Method for determining biological activity of rhTSH medicine

Abstract

The invention discloses a method for determining the biological activity of an rhTSH drug, and particularly provides two cells for co-expressing TSHR and CRE-luciferase reporter genes, and provides a method for detecting the biological activity of the rhTSH drug based on the two cells, the method is simple and convenient to operate, high in sensitivity and good in accuracy and precision, and the method can be used for detecting the biological activity of the rhTSH drug. The method has important significance in the aspects of biological activity evaluation and quality control of the rhTSH.

Description

Technical Field

[0001] The present invention belongs to the field of biological activity detection of biological drugs, and in particular, 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 through gene recombination technology. It has the same effect as human thyroid-stimulating hormone. After injection, it can quickly increase the TSH level in the patient's body, promote iodine uptake and organization, and facilitate differentiated thyroid cancer patients to quickly receive iodine-131I treatment and remove residual thyroid tissue after surgery. In addition, rhTSH can stimulate residual thyroid tissue or metastatic lesions in the body to secrete thyroglobulin (Tg), which is convenient for diagnosis and monitoring of disease progression. Compared with traditional thyroid hormone withdrawal methods, injection of rhTSH drugs can more safely and quickly increase patients' serum TSH levels, and can also greatly reduce the pain caused by patients stopping thyroid hormone. Nearly 30 years of clinical application has made it recommended by multiple thyroid cancer-related guidelines around the world.

[0003] Biological activity can reflect the correctness of the high-level 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 the mouse thyroid gland. After intravenous injection of TSH, the incremental TSH activity of I131 in the mouse blood was detected. Due to the shortcomings of animal experiments such as long cycles, large individual differences, high operational proficiency requirements, and high variability, 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 based on luminescence resonance energy transfer (LRET), which 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 highly sensitive, it is essentially a content determination method, which cannot simulate the in vivo efficacy and cannot 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 object of the present invention is to provide a method for determining the biological activity of rhTSH drugs.

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

[0006] In a first aspect, the present invention provides a cell line co-expressing TSHR and CRE-luciferase reporter genes.

[0007] Further, the cells are named HEK293-TSHR-CRE-Luc, and are deposited at the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCCNO.46053, the classification name is Human Embryonic Kidney Cell 293, and 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%, and has the strongest response to rhTSH and the highest signal-to-noise ratio. Using this cell line to measure the biological activity of rhTSH is more effective.

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

[0010] Further, the cells are named HEK293-TSHR-NFAT-Luc, and are deposited at the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCCNO.46054, the classification name is human embryonic kidney cell 293, and 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%, and has the strongest response to rhTSH and the highest signal-to-noise ratio. Using this cell line to determine the biological activity of rhTSH is more effective.

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

[0014] In the present invention, the biological activity of the rhTSH drug is determined by fitting a four-parameter curve after measuring the reporter gene signal value. The term four-parameter curve fitting is a mathematical method used to describe a set of data by fitting a curve and obtain a functional equation that can describe the data relationship so as to predict 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), i.e., the minimum and maximum values ​​of absorbance; the half-response concentration (C), i.e., the value when the drug concentration reaches half the maximum response; and the slope of the curve (B). Through these parameters, the experimental data can be understood and analyzed more accurately, thereby providing more in-depth insights into biological research.

[0015] Furthermore, the method further comprises the step of gradient dilution of the rhTSH drug before incubation.

[0016] Furthermore, 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] Furthermore, 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] Furthermore, the dilution multiple of the rhTSH drug is 3 to 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 cell seeding density is 0.8×10 5 cells / mL.

[0022] Preferably, the incubation time is 3 to 6 hours.

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

[0024] Further, 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 cell seeding density is 0.8×10 5 cells / mL.

[0026] Preferably, the incubation time is 7 to 10 hours.

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

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

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

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

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

[0032] In certain embodiments, the components of the test kit can be packaged in the form of an aqueous medium or in a lyophilized form. Suitable containers in the test kit generally include at least a vial, test tube, flask, PET bottle, syringe or other container, in which a component can be placed, and preferably, can be appropriately divided. When there is more than one component in the test kit, the test kit will generally also include a second, third or other additional container, in which additional components are placed separately. However, components of different combinations can be contained in a vial. The test kit of the present invention will generally also include a container for accommodating reactants, sealed for commercial sale. Such a container may include a plastic container of injection molding or blow molding, in which the required vial 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 comprises the following components: the cells described in the first aspect of the present invention or the second aspect of the present invention, rhTSH drug samples and reference products.

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

[0036] In some embodiments, the term "sample" refers to a sample to be identified or determined. In the present invention, rhTSH drug samples include drug samples produced in industrial production and to be tested for their biological activity.

[0037] A sixth aspect of the present invention provides applications of any of the following aspects.

[0038] Furthermore, the application includes:

[0039] 1) Use of the cell according to the first aspect or the second aspect of the present invention in the preparation of a product for detecting the biological activity of rhTSH drug;

[0040] 2) 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 response elements, that is, a short DNA sequence in the promoter region of a gene that can bind to a specific transcription factor to regulate the transcription of the gene.

[0042] In the present invention, unless otherwise specified, the terms "comprise", "include" and "contain" are open expressions, meaning that in addition to the listed elements, components and steps, other unspecified elements, components and steps may also be included.

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

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

[0045] The invention provides two 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 cells. The method is simple to operate, highly sensitive, accurate and precise, and has important significance in the biological activity evaluation and quality control of rhTSH. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0047] Figure 1Shown are the results of TSHR expression and rhTSH response in HEK293-TSHR / CRE-Luc and HEK293-TSHR / NFAT-Luc cells, wherein Figures A and C are flow cytometric analysis of the expression of hTSHR and hTSHR-luc in HEK293-TSHR / CRE-Luc and HEK293-TSHR / NFAT-Luc cell lines, and Figures B and D are dose-response curves of three HEK293-TSHR / CRE-Luc monoclonal cells (C2, C5 and C18) and three HEK293-TSHR / NFAT-Luc monoclonal cells (N4, N11 and N9) stimulated with serially diluted rhTSH, respectively;

[0048] Figure 2 The graphs show the RGA optimization results based on HEK293-TSHR / CRE-Luc cells, where Figure A shows the response curve results drawn at different cell incubation densities, Figure B shows the dose-response curve results of HEK293-TSHR / CRE-Luc cells stimulated by rhTSH at different initial concentrations, Figure C shows the dose-response curve results of different dilutions after the initial concentration was fixed at 10 μg / mL, and Figure D shows the dose-response curve results measured after incubation in a culture dish for different times (3, 4, 5, 6 h);

[0049] Figure 3 The RGA optimization results based on HEK293-TSHR / NFAT-Luc cells are shown, where Figure A is the dose-response curve results of HEK293-TSHR / NFAT-Luc cells stimulated by rhTSH at different initial concentrations, Figure B is the dose-response curve results of different dilutions after the initial concentration is fixed at 20 μg / mL, Figure C is the response curve results drawn at different cell incubation densities, and Figure D is the dose-response curve results measured after incubation in a culture dish for different times (7, 8, 9, 10 h);

[0050] Figure 4 The results of the specificity validation of the two reporter gene methods are shown, wherein Figures A and B are the relative potency of rhTSH estimated by the CRE method and the NFAT method and the SEC-HPLCE content results of rhTSH at different storage times at 60°C, respectively; Figures C and D are the dose-response results of the 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 purchased from Beijing Mega Technology Co., Ltd., hygromycin B was purchased from Invitrogen, and pLV[Exp]-Hygro-EF1A>hTSHR plasmid was purchased from Beijing Mega Technology Co., Ltd. DMEM culture medium and puromycin were purchased from Gibco.

[0060] 2. Experimental methods

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

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

[0063] 3. Experimental results

[0064] After the packaged virus infected HEK293 cells, three HEK293-TSHR / CRE-Luc monoclonal cell lines (C2, C5, C18) and three HEK293-TSHR / NFAT-Luc monoclonal cell lines (N9, N4, N11) were obtained through drug addition screening and limiting dilution method. Figure 1 As shown in A, flow cytometric analysis showed that the expression rate of TSHR on the membrane of three HEK293-TSHR / CRE-Luc cells exceeded 97%, among which the C18 monoclonal clone expressed the highest TSHR level of 98.8%. Figure 1 B The results showed that among the three HEK293-TSHR / NFAT-Luc monoclonal cells, the N9 monoclonal cell expressed the highest level of TSHR (99.4%). The above six cell lines were cultured at 8×10 4The cell density of each sample was plated and the biological activity of the same sample was determined by two reporter gene methods. Figure 1 As shown in C and 1D, all six cell lines responded to rhTSH stimulation, among which C18 and N9 monoclonal clones had the highest signal-to-noise ratio, which was consistent with the fact that TSHR expression levels were the highest in C18 and N9 monoclonal clones detected by flow cytometry. Subsequent experiments were conducted using C18 HEK293-TSHR / CRE-Luc monoclonal cell and N9 monoclonal clone.

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

[0066] Example 2 Optimization of reporter gene method

[0067] 1. Experimental Materials

[0068] Bioassay International rhTSH Standard (03 / 192) was purchased from NIBSC, and Steady-GloTM luciferase assay reagent was purchased from Promega.

[0069] 2. Experimental methods

[0070] HEK293-TSHR / CRE-Luc cells (HEK293-TSHR / NFAT-Luc cells were used for the NFAT method) were prepared into cell suspensions of different densities using DMEM medium containing 10% FBS, 100 μg / mL hygromycin B, and 1 μg / mL puromycin. The suspensions were inoculated into 96-well white cell culture plates at 100 μL / well and placed at 37°C with 5% CO. 2 After 16 to 24 hours of culture in the incubator, remove the sample and discard the culture medium. Dilute the rhTSH sample and reference product (rhTSH international standard) to different initial concentrations with the assay culture medium (DMEM culture medium containing 0.1% BSA), and continue to dilute the sample to a total of 10 (8 for the NFAT method) dilutions. Add 100 μL / well to the 96-well culture plate seeded with cells. Place the culture plate at 37°C and 5% CO 2 After incubation in the incubator for different hours, the cells were taken out, the supernatant was discarded, 100 μL of Steady-GloTM luciferase assay reagent was added to each well, and the reaction was carried out in the dark for 15 minutes. The relative luciferase units (RLU) value was read using an EnSpire microplate reader.

[0071] 3. Experimental results

[0072] 1) CRE method: 0.3×10 5 , 0.8×10 5 , 1.5×10 5 , 2.0×10 5cells / mL, and the biological activity of the same rhTSH sample was tested. Figure 2 As shown in A, when the cell density is 0.8×10 5 cells / mL, the curve R2 was 0.991, the highest S / N was 11.0, and the EC50 value was low, so the optimal cell density was determined to be 0.8×10 5 HEK293-TSHR / CRE-Luc cells were stimulated with rhTSH at initial concentrations of 0.5, 2, 10, and 20 μg / mL, respectively. Figure 2 As shown in B, when the initial concentration is 10 μg / mL, the dose-effect response curve is a typical S-shaped curve, the points on the curve are evenly distributed, the S / N is the largest, and the R2 is 0.992. Therefore, the optimal initial concentration is determined to be 10 μg / mL. The dilution multiple optimization results are shown in Figure 2 As shown in C, only the 6-fold gradient dilution of rhTSH can produce a typical S-shaped curve. The curves obtained by using the other 3 dilution multiples have no obvious upper or lower platform. Figure 2 As shown in D, S-shaped curves can be obtained when the cells are stimulated by rhTSH for 3h, 4h, 5h, and 6h. The EC50 value is smaller and the S / N is higher at 4h, so 4h is 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, as shown in Figure 3 As shown in A, when the initial concentration of rhTSH is 20 μg / mL, the HEK293-TSHR / NFAT-Luc cell dose-effect curve is a typical S-shaped curve with an R2 of 0.995. The points on the curve are evenly distributed, and 20 μg / mL is selected as the optimal initial concentration. The dilution multiple optimization results are shown in Figure 3 As shown in B, the S-shaped curve obtained by stimulating HEK293-TSHR / NFAT-Luc cells with only 5-fold gradient dilution of rhTSH has a complete upper and lower platform, and 5-fold is selected as the optimal dilution factor. Figure 3 As shown in C, the four cell densities can all obtain typical S-shaped curves, and the signal-to-noise ratio is around 3.4. 5 cells / mL, the highest R2 was 0.995 and the lowest EC50 value was 0.32, so the optimal cell density was determined to be 0.8×10 5 cells / mL. The results of stimulation time optimization are as follows Figure 3As shown in D, the S / N of the curve was the highest at 3.78 when the stimulation time was 9 h, so the optimal incubation time for rhTSH to stimulate HEK293-TSHR / NFAT-Luc cells was determined to be 9 h.

[0074] Example 3 Methodology Verification

[0075] 1. Experimental methods

[0076] 1) Specificity: Considering that TSHR, FSHR and LH / CGR are all members of the glycoprotein hormone receptor family, in order to verify that the above two reporter gene methods are specific to rhTSH, rhFSH, rhLH, rhCG, rhTSH preparation buffer and assay medium were taken together to investigate the specificity of the samples. In addition, the specificity investigation also includes the interference of possible degradation products. TSH undergoes structural changes when treated at high temperatures. In order to further verify that this method can correctly reflect the relationship between the structure and function of TSH, we took a batch of rhTSH stock solution, divided it into 1000μL per tube, and heated it in a 60℃ water bath for 2, 8, 18, 36, and 60h, respectively, and the relative potency was determined by the above two reporter gene methods. The main component content of rhTSH after being placed at 60℃ for different times was detected by molecular exclusion chromatography.

[0077] 2) Accuracy, precision and linearity: In the reporter gene method based on HEK293-TSHR / CRE-Luc cell line and HEK293-TSHR / NFAT-Luc cell line (hereinafter referred to as CRE method and NFAT method), the rhTSH international standard was dissolved in the assay medium to prepare a solution containing about 10 μg per milliliter (20 μg for the NFAT method) as a reference for method validation. A series of solutions with initial concentrations of 5, 8, 10, 12.5, and 20 μg / mL (10, 16, 20, 25, and 40 μg / mL for the NFAT method) were prepared in the assay medium as the test solutions at 5 different titer levels (50%, 80%, 100%, 125%, and 200%), and the 5 relative titer levels were evenly spaced on a logarithmic scale. Two experimenters used two cell generations to measure the relative potency of the above five test solutions within two days. Each potency level was measured 8 times and the geometric mean was calculated. The accuracy was evaluated by relative bias (RB% = (relative potency measured value / relative potency theoretical value-1) × 100%). The intermediate precision was calculated based on the geometric coefficient of variation (GCV, %) of the results of each potency level, and the chi-square test was used to calculate the 95% confidence interval upper limit of GCV. Linear regression analysis was performed with the logarithm of the relative potency measured values ​​of the five test solutions as the horizontal axis and the logarithm of the corresponding theoretical relative potency as the vertical axis.

[0078] 2. Experimental results

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

[0080] Take rhFSH, rhLH, rhCG, rhTSH preparation buffer and assay medium to stimulate HEK293-TSHR / CRE-Luc cells, as Figure 4 As shown in C, rhLH, rhTSH excipient blank (Ingredients Buffer), and assay medium were all unresponsive, while rhFSH and rhCG responded in this method, but the response values ​​did not form an upper plateau. When the reporter gene method based on HEK293-TSHR / NFAT-Luc cells was used, rhFSH, rhLH, rhCG, rhTSH excipient blank (Ingredients Buffer), and assay medium were all unresponsive, and only rhTSH was responsive ( Figure 4 D) Compared with the two methods, the NFAT method has better specificity.

[0081] 2) Accuracy: The newly established CRE and NFAT reporter gene methods were verified respectively, and the relative potency of the test solutions with 5 different potency levels (50%, 80%, 100%, 125%, 200%) was measured by 2 experimenters, and the results all passed the reliability test. As shown in Table 1 and Table 2, the relative bias of the relative potency measured by the two methods within the potency range of 50% to 200% was between -8.2% to -3.2% (CRE method) and -2.7% to 6.2% (NFAT method), respectively, which means that the measured value of the relative potency is relatively close to the theoretical value, indicating that the accuracy of the two methods is good.

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

[0083]

[0084]

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

[0086]

[0087] 3) Precision: Two experimenters measured the relative potency of the above five test solutions on the same day for two days. For one experimenter, two test solutions of the same potency level were prepared every day, two cell generations were used, and two 96-well plates were tested for each cell generation, and the geometric mean was calculated. The geometric coefficient of variation (GCV, %) was calculated based on the logarithmic value of the test results of each potency level, and the chi-square test was used to calculate the 95% confidence interval upper limit of GCV. The geometric coefficient of variation (GCV, %) of the relative potency measurement values ​​of 8 experiments at each potency level of the two methods were 4.2% to 9.8% (CRE method) and 5.2% to 7.4% (NFAT method), respectively, and the corresponding 95% confidence limits were less than 20% and 15%, respectively. The precision of the two methods was good, and the NFAT method was slightly better than the CRE method.

[0088] Table 3 Intermediate precision verification results of CRE method

[0089]

[0090] Table 4 Intermediate precision verification results of NFAT method

[0091]

[0092] 4) Linearity and range: Linear regression was performed on the natural logarithms of the theoretical values ​​of the five different potency levels and the natural logarithms of the corresponding potency measured values. The results are as follows: Figure 5 As 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 close to 1.0 and R2 is greater than 0.98, indicating that the two methods have good linearity in the potency level range of 50% to 200%.

[0093] Example 4 Application of reporter gene method

[0094] The two newly developed reporter gene methods were used to measure the biological activity of three batches of rhTSH stock solutions and three batches of rhTSH injection solutions, and each batch was measured three times in parallel. All the test results obtained by the two methods passed the reliability test. The relative standard deviation of the relative potency of three batches of stock solutions (DS1, DS2, DS3) and three batches of injection solutions (DP1, DP2, DP3) was less than 10%, indicating that the two methods have good repeatability in detecting the in vitro biological activity of rhTSH, and can be used for the relative biological activity detection of rhTSH stock solutions and preparation samples.

[0095] Table 5 Relative biological activities of rhTSH raw materials and injections determined by two reporter gene methods

[0096]

[0097] Example 5 Comparison of the results of detecting the biological activity of rhTSH in vitro using CHO-TSHR / CRE-Luc cells and HEK293-TSHR / CRE-Luc cells

[0098] 1. Experimental methods

[0099] CHO-TSHR / CRE-Luc cells were cultured at a density of 3 × 10 cells / mL using Ham's F-12 medium containing 10% FBS, 1% glutamine, and 1% penicillin-streptomycin. 5 cells / mL of cell suspension was inoculated into a 96-well white cell culture plate at 100 μL / well and placed at 37°C with 5% CO 2 After 16 to 24 hours of incubation in the incubator, remove the sample and discard the culture 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, a total of 8 dilutions, and add 100 μL / well to the 96-well culture plate seeded with cells. Place the culture plate at 37°C and 5% CO 2 After incubation in the incubator for 6 hours, remove the cells, discard the supernatant, add 80 μL Steady-GloTM luciferase reaction reagent to each well, and react for 15 minutes in the dark. Use EnSpire microplate reader to read the relative luciferase units (RLU) value. According to the above method, the in vitro biological activity of a batch of test products and rhTSH standards was determined, and the rhTSH concentration was taken as the logarithm to the base 10 as the horizontal axis, and RLU was used as the vertical axis to draw a four-parameter dose-effect curve.

[0100] HEK293-TSHR / CRE-Luc cells were cultured at a density of 8 × 10 cells / mL using DMEM medium containing 10% FBS, 100 μg / mL Hygromycin B, and 1 μg / mL puromycin. 4 cells / mL of cell suspension was inoculated into a 96-well white cell culture plate at 100 μL / well and placed at 37°C with 5% CO 2 After 16 to 24 hours of culture in the incubator, remove the cells and discard the culture medium. Dilute the rhTSH sample and reference substance (rhTSH international standard) to an initial concentration of 10 μg / mL with the assay medium (DMEM medium containing 0.1% BSA), and continue to perform 6-fold serial dilutions, a total of 10 dilutions, and add 100 μL / well to the 96-well culture plate seeded with cells. Place the culture plate at 37°C and 5% CO 2 After incubation in the incubator for 4 hours, the cells were removed, the supernatant was discarded, 100 μL Teady-GloTM luciferase reaction reagent was added to each well, and the cells were reacted in the dark for 15 minutes. The relative luciferase units (RLU) value was read using an EnSpire microplate reader. The in vitro biological activity of the rhTSH standard was determined according to the above method, and the rhTSH concentration was taken as the logarithm with the base 10 as the abscissa, and the RLU as the ordinate to draw a four-parameter dose-effect 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 in the figure. Figure 6 , 7 As shown, the signal-to-noise ratio is the ratio of the upper platform to the lower platform of the fitted four-parameter curve, that is, the D value / A value of the four-parameter equation. The signal-to-noise ratio using the CHO-TSHR / CRE-Luc method is about 6, and the signal-to-noise ratio using the HEK293-TSHR / CRE-Luc method is about 11.

[0103] Comparing the two methods, in the in vitro biological activity assay based on CHO-TSHR / CRE-Luc cells, the initial concentration of the drug was 20 μg / mL, the drug action range was 1.22-20.00 μg / mL, the number of cells per well after the cells were seeded into a 96-well plate was 30,000, the incubation time after adding rhTSH stimulation was 6 hours, and the signal-to-noise ratio of the final fitting curve was 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 genes, characterized in that: The cells are named HEK293-TSHR-CRE-Luc and deposited at the General Microbiology Center of China Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCCNO.46053, the classification 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 genes, characterized in that: The cells are named HEK293-TSHR-NFAT-Luc and deposited at the General Microbiology Center of China Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCCNO.46054, the classification 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 drugs, characterized in that: The method comprises the following steps: adding rhTSH drug to the cells according to claim 1 or claim 2, and incubating; adding enzyme reaction substrate after incubation, 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 also includes the step of gradient dilution of the rhTSH drug before incubation; Preferably, when the rhTSH drug is co-incubated with the cells according to claim 1, the initial concentration of the rhTSH drug is 0.5 to 20 μg / mL; more preferably, the initial concentration of the rhTSH drug is 10 μg / mL; Preferably, when the rhTSH drug is co-incubated with the cells according to claim 2, the initial concentration of the rhTSH drug is 4 to 40 μg / mL; more preferably, the initial concentration of the rhTSH drug is 20 μg / mL; Preferably, the dilution multiple of the rhTSH drug is 3 to 6 times; More preferably, when the rhTSH drug is co-incubated with the cells according to claim 1, the dilution factor of the rhTSH drug is 6 times; when the rhTSH drug is co-incubated with the cells according to claim 2, the dilution factor of the rhTSH drug is 5 times.

5. The method according to claim 3, characterized in that: When the rhTSH drug is co-incubated with the cells of claim 1, the seeding density of the cells is 0.3×10 5 ~2.0×10 5 cells / mL; Preferably, the cell seeding density is 0.8×10 5 cells / mL; Preferably, the incubation time is 3 to 6 hours; More preferably, the incubation time is 4 h.

6. The method according to claim 3, characterized in that When the rhTSH drug is co-incubated with the cells of claim 2, the seeding density of the cells is 0.3×10 5 ~2.0×10 5 cells / mL; Preferably, the cell seeding density is 0.8×10 5 cells / mL; Preferably, the incubation time is 7 to 10 hours; More preferably, the incubation time is 9 h.

7. A product for determining the biological activity of rhTSH drugs, characterized in that: The product comprises the cells of claim 1 or claim 2.

8. The product according to claim 7, characterized in that The product also includes a luciferase reaction substrate and a diluent; Preferably, the product type is a kit.

9. A system for evaluating the biological activity of rhTSH drugs, characterized in that: The system comprises the following components: the cells according to claim 1 or 2, rhTSH drug samples and reference products.

10. The application of any one of the following aspects, characterized in that: The applications include: 1) Use of the cell according to claim 1 or claim 2 in the preparation of a product for detecting the biological activity of rhTSH drugs; 2) Use of the system according to claim 9 in the quality control of rhTSH drugs.

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