Method for characterizing performance of inhalation vaccine

Abstract

The invention provides a method for characterizing the performance of an inhalation vaccine, which comprises the following steps of: quantitatively and jointly using an NGI cascade impactor, a polymerase chain reaction real-time fluorescent quantitative PCR (polymerase chain reaction) (Quantitative Real-time PCR, QPCR) and a droplet digital PCR (drop digital PCR, ddPCR), and establishing an integrity performance characterization method aiming at the inhalation vaccine; a plurality of influence factors of the NGI cascade impactor in characterization of the inhaled vaccine are deeply researched, and a detection environment with stable results and high repeatability is obtained; meanwhile, the optimal characterization sample dosage is determined based on multiple repeated experiments; the invention innovatively provides the combination of NGI, QPCR and ddPCR, and the method has the advantages of accurate detection and short detection time, can meet the detection of large-batch samples, and ensures the large-scale application in the industry.

Description

Technical Field

[0001] The present invention relates to the field of vaccine technology, and in particular to a method for characterizing the performance of an inhaled vaccine, and more particularly to a method for characterizing the performance of an aerosolized inhaled vaccine using an NGI cascade impactor. Background Art

[0002] Since the amount of vaccine inhaled by nebulization is low, higher requirements are placed on the performance characterization of the product. As a method of inhalation administration, the "0951 Determination of Aerodynamic Characteristics of Fine Particles in Inhaled Preparations" in Part IV of the 2020 edition of the Chinese Pharmacopoeia recommends the use of a two-stage impactor device, ACI (andersen cascade impactor) and NGI (next generation impactor) devices for the detection of aerodynamic particle size distribution (APSD) of inhaled liquid preparations. The APSD of aerosol particles determined by NGI is based on the same characteristics of their deposition in the respiratory tract. In the pulmonary conducting airways, fog particles are mostly deposited through collision, which is manifested as larger particles settling on the surface of the tracheal wall in the lungs, while smaller particles enter the next level of airway branches in the lungs with the airflow. NGI simulates the above process, so inertial collision is generally considered to be the "gold standard" for evaluating the aerodynamic particle size deposition of inhaled agents. In NGI, there are one or more nozzles or small holes with a certain particle size on each level. Below the level is the collection plate of the level, and the distance between the two is determined and can reflect the carrier gas flow rate. The particles overcome the air resistance through inertia, which is manifested as larger particles being deposited on the collection plate of that level, while smaller particles continue to move forward with the airflow and are deposited on the next collection plate of the NGI. Since the aperture of the nozzle or small hole gradually decreases as it goes deeper into the impactor, the linear velocity of the air through the successive levels increases, allowing smaller and smaller particles to impact the surface of the collection plate.

[0003] The NGI cascade impactor consists of a throat, a pre-separator and an impactor body; the impactor body includes a detachable collection cup, a bottom bracket for placing the collection cup, a sealing body with a nozzle and a lid with an inter-electrode airway; there are 7 stages in total, and a micro-orifice collector (MOC) at the end. MOC is an impact plate with 4032 small holes, and most of the particles missed by the 7th stage will be collected by MOC. The amount of drug in each collection plate is collected separately, and quantification is achieved with the help of other instruments.

[0004] Accurate quantification of the drugs distributed in each collection plate of the NGI cascade impactor is a key step in the characterization of NGI performance. The dose of inhaled new coronavirus vaccine for a single person is only 0.1ml, which is distributed in 7 collection plates through NGI. The content of inhaled new coronavirus vaccine collected in each collection plate is very low after elution, so it is necessary to develop a dedicated and highly sensitive detection method for inhaled vaccines. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for characterizing the performance of inhaled vaccines.

[0006] A method for characterizing the aerosol performance of an inhaled vaccine, comprising using NGI to characterize the performance of an inhaled vaccine, characterized in that it comprises the following steps: S1, precool the NGI impactor, connect the flow meter to adjust the flow rate, turn on the vacuum pump to adjust the flow rate, and remove the flow meter; S2, put the sample to be tested into the nebulizer cup, connect the NGI and nebulizer, and turn on the nebulizer until the sample to be tested is nebulized; S3, using the vaccine eluate to collect samples in different collection plates 1-7 respectively; S4, extracting nucleic acid from the sample obtained in step S3; S5, perform QPCR detection; Specifically, in S2, the dose of the characterization sample is no higher than 1 ml; When using NGI to characterize inhaled vaccines, the analytical testing requirements must first be met, and the NGI device carrying capacity must also be considered. After multiple experimental studies, the present invention balances the testing requirements, carrying capacity, and stability of performance characterization data. The sample dose to be tested is no higher than 1.5 ml. Specifically, S4 extracts nucleic acid from the sample obtained in step S3; extraction methods include lysis method, magnetic bead method, and column extraction method; the nucleic acid sample extracted in S4 is used for QPCR quantitative detection in the subsequent step S5, which requires the sample to be of high purity and low impurities; the OD260, OD280, and OD230 values ​​of the sample are usually tested, and the purity of the DNA is judged based on their ratio. The OD260 / OD280 ratio of pure DNA is about 1.8, and greater than 1.9 indicates that the DNA may be degraded or contaminated with RNA; less than 1.7 may indicate that there may be residual proteins or aromatic substances such as phenols. A230 is the absorption wavelength of the highest absorption peak of carbohydrates, and the OD260 / OD230 of purer nucleic acids is between 2.0 and 2.2.

[0007] The lysis method includes physical or chemical methods to lyse the sample; Physical lysis methods include: -80°C freezing extraction, ultrasonic extraction, thermal lysis; chemical lysis methods include treatment with chemical lysis agents; optional chemical lysis agents include but are not limited to: SDS lysis, Triton X-100, deoxycholate, Nonidet P40 (NP-40), etc. The chemical lysis method can dissolve cell membranes, nuclear membrane proteins and viral capsids, depolymerize nuclear proteins, and thus release DNA; after purification, the sample purity can be improved; The magnetic bead method uses the property that the active groups of magnetic particles can bind to and dissociate from nucleic acids. Lysis buffer is used to lyse samples such as cells or viruses to release nucleic acids. The free nucleic acids are then adsorbed by magnetic beads. After several washes and adsorptions, high-purity nucleic acids are obtained. The column extraction method utilizes a special silica-based adsorption material to specifically adsorb nucleic acids and filter out macromolecular products such as proteins. Finally, the nucleic acids are eluted from the adsorption membrane to obtain sample nucleic acids.

[0008] The QPCR assay in S5 specifically includes target selection, pretreatment of samples to be tested, and preparation of standards, which are quantified using the ddPCR method; Since the amount of nebulized inhaled vaccine used is extremely small, it is crucial to choose a highly sensitive, stable and rapid detection method suitable for product production.

[0009] Nucleic acids and their derivatives, nucleotides, nucleosides, purines and pyrimidines have the property of absorbing UV, and their absorption peak is at a wavelength of 260nm. The molar extinction coefficient (or absorption coefficient) of nucleic acids is used to express. It is the light absorption value (i.e., A value) of 1 mole of atomic nucleic acid phosphorus per liter of solution (table). By measuring the A260nm value of a nucleic acid solution of unknown concentration, the content of RNA or DNA in it can be calculated; the detection is accurate. However, the disadvantage is that the concentration of the inhaled vaccine collected in each collection plate of the NGI impactor is very low after elution, far below its detection limit.

[0010] Real-time fluorescence quantitative PCR (Quantitative Real-time PCR, QPCR) is a quantitative detection technology that uses fluorescent chemicals to detect the total amount of products after each polymerase chain reaction (PCR) cycle, and then quantitatively analyzes the specific nucleic acid sequence in the sample to be tested through internal or external reference methods. It can achieve high-throughput rapid detection of specific nucleic acid sequences in the sample to be tested, and requires a standard curve to be drawn with a standard product of known concentration. The quantification of nucleic acid standards is generally determined by ultraviolet quantitative determination. For low-dose samples, the ultraviolet detection method cannot achieve quantitative determination and has large errors. The deviation in the determination of standard concentration will directly affect the accuracy of quantitative detection of the sample to be tested. Droplet digital PCR (ddPCR) is based on single-molecule PCR analysis technology, that is, the template is randomly distributed in tens of thousands of nanoliter droplets. Each droplet containing the template is an independent PCR reaction system. It can achieve absolute quantitative detection of the target sample without relying on the standard curve. Compared with QPCR, ddPCR technology has the advantage of higher detection sensitivity, but the ddPCR instrument used is expensive, the cost of a single reaction is high, and the cost of large-scale detection is high and the throughput is lower than QPCR. The present invention quantifies QPCR standards through ddPCR, which can not only give play to the high-precision characteristics of ddPCR but also effectively utilize the high-throughput detection of QPCR to achieve accurate detection of low-concentration nucleic acid samples.

[0011] The beneficial effects of the present invention include: A complete performance characterization method is established for inhaled vaccines; inhaled vaccines are used in low doses, which puts higher requirements on performance characterization; the present invention conducts in-depth research on the various influencing factors of the NGI cascade impactor in the characterization of inhaled vaccines, and obtains a detection environment with stable results and high repeatability; at the same time, the present invention is based on multiple repeated experiments, and while meeting the analysis and detection needs, it is necessary to consider the NGI device carrying capacity and the stability of performance characterization data to determine the optimal characterization sample dose; the present invention innovatively proposes to combine NGI, QPCR and ddPCR. This method can detect accurately, in a short detection time, and can meet the detection of large quantities of samples, ensuring large-scale application in the industry; to meet the detection requirements of QPCR, the present invention studies a number of nucleic acid extraction methods, optimizes the extraction method, and establishes a complete method for characterizing the performance of inhaled vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 . Droplet morphology of NGI level 5, 6, and 7 collection plates at different atomization doses. DETAILED DESCRIPTION

[0013] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0014] The term "nucleic acid" refers to a polymer of nucleotides and, unless otherwise limited, includes analogs of natural nucleotides that are capable of functioning (eg, hybridizing) in a manner similar to naturally occurring nucleotides.

[0015] The term nucleic acid includes any form of DNA or RNA or PNA (a DNA analog with a polypeptide backbone in place of the sugar-phosphate backbone), including, for example, genomic DNA; complementary DNA (cDNA), which is a DNA representation of mRNA, obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced by synthesis or by amplification; mRNA; and non-coding RNA. Nucleic acids are synthesized chemically, such as solid phase-mediated chemical synthesis; can be obtained from biological sources, such as by isolation from any species that produces nucleic acids; or can be obtained by methods involving manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription; or can be obtained by a combination of these methods.

[0016] The term nucleic acid encompasses double-stranded or triple-stranded nucleic acid complexes, as well as single-stranded molecules. In double-stranded or triple-stranded nucleic acid complexes, the nucleic acid strands need not be coextensive (ie, a double-stranded nucleic acid need not be double-stranded over the entire length of both strands).

[0017] The term nucleic acid also encompasses any modification thereof, for example by methylation and / or by capping. Nucleic acid modifications may include the addition of chemical groups that introduce additional charge, polarizability, hydrogen bonding, electrostatic interactions, and functionality to individual nucleic acid bases or to the nucleic acid as a whole. Such modifications may include base modifications, such as sugar modifications at the 2' position, pyrimidine modifications at the 5' position, purine modifications at the 8' position, modifications at the amine outside the cytosine ring, displacement of 5-bromo-uracil, sugar-phosphate backbone modifications, rare base pairing combinations such as the isobases isocytidine and isoguanidine, etc.

[0018] The term "primer" refers to an oligonucleotide that is capable of hybridizing (also referred to as "annealing") to a nucleic acid and can be used as a starting point for nucleotide (RNA or DNA) polymerization under suitable conditions (i.e., in the presence of four different nucleoside triphosphates and a polymerization reagent (e.g., DNA or RNA polymerase or reverse transcriptase) in a suitable buffer and at a suitable temperature. The suitable length of a primer depends on the intended use of the primer, but the length of the primer is generally at least 7 nucleotides.

[0019] The term "QPCR" specifies quantitative real-time polymerase chain reaction (PCR), which is also known as "real-time PCR" or "kinetic polymerase chain reaction"; all terms refer to PCR with real-time signal detection.

[0020] The term "ddPCR" refers to droplet digital polymerase chain reaction. In ddPCR, one or more PCR amplifications are performed, where each reaction is separated into multiple water-oil emulsion droplets so that PCR amplification of the target sequence can occur in each individual droplet.

[0021] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1: Investigation of the sample handling method for collecting plate sample for characterization of inhaled vaccine performance Take the new coronavirus vaccine and dilute it to 5.0×10 10 VP / ml, 5.0×10 8 VP / ml, 5.0×10 6 VP / ml. The test samples were pre-treated in different ways: 50 μl of the test sample was added to the EP tube and set up as follows: chemical lysis group, physical lysis group, and commercial kit extraction group; the chemical lysis group was: SDS lysis solution or Triton X-100 was added to the EP tube; the physical lysis group used thermal lysis and freeze-thaw lysis; the commercial kit used was PrepSEQ Residual DNA Sample Preparation Kit, thermo fisher. Finally, qPCR detection was performed.

[0023] The experimental results show that chemical pyrolysis has the smallest RSD value, the shortest time, and is closest to the theoretical value, that is, the highest recovery rate.

[0024] Example 2: Investigation of quantitative methods for characterizing the performance of inhaled vaccines Take the new coronavirus vaccine and dilute it to 5.0×10 10 VP / ml, 5.0×10 9 VP / ml, 5.0×10 8 VP / ml, 5.0×10 7 VP / ml, 5.0×10 6 VP / ml. qPCR and HPLC methods were used for quantitative detection.

[0025] QPCR assay Determine the standard, quantitative plasmid reference by ddPCR method, and dilute the plasmid reference in a gradient manner; prepare the ddPCR system: (3) Droplet generation a) Add the reaction system to the 8 wells in the middle row of the DG8 cartridge; b) Add Droplet Generation Oil for Probes to each of the 8 wells in the bottom row of the DG8 cartridge and cover with a rubber pad. Gently and steadily place the Holder in the droplet generator to start generating droplets, which will be completed in about 2 minutes. c) Pipette the droplets (the top row of wells in the DG8 cartridge) and slowly inject them into the corresponding wells of the 96-well plate. d) Seal the film (4) PCR; Place the 96-well plate in the PCR instrument, set the program, and start the reaction. (5) Droplet signal reading Place the 96-well plate that has completed PCR in the Plate holder, assemble it, and then gently and steadily place it in the droplet reader and run the program; After the program is completed, analyze the results, calculate the concentration of the plasmid reference, and dilute 6 points in a 10-fold gradient as the qPCR standard curve solution.

[0026] Sample pretreatment ① Test sample group: add 50 μl of the test sample to the EP tube, and then add an equal volume of 50 μl of 1% SDS lysis buffer to the EP tube, marked as S0; ② Negative control (NEG) group: add 50 μl of sterile enzyme-free water to the EP tube, and then add an equal volume of 50 μl of 1% SDS lysis solution to the EP tube, marked as N0; Mix the above ① and ② respectively, centrifuge them for a moment and place them in a PCR instrument for lysis to obtain lysis products. PCR program settings: 95℃ 10min; 4℃ ∞.

[0027] ③ Dilute the lysis products S0 and N0 to obtain dilutions for QPCR detection Take the reagents and primers out of the refrigerator in advance to melt them, equilibrate them to room temperature, and mix them by inversion before use. Set up duplicate wells for the test sample, negative control NEG, and no-template control NTC. Set up the QPCR program.

[0028] Test conditions No template control group NTC: no amplification or Ct value should be greater than or equal to 32; Negative control group NEG: no amplification or Ct value should be greater than or equal to 32; Standard curve requirements: The slope of the standard curve is -3.1~-3.8, R 2 ≥0.98; HPLC detection method 1. Connect the instrument, prepare mobile phase A (50mM HEPES solution) and B (50mM HEPES 2M NaCl solution), and open the chromatography workstation software. 2. Equilibrate the chromatographic column. 3. Determine 2) Determination of standard and test products Ad5 virus (5 × 10 11 VP / ml) as the standard, take a series of standard solutions and samples to be tested, centrifuge at 10000rpm and 4℃ for 1min, take the supernatant for detection, and prepare 2 samples in parallel. Wash the chromatographic column with 0.5M NaOH solution between samples.

[0029] 3) Column storage After the test, the column was flushed with the mobile phase of the initial ratio for 30 min, transitioned with ultrapure water, and finally stored in a 20% ethanol solution.

[0030] 4) Results analysis The analysis function of the software was used to integrate and calculate the concentration of the sample to be tested using the area normalization method.

[0031] The results of QPCR & HPLC experiments are as follows:

[0032] The results showed that the RSD value of QPCR at different test sample concentrations was small and closest to the theoretical value.

[0033] Example 3: Characterization of inhaled vaccine performance and investigation of aerosolized dose Place a filter membrane in the MOC stage of the NGI impactor, place the NGI impactor, adapter and throat in a cooling device and cool to 5°C, the relative humidity of the experimental environment is RH40%; connect the throat to the flow meter, turn on the vacuum pump, adjust the flow rate to 15 L / min, and remove the flow meter. Take 0.05ml, 0.1ml, 0.5ml, 1.0ml, and 1.5ml of the new coronavirus vaccine into the nebulizer medicine cup respectively, connect Ultra to the nebulizer with the NGI adapter, and ensure that all connections are sealed. Open the nebulizer to nebulize the vaccine until it is finished, wait for 15 seconds, and turn off the vacuum pump. The NGI level 1-7 collection plates, MOC, throat, adapter, and Ultra+ nebulizer were washed with 10 ml of elution solution respectively. The 11 washed sample solutions were transferred to 50 ml centrifuge tubes respectively, and the number of viral particles copies / ml was detected by QPCR. The same batch of new coronavirus vaccine was used as a control, and the total number of viral particles copies / ml was detected by QPCR. The measurement results were analyzed using INHALYTIX software, and each dose was repeated 3 times. The results are shown in the table below: Main parameters of APSD with different atomization doses

[0034] The results showed that after the dose was increased to 1.5 ml, droplets in the NGI 5th, 6th, and 7th collection plates showed obvious adhesion (see Appendix). Figure 1 ), the recovery rate showed a downward trend.

Claims

1. A method for characterizing the performance of an inhaled vaccine, comprising using NGI to characterize the performance of an inhaled vaccine, characterized in that: The steps include: S1, precool the NGI impactor, connect the flow meter to adjust the flow rate, turn on the vacuum pump to adjust the flow rate, and remove the flow meter; S2, put the sample to be tested into the nebulizer medicine cup, connect the NGI and nebulizer, and turn on the nebulizer until the sample to be tested is nebulized; S3, using an eluent to collect samples in different collection plates 1-7 respectively; S4, extracting nucleic acid from the sample obtained in step S3; S5. Perform QPCR detection.

2. A method as claimed in claim 1, characterized in that: S1 NGI impactor precooling, specific cooling time 60-90min; cooling temperature 0-5℃.

3. A method according to any one of claims 1 to 2, characterized in that: The sample dosage for characterization in S2 is not higher than 1.5 ml.

4. A method according to any one of claims 1 to 3, characterized in that: S4 extracts the nucleic acid in the sample obtained in step S3; the extraction methods include lysis method, magnetic bead method, and column extraction method.

5. A method according to claim 4, characterized in that: The lysis method includes a physical method or a chemical method; the physical lysis method includes: -80°C freezing extraction, ultrasonic extraction, thermal lysis; the chemical lysis method includes treatment with a chemical lysis agent; the optional chemical lysis agents include but are not limited to: SDS lysis, Triton X-100 (Triton X-100), sodium deoxycholate (deoxycholate), ethylphenyl polyethylene glycol (Nonidet P40, NP-40).

6. A method according to any one of claims 1 to 5, characterized in that: The QPCR assay in S5 specifically includes target selection, pre-treatment of samples to be tested, and preparation of standards, which are quantified using the ddPCR method.

7. A method according to any one of claims 1 to 6, characterized in that: The inhaled vaccine includes a viral vector vaccine, an attenuated vaccine, an inactivated vaccine, a subunit vaccine, a virus-like particle vaccine, a DNA vaccine, an RNA vaccine, a PNA vaccine, or a combination of any two of the above vaccines.

8. A method according to any one of claims 1 to 7, characterized in that: The inhaled vaccine includes atomized oral inhalation or atomized nasal inhalation.

9. A method according to any one of claims 1 to 8, characterized in that: The inhalation vaccine includes a powder or a liquid formulation.

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