1 fusion proteins.
Background
Glucagon-like peptide-1 (GLP-1) is an intestinal-derived polypeptide hormone in human body, which specifically acts on GLP-1 receptor of islet cells to trigger GLP-1R-mediated cascade of signaling pathways, thereby stimulating insulin secretion in a glucose concentration-dependent manner and exerting hypoglycemic function. In order to overcome the defect of short in vivo half-life of natural GLP-1, a plurality of GLP-1 analogues or GLP-1 fusion protein hypoglycemic drugs are marketed so as to meet the clinical application of diabetes patients growing worldwide, particularly domestic.
Currently, luciferase reporter methods are widely used to determine the biological activity of GLP-1, GLP-1 analogs or GLP-1 fusion proteins. The principle of the assay is that GLP-1, GLP-1 analogues or GLP-1 fusion proteins interact with GLP-1 receptors on cell membranes to activate intracellular adenylate cyclase, resulting in increased intracellular cAMP levels, which in turn activate the luciferase signaling pathway of the cells, and when luciferase detection substrates are added, a fluorescent signal is detected. The biological activity of the corresponding GLP-1, GLP-1 analogue or GLP-1 fusion protein can be calculated according to the EC 50 value by measuring the RLU value (RELATIVE LIGHT unit ) by an enzyme-labeled instrument and analyzing the experimental data by software to draw a dose response curve.
However, the existing luciferase reporter gene assay method has long test period, more material consumption and large sample consumption, so that the test is time-consuming and material-consuming. For example, patent document CN115703766a example 13 discloses a typical gold srey (GenScript) luciferase reporter assay method, requiring overnight incubation after HEK293/CRE-Luc/GLP1R cell plating, using 10% FBS complete medium. In addition, according to the fitted curve provided by GenScript corresponding product instruction (Cat. No. M00562), the working concentration of GLP-1 (1-37) samples was estimated to be 1.0E-12 to 1.0E-6M.
There is therefore a need in the art to develop more cost effective methods to save time, materials, and/or reduce sample usage.
Disclosure of Invention
The present invention addresses the above-mentioned problems by providing an improved method for detecting the biological activity of GLP-1, GLP-1 analogs or GLP-1 fusion proteins. The 10% FBS complete medium was changed to 2% FBS assay medium while the post-plating incubation of cells was shortened to 1 hour overnight.
Through a large number of experiments, the improved method can stably and accurately obtain the dose response curve of GLP-1, GLP-1 analogues or GLP-1 fusion proteins, and has good method specificity, relative accuracy, intermediate precision and durability.
Accordingly, the present invention provides a method for detecting the biological activity of GLP-1, GLP-1 analogues or GLP-1 fusion proteins, said method being based on a luciferase reporter assay of HEK293/CRE-Luc/GLP1R cells, comprising diluting said cells with 2% FBS assay medium, plating, incubating for 1 hour and then adding a sample to be tested for activity detection.
In one aspect, the methods of the invention further comprise formulating a sample solution of the GLP-1, GLP-1 analog or GLP-1 fusion protein with 2% FBS assay medium.
In one aspect, the methods of the invention detect the chemiluminescent signal RLU value after stimulation of GLP-1, a GLP-1 analog, or a GLP-1 fusion protein by an enzyme-labeled instrument using a Luciferase detection reagent, such as a commercially available Luciferase reporter detection reagent, such as Bio-Lite TM Luciferase ASSAY SYSTEM, fire-Lumi ™ ASSAY SYSTEM, or Bright-Glo TM Luciferase ASSAY SYSTEM reagent.
In one aspect, the data analysis software of the method of the present invention may utilize GRAPHPAD PRISM or other suitable data analysis software.
In one aspect, the 2% FBS assay medium of the method of the present invention is DMEM medium supplemented with 2% FBS.
In one aspect, the cell dilution concentration in the method of the invention is 8X 10 5 cells/ml.
The methods of the invention can be used to analyze the activity of GLP-1, GLP-1 analogs, or GLP-1 fusion proteins (e.g., GLP-1/antibody fusion proteins, GLP-1/Fc fusion proteins). Preferably, the GLP-1 analog or GLP-1 fusion protein comprises liraglutide, semraglutide, duraglutide, liraglutide, abirudin, benraglutide, tasraglutide, su Palu peptide, exenatide, telpofungin and CPX101 product.
Preferably, the GLP-1 analog is liraglutide. The preferable range of the action concentration of the liraglutide sample is 2.7E-14-1.6E-9M.
Preferably, the GLP-1 analog is semaglutinin. The preferable range of the action concentration of the semaglutin sample is 1.9E-14 to 1.9E-8M.
Compared with the prior art such as a general gold-sry detection method, the method has the advantages that the material FBS is saved, the test period is greatly shortened, the required sample action concentration is lower, and the range is about 1.0E-14-1.0E-8M.
Drawings
FIG. 1a is a fitted curve of liraglutide measured after incubation overnight after cell plating; and FIG. 1b is a fitted curve of liraglutide measured 1 hour after incubation of the cell plates.
Fig. 2 is a fitted curve of semaglutin measured 1 hour after incubation of cell plating.
FIG. 3a is a fitting curve of a specificity investigation test in the verification of the method of the present invention when a buffer blank or a insulin-delta negative control is added with a liraglutide reference as a reference; and FIG. 3b shows a fitting curve of a proprietary investigation test in the method of the present invention when the reference drug Noand Node liraglutide preparation is added with the reference liraglutide reference as a control.
FIG. 4a is a graph showing the fit of the relative accuracy in the verification of the method of the present invention when Day1 is added to the verification sample for 100% S detection using a reference Liraglutide as a control; FIG. 4b is a graph showing the fit of the relative accuracy in the verification of the method of the present invention when Day2 is added to the verification sample for 100% S detection using a reference Liraglutide as a control; FIG. 4c is a graph showing the fit of the relative accuracy in the verification of the method of the present invention when Day3 is added to the verification sample for 100% S detection using the Liraglutide reference as a reference; and FIG. 4d is a graph showing the fit of the relative accuracy in the verification of the method of the present invention when Day4 is added to the verification sample for 100% S detection, using the Liraglutide reference as a reference.
FIG. 5a is a fit curve of durability in the method of the present invention when HEK293/CRE-Luc/GLP1R cells of F5 generation were used with a reference liraglutide as a control; FIG. 5b is a fit curve of durability in the method of the invention when HEK293/CRE-Luc/GLP1R cells of F7 generation were used with a reference liraglutide as a control; and FIG. 5c is a robust fit curve in the method of the present invention when HEK293/CRE-Luc/GLP1R cells of F31 generation were used with a reference liraglutide as a control.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the present invention is not limited thereto.
Description: the cells in the examples were HEK293/CRE-Luc/GLP1R cells purchased from Nanjing Kirschner Biotechnology Co., ltd (M00562), 96 Kong Quanbai plates purchased from Corning Corp, and Bio-Lite TM Luciferase ASSAY SYSTEM detection kit purchased from Vazyme Corp (DD 1201-03). The liraglutide, semex Lu Taican, is self-made by the pharmaceutical company, inc. of the pharmaceutical industry, inc. of Zhengda Tianqing.
The reagents are all commercially available products unless otherwise specified. The methods used in the examples are conventional in the art unless otherwise specified.
Solution preparation description:
2% FBS assay medium: under the aseptic condition, 2ml of FBS is absorbed, 98ml of DMEM GlutaMax basic culture medium is added, and the mixture is uniformly mixed and stored at 2-8 ℃.
Preparation of Bio-Lite TM Luciferase reagent: and taking out the Bio-Lite TM Luciferase ASSAY SYSTEM kit, balancing to room temperature, adding the kit into the Bio-Lite TM Luciferase Assay Substrate after the Bio-Lite TM Luciferase Assay Buffer is completely dissolved, uniformly mixing, packaging, and preserving at-20 ℃ in a dark place.
Biological activity calculation formula:
Biological activity calculation takes log (actual concentration of compound) as an abscissa, and chemiluminescence value RLU as an ordinate, four-parameter fitting is performed, and a specific formula is as follows:
Wherein Y is RLU value;
X is log (compound concentration) value, mol/L;
Bottom is the asymptote estimate under the curve;
top is the asymptote estimate on the curve;
HillSlope is the slope of the curve;
EC 50 is the half-maximal effect concentration, mol/L.
Example 1 incubation of comparative example overnight after cell plating
1. Cell dilution, plating and incubation
Cell collection and counting: taking out cells (HEK 293/CRE-Luc/GLP 1R), discarding culture supernatant, sucking pancreatin to clean residual culture medium, adding 5ml pancreatin into each T75 cell culture flask after removing pancreatin, digesting for 2-3 min at 37 ℃, adding 5 ml/flask analysis culture medium to elute after complete cell digestion, transferring to a sterile centrifuge tube, and centrifuging for 5min at 1000 rpm min. The supernatant was discarded, the cells were resuspended in 2% FBS assay medium, counted, and the viable cell density was adjusted to 8X 10 5 cells/ml with 2% FBS assay medium.
Cell plating and incubation: after the cell suspension was homogenized, 50. Mu.l/well was added to a 96 Kong Quanbai plate and incubated overnight at 37℃in a 5% CO 2 incubator.
2. Preparation of sample solution to be tested
The liraglutide sample was diluted to 1.1E-5M (working concentration 5.3E-6M after loading) with 2% FBS assay medium as the first compound concentration point, followed by 5-fold gradient dilution to give 12 compound solutions of different concentrations.
3. Compound stimulation
The compound solution formulated in step 2 was added at 50 μl/well to the 96 Kong Quanbai plates incubated overnight in step 1.2 duplicate wells were placed for each dilution and incubated in a 5% CO 2 incubator at 37℃for 6 hours.
4. Detection of
And taking out the Bio-Lite TM Luciferase reagent after mixing and packaging in advance, melting at room temperature, adding 100 μl/hole into the experimental hole, and incubating for 2-3 minutes at room temperature in a dark place. And reading the RLU value in a chemiluminescent detection module of the enzyme label instrument.
5. Data processing
And analyzing experimental data by GRAPHPAD PRISM software, taking the logarithm of the molar concentration of the sample as an X axis, taking the corresponding RLU value as a Y axis, fitting by using a four-parameter equation, and drawing a sample dose response curve to obtain the EC 50 value of the sample.
As a result, as shown in FIG. 1a, the fitting curve R 2 of the method is 0.9208, the fitting degree is poor, and the signal window is only 1.2 times. The EC 50 of the liraglutide sample was measured to be 1.0E-9M.
Example 2 improved Liraglutide sample Activity determination method
In view of the poor signal window of the overnight incubation method used in the comparative example of example 1, in this example, the post-plating incubation time was adjusted to 1 hour while the concentration of the effect was adjusted to give a more complete result fitting curve for the upper and lower platforms.
Referring to the experimental procedure of example 1, except that the incubation time after cell plating was adjusted to 1 hour and the effect concentration of liraglutide sample was adjusted, the sample was diluted to 3.2E-9M (effect concentration after sample addition 1.6E-9M) with 2% FBS assay medium as the first compound concentration point, followed by 3-fold gradient dilution to obtain 11 compound solutions of different concentrations. Namely, the action concentration range of the compound reference substance/the test substance is 2.7E-14-1.6E-9M.
As a result, as shown in fig. 1b, the fitting curve is approximately "S" shaped, R 2 is 0.9989, the fitting degree is good, and the signal window is increased to 336.4 times compared with the fitting curve before adjustment. The liraglutide EC 50 measured by the method is 1.4E-11M. The method is rapid, saves materials and samples, and has a good dose-response curve for detecting the liraglutide.
Example 3 improved method for determining the Activity of a sample of semaglutin
The experimental procedure of example 2 is referred to, except that the concentration of the active concentration of the semaglutin sample is adjusted. The semaglutin sample was diluted to 3.8E-8M (post-loading concentration 1.9E-8M) with 2% FBS assay medium as the first compound concentration point, followed by 4-fold gradient dilution to give 11 compound solutions of different concentrations. Namely, the action concentration range of the compound reference substance/the test substance is 1.9E-14 to 1.9E-8M.
As shown in FIG. 2, the fitting curve is approximately S-shaped, R 2 is 0.9992, the fitting degree is good, and the method measures that the semaglutin EC 50 is 3.7E-11M. The method is rapid, saves materials and samples, and has a good dose-response curve for detecting the semaglutin.
Example 4 method verification example
In the embodiment, liraglutide is taken as a test sample, and the scientificity of the measuring method is verified from four aspects of specificity, intermediate precision, relative accuracy and durability from the aspect of professional analysis.
(1) Specialization of
The self-made liraglutide reference substance is used as a reference substance, and the influence of the following three conditions on the biological activity detection is respectively examined. a) Blank control test: a buffer without liraglutide was used as a blank to examine whether it interfered with the assay of the method. b) Negative control test: insulin deluge was used as a negative control to examine whether the method responded to it. c) Reference drug comparison test: with the liraglutide formulation of norand nod company as a control, it was examined whether a dose response curve could be specifically fitted using this method.
The results are shown in fig. 3a, where only the blank or negative control was added, the RLU values measured were comparable to the plateau under the dose-response curve of liraglutide and were dose-independent, indicating that the blank buffer did not interfere with the assay of the biological activity of the sample and that the method was not responsive to insulin deluge. In contrast, as shown in fig. 3b, a dose response curve can be fitted when adding the liraglutide formulation of norand nod. The above results indicate that the method has good specificity.
(2) Intermediate precision
The self-made liraglutide reference product is used as a reference Substance (STD), the self-made liraglutide reference product is pre-diluted to 1.0E-8M by using a 2% FBS analysis medium, then the first concentrations of 64% S, 80% S, 100% S, 125% S and 156% S of verification samples are respectively obtained by dilution according to the following table 1, and then 3-time gradient dilution is continuously carried out, so that 11 verification sample solutions with different concentrations are obtained.
The different potency levels of the above-mentioned verification samples are set according to the biological activity/potency measurement method verification guidelines prescribed by the four parts of the national formulary of the pharmacopoeia Committee of the people's republic of China (edition 2020), eleventh edition, national medical science and technology Press, beijing.
TABLE 1 first concentration point dilution scheme for control and validation samples
The relative titers of control STD and validation samples 64% s, 80% s, 100% s, 125% s, 156% s were measured 4 times each using 2 cell passages on different dates by different inspectors (2, analyst1 and Analyst 2), and the biological activity and RSD values for each sample were calculated.
The theoretical value is an ideal value of biological activity obtained by theoretical calculation under ideal conditions of different titer levels, and the structure and the property of the self-made liraglutide reference substance are the same as those of the reference substance because the verification sample and the reference substance are derived from the same substance, and the verification sample with different titer levels is prepared from the reference substance according to different concentration ratios. For example, the first concentration point formulation for the validation sample 64% s prepared in table 1 above was calculated as STD concentration x 64%, and correspondingly, its biological activity should be the biological activity of the control (assigned 100%) x 64%, thus validating the sample 64% s to be 64% of the theoretical biological activity value, and so on. The measured value is the biological activity value actually measured by the test, i.e., reference EC 50/validation sample EC 50 x 100%, the average is the average of 4 measured values, and RSD is the standard deviation/average of 4 measured values x 100%.
RSD less than or equal to 20% meets the intermediate precision validation requirement, and a smaller RSD in this range represents a higher intermediate precision of the method.
TABLE 2 intermediate precision validation results of the inventive method
The results are shown in Table 2, and the intermediate precision detection results RSD of the samples 64% S, 80% S, 100% S, 125% S and 156% S are verified to be between 3.6% and 11.9%. The method is shown to have good intermediate precision.
(3) Relative accuracy
Under the method, the detection results of the intermediate precision experiment are shared, and the recovery rate of the average value of the detection results of 64% S, 80% S, 100% S, 125% S and 156% S4 times of the verification sample is examined. Comparing the self-made liraglutide reference substance with the dose-response curve of the verification sample.
Theoretical, measured and average values are as described above, and recovery is the average of 4 measured values/theoretical value x 100%.
Recovery is between 70% and 130%, meets acceptable standards for relative accuracy, and the closer to 100% represents the higher the relative accuracy of the process.
TABLE 3 results of the relative accuracy verification of the inventive method
The results are shown in Table 3, and the recovery rate of the detection result of the relative accuracy of the verification sample is between 96.2% and 105.1%, which shows that the method has good relative accuracy.
The results of 4 times of detection of representative verification samples 100% S Day1, day2, day3 and Day4 are shown in FIG. 4a (Day1), FIG. 4b (Day 2), FIG. 4c (Day 3) and FIG. 4d (Day 4), respectively, and the control and the verification sample have parallelism in the dose-response curves, and the recovery rate of the verification sample of 100% S is good.
(4) Durability of
The effect of different cell passages on the results of the 100% s assay of the validation sample was examined using cells of passages F5, F7, F31, respectively.
TABLE 4 results of durability verification of the inventive method
As shown in Table 4 and FIG. 5a (F5 times), FIG. 5b (F7 times) and FIG. 5c (F31 times), the results show that the liraglutide dose-response curves obtained with HEK293/CRE-Luc/GLP1R cells of F5, F7 and F31 times have good parallelism and RSD is 11.5%, indicating that the method has good durability.
In conclusion, the method has good specificity, intermediate precision, relative accuracy and durability, and meets the acceptable standard of biological activity method verification. The method verification (the test result is slightly less) of the semaglutin is also successfully completed by adopting the method. All technical features disclosed in this specification may be combined in any combination.
Each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the foregoing description, it will be apparent to those skilled in the art from this disclosure that many modifications can be made to the present invention without departing from its spirit or scope, as defined in 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.
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