A method for detecting encapsulated lipids and / or free lipids in LNP drugs

Abstract

The present invention provides a method for detecting encapsulated lipids and / or free lipids in LNP drugs, wherein the method comprises separating the encapsulated lipids and free lipids in the LNP drugs using a filter device with a molecular weight cutoff of 1KDa-1000KDa (preferably 3.5KDa-500KDa; more preferably 3.5KDa-100KDa), and then detecting the content of the encapsulated lipids and / or free lipids using an ultra-high performance liquid chromatography-charged aerosol detector. The method is simple to operate, has good repeatability, and can directly and accurately analyze the content of each component of free lipids and encapsulated lipids in lipid nanoparticles, meeting the requirements for quantitative analysis.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular, to a method for detecting encapsulated lipids and / or free lipids in LNP drugs. Background Art

[0002] The active molecules of nucleic acid drugs include messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotides (ASO) or plasmid DNA (pDNA), etc., which have great application potential in vaccines and in vitro or in vivo gene therapy. Pure nucleic acid drugs are easily degraded in the body and lose their activity, so they need to be delivered with the help of suitable delivery vectors. Designing suitable vectors to successfully deliver nucleic acid drug molecules to cells in the body and express them efficiently to achieve prevention or treatment purposes is one of the technologies urgently needed in the field of nucleic acid drugs. At present, the most widely used and marketed nucleic acid drugs mostly use lipid nanoparticles (LNP) as carriers. For example LNPs loaded with siRNA are used to treat polyneuropathy caused by transthyretin-mediated amyloidosis (hATTR), and its lipid components are DLin-MC3-DMA, DSPC, CHOL, and DMG-PEG. The COVID-19 vaccine launched by Pfizer-BioNTech is an LNP loaded with COVID-19 antigen mRNA, and its lipid components are ALC-0315 (A5), ALC-0159 (A9), DSPC, and CHOL.

[0003] Although LNP is the most widely used, its in vivo toxicity cannot be ignored. The cationic lipids in LNP can cause a strong immune response, which can produce organ toxicity at a certain dose and can be lethal in severe cases. Therefore, it is very important to study and characterize the in vivo distribution and content of each lipid. However, the history of large-scale industrial preparation of LNP is not long, and various processes are not yet fully mature, and the characterization of each lipid component of LNP has not yet been thoroughly studied. One of the reasons is that LNP itself has poor stability, and each lipid component is prone to oxidative decomposition to varying degrees during storage; and the structures of each lipid are similar, and the degradation products are of similar polarity, making them difficult to analyze. In summary, the development of a method for characterizing the content of various lipid components in LNP is of great guiding significance for the research and development of new lipid carriers.

[0004] After LNP preparation, there are some well-encapsulated particles and a certain amount of free lipids. During long-term storage or freeze-drying, the encapsulation rate will decrease, the particle size will increase, and a small amount of free lipids will leak out. Free lipids may cause additional in vivo toxicity and affect the stability of LNP. The content of free lipids can also reflect the stability of the nucleic acid drug LNP dosage form and the quality of the freeze-drying process. Therefore, it is necessary to detect free lipids. When free lipids are effectively separated from encapsulated lipids, the structure of the well-encapsulated LNP nucleic acid drug cannot be affected. Therefore, methods for separating and quantitatively detecting the content of free lipids are also urgently needed. At present, there are various means of conventional drug separation, and the separation methods of each component are different according to the different properties of the material components. To separate substances with different solubility, salting out, precipitation and extraction methods are usually used; to separate substances with different molecular weights or particle sizes, dialysis, ultrafiltration centrifugation and gel filtration are usually used; to separate substances with different charged properties, electrophoresis and ion exchange chromatography are usually used.

[0005] The lipid components of LNP have the characteristics of non-volatility, poor water solubility, and high temperature resistance. Different lipid molecules have similar properties, and the only difference between similar molecules is the carbon number and unsaturation of the fatty acid chain. During separation, problems such as chromatographic peak overlap and tailing are prone to occur. Therefore, in order to obtain good chromatographic separation, it is necessary to choose a suitable separation method and elution method. The main chromatographic methods used for phospholipid separation are thin layer chromatography (TLC), liquid chromatography (HPLC) and gas chromatography (GC). The TLC method is simple and the results are intuitive, but the pretreatment is cumbersome, requires a higher sampling technique, and has poor quantitative ability, so it is rarely used in key quality control processes. The GC method for separating phospholipid compounds requires derivatization, and its application is also subject to certain restrictions.

[0006] RPLC can separate different molecules in the same class of phospholipid compounds, but the separation effect between categories is poor, and lipids with stronger polarity are eluted first. NPLC can achieve better separation between categories, and lipids with weaker polarity are eluted first. Since NPLC method often uses weak polar solvents as mobile phase, it is easy to generate bubbles when mixed with relatively strong polar solutions, and the drift of retention time is inevitable.

[0007] UV detector is a commonly used detector for HPLC determination of specific compounds. After separation, the compounds to be tested are qualitatively analyzed according to the retention time of the chromatographic peak and quantitatively analyzed according to the peak area integral value. However, the commonly used lipid compounds in LNP have poor UV absorption and weak characteristic peaks, so this type of detector cannot be used. The ELSD evaporative light scattering detector can detect any volatile substance, and its detector sensitivity and reproducibility are improved compared to the UV detector. However, ELSD is very sensitive to the chemical structure, spatial positioning, physicochemical properties, sublimation characteristics, thermal stability, etc. of different components. The S-shaped response curve and very narrow linear range will bring inconvenience to the quantification.

[0008] Currently, there are no reports on the separation and detection methods of free lipids and encapsulated lipids in lipid nanoparticles. Summary of the invention

[0009] The present invention develops a new method for separating and detecting free lipids from encapsulated lipids in LNP nucleic acid drugs based on drug separation means and phospholipid detection methods, which has good separation effect, good reproducibility, and is simple and fast. In particular, the invention combines the characteristics of the independently developed LNP nucleic acid drug vaccine preparation (mRNA composite phospholipid liposome vaccine), separates free lipids from encapsulated lipids for the first time, and also establishes a UPLC-CAD method, which can effectively detect the content of each lipid (CHOL, DSPC, A5 and A9) in a mixed sample.

[0010] To achieve the above-mentioned object, the present invention provides a method for detecting encapsulated lipids and / or free lipids in LNP drugs, wherein the method comprises using a filter device with a molecular weight cutoff of 1KDa-1000KDa to separate the encapsulated lipids and free lipids in the LNP drugs, and then using an ultra-performance liquid chromatography-charged aerosol detector (UPLC-CAD) to detect the content of the encapsulated lipids and / or free lipids.

[0011] According to some specific embodiments of the present invention, the LNP drug is a drug using LNP as a carrier.

[0012] According to some specific embodiments of the present invention, the molecular weight cutoff of the filtration device is 3.5KDa-500KDa.

[0013] According to some specific embodiments of the present invention, the molecular weight cutoff of the filtration device is 3.5KDa-100KDa.

[0014] According to some specific embodiments of the present invention, the filtration device is a dialysis device.

[0015] According to some specific embodiments of the present invention, the filtration device is a dialysis bag, a dialysis bottle or a dialysis tube.

[0016] According to some specific embodiments of the present invention, the dialysis extrafluid of the dialysis device is selected from one or more combinations of water, PBS buffer, Tris buffer and HERPES buffer.

[0017] According to some specific embodiments of the present invention, the pH of each buffer is 5.0-8.0.

[0018] According to some specific embodiments of the present invention, the method includes separating the encapsulated lipids and free lipids in the LNP drug using a dialysis device, and the dialysis conditions are 2-25°C environment and oscillation for 4 to 72 hours.

[0019] According to some specific embodiments of the present invention, the dialysis condition is 2-15°C environment and shaking for 4-48 hours.

[0020] According to some specific embodiments of the present invention, the dialysis condition is 2-8°C environment and shaking for 8-48 hours.

[0021] According to some specific embodiments of the present invention, the method includes separating the encapsulated lipids and free lipids in the LNP drug using a dialysis device, including: taking the LNP drug sample to be tested, placing it in a dialysis device, and dialyzing it against enzyme-free water; after the dialysis is completed, collecting the dialysis external fluid and the dialysis internal fluid respectively; freeze-drying the collected dialysis internal fluid and dialysis external fluid to obtain freeze-dried powder, and detecting them separately using an ultra-high performance liquid chromatography-charged aerosol detector.

[0022] According to some specific embodiments of the present invention, the method includes separating the encapsulated lipids and free lipids in the LNP drug using a dialysis device, including: taking 1 mL of the LNP drug sample to be tested, placing it in a dialysis device, and dialyzing it against 25 mL of enzyme-free water; after the dialysis is completed, collecting the dialysis external fluid and the dialysis internal fluid respectively; performing a freeze-drying treatment to obtain a freeze-dried powder, and detecting them separately using an ultra-high performance liquid chromatography-charged aerosol detector.

[0023] According to some specific embodiments of the present invention, the lyophilization treatment of the collected external dialysate and internal dialysate includes freezing the collected external dialysate and internal dialysate at -90°C to -60°C for 8-24 hours, respectively, and then placing them in a freeze dryer for freeze drying for 24 to 72 hours to obtain a freeze-dried powder.

[0024] According to some specific embodiments of the present invention, the lyophilization treatment of the collected external dialysis fluid and the internal dialysis fluid comprises freezing the collected external dialysis fluid and the internal dialysis fluid at -80°C for 8-16 hours, respectively, and then placing them in a freeze dryer for freeze drying for 24 to 72 hours to obtain a lyophilized powder.

[0025] The freezer temperature for freeze drying for 24 to 72 hours in the present invention can be set according to conventional freezing processes, as long as the dialysate external fluid and the dialysate internal fluid are freeze-dried into powder. For example, the freeze dryer temperature can be set arbitrarily between -40 degrees Celsius and 35 degrees Celsius.

[0026] According to some specific embodiments of the present invention, when collecting the dialysate, the process includes flushing the inner bag of the dialysis bag with enzyme-free water, repeating the process 1-4 times, and combining the flushing solution with the dialysate.

[0027] According to some specific embodiments of the present invention, the flushing fluid and the dialysate are combined and collected into a 50 mL centrifuge tube; and the dialysate sample is divided into two 50 mL centrifuge tubes.

[0028] According to some specific embodiments of the present invention, the method further comprises freeze-drying the collected dialysate and the dialysate to obtain freeze-dried powder, dissolving the freeze-dried powder in an appropriate amount of enzyme-free water or methanol, mixing well and filtering through a 0.45 μm organic filter membrane to obtain a clear solution, which is then detected separately using an ultra-high performance liquid chromatography-charged aerosol detector.

[0029] It is understood that the amount of the appropriate amount of enzyme-free water or methanol can be determined according to the dissolution conditions, and the specific amount is sufficient to allow the freeze-dried powder to be dissolved in enzyme-free water or methanol.

[0030] The mixing can be carried out by any mixing and stirring means, such as stirring or vortex oscillation.

[0031] Nucleic acid drug LNP carriers usually contain a variety of lipid excipient components. The present invention screens dialysis bags with different molecular weight cutoffs and combines the UPLC-CAD method to detect and analyze the contents of four main excipients, CHOL, DSPC, A5 and A9, and finally obtains a new method for separating and detecting free lipids and encapsulated lipids in LNP nucleic acid drugs with good separation effect, good reproducibility, and simple and fast operation. The method is low in cost and easy to promote. The present invention can separate free lipids and encapsulated lipids and determine their contents for a variety of complex components, and can then indirectly analyze the stability and product quality of nucleic acid drug LNP carriers.

[0032] According to some specific embodiments of the present invention, the mobile phase A used for detecting the content of encapsulated lipids and / or free lipids by ultra performance liquid chromatography-charged aerosol detector is an aqueous solution of triethylamine acetate with a concentration of 0.1wt% to 1.0wt%, and the mobile phase B is a methanol solution of triethylamine acetate with a concentration of 0.1wt% to 1.0wt%.

[0033] According to some specific embodiments of the present invention, the mobile phase A is an ultrapure aqueous solution of triethylamine acetate with a concentration of 0.1 wt% to 1.0 wt%.

[0034] According to some specific embodiments of the present invention, the concentration of triethylamine acetate in the aqueous solution of triethylamine acetate and the methanol solution of triethylamine acetate is 0.25 wt % to 0.75 wt %, respectively.

[0035] According to some specific embodiments of the present invention, the concentration of triethylamine acetate in the aqueous solution of triethylamine acetate and the methanol solution of triethylamine acetate is 0.5 wt %.

[0036] According to some specific embodiments of the present invention, the use of ultra-high performance liquid chromatography-charged aerosol detector to detect encapsulated lipids and / or free lipids includes detecting encapsulated lipids and / or free lipids by gradient elution, and the gradient elution procedure includes: 0 min, B phase gradient 70% to 90%; within 0 to 90 min, the B phase gradient gradually increases to 80% to 100%; then the B phase gradient drops back to the initial gradient.

[0037] According to some specific embodiments of the present invention, the flow rate of the mobile phase for detecting encapsulated lipids and / or free lipids using ultra-high performance liquid chromatography-charged aerosol detector is 0.1-1 mL / min.

[0038] According to some specific embodiments of the present invention, the flow rate of the mobile phase for detecting encapsulated lipids and / or free lipids using ultra-high performance liquid chromatography-charged aerosol detector is 0.2-0.6 mL / min.

[0039] According to some specific embodiments of the present invention, wherein the detection of the encapsulated lipids and / or free lipids by ultra-high performance liquid chromatography-charged aerosol detector comprises detecting the encapsulated lipids and / or free lipids by gradient elution, and the procedure of gradient elution comprises:

[0040] Serial number Time (min) Flow rate (mL / min) A(％) B(％) 1 0.00 0.4 15 85 2 5.00 0.4 10 90 3 10.00 0.4 5 95 4 15.00 0.4 5 95 5 20.00 0.4 0 100 6 40.00 0.4 0 100 7 40.30 0.4 15 85 8 60.00 0.4 15 85

[0041] According to some specific embodiments of the present invention, the chromatographic conditions of the ultra-high performance liquid chromatography include: the chromatographic column is a C18 column, the column temperature is 50°C±0.5°C, and the post-column cooling temperature is 40°C±0.5°C.

[0042] According to some specific embodiments of the present invention, the detection conditions of the charged aerosol detector include: atomization temperature of 50°C±0.5°C; acquisition frequency: 5Hz; filtration constant: 3.6s.

[0043] According to some specific embodiments of the present invention, the LNP drug is a LNP nucleic acid drug.

[0044] According to some specific embodiments of the present invention, the LNP drug is an mRNA vaccine with LNP as a carrier.

[0045] According to some specific embodiments of the present invention, the LNP drug is an LNP vaccine carrying a new coronavirus antigen nucleic acid.

[0046] According to some specific embodiments of the present invention, the lipid component in the LNP drug is selected from a combination of one or more of DSPC, cholesterol (CHOL), ALC-0315 (A5) and ALC-0159 (A9).

[0047] According to some specific embodiments of the present invention, the injection volume for detecting the content of encapsulated lipids and / or free lipids by ultra-high performance liquid chromatography-charged aerosol detector is 5 to 50 μL.

[0048] According to some specific embodiments of the present invention, the injection volume for detecting the content of encapsulated lipids and / or free lipids using ultra-high performance liquid chromatography-charged aerosol detector is 10 μL.

[0049] In summary, the present invention provides a method for detecting encapsulated lipids and / or free lipids in LNP drugs. The method of the present invention has the following advantages:

[0050] The present invention establishes a simple and rapid method for separating free lipids and encapsulated lipids of LNP nucleic acid drugs, and establishes a reversed phase ultra-high performance liquid chromatography-charged aerosol detector (UPLC-CAD) method to simultaneously detect the content of each lipid component. The method is simple to operate, has good repeatability, and can directly and accurately analyze the content of each component of free lipids and encapsulated lipids in lipid nanoparticles, meeting the requirements of quantitative analysis. It is suitable for LNP nucleic acid drugs, especially the separation of free lipids and encapsulated lipids, and the determination of the content of each component in the screening and verification of the preparation process of COVID-19 nucleic acid vaccines, stability testing and product quality testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 , the standard spectrum obtained by detection in Example 1, the retention time of CHOL is 11.69 min, the retention time of A9 is 14.11 min, the retention time of A5 is 19.69 min and the retention time of DSPC is 20.56 min.

[0052] Figure 2 , Particle size distribution diagram of the sample in Example 4 before vigorous shaking.

[0053] Figure 3 , Particle size distribution diagram of the sample in Example 4 after vigorous shaking for 24 hours.

[0054] Figure 4, Particle size distribution diagram of the sample before freeze-drying in Example 5.

[0055] Figure 5 , Particle size distribution diagram of the sample after freeze-drying in Example 5.

[0056] Figure 6 , Particle size distribution diagram of the freeze-dried sample of Example 6 stored at 40°C for three months. DETAILED DESCRIPTION

[0057] The implementation process of the present invention and the beneficial effects produced are described in detail below through specific embodiments, which is intended to help readers better understand the essence and characteristics of the present invention, and is not intended to limit the scope of implementation of the present invention.

[0058] Example 1

[0059] Standard curve drawing

[0060] 1. Preparation of stock solution and standard curve solution

[0061] 1. Preparation of standard stock solution

[0062] Accurately weigh 10.0±0.1 mg of CHOL standard, dilute to 10 mL with methanol solution in a volumetric flask, mix by ultrasonication, and mark as CHOL-mother liquor.

[0063] Accurately weigh 10.0±0.1 mg of A5 standard, dilute to 10 mL with methanol solution in a volumetric flask, mix by ultrasonication, and mark as A5-mother liquor.

[0064] Accurately weigh 10.0±0.1 mg of A9 standard, dilute to 10 mL with methanol solution in a volumetric flask, mix by ultrasonication, and mark as A9-mother liquor.

[0065] Accurately weigh 10.0±0.1 mg of DSPC standard, dilute to 10 mL with methanol solution in a volumetric flask, mix by ultrasonication, and mark as DSPC-mother liquor.

[0066] The above solution should be sealed and stored at 4°C away from light. The validity period is 2 weeks.

[0067] 2. Preparation of standard mixed solution

[0068] Accurately pipette 1 mL of CHOL-mother liquor, A5-mother liquor, A9-mother liquor and DSPC-mother liquor and mix them with 1 mL of methanol to prepare mixed standard solutions of CHOL, DSPC, A5 and A9, that is, the concentrations of CHOL, DSPC, A5 and A9 in the mixed standard solution are all 200 μg / mL, marked as mixed standard, sealed and stored at 4°C away from light, with a shelf life of 2 weeks.

[0069] 3. Preparation of standard curve solution

[0070] Prepare the standard curve solution according to Table 1, seal and store at 4°C away from light, with a shelf life of 2 weeks;

[0071] Table 1 Concentration of standard series

[0072]

[0073]

[0074] 2. UPLC-CAD detection

[0075] Take 6 concentrations of standard curve samples of CHOL, DSPC, A5 and A9, with an injection volume of 10 μL, and perform UPLC-CAD detection. The specific detection conditions are as follows:

[0076] The chromatographic column is AcclaimTM C18 column. 2.1×150mm, particle size 3μm, mobile phase flow rate 0.4mL / min, column temperature 50℃±0.5℃, post-column cooling temperature: 40℃±0.5℃; mobile phase A is 0.5% TEAA in ultrapure water, mobile phase B is 0.5% TEAA in methanol, gradient elution, gradient elution program is:

[0077] Serial number Time (min) Flow rate (mL / min) A(％) B(％) 1 0.00 0.4 15 85 2 5.00 0.4 10 90 3 10.00 0.4 5 95 4 15.00 0.4 5 95 5 20.00 0.4 0 100 6 40.00 0.4 0 100 7 40.30 0.4 15 85 8 60.00 0.4 15 85

[0078] The detection conditions of the charged aerosol detector include: atomization temperature of 50°C ± 0.5°C; acquisition frequency: 5Hz; filtration constant: 3.6s.

[0079] The preparation method of TEAA is as follows: add 1 mL of triethylamine to 9 mL of ultrapure water, adjust the pH to 6.00-7.00 with glacial acetic acid, and mix by ultrasonication for 5 min for use; the preparation method of mobile phase A is as follows: add 2.5 mL of TEAA to 497.5 mL of ultrapure water, and ultrasonication for 5 min; the preparation method of mobile phase B is as follows: add 5 mL of TEAA to 995 mL of methanol, and ultrasonication for 5 min;

[0080] The representative chromatogram of the mixed standard 20-fold diluted reference solution is as follows Figure 1 As shown, it can be seen that the peak times of CHOL, A9, A5 and DSPC are 12.303 min, 14.507 min, 20.267 min and 21.417 min, respectively.

[0081] According to the test results, a cubic curve was drawn with Y as the peak area and X as the logarithm of the injection concentration. The standard curves of each component were:

[0082] CHOL:Y=0.0000003459X 3 -0.0005333373X2 +0.2727541714X+0.2517431721(coefficient of determination = 0.9998);

[0083] A9:Y=0.00001527X 3 -0.000989919X 2 +0.3588059574X+0.5684472439(coefficient of determination = 0.9991);

[0084] A5:Y=0.00002488X 3 -0.0011534282X 2 +0.3177345056X+0.5293667561(coefficient of determination = 0.9996);

[0085] DSPC:Y=0.000025821X 3 -0.0012573252X 2 +0.3409778029X+0.4772219326 (coefficient of determination = 0.9996).

[0086] The results showed that the standard curves of CHOL, A9, A5 and DSPC had a good linear relationship.

[0087] Example 2

[0088] Microfluidic preparation of LNPs

[0089] Luciferase mRNA was diluted in 100 mM, pH 4.0 citrate buffer at a final concentration of 0.134 mg / mL; each lipid component (A5: DSPC: CHOL: A9) was dissolved in ethanol at a total concentration of 10 mg / mL and a molar ratio of 46.3:9.4:42.7:1.6.

[0090] 3mL of mRNA solution and 1mL of lipid solution were loaded into two 5mL syringes respectively, installed on the microfluidic injection pump, the chip was connected to the syringe, the injection pump flow rate was set, and the start button of the injection pump was clicked to inject the chip at a flow rate ratio of 3:1. Observe the product color at the outlet of the chip, discard the first 5 drops of milky white droplets (about 100μL), and collect the rear end sample into the EP tube. The collected product was placed in a dialysis bag and dialyzed overnight (molecular weight cutoff: 100KDa) with enzyme-free water (pH7.4), and the liquid was changed 3 times during the period to obtain freshly prepared LNP.

[0091] Take 3 clean dialysis bags (100KDa), add 1mL of prepared LNP nucleic acid drug sample to each dialysis bag, place them in 50mL centrifuge tubes, add 24mL of enzyme-free water to each centrifuge tube, place them in a 4℃ refrigerator, shake and dialyze for 8h with a mixer, and change the liquid every 2 hours.

[0092] After 8 hours, all the dialysate external fluid and dialysate internal fluid were collected. When collecting the dialysate internal fluid, the inner bag of the dialysis bag needed to be rinsed with 2 mL of enzyme-free water, repeated 4 times, and the rinse fluid was collected into a 50 mL centrifuge tube; the dialysate external fluid samples were divided into 2 50 mL centrifuge tubes, and the collected samples were stored at -80°C and marked as external fluid-5, external fluid-6, external fluid-7, internal fluid-5, internal fluid-6 and internal fluid-7 respectively.

[0093] The collected samples were frozen in a -80°C refrigerator overnight, and then placed in a freeze dryer (Scientz-10N) for freeze drying for 48 h (temperature was room temperature (about 22°C)), and the freeze-dried powder was collected.

[0094] Sample treatment before injection: add 1 mL of enzyme-free water to the freeze-dried inner and outer liquid samples respectively, vortex to fully dissolve or fully disperse. Take 100 μL of the reconstitution solution of the freeze-dried dialysis bag inner liquid sample and add 900 μL of methanol, vortex to mix thoroughly, and then pass through a 0.45 μm organic filter membrane before injection; the reconstitution solution of the freeze-dried dialysis bag outer liquid is directly passed through a 0.45 μm aqueous phase filter membrane before injection; the injection volume is 10 μL, and UPLC-CAD detection is performed, and the detection conditions are the same as in Example 1.

[0095] After detection and calculation analysis, the contents of CHOL, A9, A5 and DSPC in the dialysis fluid and the dialysis fluid are shown in Table 2.

[0096] Table 2 Contents of CHOL, A9, A5 and DSPC in the external and internal dialysate

[0097]

[0098] From the results in Table 2, it can be seen that during the preparation process of the LNP nucleic acid drug sample, after dialyzing with a 100KDa dialysis bag, the free lipid part that failed to form encapsulated lipids can be removed. In the obtained LNP nucleic acid drug, no free lipids were detected after secondary dialysis. It can be considered that the LNP nucleic acid drug product prepared by the microfluidic post-dialysis method has a low free lipid content and a high quality.

[0099] Example 3

[0100] LNP was prepared by microfluidics. The preparation method was the same as in Example 2. 15% sucrose was added to the prepared LNP, and the LNP was packaged in vials for freeze-drying. The freeze-drying procedure was as follows:

[0101]

[0102]

[0103] The LNP nucleic acid drug lyophilized powder was obtained through the above procedure. 15 LNP nucleic acid drug lyophilized powder products were taken, 500 μL of enzyme-free water was added to each lyophilized powder, and after being fully dispersed, all were transferred to a 15 mL centrifuge tube to obtain the LNP nucleic acid drug reconstituted solution.

[0104] Take clean dialysis bags with molecular weight cutoff of 500Da, 3500Da, 30KDa, and 100KDa, respectively, add 1mL of LNP nucleic acid drug reconstitution solution to each dialysis bag, place them in 50mL centrifuge tubes, add 24mL of enzyme-free water to each centrifuge tube, mark them as sample-1, sample-2, sample-3, and sample-4 respectively, place them in a 4℃ refrigerator, and shake and dialyze them in a mixer for 48h;

[0105] After 48 hours, all the dialysate external fluid and dialysate internal fluid were collected. When collecting the dialysate internal fluid, the inner bag of the dialysis bag needed to be rinsed with 2 mL of enzyme-free water, repeated 4 times, and the rinse fluid was collected into a 50 mL centrifuge tube; the dialysate external fluid samples were divided into 2 50 mL centrifuge tubes, and the collected samples were stored at -80°C and annotated as external fluid-1, external fluid-2, external fluid-3, external fluid-4, internal fluid-1, internal fluid-2, internal fluid-3 and internal fluid-4 respectively.

[0106] The collected samples were frozen in a -80°C refrigerator overnight, and then placed in a freeze dryer (Scientz-10N) for freeze drying for 48 h (temperature was room temperature (about 22°C)), and the freeze-dried powder was collected.

[0107] Sample treatment before injection: add 1 mL of enzyme-free water to the freeze-dried inner and outer liquid samples respectively, vortex to fully dissolve or fully disperse. Take 100 μL of the reconstitution solution of the freeze-dried dialysis bag inner liquid sample and add 900 μL of methanol, vortex to mix thoroughly, and then pass through a 0.45 μm organic filter membrane before injection; the reconstitution solution of the freeze-dried dialysis bag outer liquid is directly passed through a 0.45 μm aqueous phase filter membrane before injection; the injection volume is 10 μL, and UPLC-CAD detection is performed, and the detection conditions are the same as in Example 1.

[0108] After detection and calculation analysis, the contents of CHOL, A9, A5 and DSPC in the dialysate and the dialysate are shown in Table 3.

[0109] Table 3 Contents of CHOL, A9, A5 and DSPC in the external and internal dialysis fluids

[0110]

[0111] Note: Sample-13 (500Da), Sample-14 (3500Da), Sample-15 (30KDa), Sample-16 (100KDa)

[0112] From the results in Table 3, it can be seen that the dialysis bags with different molecular cutoffs have different separation effects on the free lipids and encapsulated lipids in LNP nucleic acid drugs. The molecular cutoffs of 3500Da, 30KDa and 100KDa can effectively separate the free lipids and encapsulated lipids in LNP nucleic acid drugs.

[0113] Example 4

[0114] Separation and detection of free lipids from LNPs after oscillation disruption

[0115] Take a clean dialysis bag (100 KDa), add 5 mL of the prepared nucleic acid drug LNP sample (the method is the same as Example 3) into the dialysis bag, place them in 50 mL centrifuge tubes, add 45 mL of enzyme-free water to each centrifuge tube, place them in a 4 ° C refrigerator, shake and dialyze with a mixer for 8 h, change the liquid every 2 hours, and collect the dialyzate after 8 h, and note it as dialyzate liquid-8.

[0116] Take 1.5 mL of dialyzed solution-8 into three 15 mL clean centrifuge tubes respectively, shake vigorously at room temperature for 2 h, collect samples, and mark them as shaken sample-1, shaken sample-2 and shaken sample-3; the remaining pre-shake samples are placed in a 4°C refrigerator to store the average particle size, PDI, Zeta potential and individual component content to be measured.

[0117] Take 100 μL of the sample before and after vigorous shaking, add 1.4 mL of 1×TE solution to dilute, and measure the average particle size, PDI and Zeta potential of the sample. The measured results are shown in Table 4. The particle size distribution before shaking is as follows: Figure 2 As shown, the particle size distribution after 24 hours of oscillation is as follows Figure 3 shown.

[0118] Table 4 Average particle size, PDI and Zeta potential of nucleic acid drug LNP before and after vigorous oscillation

[0119] sample Average particle size (nm) PDI Zeta potential (mV) Before oscillation 74.43 0.150 -3.25 Oscillating sample-1 209.6 0.260 -3.33 Oscillating sample-2 234.8 0.330 -3.38 Oscillating sample-3 227.9 0.324 -3.29

[0120] Take 3 clean dialysis bags (100KDa), add 1mL of the vigorously shaken sample to each dialysis bag, place them in 50mL centrifuge tubes, add 24mL of enzyme-free water to each centrifuge tube, place them in a 4℃ refrigerator, and shake and dialyze them for 48h using a mixer.

[0121] After 48 hours, the dialysate inner fluid was collected. When collecting the dialysate inner fluid, 2 mL of enzyme-free water was used to rinse the inner bag of the dialysis bag. This was repeated 4 times, and the rinse fluid was collected into a 50 mL centrifuge tube. The dialysate external fluid samples were divided into 2 50 mL centrifuge tubes and stored at -80 °C. The collected samples were marked as external fluid-9, external fluid-10, external fluid-11, internal fluid-9, internal fluid-10 and internal fluid-11 respectively.

[0122] The collected samples were frozen in a -80°C refrigerator overnight, and then placed in a freeze dryer (Scientz-10N) for freeze drying for 48 h (temperature was room temperature (about 22°C)), and the freeze-dried powder was collected.

[0123] Sample treatment before injection: add 1 mL of enzyme-free water to the freeze-dried inner and outer liquid samples respectively, vortex to fully dissolve or fully disperse. Take 100 μL of the reconstitution solution of the freeze-dried dialysis bag inner liquid sample and add 900 μL of methanol, vortex to mix thoroughly, and then pass through a 0.45 μm organic filter membrane before injection; the reconstitution solution of the freeze-dried dialysis bag outer liquid is directly passed through a 0.45 μm aqueous phase filter membrane before injection; the injection volume is 10 μL, and UPLC-CAD detection is performed, and the detection conditions are the same as in Example 1.

[0124] After detection and calculation analysis, the contents of CHOL, A9, A5 and DSPC in the dialysis fluid and the dialysis fluid are shown in Table 5.

[0125] Table 5 Contents of CHOL, A9, A5 and DSPC in the external and internal dialysis fluids

[0126]

[0127] The room temperature violent oscillation experiment simulates the extreme conditions during the storage and transportation of LNP nucleic acid drugs. According to the results in Table 4, after the LNP nucleic acid drug solution was violently oscillated for 2 hours, the average particle size and PDI of the LNP nucleic acid drug changed significantly. Combined with the results in Table 5, it was found that after the LNP nucleic acid drug solution was violently oscillated for 2 hours, the content of each component of free lipid increased. This shows that during the storage and transportation of LNP nucleic acid drugs, after collision and shaking, the particle size increases and the particle distribution becomes wider, which will cause some particles to break and produce more free lipids, and the product quality will decrease.

[0128] Example 5

[0129] Part of the newly prepared LNP nucleic acid drug (the method is the same as that in Example 3) was freeze-dried to obtain 7 LNP nucleic acid drug freeze-dried powder products, 500 μL of enzyme-free water was added to each freeze-dried powder, and after being fully dispersed, all were transferred to a 15 mL centrifuge tube to obtain an LNP nucleic acid drug reconstituted solution.

[0130] Take 100 μL of the samples before lyophilization and the lyophilized solution respectively, add 1.4 mL of 1×TE solution to dilute, and measure the average particle size, PDI and Zeta potential of the samples. The measured results are shown in Table 5. The particle size distribution of the samples before lyophilization is as follows: Figure 4 As shown, the particle size distribution of the freeze-dried samples is as follows Figure 5 shown.

[0131] Table 6 Average particle size, PDI and Zeta potential of LNP nucleic acid drugs before lyophilization and lyophilized reconstituted solution

[0132] sample Average particle size (nm) PDI Zeta potential (mV) Before freeze-drying 70.27 0.078 -4.34 Lyophilized reconstitution solution 91.37 0.171 -5.49

[0133] Take three clean dialysis bags (100KDa), add 1mL of LNP nucleic acid drug reconstitution solution to each dialysis bag, place them in 50mL centrifuge tubes, add 24mL of enzyme-free water to each centrifuge tube, place them in a 4℃ refrigerator, and shake and dialyze for 48h;

[0134] After 48 hours, all the dialysate external fluid and dialysate internal fluid were collected. When collecting the dialysate internal fluid, the inner bag of the dialysis bag needed to be rinsed with 2 mL of enzyme-free water, repeated 4 times, and the rinse fluid was collected into a 50 mL centrifuge tube; the dialysate external fluid samples were divided into 2 50 mL centrifuge tubes, and the collected samples were stored at -80°C and marked as external fluid-12, external fluid-13, external fluid-14, internal fluid-12, internal fluid-13 and internal fluid-14 respectively.

[0135] The collected samples were frozen in a -80°C refrigerator overnight, and then placed in a freeze dryer (Scientz-10N) for freeze drying for 48 h (temperature was room temperature (about 22°C)), and the freeze-dried powder was collected.

[0136] Sample treatment before injection: add 1 mL of enzyme-free water to the freeze-dried inner and outer liquid samples respectively, vortex to fully dissolve or fully disperse. Take 100 μL of the reconstitution solution of the freeze-dried dialysis bag inner liquid sample and add 900 μL of methanol, vortex to mix thoroughly, and then pass through a 0.45 μm organic filter membrane before injection; the reconstitution solution of the freeze-dried dialysis bag outer liquid is directly passed through a 0.45 μm aqueous phase filter membrane before injection; the injection volume is 10 μL, and UPLC-CAD detection is performed, and the detection conditions are the same as in Example 1.

[0137] After detection and calculation analysis, the contents of CHOL, A9, A5 and DSPC in the dialysate and the dialysate are shown in Table 7.

[0138] Table 7 Contents of CHOL, A9, A5 and DSPC in the external and internal dialysate

[0139]

[0140] Combining the results of Tables 6 and 7, it can be seen that the particle size of the LNP nucleic acid drug has changed slightly before and after freeze-drying. From the content data of each component CHOL, A9, A5 and DSPC measured, it can be seen that there is some free lipid in the freeze-dried powder LNP nucleic acid drug preparation. Such a result may be caused by the liquid-solid phase change of the LNP nucleic acid drug during the freeze-drying process, which leads to a slight change in the LNP result.

[0141] Example 6

[0142] The finished LNP nucleic acid drug lyophilized powder was placed at 40°C away from light for three months, 7 LNP nucleic acid drug lyophilized powders were taken, 500 μL of enzyme-free water was added to each lyophilized powder, and after being fully dispersed, all were transferred to a 15 mL centrifuge tube to obtain the LNP nucleic acid drug reconstitution solution.

[0143] Take 100 μL of the freeze-dried reconstituted solution sample, add 1.4 mL of 1×TE solution to dilute, and measure the average particle size, PDI and Zeta potential of the sample. The measured results are shown in Table 8. The particle size distribution of the freeze-dried sample stored at 40°C for three months is as follows: Figure 6 shown.

[0144] Table 8 Average particle size, PDI and Zeta potential of lyophilized powder, reconstituted LNP nucleic acid drug at room temperature and away from light for three months

[0145] sample Average particle size (nm) PDI Zeta potential (mV) Lyophilized initial reconstitution solution 91.37 0.171 -5.49 Lyophilized powder-three-month reconstitution solution 111.2 0.159 -4.51

[0146] Take three clean dialysis bags (100KDa), add 1mL of LNP nucleic acid drug reconstitution solution to each dialysis bag, place them in 50mL centrifuge tubes, add 24mL of enzyme-free water to each centrifuge tube, place them in a 4℃ refrigerator, and shake and dialyze for 48h;

[0147] After 48 hours, all the dialysate external fluid and dialysate internal fluid were collected. When collecting the dialysate internal fluid, the inner bag of the dialysis bag needed to be rinsed with 2 mL of enzyme-free water, repeated 4 times, and the rinse fluid was collected into a 50 mL centrifuge tube; the dialysate external fluid samples were divided into 2 50 mL centrifuge tubes, and the collected samples were stored at -80°C and marked as external fluid-15, external fluid-16, external fluid-17, internal fluid-15, internal fluid-16 and internal fluid-17 respectively.

[0148] The collected samples were frozen in a -80°C refrigerator overnight, and then placed in a freeze dryer (Scientz-10N) for freeze drying for 48 h (temperature was room temperature (about 22°C)), and the freeze-dried powder was collected.

[0149] Sample treatment before injection: Add 1mL of enzyme-free water to the freeze-dried internal and external liquid samples respectively, and vortex to fully dissolve or disperse. Take 100μL of the reconstitution solution of the freeze-dried dialysis bag sample and add 900μL of methanol. Vortex and oscillate to mix thoroughly, then pass through a 0.45μm organic filter membrane before injection; the reconstitution solution of the freeze-dried dialysis bag external liquid is directly passed through a 0.45μm aqueous filter membrane before injection; the injection volume is 10μL, and UPLC-CAD detection is performed. The specific detection conditions are:

[0150] The chromatographic column is AcclaimTM C18 column. 2.1×150mm, particle size 3μm, mobile phase flow rate 0.4mL / min, column temperature 50℃±0.5℃, post-column cooling temperature: 40℃±0.5℃; mobile phase A is 0.5% TEAA in ultrapure water, mobile phase B is 0.5% TEAA in methanol, gradient elution, gradient elution program is:

[0151] Serial number Time (min) Flow rate (mL / min) A(％) B(％) 1 0.00 0.4 20 80 2 15.00 0.4 5 95 3 25.00 0.4 5 95 4 30.00 0.4 0 100 5 40.00 0.4 0 100 6 40.30 0.4 15 85 7 60.00 0.4 15 85

[0152] The peak times of CHOL, A9, A5 and DSPC were 15.74min, 19.33min, 29.02min and 30.86min, respectively.

[0153] After detection and calculation and analysis, the contents of CHOL, A9, A5 and DSPC in the dialysate and the dialysate are shown in Table 9.

[0154] Table 9 Contents of CHOL, A9, A5 and DSPC in the dialysis fluid and dialysis fluid of Example 6

[0155]

[0156] From the results in Tables 8 and 9, it can be seen that the particle size of the LNP nucleic acid drug freeze-dried powder changed slightly after being treated at 40°C away from light for three months, and the content of free lipids also increased slightly compared to the initial freeze-drying.

Claims

1. A method for detecting encapsulated lipids and / or free lipids in LNP drugs, wherein: The method comprises separating the encapsulated lipids and free lipids in the LNP drug by using a filter device with a molecular weight cutoff of 1 KDa-1000 KDa, and then detecting the content of the encapsulated lipids and / or free lipids by using an ultra-high performance liquid chromatography-charged aerosol detector; The filtering device is a dialysis device; The method comprises using a dialysis device to separate the encapsulated lipids and free lipids in the LNP drug, including: taking a sample of the LNP drug to be tested, putting it into the dialysis device, and dialyzing it with enzyme-free water; after the dialysis is completed, collecting the dialysis external fluid and the dialysis internal fluid respectively; freeze-drying the collected dialysis internal fluid and dialysis external fluid to obtain a lyophilized powder; dissolving the lyophilized powders of the dialysis internal fluid and the dialysis external fluid in an appropriate amount of enzyme-free water respectively, adding methanol to the freeze-dried dialysis bag internal fluid sample reconstitution solution, adding 900 μL of methanol to every 100 μL of reconstitution solution, mixing well, passing through a 0.45 μm organic filter membrane to obtain a clear solution, and directly passing through a 0.45 μm organic filter membrane to obtain a clear solution of the freeze-dried dialysis bag external fluid sample reconstitution solution; and using an ultra-high performance liquid chromatography-charged aerosol detector for detection respectively; The collected external dialysate and internal dialysate are freeze-dried, including freezing the collected external dialysate and internal dialysate at -90°C to -60°C for 8-24 hours, and then placing them in a freeze dryer for freeze drying for 24-72 hours to obtain freeze-dried powder; The mobile phase A used for detecting the content of encapsulated lipids and / or free lipids by ultra-high performance liquid chromatography-charged aerosol detector is an aqueous solution of triethylamine acetate with a concentration of 0.1wt% to 1.0wt%, and the mobile phase B is a methanol solution of triethylamine acetate with a concentration of 0.1wt% to 1.0wt%; Detecting the encapsulated lipids and / or free lipids by ultra-high performance liquid chromatography-charged aerosol detector includes detecting the encapsulated lipids and / or free lipids by gradient elution, and the procedure of gradient elution includes: The mobile phase flow rate for detecting the encapsulated lipids and / or free lipids using ultra-high performance liquid chromatography-charged aerosol detector is 0.1-1 mL / min.

2. The method according to claim 1, wherein: The molecular weight cut-off of the filter device is 3.5KDa-500KDa.

3. The method according to claim 1, wherein: The molecular weight cut-off of the filter device is 3.5KDa-100KDa.

4. The method according to claim 1, wherein: The dialysis equipment is a dialysis bag, a dialysis bottle or a dialysis tube.

5. The method according to claim 1, wherein: The dialysis external fluid of the dialysis device is selected from one or more combinations of water, PBS buffer, Tris buffer and HERPES buffer; the pH of the buffer is 5.0-8.

0.

6. The method according to claim 1, wherein: The method comprises using a dialysis device to separate the encapsulated lipids and free lipids in the LNP drug, and the dialysis conditions are an environment of 2-25° C. and oscillation for 4-72 hours.

7. The method according to claim 6, wherein: The dialysis conditions are 2-15°C and shaking for 4-48 hours.

8. The method according to claim 6, wherein: The dialysis conditions are 2-8°C and shaking for 8-48 hours.

9. The method according to claim 1, wherein: When collecting the dialysate, rinse the inner bag of the dialysis bag with enzyme-free water, repeat 1-4 times, and combine the rinse solution with the dialysate.

10. The method according to claim 1, wherein: The aqueous solution of mobile phase A is an ultrapure aqueous solution, and the mobile phase B is a methanol solution of triethylamine acetate with a concentration of 0.1wt% to 1.0wt%.

11. The method according to claim 1, wherein: The concentrations of triethylammonium acetate in the aqueous solution of triethylammonium acetate and the methanol solution of triethylammonium acetate are 0.25 wt % to 0.75 wt % respectively.

12. The method according to claim 1, wherein: In the aqueous solution of triethylammonium acetate and the methanol solution of triethylammonium acetate, the concentration of triethylammonium acetate is 0.5 wt %.

13. The method according to claim 1, wherein: The mobile phase flow rate for detecting the encapsulated lipids and / or free lipids using ultra-high performance liquid chromatography-charged aerosol detector is 0.2-0.6 mL / min.

14. The method according to any one of claims 1 to 13, wherein: The chromatographic conditions of the ultra-high performance liquid chromatography include: the chromatographic column is a C18 column, the column temperature is 50°C±0.5°C, and the post-column cooling temperature is 40°C±0.5°C.

15. The method according to any one of claims 1 to 13, wherein: The detection conditions of the charged aerosol detector include: atomization temperature of 50°C ± 0.5°C; acquisition frequency: 5Hz; filtration constant: 3.6s.

16. The method according to any one of claims 1 to 13, wherein: The LNP drug is an LNP nucleic acid drug.

17. A method according to any one of claims 1 to 13, wherein the LNP drug is an LNP vaccine carrying a new coronavirus antigen nucleic acid.

18. The method according to any one of claims 1 to 13, wherein: The lipid component in the LNP drug is selected from a combination of one or more of DSPC, cholesterol, ALC-0315 and ALC-0159.

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