Collagenase detection method

Abstract

The invention discloses a detection method of collagenase. According to the detection method, the collagenase is detected through an HPLC (High Performance Liquid Chromatography) method. The collagenase comprises collagenase type I and / or collagenase type II, a stationary phase in the HPLC method is alkylsilane bonded silica gel, a mobile phase A is a 0.05-0.25% trifluoroacetic acid aqueous solution, a mobile phase B is a 0.05-0.25% trifluoroacetic acid acetonitrile solution, and gradient elution is adopted. According to the method, collagenase I, collagenase II and impurities can be effectively separated, the specificity, the detection limit, the repeatability, the durability, the solution stability and other aspects completely meet the acceptance standard, the performance is good, and an effective guarantee is provided for subsequent quality control or quality evaluation of collagenase products.

Description

Technical Field

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

[0002] Collagenase, also known as collagen hydrolase, is a protease that can decompose natural collagen that binds animal tissues together under specific conditions (suitable temperature, pH and ionic strength, etc.). It can be produced by a variety of microorganisms and different animal cells. At present, collagenase is mainly extracted and purified from specific microbial fermentation broth. The most effective collagenase is secreted by the anaerobic bacterium Clostridium histolyticum. Its main components include two types of collagenase complete proteins and truncated molecular weight variants 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 proteases, 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), wherein the enzyme is selected from the group consisting of collagenase I, collagenase II, neutral protease and clostripain, and the method comprises at least one hydrophobic interaction chromatography, wherein the stationary phase is selected from polypropylene glycol or butyl agarose, and the mobile phase is selected from an aqueous solution of ammonium sulfate or potassium chloride. However, the method obtains a mixture of collagenase type I and type II with a certain purity, and it is difficult to further obtain a single component thereof.

[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, which contains collagenase I and collagenase II in a mass ratio of about 1:1 and a purity of at least 95%, and can be used to treat diseases mediated by bone collagen. It can be seen that controlling the purity and ratio of collagenase type I and collagenase type II is particularly important in preparing therapeutic collagenase preparations.

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

[0006] In order to overcome the deficiencies of the prior art, the present invention provides a method for detecting collagenase, which adopts reverse phase high performance liquid chromatography (RP-HPLC), uses alkylsilane bonded silica gel as the stationary phase, and uses trifluoroacetic acid aqueous solution and trifluoroacetic acid acetonitrile solution as the mobile phase for gradient elution. The method has simple steps and rapid reaction, can effectively detect the purity of collagenase type I and collagenase type II in collagenase products, and uses common instruments and reagents, and has low cost.

[0007] A first aspect of the present invention provides a method for detecting collagenase, wherein the method comprises detecting collagenase by HPLC.

[0008] Furthermore, the collagenase comprises collagenase type I and / or collagenase type II.

[0009] Furthermore, the collagenase may also contain one or more impurities, 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, tyrosine protease, etc.), and other impurities (such as nucleic acids, endotoxins, exotoxins, phage components, etc.).

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

[0011] Furthermore, the collagenase is obtained by pre-treating a complex (such as Clostridium histolyticum culture).

[0012] Furthermore, 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] Furthermore, in the HPLC method, collagenase is detected directly or 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] Furthermore, 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 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%, preferably an aqueous solution of trifluoroacetic acid with a volume percentage concentration of 0.05-0.25%.

[0017] Preferably, the diluent is water.

[0018] Furthermore, 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, octaalkylsilane bonded silica gel, octadecylsilane bonded silica gel, phenylsilane bonded silica gel, pentafluorophenylsilane bonded silica gel, and phenyl-hexylsilane bonded silica gel.

[0019] Preferably, the stationary phase of the chromatographic column is selected from: tetraalkylsilane bonded silica gel, octadecylsilane bonded silica gel, octadecylsilane bonded silica gel.

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

[0021] Further, in the HPLC method, the mobile phase includes a mobile phase A and a mobile phase B, the mobile phase A is a trifluoroacetic acid aqueous solution with a volume percentage concentration of 0.01-1.00%, and the mobile phase B is a trifluoroacetic acid acetonitrile solution with a volume percentage concentration of 0.01-1.00%; wherein the volume percentage concentration values ​​of the mobile phase A and the mobile phase B are specifically 0.01, 0.02, 0.04, 0.05, 0.06, 0.08, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65 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, the mobile phase A is a trifluoroacetic acid aqueous solution with a volume percentage concentration of 0.05-0.25%.

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

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

[0025]

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

[0027]

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

[0029]

[0030] Furthermore, in the HPLC method, the flow rate is 0.1-5 mL / min, 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] Further, in the HPLC method, the column temperature is 20-50°C, specifically 20, 22, 24, 25, 26, 28, 30, 32, 34, 35, 36, 38, 40, 42, 44, 45, 46, 48, 50°C, preferably 35-45°C, more preferably 35-42°C.

[0032] Furthermore, in the HPLC method, the injection volume is 1-100 μL, 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, 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] Furthermore, in the HPLC method, the detection wavelength is 200-300 nm, specifically 200, 210, 214, 220, 230, 240, 250, 260, 270, 280, 290, 300 nm, preferably 214 nm or 280 nm.

[0036] The second aspect of the present invention provides 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] Furthermore, the product is a preparation containing collagenase, including but not limited to liquid preparations, solid preparations, semi-solid preparations, and gaseous preparations.

[0038] Furthermore, the product may be a medicine, a food, or a health product.

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

[0040] Furthermore, the product may also contain one or more impurities, 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, tyrosine protease, etc.), and other impurities (such as nucleic acids, endotoxins, exotoxins, phage components, etc.).

[0041] The third aspect of the present invention provides 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 a collagenase product.

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

[0043] The fourth aspect of the present invention provides a method for quantitative detection of collagenase, wherein the detection method and collagenase have the definitions as 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 collagenase type I, collagenase type II and impurities, and the method fully meets the acceptance standards and performs well in terms of specificity, detection limit, repeatability, durability, solution stability, etc., providing effective guarantee for the quality control or quality evaluation of subsequent collagenase products and their preparation processes. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0048] Figure 3 Shown is the liquid phase detection spectrum of sample 1 in Example 1. DETAILED DESCRIPTION

[0049] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0050] The term "collagenase", also known as "collagen hydrolase", is a protease that can specifically hydrolyze the three-dimensional helical structure of natural collagen under physiological pH and temperature conditions without damaging other proteins and tissues. It 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 situation also applies to "collagenase type II", "collagenase type III", "collagenase type VI", and "collagenase type V".

[0051] The "collagenase" herein at least includes collagenase type I and / or collagenase type II. In addition, the 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 collagenase (such as collagenase type III, collagenase type VI, collagenase type V), other types of proteases (such as clostripain, neutral protease, trypsin, tyrosine protease, etc.), 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 Clostridium histolyticum culture (i.e., one or more separation and / or purification steps have been performed), and the like.

[0052] The "collagenase solution" used in the embodiment of the present invention is obtained by separating and purifying Clostridium histolyticum after fermentation, specifically comprising the following steps: Clostridium histolyticum is fermented statically using an improved culture medium to obtain low-toxic collagenase, the fermentation broth is sterile filtered, ammonium sulfate precipitated, desalted, and freeze-dried to obtain a crude collagenase, and then desalted by DEAE column chromatography and SephdexG-25 to obtain a refined collagenase solution. The collagenase solution includes collagenase type I, collagenase type II, and related impurity proteins, such as clostripain, neutral protease, trypsin, casein, etc.

[0053]

[00136] Various publications, patents, and published patent specifications are cited herein, the disclosures of which are incorporated by reference in their entireties.

[0054] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] Embodiment 1:

[0056] 1. Solution preparation

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

[0058] (2) Reference material: Collagenase standard I (source: United States Pharmacopoeia USP, batch number: F029B0, protein content: 53 mg / mL) and collagenase standard II (source: United States Pharmacopoeia USP, batch number: F029C0, protein content: 52 mg / mL) were measured in appropriate amounts and diluted with water to 1 mg / mL.

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

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

[0061] 2. Liquid phase conditions

[0062] Instrument: Agilent 1260 liquid chromatography system;

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

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

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

[0066] Chromatographic column temperature: 40°C;

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

[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 test products

[0074]

[0075]

[0076] Embodiment 2:

[0077] 1. Solution preparation

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

[0079] (2) Reference substances: Collagenase standard I (source: United States Pharmacopoeia USP, batch number: F029B0, protein content: 53 mg / mL) and collagenase standard II (source: United States Pharmacopoeia USP, batch number: F029C0, protein content: 52 mg / mL) were taken in appropriate amounts and diluted to 0.5 mg / mL with mobile phase A.

[0080] (3) Test sample: Take an appropriate amount of collagenase solution and add mobile phase A to dilute it to 0.5 mg / mL.

[0081] (4) Blank control: Same as mobile phase A.

[0082] 2. Liquid phase conditions

[0083] Instrument: Agilent 1260 liquid chromatography system;

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

[0085] Mobile phase: Mobile phase A: 0.05% trifluoroacetic acid in water;

[0086] Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile;

[0087] UV detector, detection wavelength: 214nm;

[0088] Chromatographic column temperature: 38°C;

[0089] Flow rate: 1.2 mL / 1 min;

[0090] Injection volume: 10 μL;

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

[0092] Table 3 Gradient elution gradient

[0093]

[0094]

[0095] 3. Experimental results

[0096] Table 4 Test results of the test products

[0097]

[0098] Embodiment 3:

[0099] 1. Solution preparation

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

[0101] (2) Reference substance: Collagenase standard I (source: United States Pharmacopoeia USP, batch number: F029B0, protein content: 53 mg / mL) and collagenase standard II (source: United States Pharmacopoeia USP, batch number: F029C0, protein content: 52 mg / mL) are measured in appropriate amounts and diluted with water to 1.5 mg / mL.

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

[0103] (4) Blank control: pure water.

[0104] 2. Liquid phase conditions

[0105] Instrument: Agilent 1260 liquid chromatography system;

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

[0107] Mobile phase: Mobile phase A: 0.15% trifluoroacetic acid in water;

[0108] Mobile phase B: 0.15% trifluoroacetic acid in acetonitrile;

[0109] UV detector, detection wavelength: 280nm;

[0110] Chromatographic column temperature: 42°C;

[0111] Flow rate: 0.8 mL / 1 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 products

[0118]

[0119] Embodiment 4:

[0120] 1. Solution preparation

[0121] (1) Mobile phase: Add 2.5 mL of trifluoroacetic acid to 1 L of pure water, mix and sonicate to obtain mobile phase A; add 1.5 mL of trifluoroacetic acid to 1 L of acetonitrile, mix and sonicate to obtain mobile phase B.

[0122] (2) Reference substance: Collagenase standard I (source: United States Pharmacopoeia USP, batch number: F029B0, protein content: 53 mg / mL) and collagenase standard II (source: United States Pharmacopoeia USP, batch number: F029C0, protein content: 52 mg / mL) were measured in appropriate amounts and diluted with water to 2 mg / mL.

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

[0124] (4) Blank control: pure water.

[0125] 2. Liquid phase conditions

[0126] Instrument: Agilent 1260 liquid chromatography system;

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

[0128] Mobile phase: Mobile phase A: 0.25% trifluoroacetic acid in water;

[0129] Mobile phase B: 0.15% trifluoroacetic acid in acetonitrile;

[0130] UV detector, detection wavelength: 214nm;

[0131] Chromatographic column temperature: 35°C;

[0132] Flow rate: 1.5 mL / 1 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 products

[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 products were obtained as shown in Table 9 below:

[0142] Table 9 Test results of test products

[0143]

[0144]

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

[0146] Comparative Example 2:

[0147] Compared with Example 3, the gradient elution program was changed as follows; 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 products

[0152]

[0153] As can be seen from Table 11, the change in the gradient elution program significantly reduced the separation degree and theoretical plate number, making it impossible to separate collagenase type I and type II, affecting the calculation of purity %.

[0154] Test case: Methodological investigation

[0155] The methodological 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 solution: Take an appropriate amount of collagenase solution and dilute it to 0.5 mg / mL with water.

[0158] 100% test solution (test solution): Take an appropriate amount of collagenase solution and dilute it to 1 mg / mL with water.

[0159] 150% test solution: Take an appropriate amount of collagenase solution and dilute it to 1.5 mg / mL with water.

[0160] Detection limit solution: Take an appropriate amount of collagenase solution and dilute it to 0.05 mg / mL with water.

[0161] 1. Exclusivity

[0162] Use pure water as blank and prepare 1 portion of test solution, and inject 1 needle each time.

[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 solution, and the specificity meets the standard requirements.

[0166] 2. Repeatability

[0167] Prepare three concentrations of test samples at 50%, 100% and 150%, prepare three copies for each concentration, and inject one needle into each copy. Use the area normalization method to calculate the purity RSD% of three injections with the same concentration; the purity RSD% of nine injections with different concentrations. The results are shown in the following table:

[0168] Table 13 Repeatability test results

[0169]

[0170]

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

[0172] 3. Detection limit

[0173] Prepare 1 portion of the detection limit solution and inject 1 injection. Detection limit: The detection limit of the lower verification peak type I is used as the detection limit of this method, and the S / N ratio is not less than 3. 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, which meets the signal-to-noise ratio requirement of the detection limit, so the detection limit of this method is 0.05 mg / mL.

[0174] The same method was used to measure the detection limit of type II. When the test sample concentration was 0.05 mg / mL, the average signal-to-noise ratio of the type II collagenase peak was 4.002, which met the signal-to-noise ratio requirement of 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] Changing column temperature: other chromatographic conditions remain unchanged, only the column temperature is changed: 38℃, 40℃, 42℃ respectively; prepare 100% concentration of test sample, inject 1 injection under each condition, calculate the RSD% of the purity of the 3 injections, the results are as follows:

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

[0179]

[0180] Conclusion: When the column temperature is in the range of 38℃-42℃, the theoretical plate number and separation degree of the main peak meet the requirements and the durability is good.

[0181] 4.2 Different flow rates

[0182] Changing the flow rate: other chromatographic conditions remain unchanged, only the flow rates are changed: 0.8mL / min, 1mL / min, 1.2mL / min respectively; prepare 100% concentration of the test sample, inject 1 injection under each condition, calculate the RSD% of the purity of the 3 injections, and the results are as follows:

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

[0184]

[0185] Conclusion: When the flow rate is in the range of 0.8-1.2 mL / min, the theoretical plate number and separation degree of the main peak meet the requirements and the durability is good.

[0186] 4.3. Stability of test solution

[0187] Prepare one portion of the test sample at 100% concentration and place it under sample cell conditions for 0 h, 6 h, 12 h, 18 h, and 24 h. Take one injection at each time point and calculate the RSD% of the purity of the five injections.

[0188] Table 16 Test solution stability test results

[0189]

[0190] Conclusion: When the sample was placed in the sample cell for 0-24 hours, the RSD% of the peak area percentage was ≤2%, so the test solution had good stability within 24 hours.

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

[0192] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0193] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience and preference of those skilled in the art.

[0194] In the present invention, merely listing the steps of the method in a certain order does 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, octaalkylsilane bonded silica gel, octadecylsilane bonded silica gel, phenylsilane bonded silica gel, pentafluorophenylsilane bonded silica gel, and phenyl-hexylsilane bonded silica gel.

2. The detection method according to claim 1, characterized in that: The stationary phase of the chromatographic column is selected from: tetraalkylsilane bonded silica gel, octadecylsilane bonded silica gel, and octadecylsilane bonded silica gel.

3. The detection method according to claim 1, characterized in that: In the HPLC method, the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a trifluoroacetic acid aqueous solution with a volume percentage concentration of 0.01-1.00%, and the mobile phase B is a trifluoroacetic acid acetonitrile solution with a volume percentage concentration of 0.01-1.00%; Preferably, 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 0.05-0.25%.

4. The detection method according to claim 1, characterized in that: In the HPLC method, a gradient elution program is used, and the gradient elution program is as follows: t = 0.00 min, the volume percentage concentration of mobile phase B is 10-50%; 3≤t≤7min, the volume percentage concentration of mobile phase B is 10-50%; 33≤t≤37min, the volume percentage concentration of mobile phase B is 40-80%; 34≤t≤42min, the volume percentage concentration of mobile phase B is 80-100%; 38≤t≤46min, the volume percentage concentration of mobile phase B is 80-100%; 39≤t≤51min, the volume percentage concentration of mobile phase B is 10-50%; 43≤t≤55min, the volume percentage concentration of mobile phase B is 10-50%; Preferably, the gradient elution procedure is as follows: t = 0.00 min, the volume percentage concentration of mobile phase B is 20-45%; 3≤t≤7min, the volume percentage concentration of mobile phase B is 20-45%; 33≤t≤37min, the volume percentage concentration of mobile phase B is 50-75%; 34≤t≤42min, the volume percentage concentration of mobile phase B is 90-100%; 38≤t≤46min, the volume percentage concentration of mobile phase B is 90-100%; 39≤t≤51min, the volume percentage concentration of mobile phase B is 20-45%; 43≤t≤55min, the volume percentage concentration of mobile phase B is 20-45%; More preferably, the gradient elution procedure is as follows: t = 0.00 min, the volume percentage concentration of mobile phase B is 20-45%; 3≤t≤5min, the volume percentage concentration of mobile phase B is 20-45%; 35≤t≤37min, the volume percentage concentration of mobile phase B is 50-75%; 36≤t≤42min, the volume percentage concentration of mobile phase B is 90-100%; 40≤t≤46min, the volume percentage concentration of mobile phase B is 90-100%; 41≤t≤48min, the volume percentage concentration of mobile phase B is 20-45%; 45≤t≤53min, the volume percentage concentration of mobile phase B is 20-45%.

5. 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; Preferably, the diluent is selected from: one or more of acetonitrile, trifluoroacetic acid, formic acid, and water; Preferably, the diluent is a trifluoroacetic acid aqueous solution with a volume percentage concentration of 0.01-1.00%, preferably a trifluoroacetic acid aqueous solution with a volume percentage concentration of 0.05-0.25%; Preferably, the diluent is water.

6. The detection method according to claim 1, characterized in that: In the HPLC method, the flow rate is 0.1-5 mL / min, preferably 0.5-1.5 mL / min, more preferably 0.8-1.5 mL / min; Preferably, in the HPLC method, the column temperature is 20-50°C, preferably 35-45°C, more preferably 35-42°C.

7. The detection method according to claim 1, characterized in that: In the HPLC method, the injection volume is 1-100 μL, preferably 5-50 μL; Preferably, in the HPLC method, the concentration of the test solution is 0.05-5 mg / mL, preferably 0.05-2.5 mg / mL.

8. The detection method according to claim 1, characterized in that: In the HPLC method, the detector is a UV detector; Preferably, in the HPLC method, the detection wavelength is 200-300 nm, preferably 214 nm or 280 nm.

9. The detection method according to claim 1, characterized in that: The collagenase further comprises one or more impurities, wherein the impurities are selected from: clostripain, neutral protease, trypsin, and tyrosinase; Preferably, the collagenase is obtained by pre-treating the complex, wherein the pre-treatment comprises one or more separation and / or purification steps; Preferably, the complex is a culture of Clostridium histolyticum.

10. Use of the detection method according to any one of claims 1 to 9 in the quality control or quality evaluation of collagenase products, or the quality control or quality evaluation of the preparation process of collagenase products.

Images