Method for detecting Tween 20 in protein preparation

Abstract

The invention relates to a colorimetric detection method for Tween-20 in a protein preparation, which comprises the following steps: diluting the protein preparation with a diluent for enzymolysis, carrying out chromogenic reaction by using dichloromethane and an ammonium iron thiocyanate system, and measuring absorbance by using a visible spectrophotometer. The diluent is composed of other components except protein and Tween 20 in the protein preparation; an enzyme used in the enzymolysis is protease K; the concentration of the protease K is preferably 200 mu g / ml; the enzymolysis temperature is preferably 55-57 DEG C; the enzymolysis time is preferably 3.5 to 4 hours.

Description

Technical Field

[0001] The invention relates to a method for detecting Tween 20 in a protein preparation, and more particularly to a colorimetric detection method for Tween 20 in a protein preparation. Background Art

[0002] Polysorbate 20 (Tween 20) is a common nonionic surfactant with both hydrophobic and hydrophilic properties. Its chemical composition is polyoxyethylene sorbitan fatty acid ester. Its hydrophobic properties are provided by hydrocarbon chains, and its hydrophilic properties come from the bound ethylene oxide. Proteins are easily adsorbed on the surface of containers or exist at the interface, resulting in instability or changes in their secondary structure. The protective effect of Tween 20 on proteins in solution is due to its amphiphilic molecular properties. Its hydrophobic end will rearrange at the gas-liquid interface and the solid-liquid interface, and its hydrophilic end will be exposed to the molecular layer in the water, which can compete with protein molecules and prevent them from being adsorbed on the solid interface or exposed at the gas-liquid interface, thereby effectively preventing protein denaturation and forming aggregates that lose activity. Therefore, it is widely used in protein drug preparations.

[0003] Modern pharmacological studies have shown that polysorbates have certain biological and pharmacological activities in addition to being solubilizers and stabilizers in drugs. Therefore, for the sake of drug safety, their content in drug preparations should be strictly controlled. Tween20 has no strong chromophore, and conventional HPLC-UV methods cannot accurately quantify it, so its limit control method is challenging. Currently, the commonly used methods for determining the content of polysorbates in preparations include cobalt thiocyanate colorimetry, molecular exclusion chromatography-evaporative light scattering, and HPLC-fluorescence method, which are included in the Chinese Pharmacopoeia and reported in the literature.

[0004] The current detection method of Tween 20 in protein preparations cannot be adapted to preparations with higher protein concentrations. When the protein concentration is high, the use of corresponding detection methods is likely to cause interference with the color development reaction, resulting in low test results. Summary of the invention

[0005] The invention provides a colorimetric detection method of Tween 20 in a protein preparation. The detection method has good accuracy and repeatability, good specificity, other preparation components except polysorbate 20 substantially have no interference on the detection result, and good linearity.

[0006] The present invention solves the above technical problems through the following technical solutions:

[0007] The invention provides a method for detecting Tween 20 in a protein preparation, which is characterized in that the protein preparation is firstly diluted with a diluent for enzymatic hydrolysis, a color development reaction is performed with a dichloromethane and ammonium thiocyanate iron system, and the absorbance is measured with a visible spectrophotometer.

[0008] The diluent described in the present invention is composed of other components of the protein preparation except protein and Tween 20.

[0009] The diluent in the present invention comprises citric acid, sucrose, and arginine, and the pH is adjusted to 6-8 by hydrochloric acid; further, the diluent comprises 1.5-3.5g citric acid, 30-60g sucrose, and 15-20g arginine, and the pH is adjusted to 6-8 by hydrochloric acid; further, the diluent is 2.1g citric acid, 50.0g sucrose, and 17.42g arginine, and the pH is adjusted to 7.7 by hydrochloric acid.

[0010] The protein in the protein preparation of the present invention is shown in Sequence 1; the protein concentration of the protein preparation is 20 mg / ml-120 mg / ml; preferably, the protein concentration is 20 mg / ml, 40 mg / ml, 80 mg / ml, and 120 mg / ml.

[0011] The enzyme used in the enzymatic hydrolysis of the present invention is proteinase K; the concentration of the proteinase K is preferably 200 μg / ml; the temperature of the enzymatic hydrolysis is preferably 55-57° C.; and the enzymatic hydrolysis time is preferably 3.5 h-4 h.

[0012] The pre-treatment steps of the protein preparation in the present invention are as follows:

[0013] 1) Dilute the protein preparation to 20 mg / ml with a diluent, add an enzymatic agent diluted with a buffer solution, and perform enzymatic hydrolysis in a water bath;

[0014] 2) Add dichloromethane and ferric ammonium thiocyanate solution to the solution in step 1), mix well, and discard the upper layer.

[0015] The diluent in step 1) of the present invention is composed of other components in the protein preparation except protein and Tween 20; further, the diluent contains citric acid, sucrose, and arginine, and the pH is adjusted to 6-8 by hydrochloric acid; further, the diluent contains 1.5-3.5g citric acid, 30-60g sucrose, 15-20g arginine, and the pH is adjusted to 6-8 by hydrochloric acid; further, the diluent is 2.1g citric acid, 50.0g sucrose, 17.42g arginine, and the pH is adjusted to 7.7 by hydrochloric acid.

[0016] The buffer in step 1) of the present invention is pH 8.5 Tris buffer.

[0017] The enzymatic agent in step 1) of the present invention is proteinase K; the concentration of the proteinase K is 200 μg / ml.

[0018] The time of water bath enzymolysis in step 1) of the present invention is 3.5h to 4h.

[0019] The temperature of water bath enzymolysis in step 1) of the present invention is 55-57°C.

[0020] In the step 2) of the present invention, the volume ratio of dichloromethane to ammonium ferric thiocyanate is 2:3.

[0021] The steps of the detection method described in the present invention are as follows:

[0022] 1) Take the protein preparation and dilute it to 20 mg / ml with a diluent consisting of the other components of the protein preparation except protein and Tween 20, add pH 8.5 Tris buffer containing 200 μg / ml proteinase K, mix well, and enzymolyze in a water bath at 55-57°C for 3.5h-4h;

[0023] 2) adding dichloromethane and ammonium thiocyanate iron solution to the solution of step 1), mixing well, and discarding the upper layer;

[0024] 3) Add dichloromethane and ferric ammonium thiocyanate solution to polysorbate 20 of different concentrations, measure the absorbance of the mixed solution at 510 nm with a visible spectrophotometer, and draw a standard curve with the polysorbate 20 reference substance content as the X-axis and the corresponding absorbance value at a wavelength of 510 nm as the Y-axis;

[0025] 4) Measure the absorbance of the lower layer liquid in step 2) at 510 nm using a visible spectrophotometer, and calculate the concentration of Tween 20 in the protein preparation based on the standard curve obtained in step 3).

[0026] The diluent in step 1) of the present invention is 2.1 g citric acid, 50.0 g sucrose, and 17.42 g arginine, and the pH is adjusted to 7.7 with hydrochloric acid.

[0027] The components of the pH 8.5 Tris buffer in step 1) of the present invention are Tris and sodium chloride; preferably, the ratio of Tris to sodium chloride is 1:4.83.

[0028] The detection method of the present invention has good specificity, and other preparation ingredients except polysorbate 20 have basically no interference with the detection result; and the accuracy and repeatability are good. Specific embodiments

[0029] 1. Reagent preparation

[0030] Ammonium cobalt thiocyanate solution: weigh 6.0 g of cobalt nitrate and 40.0 g of ammonium thiocyanate, dissolve in water and dilute to 200 ml.

[0031] Ammonium thiocyanate iron solution: Weigh 2.7g ferric chloride hexahydrate and 3.0g ammonium thiocyanate, dissolve in water and dilute to 100ml. pH8.5 Tris buffer: Weigh 1.21g Tris and 5.84g sodium chloride, dissolve in 800ml water, adjust pH to 8.5, add water to 1000ml, filter with a 0.45μm or less pore size filter before use.

[0032] Diluent: All components in the protein preparation except Tween 20 and protein should be filtered through a 0.2μm filter membrane before use.

[0033] Polysorbate 20 reference substance working solution: weigh 1.016 g of polysorbate 20 into a 100 ml volumetric flask, dilute to the mark with ultrafiltration buffer, and mix well.

[0034] 2. Protein preparation prescription

[0035] The prescription of the protein preparation is: fusion protein, polysorbate 20, citric acid, arginine, sucrose, and the pH is adjusted to 7.7 with hydrochloric acid, wherein the amounts of citric acid, arginine, and sucrose are 2.1 g, 17.42 g, and 50.0 g, respectively, the content of polysorbate 20 is 500 μg / ml, and the protein sequence is shown in sequence 1.

[0036] Example 1: Study on high concentration proteolysis

[0037] 1) Sample processing steps

[0038] Take 200μl of 80mg / ml and 120mg / ml protein preparations respectively in 15ml centrifuge tubes, add 800μl of proteinase K diluted with pH8.5 Tris buffer, the concentration of proteinase K is shown in Table 1, mix well, and enzymolyze in a water bath at 55-57℃ for 2h. Add 2.0ml of dichloromethane and 3.0ml of cobalt thiocyanate ammonium solution (see reagent preparation) to the test sample centrifuge tubes, mix well, and place at room temperature. Oscillate and mix once every 15 minutes, at least 4 times, let stand for at least 30 minutes before measurement, and discard the upper layer.

[0039] 2) Reference material processing steps

[0040] Accurately pipette 0μl, 20μl, 40μl, 60μl, 80μl, and 100μl of polysorbate 20 reference substance into clean 15ml centrifuge tubes, add the pre-prepared diluent in the reagent preparation to make the final volume 200μl, add 800μl of proteinase K diluted with pH8.5 Tris buffer, the concentration of proteinase K is 200μg / ml, mix well, and enzymolyze in a water bath at 55-57℃ for 2h. Add 2.0ml of dichloromethane and 3.0ml of ammonium cobalt thiocyanate solution (see reagent preparation) to each centrifuge tube, mix well, and place at room temperature. Oscillate and mix once every 15 minutes, at least 4 times, let stand for at least 30 minutes before measurement, and discard the upper layer.

[0041] 3) Detection steps:

[0042] The treated test and reference substances were measured at 620nm with dichloromethane as blank control for absorbance. Result calculation: With the polysorbate 20 reference substance content (μg) as the X-axis and its corresponding absorbance value at 620nm wavelength as the Y-axis, a linear regression was performed, and the absorbance value of the test substance was brought into the standard curve to calculate the measured value, and the polysorbate 20 content of the test substance was calculated according to the following formula: Sample polysorbate 20 content (μg / ml) = measured value × 5 × dilution multiple.

[0043] The protein concentration was 80 or 120 mg / ml, respectively. Proteinase K was unable to completely digest the excess protein into small peptides that did not interfere with the detection within a certain period of time. The interference of the protein was attempted to be eliminated by increasing the concentration of proteinase K. Table 1 shows the results of the polysorbate 20 content detection in the samples under the conditions of different concentrations of proteinase K treatment. As can be seen from Table 1, the polysorbate 20 detection values ​​in the samples with two protein concentrations (80 mg / ml and 120 mg / ml) were both low. With the increase of proteinase K concentration, although the polysorbate 20 content detection value had a certain recovery, it was still lower than the lower limit of the quality standard of 350 μg / ml. In the color development process of the cobalt thiocyanamide-dichloromethane color development system, it was found that serious emulsification occurred, and the emulsification phenomenon became more serious with the increase of concentration; for example, for the 120 mg / ml sample, even if the proteinase K concentration was increased 5 times to 1000 ug / mL, the emulsification phenomenon was still obvious.

[0044] Table 1 Detection results of polysorbate 20 in samples under different concentrations of proteinase K solution

[0045]

[0046] Example 2 Study on color reaction system

[0047] For protein preparations with a protein concentration of 80 mg / ml and 120 mg / ml, the protein concentration was diluted to 20 mg / ml with purified water before detection, and proteinase K was diluted to 200 μg / ml with pH 8.5 Tris buffer. Except that the color developing solution was changed from cobalt ammonium thiocyanate to iron ammonium thiocyanate (see reagent preparation), other conditions were the same as in Example 1, and the relevant detection results are shown in Table 2. As can be seen from Table 2, the polysorbate 20 detection values ​​of all samples were low (theoretical value 500 μg / ml).

[0048] Table 2 Results of polysorbate 20 content detection in the test samples under the ammonium thiocyanate iron-dichloromethane color reaction system

[0049] Protein concentration Polysorbate 20 (μg / ml) 80mg / ml 241 120mg / ml 131

[0050] Example 3 Water bath time inspection

[0051] On the basis of Example 2, the test sample was diluted to 20 mg / ml with a diluent (see reagent preparation), and the effects of different water bath enzymatic hydrolysis times of 2 h, 3.5 h, and 4 h in the test sample treatment step in Example 2 on the content of polysorbate 20 in the high-concentration protein preparation were investigated, and the results are shown in Table 3. The RSD% of the polysorbate 20 content detection results in the protein preparation at the water bath enzymatic hydrolysis time of 3.5 h and 4 h was 1.7% to 3.5%.

[0052] Table 3 Test results of different water bath time

[0053]

[0054] Example 4 Detection method

[0055] 1) Sample processing steps

[0056] Take protein preparation (40mg / ml, 80mg / ml, 120mg / ml) and dilute it to 20mg / ml with diluent (see reagent preparation). Take 200μl of the diluted sample in a 15ml centrifuge tube, add 800μl of proteinase K diluted to a concentration of 200ug / ml by pH8.5 Tris buffer, mix well, and enzymolyze in a water bath at 55-57℃ for 3.5h-4h. Add 2.0ml of dichloromethane and 3.0ml of ammonium thiocyanate iron solution (see reagent preparation) to the test sample centrifuge tube, mix well, and place at room temperature. Oscillate and mix once every 15 minutes, at least 4 times, let it stand for at least 30 minutes before measurement, and discard the upper layer.

[0057] 2) Reference material processing steps

[0058] Accurately pipette 0μl, 20μl, 40μl, 60μl, 80μl, and 100μl of polysorbate 20 reference substance into clean 15ml centrifuge tubes, add the pre-prepared diluent (see reagent preparation) to make the final volume 200μl, add 800μl of proteinase K diluted to a concentration of 200ug / ml by pH8.5 Tris buffer, mix well, and enzymolyze in a water bath at 55-57℃ for 3.5h-4h. Add 2.0ml of dichloromethane and 3.0ml of ammonium thiocyanate iron solution (see reagent preparation) to each centrifuge tube, mix well, and place at room temperature. Oscillate and mix once every 15 minutes, at least 4 times, let stand for at least 30 minutes before measurement, and discard the upper layer.

[0059] 3) Detection steps

[0060] The treated test and reference substances were measured at 510nm with dichloromethane as blank control. Result calculation: With the polysorbate 20 reference substance content (μg) as the X-axis and its corresponding absorbance value at 510nm wavelength as the Y-axis, a linear regression was performed, and the absorbance value of the test substance was brought into the standard curve to calculate the measured value, and the polysorbate 20 content of the test substance was calculated according to the following formula: sample polysorbate 20 content (μg / ml) = measured value × 5 × dilution multiple. The test results are shown in the following table.

[0061] Table 4 Test results of polysorbate 20 in test solution

[0062]

[0063]

[0064] Example 5 Methodology Verification

[0065] 1. Exclusive research

[0066] Sample preparation: A protein sample with a protein concentration of 122 mg / ml was prepared according to the protein preparation prescription (except Tween 20), and the protein sample was diluted to 20 mg / ml with a diluent (see reagent preparation), and tested according to the method of Example 4, and 3 batches were tested. The results are shown in Table 5. The detection value of the polysorbate 20 content of the specific sample is in the range of -2.6 μg / ml to 0.1 μg / ml, which is very small (less than 0.6%) compared to the polysorbate 20 content (500 μg / ml) in the protein preparation, so the method has good specificity.

[0067] Table 5 Specificity sample test results

[0068] sample Absorbance value Polysorbate 20 (μg / ml) Sample-1 0.0189 -2.6 Sample-2 0.0192 -2.2 Sample-3 0.0212 0.1

[0069] 2. Accuracy and repeatability study

[0070] 1. Prepare protein samples with a protein concentration of 122 mg / ml according to the protein preparation prescription (except Tween 20), and dilute the protein samples to different protein concentrations with diluents (see reagent preparation) according to Table 6; 2. The sample preparation process of medium concentration samples (500 μg / ml) and high concentration samples (780 μg / ml) is shown in Table 7. As shown in Table 8, the recovery rates of samples with different polysorbate 20 concentrations are 91% to 102%, and the repeatability RSD% is 2% to 3.7%, indicating that the accuracy and repeatability of this method are good.

[0071] Table 6 Dilution steps

[0072]

[0073] Table 7 Accuracy sample preparation process

[0074]

[0075]

[0076] Table 8 Accuracy and repeatability test results

[0077]

Claims

1. A method for detecting Tween 20 in a protein preparation, characterized in that: The protein preparation is first diluted with a diluent for enzymatic hydrolysis, and then a color reaction is carried out with a dichloromethane and ammonium thiocyanate iron system, and the absorbance is measured with a visible spectrophotometer.

2. The detection method according to claim 1, characterized in that: The diluent is composed of other components in the protein preparation except protein and Tween 20; preferably, the diluent contains citric acid, sucrose, arginine, and hydrochloric acid is used to adjust the pH to 6-8.

3. The detection method according to claim 1, characterized in that: The protein in the protein preparation is shown in Sequence 1; the protein concentration of the protein preparation is 20 mg / ml-120 mg / ml; preferably 20 mg / ml, 40 mg / ml, 80 mg / ml, 120 mg / ml.

4. The detection method according to claim 1, characterized in that: The enzyme used in the enzymatic hydrolysis is proteinase K; the concentration of the proteinase K is preferably 200 μg / ml; the temperature of the enzymatic hydrolysis is preferably 55-57° C.; and the enzymatic hydrolysis time is preferably 3.5 h to 4 h.

5. The detection method according to claim 1, characterized in that: The pre-treatment steps of the protein preparation are as follows: 1) Dilute the protein preparation to 20 mg / ml with a diluent, add an enzymatic agent diluted with a buffer solution, and perform enzymatic hydrolysis in a water bath; 2) Add dichloromethane and ammonium thiocyanate iron solution to the solution in step 1), mix well, and discard the upper layer.

6. The detection method according to claim 5, characterized in that: The buffer in step 1) is pH 8.5 Tris buffer.

7. The detection method according to claim 5, characterized in that: The enzymatic agent in step 1) is proteinase K; the concentration of proteinase K is 200 μg / ml.

8. The detection method according to claim 5, characterized in that: The time of water bath enzymolysis in step 1) is 3.5h to 4h.

9. The detection method according to claim 5, characterized in that: The temperature of the water bath enzymolysis in step 1) is 55-57°C.

10. The detection method according to claim 5, characterized in that: In the step 2), the volume ratio of dichloromethane to ferric ammonium thiocyanate is 2:

3.

11. The detection method according to claims 1-10, characterized in that: The steps of the detection method are as follows: 1) Take the protein preparation and dilute it to 20 mg / ml with a diluent containing citric acid, sucrose, arginine, and hydrochloric acid to adjust the pH to 6-8, add pH8.5 Tris buffer containing 200 μg / ml proteinase K, mix well, and enzymolyze in a water bath at 55-57°C for 3.5h-4h; 2) adding dichloromethane and ammonium thiocyanate iron solution to the solution of step 1), mixing well, and discarding the upper layer; 3) Add dichloromethane and ferric ammonium thiocyanate solution to polysorbate 20 of different concentrations, measure the absorbance of the mixed solution at 510 nm with a visible spectrophotometer, and draw a standard curve with the polysorbate 20 reference substance content as the X-axis and the corresponding absorbance value at a wavelength of 510 nm as the Y-axis; 4) Measure the absorbance of the lower layer liquid in step 2) at 510 nm using a visible spectrophotometer, and calculate the concentration of Tween 20 in the protein preparation based on the standard curve obtained in step 3).

Images