Protease residue detection method

Abstract

The invention relates to a protease residue detection method which comprises the following steps: step 1, sample preparation: collecting a corresponding sample according to the type of protease to be detected to obtain a sample to be tested; step 2, reagent preparation; 3, setting a detection method, and sampling by using a wiping sampling method; step 4, signal reading: reading a signal generated by reaction through detection equipment, recording the signal reading of each sample, and providing original data for subsequent data analysis; and 5, data analysis. The detection method disclosed by the invention is rapid and convenient, the analysis and inspection time is shortened, and the detection method also has important significance in the aspect of maintenance of the cleaning verification state.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for detecting residual protease. Background Art

[0002] Cleaning validation is to collect enough data through scientific methods to prove that the equipment can consistently achieve the expected cleaning effect after cleaning according to the prescribed method. According to the relevant requirements of GMP, enterprises must validate the cleaning methods of pharmaceutical equipment to confirm the effectiveness of the cleaning methods and confirm their cleaning effects, so as to effectively prevent contamination and cross-contamination.

[0003] The analytical method used to detect residues or contaminants should be specific, effective and sensitive to the substance being analyzed. At present, the determination of protease residues is carried out by mass spectrometry, liquid chromatography, liquid chromatography-mass spectrometry, ultraviolet-visible spectrophotometry, etc. These methods ① have low sensitivity (the detection limit is generally above mg / L level), ② the detection of a single sample takes a long time, and ③ some samples require pretreatment, which brings inevitable errors to the detection, low sensitivity, cumbersome operation, and time-consuming determination. Therefore, it is necessary to establish a new set of detection methods that can detect residual proteases and have high sensitivity. Summary of the invention

[0004] (I) Purpose of the invention

[0005] In order to overcome the above shortcomings, the purpose of the present invention is to provide a method for detecting residual protease to solve the above technical problems.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the technical solutions provided by this application are as follows:

[0008] A method for detecting residual protease comprises the following steps:

[0009] Step 1: Sample preparation: according to the type of protease to be detected, collect the corresponding samples, and process the samples according to the characteristic pretreatment method to obtain the test samples;

[0010] Step 2: Reagent preparation: prepare 5 ppm potassium hydrogen phthalate standard solution, 10% persulfate solution, 20% phosphoric acid solution and ultrapure water;

[0011] Step 3: Setting up the detection method: when testing the test sample, add an acid and an oxidant to the TOC total organic carbon analyzer. The acid is phosphoric acid, and the oxidant is sodium persulfate. The prepared solution needs to be ultrasonicated for 5 minutes before sampling to remove dissolved CO2, and the wiping sampling method is used for sampling;

[0012] Step 4: Signal reading: reading the signal generated by the reaction through the detection equipment and recording the signal reading of each sample to provide raw data for subsequent data analysis;

[0013] Step 5 data analysis, calculating the residual amount of protease based on the signal readings, including establishing a standard curve, signal calibration, calculating the residual amount and statistical analysis.

[0014] Preferably, the preparation of the 5 ppm potassium hydrogen phthalate standard solution in step 2 specifically includes the following steps: taking 0.5 ml of potassium hydrogen phthalate standard solution in a 100 ml volumetric flask, adding ultrapure water to dilute to the scale line, shaking well, ultrasonicating for 5 minutes, and then transferring to a 100 ml TOC brown injection bottle.

[0015] Preferably, the preparation of the 10% persulfate solution in step 2 specifically includes the following steps: weigh 12.5 g of sodium persulfate into a 250 ml reagent bottle, take 4.5 ml of 85% phosphoric acid and 106.5 ml of ultrapure water into the reagent bottle, shake well to dissolve, and remove dissolved CO2 by ultrasonic cleaning in an ultrasonic cleaning machine for 5 minutes.

[0016] Preferably, the preparation of the 20% phosphoric acid solution in step 2 specifically includes the following: respectively measuring 18.5 ml of 85% phosphoric acid and 94 ml of ultrapure water into a 250 ml reagent bottle, shaking well, and ultrasonically cleaning in an ultrasonic cleaning machine for 5 minutes to remove dissolved CO2.

[0017] Preferably, in step 2, the ultrapure water is placed in a special reagent bottle and ultrasonicated in an ultrasonic cleaning machine for 5 minutes to remove dissolved CO2.

[0018] Preferably, in the step 3, the Clean and Blank samples are tested first, and after the absorbance values ​​of the Reagentblank and Acidblank in the Blank samples are within an acceptable range, the linearity of the standard curve and the sample are tested, and the correlation coefficient r is greater than 0.995.

[0019] Preferably, the acceptable range is Reagentblank≤7.0Abs, Acidblank≤5.0Abs.

[0020] Preferably, the swabbing sampling method in step 3 is specifically as follows:

[0021] S1 takes three cotton swabs washed with ultrapure water and squeezes them against the wall of the test tube to remove excess water;

[0022] S2 Take one of the cotton swabs, press the cotton swab head on the sampling surface, bend it slightly, and wipe the sampling surface steadily and slowly. The wiping route is a horizontal S shape. Then use the reverse side of the cotton swab to wipe the same sampling surface. The wiping route is perpendicular to the last wiping route, which is a vertical S line. The whole surface should be covered in the process.

[0023] After S3 is wiped, the cotton part of the cotton swab is snapped off at the mouth of the test tube corresponding to the sampling point, and the head of the cotton swab falls into the stoppered test tube;

[0024] S4 takes another two cotton swabs and repeats the steps of S2 to S3, and puts them into the same sampling tube;

[0025] S5 Place the sampling cotton swab test tube in the test tube rack, add 20 ml of ultrapure water, ultrasonicate for 10 min as the test solution, mix well, and test.

[0026] Beneficial effects:

[0027] The detection method of the present invention is fast and convenient, reduces analysis and inspection time, and is also of great significance in maintaining the cleaning validation status. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the TOC standard curve diagram of the present invention. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0030] The present invention provides a method for detecting residual protease, characterized in that it comprises the following steps:

[0031] Step 1: Sample preparation: according to the type of protease to be detected, collect the corresponding samples, and process the samples according to the characteristic pretreatment method to obtain the test samples;

[0032] Step 2: Reagent preparation: prepare 5 ppm potassium hydrogen phthalate standard solution, 10% persulfate solution, 20% phosphoric acid solution and ultrapure water;

[0033] Step 3: Setting up the detection method: when testing the test sample, add an acid and an oxidant to the TOC total organic carbon analyzer. The acid is phosphoric acid, and the oxidant is sodium persulfate. The prepared solution needs to be ultrasonicated for 5 minutes before sampling to remove dissolved CO2, and the wiping sampling method is used for sampling;

[0034] Step 4: Signal reading: reading the signal generated by the reaction through the detection equipment and recording the signal reading of each sample to provide raw data for subsequent data analysis;

[0035] Step 5 data analysis, calculating the residual amount of protease based on the signal readings, including establishing a standard curve, signal calibration, calculating the residual amount and statistical analysis.

[0036] The preparation of 5ppm potassium hydrogen phthalate standard solution specifically includes the following steps: take 0.5ml of potassium hydrogen phthalate standard solution in a 100ml volumetric flask, add ultrapure water to dilute to the scale line, shake well, ultrasonicate for 5 minutes, and then transfer to a 100ml TOC brown injection bottle.

[0037] The preparation of the 10% persulfate solution in step 2 specifically includes the following steps: weigh 12.5 g of sodium persulfate into a 250 ml reagent bottle, take 4.5 ml of 85% phosphoric acid and 106.5 ml of ultrapure water into the reagent bottle, shake well to dissolve, and remove dissolved CO2 by ultrasonic cleaning for 5 minutes in an ultrasonic cleaning machine.

[0038] The preparation of 20% phosphoric acid solution specifically includes the following steps: 18.5 ml of 85% phosphoric acid and 94 ml of ultrapure water are respectively measured in a 250 ml reagent bottle, shaken well, and ultrasonicated in an ultrasonic cleaning machine for 5 minutes to remove dissolved CO2.

[0039] Ultrapure water was placed in a special reagent bottle and ultrasonicated in an ultrasonic cleaning machine for 5 min to remove dissolved CO2.

[0040] In the step 3, during the detection, the Clean and Blank tests are first performed, and after the absorbance values ​​of Reagentblank and Acidblank in Blank are within an acceptable range, the acceptable range is Reagentblank≤7.0Abs, Acidblank≤5.0Abs, and then the linearity of the standard curve and the sample are detected, and the correlation coefficient r is greater than 0.995.

[0041] The detection temperature in step 3 is room temperature, the detection time is 5-15 minutes, the sample volume is 9 ml, the dilution ratio is 1:1, the acid volume is 0.5 ml, the reagent volume is 0.8 ml, the ion chromatography purge time is 0.50 minutes, the detector scanning flow rate is 500 ml / min, the pre-purge time is 0.20 minutes, and the system flow rate is 500 ml / min.

[0042] The wiping sampling method in step 3 is specifically as follows:

[0043] S1 takes three cotton swabs washed with ultrapure water and squeezes them against the wall of the test tube to remove excess water;

[0044] S2 Take one of the cotton swabs, press the cotton swab head on the sampling surface, bend it slightly, and wipe the sampling surface steadily and slowly. The wiping route is a horizontal S shape. Then use the reverse side of the cotton swab to wipe the same sampling surface. The wiping route is perpendicular to the last wiping route, which is a vertical S line. The whole surface should be covered in the process.

[0045] After S3 is wiped, the cotton part of the cotton swab is snapped off at the mouth of the test tube corresponding to the sampling point, and the head of the cotton swab falls into the stoppered test tube;

[0046] S4 takes another two cotton swabs and repeats the steps of S2 to S3, and puts them into the same sampling tube;

[0047] S5 Place the sampling cotton swab test tube in the test tube rack, add 20 ml of ultrapure water, ultrasonicate for 10 min as the test solution, mix well, and test.

[0048] The reagents and instruments used in the present invention include: 1. Reagents: Sodium persulfate batch number E2323548 from Anage Chemical Reagent Company, 85% phosphoric acid batch number B210528 from Xilong Science Co., Ltd., 1000ppm KHP standard solution batch number 20231010 from Beijing Weiye Metrology Technology Research Institute. 2. Instruments: Total organic carbon analyzer model Fusion, electronic balance model BSA224S.

[0049] Limit of quantitation and limit of detection.

[0050] Determination of the applicable limit of detection and limit of quantitation for the test system is confirmed based on the standard deviation of the responses of an appropriate number of blanks.

[0051] Fill 10 cleaned glass vials with test water and record the measured values ​​according to the law. Take the average value of the results. Calculate the standard deviation of the 10 blank vials.

[0052] TOC test results of needle blank (ultrapure water):

[0053]

[0054] Detection limit and quantification limit concentration.

[0055] project concentration Detection limit 0.06％ Limit of Quantitation 0.18％

[0056] As can be seen from the table above, the detection limit of carbon is as low as 0.06%, which is very sensitive.

[0057] Determination of the standard curve.

[0058] With the concentration of the standard as the horizontal axis and the measured absorbance value as the vertical axis, linear regression was performed to obtain the linear equation: y = 51.828x + 6.12, and the linear correlation coefficient r 2=0.9995. This indicates that the linearity is good and meets the requirement of the validation and acceptance standard correlation coefficient r>0.995.

[0059] Precision. Precision includes repeatability and intermediate precision.

[0060] Repeated inspection.

[0061] Weigh 0.1 g of chymotrypsin and add 100 ml of water to dissolve it as solution a. Take 0.18 ml of solution a in a 100 ml volumetric flask, dilute it with ultrapure water, make up to volume, shake well, and test.

[0062] Prepare 6 solutions according to the above steps, inject each solution once, and record the TOC value with a relative standard deviation of ≤10%.

[0063]

[0064] As can be seen from the above table, the repeatability RSD% = 8.9% (n = 6), indicating that the precision is good and meets the requirement of the validation and acceptance standard precision RSD% ≤ 10%.

[0065] Investigation of intermediate precision

[0066] On different dates and by different personnel, 6 injections of test solution were respectively made, and the relative standard deviations of TOC values ​​of 12 injections of test solution by two people were compared. If the relative standard deviation is ≤20%, the intermediate precision is good.

[0067]

[0068] As can be seen from the above table, the intermediate precision RSD% = 10.3% (n = 6), indicating that the precision is good and meets the requirement of the verification and acceptance standard precision RSD% ≤ 20%.

[0069] Accuracy test

[0070] The average recovery rate of TOC in the test solution should be between 70% and 125%, and the RSD value of TOC recovery in test solutions of different concentrations should be ≤20.0%.

[0071]

[0072] As can be seen from the above table, the accuracy RSD% = 6.2% (n = 9), indicating that the accuracy is good and meets the requirement of the verification and acceptance standard precision RSD% ≤ 20%.

[0073] Investigation of sampling recovery rate

[0074] Using the wiping method, a protease solution of known concentration is applied to stainless steel and silicone in a controlled manner, dried naturally, and a suitable solvent is selected to extract the residue on the swab, and then the amount of total organic carbon in the extract is detected. The ratio of the recovered amount to the amount added to the material sample is the sampling percentage recovery rate. Since the recovery rate experiment is a manual operation, each person usually needs to repeat the recovery rate study three times, and at least two people are required to conduct a wiping recovery rate study on each material surface. The recovery rate is defined as the lowest average recovery rate of any wiping sampler.

[0075] Sample processing was carried out according to the swab sampling method.

[0076] The overall recovery rate of a single sample by the wiping method shall not be less than 70.0%, and the RSD between recoveries shall not be greater than 30.0%.

[0077]

[0078]

[0079] The swab sampling recoveries of stainless steel and silicone were 79.2% and 72.5%, respectively, which met the requirement of swab sampling recovery rate ≥ 70% of the validation acceptance standard, indicating that this method is suitable for cleaning validation testing of protease cotton swab method.

[0080] In summary, the TOC analysis method was used to verify the quantitative limit and detection limit, linear relationship, precision, accuracy, and swab sampling recovery rate of the protease cleaning validation method. The results showed that the correlation coefficient of the standard curve, precision test, accuracy test, and swab recovery rate all met the requirements.

[0081] The TOC method can produce response values ​​for all carbon-containing organic matter, rather than a certain type or class of substances. The TOC method is used to detect all carbon-containing compounds, including active components, auxiliary materials and detergents, by using its specificity for carbon in organic matter. The TOC method is used for cleaning validation of protease products, which has the advantages of not needing to explore active ingredients, consider sample factors and find cleaning markers. Limiting all organic matter contained in the protein as a whole can achieve good cleaning validation results and effectively avoid the problem of inability to determine cleaning markers. The detection method of the present invention is fast and convenient, reduces analysis and inspection time, and is also of great significance in maintaining the cleaning validation status.

[0082] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0083] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting residual protease, characterized in that: The following steps are involved: Step 1: Sample preparation: according to the type of protease to be detected, collect the corresponding samples, and process the samples according to the characteristic pretreatment method to obtain the test samples; Step 2: Reagent preparation: prepare 5 ppm potassium hydrogen phthalate standard solution, 10% persulfate solution, 20% phosphoric acid solution and ultrapure water; Step 3: Setting up the detection method: when testing the test sample, add an acid and an oxidant to the TOC total organic carbon analyzer. The acid is phosphoric acid, and the oxidant is sodium persulfate. The prepared solution needs to be ultrasonicated for 5 minutes before sampling to remove dissolved CO2, and the wiping sampling method is used for sampling; Step 4: Signal reading: reading the signal generated by the reaction through the detection equipment and recording the signal reading of each sample to provide raw data for subsequent data analysis; Step 5 data analysis, calculating the residual amount of protease based on the signal readings, including establishing a standard curve, signal calibration, calculating the residual amount and statistical analysis.

2. The method for detecting residual protease according to claim 1, characterized in that: The preparation of the 5 ppm potassium hydrogen phthalate standard solution in step 2 specifically includes the following steps: taking 0.5 ml of potassium hydrogen phthalate standard solution in a 100 ml volumetric flask, adding ultrapure water to dilute to the scale line, shaking well, ultrasonicating for 5 minutes, and then transferring to a 100 ml TOC brown injection bottle.

3. The method for detecting residual protease according to claim 1, characterized in that: The preparation of the 10% persulfate solution in step 2 specifically includes the following steps: weigh 12.5 g of sodium persulfate into a 250 ml reagent bottle, take 4.5 ml of 85% phosphoric acid and 106.5 ml of ultrapure water into the reagent bottle, shake well to dissolve, and remove dissolved CO2 by ultrasonic cleaning in an ultrasonic cleaner for 5 minutes.

4. The method for detecting residual protease according to claim 1, characterized in that: The preparation of the 20% phosphoric acid solution in step 2 specifically includes the following contents: Separately measure 18.5 ml of 85% phosphoric acid and 94 ml of ultrapure water into a 250 ml reagent bottle, shake well, and ultrasonicate in an ultrasonic cleaner for 5 minutes to remove dissolved CO2.

5. The method for detecting residual protease according to claim 1, characterized in that: In step 2, the ultrapure water is placed in a special reagent bottle and ultrasonicated in an ultrasonic cleaning machine for 5 minutes to remove dissolved CO2.

6. The method for detecting residual protease according to claim 1, characterized in that: In the step 3, during the detection, the Clean and Blank samples are tested first, and after the absorption values ​​of the Reagentblank and Acidblank in the Blank samples are within an acceptable range, the linearity of the standard curve and the sample are detected, and the correlation coefficient r is greater than 0.

995.

7. A method for detecting residual protease according to claim 6, characterized in that: The acceptable range is Reagentblank≤7.0Abs, Acidblank≤5.0Abs.

8. The method for detecting residual protease according to claim 6, characterized in that: The wiping sampling method in step 3 is specifically as follows: S1 takes three cotton swabs washed with ultrapure water and squeezes them against the wall of the test tube to remove excess water; S2 Take one of the cotton swabs, press the cotton swab head on the sampling surface, bend it slightly, and wipe the sampling surface steadily and slowly. The wiping route is a horizontal S shape. Then use the reverse side of the cotton swab to wipe the same sampling surface. The wiping route is perpendicular to the last wiping route, which is a vertical S line. The whole surface should be covered in the process. After S3 is wiped, the cotton part of the cotton swab is snapped off at the mouth of the test tube corresponding to the sampling point, and the head of the cotton swab falls into the stoppered test tube; S4 takes another two cotton swabs and repeats the steps of S2 to S3, and puts them into the same sampling tube; S5 Place the sampling cotton swab test tube in the test tube rack, add 20 ml of ultrapure water, ultrasonicate for 10 min as the test solution, mix well, and test.

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