Heparin precursor content detection method and application thereof

Abstract

The invention provides a heparin precursor content detection method and application thereof, and the detection method comprises the following steps: (S1) preparing a heparin precursor solid sample to be detected into a heparin precursor test solution, or diluting a heparin precursor liquid sample to be detected into the heparin precursor test solution; carrying out enzymolysis on the heparin precursor test solution or the heparin precursor reference substance solution by adopting heparinase III to obtain heparin precursor test solution enzymatic hydrolysate or heparin precursor reference substance enzymatic hydrolysate; determining the peak area of the enzymatic hydrolysate by adopting a high performance liquid chromatography method; (S2) performing linear regression by applying a least square method, and establishing a mathematical relationship between the peak area Y of the heparin precursor reference substance enzymatic hydrolysate and the concentration X of the heparin precursor reference substance solution, namely Y = kX + b; and calculating the concentration of the heparin precursor in the liquid sample or the content of the heparin precursor in the solid sample according to the established mathematical relationship. The liquid phase detection method provided by the invention is higher in accuracy and good in precision, and can provide a detection result more intuitively.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting heparin precursor content and application thereof. Background Art

[0002] Heparin is one of the most widely used drugs and is the first choice for anticoagulation. Traditional heparin includes heparin drugs and APIs, which are mainly extracted by physical extraction. Crude heparin is extracted from pig intestinal membranes and bovine lung tissues, and then purified to obtain pharmaceutical-grade heparin. However, this method, which has been used to this day, still has some shortcomings, such as environmental pollution and ecological hazards caused by the large-scale use of extraction reagents in the production process, unstable supply of pig small intestine raw materials, and the risk of cross-infection between humans and animals. Therefore, it is urgent to find new sources of heparin or new methods for producing heparin. The enzymatic synthesis of heparin uses oligosaccharides or heparin precursors as raw materials, and synthesizes heparin using green methods through glycosyl transfer. This will cause major changes in the heparin industry and will have a profound impact on the pharmaceutical application of heparin.

[0003] Heparin precursor (heparosan) polysaccharide belongs to glycosaminoglycan, which is a disaccharide repeating unit formed by alternating glucuronic acid GlcUA and acetylglucosamine GlcNAc. It exists in large quantities in the capsules of many bacteria, but currently heparin precursor is mainly extracted from Escherichia coli K5. Heparin precursor (heparosan) has a polysaccharide skeleton structure similar to heparin / heparan sulfate, and can be modified appropriately to obtain non-animal heparin or its analogs. Therefore, heparin precursor has become an ideal raw material for enzymatic synthesis of heparin. The structural formula of heparin precursor is shown below:

[0004] .

[0005] The efficient and low-cost use of microbial fermentation to obtain heparin precursors with controllable molecular weight has important application value and strategic significance. On the one hand, heparin precursors have potential applications in ophthalmology, plastic surgery, dermatology, and coatings for medical devices; heparin precursors and heparin precursor derivatives are also good candidates for high-quality biomaterials for tissue engineering gels and scaffolds and drug delivery carriers; heparin precursors can also be made into drug carriers to enhance the targeting of anticancer drugs, and have important application prospects in the field of pharmaceutical development.

[0006] At present, there are few reports on the detection and quality control methods of heparin precursor content. The sulfuric acid-carbazole method is generally used for the detection of heparin precursor content, which is suitable for the determination of uronic acid content in acidic polysaccharides. Acidic polysaccharides are hydrolyzed into gluconic acid under the action of concentrated sulfuric acid, and react with carbazole in the presence of sulfuric acid to generate a purple-red compound with a carbonyl group, which has a maximum absorption at a wavelength of 530 nm. Heparin precursor (heparosan) polysaccharide is composed of acetylglucosamine and glucuronic acid, of which the percentage of glucuronic acid content is 45.56%. By establishing a standard curve, the content of heparin precursor (heparosan) in the sample can be detected according to the content of glucuronic acid.

[0007] The sulfuric acid-carbazole method is used to measure the content of heparin precursor. The biggest problem of this determination method is poor specificity. It is easily interfered by impurities (heparin precursor analogs, residual culture medium) in reagents or samples. It has the disadvantages of cumbersome operation, harsh reaction reagents and reaction conditions that are not friendly to personnel, poor reproducibility, many interference factors, and poor specificity. The specificity and accuracy of this method need to be improved.

[0008] Quality control of heparin precursors has always been the focus and difficulty in the development, production and utilization of heparin precursors. The current content determination and quality detection methods of heparin precursors have many shortcomings. Therefore, it is urgent to develop a quality control and quality detection method for heparin precursors to ensure the accuracy of quality detection results of heparin precursors during development and application. Summary of the invention

[0009] In view of the shortcomings of the prior art, the present invention aims to provide a method for detecting the content of heparin precursor and its application. The detection method has mild operating conditions, simple operation and accurate results. In particular, the detection method can simultaneously obtain the content and composition results of heparin precursor.

[0010] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0011] In a first aspect, the present invention provides a method for detecting the content of heparin precursor, the detection method comprising:

[0012] (S1) preparing a solid sample of the heparin precursor to be tested into a heparin precursor test solution, or diluting a liquid sample of the heparin precursor to be tested into a heparin precursor test solution; enzymolyzing the heparin precursor test solution or the heparin precursor reference solution with heparinase III to obtain a heparin precursor test solution or a heparin precursor reference solution; and determining the peak area of ​​the solution with high performance liquid chromatography;

[0013] (S2) using the least square method for linear regression, establishing a mathematical relationship between the peak area Y of the heparin precursor reference substance enzymatic hydrolyzate and the concentration X of the heparin precursor reference substance solution, Y = kX + b;

[0014] (S3) Calculating the concentration of the heparin precursor in the heparin precursor liquid sample to be tested according to the established mathematical relationship; the calculation formula for the concentration of the heparin precursor in the heparin precursor liquid sample to be tested is:

[0015] ;

[0016] In the above formula, A is the peak area of ​​the main peak of the heparin precursor test sample enzymatic hydrolyzate, and the main peak is the peak of the product ΔIVa; k is the slope of the linear equation; b is the intercept of the linear equation; N is the dilution multiple of the heparin precursor test sample solution when the heparin precursor sample to be tested is a liquid sample;

[0017] Or, the heparin precursor content in the solid heparin precursor sample to be tested is calculated according to the established mathematical relationship; the calculation formula for the heparin precursor content in the solid heparin precursor sample to be tested is:

[0018] ;

[0019] In the above formula, A is the peak area of ​​the main peak of the heparin precursor test sample enzymatic hydrolyzate, and the main peak is the peak of the product ΔIVa; k is the slope of the linear equation; b is the intercept of the linear equation; m is the mass of the heparin precursor sample to be tested in mg when the heparin precursor sample to be tested is a solid sample; V is the volume of the heparin precursor test solution prepared in mL when the heparin precursor sample to be tested is a solid sample.

[0020] The composition characteristics of the β-D-glucuronic acid and N-acetyl-α-D-glucosamine disaccharide repeating units in the heparin precursor enable it to be specifically and completely degraded into disaccharide units by heparinase III, and the product component is qualitatively identified as ΔIVa. Based on the specificity of this enzyme, the present invention has developed a liquid phase detection method for accurately quantifying the content of heparin precursor. The structure of ΔIVa is shown below.

[0021] .

[0022] Preferably, in step (S1), the heparin precursor reference solution is prepared by dissolving the heparin precursor reference in a solvent; the gradient concentration range of the heparin precursor reference solution is 0.1-10.0 mg / mL; preferably 0.5-2.0 mg / mL.

[0023] In the present invention, the solvent in the heparin precursor reference solution is purified water or buffer; the 0.1-10 mg / mL gradient concentration is selected from a combination of 3 or more concentrations of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 5.0 mg / mL or 10.0 mg / mL. The gradient concentration is further preferably 0.1 mg / mL, 5.0 mg / mL and 10.0 mg / mL, or 0.1 mg / mL, 1.0 mg / mL, 5.0 mg / mL and 10.0 mg / mL, or 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 5.0 mg / mL and 10.0 mg / mL, or 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 5.0 mg / mL and 10.0 mg / mL.

[0024] In a specific embodiment of the present invention, the solvent is purified water. In the present invention, the heparin precursor reference substance is a calibrated heparin precursor.

[0025] Preferably, in step (S1), the concentration of the heparin precursor test solution is 0.1-10.0 mg / mL, preferably 0.5-2.0 mg / mL. 0.1-10.0 mg / mL can be, for example, 0.1 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL, 6.0 mg / mL, 7.0 mg / mL, 8.0 mg / mL, 9.0 mg / mL or 10.0 mg / mL.

[0026] In the present invention, the solvent in the preparation of the heparin precursor test solution is purified water or buffer; in a specific embodiment of the present invention, the solvent is water.

[0027] Preferably, the enzymatic hydrolysis step comprises: mixing sodium calcium acetate solution, heparin precursor reference solution or heparin precursor test solution and heparinase III, enzymatic hydrolysis, terminating the reaction in a water bath after the enzymatic hydrolysis reaction is complete, and centrifuging to obtain the supernatant as the sample to be tested.

[0028] In the present invention, the temperature of the water bath is 80-100° C., for example, 80° C., 85° C., 90° C., 95° C. or 100° C., etc. In the present invention, the enzyme is inactivated by heating in a water bath.

[0029] In the present invention, the enzymatic hydrolysis is carried out at room temperature (20-30°C) under mild conditions; the enzymatic hydrolysis reaction is carried out under pH 5.0-7.0, for example, it can be 5.0, 5.5, 6.0, 6.5 or 7.0, and preferably under pH 5.8-6.5. The reaction is carried out until the heparin precursor in each solution is completely hydrolyzed to obtain an enzymatic hydrolyzate.

[0030] In the present invention, the sodium calcium acetate solution in the enzymatic hydrolysis reaction system is a buffer solution, which provides a buffer system for the enzyme; the pH range of the buffer solution is 6.0-7.5, for example, it can be 6.0, 6.5, 7.0 or 7.5, and more preferably 6.5-7.0; other buffer solutions in the same pH range are also feasible.

[0031] In a specific embodiment of the present invention, a pH 7.0 sodium calcium acetate solution is used as a buffer solution, and the preparation method of the pH 7.0 sodium calcium acetate solution is as follows:

[0032] pH 7.0 sodium calcium acetate solution: Take 10 mg bovine serum albumin and 32 mg calcium acetate, weigh accurately, and place in a 100 mL volumetric flask. Add 60 mL purified water to dissolve, add 580 µL acetic acid, and mix by ultrasonic. Adjust the pH to 7.0 with 2 mol / L sodium hydroxide, add purified water to 100 mL, and then filter with a filter membrane with a pore size of ≤0.45 µm to obtain pH 7.0 sodium calcium acetate solution.

[0033] Preferably, the dosage ratio of heparinase III to heparin precursor in the enzymatic hydrolysis reaction system is greater than or equal to 0.1 U / mg; preferably 0.1-50 U / mg, further preferably 0.2-2 U / mg, for example, it can be 0.1 U / mg, 0.2 U / mg, 0.3 U / mg, 0.5 U / mg, 1 U / mg, 2 U / mg, 5 U / mg, 8 U / mg, 10 U / mL or 50 U / mg, etc.

[0034] In the present invention, the dosage of the heparinase III is excessive, and the excessive heparinase III can ensure that the heparin precursor is fully enzymatically hydrolyzed, which is beneficial to the accuracy of subsequent detection.

[0035] Preferably, the enzymatic hydrolysis time is 3-6 h, for example, 3 h, 4 h, 5 h or 6 h, etc., preferably 5-6 h.

[0036] Preferably, the heparinase III is added in the form of an enzyme stock solution or a heparinase III dilution solution.

[0037] Preferably, the diluent used in the heparinase III diluent is potassium phosphate buffer, and the concentration of the heparinase III diluent is 0.1-4 U / mL, for example, it can be 0.1 U / mL, 0.5 U / mL, 1 U / mL, 2 U / mL, 3 U / mL or 4 U / mL.

[0038] In a specific embodiment of the present invention, the enzymatic hydrolysis comprises the following steps: 50 μL of pH 7.0 sodium calcium acetate solution, 20 μL of the sample to be tested and 50 μL of heparinase III solution are pipetted and mixed evenly by repeated pipetting, and reacted at room temperature for 5-6 h. After the enzymatic hydrolysis reaction is complete, the reaction is terminated by heating to boiling for 1-3 min, centrifuging at 11000 rpm for 10 min, and taking the supernatant as the sample to be tested.

[0039] Preferably, in (S1), the detector used in the high performance liquid chromatography is a UV / visible detector.

[0040] Preferably, in (S1), the chromatographic column used in the high performance liquid chromatography is a strong anion exchange column.

[0041] Preferably, in (S1), the chromatographic column is a SAX chromatographic column or a chromatographic column with equivalent performance.

[0042] Preferably, in (S1), the chromatographic column is Waters Spherisorb SAX, 5.0 μm, 4.6 mm×250 mm, or a chromatographic column with equivalent performance.

[0043] The chromatographic column of the present invention has no interference from foreign peaks during detection and has good detection effect.

[0044] Preferably, in (S1), the column temperature of the HPLC is 38-42°C, for example, 38°C, 39°C, 40°C, 41°C or 42°C.

[0045] Preferably, in (S1), the injection volume of the HPLC is 8-12 μL, for example, 8 μL, 9 μL, 10 μL, 11 μL or 12 μL.

[0046] Preferably, in (S1), the mobile phase used in the high performance liquid chromatography includes mobile phase A and mobile phase B; the mobile phase A is a sodium dihydrogen phosphate solution, and the mobile phase B is a sodium dihydrogen phosphate solution containing sodium perchlorate.

[0047] Preferably, the volume concentration of the sodium dihydrogen phosphate solution in the mobile phase A is 0.02%-0.03%, for example, it can be 0.02%, 0.025% or 0.03%, etc.; preferably, it is 0.028%.

[0048] Preferably, the mobile phase B is a 0.02%-0.03% sodium dihydrogen phosphate solution containing 10%-18% sodium perchlorate; preferably, 0.028% sodium dihydrogen phosphate containing 14% sodium perchlorate.

[0049] Among them, 0.02%-0.03% sodium dihydrogen phosphate can be, for example, 0.02%, 0.025% or 0.03%, etc.; 10%-18% sodium perchlorate can be, for example, 10%, 12%, 14%, 16% or 18%, etc.

[0050] Preferably, the flow rate of the mobile phase is 1.0-2.0 mL / min, for example, 1.0 mL / min, 1.2 mL / min, 1.4 mL / min, 1.5 mL / min, 1.6 mL / min, 1.8 mL / min or 2.0 mL / min, etc.; preferably, it is 1.4 mL / min.

[0051] Preferably, in (S1), the high performance liquid chromatography adopts gradient elution; the procedure of the gradient elution is:

[0052] At 0 min, the volume fraction of mobile phase B was 6.0-8.0%, and the rest was mobile phase A;

[0053] From 0 to 10 minutes, the volume fraction of mobile phase B changed uniformly from 6.0-8.0% to 40.0-42.0%, and the rest was mobile phase A.

[0054] Among them, the volume fraction of 6.0-8.0% can be, for example, 6.0%, 6.5%, 7.0%, 7.5% or 8.0%, and the volume fraction of 40.0-42.0% can be, for example, 40%, 40.5%, 41%, 41.5% or 42.0%.

[0055] In the present invention, before gradient elution, a column balancing step is also included, wherein the column balancing includes balancing the column using a gradient in which the volume fraction of mobile phase B is 6.0-8.0% and the rest is mobile phase A. After gradient elution, a column regeneration step is also included, wherein the column regeneration includes uniformly changing the volume fraction of mobile phase B from 40-41% to 100%, and the rest is mobile phase A.

[0056] In a second aspect, the present invention provides a method for qualitatively detecting a heparin precursor, the method comprising: using heparinase III to enzymatically hydrolyze a glycosaminoglycan sample to be tested, and measuring the enzymatic hydrolysate by high performance liquid chromatography, and the collected spectrum contains only the characteristic peak of ΔⅣa, indicating that the glycosaminoglycan sample is a heparin precursor.

[0057] Preferably, the chromatographic conditions of the HPLC include: the detector is a UV / visible detector; the chromatographic column used is a strong anion exchange column; the mobile phase used includes mobile phase A and mobile phase B; the mobile phase A is a sodium dihydrogen phosphate solution, and the mobile phase B is a sodium dihydrogen phosphate solution containing sodium perchlorate; the volume concentration of the sodium dihydrogen phosphate solution in the mobile phase A is 0.02%-0.03%; the mobile phase B is a 0.02%-0.03% sodium dihydrogen phosphate solution containing 10%-18% sodium perchlorate; the flow rate of the mobile phase is 1.0-2.0 mL / min; the column temperature of the HPLC is 38-42°C; the injection volume of the HPLC is 8-12 μL; the HPLC uses gradient elution; the procedure of the gradient elution is:

[0058] At 0 min, the volume fraction of mobile phase B was 6.0-8.0%, and the rest was mobile phase A;

[0059] From 0 to 10 minutes, the volume fraction of mobile phase B changed uniformly from 6.0-8.0% to 40.0-42.0%, and the rest was mobile phase A.

[0060] Preferably, the chromatographic column is a SAX chromatographic column or a chromatographic column with equivalent performance.

[0061] Preferably, the chromatographic column is Waters Spherisorb SAX, 5.0 μm, 4.6 mm×250 mm, or a chromatographic column with equivalent performance.

[0062] The present invention also provides information on the structural composition of heparin precursors. In addition to quantitative analysis, the spectrum collected by the method can also be qualitatively analyzed based on retention time based on external standard substances. After the glycosaminoglycan derivatives undergo enzymatic hydrolysis by heparinase III, disaccharide structure fragments such as ΔⅣa and ΔⅣs may be produced. If only ΔⅣa is produced, it can be qualitatively shown that the detected glycosaminoglycan is a heparin precursor, that is, the method can qualitatively judge the heparin precursor.

[0063] In a third aspect, the present invention provides the use of the method for detecting the content of heparin precursor described in the first aspect or the method for qualitatively detecting heparin precursor described in the second aspect in the detection of heparin precursor.

[0064] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0065] Compared with the prior art, the present invention has the following beneficial effects:

[0066] (1) The present invention provides a method for detecting the content and controlling the quality of a heparin precursor, and in particular, provides a method for controlling the quality of a heparin precursor by high performance liquid chromatography. The quality control method can be used to control the quality of a crude heparin precursor in the preparation process and a finished heparin precursor product obtained. The quality control method for a heparin precursor has mild operating conditions and high sensitivity, can efficiently control the quality of a crude heparin precursor in the preparation process and a finished heparin precursor product, and is easy to apply to large-scale industrial production.

[0067] (2) The heparin precursor detection method provided by the present invention has high separation degree and good adaptability, and can quickly quantify and detect whether there are impurity analogs. It can overcome the interference of other substances in the reaction system on the reaction results. The high-performance liquid chromatography analysis method of the present invention is simpler, the detection results are more objective, and it is suitable for large-scale industrial production.

[0068] (3) In the experiment of determining the content of heparin precursor by carbazole sulfate method, it can be seen from the experimental results that the purified water group and the blank culture medium group in the control experimental group also have absorbance, indicating that the determination of heparin precursor by carbazole sulfate method will naturally have errors. The heparin precursor liquid phase detection method provided by the present invention can effectively overcome the influence of blank culture medium and carbazole and other reagents introduced during the experiment on the detection results. The heparin precursor liquid phase detection method provided by the present invention has higher accuracy, better precision, and can provide more intuitive detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 This is the size exclusion chromatogram of the heparin precursor prepared in Example 1.

[0070] Figure 2 The heparin precursor prepared in Example 1 1 H-NMR spectrum.

[0071] Figure 3 This is the HSQC spectrum of the heparin precursor prepared in Example 1.

[0072] Figure 4 The HPLC chromatogram of the blank enzymatic solution described in Example 2.

[0073] Figure 5 This is a chromatogram of the enzymatic hydrolyzate of the heparin precursor reference substance (heparin precursor concentration is 2 mg / mL) determined by the method of Example 2.

[0074] Figure 6 This is the HPLC chromatogram of the ΔIVa standard solution (1 mg / mL) described in Example 2.

[0075] Figure 7 It is the HPLC chromatogram of blank solution 1 described in Example 4.

[0076] Figure 8 It is the HPLC chromatogram of blank solution 2 described in Example 4.

[0077] Fig. 9 It is the HPLC chromatogram of blank solution 3 described in Example 4.

[0078] Fig.10 It is the linear curve of the linear regression equation of the heparin precursor in Example 7.

[0079] Fig.11 This is the chromatogram of sample 1 to be tested in Example 9.

[0080] Fig.12 This is the chromatogram of sample 2 to be tested in Example 9.

[0081] Fig.13 It is the linear curve of the linear regression equation of uronic acid in Comparative Example 1. DETAILED DESCRIPTION

[0082] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0083] In the present invention, the term "system suitability experiment" has the meaning defined in the Chinese Pharmacopoeia, and the suitability experiment of the chromatographic system generally includes five parameters, namely, the number of theoretical plates, separation, sensitivity, tailing factor and repeatability. The evaluation index of the system suitability of the present invention mainly includes injection repeatability (precision). The acceptable standard of system suitability in the present invention is: the RSD of the heparinase peak area of ​​6 injections of the standard solution shall not be greater than 1.0%, and the RSD of the retention time shall not be greater than 1.0%.

[0084] In the present invention, the FF column refers to a column filled with DEAE Sepharose Fast Flow weak anion exchange resin as a filler.

[0085] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0086] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0087] All reagents and solvents used in the present invention are commercially available and can be used without further purification. The sources of the reagents used in the present invention are specifically shown in Table 1 below.

[0088] Table 1

[0089]

[0090] The present invention uses the following abbreviations: mg / mL represents milligrams per milliliter; r represents the linear correlation coefficient, and the larger the linear correlation coefficient, the better the quantitative ability; RSD represents the relative standard deviation; BSA represents bovine serum albumin; M represents mole per liter (mol / L); mM represents millimole per liter (mmol / L); PBS represents phosphate buffered saline; TSP represents 3-(trimethylsilyl) deuterated propionate sodium salt (2,2,3,3-D4-3-(trimethylsilyl)propionic acid sodium salt); ΔIVa represents a standard disaccharide substance formed by glucuronic acid GlcUA and acetylglucosamine GlcNAc connected by a glycosidic bond; δ represents the chemical shift value of the atom, and the unit is ppm; δ H Represents the chemical shift value of H atoms, in ppm; δ C Represents the chemical shift value of the C atom, in ppm.

[0091] Example 1

[0092] Preparation of heparin precursor reference substance

[0093] (1) Fermentation of E. coli Nissle 1917

[0094] Pick the colonies on the cultured plate into a shake flask containing glucose synthetic medium, and culture in a shaker at 37°C and 200 rpm for 6-10 h. Take the obtained first-level seed solution and add it to 100 mL of glucose synthetic medium at a 3% (V / V) inoculation rate. Culture in a shaker at 37°C and 200 rpm for 6-10 h to obtain the second-level seed solution. Take the second-level seed solution and inoculate it into a fermenter at a 4% (V / V) inoculation rate, and ferment it at 37°C, 450 rpm, and a ventilation rate of 3-4 L / min for 24 h, then harvest the bacteria and obtain the fermentation liquid.

[0095] (2) Preparation of crude extract

[0096] Place the fermentation liquid from step (1) into a 2.0 L centrifuge cup and set the centrifuge speed to 3800 rpm for 60 min. After centrifugation, if the supernatant is still turbid, repeat the centrifugation 1-2 times until the supernatant is relatively clear.

[0097] Centrifuge the fermentation broth at 3800 rpm for 60 min. If the supernatant is still turbid, repeat the centrifugation 1-2 times until the supernatant is clear. Use a 0.45 μm filter membrane to remove insoluble particles in the supernatant to protect the ultrafiltration membrane in the next step.

[0098] The supernatant obtained in the previous step was ultrafiltered (the molecular weight cutoff of the ultrafiltration membrane was 1000 Da), and 2.4 L of purified water was slowly added. The ultrafiltration was performed while adding purified water until the volume was concentrated to 800 mL. The ultrafiltration was terminated to obtain a crude heparin precursor extract.

[0099] (3) FF column purification

[0100] The crude extract obtained in step (2) was separated on an agarose resin FF column (column size 2.5×20 cm), and 225 mL of 50 mM sodium chloride solution (containing 20 mM CH 3 COONa, pH 4.0) to balance the agarose resin FF column. Take 1 L of 50 mM sodium chloride solution (containing 20 mM CH 3 COONa, pH 4.0), 1 L 1000 mM sodium chloride solution (containing 20 mM CH 3 COONa, pH 4.0). During the elution process, the flow rate was controlled, and one tube was collected every 2-5 min, with 4-10 mL in each tube. The ultraviolet absorption at A206 nm (heparin precursor signal peak), A260 nm (nucleic acid signal peak) and A280 nm (protein signal peak) was detected online using an ultraviolet detector. The active eluate at A206 nm was collected and combined to obtain the FF column purified solution.

[0101] (4) Ultrafiltration desalination and freeze drying

[0102] The FF column purified liquid obtained in step (3) is ultrafiltered (using a centrifugal ultrafiltration tube and the molecular weight cutoff of the ultrafiltration membrane is 1000 Da) to remove salt until the salt is completely removed to obtain an ultrafiltration concentrate. 30 mL of ethanol is added, stirred thoroughly to precipitate, and centrifuged. The precipitate after centrifugation is collected and dried to obtain the heparin precursor.

[0103] (5) Characterization of heparin precursor structure

[0104] Molecular weight detection: Take the heparin precursor obtained in step (4) and use size exclusion chromatography to detect the molecular weight of the heparin precursor. The molecular weight of the heparin precursor obtained in step (4) is 68053 Da. The molecular weight collection spectrum is shown in Figure 1. The literature reports that the weight average molecular weight of the heparin precursor prepared by fermentation of Escherichia coli E. coli Nissle 1917 is 68000 Da (see PDatta, F Li, P Brodfuerer, JS Dordick, RJ Linhardt. High density fermentation of probiotic E. coli Nissle 1917 towards heparosan production, characterization, and modification, Microbiology and Biotechnology. 2021, 105(3): 1051–1062). The weight average molecular weight of the heparin precursor obtained in step (4) of this example is consistent with that reported in the literature, indicating that the product prepared in step (4) of the example is a heparin precursor.

[0105] NMR structural characterization: Weigh 15 mg of the heparin precursor obtained in step (4) of this example, add 1.0 mL of a pH 7.0 PBS heavy aqueous solution (containing 0.002% (W / V) TSP), and after complete dissolution, pipette 0.6 mL into a NMR tube and sonicate for 5 min. NMR detection of the above heparin precursor sample.

[0106] 1 The H-NMR experimental parameters are shown in Table 2, and the spectrum is collected as follows Figure 2 The HSQC experimental parameters are shown in Table 3, and the spectrum collected is as follows Figure 3 . 13 The C-NMR experimental parameters are shown in Table 3.

[0107] Table 2

[0108]

[0109] Table 3

[0110]

[0111] pass Figure 2 and Figure 3 By comparison, 1In the H NMR (600 MHz) spectrum, δ5.38 (hydrogen atom peak at position 1 of GlcNAc), δ4.48 (hydrogen atom peak at position 1 of GlcA), δ3.83 (superimposed peak of hydrogen atom signals at positions 2, 3, 5 and 6 of GlcNAc), δ3.74 (hydrogen atom peak at position 5 of GlcA), δ3.68 (superimposed peak of hydrogen atom signals at positions 3 and 4 of GlcA and hydrogen atom signals at position 4 of GlcNAc), δ3.35 (hydrogen atom peak at position 2 of GlcA), and δ2.03 (methyl signal peak of GlcNAc).

[0112] In the HSQC spectrum, δ H 2.03, δ C 104.70, suggesting the presence of the carbon-hydrogen structure of the N-methyl group in GlcNAc; δ H 4.48, δ C 105.20, suggesting the presence of a carbon-hydrogen structure at position 1 of GlcA; δ H 5.38, δ C 99.61 correlation, suggesting the presence of a GlcNAc carbon-hydrogen structure at position 1; δ H 3.68, δ C 81.18, suggesting the presence of a carbon-hydrogen structure at position 3 of GlcA and a carbon-hydrogen structure at position 4 of GlcNAc; δ H 3.74, δ C 78.96, suggesting the presence of a carbon-hydrogen structure at position 5 of GlcA; δ H 3.68, δ C 78.99 correlation, suggesting the presence of a carbon-hydrogen structure at position 4 of GlcA; δ H 3.35, δ C 76.25, suggesting the presence of a carbon-hydrogen structure at position 2 of GlcA; δ H 3.83, δ C 73.46, suggesting the presence of a GlcNAc carbon-hydrogen structure at position 5; δ H 3.85, δ C 71.89, suggesting the presence of a GlcNAc carbon-hydrogen structure at position 3; δ H 3.84, δ C 62.12, suggesting the presence of a GlcNAc carbon-hydrogen structure at position 6; δ H 3.86, δ C 56.04, suggesting the presence of a carbon-hydrogen structure at position 2 of GlcNAc.

[0113] In summary, the present invention Figure 2 In 1 H-NMR spectrum and Figure 3The HSQC spectrum is compared with the standard spectrum of the heparin precursor reported in the literature (see Li X, Yu Y, Tang J, Gong B, Li W, Chen T, Zhou X. The construction of a dual-functional strain that produces both polysaccharides andsulfotransferases. Biotechnol Lett. 2021 Sep;43(9):1831-1844, Fig.5b). The chemical shift and peak shape of the main signal peak are consistent with the spectrum reported in the literature. The structure was assigned by HSQC, which is consistent with the structure of the heparin precursor reported in the literature. It shows that the product prepared in this example is a heparin precursor.

[0114] Example 2

[0115] Heparin precursor content detection method

[0116] 1. Instruments and analytical conditions

[0117] High performance liquid chromatograph (Waters e2695), Waters 2489 UV / visible detector, chromatographic column Waters Spherisorb SAX, 5.0 μm, 4.6 mm×250 mm, mobile phase A was 0.028% (w / w) sodium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid), mobile phase B was 0.028% (w / w) sodium dihydrogen phosphate solution containing 14% (w / w) sodium perchlorate (pH adjusted to 3.0 with phosphoric acid), detection wavelength was 232 nm, column temperature was 40°C, flow rate was 1.4 mL / min, injection volume was 20 μL, and running time was 15 min. The elution gradient is shown below.

[0118] At 0 min, the volume fraction of mobile phase B was 6.0%, and the rest was mobile phase A;

[0119] From 0 to 10 min, the volume fraction of mobile phase B changed from 6.0% to 40.0% at a constant rate, and the rest was mobile phase A;

[0120] At 10-12 minutes, the volume fraction of mobile phase B changed from 40.0% to 100.0% at a constant rate, and the rest was mobile phase A.

[0121] 2. Preparation of solvent, standard solution, reference solution and test solution

[0122] Heparinase diluent: It is a potassium phosphate buffer with a pH of 7.0. The preparation method is as follows: take 68 mg of potassium dihydrogen phosphate and 10 mg of bovine serum albumin, accurately weigh them, and place them in a 50 mL volumetric flask; add 30 mL of purified water and sonicate to dissolve them, adjust the pH to 7.0 with potassium hydroxide, and finally dilute to the scale with purified water, shake well, and the heparinase diluent is obtained.

[0123] Heparinase III solution: Dissolve heparinase III with heparinase diluent and dilute to an activity of 2.0 IU / mL to obtain heparinase III solution. Store in a -20℃ refrigerator and take out before use.

[0124] pH 7.0 sodium calcium acetate solution: Take 10 mg of bovine serum albumin and 32 mg of calcium acetate, weigh accurately, and place in a 100 mL volumetric flask. Add 60 mL of purified water to dissolve, add 580 µL of acetic acid, and mix by ultrasonic. Adjust the pH to 7.0 with 2 mol / L sodium hydroxide, add purified water to 100 mL, and then filter with a filter membrane with a pore size of ≤0.45 µm to obtain pH 7.0 sodium calcium acetate solution.

[0125] Mobile phase A: Mobile phase A is 0.028% (w / w) sodium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid). The preparation method is as follows: weigh 0.280 g of sodium dihydrogen phosphate and dissolve it in 950 mL of purified water, adjust the pH to 3.0 with phosphoric acid, and dilute to 1000 mL with purified water. Filter with a filter membrane with a pore size of ≤0.45 µm.

[0126] Mobile phase B: Mobile phase B is a 0.028% (w / w) sodium dihydrogen phosphate solution containing 14% (w / w) sodium perchlorate (pH adjusted to 3.0 with phosphoric acid). The preparation method is as follows: weigh 140 g of sodium perchlorate and dissolve it in 950 mL of mobile phase A, adjust the pH to 3.0 with phosphoric acid, and dilute to 1000 mL with mobile phase A. Filter with a filter membrane with a pore size of ≤0.45 µm.

[0127] ΔIVa standard solution: It is a solution containing 1 mg / mL of standard disaccharide substance ΔIVa. The preparation method is: accurately weigh 1 mg of ΔIVa standard, transfer 1 mL of purified water with a pipette, and fully dissolve it.

[0128] Preparation of heparin precursor reference solution: Take about 500 mg of heparin precursor reference, accurately weigh, place in a 50 mL volumetric flask, add solvent and ultrasonically dissolve and dilute to the scale, shake well to obtain a 10.0 mg / mL heparin precursor reference solution.

[0129] Take 5 mL, 2 mL, and 1 mL of 10.0 mg / mL heparin precursor reference solution in turn, place them in 10 mL volumetric flasks, dilute to the mark with solvent, and shake well to obtain 5.0 mg / mL, 2.0 mg / mL, and 1.0 mg / mL heparin precursor reference solutions.

[0130] Take 1 mL of 5.0 mg / mL and 1.0 mg / mL heparin precursor reference solution respectively, place them in 10 mL volumetric flasks, add solvent to dilute to the scale, shake well, and obtain 0.5 mg / mL and 0.1 mg / mL heparin precursor reference solution.

[0131] Preparation of heparin precursor reference substance enzymatic hydrolysate: Take 20 μL of heparin precursor reference substance solution, add 70 μL pH7.0 sodium calcium acetate solution and 50 μL heparinase III solution, mix well, and react at room temperature for 5-6 hours. After the enzymatic hydrolysis reaction is complete, place in a 100℃ water bath for 3 minutes to terminate the reaction. Centrifuge at 11000 rpm for 10 minutes, and the resulting supernatant is the heparin precursor reference substance enzymatic hydrolysate.

[0132] Preparation of heparin precursor test solution:

[0133] Test sample solution: If the sample to be tested is a liquid sample, filter it with a filter membrane with a pore size of ≤0.45 µm and further dilute it with purified water to obtain the test sample solution.

[0134] If it is a solid sample, prepare it into a 2 mg / mL solution with purified water and filter it with a filter membrane with a pore size of ≤0.45 µm to obtain the test solution.

[0135] Preparation of heparin precursor test sample enzymatic hydrolyzate: Take 20 μL of heparin precursor test sample solution, add 70 μL pH7.0 sodium calcium acetate solution and 50 μL heparinase III solution, mix well, and react at room temperature for 5-6 hours. After the enzymatic hydrolysis reaction is complete, place in a 100℃ water bath for 3 minutes to terminate the reaction. Centrifuge at 11000 rpm for 10 minutes, and the resulting supernatant is the heparin precursor test sample enzymatic hydrolyzate.

[0136] Preparation of blank enzymatic solution: Take 20 μL of purified water, add 50 μL of pH 7.0 sodium calcium acetate solution and 50 μL of heparinase III solution, mix well, react at room temperature for 5-6 hours, heat to boiling for 1-3 minutes to terminate the reaction, centrifuge at 11000 rpm for 10 minutes, and the obtained supernatant is the blank enzymatic solution.

[0137] 3. Experimental process and results

[0138] Take 20 μL of blank enzymatic solution and inject it into HPLC to record the chromatogram. Figure 4; The blank enzymatic solution should have no interference.

[0139] Take 20 µL of heparin precursor hydrolyzate (heparin precursor concentration is 2 mg / mL) and inject 6 times continuously to examine the injection precision. Record the chromatogram. Figure 5 The acceptance criteria for injection precision are: the RSD of the peak area of ​​the main peak of the 6-shot heparin precursor reference enzymatic hydrolysate should not be greater than 1.0%, and the RSD of the retention time should not be greater than 1.0%.

[0140] Take 20 μL of ΔIVa standard solution and inject it into the HPLC instrument. Record the chromatogram. Figure 6 In subsequent experiments, the retention time of the main peak of the heparin precursor hydrolyzate should be within the range of ±1.0% of the retention time of the main peak of the ΔIVa standard solution.

[0141] Take 20 µL of each concentration of heparin precursor reference substance enzymatic hydrolysate, inject it into the high performance liquid chromatograph, record the chromatogram, peak area and retention time of the main peak in each corresponding spectrum. Use the least squares method for linear regression to establish the mathematical relationship between the peak area and the reference substance solution concentration, Y = kX + b.

[0142] Take 20 μL of the enzymatic hydrolyzate of the heparin precursor test sample and inject it into the high performance liquid chromatograph, and record the chromatogram, peak area and retention time of the main peak in each corresponding spectrum.

[0143] The calculation is performed using the mathematical relationship Y=kX+b in the previous steps.

[0144] When the heparin precursor sample to be tested is a liquid sample, the heparin precursor concentration C of the heparin precursor sample to be tested is calculated according to the following formula ①: 肝素前体 When the heparin precursor sample to be tested is a solid sample, the heparin precursor content in the heparin precursor sample to be tested is calculated according to the following formula ②.

[0145] ① The formula for calculating the heparin precursor concentration in the heparin precursor sample to be tested is:

[0146] .

[0147] ② The calculation formula for the heparin precursor content in the heparin precursor sample to be tested is:

[0148] .

[0149] In the above formula: C 肝素前体is the heparin precursor concentration in the heparin precursor sample to be tested, mg / mL; A is the peak area of ​​the main peak of the heparin precursor test sample enzymatic hydrolyzate (the main peak of the heparin precursor test sample enzymatic hydrolyzate is the peak of the product ΔIVa); k is the slope of the linear equation; b is the intercept of the linear equation; N is the dilution multiple of the heparin precursor test solution when the heparin precursor sample to be tested is a liquid sample; m is the mass of the heparin precursor sample to be tested when the heparin precursor sample to be tested is a solid sample, mg; V is the volume of the prepared heparin precursor test solution when the heparin precursor sample to be tested is a solid sample, mL.

[0150] Example 3

[0151] System suitability test of detection method

[0152] The purpose of this experiment is to confirm whether the detection method of Example 2 meets the requirements of system suitability. The acceptable standards for the system suitability investigation experiment are: the RSD of the peak area of ​​6 injections of heparin precursor reference substance enzymatic hydrolysate should be ≤1.0%, and the RSD of the retention time should be ≤1.0%.

[0153] 1. Instruments and analytical conditions

[0154] The instruments and analytical conditions were the same as those in Example 2.

[0155] 2. Solution preparation

[0156] The specific preparation method is as follows: prepare the reference solution of heparin precursor and the blank solution according to Example 2.

[0157] 3. Experimental process

[0158] Take 20 μL of blank enzymatic hydrolysate and inject it into the HPLC to record the chromatogram. The blank enzymatic hydrolysate should have no interference. Take 20 μL of heparin precursor reference enzymatic hydrolysate (heparin precursor concentration 1.98 mg / mL) and inject it into the HPLC. Inject 6 injections in a row to examine the injection precision. The specific results are shown in Table 4 below.

[0159] Table 4

[0160]

[0161] From the experimental results in Table 4, it can be seen that the blank enzymatic hydrolysate has no interference at the peak position of the heparin precursor enzymatic hydrolysate. The RSDs of the peak area and retention time of the 6-shot heparin precursor reference solution were 0% and 0.1%, respectively, which were significantly better than the acceptable standards (the RSD of the peak area of ​​the heparin precursor reference enzymatic hydrolysate should be ≤1.0%, and the RSD of the retention time should be ≤1.0%).

[0162] Example 4

[0163] Investigation on the specificity of the method for determining the content of heparin precursor

[0164] The purpose of this experiment is to verify that the heparin precursor content determination method is not interfered by the blank solution and the culture medium, so as to prove that the heparin precursor content test method has good specificity.

[0165] 1. Instruments and analytical conditions

[0166] The instruments and analytical conditions were the same as those in Example 2.

[0167] 2. Solution preparation

[0168] Blank solution 1: It is a commonly used elution solvent for the purification of heparin precursors, which is a 1000 mM sodium chloride solution.

[0169] Blank solution 2: Heparin precursor culture medium, containing 16 g peptone, 10 g yeast and 5 g sodium chloride per liter of culture medium. The preparation method of heparin precursor culture medium is as follows: 16 g peptone, 10 g yeast and 5 g sodium chloride are dissolved in 600 mL purified water, transferred to a 1000 mL volumetric flask, and water is added to make up to volume to obtain heparin precursor culture medium.

[0170] Blank solution 3: pH 7.0 is sodium calcium acetate solution, which is the solution used for enzymatic treatment of heparin precursor samples. The preparation method of pH 7.0 sodium calcium acetate solution is as follows: dissolve 10 mg of bovine serum albumin and 32 mg of calcium acetate in 60 mL of purified water. Add 580 µL of acetic acid and adjust the pH to 7.0 with 2 mol / L sodium hydroxide. Transfer to a 100 mL volumetric flask and add water to the fixed volume. Filter with a filter membrane with a pore size of ≤0.45 µm.

[0171] Preparation of standard solution and solvent: Prepare standard solution and solvent according to the method in Example 2.

[0172] 3. Experimental process and results

[0173] After the system suitability was verified as qualified according to the method of Example 3, 20 μL of each blank solution was injected. The blank solution should have no interference at the peak position of the heparin precursor hydrolyzate. The specific results are shown in Table 5 below, and the relevant specific chromatograms are shown in Figure 7-Figure 9 .

[0174] One injection each of blank solution 1, blank solution 2, blank solution 3 and heparin precursor control solution was injected for analysis.

[0175] 4. Acceptable standards

[0176] No peaks appeared at the retention time of the heparinase precursor control solution for blank solution 1, blank solution 2, and blank solution 3. Table 5 shows the blank solution interference test results.

[0177] Table 5

[0178]

[0179] Through the above tests and results, it can be seen that blank solution 1, blank solution 2 and blank solution 3 do not produce peaks at the retention time of 3.386min (the retention time of the main peak of heparin precursor hydrolysate), indicating that blank solution 1, blank solution 2 and blank solution 3 have no interference at the peak position of heparin precursor hydrolysate, proving that the method has good specificity.

[0180] Example 5

[0181] Study on the Solution Stability of Heparin Procuratorial Detection Method

[0182] The purpose of this experiment is to determine the extent to which the test results of the heparin precursor reference substance enzymatic hydrolyzate used in the content determination method of the present invention are not affected after being placed for a period of time under certain conditions.

[0183] The storage temperature and storage time of the prepared heparin precursor reference substance were investigated.

[0184] Acceptable criteria: peak area RSD of all samples ≤ 1.0%.

[0185] 1. Instruments and analytical conditions

[0186] The instruments and analytical conditions were the same as those in Example 2.

[0187] 2. Solution preparation

[0188] The specific preparation is based on the preparation of the reference sample hydrolysate and the test sample hydrolysate in Example 1.

[0189] 3. Experimental process and results

[0190] The reference solution after enzymatic hydrolysis for 0 h, the enzymatic hydrolysis heparin precursor solution after being placed at room temperature for 24 h, room temperature for 48 h, 4°C for 24 h, and 4°C for 48 h were injected for analysis, and the peak area was recorded.

[0191] After the system suitability was verified as qualified according to the method in Example 3, 10 μL of each heparin precursor hydrolyzate placed at room temperature and 4°C was taken, and two injections were made at 0 h, 24 h and 48 h, respectively, to examine the peak area changes of the main peaks of each enzyme solution. Compared with 0 h, the peak area RSD of each heparin precursor hydrolyzate was ≤1.0%, and the stability results of the heparin precursor solution (2.0 mg / mL) are shown in Table 6.

[0192] Table 6

[0193]

[0194] The above tests and results show that the heparin precursor reference substance enzymatic hydrolysate is stable within 48 hours at room temperature and 4°C, indicating that the heparin precursor reference substance enzymatic hydrolysate of the present invention can be effectively and accurately used for testing the sample content within 48 hours.

[0195] Example 6

[0196] Experimental study on the repeatability of the method for determining the content of heparin precursor

[0197] This embodiment is to determine the reproducibility of multiple measurement results of the same batch of samples by the same operator.

[0198] 1. Instruments and analytical conditions

[0199] The instruments and analytical conditions were the same as those in Example 2.

[0200] 2. Solution preparation

[0201] For specific preparation, refer to Example 2 for preparing the heparin precursor reference substance enzymatic hydrolyzate and solvent.

[0202] Prepare 6 sets of reference enzymatic hydrolysate solutions with a heparin precursor concentration of 2.0 mg / mL.

[0203] 3. Experimental process and results

[0204] After the system suitability was verified as qualified according to the method of Example 3, 20 μL of each of the 6 heparin precursor test sample hydrolyzates was injected twice, and the content of each reference solution was calculated. The RSD of the content determination results of the 6 reference solution solutions should be ≤1.0%. The repeatability results and chromatograms are shown in Table 7 below.

[0205] Table 7

[0206]

[0207] Through the test and the above results, it can be seen that the RSD of the enzymatic hydrolyzate content of the heparin precursor test sample is 0, which is far less than the acceptable limit of 2.0%. The analytical repeatability of this method meets the acceptance standard, proving that this method has good repeatability.

[0208] Example 7

[0209] Linearity test of heparin precursor detection method

[0210] The purpose of this experiment is to determine the linear relationship between the measurement result and the concentration of the solution tested by the detection method of the present invention within a preset range.

[0211] The concentrations of heparin precursors in the linear experiment of this method were set to 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 5.0 mg / mL and 10.0 mg / mL. Heparin precursor hydrolysate was sampled and analyzed, and a standard curve was drawn with sample concentration as the abscissa and peak area of ​​heparin precursor hydrolysate as the ordinate, and the linearity between sample concentration and peak area was calculated.

[0212] 1. Instruments and analytical conditions

[0213] The instruments and analytical conditions were the same as those in Example 2.

[0214] 2. Solution preparation

[0215] Preparation of heparin precursor reference substance enzymatic hydrolysate: Prepare heparinase III with potassium phosphate buffer to a concentration of about 2 U / mL. According to Table 8 below, take 20 μL of heparin precursor reference substance solution, 50 μL of pH 7.0 sodium calcium acetate solution and 50 μL of enzyme III solution, pipette repeatedly to mix them evenly, and react at room temperature for 5-6 hours. After confirming that the enzymatic hydrolysis is complete, heat to boiling for 3 minutes to terminate the reaction, centrifuge at 11000 rpm for 10 minutes, and obtain the corresponding concentration of heparin precursor reference substance enzymatic hydrolysate, and collect the peak area by liquid phase detection.

[0216] Table 8

[0217]

[0218] 3. Experimental process and results

[0219] After the system suitability was verified as qualified according to the method of Example 3, the linear solutions of each concentration of heparin precursor hydrolyzate (0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 5.0 mg / mL and 10.0 mg / mL) were injected one injection each, and the peak area was recorded. A linear regression equation was made with the peak area versus the heparin precursor concentration (C, mg / mL). The correlation coefficient r should be ≥0.98. The linear results of heparin precursor are shown in Table 9 below, and the linear curve is shown in Fig.10 .

[0220] Table 9

[0221]

[0222] The above tests and results show that heparin precursor has a good linear relationship with the peak area in the concentration range of 0.1 mg / mL-10.0 mg / mL. The correlation coefficient r of the linear regression curve is 0.999, and the quantitative ability of the detection method is high.

[0223] Example 8

[0224] Preparation of heparin precursor test sample

[0225] The fermentation liquid obtained in step (1) of Example 1 is used as the heparin precursor sample 1 to be tested; the crude heparin precursor extract obtained in step (2) of Example 1 is used as the heparin precursor sample 2 to be tested.

[0226] Example 9

[0227] Detection of heparin precursor concentration using heparin precursor detection method

[0228] According to the method of Example 2, the standard curve equation is drawn, as shown in Table 10 below; the HPLC spectra of the test samples 1 and 2 described in Example 8 are respectively shown in Fig.11 , Fig.12 .

[0229] Using the method of Example 2, the heparin precursor concentration in the heparin precursor sample to be tested is determined according to Formula ① in Example 2, as shown in Table 11 below.

[0230] Through Table 11 and Fig.11 , Fig.12 It can be seen that the sample 1 and the sample 2 have a high content of heparin precursor. The components of the enzymatic hydrolysate are located based on the relative retention time, and the retention time of the main peak of the enzymatic hydrolysate is 3.39 min.

[0231] Table 10

[0232]

[0233] Table 11

[0234]

[0235] The heparin precursor detection method can not only quantify the content of heparin precursor, but also qualitatively determine whether the sample is a heparin precursor by detecting the disaccharide composition after enzymatic hydrolysis of the heparin precursor. The above detection method has good sensitivity within the linear range and the detection result is reliable.

[0236] Example 10

[0237] This embodiment provides a method for detecting the content of heparin precursor. The difference between the method and embodiment 2 is that the chromatographic conditions are: the high performance liquid chromatograph is Waters 2695, and the elution gradient is as follows:

[0238] At 0 min, the volume fraction of mobile phase B was 8.0%, and the rest was mobile phase A;

[0239] From 0 to 10 min, the volume fraction of mobile phase B changed from 8.0% to 42.0% at a constant rate, and the rest was mobile phase A;

[0240] At 10-12 minutes, the volume fraction of mobile phase B changed from 42.0% to 100.0% at a constant rate, and the rest was mobile phase A.

[0241] Take 20 µL of heparin precursor reference enzymatic hydrolysate (heparin precursor concentration is 2 mg / mL) and inject 6 times in succession to examine the injection precision. The injection precision acceptance standard is: the RSD of the peak area of ​​the main peak of the 6 injections of heparin precursor reference enzymatic hydrolysate should not be greater than 1.0%, and the RSD of the retention time should not be greater than 1.0%.

[0242] After the system injection precision was qualified, 20 μL of ΔIVa standard solution was injected into the HPLC. The main peak of ΔIVa standard solution was good, and the retention time of the main peak was 2.798 min.

[0243] Embodiment 11

[0244] This embodiment provides a method for detecting the content of heparin precursor. The difference between the method and embodiment 2 is that the chromatographic conditions are: the high performance liquid chromatograph is Dionex U3000, and the elution gradient is as follows:

[0245] At 0 min, the volume fraction of mobile phase B was 7.0%, and the rest was mobile phase A;

[0246] From 0 to 10 min, the volume fraction of mobile phase B changed from 7.0% to 41.0% at a constant rate, and the rest was mobile phase A;

[0247] At 10-12 minutes, the volume fraction of mobile phase B changed from 41.0% to 100.0% at a constant rate, and the rest was mobile phase A.

[0248] Take 20 µL of heparin precursor reference enzymatic hydrolysate (heparin precursor concentration is 2 mg / mL) and inject 6 times in succession to examine the injection precision. The injection precision acceptance standard is: the RSD of the peak area of ​​the main peak of the 6 injections of heparin precursor reference enzymatic hydrolysate should not be greater than 1.0%, and the RSD of the retention time should not be greater than 1.0%.

[0249] After the system injection precision was qualified, 20 μL of ΔIVa standard solution was injected into the HPLC. The main peak of ΔIVa standard solution was good, and the retention time of the main peak was 3.092 min.

[0250] Comparative Example 1

[0251] Detection of heparin precursor content in samples by carbazole sulfate method

[0252] Experimental purpose: To detect the heparin precursor content in the heparin precursor test sample 1 and the heparin precursor test sample 2 described in Example 8 according to the carbazole sulfate method.

[0253] 1. Instrument samples

[0254] Microplate reader, water bath, 96-well plate, pipette.

[0255] Test sample 1: The fermentation broth obtained in step (1) of the preparation method of Example 1 is used as the heparin precursor test sample 1.

[0256] Test sample 2: The crude heparin precursor extract obtained in step (2) of the preparation method of Example 1 is used as the heparin precursor test sample 2.

[0257] 2. Experimental process

[0258] Preparation of glucuronic acid standard curve: weigh 20 mg of glucuronic acid and dissolve it in a 100 mL volumetric flask, mix it upside down, add water to the scale, and save it for later use; take out 1.0 mL, 2.5 mL, 5.0 mL, and 10.0 mL from the prepared 100 mL standard solution, add them to a 10 mL volumetric flask, mix it upside down, and add water to the scale to obtain 20 mg / L, 50 mg / L, 100 mg / L, and 200 mg / L glucuronic acid standard solutions. Then determine the OD value by the carbazole sulfate method. 530 Value; in OD 530 The value is the ordinate, and the corresponding glucuronic acid content is the abscissa for linear regression. The standard curve equation is shown in Table 12 below, and the linear curve is shown in Fig.13 .

[0259] Experimental process of the experimental group: Take 1 mL of sample 1 and sample 2 respectively, and slowly add borax sulfuric acid solution in an ice bath. After gently shaking, immediately insert into ice, repeat several times, take out after the heat release is completed, heat in boiling water for 10 min, and quickly put into cold water to cool down. After cooling to room temperature, add 0.2 mL of carbazole test solution, invert and mix, and quickly put into cold water. After all are added quickly, heat in boiling water for 15 minutes. After the heating is completed, quickly cool to room temperature and measure OD 530 To ensure the OD 530 If the value is within the range of the standard curve, the obtained heparin precursor (Heparosan) polysaccharide separation extract can be appropriately diluted.

[0260] Experimental process of the control group: Take 1 mL of purified water and 1 mL of blank culture medium respectively, and slowly add the same volume of borax sulfuric acid solution as the experimental group. Shake gently and immediately put into ice, repeat several times. Heat in boiling water for 10 minutes, quickly put into cold water to cool after heating, and after cooling to room temperature, add 0.2 mL of carbazole test solution, invert and mix, and quickly put into cold water. After all are added quickly, heat in boiling water for 15 minutes. After heating, quickly cool to room temperature and measure OD 530To ensure the OD 530 If the value is within the range of the standard curve, the obtained heparin precursor (Heparosan) polysaccharide separation extract can be appropriately diluted.

[0261] 3. Experimental results

[0262] Result calculation: Heparin precursor is composed of acetylglucosamine and glucuronic acid, of which the percentage of glucuronic acid content to heparin precursor content is 46.56%. Therefore, the content of heparin precursor can be detected according to the content of glucuronic acid. The concentration of glucuronic acid in the fermentation broth can be converted according to the glucuronic acid standard curve, and then the content of heparin precursor can be converted, that is, heparin precursor concentration (mg / mL) = glucuronic acid concentration (mg / mL) × dilution factor / 0.4656. According to the above calculation formula, the heparin precursor concentrations of the test sample 1, the test sample 2, and the control group were calculated. The specific results are shown in Table 13 below.

[0263] Table 12

[0264]

[0265] Table 13

[0266]

[0267] From Table 13 and the experimental results, it can be seen that the purified water group and the blank culture medium group in the control experimental group also have absorbance, indicating that the carbazole sulfate method for determining heparin precursors will naturally have errors.

[0268] In summary, the heparin precursor liquid phase detection method provided by the present invention can effectively overcome the influence of blank culture medium and reagents such as carbazole introduced during the experiment on the detection results. The heparin precursor liquid phase detection method provided by the present invention has higher accuracy, good precision, and can provide more intuitive detection results.

[0269] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for detecting the content of heparin precursor, characterized in that: The detection method comprises: (S1) preparing a solid sample of the heparin precursor to be tested into a heparin precursor test solution, or diluting a liquid sample of the heparin precursor to be tested into a heparin precursor test solution; enzymolyzing the heparin precursor test solution or the heparin precursor reference solution with heparinase III to obtain a heparin precursor test solution or a heparin precursor reference solution; and determining the peak area of ​​the solution with high performance liquid chromatography; (S2) using the least square method for linear regression, establishing a mathematical relationship between the peak area Y of the heparin precursor reference substance enzymatic hydrolyzate and the concentration X of the heparin precursor reference substance solution, Y = kX + b; (S3) Calculating the concentration of the heparin precursor in the heparin precursor liquid sample to be tested according to the established mathematical relationship; the calculation formula for the concentration of the heparin precursor in the heparin precursor liquid sample to be tested is: ； In the above formula, A is the peak area of ​​the main peak of the heparin precursor test sample enzymatic hydrolyzate, and the main peak is the peak of the product ΔIVa; k is the slope of the linear equation; b is the intercept of the linear equation; N is the dilution multiple of the heparin precursor test sample solution when the heparin precursor sample to be tested is a liquid sample; Or, the heparin precursor content in the solid heparin precursor sample to be tested is calculated according to the established mathematical relationship; the calculation formula for the heparin precursor content in the solid heparin precursor sample to be tested is: ； In the above formula, A is the peak area of ​​the main peak of the heparin precursor test sample enzymatic hydrolyzate, and the main peak is the peak of the product ΔIVa; k is the slope of the linear equation; b is the intercept of the linear equation; m is the mass of the heparin precursor sample to be tested in mg when the heparin precursor sample to be tested is a solid sample; V is the volume of the heparin precursor test solution prepared in mL when the heparin precursor sample to be tested is a solid sample.

2. The method for detecting the heparin precursor content according to claim 1, characterized in that: In step (S1), the heparin precursor reference solution is prepared by dissolving the heparin precursor reference in a solvent; the gradient concentration range of the heparin precursor reference solution is 0.1-10.0 mg / mL; the concentration of the heparin precursor test solution is 0.1-10.0 mg / mL.

3. The method for detecting the content of heparin precursor according to claim 1, characterized in that: The enzymatic hydrolysis step comprises: mixing sodium calcium acetate solution, heparin precursor reference solution or heparin precursor test solution and heparinase III, enzymatic hydrolysis, and after the enzymatic hydrolysis reaction is complete, terminating the reaction in a 80-100° C. water bath, and centrifuging to obtain the supernatant as the sample to be tested.

4. The method for detecting the heparin precursor content according to claim 3, characterized in that: The ratio of heparinase III to heparin precursor in the enzymatic hydrolysis reaction system is greater than or equal to 0.1 U / mg; and the enzymatic hydrolysis time is 3-6 h.

5. The method for detecting the heparin precursor content according to claim 3, characterized in that: The heparinase III is added in the form of enzyme stock solution or heparinase III diluent; the diluent used in the heparinase III diluent is potassium phosphate buffer, and the concentration of the heparinase III diluent is 0.1-4 U / mL.

6. The method for detecting the content of heparin precursor according to claim 1, characterized in that: In (S1), the detector used by the high performance liquid chromatography is a UV / visible detector; the chromatographic column used by the high performance liquid chromatography is a strong anion exchange column; and the column temperature of the high performance liquid chromatography is 38-42°C.

7. The method for detecting the content of heparin precursor according to claim 1, characterized in that: In (S1), the mobile phase used in the high performance liquid chromatography includes mobile phase A and mobile phase B; the mobile phase A is a sodium dihydrogen phosphate solution, and the mobile phase B is a sodium dihydrogen phosphate solution containing sodium perchlorate; The volume concentration of sodium dihydrogen phosphate solution in the mobile phase A is 0.02%-0.03%; The mobile phase B is a 0.02%-0.03% sodium dihydrogen phosphate solution containing 10%-18% sodium perchlorate; The flow rate of the mobile phase is 1.0-2.0 mL / min.

8. The method for detecting the content of heparin precursor according to claim 1, characterized in that: In (S1), the high performance liquid chromatography adopts gradient elution; the procedure of the gradient elution is: At 0 min, the volume fraction of mobile phase B was 6.0-8.0%, and the rest was mobile phase A; From 0 to 10 minutes, the volume fraction of mobile phase B changed uniformly from 6.0-8.0% to 40.0-42.0%, and the rest was mobile phase A.

9. A method for qualitatively detecting heparin precursor, characterized in that: The method comprises: using heparinase III to enzymatically hydrolyze a glycosaminoglycan sample to be tested, and using high performance liquid chromatography to measure the enzymatic hydrolyzate. The collected spectrum contains only the characteristic peak of ΔⅣa, indicating that the glycosaminoglycan sample is a heparin precursor.

10. Use of the method for detecting the content of heparin precursor according to any one of claims 1 to 8 or the method for qualitatively detecting heparin precursor according to claim 9 in detecting heparin precursor.

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