A method for determining the biological activity of rhTSH drug

Abstract

The present invention discloses a method for determining the biological activity of rhTSH drug. Specifically, the present invention provides two strains of cells co-expressing TSHR and CRE-luciferase reporter gene, and provides a method for detecting the biological activity of rhTSH drug based on the two kinds of cells. This method is simple to operate, has high sensitivity, and good accuracy and precision, and is of great significance in the biological activity evaluation and quality control of rhTSH.

Description

Technical Field

[0001] The present invention belongs to the field of biological activity detection of biopharmaceuticals. Specifically, the present invention relates to a method for determining the biological activity of rhTSH drugs. Background Art

[0002] Recombinant human thyroid-stimulating hormone (rhTSH) is a recombinant protein expressed in vitro by genetic recombination technology and has the same effect as human thyroid-stimulating hormone. After injection, it can rapidly increase the TSH level in patients, promote iodine uptake and organification, and facilitate the rapid acceptance of iodine-131I treatment by patients with differentiated thyroid cancer to remove residual thyroid tissue after surgery. In addition, rhTSH can stimulate the secretion of thyroglobulin (Tg) by residual thyroid tissue or metastases in the body, facilitating the diagnosis and monitoring of disease progression. Compared with the traditional thyroid hormone withdrawal method, injecting rhTSH drugs can more safely and rapidly increase the serum TSH level in patients and greatly reduce the pain caused by patients stopping taking thyroid hormones. Its clinical application for nearly 30 years has obtained recommendations from multiple global thyroid cancer-related guidelines.

[0003] Biological activity can reflect the correctness of the higher-order structure of therapeutic proteins, the integrity of biological functions, and the batch-to-batch consistency of products, and is a key quality attribute of rhTSH drugs. The earliest TSH activity assay was based on the uptake of I131 by mouse thyroid glands. After intravenous injection of TSH, the increase in I131 in the blood of mice was detected to indicate TSH activity. Due to the disadvantages of long cycle, large individual differences, high requirements for operation proficiency, and high variability in animal experiments, it has become a consensus to vigorously develop in vitro bioactivity analysis methods to supplement or even replace in vivo animal analysis methods. In 2022, Liu et al. developed a TSH activity assay method based on luminescence resonance energy transfer (LRET), and evaluated TSH activity by detecting the luminescence (UCL) intensity (I545 nm) generated by the binding of TSH to donors and receptors. Although this method is simple to operate, highly specific, and sensitive, it is still essentially a content determination method, unable to simulate in vivo drug efficacy and truly reflect the biological activity of TSH. Summary of the Invention

[0004] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a method for determining the biological activity of rhTSH drugs.

[0005] In order to achieve the above purpose, the present invention provides the following technical solutions:

[0006] In the first aspect of the present invention, a cell line co-expressing TSHR and CRE-luciferase reporter gene is provided.

[0007] Furthermore, the cell is named HEK293-TSHR-CRE-Luc and is deposited in the China General Microbiological Culture Collection Center, with the address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO.46053, and the taxonomic name is human embryonic kidney cell 293. The deposit date is October 9, 2024.

[0008] In a specific embodiment of the present invention, the C18 cell line has the highest positive rate of TSHR expression, reaching 98.8%. At the same time, it has the strongest response ability to rhTSH and the highest signal-to-noise ratio. Using this cell to measure the biological activity of rhTSH has a better effect.

[0009] The second aspect of the present invention provides a cell line co-expressing TSHR and NFAT-luciferase reporter gene.

[0010] Furthermore, the cell is named HEK293-TSHR-NFAT-Luc and is deposited in the China General Microbiological Culture Collection Center, with the address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO.46054, and the taxonomic name is human embryonic kidney cell 293. The deposit date is October 9, 2024.

[0011] In a specific embodiment of the present invention, the N9 cell line has the highest positive rate of TSHR expression, reaching 99.4%. At the same time, it has the strongest response ability to rhTSH and the highest signal-to-noise ratio. Using this cell to measure the biological activity of rhTSH has a better effect.

[0012] The third aspect of the present invention provides a method for detecting the in vitro biological activity of rhTSH drugs.

[0013] Furthermore, the method includes the following steps: adding the rhTSH drug to the cells described in the first aspect or the second aspect of the present invention and incubating; after incubation, adding an enzyme reaction substrate, and determining the in vitro biological activity of the rhTSH drug according to the measured reporter gene signal value.

[0014] In the present invention, after measuring the reporter gene signal value, a four-parameter curve is fitted to determine the biological activity of the rhTSH drug. The term "fitting a four-parameter curve" is a mathematical method used to describe a set of data by fitting a curve and obtaining a functional equation that can describe the data relationship for predicting future data. The four-parameter curve is usually defined by four parameters, which have practical significance in the biological context. For example, in detecting the biological activity of a drug, these four parameters may represent: the lower limit of absorbance (D), the upper limit of absorbance (A), that is, the minimum and maximum values of absorbance; the half-response concentration (C), that is, the value when the drug concentration reaches half of the maximum response; and the slope of the curve (B). Through these parameters, experimental data can be understood and analyzed more accurately, thus providing deeper insights for biological research.

[0015] Further, the method further includes the step of gradient diluting the rhTSH drug before incubation.

[0016] Further, when the rhTSH drug is co-incubated with the cells described in the first aspect of the present invention, the initial concentration of the rhTSH drug is 0.5 - 20 μg / mL; preferably, the initial concentration of the rhTSH drug is 10 μg / mL.

[0017] Further, when the rhTSH drug is co-incubated with the cells described in the second aspect of the present invention, the initial concentration of the rhTSH drug is 4 - 40 μg / mL; preferably, the initial concentration of the rhTSH drug is 20 μg / mL.

[0018] Further, the dilution factor of the rhTSH drug is 3 - 6 times.

[0019] Further, when the rhTSH drug is co-incubated with the cells described in the first aspect of the present invention, the dilution factor of the rhTSH drug is 6 times; when the rhTSH drug is co-incubated with the cells described in the second aspect of the present invention, the dilution factor of the rhTSH drug is 5 times.

[0020] Further, when the rhTSH drug is co-incubated with the cells described in the first aspect of the present invention, the seeding density of the cells is 0.3×10 5 ~2.0×10 5 cells / mL.

[0021] Preferably, the seeding density of the cells is 0.8×10 5 cells / mL.

[0022] Preferably, the incubation time is 3 - 6 h.

[0023] More preferably, the incubation time is 4 h.

[0024] Furthermore, when the rhTSH drug is co-incubated with the cells described in the second aspect of the present invention, the seeding density of the cells is 0.3×10 5 ~2.0×10 5 cells / mL.

[0025] Preferably, the seeding density of the cells is 0.8×10 5 cells / mL.

[0026] Preferably, the incubation time is 7 - 10 h.

[0027] More preferably, the incubation time is 9 h.

[0028] The fourth aspect of the present invention provides a product for measuring the biological activity of rhTSH drugs.

[0029] Furthermore, the product includes the cells described in the first aspect or the second aspect of the present invention.

[0030] Furthermore, the product further includes a luciferase reaction substrate and a diluent.

[0031] Preferably, the product type is a kit.

[0032] In some embodiments, the components of the kit can be packaged in the form of an aqueous medium or in a lyophilized form. Suitable containers in the kit generally include at least one vial, test tube, flask, bottle, syringe or other container, in which one component can be placed, and preferably, can be appropriately aliquoted. When there is more than one component in the kit, the kit will generally also include a second, third or other additional containers in which the additional components are separately placed. However, different combinations of components can be included in one vial. The kit of the present invention will generally also include a container for containing the reactants, sealed for commercial sale. Such a container can include a molded or blow-molded plastic container in which the required vials can be retained.

[0033] The fifth aspect of the present invention provides a system for evaluating the biological activity of rhTSH drugs.

[0034] Furthermore, the system includes the following components: the cells described in the first aspect or the second aspect of the present invention, an rhTSH drug sample and a reference product.

[0035] In some embodiments, the term "system" refers to a drug quality control system, which refers to controlling various factors in the drug production process through scientific management methods to ensure that the drug quality meets the predetermined quality standards and requirements.

[0036] In some embodiments, the term "sample" refers to a test sample to be identified or measured. In the present invention, the rhTSH drug sample includes a drug sample produced in industrial production to be detected for its biological activity.

[0037] The sixth aspect of the present invention provides an application in any of the following aspects.

[0038] Furthermore, the application includes:

[0039] 1) The application of the cells described in the first aspect or the second aspect of the present invention in the preparation of a product for detecting the biological activity of rhTSH drugs;

[0040] 2) The application of the system described in the fifth aspect of the present invention in the quality control of rhTSH drugs.

[0041] In the present invention, the terms "CRE" and "NFAT" are both response elements, that is, a short DNA sequence in the gene promoter region that can bind to specific transcription factors and regulate gene transcription.

[0042] In the present invention, unless otherwise specified, the terms "comprising", "including" and "containing" are open expressions, meaning that in addition to the listed elements, components and steps, other unspecified elements, components and steps can also be covered.

[0043] Unless otherwise defined, all technical terms in the context of the present invention have the same meaning as understood by those of ordinary skill in the art.

[0044] The advantages and beneficial effects of the present invention are as follows:

[0045] The present invention provides two kinds of cells for rapidly detecting the biological activity of rhTSH, and provides a method for detecting the biological activity of rhTSH drugs based on the two kinds of cells. This method is simple to operate, has high sensitivity, good accuracy and precision, and is of great significance in the biological activity evaluation and quality control of rhTSH. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

[0047] Figure 1Shown are the results of TSHR expression and the response to rhTSH in HEK293-TSHR / CRE-Luc and HEK293-TSHR / NFAT-Luc cells. Among them, Figures A and C are the expression profiles of hTSHR and hTSHR-luc in HEK293-TSHR / CRE-Luc and HEK293-TSHR / NFAT-Luc cell lines analyzed by flow cytometry, respectively. Figures B and D are the dose-response curve results obtained by stimulating three HEK293-TSHR / CRE-Luc monoclonal cells (C2, C5, and C18) and three HEK293-TSHR / NFAT-Luc monoclonal cells (N4, N11, and N9) with serially diluted rhTSH, respectively.

[0048] Figure 2 Shown is the result graph of RGA optimization based on HEK293-TSHR / CRE-Luc cells. Among them, Figure A is the result of the reaction curve plotted at different cell incubation densities. Figure B is the dose-response curve result of stimulating HEK293-TSHR / CRE-Luc cells with rhTSH at different initial concentrations. Figure C is the dose-response curve result when the initial concentration is fixed at 10 μg / mL and serially diluted by different multiples. Figure D is the dose-response curve result measured after incubating in a culture dish for different times (3, 4, 5, 6 h).

[0049] Figure 3 Shown is the result graph of RGA optimization based on HEK293-TSHR / NFAT-Luc cells. Among them, Figure A is the dose-response curve result of stimulating HEK293-TSHR / NFAT-Luc cells with rhTSH at different initial concentrations. Figure B is the dose-response curve result when the initial concentration is fixed at 20 μg / mL and serially diluted by different multiples. Figure C is the result of the reaction curve plotted at different cell incubation densities. Figure D is the dose-response curve result measured after incubating in a culture dish for different times (7, 8, 9, 10 h).

[0050] Figure 4 Shown are the result graphs of the specificity verification of two reporter gene methods. Among them, Figures A and B are the result graphs of the relative potency of rhTSH estimated by the CRE method and the SEC-HPLC content of rhTSH stored at 60 °C for different times, respectively. Figures C and D are the dose-response results of component buffer, assay medium, rhFSH, rhTSH, rhCG, and rhLH with the CRE method (C) and the NFAT method (D), respectively.

[0051] Figure 5The linear result graphs of two reporter genes are shown, where A and B are the linear result graphs of the measured value and expected potency of rhTSH after analyzing samples with five different relative bioactivity levels by the CRE method and the NFAT method, respectively;

[0052] Figure 6 Shown is the dose response curve result of detecting the biological activity of rhTSH in vitro using CHO-TSHR-CRE-Luc cells;

[0053] Figure 7 Shown is a dose-response curve of the in vitro biological activity of rhTSH detected using HEK293-TSHR-CRE-Luc cells. DETAILED DESCRIPTION

[0054] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. Those skilled in the art can understand that: various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0055] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0056] In the present invention, the experimental data were analyzed using SoftMax Pro software and plotted using GraphPad Prism software. The rhTSH concentration was taken as the logarithm with a base of 10 as the abscissa, and RLU was taken as the ordinate to plot a four-parameter dose-effect curve. The relative biological activity was expressed as the ratio of the 50% maximum effect concentration (EC50) of the reference to the concentration of the sample in the constraint model. The signal-to-noise ratio (S / N) was expressed as the ratio of the upper asymptote to the lower asymptote. The results were tested for reliability using analysis of variance and F-test. The criteria were: R2 of the fitted four-parameter curve was ≥0.98, the regression term should be very significant (P<0.01), the deviation from parallelism should not be significant (P value ≥0.01), and the relative biological activity could be calculated only after the results of the reliability test were passed.

[0057] Example 1 Construction of HEK293-TSHR / CRE-Luc and HEK293-TSHR / NFAT-Luc cell lines

[0058] 1. Experimental Materials

[0059] The HEK293 / CRE-Luc stable cell line and HEK293 / NFAT-Luc stable cell line used in the present invention were both purchased from Beijing Miga Technology Co., Ltd. Hygromycin B was purchased from Invitrogen Corporation, and the pLV[Exp]-Hygro-EF1A>hTSHR plasmid was purchased from Beijing Miga Technology Co., Ltd. DMEM medium and puromycin were both purchased from Gibco Corporation.

[0060] 2. Experimental methods

[0061] 1) Construction of cell lines: The pLV[Exp]-Hygro-EF1A>hTSHR plasmid was packaged into lentivirus and then used to infect the HEK293 / CRE-Luc stable cell line and HEK293 / NFAT-Luc stable cell line respectively. The above cells were screened with 100 μg / mL Hygromycin B (Hygromycin B) to obtain stably transfected cells, and monoclonal cell lines were obtained by the limiting dilution method. The cell lines were named HEK293-TSHR / CRE-Luc and HEK293-TSHR / NFAT-Luc and were stored in our laboratory. Finally, flow cytometry was used to analyze the expression of TSHR on the cell surface to determine positive monoclonal cells.

[0062] 2) Cell culture: The HEK293-TSHR / CRE-Luc cell line and HEK293-TSHR / NFAT-Luc cell line were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 μg / mL Hygromycin B and 1 μg / mL puromycin. To maintain good condition, both cell lines should be passaged every 3 - 4 days at a passage density of 200,000 - 300,000 cells / mL.

[0063] 3. Experimental results

[0064] After packaging the virus and infecting HEK293 cells, 3 monoclonal cell lines of HEK293-TSHR / CRE-Luc (C2, C5, C18) and 3 monoclonal cell lines of HEK293-TSHR / NFAT-Luc (N9, N4, N11) were obtained through drug screening and the limiting dilution method. As Figure 1 shown in A, flow cytometry analysis showed that the expression rate of TSHR on the cell membranes of the 3 HEK293-TSHR / CRE-Luc cell lines exceeded 97%, and among them, the C18 monoclonal cell line had the highest expression level of TSHR, which was 98.8%. Figure 1 The results in B showed that among the 3 HEK293-TSHR / NFAT-Luc monoclonal cell lines, the N9 monoclonal cell line had the highest expression level of TSHR (99.4%). The above 6 cell lines were seeded at 8×10 4Plated at a certain cell density, the biological activities of the same sample were measured using two reporter gene methods respectively. As Figure 1 shown in Figures 2C and 2D, 6 cell lines responded to rhTSH stimulation. Among them, the signal-to-noise ratios obtained from monoclonal C18 and N9 were the highest, which was consistent with the highest expression levels of TSHR detected by flow cytometry in monoclonal C18 and N9. In subsequent experiments, monoclonal C18 HEK293-TSHR / CRE-Luc cells and

[0065] monoclonal N9 HEK293-TSHR / NFAT-Luc cells were selected.

[0066] Example 2 Optimization of the Reporter Gene Method

[0067] 1. Experimental Materials

[0068] The international rhTSH standard for bioassay (03 / 192) was purchased from NIBSC, and the Steady-GloTM luciferase assay reagent was purchased from Promega.

[0069] 2. Experimental Methods

[0070] Take HEK293-TSHR / CRE-Luc cells (HEK293-TSHR / NFAT-Luc cells were used for the NFAT method), and make cell suspensions with different densities using DMEM medium containing 10% FBS, 100 μg / mL hygromycin B, and 1 μg / mL puromycin. Inoculate 100 μL per well into a 96-well white cell culture plate, and place it in an incubator at 37 °C and 5% CO2 for 16 - 24 hours, then take it out and discard the medium. Dilute the rhTSH sample and reference product (international rhTSH standard) to different initial concentrations with the assay medium (DMEM medium containing 0.1% BSA), and continue to perform gradient dilution downward, with a total of 10 (8 for the NFAT method) dilution degrees, and add 100 μL per well to the 96-well culture plate already inoculated with cells. Take out the culture plate after incubating in an incubator at 37 °C and 5% CO2 for different hours, discard the supernatant, add 100 μL of Steady-GloTM luciferase assay reagent to each well, and react in the dark for 15 min. Use an EnSpire microplate reader to read the Relative luciferase units (RLU) value.

[0071] 3. Experimental Results

[0072] 1) CRE method: Respectively at 0.3×10 5 , 0.8×10 5 , 1.5×10 5 , 2.0×10 5Plated at a cell density of cells / mL, the biological activity of the same rhTSH sample was detected. As Figure 2 shown in A, when the cell density was 0.8×10 5 cells / mL, the curve R2 was 0.991, the S / N was up to 11.0 at most, and the EC50 value was relatively low. Therefore, the optimal cell density was determined to be 0.8×10 5 cells / mL. HEK293-TSHR / CRE-Luc cells were stimulated with rhTSH at initial concentrations of 0.5, 2, 10, and 20 μg / mL respectively. As Figure 2 shown in B, when the initial concentration was 10 μg / mL, the dose-response curve showed a typical S shape, the points on the curve were evenly distributed, the S / N was the largest, and R2 was 0.992. Therefore, the optimal initial concentration was determined to be 10 μg / mL. The optimization results of the dilution factor are shown in Figure 2 C. Only the rhTSH stimulated cells with a 6-fold gradient dilution could obtain a typical S-shaped curve. For the curves obtained with the other three dilution factors, there was no obvious upper or lower plateau. The optimization results of the drug stimulation time are shown in Figure 2 D. S-shaped curves could be obtained when HEK293-TSHR / CRE-Luc cells were stimulated with rhTSH for 3 h, 4 h, 5 h, and 6 h. When the stimulation time was 4 h, the EC50 value was relatively small and the S / N was relatively high. 4 h was selected as the optimal drug stimulation time.

[0073] 2) NFAT method: HEK293-TSHR / NFAT-Luc cells were stimulated with rhTSH at initial concentrations of 4, 8, 20, and 40 μg / mL respectively. As Figure 3 shown in A, when the initial concentration of rhTSH was 20 μg / mL, the dose-response curve of HEK293-TSHR / NFAT-Luc cells showed a typical S shape, R2 was 0.995, and the points on the curve were relatively evenly distributed. 20 μg / mL was selected as the optimal initial concentration. The optimization results of the dilution factor are shown in Figure 3 B. Only the S-shaped curve obtained by stimulating HEK293-TSHR / NFAT-Luc cells with a 5-fold gradient dilution of rhTSH had complete upper and lower plateaus. 5-fold was selected as the optimal dilution factor. The optimization results of the cell density are shown in Figure 3 C. Typical S-shaped curves could be obtained with cells at four densities, and the signal-to-noise ratios were all around 3.4. When the cell density was 0.8×10 5 cells / mL, R2 was up to 0.995 at most, and the EC50 value was as low as 0.32 at least. Therefore, the optimal cell density was determined to be 0.8×10 5 cells / mL. The optimization results of the stimulation time are shown in Figure 3As shown in D, when the stimulation time was 9 h, the highest S / N of the curve was 3.78. Therefore, the optimal incubation time for rhTSH to stimulate HEK293-TSHR / NFAT-Luc cells was determined to be 9 h.

[0074] Methodology verification of Example 3

[0075] 1. Experimental method

[0076] 1) Specificity: Considering that TSHR, FSHR, and LH / CGR belong to the glycoprotein hormone receptor family, to verify the specificity of the above two reporter gene methods for rhTSH, rhFSH, rhLH, rhCG, rhTSH preparation buffer, and assay medium were used together for the specificity investigation of samples. In addition, the specificity investigation also included the interference of possible degradation products. TSH undergoes structural changes during high-temperature treatment. To further verify that this method can correctly reflect the relationship between the structure and function of TSH, we took 1 batch of rhTSH stock solution, aliquoted it into 1000 μL per tube, and placed it in a 60 °C water bath for 2, 8, 18, 36, and 60 h respectively, and measured the relative potency by the above two reporter gene methods. The content of the main component of rhTSH after being placed at 60 °C for different times was detected by size exclusion chromatography.

[0077] 2) Accuracy, precision, and linearity: In the reporter gene methods based on HEK293-TSHR / CRE-Luc cell line and HEK293-TSHR / NFAT-Luc cell line (hereinafter referred to as CRE method and NFAT method), rhTSH international standard was taken and dissolved in the assay medium to prepare a solution containing approximately 10 μg / mL (20 μg / mL for NFAT method), which was used as the reference product for method verification. Serial solutions with initial concentrations of 5, 8, 10, 12.5, 20 μg / mL (10, 16, 20, 25, 40 μg / mL for NFAT method) were prepared with the assay medium as the test solutions at 5 different potency levels (50%, 80%, 100%, 125%, 200%). The 5 relative potency levels were evenly spaced on a logarithmic scale. Two experimenters used 2 cell passages and measured the relative potency of the above 5 test solutions within 2 days. Each potency level was measured 8 times, and the geometric mean was calculated. The accuracy was evaluated by the relative bias (RB% = (measured value of relative potency / theoretical value of relative potency - 1) × 100%). The intermediate precision was calculated according to the geometric coefficient of variation (GCV, %) of the measurement results at each potency level, and the upper limit of the 95% confidence interval of GCV was calculated by the chi-square test. Taking the logarithm of the measured value of the relative potency of the 5 test solutions as the abscissa and the logarithm of the corresponding theoretical relative potency as the ordinate, a linear regression analysis was performed.

[0078] 2. Experimental results

[0079] 1) Specificity: After placing rhTSH at 60 °C for 0, 2, 8, 18, 36, and 60 h, its biological activity was detected by two reporter gene methods respectively. As Figure 4 shown in A, when using the CRE method, compared with the untreated reference product, the relative potency of the treated samples decreased with the prolongation of the treatment time, which were 86.04%, 79.29%, 78.41%, 62.10%, and 53.93% respectively; the relative potencies of the heat-damaged samples measured by using HEK293-TSHR / NFAT-Luc cells were 97.17%, 66.14%, 61.64%, 61.26%, and 46.42% respectively ( Figure 4 B); correspondingly, the contents of rhTSH detected by size exclusion chromatography were 99.44%, 95.36%, 88.58%, 85.43%, 81.24%, and 78.16% respectively. The above results indicate that both methods can sensitively detect the biological activity of heat-damaged rhTSH samples. The decrease in the main component content may be one of the reasons for the decrease in activity. Compared with the physical and chemical determination method, the reporter gene method can more sensitively reflect the sample stability.

[0080] Take rhFSH, rhLH, rhCG, rhTSH preparation buffer, and assay medium to stimulate HEK293-TSHR / CRE-Luc cells. As Figure 4 shown in C, rhLH, rhTSH excipient blank (Ingredients Buffer), and assay medium showed no reactivity. rhFSH and rhCG had responses in this method, but the response values did not form a plateau. When using the reporter gene method based on HEK293-TSHR / NFAT-Luc cells, rhFSH, rhLH, rhCG, rhTSH excipient blank (Ingredients Buffer), and assay medium showed no reactivity, and only rhTSH had a response ( Figure 4 D). Comparing the two methods, the NFAT method has better specificity.

[0081] 2) Accuracy: The newly established CRE and NFAT reporter gene methods were verified respectively. Two experimental personnel measured the relative potencies of 5 test solutions with different potency levels (50%, 80%, 100%, 125%, 200%) respectively. The results all passed the reliability test. As shown in Table 1 and Table 2, the relative biases of the relative potencies measured by the two methods in the range of 50% - 200% potency were between -8.2% and -3.2% (CRE method) and between -2.7% and 6.2% (NFAT method) respectively. This represents that the measured values of the relative potencies are relatively close to the theoretical values, indicating that the accuracies of the two methods are good.

[0082] Table 1 Accuracy verification results of detecting the relative potency of rhTSH by the CRE method

[0083]

[0084]

[0085] Table 2 Verification results of the accuracy of detecting the relative potency of rhTSH by the NFAT method

[0086]

[0087] 3) Precision: Two experimenters separately determined the relative potencies of the above 5 test solutions on the same day for 2 days. For one experimenter, 2 test solutions of the same potency level need to be prepared every day, using 2 cell passages, and 2 96-well plates are tested for each cell passage respectively, and the geometric mean is calculated. Calculate the geometric coefficient of variation (GCV, %) based on the logarithms of the determination results at each potency level, and calculate the upper limit of the 95% confidence interval of GCV using the chi-square test. The geometric coefficients of variation (GCV, %) of the relative potency determination values of 8 experiments at each potency level for the two methods are respectively in the range of 4.2% - 9.8% (CRE method) and 5.2% - 7.4% (NFAT method), and the corresponding upper limits of 95% confidence are less than 20% and 15% respectively. The precisions of the two methods are good, and the NFAT method is slightly better than the CRE method.

[0088] Table 3 Verification results of the intermediate precision of the CRE method

[0089]

[0090] Table 4 Verification results of the intermediate precision of the NFAT method

[0091]

[0092] 4) Linearity and range: Perform a linear regression of the natural logarithm of the theoretical values at 5 different potency levels against the natural logarithm of their corresponding potency determination values. The results are as Figure 5 shown. The linear regression equations fitted by the CRE method and the NFAT method are Y = 0.982X - 0.059 (R2 = 0.9989) and Y = 0.996X + 0.019 (R2 = 0.9959) respectively. The slopes are both close to 1.0, and R2 is greater than 0.98, indicating that both methods have good linearity in the potency level range of 50% - 200%.

[0093] Example 4 Application of the reporter gene method

[0094] The biological activities of 3 batches of rhTSH bulk solution and 3 batches of rhTSH injection were determined respectively by the above two newly established reporter gene methods, and each batch was determined in parallel 3 times. All the test results obtained by the two methods passed the reliability test. The relative standard deviations of the relative potencies of the 3 batches of bulk solutions (DS1, DS2, DS3) and 3 batches of injections (DP1, DP2, DP3) were less than 10% after repeated determination 3 times, indicating that the two methods had good repeatability in detecting the in vitro biological activity of rhTSH and could both be used for the relative biological activity detection of rhTSH bulk solution and preparation samples.

[0095] Table 5 Relative Biological Activities of rhTSH API and Injection Determined by Two Reporter Gene Methods

[0096]

[0097] Comparison of Results of Detecting In Vitro Biological Activity of rhTSH by Using CHO-TSHR / CRE-Luc Cells and HEK293-TSHR / CRE-Luc Cells in Example 5

[0098] 1. Experimental Method

[0099] Take CHO-TSHR / CRE-Luc cells and prepare a cell suspension with a density of 3×10 5 cells / mL in Ham’s F-12 medium containing 10% FBS, 1% glutamine and 1% penicillin-streptomycin. Inoculate 100 μL / well into a 96-well white cell culture plate, place it in an incubator at 37°C and 5% CO2 for 16 - 24 hours, then take it out and discard the medium. Dilute the rhTSH sample and reference product to an initial concentration of 20 μg / mL with the assay medium (HBSS containing 0.4% BSA), and continue to perform 4-fold serial dilutions, with a total of 8 dilution degrees. Add 100 μL / well to the 96-well culture plate seeded with cells. Take out the culture plate after incubating in an incubator at 37°C and 5% CO2 for 6 h, discard the supernatant, add 80 μL of Steady-GloTM luciferase reaction reagent to each well, and react in the dark for 15 min. Use an EnSpire microplate reader to read the Relative luciferase units (RLU) value. Determine the in vitro biological activities of a batch of test samples and rhTSH reference standard according to the above method. Take the logarithm of the rhTSH concentration with base 10 as the abscissa and RLU as the ordinate to plot a four-parameter dose-response curve.

[0100] Take HEK293-TSHR / CRE-Luc cells and prepare a cell suspension with a density of 8×10 4A cell suspension of cells / mL was inoculated into a 96-well white cell culture plate at 100 μL / well, incubated in a 37 °C, 5% CO2 incubator for 16 - 24 hours, and then removed. The culture medium was discarded. The rhTSH sample and the reference product (rhTSH international standard) were diluted to an initial concentration of 10 μg / mL with the assay medium (DMEM medium containing 0.1% BSA), and then serially diluted 6-fold downwards for a total of 10 dilution steps, and 100 μL / well was added to the 96-well culture plate seeded with cells. The culture plate was removed after incubation in a 37 °C, 5% CO2 incubator for 4 h, the supernatant was discarded, and 100 μL of Steady-GloTM luciferase reaction reagent was added to each well and reacted in the dark for 15 min. The Relativeluciferase units (RLU) value was read using an EnSpire microplate reader. The in vitro biological activity of the rhTSH standard product was determined according to the above method. The logarithm to the base 10 of the rhTSH concentration was used as the abscissa, and the RLU was used as the ordinate to plot a four-parameter dose-response curve.

[0101] 2. Experimental results

[0102] The results of detecting the in vitro biological activity of rhTSH using CHO-TSHR / CRE-Luc cells and HEK293-TSHR / CRE-Luc cells are shown respectively as Figure 6 and 7 shown. The signal-to-noise ratio is the ratio of the upper plateau to the lower plateau of the fitted four-parameter curve, that is, the D value / A value of the four-parameter equation. The calculated signal-to-noise ratio using the CHO-TSHR / CRE-Luc method is approximately 6, and the signal-to-noise ratio using the HEK293-TSHR / CRE-Luc method is approximately 11.

[0103] Comparing the two methods, in the in vitro biological activity determination method based on CHO-TSHR / CRE-Luc cells, the initial concentration of the drug is 20 μg / mL, the drug action range is 1.22 - 20.00 μg / mL, the number of cells per well is 30000 after the cells are seeded into the 96-well plate, the incubation time after adding rhTSH stimulation is 6 hours, and the signal-to-noise ratio of the final fitted curve is 6.

[0104] However, in the in vitro biological activity assay based on HEK293-TSHR / CRE-Luc cells, the initial concentration of the drug was 10 μg / mL, the drug action range was 0.99 ng / mL to 20.00 μg / mL, and the detection sensitivity was higher than that of the activity assay based on CHO-TSHR / CRE-Luc cells. After the cells were inoculated into a 96-well plate, the number of cells per well was 8,000, and the incubation time after adding rhTSH stimulation was 4 hours. The required amount of cells was less and the detection efficiency was higher. The signal-to-noise ratio of the final fitting curve of this method was 11, which was nearly 1 times higher than that of the activity assay based on CHO-TSHR / CRE-Luc cells, and it could more sensitively reflect the changes in signal values ​​caused by small changes in drug concentration.

[0105] The CHO-TSHR / CRE-Luc cells used in the present invention are from Genzyme Corporation, and the method validation data submitted for testing show that the intermediate precision CV% of the method is 21.8%, which is much higher than the HEK293-TSHR / CRE-Luc cell activity assay method developed by us (GCV% is less than 10%). This intermediate precision is also consistent with the 22% described in the literature published by the company (Rebecca A. et al, Comparison of Two In Vitro Methods for the Measurement of Recombinant Human TSH Bioactivity, Biologicals, Volume 30, Issue 3, 2002, Pages 245-254, ISSN 1045-1056, https: / / doi.org / 10.1006 / biol.2002.0338).

[0106] In summary, of the two methods, the in vitro biological activity assay based on HEK293-TSHR / CRE-Luc cells requires fewer cells and lower drug concentrations, has higher sensitivity, detection efficiency and precision, and performs better.

[0107] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A cell line co-expressing TSHR and CRE-luciferase reporter gene, characterized in that, The cell is named HEK293-TSHR-CRE-Luc, and is deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO.46053, and the taxonomic name is human embryonic kidney cell 293, and the deposit date is October 9, 2024.

2. A cell line co-expressing TSHR and NFAT-luciferase reporter gene, characterized in that, The cell is named HEK293-TSHR-NFAT-Luc, and is deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO.46054, and the taxonomic name is human embryonic kidney cell 293, and the deposit date is October 9, 2024.

3. A method for detecting the in vitro biological activity of rhTSH drug, characterized in that, The method comprises the following steps: adding the rhTSH drug into the cell according to claim 1 or claim 2, and incubating; after incubation, adding an enzyme reaction substrate, and determining the in vitro biological activity of the rhTSH drug according to the measured reporter gene signal value.

4. The method according to claim 3, characterized in that, The method further comprises the step of gradient diluting the rhTSH drug before incubation.

5. The method according to claim 4, characterized in that, When the rhTSH drug is co-incubated with the cell according to claim 1, the initial concentration of the rhTSH drug is 0.5-20 μg / mL.

6. The method according to claim 5, wherein When the rhTSH drug is co-incubated with the cell according to claim 1, the initial concentration of the rhTSH drug is 10 μg / mL.

7. According to the method of claim 4, when the rhTSH drug is co-incubated with the cell according to claim 2, the initial concentration of the rhTSH drug is 4-40 μg / mL.

8. The method according to claim 7, wherein When the rhTSH drug is co-incubated with the cell according to claim 2, the initial concentration of the rhTSH drug is 20 μg / mL.

9. The method according to claim 4, characterized in that The dilution factor of the rhTSH drug is 3-6 times.

10. The method according to claim 9, characterized in that, When the rhTSH drug is co-incubated with the cell according to claim 1, the dilution factor of the rhTSH drug is 6 times; when the rhTSH drug is co-incubated with the cell according to claim 2, the dilution factor of the rhTSH drug is 5 times.

11. The method according to claim 3, wherein When the rhTSH drug is co-incubated with the cells described in claim 1, the seeding density of the cells is 0.3×10 5 ~2.0×10 5 cells / mL.

12. The method according to claim 11, wherein When the rhTSH drug is co-incubated with the cells described in claim 1, the seeding density of the cells is 0.8×10 5 cells / mL.

13. The method according to claim 3, characterized in that, When the rhTSH drug is co-incubated with the cell according to claim 1, the incubation time is 3-6 h.

14. The method according to claim 13, wherein When the rhTSH drug is co-incubated with the cell according to claim 1, the incubation time is 4 h.

15. The method according to claim 3, wherein When the rhTSH drug is co-incubated with the cells described in claim 2, the seeding density of the cells is 0.3×10 5 ~2.0×10 5 cells / mL.

16. The method according to claim 15, wherein When the rhTSH drug is co-incubated with the cells described in claim 2, the seeding density of the cells is 0.8×10 5 cells / mL.

17. The method according to claim 3, characterized in that, When the rhTSH drug is co-incubated with the cell according to claim 2, the incubation time is 7-10 h.

18. The method according to claim 17, wherein When the rhTSH drug is co-incubated with the cell according to claim 2, the incubation time is 9 h.

19. A product for determining the biological activity of rhTSH drug, characterized in that, The product comprises the cell according to claim 1 or claim 2.

20. The product according to claim 19, characterized in that, The product further comprises a luciferase reaction substrate and a diluent.

21. The product according to claim 19, wherein The product type is a kit.

22. A system for evaluating the biological activity of rhTSH drug, characterized in that, The system comprises the following components: the cell according to claim 1 or 2, the rhTSH drug sample and the reference product.

23. Application of any of the following aspects, characterized in that, The application comprises: 1) The application of the cell according to claim 1 or claim 2 in the preparation of a product for detecting the biological activity of the rhTSH drug; 2) Use of the system according to claim 22 in the quality control of rhTSH drug.

Images