Method for detecting purity of carbonic anhydrase

Abstract

The invention relates to a carbonic anhydrase purity detection method, and belongs to the technical field of enzyme detection and analysis. The carbonic anhydrase purity detection method provided by the invention comprises the following steps: preparing a to-be-detected sample solution, analyzing by adopting reversed-phase liquid chromatography to obtain a chromatogram, and calculating to obtain the carbonic anhydrase purity, wherein the mobile phases respectively adopt an aqueous solution containing sodium chloride and formic acid and a mixed solution of the aqueous solution containing sodium chloride and formic acid and acetonitrile, and elution separation is carried out under specific elution conditions. By adopting the detection method disclosed by the invention, the purity of carbonic anhydrase in a sample can be rapidly detected within 20 minutes, the result accuracy is high, the precision of the method is good, the durability is good, bovine serum albumin, recombinant carbonic anhydrase and bovine hemoglobin can be well separated, the detection of carbonic anhydrase of various different sources is realized, and the detection method has a wide application prospect. The method has a very high application value in the quality control of carbonic anhydrase products.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme detection and analysis, and in particular to a method for detecting the purity of carbonic anhydrase. Background Art

[0002] Carbonic anhydrase is a Zn-containing 2+ Metalloenzymes are one of the most active enzymes in nature, with a reaction constant of up to 106s. -1 Because of its sensitivity to heavy metals, its ability to catalyze the reversible hydration reaction of CO2 and its esterase activity, and its multiple physiological functions in the body, participation in multiple ion exchanges, and maintenance of the body's internal environmental homeostasis, it has been widely used in environmental testing, CO2 capture and conversion, biosensors, and clinical medicine.

[0003] Purity detection of carbonic anhydrase is an important part of the quality control of carbonic anhydrase products. Polyacrylamide gel electrophoresis and high performance liquid chromatography are two commonly used detection methods. Polyacrylamide gel electrophoresis separates proteins according to the different mobilities produced by the different relative molecular masses of protein molecules, and identifies the purity of carbonic anhydrase by the presence of impurities in the protein bands and the depth of the color of the target bands. The traditional polyacrylamide gel electrophoresis operation steps are cumbersome, time-consuming, and there are many interference factors in the detection. It requires a high level of operation for personnel, and it is impossible to detect impurities with a molecular weight similar to that of carbonic anhydrase. High performance liquid chromatography uses the different polarity characteristics of protein molecules and the difference in the stationary phase to make their retention time in the chromatographic column different, so as to achieve the effect of chromatographic peak separation, and the purity is identified by the peak shape and peak area of ​​the chromatogram. It has the advantages of simple operation, high resolution, high sensitivity, good repeatability, and more accurate analysis results.

[0004] The existing high performance liquid chromatography method for detecting the purity of carbonic anhydrase refers to the article "Identification and Application of Mass Spectrometry for Protein Coupling Sites on Media Surfaces", selects acetonitrile and trifluoroacetic acid as mobile phases, and adopts gradient elution, which can better separate carbonic anhydrase and detect its purity. However, this method lacks methodological investigations such as specificity, precision, and durability, and needs further verification. In addition, the detection time is as long as 60 minutes, and it has the disadvantages of long time consumption, high detection cost, and low efficiency, and cannot meet the needs of large-scale carbonic anhydrase purity detection.

[0005] Therefore, developing a liquid chromatography method that is efficient, accurate, widely applicable, and can achieve good separation effects to meet the purity detection requirements of carbonic anhydrase is an urgent need for the quality control of carbonic anhydrase products and has high application value in the industry. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for detecting the purity of carbonic anhydrase. The detection method provided by the present invention has good specificity, precision, and durability, and the results are accurate and reliable. At the same time, the detection time is short and the efficiency is high, which can better meet the actual application needs.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides a method for detecting the purity of carbonic anhydrase, comprising the following steps:

[0009] (1) Dissolving a sample to be tested in a solvent to obtain a sample solution, and analyzing the sample solution by reverse phase liquid chromatography to obtain a chromatogram; the elution conditions of the reverse phase liquid chromatography are as follows:

[0010] At 0-10 min, the volume ratio of mobile phase A to mobile phase B was controlled to change from 30:70 to 0:100 for gradient elution; at 10.01-20 min, the volume ratio of mobile phase A to mobile phase B was controlled to be 30:70 for isocratic elution;

[0011] The mobile phase A is an aqueous solution with a sodium chloride concentration of 0.1-0.15 mol / L and a formic acid volume content of 0.02-0.07%; the mobile phase B is a mixture of solution B and acetonitrile, wherein solution B is an aqueous solution with a sodium chloride concentration of 0.1-0.15 mol / L and a formic acid volume content of 0.05-0.15%, and the volume ratio of solution B to acetonitrile is 1:(0.9-1.1);

[0012] (2) Analyze the chromatogram to obtain the purity of carbonic anhydrase in the sample to be tested.

[0013] The present invention aims at the quality control detection problem of carbonic anhydrase products, and provides a detection method for the purity of carbonic anhydrase with reversed phase liquid chromatography as the core. The present invention optimizes the mobile phase system of liquid chromatography elution separation, and adopts a mobile phase A as an aqueous solution containing 0.1-0.15mol / L sodium chloride solution and 0.02-0.07% v / v formic acid; the mobile phase B is a mixed solution of an aqueous solution containing 0.1-0.15mol / L sodium chloride solution, 0.05-0.15% v / v formic acid and acetonitrile. According to the above-mentioned gradient elution method, it only takes less than 20 minutes to completely elute and separate the carbonic anhydrase, thereby analyzing and obtaining its purity.

[0014] The mobile phase and elution conditions adopted by the present invention can achieve the complete separation of recombinant carbonic anhydrase from bovine hemoglobin and bovine serum albumin, eliminate the interference of impurities, have strong specificity for carbonic anhydrase, high accuracy, and meet the basic requirements of carbonic anhydrase purity detection; at the same time, the influence of solvent peak on the detection result can be avoided, and the detection result can be more accurate. In addition, the method has excellent resolution effect on carbonic anhydrase from different sources, and can separate carbonic anhydrase extracted from different sources (such as bovine carbonic anhydrase and porcine carbonic anhydrase) according to the difference in retention time, and has wide applicability.

[0015] The carbonic anhydrase purity detection method provided by the invention has good precision and durability. When the mobile phase changes within a limited range or other detection conditions change to a certain extent, the detection result of the carbonic anhydrase purity is basically not affected, and the RSD of the main peak purity under the conditions of various parameter changes is less than 2%. At the same time, the detection accuracy of the method of the invention is high and the specificity is good, and the complete separation of recombinant carbonic anhydrase and impurities can be achieved, as well as the separation and detection of carbonic anhydrase samples extracted from different sources can be achieved.

[0016] The method for detecting the purity of carbonic anhydrase provided by the present invention can obtain relatively accurate and reliable detection data, meet the quality control requirements of carbonic anhydrase products, and has the advantages of simple operation, low detection cost, short time consumption, high detection efficiency, etc., and has high practical application value.

[0017] Preferably, in step (1), the ratio of the mass of the sample to be tested to the volume of the sample solution is (0.5-1.5) mg: (0.8-1.2) mL.

[0018] Preferably, in step (1), the solvent is water.

[0019] Preferably, the reverse phase liquid chromatography method uses C 18 Reverse phase chromatography column, the specification of the chromatography column is 4.6mm×250mm, the particle size is 5μm, the pore size is There is no special requirement for the chromatographic column used in the reverse phase liquid chromatography method of the present invention, as long as it has the same performance.

[0020] Preferably, the detection wavelength of the reverse phase liquid chromatography is 220-280 nm.

[0021] Preferably, in the reverse phase liquid chromatography, the injection volume of the sample solution is 10-20 μL.

[0022] Preferably, the elution flow rate of the reverse phase liquid chromatography is 0.8-1.2 mL / min.

[0023] Preferably, in the reverse phase liquid chromatography, the column temperature of the chromatographic column is 25-35°C.

[0024] When the detection method of carbonic anhydrase purity of the present invention is changed within the above-mentioned condition range, the main peak purity of carbonic anhydrase in the chromatogram is basically not affected, and relatively accurate and reliable results can be obtained, and the method has good durability.

[0025] Further preferably, in the reverse phase liquid chromatography, the detection wavelength is 280 nm, the injection volume of the sample solution is 20 μL, the elution flow rate is 1.0 mL / min, and the column temperature of the chromatographic column is 30° C.

[0026] Preferably, in step (2), the chromatogram is analyzed using an area normalization method.

[0027] The invention adopts an area normalization method, can efficiently analyze carbonic anhydrase products with higher purity, and is suitable for the rapid detection demand for quality control of carbonic anhydrase products in large-scale production.

[0028] In a second aspect, the present invention provides use of the above-mentioned method for detecting the purity of carbonic anhydrase in quality control of carbonic anhydrase products.

[0029] Compared with the traditional polyacrylamide gel electrophoresis method, the method provided by the present invention is simple to operate, short in time, high in resolution, less in interference factors, good in precision and repeatability, can be tested by continuous sampling, and the detection accuracy and detection efficiency are significantly better; compared with the existing high performance liquid chromatography technology for detecting carbonic anhydrase, the detection time required by the present invention is greatly shortened, the detection efficiency is significantly improved, and the consumption of mobile phase is saved, and the detection time cost is reduced. The detection method of carbonic anhydrase purity provided by the present invention is applied to large-scale production quality control, can well separate impurities in the product, has high detection efficiency, and can realize rapid detection of high impurity content purity defective products.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The detection method of carbonic anhydrase purity of the present invention provides an optimized reverse phase liquid chromatography method, realizes efficient detection of carbonic anhydrase purity, and the method has high durability and good precision; by controlling the specific mobile phase elution condition, the complete separation of recombinant carbonic anhydrase and impurities, and the separation and detection of carbonic anhydrase from different extraction sources are realized, and the specificity is strong and the accuracy is high. Compared with the traditional polyacrylamide gel electrophoresis method and the existing high performance liquid chromatography technology for detecting carbonic anhydrase, the method provided by the present invention is simple to operate, short in time, high in resolution, few in interference factors, good in precision and repeatability, can be detected by continuous sampling, has high detection accuracy and better detection efficiency, and has high application value in the quality control and detection of carbonic anhydrase products. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1A chromatogram of high-purity carbonic anhydrase detected by the method for detecting the purity of carbonic anhydrase of the present invention;

[0033] Figure 2 A chromatogram of bovine serum albumin detected by the method for detecting the purity of carbonic anhydrase of the present invention;

[0034] Figure 3 A chromatogram of bovine hemoglobin detected by the method for detecting the purity of carbonic anhydrase of the present invention;

[0035] Figure 4 A chromatogram of bovine serum albumin + recombinant carbonic anhydrase detected by the carbonic anhydrase purity detection method of the present invention;

[0036] Figure 5 A chromatogram of bovine hemoglobin + recombinant carbonic anhydrase detected by the carbonic anhydrase purity detection method of the present invention;

[0037] Figure 6 A chromatogram of bovine serum albumin+bovine hemoglobin+recombinant carbonic anhydrase detected by the carbonic anhydrase purity detection method of the present invention;

[0038] Figure 7 A chromatogram of bovine carbonic anhydrase detected by the method for detecting the purity of carbonic anhydrase of the present invention;

[0039] Figure 8 A chromatogram of porcine carbonic anhydrase detected by the method for detecting the purity of carbonic anhydrase of the present invention;

[0040] Fig. 9 A chromatogram of recombinant carbonic anhydrase detected by the method for detecting the purity of carbonic anhydrase of the present invention;

[0041] Fig.10 A chromatogram of a mixed sample of bovine carbonic anhydrase + porcine carbonic anhydrase + recombinant carbonic anhydrase detected by the carbonic anhydrase purity detection method of the present invention;

[0042] Fig.11 is a chromatogram of a blank solvent obtained by the detection method of the comparative example;

[0043] Fig.12 The chromatogram of the recombinant carbonic anhydrase obtained by the detection method of the comparative example;

[0044] Fig.13 is a chromatogram of bovine serum albumin obtained by the detection method of the comparative example;

[0045] Fig.14 is a chromatogram of bovine hemoglobin obtained by the detection method of the comparative example;

[0046] Fig.15The summary diagram of the chromatogram is obtained by adopting the detection method of the comparative example. DETAILED DESCRIPTION

[0047] In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention will be further described below in conjunction with specific examples. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0048] Experimental Example 1

[0049] In order to explore the accuracy of the detection method of the purity of carbonic anhydrase of the present invention, the detection method of the purity of carbonic anhydrase of the present invention is used to detect high-purity commercially available samples, comprising the following steps:

[0050] (1) Pretreatment and mobile phase preparation

[0051] Prepare the sample solution; weigh an appropriate amount of a commercially available high-purity recombinant carbonic anhydrase product (labeled purity > 99%), dissolve it in ultrapure water and dilute it into a sample solution with a protein concentration of 1 mg / mL, filter it through a 0.45 μm microporous filter membrane, and set aside.

[0052] Prepare mobile phase A: prepare 500 mL of 0.1 mol / L sodium chloride solution, add 0.25 mL of formic acid, mix well, filter through a 0.45 μm microporous membrane, and sonicate for 10 min.

[0053] Prepare mobile phase B: prepare 500 mL of 0.1 mol / L sodium chloride aqueous solution, add 0.5 mL of formic acid, mix with 500 mL of acetonitrile, pass through a 0.45 μm microporous filter membrane, and ultrasonicate for 10 min.

[0054] (2) Reversed-phase liquid chromatography analysis

[0055] Adopt Elite C 18 Column (Cat. No. E3322624), size 4.6 mm × 250 mm, particle size 5 μm, pore size The liquid chromatography conditions were set as follows: detection wavelength 280 nm, mobile phase flow rate 1.0 mL / min, sample injection volume 20 μL, column temperature 30 °C, elution program settings as shown in Table 1, and the chromatogram was obtained. Figure 1 .

[0056] (3) The chromatogram was automatically integrated by the instrument workstation and the area normalization method was used to calculate the purity of carbonic anhydrase. The results of three injection tests are shown in Table 2.

[0057] like Figure 1As shown in Table 2, the retention time of the main peak is basically the same, and the main peak is well separated from the impurity peak; as shown in Table 2, the sample was injected 3 times, and the RSD of the main peak purity was less than 2%. The purity of the commercially purchased carbonic anhydrase used in this experimental example is marked as >99%, and the average value of the test result is 99.65%, with very small deviation. The purity of the carbonic anhydrase detected by the detection method of the carbonic anhydrase purity of the present invention is relatively high in accuracy.

[0058] Table 1 Elution program

[0059] Time (min) Mobile phase A (%) Mobile phase B (%) 0.00 30 70 10.00 0 100 10.01 30 70 20.00 30 70

[0060] Table 2 Purity test results of commercially available samples with known purity

[0061]

[0062] Experimental Example 2

[0063] In order to explore the specificity of the detection method of carbonic anhydrase purity of the present invention, the detection method of carbonic anhydrase purity of the present invention was used to detect bovine serum albumin sample solution, bovine hemoglobin sample solution, bovine serum albumin + recombinant carbonic anhydrase sample solution, bovine hemoglobin solution + recombinant carbonic anhydrase sample solution, and bovine serum albumin + bovine hemoglobin + recombinant carbonic anhydrase mixed solution. The specific steps are different from those of Experimental Example 1 in that the sample solution is prepared using the following steps:

[0064] Recombinant carbonic anhydrase solution: Weigh an appropriate amount of the recombinant carbonic anhydrase sample, dissolve it in ultrapure water and dilute it to a solution with a protein concentration of 1 mg / mL.

[0065] Bovine serum albumin sample solution: Weigh 10 mg of bovine serum albumin standard, dilute to 10 mL with ultrapure water to make a solution with a protein concentration of 1 mg / mL, and filter through a 0.45 μm microporous filter membrane.

[0066] Bovine hemoglobin sample solution: Take an appropriate amount of purified bovine hemoglobin sample, dissolve and dilute it with ultrapure water to a solution with a protein concentration of 1 mg / mL, and pass it through a 0.45 μm microporous filter membrane.

[0067] The recombinant carbonic anhydrase solution was mixed with a bovine serum albumin solution and a bovine hemoglobin solution at a volume ratio of 1:1, and filtered through a 0.45 μm microporous filter membrane to obtain a bovine serum albumin + recombinant carbonic anhydrase sample solution and a bovine hemoglobin solution + recombinant carbonic anhydrase sample solution, respectively;

[0068] The recombinant carbonic anhydrase solution, bovine serum albumin solution and bovine hemoglobin solution were mixed in a volume ratio of 1:1:1, and filtered through a 0.45 μm microporous filter membrane to obtain a mixed solution of bovine serum albumin+bovine hemoglobin+recombinant carbonic anhydrase.

[0069] The bovine serum albumin chromatogram obtained by the detection method of carbonic anhydrase purity of the present invention is as follows: Figure 2 , the chromatogram of bovine hemoglobin is as follows Figure 3 , the chromatogram of bovine serum albumin + recombinant carbonic anhydrase is as follows Figure 4 , the chromatogram of bovine hemoglobin + recombinant carbonic anhydrase is as follows Figure 5 , the chromatogram of bovine serum albumin + bovine hemoglobin + recombinant carbonic anhydrase is as follows Figure 6 .

[0070] Compare Figure 1 and Figure 2-6 It can be seen that when a single component is detected, the retention time of the recombinant carbonic anhydrase is 10.496 min, the retention time of bovine serum albumin is 8.577 min, and the retention time of bovine hemoglobin is 11.941 min, and there is a certain difference in their retention times; when prepared as a mixed sample for detection, it can be seen that in the chromatograms of the components of recombinant carbonic anhydrase+bovine serum albumin, recombinant carbonic anhydrase+bovine hemoglobin, and recombinant carbonic anhydrase+bovine serum albumin+bovine hemoglobin, the recombinant carbonic anhydrase, bovine serum albumin and bovine hemoglobin can be well separated without interference with each other. The detection method of the purity of the carbonic anhydrase of the present invention has good specificity and can eliminate the interference of impurities.

[0071] Experimental Example 3

[0072] In order to explore the separation effect of the carbonic anhydrase purity detection method of the present invention on carbonic anhydrase extracted from different sources, the carbonic anhydrase purity detection method of the present invention was used to detect bovine carbonic anhydrase sample solution, porcine carbonic anhydrase sample solution, recombinant carbonic anhydrase sample solution, and a mixed sample solution of recombinant carbonic anhydrase + porcine carbonic anhydrase + bovine carbonic anhydrase. The specific steps are different from those of Experimental Example 1 in that the sample solution is prepared using the following steps:

[0073] Recombinant carbonic anhydrase solution: Weigh an appropriate amount of the lyophilized recombinant carbonic anhydrase sample, dissolve and dilute it with ultrapure water to a solution with a protein concentration of 1 mg / mL, filter it through a 0.45 μm microporous filter membrane, and set aside.

[0074] Bovine carbonic anhydrase: Weigh an appropriate amount of freeze-dried bovine carbonic anhydrase sample, dissolve and dilute with ultrapure water to a solution with a protein concentration of 1 mg / mL, filter through a 0.45 μm microporous filter membrane, and set aside.

[0075] Porcine carbonic anhydrase: Weigh an appropriate amount of freeze-dried porcine carbonic anhydrase sample, dissolve and dilute with ultrapure water to a solution with a protein concentration of 1 mg / mL, filter through a 0.45 μm microporous filter membrane, and set aside.

[0076] Mixed sample of bovine carbonic anhydrase + porcine carbonic anhydrase + recombinant carbonic anhydrase: Mix the above-prepared bovine carbonic anhydrase solution, porcine carbonic anhydrase solution and recombinant carbonic anhydrase solution in a volume ratio of 1:1:1, filter through a 0.45 μm microporous filter membrane and set aside.

[0077] The chromatogram of bovine carbonic anhydrase obtained by detecting the above sample solution using the carbonic anhydrase purity detection method of the present invention is as follows: Figure 7 The chromatogram of porcine carbonic anhydrase is as follows: Figure 8 The chromatogram of recombinant carbonic anhydrase is shown in Fig. 9 The chromatogram of the mixed sample of bovine carbonic anhydrase + porcine carbonic anhydrase + recombinant carbonic anhydrase is as follows Fig.10 .

[0078] Depend on Figure 7-9 It can be seen that when a single component is detected, the retention time of bovine carbonic anhydrase is 11.605min, the retention time of porcine carbonic anhydrase is 10.593min, and the retention time of recombinant carbonic anhydrase is 10.223min; Fig.10 It can be seen that under the detection method of the purity of carbonic anhydrase of the present invention, the retention times of carbonic anhydrases from three different extraction sources have certain differences. Different types of bovine carbonic anhydrase, porcine carbonic anhydrase and recombinant carbonic anhydrase can be separated from the chromatogram, which is suitable for the detection of carbonic anhydrase prepared by recombinant or extraction method.

[0079] Experimental Example 4

[0080] In order to explore the precision of the detection method of the purity of carbonic anhydrase of the present invention, the following test was performed using the detection method of the purity of carbonic anhydrase of the present invention:

[0081] 1. The commercially available recombinant carbonic anhydrase sample was detected by referring to the method in Experimental Example 1, and the sample was injected 6 times continuously. The results are shown in Table 3.

[0082] 2. Referring to the method in Experimental Example 1, different experimenters measured commercially available recombinant carbonic anhydrase samples and injected them 6 times in succession. The results are shown in Table 4.

[0083] From Table 3 and Table 4, we can see that:

[0084] The recombinant carbonic anhydrase sample solution was continuously injected 6 times, and the purity average value obtained was 97.495%, and the RSD value was 0.16% (less than 2%), the instrument had good precision, and the method had good repeatability. The RSD of the recombinant carbonic anhydrase purity measured by different experimenters was 0.16% and 0.05% respectively, both less than 2%, and the intermediate precision was 0.15% (less than 2%). The chromatographic conditions of the detection method of the carbonic anhydrase purity of the present invention are stable, the precision is good, and the methodological requirements are met.

[0085] Table 3 Precision experimental data 1

[0086]

[0087]

[0088] Table 4 Precision experimental data 2

[0089]

[0090] Experimental Example 5

[0091] In order to explore the durability of the detection method for the purity of carbonic anhydrase of the present invention, on the basis of the detection steps and conditions of Experimental Example 1, the same batch of recombinant carbonic anhydrase samples were detected by changing the mobile phase preparation ratio, flow rate, column temperature and detection wavelength, and the tolerance of the detection method of the present invention to the results not being affected when the detection conditions were changed was evaluated, so as to examine the durability of the method developed by the present invention and provide a basis for the method to be used for routine inspection of the purity of carbonic anhydrase.

[0092] The nine different test conditions used in the durability experiment are shown in Table 5, and the purity results of the same batch of recombinant carbonic anhydrase samples tested under different durability test conditions are shown in Table 6.

[0093] As shown in Tables 5 and 6, when the sodium chloride concentration of mobile phase A in the method for detecting the purity of carbonic anhydrase of the present invention is 0.1-0.15 mol / L, 0.10-0.35 mL of formic acid is added, the sodium chloride concentration of mobile phase B is 0.1-0.15 mol / L, 0.25-0.75 mL of formic acid is added, and the mobile phase flow rate is 0.8-1.2 mL / min, the column temperature is 25-35° C., and the detection wavelength is 220-280 nm, when the parameters of each condition vary within the range, the RSD of the main peak purity of the recombinant carbonic anhydrase is less than 2%; the main peak purity of the recombinant carbonic anhydrase under different chromatographic conditions is substantially unaffected, and the purity is all within 97.42%±0.57, and the RSD under different parameters is less than 2%. The detection method for the purity of carbonic anhydrase provided by the present invention has only slight and insignificant changes in the detection results under parameter fluctuations within the limited condition range, and has good durability.

[0094] Table 5 Durability test conditions of Experimental Example 5

[0095]

[0096]

[0097] Table 6 Test results of different durability test conditions in Experimental Example 5

[0098]

[0099]

[0100] Example 1

[0101] An embodiment of a method for detecting the purity of carbonic anhydrase of the present invention, the method for detecting the purity of carbonic anhydrase described in this embodiment comprises the following steps:

[0102] (1) Pretreatment and mobile phase preparation

[0103] Prepare sample solution; weigh appropriate amount of recombinant carbonic anhydrase products from 3 different production batches, dissolve and dilute with ultrapure water to a sample solution with a protein concentration of 1 mg / mL, filter through a 0.45 μm microporous filter membrane, and set aside.

[0104] Prepare mobile phase A: prepare 500 mL of 0.1 mol / L sodium chloride solution, add 0.25 mL of formic acid, mix well, filter through a 0.45 μm microporous membrane, and ultrasonically vibrate for 10 min.

[0105] Prepare mobile phase B: prepare 500 mL of 0.1 mol / L sodium chloride aqueous solution, add 0.5 mL of formic acid, mix with 500 mL of acetonitrile, filter through a 0.45 μm microporous membrane, and ultrasonically vibrate for 10 min.

[0106] (2) Reversed-phase liquid chromatography analysis

[0107] Adopt Elite C 18 Column (Cat. No. E3322624), size 4.6 mm × 250 mm, particle size 5 μm, pore size The liquid chromatography conditions were set as follows: detection wavelength 280 nm, mobile phase flow rate 1.0 mL / min, sample injection volume 20 μL, column temperature 30° C., elution program settings were the same as in Experimental Example Table 1, and a chromatogram was obtained.

[0108] (3) The chromatogram was automatically integrated by the instrument workstation and the area normalization method was used to calculate the purity of the carbonic anhydrase. Three parallel tests were performed on each batch of recombinant carbonic anhydrase samples. The results are shown in Table 7.

[0109] The retention time of the main peak of the three batches of recombinant carbonic anhydrase was consistent, and the main peak was well separated from the impurity peak. The RSD of the main peak purity of the three batches was less than 2%. The method had good stability and was suitable for purity detection of different batches of recombinant carbonic anhydrase.

[0110] Table 7 Purity test results of different batches of recombinant carbonic anhydrase

[0111]

[0112] Example 2

[0113] An embodiment of a method for detecting the purity of carbonic anhydrase of the present invention. The purity detection of carbonic anhydrase described in this embodiment is different from that in Example 1 only in that the purity detection is performed on recombinant carbonic anhydrase, porcine carbonic anhydrase and bovine carbonic anhydrase samples from different extraction sources, respectively. The results are shown in Table 8.

[0114] It can be seen that the main peak purity RSDs of the recombinant carbonic anhydrase, porcine carbonic anhydrase and bovine carbonic anhydrase are all less than 2%. The detection method provided by the present invention is suitable for the purity detection of carbonic anhydrase from different sources.

[0115] Table 8 Purity test results of carbonic anhydrase from different sources

[0116]

[0117] Comparative Example 1

[0118] Comparative Example 1 uses the same sample preparation method and chromatographic column, detection wavelength, flow rate, injection volume, and column temperature conditions as the detection method for carbonic anhydrase purity in Example 1; the difference from Example 1 is that different mobile phases and elution conditions are used, specifically replaced by:

[0119] Mobile phase A: Prepare 450 mL of 0.05 mol / L sodium chloride, add 0.15 mL of trifluoroacetic acid and 45 mL of acetonitrile, mix and shake well, pass through a 0.45 μm microporous filter membrane, and ultrasonicate for 10 min.

[0120] Mobile phase B: prepare 500 mL of 0.05 mol / L sodium chloride, add 0.3 mL of trifluoroacetic acid, mix with 500 mL of acetonitrile, shake well, pass through a 0.45 μm microporous filter membrane, and ultrasonicate for 10 min.

[0121] The elution program used is shown in Table 9.

[0122] The chromatogram of the blank solvent obtained by the above-mentioned conditions is as follows: Fig.11 The chromatogram of the recombinant carbonic anhydrase detected is as follows Fig.12 The chromatogram of bovine serum albumin obtained by detection is as follows Fig.13 The chromatogram of bovine hemoglobin obtained by detection is as follows Fig.14 , Fig.15 The above chromatograms are summarized. Fig.15 It can be seen that there is no solvent peak within the elution time of bovine serum albumin and bovine hemoglobin, but the retention time of recombinant carbonic anhydrase is very close to the retention time of the solvent peak, which affects the purity analysis of the sample during the detection process.

[0123] Table 9 Elution procedure of comparative example

[0124] Time (min) Mobile phase A (%) Mobile phase B (%) 0.00 50 50 11.00 0 100 11.50 50 50 20.00 50 50

[0125] Based on the results of the above experimental examples, embodiments and comparative examples, the method for detecting the purity of carbonic anhydrase of the present invention eliminates interference factors by controlling specific mobile phase elution conditions, realizes the complete separation of recombinant carbonic anhydrase and impurities, and separates and detects carbonic anhydrase from different extraction sources, has strong specificity, high accuracy, good precision, and high durability within a limited range. It has significant advantages over traditional polyacrylamide gel electrophoresis and existing high performance liquid chromatography technology for detecting carbonic anhydrase, and has high application value in quality control and detection of carbonic anhydrase products.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting the purity of carbonic anhydrase, characterized in that: The following steps are involved: (1) Dissolving a sample to be tested in a solvent to obtain a sample solution, and analyzing the sample solution by reverse phase liquid chromatography to obtain a chromatogram; the elution conditions of the reverse phase liquid chromatography are as follows: At 0-10 min, the volume ratio of mobile phase A to mobile phase B was controlled to change from 30:70 to 0:100 for gradient elution; at 10.01-20 min, the volume ratio of mobile phase A to mobile phase B was controlled to be 30:70 for isocratic elution; The mobile phase A is an aqueous solution with a sodium chloride concentration of 0.1-0.15 mol / L and a formic acid volume content of 0.02-0.07%; the mobile phase B is a mixture of solution B and acetonitrile, wherein solution B is an aqueous solution with a sodium chloride concentration of 0.1-0.15 mol / L and a formic acid volume content of 0.05-0.15%, and the volume ratio of solution B to acetonitrile is 1:(0.9-1.1); (2) Analyze the chromatogram to obtain the purity of carbonic anhydrase in the sample to be tested.

2. The method for detecting the purity of carbonic anhydrase according to claim 1, characterized in that: In the step (1), the ratio of the mass of the sample to be tested to the volume of the sample solution is (0.5-1.5) mg: (0.8-1.2) mL.

3. The method for detecting the purity of carbonic anhydrase according to claim 1, characterized in that: In the step (1), the solvent is water.

4. The method for detecting the purity of carbonic anhydrase according to claim 1, characterized in that: The reverse phase liquid chromatography method uses C 18 Reverse phase chromatography column, the specification of the chromatography column is 4.6mm×250mm, the particle size is 5μm, the pore size is 5. The method for detecting the purity of carbonic anhydrase according to claim 1, characterized in that: The detection wavelength of the reverse phase liquid chromatography is 220-280 nm.

6. The method for detecting the purity of carbonic anhydrase according to claim 1, characterized in that: In the reverse phase liquid chromatography, the injection volume of the sample solution is 10-20 μL.

7. The method for detecting the purity of carbonic anhydrase according to claim 1, characterized in that: The elution flow rate of the reverse phase liquid chromatography is 0.8-1.2 mL / min.

8. The method for detecting the purity of carbonic anhydrase according to claim 1, characterized in that: In the reverse phase liquid chromatography, the column temperature of the chromatographic column is 25-35°C.

9. The method for detecting the purity of carbonic anhydrase according to claim 1, characterized in that: In the step (2), the chromatogram is analyzed using an area normalization method.

10. Use of the method for detecting the purity of carbonic anhydrase according to any one of claims 1 to 9 in the quality control of carbonic anhydrase products.

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