A method for determining sodium dextran sulfate in biological products

Abstract

This invention belongs to the field of biodetection technology, specifically relating to a method for determining sodium dextran sulfate in biological products. The method includes: establishing a standard curve; pretreating the biological product to obtain a test solution; detecting the test solution using high-performance liquid chromatography-electro-spray detector; and calculating the content of sodium dextran sulfate in the test solution based on the detection results. This invention achieves direct detection without derivatization or colorimetric treatment of sodium dextran sulfate, and has advantages such as simple operation, high detection efficiency, good accuracy, high sensitivity, and wide linear range. It is suitable for determining the residual amount of sodium dextran sulfate in various biological matrices such as antibodies, antibody purification solutions, and protein products.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for determining sodium dextran sulfate in biological products. Background Technology

[0002] Dextran sulfate sodium (DSS), also known as sodium dextran sulfate ester, is a polyanionic derivative of dextran. In the production of biopharmaceuticals (such as monoclonal antibodies, fusion proteins, and vaccines), DSS is often used as an anti-clustering agent. It can promote the suspension of single cells by altering the cell surface charge, supporting high-density cell culture and thus increasing protein expression. However, studies have shown that DSS has certain toxicity, capable of disrupting the integrity of the colonic epithelial barrier and inducing inflammatory responses. Therefore, according to Good Manufacturing Practices (GMP) for pharmaceuticals, the residual amount of DSS in biopharmaceuticals must be strictly controlled below safe limits, and accurate determination of its residual amount is crucial for drug quality control.

[0003] However, the detection of DSS faces the following technical challenges: First, the lack of chromophores and fluorescent groups in the DSS molecular structure prevents it from responding to conventional high-performance liquid chromatography-ultraviolet detectors or diode array detectors, and also makes direct detection by fluorescence detectors difficult. To address this issue, two types of indirect detection methods have been developed in the existing technology. The first type involves derivatizing DSS, for example, by reacting DSS with fluorescently labeled reagents to introduce detectable groups, followed by quantification through chromatographic separation and fluorescence detection. This type of method is cumbersome, involving the addition of derivatizing reagents and strict control of reaction conditions. The derivatization efficiency is unstable, easily leading to poor repeatability and low accuracy of detection results, and the reaction time is long, resulting in low detection efficiency. The second type of method utilizes the electrostatic binding of the polyanionic framework of DSS with cationic dyes to form a complex with ultraviolet absorption, followed by quantification by measuring absorbance using an enzyme-linked immunosorbent assay (ELISA) reader. Although this type of colorimetric reaction method is relatively simple to operate, it has inherent defects: the stability of the colorimetric complex is greatly affected by environmental factors such as ionic strength and pH, and the linear range and reproducibility of the standard curve are limited. In summary, there is an urgent need in the existing technology for a direct DSS detection method that is easy to operate, highly accurate, has good repeatability, and does not require derivatization or color development. Summary of the Invention

[0004] Based on this, the present invention provides a method for determining sodium dextran sulfate in biological products. The present invention uses high-performance liquid chromatography-electro-spray detector to detect sodium dextran sulfate in biological products, eliminating the need for a derivatization step. This effectively solves the problems of poor repeatability and complex operation caused by derivatization of sodium dextran sulfate, providing a simple, accurate, and repeatable method for its determination.

[0005] To achieve the above objectives, the present invention includes the following technical solutions:

[0006] This invention provides a method for determining sodium dextran sulfate in biological products, comprising the following steps: S1: Preparation of test solution: Add a precipitant to the test sample to precipitate, centrifuge, take the supernatant, dilute with water, filter through a filter membrane, and take the filtrate as the test solution; wherein the test sample is a biological product; S2: Determination of the test solution: The peak area of ​​sodium dextran sulfate in the test solution was determined by isocratic elution with ammonium acetate solution as the mobile phase using high performance liquid chromatography-electro-fogging detector (HPLC-CAD). S3: Content Calculation: Calculate the content of sodium dextran sulfate in the test sample based on the test results.

[0007] Step S1 involves adding a precipitant and centrifuging to settle large molecular impurities such as proteins and peptides in the biological matrix, eliminating their contamination of the chromatographic column, peak interference, and baseline noise. The target analyte, sodium dextran sulfate, is then filtered through a membrane to remove small particles and suspended solids, resulting in a clear solution suitable for liquid chromatography detection and preventing clogging of the column and tubing. Step S2 utilizes high-performance liquid chromatography (HPLC) for component separation, combined with the specific response of an electro-cavitation detector (CAD) to quantitatively acquire the chromatographic peak signal of sodium dextran sulfate. Based on the ionic characteristics of sodium dextran sulfate, this scheme uses ammonium acetate solution as the mobile phase, achieving effective separation of the target analyte from residual impurities through isocratic elution. Since the CAD detector has a stable response to non-volatile, ionic compounds, its use effectively compensates for the lack of characteristic absorption of sodium dextran sulfate by ultraviolet detectors, accurately acquiring the peak area of ​​the target analyte and providing reliable raw data for quantification. Step S3, based on the principles of chromatographic quantification and combined with a standard curve, calculates the actual content of sodium dextran sulfate in the biological product.

[0008] Furthermore, the volume ratio of the biological product sample to the precipitant is 1:(1~5).

[0009] Furthermore, the precipitant is a saturated ammonium sulfate solution.

[0010] Furthermore, the high-performance liquid chromatography separation uses a TSKgel G3000PWxl gel column, 7.8mm×300mm, 7μm, with the column temperature set to 35℃.

[0011] Furthermore, the concentration of the ammonium acetate solution is 45-55 mmol / L, and the flow rate is 0.3-0.5 mL / min.

[0012] Furthermore, the injection volume for the high-performance liquid chromatography is 20 μL.

[0013] Furthermore, the atomization temperature of the electro-fog detector is 70℃, and the sampling frequency is 5Hz.

[0014] Furthermore, the content of sodium dextran sulfate was calculated using the standard curve method.

[0015] Furthermore, the establishment of the standard curve in the standard curve method includes preparing a series of diglucan sulfate sodium reference solutions with gradient concentrations, measuring them using high performance liquid chromatography-electro-fogging detector, and establishing a standard curve with concentration as the abscissa and peak area as the ordinate.

[0016] Furthermore, the sodium dextran sulfate reference solution is obtained by diluting a certain amount of the reference standard stock solution with a reference standard diluent; wherein the reference standard stock solution is obtained by diluting sodium dextran sulfate reference standard; and the reference standard diluent is obtained by diluting a saturated ammonium sulfate solution.

[0017] Beneficial effects: This invention uses a CAD detector to directly detect sodium dextran sulfate without chromophores, eliminating the need for any derivatization or colorimetric treatment. Samples can be directly injected for detection after simple pretreatment, significantly simplifying experimental procedures, shortening the detection cycle, and reducing detection costs. It is suitable for rapid analysis of large batches of samples. Simultaneously, the use of a gel chromatography column effectively separates sodium dextran sulfate from proteins and other impurities in biological products, avoiding matrix interference on the detection results. This method is applicable to various biological product matrices and exhibits good matrix adaptability. Attached Figure Description

[0018] Figure 1 This is the linear regression curve for Example 1.

[0019] Figure 2 The chromatogram is of the sodium dextran sulfate reference solution from Example 1.

[0020] Figure 3 This is the chromatogram of the blank control solution from Example 1.

[0021] Figure 4 This is the chromatogram of the blank test solution from Example 1.

[0022] Figure 5 This is the chromatogram of the spiked test solution from Example 1. Detailed Implementation

[0023] This invention provides a method for determining sodium dextran sulfate in biological products, comprising the following steps: S1: Preparation of test solution: Add saturated ammonium sulfate solution as a precipitant to the test sample at a volume ratio of 1:1, centrifuge, take the supernatant, dilute with water, filter through a 0.22μm aqueous phase filter membrane, and take the filtrate as the test solution; wherein the test sample is a biological product; S2: Determination of the test solution: High-performance liquid chromatography-electro-atomization detector (HPLC-EAD) was used with ammonium acetate solution as the mobile phase for isocratic elution. The peak area of ​​sodium dextran sulfate in the test solution was determined. The chromatographic column was a TSKgel G3000PWxl gel column (7.8 mm × 300 mm, 7 μm), the column temperature was set to 35℃, the ammonium acetate solution concentration was 50 mmol / L, the flow rate was 0.4 mL / min, and the injection volume was 20 μL. The electro-atomization detector nebulization temperature was 70℃, and the acquisition frequency was 5 Hz. S3: Content Calculation: A certain amount of the reference standard stock solution was diluted with the reference standard diluent to prepare a series of dilution solutions of sodium dextran sulfate at varying concentrations. High-performance liquid chromatography with an electro-fogging detector was used for determination. A standard curve was established with concentration on the x-axis and peak area on the y-axis. The concentration of sodium dextran sulfate in the test solution was obtained using the standard curve method based on the peak area of ​​the sodium dextran sulfate in the test solution. To obtain the measured concentration, the concentration of sodium dextran sulfate in the test sample was calculated using the dilution factor and solution concentration. Combined with the amount of test sample weighed, the content of sodium dextran sulfate in the test sample could be calculated. The reference standard stock solution was obtained by diluting the sodium dextran sulfate reference standard. The reference standard diluent was obtained by diluting a saturated ammonium sulfate solution.

[0024] The detection method of this invention was obtained after extensive experimental screening and verification. The invention will be further described below with reference to the accompanying drawings and specific embodiments, but these embodiments do not limit the invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field.

[0025] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0026] Example 1 (1) Solution preparation Reference stock solution: Take 500 mg of sodium dextran sulfate reference standard, place it in a 10 mL volumetric flask, add water to dissolve and dilute to the mark, mix well, and the solution is ready.

[0027] Reference standard diluent: Take 25 mL of saturated ammonium sulfate solution, place it in a 100 mL volumetric flask, dilute with water to the mark, mix well, and the solution is ready.

[0028] Sodium dextran sulfate reference solution: Take a certain amount of the reference solution and dilute it with the reference diluent to obtain the solution.

[0029] Blank control solution: Take 0.5 mL of water, place it in a 1.5 mL centrifuge tube, add 0.5 mL of saturated ammonium sulfate solution, vortex for 2 min, centrifuge at 8000 r / min for 5 min, transfer 0.5 mL of supernatant, add 0.5 mL of water, mix well, filter through a 0.22 μm aqueous phase filter membrane, and take the filtrate.

[0030] Blank test solution: Take 0.5 mL of blank test sample, place it in a 1.5 mL centrifuge tube, add 0.5 mL of saturated ammonium sulfate solution, vortex for 2 min, centrifuge at 8000 r / min for 5 min, transfer 0.5 mL of supernatant, add 0.5 mL of water, mix well, filter through a 0.22 μm aqueous phase filter membrane, and collect the filtrate. The blank test sample is a blank biological product.

[0031] Spiked test solution: Take 0.5 mL of the spiked test sample, place it in a 1.5 mL centrifuge tube, add 0.5 mL of saturated ammonium sulfate solution, vortex for 2 min, centrifuge at 8000 r / min for 5 min, transfer 0.5 mL of the supernatant, add 0.5 mL of water, mix well, filter through a 0.22 μm aqueous phase filter membrane, and collect the filtrate. The spiked test sample is prepared by adding an appropriate amount of reference stock solution to a blank biological product to achieve the required concentration.

[0032] Test solution: Take 0.5 mL of the test sample, place it in a 1.5 mL centrifuge tube, add 0.5 mL of saturated ammonium sulfate solution, vortex for 2 min, centrifuge at 8000 r / min for 5 min, transfer 0.5 mL of the supernatant, add 0.5 mL of water, mix well, filter through a 0.22 μm aqueous phase filter membrane, and collect the filtrate. The test sample is a biological product. This embodiment uses a spiked test sample for method validation; the test solution should be prepared according to this method for actual sample testing.

[0033] (2) System suitability test A 250 μg / mL sodium dextran sulfate standard solution was prepared as the system suitability solution and injected for determination. The experimental results are shown in Table 1. After six consecutive injections of the system suitability solution, the relative standard deviation of the retention time (n=6) of sodium dextran sulfate was 0.32%, and the relative standard deviation of the peak area (n=6) was 1.56%, which met the validation requirements.

[0034] Table 1 System Applicability Test Results

[0035] (3) Validation of detection limit and quantitation limit A 12.5 μg / mL sodium dextran sulfate reference solution was prepared as the limit of detection (LOD) solution, and a 25.0 μg / mL sodium dextran sulfate reference solution was prepared as the limit of quantitation (LOQ) solution. These solutions were injected separately for detection, and the signal-to-noise ratio (S / N) was calculated. The LOD solution was measured three times, and the LOQ solution was measured six times to examine the S / N and peak area.

[0036] The results of the detection limit experiment are shown in Table 2. The detection limit of this method for sodium dextran sulfate is 12.5 μg / mL. After three consecutive injections, the signal-to-noise ratio (SNR) was within the range of 10.5 to 19.6, which meets the requirement of an SNR of not less than 3. Based on the dilution factor (4 times) of the test sample during the actual test, the detection limit of this method is 50 μg / mL. The results of the quantitation limit experiment are shown in Table 3. The quantitation limit of this method for sodium dextran sulfate is 25.0 μg / mL. After six consecutive injections, the relative standard deviation of the peak area was 1.95%, and the SNR was within the range of 24.3 to 41.4, which meets the requirement of an SNR of not less than 10. Based on the dilution factor (4 times) of the test sample during the actual test, the quantitation limit of this method is 100 μg / mL.

[0037] Table 2 Detection Limit Experiment Results

[0038] Table 3 Results of Limit of Quantitation Experiment

[0039] (4) Linear Dextran sulfate sodium reference solutions with concentrations of 25.0 μg / mL, 50.0 μg / mL, 125 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 375 μg / mL, and 500 μg / mL were prepared and injected for detection. A linear regression was performed on the peak area y of the measured dextran sulfate sodium solution against the concentration x of the dextran sulfate sodium solution. The correlation coefficient r was used to examine the linear relationship. The linear regression curves are shown below. Figure 1 Experimental results show that sodium dextran sulfate exhibits a good linear relationship between concentration and peak area within the concentration range of 25.0 μg / mL to 500 μg / mL, with the linear equation being y = 0.01x + 0.43 and a correlation coefficient r of 0.9960.

[0040] (5) Repeatability Six spiked test solutions were prepared in parallel, and each was injected once. The concentration of sodium dextran sulfate in the spiked test solutions was calculated using the linear equation described above based on the obtained peak area results. The converted concentration of sodium dextran sulfate in the spiked test solutions was then calculated, and the relative standard deviation of the six determinations was examined. The experimental results show that the relative standard deviation of sodium dextran sulfate in the spiked test solutions using this method is 1.77%, indicating good repeatability.

[0041] Table 4 Results of Repeatability Experiments

[0042] (6) Accuracy Three spiked test solutions (9 in total) were prepared using the spiked sample at DSS concentrations of 500 μg / mL, 1000 μg / mL, and 1500 μg / mL. The recoveries and relative standard deviations (RSDs) of these 9 spiked test solutions were investigated. The results showed that the recoveries of sodium dextran sulfate at the three concentration levels ranged from 112.0% to 117.6%, with an average recovery of 114.7% (n=9) and a RSD of 1.37% (n=9). This method demonstrated good accuracy at all concentration levels.

[0043] Table 5 Accuracy Experiment Results

[0044] (7) Specificity test Inject 250 μg / mL sodium dextran sulfate reference solution, blank control solution, blank test solution, and spiked test solution into the sample for analysis. Compare the chromatograms. The results are as follows: Figures 2-4 . Figure 2 This is the chromatogram of the sodium dextran sulfate reference solution. Figure 3 This is the chromatogram of the blank control solution. Figure 4 The image shows the chromatogram of the blank test solution. The results indicate that neither the blank control solution nor the blank test solution exhibited a significant interfering peak at the DSS elution position of the control solution, while the spiked test solution showed the same target peak at that position as the control solution, thus confirming that this method can specifically detect sodium dextran sulfate.

[0045] (8) Applicability of different biopharmaceutical matrices A certain amount of blank antibody, antibody purification solution, and protein product were used as blank biological products. Each blank biological product was divided into three portions, and an appropriate amount of reference stock solution was added to each portion to prepare spiked test samples with DSS concentrations of 500 μg / mL, 1000 μg / mL, and 1500 μg / mL. Spiked test sample solutions were prepared and analyzed using high-performance liquid chromatography-electrospray detector with a 50 mmol / L ammonium acetate solution as the mobile phase, a flow rate of 0.4 mL / min, and a TSK gel G3000 PWxl gel chromatography column (7.8 mm × 300 mm) set to 35℃. Using a 7 μm stationary phase, isocratic elution was performed. An electro-negation detector with a nebulization temperature of 70℃ and a sampling frequency of 5Hz was used to measure the peak area of ​​sodium dextran sulfate in the spiked test solution with different concentrations and different biological product matrices. Based on the obtained peak area results, the measured concentration of sodium dextran sulfate in the spiked test solution was calculated using the above linear equation. The concentration and content of sodium dextran sulfate in the spiked test solution were calculated by using the measured concentration of the spiked test solution, the dilution factor (4 times), and the amount of spiked test sample weighed (0.5 mL).

[0046] Data analysis was used to obtain the repeatability and accuracy of spiked test samples under different biopharmaceutical matrices. Repeatability was expressed as the relative standard deviation of the measured concentration, and accuracy was expressed as the recovery rate. The results are shown in Table 6. The experimental results show that the relative standard deviation of this method is less than 6% for biopharmaceuticals under different matrices, and the recovery rate is 88.1%~117.6%, which shows good repeatability and accuracy.

[0047] Table 6. Results of sodium dextran sulfate determination under different matrices

[0048] In summary, the method for detecting sodium dextran sulfate using high-performance liquid chromatography-electro-fogging detector established in this invention can achieve direct detection without derivatization or colorimetric treatment of DSS. Furthermore, the method validation results show that the method has good system stability, high sensitivity, wide linear range, good repeatability, high accuracy, and wide matrix adaptability, which can meet the detection requirements of sodium dextran sulfate residue in biological products and has good application prospects.

[0049] The above description is merely a preferred embodiment of the present invention and is only used to illustrate the technical solution of the present invention. It is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the technical solution of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the technical solution of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for determining sodium dextran sulfate in biological products, characterized in that, Includes the following steps: S1: Preparation of test solution: Add a precipitant to the test sample to precipitate, centrifuge, take the supernatant, dilute with water, filter through a filter membrane, and take the filtrate as the test solution; wherein the test sample is a biological product; S2: Determination of the test solution: The peak area of ​​sodium dextran sulfate in the test solution was determined by isocratic elution with ammonium acetate solution as the mobile phase using high performance liquid chromatography-electro-spray detector. S3: Content Calculation: Calculate the content of sodium dextran sulfate in the test sample based on the test results.

2. The method for determining sodium dextran sulfate in biological products according to claim 1, characterized in that, The volume ratio of the biological product sample to the precipitant is (1:1~5).

3. The method for determining sodium dextran sulfate in biological products according to claim 1, characterized in that, The precipitant is a saturated ammonium sulfate solution.

4. The method for determining sodium dextran sulfate in biological products according to claim 1, characterized in that, The high-performance liquid chromatography separation used a TSKgel G3000PWxl gel column, 7.8 mm × 300 mm, 7 μm, with the column temperature set at 35 °C.

5. The method for determining sodium dextran sulfate in biological products according to claim 4, characterized in that, The ammonium acetate solution has a concentration of 45-55 mmol / L and a flow rate of 0.3-0.5 mL / min.

6. The method for determining sodium dextran sulfate in biological products according to claim 4, characterized in that, The injection volume for the high-performance liquid chromatography was 20 μL.

7. The method for determining sodium dextran sulfate in biological products according to claim 1, characterized in that, The atomization temperature of the electro-fogging detector is 70℃, and the sampling frequency is 5Hz.

8. The method for determining sodium dextran sulfate in biological products according to claim 1, characterized in that, The content of sodium dextran sulfate was calculated using the standard curve method.

9. The method for determining sodium dextran sulfate in biological products according to claim 8, characterized in that, The establishment of the standard curve in the standard curve method includes preparing a series of diglucan sulfate sodium reference solutions with gradient concentrations, measuring them using high performance liquid chromatography-electro-fogging detector, and establishing a standard curve with concentration as the abscissa and peak area as the ordinate.

10. The method for determining sodium dextran sulfate in biological products according to claim 9, characterized in that, The sodium dextran sulfate reference solution is obtained by diluting a certain amount of the reference standard stock solution with the reference standard diluent; wherein the reference standard stock solution is obtained by diluting sodium dextran sulfate reference standard; and the reference standard diluent is obtained by diluting a saturated ammonium sulfate solution.

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