Method for detecting biological activity of active factors in drug-containing gel product

Abstract

The invention discloses a biological activity detection method for active factors in a drug-containing gel product, which comprises the following steps: S1, diluting the drug-containing gel product by using a culture solution containing a stabilizer to obtain a gel product solution; the culture solution is prepared from a nonionic surfactant, fetal calf serum and an RPMI-1640 culture medium; s2, oscillating and swelling, so that the active factors are uniformly distributed in the gel product solution; s3, re-diluting the gel product solution with the culture solution; and S4, detecting the biological activity of the active factors in the drug-containing gel product by using a CCK8 method. According to the method, swelling of the drug-containing gel product can be accelerated through dilution and oscillation, so that the active factors are uniformly distributed, dilution and sample loading are facilitated, the surfactant can protect the biological activity of the released active factors, and protein inactivation caused by too long time from dilution, earlier-stage treatment to detection of the drug-containing gel product is avoided. The method is simple to operate, has relatively high sensitivity, repeatability, specificity and accuracy, and is suitable for biological activity detection of various gel products containing active factors.

Description

Technical Field

[0001] The present invention relates to the technical field of active factor detection, and in particular to a method for detecting the biological activity of active factors in a drug-containing gel product. Background Art

[0002] During the drug development process, biological activity assays are crucial evaluation indicators for drug screening and quality control. Method validation must be completed and meet specificity, precision, accuracy, and other validation requirements to ensure the reliability of biological activity assay results and guide drug screening and quality control. Therefore, establishing effective and reliable biological activity assays is crucial for the research and development, quality control, and clinical application of active factors in drug-containing gel products.

[0003] Sodium hyaluronate has a very high viscosity in aqueous solution, and its content is greater than 1% of the aqueous solution in a gel-like state, which can provide mechanical protection for tissues and cell layers, and has thickening, moisturizing, and lubricating effects. In addition, sodium hyaluronate can also be used as a carrier for drugs, bringing peptide growth factors and other structural proteins to the site of action, increasing the retention time of the drug, thereby improving the bioavailability of the drug, and is widely used in products such as wound repair, ophthalmology, orthopedics, gynecology, and beauty. However, drug-containing sodium hyaluronate products have a higher viscosity, and the active factor is wrapped by sodium hyaluronate, and direct dilution and sampling are more difficult. In addition, the active factor has natural instability, which takes a long time to detect biological activity and is easily affected by external conditions (such as temperature, buffer system, ionic strength, etc.), resulting in changes in protein activity, and it is impossible to accurately measure the biological activity of the protein in the gel product.

[0004] Currently, the Pharmacopoeia of the People's Republic of China, Part IV, Section 3528, specifies that the biological activity assay for human epidermal growth factor (hEGF) uses the MTT colorimetric assay. The formazan product produced by MTT reduction is insoluble in water and must be dissolved before testing. This increases the testing workload and can affect the accuracy of experimental results. Furthermore, when using MTT to test gel products containing hEGF, the gel material can also affect the test results, leading to significant deviations. Summary of the Invention

[0005] The purpose of the present invention is to address the above-mentioned shortcomings of existing detection methods and provide a method for detecting the biological activity of active factors in medicated gel products. The method is rapid, simple, stable, accurate, specific and has wide applicability. The method can meet the requirements of specificity, accuracy, precision, linearity and range in the method validation process, and is of great significance to the research and development and quality control of active factors in medicated gel products.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a method for detecting the biological activity of active factors in a drug-containing gel product, the method comprising the following steps:

[0008] S1. diluting the drug-containing gel product with a culture medium containing a stabilizer to obtain a gel product solution;

[0009] Wherein: the culture medium containing the stabilizer is composed of a non-ionic surfactant, fetal bovine serum and RPMI-1640 culture medium;

[0010] S2. Oscillating and swelling the gel product solution to uniformly distribute the active factors in the gel product solution;

[0011] S3, diluting the gel product solution again with the culture solution;

[0012] S4. Use CCK8 method to detect the biological activity of active factors in drug-containing gel products.

[0013] The detection method of the present invention is particularly suitable for detecting the biological activity of human epidermal growth factor (hEGF) in hEGF gel products.

[0014] Specifically, the drug-containing gel product includes a combination of at least one of sodium hyaluronate, chitosan, alginate, and gelatin and active factors; the active factors include proteins, polypeptides, amino acid factors, and related nucleic acid factors.

[0015] Furthermore, a method for detecting the biological activity of active factors in a drug-containing gel product: the culture medium containing a stabilizer in step S1 is composed of the following components in mass fractions:

[0016] 0.005-0.1 wt% of nonionic surfactant, 0.4-4.0 wt% of fetal bovine serum and the balance of RPMI-1640 culture medium.

[0017] Preferably, the culture solution containing the stabilizer is composed of the following components in mass fractions: 0.02-0.08 wt% of a non-ionic surfactant, 1.2-2.0 wt% of fetal bovine serum, and the remainder of RPMI-1640 culture medium.

[0018] Furthermore, a method for detecting the biological activity of active factors in a drug-containing gel product: the non-ionic surfactant is selected from any one of polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 80 (Tween 80), and poloxamer, or a mixture of several of them.

[0019] Furthermore, a method for detecting the biological activity of active factors in a drug-containing gel product: the mass-to-volume ratio of the drug-containing gel product to the culture medium in step S1 is 0.05-1.0 g / mL. Preferably, the mass-to-volume ratio of the drug-containing gel product to the culture medium is 0.1-0.5 g / mL.

[0020] Furthermore, a method for detecting the biological activity of active factors in a drug-containing gel product: in step S2, the oscillation temperature is 10-30°C, the oscillation speed is 100-300 rpm, and the oscillation time is 6-24 hours. Preferably, the oscillation temperature is 20°C, the oscillation speed is 200 rpm, and the oscillation time is 12 hours.

[0021] Furthermore, a method for detecting the biological activity of active factors in a drug-containing gel product: Step S3 is diluted to 40-60 IU / mL.

[0022] Furthermore, a method for detecting the biological activity of active factors in a medicated gel product is provided: this method is suitable for detecting the biological activity of active factors in a medicated gel product using sodium hyaluronate as a gel matrix.

[0023] Specifically, the present invention provides a method for diluting a gel product by adding a culture solution containing a non-ionic surfactant such as Tween 80, using oscillation swelling to uniformly distribute the active factors, and using the CCK8 method to detect its biological activity. In the method of the present invention, dilution and oscillation can accelerate the swelling of the drug-containing gel product to uniformly distribute the active factors, facilitating dilution and loading. Tween 80 can protect the biological activity of the released active factors, avoiding protein inactivation caused by the dilution of the drug-containing gel product, the long pre-processing and detection time. In addition, the biological activity of the active factors in the drug-containing gel product is detected by the CCK8 method. This method is simple to operate, has high sensitivity, repeatability, specificity and accuracy, and is suitable for the biological activity detection of a variety of gel products containing active factors.

[0024] Beneficial effects of the present invention:

[0025] (1) The detection method of the present invention performs sample pretreatment on the drug-containing gel product, and pre-oscillates, dilutes and swells the test gel sample to make the protein evenly distributed, thereby increasing the accuracy of the sample biological activity detection while maintaining the biological activity of the protein.

[0026] (2) The method of the present invention uses a preferred culture medium to dilute the gel product and oscillate to swell it, so that the active factors are evenly distributed. On the one hand, the viscous gel product can be diluted to evenly disperse the protein, thereby increasing the accuracy of dilution and loading. On the other hand, the non-ionic surfactant in the culture medium can protect the active protein in the diluted gel product, thereby avoiding the problem of protein inactivation caused by the dilution of the drug-containing gel product, the early processing and the detection time being too long.

[0027] (3) The present invention addresses the problem of large deviation in the detection results of existing methods for detecting the biological activity of active factors in drug-containing gel products, and provides a rapid, simple, stable, accurate, specific and widely applicable detection method. This method can meet the requirements of specificity, accuracy, precision, linearity and range in the method validation process, and will be of great significance to the research and development and quality control of gel products containing human epidermal growth factor (hEGF). BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 This is a diagram for verifying the specificity of the biological activity detection method of the present invention;

[0030] Figure 2 This is the linear regression equation fitting graph. DETAILED DESCRIPTION

[0031] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is in no way intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] Example 1

[0033] Provided is a method for detecting the biological activity of active factors in a medicated gel product, the method comprising the following specific steps:

[0034] S1. Dilute 1.0 g of the drug-containing gel product with 4.0 mL of a culture medium containing a stabilizer to obtain a gel product solution;

[0035] Wherein: the culture medium containing stabilizer is composed of the following components by mass fraction:

[0036] Tween 80 (non-ionic surfactant) 0.02wt%, fetal bovine serum 1.2wt% and the remainder RPMI-1640 medium;

[0037] S2. Shake and swell in an oscillating incubator at 20°C and 200 rpm for 12 hours to evenly distribute the active factors in the gel product solution;

[0038] S3. Continue to dilute the gel product solution with the above culture medium to contain 50 IU per 1 mL, and then make 4-fold serial dilutions in a 96-well cell culture plate, for a total of 8 dilutions to obtain the test solution; then, the test solution is placed at 4°C for 0, 1, 3, 5, 7, 10, and 10 days, and then immediately tested;

[0039] S4. Use CCK8 assay to detect the biological activity of active factors in drug-containing gel products;

[0040] The specific test is as follows: BALB / c 3T3 cell lines were cultured in complete culture medium at 37°C and 5% carbon dioxide. The culture medium in the culture flask was discarded, the cells were digested and collected, and the complete culture medium was used to prepare a mixture containing 4.0×10 4 The cell suspension of cells was then inoculated into a 96-well cell culture plate, 100 μL per well, and then cultured at 37°C, 5% carbon dioxide for 4 to 24 hours. The maintenance solution was discarded, and the standard solution and the test solution were added, 100 μL per well, and cultured at 37°C, 5% carbon dioxide for 64 to 72 hours; after adding 100 μL of a mixed solution of CCK8 and the culture solution to each well, the plate was placed in a 37°C, 5% CO2 incubator in the dark for incubation for 1.5 hours, and then placed in a microplate reader for measurement at 450 nm as the detection wavelength and 650 nm as the reference wavelength. The test results are shown in Table 1 below:

[0041] The preparation of the standard solution is as follows: take the human epidermal growth factor standard, reconstitute it according to the instructions, dilute it with the above culture medium to contain 50 IU per 1 mL, and then make a 4-fold serial dilution in a 96-well cell culture plate, for a total of 8 dilutions.

[0042] Specifically, polysorbate 80 (Tween 80) used in Example 1 above is a nonionic surfactant that stabilizes proteins primarily through interfacial competition. Tween 80 has a much higher surface activity than proteins and can competitively occupy interfacial positions, inhibiting protein adsorption to the air-liquid interface. This, in turn, reduces oxidation or aggregation caused by contact with air and effectively prevents protein aggregation and denaturation during the manufacturing process, sample handling, and storage. Therefore, by adding Tween 80 to the diluent (culture fluid) to protect the protein activity in the diluted gel product, the problem of protein inactivation caused by prolonged waiting time between dilution and detection can be avoided, thereby improving detection accuracy.

[0043] Example 2

[0044] Provided is a method for detecting the biological activity of active factors in a medicated gel product, the method comprising the following specific steps:

[0045] S1. Dilute 1.0 g of the drug-containing gel product with 10.0 mL of a culture medium containing a stabilizer to obtain a gel product solution;

[0046] Wherein: the culture medium containing stabilizer is composed of the following components by mass fraction:

[0047] Tween 40 (non-ionic surfactant) 0.08wt%, fetal bovine serum 0.6wt% and the remainder RPMI-1640 medium;

[0048] S2. Shake and swell in an oscillating incubator at 10°C and 300 rpm for 24 hours to ensure that the active factors are evenly distributed in the gel product solution;

[0049] S3. Continue to dilute the gel product solution with the above culture medium to contain 40 IU per 1 mL, and then make 4-fold serial dilutions in a 96-well cell culture plate, for a total of 8 dilutions to obtain the test solution; then, the test solution is placed at 4°C for 0, 1, 3, 5, 7, 10, and 10 days, and then immediately tested;

[0050] S4. Use CCK8 method to detect the biological activity of active factors in drug-containing gel products.

[0051] Example 3

[0052] Provided is a method for detecting the biological activity of active factors in a medicated gel product, the method comprising the following specific steps:

[0053] S1. Dilute 1.0 g of the drug-containing gel product with 2.0 mL of culture medium containing a stabilizer to obtain a gel product solution;

[0054] Wherein: the culture medium containing stabilizer is composed of the following components by mass fraction:

[0055] Poloxamer (non-ionic surfactant) 0.1 wt%, fetal bovine serum 3.0 wt% and the remainder RPMI-1640 medium;

[0056] S2. Shake and swell in an oscillating incubator at 30°C and 100 rpm for 8 hours to evenly distribute the active factors in the gel product solution;

[0057] S3. Continue to dilute the gel product solution with the above culture medium to contain 60 IU per 1 mL, and then make 4-fold serial dilutions in a 96-well cell culture plate, for a total of 8 dilutions to obtain the test solution; then, the test solution is placed at 4°C for 0, 1, 3, 5, 7, 10, and 10 days, and then immediately tested;

[0058] S4. Use CCK8 method to detect the biological activity of active factors in drug-containing gel products.

[0059] Comparative Example 1

[0060] The difference between Comparative Example 1 and Example 1 is that the culture medium used in the preparation of the test solution does not contain Tween 80, and the rest is the same as Example 1; the test results of Comparative Example 1 are shown in Table 1 below.

[0061] Comparative Example 2

[0062] The difference between Comparative Example 2 and Example 1 is that the assay method was changed from CCK8 assay to MTT assay, and the specific steps were as follows: 20 μL of MTT solution was added to each well, and the cells were cultured at 37° C. and 5% carbon dioxide for 5 hours. After discarding the liquid in the culture plate, 100 μL of dimethyl sulfoxide was added to each well, mixed, and the absorbance was measured at a wavelength of 570 nm on a microplate reader with a reference wavelength of 630 nm. The measurement results were recorded. The results are shown in Table 1 below.

[0063] Table 1 shows the biological activity results of hEGF under different conditions

[0064]

[0065]

[0066] The test data of Example 1 and Comparative Example 1 above show that gel samples diluted with culture medium containing Tween 80 can effectively prevent the reduction of protein activity. When the number of test samples is large, the addition of Tween 80 can avoid the problem of reduced activity results due to long sample waiting time.

[0067] In addition, it can be seen from the test data of Example 1 and Comparative Example 2 above that the CCK8 detection method adopted in the present invention has good repeatability and small deviation in the test results, and can meet the requirements of specificity, accuracy, precision, etc. in the method validation process. It is a detection method for the biological activity of drug-containing gel products with high sensitivity, good accuracy, high precision and good stability. It can quickly and efficiently detect the activity of active factors in gel products, which will be of great significance to the research and development and quality control of gel products containing active factors.

[0068] The biological activity detection method of hEGF in the hEGF-containing gel product provided by the present invention was verified:

[0069] (1) Specificity verification: The hEGF standard solution of the China National Institute for Food and Drug Control was used as the reference substance, and the hEGF was added to the HA blank matrix solution as the test sample for biological activity detection. The curves of the blank matrix spiked solution and the standard solution were examined to be similar and consistent in potency, thereby evaluating the specificity of the CCK8 method for the biological activity detection of hEGF HA products.

[0070] Acceptance criteria: The proliferation curve of the standard solution conforms to the "S" curve, and the linear R 2 ≥0.95; the curves of the blank matrix spiked solution should be similar and consistent with those of the standard solution, and should be between 70.0% and 160.0% of the labeled value.

[0071] The results are as follows Figure 1 As shown in the figure, the linearity of hEGF standard solution and hEGF sample containing HA blank matrix solution conforms to the "S" curve, and the linear R 2 ≥0.95. Furthermore, the relative potency of the spiked solution calculated based on the hEGF standard solution was 103% of the labeled amount, indicating that the curves obtained for the blank matrix spiked solution should be similar and consistent in potency with those obtained for the standard solution, indicating that the method has good specificity.

[0072] (2) Accuracy verification: Cell viability testing was repeated at activity titer levels of 60%, 80%, 100%, 135%, and 175%. Relative titer determination was performed for each titer level by two analysts on different days using four cell generations. Each analyst performed two independent determinations for each titer level using each cell generation in each test.

[0073] Acceptable criteria: The relative bias of the relative potency determination value of each potency level should be within the range of ±20%, and the quality standard range of the relative potency (recovery rate) of each potency level should be between 70% and 160%.

[0074] The results are shown in Tables 2 and 3. Relative titer determinations were performed by two analysts on different days using four cell passages for each titer level. The recoveries were all within the range of 80% to 130%, the relative bias was within the range of ±20%, and the relative accuracy met the acceptable standards, indicating that the method meets the accuracy verification requirements.

[0075] Table 2 is a summary of the biological activity assay results

[0076]

[0077] Table 3 shows the confidence intervals of relative potency values ​​at different potency levels

[0078]

[0079] (3) Precision verification: Cell viability testing was repeated at activity titer levels of 60%, 80%, 100%, 135%, and 175%. Relative titer determination was performed for each titer level by two analysts on different days using four cell generations. Each analyst performed two independent determinations for each titer level and each cell generation in each test.

[0080] Acceptability criteria: The relative bias of each potency level relative to the potency measurement value should be within the range of ±20%, and the geometric coefficient of variation (GCV, %) of each potency level relative to the potency measurement value should not be greater than 20%. The results are shown in Table 4:

[0081] Table 4 shows the geometric standard deviation, geometric coefficient of variation and upper confidence limit of relative potency determination at different potency levels

[0082] potency level Number of trials GSD <![CDATA[CI GSD ]]> GCV <![CDATA[CI GCV ]]> 60％ 8 1.103 1.018 10.3％ 1.8％ 80％ 8 1.075 1.013 7.5％ 1.3％ 100％ 8 1.119 1.020 11.9％ 2.0％ 135％ 8 1.055 1.010 5.5％ 1.0％ 175％ 8 1.044 1.008 4.4％ 0.8％

[0083] It can be seen from Table 4 that the geometric coefficient of variation (GCV, %) is no more than 12%, which shows that the method of the present invention has high precision.

[0084] (4) Linear validation: Plot the logarithm of the theoretical potency value (abscissa) against the logarithm of the corresponding measured potency value (ordinate), and perform linear regression using the least squares method.

[0085] Acceptable criteria: The correlation coefficient of the linear regression equation should be no less than 0.98, and the slope of the regression equation should be within the range of 0.80 to 1.25. Figure 2 As shown, the fitted linear regression equation is y=0.9114x+0.0378, the slope of the regression equation is 0.9114, and the correlation coefficient is 0.9894.

[0086] The above preferred embodiments of the present invention are only used to explain the present invention and are not used to limit the present invention. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for detecting the biological activity of active factors in a drug-containing gel product, characterized in that: The method comprises the following steps: S1. diluting the drug-containing gel product with a culture medium containing a stabilizer to obtain a gel product solution; Wherein: the culture medium containing the stabilizer is composed of a non-ionic surfactant, fetal bovine serum and RPMI-1640 culture medium; S2. Oscillating and swelling the gel product solution to uniformly distribute the active factors in the gel product solution; S3, diluting the gel product solution again with the culture solution; S4. Use CCK8 method to detect the biological activity of active factors in drug-containing gel products.

2. The method for detecting the biological activity of active factors in a drug-containing gel product according to claim 1, characterized in that: The culture solution containing the stabilizer in step S1 is composed of the following components by mass fraction: 0.005-0.1 wt% of nonionic surfactant, 0.4-4.0 wt% of fetal bovine serum and the balance of RPMI-1640 culture medium.

3. The method for detecting the biological activity of active factors in a drug-containing gel product according to claim 1 or 2, characterized in that: The nonionic surfactant is selected from any one of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 and poloxamer, or a mixture of several of them.

4. The method for detecting the biological activity of active factors in a drug-containing gel product according to claim 1, characterized in that: The mass volume ratio of the drug-containing gel product to the culture medium in step S1 is 0.05 to 1.0 g / mL.

5. The method for detecting the biological activity of active factors in a drug-containing gel product according to claim 1, characterized in that: In step S2, the oscillation temperature is 10-30° C., the oscillation speed is 100-300 rpm, and the oscillation time is 6-24 hours.

6. The method for detecting the biological activity of active factors in a drug-containing gel product according to claim 1, characterized in that: Step S3: dilute to 40-60 IU / mL.

7. The method for detecting biological activity of active factors in a drug-containing gel product according to claim 1, characterized in that: This detection method is suitable for the biological activity detection of active factors in medicated gel products with sodium hyaluronate as the gel matrix.

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