A method for detecting free protein content in a polysaccharide-protein conjugate
Patent Information
- Application Number
- CN202311471647.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-07
- Publication Date
- 2026-09-29
- Estimated Expiration
- 2043-11-07
AI Technical Summary
凝胶色谱分离无法直观的反应溶液中各组分的分离情况；另外由于游离蛋白浓度很低，低于Lowry法的检测限，难以测出游离蛋白的准确结果
[0018]本发明扩宽了毛细管电泳的实际应用范围；提供了一种针对大分子复杂样品中的蛋白组分的高选择性分离检测系统；建立了性质相近、不易常规分离的组分的色谱分离检测方法；实现了多糖蛋白结合疫苗原液和成品中游离载体蛋白的含量测定。
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Figure CN117368295B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of vaccine quality testing, and more particularly to a method for detecting the content of free protein in polysaccharide-protein conjugates. Background Technology
[0002] Conjugate vaccines are polysaccharide-protein conjugate vaccines prepared by chemically binding polysaccharides to a carrier protein. They are used to enhance the immunogenicity of polysaccharide antigens in bacterial vaccines, such as Haemophilus influenzae type b conjugate vaccines, meningococcal conjugate vaccines, and pneumococcal conjugate vaccines.
[0003] In polysaccharide-protein conjugate vaccines, the content of both bound and free proteins needs to be examined. Controlling the proportion of free protein is one of the important indicators for evaluating the quality of conjugate vaccines.
[0004] Existing techniques employ gel chromatography to separate bound and free proteins, collect the separated free protein fraction solution, and then use the Lowry method to determine the free protein concentration, thereby calculating the free protein content in the sample. However, gel chromatography cannot directly reflect the separation of each component in the solution; furthermore, because the free protein concentration is very low, below the detection limit of the Lowry method, it is difficult to obtain accurate results for free protein determination. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides a method for detecting the content of free protein in polysaccharide-protein conjugate vaccines. This invention separates the binding protein and the free carrier protein using capillary electrophoresis-laser induced fluorescence (CE-LIF) and accurately calculates the content of the free carrier protein.
[0006] To solve the above technical problems, the technical solution of the present invention is as follows: a method for detecting the content of free protein in polysaccharide-protein conjugates, the method being operated as follows: adding protein labeling solution and Chromeo P503 fluorescent reagent to the pretreated polysaccharide-protein conjugates for labeling, adding deionized water to terminate the reaction after labeling, centrifuging to remove air bubbles, and then injecting the sample for capillary electrophoresis-laser induced fluorescence detection of the content of free protein;
[0007] The protein labeling solution consists of 30-80 mM sodium tetraborate and 0.8-1.5% SDS. The reason is that the peak response value increases with the increase of sodium tetraborate concentration. When the sodium tetraborate concentration in the labeling solution is higher than 80 mM, the peak drift is easily caused because the concentration of the sample solvent medium is higher than the concentration of the running buffer.
[0008] The polysaccharide-protein conjugate is either a finished polysaccharide-protein conjugate vaccine or a polysaccharide-protein conjugate stock solution.
[0009] As a preferred embodiment, the pretreatment method for the polysaccharide-protein conjugate vaccine is as follows: centrifuge the polysaccharide-protein conjugate vaccine solution at 7000-8000g for 10-20min to fully remove the precipitate and obtain the supernatant.
[0010] Take the supernatant or the original polysaccharide-protein conjugate solution and add it to the protein labeling solution and Chromeo P503 fluorescent reagent at a volume ratio of 0.8-1.2:2-4:0.08-0.15. Label at 50℃-70℃ for 10-40 minutes, then add deionized water to terminate the reaction. Centrifuge at 8000-12000 rpm for 5-20 minutes to remove air bubbles, and add the sample to a sample vial for instrumental analysis. If there are many bubble peaks, the centrifugation time can be extended.
[0011] Preferably, the capillary used in the capillary electrophoresis-laser induced fluorescence detection is an uncoated fused silica capillary column.
[0012] Preferably, the excitation wavelength of the laser-induced fluorescence detection is 488 nm; the emission wavelength of the laser-induced fluorescence detection is 600 nm.
[0013] Preferably, the capillary electrophoresis background electrolyte (BGE) is a mixture of 15mM to 60mM sodium tetraborate solution and 0.025M to 0.1M SDS solution, filtered through a filter membrane, and centrifuged at 7000g-8000g for 10 to 20 min to remove air bubbles before use.
[0014] Preferably, the sample introduction method for capillary electrophoresis-laser induced fluorescence detection is: 0.8-1.2 psi, 15-25 s; capillary temperature is: 15-25℃; separation voltage is 20-30 kV.
[0015] Preferably, the free protein in the polysaccharide-protein conjugate is tetanus toxoid (TT) or a non-toxic variant of diphtheria toxoid (CRM197); the polysaccharide-protein conjugate is GBS streptococcal capsular polysaccharide-protein conjugate, pneumococcal capsular polysaccharide-protein conjugate, or meningococcal polysaccharide-protein conjugate.
[0016] Preferably, the total protein concentration in the polysaccharide-protein conjugate is not less than 400 μg / mL.
[0017] Preferably, the capillary electrophoresis-laser induced fluorescence detection uses the standard curve method: a standard curve is plotted using a carrier protein tetanus toxoid standard (TT) or a diphtheria toxoid non-toxic variant (CRM197) standard. The tetanus toxoid standard (TT) or diphtheria toxoid non-toxic variant (CRM197) is gradually diluted with ultrapure water to solutions with concentrations of 5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL to plot a working curve. The concentration of free protein in the sample is calculated based on the standard curve, and then the content of free protein in the sample as a percentage of total protein is calculated.
[0018] This invention broadens the practical application scope of capillary electrophoresis; provides a highly selective separation and detection system for protein components in complex macromolecular samples; establishes a chromatographic separation and detection method for components with similar properties that are not easily separated by conventional methods; and realizes the determination of the content of free carrier protein in polysaccharide-protein conjugated vaccine stock solution and finished product.
[0019] This invention establishes a method for determining the free carrier proteins tetanus toxoid (TT) and diphtheria toxoid non-toxic variant (CRM197) in polysaccharide-protein conjugate vaccines using capillary electrophoresis-laser-induced fluorescence (CE-LIF), coupled with Chromeo P503 fluorescent reagent and uncoated fused silica capillaries. The changes in sample peak patterns under different sample processing and separation conditions were investigated, and the optimal sample processing conditions for polysaccharide-protein conjugates were determined as follows: Add protein labeling solution (self-made) and Chromeo P503 fluorescent reagent to the sample; after labeling, add deionized water to terminate the reaction; and then inject the sample for detection. Capillary electrophoresis can effectively determine the free carrier proteins from process samples to finished products.
[0020] Compared with the prior art, the present invention has the following advantages: The method uses fluorescent labeling to first label the protein components in the sample, then separates them by micellar electrokinetic capillary electrophoresis, and finally uses a LIF detector to detect the free carrier protein (especially the non-toxic variant of tetanus toxoid and diphtheria toxoid). The advantages of this method are as follows: (1) Good selectivity. The protein labeling method is used to process the sample to be tested, which effectively eliminates the interference of other impurities (such as polysaccharides) besides the protein components; (2) The separation of each component in the sample solution can be visually reflected by the chromatogram; (3) The detection sensitivity of protein is improved by replacing the Lowry method with CE-LIF; (4) It is easy to automate, reduce operational errors, and improve repeatability and quantitative accuracy; (5) The sample volume is small. Only a few microliters of solution sample are needed for one analysis, which can realize the detection and analysis of trace samples; (6) The analysis speed is fast. The analysis of a sample can be completed within tens of minutes. Attached Figure Description
[0021] Figure 1 The image shows the capillary electrophoresis-laser induced fluorescence (CE-LIF) pattern for Example 1. a: TT standard, b: polysaccharide-protein conjugate vaccine, c: sample buffer.
[0022] Figure 2 Here is the capillary electrophoresis-laser induced fluorescence (CE-LIF) pattern for Example 2: a: TT standard, b: polysaccharide-protein conjugate vaccine stock solution, c: sample buffer.
[0023] Figure 3 Example 3 shows the capillary electrophoresis-laser induced fluorescence (CE-LIF) pattern: a: CRM197 standard, b: polysaccharide-protein conjugate vaccine stock solution, c: sample buffer.
[0024] Figure 4 Spectra of different protein labeling solutions;
[0025] Figure 5 Spectra of different labeling temperatures and labeling times;
[0026] Figure 6 Capillary electrophoresis-laser induced fluorescence (CE-LIF) spectra with background electrolyte of 30 mM sodium tetraborate + 0.025 M SDS: a: TT standard, b: polysaccharide-protein conjugate vaccine, c: sample buffer.
[0027] Figure 7 Capillary electrophoresis-laser induced fluorescence (CE-LIF) spectra with background electrolyte of 45 mM sodium tetraborate + 0.025 M SDS: a: TT standard, b: polysaccharide-protein conjugate vaccine, c: sample buffer.
[0028] Figure 8 Capillary electrophoresis-laser induced fluorescence (CE-LIF) spectra of 60 mM sodium tetraborate + 0.05 M SDS background electrolyte: a: TT standard, b: polysaccharide-protein conjugate vaccine, c: sample buffer.
[0029] Figure 9 Capillary electrophoresis-laser induced fluorescence (CE-LIF) spectra of 15 mM sodium tetraborate + 0.075 M SDS background electrolyte, a: TT standard, b: polysaccharide-protein conjugate vaccine, c: sample buffer;
[0030] Figure 10Capillary electrophoresis-laser induced fluorescence (CE-LIF) spectra of 60 mM sodium tetraborate + 0.075 M SDS as background electrolyte; a: TT standard, b: polysaccharide-protein conjugate vaccine, c: sample buffer;
[0031] Figure 11 Capillary electrophoresis-laser induced fluorescence (CE-LIF) spectra of 30 mM sodium tetraborate + 0.1 M SDS with background electrolyte: a: TT standard, b: polysaccharide-protein conjugate vaccine, c: sample buffer. Detailed Implementation
[0032] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.
[0033] Example 1: Detection of Free Protein Content in GBS Streptococcus Capsular Polysaccharide Conjugate Vaccine
[0034] (1) Sampling and Sample Preparation
[0035] Protein labeling solution preparation: Sodium tetraborate and SDS were added to deionized water to prepare a protein labeling solution with a sodium tetraborate concentration of 40 mM and an SDS concentration of 1%.
[0036] Take 1 mL of polyvalent GBS streptococcal capsular polysaccharide-tetanus toxoid conjugate vaccine and centrifuge at 7500g for 15 min to thoroughly remove the precipitate. Accurately transfer 20 μL of the supernatant to a 1.5 mL centrifuge tube, add 60 μL of the prepared protein labeling solution, and then add 2 μL of Chromeo P503 fluorescent probe. Vortex mix. Briefly centrifuge and react at 60℃ for 20 min. After that, remove the sample and add 118 μL of ultrapure water to stop the reaction. Centrifuge at 10000 rpm for 5 min to remove air bubbles, and add 100 μL to a sample vial for instrumental analysis.
[0037] (1) Capillary electrophoresis-laser induced fluorescence (CE-LIF)
[0038] ① The instrument and equipment used were the Beckman Coulter (AB SCIEX) PA 800 high-performance capillary electrophoresis system (equipped with 32Karat software for system operation and data processing) for data analysis.
[0039] ② Uncoated capillary fused silica capillary column, with a total capillary length of 30-100cm (detection length of 20-90cm), an inner diameter of 50μm or 75μm. In this embodiment, the total length is 60cm, the detection length is 50cm, the inner diameter is 50μm, the outer layer is coated with a polyimide protective layer, and the inner wall is uncoated.
[0040] ③ Background electrolyte 52mM sodium tetraborate solution + 0.1M SDS solution: Take 10mL of 70mM sodium tetraborate solution + 1.67mL of 0.8M SDS solution + 1.67mL of deionized water, mix well, filter through a 0.45μm filter membrane, and centrifuge at 7500g for 10-20min to remove air bubbles.
[0041] ④ New capillary column flushing method
[0042] A. New Capillary Pre-Balancing Procedure
[0043] Rinse with 0.1 mol / L NaOH at 20 psi for 10 minutes; rinse with deionized water at 20 psi for 10 minutes; rinse with background electrolyte at 20 psi for 10 minutes; separate with 30 kV voltage for 10 minutes.
[0044] B. Sample Room Washing Procedure
[0045] Rinse with 0.1 mol / L NaOH at 20 psi for 4 minutes; rinse with deionized water at 20 psi for 2 minutes; rinse with background electrolyte at 20 psi for 5 minutes.
[0046] ⑤ Electrophoresis parameters
[0047] Operating voltage: 25kV
[0048] Capillary temperature: 20℃
[0049] Sample cell temperature: 20℃
[0050] Injection: 1.0 psi, 20 sec
[0051] Laser-induced fluorescence detection excitation wavelength: 488nm
[0052] Laser-induced fluorescence detection emission wavelength: 600nm
[0053] ⑥ Construction of standard curve: The tetanus toxoid standard was gradually diluted with ultrapure water to obtain solutions with concentrations of 5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL to construct a working curve; the concentration of free protein in the sample was calculated based on the standard curve, and then the content of free protein in the sample as a percentage of total protein was calculated.
[0054] ⑦ Data processing: Data processing was performed using the 32Karat software that comes with CE, and the results were analyzed using Excel.
[0055] (3) Experimental results: The capillary electrophoresis-laser induced fluorescence (CE-LIF) pattern of the polysaccharide-protein conjugate vaccine is shown in the figure below. Figure 1As shown: Peak ① is the bound protein peak, and peak ② is the free carrier protein TT peak. The free protein content in the multivalent GBS streptococcal capsular polysaccharide-tetanus toxoid conjugate vaccine was detected to be 1.57%, less than 5%, which meets the pharmacopoeia requirements.
[0056] Example 2: Detection of free protein content in GBS streptococcal capsular polysaccharide-tetanus toxoid conjugate stock solution
[0057] (1) Sampling and Sample Preparation
[0058] Preparation of protein labeling solution: Sodium tetraborate and SDS were added to deionized water to prepare a protein labeling solution with a sodium tetraborate concentration of 60 mM and an SDS concentration of 1%.
[0059] Accurately transfer 20 μL of the stock solution of type II GBS streptococcal capsular polysaccharide-tetanus toxoid conjugate into a 1.5 mL centrifuge tube. Add 50 μL of the prepared protein labeling solution, followed by 2.5 μL of Chromeo P503 fluorescent probe. Vortex mix thoroughly. Centrifuge briefly, and react at 55 °C for 20 minutes. Then, remove the sample and add 118 μL of ultrapure water to stop the reaction. Centrifuge at 10,000 rpm for 5 minutes to remove air bubbles. Add 100 μL of the solution to a sample vial for instrumental analysis.
[0060] (2) Capillary electrophoresis-laser induced fluorescence (CE-LIF) is equivalent to Example 1.
[0061] (3) The experimental results are as follows: The capillary electrophoresis-laser induced fluorescence (CE-LIF) spectrum of the polysaccharide-protein conjugate vaccine stock solution is shown in the figure. Figure 2 As shown: Peak ① is the bound protein peak, and peak ② is the free carrier protein TT peak. The free protein content in the GBS streptococcal capsular polysaccharide-tetanus toxoid conjugate is 1.46%, which is less than 5%, and meets the requirements of the pharmacopoeia.
[0062] Example 3: Detection of free protein CRM197 in the stock solution of 13-valent pneumococcal polysaccharide-protein conjugate vaccine
[0063] (1) Sampling and Sample Preparation
[0064] Preparation of protein labeling solution: Sodium tetraborate and SDS were added to deionized water to prepare a protein labeling solution with a sodium tetraborate concentration of 60 mM and an SDS concentration of 1.2%.
[0065] Accurately transfer 20 μL of the pneumococcal polysaccharide-protein conjugate stock solution into a 1.5 mL centrifuge tube, add 50 μL of the prepared labeling solution, and then add 2 μL of the Chromeo P503 fluorescent probe. Mix well using a vortex mixer. Centrifuge briefly, react at 60 °C for 20 minutes, then remove the sample and add 118 μL of ultrapure water to stop the reaction. Centrifuge at 10,000 rpm for 5 minutes to remove air bubbles, and add 100 μL to a sample vial for instrumental analysis.
[0066] (2) Capillary electrophoresis-laser induced fluorescence (CE-LIF) is equivalent to Example 1.
[0067] (3) The experimental results are as follows: The capillary electrophoresis-laser induced fluorescence (CE-LIF) spectrum of the pneumococcal polysaccharide conjugate vaccine stock solution is shown in the figure. Figure 3 As shown: Peak ① is the peak of the bound protein, and peak ② is the peak of the free carrier protein CRM197; the content of free protein is 1.12%, which is less than 5%, and meets the requirements of the pharmacopoeia.
[0068] Example 4: Screening under different detection conditions
[0069] Screening of protein labeling solutions: GBS streptococcal capsular polysaccharide-tetanus toxoid conjugate stock solution was labeled with different protein labeling solutions at 60℃ for 20 min. The effect of protein labeling solutions on the detection results was studied. Grouping is shown in Table 1. Detection results are as follows: Figure 4 As shown. By Figure 4 Analysis shows that the sodium tetraborate + 1% SDS system has a greater impact on the peak response than other labeling solutions. Furthermore, the peak response value of the protein is significantly higher when the labeling solution is 30-80 mM sodium tetraborate + 1% SDS than other labeling solutions. When the sodium tetraborate concentration in the labeling solution is higher than 80 mM, the higher concentration of sodium tetraborate in the sample solvent compared to the running buffer can easily lead to peak retention time drift. Therefore, the preferred protein labeling solution is 30-80 mM sodium tetraborate + 1% SDS.
[0070] Table 1 Formulations of different labeling solutions
[0071]
[0072]
[0073] Screening of labeling temperature and labeling time: GBS streptococcal capsular polysaccharide-tetanus toxoid conjugate stock solution was labeled with 40 mM sodium tetraborate solution and 1% SDS protein labeling solution. The effects of labeling temperature and labeling time on the detection results were studied. Grouping is shown in Table 2. Figure 5Analysis shows that labeling temperature and labeling time have a certain influence on the peak response. The peak response value increases with increasing labeling temperature and increases with increasing labeling time. However, excessively high temperature will cause protein denaturation and structural instability. Therefore, the labeling temperature should preferably not exceed 70℃.
[0074] Table 2. Effects of labeling temperature and labeling time on peak area.
[0075]
[0076]
[0077] Screening of background electrolytes: The GBS streptococcal capsular polysaccharide-tetanus toxoid conjugate stock solution was labeled with 40 mM sodium tetraborate solution and 1% SDS protein labeling solution at 60℃ for 20 min. Different background electrolytes were used for separation. The preparation process of the background electrolytes is as follows: sodium tetraborate concentration (before mixing) 0.4M~0.8M and SDS concentration (before mixing) 0.2~0.8M were mixed.
[0078] Test results as follows Figures 6-11 As shown. Combined with Figures 6-11 Analysis revealed that the separation degree between free and bound proteins was affected by the concentrations of sodium tetraborate and SDS in the background electrolyte. The separation degree increased with increasing sodium tetraborate concentration, and also with increasing SDS concentration. When the background electrolyte was a mixture of 15mM–60mM sodium tetraborate solution and 0.025M–0.1M MSDS solution, the separation degree between free and bound proteins was sufficient for the detection and quantification of free proteins.
[0079] It should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Equivalent substitutions or alternatives made on the basis of the above shall all fall within the scope of protection of the present invention.
Claims
1. A method for detecting the content of free protein in polysaccharide-protein conjugates, characterized in that, The method is as follows: protein labeling solution and Chromeo P503 fluorescent reagent are added to the pretreated polysaccharide-protein conjugate for labeling. After labeling, deionized water is added to terminate the reaction. After centrifugation, air bubbles are removed and the sample is injected for capillary electrophoresis-laser induced fluorescence detection of the free protein content. The protein labeling solution consists of the following components: a solution of 30-80 mM sodium tetraborate and 0.8-1.5% SDS. The polysaccharide-protein conjugate is a finished polysaccharide-protein conjugate vaccine or a polysaccharide-protein conjugate stock solution; The pretreatment method for the polysaccharide-protein conjugate vaccine is as follows: The polysaccharide-protein conjugate vaccine solution is centrifuged at 7000-8000g for 10-20 min to thoroughly remove the precipitate, obtaining a supernatant. The supernatant or the original polysaccharide-protein conjugate solution is taken and added to protein labeling solution and Chromeo P503 fluorescent reagent at a volume ratio of 0.8-1.2:2-4:0.08-0.
15. Labeling is performed at 50℃-70℃ for 10-40 min, followed by the addition of deionized water to terminate the reaction. After centrifugation at 8000-12000rpm for 5-20 min to remove air bubbles, the sample is added to a sample vial for instrumental analysis. The capillary used in the capillary electrophoresis-laser induced fluorescence detection is an uncoated fused silica capillary column.
2. The method for detecting the content of free protein in polysaccharide-protein conjugates according to claim 1, characterized in that, The excitation wavelength for laser-induced fluorescence detection is 488 nm; the emission wavelength for laser-induced fluorescence detection is 600 nm.
3. The method for detecting the content of free protein in polysaccharide-protein conjugates according to claim 1, characterized in that, The background electrolyte for capillary electrophoresis is a mixture of 15mM~60mM sodium tetraborate solution and 0.025M~0.1M SDS solution. After filtration through a filter membrane and centrifugation at 7000g-8000g for 10~20min to remove air bubbles, it is used.
4. The method for detecting the content of free protein in polysaccharide-protein conjugates according to claim 1, characterized in that, The sample introduction method for capillary electrophoresis-laser induced fluorescence detection is: 0.8-1.2 psi, 15-25 s; capillary temperature is: 15-25℃; separation voltage is 20-30 kV.
5. The method for detecting the content of free protein in polysaccharide-protein conjugates according to any one of claims 1 to 4, characterized in that, The free protein in the polysaccharide-protein conjugate is a non-toxic variant of tetanus toxoid or diphtheria toxoid; the polysaccharide-protein conjugate is a GBS streptococcal capsular polysaccharide-protein conjugate, a pneumococcal capsular polysaccharide-protein conjugate, or a meningococcal polysaccharide-protein conjugate.
6. The method for detecting the content of free protein in polysaccharide-protein conjugates according to claim 5, characterized in that, The total protein concentration in the polysaccharide-protein conjugate is not less than 400 μg / mL.
7. The method for detecting the content of free protein in polysaccharide-protein conjugates according to claim 5, characterized in that, The capillary electrophoresis-laser induced fluorescence detection uses the standard curve method: a standard curve is plotted using tetanus toxoid standard or diphtheria toxoid non-toxic variant standard. The tetanus toxoid standard or diphtheria toxoid non-toxic variant standard is gradually diluted with ultrapure water to concentrations of 5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL to plot working curves. The concentration of free protein in the sample is calculated based on the standard curve, and then the proportion of free protein in the total protein in the sample is calculated.