CN116448902A - Identification method of unfractionated heparin or low molecular heparin species source - Google Patents

Identification method of unfractionated heparin or low molecular heparin species source Download PDF

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CN116448902A
CN116448902A CN202310228792.7A CN202310228792A CN116448902A CN 116448902 A CN116448902 A CN 116448902A CN 202310228792 A CN202310228792 A CN 202310228792A CN 116448902 A CN116448902 A CN 116448902A
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heparin
sample
iisglu
species
percentage
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金永生
靳彩娟
陆晓华
周华
姚瑶
肖洋
钱建根
姚亦明
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Suzhou Ronnsi Pharma Co ltd
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Abstract

The invention discloses a species source identification method of unfractionated heparin or low molecular heparin, which is used for identifying species sources through the content and proportion of oligosaccharides delta I S and delta II A-II Sglu in heparin. The heparin oligosaccharide can be analyzed by using SAX-HPLC, IPRP-HPLC, IC, CE or 2D-NMR technology, the method is preferably a technology of heparinase enzymolysis-SAX-HPLC analysis, specifically, the heparinase is firstly utilized to thoroughly carry out enzymolysis on a sample to be detected, then SAX-HPLC is utilized to analyze the oligosaccharide of a degraded (or reduced) sample, the percentage content and the ratio of DeltaI) and DeltaIIA-II Sglu (or reduced) are obtained according to the characteristic signals of each oligosaccharide, and the species source of heparin is identified according to a standard comparison table. The analysis method is accurate, practical, simple and easy to operate, and is particularly suitable for identifying the seed sources of unfractionated heparin or low-molecular heparin with DNA broken by purification.

Description

Identification method of unfractionated heparin or low molecular heparin species source
Technical Field
0001. The invention relates to the technical field of biological medicine, in particular to a method for identifying the source of unfractionated heparin or low-molecular heparin species.
Background
0002. Heparin (Heparin) and low molecular Heparin (Low Molecular Weight Heparin, LMWH) are the most widely and most important anticoagulants used clinically, mainly extracted from intestinal mucosa or lungs of pigs, cattle and sheep. At present, the main source of the medical heparin is porcine intestinal mucosa heparin, but bovine and sheep heparin is still widely used in some countries and regions (south america, southeast asia and south asia). Unfractionated heparin (Unfractionated Heparin, UFH) refers to natural macromolecular heparin, and low-molecular heparin is small-molecular heparin prepared by depolymerizing the natural macromolecular heparin, and is a clinically main heparin preparation application form.
0003. It is known that heparin from different heparin species differ in sugar chain composition and physicochemical properties, and that in the patent application publications CN106243246B and CN105131153a, there are published unfractionated heparin from various sources and correspondingly prepared low molecular heparin (Enoxaparin), which differ very significantly in oligosaccharide structure composition. The unfractionated heparin and low molecular heparin (enoxaparin sodium) of sheep source (intestinal mucosa) are similar to the pig intestinal mucosa heparin or low molecular heparin (enoxaparin) regulated by mainstream pharmacopoeia in terms of physicochemical properties and biological activity, and can meet the requirements of other indexes except for species sources in pharmacopoeia, while the heparin of bovine source (bovine lung heparin and bovine intestinal mucosa heparin) is greatly different from the pig source heparin, especially the factor (anti-Xa and anti-IIa) activity can not meet the requirements of pharmacopoeia on potency. Thus, heparin and low molecular heparin of porcine, bovine and ovine origin should be "similar but not identical" active pharmaceutical ingredients.
0004. In the pharmaceutical industry, there are various methods for controlling heparin sources: firstly, from the perspective of a supply chain, heparin is produced by using only pig intestinal mucosa of legal edible sources; secondly, from the aspect of material control, the content of pig DNA and ruminant DNA in the crude heparin raw material is measured by utilizing a fluorescence quantitative PCR (Quantitative Real-time PCR, Q-PCR) technology, and the ruminant DNA is required to be less than 0.5 percent; thirdly, controlling a heparin production link to prevent confusion and pollution; fourth, in the final product stage, precise analysis techniques such as nuclear magnetic resonance are used to identify the fine differences of heparin species.
0005. In the above, we have found that, with respect to the control of the identification of heparin species, the most advantageous analysis technique is to analyze the species DNA source of the material using Q-PCR technique in the crude heparin raw material stage, and then to identify the fine differences in disaccharide species using sophisticated precision analysis equipment in the final product stage. However, the precondition of Q-PCR is that the DNA in the material cannot be destroyed, and some crude heparin materials are produced, and manufacturers may inadvertently or intentionally use chemicals such as hydrogen peroxide to treat the materials, which can destroy the DNA of the species and thus cannot be analyzed by Q-PCR techniques. The medical unfractionated heparin and low molecular heparin as products have been oxidized, so the Q-PCR method is no longer suitable for heparin species source identification of these heparin end products. In addition, the nuclear magnetic resonance technology is not only noble in instruments and equipment, but also heparin or low molecular heparin is mainly piled up as a macromolecular polysaccharide composition, hydrogen/carbon signals of the macromolecular polysaccharide composition are difficult to distinguish, on one hand, the purity of an analysis sample is required to be dependent, on the other hand, the difference of heparin of each species is small on the characteristic nitrogen-acetyl methyl hydrogen signal and the hydrogen signal of a sulfation area, so that the nuances are limited, and the provided information is low in precision.
0006. In summary, the prior art analysis techniques are not suitable for heparin seed source analysis of purified unfractionated heparin or low molecular heparin products, and therefore, there is a great need for accurate and efficient analysis techniques for use as heparin seed source identification methods.
Disclosure of Invention
0007. The technical problems to be solved by the invention are as follows: the identification method can accurately and efficiently identify the source of unfractionated heparin or low-molecular heparin species.
0008. In order to solve the problems, the invention adopts the following technical scheme: the identification method of the unfractionated heparin or low molecular heparin species sources is to identify and judge the sample species sources according to the percentage of delta I S, the percentage of delta II A-II Sglu, the ratio of delta I S to delta II A-II Sglu which are reserved with heparin related characteristics in the table 1 and the samples, and the identification method comprises the following steps:
TABLE 1 species Source judgment criteria for unfractionated heparin or Low molecular heparin
(1) The Δis percentage of retained heparin affinity in the sample is compared to each unfractionated heparin or low molecular heparin species source category under the Δis percentage entry in table 1:
the DeltaIS percentage of the heparin related characteristics preserved in the sample is within the range of 44.0-86.0%, and the unfractionated heparin or low molecular heparin species sources corresponding to the sample are found out;
the Δis percentage of the heparin related characteristics in the sample is outside the range of 44.0% -86.0%, and the sample is identified and judged not to belong to unfractionated heparin or low molecular heparin species;
(2) Under the category of the unfractionated heparin or low-molecular heparin species corresponding to the found sample, comparing the ΔIIA-IISglu percentage and the ΔI S/ΔIIA-IISglu ratio of the residual heparin related characteristics in the sample with the ΔIIA-IISglu percentage and the ΔI S/ΔIIA-IISglu ratio of the unfractionated heparin or low-molecular heparin species corresponding to the sample in the table 1 respectively:
the DeltaIIA-IISglu percentage of the heparin related characteristics in the sample is within the DeltaIIA-IISglu percentage range of the unfractionated heparin or the low molecular heparin species corresponding to the sample in the table 1, or the DeltaIS/DeltaIIA-IISglu ratio of the heparin related characteristics in the sample is within the DeltaIS/DeltaIIA-IISglu ratio range of the unfractionated heparin or the low molecular heparin species corresponding to the sample in the table 1, and the unfractionated heparin or the low molecular heparin species corresponding to the sample found in the step (1) is identified and judged to have accurate sources;
the ΔIIA-IISglu percentage of the sample with the heparin affinity is outside the range of the ΔIIA-IISglu percentage of the class of unfractionated heparin or low molecular heparin species corresponding to the sample in Table 1, and the ΔIS/ΔIIA-IISglu ratio of the sample with the heparin affinity is outside the range of the ΔIS/ΔIIA-IISglu ratio of the class of unfractionated heparin or low molecular heparin species corresponding to the sample in Table 1, and the sample is identified as heparin from a non-pure species.
0009.ΔiS (one of the standard disaccharides in heparin analysis, ΔUA2S- (1→4) α -GlcN (NS, 6S)) and ΔIIA-IISglu (ΔUA- (1→4) α -GlcNAc (6S) - (1→4) β -GlcA- (1→4) α -GlcN (NS, 6S)) are among the most important oligosaccharides in heparin sugar chains, and the non-reducing 4,5 unsaturated uronic acid structure of the non-reducing end is formed by enzymatic hydrolysis or degradation of heparin sugar chains during sample analysis.
0010. The structural formula of Δis is:
ΔUA2S-(1→4)α-GlcN(NS,6S)。
0011. the structural formula of the delta IIA-II Sglu is as follows:
ΔUA-(1→4)α-GlcNAc(6S)-(1→4)β-GlcA-(1→4)α-GlcN(NS,6S)。
0012. it is known that heparin or low molecular heparin has a structure in which disaccharide repeating units are mainly composed of glucosamine (D-glucosamine, glcN) and one of two kinds of uronic acid (iduronic, idoA) and glucuronic acid (D-glucuronic, glcA), and sulfate groups are randomly modified at different positions of the sugar units. All heparin structures can be represented by one tetrasaccharide unit, a and B as shown below, while C shows the pentasaccharide structure that binds to antithrombin, a core structure necessary to maintain anticoagulant activity.
0013. All heparin structures were:
0014. as described above, these structures are frequently different depending on the number and types of sugar units, the number and positions of modification of sulfate groups, and the like. The major part of heparin sugar chains is a 'regular region' with ordered structure, namely a trisulfated disaccharide repeating unit with regular structure, namely I2S-GlcNS6S (2-oxo-sulfate-iduronic acid-nitrogen-sulfate-6-oxo sulfate-glucosamine, and delta I2S structure is formed when the oligosaccharide is analyzed by enzymolysis), and is shown in A; and B shows the low sulfation region, which is a structurally disordered "irregularity", mainly some units containing IdoA and GlcA followed by N-acetyl-GlcN or N-sulfonyl-GlcN (GlcNAc, glcNS) [ R.J.Linhardt, J.Med.Chem.46, (2003) 2551-2564 ], part B being smaller in heparin oligosaccharide chains. In addition, C shows the pentasaccharide structure bound to antithrombin, which is the core site for heparin anticoagulation, and is shown in bold as necessary to maintain anticoagulation activity, if removed, the anticoagulation activity is reduced by 95%, and in italics, the anticoagulation activity is reduced by 25-50% after removal; the structure of C is GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S, which is a partial structure of ΔIIA-IISglu (the last three sugar units) and a trisulfated disaccharide repeating unit-I2S-GlcNS 6S (the structure of ΔI2S is formed during enzymolysis), and the ΔIIA-IISglu sequence is the main component in C, and the content of the sequence can reflect the size of anticoagulation activity.
0015. Different low molecular heparin forms special chain end structures at two ends of the produced low molecular heparin sugar chain due to different depolymerization modes, such as enoxaparin has two typical characteristics of a 1, 6-anhydride ring structure at the reducing end of the sugar chain and a 4, 5-unsaturated uronic acid structure at the non-reducing end, and other low molecular heparin such as Nadroparin (Nadroparin), dalteparin (Dalteparin), tinzaparin (Tinzaparin), pamaparin (bermiparin) and the like also forms special structural characteristics at two ends of each sugar chain. However, in contrast to the two ends of the sugar chain, ΔIS and ΔIIA-IISglu are mainly located inside the sugar chain, and in general, both ΔIS and ΔIIA-IISglu can represent the constituent features of heparin samples derived from heparin relatives, so that the identification of the origin of heparin species can be performed by analyzing both.
0016. Above, Δis a main component of heparin sugar chain, is located in "regular region" with ordered structure, ΔIIA-IISglu is a main component of heparin anticoagulation active site, is located in core pentasaccharide structure, and belongs to "irregular region" with disordered structure. They are mainly located in the interior of heparin sugar chains, and in unfractionated heparin or low molecular heparin which are produced as end products, the sugar chain internal structures are brought by kindred heparin, namely heparin products of different species can all keep the sugar chain composition characteristics of raw heparin (kindred heparin). In addition, heparin oligosaccharides are various, not only ΔiS and ΔIIA-IISglu examined by the method, but also other numerous oligosaccharide units reflect the characteristics of heparin relatives, but besides ΔiS which is an ordered "regular area" of heparin structure, other oligosaccharide units are mainly located in an "irregular area" of disordered structure, and only ΔIIA-IISglu can reflect the content of the core pentasaccharide structure of heparin most. The inventors have performed a summary comparison analysis of oligosaccharide unit data, with both oligosaccharides being most accurate and efficient for seed source identification.
0017. In addition, ΔIS and ΔIIA-IISglu may be subjected to sodium borohydride reduction prior to testing, which is described in U.S. pharmacopoeia appendix <207>1, 6-anhydride derivative examination, and shows that the percentage of all disaccharides and/or tetrasaccharides is consistent before and after the reduction process and does not change as a result of the reduction reaction.
0018. Currently, the ΔIS percentage, ΔIIA-IISglu percentage, and the ratio of both ΔIS and ΔIIA-IISglu may be determined using a variety of analytical techniques. One method is to degrade the heparin sample, and then analyze the heparin sample by using the technologies such as enzymolysis-strong anion exchange-high performance liquid chromatography (SAX-HPLC), enzymolysis-Ion Chromatography (IC), enzymolysis-reversed phase ion pair-high performance liquid chromatography (IPRP-HPLC), enzymolysis-Capillary Electrophoresis (CE) and the like; the other method is to directly use two-dimensional nuclear magnetic resonance (2D-NMR) technique without degrading the sample, and calculate the sample by using the difference between the sugar unit connection and the modification mode.
0019. The method adopts enzymolysis-strong anion exchange-high performance liquid chromatography. When the enzymolysis-strong anion exchange-high performance liquid chromatography analysis mode is adopted, the sample is subjected to enzymolysis by heparinase, the degradation liquid is reduced by sodium borohydride, and then the enzymolysis-strong anion exchange-high performance liquid chromatography analysis is carried out, and according to the characteristic peak signals of each heparin oligosaccharide, the DeltaIS and DeltaIIA-IISglu of the sample are obtained, wherein the DeltaIS and DeltaIIA-IISglu of the sample are the reduced DeltaIS and DeltaIIA-IISglu.
0020. Further, the method for identifying the source of unfractionated heparin or low molecular heparin species, wherein the degradation solution is reduced by sodium borohydride, comprises the following steps: adding sodium borohydride solution into the degradation product solution, and reducing at room temperature for not less than 1 hour; the concentration of the sodium borohydride solution is 80-100mg/mL, and the sodium borohydride solution is prepared for use at present.
0021. Further, in the aforementioned method for identifying the source of unfractionated heparin or low molecular heparin species, when an enzymolysis-strong anion exchange-high performance liquid chromatography analysis mode is adopted, a mode that degradation liquid is not reduced by sodium borohydride may also be adopted: and carrying out enzymolysis on the sample by heparinase, reducing the degradation liquid without sodium borohydride, and then carrying out enzymolysis-strong anion exchange-high performance liquid chromatography analysis, thereby obtaining DeltaIS and DeltaIIA-II Sglu of the sample according to the characteristic peak signals of each heparin oligosaccharide.
0022. Further, the method for identifying the source of unfractionated heparin or low molecular heparin species, wherein the step of enzymatic hydrolysis of the sample with heparinase comprises: adding heparanase into unfractionated heparin or low molecular heparin sample solution with concentration of 5-50mg/mL, and performing enzymolysis at room temperature for at least 24 hours.
0023. Further, a method of identifying a source of unfractionated heparin or low molecular heparin species as described above, wherein the heparanase comprises one or more combinations of heparanase I, heparanase II and heparanase III.
0024. In the case of enzymolysis-strong anion exchange-high performance liquid chromatography, the chromatographic conditions are shown in Table 2, and the mobile phase gradient is shown in Table 3.
0025. TABLE 2 chromatographic conditions table
0026. TABLE 3 Mobile phase gradient Meter
0027. Further, the aforementioned method for identifying the origin of unfractionated heparin or low molecular heparin species, wherein the manner of obtaining Δis and Δiia-ii Sglu of the sample according to the characteristic peak signals of the respective heparin oligosaccharides is as follows: based on the control profile, ΔIS and ΔIIA-IISglu were identified from heparin oligosaccharides or derivatives.
0028. The beneficial effects of the invention are as follows: the oligosaccharide unit based on unfractionated heparin or low molecular heparin provided by the invention retains the characteristics of related heparin, analyzes the percentage and the ratio of DeltaI S and DeltaII A-II Sglu or reduced DeltaI S and reduced DeltaII A-II Sglu in a sample to be tested, classifies and identifies the species source, can be used for accurately identifying the source of heparin species, especially identifying the species source of unfractionated heparin or low molecular heparin with DNA broken by purification, and fills the blank of the analysis method in the field. The method is accurate, practical, simple and easy to operate, and is suitable for heparin production enterprises, laboratories and scientific research institutions.
Drawings
0029. FIG. 1 is a diagram showing comparison of SAX-HPLC patterns of oligosaccharide control and heparin test sample analysis.
0030. FIG. 2 is a schematic diagram showing comparison of oligosaccharide SAX-HPLC profiles of unfractionated heparin samples from different species.
0031. FIG. 3 is a schematic representation of oligosaccharide normalized cluster analysis of unfractionated heparin from different species sources.
Detailed Description
0032. The technical scheme of the invention is further described in detail below with reference to the attached drawings and the preferred embodiments.
0033. For convenience of description of the present invention, since the percentage of oligosaccharide units (including ΔIS and ΔIIA-IISglu) in heparin samples can be analyzed by various methods, particularly SAX-HPLC, IC, IPRP-HPLC or CE techniques after enzymatic hydrolysis, which are all known literature methods, the present invention is exemplified in the following examples by SAX-HPLC analysis techniques only. It should be noted that the analysis method of the present invention for analyzing the percentage of Δis to Δiia-ii Sglu and the ratio of Δis to Δiia-ii Sglu in heparin samples is not limited to the following specific examples, and any implementation based on the present invention is intended to be within the scope of the present invention.
0034. [ term/definition/abbreviation ]
Δis: is DeltaUA 2S- (1.fwdarw.4) alpha-GlcN (NS, 6S)), one of the standard disaccharides in heparin analysis;
ΔIIA-IISglu: is DeltaUA- (1.fwdarw.4) alpha-GlcNAc (6S) - (1.fwdarw.4) beta-GlcA- (1.fwdarw.4) alpha-GlcN (NS, 6S)), a heparin tetrasaccharide;
heparin: heparin;
LWMH: low molecular heparin, low Molecular Weight Heparin;
UFH: unfractionated heparin, unfractionated Heparin;
GlcA: glucuronic Acid, D-glucuronic Acid;
GlcN: glucosamine, D-glucosamine;
IdoA, L-iduronic Acid: iduronic acid;
ManN: mannuronic Acid, mannuronic Acid;
Q-PCR: quantitative polymerase chain reaction;
DNA: deoxyribonucleic acid;
CE: capillary electrophoresis;
SAX-HPLC: strong anion exchange-high performance liquid chromatography;
IPRP-HPLC: reversed phase ion pair-high performance liquid chromatography;
2D-NMR: two-dimensional nuclear magnetic resonance techniques.
0035. This example uses an enzymolysis-SAX-HPLC analysis method.
0036. The enzymolysis-SAX-HPLC analysis method specifically comprises the following steps:
0037. the heparin sample to be detected is hydrolyzed by heparinase, then the degradation liquid is filtered by 0.22 micron, oligosaccharide analysis is carried out by SAX-HPLC, and the characteristic DeltaIS percentage, deltaIIA-IISglu percentage and the ratio of the two are determined.
0038. (1) And (3) preparation of a reagent:
solution a: weighing 136mg of potassium dihydrogen phosphate, using about 40mL of ultrapure water solution, regulating the pH to 7.0 by using 2mol/L of sodium hydroxide solution, adding 20mg of bovine albumin, shaking uniformly, using ultrapure water to fix the volume to 100mL, and filtering to obtain 10mM of potassium dihydrogen phosphate solution;
solution B: 0.57mL of glacial acetic acid was removed in a beaker, about 80mL of ultrapure water was added, 32mg of calcium acetate was added, and the mixture was stirred to dissolve the calcium acetate. Adjusting pH to 7.0 with 2mol/L sodium hydroxide solution, adding 10mg of bovine albumin, shaking, adding ultrapure water to 100mL, and filtering to obtain 100mM sodium acetate solution;
heparinase solution: according to the packing specifications of heparinase I, heparinase II and heparinase III, respectively dissolving and diluting by using the solution A;
mobile phase a: 280mg of sodium dihydrogen phosphate is weighed and dissolved in about 950mL of ultrapure water, the pH is adjusted to 3.0 by phosphoric acid, the volume of water is fixed to 1000mL, and the solution is filtered by a 0.22 mu m filter membrane;
mobile phase B: 140g of sodium perchlorate monohydrate is weighed and dissolved in 950mL of mobile phase A, the pH is regulated to 3.0 by phosphoric acid, the mobile phase A is used for constant volume to 1000mL, and the solution is filtered by a 0.22 mu m filter membrane;
oligosaccharide Δis control solution: dissolving with ultrapure water to prepare 0.1mg/mL of DeltaIS reference substance solution, subpackaging, and freezing below-20deg.C;
oligosaccharide ΔIIA-IISglu control solution: dissolving with ultrapure water to prepare 0.1mg/mL of DeltaIIA-II Sglu reference substance solution, subpackaging, and freezing below-20 ℃;
test solution: weigh-20 mg unfractionated heparin or low molecular heparin for sample, add 1mL solution B, shake and dissolve.
0039. (2) Heparanase degradation:
20 mu L of the sample solution, 90 mu L of the solution B and 90 mu L of the heparinase mixed solution (each of 0.4IU/ml of I, II and III is mixed according to the ratio of 1:1:1) are added into a centrifugal tube, and the mixture is uniformly mixed by vortex and subjected to enzymolysis for 48 hours at room temperature.
(3) Chromatographic conditions for SAX-HPLC: chromatographic conditions and mobile phase gradient tables are shown in tables 4 and 5.
0040. Table 4 hplc chromatography conditions table
0041. TABLE 5 mobile phase gradient table
Time [ (time ] min Mobile phase A% Mobile phase B%
0 97 3
20 65 35
40 20 80
60 0 100
70 97 3
0043. (4) Analysis:
and respectively carrying out sample injection analysis on the oligosaccharide DeltaIS reference substance solution, the oligosaccharide DeltaIIA-IISglu reference substance solution and the test sample solution to obtain DeltaIS percentage, deltaIIA-IISglu percentage and ratio data of the DeltaIS percentage and the DeltaIIA-IISglu percentage in the test sample. SAX-HPLC chromatograms of the control and test analyses are shown in FIG. 1.
Example 1
0043. Heparinase enzymolysis-SAX-HPLC analysis of unfractionated heparin from different species was performed with reference to the above method.
0044. Test sample: randomly labeling four unfractionated heparins from different sources, encoded as samples 1 through 4, each from bovine pulmonary heparin, bovine intestinal mucosal heparin, sheep intestinal mucosal heparin, porcine intestinal mucosal heparin; samples 1 through 4 were submitted to blind testing by the analyst.
0045. In heparinase enzymolysis-SAX-HPLC analysis, a sample is subjected to enzymolysis by using heparinase, a degradation solution is not reduced by using sodium borohydride, enzymolysis-strong anion exchange-high performance liquid chromatography analysis is performed, and DeltaIS and DeltaIIA-IISglu of the sample are obtained according to characteristic peak signals of each heparin oligosaccharide. The step of enzymolysis of the sample by heparinase is as follows: adding heparanase into unfractionated heparin or low molecular heparin sample solution with concentration of 5-50mg/mL, and performing enzymolysis at room temperature for at least 24 hours.
0046. The heparanase comprises one or more of heparanase I, heparanase II and heparanase III.
0047. Analysis results:
the disaccharide/oligosaccharide profile comparison of unfractionated heparin from different species is shown in FIG. 2. The results show that there is a large difference in oligosaccharide unit composition from species to species, which is manifested by a large percentage difference of several oligosaccharide peaks. Furthermore, the data of the ΔIS percentage, ΔIIA-IISglu percentage, and the ratio of both ΔIS and ΔIIA-IISglu obtained in samples 1 to 4 are shown in Table 6.
0048. TABLE 6 comparison of characteristic saccharide units of unfractionated heparin from different species
0049. The method for identifying the source of unfractionated heparin or low molecular heparin species is adopted to identify samples 1-4 in table 6 to determine the source of the sample species, and the method comprises the following steps:
(1) The Δis percentage of retained heparin affinity in the sample is compared to each unfractionated heparin or low molecular heparin species source category under the Δis percentage entry in table 1:
the DeltaIS percentage of the heparin related characteristics preserved in the sample is within the range of 44.0-86.0%, and the unfractionated heparin or low molecular heparin species sources corresponding to the sample are found out;
the Δis percentage of retained heparin affinity in the sample is outside the range of 44.0% -86.0%, and the sample is identified as not belonging to unfractionated heparin or low molecular heparin species.
0050. The Δis percentage of sample 1 is 78.69% and the corresponding range in table 1 is 76.0% -86.0% for bovine lung.
0051. The Δis percentage of sample 2 is 52.43% and the corresponding range in table 1 is 44.0% -54.0% of bovine intestinal mucosa.
0052. The Δis percentage of sample 3 was 66.26% and the corresponding range in table 1 was 64.0% -76.0% of the sheep intestinal mucosa.
0053. The Δis percentage of sample 4 is 63.31% and the corresponding range in table 1 is 54.0% -64.0% of porcine intestinal mucosa.
0054. (2) Under the category of the unfractionated heparin or low-molecular heparin species corresponding to the found sample, comparing the ΔIIA-IISglu percentage and the ΔI S/ΔIIA-IISglu ratio of the residual heparin related characteristics in the sample with the ΔIIA-IISglu percentage and the ΔI S/ΔIIA-IISglu ratio of the unfractionated heparin or low-molecular heparin species corresponding to the sample in the table 1 respectively:
the DeltaIIA-IISglu percentage of the heparin related characteristics in the sample is within the DeltaIIA-IISglu percentage range of the unfractionated heparin or the low molecular heparin species corresponding to the sample in the table 1, or the DeltaIS/DeltaIIA-IISglu ratio of the heparin related characteristics in the sample is within the DeltaIS/DeltaIIA-IISglu ratio range of the unfractionated heparin or the low molecular heparin species corresponding to the sample in the table 1, and the unfractionated heparin or the low molecular heparin species corresponding to the sample found in the step (1) is identified and judged to have accurate sources;
the ΔIIA-IISglu percentage of the sample with the heparin affinity is outside the range of the ΔIIA-IISglu percentage of the class of unfractionated heparin or low molecular heparin species corresponding to the sample in Table 1, and the ΔIS/ΔIIA-IISglu ratio of the sample with the heparin affinity is outside the range of the ΔIS/ΔIIA-IISglu ratio of the class of unfractionated heparin or low molecular heparin species corresponding to the sample in Table 1, and the sample is identified as heparin from a non-pure species.
0055. The ΔIIA-IISglu percentage of sample 1 was 0.58%, and it was determined that sample 1 belongs to bovine lung by comparing the ΔIIA-IISglu percentage range of 0.3% -0.8% with the ΔIIA-IISglu percentage range of 0.3% -0.8% in Table 1.
0056. The ΔIIA-IISglu percentage of sample 2 was 0.67%, and it was judged that sample 1 belongs to the bovine intestinal mucosa by comparing the ΔIIA-IISglu percentage range of 0.3% -1.0% with the ΔIIA-IISglu percentage range of 0.3% -1.0% in Table 1.
0057. The ΔIIA-IISglu percentage of sample 3 was 1.84%, and it was judged that sample 1 belongs to the sheep intestinal mucosa by comparing the ΔIIA-IISglu percentage range of 1.6% -2.2% with the ΔIIA-IISglu percentage range of 1.6% -2.2% in Table 1.
0058. The ΔIIA-IISglu percentage of sample 4 was 2.38%, and it was judged that sample 1 belongs to the porcine intestinal mucosa by comparing the ΔIIA-IISglu percentage range of 2.2% -2.6% with the ΔIIA-IISglu percentage range of 2.2% -2.6% of the porcine intestinal mucosa in Table 1.
0059. This result is consistent with the source of material prior to blind marking.
Example two
0060. The heparinase enzymolysis-SAX-HPLC analysis of low molecular heparin from different species is different from the first embodiment in that sodium borohydride is used for reduction after enzymolysis of a test sample, and similarly, reduced oligosaccharides are also used as a reference substance for judging the positions of reduced ΔIS and reduced ΔIIA-IISglu, and in addition, the relative retention time of the peaks of the two can be judged according to the characteristics of a heparin map (the ΔIS is the maximum main peak signal).
0061. Test sample: randomly labeling four low molecular heparin from different sources, namely sample 5 to sample 8, wherein the low molecular heparin is respectively from sheep intestinal mucosa enoxaparin, niu Fei enoxaparin, bovine intestinal mucosa enoxaparin and pig intestinal mucosa enoxaparin; samples 5 through 9 were submitted to blind testing by the analyst.
0062. In heparinase enzymolysis-SAX-HPLC analysis, a sample is subjected to enzymolysis by using heparinase, a degradation solution is reduced by using sodium borohydride, and then enzymolysis-strong anion exchange-high performance liquid chromatography analysis is performed, so that DeltaIS and DeltaIIA-II Sglu of the sample are obtained according to characteristic peak signals of each heparin oligosaccharide.
0063. The degradation liquid is reduced by sodium borohydride as follows: adding sodium borohydride solution into the degradation product solution, and reducing at room temperature for not less than 1 hour; the concentration of the sodium borohydride solution is 80-100mg/mL.
0064. The step of enzymolysis of the sample by heparinase is as follows: adding heparanase into unfractionated heparin or low molecular heparin sample solution with concentration of 5-50mg/mL, and performing enzymolysis at room temperature for at least 24 hours.
0065. The heparanase comprises one or more of heparanase I, heparanase II and heparanase III.
0066. Analysis results:
SAX-HPLC analysis data measured after reduction are shown in Table 7.
0067. TABLE 7 comparison of characteristic sugar units of low molecular weight heparin (enoxaparin) of different origins
0068. According to Table 1. The species source judgment criteria for unfractionated heparin or low molecular heparin, samples 5-8 in Table 7 were identified to determine the sample species source, the steps of the identification method were:
(1) The Δis percentage of retained heparin affinity in the sample is compared to each unfractionated heparin or low molecular heparin species source category under the Δis percentage entry in table 1:
the DeltaIS percentage of the heparin related characteristics preserved in the sample is within the range of 44.0-86.0%, and the unfractionated heparin or low molecular heparin species sources corresponding to the sample are found out;
the Δis percentage of retained heparin affinity in the sample is outside the range of 44.0% -86.0%, and the sample is identified as not belonging to unfractionated heparin or low molecular heparin species.
0069. The Δis percentage of sample 5 was 71.26% and the corresponding range in table 1 was 64.0% -76.0% of the sheep intestinal mucosa.
0070. The Δis percentage of sample 6 is 81.42% and the corresponding range in table 1 is 76.0% -86.0% for bovine lung.
0071. The Δis percentage of sample 7 was 47.45% and the corresponding range in table 1 was 44.0% -54.0% of bovine intestinal mucosa.
0072. The Δis percentage of sample 8 is 58.45% and the corresponding range in table 1 is 54.0% -64.0% of porcine intestinal mucosa.
0073. (2) Under the category of the unfractionated heparin or low-molecular heparin species corresponding to the found sample, comparing the ΔIIA-IISglu percentage and the ΔI S/ΔIIA-IISglu ratio of the residual heparin related characteristics in the sample with the ΔIIA-IISglu percentage and the ΔI S/ΔIIA-IISglu ratio of the unfractionated heparin or low-molecular heparin species corresponding to the sample in the table 1 respectively:
the DeltaIIA-IISglu percentage of the heparin related characteristics in the sample is within the DeltaIIA-IISglu percentage range of the unfractionated heparin or the low molecular heparin species corresponding to the sample in the table 1, or the DeltaIS/DeltaIIA-IISglu ratio of the heparin related characteristics in the sample is within the DeltaIS/DeltaIIA-IISglu ratio range of the unfractionated heparin or the low molecular heparin species corresponding to the sample in the table 1, and the unfractionated heparin or the low molecular heparin species corresponding to the sample found in the step (1) is identified and judged to have accurate sources;
the ΔIIA-IISglu percentage of the sample with the heparin affinity is outside the range of the ΔIIA-IISglu percentage of the class of unfractionated heparin or low molecular heparin species corresponding to the sample in Table 1, and the ΔIS/ΔIIA-IISglu ratio of the sample with the heparin affinity is outside the range of the ΔIS/ΔIIA-IISglu ratio of the class of unfractionated heparin or low molecular heparin species corresponding to the sample in Table 1, and the sample is identified as heparin from a non-pure species.
0074. The ΔIIA-IISglu percentage of sample 5 was 1.78%, and it was judged that sample 5 belongs to the sheep intestinal mucosa by comparing the ΔIIA-IISglu percentage range of 1.6% -2.2% with the ΔIIA-IISglu percentage range of 1.6% -2.2% in Table 1.
0075. Sample 6 was judged to belong to bovine lung by comparing the ΔIIA-IISglu percentage of sample 6 of 0.61% with the ΔIIA-IISglu percentage range of 0.3% -0.8% for bovine lung in Table 1, with 0.61% within 0.3% -0.8%.
0076. The ΔIIA-IISglu percentage of sample 7 was 0.71%, and it was judged that sample 7 belongs to the bovine intestinal mucosa by comparing the ΔIIA-IISglu percentage range of 0.3% -1.0% with the ΔIIA-IISglu percentage range of 0.3% -1.0% in Table 1.
0077. Sample 8 was judged to belong to porcine intestinal mucosa by comparing the ΔIIA-IISglu percentage of sample 8 with the ΔIIA-IISglu percentage range of 2.2% -2.6% in Table 1, and the ΔIIA-IISglu percentage range of 2.28% -2.6%.
0078. This result is consistent with the source of material prior to blind marking.
0079. Characteristic oligosaccharide data for batches of unfractionated heparin or low molecular heparin were summarized in the examples and subjected to standardized clustering analysis, see fig. 3.
0080. The results show that there is little variation between intraspecies batches of unfractionated heparin or low molecular heparin from each species, and that they are very significantly different from unfractionated heparin or low molecular heparin from other species. The data of the content and proportion of the characteristic oligosaccharides are indirectly reflected by the fact that the data are derived from the characteristic oligosaccharides delta IS and delta II A-II Sglu, and the characteristic oligosaccharides are suitable for identifying unfractionated heparin or low-molecular heparin sources.
0081. In summary, the oligosaccharide unit based on unfractionated heparin or low molecular heparin provided by the invention retains the characteristics of related heparin, analyzes the percentage and the ratio of ΔI S and ΔIIA-IISglu or reduced ΔI S and reduced ΔIIA-IISglu in a sample to be tested, classifies and identifies the species source, can be used for accurately identifying the source of heparin species, particularly identifying the species source of unfractionated heparin or low molecular heparin with DNA broken by purification, and fills the blank of the analysis method in the field. The method is accurate, practical, simple and easy to operate, and is suitable for heparin production enterprises, laboratories and scientific research institutions.
0082. The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.

Claims (9)

1. A method for identifying a source of unfractionated heparin or low molecular heparin species, characterized by: the method for identifying and judging the source of the sample species comprises the following steps of:
(1) Comparing the Δi S percentage of the samples that retain heparin affinity characteristics with the respective unfractionated heparin or low molecular heparin species source categories under the Δi percentage entry in the unfractionated heparin or low molecular heparin species source judgment standard table:
the DeltaIS percentage of the heparin related characteristics preserved in the sample is within the range of 44.0-86.0%, and the unfractionated heparin or low molecular heparin species sources corresponding to the sample are found out;
the Δis percentage of the heparin related characteristics in the sample is outside the range of 44.0% -86.0%, and the sample is identified and judged not to belong to unfractionated heparin or low molecular heparin species;
(2) Under the condition that the found sample corresponds to the class of unfractionated heparin or low-molecular heparin species, the ΔIIA-IISglu percentage and the ΔI S/ΔIIA-IISglu ratio of the heparin related characteristics in the sample are respectively compared with the ΔIIA-IISglu percentage and the ΔI S/ΔIIA-IISglu ratio under the condition that the sample corresponds to the class of unfractionated heparin or low-molecular heparin species:
the DeltaIIA-IISglu percentage of the heparin related characteristics in the sample is within the DeltaIIA-IISglu percentage range of the unfractionated heparin or low molecular heparin species corresponding to the sample, or the DeltaIS/DeltaIIA-IISglu ratio of the heparin related characteristics in the sample is within the DeltaIS/DeltaIIA-IISglu ratio range of the unfractionated heparin or low molecular heparin species corresponding to the sample, and the unfractionated heparin or low molecular heparin species corresponding to the sample found in the step (1) is accurate in source identification and judgment;
the DeltaIIA-IISglu percentage of the heparin related features in the sample is outside the DeltaIIA-IISglu percentage range of the unfractionated heparin or low-molecular heparin species corresponding to the sample, and the DeltaIS/DeltaIIA-IISglu ratio of the heparin related features in the sample is outside the DeltaIS/DeltaIIA-IISglu ratio range of the unfractionated heparin or low-molecular heparin species corresponding to the sample, and the sample is identified and judged to be heparin from a non-pure product species.
2. The method of claim 1, wherein the method comprises the steps of: the three data of the percentage of DeltaIS, the percentage of DeltaIIA-IISglu, and the ratio of DeltaIS to DeltaIIA-IISglu, which are retained in the sample, are obtained by adopting any one of enzymolysis-strong anion exchange-high performance liquid chromatography, enzymolysis-reversed phase ion pair-high performance liquid chromatography, enzymolysis-capillary electrophoresis-high performance liquid chromatography, enzymolysis-ion chromatography and two-dimensional nuclear magnetic resonance spectroscopy.
3. A method of identifying a source of unfractionated heparin or low molecular heparin species according to claim 2, wherein: and carrying out enzymolysis-strong anion exchange-high performance liquid chromatography analysis on the sample by using heparinase, reducing the degradation liquid by using sodium borohydride, and obtaining ΔIS and ΔIIA-II Sglu of the sample according to characteristic peak signals of each heparin oligosaccharide.
4. A method of identifying a source of unfractionated heparin or low molecular heparin species according to claim 3, wherein: the degradation liquid is reduced by sodium borohydride as follows: adding sodium borohydride solution into the degradation product solution, and reducing at room temperature for not less than 1 hour; the concentration of the sodium borohydride solution is 80-100mg/mL.
5. A method of identifying a source of unfractionated heparin or low molecular heparin species according to claim 2, wherein: and carrying out enzymolysis-strong anion exchange-high performance liquid chromatography analysis on the sample by using heparinase without reducing the degradation liquid by using sodium borohydride, and obtaining ΔIS and ΔIIA-II Sglu of the sample according to the characteristic peak signals of each heparin oligosaccharide.
6. The method of claim 3, 4 or 5, wherein the method comprises the steps of: the step of enzymolysis of the sample by heparinase is as follows: adding heparanase into unfractionated heparin or low molecular heparin sample solution with concentration of 5-50mg/mL, and performing enzymolysis at room temperature for at least 24 hours.
7. The method of claim 6, wherein the method comprises the steps of: the heparanase comprises one or more of heparanase I, heparanase II and heparanase III.
8. The method of claim 3, 4 or 5, wherein the method comprises the steps of: when the enzymolysis-strong anion exchange-high performance liquid chromatography analysis mode is adopted, the detector is an ultraviolet detector; the wavelength is 232nm; the chromatographic column is an anion exchange chromatographic column, L14A, and the particle size of the filler is 5 μm and 4×250mm; the pre-column is a strong anion exchange chromatographic column, L14A, and the particle size of the filler is 5 mu m and 4.6X10 mm; the column temperature is 15-35 ℃; the mobile phase is a mobile phase A and a mobile phase B for gradient elution; the flow rate is 0.4-1.0mL/min; the sample injection amount is 5-25 mu L; wherein the mobile phase gradient is shown in the following table;
9. the method of claim 3, 4 or 5, wherein the method comprises the steps of: the samples ΔIS and ΔIIA-IISglu were obtained according to the characteristic peak signals of the respective heparin oligosaccharides in the following manner: based on the control profile, ΔIS and ΔIIA-IISglu were identified from heparin oligosaccharides or derivatives.
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