CN114778712A - Polyethylene glycol lipid and detection method for content of lipid nanoparticles containing polyethylene glycol lipid - Google Patents
Polyethylene glycol lipid and detection method for content of lipid nanoparticles containing polyethylene glycol lipid Download PDFInfo
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- CN114778712A CN114778712A CN202210278380.XA CN202210278380A CN114778712A CN 114778712 A CN114778712 A CN 114778712A CN 202210278380 A CN202210278380 A CN 202210278380A CN 114778712 A CN114778712 A CN 114778712A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention discloses a polyethylene glycol lipid and a detection method of the content of lipid nanoparticles containing the lipid, the method is to detect by high performance liquid chromatography, the high performance liquid chromatography adopts reversed phase chromatography, the detector of the high performance liquid chromatography is an electrospray detector, the detection method can quickly and conveniently separate polyethylene glycol lipid samples and impurities, thereby effectively controlling the product quality of the polyethylene glycol lipid, and the method completely meets the standard in the aspects of system applicability, specificity, detectability, quantitativity, linear range, recovery rate, repeatability and the like and has higher durability.
Description
Technical Field
The invention relates to the technical field of chemical analysis, in particular to a method for detecting lipid nanoparticles by a high performance liquid chromatography tandem mass spectrometry technology.
Background
The central principle of mRNA vaccines is to encode the genetic information of the antigen in mRNA, which is then delivered into the cytoplasm of the host cell, expressed in vivo and induces an antigen-specific immune response. One of the challenges in developing mRNA vaccines is their poor stability, because the single-chain structure of mRNA itself makes mRNA extremely unstable and easily degradable, and since it carries negative charges, it is difficult to deliver mRNA across cell membrane with negative charges on its surface, so special modification or coating of delivery system is needed to achieve intracellular expression of mRNA, change the intracellular biodistribution, cell targeting and uptake mechanism of mRNA, promote mRNA delivery, and exert the effect of vaccine.
LNPs are one of the most widely used delivery systems in nucleic acid drug research, and LNPs can deliver nucleic acids safely and efficiently, and have many advantages over other types of nucleic acid drug delivery systems, such as high encapsulation efficiency of nucleic acids, efficient transfection of cells, high tissue penetration, low cytotoxicity and immunogenicity, and better drug delivery, which make LNPs excellent nucleic acid delivery systems. The current mRNA vaccines are essentially using LNP technology.
LNP in mRNA vaccines consists of four major components: neutral phospholipids, cholesterol, polyethylene glycol lipids, and ionizable lipids. Wherein the polyethylene glycol lipid is used for controlling particle size and acting as a steric barrier to stabilize and prevent aggregation of LNP microparticles during storage.
M-DMG, one of the polyethylene glycol lipids, is also particularly important in LNP delivery systems. However, in the prior art, there is no suitable detection method for the above-mentioned polymers.
M-DMG is a less polar, large molecule compound that elutes poorly on a C18 column, and some known and unknown impurities that are mixed with this compound are more poorly eluted. Therefore, in reverse phase chromatography using a C18 column as a main detection means, the substance is not easily detected and separated; since the material does not absorb ultraviolet light, it cannot be detected by a selective detector, an ultraviolet detector. At present, no report exists on a specific determination method of the polymer M-DMG, and therefore, the specific determination method for establishing the polymer M-DMG has important significance in the aspect of quality control of polyethylene glycol lipid, lipid nanoparticle, nucleic acid pharmaceutical composition and preparation thereof.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the polyethylene glycol lipid and the detection method of the content of the lipid nanoparticles containing the polyethylene glycol lipid, which can effectively separate products and impurities, have good chromatographic peak shape, can effectively react the purity of the products, and have good separation degree and repeatability and high column efficiency.
The invention provides a method for detecting the content of polyethylene glycol lipid, which is to detect the polyethylene glycol lipid through a high performance liquid chromatography, wherein a detector of the high performance liquid chromatography is an electrospray detector, namely a Charged Aerosol Detector (CAD).
The polyethylene glycol lipid may be selected from: 2- [ (polyethylene glycol) -2000] -N, N-tetracosanyl acetamide (ALC-0159)1, 2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (M-DMG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE), PEG-distearoyl glycerol (PEG-DSG), PEG-dipalmitoyl, one of PEG-dioleyl, PEG-distearyl, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), and PEG-1, 2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).
Preferably, the polyethylene glycol lipid has a structural formula of
n is the degree of polymerization of the polyethylene glycol residue, L1And L2Each independently is C4-C20Alkyl or C4-C20An alkenyl group.
More preferably, said L1And L2Each independently is C4-C20Alkyl (e.g. C)4Alkyl radical, C5Alkyl radical, C6Alkyl radical, C7Alkyl radical, C8Alkyl radical, C9Alkyl radical, C10Alkyl radical, C11Alkyl radical, C12Alkyl radical, C13Alkyl radical, C14Alkyl radical, C15Alkyl radical, C16Alkyl radical, C17Alkyl radical, C18Alkyl radical, C19Alkyl radical, C20Alkyl), particularly preferably, said L1And L2Each independently is C10-C18Alkyl, most preferably, said L1And L2Each independently is C13An alkyl group.
In one embodiment of the present invention, the polyethylene glycol lipid is M-DMG, which has the structural formula
The molecular weight is 1500-2500 daltons, preferably 2000 daltons; n is the degree of polymerization of the polyethylene glycol residue.
The chromatographic conditions are as follows:
the high performance liquid chromatography adopts reversed phase chromatography.
The stationary phase of the chromatographic column of the high performance liquid chromatography is butyl silane bonded silica gel.
The particle size of the stationary phase is 2-15 μm (such as 2 μm, 3 μm, 5 μm, 10 μm, 15 μm), preferably, the particle size of the stationary phase is 3-10 μm, and more preferably, the particle size of the stationary phase is 5 μm.
The aperture of the stationary phase is
(e.g. using
) Preferably, the pore size of the stationary phase is
More preferably, the pore size of the stationary phase is
The length of the chromatographic column is 100-300 mm (such as 100mm, 150mm, 220mm, 230mm, 240mm, 250mm, 260mm, 270mm, 280mm, 290mm and 300mm), and preferably the length of the chromatographic column is 250 mm.
The diameter of the chromatographic column is 4-8 mm (such as 4.6mm and 7.8mm), preferably, the diameter of the chromatographic column is 4.6 mm.
The mobile phase of the high performance liquid chromatography comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is trifluoroacetic acid aqueous solution, the mobile phase B is acetonitrile or methanol, and the mobile phase A + the mobile phase B is 100%.
Further, the mobile phase A is 0.01-0.5% trifluoroacetic acid aqueous solution, preferably, the mobile phase A is 0.1% trifluoroacetic acid aqueous solution; the mobile phase B is acetonitrile.
The volume ratio of trifluoroacetic acid to water in the mobile phase A is 0.1-1: 100, and preferably the volume ratio of trifluoroacetic acid to water in the mobile phase A is 0.1-100.
The flow rate of the mobile phase is 0.5-1.5 mL/min (such as 0.8, 0.9, 1.0, 1.1, 1.2mL/min), preferably, the flow rate of the mobile phase is 1.0 mL/min.
The column temperature of the chromatography column is 30-45 ℃ (e.g., 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 45 ℃), preferably, the column temperature of the chromatography column is 35-40 ℃.
In one embodiment of the invention, the column temperature of the chromatography column is 35 ℃.
In one embodiment of the invention, the column temperature of the chromatography column is 40 ℃.
The sample injection amount of the high performance liquid chromatography is 5-15 muL (such as 5, 8, 9, 10, 11, 12 and 15 muL), and preferably, the sample injection amount of the high performance liquid chromatography is 8-12 muL.
In one embodiment of the present invention, the sample volume of the high performance liquid chromatography is 10. mu.L.
The high performance liquid chromatography adopts a gradient elution program which comprises the following steps:
volume concentration of mobile phase at 0 min elution time: a mobile phase A: 85-95%, mobile phase B: the balance;
when the elution time is 0-2 minutes, the volume concentration of the mobile phase is as follows: a mobile phase A: the gradient change is 25-35% from 85-95%, and the mobile phase B: the balance;
when the elution time is 2-18 minutes, the volume concentration of the mobile phase is as follows: mobile phase A: the gradient change is 2-8% from 25-35%, and the mobile phase B: the balance;
when the elution time is 18-22 minutes, maintaining isocratic elution, and the volume concentration of a mobile phase is as follows: a mobile phase A: 2-8%, mobile phase B: the balance;
when the elution time is 22-23 minutes, the volume concentration of the mobile phase is as follows: a mobile phase A: the gradient change is 85-95% from 2-8%, and the mobile phase B: the balance;
when the elution time is 23-30 minutes, keeping isocratic elution, and obtaining the following mobile phase by volume concentration: mobile phase A: 85-95%, mobile phase B: and (4) the balance.
In one embodiment of the invention, the gradient elution procedure is as follows:
volume concentration of mobile phase at 0 min elution time: mobile phase A: 90%, mobile phase B: the balance;
when the elution time is 0-2 minutes, the volume concentration of the mobile phase is as follows: mobile phase A: from a 90% gradient change to 30%, mobile phase B: the balance;
when the elution time is 2-18 minutes, the volume concentration of the mobile phase is as follows: a mobile phase A: gradient change from 30% to 5%, mobile phase B: the balance;
when the elution time is 18-22 minutes, keeping isocratic elution, and obtaining the following mobile phase by volume concentration: mobile phase A: 5%, mobile phase B: the balance;
when the elution time is 22-23 minutes, the volume concentration of the mobile phase is as follows: a mobile phase A: change from 5% gradient to 90%, mobile phase B: the balance;
when the elution time is 23-30 minutes, maintaining isocratic elution, and the volume concentration of the mobile phase is as follows: mobile phase A: 90%, mobile phase B: and (4) the balance.
Further, the parameters of the electrospray detector are as follows:
temperature: 30 ℃ to 55 ℃ (e.g., 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃);
a Filter: 0.1sec to 10sec (e.g., 0.1sec, 0.2sec, 0.5sec, 1.0sec, 2.0sec, 3.6sec, 5.0sec, 10.0 sec);
range: 1pA to 500pA (for example, 1pA, 2pA, 5pA, 10pA, 20pA, 50pA, 100pA, 200pA, 500 pA).
Further, the high performance liquid chromatograph in the high performance liquid chromatograph may be shimadzu LC-16 high performance liquid chromatograph.
Further, the chromatographic column of the high performance liquid chromatograph can be a ChromeCore 300(C4, 4.6X 250 mm; 5 mu m) chromatographic column.
Furthermore, the method for detecting the content of the polymer M-DMG is an external standard method.
The second aspect of the invention provides a method for detecting the content of lipid nanoparticles, wherein the lipid nanoparticles comprise polyethylene glycol lipid, the detection method is to detect the polyethylene glycol lipid by high performance liquid chromatography, and a detector of the high performance liquid chromatography is an electrospray detector, also known as a Charged Aerosol Detector (CAD).
Further, the lipid nanoparticle also comprises a steroid lipid, a cationic lipid and a neutral lipid.
Further, the steroidal lipid is selected from the group consisting of avenasterol, beta-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, echinosterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol; one of lanosterol, photosterol, fucosterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid.
Further, the cationic lipid is selected from one of (2, 3-dioleoxypropyl) trimethylammonium chloride (DOTAP), N- [1- (2, 3-dioleoyl chloride) propyl ] -N, N-trimethylamine chloride (DOTMA), dimethyl-2, 3-dioleyloxypropyl-2- (2-sperminoylamido) ethylammonium (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), and dimethyldioctadecylammonium bromide (DDAB).
Further, the neutral lipid is selected from 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1, 2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho- (1' -rac-glycerol) (DOPG), oleoyl phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE).
The polyethylene glycol lipid may be selected from: 2- [ (polyethylene glycol) -2000] -N, N-tetracosanyl acetamide (ALC-0159)1, 2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (M-DMG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE), PEG-distearoyl glycerol (PEG-DSG), PEG-dipalmitoyl, one of PEG-dioleyl, PEG-distearyl, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), and PEG-1, 2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).
Preferably, the polyethylene glycol lipid has a structural formula of
n is the degree of polymerization of the polyethylene glycol residue, L1And L2Each independently is C4-C20Alkyl or C4-C20An alkenyl group.
More preferably, said L1And L2Each independently is C4-C20Alkyl (e.g. C)4Alkyl radical, C5Alkyl radical, C6Alkyl radical, C7Alkyl radical, C8Alkyl radical, C9Alkyl radical, C10Alkyl radical, C11Alkyl radical, C12Alkyl radical, C13Alkyl radical, C14Alkyl radical, C15Alkyl radical, C16Alkyl radical, C17Alkyl radical, C18Alkyl radical, C19Alkyl radical, C20An alkyl group),particularly preferably, said L1And L2Each independently is C10-C18Alkyl, most preferably, said L1And L2Each independently is C13An alkyl group.
In one embodiment of the present invention, the polyethylene glycol lipid is M-DMG, which has the structural formula
The molecular weight is 1500-2500 daltons, preferably 2000 daltons; and n is the polymerization degree of polyethylene glycol residue.
The chromatographic conditions are as follows:
the high performance liquid chromatography adopts reversed phase chromatography.
The stationary phase of the chromatographic column of the high performance liquid chromatography is butylsilane bonded silica gel.
The particle size of the stationary phase is 2-15 μm (such as 2 μm, 3 μm, 5 μm, 10 μm, 15 μm), preferably, the particle size of the stationary phase is 3-10 μm, and more preferably, the particle size of the stationary phase is 5 μm.
The aperture of the stationary phase is
(e.g. using
) Preferably, the pore size of the stationary phase is
More preferably, the pore size of the stationary phase is
The length of the chromatographic column is 100-300 mm (such as 100mm, 150mm, 220mm, 230mm, 240mm, 250mm, 260mm, 270mm, 280mm, 290mm, 300mm), preferably the length of the chromatographic column is 250 mm.
The diameter of the chromatographic column is 4-8 mm (such as 4.6 and 7.8mm), and preferably the diameter of the chromatographic column is 4.6 mm.
The mobile phase of the high performance liquid chromatography comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is trifluoroacetic acid aqueous solution, the mobile phase B is acetonitrile or methanol, and the mobile phase A + the mobile phase B is 100%.
Further, the mobile phase A is 0.01-0.5% trifluoroacetic acid aqueous solution, preferably, the mobile phase A is 0.1% trifluoroacetic acid aqueous solution; the mobile phase B is acetonitrile.
The volume ratio of trifluoroacetic acid to water in the mobile phase A is 0.1-1: 100, and preferably the volume ratio of trifluoroacetic acid to water in the mobile phase A is 0.1-100.
The flow rate of the mobile phase is 0.5-1.5 mL/min (such as 0.8, 0.9, 1.0, 1.1, 1.2mL/min), preferably, the flow rate of the mobile phase is 1.0 mL/min.
The column temperature of the chromatography column is 30-45 ℃ (e.g., 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 45 ℃), preferably, the column temperature of the chromatography column is 35-40 ℃.
In one embodiment of the invention, the column temperature of the chromatography column is 35 ℃.
In one embodiment of the invention, the column temperature of the chromatography column is 40 ℃.
The sample injection amount of the high performance liquid chromatography is 5-15 muL (such as 5, 8, 9, 10, 11, 12 and 15 muL), and preferably, the sample injection amount of the high performance liquid chromatography is 8-12 muL.
In one embodiment of the invention, the sample size of the high performance liquid chromatography is 10. mu.L.
The high performance liquid chromatography adopts a gradient elution program which comprises the following steps:
volume concentration of mobile phase at 0 min elution time: a mobile phase A: 85-95%, mobile phase B: the balance;
when the elution time is 0-2 minutes, the volume concentration of the mobile phase is as follows: a mobile phase A: the gradient change is 25-35% from 85-95%, and the mobile phase B: the balance;
when the elution time is 2-18 minutes, the volume concentration of the mobile phase is as follows: mobile phase A: the gradient change is 2-8% from 25-35%, and the mobile phase B: the balance;
when the elution time is 18-22 minutes, maintaining isocratic elution, and the volume concentration of a mobile phase is as follows: mobile phase A: 2-8%, mobile phase B: the balance;
when the elution time is 22-23 minutes, the volume concentration of the mobile phase is as follows: mobile phase A: the gradient change is 85-95% from 2-8%, and the mobile phase B: the balance;
when the elution time is 23-30 minutes, maintaining isocratic elution, and the volume concentration of the mobile phase is as follows: mobile phase A: 85-95%, mobile phase B: and (4) the balance.
In one embodiment of the invention, the gradient elution procedure is as follows:
volume concentration of mobile phase at 0 min elution time: mobile phase A: 90%, mobile phase B: the balance;
when the elution time is 0-2 minutes, the volume concentration of the mobile phase is as follows: mobile phase A: from a 90% gradient change to 30%, mobile phase B: the balance;
when the elution time is 2-18 minutes, the volume concentration of the mobile phase is as follows: mobile phase A: gradient change from 30% to 5%, mobile phase B: the balance;
when the elution time is 18-22 minutes, keeping isocratic elution, and obtaining the following mobile phase by volume concentration: mobile phase A: 5%, mobile phase B: the balance;
when the elution time is 22-23 minutes, the volume concentration of the mobile phase is as follows: mobile phase A: change from 5% gradient to 90%, mobile phase B: the balance;
when the elution time is 23-30 minutes, maintaining isocratic elution, and the volume concentration of the mobile phase is as follows: mobile phase A: 90%, mobile phase B: and the balance.
Further, the parameters of the electrospray detector are as follows:
temperature: 30 to 55 ℃ (e.g., 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃);
and (3) Filter: 0.1sec to 10sec (e.g., 0.1sec, 0.2sec, 0.5sec, 1.0sec, 2.0sec, 3.6sec, 5.0sec, 10.0 sec);
range: 1pA to 500pA (for example, 1pA, 2pA, 5pA, 10pA, 20pA, 50pA, 100pA, 200pA, 500 pA).
Further, the high performance liquid chromatograph in the high performance liquid chromatograph may be shimadzu LC-16 high performance liquid chromatograph.
Further, the column of the high performance liquid chromatograph may be a ChromeCore 300(C4, 4.6X 250 mm; 5 μm) column.
Further, the method for detecting the content of the lipid nanoparticles is an external standard method.
The third aspect of the invention provides the use of the detection method of the first or second aspect of the invention in the quality assessment of the polymer M-DMG.
In a fourth aspect, the invention provides the use of the detection method of the first or second aspect in the quality evaluation of polyethylene glycol lipid, preferably, the polyethylene glycol lipid comprises the polymer M-DMG.
In a fifth aspect, the invention provides an application of the detection method of the first or second aspect in quality evaluation of lipid nanoparticles, wherein the lipid nanoparticles comprise polyethylene glycol lipid.
Further, the lipid nanoparticle also comprises a steroid lipid, a cationic lipid and a neutral lipid.
Further, the steroidal lipid is selected from the group consisting of avenasterol, beta-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, echinosterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol; one of lanosterol, photosterol, fucosterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid.
Further, the cationic lipid is selected from one of (2, 3-dioleoxypropyl) trimethylammonium chloride (DOTAP), N- [1- (2, 3-dioleoyl chloride) propyl ] -N, N-trimethylamine chloride (DOTMA), dimethyl-2, 3-dioleyloxypropyl-2- (2-sperminoylamido) ethylammonium (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), and dimethyldioctadecylammonium bromide (DDAB).
Further, the neutral lipid is selected from 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1, 2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine DOPE), 1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho- (1' -rac-glycerol) (DOPG), oleoyl phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE).
The sixth aspect of the present invention provides an application of the detection method of the first aspect or the second aspect of the present invention in quality evaluation of a nucleic acid pharmaceutical composition and a preparation thereof, where the nucleic acid pharmaceutical composition and the preparation thereof include lipid nanoparticles, and the lipid nanoparticles include polyethylene glycol lipid.
The detection method can quickly and conveniently separate the polyethylene glycol lipid sample and impurities, so that the product quality of the polyethylene glycol lipid is effectively controlled, and the method completely meets the standard in the aspects of system applicability, specificity, detectability, quantitativity, linear range, recovery rate, repeatability and the like and has higher durability.
Drawings
FIG. 1 is a liquid chromatogram of a 100% sample solution.
FIG. 2 is a liquid chromatogram of a blank solution.
Fig. 3 is a liquid chromatogram of impurity 1.
Fig. 4 is a liquid chromatogram of impurity 2.
Fig. 5 is a liquid chromatogram of impurity 3.
Fig. 6 is a liquid chromatogram of impurity 4.
Fig. 7 is a liquid chromatogram of impurity 5.
FIG. 8 is a graph showing the linear relationship between control solutions 1 to 6.
FIG. 9 is a graph showing the linear relationship between control solutions G1-G5.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below with reference to embodiments of the present invention, and it should be apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
The term "cationic lipid" according to the present invention refers to a lipid molecule capable of being positively charged.
The term "neutral lipid" as used herein refers to an uncharged, non-phosphoglyceride lipid molecule.
The term "pegylated lipid" according to the present invention refers to a molecule comprising a lipid moiety and a pegylated moiety.
The term "lipid nanoparticle" of the present invention refers to a particle having at least one nanoscale dimension, which comprises at least one lipid.
The term "C" according to the invention4-C20Alkyl "includes C4Straight chain/branched alkyl, C5Straight chain/branched alkyl, C6Straight chain/branched alkyl, C7Straight chain/branched alkyl, C8Straight chain/branched alkyl, C9Straight chain/branched alkyl, C10Straight chain/branched alkyl, C11Straight chain/branched alkyl, C12Straight chain/branched alkyl, C13Straight chain/branched alkyl, C14Straight chain/branched alkyl, C15Straight chain/branched alkyl, C16Straight chain/branched alkyl, C17Straight chain/branched alkyl, C18Straight chain/branched alkyl, C19Straight chain/branched alkyl, C20Straight chain/branched alkyl.
The polymer M-DMG-2000 described in the examples of this application has the structural formula
The molecular weight is 2000 daltons; n is the polymerization degree of polyethylene glycol residue and is derived from M-DMG-2000 sold by Kerkin technology.
According to the detection method, 1mL of trifluoroacetic acid is measured in 0.1% trifluoroacetic acid aqueous solution and added into 1000 liters of purified water to prepare 0.1% trifluoroacetic acid aqueous solution. Diluent (b): acetonitrile is used as a diluent.
Example 1
HPLC detection of M-DMG-2000:
the instrument comprises the following steps: shimadzu LC-16 high performance liquid chromatograph detector: thermo Veo RS detector column: butylsilane bonded silica gel as filler (ChromeCore 300C 44.6X 250 mm; 5 μm)
Mobile phase A: as mobile phase a, 0.1% aqueous trifluoroacetic acid was used.
And (3) mobile phase B: acetonitrile is used as mobile phase B
Range was detected using an electrospray detector (CAD): 500 pA; a filter: 10 sec; tem 35 deg.C
Gradient elution was performed as in table 1 below:
elution procedure
TABLE 1
| Time | Mobile phase A | |
| 0 | 90 | 10 |
| 2 | 30 | 70 |
| 18 | 5 | 95 |
| 22 | 5 | 95 |
| 23 | 90 | 10 |
| 30 | 90 | 10 |
Column temperature: 40 deg.C
Flow rate: 1.0mL/min
Sample injection amount: 10 μ L
A workstation: LC-Solution
100% test solution: precisely weighing 20mg of the M-DMG-2000 to-be-detected sample, placing the M-DMG-2000 to-be-detected sample into a 10mL measuring flask, adding acetonitrile to dissolve the M-DMG-2000 to-be-detected sample, and shaking up to a constant volume to obtain a sample solution.
1% test article solution: precisely measuring 1mL of the 100% test sample solution, placing the 100mL test sample solution in a 100mL capacity product, adding acetonitrile to a constant volume to a scale, and shaking up to obtain a 1% test sample solution.
Blank solution: pure acetonitrile
Precisely measuring the 100% sample solution, 1% sample solution and blank solution, respectively, 10 μ L injecting into a liquid chromatograph, recording chromatogram for 30 min, and calculating sample purity according to high and low concentration method.
Wherein, the liquid chromatogram of 100% sample solution is shown in figure 1 and table 2; the liquid chromatogram of the blank solution is shown in FIG. 2, which indicates that the blank solution is not interfered; the liquid chromatogram of the impurities 1-5 is shown in FIGS. 3-7, and the peak tables corresponding to the impurities 1-5 are shown in tables 3-7, which indicates that the impurities do not interfere with the main peak of the sample.
TABLE 2
| Peak number | Retention time | Name of Compound | Area of | Height | Area% | S/N |
| 1 | 93743 | 89080 | 6338 | 0.503 | 64.15 | |
| 2 | 15.479 | 25380 | 1769 | 0.143 | 17.90 | |
| 3 | 16.458 | M-DMG-2000 | 17594642 | 420879 | 99.354 | 4259.47 |
| Total of | 17709102 | 428986 | 100.000 |
TABLE 3
| Number of peak | Retention time | Area of | Area% | Height | Degree of separation (USP) | Theoretical towerPlate number (USP) | Concentration of |
| 1 | 7.629 | 5346529 | 100.000 | 682763 | -- | 25599 | 0.000 |
| Total of | 5346529 | 100.000 | 682763 |
TABLE 4
| Peak number | Retention time | Area of | Area% | Height | Degree of separation (USP) | Number of theoretical plate (USP) | Concentration of |
| 1 | 7.505 | 4760449 | 100.000 | 670622 | -- | 22889 | 0.000 |
| Total of | 4760449 | 100.000 | 670622 |
TABLE 5
| Number of peak | Retention time | Area of | Area% | Height | Degree of separation (USP) | Theoretical plate number (USP) | Concentration of |
| 1 | 7.559 | 6428914 | 100.000 | 725853 | -- | 19254 | 0.000 |
| In total | 6428914 | 100.000 | 725853 |
TABLE 6
| Number of peak | Retention time | Area of | Area% | Height | Degree of separation (USP) | Theoretical plate number (USP) | Concentration of |
| 1 | 9.774 | 8330882 | 100.000 | 456778 | -- | 6820 | 100.000 |
| In total | 8330882 | 100.000 | 456778 |
TABLE 7
| Peak number | Retention time | Area of | Area% | Height | Degree of separation (USP) | Number of theoretical plate (USP) | Concentration of |
| 1 | 10.226 | 10996754 | 100.000 | 468162 | -- | 4436 | 100.000 |
| In total | 10996754 | 100.000 | 468162 |
Example 2
The sample detection adopts a self-contrast method to calculate the purity, and the high concentration and the low concentration are linearly verified.
Preparing a reference substance stock solution and a reference substance solution:
(1) control stock solution 1 (about 2mg/mL)
Precisely weighing about 20mgM-DMG-2000, placing in a 10mL measuring flask, adding acetonitrile to dissolve, fixing the volume to the scale, and shaking up.
(2) Control stock solution 2 (about 0.2mg/mL)
Precisely measuring 1mL of reference stock solution 1, placing in a 10mL measuring flask, adding diluent to a constant volume to reach a scale, and shaking up.
(3) Control solution 1 (about 0.1mg/mL)
Precisely measuring 5mL of reference substance stock solution 2, placing the reference substance stock solution in a 10mL measuring flask, adding a diluent to a constant volume to be scaled, and shaking up.
(4) Control solution 2 (about 0.05mg/mL)
Precisely measuring 5mL of reference solution 1, placing in a 10mL measuring flask, adding diluent to a constant volume to a scale, and shaking up.
(5) Control solution 3 (about 0.02mg/mL)
Precisely measuring 2mL of reference solution 1, placing in a 10mL measuring flask, adding diluent to a constant volume to a scale, and shaking up.
(6) Control solution 4 (about 0.01mg/mL)
Precisely measuring 1mL of reference solution 1, placing in a 10mL measuring flask, adding diluent to constant volume to reach the scale, and shaking up.
(7) Control solution 5 (about 0.005mg/mL)
Precisely measuring 5mL of the reference solution 4, placing the reference solution in a 10mL measuring flask, adding a diluent to a constant volume to a scale, and shaking up.
(8) Control solution 6(LOQ) (about 0.002mg/mL)
Precisely measuring 2mL of the reference solution 4, placing the reference solution in a 10mL measuring flask, adding the diluent to a constant volume to be calibrated, and shaking up.
(9) Control solution G-1 (about 3mg/mL)
Accurately weighing about 15mgM-DMG-2000, placing in a 5mL measuring flask, adding acetonitrile to dissolve, fixing the volume to the scale, and shaking up.
(10) Control solution G-2 (about 2.4mg/mL)
Accurately weighing about 12mgM-DMG-2000, placing in a 5mL measuring flask, adding acetonitrile to dissolve, metering to a scale, and shaking up.
(11) Control solution G-3 (about 2mg/mL)
Accurately weighing about 20mgM-DMG-2000, placing in a 10mL measuring flask, adding acetonitrile to dissolve, metering to a scale, and shaking up.
(12) Control solution G-4 (about 1.5mg/mL)
Precisely measuring 2.5mL of reference solution G-1, placing in a 5mL measuring flask, adding diluent to a constant volume to reach a scale, and shaking up.
(13) Control solution G-5 (about 1.2mg/mL)
Precisely measuring 2.5mL of reference solution G-2, placing in a 5mL measuring flask, adding diluent to constant volume to reach a scale, and shaking up.
Preparing a repetitive solution:
precisely weighing about 20mgM-DMG-2000 parts, respectively placing into 10mL measuring bottles, adding acetonitrile to dissolve, fixing the volume to the scale, and shaking up.
The results of the repeatability experiments are shown in table 8:
TABLE 8
And (4) conclusion: 5 sample solutions were prepared, respectively, and the RSD of the area/concentration ratio was 1.15%, indicating that the reproducibility of the analysis method was good.
And (3) preparing a recovery sample:
the samples were weighed according to table 9 below, dissolved in acetonitrile and diluted to volume. As a recovery solution.
TABLE 9
| Item | Weighing sample | Number of parts | Dilution volume |
| Reference substance | 20mg | 1 | 10ml |
| 80％ | 16mg | 3 | |
| 100％ | 20mg | 3 | 10ml |
| 120％ | 24mg | 3 | 10ml |
The results of the recovery experiments are shown in table 10:
watch 10
And (4) conclusion: samples with the concentrations of 80%, 100% and 120% are respectively prepared, the recovery rate is 92.0% -104.5%, and the RED is less than 10%, which shows that the method has good accuracy.
The results of the control solutions 1 to 6 are shown in Table 11, and a linear relationship chart obtained by plotting the results of Table 11 is shown in FIG. 8.
TABLE 11
| Serial number | 1 | 2 | 3 | 4 | 5 | 6 |
| Control concentration (mg/mL) | 0.102 | 0.051 | 0.0204 | 0.0102 | 0.0051 | 0.00204 |
| Peak area | 1331233 | 702879 | 281260 | 126374 | 53518 | 18758 |
And (4) conclusion: M-DMG-2000 has a linear response in the range of 0.002-0.1mg/mL, R20.998, indicating that the linearity of the analytical method is good.
The results of the control solutions G1-G5 are shown in Table 12, and the results in Table 12 are plotted to obtain a linear relationship chart shown in FIG. 9.
TABLE 12
| Serial number | 1 | 2 | 3 | 4 | 5 |
| Control concentration (mg/mL) | 3.16 | 2.53 | 2.11 | 1.67 | 1.1.05 |
| Peak area | 20961356 | 17881366 | 16084820 | 13733786 | 9940340 |
And (4) conclusion: M-DMG-2000 is in the range of 1-3mg/mL, the response is linear, R2A value of 0.995 indicates good linearity for this analytical method.
Claims (14)
1. The detection method of the content of the polyethylene glycol lipid is characterized in that the structural formula of the polyethylene glycol lipid is shown in the specification
n is the degree of polymerization of the polyethylene glycol residue, L1And L2Each independently is C4-C20Alkyl or C4-C20The method comprises the step of detecting through high performance liquid chromatography, wherein the high performance liquid chromatography adopts reversed phase chromatography, the detector of the high performance liquid chromatography is an electrospray detector, the stationary phase of a chromatographic column of the high performance liquid chromatography is butylsilane bonded silica gel, and the aperture of the stationary phase is
2. A polyethylene glycol lipid content of claim 1The detection method of (1), wherein L is1And L2Each independently is C4-C20Alkyl, preferably said L1And L2Each independently is C10-C18Alkyl, more preferably, said L1And L2Each independently is C13An alkyl group.
3. The method for detecting the content of the polyethylene glycol lipid according to claim 1, wherein the polyethylene glycol lipid is M-DMG with the structural formula
The molecular weight is 1500-2500 daltons, preferably 2000 daltons.
4. The method for detecting the content of polyethylene glycol lipid according to claim 1, wherein the pore size of the stationary phase is
Preferably, the pore size of the stationary phase is
5. The method for detecting the content of polyethylene glycol lipid according to claim 1, wherein the particle size of the stationary phase is 2 to 15 μm, preferably the particle size of the stationary phase is 3 to 10 μm, and more preferably the particle size of the stationary phase is 5 μm.
6. The method for detecting the content of polyethylene glycol lipid according to claim 1, wherein the mobile phase of the high performance liquid chromatography comprises a mobile phase A and a mobile phase B, the mobile phase A is aqueous trifluoroacetic acid, the mobile phase B is acetonitrile or methanol, preferably, the mobile phase A is 0.01-0.5% aqueous trifluoroacetic acid, the mobile phase B is acetonitrile, more preferably, the mobile phase A is 0.1% aqueous trifluoroacetic acid, and the mobile phase B is acetonitrile.
7. The method for detecting the content of polyethylene glycol lipid according to claim 1, wherein the flow rate of the mobile phase is 0.5-1.5 mL/min, preferably 1.0 mL/min.
8. The method for detecting the content of polyethylene glycol lipid according to claim 1, wherein the column temperature of the chromatographic column is 30-45 ℃, preferably 35-40 ℃.
9. The method for detecting the content of polyethylene glycol lipid according to claim 1, wherein the high performance liquid chromatography adopts a gradient elution procedure, and the gradient elution procedure is as follows:
volume concentration of mobile phase at 0 minute of elution time: a mobile phase A: 85-95%, mobile phase B: the balance;
when the elution time is 0-2 minutes, the volume concentration of the mobile phase is as follows: mobile phase A: the gradient change is 25-35% from 85-95%, and the mobile phase B: the balance;
when the elution time is 2-18 minutes, the volume concentration of the mobile phase is as follows: a mobile phase A: the gradient change is 2-8% from 25-35%, and the mobile phase B: the balance;
when the elution time is 18-22 minutes, maintaining isocratic elution, and the volume concentration of a mobile phase is as follows: mobile phase A: 2-8%, mobile phase B: the balance;
when the elution time is 22-23 minutes, the volume concentration of the mobile phase is as follows: mobile phase A: the gradient change is 85-95% from 2-8%, and the mobile phase B: the balance;
when the elution time is 23-30 minutes, maintaining isocratic elution, and the volume concentration of the mobile phase is as follows: mobile phase A: 85-95%, mobile phase B: and (4) the balance.
10. The method for detecting the content of polyethylene glycol lipid according to claim 1, wherein the high performance liquid chromatography adopts a gradient elution procedure, and the gradient elution procedure is as follows:
volume concentration of mobile phase at 0 min elution time: mobile phase A: 90%, mobile phase B: the balance;
when the elution time is 0-2 minutes, the volume concentration of the mobile phase is as follows: mobile phase A: from a 90% gradient change to 30%, mobile phase B: the balance;
when the elution time is 2-18 minutes, the volume concentration of the mobile phase is as follows: mobile phase A: gradient change from 30% to 5%, mobile phase B: the balance;
when the elution time is 18-22 minutes, keeping isocratic elution, and obtaining the following mobile phase by volume concentration: mobile phase A: 5%, mobile phase B: the balance;
when the elution time is 22-23 minutes, the volume concentration of the mobile phase is as follows: mobile phase A: gradient change from 5% to 90%, mobile phase B: the balance;
when the elution time is 23-30 minutes, maintaining isocratic elution, and the volume concentration of the mobile phase is as follows: a mobile phase A: 90%, mobile phase B: and the balance.
11. A method for detecting the content of lipid nanoparticles is characterized in that the lipid nanoparticles contain polyethylene glycol lipid, the detection method is to detect the polyethylene glycol lipid by high performance liquid chromatography-mass spectrometry, and the structural formula of the polyethylene glycol lipid is shown in the specification
n is the degree of polymerization of the polyethylene glycol residue, L1And L2Each independently is C4-C20Alkyl or C4-C20The high performance liquid chromatography adopts reversed phase chromatography, the mass spectrum adopts electrospray mass spectrum, the stationary phase of a chromatographic column of the high performance liquid chromatography is butylsilane bonded silica gel, and the aperture of the stationary phase is
12. Use of the method for detecting content of pegylated lipid according to claim 1 or the method for detecting content of lipid nanoparticle according to claim 11 in quality evaluation of nucleic acid pharmaceutical composition and preparation thereof, wherein the nucleic acid pharmaceutical composition and preparation thereof comprises lipid nanoparticles, and the lipid nanoparticles comprise pegylated lipid.
13. Use of the method for detecting content of pegylated lipid according to claim 1 or the method for detecting content of lipid nanoparticle according to claim 11 in the quality evaluation of lipid nanoparticle, wherein the lipid nanoparticle comprises pegylated lipid.
14. The use of the method for detecting a content of a polyglycol lipid according to claim 1 or the method for detecting a content of a lipid nanoparticle according to claim 11 in the evaluation of the quality of a polyglycol lipid, wherein the polyglycol lipid contains the polymer M-DMG.
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