CN114405298B - Method for purifying polyethylene glycol conjugated lipids - Google Patents

Method for purifying polyethylene glycol conjugated lipids Download PDF

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CN114405298B
CN114405298B CN202111613752.1A CN202111613752A CN114405298B CN 114405298 B CN114405298 B CN 114405298B CN 202111613752 A CN202111613752 A CN 202111613752A CN 114405298 B CN114405298 B CN 114405298B
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CN114405298A (en
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王秀莲
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Suzhou Aibo Biotechnology Co ltd
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01D11/0492—Applications, solvents used
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
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    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
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Abstract

The present invention relates to a method for purifying polyethylene glycol conjugated lipids. The method comprises i) dissolving crude polyethylene glycol conjugated lipid in a solvent; and ii) carrying out reversed-phase high performance liquid chromatography separation on the solution obtained in i), wherein a chromatographic column used for the reversed-phase high performance liquid chromatography is selected from C8 and C18 chromatographic columns, a mobile phase is mobile phase A, mobile phase B or a mixture thereof, the mobile phase A is acetonitrile, the mobile phase B is methanol, the volume ratio of the mobile phase A to the mobile phase B in the mixture is 5:95 to 95:5, the reversed-phase high performance liquid chromatography is eluted in a gradient elution mode, wherein the ratio of the mobile phase B in the mobile phase is 5% -20% when a first gradient starts, the mobile phase is mobile phase A or a mixture of the mobile phase A and the mobile phase B in a range of 10:90 to 90:10 when the first gradient ends, and the first gradient running time is 1min-20min. The method can obtain the product with higher purity and is easy for commercial production.

Description

Method for purifying polyethylene glycol conjugated lipids
Technical Field
The invention relates to the technical field of organic chemical synthesis and medicine. In particular, the invention relates to a method for purifying polyethylene glycol conjugated lipids.
Background
Nucleic acid isogenic drugs have been developed extensively recently due to their own advantages. However, nucleic acid has the disadvantages of unstable structure, easy degradation, strong negative charge, difficult entering cells and the like, and thus the application of the nucleic acid is limited. Efficient delivery of nucleic acids has become one of the major challenges for nucleic acid applications. Currently, nucleic acid delivery vectors commonly used in research and clinical applications are broadly divided into two categories, viral vectors and non-viral vectors. Viral vectors have high efficacy but greater toxicity, so non-viral vectors, particularly liposome technology, are becoming a major means of nucleic acid delivery.
The common liposome is a cationic liposome compound, but the cationic liposome compound has positive charges on the surface, so that the cationic liposome compound is easy to generate nonspecific adsorption with serum proteins in blood plasma to form large-size aggregates, and the aggregates are easy to be cleared by reticuloendothelial systems, so that the blood circulation time is short, the stability is poor, and the operation efficiency is low. For this purpose, it is necessary to surface-modify cationic liposomes to prepare long-circulating cationic liposomes. The long-circulating cationic liposome-modifying agents commonly used today are polyethylene glycol (PEG) -based lipid molecules such as 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) molecules. PEG forms a hydration layer on the surface of the modified cationic liposome through the action of hydrogen bond and water molecules in the solvent to mask the positive charge on the surface of the cationic liposome, thereby achieving the effects of inhibiting protein adsorption and reducing phagocytic system recognition.
The polyethylene glycol lipid commonly used at present is DMG-PEG2000, and the specific structure is shown in formula 1. And DMG-PEG2000 has been approved for clinical emergency use abroad as one of the four components of LNP in the Moderna company mRNA-1273 product for the prevention of novel coronavirus infection.
CN 112996854A discloses that high purity PEG lipids exhibit excellent physical and biological properties, especially when used in Lipid Nanoparticle (LNP) formulations. LNP (lipid nanoparticles) comprising high purity PEG lipids have improved physical and biological properties including less easy recognition and clearance by the body. The acceptable range of chemical purity was estimated to be greater than or equal to 87% based on manufacturability evaluation. The lower limit is based on demonstrated ability to treat the formulation and minimization of IgM immune response. There is therefore a need to prepare PEG lipids of higher purity.
However, no method for synthesizing DMG-PEG2000 with higher purity has been disclosed. Therefore, there is a need to provide a purification process for DMG-PEG2000 which can yield a product of higher purity and which is easy to produce commercially.
Disclosure of Invention
It is an object of the present invention to provide a method for purifying polyethylene glycol conjugated lipid molecules, which can give products of higher purity and which are easy to produce commercially.
Thus, according to one aspect of the present invention there is provided a method of purifying a polyethylene glycol conjugated lipid molecule comprising the steps of:
i) Dissolving polyethylene glycol conjugated lipid crude product in solvent; and
Ii) subjecting the solution obtained in i) to reversed-phase high performance liquid chromatography,
Wherein the chromatographic column used for the reversed-phase high-performance liquid chromatography is selected from C8 and C18 chromatographic columns, the mobile phase is mobile phase A, mobile phase B or a mixture thereof, the mobile phase A is acetonitrile, the mobile phase B is methanol, the volume ratio of the mobile phase A to the mobile phase B in the mixture is 5:95 to 95:5,
The reversed-phase high performance liquid chromatography is eluted by adopting a gradient elution mode, wherein the mobile phase is a mobile phase A or a mixture of the mobile phase A and the mobile phase B from 50:50 to 90:10 when the first gradient starts, the mobile phase is a mobile phase A or a mixture of the mobile phase A and the mobile phase B from 10:90 to 90:10 when the first gradient stops, and the running time of the first gradient is 1min-20min.
According to a second aspect of the present invention, a method for purifying polyethylene glycol conjugated lipids comprises the steps of:
i) Dissolving polyethylene glycol conjugated lipid crude product in solvent;
ii) subjecting the solution obtained in i) to normal phase silica gel column chromatography separation wherein the mobile phase is a mixture of dichloromethane and methanol or dichloromethane, and
Iii) And (3) carrying out reversed-phase high performance liquid chromatography on the normal phase chromatography product obtained in the ii), wherein the chromatographic column is a C18 chromatographic column, and the mobile phase is methanol.
The method can obtain a product with higher purity and is easy for commercial production, and the purity of the obtained product can exceed 93 percent, and in some cases, can even reach more than 99 percent.
Detailed Description
Various aspects, as well as further objects, features, and advantages of the present invention will be more fully apparent hereinafter.
According to one aspect of the present invention there is provided a method of purifying a polyethylene glycol conjugated lipid molecule comprising the steps of:
i) Dissolving polyethylene glycol conjugated lipid crude product in solvent; and
Ii) subjecting the solution obtained in i) to reversed-phase high performance liquid chromatography,
Wherein the chromatographic column used for the reversed-phase high-performance liquid chromatography is selected from C8 and C18 chromatographic columns, the mobile phase is mobile phase A, mobile phase B or a mixture thereof, the mobile phase A is acetonitrile, the mobile phase B is methanol, the volume ratio of the mobile phase A to the mobile phase B in the mixture is 5:95 to 95:5,
The reversed-phase high performance liquid chromatography is eluted by adopting a gradient elution mode, wherein the mobile phase is a mobile phase A or a mixture of the mobile phase A and the mobile phase B from 50:50 to 90:10 when the first gradient starts, the mobile phase is a mobile phase A or a mixture of the mobile phase A and the mobile phase B from 10:90 to 90:10 when the first gradient stops, and the running time of the first gradient is 1min-20min.
Unless otherwise indicated herein, the term "lipid" refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by generally poor solubility in water but are soluble in many non-polar organics.
Although lipids generally have poor solubility in water, certain classes of lipids (e.g., lipids modified with polar groups such as DMG-PEG 2000) have limited water solubility and can be dissolved in water under certain conditions.
Known types of lipids include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides and phospholipids. Lipids can generally be divided into at least three classes: (1) "simple lipids", including fats and oils and waxes; (2) "Compound lipids" including phospholipids and glycolipids (such as DMPE-PEG 2000); (3) "derived lipids", such as steroids, and the like. In addition, lipids also include lipid compounds.
The term "polymer conjugated lipid" refers to a molecule comprising both a lipid moiety and a polymer moiety.
An example of a polymer conjugated lipid is a pegylated lipid (PEG-lipid), wherein the polymer moiety comprises polyethylene glycol.
The lipid may be a neutral lipid or a charged lipid.
The term "neutral lipid" encompasses any lipid molecule that exists in an uncharged form or a neutral zwitterionic form at a selected pH. In some embodiments, the selected useful pH value or range corresponds to the pH condition of the environment in which the lipid is intended to be used, e.g., physiological pH. As non-limiting examples, neutral lipids include, but are not limited to, phosphatidylcholine, such as 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 11, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), phosphatidylethanolamine such as 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2- (((2, 3-bis (oleoyloxy) propyl)) phosphodimethylammonium) ethyl hydrogen (DOCP), sphingomyelin (SM), ceramides, steroids such as sterols and derivatives thereof. Neutral lipids may be synthetic or derived (isolated or modified) from natural sources or compounds.
The term "charged lipid" encompasses any lipid molecule that exists in a positively or negatively charged form at a selected pH or range. In some embodiments, the selected pH value or range corresponds to the pH condition of the intended use environment of the lipid, e.g., physiological pH. As non-limiting examples, charged lipids include, but are not limited to, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethane carbamoylsterols (e.g., DC-Chol), 1, 2-dioleoyl-sn-glycero-3-phosphate-L-serine sodium salt (DOPS-Na), 1, 2-dioleoyl-sn-glycero-3-phosphate- (1' -rac-glycero) sodium salt (DOPG-Na), and 1, 2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPA-Na). Charged lipids provided herein may be synthetic or derived (isolated or modified) from natural sources or compounds.
Preferably, the polyethylene glycol conjugated lipid is selected from the group consisting of DMG-PEG1000, DMG-PEG2000, DLG-PEG2000 and DSG-PEG2000.
DMG-PEG1000 has the following structure:
DMG-PEG2000 has the following structure:
DLG-PEG2000 has the following structure:
DSG-PEG2000 has the following structure:
c18 column refers to an alkane bonded to a silica gel containing 18 carbon atoms.
By C8 column is meant that the alkane bonded to the silica gel contains 8 carbon atoms.
Preferably, the solvent is selected from ethanol, dichloromethane and acetonitrile.
In some embodiments, in step i), the crude product is sonicated and filtered after being dissolved in a solvent.
Preferably, the chromatographic column is a C18 chromatographic column with a particle size of 10-40 μm.
Preferably, the chromatographic column C8 has a particle size of 10-40 μm.
In some embodiments, the chromatographic column is selected from Waters sunfire C18.19X250 mm 10 μm, welch ultimate XB-C18.sub.20-40 μm80G and WELFLASH C g of 20-40 μm 120g.
Preferably, the purification is performed at normal temperature.
Preferably, the gradient elution of the reversed phase high performance liquid chromatography comprises at least 3 gradients, more preferably comprises 4-6 gradients.
Preferably, the total running time of the reversed phase high performance liquid chromatography is 15-100min.
Preferably, the first gradient run time is 1min-17min.
Preferably, in step i), normal phase high performance liquid chromatography is performed after dissolving the crude product in a solvent, wherein the chromatographic column used is a silica gel column and the mobile phase is a mixture of dichloromethane and methanol or dichloromethane, the volume ratio of dichloromethane to methanol in the mixture being from 70:30 to 90:10.
Preferably, the particle size of the normal phase silica gel column is 10 to 150. Mu.m, more preferably 20 to 150. Mu.m.
Preferably, the elution gradient of normal phase high performance liquid chromatography is:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 100 0
2 100 0
52 90 10
67 90 10
82 70 30
117 70 30。
Preferably, the elution gradient of the reversed phase high performance liquid chromatography is as shown in any one of tables 1 to 9:
TABLE 1
Time (min) Mobile phase a (%) Mobile phase B (%)
0 90 10
1 70 30
13 10 90
15 5 95
16 5 95
18 95 5
TABLE 2
TABLE 3 Table 3
Time (min) Mobile phase a (%) Mobile phase B (%)
0 100 0
15 100 0
25 80 20
45 50 50
70 15 85
85 0 100
95 0 100
TABLE 4 Table 4
Time (min) Mobile phase a (%) Mobile phase B (%)
0 100 0
16.3 100 0
25.9 81 19
48.4 47 53
71.5 16 84
89 0 100
99 0 100
TABLE 5
Time (min) Mobile phase a (%) Mobile phase B (%)
0 90 10
1 90 10
13 70 30
15 5 95
16 5 95
18 95 5
TABLE 6
TABLE 7
Time (min) Mobile phase a (%) Mobile phase B (%)
0 50 50
1 20 80
13 15 85
15 5 95
16 95 5
TABLE 8
Time (min) Mobile phase a (%) Mobile phase B (%)
0 50 50
1 20 80
13 15 85
15 5 95
16 5 95
18 95 5
TABLE 9
Time (min) Mobile phase a (%) Mobile phase B (%)
0 50 50
1 10 90
13 9 91
15 5 95
16 5 95
18 95 5。
According to a second aspect of the present invention, a method for purifying polyethylene glycol conjugated lipids comprises the steps of:
i) Dissolving polyethylene glycol conjugated lipid crude product in solvent;
ii) subjecting the solution obtained in i) to normal phase silica gel column chromatography separation wherein the mobile phase is a mixture of dichloromethane and methanol or dichloromethane, and
Iii) And (3) carrying out reversed-phase high performance liquid chromatography on the normal phase chromatography product obtained in the ii), wherein the chromatographic column is a C18 chromatographic column, and the mobile phase is methanol.
Preferably, in step ii) the elution is performed with dichloromethane and then with a mixture of dichloromethane and methanol in which the ratio of dichloromethane to methanol is from 20:1 to 40:1.
Preferably, the particle size of the normal phase silica gel column is 10 to 150. Mu.m, preferably 75 to 150. Mu.m.
Preferably, the polyethylene glycol conjugated lipid is selected from the group consisting of DMG-PEG1000, DMG-PEG2000, DLG-PEG2000 and DSG-PEG2000.
Preferably, the solvent is selected from ethanol, dichloromethane and acetonitrile.
Preferably, the particle size of the C18 column is 10-40. Mu.m, preferably 20-35. Mu.m.
In some embodiments, the C18 chromatographic column is BUCHI, pure C-815 Flash C18 spherical 20-35 μm40g。
Preferably, the purification is performed at normal temperature.
The descriptions of the various features of the application may be combined with each other without contradiction to each other and are within the scope of the application as claimed.
The terms "comprising" and "including" as used in the present application encompass the situation in which other elements not explicitly mentioned are also included or included as well as the situation in which they consist of the elements mentioned.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. To the extent that the definitions of terms in this specification are inconsistent with the ordinary understanding of those skilled in the art to which this invention pertains, the definitions described herein control.
Unless otherwise indicated, all numbers expressing quantities of parameters and so forth used in the specification and claims are to be understood as being modified by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties to be obtained.
Examples
The present invention is described in detail below with reference to specific embodiments, but the scope of the present invention is not limited thereto, and any changes or substitutions easily conceivable by those skilled in the art within the technical scope of the present invention should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Preparation example 1: synthesis of DMG-PEG2000
Step 1: synthesis of Compound 3
Compound 1 (10.0 g,5.0mmol,1.0 eq) was dissolved in tetrahydrofuran (100.0 mL) under nitrogen, then NaH (0.4 g,10.0mmol,2.0 eq) was added and reacted at room temperature for 2 hours. Compound 2 (2.7 g,20.0mmol,4.0 eq) was dissolved in tetrahydrofuran (50.0 mL) and added dropwise to the reaction system, followed by stirring at room temperature for 48 hours. HPLC (ELSD, evaporative light scattering detector) showed complete reaction, the reaction was poured into ice water, extracted with dichloromethane, the organic phase dried over anhydrous sodium sulfate, and spin-dried to give compound 3.
And a second step of: synthesis of Compound 4
Compound 3 (10.0 g,4.88mmol,1.0 eq) was dissolved in water (100.0 mL), perchloric acid (2.0 mL) was added and stirred at 100deg.C for 16 hours. HPLC showed the reaction to be complete, ph=8-9 was adjusted with saturated aqueous sodium bicarbonate, extracted with dichloromethane, the organic phase dried over anhydrous sodium sulfate and spin-dried to give compound 4. And a third step of: synthesis of Compound DMG-PEG2000
Compound 4 (10.0 g,4.8mmol,1.0 eq) and myristic acid (10.9 g,48.0mmol,10.0 eq) were dissolved in dichloromethane (100.0 mL) and EDCI (1-ethyl-3 (3-dimethylpropylamine) carbodiimide) (4.7 g,24.0mmol,5.0 eq), DMAP (4-dimethylaminopyridine) (350.0 mg,2.4mmol,0.5 eq) and DIEA (N, N-diisopropylethylamine) (6.2 g,48.0mmol,10.0 eq) were added and stirred at 70℃for 24 hours. CAD showed complete reaction, the reaction was poured into 100 ml of ice water, extracted with dichloromethane, the organic phase dried over anhydrous sodium sulfate and spin-dried to give crude DMG-PEG2000 (CAD: 67%).
The crude DMG-PEG2000 product referred to in the examples below was obtained according to this example at a purity of 67% unless explicitly stated otherwise.
Comparative example 1
100Mg of the crude DMG-PEG2000 obtained in the preparation example was dissolved in 5mL of ethanol, the solution was packed in a 5cm dialysis bag, placed in 1L of ethanol, stirred with a stirrer for 48 hours (24 hours for fresh ethanol), the ethanol solution containing the product in the dialysis bag was taken out, dried by spinning, and lyophilized to obtain the product (50 mg, CAD: 88%).
Comparative example 2
100Mg of the crude DMG-PEG2000 obtained in the preparation example was dissolved in 5mL of acetonitrile, filled into a 5cm dialysis bag, placed into 1L of acetonitrile, stirred with a stirrer for 48 hours (24 hours for fresh acetonitrile), the acetonitrile solution containing the product in the dialysis bag was taken out, dried by spin, and lyophilized to obtain the product (30 mg, CAD: 87%).
Comparative example 3
100Mg of the crude DMG-PEG2000 obtained in the preparation example was dissolved in 5mL of deionized water, filled into a 5cm dialysis bag, placed into 1L of deionized water, stirred for 48 hours (24 hours for fresh deionized water), taken out of the dialysis bag, and directly lyophilized to obtain the product (90 mg, CAD: 67%).
Comparative example 4
Soaking 10g of Sephadex LH-20 in 100ml of ethanol, stirring overnight, washing away impurities, filtering, and spin-drying.
The soaked sephadex LH-20 was packed in a glass column and rinsed once with DCM/methanol=20/1 for use.
100Mg of crude DMG-PEG2000 obtained in the preparation example was taken and dissolved in 2ml of methylene chloride, and the mixture was spread on a column uniformly, washed with DCM/methanol=1/0 to 20/1, and TLC (DCM/methanol=20/1, developer: potassium permanganate developer) showed poor purification and separation was not achieved.
Comparative example 5
500Mg of the crude DMG-PEG2000 obtained in the preparation example was taken and dissolved in 3ml of ethyl acetate, n-hexane was slowly added dropwise, and no solid was precipitated during the dropwise addition process, so that a clean product was not obtained.
Comparative example 6
500Mg of the crude DMG-PEG2000 obtained in the preparation example was taken and dissolved in 2ml of ethanol under heat, after which the crude DMG-PEG2000 was cooled down slowly without crystals being precipitated. No crystals precipitated when the system was left in an environment of-20 ℃.
Comparative example 7
500Mg of the crude DMG-PEG2000 obtained in the preparation example was taken and dissolved in 2ml of isopropanol under heat, and after dissolution was completed, the crude DMG-PEG2000 was cooled down slowly without precipitation of crystals. No crystals precipitated when the system was left in an environment of-20 ℃.
Comparative example 8
500Mg of crude DMG-PEG2000 obtained in the preparation example was stirred with 10ml of petroleum ether and the product was found to become milky and impossible to filter.
Comparative example 9
3.5G of crude DMG-PEG2000 obtained in the preparation example was dissolved in 10mL of methylene chloride and sonicated, and filtered to give 15mL of a clear solution. Purification by normal phase high performance liquid chromatography using the following chromatographic conditions: the chromatographic column is Agela-35 μm104G; the mobile phase is A: dichloromethane, B: methanol; the flow rate is 14mL/min; the detection wavelength is 210nm; the sample injection amount was 15mL. Elution gradients are as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 100 0
2 100 0
52 90 10
67 90 10
82 70 30
117 70 30
The fractions were collected and concentrated and the CAD assay purity was 66.59%.
Comparative example 10
8.9G of crude DMG-PEG2000 obtained in the preparation example was dissolved in 33mL of methylene chloride and sonicated, and filtered to give 41mL of a clear solution. Purification by normal phase high performance liquid chromatography using the following chromatographic conditions: the chromatographic column is AGELA SPHERICAL-35 μm104G; the mobile phase is A: dichloromethane, B: methanol; the flow rate is 50mL/min; the detection wavelength is 210nm; the sample loading was 41mL. Elution gradients are as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 100 0
2 100 0
52 90 10
67 90 10
82 70 30
117 70 30
The fractions were collected to give 3.9g of crude product with a CAD purity of 65.58%.
Comparative example 11
12G of the crude DMG-PEG2000 obtained in the preparation example was taken and dissolved in 200mL of methylene chloride, and stirred until the solid was completely dissolved for use.
Purifying the crude product by normal phase silica gel column chromatography (silica gel column particle diameter: 100-200 mesh, or 75-150 μm mesh), eluting with pure dichloromethane, and color-developing and purifying by phosphomolybdic acid baking plate to obtain an obvious impurity. Then a portion of the impurities was eluted with DCM/methanol=40/1 followed by washing with DCM/methanol=20/1, and the product eluted (the latter two steps were developed with potassium permanganate developer) to give 5.5g of the product with a CAD detection purity of 77%. And the purification of normal phase is continued for many times, and the purity can only reach 90 percent at most.
Comparative example 12
25Mg of the sample (crude DMG-PEG2000 and standard) was dissolved in ethanol until clear and transparent. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the column was Waters sunfire C, 19 x 250mm 10 μm; the mobile phase is A:0.1% tfa (triethanolamine) in water, B: acetonitrile, elution gradient as follows:
no target peak was detected.
Comparative example 13
The mobile phase B gradient of comparative example 12 was increased and the elution gradient was as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 50 50
2 10 90
11 10 90
14 5 95
16 5 95
18 95 5
No target peak was detected.
Comparative example 14
25Mg of the sample (crude DMG-PEG2000 and standard) was dissolved in ethanol until clear and transparent. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the chromatographic column is Waters sunfire C, 19, 250mm and 10 μm; the mobile phase is A:0.1% tfa in water, B: CH 3 OH, elution gradient as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 50 50
1 10 90
11 5 95
13 5 95
18 95 5
30 95 5
No target peak was detected.
Comparative example 15
25Mg of the sample (crude DMG-PEG2000 and standard) was dissolved in ethanol until clear and transparent. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the chromatographic column is Welch Ultimate XB-C4 (packing 70 g) 10 μm; the mobile phase is A:0.1% tfa in water, B: acetonitrile, elution gradient as follows:
no target peak was detected.
Comparative example 16
25Mg of the sample (crude DMG-PEG2000 and standard) was dissolved in ethanol until clear and transparent. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the chromatographic column is Welch Ultimate XB-C4 (packing 70 g) 10 μm; the mobile phase is A:0.1% tfa in water, B: methanol, elution gradient is as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 50 50
1 10 90
11 10 90
16 10 90
20 5 95
25 5 95
30 95 5
No target peak was detected.
Comparative example 17
25Mg of the sample (crude DMG-PEG2000 and standard) was dissolved in ethanol until clear and transparent. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the column was Waters sunfire C, 19 x 250mm10 μm; the mobile phase is A:0.1% tfa in water, B: CH 3 CN, elution gradient as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 50 50
2 13 87
11 10 90
14 5 95
16 5 95
18 95 5
30 95 5
No target peak was detected.
Comparative example 18
0.8G of DMG-PEG2000 crude product is taken and dissolved by 6mL of acetonitrile, and a clear solution is obtained by filtration after ultrasonic treatment. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the chromatographic column is Welch ultimate XB-C18-40 μm100G; mobile phase A is water, mobile phase B1 is acetonitrile, and B2 is dichloromethane; the flow rate is 14mL/min; the detection wavelength was 210nm. Elution gradients are as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 95 5
15 95 5
25 80 20
45 50 50
70 15 85
85 0 100
160 0 100(B1)
160.01 0 100(B2)
No target product was obtained.
Comparative example 19
0.5G of DMG-PEG2000 crude product is taken and dissolved by 4mL of acetonitrile, and a clear solution is obtained by filtration after ultrasonic treatment. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the chromatographic column is Welch ultimate XB-C18-40 μm100G; mobile phase A is acetonitrile, mobile phase B is dichloromethane; the flow rate is 14mL/min; the detection wavelength was 210nm. Elution gradients are as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 100 0
15 100 0
60 80 20
85 50 50
100 15 85
115 0 100
125 0 100
The separation effect is poor.
Inventive example 1
2.0G of the crude product (DMG-PEG 2000, purity 77%) after column chromatography (performed according to comparative example 11) was purified by reverse phase high performance liquid chromatography using the following chromatographic conditions: the chromatographic column is BUCHI, pure C-815 Flash C18 spherical 20-35 μm40G, methanol as mobile phase and flow rate of 45mL/min. The fractions were collected and concentrated to give 900mg of final product with a purity of 97.8% in CAD detection.
Inventive example 2
25Mg of crude product (DMG-PEG 2000) was taken and added with ethanol to give a clear and transparent product. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the column was Waters sunfire C, 19 x 250mm 10 μm; the mobile phase is A: acetonitrile, B: CH 3 OH, flow rate 20mL/min, elution gradient as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 90 10
1 70 30
13 10 90
15 5 95
16 5 95
18 95 5
And collecting fractions to obtain a target product, wherein the CAD detection purity is 97.77%.
Inventive example 3
25Mg of crude product (DMG-PEG 2000) was taken and dissolved in ethanol until clear and transparent. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the column was Waters sunfire C, 19 x 250mm10 μm; the mobile phase is A: acetonitrile, B: CH 3 OH, flow rate 20mL/min, elution gradient as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 50 50
1 15 85
13 10 90
15 5 95
16 5 95
18 95 5
Collecting the fraction to obtain the target product, wherein the CAD detection purity is 96.2%.
Inventive example 4
2.3G of crude product (DMG-PEG 2000) after normal phase separation (according to comparative example 9) were taken and dissolved with 16mL of acetonitrile, sonicated and filtered to give a clear solution. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the chromatographic column is Welch ultimate XB-C18-40 μm80G; the mobile phase is (A: acetonitrile, B: methanol); the flow rate is 14mL/min; the detection wavelength was 210nm. Elution gradients are as follows:
And collecting the target product to finally obtain 0.81g of the product, wherein the CAD detection purity is 98.4%.
Inventive example 5
The normal phase was separated (as in comparative example 9) and concentrated to give 0.48g of crude product (DMG-PEG 2000) which was dissolved in 5mL of acetonitrile, sonicated and filtered to give a clear solution. The separation is carried out by reversed phase high performance liquid chromatography, and the following chromatographic conditions are adopted: the chromatographic column is WELFLASH C, 20-40 μm and 120g; the mobile phase is (A: acetonitrile, B: methanol); the flow rate is 50mL/min; the detection wavelength was 210nm. Elution gradients are as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 100 0
16.3 100 0
25.9 81 19
48.4 47 53
71.5 16 84
89 0 100
99 0 100
And collecting fractions with CAD detection purity of more than 90 percent, and concentrating to obtain a final product, wherein the CAD detection purity is more than 93 percent.
Comparative example 20
25Mg of the sample (crude DMG-PEG2000 and standard) was dissolved in ethanol until clear and transparent. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the chromatographic column is Welch Ultimate XB-C4 (packing 70 g) 10 μm; the mobile phase is A: acetonitrile, B: methanol, elution gradient is as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 50 50
1 10 90
13 10 90
15 5 95
16 5 95
18 95 5
Collecting the fraction to obtain the target product, wherein the CAD detection purity is 85.23%.
Comparative example 21
25Mg of the sample (crude DMG-PEG2000 and standard) was dissolved in ethanol until clear and transparent. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the column was Waters sunfire C, 19 x 250mm10 μm; the mobile phase is A: acetonitrile, B: methanol, elution gradient is as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 95 5
1 90 10
13 80 20
15 5 95
16 5 95
18 95 5
The separation effect is poor.
Inventive example 6
25Mg of the sample (crude DMG-PEG2000 and standard) was dissolved in ethanol until clear and transparent. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the column was Waters sunfire C, 19 x 250mm10 μm; the mobile phase is A: acetonitrile, B: methanol, elution gradient is as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 90 10
1 90 10
13 70 30
15 5 95
16 5 95
18 95 5
And collecting fractions to obtain a target product, wherein the CAD detection purity is 99.41%.
Inventive example 7
25Mg of the sample (crude DMG-PEG2000 and standard) was dissolved in ethanol until clear and transparent. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the column was Waters sunfire C, 19 x 250mm10 μm; the mobile phase is A: acetonitrile, B: methanol, elution gradient is as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 50 50
1 10 90
13 10 90
15 5 95
16 5 95
18 95 5
Collecting the fraction to obtain the target product, wherein the purity of the CAD detection is 97.62%.
Preparation example 2: synthesis of DMG-PEG1000
The preparation process is as follows:
DMG-PEG1000 was prepared with reference to preparation example 1, except that 10.0g of compound 5 was used instead of compound 1 in preparation example 1.
The crude DMG-PEG1000 product mentioned in the following examples was obtained according to the present example in a purity of 64.8%.
Inventive example 8
250Mg of the crude product (DMG-PEG 1000) after column chromatography (performed according to comparative example 11) was taken and dissolved in ethanol until clear and transparent. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the column was Waters sunfire C, 19 x 250mm 10 μm; the mobile phase is A: acetonitrile, B: methanol, elution gradient is as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 50 50
1 20 80
13 15 85
15 5 95
16 95 5
The fractions were collected to give 25mg of the desired product with a CAD detection purity of 98.8%.
Preparation example 3: synthesis of DLG-PEG2000
DSG-PEG2000 was prepared with reference to preparation example 1, except that 324mg of lauric acid was used instead of myristic acid in preparation example 1.
The crude DLG-PEG2000 product mentioned in the following examples was a crude product of 78.09% purity obtained according to the present example.
Inventive example 9
250Mg of the crude product (crude DLG-PEG 2000) after column chromatography (carried out according to comparative example 11) was taken and dissolved in ethanol until clear and transparent. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the column was Waters sunfire C, 19 x 250mm 10 μm; the mobile phase is A: acetonitrile, B: methanol, elution gradient is as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 50 50
1 20 80
13 15 85
15 5 95
16 5 95
18 95 5
The fractions were collected to give 88mg of the desired product with a CAD detection purity of 98.98%.
Preparation example 4: synthesis of DSG-PEG2000
DSG-PEG2000 was prepared with reference to preparation example 1, except that 427mg of stearic acid was used instead of myristic acid in preparation example 1.
The crude DSG-PEG2000 product mentioned in the following examples was obtained according to this example with a purity of 28.51%.
Inventive example 10
250Mg of the crude product (crude DSG-PEG 2000) after column chromatography (performed according to comparative example 11) was taken and dissolved in ethanol until clear and transparent. Purification by reverse phase high performance liquid chromatography using the following chromatographic conditions: the column was Waters sunfire C, 19 x 250mm 10 μm; the mobile phase is A: acetonitrile, B: methanol, elution gradient is as follows:
Time (min) Mobile phase a (%) Mobile phase B (%)
0 50 50
1 10 90
13 9 91
15 5 95
16 5 95
18 95 5
The fractions were collected to give 54mg of the desired product with a CAD detection purity of 96.95%.
While particular embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are merely illustrative, and that many changes and modifications may be made to these embodiments without departing from the principles and spirit of the invention. Accordingly, the scope of the invention is defined by the appended claims.

Claims (22)

1. A method of purifying a polyethylene glycol conjugated lipid, the method comprising the steps of:
i) Dissolving polyethylene glycol conjugated lipid crude product in solvent; and
Ii) subjecting the solution obtained in i) to reversed-phase high performance liquid chromatography,
Wherein the chromatographic column used for the reversed-phase high-performance liquid chromatography is selected from C8 and C18 chromatographic columns, the mobile phase is mobile phase A, mobile phase B or a mixture thereof, the mobile phase A is acetonitrile, the mobile phase B is methanol, the volume ratio of the mobile phase A to the mobile phase B in the mixture is 5:95 to 95:5,
The reversed-phase high performance liquid chromatography is eluted by adopting a gradient elution mode, wherein the mobile phase is a mobile phase A or a mixture of the mobile phase A and the mobile phase B from 50:50 to 90:10 when the first gradient starts, the mobile phase is a mobile phase A or a mixture of the mobile phase A and the mobile phase B from 10:90 to 90:10 when the first gradient stops, and the running time of the first gradient is 1min-20min.
2. The method of claim 1, wherein the polyethylene glycol-based conjugated lipid is selected from the group consisting of DMG-PEG1000, DMG-PEG2000, DLG-PEG2000, and DSG-PEG2000.
3. The process according to claim 1 or 2, characterized in that the solvent is selected from ethanol, dichloromethane and acetonitrile.
4. A process according to claim 1 or 2, characterized in that in step i) the crude product is sonicated and filtered after being dissolved in a solvent.
5. The method according to claim 1 or 2, wherein the chromatography column is a C18 chromatography column having a particle size of 10-40 μm.
6. The method according to claim 1 or 2, wherein the chromatography column is a C8 chromatography column having a particle size of 10-40 μm.
7. The method according to claim 1 or 2, wherein the purification is performed at ambient temperature.
8. The method according to claim 1 or 2, wherein the gradient elution of the reversed phase high performance liquid chromatography comprises at least 3 gradients.
9. The method according to claim 1 or 2, wherein the gradient elution of the reversed phase high performance liquid chromatography comprises 4-6 gradients.
10. The method according to claim 1 or 2, characterized in that the total run time of the reversed phase high performance liquid chromatography is 15-100min.
11. The method according to claim 1 or 2, characterized in that the first gradient run time is 1min-17min.
12. The process according to claim 1 or 2, characterized in that in step i) normal phase high performance liquid chromatography is performed after dissolution of the crude product in a solvent, wherein the column used is a silica gel column, the mobile phase is a mixture of dichloromethane and methanol or dichloromethane, the volume ratio of dichloromethane and methanol in the mixture being from 70:30 to 90:10.
13. The method according to claim 12, wherein the particle size of the normal phase silica gel column is 10-150 μm.
14. The method according to claim 12, wherein the particle size of the normal phase silica gel column is 20-35 μm.
15. The method of claim 12, wherein the elution gradient of normal phase high performance liquid chromatography is:
Time (min) Mobile phase a (%) Mobile phase B (%) 0 100 0 2 100 0 52 90 10 67 90 10 82 70 30 117 70 30
。
16. The method according to claim 1 or 2, wherein the elution gradient of the reversed phase high performance liquid chromatography is as shown in any one of tables 1 to 9:
TABLE 1
Time (min) Mobile phase a (%) Mobile phase B (%) 0 90 10 1 70 30 13 10 90 15 5 95 16 5 95 18 95 5
TABLE 2
Time (min) Mobile phase a (%) Mobile phase B (%) 0 50 50 1 15 85 13 10 90 15 5 95 16 5 95 18 95 5
TABLE 3 Table 3
Time (min) Mobile phase a (%) Mobile phase B (%) 0 100 0 15 100 0 25 80 20 45 50 50 70 15 85 85 0 100 95 0 100
TABLE 4 Table 4
Time (min) Mobile phase a (%) Mobile phase B (%) 0 100 0 16.3 100 0 25.9 81 19 48.4 47 53 71.5 16 84 89 0 100 99 0 100
TABLE 5
Time (min) Mobile phase a (%) Mobile phase B (%) 0 90 10 1 90 10 13 70 30 15 5 95 16 5 95 18 95 5
TABLE 6
Time (min) Mobile phase a (%) Mobile phase B (%) 0 50 50 1 10 90 13 10 90 15 5 95 16 5 95 18 95 5
TABLE 7
Time (min) Mobile phase a (%) Mobile phase B (%) 0 50 50 1 20 80 13 15 85 15 5 95 16 95 5
TABLE 8
Time (min) Mobile phase a (%) Mobile phase B (%) 0 50 50 1 20 80 13 15 85 15 5 95 16 5 95 18 95 5
TABLE 9
Time (min) Mobile phase a (%) Mobile phase B (%) 0 50 50 1 10 90 13 9 91 15 5 95 16 5 95 18 95 5
。
17. A method of purifying a polyethylene glycol conjugated lipid, the method comprising the steps of:
i) Dissolving polyethylene glycol conjugated lipid crude product in solvent;
ii) subjecting the solution obtained in i) to normal phase silica gel column chromatography separation comprising eluting with dichloromethane first and then with a mixture of dichloromethane and methanol in a ratio of dichloromethane to methanol of from 20:1 to 40:1, and
Iii) And (3) carrying out reversed-phase high performance liquid chromatography on the normal phase chromatography product obtained in the ii), wherein the chromatographic column is a C18 chromatographic column, and the mobile phase is methanol.
18. The method of claim 17, wherein the polyethylene glycol-based conjugated lipid is selected from the group consisting of DMG-PEG1000, DMG-PEG2000, DLG-PEG2000, and DSG-PEG2000.
19. The method according to claim 17 or 18, wherein the solvent is selected from ethanol, dichloromethane and acetonitrile.
20. The method of claim 17 or 18, wherein the C18 chromatographic column has a particle size of 10-150 μm.
21. The method of claim 17 or 18, wherein the C18 chromatographic column has a particle size of 75-150 μm.
22. The method according to claim 17 or 18, wherein the purification is performed at ambient temperature.
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