Analysis method of O-sugar chain of ulinastatin

Abstract

The invention provides a method for analyzing an O sugar chain of glycoprotein. Specifically, the invention provides an analysis method of ulinastatin O-sugar chain, which comprises the following steps: (1) carrying out enzyme digestion on a sample by using streptasin E to obtain an enzyme-digested sample; (2) reacting fluorescent molecules with the sample subjected to enzyme digestion to obtain a fluorescently-labeled sample; and (3) carrying out ultrahigh pressure liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS) analysis on the fluorescence labeled sample, and determining the structure of the O carbohydrate chain according to the obtained mass spectrum data. The method provided by the invention can simply, conveniently, quickly and accurately confirm the specific glycoform and relative proportion of the ulinastatin O sugar chain, can be used for daily detection of the ulinastatin O sugar chain, and provides an important basis for structural research and quality control of ulinastatin.

Description

Technical Field

[0001] The present invention relates to the technical field of protein analysis, in particular to an analysis method for O-sugar chains of ulinastatin. Background Art

[0002] Glycosylation is a common protein modification translation, which has important effects on protein structure and function, including protein folding, stability, molecular recognition and immunogenicity. More than half of natural proteins are glycosylated at the hydroxyl group of appropriate serine / threonine residues or at the amide group of asparagine in asparagine-serine / threonine complexes.

[0003] There are two main types of glycosylation that are most studied at present: N-glycosylation and O-glycosylation. N-glycosylation occurs on the side chain of asparagine. The analytical methods for protein N-glycosylation are relatively mature. In comparison, the identification and analysis of protein O-glycosylation still have great technical bottlenecks. O-glycosylation occurs on the side chain of serine or threonine. There is no characteristic modification sequence of proteins, and there is a lack of efficient and specific O-glycosylation enzymes. Therefore, most researchers currently use chemical methods to release O-linked sugar chains from peptides. The commonly used chemical methods are β-elimination reaction and hydrazine hydrolysis under alkaline conditions. Hydrazine hydrolysis can be used to release N-linked sugar chains and O-linked sugar chains, but because the reaction conditions of this method are relatively violent and harsh, it is easy to cause explosions and hydrazine is a highly toxic substance. Therefore, hydrazine hydrolysis has not been widely used. The β-elimination reaction refers to the fact that glycoproteins are unstable in alkaline environments and are prone to β-elimination, resulting in the cleavage of glycopeptide bonds, thereby dissociating O-linked sugar chains from the corresponding glycoproteins. However, the hemiacetal structure of sugar chains under non-reducing conditions is prone to peeling reactions. In order to prevent the peeling reaction, an excess of reducing agent is added to reduce the hemiacetal structure of the sugar chain to a relatively stable sugar alcohol structure to protect the reducing end of the sugar chain. Therefore, most published literature uses the β-elimination method to release O-linked sugar chains. In addition, the structure of O-sugar chains is complex and diverse. The high molecular weight and easy breakage of glycosidic bonds of sugar chains make it impossible to achieve it by setting a fixed molecular weight modification during the search (search database) process. Multiple factors make the analysis of O-glycosylation very difficult.

[0004] Ulinastatin is a urinary trypsin inhibitor (UTI) present in human serum and urine. It is one of the main inhibitors of the trypsin family of serine proteases. It can inhibit proteolytic activity in a variety of tissues and can also exert local anti-inflammatory effects. The peptide has two Kunitz-type domains and therefore plays a key role in inflammation and innate immunity. Due to the complexity of ulinastatin O-linked sugars, it is very difficult to analyze directly at the level of ulinastatin intact protein or peptide segments, and no relevant research data has been reported.

[0005] Therefore, there is an urgent need in the art to provide an analysis method for the O-sugar chain of ulinastatin. Summary of the invention

[0006] The purpose of the present invention is to provide a fluorescently labeled ulinastatin O-sugar chain for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry characterization, so as to provide an important basis for the structural research and quality control of ulinastatin.

[0007] The present invention provides an analysis method for ulinastatin O-sugar chains, comprising the following steps:

[0008] (1) digesting the sample with pronase E to obtain a digested sample;

[0009] (2) reacting the fluorescent molecule with the enzyme-digested sample to obtain a fluorescently labeled sample;

[0010] (3) The fluorescently labeled sample is subjected to ultra-high pressure liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS) analysis, and the structure of the O-sugar chain is determined by the obtained mass spectrometry data.

[0011] In another preferred embodiment, the mass ratio of pronase E to ulinastatin is 1:0.5-2, preferably 1:0.6-1.5, such as 1:0.8, 1:1 or 1:1.2;

[0012] In another preferred embodiment, the enzyme cleavage temperature is 45±10°C, preferably 45±5°C, ±3°C, ±2°C or ±1°C;

[0013] In another preferred embodiment, the enzyme digestion time is 6 to 24 hours, preferably 10 to 20 hours, such as 12 hours, 15 hours or 18 hours;

[0014] In another preferred embodiment, the pH of the enzymatic cleavage is 7.5±0.5, preferably 7.5±0.3, ±0.2 or ±0.1.

[0015] In another preferred embodiment, the enzyme cleavage reaction can be terminated by heating the reaction solution of step (1) to 100±10°C (±5°C, ±3°C, ±2°C or ±1), and then centrifuging to remove the precipitate.

[0016] In another preferred embodiment, the method further comprises step (1-a), wherein the sample obtained in step (1) after enzymatic digestion is subjected to chromatographic purification, using a polymethacrylic acid resin anion exchange column (such as Super Q-650M), eluted, and then ultrafiltered to remove salt to obtain a purified sample. The purified sample is usually concentrated or dried before step (2).

[0017] In another preferred embodiment, the step (1-a) comprises a chromatographic purification step:

[0018] Equilibration: equilibration with buffer A (e.g. 4-6 column volumes);

[0019] Loading: Load the supernatant of the digested sample obtained in step (1) onto an anion exchange column;

[0020] Wash: Wash with buffer A for 8-12 column volumes, preferably 9-11 volumes;

[0021] Elution: 8-12 column volumes, preferably 9-11 volumes, of buffer B;

[0022] Buffer A: 50±5mM NaCl; Buffer B: 1.5±0.1M NaCl.

[0023] In another preferred embodiment, in the step (1-a), ultrafiltration desalination includes collecting the eluate and then performing ultrafiltration desalination using an ultrafiltration tube with a molecular weight cutoff of 3KD.

[0024] In another preferred embodiment, the fluorescent molecule is rhodamine B isothiocyanate, fluorescein isothiocyanate (such as 5-fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, 7-fluorescein isothiocyanate).

[0025] In another preferred embodiment, the reaction in step (2) has one or more technical features selected from the following group:

[0026] The mass ratio of the fluorescent molecule to the O sugar chain is 1:1-3, preferably 1:1-2, such as 1:1.2, 1:1.5 or 1:1.8;

[0027] The reaction temperature is 37±10°C, preferably 37±5°C, ±3°C, ±2°C or ±1°C;

[0028] The reaction time is 6 to 24 hours, preferably 10 to 20 hours, such as 12 hours, 15 hours or 18 hours;

[0029] The solvent for the reaction is an organic solvent, such as DMSO.

[0030] In another preferred embodiment, the method further comprises step (2-a), adding deionized water to terminate the reaction, and then removing the reaction solvent by ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 3KD.

[0031] In another preferred embodiment, the method further comprises step (2-b), wherein the fluorescently labeled sample obtained in step (2) is chromatographically purified using a gel filtration column (such as Superdex 200Increase 10 / 300GL), eluted, and then ultrafiltered to remove salt to obtain a purified sample, and the purified sample is subjected to step (3).

[0032] In another preferred embodiment, in the step (2-b), ultrafiltration desalination includes collecting the eluate and then performing ultrafiltration desalination using an ultrafiltration tube with a molecular weight cutoff of 3KD.

[0033] In another preferred embodiment, the step (2-b) comprises a chromatographic purification step:

[0034] Equilibration: Buffer C equilibration (e.g. 4-6 column volumes);

[0035] Loading: Loading the sample obtained in step (2) or (2-a) onto a gel filtration column;

[0036] Elution: flushing with equilibrium solution (e.g. 4-6 column volumes);

[0037] Buffer C: 10±1 mM phosphate buffer + 1±0.1 M NaCl pH = 7.4±0.2.

[0038] In another preferred embodiment, in step (2-b), the ultrafiltration includes collecting the peak containing the fluorescently labeled O sugar chain and then performing ultrafiltration to remove salt using an ultrafiltration tube with a molecular weight cutoff of 3KD. The obtained sample is usually concentrated or dried before proceeding to step (3).

[0039] In another preferred embodiment, the chromatographic conditions used in the UPLC-Q-TOF-MS / MS analysis in step (3) are: Chromatographic column information: Waters Acquity TM UPLC Glycan BEH Amide, 1.7μm, 2.1mm×150mm; wavelength: 280±5nm; column temperature: 40±2℃; flow rate: 0.2ml / min; gradient elution: elution time 0-20min, mobile phase A (0.1% ammonia aqueous solution) 10%-80%, mobile phase B (0.1% ammonia acetonitrile solution 20%-90%).

[0040] In another preferred embodiment, the injection volume is 1-5 μl, such as 2 μl.

[0041] In another preferred embodiment, the sample solvent is water.

[0042] In another preferred embodiment, the sample concentration is 1-3 mg / mL.

[0043] In another preferred embodiment, the ion source of the mass spectrometer is ESI, and the spray voltage is preferably 3.0-4.0 KV.

[0044] In another preferred embodiment, the mass spectrometry conditions used in the UPLC-Q-TOF-MS / MS analysis described in step (3) are: negative charge mode, spray voltage: 3.3±0.2KV; capillary temperature: 300±10°C; auxiliary gas heater temperature: 280±10°C; scan type: Full Mass / DD-MS2 (TOPN); resolution: MS: 70,000±100; MS / MS: 17500±100; scan range: 300~3000m / z.

[0045] In another preferred embodiment, the fluorescently labeled sample can be subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescent color development, which can assist in the analysis during the method of the present invention.

[0046] In another preferred embodiment, when the O-glycoform types of the obtained mass spectrum mainly fall into the O-glycoform types listed in Table 7, and the five most distributed glycoforms are: dp33-5-Ser; dp31-5-Ser; dp33-6-Ser; dp35-5-Ser;

[0047] dp-35-6-Ser, it can be judged that the sample meets the characteristics of ulinastatin O-sugar chain.

[0048] In a second aspect, the present invention provides a method for releasing / preparing ulinastatin O-sugar chains, comprising the steps of: enzymatically cleaving ulinastatin with pronase E to obtain ulinastatin O-sugar chains.

[0049] In another preferred embodiment, the enzymatic cleavage has one or more technical features selected from the following group: the mass ratio of pronase E to ulinastatin is 1:0.5-2, preferably 1:0.6-1.5, such as 1:0.8, 1:1 or 1:1.2;

[0050] In another preferred embodiment, the mass ratio of pronase E to ulinastatin is 1:0.5-2, preferably 1:0.6-1.5, such as 1:0.8, 1:1 or 1:1.2;

[0051] In another preferred embodiment, the enzyme cleavage temperature is 45±10°C, preferably 45±5°C, ±3°C, ±2°C or ±1°C;

[0052] In another preferred embodiment, the enzyme digestion time is 6 to 24 hours, preferably 10 to 20 hours, such as 12 hours, 15 hours or 18 hours;

[0053] In another preferred embodiment, the pH of the enzymatic cleavage is 7.5±0.5, preferably 7.5±0.3, ±0.2 or ±0.1.

[0054] In another preferred embodiment, the enzyme cleavage reaction can be terminated by heating the reaction solution of step (1) to 100±10°C (±5°C, ±3°C, ±2°C or ±1), and then centrifuging to remove the precipitate.

[0055] In another preferred embodiment, the sample after enzyme digestion can be purified by chromatography, such as using a polymethacrylic acid resin anion exchange column (such as Super Q-650M), eluting, and then ultrafiltration to remove salts to obtain a purified sample.

[0056] In another preferred embodiment, the step (1-a) comprises a chromatographic purification step:

[0057] Equilibration: equilibration with buffer A (e.g. 4-6 column volumes);

[0058] Loading: Load the supernatant of the digested sample obtained in step (1) onto an anion exchange column;

[0059] Wash: Wash with buffer A for 8-12 column volumes, preferably 9-11 volumes;

[0060] Elution: 8-12 column volumes, preferably 9-11 volumes, of buffer B;

[0061] Buffer A: 50±5mM NaCl; Buffer B: 1.5±0.1M NaCl.

[0062] In another preferred embodiment, the ultrafiltration desalination comprises collecting the eluate and then performing ultrafiltration desalination using an ultrafiltration tube with a molecular weight cutoff of 3KD, followed by drying to obtain the ulinastatin O-sugar chain solid.

[0063] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 is a schematic diagram of the structure of ulinastatin;

[0065] Figure 2 is the chemical structure of chondroitin sulfate A;

[0066] Figure 3SEC-HPLC diagram of sample 1 and sample 2 of Example 3;

[0067] Figure 4 The SDS-PAGE images of samples 1 and 2 of Example 2 and Example 3 are shown;

[0068] Figure 5 This is the total ion current diagram of the O-sugar chain of ulinastatin (the sample batch numbers from top to bottom are: sample 1, sample 2, sample 3).

[0069] Figure 6 This is the distribution diagram of the O-glycoform ratio of ulinastatin. DETAILED DESCRIPTION

[0070] The inventors have conducted extensive and in-depth research, and through a large number of screenings and tests, provided a method for analyzing the O-sugar chains of glycoproteins. The present invention uses pronase E to digest ulinastatin, obtains O-glycans with only one serine residue, and then uses rhodamine B to label it. The complexity of the sample is reduced, and the accurate molecular weight of the O-sugar chain can be detected by high-resolution mass spectrometry, which is matched with the molecular weight of the theoretical structure, thereby inferring the structure of the O-sugar chain, providing an important basis for structural research and quality control of ulinastatin. The present invention was completed on this basis.

[0071] the term

[0072] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0073] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0074] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0075] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0076] Ulinastatin

[0077] like Figure 1As shown, ulinastatin is an acidic glycoprotein consisting of a peptide chain and two glycoconjugate parts. Among them: the peptide chain consists of 143 amino acids, with Kunitz domains I (~8.46kDa) and II (~5.90kDa) connected to the N-terminal peptide tail, each Kunitz domain contains 3 disulfide bonds (Cys-Cys), a polypeptide, and an N-terminus and a C-terminus each having 10-25 amino acid residues. Ulinastatin can inhibit trypsin serine protease by binding through either of its two Kunitz domains. There are glycoside chains on the serine at position 10 and the asparagine at position 45 of the peptide chain. Both O- and N-linked glycoside chains are connected in domain I of ulinastatin. The main feature of the O-linked glycoside is the chondroitin sulfate chain (~11.70kDa), and the N-linked glycoside chain has a double-line structure (~2.40kDa). Ulinastatin O-linked sugar is chondroitin sulfate A glycosaminoglycan, its structure is as follows Figure 2 As shown in the figure, its basic structure is GalNAc-(GlcA-GalNAc±4S)n-GlcA-Gal4S-Gal-Xyl-Ser (GalNAc: N-acetylgalactosamine; GalNAc-4S: 4-sulfated N-acetylgalactosamine; GlcA: glucuronic acid; Gal4S: 4-sulfated galactose; Gal: galactose; Xyl: xylose; Ser: serine). The structure contains repeated disaccharide units, mainly composed of repeated disaccharide units of GlcA and GalNAc. GalNAc may be sulfated. The heterogeneity of O-linked sugars is caused by the difference in the number of repeated disaccharide units n and the number of sulfations.

[0078] Pronase E is a mixture of proteolytic enzymes extracted from Streptomyces griseus. It is able to break down proteins into individual amino acids, making it suitable for a wide range of protein degradation. Pronase E contains at least three protease activities, including extracellular serine proteases. In general, serine proteases have broad substrate specificity, which is regulated by an active site consisting of an aspartic acid, a histidine, and a serine on the molecule. Pronase E also contains at least three casein hydrolysis activities and one aminopeptidase activity. The three casein hydrolases are named Streptomyces griseus Protease A, Steptomycesgtniseus Protease B, and Streptomyces gfiseus Typsin.

[0079] The present invention unexpectedly discovered that by enzymatically cleaving ulinastatin with pronase E, O-glycan with only one serine residue can be obtained, which is then labeled with rhodamine B. The complexity of the sample is reduced, and the accurate molecular weight of the O-glycan chain can be detected by high-resolution mass spectrometry and matched with the molecular weight of the theoretical structure, thereby inferring the structure of the O-glycan chain, providing an important basis for structural research and quality control of ulinastatin.

[0080] Release / preparation method of ulinastatin O-sugar chain

[0081] The invention provides a method for releasing / preparing ulinastatin O-sugar chains, comprising the steps of: enzymatically cleaving ulinastatin with pronase E to obtain ulinastatin O-sugar chains.

[0082] In another preferred embodiment, the enzymatic cleavage has one or more technical features selected from the following group: the mass ratio of pronase E to ulinastatin is 1:0.5-2, preferably 1:0.6-1.5, such as 1:0.8, 1:1 or 1:1.2;

[0083] In another preferred embodiment, the mass ratio of pronase E to ulinastatin is 1:0.5-2, preferably 1:0.6-1.5, such as 1:0.8, 1:1 or 1:1.2;

[0084] In another preferred embodiment, the enzyme cleavage temperature is 45±10°C, preferably 45±5°C, ±3°C, ±2°C or ±1°C;

[0085] In another preferred embodiment, the enzyme digestion time is 6 to 24 hours, preferably 10 to 20 hours, such as 12 hours, 15 hours or 18 hours;

[0086] In another preferred embodiment, the pH of the enzymatic cleavage is 7.5±0.5, preferably 7.5±0.3, ±0.2 or ±0.1.

[0087] In another preferred embodiment, the enzyme cleavage reaction can be terminated by heating the reaction solution of step (1) to 100±10°C (±5°C, ±3°C, ±2°C or ±1), and then centrifuging to remove the precipitate.

[0088] In another preferred embodiment, the sample after enzyme digestion can be purified by chromatography, such as using a polymethacrylic acid resin anion exchange column (such as Super Q-650M), eluting, and then ultrafiltration to remove salts to obtain a purified sample.

[0089] In another preferred embodiment, the step (1-a) comprises a chromatographic purification step:

[0090] Equilibration: equilibration with buffer A (e.g. 4-6 column volumes);

[0091] Loading: Load the supernatant of the digested sample obtained in step (1) onto an anion exchange column;

[0092] Wash: Wash with buffer A for 8-12 column volumes, preferably 9-11 volumes;

[0093] Elution: 8-12 column volumes, preferably 9-11 volumes, of buffer B;

[0094] Buffer A: 50±5mM NaCl; Buffer B: 1.5±0.1M NaCl.

[0095] In another preferred embodiment, the loading amount of the chromatographic purification is about 5-20 mg of ulinastatin digestion solution / ml of column filler, preferably 10-15 mg of ulinastatin digestion solution / ml of column filler.

[0096] In another preferred embodiment, the ultrafiltration desalination comprises collecting the eluate and then performing ultrafiltration desalination using an ultrafiltration tube with a molecular weight cutoff of 3KD, followed by drying to obtain the ulinastatin O-sugar chain solid.

[0097] Analysis Method of O-Glycans in Ulinastatin

[0098] The present invention provides an analysis method for ulinastatin O-sugar chains, comprising the following steps:

[0099] (1) digesting the sample with pronase E to obtain a digested sample;

[0100] (2) reacting the fluorescent molecule with the enzyme-digested sample to obtain a fluorescently labeled sample;

[0101] (3) The fluorescently labeled sample is subjected to ultra-high pressure liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS) analysis, and the structure of the O-sugar chain is determined by the obtained mass spectrometry data.

[0102] In another preferred embodiment, the enzymatic cleavage in step (1) has one or more technical features selected from the following group: the mass ratio of pronase E to ulinastatin is 1:0.5-2, preferably 1:0.6-1.5, such as 1:0.8, 1:1 or 1:1.2;

[0103] In another preferred embodiment, the enzyme cleavage temperature is 45±10°C, preferably 45±5°C, ±3°C, ±2°C or ±1°C;

[0104] In another preferred embodiment, the enzyme digestion time is 6 to 24 hours, preferably 10 to 20 hours, such as 12 hours, 15 hours or 18 hours;

[0105] In another preferred embodiment, the pH of the enzymatic cleavage is 7.5±0.5, preferably 7.5±0.3, ±0.2 or ±0.1.

[0106] In another preferred embodiment, the enzyme cleavage reaction can be terminated by heating the reaction solution of step (1) to 100±10°C (±5°C, ±3°C, ±2°C or ±1), and then centrifuging to remove the precipitate.

[0107] In another preferred embodiment, the method further comprises step (1-a), wherein the sample obtained in step (1) after enzymatic digestion is subjected to chromatographic purification, using a polymethacrylic acid resin anion exchange column (such as Super Q-650M), eluted, and then ultrafiltered to remove salt to obtain a purified sample. The purified sample is usually concentrated or dried before step (2).

[0108] In another preferred embodiment, the step (1-a) comprises a chromatographic purification step:

[0109] Equilibration: equilibration with buffer A (e.g. 4-6 column volumes);

[0110] Loading: Load the supernatant of the digested sample obtained in step (1) onto an anion exchange column;

[0111] Wash: Wash with buffer A for 8-12 column volumes, preferably 9-11 volumes;

[0112] Elution: 8-12 column volumes, preferably 9-11 volumes, of buffer B;

[0113] Buffer A: 50±5mM NaCl; Buffer B: 1.5±0.1M NaCl.

[0114] In another preferred embodiment, the sample loading amount for the chromatographic purification is about 5-20 mg of sample digestion solution / ml of column packing, preferably 10-15 mg of sample digestion solution / ml of column packing.

[0115] In another preferred embodiment, in the step (1-a), ultrafiltration desalination includes collecting the eluate and then performing ultrafiltration desalination using an ultrafiltration tube with a molecular weight cutoff of 3KD.

[0116] In another preferred embodiment, the fluorescent molecule is rhodamine B isothiocyanate, fluorescein isothiocyanate (such as 5-fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, 7-fluorescein isothiocyanate).

[0117] In another preferred embodiment, the reaction in step (2) has one or more technical features selected from the following group:

[0118] The mass ratio of the fluorescent molecule to the O sugar chain is 1:1-3, preferably 1:1-2, such as 1:1.2, 1:1.5 or 1:1.8;

[0119] The reaction temperature is 37±10°C, preferably 37±5°C, ±3°C, ±2°C or ±1°C;

[0120] The reaction time is 6 to 24 hours, preferably 10 to 20 hours, such as 12 hours, 15 hours or 18 hours;

[0121] The solvent for the reaction is an organic solvent, such as DMSO.

[0122] In another preferred embodiment, the method further comprises step (2-a), adding deionized water to terminate the reaction, and then removing the reaction solvent by ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 3KD.

[0123] In another preferred embodiment, the method further comprises step (2-b), wherein the fluorescently labeled sample obtained in step (2) is chromatographically purified using a gel filtration column (such as Superdex 200Increase 10 / 300GL), eluted, and then ultrafiltered to remove salt to obtain a purified sample, and the purified sample is subjected to step (3).

[0124] In another preferred embodiment, in the step (2-b), ultrafiltration desalination includes collecting the eluate and then performing ultrafiltration desalination using an ultrafiltration tube with a molecular weight cutoff of 3KD.

[0125] In another preferred embodiment, the step (2-b) comprises a chromatographic purification step:

[0126] Equilibration: Buffer C equilibration (e.g. 4-6 column volumes);

[0127] Loading: Loading the sample obtained in step (2) or (2-a) onto a gel filtration column;

[0128] Elution: flushing with equilibrium solution (e.g. 4-6 column volumes);

[0129] Buffer C: 10±1 mM phosphate buffer + 1±0.1 M NaCl pH = 7.4±0.2.

[0130] In another preferred embodiment, in step (2-b), the ultrafiltration includes collecting the peak containing the fluorescently labeled O sugar chain and then performing ultrafiltration to remove salt using an ultrafiltration tube with a molecular weight cutoff of 3KD. The obtained sample is usually concentrated or dried before proceeding to step (3).

[0131] In another preferred embodiment, the chromatographic conditions used in the UPLC-Q-TOF-MS / MS analysis in step (3) are: Chromatographic column information: Waters Acquity TMUPLC Glycan BEH Amide, 1.7μm, 2.1mm×150mm; wavelength: 280±5nm; column temperature: 40±2℃; flow rate: 0.2ml / min; gradient elution: elution time 0-20min, mobile phase A (0.1% ammonia aqueous solution) 10%-80%, mobile phase B (0.1% ammonia acetonitrile solution 20%-90%).

[0132] In another preferred embodiment, the injection volume is 1-5 μl, such as 2 μl.

[0133] In another preferred embodiment, the sample solvent is water.

[0134] In another preferred embodiment, the sample concentration is 1-3 mg / mL.

[0135] In another preferred embodiment, the ion source of the mass spectrometer is ESI, and the spray voltage is preferably 3.0-4.0 KV.

[0136] In another preferred embodiment, the mass spectrometry conditions used in the UPLC-Q-TOF-MS / MS analysis described in step (3) are: negative charge mode, spray voltage: 3.3±0.2KV; capillary temperature: 300±10°C; auxiliary gas heater temperature: 280±10°C; scan type: Full Mass / DD-MS2 (TOPN); resolution: MS: 70,000±100; MS / MS: 17500±100; scan range: 300~3000m / z.

[0137] In another preferred embodiment, the fluorescently labeled sample can be subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescent color development, which can assist in the analysis during the method of the present invention.

[0138] In another preferred embodiment, when the O-glycoform types of the obtained mass spectrum mainly fall into the O-glycoform types listed in Table 4, and the five most distributed glycoforms are: dp33-5-Ser; dp31-5-Ser; dp33-6-Ser; dp35-5-Ser;

[0139] dp-35-6-Ser, it can be judged that the sample meets the characteristics of ulinastatin O-sugar chain.

[0140] The main advantages of the present invention include:

[0141] 1) Compared with the traditional chemical method for releasing O-sugar chains, the method of the present invention can obtain complete O-sugar chains by digesting the sample with pronase E;

[0142] 2) The method of the present invention can simply, quickly and accurately identify the specific glycoforms and relative proportions of the O-sugar chain of ulinastatin, and can be used for routine detection of the O-sugar chain of ulinastatin, providing an important basis for structural research and quality control of ulinastatin.

[0143] 3) The method of the present invention has high accuracy, simple operation, and is economical and applicable.

[0144] The present invention will be further described below in conjunction with specific implementation. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0145] Ulinastatin: Jiangsu Aidi Pharmaceutical Co., Ltd.

[0146] Pronase E: Shanghai Myril Biochemical Technology Co., Ltd.

[0147] Example 1 Investigation of the amount of pronase E and the enzyme cleavage time

[0148] Take 30 mg of ulinastatin and dilute it to 5 mL with 50 mM Tris-HCl (pH = 7.5). Weigh 10-50 mg of pronase E and dissolve it in 1 mL of 50 mM Tris-HCl (pH = 7.5). Then mix the two solutions in a 45°C shaker and digest for 4 hours, 6 hours, 18 hours, and 24 hours. Optimize the amount of pronase E and the digestion time, and confirm whether the digestion is complete by SDS-PAGE.

[0149] Table 1 Investigation of the dosage of pronase E and enzyme cleavage time

[0150]

[0151] As can be seen from Table 1, when the mass ratio of pronase E to ulinastatin is 2:3-5:3 and the enzymatic cleavage time is 6-24 h, the enzymatic cleavage can be complete to obtain the O-sugar chain.

[0152] Example 2

[0153] 1.1. Release of O-glycans

[0154] 1) Take 30 mg of ulinastatin and dilute it to 5 mL with 50 mM Tris-HCl (pH = 7.5). Weigh 20 mg of pronase E and dissolve it in 1 mL of 50 mM Tris-HCl (pH = 7.5). Then mix the two solutions and digest them in a shaker at 45°C for 18 hours;

[0155] 2) The reaction solution was placed in a 100°C water bath for 5 min to terminate the reaction, and the precipitate was removed by centrifugation;

[0156] 1.2. Purification of O-glycans

[0157] Chromatographic conditions: Column: Super Q-650M 2mL; Buffer A: 50mM NaCl; Buffer B: 1.5M NaCl;

[0158] Equilibration: 5 column volumes of buffer A.

[0159] Sample loading: The supernatant after centrifugation is directly loaded.

[0160] Rinse: Buffer A

[0161] Elution: Buffer B

[0162] Ultrafiltration: After the eluate is collected, ultrafiltration is performed to remove salt using an ultrafiltration tube with a molecular weight cutoff of 3KD. Finally, freeze-drying is performed to obtain a white powder.

[0163] The experimental steps are consistent with those in Example 2

[0164] The purification conditions of O-glycans are shown in Table 2.

[0165] Table 2O Optimization of sugar chain purification conditions

[0166]

[0167] The results showed that when the amount of flushing and the amount of eluent used both reached 10 column volumes, the purity and yield of O-sugar chains were the highest.

[0168] Comparative Example 1

[0169] This comparative example adopts the chemical method "β-elimination method" in the prior art to release the O-linked sugar chain.

[0170] 1.1. Release of O-glycans

[0171] 1) Take 30 mg of ulinastatin and dilute it to 5 mL with 0.5 M NaOH solution containing 0.3 M NaBH4. Then react in a 4°C water bath for 24 hours. After the reaction is completed, neutralize with 1 M HCl;

[0172] 2) Place the reaction solution in a 100°C water bath for 5 min to terminate the reaction, and centrifuge to remove the precipitate.

[0173] 1.2. Purification of O-glycans

[0174] Chromatographic conditions: Column: Super Q-650M 2mL; Buffer A: 50mM NaCl; Buffer B: 1.5M NaCl;

[0175] Equilibration: 5 column volumes of buffer A.

[0176] Sample loading: The supernatant after centrifugation is directly loaded.

[0177] Wash: 10 column volumes of buffer A

[0178] Elution: 10 column volumes of buffer B

[0179] Ultrafiltration: After the eluate is collected, ultrafiltration is performed to remove salt using an ultrafiltration tube with a molecular weight cutoff of 3KD. Finally, lyophilization is performed to obtain 2 mg of white powder.

[0180] Compared with Comparative Example 1, the obtained O-sugar chain in Experiment 7 of Example 2 increased from 2 mg to 6 mg, and the yield increased by 2 times. This shows that the method of the present invention has high efficiency in releasing O-sugar chains, which can greatly reduce the sample dosage and reduce the detection limit.

[0181] Example 3O Fluorescent labeling and purification of sugar chains

[0182] 1) Weigh 3 mg of freeze-dried O-sugar chain and 1 mg of rhodamine B isothiocyanate, dissolve them in 2 mL of anhydrous DMSO, and then react in a shaker at 37°C for 18 hours.

[0183] 2) After the reaction is completed, deionized water is added to terminate the reaction. Then, DMSO is removed by ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 3KD.

[0184] 1.4. Purification of fluorescently labeled O-glycans

[0185] Chromatographic conditions: Column: Superdex 200Increase 10 / 300GL; Buffer: 10 mM phosphate buffer + 1 M NaCl pH = 7.4; Flow rate: 1 mL / min

[0186] Equilibration: Equilibrate with buffer for 5 column volumes.

[0187] Sample loading: The ultrafiltration solution was directly loaded.

[0188] Elution: 5 column volumes of equilibrium solution

[0189] Collection: The peak containing fluorescently labeled O sugar chains was collected and then ultrafiltered to remove salt using an ultrafiltration tube with a molecular weight cutoff of 3KD. Finally, lyophilization was performed to obtain 2 mg of red powder.

[0190] Example 4 SEC-HPLC purity detection of fluorescently labeled O-sugar chains.

[0191] The fluorescently labeled O-sugar chain purified sample of Example 3 was evaluated by SEC-HPLC. A Sepax SRT-SEC-300 (7.8*300mm, 5μm) column was used, with a mobile phase of 0.15mol / L phosphate buffer and acetonitrile in a volume ratio of 9:1, a flow rate of 1ml / min, and SEC-HPLC analysis was performed. The detection wavelength was 280nm, the injection volume was 20μL, and the analysis time was 20min. The analysis results of the fluorescently labeled O-sugar chain purified sample of Example 3 are as follows: Figure 3 As shown, they all show a single peak, and the HPLC purity of the obtained O-sugar chain sample can reach more than 98%.

[0192] Example 50 SDS-PAGE of sugar chains

[0193] The fluorescently labeled O-glycan purified samples were subjected to SDS-PAG electrophoresis at 200 V and 25 mA using 10% isocratic gel and Alcian blue was used for color development. The results are shown in Figure 2. Figure 4 shown.

[0194] When using fluorescence color development, only the O sugar chain labeled with rhodamine B showed a signal, and the O sugar chain without rhodamine B had no signal. After using Alcian blue color development, the O sugar chain labeled with rhodamine B and the O sugar chain without rhodamine B showed signals at 10-15KDa. Its molecular weight is larger than the 5-8KDa reported in the literature, which may be due to the extremely strong hydrophilicity of the chondroitin sulfate chain, which makes its apparent molecular weight larger. At the same time, the peak position of the O sugar chain labeled with rhodamine B is basically the same as that of the O sugar chain without rhodamine B, indicating that the labeling of rhodamine B has little effect on the molecular weight of the O sugar chain.

[0195] Example 6 Fluorescently labeled O-glycan mass spectrometry analysis

[0196] 5.1UPLC-Q-TOF-MS / MS test method

[0197] Sample preparation:

[0198] Take 1 mg of fluorescently labeled O-sugar chain, add 500 μL of pure water to dissolve, centrifuge at 13000 g for 1 min, and take the supernatant solution and place it in the inner tube of the injection vial.

[0199] Chromatographic column: Waters Acquity TM UPLC Glycan BEH Amide, 1.7μm, 2.1mm×150mm

[0200] Mobile phase: A. Water + 0.1% ammonia; B. Acetonitrile + 0.1% ammonia;

[0201] Flow rate: 0.2 mL / min; injection volume: 2 μL; column temperature: 40 °C;

[0202] The elution conditions are shown in Table 3.

[0203] Table 3 Chromatographic conditions settings

[0204]

[0205] 5.2Q-TOF-MS conditions:

[0206] Table 4 Mass spectrometry condition settings

[0207]

[0208]

[0209] 5.3 Optimization of spray voltage conditions

[0210] The ESI ion source parameters that have a significant impact on the detection sensitivity of compounds were optimized, mainly the spray voltage (kV) (initial value 2.5 kV, interval 0.5, a total of 12 groups, namely 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 kV). The settings of other parameters are shown in Table 4.

[0211] In high-resolution mass spectrometry, electrospray ionization (ESI) is a commonly used ion source. By applying high voltage, the sample solution is atomized into charged droplets. These droplets then undergo evaporation and Coulomb explosion processes to produce charged analyte ions, which are ultimately transmitted to the mass analyzer of the mass spectrometer. As shown in Table 4, the parameters of the ion source include three categories: voltage, temperature, and gas. Among them, the voltage has the most significant effect on the ionization efficiency. Therefore, the optimization of the ESI ion source parameters is mainly focused on the spray voltage. For the spray voltage, a voltage that is too low may not be enough to produce ions and it is difficult to bring a large number of ions from the needle port into the cone hole, while a voltage that is too high may lead to the generation of too many fragment ions. That is, selecting a suitable spray voltage can make the fluorescently labeled O-sugar chain have a high peak area. As can be seen from Table 5, when the spray voltage increases from 2.5 kV to 4 kV, the peak area of ​​the fluorescently labeled O-sugar chain increases significantly. This should be because the higher voltage allows more ions to enter the mass spectrometer. After 4 kV, the peak area of ​​the fluorescently labeled O-sugar chain continued to decrease with the increase of the spray voltage. This may be because the high voltage caused the decomposition of the analyte - too many ions entered the mass spectrometer, collided with the solvent or its own ions, and split.

[0212] In summary, the spray voltage is preferably between 3.0 and 4.0 KV.

[0213] Table 5 Optimization of spray voltage conditions

[0214]

[0215]

[0216] 5.4 Mass spectrometry data processing methods

[0217] The primary mass spectra of the test sample at 8 to 12 minutes were exported in Xcalibur for deconvolution. The structure of the fluorescently labeled ulinastatin O sugar chain is GalNAc-(GlcA-GalNAc±4S)n-GlcA-Gal4S-Gal-Xyl-RhBITC (rhodamine B isothiocyanate)-Ser, of which GalNAc-GlcA-Gal4S-Gal-Xyl-RhBITC (rhodamine B isothiocyanate) is a common structure with a molecular weight of 1451.301Da, which is set as an inherent modification on S. GlcA-GalNAc is an unfixed number with a molecular weight of 379.115Da, and GlcAGalNAc1 to GlcAGalNAc25 are set as variable modifications; 4S is an unfixed number with a molecular weight of 79.9568Da, and SO3_1 to SO3_7 are set as variable modifications.

[0218] The search parameters of Thermo BioPharma Finder 4.1 (version number: 4.1.53.14) are shown in Table 6 below.

[0219]

[0220] Thermo Q Exactive Plus high-resolution mass spectrometry was used to detect three batches of fluorescently labeled ulinastatin O-glycans. The total ion currents of samples 1-3 are shown in Figure 5 .

[0221] From Table 7 below and Figure 6 It can be seen that 38, 23 and 31 O-glycoforms were identified in samples 1-3, respectively, and a total of 46 O-glycoforms were identified. The five most distributed O-glycoforms in samples 1-3 are: dp33-5-Ser; dp31-5-Ser; dp33-6-Ser; dp35-5-Ser; dp-35-6-Ser.

[0222] Table 7 List of O-glycoform proportions of ulinastatin

[0223]

[0224]

[0225]

[0226] Note: The relationship between Name and Modification is: in dpx-y-Ser, x is (the number of GlcAGalNAc*2+5); y is (the number of SO3+1).

[0227] In summary, by using a suitable amount of pronase E and an appropriate time for enzyme cleavage, and then optimizing the conditions for purification of the O sugar chain, the method of the present invention has a high release efficiency of the O sugar chain, can greatly reduce the sample dosage, and reduce the detection limit. In addition, the HPLC purity of the O sugar chain sample that should be labeled obtained by the present invention can reach more than 98%, and the purity is relatively high. Finally, combined with UPLC-Q-TOF-MS / MS analysis, the specific glycoform and relative proportion of the O sugar chain of ulinastatin can be easily, quickly and accurately confirmed. The method of the present invention can be used for the daily detection of the O sugar chain of ulinastatin, providing an important basis for the structural research and quality control of ulinastatin.

[0228] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for analyzing the O-sugar chain of ulinastatin, comprising the following steps: (1) digesting the sample with pronase E to obtain a digested sample; (2) reacting the fluorescent molecule with the enzyme-digested sample to obtain a fluorescently labeled sample; (3) The fluorescently labeled sample is subjected to ultra-high pressure liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS) analysis, and the structure of the O-sugar chain is determined by the obtained mass spectrometry data.

2. The method according to claim 1, wherein the enzymatic cleavage in step (1) has one or more technical features selected from the following group: the mass ratio of pronase E to ulinastatin is 1:0.5-2, preferably 1:0.6-1.5, such as 1:0.8, 1:1 or 1:1.2; The enzyme cleavage temperature is 45±10°C, preferably 45±5°C, ±3°C, ±2°C or ±1°C; The enzyme digestion time is 6 to 24 hours, preferably 10 to 20 hours, such as 12 hours, 15 hours or 18 hours; The pH of the enzyme digestion is 7.5±0.5, preferably 7.5±0.3, ±0.2 or ±0.

1.

3. The method according to claim 1, characterized in that The method further comprises step (1-a), wherein the sample obtained in step (1) after enzymatic cleavage is subjected to chromatographic purification, using a polymethacrylic acid resin anion exchange column (such as Super Q-650M) for elution, and then ultrafiltration for desalting to obtain a purified sample.

4. The method according to claim 3, characterized in that The step (1-a) comprises a chromatographic purification step: Equilibration: equilibration with buffer A (e.g. 4-6 column volumes); Loading: Load the supernatant of the digested sample obtained in step (1) onto an anion exchange column; Wash: Wash with buffer A for 8-12 column volumes, preferably 9-11 volumes; Elution: 8-12 column volumes, preferably 9-11 volumes, of buffer B; Buffer A: 50±5mM NaCl; Buffer B: 1.5±0.1M NaCl.

5. The method according to claim 1, characterized in that The fluorescent molecule is rhodamine B isothiocyanate, fluorescein isothiocyanate (such as 5-fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, 7-fluorescein isothiocyanate).

6. The method according to claim 1, characterized in that The reaction in step (2) has one or more technical features selected from the group consisting of: The mass ratio of the fluorescent molecule to the O sugar chain is 1:1-3, preferably 1:1-2, such as 1:1.2, 1:1.5 or 1:1.8; The reaction temperature is 37±10°C, preferably 37±5°C, ±3°C, ±2°C or ±1°C; The reaction time is 6 to 24 hours, preferably 10 to 20 hours, such as 12 hours, 15 hours or 18 hours; The solvent for the reaction is an organic solvent, such as DMSO.

7. The method according to claim 1, characterized in that The method further comprises a step (2-b), wherein the fluorescently labeled sample obtained in step (2) is subjected to chromatographic purification, using a gel filtration column (such as Superdex 200Increase10 / 300GL), eluted, and then subjected to ultrafiltration and desalting to obtain a purified sample, and the purified sample is subjected to step (3).

8. The method according to claim 1, characterized in that Chromatographic conditions used in the UPLC-Q-TOF-MS / MS analysis described in step (3): Chromatographic column information: Waters Acquity TM UPLC Glycan BEH Amide, 1.7μm, 2.1mm×150mm; Wavelength: 280±5nm; Column temperature: 40±2°C; flow rate: 0.2 ml / min; gradient elution: elution time 0-20 min, mobile phase A (0.1% ammonia aqueous solution) 10%-80%, mobile phase B (0.1% ammonia acetonitrile solution 20%-90%).

9. The method according to claim 1, characterized in that: The mass spectrometry conditions used in the UPLC-Q-TOF-MS / MS analysis described in step (3) are: negative charge mode, spray voltage: 3.3±0.2KV; capillary temperature: 300±10°C; auxiliary gas heater temperature: 280±10°C; scan type: FullMass / DD-MS2 (TOPN); Resolution: MS: 70,000±100; MS / MS: 17500±100; Scanning range: 300~3000m / z.

10. A method for releasing / preparing ulinastatin O-sugar chains, comprising the steps of: enzymatically cleaving ulinastatin with pronase E to obtain ulinastatin O-sugar chains.

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