Method for determining biological activity of recombinant human endostatin

Abstract

The embodiment of the invention relates to a method for determining the biological activity of recombinant human endostatin. The method comprises the following steps: carrying out mixed culture on a subculture cell which is subjected to L929 cell culture subculture and is used as an effector cell and recombinant human endostatin to be detected; cracking the cells subjected to mixed culture to obtain a lysis solution; the method comprises the following steps of: preparing a lysate, measuring the absorbance of the lysate, performing mixed culture on the basis of a measured result, a human endostatin standard substance and effector cells, and obtaining the biological activity of the recombinant human endostatin to be measured by utilizing a relative biological activity method. The biological activity of the recombinant human endostatin to be detected can be obtained, and the detection result is reliable.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular, to a method for determining the biological activity of recombinant human endostatin. Background Art

[0002] Recombinant human endostatin is China's first anti-angiogenic targeted drug, used to treat patients with stage III / IV non-small cell lung cancer who are receiving initial or retreatment treatment, and has shown good efficacy. Biological activity is one of the key quality attributes for evaluating the effectiveness of such biotechnology drugs and is an important indicator for preclinical quality control of drugs. Cell assays can generally better reflect the biological activity of biological products and are often used to determine the activity of various biological products.

[0003] The existing method for determining the biological activity of recombinant human endostatin is the HUVEC cell proliferation inhibition method, which uses primary HUVEC cells as effector cells and limits the cell detection generation to less than 5 generations. However, compared with chemical methods, the biological activity determination method has the disadvantage of large variation due to its dependence on biological factors; and compared with the use of passage cells, the method using primary cells has greater variation. The current method for determining the biological activity of recombinant human endostatin has poor stability of the test results. Summary of the invention

[0004] The present application is provided to solve the above-mentioned defects existing in the prior art. A method for determining the biological activity of recombinant human endostatin is needed, which can obtain the biological activity of the recombinant human endostatin to be tested by using the relative biological activity method based on the inhibitory effect of the specific S-shaped curve, with small variation and stable test results.

[0005] In a first aspect of the present application, a method for determining the biological activity of recombinant human endostatin is provided, the method comprising: using L929 cell culture passaged cells as effector cells for mixed culture with the recombinant human endostatin to be tested; lysing the cells after the mixed culture to obtain a lysate; measuring the absorbance of the lysate, and obtaining the biological activity of the recombinant human endostatin to be tested by using a relative biological activity method based on the result of the measurement and the absorbance measurement result of the mixed culture of a human endostatin standard and the effector cells.

[0006] The methods for determining the biological activity of recombinant human endostatin provided in various embodiments of the present application can inhibit the proliferation of L929 cell culture-transferred cells by using recombinant human endostatin, and have exclusive inhibitory properties on L929 cell culture-transferred cells, and the inhibitory concentration-absorbance curve is an S-shaped curve, which can obtain the biological activity of the recombinant human endostatin to be tested, has the advantage of small variation, and the detection results are stable and reliable, and have good practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In the drawings, which are not necessarily drawn to scale, the same reference numerals may describe similar parts in different views. The same reference numerals with letter suffixes or different letter suffixes may represent different instances of similar parts. The accompanying drawings generally illustrate various embodiments by way of example and not limitation, and together with the specification and claims, are used to illustrate the embodiments applied for. When appropriate, the same reference numerals are used throughout the drawings to refer to the same or similar parts. Such embodiments are illustrative and are not intended to be exhaustive or exclusive embodiments of the present apparatus or method.

[0008] Figure 1 The inhibition curve of the recombinant human endostatin on L929 cells in Experimental Example 1 of the present application is shown; Figure 2 A linear curve showing the potency measurement value and the potency theoretical value of Experimental Example 2 of the present application; Figure 3 The dose-effect curve to be tested and the dose-effect curve of the standard product of Comparative Example 1 of the present application are shown. DETAILED DESCRIPTION

[0009] In order to enable those skilled in the art to better understand the technical solution of the present application, the present application is described in detail below in conjunction with the accompanying drawings and specific implementation examples. The embodiments of the present application are further described in detail below in conjunction with the accompanying drawings and specific implementation examples, but are not intended to limit the present application.

[0010] The words "first", "second" and similar words used in this application do not indicate any order, quantity or importance, but are only used to distinguish. The words "include" or "comprise" and similar words mean that the elements before the word include the elements listed after the word, and do not exclude the possibility of including other elements.

[0011] According to an embodiment of the present application, a method for determining the biological activity of recombinant human endostatin is provided, the method comprising: using L929 cell culture passaged cells as effector cells for mixed culture with the recombinant human endostatin to be tested; lysing the cells after the mixed culture to obtain a lysate; measuring the absorbance of the lysate, and obtaining the biological activity of the recombinant human endostatin to be tested by using a relative biological activity method based on the result of the measurement and the absorbance measurement result of the mixed culture of a human endostatin standard and the effector cells.

[0012] By studying the effect of recombinant human endostatin on effector cells after L929 cell culture, it was unexpectedly found that it can inhibit the proliferation of effector cells, and the reaction curve is similar to the inhibitory S-shaped curve. Therefore, using L929 cell culture and passage cells as effector cells to determine the biological activity of recombinant human endostatin has the advantages of accuracy and small variation. The test results are stable and reliable, simple to operate, short test cycle, high accuracy, good repeatability, and can be used for standard calibration, product quality control and other inspections.

[0013] Due to the inhibitory effect of recombinant human endostatin, the absorbance of the cultured L929 cells after lysis is different. Based on the absorbance measurement results of the recombinant human endostatin to be tested and the absorbance measurement results of the standard, the relative biological activity of the recombinant human endostatin to be tested can be calculated using the formula of relative biological activity, that is, the biological activity of the recombinant human endostatin to be tested. The above method relies on the specificity between L929 cells and the recombinant human endostatin to be tested, which can solve the complex variability brought by the biological activity determination and reduce the variability. The variability obtained after multiple measurements is within 10%, and for different concentrations of the recombinant human endostatin solutions to be tested, it can have a small variability. The operation steps are simple and quick, and the biological activity of the recombinant human endostatin to be tested is relatively stable, that is, the results obtained by multiple measurements meet the deviation range, and the biological activity is close to the theoretical value.

[0014] In some embodiments, L929 cells are cultured and subcultured using DMEM medium containing 8-12% FBS (fetal bovine serum) and 0.8-1.2% PS (penicillin and streptomycin); the culture and subculture temperature is 30-40° C.; the culture and subculture are performed in an incubator, and the mass fraction of carbon dioxide in the incubator is 4-6%.

[0015] In some embodiments, L929 healthy cells obtained after culture and passage are obtained, washed, digested with trypsin, and after terminating the digestion, the supernatant is discarded to obtain treated L929 cells; the treated L929 cells are suspended with a culture medium of DMEM culture medium to obtain an L929 cell suspension. In some embodiments, PBS (phosphate buffered saline) is used for washing. In some embodiments, L929 healthy cells obtained after culture and passage are digested with a 0.25% mass fraction of trypsin solution. In order to enable recombinant human endostatin to better exert its inhibitory effect on the cultured passaged L929 cells, serum-containing DMEM culture medium can be used to terminate the digestion. The solvent of the trypsin solution can generally be PBS.

[0016] In some embodiments, the effector cells are mixed with the recombinant human endostatin to be tested, and the inoculation density of the effector cells is 5000-15000 cells / culture unit. In some embodiments, the L929 cell suspension is inoculated with a cell density of (0.5-1.5)*10 5 For a 96-well plate, the culture unit can be one well, and 50 μl of L929 cell suspension can be inoculated into each well.

[0017] In some embodiments, the effector cells are mixed and cultured with the recombinant human endostatin to be tested, further comprising: obtaining recombinant human endostatin solutions of different concentrations prepared by the recombinant human endostatin to be tested; the recombinant human endostatin solutions of different concentrations to be tested are cultured with the effector cells respectively to obtain the cultured products corresponding to the recombinant human endostatin solutions of different concentrations to be tested. The recombinant human endostatin solutions of different concentrations are mixed and cultured with the L929 cell suspension of the same concentration respectively to obtain the inhibitory effect of the recombinant human endostatin solutions of different concentrations on L929 cells.

[0018] The recombinant human endostatin to be tested is first mixed with the culture fluid of the DMEM culture medium, and then diluted to obtain recombinant human endostatin solutions of different concentrations.

[0019] In some embodiments, the inoculation volume ratio of the L929 cell suspension to the recombinant human endostatin solution to be tested is 0.8-1.2: 1. For a 96-well plate, the culture unit may be one well, and 50 μl of the recombinant human endostatin solution to be tested may be inoculated into each well.

[0020] In some embodiments, the effector cells and the recombinant human endostatin to be tested are mixed and cultured for 48 to 72 hours. In some embodiments, the culture is carried out in an incubator to provide conditions for the mixed culture of the effector cells and the recombinant human endostatin to be tested.

[0021] In some embodiments, the cells after mixed culture are lysed to obtain cell lysate, including: adding a staining solution to the culture corresponding to the recombinant human endostatin solutions to be tested at different concentrations, and incubating after adding the staining solution; after staining and incubation, adding a lysing agent, lysing, and obtaining lysates corresponding to the recombinant human endostatin solutions to be tested at different concentrations. In some embodiments, the volume ratio of the staining solution to the volume of the recombinant human endostatin solution to be tested is 1-3:5; the concentration of the staining solution is 4-6 mg / ml. In some embodiments, the incubation time after staining is 4-6 hours.

[0022] In some embodiments, after the staining and incubation, the supernatant is removed, and then the solid is washed, and then the solid is mixed with a lysing agent for lysing. In some embodiments, the lysing agent is a DMSO (dimethyl sulfoxide) solution. In some embodiments, the volume ratio of the lysing agent to the volume of the recombinant human endostatin solution is 1.5-2.5:1; the DMSO in the lysing agent is analytical grade (stock solution).

[0023] In some embodiments, the absorbance of the lysate is measured, and the biological activity of the recombinant human endostatin to be tested is obtained based on the result of the measurement using a relative biological activity method, including: measuring the absorbance of the lysates corresponding to different concentrations of the recombinant human endostatin solution to be tested to obtain an absorbance-concentration curve; performing four-parameter curve fitting based on the absorbance-concentration curve to obtain a dose-effect curve of the recombinant human endostatin to be tested; and obtaining the biological activity of the recombinant human endostatin to be tested using a relative biological activity method based on the dose-effect curve of the recombinant human endostatin solution to be tested and the dose-effect curve corresponding to the standard.

[0024] In some embodiments, the relative biological activity is calculated by formula (1).

[0025] Relative biological activity (IU / ml) = (1) Where Pr is the biological activity of the standard, IU / ml Ds is the pre-dilution multiple of the recombinant human endostatin solution to be tested; Dr is the pre-dilution multiple of the standard; Es is the dilution multiple of the recombinant human endostatin solution to be tested equivalent to the half-effective amount of the standard; Er is the dilution multiple of the half-effective dose of the standard.

[0026] First, the concentration of the recombinant human endostatin standard corresponding to the half-effective amount of the standard can be obtained through the dose-effect curve of the standard, and then the dilution factor Er can be obtained; then, based on the dose-effect curve to be tested, the concentration of the recombinant human endostatin to be tested corresponding to the half-effective amount of the standard can be obtained, and then the dilution factor Es can be obtained. The pre-dilution factor of the recombinant human endostatin solution to be tested and the pre-dilution factor of the standard can be obtained through the solution configuration process during the determination process. Taking the pre-dilution factor of the recombinant human endostatin solution to be tested as 2 and the pre-dilution factor of the standard as 2 as an example, the relative biological activity of the recombinant human endostatin to be tested can be obtained.

[0027] The absorbance measurement result after the human endostatin standard product is mixed and cultured with the effector cells includes a dose-effect curve corresponding to the standard product. In some embodiments, the dose-effect curve corresponding to the standard product is obtained by the following steps: obtaining recombinant human endostatin standard product solutions of different concentrations; culturing recombinant human endostatin standard product solutions of different concentrations with effector cells respectively to obtain cultures corresponding to different concentrations; lysing the cells after the mixed culture to obtain a lysate; measuring the absorbance of the lysate to obtain an absorbance-concentration curve corresponding to the standard product; performing four-parameter curve fitting based on the absorbance-concentration curve corresponding to the standard product to obtain a dose-effect curve of the recombinant human endostatin standard product.

[0028] Recombinant human endostatin standard solutions of different concentrations were mixed and cultured with L929 cell suspensions of the same concentration to obtain the inhibitory effects of recombinant human endostatin standard solutions of different concentrations on L929 cells.

[0029] In some embodiments, the cells after mixed culture are lysed to obtain cell lysate, including: adding a staining solution to the culture corresponding to the recombinant human endostatin standard solution of different concentrations, and incubating after adding the staining solution; after staining and incubation, adding a lysing agent for lysis to obtain lysate corresponding to the recombinant human endostatin standard solution of different concentrations. In some embodiments, the volume ratio of the staining solution to the volume of the recombinant human endostatin standard solution is 1-3:5; the concentration of the staining solution is 4-6 mg / ml. In some embodiments, the incubation time after staining is 4-6 hours.

[0030] In some embodiments, after the staining and incubation, the supernatant is removed, and then the solid is washed, and then the solid is mixed with a lysing agent for lysing. In some embodiments, the lysing agent is a DMSO (dimethyl sulfoxide) solution. In some embodiments, the volume ratio of the lysing agent to the volume of the recombinant human endostatin solution is 1.5-2.5:1; the DMSO in the lysing agent is analytical grade (stock solution).

[0031] In some embodiments, the number of different concentrations of the recombinant human endostatin to be tested is 8-10, so as to accurately obtain the inhibition curve of the recombinant human endostatin on L929 cells.

[0032] Experimental Example 1 The specificity experiment of L929 cells for recombinant human endostatin includes the following steps: (1) L929 cell culture: Purchased L929 cells were cultured and passaged in DMEM (dulbecco's modified eagle medium) containing 10% FBS (fetal bovine serum) and 1% PS (penicillin-streptomycin) in a 37°C, 5% carbon dioxide incubator. (2) Take healthy L929 cells in the logarithmic growth phase after subculturing, discard the culture medium, wash 2-3 times with PBS, add 0.02% mass fraction EDTA (ethylenediaminetetraacetic acid) solution and 0.25% mass fraction trypsin solution to the obtained L929 healthy cells for digestion, add serum-containing culture medium to terminate digestion, blow until the cells fall off, centrifuge, discard the supernatant, resuspend with the above culture medium until the cell suspension is uniform, count the cell density, and adjust the cell density to 1*10 with complete culture medium. 5 cells / ml, 50 μl / well (i.e., the number of cells inoculated is 5000 / well); the solvent of EDTA solution and trypsin solution can be PBS.

[0033] (3) Take the recombinant human endostatin lyophilized powder, reconstitute it with DMEM culture medium (DMEM culture medium containing 10% FBS and 1% PS) to prepare a 160,000 IU / ml solution, perform a 2-fold serial dilution to prepare 8 concentrations, add 50 μl / well of the 96-well plate that has been seeded with cells, and replicate for 2 wells for each concentration; (4) Incubate the 96-well plate in a carbon dioxide incubator (37°C, 5% carbon dioxide) for 72 h.

[0034] (5) Remove the culture plate, add 20 μl / well of 5.0 mg / ml MTT (thiazolyl blue) solution, and continue incubation for 5 h.

[0035] (6) Remove the culture plate, gently aspirate the supernatant in each well, wash once with PBS buffer in the same way, discard the washing solution, add 100 μl of DMSO (dimethyl sulfone) solution to each well for lysis, and mix well.

[0036] (7) Use an ELISA reader to measure the absorbance of each well at 490 nm and record the absorbance value. Use the logarithmic value of the concentration of recombinant human endostatin as the horizontal axis and the absorbance value as the vertical axis to obtain the following: Figure 1The results shown by Figure 1 The inhibitory S-shaped curve of recombinant human endostatin on L929 cells can be obtained.

[0037] The same type of drug bevacizumab was tested, and bevacizumab replaced recombinant human endostatin. The process was performed according to steps (1) to (7), and DMEM culture medium containing 10% FBS and 1% PS was selected as a blank control. The results are as follows Figure 1 As shown, through Figure 1 It can be obtained that L929 cells have good specificity for recombinant human endostatin. Figure 1 The vertical axis ABS in the graph represents absorbance.

[0038] Experimental Example 2 Result verification includes the following steps: (1) L929 cell culture: Purchased L929 cells were cultured and passaged in DMEM containing 10% FBS and 1% PS in an incubator at 37°C and 5% CO2. (2) Take the healthy L929 cells after subculturing, discard the culture medium, wash 2-3 times with PBS, digest with trypsin containing EDTA, add 3 times the amount of culture medium containing serum to terminate the digestion, centrifuge, discard the supernatant, resuspend with the above culture medium, count the cell density, and adjust the cell suspension to a uniform cell density of 1*10 5 cells / ml, 50 μl / well (i.e., the number of cells inoculated is 5000 / well); (3) Take human endostatin, reconstitute it with DMEM culture medium (DMEM culture medium containing 10% FBS and 1% PS) to a solution of 160,000 IU / ml, perform a 2-fold serial dilution to make 8 concentrations, add 50 μl / well of a 96-well plate that has been seeded with cells, and duplicate wells for each concentration; this is used as the standard series.

[0039] (4) Recombinant human endostatin was reconstituted in DMEM culture medium (DMEM culture medium containing 10% FBS and 1% PS) into five test solutions with different titers of 320,000 IU / ml, 240,000 IU / ml, 160,000 IU / ml, 120,000 IU / ml and 80,000 IU / ml, respectively. The above solutions were used as the initial concentrations for two-fold serial dilutions to prepare eight concentrations, which were added to a 96-well plate seeded with cells at 50 μl / well, with two replicates for each concentration; these were used as five test solution series with relative titers of 200%, 150%, 100%, 75% and 50%.

[0040] (5) Incubate the 96-well plate in a carbon dioxide incubator (37°C, 5% carbon dioxide) for 72 h.

[0041] (6) Remove the culture plate, add 20 μl / well of 5.0 mg / ml MTT solution, and continue incubation for 5 h.

[0042] (7) Remove the culture plate, gently aspirate the supernatant in each well, wash 1-2 times with PBS buffer in the same way, discard the washing solution, add 100 μl of DMSO solution to each well for lysis, and mix well.

[0043] (8) Use an ELISA reader to measure the absorbance of each well at 490 nm and record the absorbance value. Use the logarithmic value of the recombinant human endostatin concentration as the abscissa and the absorbance value as the ordinate to perform four-parameter curve fitting to obtain the dose-effect curve of the test sample and the dose-effect curve of the standard sample.

[0044] The above test steps are performed by different operators at different times using the same method, and the relative biological activities of the test solutions obtained are statistically analyzed. The reliability of the method is verified in terms of linearity, relative accuracy and intermediate precision in accordance with the requirements of the Chinese Pharmacopoeia.

[0045] According to the results obtained by testing the analytes at 5 different titer levels according to this method, linear fitting was performed, and the linear results obtained were as follows: Figure 2 As shown, Figure 2 Each coordinate point in the graph corresponds to a concentration of the recombinant human endostatin to be tested. The logarithm of the theoretical potency value (theoretical potency of the recombinant human endostatin) is used as the abscissa, and the logarithm of the measured potency value (measured potency of the recombinant human endostatin to be tested) is used as the ordinate to plot the graph. The least squares method is used for linear regression. The results are as follows: Figure 2 The fitted linear regression equation is y=0.9743x+0.9857, and the correlation coefficient R 2 =0.982 (>0.98); meets the requirements. The slope of the regression equation is 0.9473, which is within the range of 0.80~1.25 and meets the requirements.

[0046] The relative accuracy is shown in Appendix 1, from which we can obtain ① the relative bias of the relative potency measurement values ​​of each potency level is within the range of ±12%, which is in line with the regulations.

[0047] Appendix 1. Relative bias of relative potency values ​​at each potency level The results of intermediate precision are shown in Appendix 2, from which we can obtain the geometric coefficient of variation (GCV, %) of the relative potency measurement values ​​of each potency level, which are all less than 20%, which is in compliance with the regulations.

[0048] Appendix 2. Geometric coefficient of variation of relative potency values ​​at each potency level The repeatability is shown in Appendix 3. From Appendix 3, we can see that taking the relative potency measurement values ​​of potency levels of 100% and 150% as an example, the variability of the measurement results is within 10%, and the repeatability is good.

[0049] Appendix 3 Variation of results of repeated measurements Example 1 A method for determining the biological activity of recombinant human endostatin, comprising the following steps: (1) L929 cell culture: Purchased L929 cells were cultured and passaged in DMEM containing 10% FBS and 1% PS in an incubator at 37°C and 5% CO2. (2) Take the healthy L929 cells after subculturing, discard the culture medium, wash 2-3 times with PBS, digest with trypsin containing EDTA, add 3 times the amount of culture medium containing serum to terminate the digestion, centrifuge, discard the supernatant, resuspend with the above culture medium, count the cell density, and adjust the cell suspension to a uniform cell density of 1*10 5 cells / ml, 50 μl / well (i.e., the number of cells inoculated is 5000 / well); (3) Take human endostatin standard and use DMEM culture medium (DMEM culture medium containing 10% FBS and 1% PS) to reconstitute it into a solution of 80,000 IU / ml. Perform a 2-fold serial dilution to make 8 concentrations. Add 50 μl / well of a 96-well plate that has been seeded with cells, and duplicate wells for each concentration; this is used as the standard series.

[0050] (4) Take the human endostatin test product and dilute it 2-fold into 8 concentrations in DMEM culture medium (DMEM culture medium containing 10% FBS and 1% PS). Add 50 μl / well to a 96-well plate that has been seeded with cells, and replicate for each concentration in duplicate.

[0051] (5) Incubate the 96-well plate in a carbon dioxide incubator (37°C, 5% carbon dioxide) for 72 h.

[0052] (6) Remove the culture plate, add 20 μl / well of 5.0 mg / ml MTT solution, and continue incubation for 5 h.

[0053] (7) Remove the culture plate, gently aspirate the supernatant in each well, wash 1-2 times with PBS buffer in the same way, discard the washing solution, add 100 μl of DMSO solution to each well for lysis, and mix well.

[0054] (8) Use an ELISA reader to measure the absorbance of each well at 490 nm and record the absorbance value. Use the logarithmic value of the recombinant human endostatin concentration as the abscissa and the absorbance value as the ordinate to perform four-parameter curve fitting to obtain the dose-effect curve of the test sample and the dose-effect curve of the standard sample.

[0055] (9) Calculation of relative biological activity: Based on the dose-effect curve of the test drug and the dose-effect curve of the standard, a computer program or a four-parameter regression method is used to process and calculate the relative biological activity of the test drug according to the following formula: Relative biological activity (IU / ml) = Where Pr is the biological activity of the standard, IU / ml Ds is the pre-dilution multiple of the test product; Dr is the pre-dilution multiple of the standard; Es is the dilution multiple of the test sample equivalent to the half-effective dose of the standard sample; Er is the dilution multiple of the half-effective dose of the standard.

[0056] The test results are shown in Table 4. The theoretical value of the sample to be tested in this embodiment is 80,000 IU / ml.

[0057] Appendix 4 Relative biological activity of the samples to be tested Therefore, the method for determining the biological activity of recombinant human endostatin of the present application has good accuracy.

[0058] Comparative Example 1 (1) The purchased L929 cells were suspended in DMEM medium containing 10% FBS and 1% PS, inoculated in a 96-well plate, and the cell density was adjusted to 1*10 5 cells / ml, 50 μl / well (i.e., the number of cells inoculated is 5000 / well); (2) Take human endostatin standard, reconstitute it in DMEM culture medium to a solution of 80,000 IU / ml, perform 2-fold serial dilution to make 8 concentrations, add 50 μl / well of a 96-well plate that has been seeded with cells, and duplicate wells for each concentration; this is used as the standard series.

[0059] (3) Take the human endostatin test product, dilute it 2-fold into 8 concentrations in DMEM culture medium, add 50 μl / well of a 96-well plate that has been seeded with L929 cells, and replicate for each concentration in duplicate.

[0060] (4) Incubate the 96-well plate in a carbon dioxide incubator (37°C, 5% carbon dioxide) for 72 h.

[0061] (5) Remove the culture plate, add 20 μl / well of 5.0 mg / ml MTT solution, and continue incubation for 5 h.

[0062] (6) Remove the culture plate, gently aspirate the supernatant in each well, wash 1-2 times with PBS buffer in the same way, discard the washing solution, add 100 μl of DMSO solution to each well for lysis, and mix well.

[0063] (7) Use an ELISA reader to measure the absorbance of each well at 490 nm and record the absorbance value. Use the logarithmic value of the recombinant human endostatin concentration as the abscissa and the absorbance value as the ordinate to perform four-parameter curve fitting to obtain the dose-effect curve of the test sample and the dose-effect curve of the standard sample.

[0064] Get as Figure 3 The results shown, Figure 3 The vertical axis ABS represents absorbance, and the horizontal axis represents concentration. Figure 3 It can be seen that the trend lines of the standard and the test products are roughly consistent, and the degree of cell inhibition is roughly positively correlated with the concentration. Attempting to perform statistics through four-parameter fitting found that there is a large deviation between each point and the four-parameter fitting curve, so the cell state is not suitable for biological activity determination, and the cells need to be subcultured.

[0065] The L929 cells in Experimental Example 1-Experimental Example 2, Example 1 and Comparative Example 1 were purchased from Wuhan Punosai Life Science Technology Co., Ltd. They were cryopreserved tube cells with cell product number CL-0137, release date 2024.03.12, cell density>75%, cell viability>95%, and the conclusion of the L929 cell STR identification report was that there was no human cell contamination.

[0066] The DMEM culture medium in Experimental Examples 1-2, Example 1 and Comparative Example 1 was purchased from Cytiva.

[0067] In addition, although exemplary embodiments have been described herein, the scope includes any and all embodiments based on the present application with equivalent elements, modifications, omissions, combinations (e.g., various embodiments intersecting schemes), adaptations or changes. The elements in the claims are to be interpreted broadly based on the language adopted in the claims, and are not limited to the examples described in this specification or during the practice of this application, and the examples are to be interpreted as non-exclusive. Therefore, this specification and examples are intended to be considered as examples only, and the true scope and spirit are indicated by the following claims and the full scope of their equivalents.

[0068] The above description is intended to be illustrative rather than restrictive. For example, the above examples (or one or more schemes thereof) may be used in combination with each other. For example, a person of ordinary skill in the art may use other embodiments when reading the above description. In addition, in the above-mentioned specific embodiments, various features may be grouped together to simplify the present application. This should not be interpreted as an intention that a feature of an application that does not require protection is necessary for any claim. On the contrary, the subject matter of the present application may be less than all the features of an embodiment of a particular application. Thus, the following claims are incorporated herein in the specific embodiments as examples or embodiments, wherein each claim is independently a separate embodiment, and it is considered that these embodiments may be combined with each other in various combinations or arrangements. The scope of the present invention should be determined with reference to the appended claims and the full scope of equivalent forms granted by these claims.

[0069] The above embodiments are only exemplary embodiments of the present application and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. A method for determining the biological activity of recombinant human endostatin, It is characterized in that The method comprises: using L929 cell culture passaged cells as effector cells for mixed culture with recombinant human endostatin to be tested; lysing the mixed cultured cells to obtain a lysate; measuring the absorbance of the lysate, and obtaining the biological activity of the recombinant human endostatin to be tested by using a relative biological activity method based on the measurement result and the absorbance measurement result of a human endostatin standard product after mixed culture with the effector cells.

2. The method according to claim 1, It is characterized in that When the effector cells are mixed with the recombinant human endostatin to be tested, the inoculation density of the effector cells is 5000-15000 per culture unit.

3. The method according to claim 1, It is characterized in that The effector cells are mixed with the recombinant human endostatin to be tested, and the incubation time is 48 to 72 hours.

4. The method according to claim 1, It is characterized in that L929 cells were cultured and subcultured using DMEM culture medium containing 8-12% FBS and 0.8-1.2% PS; the culture and subculture temperature was 30-40° C.; the culture and subculture were carried out in an incubator, and the mass fraction of carbon dioxide in the incubator was 4-6%.

5. The method according to claim 1, It is characterized in that The effector cells are mixed and cultured with the recombinant human endostatin to be tested, and the method also includes: obtaining recombinant human endostatin solutions of different concentrations prepared with the recombinant human endostatin to be tested; and culturing the recombinant human endostatin solutions of different concentrations with the effector cells respectively to obtain cultured products corresponding to the recombinant human endostatin solutions of different concentrations.

6. The method according to claim 5, It is characterized in that The mixed cultured cells are lysed to obtain cell lysate, comprising: adding a staining solution to the cultured cells corresponding to the recombinant human endostatin solutions to be tested at different concentrations, and incubating after adding the staining solution; after the staining and incubation, adding a lysing agent to perform lysing to obtain lysate corresponding to the recombinant human endostatin solutions to be tested at different concentrations.

7. The method according to claim 6, It is characterized in that After adding the staining solution, the incubation time is 4-6 hours.

8. The method according to claim 5, It is characterized in that The absorbance of the lysate is measured, and the biological activity of the recombinant human endostatin to be tested is obtained by using a relative biological activity method based on the measured result, including: measuring the absorbance of the lysates corresponding to the recombinant human endostatin solutions of different concentrations to obtain an absorbance-concentration curve; performing four-parameter curve fitting based on the absorbance-concentration curve to obtain a dose-effect curve of the recombinant human endostatin to be tested; and obtaining the biological activity of the recombinant human endostatin to be tested by using a relative biological activity method based on the dose-effect curve of the recombinant human endostatin to be tested and the dose-effect curve corresponding to the standard.

9. The method according to claim 8, It is characterized in that Based on the dose-effect curve of the recombinant human endostatin to be tested and the dose-effect curve corresponding to the standard, the biological activity of the recombinant human endostatin to be tested is obtained by using the relative biological activity method, including: calculating the relative biological activity by formula (1): Relative biological activity (IU / ml) = (1) Where Pr is the biological activity of the standard, IU / ml Ds is the pre-dilution multiple of the recombinant human endostatin to be tested; Dr is the pre-dilution multiple of the standard; Es is the dilution multiple of the recombinant human endostatin to be tested equivalent to the half-effective dose of the standard; Er is the dilution multiple of the half-effective dose of the standard.

10. The method according to claim 5, It is characterized in that The number of the recombinant human endostatin solutions with different concentrations prepared by the recombinant human endostatin to be tested is 8-10.

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