CN115728406A - Liquid chromatography separation method of thio-oligonucleotide and application thereof - Google Patents
Liquid chromatography separation method of thio-oligonucleotide and application thereof Download PDFInfo
- Publication number
- CN115728406A CN115728406A CN202210939373.XA CN202210939373A CN115728406A CN 115728406 A CN115728406 A CN 115728406A CN 202210939373 A CN202210939373 A CN 202210939373A CN 115728406 A CN115728406 A CN 115728406A
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- proportion
- tris solution
- concentration
- liquid chromatography
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000004811 liquid chromatography Methods 0.000 title claims abstract description 31
- 238000000926 separation method Methods 0.000 title claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 30
- 238000000746 purification Methods 0.000 claims abstract description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 82
- 239000000243 solution Substances 0.000 claims description 59
- 239000007983 Tris buffer Substances 0.000 claims description 55
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 54
- 239000011780 sodium chloride Substances 0.000 claims description 41
- 238000010828 elution Methods 0.000 claims description 28
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 24
- 108091034117 Oligonucleotide Proteins 0.000 claims description 13
- 238000005349 anion exchange Methods 0.000 claims description 9
- 230000000630 rising effect Effects 0.000 claims description 8
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 238000013375 chromatographic separation Methods 0.000 claims 4
- 239000007788 liquid Substances 0.000 claims 4
- 229920000642 polymer Polymers 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 230000014759 maintenance of location Effects 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 28
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 15
- 229910021642 ultra pure water Inorganic materials 0.000 description 13
- 239000012498 ultrapure water Substances 0.000 description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 12
- 108020004707 nucleic acids Proteins 0.000 description 8
- 150000007523 nucleic acids Chemical class 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 8
- 239000012266 salt solution Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000005303 weighing Methods 0.000 description 5
- 238000007865 diluting Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- 238000005251 capillar electrophoresis Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000012982 microporous membrane Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical class NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Images
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention relates to the technical field of purification of thio-oligonucleotide, in particular to a liquid chromatography separation method of a thio-oligonucleotide polymer and application thereof. The liquid chromatography separation method for the thiooligonucleotide provided by the invention is simple and convenient to operate and low in cost, and the detection result has the advantages that the target peak retention time error is less than 1min, the peak area RE is less than 10%, the purity RE is less than 3%, the purification requirement of the thiooligonucleotide is met, the stability is good, the repeatability is high, and the method is suitable for practical production, popularization and application.
Description
Technical Field
The invention relates to the technical field of purification of a thio-oligonucleotide polymer, in particular to a liquid chromatography separation method of a thio-oligonucleotide polymer and application thereof.
Background
The sample of oligonucleotide fragments (e.g., CPG-ODN) are Oligodeoxynucleotides (ODN) that contain unmethylated cytosine guanine dinucleotides (CpG). The CPG-ODN obtained by the chemical synthesis method is a single-stranded deoxyoligonucleotide high molecular polymer modified by sulfo, the difference of hydrophobicity of the polymer is gradually reduced along with the increase of the chain length of the product, so that the separation and analysis of the CPG-ODN by the conventional high performance liquid chromatography become extremely difficult, and in the prior art, the reported method for detecting the purity of the CPG-ODN comprises (1) the purity of the CPG-ODN is detected by a colorimetric method, such as an MTT colorimetric method provided by Wangchua, liujing and the like to establish a B-type CPG-ODN activity detection standard, and the national Standard of cell and molecular immunology impurities 2008, no. 01, page 69-71). (2) The purity of CPG-ODN is detected by ELISA method, for example, CN100526876C provides an ELISA detection kit for detecting human unmethylated oligodeoxynucleotide, CPG-ODN is modified and hydrolyzed, and then a reverse phase-high performance liquid chromatography is combined to detect a hydrolyzed sample, thereby determining the purity of CPG-ODN. And (3) detecting the purity of the CPG-ODN by a capillary electrophoresis method.
The methods have the defects of poor stability and low repeatability, and meanwhile, a capillary electrophoresis apparatus which is one of main detection devices used by the capillary electrophoresis method is expensive in price and low in popularization rate, so that the method is difficult to popularize and apply in actual production.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a liquid chromatography separation method of a thio-oligonucleotide and apply the method to purification of the thio-oligonucleotide, so as to relieve the technical problems of poor stability and low repeatability in the prior art, and the method is suitable for popularization and application.
In order to solve the technical problems and achieve the purpose, the invention provides the following technical scheme:
in a first aspect, the present invention provides a method for separating a thiooligonucleotide by liquid chromatography, wherein the liquid chromatography is anion exchange high performance liquid chromatography, and comprises the following chromatographic conditions:
taking a Tris solution as a mobile phase A, taking a Tris solution containing NaCl as a mobile phase B, wherein the flow rate of the mobile phase A and the mobile phase B is 0.8-1.2 ml/min, and the temperature of a chromatographic column: 48-52 ℃, detection wavelength: 260nm;
the mobile phase gradient setting includes any one of the following (a) to (c):
(a) The initial proportion of the mobile phase B is 0%, and the elution stages of the proportion of the mobile phase B rising from 0% to 100% are at least two;
(b) The initial proportion of the mobile phase B is 20 percent, the elution stage of the proportion of the mobile phase B is at least one when the proportion of the mobile phase B is increased from 20 percent to 100 percent, and the time for increasing the proportion of the mobile phase B from 20 percent to 100 percent is 20-30 min;
(c) The initial proportion of the mobile phase B is 50%, the elution stage of the proportion of the mobile phase B is at least one when the proportion of the mobile phase B is increased from 50% to 100%, and the time for increasing the proportion of the mobile phase B from 50% to 100% is 29-31 min.
In an alternative embodiment, the concentration of Tris solution in mobile phase A and mobile phase B is 19-21 mM, and the concentration of NaCl in mobile phase B is 3.9-4.1M.
In alternative embodiments, the concentration of the Tris solution in mobile phase A and mobile phase B is 20mM and the concentration of NaCl in mobile phase B is 4M.
In an alternative embodiment, the flow rate of mobile phase a and mobile phase B is 1ml/min, the column temperature: at 50 ℃.
In alternative embodiments, the chromatographic conditions are:
taking a Tris solution as a mobile phase A, taking a Tris solution containing NaCl as a mobile phase B, wherein the concentration of the Tris solution is 20mM, the concentration of NaCl in the mobile phase B is 4M, the flow rate of the mobile phase A and the mobile phase B is 1ml/min, the temperature of a chromatographic column is 50 ℃, and the detection wavelength: 260nm;
the initial proportion of the mobile phase B is 0%, the elution stages of the proportion of the mobile phase B rising from 0% to 100% are two, and after the proportion of the mobile phase B rises to 100% at least once, the stable proportion of 100% is kept for 10-12 min, preferably 10min.
In alternative embodiments, the chromatographic conditions are:
taking a Tris solution as a mobile phase A, taking a Tris solution containing NaCl as a mobile phase B, wherein the concentration of the Tris solution is 20mM, the concentration of NaCl in the mobile phase B is 4M, the flow rate of the mobile phase A and the mobile phase B is 1ml/min, the temperature of a chromatographic column is 50 ℃, and the detection wavelength: 260nm;
the initial proportion of the mobile phase B is 20%, the elution stage of the proportion of the mobile phase B from 20% to 100% is one, and the time for the proportion of the mobile phase B to rise from 20% to 100% is 25-26 min, preferably 26min.
In alternative embodiments, the chromatographic conditions are:
taking a Tris solution as a mobile phase A, taking a Tris solution containing NaCl as a mobile phase B, wherein the concentration of the Tris solution is 20mM, the concentration of NaCl in the mobile phase B is 4M, the flow rates of the mobile phase A and the mobile phase B are 1ml/min, the temperature of a chromatographic column is 50 ℃, and the detection wavelength is as follows: 260nm;
the initial proportion of the mobile phase B is 50%, the elution stage of the proportion of the mobile phase B rising from 50% to 100% is one, and the time for rising the proportion of the mobile phase B from 50% to 100% is 25-26 min, preferably 25.8min.
In a second aspect, the present invention provides a method for separating a thiooligonucleotide by liquid chromatography, wherein the liquid chromatography is anion exchange high performance liquid chromatography, and comprises the following chromatographic conditions:
taking Tris solution containing acetonitrile as a mobile phase A, taking Tris solution containing NaCl as a mobile phase B, wherein the flow rate of the mobile phase A and the mobile phase B is 0.8-1.2/min, and the temperature of a chromatographic column is as follows: 48-52 ℃, detection wavelength: 260nm;
the concentration of the Tris solution is 20mM, the volume fraction of acetonitrile in the mobile phase A is 4% -6%, preferably 5%, the concentration of NaCl in the mobile phase B is 4M, the initial proportion of the mobile phase B is 25%, the elution stage of the proportion of the mobile phase B which is increased from 25% to 100% is at least one, and the time for increasing the proportion of the mobile phase B from 25% to 100% is 25-26 min, preferably 25.8min.
In a third aspect, the present invention provides the use of a liquid chromatography method for the purification of a thioacid oligonucleotide according to any one of the preceding embodiments.
In alternative embodiments, the thioacid oligonucleotide comprises a thioacid oligonucleotide comprising 22 to 24 nucleotides, such as 22, 23 or 24 nucleotides.
The liquid chromatography separation method of the thio-oligonucleotide provided by the invention is simple and convenient to operate and low in cost, and through verification, the retention time error of a target peak in a detection result is less than 1min, the peak area RE is less than 10%, the purity RE is less than 3%, the purification requirement of the oligonucleotide is met, the stability is good, the repeatability is high, and the method is suitable for practical production, popularization and application.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the results of chromatographic detection in example 1 of the present invention;
FIG. 2 shows the results of chromatographic detection in example 2 of the present invention;
FIG. 3 shows the result of chromatographic detection in example 3 of the present invention;
FIG. 4 shows the result of chromatographic detection in example 4 of the present invention;
FIG. 5 shows the results of chromatographic detection in example 5 of the present invention;
FIG. 6 shows the results of continuous sample injection 6-pin chromatography in example 5 of the present invention;
FIG. 7 is a calibration curve obtained in the linear evaluation of the effect example of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.
In a first aspect, the present invention provides a method for separating a thiooligonucleotide by liquid chromatography, wherein the liquid chromatography is anion exchange high performance liquid chromatography, and comprises the following chromatographic conditions:
taking a Tris solution as a mobile phase A, taking a Tris solution containing NaCl as a mobile phase B, wherein the flow rate of the mobile phase A and the mobile phase B is 0.8-1.2 ml/min, and the temperature of a chromatographic column: 48-52 ℃, detection wavelength: 260nm;
the mobile phase gradient setting includes any one of the following (a) to (c):
(a) The initial proportion of the mobile phase B is 0%, and the elution stages of the mobile phase B rising from 0% to 100% are at least two;
(b) The initial proportion of the mobile phase B is 20 percent, the elution stage of the proportion of the mobile phase B is at least one when the proportion of the mobile phase B is increased from 20 percent to 100 percent, and the time for increasing the proportion of the mobile phase B from 20 percent to 100 percent is 20-30 min;
(c) The initial proportion of the mobile phase B is 50%, the elution stage of the proportion of the mobile phase B from 50% to 100% is at least one, and the time for the proportion of the mobile phase B to rise from 50% to 100% is 29-31 min.
In an alternative embodiment, the concentration of the Tris solution in mobile phase A and the concentration of the Tris solution in mobile phase B are 19-21 mM, and the concentration of the NaCl in mobile phase B is 3.9-4.1M.
In an alternative embodiment, the concentration of the Tris solution in mobile phase A and mobile phase B is 20mM, and the concentration of NaCl in mobile phase B is 4M.
In an alternative embodiment, the flow rate of mobile phase a and mobile phase B is 1ml/min, the column temperature: at 50 ℃.
In alternative embodiments, the chromatographic conditions are:
taking a Tris solution as a mobile phase A, taking a Tris solution containing NaCl as a mobile phase B, wherein the concentration of the Tris solution is 20mM, the concentration of NaCl in the mobile phase B is 4M, the flow rates of the mobile phase A and the mobile phase B are 1ml/min, the temperature of a chromatographic column is 50 ℃, and the detection wavelength is as follows: 260nm;
the initial proportion of the mobile phase B is 0%, the elution stages of the proportion of the mobile phase B rising from 0% to 100% are two, and after the proportion of the mobile phase B rises to 100% at least once, the stable proportion of 100% is kept for 10-12 min, preferably 10min.
In alternative embodiments, the chromatographic conditions are:
taking a Tris solution as a mobile phase A, taking a Tris solution containing NaCl as a mobile phase B, wherein the concentration of the Tris solution is 20mM, the concentration of NaCl in the mobile phase B is 4M, the flow rate of the mobile phase A and the mobile phase B is 1ml/min, the temperature of a chromatographic column is 50 ℃, and the detection wavelength: 260nm;
the initial proportion of the mobile phase B is 20%, the elution stage of the proportion of the mobile phase B from 20% to 100% is one, and the time for the proportion of the mobile phase B to rise from 20% to 100% is 25-26 min, preferably 26min.
In alternative embodiments, the chromatographic conditions are:
taking a Tris solution as a mobile phase A, taking a Tris solution containing NaCl as a mobile phase B, wherein the concentration of the Tris solution is 20mM, the concentration of NaCl in the mobile phase B is 4M, the flow rate of the mobile phase A and the mobile phase B is 1ml/min, the temperature of a chromatographic column is 50 ℃, and the detection wavelength: 260nm;
the initial proportion of the mobile phase B is 50%, the elution stage of the proportion of the mobile phase B is one, and the time for the proportion of the mobile phase B to rise from 50% to 100% is 25-26 min, preferably 25.8min.
In a second aspect, the present invention provides a method for separating a thiooligonucleotide by liquid chromatography, wherein the liquid chromatography is anion exchange high performance liquid chromatography, and comprises the following chromatographic conditions:
taking Tris solution containing acetonitrile as a mobile phase A, taking Tris solution containing NaCl as a mobile phase B, wherein the flow rate of the mobile phase A and the mobile phase B is 0.8-1.2 ml/min, and the temperature of a chromatographic column is as follows: 48-52 ℃, detection wavelength: 260nm;
the concentration of the Tris solution is 20mM, the volume fraction of acetonitrile in the mobile phase A is 4-6%, preferably 5%, the concentration of NaCl in the mobile phase B is 4M, the initial proportion of the mobile phase B is 25%, the elution stage of the proportion of the mobile phase B from 25% to 100% is at least one, and the time for the proportion of the mobile phase B from 25% to 100% is 25-26 min, preferably 25.8min.
In a third aspect, the present invention provides the use of a method of liquid chromatography of a thiocPG-ODN as described in any of the preceding embodiments for the purification of a thio-oligonucleotide.
In alternative embodiments, the thioacid oligonucleotide comprises a thioacid oligonucleotide comprising 22 to 24 nucleotides, such as 22, 23 or 24 nucleotides. Including but not limited to a thioated oligonucleotide comprising 24 nucleotides, such as that denoted by sample 1 having the nucleotide sequence 5-.
It should be noted that the liquid chromatography separation effect difference caused by the specific compositions of different kinds of thiooligonucleotides is not large, the liquid chromatography separation method provided by the present invention can be applied to the ion chromatography separation and purification of thiooligonucleotides, and the above sample 1 or sample 2 is only described as an example in the embodiment of the present invention, and should not be understood as being applicable to the restriction of the kinds of thiooligonucleotides.
Some embodiments of the invention are described in detail below with reference to the accompanying drawings. The embodiments and features of the embodiments described below can be combined with each other without conflict.
The following examples refer to materials and reagents, experimental conditions and methods as follows:
1. reagent consumable
Sodium chloride, tris, hydrochloric acid and acetonitrile.
A chromatographic column: strong anion exchange column
2. Steps of the detection method
Mobile phase A: weighing 2.42g plus or minus 0.02g Tris, adding about 900mL ultrapure water for dissolving, adjusting pH to 8.0 plus or minus 0.02 by dilute hydrochloric acid, diluting to 1000mL by ultrapure water, and filtering by a 0.22 mu m microporous filter membrane.
Mobile phase B: weighing 2.42g + -0.02 g Tris and 233g + -0.2 g NaCl, adding about 900mL ultrapure water for dissolving, adjusting pH to 8.00 + -0.02 with dilute hydrochloric acid, adding ultrapure water for diluting to 1000mL, and filtering with 0.22 μm microporous membrane.
Dilute hydrochloric acid: 234mL of hydrochloric acid is measured and diluted to 1000mL by adding ultrapure water, thus obtaining the product.
0.9% sodium chloride solution: 9.0 g. + -. 0.05g NaCl was weighed out, dissolved in ultrapure water and diluted to 1000mL.
Example 1
The embodiment provides a liquid chromatography separation method of thio-CPG-ODN, and the chromatographic conditions are as follows:
mobile phase gradiometer:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0.0 | 100 | 0 |
| 4.2 | 100 | 0 |
| 21.0 | 0 | 100 |
| 25.2 | 100 | 0 |
| 33.6 | 100 | 0 |
| 40.0 | 0 | 100 |
| 50.0 | 0 | 100 |
| 54.0 | 100 | 0 |
| 63.0 | 100 | 0 |
The sample (containing 24 nucleic acid thiooligonucleotides, sample 1) is repeatedly treated five times by adopting the chromatographic conditions, and the result is shown in fig. 1, as can be seen from fig. 1, unknown peaks appear after the main peak of the 5-pin sample is continuously injected, the repeatability is good, and the unknown peaks do not exist in front of the main peak, which indicates that the main component can be eluted by the elution strength or the elution time of the salt solution, and the separation of the nucleic acid sample can be realized, but as can be seen from fig. 1, the main component elution is not complete.
Example 2
This example provides a liquid chromatography method for separating thioacid oligonucleotides, the chromatographic conditions are as follows:
mobile phase gradiometer:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0.0 | 80 | 20 |
| 4.2 | 80 | 20 |
| 21.0 | 0 | 100 |
| 33.0 | 80 | 20 |
| 45.0 | 80 | 20 |
The chromatographic conditions were used to treat a thiooligonucleotide sample (thiooligonucleotide containing 24 nucleic acids, sample 1) and the results are shown in FIG. 2. This example improves the initial ratio of the salt solution compared to example 1. It can be seen that by increasing the initial ratio of the salt solution, no peak shift is known, indicating that the elution of the main component cannot be completed due to insufficient elution strength of the salt solution.
Example 3
This example provides a method for liquid chromatography of thio-compounds, the chromatographic conditions are as follows:
mobile phase gradiometer:
the chromatographic conditions were used to treat a thiooligonucleotide sample (a thiooligonucleotide containing 24 nucleic acids, sample 1) and the results are shown in FIG. 3. In this example, compared to example 2, the initial ratio of the salt solution was increased while the elution time was increased, i.e., the rate of the salt solution was slowed from 20% to 100%, and as can be seen from FIG. 3, the unknown peak was smaller and the main peak area was larger.
The results of comparative example 2 and example 3 are as follows:
from the comparison, it can be seen that the longer the elution time of the salt solution, the larger the main peak area, the smaller the unknown peak area, but the less the total peak area change, and the two chromatographic peaks are the same substance from the side.
Example 4
This example provides a liquid chromatography method for separating thio-oligonucleotide, the chromatographic conditions are as follows:
mobile phase gradiometer:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0.0 | 50 | 50 |
| 4.2 | 50 | 50 |
| 30.0 | 0 | 100 |
| 38.0 | 50 | 50 |
| 48.0 | 50 | 50 |
The results of treating a thiooligonucleotide sample (a thiooligonucleotide containing 24 nucleic acids, sample 1) with the chromatographic conditions are shown in FIG. 4. It can be seen from FIG. 4 that after the initial ratio of the salt solution is increased from 20% to 50%, the main component is completely eluted, the peak area is significantly increased, and no significant change is observed compared with the total peak area of the previous main peak and the unknown peak, but the peak shape is not good.
Example 5
This example provides a liquid chromatography method for separating thioacid oligonucleotides, the chromatographic conditions are as follows:
mobile phase gradiometer:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0.0 | 75 | 25 |
| 4.2 | 75 | 25 |
| 30.0 | 0 | 100 |
| 38.0 | 75 | 25 |
| 48.0 | 75 | 25 |
The results of processing the thiooligonucleotide sample (containing 24 nucleic acid thiooligonucleotides, sample 1) by the chromatographic conditions are shown in fig. 5, and the detection result is shown in fig. 6 after continuous sample introduction for 6 needles, as can be seen from fig. 5 and 6, the main components are completely eluted without residue, the repeatability is better, but the tailing phenomenon exists, according to the literature report, the tailing phenomenon in oligonucleotide chromatography and analysis based on anion exchange is a more common phenomenon, because the full-thio modification can increase the viscosity of nucleic acid molecules and increase the separation difficulty, and meanwhile, the nucleic acid analysis conditions based on anion exchange require higher column temperature (50 ℃) and higher pH (8.0), the tailing phenomenon is easily caused, but the analysis of the sample cannot be influenced, and the method is verified by the analysis methods of items such as precision, linearity, accuracy and durability, and the like, and passes the verification requirements.
Effects of the invention
The detection effect of the detection method provided in example 5 above was verified using sample thioaligonucleotides, with the following detection steps:
mobile phase A: weighing 2.42g plus or minus 0.02g Tris, adding about 900mL of ultrapure water for dissolving, adjusting the pH to 8.0 plus or minus 0.02 with dilute hydrochloric acid, adding 50mL of acetonitrile, diluting with ultrapure water to 1000mL, and filtering with a 0.22 μm microporous membrane.
Mobile phase B: weighing 2.42g plus or minus 0.02g Tris and 233g plus or minus 0.2g NaCl, adding about 900mL ultrapure water for dissolving, adjusting the pH value to 8.00 plus or minus 0.02 by using dilute hydrochloric acid, adding ultrapure water for diluting to 1000mL, and filtering by using a 0.22 mu m microporous filter membrane.
Dilute hydrochloric acid: 234mL of hydrochloric acid is measured and diluted to 1000mL by adding ultrapure water, thus obtaining the product.
0.9% sodium chloride solution: 9.0 g. + -. 0.05g NaCl was weighed, dissolved in ultrapure water and diluted to 1000mL.
Sample solution of ZJ 001: weighing a proper amount of ZJ001 freeze-dried powder, dissolving the ZJ001 freeze-dried powder by using 0.9% sodium chloride solution, determining that the content of the ZJ001 freeze-dried powder is within the range of 0.5 mg/mL-2.5 mg/mL, centrifuging a sample at 10000rpm for 10min, and taking supernatant and filling the supernatant into a sample bottle.
The column was equilibrated with the initial ratio of mobile phase for more than 60min until the pressure and baseline stabilized and the sample sequence was run.
After the sequence was completed, the column was washed with 20mM Tris pH8.0 for 60min or more, the column was removed, and the system line was then washed with ultrapure water for at least 4 hours.
And obtaining the peak area% of the main peak according to an area normalization method, wherein the peak area% is the purity of the ZJ001 sample.
The test effect was evaluated as follows:
(1) The specificity is as follows: no target peak exists in the blank buffer solution, and a target peak exists in the test solution; in the sample impurity adding process, the target peak and the impurity peak reach the base line separation, and the result meets the requirement of specificity.
(2) Precision (reproducibility): 6 portions of the sample were prepared in parallel by one experimenter according to the above operations, and 6-pin peak areas RSD% and purities RSD% were examined, and the results are as follows, all of which meet the requirement of reproducibility.
(3) Precision (intermediate precision): two analysts prepare 3 parts in parallel according to the preparation method on different dates respectively, and the results of 6-pin peak area RSD% and purity RSD% are both in accordance with the intermediate precision requirement.
(4) Linearity: within the range of 500-2500 mu g/mL, the method meets the linear verification requirement.
(5) Accuracy of
(6) Durability
And (3) stability of the test solution: the test solution is respectively placed for 0h, 12h, 24h, 48h and 72h at the temperature of 2-8 ℃.
Fine adjustment of chromatographic conditions: (this item is to verify the robustness of the method)
And (4) conclusion: all the verification contents meet the acceptable standard, which indicates that the method is suitable for IEX HPLC purity and stability analysis and detection of the special sample thiooligonucleotide.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. The liquid chromatography separation method of the thio-oligonucleotide is characterized in that the liquid chromatography is anion exchange high performance liquid chromatography and comprises the following chromatographic conditions:
taking a Tris solution as a mobile phase A, taking a Tris solution containing NaCl as a mobile phase B, wherein the flow rate of the mobile phase A and the mobile phase B is 0.8-1.2 ml/min, and the temperature of a chromatographic column is as follows: 48-52 ℃, detection wavelength: 260nm;
the mobile phase gradient setting includes any one of the following (a) to (c):
(a) The initial proportion of the mobile phase B is 0%, and the elution stages of the proportion of the mobile phase B rising from 0% to 100% are at least two;
(b) The initial proportion of the mobile phase B is 20 percent, the elution stage of the proportion of the mobile phase B is at least one when the proportion of the mobile phase B is increased from 20 percent to 100 percent, and the time for increasing the proportion of the mobile phase B from 20 percent to 100 percent is 20-30 min;
(c) The initial proportion of the mobile phase B is 50%, the elution stage of the proportion of the mobile phase B from 50% to 100% is at least one, and the time for the proportion of the mobile phase B to rise from 50% to 100% is 29-31 min.
2. The liquid chromatography separation method of claim 1, wherein the concentration of the Tris solution in the mobile phase A and the concentration of the NaCl solution in the mobile phase B are 19 to 21mM, and 3.9 to 4.1M.
3. The liquid chromatography separation method of claim 2, wherein the concentration of the Tris solution in the mobile phase a and the mobile phase B is 20mM, and the concentration of NaCl in the mobile phase B is 4M.
4. The liquid chromatography separation method according to claim 2, wherein the flow rate of the mobile phase a and the mobile phase B is 1ml/min, the column temperature: at 50 ℃.
5. The liquid chromatographic separation method of claim 1, wherein the chromatographic conditions are:
taking a Tris solution as a mobile phase A, taking a Tris solution containing NaCl as a mobile phase B, wherein the concentration of the Tris solution is 20mM, the concentration of NaCl in the mobile phase B is 4M, the flow rate of the mobile phase A and the mobile phase B is 1ml/min, the temperature of a chromatographic column is 50 ℃, and the detection wavelength: 260nm;
the initial proportion of the mobile phase B is 0%, the elution stages of the proportion of the mobile phase B rising from 0% to 100% are two, and after the proportion of the mobile phase B rises to 100% at least once, the stable proportion of 100% is kept for 10-12 min, preferably 10min.
6. The liquid chromatographic separation method of claim 1, wherein the chromatographic conditions are:
taking a Tris solution as a mobile phase A, taking a Tris solution containing NaCl as a mobile phase B, wherein the concentration of the Tris solution is 20mM, the concentration of NaCl in the mobile phase B is 4M, the flow rates of the mobile phase A and the mobile phase B are 1ml/min, the temperature of a chromatographic column is 50 ℃, and the detection wavelength is as follows: 260nm;
the initial proportion of the mobile phase B is 20%, the elution stage of the proportion of the mobile phase B from 20% to 100% is one, and the time for the proportion of the mobile phase B to rise from 20% to 100% is 25-26 min, preferably 26min.
7. The liquid chromatographic separation method of claim 1, wherein the chromatographic conditions are:
taking a Tris solution as a mobile phase A, taking a Tris solution containing NaCl as a mobile phase B, wherein the concentration of the Tris solution is 20mM, the concentration of NaCl in the mobile phase B is 4M, the flow rate of the mobile phase A and the mobile phase B is 1ml/min, the temperature of a chromatographic column is 50 ℃, and the detection wavelength: 260nm;
the initial proportion of the mobile phase B is 50%, the elution stage of the proportion of the mobile phase B is one, and the time for the proportion of the mobile phase B to rise from 50% to 100% is 25-26 min, preferably 25.8min.
8. The liquid chromatography separation method of the thio-oligonucleotide is characterized in that the liquid chromatography is anion exchange high performance liquid chromatography and comprises the following chromatographic conditions:
taking Tris solution containing acetonitrile as a mobile phase A, taking Tris solution containing NaCl as a mobile phase B, wherein the flow rate of the mobile phase A and the mobile phase B is 0.8-1.2 ml/min, and the temperature of a chromatographic column is as follows: 48-52 ℃, detection wavelength: 260nm;
the concentration of the Tris solution is 20mM, the volume fraction of acetonitrile in the mobile phase A is 4-6%, preferably 5%, the concentration of NaCl in the mobile phase B is 4M, the initial proportion of the mobile phase B is 25%, the elution stage of the proportion of the mobile phase B from 25% to 100% is at least one, and the time for the proportion of the mobile phase B from 25% to 100% is 25-26 min, preferably 25.8min.
9. Use of the thiooligonucleotide of any one of claims 1 to 8 for its liquid chromatographic separation for purification of thiooligonucleotides.
10. The use of claim 9, wherein said thioacid oligonucleotide comprises a thioacid oligonucleotide comprising 22 to 24 nucleotides.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210939373.XA CN115728406A (en) | 2022-08-05 | 2022-08-05 | Liquid chromatography separation method of thio-oligonucleotide and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210939373.XA CN115728406A (en) | 2022-08-05 | 2022-08-05 | Liquid chromatography separation method of thio-oligonucleotide and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115728406A true CN115728406A (en) | 2023-03-03 |
Family
ID=85292693
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210939373.XA Pending CN115728406A (en) | 2022-08-05 | 2022-08-05 | Liquid chromatography separation method of thio-oligonucleotide and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115728406A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5183885A (en) * | 1991-10-18 | 1993-02-02 | Applied Biosystems, Inc. | Method for chromatographic separation of synthetic phosphorothioate oligonucleotides |
| JP2021525086A (en) * | 2018-05-30 | 2021-09-24 | アクソーラボズ ゲーエムベーハー | Methods for Detecting Oligonucleotide Conjugates |
| US20220049240A1 (en) * | 2018-09-11 | 2022-02-17 | Amgen Inc. | Purification methods for guanine-rich oligonucleotides |
-
2022
- 2022-08-05 CN CN202210939373.XA patent/CN115728406A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5183885A (en) * | 1991-10-18 | 1993-02-02 | Applied Biosystems, Inc. | Method for chromatographic separation of synthetic phosphorothioate oligonucleotides |
| JP2021525086A (en) * | 2018-05-30 | 2021-09-24 | アクソーラボズ ゲーエムベーハー | Methods for Detecting Oligonucleotide Conjugates |
| US20220049240A1 (en) * | 2018-09-11 | 2022-02-17 | Amgen Inc. | Purification methods for guanine-rich oligonucleotides |
Non-Patent Citations (6)
| Title |
|---|
| A.J.BOURQUE 等: "Quantitative analysis of phosphorothioate oligonucleotides in biological fluids using fast anion-exchange chromatography", JOURNAL OF CHROMATOGRAPHY, vol. 617, 31 December 1993 (1993-12-31), pages 43 - 49, XP000770928, DOI: 10.1016/0378-4347(93)80419-5 * |
| BOB MILLEY 等: "Optimization, Production, and Characterization of a CpG-oligonucleotide-Ficoll Conjugate Nanoparticle Adjuvant for Enhanced Immunogenicity of Anthrax Protective Antigen", BIOCONJUGATE CHEM., vol. 27, 31 December 2016 (2016-12-31), pages 1293 - 1304 * |
| 冯指泉 等: "离子交换色谱法测定反义寡核苷酸药物CT102的含量", 国际药学研究杂志, no. 04, 30 August 2013 (2013-08-30), pages 485 - 490 * |
| 张宏燕 等: "硫代反义寡核苷酸药物FT19有关物质的分析", 色谱, vol. 26, no. 05, 31 December 2008 (2008-12-31), pages 540 - 543 * |
| 张嵬 等: "硫代反义寡核苷酸药物癌泰得纯化工艺的建立", 中国药学杂志, no. 15, 8 March 2006 (2006-03-08), pages 1188 - 1192 * |
| 李麒麟 等: "用IE-HPLC、PAGE分析合成的硫代寡核苷酸相关物质"n-1"、"n-2"和"n-3"", 药物分析杂志, vol. 22, no. 05, 31 December 2002 (2002-12-31), pages 371 - 375 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Rosenberg et al. | Interactions of nucleotides, polynucleotides, and nucleic acids with dihydroxyboryl-substituted celluloses | |
| Fritz et al. | Studies on polynucleotides. 146. High-pressure liquid chromatography in polynucleotide synthesis | |
| Enmark et al. | Investigation of factors influencing the separation of diastereomers of phosphorothioated oligonucleotides | |
| CN111272895B (en) | Method for simultaneously and rapidly detecting amino sugar, neutral sugar and uronic acid in seaweed | |
| CN110709696B (en) | Method for separating and analyzing mixture of oligonucleotides | |
| CN114594167A (en) | Method for detecting content of mono-oligosaccharide and fingerprint of compound sophora flavescens injection | |
| CN117677710A (en) | Assays for quantitative assessment of mRNA capping efficiency | |
| CN115728406A (en) | Liquid chromatography separation method of thio-oligonucleotide and application thereof | |
| Zunino | Studies on the mode of interaction of daunomycin with DNA | |
| CN116426610B (en) | Method for accurately quantifying mRNA capping efficiency by LC-MS method | |
| US20110137022A1 (en) | Method for the analysis of oligonucleotides | |
| CN108426965B (en) | Method for detecting isomaltose, isomaltotriose, maltose and panose in yellow wine | |
| CN101482544B (en) | Method for detecting cefuroxime sodium polymer impurities | |
| CN115856189A (en) | Oligonucleotide compound concentration liquid-phase mass spectrometry combined detection method | |
| CN104195235B (en) | Detect method and the application of CpG ODN sequence purity | |
| CN113866318A (en) | Method for detecting (6-aminopyridine-2-yl) (1-methylpiperidine-4-yl) methanone dihydrochloride | |
| Kennedy et al. | Analysis of ribonucleotides and deoxyribonucleotides using high-pressure liquid column chromatography | |
| Stanley Jr | [48] Fractionation of oligoribonucleotides according to degree of polymerization | |
| CN112400017A (en) | Method for detecting oligonucleotide conjugates | |
| CN116858978B (en) | Method for simultaneously detecting insulin aspart and insulin deglutition and plasma sample processing method thereof | |
| CN111198235A (en) | Method for detecting content of isosinensetin in plasma | |
| CN110146603A (en) | A kind of analysis method measuring Oxcarbazepine dissolution of sustained-release tablets | |
| EP3673990A1 (en) | Chromatography packing for separation and/or detection of methylated dna | |
| CN115327021B (en) | Ion chromatography for determining ferric citrate and organic acid impurities thereof | |
| CN112816584B (en) | Bioanalysis method of fidaxomicin and metabolite OP-1118 thereof in human plasma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |