A method for determining the biological activity of recombinant teriparatide

Abstract

The present invention discloses a method for detecting the biological activity of recombinant teriparatide. Specifically, rat osteosarcoma (UMR-106) cells are selected as test cells, and the biological activity is reflected by measuring the cAMP level of the cells under the action of recombinant teriparatide. The method established in the present invention has high curve fitting, good accuracy, high repeatability, and strong durability, and is well suited for detecting the biological activity of recombinant teriparatide.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection of biopharmaceuticals, and in particular relates to a method for determining the biological activity of recombinant teriparatide. Background Art

[0002] Recombinant teriparatide (chemically known as recombinant human parathyroid hormone 1-34, rhPTH(1-34)) is a peptide consisting of the N-terminal 1-34 amino acid residues of recombinant human parathyroid hormone. It is obtained by transforming a recombinant plasmid carrying the gene encoding it into Escherichia coli through genetic recombination technology, followed by cell culture, isolation, and purification. Recombinant teriparatide is a bone-forming, anti-osteoporotic drug that effectively improves bone microarchitecture and increases bone strength, while promoting bone healing and reducing the risk of vertebral and non-vertebral fractures. It is suitable for the treatment of osteoporosis in postmenopausal women at high risk of fracture.

[0003] The determination of the biological activity of polypeptide drugs is an important indicator for evaluating the effectiveness of drugs, and it is indispensable in product development, production process control and quality control. The methods for determining the biological activity of recombinant teriparatide that have been reported so far mainly include radioimmunoassay, chemiluminescence method, and cell proliferation method. The above detection methods are relatively cumbersome to operate, and large instruments are required for detection. The cell proliferation method also requires strict control of cell density. The method disclosed in the present invention is a biological activity determination method based on UMR-106 cells. This method is accurate, stable and reliable, has high precision, and has good durability to changes in cell generations. It can better meet the process control, release inspection and other inspection requirements of recombinant teriparatide drugs. Summary of the Invention

[0004] The purpose of the present invention is to optimize the shortcomings of the current recombinant teriparatide biological activity detection technology and conduct methodological verification to provide a recombinant teriparatide biological activity detection method with high accuracy, good precision and strong durability. The method can not only detect pure recombinant teriparatide, but also be applicable to recombinant teriparatide preparations with complex formulas, and can be better applied to the biological activity detection of recombinant teriparatide.

[0005] To achieve the purpose of the present invention, a method for detecting the biological activity of recombinant teriparatide is provided, which comprises the following steps:

[0006] (1) Take UMR-106 cells in good growth state in logarithmic growth phase and prepare 1.0×

[0007] 10 5 ~3.0×10 5 cells / ml cell suspension, 100 μl / well was plated onto cell culture plates and cultured for 16-24 h;

[0008] (2) diluting the recombinant teriparatide reference and test samples in a serial dilution, wherein the starting concentration of the recombinant teriparatide reference and test samples is 0.5 to 0.7 μg / ml, the dilution multiple is 3.5 to 5, and a total of 8 to 9 concentration gradients;

[0009] (3) adding the reference sample and the test sample of each concentration gradient in step (2) to the cell culture plate at 100 μl / well, and incubating with the cells for 15 to 30 minutes;

[0010] (4) After incubation, discard the supernatant and wash the cells with pre-chilled PBS;

[0011] (5) Lyse cells, detect cAMP content, and calculate biological activity.

[0012] In some embodiments, the cell suspension is 2.0×10 5 ~3.0×10 5 cells / ml, preferably 2.0×10 5 cells / ml. In some embodiments, the culture time is 20h. In some preferred embodiments, the cell suspension is 1.0×10 5 ~3.0×10 5 cells / ml, the culture time is 20h; or the cell suspension is 2.0×10 5 cells / ml, and the culture time is 16 to 24 hours.

[0013] In some embodiments, the starting concentration is 0.5 μg / ml, the dilution factor is 3.5, and there are 9 concentration gradients in total; or the starting concentration is 0.5 μg / ml, the dilution factor is 4, and there are 8 concentration gradients in total; or the starting concentration is 0.7 μg / ml, the dilution factor is 5, and there are 8 concentration gradients in total; the starting concentration is preferably 0.5 μg / ml, the dilution factor is preferably 3.5, and there are preferably 9 concentration gradients in total.

[0014] In some embodiments, lysing the cells comprises adding 150 μl / well of cell lysis buffer and freezing and thawing once. In some embodiments, the cell lysis buffer comprises 5 mM Tris, 1% SDS, and the pH of the Tris is 7.5.

[0015] In some embodiments, the confluence of the cells is controlled between 80% and 90%, preferably about 85%; the culture medium is a complete culture medium comprising DEME medium and 10% fetal bovine serum; and in some embodiments, the cell viability of the cell suspension is ≥90%.

[0016] In some embodiments, the recombinant teriparatide reference and test samples are diluted in a sample diluent comprising phosphate-buffered saline (PBS) and 3-isobutyl-1-methylxanthine (IBMX) at a concentration of 1 mmol / L. IBMX is a nonselective inhibitor of cAMP and cGMP phosphodiesterase (PDE) that can enhance intracellular cAMP levels. Using PBS instead of conventional culture medium as the diluent can further reduce costs while maintaining assay performance.

[0017] In some embodiments, the incubation is preferably 15 minutes. The culture or incubation conditions include 37° C. and 5% CO 2 .

[0018] In some embodiments, detecting the cAMP content comprises detecting the cAMP content by a competitive ELISA method. The competitive ELISA method comprises coating a cAMP antibody on an ELISA plate, adding cell lysate, then adding cAMP labeled with horseradish peroxidase (HRP), developing color, then stopping the assay, and measuring the OD value at 450 nm.

[0019] In some embodiments, the calculation of biological activity includes fitting a four-parameter equation curve with the dilution multiple of the reference or test sample as the X-axis and the corresponding cAMP content as the Y-axis to calculate the half dilution multiple (EC 50 ) value, the relative biological activity of the test article = the test article EC 50 / Standard EC 50 × 100%. In some embodiments, the cAMP content is expressed as OD 450 The four-parameter equation curve is as follows:

[0020] y=A2+(A1-A2) / (1+(x / X0)^p)

[0021] x: dilution factor corresponding to the concentration of test sample and reference sample;

[0022] y: OD value;

[0023] A1: The minimum value of y when x approaches infinity or approaches infinity;

[0024] A2: The maximum value of y when x approaches infinity or is infinitely small;

[0025] p: slope of the four-parameter curve;

[0026] X0(EC 50 ): half-effective dilution multiple.

[0027] In some embodiments, the test sample includes recombinant teriparatide injection, which contains recombinant teriparatide stock solution, glacial acetic acid, anhydrous sodium acetate, mannitol, m-cresol, hydrochloric acid, sodium hydroxide, and water for injection.

[0028] In some preferred embodiments, the detection method comprises the following steps:

[0029] (1) Take UMR-106 cells in good growth state in logarithmic growth phase and prepare 2.0×

[0030] 10 5 cells / ml of cell suspension, 100 μl / well was plated onto a cell culture plate and cultured for 20 h; the confluence of the cells was controlled at about 85%, and the cell viability of the cell suspension was ≥90%;

[0031] (2) diluting the recombinant teriparatide reference substance and the test substance in a serial dilution ratio with a sample diluent, wherein the starting concentration of the recombinant teriparatide reference substance and the test substance is 0.5 μg / ml, the dilution factor is 3.5, and there are 9 concentration gradients in total. The sample diluent contains PBS and 1 mmol / L IBMX;

[0032] (3) adding the reference sample and the test sample of each concentration gradient in step (2) to the cell culture plate at 100 μl / well and incubating with the cells for 15 minutes;

[0033] (4) After incubation, discard the supernatant and wash the cells with pre-chilled PBS;

[0034] (5) Add 150 μl / well of cell lysis buffer, place at ≤-70°C and freeze-thaw at 37°C once, the cell lysis buffer contains 5 mM Tris, 1% SDS, and the pH of Tris is 7.5. Detect the cAMP content, with the dilution factor of the reference or test sample as the X-axis and the corresponding cAMP content as the Y-axis, fit the four-parameter equation curve, and calculate the half dilution factor (EC 50 ) value, the relative biological activity of the test article = the test article EC 50 / Standard EC 50 ×100%; the four-parameter equation curve is as follows:

[0035] y=A2+(A1-A2) / (1+(x / X0)^p)

[0036] x: dilution factor corresponding to the concentration of test sample and reference sample;

[0037] y: OD value;

[0038] A1: The minimum value of y when x approaches infinity or approaches infinity;

[0039] A2: The maximum value of y when x approaches infinity or is infinitely small;

[0040] p: slope of the four-parameter curve;

[0041] X0(EC 50 ): half-effective dilution multiple.

[0042] In another aspect, the present invention provides the use of the method for detecting the biological activity of recombinant teriparatide in the quality control of the production process of recombinant teriparatide drugs.

[0043] The present invention has the following advantages and beneficial effects compared to the prior art:

[0044] 1. The present invention provides a method for detecting the biological activity of recombinant teriparatide. By optimizing the cell density, lysis method and sample dilution method, the accuracy of the detection results is improved, and the method has good precision and durability. The relative bias (RB) and geometric coefficient of variation (GCV) of each group of samples are less than 5%, and there is a good linear relationship between the theoretical activity value and the measured value. 2 =0.999, with high tolerance to cell generations and flexible culture time, which is more convenient in practical applications.

[0045] 2. The present invention provides a method for detecting the biological activity of recombinant teriparatide, which can achieve accurate detection of complex recombinant teriparatide preparations, especially recombinant teriparatide injection. The method has strong anti-interference ability and has a wider range of applicability in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a four-parameter curve fitting diagram for the experiment on different cell plating densities in Example 1.

[0047] Figure 2 This is a four-parameter curve fitting diagram for the study using cell lysis buffer 1-2 and lysis method 1 in Example 2.

[0048] Figures 3 to 8 The four-parameter curve fitting diagrams of experimental groups 1-6 in Example 3 are shown in order.

[0049] Figure 9 This is the linear regression equation of the logarithmic values ​​of the theoretical and measured values ​​of biological activity for the relative accuracy evaluation in Example 4.

[0050] Note: In all four-parameter fitting curves, the starting concentration is plotted as a dilution factor of 1. DETAILED DESCRIPTION

[0051] To illustrate the purpose, technical methods and advantages of the present invention in detail, the present invention is described in detail and clearly with reference to specific embodiments and accompanying drawings.

[0052] Experimental materials: UMR-106 cells (Chinese Academy of Sciences); fetal bovine serum (FBS) (GIBCO, cat. no. 10099141C), DMEM medium (GIBCO, cat. no. 11965-092), trypsin (GIBCO, cat. no. 25200-056); cAMP ELISA kit (GenScript, cat. no. L00460); recombinant teriparatide national standard (China National Institute for Food and Drug Control, 410019-201801); recombinant teriparatide international standard (WHO, 15 / 304); two batches of recombinant teriparatide test products: recombinant teriparatide stock solution and teriparatide injection (Sinlitai (Suzhou) Pharmaceutical Co., Ltd.); complete culture medium: DMEM medium containing 10% FBS; sample diluent: PBS (containing 1 mmol / L IBMX).

[0053] The ingredients of the teriparatide injection are recombinant teriparatide stock solution, glacial acetic acid, sodium acetate (anhydrous), mannitol, m-cresol, hydrochloric acid, sodium hydroxide, and water for injection.

[0054] Experimental instruments: multifunctional microplate reader, carbon dioxide incubator (Thermo Fisher).

[0055] Example 1: Cell density screening research experiment

[0056] Cell culture: UMR-106 cells were revived and subcultured using complete culture medium in a 37.0° C., 5.0% CO 2 incubator. The confluence of the subcultured cells was controlled at approximately 85%.

[0057] Cell treatment: Take UMR-106 cells with good growth status, discard the culture supernatant, aspirate 3.0-5.0 ml PBS to wash the residual culture medium, discard the supernatant, add 1.0-3.0 ml 0.25% trypsin, digest at room temperature, observe under a microscope until about 80% of the cells become round, immediately add 4-10 ml complete culture medium to terminate the digestion, gently pipette the cells on the flask wall to make a cell suspension, collect the cell suspension and place it in a sterile centrifuge tube, centrifuge at 800-1000 rpm for 3-5 minutes, discard the supernatant, resuspend with complete culture medium, count, confirm that the cell viability is ≥90%, and then dilute with complete culture medium to 0.5×10 5 , 1.0×10 5 , 2.0×10 5 , 3.0×10 5 cells / ml, 100 μl per well, plated into 96-well cell culture plates, and cultured in a 37.0°C, 5% CO2 incubator for 20 h.

[0058] Sample processing: The recombinant teriparatide international standard was serially diluted with sample diluent, starting at 0.6 μg / ml, and diluted 5-fold to prepare 9 concentration points. The sample at each concentration point was added to a 96-well cell culture plate at 100 μl / well and incubated with the cells. Duplicate wells were set for each concentration point and incubated in a 37.0°C, 5.0% CO2 incubator for 15 min. After that, the cell culture plate was removed, the supernatant was discarded, and the plate was washed once with 2-8°C pre-cooled PBS, 300 μl / well.

[0059] Cell lysis: After discarding the supernatant, add 150 μl / well of cell lysis buffer and freeze-thaw once in a ≤-70℃ refrigerator and a 37℃ CO2 incubator.

[0060] cAMP detection: Follow the instructions of the kit.

[0061] Data analysis: The dilution factor corresponding to the sample concentration was used as the X-axis, and the corresponding OD value was used as the Y-axis. The ORIGIN software was used to fit a four-parameter equation curve to calculate the half-effective dilution factor. The four-parameter equation curve is as follows:

[0062] y=A2+(A1-A2) / (1+(x / X0)^p)

[0063] x: dilution factor corresponding to sample concentration;

[0064] y: OD value;

[0065] A1: The minimum value of y when x approaches infinity or approaches infinity;

[0066] A2: The maximum value of y when x approaches infinity or is infinitely small;

[0067] p: slope of the four-parameter curve;

[0068] X0(EC 50 ): half-effective dilution multiple.

[0069] The experimental results are as follows Figure 1 As shown, it can be seen that the low density point 0.5×10 5 cells / ml, the lower platform signal value is high, and the number of lower platform points is small. The curve distribution of the other three concentration points is generally better than 0.5×10 5 cells / ml, the curve fitting degree is high and close, so the cell plating density range is selected as 1.0×10 5 ~3.0×10 5Considering the influence of culture time on cell density, in order to further improve the flexibility of the method, the cell plating density range of 1.0×10 5 ~3.0×10 5 cells / ml, the culture time was controlled within 16h~24h to affect the detection. The results showed that 2.0×10 5 cells / ml can be used as the optimal plating density, at which the cell density after 16 to 24 hours of culture can be controlled within a reasonable range, ensuring that the cells maintain an optimal growth state in the culture plate.

[0070] Example 2: Lysis solution and lysis method screening research experiment

[0071] Cell culture and treatment: refer to the method of Example 1, wherein the cell plating density is 2.0×10 5 cells / ml.

[0072] Sample processing: The recombinant teriparatide national standard was serially diluted with sample diluent, and 0.5 μg / ml was used as the starting concentration. The sample was diluted 4-fold to prepare 8 concentration points for biological activity detection. The sample of each concentration point was added to a 96-well cell culture plate at 100 μl / well and incubated with the cells. Duplicate wells were set for each concentration point and incubated in a 37.0°C, 5.0% CO2 incubator for 15 min. After that, the cell culture plate was removed, the supernatant was aspirated and discarded, and the plate was washed once with 2-8°C pre-cooled PBS, 300 μl / well.

[0073] Cell Lysis: After discarding the supernatant, add 150 μl / well of each of Lysis Buffers A to I, as well as commercial Lysis Buffer 2 (Biyuntian, Cat. No. P0013G), and Lysis Buffer 3 (Biyuntian, Cat. No. P0013J). The compositions of Lysis Buffers A to I are shown in Table 1. Incubate at 37°C in a CO2 incubator for 30 min.

[0074] cAMP detection and data analysis: The same method as in Example 1 was used.

[0075] Table 1 Lysate components

[0076]

[0077] The results of cAMP content detection in cells treated with samples at the third concentration point are used as an example. As shown in Table 2, it can be seen that after cells are treated with samples at the same concentration, the amount of cAMP released by different lysis buffers varies significantly. Lysis buffer H releases the largest amount of cAMP, which can more truly reflect the stimulatory effect of recombinant teriparatide on cells. Therefore, lysis buffer H (5 mM Tris (pH 7.5), 1% SDS) is preferred for cell lysis.

[0078] Table 2 cAMP content detection results

[0079] Lysate cAMP concentration (pmol / ml) A 60.92 B 30.26 C 5.27 D 77.79 E 71.71 F 7.12 G 103.63 H 124.05 I 19.15 Lysate 2 103.25 Lysate 3 88.57

[0080] In addition, the effects of different lysis methods used in lysis buffer 1 (i.e., lysis buffer H) and lysis buffer 2 on the detection were also tested. Lysis method 1: freeze and thaw once in a ≤-70°C refrigerator and a 37°C CO2 incubator; lysis method 2: lysis at 37°C for 30 minutes. The cAMP content of cells after treatment with samples of the same concentration is shown in Table 3 below, which shows that when lysis buffer 1 is used, lysis method 1 produces more cAMP than lysis method 2, and freezing and thawing has no obvious effect on the effect of lysis buffer 2. The results of biological activity detection using lysis buffer 1 and lysis buffer 2 under method 1 are shown in the following table. Figure 2 As shown in the figure, the absorbance values ​​of the upper and lower platforms at each concentration point of lysate 1 are significantly lower than those of lysate 2, which also shows that lysate 1 releases more cAMP and lyses cells more fully. In addition, the curve fitting degree of lysate 1 is also better than that of lysate 2 (R 2 0.999 and 0.998, respectively). Therefore, it is preferred to perform the test by freezing and thawing the lysate 1 once.

[0081] Table 3 cAMP content detection results

[0082]

[0083] Example 3: Sample initial concentration and dilution scheme research experiment

[0084] Cell culture and treatment: The same method as in Example 2 was used.

[0085] Sample treatment: refer to the method of Example 2, wherein the recombinant teriparatide national standard is diluted stepwise with the sample diluent according to the experimental groups in Table 4.

[0086] Cell lysis: After discarding the supernatant, add 150 μl / well of cell lysis buffer 1 (5 mM Tris (pH 7.5), 1% SDS) and freeze-thaw once in a ≤-70°C refrigerator and a 37°C CO2 incubator.

[0087] cAMP detection and data analysis: The same method as in Example 1 was used.

[0088] The sample starting concentration and dilution ratio were determined based on the criteria of ≥2 upper platform points, ≥1 lower platform points, and ≥3 linear region points on the four-parameter curve. Figures 3 to 8 As shown, it can be seen that the number of upper platform points in experimental group 1 and experimental group 2 is relatively small, and the number of lower platform points in experimental group 6 is too small. The concentration points of the fitting curves of the above experimental groups are unevenly distributed, while the starting concentrations of the samples in experimental groups 3-5 are moderate and the concentration points are reasonably set, all of which show a relatively good "S"-shaped response curve. Moreover, when the sample is processed with experimental group 5 (the starting concentration of the sample is 0.5 μg / ml, and the concentration points are 3.5 times the gradient dilution with 9 points), the concentration points of the upper and lower platforms are most balanced, and the curve symmetry and stability are the best.

[0089] Table 4 Sample starting concentration and dilution scheme

[0090]

[0091]

[0092] Example 4: Evaluation of relative accuracy and precision of the method

[0093] This example evaluates the relative accuracy and precision of the established method. Comparative Examples 1 and 2 are also provided. Comparative Example 1 is performed according to the biological activity assay described in Example 17 of patent document CN 116410334 A, and Comparative Example 2 is performed according to the rhPTH drug activity assay described in Example 4 of patent document CN108318677A. The relative biological activity is calculated according to the formula of the present invention.

[0094] Relative accuracy refers to the degree of closeness of the measured relative activity to the true value or reference value within a specified range and is generally expressed as relative bias (RB, %). The experimental design was as follows: Two experimenters (A and B) prepared a total of five samples with starting concentrations of 0.25 μg / ml, 0.375 μg / ml, 0.5 μg / ml, 0.75 μg / ml, and 1.0 μg / ml, respectively, with theoretical activities of 50%, 75%, 100%, 150%, and 200%, respectively. Using a recombinant teriparatide national standard at a starting concentration of 0.5 μg / ml as a reference, biological activity was assayed according to the method in Example 1 and the preferred parameters listed in Table 5.

[0095] Precision refers to the degree of similarity between the results obtained from multiple samplings of the same homogeneous sample under specified assay conditions. The precision of relative activity assays is generally expressed as the geometric standard deviation (GSD) or geometric coefficient of variation (GCV, %). The experimental design was as follows: Two experimenters (A and B) each performed an experiment on separate days, preparing samples with a starting concentration of 0.5 μg / ml. These samples were prepared in triplicate and biological activity assayed according to the method in Example 1 and the preferred parameters listed in Table 5.

[0096] Table 5 Preferred parameters of the method

[0097] Plating density (cells / ml) <![CDATA[2.0×10 5 ]]> Dilution multiple 3.5 Number of points involved in fitting curve 9 Lysate 5 mM Tris (pH 7.5), 1% SDS

[0098] The relative accuracy test results are shown in Table 6 below. The relative bias of the method of the present invention for different samples is within the range of ±5%, which is relatively small. The logarithm of the theoretical activity value (abscissa) is used to make a linear regression with the logarithm of the corresponding measured activity value (ordinate). The logarithm is the ln value. Figure 9 As shown, the R of the fitting curve 2 =0.999, indicating that the method of the present invention has a very high accuracy.

[0099] The results of the precision experiment are shown in Table 7 below. The geometric coefficient of variation (GCV) is within 5%, indicating that the method of the invention has high precision.

[0100] Table 6 Relative accuracy evaluation experimental results

[0101]

[0102] Table 7 Precision test results

[0103]

[0104] Example 5: Method Robustness Assessment

[0105] This example evaluates the robustness of the established method, and comparative examples 1 and 2 are provided. Comparative example 1 is performed according to the biological activity assay described in Example 17 of patent document CN 116410334 A, and comparative example 2 is performed according to the rhPTH drug activity assay described in Example 4 of patent document CN108318677A. The relative biological activity is calculated according to the formula of the present invention.

[0106] The method established by the present invention evaluates durability according to the following steps:

[0107] 1. Cell processing

[0108] Cells of three different passages (P12, P19, and P27) were plated and diluted to 2.0 × 10 5 cells / ml, 100 μl per well was plated into a 96-well cell culture plate and cultured for 18-20 h.

[0109] 2. Sample processing

[0110] Recombinant teriparatide national standard was used as a reference substance, and recombinant teriparatide stock solution and injection (one batch each) were used as test samples. Diluted according to the three treatments in Experimental Groups 3, 4, and 5 in Table 2 of Example 3, 100 μl / well was added to a cell culture plate in duplicate. After incubation for 15 minutes, the plate was removed, the supernatant discarded, and 300 μl / well of PBS pre-chilled at 2-8°C was added and washed once. After discarding the supernatant, 150 μl / well of cell lysis buffer 1 (5 mM Tris (pH 7.5), 1% SDS) was added. The plates were then placed in a refrigerator at ≤-70°C and then frozen and thawed once in a 37°C CO2 incubator.

[0111] 3. cAMP detection: Follow the instructions of the kit.

[0112] Comparative Examples 1 and 2 also used cells of three different generations (P12, P19, and P27) for detection.

[0113] The experimental results are shown in Table 8 below. Using the method of the present invention, the results of biological activity detection of each test sample using three different cell passages were relatively close. In particular, the test results under experimental group 5 were more stable, indicating that the method of the present invention is not affected by cell passage and has good durability. The test results of Comparative Examples 1 and 2 at different cell passages fluctuated greatly, which was more significantly affected by the unstable cell state caused by cell passage, and their test results were unreliable.

[0114] Table 8 Test results of different cell generations

[0115]

Claims

1. A method for detecting the biological activity of recombinant teriparatide, comprising the following steps: (1) Take UMR-106 cells in good growth state in logarithmic growth phase and prepare 2.0×10 5 cells / ml cell suspension, 100 μl / well was plated on a cell culture plate and cultured for 20 h; (2) The recombinant teriparatide reference substance and the test substance were diluted in series with the sample diluent. The test substance was the recombinant teriparatide injection, which consisted of recombinant teriparatide stock solution, glacial acetic acid, anhydrous sodium acetate, mannitol, m-cresol, hydrochloric acid, sodium hydroxide, and water for injection. The sample diluent consisted of phosphate buffer and 1 mmol / L 3-isobutyl-1-methylxanthine. The starting concentration of the recombinant teriparatide reference substance and the test substance was 0.5 μg / ml, the dilution factor was 3.5, and there were 9 concentration gradients in total. (3) adding the reference sample and the test sample of each concentration gradient in step (2) to the cell culture plate at 100 μl / well and incubating with the cells for 15 minutes; (4) After incubation, discard the supernatant and wash the cells with pre-chilled phosphate buffer; (5) Add 150 μl / well of cell lysis buffer, which consists of 5 mM Tris and 1% SDS, with a pH of 7.5, and freeze-thaw once at ≤-70°C and 37°C. Detect cAMP content and calculate biological activity. The confluence of the cells is controlled between 80% and 90%, and the cell viability of the cell suspension is ≥90%; The detection of cAMP content includes detecting the cAMP content by competitive ELISA, coating a cAMP antibody on an enzyme-labeled plate, adding cell lysate, then adding cAMP labeled with horseradish peroxidase (HRP), developing color and then stopping, and measuring the OD value at 450 nm; The dilution factor corresponding to the concentration of the test or reference substance is used as the X-axis and the corresponding OD value is used as the Y-axis. Fit the four-parameter equation curve and calculate the half-effective dilution factor EC50. The four-parameter equation is as follows: <h2 style=";text-align:left;direction:ltr">y = A2 + (A1 - A2) / (1 + (x / X0)<h2 style=";text-align:left;direction:ltr"> p <h2 style=";text-align:left;direction:ltr"> ) x: dilution factor corresponding to the concentration of the test or reference product; y: OD value; A1: The minimum value of y when x approaches infinity; A2: The maximum value of y when x approaches infinity; p: slope of the four-parameter curve; X0: half-effective dilution multiple EC50; The relative biological activity of the test article = EC50 of the test article / EC50 of the reference article × 100%.

2. Use of the method for detecting the biological activity of recombinant teriparatide according to claim 1 in the quality control of the production process of recombinant teriparatide drugs.

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