A method for detecting collagenase

Abstract

The present invention discloses a method for detecting collagenase. The detection method is to detect collagenase by HPLC method. The collagenase contains type I collagenase and / or type II collagenase. In the HPLC method, the stationary phase is alkylsilyl-bonded silica gel, mobile phase A is an aqueous solution of 0.05-0.25% trifluoroacetic acid, and mobile phase B is an acetonitrile solution of 0.05-0.25% trifluoroacetic acid, and gradient elution is adopted. This method can effectively separate type I collagenase, type II collagenase and impurities, and fully meets the acceptance criteria and shows good performance in terms of specificity, detection limit, repeatability, durability, solution stability, etc., providing an effective guarantee for the quality control or quality evaluation of subsequent collagenase products.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry, and particularly relates to a detection method for collagenase. Background Art

[0002] Collagenase, also known as collagen hydrolase, is a protease that can decompose the natural collagen that binds animal tissues under specific conditions (suitable temperature, pH, ionic strength, etc.). It can be produced by a variety of microorganisms and different animal cells. Currently, collagenase is mainly extracted and purified from specific microbial fermentation broths. The most effective collagenase is the one secreted by the anaerobic bacterium Clostridium histolyticum. Its main components include two types of complete proteins and truncated molecular weight variants of collagenase expressed by collagenase type I and collagenase type II genes, and may also include a small amount of other types of proteolytic enzymes, such as neutral protease, Clostripain, etc.

[0003] Patent document CN113474355A discloses a method for purifying at least one enzyme from a mixture of substances (such as the culture supernatant of Clostridium histolyticum). The enzyme is selected from the group consisting of collagenase I, collagenase II, neutral protease, and Clostripain. The method includes at least one hydrophobic interaction chromatography, the stationary phase of which is selected from polypropylene glycol or butyl agarose, and the mobile phase is selected from aqueous solutions of ammonium sulfate or potassium chloride. However, the mixture obtained by this method is a mixture of collagenase type I and type II with a certain purity, and it is difficult to further obtain its single component.

[0004] In therapeutic collagenase preparations, different ratios of collagenase type I and collagenase type II have different biological effects. For example, patent document CN101400788A discloses a collagenase composition that contains collagenase I and collagenase II, and their mass ratio is about 1:1, and its purity is at least higher than 95%. It can be used to treat diseases mediated by bone collagen. Thus, it is particularly important to control the purity and ratio of collagenase type I and collagenase type II in the preparation of therapeutic collagenase preparations.

[0005] Therefore, developing a detection method for collagenase is of great significance for the quality control or quality evaluation of subsequent collagenase products and their preparation processes. Summary of the Invention

[0006] To overcome the deficiencies of the prior art, the present invention provides a method for detecting collagenase, which uses reverse-phase high-performance liquid chromatography (RP-HPLC), with alkylsilyl-bonded silica gel as the stationary phase, and gradient elution is carried out with trifluoroacetic acid aqueous solution and trifluoroacetic acid acetonitrile solution as the mobile phases. This method has simple steps and rapid reactions, can effectively detect the purity of type I and type II collagenase in collagenase products, and the instruments and reagents used are all common, with relatively low costs.

[0007] In the first aspect of the present invention, there is provided a method for detecting collagenase, and the detection method is to detect collagenase by HPLC method.

[0008] Further, the collagenase contains type I collagenase and / or type II collagenase.

[0009] Further, the collagenase may also contain one or more impurities, including but not limited to, other types of collagenase (such as type III collagenase, type VI collagenase, type V collagenase), other types of proteases (such as clostripain, neutral protease, trypsin, caseinase, etc.), and other impurities (such as nucleic acids, endotoxins, exotoxins, phage components, etc.).

[0010] Preferably, the impurities are selected from: clostripain, neutral protease, trypsin, caseinase.

[0011] Further, the collagenase is obtained by pretreating a complex (such as Clostridium histolyticum culture).

[0012] Further, the pretreatment includes one or more separation and / or purification steps, including but not limited to, precipitation, chromatography (such as ion exchange chromatography, gel chromatography, hydrophobic interaction chromatography, etc.), electrophoresis, centrifugation, desalting, drying, filtration, etc.

[0013] Further, in the HPLC method, the collagenase is directly detected or the collagenase is dissolved in a diluent for detection.

[0014] In some embodiments of the present invention, in the HPLC method, the collagenase is dissolved in a diluent for detection.

[0015] Further, the diluent is selected from: one or more of acetonitrile, trifluoroacetic acid, formic acid, and water.

[0016] Preferably, the diluent is an aqueous solution of trifluoroacetic acid with a volume percentage concentration of 0.01 - 1.00%, specifically such as 0.01, 0.02, 0.04, 0.05, 0.06, 0.08, 0.10, 0.12, 0.14, 0.15, 0.16, 0.18, 0.20, 0.22, 0.24, 0.25, 0.26, 0.28, 0.30, 0.32, 0.34, 0.36, 0.38, 0.40, 0.42, 0.44, 0.46, 0.48, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00%, and preferably an aqueous solution of trifluoroacetic acid with a volume percentage concentration of 0.05 - 0.25%.

[0017] Preferably, the diluent is water.

[0018] Further, in the HPLC method, the stationary phase of the chromatographic column is alkylsilyl-bonded silica gel, which is selected from: tetraalkylsilyl-bonded silica gel, octadecylsilyl-bonded silica gel, octadecylsilyl-bonded silica gel, phenylsilyl-bonded silica gel, pentafluorophenylsilyl-bonded silica gel, phenyl-hexylsilyl-bonded silica gel.

[0019] Preferably, the stationary phase of the chromatographic column is selected from: tetraalkylsilyl-bonded silica gel, octadecylsilyl-bonded silica gel, octadecylsilyl-bonded silica gel.

[0020] In some embodiments of the present invention, the specifications of the chromatographic column are 250×4.6mm, 5μm.

[0021] Further, in the HPLC method, the mobile phase includes mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of trifluoroacetic acid with a volume percentage concentration of 0.01 - 1.00%, and mobile phase B is an acetonitrile solution of trifluoroacetic acid with a volume percentage concentration of 0.01 - 1.00%; wherein, the volume percentage concentration values of mobile phase A and mobile phase B are specifically such as 0.01, 0.02, 0.04, 0.05, 0.06, 0.08, 0.10, 0.12, 0.14, 0.15, 0.16, 0.18, 0.20, 0.22, 0.24, 0.25, 0.26, 0.28, 0.30, 0.32, 0.34, 0.36, 0.38, 0.40, 0.42, 0.44, 0.46, 0.48, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00%.

[0022] Preferably, mobile phase A is an aqueous solution of trifluoroacetic acid with a volume percentage concentration of 0.05 - 0.25%.

[0023] Preferably, the mobile phase B is an acetonitrile solution of trifluoroacetic acid with a volume percentage concentration of 0.05 - 0.25%.

[0024] Furthermore, in the HPLC method, a gradient elution program is adopted, and the gradient elution program is as follows:

[0025]

[0026] Preferably, the gradient elution program is as follows:

[0027]

[0028] More preferably, the gradient elution program is as follows:

[0029]

[0030] Furthermore, in the HPLC method, the flow rate is 0.1 - 5 mL / min, specifically such as 0.1, 0.5, 0.8, 1.0, 1.2, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 mL / min, preferably 0.5 - 1.5 mL / min, and more preferably 0.8 - 1.5 mL / min.

[0031] Furthermore, in the HPLC method, the column temperature is 20 - 50 °C, specifically such as 20, 22, 24, 25, 26, 28, 30, 32, 34, 35, 36, 38, 40, 42, 44, 45, 46, 48, 50 °C, preferably 35 - 45 °C, and more preferably 35 - 42 °C.

[0032] Furthermore, in the HPLC method, the injection volume is 1 - 100 μL, specifically such as 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100 μL, preferably 5 - 50 μL.

[0033] Furthermore, in the HPLC method, the concentration of the test solution is 0.05 - 5 mg / mL, specifically such as 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 mg / mL, preferably 0.05 - 2.5 mg / mL.

[0034] Furthermore, in the HPLC method, the detector is an ultraviolet detector.

[0035] Further, in the HPLC method, the detection wavelength is 200 - 300 nm, specifically such as 200, 210, 214, 220, 230, 240, 250, 260, 270, 280, 290, 300 nm, and preferably 214 nm or 280 nm.

[0036] In the second aspect of the present invention, there is provided an application of the detection method described in the first aspect of the present invention in the quality control or quality evaluation of collagenase products.

[0037] Further, the product is a preparation containing collagenase, including but not limited to, liquid preparations, solid preparations, semi-solid preparations, and gas preparations.

[0038] Further, the product can be a drug, food, or health product.

[0039] Further, the product contains type I collagenase and / or type II collagenase.

[0040] Further, the product may also contain one or more impurities, including but not limited to, other types of collagenase (such as type III collagenase, type VI collagenase, type V collagenase), other types of proteases (such as clostripain, neutral protease, trypsin, caseinase, etc.), and other impurities (such as nucleic acids, endotoxins, exotoxins, phage components, etc.).

[0041] In the third aspect of the present invention, there is provided an application of the detection method described in the first aspect of the present invention in the quality control or quality evaluation of the preparation process of collagenase products.

[0042] Further, the product has the definition described in the second aspect of the present invention.

[0043] In the fourth aspect of the present invention, there is provided a quantitative detection method for collagenase, and the detection method and collagenase have the definitions described in the first aspect of the present invention.

[0044] The present invention has the following beneficial effects:

[0045] The detection method of the present invention can effectively separate type I collagenase, type II collagenase, and impurities, and this method fully meets the acceptance criteria and performs well in terms of specificity, detection limit, repeatability, durability, solution stability, etc., providing an effective guarantee for the subsequent quality control or quality evaluation of collagenase products and their preparation processes. Description of the Drawings

[0046] Figure 1 Shown is the liquid phase detection chromatogram of the blank control in Example 1.

[0047] Figure 2 Shown is the liquid phase detection chromatogram of the reference substance in Example 1.

[0048] Figure 3 The following is the liquid phase detection chromatogram of Test Sample 1 in Example 1. Detailed implementation mode

[0049] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains.

[0050] The term "collagenase", also known as "collagen hydrolase", is a protease that can specifically hydrolyze the triple helix structure of natural collagen under physiological pH and temperature conditions without damaging other proteins and tissues, and can be divided into collagenase type I, collagenase type II, collagenase type III, collagenase type VI, and collagenase type V. In this article, "collagenase type I", "collagenase I", and "type I collagenase" can be used interchangeably, and the same applies to "collagenase type II", "collagenase type III", "collagenase type VI", and "collagenase type V".

[0051] The "collagenase" in this article at least includes collagenase type I and / or collagenase type II. In addition, the said collagenase may also contain one or more impurities. The impurities refer to substances different from any one of collagenase type I and collagenase type II, including but not limited to other types of collagenases (such as collagenase type III, collagenase type VI, collagenase type V), other types of proteases (such as clostripain, neutral protease, trypsin, caseinase, etc.), and other impurities (such as nucleic acids, endotoxins, exotoxins, phage components, etc.). The collagenase can be obtained from a complex, and the complex can be an untreated Clostridium histolyticum culture or a partially purified (i.e., having undergone one or more separation and / or purification steps) Clostridium histolyticum culture, etc.

[0052] The "collagenase solution" used in the examples of the present invention is obtained by separating and purifying Clostridium histolyticum after fermentation, and specifically includes the following steps: Clostridium histolyticum uses an improved medium for static fermentation to obtain collagenase with low toxicity. The fermentation broth is subjected to sterile filtration, ammonium sulfate precipitation, desalting, and freeze-drying to obtain a crude collagenase product, and then DEAE column chromatography and Sephdex G-25 desalting are carried out to obtain a refined collagenase solution. This collagenase solution includes collagenase type I, collagenase type II, and related impurity proteins, such as clostripain, neutral protease, trypsin, caseinase, etc.

[0053] The public contents of various publications, patents, and published patent specifications cited herein are incorporated herein by reference in their entirety.

[0054] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention.

[0055] Example 1:

[0056] 1. Solution preparation

[0057] (1) Mobile phase: Take 1 mL of trifluoroacetic acid and add 1 L of pure water, mix and ultrasonicate to obtain mobile phase A; take 1 mL of trifluoroacetic acid and add 1 L of acetonitrile, mix and ultrasonicate to obtain mobile phase B.

[0058] (2) Reference substance: Appropriate amounts of collagenase reference standard I (source: USP, batch number: F029B0, protein content: 53 mg / mL) and collagenase reference standard II (source: USP, batch number: F029C0, protein content: 52 mg / mL) are respectively measured and diluted with water to 1 mg / mL.

[0059] (3) Test sample: Take an appropriate amount of collagenase solution and dilute it with water to 1 mg / mL.

[0060] (4) Blank control: Pure water.

[0061] 2. Liquid phase conditions

[0062] Instrument: Agilent 1260 liquid chromatography system;

[0063] Chromatographic column: Waters Symmetry300 C4 250×4.6 mm 5 μm; Mobile phase: Mobile phase A: 0.1% trifluoroacetic acid aqueous solution;

[0064] Mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution;

[0065] UV detector, detection wavelength: 280 nm;

[0066] Column temperature of the chromatographic column: 40 °C;

[0067] Flow rate: 1 mL / 1 min;

[0068] Injection volume: 50 μL;

[0069] The gradient elution program is shown in Table 1.

[0070] Table 1 Gradient elution program

[0071] Time min Mobile phase (volume A%) B% (volume B%) 0.00 70 30 5.00 70 30 35.00 40 60 36.00 0 100 40.00 0 100 41.00 70 30 45.00 70 30

[0072] 3. Experimental Results

[0073] Table 2 Test Results of the Test Substances

[0074]

[0075]

[0076] Example 2:

[0077] 1. Solution Preparation

[0078] (1) Mobile Phase: Take 0.5 mL of trifluoroacetic acid and add 1 L of pure water, mix and sonicate to obtain Mobile Phase A; take 0.5 mL of trifluoroacetic acid and add 1 L of acetonitrile, mix and sonicate to obtain Mobile Phase B.

[0079] (2) Reference Substances: Appropriate amounts of Collagenase Reference Standard I (Source: USP, Batch No.: F029B0, Protein Content: 53 mg / mL) and Collagenase Reference Standard II (Source: USP, Batch No.: F029C0, Protein Content: 52 mg / mL) are respectively measured and diluted to 0.5 mg / mL with Mobile Phase A.

[0080] (3) Test Substances: Take an appropriate amount of the collagenase solution and dilute it to 0.5 mg / mL with Mobile Phase A.

[0081] (4) Blank Control: The same as Mobile Phase A.

[0082] 2. HPLC Conditions

[0083] Instrument: Agilent 1260 series HPLC system;

[0084] Chromatographic Column: HICHROM C4 250×4.6 mm 5 μm;

[0085] Mobile Phase: Mobile Phase A: 0.05% trifluoroacetic acid aqueous solution;

[0086] Mobile Phase B: 0.05% trifluoroacetic acid acetonitrile solution;

[0087] UV Detector, Detection Wavelength: 214 nm;

[0088] Column Temperature: 38°C;

[0089] Flow Rate: 1.2 mL / min;

[0090] Injection Volume: 10 μL;

[0091] The gradient elution program is shown in Table 3.

[0092] Table 3 Gradient Elution Program

[0093]

[0094]

[0095] 3. Experimental Results

[0096] Table 4 Test Results of Test Substances

[0097]

[0098] Example 3:

[0099] 1. Solution Preparation

[0100] (1) Mobile Phase: Take 1.5 mL of trifluoroacetic acid and add 1 L of pure water, mix and sonicate to obtain Mobile Phase A; take 1.5 mL of trifluoroacetic acid and add 1 L of acetonitrile, mix and sonicate to obtain Mobile Phase B.

[0101] (2) Reference Substances: Appropriate amounts of Collagenase Standard I (Source: USP, Batch No.: F029B0, Protein Content: 53 mg / mL) and Collagenase Standard II (Source: USP, Batch No.: F029C0, Protein Content: 52 mg / mL) are each measured and diluted with water to 1.5 mg / mL.

[0102] (3) Test Substance: Take an appropriate amount of collagenase solution and dilute it with water to 1.5 mg / mL.

[0103] (4) Blank Control: Pure water.

[0104] 2. HPLC Conditions

[0105] Instrument: Agilent 1260 series HPLC system;

[0106] Chromatographic Column: HICHROM C18 250×4.6 mm 5 μm;

[0107] Mobile Phase: Mobile Phase A: 0.15% trifluoroacetic acid aqueous solution;

[0108] Mobile Phase B: 0.15% trifluoroacetic acid acetonitrile solution;

[0109] UV Detector, Detection Wavelength: 280 nm;

[0110] Column Temperature: 42 °C;

[0111] Flow Rate: 0.8 mL / min;

[0112] Injection Volume: 30 μL;

[0113] The gradient elution program is shown in Table 5.

[0114] Table 5 Gradient Elution Gradient

[0115] Time min Mobile phase (volume A%) B% (volume B%) 0.00 80 20 5.00 80 20 37.00 50 50 42.00 10 90 46.00 10 90 48.00 80 20 53.00 80 20

[0116] 3. Experimental Results

[0117] Table 6 Test Results of Test Substances

[0118]

[0119] Example 4:

[0120] 1. Solution Preparation

[0121] (1) Mobile Phase: Take 2.5 mL of trifluoroacetic acid and add 1 L of purified water, mix and sonicate to obtain Mobile Phase A; take 1.5 mL of trifluoroacetic acid and add 1 L of acetonitrile, mix and sonicate to obtain Mobile Phase B.

[0122] (2) Reference Substances: Appropriate amounts of Collagenase Standard I (Source: USP, Lot No.: F029B0, Protein Content: 53 mg / mL) and Collagenase Standard II (Source: USP, Lot No.: F029C0, Protein Content: 52 mg / mL) are each measured and diluted with water to 2 mg / mL.

[0123] (3) Test Substances: Take an appropriate amount of collagenase solution and dilute it with water to 2.5 mg / mL.

[0124] (4) Blank Control: Purified water.

[0125] 2. HPLC Conditions

[0126] Instrument: Agilent 1260 series HPLC system;

[0127] Chromatographic Column: HICHROM C4 250×4.6 mm 5 μm;

[0128] Mobile Phase: Mobile Phase A: 0.25% aqueous trifluoroacetic acid solution;

[0129] Mobile Phase B: 0.15% trifluoroacetic acid acetonitrile solution;

[0130] UV Detector, Detection Wavelength: 214 nm;

[0131] Column Temperature: 35°C;

[0132] Flow Rate: 1.5 mL / min;

[0133] Injection Volume: 5 μL;

[0134] The gradient elution program is shown in Table 7.

[0135] Table 7 Gradient Elution Gradient

[0136] Time min Mobile phase (volume A%) B% (volume B%) 0.00 55 45 4.00 55 45 37.00 25 75 40.00 5 95 44.00 5 95 47.00 55 45 52.00 55 45

[0137] 3. Experimental Results

[0138] Table 8 Test Results of Test Samples

[0139]

[0140] Comparative Example 1:

[0141] Compared with Example 3, the chromatographic column was changed to: ZORBAX NH2 250×4.6mm 5μm; other operations remained unchanged, and the test results of the test samples are shown in Table 9 below:

[0142] Table 9 Test Results of Test Samples

[0143]

[0144]

[0145] Using the ZORBAX NH2 250×4.6mm 5μm chromatographic column, the peak separation effect was poor, the resolution was less than 1.5, effective separation could not be achieved, and the peak shape was poor, affecting the calculation of the peak area%, that is, the purity of type I and type II collagenase could not be accurately detected.

[0146] Comparative Example 2:

[0147] Compared with Example 3, the gradient elution program was changed as shown below; other operations remained unchanged.

[0148] The gradient elution program is shown in Table 10.

[0149] Table 10 Gradient Elution Gradient

[0150] Time min Mobile phase (volume A%) B% (volume B%) 0.00 40 60 3.00 40 60 35.00 10 90 38.00 30 70 42.00 30 70 45.00 40 60 50.00 40 60

[0151] Table 11 Test Results of Test Samples

[0152]

[0153] It can be seen from Table 11 that with the change of the gradient elution program, the resolution and the number of theoretical plates decreased significantly, the separation of type I and type II collagenase could not be achieved, and the calculation of the purity% was affected.

[0154] Test Example: Methodology Investigation

[0155] The methodology investigation was carried out using the following prepared solution and the same liquid phase conditions as in Example 1.

[0156] Blank control: pure water.

[0157] 50% Test Sample Solution: Dilute an appropriate amount of collagenase solution with water to 0.5 mg / mL.

[0158] 100% Test Sample Solution (Test Sample Solution): Dilute an appropriate amount of collagenase solution with water to 1 mg / mL.

[0159] 150% Test Sample Solution: Dilute an appropriate amount of collagenase solution with water to 1.5 mg / mL.

[0160] Limit of Detection Solution: Dilute an appropriate amount of collagenase solution with water to 0.05 mg / mL.

[0161] 1. Specificity

[0162] Using pure water as the blank, prepare 1 portion of the test sample solution and inject it once.

[0163] Table 12 Specificity Test Results

[0164]

[0165] Conclusion: The chromatographic peak of the blank excipient solution has no interference with the main peak in the chromatogram of the test sample solution, and the specificity meets the standard requirements.

[0166] 2. Repeatability

[0167] Prepare test samples at 50%, 100%, and 150% concentrations, prepare 3 portions for each concentration, and inject each portion once. Calculate the RSD% of the purity for 3 injections at the same concentration using the area normalization method; calculate the RSD% of the purity for 9 injections at different concentrations. The results are shown in the following table:

[0168] Table 13 Repeatability Test Results

[0169]

[0170]

[0171] Conclusion: The RSD% of the purity for 3 injections at the same concentration in the test sample solution ≤ 2.0%, and the RSD of the purity for 9 portions of the test sample solution ≤ 2.0%. Conclusion: The RSD% of the peak area% for test sample solutions at different concentrations ≤ 2%, meeting the standard requirements, so the repeatability is good.

[0172] 3. Limit of Detection

[0173] Prepare 1 portion of the limit of detection solution and inject it once. Limit of Detection: Use the lower verified peak type I detection limit as the limit of detection for this method, and an S / N ratio of not less than 3 is considered the limit of detection. When the test sample concentration is 0.05 mg / mL, the average signal-to-noise ratio of the type I collagenase peak is 3.660, meeting the signal-to-noise ratio requirement for the limit of detection. Therefore, the limit of detection for this method is 0.05 mg / mL.

[0174] The detection limit of type II was measured by the same method. When the concentration of the test sample was 0.05 mg / mL, the average signal-to-noise ratio of the type II collagenase peak was 4.002, meeting the signal-to-noise ratio requirement for the detection limit. Therefore, the detection limit of this method was 0.05 mg / mL.

[0175] 4. Durability

[0176] 4.1 Different column temperatures

[0177] Change the column temperature: Keep other chromatographic conditions unchanged and only change the column temperature to 38 °C, 40 °C, and 42 °C respectively; prepare 1 portion of the test sample with a concentration of 100%, inject 1 injection for each condition, and calculate the RSD% of the purity of 3 injections. The results are shown below:

[0178] Table 14 Durability test results at different column temperatures

[0179]

[0180] Conclusion: Within the range of 38 °C - 42 °C for the column temperature, the number of theoretical plates and the resolution of the main peak both meet the requirements, and the durability is good.

[0181] 4.2 Different flow rates

[0182] Change the flow rate: Keep other chromatographic conditions unchanged and only change the flow rate to 0.8 mL / min, 1 mL / min, and 1.2 mL / min respectively; prepare 1 portion of the test sample with a concentration of 100%, inject 1 injection for each condition, and calculate the RSD% of the purity of 3 injections. The results are shown below:

[0183] Table 15 Durability test results at different flow rates

[0184]

[0185] Conclusion: Within the range of 0.8 - 1.2 mL / min for the flow rate, the number of theoretical plates and the resolution of the main peak both meet the requirements, and the durability is good.

[0186] 4.3 Stability of the test sample solution

[0187] Prepare 1 portion of the test sample with a concentration of 100%, place it under the sample cell conditions for 0 h, 6 h, 12 h, 18 h, and 24 h, inject 1 injection for each time point, and calculate the RSD% of the purity of 5 injections.

[0188] Table 16 Stability test results of the test sample solution

[0189]

[0190] Conclusion: When the sample was placed in the sample cell for 0 - 24 h, the RSD% of the peak area percentage ≤ 2%. Therefore, the test solution was stable within 24 h.

[0191] In summary, the detection method of the present invention can effectively separate type I collagenase, type II collagenase and impurities in the collagenase solution, and this method fully meets the acceptance criteria and performs well in terms of specificity, detection limit, repeatability, durability, etc.

[0192] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0193] The foregoing embodiments and methods described in the present invention may vary based on the capabilities, experience and preferences of those skilled in the art.

[0194] The steps of the method listed only in a certain order in the present invention do not constitute any limitation on the order of the method steps.

Claims

1. A method for detecting collagenase, wherein the method is to detect collagenase by HPLC; The collagenase comprises collagenase type I and / or collagenase type II; In the HPLC method, the stationary phase of the chromatographic column is an alkylsilane bonded silica gel, which is selected from: tetraalkylsilane bonded silica gel, octadecylsilane bonded silica gel; The mobile phase includes mobile phase A and mobile phase B, wherein the mobile phase A is a trifluoroacetic acid aqueous solution with a volume percentage concentration of 0.05-0.25%; the mobile phase B is a trifluoroacetic acid acetonitrile solution with a volume percentage concentration of 0.05-0.15%; A gradient elution program was used, and the gradient elution program was as follows: t = 0.00 min, the volume percentage concentration of mobile phase B is 20-45%; 3≤t≤5 min, the volume percentage concentration of mobile phase B is 20-45%; 35≤t≤37 min, the volume percentage concentration of mobile phase B is 50-75%; 36≤t≤42 min, the volume percentage concentration of mobile phase B is 90-100%; 40≤t≤46 min, the volume percentage concentration of mobile phase B is 90-100%; 41≤t≤48 min, the volume percentage concentration of mobile phase B is 20-45%; 45≤t≤53 min, the volume percentage concentration of mobile phase B is 20-45%; The detector is an ultraviolet detector with a detection wavelength of 214nm or 280nm.

2. The detection method according to claim 1, characterized in that: In the HPLC method, collagenase is directly detected or dissolved in a diluent for detection.

3. The detection method according to claim 2, characterized in that: The diluent is selected from: one or more of acetonitrile, trifluoroacetic acid, formic acid and water.

4. The detection method according to claim 2, characterized in that: The diluent is a trifluoroacetic acid aqueous solution with a volume percentage concentration of 0.01-1.00%.

5. The detection method according to claim 4, characterized in that: The diluent is a trifluoroacetic acid aqueous solution with a volume percentage concentration of 0.05-0.25%.

6. The detection method according to claim 2, characterized in that: The diluent is water.

7. The detection method according to claim 1, characterized in that: In the HPLC method, the flow rate is 0.1-5 mL / min.

8. The detection method according to claim 7, characterized in that: The flow rate is 0.5-1.5 mL / min.

9. The detection method according to claim 8, characterized in that: The flow rate is 0.8-1.5 mL / min.

10. The detection method according to claim 1, characterized in that: In the HPLC method, the column temperature is 20-50°C.

11. The detection method according to claim 10, characterized in that: The column temperature is 35-45°C.

12. The detection method according to claim 11, characterized in that: The column temperature is 35-42°C.

13. The detection method according to claim 1, characterized in that: In the HPLC method, the injection volume is 1-100 μL.

14. The detection method according to claim 13, characterized in that: The injection volume is 5-50 μL.

15. The detection method according to claim 1, characterized in that: In the HPLC method, the concentration of the test solution during detection is 0.05-5 mg / mL.

16. The detection method according to claim 15, characterized in that: The concentration of the test solution is 0.05-2.5 mg / mL.

17. The detection method according to claim 1, characterized in that: The collagenase further comprises one or more impurities, and the impurities are selected from: clostripain, neutral protease, trypsin, and tyrosine protease.

18. The detection method according to claim 1, characterized in that: The collagenase is obtained by pre-treating the complex, wherein the pre-treatment includes one or more separation and / or purification steps.

19. The detection method according to claim 18, characterized in that: The complex is a culture of Clostridium histolyticum.

20. Use of the detection method according to any one of claims 1 to 19 in quality control or quality evaluation of collagenase products, or quality control or quality evaluation of a preparation process of collagenase products.

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