Method for detecting biological activity of dimer Fc peptide fusion protein for stimulating platelet generation
The reporter gene method combining TD-Luc cells with luciferase substrates simplifies the activity detection of dimeric Fc peptide fusion proteins that stimulate platelet production, solves the problems of cumbersome operations and long cycles in existing technologies, and realizes simple and accurate activity detection, which is suitable for large-scale quality control.
Patent Information
- Application Number
- CN202410260584.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-07
- Publication Date
- 2025-09-09
AI Technical Summary
In the prior art, the activity detection method of the dimeric Fc peptide fusion protein that stimulates platelet production is complicated, requires high experimental conditions and has a long cycle, which makes it difficult to meet the needs of large-scale quality control.
The reporter gene method combining TD-Luc cells and luciferase substrate was used. After incubating the dimeric Fc peptide fusion protein that stimulates platelet production with the cell surface receptor, its biological activity was detected by chemiluminescence reaction, and the data were analyzed using a four-parameter mathematical model.
This enables simple and quantitative activity detection, improves the accuracy of the detection method, and is suitable for large-scale quality control applications.
Smart Images
Figure CN120609809A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of protein activity detection, and in particular to a method for detecting the biological activity of a dimeric Fc peptide fusion protein that stimulates platelet production. Background Art
[0002] The dimeric Fc peptide fusion protein (peptide body) that stimulates platelet production is produced in Escherichia coli through recombinant DNA technology. It can simulate the effect of the human body's natural thrombopoietin. When it binds to the TPO receptor, it promotes the growth of bone marrow megakaryocyte colony-forming cells (CFU-Meg) and leads to increased platelet production through the JAK2 and STAT5 kinase pathways.
[0003] The product of the proto-oncogene c-Mpl is a cell surface receptor for TPO. Binding of the protein to the TPO receptor leads to homodimerization, inducing activation of Janus kinase 2 (JAK2). The phosphotyrosine sites on the receptor serve as docking sites, allowing the binding of various signaling molecules (including SHC, GRB2, SOS, VAV, and CBL), initiating intracellular signaling. The protein activates multiple signaling pathways, including JAK / signal transducer and activator of transcription (STAT), mitogen-activated protein kinase (MAPK) / extracellular signal-regulated kinase (ERK), and phosphoinositide 3-kinase (PI3K) / ATK. The JAK / STAT and MAPK / ERK signaling pathways lead to the proliferation and maturation of megakaryocyte progenitor cells, while the PI3K / AKT pathway is important for the regulation of cellular processes.
[0004] The amino acid sequence of the dimeric Fc-peptide fusion protein (peptibody) that stimulates platelet production is not similar to that of endogenous thrombopoietin. It is a human TPO mimetic peptide produced by recombinant DNA technology. The peptibody molecule has two identical single-chain subunits, each composed of 269 amino acid residues. Each subunit consists of an IgG1 Fc domain covalently linked to two polypeptides that interact with the thrombopoietin receptor c-MPL, each composed of 14 amino acids.
[0005] The Fc constant region of the dimeric Fc-peptide fusion protein (peptibody) that stimulates platelet production can extend the half-life of the drug, thereby extending the dosing interval. In addition, because the dimeric Fc-peptide fusion protein (peptibody) that stimulates platelet production does not have endogenous TPO homology sequences, it will not cause cross-reactions and produce neutralizing antibodies.
[0006] As a biological product, a good activity detection method is an important part of its quality control. Currently, the research on the activity detection method of the dimeric Fc peptide fusion protein (peptibody) that stimulates platelet production mainly relies on cell culture. However, cell culture methods are cumbersome, require high experimental conditions, and have a long culture cycle. Therefore, a simple, quantitative, and rapid activity detection method is needed. Summary of the Invention
[0007] The object of the present invention is to provide a method for detecting the biological activity of a dimeric Fc peptide fusion protein that stimulates platelet production. The method is simple and suitable for application in large-scale quality control.
[0008] In order to achieve the purpose of the present invention, the present invention provides the following technical solutions:
[0009] The first aspect of the present invention provides a method for detecting the biological activity of a dimeric Fc peptide fusion protein that stimulates thrombopoietin, comprising: incubating a dimeric Fc peptide fusion protein standard that stimulates thrombopoietin and a sample to be tested with the same gradient concentration of the dimeric Fc peptide fusion protein with a TD-Luc cell suspension for 5 to 7 hours, then adding a luciferase substrate, mixing, measuring the chemiluminescence values of the standard and the sample to be tested, analyzing the chemiluminescence values using a four-parameter method, and calculating the activity of the sample to be tested; the TD-Luc cells are cells expressing thrombopoietin receptors and luciferase; the gradient concentration range is 0.00128 ng / mL to 100 ng / mL, the gradient concentration is 8 concentrations, and the dilution multiple of the gradient concentration is 4 to 5 times.
[0010] The present invention provides a detection method based on the positive correlation between the number of TD-Luc cells and the biological activity of a dimeric Fc peptide fusion protein that stimulates platelet production. The detection method of the present invention belongs to the reporter gene method, and its detection principle is as follows: the dimeric Fc peptide fusion protein that stimulates platelet production binds to the thrombopoietin receptor expressed on the surface of TD-Luc cells, inducing TD-Luc cell proliferation. Therefore, the number of TD-Luc cells is positively correlated with the biological activity of the dimeric Fc peptide fusion protein (peptide body) that stimulates platelet production. By utilizing the characteristics of the luciferase expressed by TD-Luc cells and the luciferase substrate to produce a chemiluminescent reaction, the luminescence value (chemiluminescence value) that can reflect the number of TD-Luc cells is measured, thereby detecting the biological activity of the dimeric Fc peptide fusion protein that stimulates platelet production.
[0011] In a preferred embodiment, the gradient concentration range is 0.0061 ng / mL to 100 ng / mL, and the dilution multiple of the gradient concentration is 4 times.
[0012] In a preferred embodiment, the gradient concentration range is 0.00128 ng / mL to 100 ng / mL, and the dilution multiple of the gradient concentration is 5 times.
[0013] In the detection method of the present invention, in order to ensure that the test sample and the standard have the same starting concentration, the test sample stock solution and the standard stock solution need to be pre-diluted, and the pre-dilution multiple is determined based on the specific concentrations of the test sample stock solution and the standard stock solution. Based on the pre-dilution, the test sample and the standard are further diluted to obtain the same gradient concentration of the test sample and the standard. Preferably, phenol red-free RPMI 1640 medium containing 5% (volume ratio) FBS is used for dilution. More preferably, the starting concentration of the test sample and the standard is 100 ng / mL.
[0014] In the detection method of the present invention, incubation is performed at 37° C. and 5% CO 2 .
[0015] In the detection method of the present invention, TD-Luc cells are cells that can express thrombopoietin receptors and luciferase. Plasmids containing thrombopoietin receptor genes and luciferase genes can be transfected into host cells by conventional genetic engineering techniques, thereby causing the host cells to express thrombopoietin receptors and luciferase. The host cells are host cells commonly used in the reporter gene method for detecting the activity of biological products in the prior art. Preferably, TD-Luc cells are Ba / F3 cells that express thrombopoietin receptors and luciferase.
[0016] In a preferred embodiment, the density of the TD-Luc cell suspension is 1×10 5 More preferably, the TD-Luc cell suspension is obtained by suspending TD-Luc cells in the logarithmic growth phase in phenol red-free RPMI 1640 medium containing 5% (volume ratio) FBS; even more preferably, the TD-Luc cells are washed with DPBS culture medium before suspension.
[0017] In a preferred embodiment, the volume ratio of the TD-Luc cell suspension to the standard or the sample to be tested is 1:1; the volume ratio of the TD-Luc cell suspension to the luciferase substrate is 1:1 or 1:2. Preferably, the volume ratio of the TD-Luc cell suspension to the luciferase substrate is 1:2. More preferably, the volume of the TD-Luc cell suspension is 60 μL, the volume of the standard and the sample to be tested is 60 μL, and the volume of the luciferase substrate is 120 μL.
[0018] In a preferred embodiment, the cells in the TD-Luc cell suspension are starved and then incubated with the standard and the sample to be tested, respectively.
[0019] The dimeric Fc peptide fusion protein that stimulates thrombopoietin of the present invention is a peptide thrombopoietin receptor agonist such as romiplostim, etc. Preferably, the dimeric Fc peptide fusion protein that stimulates thrombopoietin is romiplostim.
[0020] In a preferred embodiment, the four-parameter method comprises:
[0021] 1) drawing a four-parameter regression curve of the standard and the sample according to the chemiluminescence values of the standard and the sample;
[0022] 2) Calculating the activity of the sample to be tested based on the four-parameter regression curve and the four-parameter mathematical model; the four-parameter mathematical model is the following calculation formula: Among them, P t is the activity of the standard (ng / mL); D S D is the pre-dilution multiple of the sample to be tested; r E is the pre-dilution multiple of the standard; s E is the dilution multiple of the sample to be tested equivalent to the half-effective dose of the standard; r The dilution factor is the half-effective dose of the standard.
[0023] The drawing and calculation are completed by the software SoftMax Pro.
[0024] The second aspect of the present invention provides the use of the above-mentioned detection method in the quality control of the dimeric Fc peptide fusion protein that stimulates thrombopoiesis.
[0025] The beneficial effects of the present invention are as follows: (1) The present invention improves the four-parameter regression curve by optimizing the experimental conditions; and at the same time, by using a four-parameter mathematical model to calculate the biological activity, the accuracy of the detection method is improved. (2) The detection method is simple to operate and is suitable for application in large-scale quality control. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is the standard curve diagram of Example 1.
[0027] Figure 2 This is the standard curve diagram of Example 2.
[0028] Figure 3 This is the standard curve diagram of Example 3.
[0029] Figure 4 This is the standard curve diagram of Example 4.
[0030] Figure 5 This is the standard curve diagram of Example 5.
[0031] Figure 6This is the standard curve diagram of Example 6. DETAILED DESCRIPTION
[0032] The present invention will be further described below with reference to specific embodiments.
[0033] Unless otherwise specified, the equipment, materials, and reagents used in the following examples were obtained from commercial sources.
[0034] The TD-Luc cells used in the following examples were Ba / F3-SGG-hTR cells constructed according to the method described in "Development and validation of a novel reporter gene assay for determination of recombinant human thrombopoietin" (International Immunopharmacology, Volume 99, October 2021, 107982). The luciferase substrate solution used was Bright-Luciferase Assay Substrate.
[0035] Example 1 Detection of the activity of dimeric Fc peptide fusion protein stimulating platelet production (Condition 1)
[0036] 1.1 Sample preparation
[0037] The romiplostim stock solution was diluted with phenol red-free RPMI 1640 medium containing 5% (volume ratio) FBS, with a starting dilution concentration of 100 ng / mL, and serially diluted 4-fold for a total of 8 dilutions (0.0061 ng / mL to 100 ng / mL).
[0038] 1.2 Activity Detection
[0039] (1) TD-Luc cells in the logarithmic growth phase were washed three times with DPBS preheated at 37°C, centrifuged at 1000 rpm for 5 min, and suspended in phenol red-free RPMI 1640 medium containing 5% (volume ratio) FBS to an appropriate density. The cells were seeded into a 96-well cell culture plate at 60 μL / 6000 cells per well, and the cells were starved and cultured overnight.
[0040] (2) The sample was added to a 96-well cell culture plate inoculated with TD-Luc cells in step (1), 60 μL per well, and duplicate wells were incubated at 37°C, 5% CO2 for 5 h. 120 μL of luciferase substrate solution was added to each well, and the microplate was shaken on a microplate shaker for 5 min. After the sample in the well plate was mixed, the chemiluminescence value was measured on a microplate reader.
[0041] 1.3 Data Analysis
[0042] The standard curve of the sample was drawn by SoftMax Pro software using the 4-Parameter method. Figure 1 As shown, the standard curve fitting coefficient R 2 The EC50 value was 0.805 ng / mL.
[0043] Example 2 Detection of the activity of dimeric Fc peptide fusion protein stimulating platelet production (condition 2)
[0044] 2.1 Sample preparation
[0045] The romiplostim stock solution was diluted with phenol red-free RPMI 1640 medium containing 5% (volume ratio) FBS, with a starting dilution concentration of 100 ng / mL, and serially diluted 4-fold for a total of 8 dilutions (0.0061 ng / mL to 100 ng / mL).
[0046] 2.2 Activity Detection
[0047] (1) TD-Luc cells in the logarithmic growth phase were washed three times with DPBS preheated at 37°C, centrifuged at 1000 rpm for 5 min, and suspended in phenol red-free RPMI 1640 medium containing 5% (volume ratio) FBS to an appropriate density. The cells were seeded into a 96-well cell culture plate at 60 μL / 6000 cells per well, and the cells were starved and cultured overnight.
[0048] (2) The sample was added to a 96-well cell culture plate inoculated with TD-Luc cells in step (1), 60 μL per well, and duplicate wells were incubated at 37°C, 5% CO2 for 6 h. 120 μL of luciferase substrate solution was added to each well, and the microplate was shaken on a microplate shaker for 5 min. After the sample in the well plate was mixed, the chemiluminescence value was measured on a microplate reader.
[0049] 2.3 Data Analysis
[0050] The standard curve of the sample was drawn by SoftMax Pro software using the 4-Parameter method. Figure 2 As shown, the standard curve fitting coefficient R 2 The EC50 value was 0.714 ng / mL.
[0051] Example 3 Activity Detection of Dimeric Fc Peptide Fusion Proteins Stimulating Platelet Production (Condition 3)
[0052] 3.1 Sample preparation
[0053] The romiplostim stock solution was diluted with phenol red-free RPMI 1640 medium containing 5% (volume ratio) FBS, with a starting dilution concentration of 100 ng / mL, and serially diluted 5-fold for a total of 8 dilutions (0.00128 ng / mL to 100 ng / mL).
[0054] 3.2 Activity Detection
[0055] (1) TD-Luc cells in the logarithmic growth phase were washed three times with DPBS preheated at 37°C, centrifuged at 1000 rpm for 5 min, and suspended in phenol red-free RPMI 1640 medium containing 5% (volume ratio) FBS to an appropriate density. The cells were seeded into a 96-well cell culture plate at 60 μL / 6000 cells per well, and the cells were starved and cultured overnight.
[0056] (2) The sample was added to a 96-well cell culture plate inoculated with TD-Luc cells in step (1), 60 μL per well, and duplicate wells were incubated at 37°C, 5% CO2 for 5 h. 120 μL of luciferase substrate solution was added to each well, and the microplate was shaken on a microplate shaker for 5 min. After the sample in the well plate was mixed, the chemiluminescence value was measured on a microplate reader.
[0057] 3.3 Data Analysis
[0058] The standard curve of the sample was drawn by SoftMax Pro software using the 4-Parameter method. Figure 3 As shown, the standard curve fitting coefficient R 2 The EC50 value was 3.617 ng / mL.
[0059] Example 4 Detection of the Activity of Dimeric Fc Peptide Fusion Proteins Stimulating Thrombopoiesis (Condition 4)
[0060] 4.1 Sample preparation
[0061] The romiplostim stock solution was diluted with phenol red-free RPMI 1640 medium containing 5% (volume ratio) FBS, with a starting dilution concentration of 100 ng / mL, and serially diluted 5-fold for a total of 8 dilutions (0.00128 ng / mL to 100 ng / mL).
[0062] 4.2 Activity Detection
[0063] (1) TD-Luc cells in the logarithmic growth phase were washed three times with DPBS preheated at 37°C, centrifuged at 1000 rpm for 5 min, and suspended in phenol red-free RPMI 1640 medium containing 5% (volume ratio) FBS to an appropriate density. The cells were seeded into a 96-well cell culture plate at 60 μL / 6000 cells per well, and the cells were starved and cultured overnight.
[0064] (2) The sample was added to a 96-well cell culture plate inoculated with TD-Luc cells in step (1), 60 μL per well, and duplicate wells were incubated at 37°C, 5% CO2 for 6 h. 120 μL of luciferase substrate solution was added to each well, and the microplate was shaken on a microplate shaker for 5 min. After the sample in the well plate was mixed, the chemiluminescence value was measured on a microplate reader.
[0065] 4.3 Data Analysis
[0066] The standard curve of the sample was drawn by SoftMax Pro software using the 4-Parameter method. Figure 4 As shown, the standard curve fitting coefficient R 2 The EC50 value was 3.799 ng / mL.
[0067] Example 5 Activity Detection of Dimeric Fc Peptide Fusion Proteins Stimulating Platelet Production (Condition 5)
[0068] 5.1 Sample preparation
[0069] The romiplostim stock solution was diluted with phenol red-free RPMI 1640 medium containing 5% (volume ratio) FBS, with a starting dilution concentration of 100 ng / mL, and serially diluted 4-fold for a total of 8 dilutions (0.0061 ng / mL to 100 ng / mL).
[0070] 5.2 Activity Detection
[0071] (1) TD-Luc cells in the logarithmic growth phase were washed three times with DPBS preheated at 37°C, centrifuged at 1000 rpm for 5 min, and suspended in phenol red-free RPMI 1640 medium containing 5% (volume ratio) FBS to an appropriate density. The cells were seeded into a 96-well cell culture plate at 60 μL / 6000 cells per well, and the cells were starved and cultured overnight.
[0072] (2) The sample was added to a 96-well cell culture plate inoculated with TD-Luc cells in step (1), 60 μL per well, and duplicate wells were incubated at 37°C, 5% CO2 for 7 h. 120 μL of luciferase substrate solution was added to each well, and the microplate was shaken on a microplate shaker for 5 min. After the sample in the well plate was mixed, the chemiluminescence value was measured on a microplate reader.
[0073] 5.3 Data Analysis
[0074] The standard curve of the sample was drawn by SoftMax Pro software using the 4-Parameter method. Figure 5 As shown, the standard curve fitting coefficient R 2 The EC50 value was 0.919 ng / mL.
[0075] According to the optimization process of Examples 1-5, the fitting coefficients R of Examples 3 and 4 are as follows: 2 The fitting coefficient R of Example 1 is 0.999, but the platform is missing. 2 The fitting coefficient R of Example 2 and Example 5 is 0.989. 2 The results are all 1.000, which shows that increasing the incubation time can improve the standard curve and more accurately measure and calculate the activity value of the sample. Since the four-parameter fit is good for incubation times of 6 h and 7 h, considering the experimental time, the preferred experimental conditions of Example 2 are selected as the conditions for quantitative detection of romiplostim activity.
[0076] Example 6 Activity Detection of Dimeric Fc Peptide Fusion Proteins Stimulating Platelet Production
[0077] 6.1 Preparation of standards and test samples
[0078] Standard: Commercially available romiplostim for injection (purchased from Kyowa KIRIN, batch number: 22Y01RA, 0.5 mg, mass concentration 500,000 ng / mL) was diluted with phenol red-free RPMI 1640 medium containing 5% (volume ratio) FBS, with a starting dilution concentration of 100 ng / mL (pre-dilution multiple of 5000 times), and a 4-fold serial dilution was performed for a total of 8 dilutions (0.0061 ng / mL to 100 ng / mL).
[0079] Test sample: Dilute the romiplostim stock solution with phenol red-free RPMI 1640 medium containing 5% FBS (volume ratio), starting at 100 ng / mL (pre-dilution factor 5000-fold), and make 4-fold serial dilutions for a total of 8 dilutions (0.0061 ng / mL to 100 ng / mL).
[0080] 6.2 Activity Detection
[0081] (1) TD-Luc cells in the logarithmic growth phase were washed three times with DPBS preheated at 37°C, centrifuged at 1000 rpm for 5 min, and suspended in phenol red-free RPMI 1640 medium containing 5% (volume ratio) FBS to an appropriate density. The cells were seeded into a 96-well cell culture plate at 60 μL / 6000 cells per well, and the cells were starved and cultured overnight.
[0082] (2) The sample was added to a 96-well cell culture plate inoculated with TD-Luc cells in step (1), 60 μL per well, and duplicate wells were incubated at 37°C, 5% CO2 for 6 h. 120 μL of luciferase substrate solution was added to each well, and the microplate was shaken on a microplate shaker for 5 min. After the sample in the well plate was mixed, the chemiluminescence value was measured on a microplate reader.
[0083] 6.3 Data Analysis
[0084] The standard curve of the standard and the sample to be tested was drawn by the SoftMax Pro software using the 4-Parameter method. Figure 6 As shown, the curves of the standard and the sample fit well, and the fitting coefficient R 2 The EC50 values were 0.998 and 1.000 respectively, and the EC50 values were 0.700 and 0.707 respectively, and the reaction curves were consistent.
[0085] The activity of the sample was calculated according to the following formula:
[0086] Wherein, Pt is the activity of the standard (ng / mL), D S is the pre-dilution multiple of the sample, Dr is the pre-dilution multiple of the standard, Es is the dilution multiple of the sample to be tested equivalent to the half-effective amount of the standard, and Er is the dilution multiple of the standard to be tested equivalent to the half-effective amount. Es and Er are calculated by the computer software SoftMax Pro.
[0087] The computer software SoftMax Pro automatically calculates the test results according to the above formula, and the sample activity
[0088] =494653ng / mL.
Claims
1. A method for detecting the biological activity of a dimeric Fc peptide fusion protein that stimulates platelet production, characterized in that: include: The same gradient concentration of a platelet-stimulating dimeric Fc peptide fusion protein standard and a test sample were incubated with a TD-Luc cell suspension for 5 to 7 hours, and then luciferase substrate was added. After mixing, the chemiluminescence values of the standard and the test sample were measured. The chemiluminescence values were analyzed using a four-parameter method to calculate the activity of the test sample. The TD-Luc cells express thrombopoietin receptors and luciferase. The gradient concentration range was 0.00128 ng / mL to 100 ng / mL, the gradient concentration was 8 concentrations, and the dilution multiple of the gradient concentration was 4 to 5 times.
2. The detection method according to claim 1, wherein The gradient concentration range is 0.0061 ng / mL to 100 ng / mL, and the dilution multiple of the gradient concentration is 4 times.
3. The detection method according to claim 1, wherein The gradient concentration range is 0.00128 ng / mL to 100 ng / mL, and the dilution multiple of the gradient concentration is 5 times.
4. The detection method according to any one of claims 1 to 3, characterized in that: The dilution was performed using phenol red-free RPMI 1640 medium containing 5% (volume ratio) FBS.
5. The detection method according to claim 1, wherein The cell density in the TD-Luc cell suspension was 1×10 5 cells / mL; preferably, the TD-Luc cell suspension is obtained by suspending TD-Luc cells in the logarithmic growth phase in phenol red-free RPMI 1640 culture medium containing 5% (volume ratio) FBS; more preferably, the TD-Luc cells are washed with DPBS culture medium before suspension.
6. The detection method according to claim 1, characterized in that The volume ratio of the TD-Luc cell suspension to the standard or the sample to be tested is 1:1; and / or the volume ratio of the TD-Luc cell suspension to the luciferase substrate is 1:1 or 1:
2.
7. The detection method according to claim 1, characterized in that The cells in the TD-Luc cell suspension were starved and then incubated with the standard and the sample to be tested.
8. The detection method according to claim 1, wherein The dimeric Fc peptide fusion protein that stimulates platelet production is romiplostim.
9. The detection method according to claim 1, wherein The four-parameter method includes: 1) drawing a four-parameter regression curve of the standard and the sample according to the chemiluminescence values of the standard and the sample; 2) Calculate the activity of the sample to be tested based on the four-parameter regression curve and the four-parameter mathematical model, wherein the four-parameter mathematical model is the following calculation formula: Wherein, Pt is the activity of the standard (ng / mL); D S is the pre-dilution multiple of the sample to be tested; Dr is the pre-dilution multiple of the standard; Es is the dilution multiple of the sample to be tested equivalent to the half-effective amount of the standard; Er is the dilution multiple of the standard equivalent to the half-effective amount.
10. Use of the detection method according to any one of claims 1 to 9 in controlling the quality of a dimeric Fc peptide fusion protein that stimulates platelet production.