LHCG receptor expressing cell line

Abstract

The present invention relates to novel cell lines for the expression of the human LHCG receptor and for the expression of the human LHCG receptor and a luciferase reporter gene. Methods for producing such cell lines are also described. The cell lines of the present invention, which express the LHCG receptor and a luciferase reporter gene, can be used in methods for determining the presence and biological activity of r-hLH and r-hCG samples.

Description

[Technical Field]

[0001] Described herein are novel cell lines useful for the development of bioassays related to the production of recombinant human luteinizing hormone (r-hLH) and recombinant human chorionic gonadotropin (r-hCG). The first cell line expresses the human LHCG receptor (HEK 293T LHCGR), and the second cell line expresses both the human LHCG receptor and a luciferase reporter gene (HEK 293T LHCGR luciferase) under the control of a cAMP response element (CRE). [Background technology]

[0002] Bioassays are used to assess the efficacy of drugs before commercial products reach the market. Estimating drug efficacy requires assays that reflect or mimic the known mechanism of action (or part of it) that occurs in humans in order to reliably assess the product's biological activity.

[0003] In vivo potency testing is a method for estimating potency by administering a standard and test substance to animals and assessing the relationship between product concentration and the resulting response. Cell-based (or in vitro) potency testing, on the other hand, envisions the use of cell lines that respond to specific ligands or infectious agents. Cell lines vary in nature and include tumor-derived cell lines, immortalized cell lines for factor-dependent cell lines, or genetically engineered cell lines transfected with the appropriate target. One of the main requirements is that these cell lines be stable to ensure assay reproducibility over time and reliable results. The cellular response to the target protein is related to the drug's mechanism of action and duration of exposure. Drug responses include cell proliferation, cell killing, cell differentiation, secretion of cytokines and other molecules, activation of intracellular responses, and enzyme activation. Summary of the Invention [Problem to be solved by the invention]

[0004] Currently, in vivo animal bioassays are used to determine the presence and potency of recombinant human LH (r-hLH) and recombinant human CG (r-hCG). There are no cell-based alternatives available for the testing required for the production of these biological molecules. Therefore, there is a need for cell lines that can be used in bioassays to determine the presence and potency of r-hLH or r-hCG in samples. [Means for solving the problem]

[0005] The present invention provides a solution to the lack of a cell-based potency assay for determining the potency of r-hLH or r-hCG samples. This solution provides a novel mammalian cell line engineered to highly express the human LHCG receptor and coupled to a reporter. Thus, the biological activity of r-hLH or r-hCG can be quantified by binding of r-hLH or r-hCG to the human LHCG receptor on the novel cells of the invention.

[0006] According to one embodiment, a mammalian cell line for expressing the human LHCG receptor is provided, the cell line comprising: a) a recombinant human LHCG receptor comprising a nucleic acid molecule encoding the human LHCG receptor under the control of a promoter for protein expression; and b) a reporter gene comprising a nucleic acid molecule encoding a luciferase reporter gene under the control of a cAMP response element (CRE).

[0007] According to another embodiment, there is provided a method for producing a human LHCG receptor in a mammalian cell, the method comprising the steps of: a) transfecting a mammalian cell with an isolated nucleic acid molecule encoding an LHCG receptor under the control of a protein expression promoter; b) adding a first selection factor to the transfected cells of step a) to induce a stably expressing human LHCG receptor cell line (HEK 293-T LHCGR); c) transfecting the mammalian cell of step b) with an isolated nucleic acid molecule encoding a luciferase reporter gene under the control of a cAMP response element (CRE) to obtain a transfected cell; d) adding a second selection factor to the stably expressing human LHCG receptor cells of step c) to induce a cell stably expressing the human LHCG receptor and the luciferase reporter; and e) harvesting the cell stably expressing the human LHCG receptor and the luciferase reporter (HEK 293-T LHCGR luciferase).

[0008] According to yet another embodiment, there is provided a method for quantifying the biological activity of an r-hLH or r-hCG sample, the method comprising the steps of: a) contacting the sample with a cell culture of mammalian cells expressing a human LHCG receptor and a luciferase reporter; b) adding a luminescent substrate for the luciferase enzyme to the cell culture; c) detecting luminescence from the cells; d) quantifying the amount of luminescence; and e) determining the potency of the r-hLH or r-hCG sample. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows the initial transfection efficiency assessment. A) Responses of HEK 293-T LHCGR and HEK 293-T WT to r-hLH stimulation. B) Responses of HEK 293-T LHCGR to r-hFSH stimulation.

[0010] [Figure 2]Figure 2 shows LHR staining of HEK 293-T WT and HEK 293-T LHCGR luciferase. Data were acquired using a BD FACS Aria Fusion and analyzed using Flowjo software.

[0011] [Figure 3] Figure 3 shows the behavior of cells over a defined range of passages (p. 17-p. 41). A) Cell viability analysis, B) Cell doubling analysis. (A-B) Data are presented as the mean ± SD of three banks. Statistics were performed using linear regression analysis (A-B: no significant difference). C) Real-time cell proliferation analysis using IncuCyte. Growth curves (12,500 cells / well) for p. 17 and p. 41 are overlaid. Data are presented as the mean ± SD of different replicate experiments.

[0012] [Figure 4] Figure 4 shows monitoring of cell transfection stability during a defined range of subcultures (p. 17-p. 41). A) FRET analysis for quantification of LHCG receptor activity. A four-parameter dose-response curve was generated and the EC50 was monitored from passages 17 to 41. Data are presented as the mean ± SD of triplicate banks. Statistics were performed using linear regression analysis (not significant). B) Luciferase reporter gene transcription activation with 4 μM forskolin (passages 17 to 41). Data are presented as the mean ± SD of triplicate banks. Statistics were performed using linear regression analysis (p=0.0013).

[0013] [Figure 5] Figure 5 shows representative RGA dose-response curves for potency quantification of r-hLH (panel A) and r-hCG (panel B) samples. DETAILED DESCRIPTION OF THE INVENTION

[0014] Currently, potency testing for each new drug batch release of Lutropin Alfa and Chorionic Gonadotropin Alfa is performed using an in vivo method in rats for quality control purposes, and this in vivo method must be performed in accordance with regulatory authorities and guidelines.

[0015] Given current policy and ethical considerations, reliance on in vivo assays should be reduced as much as possible and replaced with alternative in vitro methods. Alternative methods for testing r-hLH (e.g., lutropin alfa) and r-hCG (e.g., chorionic gonadotropin alfa) include in vitro cell-based methods. In vitro cell-based assays for testing r-hLH and r-hCG require appropriate cell lines expressing the LHCG receptor, which binds to r-hLH or r-hCG compositions. Currently, there are no cell lines on the market suitable for developing in vitro cell-based assays that can meet the routine requirements of quality control laboratories. Development of in vitro methods will consider simple procedures, the use of simple and stable readouts, and the use of commercially available alternative quantification reagents.

[0016] The present invention provides such cells and methods for producing such cell lines, as well as methods for in vitro testing of r-hLH or r-hCG from production batches using the novel cell lines.

[0017] The cell line of the present invention is based on the human embryonic kidney (HEK) cell line, more specifically HEK 293-T.

[0018] To comply with guidelines and health authority requirements, cell line development and in vitro methods were performed according to the mechanisms of action of r-hLH and r-hCG.

[0019] Cell line development began with HEK 293-T cells. The human-derived cell line, HEK 293-T, was stably transfected with the human LHCG receptor. A second transfection was then performed to express a luciferase reporter gene into HEK 293-T LHCGR.

[0020] The newly created cells faithfully reproduce part of the mechanism of action of r-hLH and r-hCG in the body: the first step in the metabolic cascade produced by receptor binding to the hormones.

[0021] The transfection process resulted in the generation of a new cell line of HEK 293 T cells expressing the human LHCG receptor and a luciferase reporter (HEK 293-T LHCGR luciferase). The transfected cells were selected and expanded, and various cell banks were subsequently cryopreserved. After cell bank generation, two different functional assays were developed to establish a method for preliminarily assessing transfection efficiency from a functional standpoint.

[0022] We developed the first functional assay to quantify cAMP after cell stimulation using a FRET kit, which allowed us to evaluate LHCG receptor activation in HEK 293-T LHCGR cells. This assay allowed us to clearly evaluate the difference between transfected and non-transfected cells and confirm LHCG receptor expression only in transfected cells.

[0023] A second functional assay was developed to assess reporter gene transcription following intracellular cAMP accumulation by the inserted vector in HEK 293-T LHCGR cells. This assay was performed by stimulating cells with forskolin to activate adenylate cyclase. This stimulation increases intracellular cAMP, allowing us to assess reporter gene transcription in transfected cells independently of LHCG receptor activation. Again, differences in luminescence output following cell stimulation were observed between transfected and non-transfected cells. This assay confirmed the presence and functionality of the luciferase reporter gene vector in the HEK 293-T LHCGR luciferase cell line.

[0024] Characterization studies of HEK 293-T LHCGR luciferase were performed over a defined range of passages, from passage 17 to passage 41. Cell behavior was monitored by assessing specific parameters, including LHCG receptor expression at the cell membrane, cell cycle, cell viability, and cell proliferation (including cell doubling time and real-time cell growth monitoring). Additionally, previously developed functional assays were incorporated into the characterization studies to monitor stability over time in both the first and second transfections.

[0025] Expression of the LHCG receptor in HEK 293-T LHCGR luciferase membranes was assessed compared to untransfected cells. These experiments confirmed the presence of the LHCG receptor in transfected cells, with the percentage of LHCGR-positive cells being approximately 70%.

[0026] Phenotypic and functional characterization of the HEK 293-T LHCGR luciferase cell line revealed similar behavior among the three different cell banks (MCB, WCB1, and WCB2) generated in our laboratory. Indeed, the results obtained with the HEK 293-T LHCGR luciferase cell line demonstrated stable cell viability and proliferation over the selected passage range.

[0027] The stability of receptor expression at the cell membrane was indirectly verified over a defined passage range using the developed functional assay to assess the cAMP pathway activated by LH interaction with its receptor. Stimulation of HEK 293T LHCGR luciferase cells with scalar doses of r-hLH yielded clear curves. Furthermore, the EC50 values ​​remained stable for at least 24 passages in all cell banks, confirming the stability and functionality of the transfection over time.

[0028] In a second transfection stability assessment, HEK 293-T LHCGR luciferase was compared with and without stimulation with 4 μM forskolin. Furthermore, over the 24 passages analyzed, the cellular response to forskolin was maintained, despite a gradual decrease in fold change observed. The fold change is the ratio of the signal generated by forskolin stimulation to the unstimulated sample. Nevertheless, the smallest fold change detected was greater than 10-fold, which is considered acceptable for the purposes of the assay.

[0029] In addition to all the work related to the development and characterization of the cell lines, we developed an in vitro assay for the titer determination of r-hLH and r-hCG samples. The developed method is very fast and simple, and these characteristics ensure optimal application of the assay in quality control routines. Preliminary linearity evaluations also demonstrated that the method works properly. Indeed, all results met theoretical acceptance criteria. [Example]

[0030] Example: older sister HEK 293 T cell culture. The recommended cell culture medium is Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cell doubling time is approximately 24-30 hours. The cells were cultured at 37±1°C in a humidified atmosphere of 5±1% CO2. All of these cell characteristics are listed in the cell data sheet provided by the supplier. The cell passage conditions were also confirmed in our laboratory.

[0031] Transfection of HEK 293T cells. HEK 293T cell lines were transfected with the LHCG receptor using a suitable commercially available vector (provided by Vector Builder) containing a nucleic acid molecule encoding the human LHCG receptor. This vector is a mammalian gene expression vector and is 8097 bp in length. For stable transfection, hygromycin B was used as a selection marker. Prior to transfection, a bactericidal curve assay was used to determine the minimum hygromycin B concentration capable of inhibiting HEK 293T cell proliferation over a 10-day period. 200 μg / mL was determined to be the optimal concentration and was used in all transfection experiments.

[0032] The vector encoding the LHCG receptor was first transfected into HEK 293-T cells using electroporation. The commercially available reagents used for transfection were included in the Amaxa 4D kit (Lonza, V4XC-2024).

[0033] OptiMEM medium (Life Technologies, 31985) was used for all transfections. Transfections were performed using varying amounts of vector DNA and various experimental conditions using electroporation. The transfection process and various experimental conditions were performed according to the supplier's recommended protocol. 24 hours after transfection, an antibiotic selection solution was added to the cells to select only cell populations resistant to the antibiotic concentrations identified in the kill curve experiments. The resistant cells were expanded, and different cell banks belonging to each transfection condition were frozen. A common approach to constructing cell banks is to freeze a master cell bank (MCB). After expanding the MCB, a working cell bank (WCB) is constructed. The goal of bank construction is to ensure sufficient numbers of cells during assay development and execution. Bank construction depends on the growth characteristics of the cells, and the size of the bank depends on the number of cells required for each assay and the frequency of assay execution.

[0034] Initial transfection efficiency assessment. To evaluate transfection efficiency, transfected cells were stimulated with r-hLH, and cAMP production was quantified using a commercially available kit based on fluorescence resonance energy transfer (FRET). The FRET kit used for cAMP quantification was manufactured by Cisbio. The kit contains cAMP conjugated to d2 (acceptor) and an anti-cAMP antibody conjugated to cryptate (donor, europium). FRET was detected using a plate reader equipped with homogeneous time-resolved fluorescence (HTRF) detection technology. All experiments were performed according to the data sheet provided by the kit supplier.

[0035] To interpret the data, the ratio of acceptor to donor luminescence signals must be calculated for each well using the following formula: Ratio = (signal 665nm) / (signal 620nm) x 10000.

[0036] All raw data (ratios) obtained with the FRET kit were analyzed and graphed using the statistical software GraphPad Prism. Statistical analysis included ratio logarithmic transformation and the generation of 4PL curves.

[0037] Cells were seeded at the same concentration in a 96-well plate and stimulated with different amounts of r-hLH to generate a dose-response curve. As a negative control, non-transfected HEK 293-T cells were also stimulated. After 30 minutes of incubation, the FRET reagent was added to each well of the plate. The plate was again incubated for 60 minutes under shaking, and the results were finally acquired using a plate reader. Figure 1, panel A, demonstrates the differences in the responses produced by the two cell lines. The transfected cells responded to r-hLH stimulation with a clear dose-response curve. In contrast, the non-transfected HEK 293-T cells did not show a dose-dependent response after stimulation, confirming the absence of receptors on the cell membrane.

[0038] Furthermore, the response specificity of HEK 293-T LHCGR cells was verified by stimulating the cells with r-hFSH, a hormone analogue of r-hLH. Figure 1, panel B, shows that HEK 293-T LHCGR cells failed to respond to r-hFSH stimulation, confirming that HEK 293-T LHCGR cells are unable to increase intracellular cAMP accumulation after r-hFSH stimulation.

[0039] Transfection of HEK 293T LHCGR cells. To generate a reporter gene cell line, HEK 293-T LHCGR cells already transfected with the LHCG receptor vector were transfected a second time. The commercially available vector selected (Luc2P / CRE CS188015, Promega) contained a cAMP response element (CRE) that drives transcription of the luciferase reporter gene luc2P (Photinus pyralis). The vector backbone contained a mammalian selectable marker for puromycin resistance. Prior to transfection, a kill curve assay was performed to determine the minimum concentration of puromycin required to inhibit the growth of HEK 293-T LHCGR cells over a 10-day period. 0.5 μg / mL was determined to be the optimal concentration and was used in all transfection experiments.

[0040] A vector encoding a luciferase reporter gene was transfected into HEK 293-T LHCGR cells using FuGENE HD transfection reagent (Promega, E2311). OptiMEM medium (Life Technologies, 31985) was used for transfection. Various transfection conditions were tested, including varying the amount of vector DNA and the ratio of reagent to vector DNA.

[0041] The transfection process and various experimental conditions were carried out according to the supplier's recommended protocol. 24 hours after transfection, antibiotic selection agents were added to the cells to select only cell populations resistant to the antibiotic concentrations identified in the kill curve experiments. Resistant cells were expanded and different cell banks belonging to each transfection technique were frozen.

[0042] Evaluation of the efficiency of the second transfection. To evaluate the efficiency of the second transfection, a reporter gene assay (RGA) was performed. This assay is described in the Promega vector datasheet. HEK 293-T LHCGR luciferase cells were stimulated with forskolin. As described in the literature, forskolin is a diterpene that activates adenylate cyclase (AC), leading to an increase in intracellular cAMP in various cell types. The RGA assay, utilizing the action of forskolin, allows us to evaluate reporter gene transcription in response to cAMP accumulation without the LHCG receptor. Luminescence emitted after reporter gene transcription was quantified using commercially available reagents.

[0043] HEK 293-T LHCGR luciferase and untransfected HEK 293-T cells were seeded in a 96-well plate and stimulated with different concentrations of forskolin. After incubation, the ONE-Glo EX Luciferase Assay System (Promega, E8120) was added to each well and shaken for 10 minutes before reading the results on a plate reader.

[0044] Table 1 shows the luminescence values ​​(relative luminescence units, RLU) obtained for each well after plate reading. As expected, untransfected HEK 293-T (WT) cells showed no luminescence response to forskolin stimulation due to the lack of the receptor pathway. The HEK 293-T LHCGR luciferase response was shown to be dose-dependent. The highest RLU value was obtained at 40 μM forskolin. Even at 4 μM, the luminescence value was considered adequate for assay purposes. At 400 μM, the cells reached a plateau of maximum response, resulting in a lower response than at 40 μM. These data confirm the success of the second transfection.

[0045] [Table 1]

[0046] HEK 293-T LHCGR Luciferase: Cell Line Characterization. Cell line characterization includes all tasks related to the evaluation of determined cell properties. The purpose of this study was to monitor and evaluate various parameters to investigate cell line behavior over time. Cell line behavior evaluation is important for monitoring cell viability, proliferation rate, target receptor expression, and functional performance over a certain number of cell passages. The parameters monitored for HEK 293-T LH Luciferase cells were: Presence or absence of LHCG receptor expression on the cell membrane Cell viability Cell proliferation (doubling time and real-time cell proliferation verification), First transfection stability Second transfection stability.

[0047] To ensure analytical consistency and to obtain results that were representative of the cell line and not tied to a specific cell bank, three vials from three different cell banks (MCB, WCB1, and WCB2) were tested. Cells were monitored over time, from passage 17 to passage 41.

[0048] Each parameter was evaluated over a defined passage number range. Variation among triplicate vials was calculated using the coefficient of variation (CV%). Triplicate vials were analyzed together, and the mean and standard deviation (SD) were calculated. Data were analyzed using linear regression analysis with a significance level of 95% (p-value <0.05). All statistical analyses were performed using Graph Pad Prism.

[0049] Evaluation of LHCG receptor expression on the cell membrane. The percentage of transfected cells expressing human LHCG receptor on the cell membrane was evaluated. For this purpose, cells were stained and analyzed using a FACS Aria Fusion instrument (BD Biosciences). Data analysis of the staining experiments was performed using FlowJo software. The cell lines used for staining were HEK 293-T LHCGR luciferase MCB and untransfected HEK-293-T WT. An LHR-conjugated primary antibody (LHCGR / 1417[PE], Novusbio) was used for staining. A staining protocol was developed using cell fixation and permeabilization prior to antibody addition.

[0050] Fixation buffer (FB) was prepared by diluting methanol 1:1 with PBS 1X and stored at room temperature. Two wash buffers (WB1 and WB2) were used, each containing PBS 1X and 2% fetal bovine serum. WB2 also contained 0.1% Tween® 20 reagent. 1 × 10 6 The cells were resuspended in WB1, transferred to tubes, and centrifuged at 300xg for 6 minutes. After removing the supernatant, 1 mL of FB was added to each tube and incubated at -20°C for 10 minutes. After incubation, the cells were washed with 2 mL of WB2. Staining was performed in two steps using an LHR-labeled primary antibody. In the first step, FcR blocking reagent (Miltenyi Biotec, 130-059-901) was diluted in WB2 and added to each tube. The tubes were incubated at room temperature for 5 minutes, followed by the addition of the antibody. The antibody was diluted in WB2 and incubated at room temperature for 30 minutes. After incubation, the cells were washed with WB2 and resuspended in WB2. Results were acquired using a FACS instrument.

[0051] As shown in Figure 2, slight differences were observed between non-transfected cells (HEK 293-T WT) and transfected cells (HEK 293-T LHCGR luciferase). In fact, 71% of LHCG receptor-positive cells were stained in HEK 293-T LHCGR luciferase, while only 0.89% of cells were stained positive in HEK 293-T WT. This staining experiment confirmed that the initial transfection was successful.

[0052] Viability of HEK 293-T LHCGR luciferase cells. Cell line viability measurements were performed using a Nucleocounter (Chemometec). Results showed that post-thaw viability exceeded 90% and remained stable across the range of passage numbers analyzed (Figure 3A). The mean and standard deviation for the three banks were calculated and are shown in Figure 3A. The mean viability was estimated to be approximately 96.55% ± 1.61% (mean ± standard deviation), which decreased slightly across the range of passage numbers, but no significant downward trend was observed.

[0053] Growth evaluation of HEK 293-T LHCGR luciferase. Cell proliferation evaluation allowed us to calculate the cell doubling time, allowing us to determine the optimal subculture conditions in terms of the seeded cell number. For all subcultures, the total cell number was measured using a NucleoCounter (Chemometec), and the doubling time was calculated using the following formula: Doubling time = (period (hours) x log(2)) / (log(final concentration) - log(initial concentration))

[0054] The mean and standard deviation (SD) of the three banks were calculated and are shown in Figure 3B. The doubling time was estimated to be approximately 17.71 ± 1.01 hours (mean ± SD), and a slight tendency for it to decrease within the range of passage numbers was observed, but this was not significant.

[0055] For real-time proliferation assessment, cells were seeded in 96-well plates at the following concentrations: 100,000 cells / well, 50,000 cells / well, 25,000 cells / well, 12,500 cells / well, 6,250 cells / well, and 3,125 cells / well. Cells were cultured for 4 days in an IncuCyte (Sartorius) system. Images were acquired from each well every hour. Confluence masks were created using the IncuCyte analysis tool, and the area occupied by the cell images (% confluence) was calculated over time to generate cell proliferation curves. The overlap of the growth curves (12,500 cells / well) at passages 17 and 41 confirmed that cell proliferation did not change across the range of passage numbers (Figure 3C).

[0056] First, transfection stability. The transfection stability of the LHCG receptor was indirectly assessed by quantifying cAMP produced after r-hLH interacted with its receptor. HEK 293T LHCGR luciferase cells were stimulated with a scalar amount of r-hLH in a 96-well plate, and the amount of cAMP present in each well was quantified using a commercially available FRET kit. For each test, a four-parameter dose-response curve was generated using GraphPad Prism (v.8) software. EC50 was selected as the parameter to monitor over time. The mean values ​​and CV% across the three banks were calculated and are shown in Figure 4A. As shown in Figure 4A, the EC50 remained stable throughout the analyzed period.

[0057] Stability of the second transfection. HEK 293-T LHCGR luciferase cells were stimulated with 4 μM forskolin or left unstimulated. Results were reported as the fold change of treated cells relative to the unstimulated control. The %CVs for the fold changes obtained in the three banks were calculated and were all less than 25%. The mean and standard deviations for the three banks were calculated and plotted in Figure 4B. With increasing passage number, luminescence values ​​gradually decreased, indicating a significant decrease in fold change (Figure 4B).

[0058] Reporter Gene Assay: Method Development. A reporter gene assay for evaluating the potency of r-hLH and r-hCG was constructed using the HEK 293-T LHCGR luciferase cell line. The developed method reproduced part of the mechanism of action of r-hLH and r-hCG, namely, the first step in the metabolic cascade triggered by receptor binding of the hormone. The assay principle involved seeding cells at a uniform concentration (40,000 cells / well) into each well of a 96-well plate and stimulating them with an initial dose of 200 ng / mL r-hLH or 250 ng / mL r-hCG via a 1:10 dilution series. After a 4-hour incubation, a commercially available luminescent substrate was added to each well. After shaking, the cells were read using a plate reader. All data were analyzed using GraphPad Prism software, and the RLU values ​​obtained in each well were interpolated using the logarithmically transformed nanograms / mL value of the hormone used at each dose.

[0059] Figure 5 shows the dose-response curves obtained for r-hLH (Panel A) and r-hCG (Panel B). Transfected cells responded to stimulation with both hormones, producing well-defined dose-response curves.

[0060] Two preliminary linearity studies were performed using r-hLH and r-hCG samples to evaluate the performance of the analytical method. The feasibility of linearity was evaluated by testing different theoretical relative potency levels of 60%, 77%, 100%, 120%, and 144% in triplicate. These different potency levels were calculated based on an r-hLH concentration of 200 ng / mL and an r-hCG concentration of 250 ng / mL. The starting concentrations of both samples were used as the standard values. Relative potency (RP%) results were calculated for each sample. Relative potency (RP%) RP% = ((EC50 standard value) / (EC50 sample value)) x 100

[0061] The linearity results were statistically analyzed using JMP software. JMP analysis generated a graph displaying the logarithm of the theoretical relative potency (60%-77%-100%-120%-144%) of the test samples on the X-axis and the logarithm of the measured relative potency of the test samples on the Y-axis. A common method for assessing the fit of the resulting regression line is to evaluate various parameters, such as R2, intercept confidence interval, and slope confidence interval. Thanks to the experience gained from multiple method validations in the laboratory, we were able to define approximate acceptance criteria for all parameters (Table 2).

[0062] [Table 2]

Claims

1. The following: a. a human LHCG receptor comprising a nucleic acid molecule encoding the human LHCG receptor under the control of a protein expression promoter; and b. a reporter gene comprising a nucleic acid molecule encoding a luciferase reporter gene under the control of a cAMP response element (CRE); A mammalian cell for expressing the human LHCG receptor and a luciferase reporter gene comprising:

2. The mammalian cell of claim 1 , wherein the mammalian cell is a mammalian cell culture.

3. The mammalian cell of claim 2 , wherein the mammalian cell culture is an adherent cell culture.

4. The mammalian cell according to any one of claims 1 to 3, wherein the promoter for the protein expression is a strong promoter for high-level protein expression, such as CAG.

5. a. The nucleic acid molecule encoding the human LHCG receptor is a vector comprising the LHCG receptor gene and a puromycin resistance gene; b. A mammalian cell described in any one of claims 1 to 3, wherein the nucleic acid molecule encoding the luciferase reporter gene is a vector comprising the luciferase reporter gene under the control of a cAMP response element (CRE) and a puromycin resistance gene.

6. The mammalian cell according to any one of claims 1 to 5, wherein the mammalian cell is a human embryonic kidney (HEK) 293 cell.

7. The mammalian cell of claim 6, wherein the HEK293 cell is a HEK293T cell.

8. 1. A method for producing a human LHCG receptor and a luciferase reporter gene in mammalian cells, comprising: a. introducing into a mammalian cell an isolated nucleic acid molecule encoding an LHCG receptor under the control of a protein expression promoter; b. adding the first selection agent to the cells transfected in step a) to induce a stable human LHCG receptor-expressing cell line; c. introducing into the mammalian cells transfected in step b) an isolated nucleic acid molecule encoding a luciferase reporter gene under the control of a cAMP response element (CRE) to obtain transfected cells; d. adding a second selection agent to the stably expressing human LHCG receptor cells in step c) to induce cells stably expressing the human LHCG receptor and the luciferase reporter; and e. A method comprising harvesting cells stably expressing the human LHCG receptor and a luciferase reporter.

9. 9. The method of claim 8, wherein the first selection agent is hygromycin and the second selection agent is puromycin.

10. 10. The method of claim 8 or 9, wherein the promoter for protein expression is a strong promoter for high-level protein expression, such as CAG.

11. 1. A method for determining the biological activity of a r-hLH or r-hCG sample, comprising: a. contacting a sample with a cell culture of mammalian cells according to any one of claims 1 to 7; b. adding a luminescent substrate for the luciferase enzyme to the cell culture; c. detecting luminescence from the cells; d. quantitating the amount of light emitted; and e. A method comprising determining the biological activity of a r-hLH or r-hCG sample.

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