A method for separating BDDE-modified hyaluronic acid oligosaccharides

Abstract

The present invention discloses a method for separating BDDE-modified hyaluronic acid oligosaccharides. The LC-HRMS combination is used to separate the enzymatically hydrolyzed oligosaccharides of BDDE-crosslinked hyaluronic acid. By optimizing the chromatographic type and separation conditions, effective separation of different forms of modified oligosaccharides in the enzymatic hydrolysate can be achieved, the influence of ionization suppression on the oligosaccharide quantification result is reduced, and more accurate oligosaccharide quantification detection is realized. Compared with the existing GPC chromatographic column, the diol-based hydrophilic chromatographic column (Luna-Hilic) and the silica gel hydrophilic chromatographic column (BEH-Hilic) commonly used for oligosaccharide separation, the amide chromatographic column (BEH-Amide) of the present invention has better separation degree and detection sensitivity for representative oligosaccharide targets, laying a foundation for the accurate analysis of the modification degree.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology. Specifically, it relates to a method for separating BDDE-modified hyaluronic acid oligosaccharides, and also relates to a method for determining the modification degree of BDDE-crosslinked hyaluronic acid. Background Art

[0002] Hyaluronic acid (HA) is a linear polysaccharide naturally present in the intercellular matrix of animal cells. It is formed by the repeated connection of disaccharide units composed of glucuronic acid and N-acetylglucosamine, and its chemical structure is (-4GlcAβ1-3GlcNAcβ1-) n , with relative molecular weights ranging from tens of thousands to millions. Due to the high viscosity, strong water retention ability and good biocompatibility of HA solution, its applications in the medical and aesthetic fields have become increasingly widespread in recent years: high molecular weight HA is involved in ophthalmic surgery, joint mucus supplementation, dermal filling and other processes.

[0003]

[0004] Exogenous HA will be degraded by hyaluronidase in the human body. To improve the mechanical properties of HA and extend the duration of its action in the human body, HA sugar chains can be crosslinked by a covalent crosslinking agent to form a three-dimensional network structure, which swells in water to form an HA hydrogel. HA hydrogel has become an important functional material, and its mechanical and physical properties depend on the crosslinking degree and crosslinking method. Research shows that excessive modification and crosslinking degree may affect the biocompatibility and safety of HA hydrogel. To analyze the corresponding relationship between the structure and function of HA hydrogel and establish the quality control standards for related products, it is of great significance to study the accurate characterization method of crosslinking parameters.

[0005] 1,4-Butanediol diglycidyl ether (BDDE) is the most commonly used crosslinking agent at present. The HA hydrogel obtained by crosslinking with BDDE has been proven to have high biocompatibility and low toxicity. Under alkaline conditions, the epoxy structure of BDDE undergoes a nucleophilic reaction with the hydroxyl groups on the HA molecule to form a 1,4-butanediol bis(propane-2,3-diol) ether (BDPE) derivative, and the reaction formula is as follows. There are two binding modes between BDDE and HA. One is that both ends of BDDE are bound to HA, which is called crosslinking modification. The other is that only one end of BDDE is bound to HA and the other end is free, which is called pendant modification. For HA derivatives prepared with BDDE crosslinking agent, although they have the same starting materials, the physical properties of the obtained products may vary greatly. The differences in their physical properties can be attributed to the differences in key structural parameters, such as i) the molecular weight and polydispersity of the starting material (HA); ii) the degree of modification; iii) the substitution position of the crosslinking agent on HA, etc.

[0006]

[0007] The HA crosslinked and modified by BDDE has a large molecular weight and poor solubility in water. When characterizing its degree of modification, it is usually degraded into oligosaccharide fragments by using tool enzymes (such as hyaluronidase or chondroitinase ABC, etc.). Affected by the BDDE crosslinking agent, the tool enzymes cannot completely degrade the crosslinked HA into HA disaccharide, and there will be various oligosaccharide fragments with different molecular weights, degrees of modification and modification modes in the product (as Figure 1 shown). For example, 2-B is used to represent the disaccharide with pendant modification; 2-B-2 is used to represent the disaccharide with crosslinking modification. By relatively quantitatively analyzing different oligosaccharide structures, the degree of modification and modification mode of the product can be characterized. There is currently no patent related to the characterization of the oligosaccharide structure of crosslinked HA. Yang Biao et al. (Yang Biao, Guo Xueping, Zang Hengchang, et al. Research progress on the determination method of the degree of modification of crosslinked hyaluronic acid gel [J]. Pharmaceutical Biotechnology, 2015(2):4.) used the method of coupling size exclusion chromatography and mass spectrometry (GPC-MS) to relatively quantitatively analyze the oligosaccharide fragments. However, this method has the following defects, resulting in inaccurate oligosaccharide quantification and degree of modification characterization results:

[0008] 1) The GPC chromatographic column separates according to the different molecular weights of the target substances. Some of the oligosaccharide fragments generated after the enzymatic hydrolysis of crosslinked HA have similar molecular weights, and these fragments cannot be effectively separated by GPC, and there is an overlapping phenomenon of elution time. During the electrospray process, the oligosaccharides with overlapping elution times will cause ionization mutual inhibition, resulting in the quantitative results of the relevant structures being lower than the actual values.

[0009] 2) In the paper, low-resolution mass spectrometry was used as the detector, and only the mass-to-charge ratio (m / z) of the target oligosaccharide with one decimal place could be determined. This may lead to the quantitative analysis of the target oligosaccharide being interfered by impurities with similar m / z, affecting the accuracy of the quantitative results.

[0010] 3) Due to the existence of different cross-linking methods such as pendant modification and cross-linking modification, and the uncertain binding sites of BDPE on the sugar chain, the types and structures of the oligosaccharide products after enzymatic hydrolysis are numerous and complex. The paper only performed relative quantitative analysis on some of the structures (such as 2-B, 4-B, 6-B, 2-B-2, 4-B-2, etc.) and calculated the modification degree of BDDE-crosslinked HA based on this. Such a calculation method is not comprehensive.

[0011] 4) Oligosaccharides may form different forms of adducts during the ionization process, such as ammonium adducts, sodium adducts, dimers, trimers, etc. In addition, in the negative ion detection mode, oligosaccharides may lose different numbers of H atoms to form ions with different valences (-1, -2, -3, etc.). There are significant differences in the ion adducts and their valences produced by different oligosaccharide structures. In the paper, only monomers with a specific valence and no other ions added were used as the quantitative standard, and the quantitative method has significant defects.

[0012] 5) When the paper performed relative quantitative analysis on different oligosaccharides, the monoisotopic peak of each structure was used as the quantitative basis. However, isotopes of elements such as C, H, and O are widely present in nature. For oligosaccharides with different molecular weights, the proportion of the monoisotopic peak in all isotope peaks is significantly different. For example, the monoisotopic peak of 2-B accounts for 73.2% of all isotope peaks, while the monoisotopic peak of 8-B-6 only accounts for 23.9% of all isotope peaks. Therefore, using the monoisotopic peak as the quantitative standard will result in a lower integration result for oligosaccharides with larger molecular weights, affecting the accuracy of the modification degree analysis results. Summary of the Invention

[0013] Aiming at the deficiencies in separation methods and other aspects in the determination of the modification degree of cross-linked HA in the prior art, the present invention provides a separation method for BDDE-modified hyaluronic acid oligosaccharides. This method uses LC-HRMS coupling to separate the enzymatically hydrolyzed oligosaccharides of BDDE-crosslinked hyaluronic acid, and can effectively separate various oligosaccharides during enzymatic hydrolysis, providing a strong guarantee for the structure-activity relationship research and quality control of related products.

[0014] The specific technical solution of the present invention is as follows:

[0015] A method for separating BDDE-modified hyaluronic acid oligosaccharides, the separation method comprising the following steps: separating the modified hyaluronic acid oligosaccharides using high performance liquid chromatography-high resolution mass spectrometry, wherein, in the high performance liquid chromatography-high resolution mass spectrometry, an amide chromatographic column is used, and gradient elution is performed with mobile phase A and mobile phase B, mobile phase A is an aqueous solution of ammonium formate or ammonium acetate, and mobile phase B is an aqueous acetonitrile solution of ammonium formate or an aqueous acetonitrile solution of ammonium acetate.

[0016] Further, in the above separation method, in mobile phase A, the concentration of ammonium formate or ammonium acetate in the aqueous solution of ammonium formate or ammonium acetate is 5-10 mM.

[0017] Further, in the above separation method, in mobile phase B, in the aqueous acetonitrile solution of ammonium formate or the aqueous acetonitrile solution of ammonium acetate, the concentration of ammonium formate or ammonium acetate is 5-10 mM, and the concentration of acetonitrile in the aqueous acetonitrile solution is 90-95 wt%.

[0018] Further, the mentioned gradient elution conditions are:

[0019] First, use mobile phase B;

[0020] Subsequently, the volume fraction of mobile phase A is increased from 0% to 30-40%;

[0021] Subsequently, the volume fraction of mobile phase A remains unchanged;

[0022] Subsequently, the volume fraction of mobile phase A is increased from 30-40% to 100%.

[0023] Using the above gradient elution conditions can well achieve the separation of oligosaccharides. After the separation of oligosaccharides is completed, the volume fraction of mobile phase A can be gradually reduced and the volume fraction of mobile phase B can be increased for column cleaning, which can be conventionally selected by those skilled in the art.

[0024] For example, the mentioned gradient elution conditions are:

[0025] First, use mobile phase B;

[0026] Subsequently, the volume fraction of mobile phase A is increased from 0% to 30-40%;

[0027] Subsequently, the volume fraction of mobile phase A remains unchanged;

[0028] Subsequently, the volume fraction of mobile phase A is increased from 30-40% to 100%;

[0029] Subsequently, the volume fraction of mobile phase A is reduced from 100% to 0%.

[0030] In a specific embodiment of the present invention, the gradient elution conditions are:

[0031] From 0 to 3 min, the volume fraction of mobile phase A is 0%;

[0032] From 3 to 45 min, the volume fraction of mobile phase A increases from 0% to 35%;

[0033] From 45 to 50 min, the volume fraction of mobile phase A is 35%;

[0034] From 50 to 60 min, the volume fraction of mobile phase A increases from 35% to 100%.

[0035] In a specific embodiment of the present invention, the gradient elution conditions are as follows:

[0036] Table 1

[0037] Serial number Time (min) A (volume%) B (volume%) 1 0 0 100 2 3 0 100 3 45 35 65 4 50 35 65 5 60 100 0 6 70 100 0 7 70.01 0 100 8 100 0 100

[0038] Furthermore, in the above determination method, the column temperature is 22 - 40 °C, for example, it can be 22 °C, 23 °C, 24 °C, 25 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C.

[0039] Furthermore, in the above determination method, the flow rate is 0.2 - 0.3 mL·min⁻¹, for example, it can be 0.2 mL·min -1 , 0.3 mL·min -1 .

[0040] Furthermore, in the above determination method, the injection volume is 3 - 5 μL, for example, it can be 3 μL, 4 μL, 5 μL.

[0041] Furthermore, in the above determination method, the amide chromatographic column is a BEH Amide chromatographic column.

[0042] Furthermore, in the above determination method, the high-resolution mass spectrometry conditions are as follows: negative ion scanning mode; negative ion scanning mode; sheath gas flow rate 10 - 20 arb; auxiliary gas flow rate: 0 - 5 arb; spray voltage: 3 - 4 kV; capillary temperature: 250 - 350 °C; S-Lens RF Level: 30 - 70% V.

[0043] For example, the sheath gas flow rate can be 10 arb, 11 arb, 12 arb, 13 arb, 14 arb, 15 arb, 16 arb, 17 arb, 18 arb, 19 arb, 20 arb; the auxiliary gas flow rate can be 0 arb, 1 arb, 2 arb, 3 arb, 4 arb, 5 arb; the spray voltage can be 3 KV, 4 KV; the capillary temperature can be 250 °C, 260 °C, 270 °C, 280 °C, 290 °C, 300 °C, 310 °C, 320 °C, 330 °C, 340 °C, 350 °C; the S-Lens RF Level can be 30% V, 40% V, 50% V, 60% V, 70% V.

[0044] Furthermore, in the above determination method, the sample injected into the high performance liquid chromatography is an HA oligosaccharide solution, which is obtained by subjecting BDDE-crosslinked hyaluronic acid to enzymatic hydrolysis, post-treatment and other steps. The concentration of the HA oligosaccharide solution is controlled within the linear range of the response of each structure. Preferably, the concentration of the oligosaccharide solution is 0.8 - 1.2 mg / ml. For example, the concentration of the oligosaccharide solution can be 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.1 mg / ml, 1.2 mg / ml.

[0045] Furthermore, the enzymatic hydrolysis of the HA oligosaccharide solution uses hyaluronidase, and the hyaluronidase can be any enzyme capable of enzymatically hydrolyzing hyaluronic acid reported in the prior art, such as hyaluronidase synthesized by microorganisms, hyaluronidase extracted by chemical methods, etc. In view of the low production cost of the microbial method, hyaluronidase synthesized by the microbial method is preferably used, and more preferably the hyaluronidase disclosed in CN103255076A and CN102876748A. This hyaluronidase has high enzyme activity and high purity, can improve efficiency, and reduce the introduction of impurities. For the specific enzymatic hydrolysis method, the present invention is not limited, and it can be carried out with reference to the methods in the prior art, such as the methods disclosed in CN105699578A, the literature Yang Biao. Determination of the modification degree of BDDE-crosslinked hyaluronic acid gel [D]. Shandong University, 2015., etc.

[0046] Furthermore, BDDE-crosslinked hyaluronic acid refers to the crosslinked hyaluronic acid product obtained by crosslinking hyaluronic acid using BDDE as a crosslinking agent. After enzymatic hydrolysis of BDDE-crosslinked hyaluronic acid by hyaluronidase, an HA oligosaccharide solution can be obtained through one or more of the post-treatment steps such as adjusting the pH, purification, centrifugation, concentration, and volume determination.

[0047] The present invention also provides a method for determining the modification degree of BDDE-crosslinked hyaluronic acid, and the determination method includes the following steps:

[0048] (1) Enzymatically hydrolyze the crosslinked hyaluronic acid to obtain modified hyaluronic acid oligosaccharides;

[0049] (2) Separate the modified hyaluronic acid oligosaccharides by using the separation method of the above-mentioned BDDE-modified hyaluronic acid oligosaccharides;

[0050] (3) After high performance liquid chromatography-high resolution mass spectrometry analysis, according to the chemical formula of the detected ions and their corresponding peak areas, calculate the sum of the peak areas of the total isotope peaks according to the proportion of the single isotope peaks of each ion; classify the obtained oligosaccharide structures, and classify the oligosaccharides with the same structure but different valence state ions and the same structure but different adduct form ions into oligosaccharides of the same structure, calculate the peak areas of the classified oligosaccharides, and calculate the modification degree according to the formula of the modification degree;

[0051] Modification degree = ∑(peak area of oligosaccharide structure containing BDDE * number of BDDE in this oligosaccharide) / ∑(peak area of each oligosaccharide structure * number of repetitions of HA disaccharide in this oligosaccharide).

[0052] Furthermore, the oligosaccharides with the same structure but different valence state ions refer to the oligosaccharides in the ionic state formed by losing different numbers of H atoms during the ionization process, and the oligosaccharides with the same structure but different adduct form ions refer to the oligosaccharide adducts in the ionic state obtained by combining with different adducts during the ionization process.

[0053] Furthermore, the high performance liquid chromatography-high resolution mass spectrometry analysis system of the present invention has high separation degree and better accuracy of the quantitative method, can separate oligosaccharides with different structures, and after ion classification, a total of 23 kinds of oligosaccharide ions can be detected. The accurate separation and quantitative analysis of the oligosaccharide structure after degradation greatly improves the accuracy of calculating the modification degree.

[0054] In a specific embodiment of the present invention, a method for determining the modification degree is provided, including the following steps:

[0055] 1. Dissolve hyaluronidase in an enzymatic hydrolysis buffer to obtain a hyaluronidase solution;

[0056] 2. Add the hyaluronidase solution to crosslinked hyaluronic acid and enzymatically hydrolyze until the end point to obtain an enzymatic hydrolysate;

[0057] 3. Ultrafilter the enzymatic hydrolysate to control the concentration of oligosaccharides to obtain a test solution;

[0058] 4. Perform high performance liquid chromatography-high resolution mass spectrometry analysis on the test solution;

[0059] 5. Process the obtained analysis results to obtain the peak areas of oligosaccharides with different structures;

[0060] 6. Calculate the modification degree according to the modification degree formula.

[0061] Further, in step 1, the enzymatic hydrolysis buffer is a buffer suitable for various different hyaluronidases and can be selected according to reports in the prior art. Different hyaluronidases may require different enzymatic hydrolysis buffers. The main purpose of the enzymatic hydrolysis buffer is to maintain the environment for enzymatic hydrolysis, enabling the full performance of the enzyme without affecting the enzymatic hydrolysis.

[0062] Further, in step 2, the temperature, pH, time, etc. of the enzymatic hydrolysis can be selected from the prior art according to different enzyme situations, as long as the crosslinked hyaluronic acid can be enzymatically hydrolyzed to the end point. The enzymatic hydrolysis conditions will not affect the determination of the subsequent modification degree.

[0063] Further, in order to ensure that the responses of each structure are within the linear range, unsaturated disaccharides and 2-B oligosaccharides need to be diluted 100 times before detection (concentration 8 - 12 μg / ml), and other structural oligosaccharides are detected in the range of 0.8 - 1.2 mg / ml to ensure sufficient detection sensitivity.

[0064] The present invention uses high-performance liquid chromatography-high-resolution mass spectrometry analysis technology to separate and analyze the enzymatically hydrolyzed oligosaccharides of BDDE-crosslinked hyaluronic acid, which can effectively separate various enzymatically hydrolyzed oligosaccharides. Further analysis of the separation results can accurately determine the modification degree of BDDE-crosslinked hyaluronic acid. By selecting the analysis conditions, the present invention can achieve the full separation of the target oligosaccharide structure, improving the accuracy of the modification degree calculation. In addition, the data processing method and the modification degree calculation method are optimized. For example, it is determined what ions to extract during the data processing process, and the total isotope content is converted from the content of the monoisotopic peak, etc. The obtained modification degree calculation formula is more accurate, further improving the accuracy of the modification degree. Compared with the prior art, the present invention has the following beneficial effects:

[0065] 1. By optimizing the chromatographic type and separation conditions, the present invention realizes more sufficient separation of each component and reduces the influence of ionization suppression on the quantitative results of oligosaccharides. Compared with the existing GPC chromatographic column, the diol-based hydrophilic chromatographic column (Luna-Hilic) and the silica hydrophilic chromatographic column (BEH-Hilic) commonly used for oligosaccharide separation, the amide chromatographic column (BEH-Amide) of the present invention has better separation degree and detection sensitivity for representative oligosaccharide targets.

[0066] 2. The present invention uses high-resolution mass spectrometry (HRMS) instead of low-resolution mass spectrometry as the detector. By measuring more precise m / z (mass deviation less than 5 ppm, accurate to 4 decimal places), the interference caused by possible impurities to the quantitative results is excluded.

[0067] 3. Based on the structural characteristics of BDDE-crosslinked HA, the present invention draws the possible oligosaccharide structures and integrates all 23 oligosaccharide structures detected in the mass spectrometry, achieving a comprehensive coverage of the oligosaccharide structures and ensuring the accuracy of the modification degree analysis results.

[0068] 4. The present invention precisely analyzes the mass spectrometry corresponding to each structure, combines and calculates different valence states and different adduct ions generated by the same structure to eliminate the influence of the ionization differences of different oligosaccharide structures on the quantitative results.

[0069] 5. According to the chemical formulas of the ions participating in the quantification, the present invention calculates the ratio of the monoisotopic peak to all isotope peaks and performs subsequent modification degree calculations based on the peak areas of all isotope peaks. Such a data processing method is the key to ensuring the accuracy of the modification degree analysis results. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 It is a schematic diagram of the enzymatic hydrolysis of BDDE-crosslinked HA.

[0071] Figure 2 It is the separation of representative oligosaccharides by a GPC chromatographic column.

[0072] Figure 3 It is the separation of representative oligosaccharides by a Luna-HILIC chromatographic column.

[0073] Figure 4 It is the separation of representative oligosaccharides by a BEH-Amide chromatographic column.

[0074] Figure 5 It is the separation of representative oligosaccharides by the mobile phase of Comparative Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0075] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention is provided in conjunction with specific examples and drawings.

[0076] In the following examples, the hyaluronidase used is from Bloomage Biotechnology Co., Ltd.

[0077] In the following examples, the preparation method of the crosslinked HA used is as follows: starting from sodium hyaluronate, using 1,4-butanediol diglycidyl ether, etc. as crosslinking agents to crosslink with HA under specified conditions to form a water-insoluble gel, and obtaining a hydrogel or its dry powder through purification, granulation and / or precipitation, and drying processes.

[0078] Example 1

[0079] 1. Solution preparation

[0080] Enzymatic digestion buffer: Weigh accurately about 2.74 g of sodium dihydrogen phosphate dihydrate and about 0.35 g of disodium hydrogen phosphate anhydrous, place them in the same 100-ml volumetric flask, dissolve with water and quantitatively dilute to the mark, shake well. Accurately measure 10 ml and transfer it to a 100-ml volumetric flask, quantitatively dilute to the mark with water, shake well. Adjust the pH to 6.6 with 1 mol / L sodium hydroxide solution, sterilize at 121 °C for 10 minutes, cool, and obtain the phosphate buffer with a pH of 6.6.

[0081] Hyaluronidase (10,000 units) solution: Take 1 vial of hyaluronidase (50,000 units), transfer it to a 5-ml volumetric flask, quantitatively dilute to the mark with the enzymatic digestion buffer, and shake well.

[0082] Enzymatic digestion solution: Weigh accurately about 10 mg of cross-linked HA, place it in a 20-ml headspace vial, add 1 ml of enzymatic digestion buffer and 0.5 ml of water, seal and crimp the cap, sterilize in an oven at 121 °C for 10 minutes, cool. Use a syringe to take 1 ml of hyaluronidase (10,000 units) and inject it into the headspace vial. Place it in a water bath thermostatic oscillator, set the temperature at 42 °C and the rotation speed at 190 rpm, digest for 24 h, transfer it to a 5-ml volumetric flask, wash the headspace vial once with 0.5 ml of 0.1 mol / L hydrochloric acid solution and once with an appropriate amount of water, combine the washing solutions into the same volumetric flask, quantitatively dilute to the mark with water, shake well, and filter to obtain the solution.

[0083] 2. Sample purification

[0084] 2.1 Transfer the above enzymatic digestion solution to an ultrafiltration centrifugal tube (500 μl, cut-off molecular weight 10 kDa), centrifuge at 5000 rcf for 20 min. Add 400 μl of H2O to the upper tube, centrifuge at 5000 rcf for 20 min, and retain the filtrate in the bottom tube. The recovery rate of HA oligosaccharides is improved through the above two centrifugations, and the final sample concentration is about 1 mg / ml.

[0085] 2.2 Since the contents of unsaturated disaccharides and 2-B oligosaccharides in the sample are relatively high, and their responses exceed the linear range of the instrument at this concentration. Therefore, on the basis of 2.1, the sample solution is diluted 1:100 (the diluted sample concentration is about 10 μg / ml), and the linearity of unsaturated disaccharides and 2-B oligosaccharides is good (R 2 > 0.99) at this concentration. Multiply the peak areas of unsaturated disaccharides and 2-B oligosaccharides obtained under this analysis condition by 100 for subsequent modification degree calculation.

[0086] 3. UPLC-HRMS analysis

[0087] High-performance liquid chromatography conditions:

[0088] Chromatographic column: BEH Amide chromatographic column, (2.1 mm × 150 mm, 1.9 μm);

[0089] Mobile phase A: 5 mM ammonium acetate aqueous solution;

[0090] Mobile phase B: 95 wt% acetonitrile aqueous solution containing 5 mM ammonium acetate;

[0091] Column temperature: 26 °C;

[0092] Flow rate: 0.2 mL / min -1 ;

[0093] Injection volume: 5 μL;

[0094] The elution gradient is shown in Table 1 below:

[0095] Table 1 Gradient elution program

[0096] Serial number Time (min) A (volume%) B (volume%) 1 0 0 100 2 3 0 100 3 45 35 65 4 50 35 65 5 60 100 0 6 70 100 0 7 70.01 0 100 8 100 0 100

[0097] High-performance mass spectrometry conditions (LTQ-Orbitrap, Thermo Fisher Scientific):

[0098] Negative ion scanning mode; sheath gas flow rate 15 arb; auxiliary gas flow rate: 3 arb; spray voltage: 3.5 kV; capillary temperature: 275.00 °C; S-Lens RF Level: 50% V.

[0099] 4. Data processing

[0100] All ions that can be detected in the mass spectrometry and participate in the subsequent modification degree calculation are shown in Table 2.

[0101] Table 2 Oligosaccharides obtained after enzymatic hydrolysis of BDDE-crosslinked HA and their structural information

[0102]

[0103]

[0104] Extract the m / z corresponding to the monoisotopic peak of the ions in the above table, obtain the EIC chromatogram and perform integration processing. According to the proportion of each monoisotopic peak in all isotopic peaks, calculate the peak areas of all isotopic peaks. On this basis, for the same structure, the peak areas of ions with different valences and different adduct forms are combined. For example:

[0105] A 4-B = ∑A 4-B(-1) + A 4-B+H3PO4 + A 4-B+Na + A 4-B(-2)

[0106] According to the above method, the ions in Table 2 belong to 23 oligosaccharide structures. The 23 oligosaccharides after classification and their peak areas are shown in Table 3 below. The peak areas of unsaturated disaccharides and 2-B are obtained by converting the peak areas of the samples diluted 1:100 to ensure that the responses of all structures are within the linear range of the instrument (R 2 > 0.99).

[0107] Table 3 23 Oligosaccharides after Classification and Their Peak Areas

[0108] Structure Peak area Saturated disaccharide 16804062 Unsaturated disaccharide (2) 3145900800 2-B 33917064 B-2-B 28043 2-B-2 12067305 Unsaturated trisaccharide (3) 2487219 3-B 85466 4-B 14026869 B-4-B 131151 4-B-2 25463608 4-B-4 / 6-B-2 2461148 4-B-2-B 332513 4-B-4-B / 6-B-2-B 278132 6-B 335705 B-6-B 80506 6-B-4 / 8-B-2 366798 6-B-4-B / 8-B-2-B 108822 6-B-6 / 8-B-4 79931 6-B-6-B / 8-B-4-B 17866 8-B 13666 B-8-B 81475 8-B-6 6883 8-B-6-B 11023

[0109] 5. Degree of Modification Calculation

[0110] The degree of modification (MoD) of BDDE-crosslinked HA is the stoichiometric ratio of all BDDE crosslinker molecules participating in the modification to all disaccharide units. The calculation formula is: degree of modification = ∑ peak area of oligosaccharide structures containing BDDE * number of BDDE in this oligosaccharide / ∑ peak area of each oligosaccharide structure * number of repetitions of HA disaccharide in this oligosaccharide.

[0111] According to the above formula for calculating the degree of modification and combining the oligosaccharide conditions in Tables 2 and 3, the formula for the degree of modification (MOD) is obtained as follows:

[0112]

[0113] After calculation, the degree of modification is 3.68%.

[0114] Example 2 Screening of Chromatographic Columns

[0115] The enzymatically hydrolyzed oligosaccharide solution in Example 1 was analyzed by high performance liquid chromatography-high resolution mass spectrometry. The analysis method was the same as that in Example 1, except that the chromatographic columns used for high performance liquid chromatography were: GPC chromatographic column, diol-based hydrophilic chromatographic column (Luna-Hilic), and amide chromatographic column (BEH-Amide). The separation of representative oligosaccharides is as Figure 2-4 shown. It can be seen from the figure that the amide chromatographic column (BEH-Amide) of the present invention has better separation and detection sensitivity for representative oligosaccharide targets.

[0116] After analysis and detection using different chromatographic columns, the degree of modification was calculated according to the method in Example 1, and the results are shown in Table 4 below.

[0117] Table 4

[0118] Chromatographic column Degree of modification GPC chromatographic column 2.57％ Diol-based hydrophilic chromatographic column (Luna-Hilic) 1.67％ Amide chromatographic column (BEH-Amide) 3.68％

[0119] As can be seen from the data in the above table, the GPC chromatographic column and the diol-based hydrophilic chromatographic column (Luna-Hilic) have low resolution and cannot effectively separate structurally similar oligosaccharides, resulting in low detection results. Compared with the amide chromatographic column (BEH-Amide) of the present invention, they are 30.2% and 54.6% lower respectively.

[0120] Example 3

[0121] Perform high performance liquid chromatography-high resolution mass spectrometry analysis on the oligosaccharide solution after enzymatic hydrolysis in Example 1. The analysis method is the same as that in Example 1, except that the high performance liquid chromatography conditions used are as follows:

[0122] Chromatographic column: BEH Amide chromatographic column, (2.1 mm × 150 mm, 1.9 μm);

[0123] Mobile phase A: 10 mM ammonium formate aqueous solution;

[0124] Mobile phase B: 90 wt% acetonitrile aqueous solution containing 10 mM ammonium formate;

[0125] Column temperature: 30 °C;

[0126] Flow rate: 0.2 mL·min -1 ;

[0127] Injection volume: 3 μL;

[0128] Elution gradient is the same as that in Example 1.

[0129] Finally, the obtained modification degree is 3.72%.

[0130] Example 4

[0131] Perform high performance liquid chromatography-high resolution mass spectrometry analysis on the oligosaccharide solution after enzymatic hydrolysis in Example 1. The analysis method is the same as that in Example 1, except that the high resolution mass spectrometry conditions used are as follows:

[0132] Negative ion scanning mode; sheath gas flow rate 10 arb; auxiliary gas flow rate: 1 arb; spray voltage: 4 KV; capillary temperature: 350 °C; S-Lens RF Level: 30% V.

[0133] Finally, the obtained modification degree is 3.45%.

[0134] Example 5

[0135] Perform high performance liquid chromatography-high resolution mass spectrometry analysis on the oligosaccharide solution after enzymatic hydrolysis in Example 1. The analysis method is the same as that in Example 1, except that the high resolution mass spectrometry conditions used are as follows:

[0136] Negative ion scanning mode; sheath gas flow rate: 20 arb; auxiliary gas flow rate: 5 arb; spray voltage: 3 kV; capillary temperature: 250 °C; S-Lens RF Level: 70% V.

[0137] Finally, the obtained modification degree was 3.50%.

[0138] Comparative Example 1

[0139] Perform high performance liquid chromatography-high performance mass spectrometry analysis on the oligosaccharide solution after enzymatic hydrolysis in Example 1. The chromatographic column is the same as that in Example 1, except that the mobile phases used in high performance liquid chromatography are as follows: 1. Mobile phase A: 0.15 wt% formic acid aqueous solution, mobile phase B: 95 wt% acetonitrile aqueous solution containing 0.15 wt% formic acid; 2. Mobile phase A: 20 mM ammonium acetate aqueous solution, mobile phase B: 20 mM ammonium acetate 95 wt% acetonitrile aqueous solution.

[0140] The separation of representative oligosaccharides in the first group of mobile phases is as Figure 5 shown. By Figure 4 comparing with Figure 5 it can be seen that this mobile phase has poor separation effect on oligosaccharides and cannot fully separate each oligosaccharide, resulting in inaccurate calculation results of the modification degree.

[0141] The separation of representative oligosaccharides in the second group of mobile phases is similar to Figure 4 that, but due to the high concentration of ammonium acetate, the detection sensitivity decreases by more than 50%, which is not conducive to the accurate characterization of the modification degree.

[0142] Comparative Example 2

[0143] Perform high performance liquid chromatography-high performance mass spectrometry analysis on the oligosaccharide solution after enzymatic hydrolysis in Example 1. The chromatographic conditions are the same as those in Example 1, except that the mobile phase used is 5 mM ammonium acetate aqueous solution and gradient elution is not used. As a result, each component cannot be separated or eluted, resulting in inaccurate calculation results of the modification degree.

[0144] Comparative Example 3

[0145] According to the calculation method in Yang Biao's paper, only consider modification fragments such as 2-B, 4-B, 6-B, 2-B-2, 4-B-2, etc., and use the monoisotopic peaks of each structure for modification degree calculation. The calculated modification degree is 2.59%, which is 29.6% lower than the actual modification degree (3.68%) of the sample.

Claims

1. A method for separating BDDE-modified hyaluronic acid oligosaccharides, characterized in that, The separation method includes the following steps: Separate the modified hyaluronic acid oligosaccharides using high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS), wherein, in the HPLC-HRMS, an amide chromatographic column is used, and gradient elution is performed with mobile phase A and mobile phase B, mobile phase A is an aqueous solution of ammonium formate or ammonium acetate, wherein the concentration of ammonium formate or ammonium acetate is 5-10 mM, mobile phase B is an aqueous acetonitrile solution of ammonium formate or an aqueous acetonitrile solution of ammonium acetate, wherein the concentration of ammonium formate or ammonium acetate is 5-10 mM, and the acetonitrile concentration in the aqueous acetonitrile solution is 90-95 wt%, The gradient elution conditions are as follows: First, use mobile phase B; Subsequently, the volume fraction of mobile phase A is increased from 0% to 30-40%; Subsequently, the volume fraction of mobile phase A remains unchanged; Subsequently, the volume fraction of mobile phase A is increased from 30-40% to 100%.

2. The separation method according to claim 1, characterized in that, The column temperature of the chromatographic column in liquid chromatography is 22-40 °C.

3. The separation method according to claim 1, characterized in that, The flow rate used in liquid chromatography is 0.2 - 0.3 mL / min -1 .

4. The separation method according to claim 1, characterized in that, The amide chromatographic column is a BEH Amide chromatographic column.

5. The separation method according to claim 1, characterized in that, The mass spectrometry conditions for HPLC-HRMS are: negative ion scanning mode; sheath gas flow rate 10-20 arb; auxiliary gas flow rate: 0-5 arb; spray voltage: 3-4 KV; capillary temperature: 250-350 °C; S-Lens RF Level: 30-70 %V.

6. A method for determining the modification degree of BDDE-crosslinked hyaluronic acid, characterized in that, The determination method includes the following steps: (1) Enzymatically crosslink hyaluronic acid to obtain modified hyaluronic acid oligosaccharides; (2) Separate the modified hyaluronic acid oligosaccharides using the separation method of BDDE-modified hyaluronic acid oligosaccharides according to any one of claims 1-5; (3) After HPLC-HRMS analysis, according to the chemical formula of the detected ions and their corresponding peak areas, calculate the sum of the peak areas of the full isotope peaks according to the proportion of the single isotope peaks of each ion; classify the obtained oligosaccharide structures, and classify the oligosaccharides with the same structure but different valence ions and the same structure but different adduct forms of ions into oligosaccharides of the same structure, calculate the peak areas of the classified oligosaccharides, and calculate the modification degree according to the formula of the modification degree; Modification degree = ∑(peak area of oligosaccharide structure containing BDDE * number of BDDE in this oligosaccharide) / ∑(peak area of each oligosaccharide structure * number of repetitions of HA disaccharide in this oligosaccharide).

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