Method for detecting content of inactivator C11-15 secondary alcohol polyoxyethylene ether
Through the RP-UPLC-ELSD method and gradient elution technology, combined with XBridge BEH C8 chromatography column, the problem of 13-S9 residue detection in biopharmaceuticals was solved, and fast and accurate detection was achieved, ensuring the safety and effectiveness of the drug. sex.
Patent Information
- Application Number
- CN202411637214.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-15
- Publication Date
- 2025-02-18
AI Technical Summary
There is a lack of fast, accurate and stable detection methods in the prior art to detect the residual amount of 13-S9 inactivated agent in the biopharmaceutical production process, resulting in the impact of the safety and effectiveness of the drug.
The detection was performed by RP-UPLC-ELSD method. Water and methanol were used as mobile phases through gradient elution, and XBridge BEH C8 chromatography column was selected to achieve accurate measurement of the residual amount of 13-S9.
This method can quickly, accurately and linearly detect the residual amount of 13-S9 in the recombinant protein vaccine stock solution, with good specificity and no interference from other components, and meet the detection needs of biopharmaceutical production.
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Figure CN119470707A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biopharmaceutical analysis, and particularly relates to a method for detecting the content of C11-15 secondary alcohol polyoxyethylene ether, an inactivator. Background Art
[0002] 13-S9, also known as C11-15 secondary alcohol polyoxyethylene ether, is a non-ionic surfactant. 13-S9 can be used as an inactivator for cell lysis. It can effectively destroy the cell membrane and release the internal components of the cell. Therefore, it is often used in biological experiments and biopharmaceutical production processes to extract proteins or other biological molecules. However, the residue of the inactivator may have a significant impact on the safety and effectiveness of the drug. If the inactivator is not completely removed, harmful substances may appear in the drug, causing adverse reactions. In addition, the residue of the inactivator may also interfere with the stability and biological activity of the drug and reduce the therapeutic effect. In order to ensure the safety of the drug, pharmaceutical companies must follow strict cleaning validation procedures to ensure that the inactivator can be effectively removed after production.
[0003] Triton before 2021 TM X-100 is the main inactivator used in the biomanufacturing process. Due to the harm of its degradation product 4-tert-octylphenol to aquatic ecology, the European Chemicals Agency banned its production and use in Europe in 2021. 13-S9 as Triton TM A safe alternative to X-100 that can effectively meet a variety of process requirements such as virus inactivation and cell lysis, thereby ensuring virus safety while reducing environmental impact. The permissible level of 13-S9 has not been clearly defined, but can be found in Triton TM X-100 must be below the limit of 25 μg / mL.
[0004] at present There are few studies on the detection of 13-S9 residues, and the detection method is not mature. The 13-S9 standard itself is a mixture. In liquid chromatography, the overall content of the reaction substance is often quantitatively detected by adjusting the chromatographic conditions to merge multiple chromatographic peaks of multiple components into a single chromatographic peak. However, various excipients and stabilizers (such as Tween 80) often added during the production of biological products will interfere with the detection. At present, no relevant patents have been found for the content of 13-S9 in biological products. The inventors of the present invention have used chromatographic columns with different matrices to carry out liquid phase method development experiments, but none of them can meet the detection requirements. Therefore, a rapid, accurate and stable detection method is urgently needed. 13-S9 Residue Method. Summary of the invention
[0005] In order to solve at least one of the above problems existing in the prior art, the present invention provides a A method for detecting 13-S9 content, the method comprising the operation of detecting the sample to be tested by RP-UPLC-ELSD method; the mobile phase used in the RP-UPLC-ELSD method comprises mobile phase A and mobile phase B; wherein mobile phase A is water and mobile phase B is methanol; the method uses the mobile phase for gradient elution.
[0006] The present invention provides The detection method of 13-S9 content can be used to detect the content of recombinant protein vaccine stock solution. The detection method is fast, accurate, linear, and specific, and is not interfered by other components in the recombinant protein vaccine stock solution. The detection of 13-S9 residues has important guiding significance. BRIEF DESCRIPTION OF THE DRAWINGS
[0007] Figure 1 In Example 1 of the present invention, Oasis MAX (2.1×20mm, 30μm, ) chromatogram obtained when the chromatographic column is used.
[0008] Figure 2 This is the chromatogram obtained when using an Acclaim Surfactant Plus (4.6*150mm, 3μm) chromatographic column in Example 1 of the present invention.
[0009] Figure 3 This is the chromatogram obtained when the XBridge C18 chromatographic column is used in Example 1 of the present invention.
[0010] Figure 4 This is the chromatogram obtained when the XBridge BEH C8 chromatographic column is used in Example 1 of the present invention.
[0011] Figure 5 In Example 2 of the present invention, the test solution 1, the test solution 2, the spiked sample solution, the blank solution, Chromatogram of 13-S9 standard solution detected by RP-UPLC-ELSD.
[0012] Figure 6 In Example 2 of the present invention, The standard curve of 6 standard solutions of 13-S9 was drawn after RP-UPLC-ELSD detection.
[0013] Figure 7 This is a chromatogram of the spiked sample solution in Example 2 of the present invention.
[0014] Figure 8 1 is a chromatogram of six repeated samples in Example 2 of the present invention (the chromatograms of the six repeated samples almost completely overlap). DETAILED DESCRIPTION
[0015] To make the technical solution, purpose and advantages of the present invention clearer, the present invention is further described in detail below through specific implementation examples. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.
[0016] The present invention provides a The method for detecting the content of 13-S9 is particularly suitable for the recombinant protein vaccine stock solution. Detection of 13-S9 residues.
[0017] A sort of A method for detecting the content of 13-S9, the method comprising the operation of detecting the sample to be tested by using RP-UPLC-ELSD (reverse phase high performance liquid chromatography-evaporative light scattering) method.
[0018] According to the present invention The RP-UPLC-ELSD uses a reverse matrix column, such as a C4 column, a C8 column, or a C18 column, and more particularly, an XBridge BEH C8 (3.0×75 mm, 2.5 μm, ) chromatographic column.
[0019] The present invention provides In the method for detecting the content of 13-S9, the 13-S9 In the method provided by the present invention, three chromatographic peaks appear, and a single chromatographic peak that meets the method-specific detection requirements can be selected as the quantitative peak.
[0020] According to the present invention The method for detecting the content of 13-S9, wherein the sample to be tested can be prepared by using Recombinant protein vaccine of 13-S9. 13-S9 is generally used as an inactivating agent in the preparation of recombinant protein vaccines.
[0021] According to the present invention The method for detecting the content of 13-S9, the mobile phase used in the RP-UPLC-ELSD method includes mobile phase A and mobile phase B; wherein mobile phase A is water, mobile phase B is methanol, and mobile phase A+mobile phase B=100% (by volume). The method uses the mobile phase for gradient elution.
[0022] The process and parameters of the gradient elution may include:
[0023] At 0.0-5.0min, the volume ratio of mobile phase B is maintained at 5-20%, preferably 10%;
[0024] At 5.0-5.1 min, the volume ratio of mobile phase B changes linearly from 5-20% to 40-70%, preferably from 10% to 60%;
[0025] 5. During 1-10 min, the volume ratio of mobile phase B is maintained at 40-70%, preferably 60%;
[0026] At 10.0-18.0 min, the volume ratio of mobile phase B changes linearly from 40-70% to 100%, preferably from 60% to 100%;
[0027] At 18.0-21.0 min, the volume ratio of mobile phase B was maintained at 100%;
[0028] At 21.0-21.1 min, the volume ratio of mobile phase B changes linearly from 100% to 5-20%, preferably from 100% to 10%;
[0029] At 21.1-25 min, the volume ratio of mobile phase B is restored to the same value as that at 0.0-5.0 min (ie, the ratio of mobile phase B is maintained at 5-20%; preferably 10%).
[0030] According to the present invention The detection method of 13-S9 content, the instruments and parameters used in the RP-UPLC-ELSD method include: the detector is an evaporative light scattering detector, the detector parameters are: nebulizing gas pressure 30psi, nebulizer mode heating (power 70%), drift tube temperature 60°C, gain value 500; chromatographic column temperature is 30°C-50°C, preferably 40°C; sample plate temperature is 10-18°C, preferably 10°C; flow rate is 0.4-0.6mL / min, preferably 0.4mL / min.
[0031] According to the present invention 13-S9 content detection method, the method also includes setting 13-S9 standard solution operation. The concentration linear range of 13-S9 standard solution is 5 μg / mL-200 μg / mL, for example, it can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160 , 165, 170, 175, 180, 185, 190, 195, 200 μg / mL (preferably 10 μg / mL-100 μg / mL) 5 or 6 concentration values between any two values of 10 μg / mL and 100 μg / mL can be used as a standard curve; for example, the following set of standard concentrations can be selected: 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL. High-purity water can be used to prepare the 13-S9 standard solution.
[0032] According to the present invention The method for detecting the content of 13-S9 further comprises the operation of setting a blank control, wherein the blank solution used in the blank control is water.
[0033] If necessary, the sample to be tested can be diluted according to the actual situation, and then the chromatographic column and method provided by the present invention can be used for operation, and other components in the sample can be separated from the sample after gradient elution. 13-S9 A certain chromatographic peak was effectively separated.
[0034] According to the present invention The method for detecting the content of 13-S9 further comprises setting a spiked sample solution. The spiked sample is a solution containing a known amount of 13-S9 standard solution.
[0035] According to the present invention A method for detecting the content of 13-S9, the method comprising using RP-UPLC-ELSD to detect the The 13-S9 standard solution is tested, and the linear equation of the standard curve is obtained according to the test result; then the sample solution to be tested is tested, and the test result is substituted into the linear equation to calculate 13-S9 content (residual amount). Specifically, the above The 13-S9 standard solution, the sample solution to be tested, the blank control solution, and the spiked sample solution were detected by RP-UPLC-ELSD to obtain the corresponding chromatograms and peak areas. The logarithm of the standard concentration of 13-S9 is the abscissa X, and the logarithm of the peak area is the ordinate Y, a standard curve is drawn, and the linear equation of the standard curve is calculated; the peak areas of the sample solution to be tested and the spiked sample solution detected by RP-UPLC-ELSD are substituted into the linear equation of the standard curve, and the peak areas of the sample solution to be tested and the spiked sample solution are calculated. 13-S9 content.
[0036] In the RP-UPLC-ELSD method, the sample injection volume is 20-35 μL; the chromatographic column temperature is 30-50° C.; and the flow rate is 0.4-0.6 mL / min.
[0037] In order to improve the peak shape and effectively separate the recombinant protein vaccine 13-S9 and various components (such as Tween 80), the inventors of the present invention investigated the effects of different matrix chromatographic columns on the The peaks of 13-S9 and each component (such as Tween 80) are as described in Example 1. In order to reduce the contamination of the detector, the switching valve is adjusted to set each component to enter the detector from 10.0min to 21.0min, and the rest of the time is switched to waste liquid. In the RP-UPLC-ELSD method:
[0038] As an inactivating agent used in biopharmaceutical production, the residual amount of 13-S9 must be strictly controlled within a certain limit standard. There is no rapid, stable, and highly specific detection method for 13-S9 residue detection. The method for detecting the content of 13-S9 is particularly suitable for the recombinant protein vaccine stock solution. Detection of 13-S9 residues.
[0039] The present invention obtains the sample solution to be tested, the blank solution and the different concentrations of 13-S9 standard solution was injected into ultra-high performance liquid chromatography and detected by evaporative light scattering detector; 13-S9 standard solution chromatogram and blank solution chromatogram confirmed The characteristic peak of 13-S9 was selected as the chromatographic peak in the standard solution that was different from the chromatogram of the recombinant protein vaccine stock solution. 13-S9 quantitative peak, with different concentrations of The logarithmic value and The linear equation is established based on the logarithmic value of the peak area of the 13-S9 quantitative peak, and the Substitute the logarithmic value of the peak area of 13-S9 quantitative peak into the linear equation to calculate the content of the sample solution to be tested. 13-S9 residual amount. The method provided by the present invention can be used to The detection method is fast, accurate, linear, and specific, and is not interfered by other components in the recombinant protein vaccine stock solution. The detection of 13-S9 residues is of guiding significance.
[0040] The various reagents, materials, etc. used in the following examples, unless otherwise specified, are all products that can be obtained from commercial channels; the various tests and detection methods used in the following examples, unless otherwise specified, are all conventional tests and detection methods in the art and can be obtained from textbooks, reference books or academic journals.
[0041] In the following examples, the ultra-high performance liquid chromatograph with an evaporative light scattering detector was purchased from Waters, model Acquity Premier; the chromatographic column used was XBridge BEH C8 ( 2.5 μm, 3 mm × 75 mm), purchased from Waters, catalog number 186006046; 13-S9 standard was purchased from Merck, catalog number 1.08694.0000.
[0042] Example 1
[0043] This example is used to illustrate the invention using XBridge BEH C8 (3.0×75mm, 2.5μm, ) chromatographic column can be used to accurately detect the recombinant protein vaccine stock solution 13-S9 residual amount.
[0044] Detection of recombinant protein vaccine stock solution There is no literature report on the liquid phase method for the residual amount of 13-S9. The inventors have tried many liquid phase methods, but failed to obtain the ideal detection effect. The following is an example of using different matrix chromatographic columns:
[0045] In this embodiment, the test sample solution 1 (referred to as "test sample 1") is a recombinant protein vaccine stock solution preparation buffer.
[0046] In this embodiment, the blank solution is high-purity water.
[0047] In this embodiment, the different matrix chromatographic columns include a mixed matrix chromatographic column and a reverse phase matrix chromatographic column, wherein the mixed matrix chromatographic column can be Oasis MAX (2.1×20mm, 30μm, ) and Acclaim Surfactant Plus (4.6*150mm, 3μm); the reverse phase matrix column can be XBridge BEH C8 (3.0×75mm, 2.5μm, ) and XBridgeC18 (4.6×50mm, 3.5μm).
[0048] 100 μg / mL Preparation of 13-S9 standard solution:
[0049] Accurately weigh the appropriate amount 13-S9 was placed in a volumetric flask, dissolved in high-purity water and diluted to volume, and then shaken to make a 1 mg / mL 13-S9 standard solution stock solution. Then dilute to 100 μg / mL with high-purity water as diluent to obtain 100 μg / mL standard solution.
[0050] The 13-S9 standard itself is a mixture. In order to better reflect the overall content of the substance in liquid chromatography, the inventor intends to adjust the chromatographic conditions to merge multiple chromatographic peaks of multiple components into a single chromatographic peak for quantitative detection. By adjusting the experimental parameters, chromatographic columns with different matrices can Multiple chromatographic peaks of multiple components in 13-S9 are merged into a single chromatographic peak, thus achieving quantitative detection 13-S9. However, since the recombinant protein vaccine stock solution contains other components (such as Tween 80, etc.), An interference peak appears at the 13-S9 peak position, interfering with the recombinant protein vaccine stock solution The detection of 13-S9 residues cannot achieve the desired detection effect.
[0051] Effect of different matrix chromatographic columns on the recombinant protein vaccine stock solution Comparison of detection spectra of 13-S9 residues Figure 1-4 Different matrix chromatographic columns have different effects on the recombinant protein vaccine stock solution. The detection effect of 13-S9 residue is shown in Table 1.
[0052] Table 1 Effects of different matrix chromatographic columns on the recombinant protein vaccine stock solution Impact of 13-S9 residue on detection
[0053]
[0054] Depend on Figure 1-4 And Table 1 shows:
[0055] Oasis MAX (2.1×20mm, 30μm, ) chromatographic column, such as Figure 1 As shown, 13-S9 showed a single chromatographic peak in the liquid phase system. This chromatographic peak overlapped with the chromatographic peaks of other components in the buffer solution of the recombinant protein vaccine bulk preparation. There was interference in the detection and the detection requirements for method specificity were not met.
[0056] When using Acclaim Surfactant Plus (4.6*150mm, 3μm) chromatographic column, Figure 2 As shown, A single chromatographic peak appeared in 13-S9, which overlapped with the chromatographic peaks of other components in the buffer solution of the recombinant protein vaccine bulk preparation. There was interference in the detection and the test requirements for method specificity were not met.
[0057] When using XBridge C18 (4.6×50mm, 3.5μm) chromatographic column, Figure 3 As shown, by adjusting the liquid phase elution gradient, A single chromatographic peak appeared in 13-S9, but this chromatographic peak overlapped with the chromatographic peaks of other components in the buffer solution of the recombinant protein vaccine bulk preparation, resulting in interference in the detection and failing to meet the test requirements for method specificity.
[0058] When XBridge BEH C8 (3.0×75mm, 2.5μm, ) chromatographic column, such as Figure 4 As shown, by adjusting the liquid phase elution gradient, 13-S9 has three chromatographic peaks ( Figure 4 Peak 1, Peak 2, Peak 3) are shown in the figure, and Peak 1 can be effectively separated from other components (Tween 80, etc.) in the recombinant protein vaccine stock solution preparation buffer, thereby achieving the recombinant protein vaccine stock solution. Purpose of detecting 13-S9 residues.
[0059] Example 2
[0060] This embodiment is used to illustrate the use of the present invention. Detection method of 13-S9 content in recombinant protein vaccine stock solution Applicable when 13-S9 residual amount is present.
[0061] In this embodiment, the test solution 1 is the same as that in Example 1, both of which are recombinant protein vaccine stock solution preparation buffer. The test solution 2 is the final product of the recombinant protein vaccine stock solution. The blank solution is high-purity water.
[0062] Preparation of 13-S9 standard solution:
[0063] Accurately weigh the appropriate amount 13-S9 was placed in a volumetric flask, dissolved in high-purity water and diluted to volume, and then shaken to make a 1 mg / mL 13-S9 standard solution stock solution. Then use high-purity water as diluent to dilute in steps to 10μg / mL, 20μg / mL, 40μg / mL, 60μg / mL, 80μg / mL, 100μg / mL as 6 standard curve concentration points for use.
[0064] Preparation of spiked sample solution:
[0065] After vortexing the test solution 2 for 30 seconds, take three 197 μL portions of the test solution 2 in parallel and add 3 μL of 1 mg / mL 13-S9 standard solution stock solution was prepared into a spiked amount (i.e. The final concentration of 13-S9 was 15 μg / mL in the spiked sample.
[0066] The chromatographic conditions are shown in Table 2:
[0067] Table 2 RP-UPLC-ELSD chromatographic parameters
[0068]
[0069] Exclusive properties:
[0070] The 10 μg / mL 13-S9 standard solution, blank solution, test solution 1, test solution 2, and spiked sample solution are injected into the instrument for detection, and the corresponding chromatogram is recorded. Figure 5 As shown, blank solution and test solution 1 pair There was no interference at the peak 1 position (retention time 16.375 min) of the quantitative peak of the 13-S9 standard solution, indicating that the method of the present invention has good specificity.
[0071] Comparing the chromatograms of test solution 2 and standard solution, the test solution 2 The quantitative peak of 13-S9 ( Figure 5 The peak 1) in the position does not appear, indicating There is no residue of 13-S9 in the final product of the recombinant protein vaccine stock solution.
[0072] Comparing the chromatograms of spiked samples and standard solutions, the spiked samples The quantitative peak position of 13-S9 standard solution is ( Figure 5 Peak 1) in the sample indicates that the recombinant protein vaccine stock solution can be detected 13-S9, meets the requirements of recombinant protein vaccine stock solution The detection requirements of 13-S9 residues are that this method has high specificity.
[0073] Standard curve:
[0074] The prepared 13-S9 standard solution is injected into the instrument for detection, and the corresponding chromatogram and the standard solution are recorded. 13-S9 quantitative peak 1 peak area. With the logarithm of the standard solution concentration as the abscissa X and the logarithm of the quantitative peak 1 area as the ordinate Y, a standard curve was drawn to obtain the linear equation: Y = 1.78X + 2.71, and the linear correlation coefficient R 2 =0.999 (e.g. Figure 6 This shows that this method The linear correlation was good within the range of 10 μg / mL-100 μg / mL of 13-S9 concentration.
[0075] Table 3 Results of liquid phase separation related parameters of standard solution
[0076] Standard concentration (μg / mL) Peak 1 retention time (min) Peak 1 area Signal-to-Noise Ratio (S / N) 10 16.391 30549 25 20 16.391 107531 87 40 16.391 376186 324 60 16.386 775444 509 80 16.381 1250596 747 100 16.375 1843257 1153
[0077] Accuracy:
[0078] The prepared 13-S9 spiked sample solution is injected into the instrument for detection and the corresponding chromatogram is recorded (e.g. Figure 7 as shown) and the spiked sample solution 13-S9 peak area, calculated recovery rate, the results are shown in Table 4 below.
[0079] Table 4 Spiked sample solution 13-S9 residue test results
[0080]
[0081] Depend on Figure 7 As can be seen from Table 4, the recoveries of the three spiked samples were between 96% and 105%, meeting the requirement of 80% to 120%, indicating that the accuracy of this method was good.
[0082] Repeatability:
[0083] Prepare 6 copies of 60 μg / mL in parallel 13-S9 standard solution is injected into the instrument for detection, and the corresponding chromatogram is recorded (such as Figure 8 shown) and 13-S9 peak area, calculated RSD (RSD represents relative standard deviation) of retention time and peak area, and the results are shown in Table 5.
[0084] Table 5 Repeatability test results
[0085] sample Retention time (min) area 1 16.385 1139631 2 16.385 1170393 3 16.383 1135490 4 16.382 1138127 5 16.385 1136579 6 16.381 1156098 average value 16.384 1146053 Standard Deviation 0.001607 12901.77527 Relative standard deviation% 0.01 1.13
[0086] As can be seen from Table 5, the RSDs of retention time and peak area are 0.01% and 1.13%, respectively, both less than 2%. This shows that the method provided by the present invention has good repeatability and high reliability.
[0087] For the final product The permissible level of 13-S9 has not been clearly defined, but can be found in Triton TM X-100 must be below the limit of 25 μg / mL. The signal-to-noise ratio (S / N) of the 13-S9 standard curve with a concentration of 10 μg / mL is 25, which meets the requirement of the quantitative limit signal-to-noise ratio (S / N) greater than 10 in the 2020 edition of the Chinese Pharmacopoeia "9101 Validation Guidelines for Analytical Methods". 10 μg / mL can be set as the quantitative lower limit of the method provided by the present invention. Therefore, the sensitivity of the current detection method can be used in the recombinant protein vaccine stock solution. 13-S9 residue assessment.
[0088] Example 3
[0089] This embodiment is used to illustrate the use of the present invention. 13-S9 content detection method, detection of recombinant protein vaccine Residue of 13-S9.
[0090] In this embodiment, except that the sample to be tested is a sample in the process of preparing the recombinant protein vaccine stock solution (i.e., test solution 3), the other reagents used are the same as those in Example 2, including blank solution, 13-S9 standard solution etc. are the same as those in Example 2.
[0091] The RP-UPLC-ELSD chromatographic parameters and gradient elution program used are the same as those in Example 2, as shown in Table 2.
[0092] Substitute the peak area value obtained by RP-UPLC-ELSD detection of the sample solution (test solution 3) into the linear equation (Y = 1.65X + 3.01, linear correlation coefficient R 2 =1.000), the above sample solution is obtained The content of 13-S9 in the recombinant protein vaccine test solution 3 was calculated. The content of 13-S9, the specific results are shown in Table 6 (3 parallel tests).
[0093] Table 6 Test solution 3 13-S9 content test results
[0094]
[0095] As can be seen from the above, the method of the present invention can accurately detect the content of the recombinant protein vaccine test solution. 13-S9 content.
[0096] Example 4
[0097] This embodiment is used to illustrate the use of the present invention. 13-S9 content detection method, detection of recombinant protein vaccine 13-S9 residual amount.
[0098] In this embodiment, the sample to be tested is the test solution 3 which is the same as that in Example 3, and the other reagents used are the same as those in Example 2, including blank solution, 13-S9 standard solution etc. are the same as those in Example 2.
[0099] The RP-UPLC-ELSD chromatographic parameters used are the same as those in Example 2, as shown in Table 2, except for the gradient elution program shown in Table 7.
[0100] Table 7 Gradient elution program of RP-UPLC-ELSD chromatography
[0101]
[0102] Substitute the peak area value obtained by RP-UPLC-ELSD detection of the sample solution into the linear equation (Y = 1.66X + 2.93, linear correlation coefficient R) obtained in the same manner as Example 2 except that the gradient elution program is changed to that shown in Table 7. 2 =0.999), it is concluded that the above-mentioned sample solution The content of 13-S9 in the recombinant protein vaccine test solution 3 was calculated. The content of 13-S9, the specific results are shown in Table 8 (3 parallel tests).
[0103] Table 8 Test solution 3 13-S9 content test results
[0104]
[0105] As can be seen from the above, the method of the present invention can accurately detect the content of the recombinant protein vaccine test solution. 13-S9 content.
[0106] Example 5
[0107] This embodiment is used to illustrate the use of the present invention. 13-S9 content detection method, detection of recombinant protein vaccine 13-S9 residual amount.
[0108] In this embodiment, the sample to be tested is the test solution 3 which is the same as that in Example 3, and the other reagents used are the same as those in Example 2, including blank solution, 13-S9 standard solution etc. are the same as those in Example 2.
[0109] The RP-UPLC-ELSD chromatographic parameters used are the same as those in Example 2, as shown in Table 2, except for the gradient elution program shown in Table 9.
[0110] Table 9 Gradient elution program of RP-UPLC-ELSD chromatography
[0111]
[0112] Substitute the peak area value obtained by RP-UPLC-ELSD detection of the sample solution into the linear equation (Y = 1.66X + 3.0, linear correlation coefficient R) obtained in the same manner as Example 2 except that the gradient elution program is changed to that shown in Table 9. 2 =1.000), the above sample solution is obtained The content of 13-S9 in the recombinant protein vaccine test solution 3 was calculated. The content of 13-S9, the specific results are shown in Table 10 (3 parallel tests).
[0113] Table 10 Test solution 3 13-S9 content test results
[0114]
[0115] As can be seen from the above, the method of the present invention can accurately detect the content of the recombinant protein vaccine test solution. 13-S9 content.
[0116] Example 6
[0117] This embodiment is used to illustrate the use of the present invention. 13-S9 content detection method, detection of recombinant protein vaccine 13-S9 residual amount.
[0118] In this embodiment, the sample to be tested is the test solution 3 which is the same as that in Example 3, and the other reagents used are the same as those in Example 2, including blank solution, 13-S9 standard solution etc. are the same as those in Example 2.
[0119] The RP-UPLC-ELSD chromatographic parameters used are the same as those in Example 2, as shown in Table 2, except that the gradient elution program is shown in Table 11 and the chromatographic column is replaced with an XBridge C18 (4.6×50 mm, 3.5 μm) chromatographic column.
[0120] Table 11 Gradient elution program of RP-UPLC-ELSD chromatography
[0121]
[0122] Substitute the peak area value obtained by RP-UPLC-ELSD detection of the sample solution into the linear equation (Y = 1.49X + 3.31, linear correlation coefficient R) obtained in the same manner as Example 2 except that the gradient elution program is changed to that shown in Table 11. 2 =0.999), it is concluded that the above-mentioned sample solution The content of 13-S9 in the recombinant protein vaccine test solution 3 was calculated. The content of 13-S9, the specific results are shown in Table 12 (3 parallel tests).
[0123] Table 12 Test solution 3 13-S9 content test results
[0124]
[0125] As can be seen from the above, the method of the present invention can accurately detect the content of the recombinant protein vaccine test solution. 13-S9 content.
[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A A method for detecting the content of 13-S9, the method comprising the operation of detecting the sample to be tested by using the RP-UPLC-ELSD method; the mobile phase used in the RP-UPLC-ELSD method comprises mobile phase A and mobile phase B; wherein, Mobile phase A is water, and mobile phase B is methanol; the method uses the mobile phases for gradient elution.
2. The method according to claim 1, characterized in that: The chromatographic column used in the RP-UPLC-ELSD is a reverse matrix chromatographic column; Preferably, a C4 chromatographic column, a C8 chromatographic column and a C18 chromatographic column; The further preferred one is XBridge BEH C8 (3.0×75mm, 2.5μm, ) chromatographic column.
3. The method according to claim 2, characterized in that: The process and parameters of the gradient elution include: At 0.0-5.0min, the volume ratio of mobile phase B is maintained at 5-20%, preferably 10%; At 5.0-5.1 min, the volume ratio of mobile phase B changes linearly from 5-20% to 40-70%, preferably from 10% to 60%; 5. During 1-10 min, the volume ratio of mobile phase B is maintained at 40-70%, preferably 60%; At 10.0-18.0 min, the volume ratio of mobile phase B changes linearly from 40-70% to 100%, preferably from 60% to 100%; At 18.0-21.0 min, the volume ratio of mobile phase B was maintained at 100%; At 21.0-21.1 min, the volume ratio of mobile phase B changes linearly from 100% to 5-20%, preferably from 100% to 10%; During 21.1-25 min, the volume ratio of mobile phase B returned to the same value as during 0.0-5.0 min.
4. The method according to claim 3, characterized in that: The sample to be tested was prepared using Recombinant protein vaccine of 13-S9.
5. The method according to claim 3 or 4, characterized in that: The method further comprises setting 13-S9 standard solution operation; Preferably, the The linear range of the concentration of the 13-S9 standard solution is 5 μg / mL-200 μg / mL, for example, it can be between any two values of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 μg / mL, preferably 5 or 6 concentration values between 10 μg / mL-100 μg / mL; Preferably, the The concentrations of 13-S9 standard solution include: 10μg / mL, 20μg / mL, 40μg / mL, 60μg / mL, 80μg / mL, and 100μg / mL.
6. The method according to claim 5, characterized in that: The method also includes the operation of setting a blank control; The blank solution used in the blank control was water.
7. The method according to claim 6, characterized in that: The method also includes setting up an operation of adding a spiked sample solution; The spiked sample is a sample containing a known amount of 13-S9 standard solution.
8. The method according to claim 7, characterized in that: The method comprises using RP-UPLC-ELSD to The 13-S9 standard solution is tested, and the linear equation of the standard curve is obtained according to the test result; then the sample solution to be tested is tested, and the test result is substituted into the linear equation to calculate 13-S9 content.
9. The method according to claim 8, characterized in that: The method comprises the The 13-S9 standard solution, the sample solution to be tested, the blank control solution, and the spiked sample solution were detected by RP-UPLC-ELSD to obtain the corresponding chromatograms and peak areas. The logarithm of the standard concentration of 13-S9 is the abscissa X, and the logarithm of the peak area is the ordinate Y, a standard curve is drawn, and the linear equation of the standard curve is calculated; the peak areas of the sample solution to be tested and the spiked sample solution detected by RP-UPLC-ELSD are substituted into the linear equation of the standard curve, and the peak areas of the sample solution to be tested and the spiked sample solution are calculated. 13-S9 content.
10. The method according to claim 9, characterized in that: In the RP-UPLC-ELSD method: The sample injection volume was 20 to 35 μL; The column temperature is 30-50°C; The flow rate is 0.4~0.6mL / min.