Method for detecting content of polysorbate 20 in protein freeze-drying preparation

Abstract

The invention belongs to the technical field of protein freeze-drying preparations, and particularly relates to a method for detecting the content of polysorbate 20 in a protein freeze-drying preparation, which comprises the following steps: (1) preparing standard substance solutions: respectively taking polysorbate 20 standard stock mother solutions with different volumes, and diluting with water to prepare 1-5 standard substance solutions; (2) preparing a test sample solution: taking a proper amount of a test sample, and preparing the test sample solution with the theoretical concentration of polysorbate 20 being 10-100mu g / ml; (3) measuring the content of polysorbate 20; and (4) data processing: fitting a linear regression equation of the concentration and absorbance of the polysorbate 20 by using the data obtained from the standard solutions 1-5, and calculating the concentration of the polysorbate 20 in the test solution. The method has the beneficial effects that the linearity (R2) of a standard curve can reach 0.999 and is far higher than 0.98 required by pharmacopoeia; and the absorbance values of the standard solution and the test solution with the same concentration are close to each other.

Description

Technical Field

[0001] The invention belongs to the technical field of protein freeze-dried preparations, and particularly relates to a method for detecting the content of polysorbate 20 in a protein freeze-dried preparation. Background Art

[0002] Polysorbate 20, also known as Tween-20, is a polymer formed by the addition polymerization of dehydrated sorbitan and ethylene oxide and esterification with monolauric acid. It is one of the commonly used excipients in pharmaceutical preparations and a common non-ionic surfactant in biological products. Compared with ionic surfactants (such as sodium dodecyl sulfate), its molecules contain both hydrophilic and lipophilic groups, can form micelles in water, have a solubilizing effect on insoluble components, can avoid strong interactions with protein molecules, have good biocompatibility, low toxicity and good biological tolerance, so it is often used as an emulsifier, solubilizer, stabilizer, diffusant, etc. According to the results of clinical and non-clinical safety tests at home and abroad, for biological preparations, its addition amount is generally not higher than 0.05%, which is safer. However, for protein drugs, the content of polysorbate is not the lower the better. If the content of polysorbate is too low, the stabilization effect may not be achieved. Therefore, strict control is required during the production of pharmaceutical preparations to keep the content of polysorbate 20 within the standard range. Due to the similarity in structure and properties, the detection of polysorbate 20 can refer to polysorbate 80 (both are products of the polyoxyethylene sorbitan fatty acid ester series).

[0003] In existing studies, the main methods for determining the content of polysorbate are spectrophotometry, fluorescence colorimetry, and molecular exclusion-evaporative light scattering (SEC-ELSD). Among them, fluorescence and SEC-ELSD have high requirements for experimental conditions, which is not conducive to promotion, while spectrophotometry has high sensitivity and is easy to operate, and has the widest application range. The spectrophotometry method can refer to the "3203 Polysorbate-80 Residue Determination Method" stipulated in the third part of the 2020 edition of the "Chinese Pharmacopoeia". The principle is to use the polyethoxy and ammonium cobalt thiocyanate in polysorbate to react to generate a blue complex. The blue complex is soluble in dichloromethane and has specific absorption at 620nm. Within a certain concentration range, the size of the absorption value is positively correlated with the concentration. However, this method requires washing, precipitation, and separation of proteins in the sample. The steps are cumbersome, and air purge concentration is required, which makes the sample easy to lose during the processing process, takes too long, and the accuracy of the test results needs to be improved.

[0004] Chinese invention patent CN2023117470792B discloses a method based on the pharmacopoeia method, which is improved by expanding the concentration range of the standard curve, increasing the volume of the reference substance and the sample, and using a shaker to make the ethanol-sodium chloride solution mix more fully. The method is used to detect the content of polysorbate-80 in protein freeze-dried preparations. However, this method still requires precipitation and separation of the protein in the sample, and the air purge method is used to concentrate the sample. The shaking time of the shaker is as long as 16 to 20 hours, and there are still problems such as cumbersome steps and long time consumption. In Example 1, the absorbance values ​​of the standard solutions of various concentrations used in the method for drawing the standard curve were low, resulting in poor linearity. When the standard curve was established using standard solutions of concentrations of 0 mg / ml (0 μg / ml), 0.010 mg / ml (10 μg / ml), 0.025 mg / ml (25 μg / ml), 0.050 mg / ml (50 μg / ml), 0.075 mg / ml (75 μg / ml), and 0.1 mg / ml (100 μg / ml), the correlation coefficient (R 2 ) is 0.93342~0.98435, which cannot meet R 2 The pharmacopoeia standard is greater than 0.98, and the absorbance values ​​of the control solutions of the same concentration are low and vary greatly. For example, the absorbance values ​​of the corresponding solutions of 0.1 mg / ml (100 μg / ml) are 0.0607, 0.0848, 0.1343, 0.0690, and 0.0826, respectively. In Example 2, improvements were made to increase the concentration range of the control solution (the highest concentration was increased from 0.1 mg / ml to 0.4 mg / ml), and the volume of the control solution and the sample was increased to improve the linearity of the standard curve and the repeatability of the detection data. It is generally believed that when performing spectrophotometric determination, it is more appropriate for the concentration of the sample to be tested to fall near the middle position of the standard curve. Therefore, the above method increases the maximum concentration of the standard curve to 400 μg / ml, which is no longer suitable for detecting samples with lower concentrations (for example, samples below 50 μg / ml). And the optimized detection method still has the problem of low absorbance values ​​of the control solution and the sample. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for detecting the content of polysorbate 20 in a lyophilized protein preparation.

[0006] The present invention relates to a method for detecting the content of polysorbate 20 in a lyophilized protein preparation, comprising the following steps: (1) Preparation of standard solution: Different volumes of polysorbate 20 standard stock solution were taken and diluted with water to prepare standard solutions 1-5, with concentrations of 10 μg / ml, 25 μg / ml, 50 μg / ml, 75 μg / ml and 100 μg / ml, respectively; (2) Preparation of test solution: Take an appropriate amount of the test sample to prepare a test sample solution with a theoretical concentration of polysorbate 20 of 10 to 100 μg / ml; (3) Determination of polysorbate 20 content: Add 2 ml of blank solution, standard solution 1-5 and test solution to each centrifuge tube, add 100 μl of proteinase K mixture, and water bath at 55°C for 1 hour; add 2 ml of ammonium cobalt thiocyanate reagent and 1-2 ml of dichloromethane to each centrifuge tube after water bath; mix upside down at least 60 times, let stand for 15 minutes, and repeat this operation 3 times; mix upside down at least 60 times again, let stand for 30 minutes, take the lower organic phase, and measure the absorbance at a wavelength of 620 nm by spectrophotometry; (4) Data processing: After fitting the linear regression equation of polysorbate 20 concentration and absorbance using the data obtained from standard solutions 1-5, the polysorbate 20 concentration in the test solution was calculated.

[0007] Preferably, in step (1), the preparation method of the polysorbate 20 standard stock solution is as follows: Accurately weigh 0.100 g of polysorbate 20 standard, dissolve it in an appropriate amount of water, and then make up to 100 ml to prepare a 0.1% (w / v) polysorbate 20 standard stock solution.

[0008] Preferably, in step (1), the standard solutions 1-5 are prepared by: 0.10 ml, 0.25 ml, 0.50 ml, 0.75 ml and 1.00 ml of the polysorbate 20 standard stock solution were measured respectively, and the volume was made up to 10 ml to prepare standard solutions 1-5.

[0009] Preferably, the step (3) is specifically as follows: Add 2 ml of blank solution, standard solution 1-5 and test solution to each centrifuge tube, add 100 μl of proteinase K mixture, and incubate in a water bath at 55°C for 1 h. Add 2 ml of ammonium cobalt thiocyanate reagent and 1 ml of dichloromethane to each centrifuge tube after the water bath, mix by inverting at least 60 times, let stand for 15 min, repeat this operation 3 times, and then mix by inverting at least 60 times, let stand for 30 min, take out the lower organic phase, and measure the absorbance at a wavelength of 620 nm by spectrophotometry.

[0010] Preferably, in step (3), the proteinase K mixture is prepared by: Measure TE solution, proteinase K and water in a volume ratio of 5:2:3, vortex to mix, and use immediately.

[0011] Preferably, the TE solution is prepared as follows: Weigh 4.846 g of tris(hydroxymethyl)aminomethane) and 14.890 g of disodium ethylenediaminetetraacetate, dissolve in appropriate amount of water, adjust the pH to 7.8 with 10 mol / L sodium hydroxide and 6 mol / L hydrochloric acid, add water to 100 ml, and store at 2-8°C.

[0012] Compared with the prior art, the advantages and positive effects of the present invention are: (1) Linearity of standard curve (R 2 ) can reach 0.999, which is much higher than the 0.98 required by the pharmacopoeia. The absorbance values ​​of the standard solution and the test solution of the same concentration are relatively close, so the test results are more stable and accurate. (2) The operation steps are simple. During the test, the lower organic phase is directly removed with a pipette, and the test can be quickly completed using a common UV-visible spectrophotometer. There is no need to wash, precipitate, or centrifuge the protein, nor is there any need to use air purge to concentrate the sample. There is no need for long-term (16 to 20 hours) shaking. The entire experimental process only takes 4 to 5 hours. (3) This method is capable of detecting low concentrations of polysorbate 20, with the detection concentration of polysorbate 20 ranging from 10 μg / ml to 100 μg / ml. This method can also be applied to the detection of polysorbate 40, 60, and 80 in protein freeze-dried preparations, with the detection concentrations also ranging from 10 μg / ml to 100 μg / ml. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solution of the embodiment of the utility model, the following is a brief introduction to the drawings required for describing the embodiment: Figure 1 The standard curve diagram of the polysorbate 20 standard solution provided in Example 1; Figure 2 The standard curve diagram of the polysorbate 20 standard solution provided in Example 3; Figure 3 The standard curve diagram of the polysorbate 20 standard solution provided in Example 4; Figure 4 This is a standard curve diagram of the polysorbate 20 standard solution provided in Example 5. DETAILED DESCRIPTION

[0014] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described below in conjunction with embodiments and drawings.

[0015] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments of the following disclosure.

[0016] Example 1 Experiment name: Method validation of polysorbate 20 content detection method (1) Name and purity of reagents used Polysorbate 20 standard (USP), polysorbate 20 (for injection), ammonium thiocyanate (analytical grade), cobalt nitrate hexahydrate (analytical grade), sodium chloride (analytical grade), hydrochloric acid (analytical grade), histidine (pharmaceutical grade), mannitol (analytical grade), sucrose (analytical grade), disodium ethylenediaminetetraacetate (analytical grade), dichloromethane (analytical grade), tris(hydrogenomethylaminomethane) (analytical grade), proteinase K (commercial reagent).

[0017] (2) Solution preparation method Ammonium cobalt thiocyanate reagent: weigh 50g ammonium thiocyanate, 15g cobalt nitrate hexahydrate, and 25g sodium chloride, dissolve in appropriate amount of water, stir thoroughly to dissolve (can be stirred overnight), add water to 250ml; 10mol / L sodium hydroxide solution: weigh 40g of sodium hydroxide and dissolve it in an appropriate amount of water. After dissolution, continue to add water to 100ml; 6mol / L hydrochloric acid: Take 5ml of concentrated hydrochloric acid, add water to 10ml, and mix well; Dilution solution (blank solution): weigh 1.55g L-histidine, 40g D-mannitol, and 20g sucrose, dissolve in appropriate amount of water, adjust pH to 5.0 with 10mol / L sodium hydroxide and 6mol / L hydrochloric acid, add water to 1000ml, and store at 2-8℃; TE solution: weigh 4.846g of Tris (tris(hydroxymethylaminomethane)) and 14.890g of disodium ethylenediaminetetraacetate, dissolve in appropriate amount of water, adjust pH to 7.8 with 10mol / L sodium hydroxide and 6mol / L hydrochloric acid, add water to 100ml, and store at 2-8℃; Proteinase K mixed solution: Each sample requires 50 μL TE solution, 20 μL proteinase K, and 30 μL water. Therefore, the total volume = (number of samples + 1) × 100 μL, that is, 800 μL TE solution, 320 μL proteinase K, and 480 μL water are pipetted, vortexed to mix, and used immediately. (3) Articles and utensils used Volumetric flask, micropipette, disposable filter, centrifuge tube (50ml, 15ml, 1.5ml), test tube rack, lens paper, quartz cuvette (3mm slit).

[0018] (4) Instruments used (5) Preparation of standard stock solution Accurately weigh 0.100 g of polysorbate 20 standard, dissolve it with an appropriate amount of water for injection, and then make up to 100 ml to obtain a 0.1% (w / v) polysorbate 20 standard stock solution; prepare standard solutions 1-5 according to Table 1.

[0019] Table 1 Preparation parameters of standard solutions 1-5 (6) Preparation of test solution Preparation of high-concentration samples (samples containing polysorbate 20 100 μg / ml): take 60 mg of protein (thrombopoietin peptidomimetic stock solution LY20230702, concentration of 4.34 mg / ml, volume of 13.8 ml), 4.8 g of mannitol, 2.4 g of sucrose, 0.1706 g of histidine, and 12 mg of polysorbate 20, dissolve in water for injection, adjust the pH to 5.0 with dilute hydrochloric acid, make up to 120 ml with water for injection, filter and sterilize, and divide into portions for later use.

[0020] Preparation of medium-concentration samples (samples containing polysorbate 20 50μg / ml): take 60mg of protein (thrombopoietin peptidomimetic stock solution LY20230702, concentration of 4.34mg / ml, volume of 13.8ml), 4.8g of mannitol, 2.4g of sucrose, 0.1706g of histidine, and 6mg of polysorbate 20, dissolve in water for injection, adjust the pH to 5.0 with dilute hydrochloric acid, make up to 120ml with water for injection, filter and sterilize, and divide into portions for later use.

[0021] Preparation of low-concentration samples (samples containing polysorbate 20 20μg / ml): take 60mg of protein (thrombopoietin peptidomimetic stock solution LY20230702, concentration of 4.34mg / ml, volume of 13.8ml), 4.8g of mannitol, 2.4g of sucrose, 0.1706g of histidine, and 2.4mg of polysorbate 20, dissolve in water for injection, adjust the pH to 5.0 with dilute hydrochloric acid, make up to 120ml with water for injection, filter and sterilize, and divide into portions for later use.

[0022] (7) Determination Add 2 ml of blank solution, standard solution 1-5 and test solution to each centrifuge tube, add 100 μl of proteinase K mixture, and incubate in a water bath at 55°C for 1 h; add 2 ml of ammonium cobalt thiocyanate reagent and 2 ml of dichloromethane to each centrifuge tube after the water bath; mix by inverting at least 60 times, let stand for 15 min, and repeat this operation 3 times; mix by inverting at least 60 times again, let stand for 30 min, take out the lower organic phase, and measure the absorbance at a wavelength of 620 nm by spectrophotometry.

[0023] (8) Results and analysis Data processing: The linear regression equation was made by taking the polysorbate 20 standard concentration of the standard curve and corresponding to its absorbance. The concentration of polysorbate 20 in the test solution was calculated based on the drawn standard curve. The experimental results are shown in Tables 2, 3 and Figure 1 shown.

[0024] Experimental establishment criteria: determination coefficient R of the standard curve 2 It should be greater than 0.990, the recovery rate should be 80~115%, and the RSD should be less than 6%.

[0025] Table 2 Data summary of standard solutions Table 3 Data summary of test solution From the above results, it can be seen that under this experimental condition, the R 2 The recovery rates of low, medium and high concentration samples were between 96.43% and 103.21%, and the RSDs were 0.87%, 0.98% and 0.78%, respectively, which were all higher than the experimental standard (the determination coefficient of the standard curve R 2 The value should be greater than 0.990, the recovery rate should be 80-115%, and the RSD should be less than 6%). This indicates that the experimental system meets the requirements for method validation.

[0026] Example 2 The difference between this embodiment and embodiment 1 is that: Experiment Name: Validation of polysorbate 20 content detection method (1) Instruments used (2) The standard curve equation for standard solutions 1-5 is: y=0.00163316x-0.000254065; R 2 =0.99983; the experimental results of the test solution are shown in Table 4.

[0027] Table 4 Data summary of test solution From the above data, we can see that the R 2 The recovery rates of low, medium and high concentration samples were between 96.75% and 103.00%, and the RSDs were 1.10%, 3.21% and 1.57%, respectively, which were all higher than the experimental standard (the determination coefficient of the standard curve R 2 The recovery rate should be 80-115% and the RSD should be less than 6%).

[0028] The instrument used in this experiment is a Shimadzu UV-1900 UV-Vis spectrophotometer, which has similar effects to Example 1 (using a Thermo Fisher Evolution One UV-Vis spectrophotometer), indicating that this method can obtain good results using different instruments.

[0029] Example 3 The difference between this embodiment and embodiment 1 is that: Experimental name: Detection of the content of thrombopoietin peptidomimetic polysorbate 20 for injection (1) Preparation of test solution: Take 8 samples (i.e., finished products) from three batches (LC20230901, LC20231102, and LC20231203), add 580 μL of water to each finished product, fully dissolve, and combine 4 samples into one test solution, i.e., 2 parallel samples for each batch; (2) Experimental establishment criteria: correlation coefficient R of the standard curve 2 should be greater than 0.990, and the polysorbate 20 content of the preparation sample should be 20μg / ml~60μg / ml (theoretical value is 40μg / ml); the experimental results are shown in Tables 5, 6 and Figure 2 shown.

[0030] Table 5 Data summary of standard solution Table 6 Data summary of test solution From the above data, we can see that the standard curve R 2 =0.99972, the polysorbate 20 test results of 6 parallel samples from three batches were 35.24860-39.46936 μg / ml, with an average value of 36.77 μg / ml (close to the theoretical value of 40 μg / ml), and an RSD of 3.72%, all of which met the experimental establishment criteria.

[0031] Example 4 The difference between this embodiment and embodiment 1 is that: Experiment name: Optimization of polysorbate 20 content detection method (reducing the volume of dichloromethane by half) (1) Experimental purpose: To optimize the polysorbate 20 content detection method to improve the accuracy of low-concentration sample detection.

[0032] (2) Preparation of test solution Preparation of low-concentration samples (samples containing polysorbate 20 20 μg / ml): weigh 4.8 g of mannitol, 2.4 g of sucrose, 0.1706 g of histidine, and 2.4 mg of polysorbate 20, dissolve in water for injection, adjust the pH to 5.0 with dilute hydrochloric acid, make up to 120 ml with water for injection, filter and sterilize, and divide into packages for later use; batch number is 240927-007-02.

[0033] (3) Determination Add 2 ml of blank solution (diluted solution), standard solutions 1-5 and test solution to each centrifuge tube, add 100 μl of proteinase K mixture, and incubate in a water bath at 55°C for 1 hour; add 2 ml of ammonium cobalt thiocyanate reagent and 1 ml of dichloromethane to each centrifuge tube after the water bath; mix by inverting at least 60 times, let stand for 15 minutes, and repeat this operation 3 times; mix by inverting at least 60 times again, let stand for 30 minutes, take out the lower organic phase, and measure the absorbance at a wavelength of 620 nm by spectrophotometry.

[0034] (4) Experimental results and analysis The concentration (μg / ml) of each sample was measured, and the recovery rate and RSD (%) were calculated. The data are summarized in Tables 7, 8 and Figure 3 .

[0035] Table 7 Data summary of standard solution Table 8 Data summary of test solution From the above table, we can see that the standard curve R 2 =0.99946, the recovery rate was 96.38%~100.86%, and the RSD of 6 parallel samples was 1.69%, all of which met the experimental establishment criteria; and when other experimental conditions remained unchanged, the volume of the organic solvent (dichloromethane) used for extraction was halved, resulting in an increase in the absorbance of the standard curve and the test sample (about twice the original), further improving the accuracy of the detection.

[0036] Example 5 The difference between this embodiment and embodiment 1 is that: Experimental name: Detection of polysorbate 20 content in thrombopoietin peptidomimetic for injection (1) Experimental purpose: To detect the content of polysorbate 20 in three batches of thrombopoietin peptidomimetics for injection (2) Preparation of test solution Take 12 vials of thrombopoietin peptidomimetics for injection with batch numbers LC20230901, LC20231102, and LC20231203, respectively, add 0.58 ml of water to each vial, fully dissolve, and combine four vials into one sample, that is, measure three parallels for each batch.

[0037] (3) Determination Add 2 ml of blank solution (diluted solution), standard solutions 1-5 and test solution (three parallels for each batch) to each centrifuge tube, add 100 μl of proteinase K mixture, and water bath at 55°C for 1 hour; add 2 ml of ammonium cobalt thiocyanate reagent and 1 ml of dichloromethane to the centrifuge tube after the water bath; mix by inverting at least 60 times, let stand for 15 minutes; repeat this operation 3 times; mix by inverting at least 60 times again, let stand for 30 minutes, take out the lower organic phase for detection, conduct the experiment according to the spectrophotometric method, use the blank solution as the control, and measure the absorbance at 620 nm.

[0038] (4) Experimental establishment criteria: correlation coefficient R of the standard curve 2 It should be greater than 0.990; the RSD% of the test values ​​of three parallel samples in each batch should be less than 6%.

[0039] (5) Experimental results and analysis The concentration (μg / ml) of each sample was measured, and the average value and RSD (%) were calculated. The data are summarized in Tables 9, 10 and Figure 4 .

[0040] Table 9 Data summary of standard solution Table 10 Data summary of test solution From the above data, we can see that the standard curve R 2=0.99937, which met the experimental establishment standard; the average value of three parallel samples in batch LC20230901 was 34.07μg / ml, and the intra-batch RSD was 0.52%; the average value of three parallel samples in batch LC20231102 was 36.70μg / ml, and the intra-batch RSD was 2.77%; the average value of three parallel samples in batch LC20231203 was 36.38μg / ml, and the intra-batch RSD was 1.02%; the polysorbate 20 content test results of 9 parallel samples in three batches were 33.92346μg / ml~37.44229μg / ml, with an average value of 35.72μg / ml and an RSD of 3.72%, all of which met the quality standards.

[0041] The above description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes for application in other fields. However, any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for detecting the content of polysorbate 20 in a lyophilized protein preparation, characterized in that: The following steps are involved: (1) Preparation of standard solution: Different volumes of polysorbate 20 standard stock solution were taken and diluted with water to prepare standard solutions 1-5, with concentrations of 10 μg / ml, 25 μg / ml, 50 μg / ml, 75 μg / ml and 100 μg / ml, respectively; (2) Preparation of test solution: Take an appropriate amount of the test sample to prepare a test sample solution with a theoretical concentration of polysorbate 20 of 10 to 100 μg / ml; (3) Determination of polysorbate 20 content: Add 2 ml of blank solution, standard solution 1-5 and test solution to each centrifuge tube, add 100 μl of proteinase K mixture, and water bath at 55°C for 1 hour; add 2 ml of ammonium cobalt thiocyanate reagent and 1-2 ml of dichloromethane to each centrifuge tube after water bath; mix upside down at least 60 times, let stand for 15 minutes, and repeat this operation 3 times; mix upside down at least 60 times again, let stand for 30 minutes, take the lower organic phase, and measure the absorbance at a wavelength of 620 nm by spectrophotometry; (4) Data processing: After fitting the linear regression equation of polysorbate 20 concentration and absorbance using the data obtained from standard solutions 1-5, the polysorbate 20 concentration in the test solution was calculated.

2. The method for detecting the content of polysorbate 20 in a lyophilized protein preparation according to claim 1, characterized in that: In the step (1), the preparation method of the polysorbate 20 standard stock solution is as follows: Accurately weigh 0.100 g of polysorbate 20 standard, dissolve it in an appropriate amount of water, and then make up to 100 ml to prepare a 0.1% (w / v) polysorbate 20 standard stock solution.

3. The method for detecting the content of polysorbate 20 in a lyophilized protein preparation according to claim 1, characterized in that: In step (1), the preparation method of the standard solutions 1-5 is as follows: 0.10 ml, 0.25 ml, 0.50 ml, 0.75 ml and 1.00 ml of the polysorbate 20 standard stock solution were measured respectively, and the volume was made up to 10 ml to prepare standard solutions 1-5.

4. The method for detecting the content of polysorbate 20 in a lyophilized protein preparation according to claim 1, characterized in that: The step (3) is specifically as follows: Add 2 ml of blank solution, standard solution 1-5 and test solution to each centrifuge tube, add 100 μl of proteinase K mixture, and incubate in a water bath at 55°C for 1 h. Add 2 ml of ammonium cobalt thiocyanate reagent and 1 ml of dichloromethane to each centrifuge tube after the water bath, mix by inverting at least 60 times, let stand for 15 min, repeat this operation 3 times, and then mix by inverting at least 60 times, let stand for 30 min, take out the lower organic phase, and measure the absorbance at a wavelength of 620 nm by spectrophotometry.

5. The method for detecting the content of polysorbate 20 in a lyophilized protein preparation according to claim 1, characterized in that: In step (3), the proteinase K mixture is prepared by: Measure TE solution, proteinase K and water in a volume ratio of 5:2:3, vortex to mix, and use immediately.

6. The method for detecting the content of polysorbate 20 in a lyophilized protein preparation according to claim 4, characterized in that: The configuration method of the TE solution is: Weigh 4.846 g of tris(hydroxymethyl)aminomethane) and 14.890 g of disodium ethylenediaminetetraacetate, dissolve in appropriate amount of water, adjust the pH to 7.8 with 10 mol / L sodium hydroxide and 6 mol / L hydrochloric acid, add water to 100 ml, and store at 2-8°C.

Images