Characterization of molecule delivery particles by interferometric scattering microscopy

Abstract

The purpose of the present invention is to characterize molecule delivery particles by interferometric scattering microscopy.　The present invention relates to a method for characterizing molecule delivery particles by interferometric scattering microscopy, wherein a characterizing step comprises determining the molecular weight of the molecule delivery particles and / or the count number thereof on the basis of the interferometric scattered light intensity derived from the molecule delivery particles which desorb from a translucent substrate.

Description

Characterization of molecular delivery particles using interference scattering microscopy

[0001] This patent application claims priority and interest under the Paris Convention and priority under Article 41 of the Japanese Patent Act, based on Japanese Patent Application No. 2024-182587 (filed on 18 October 2024), and by reference herein the entire contents of the said application are incorporated herein by reference.

[0002] The present invention relates to a method for characterizing molecular delivery particles by interference scattering microscopy. More specifically, the present invention relates to a method in which the characterization step uses the interference scattering light intensity derived from molecular delivery particles de-adsorbed from a translucent substrate in interference scattering microscopy, a method for producing a composition comprising a first molecular delivery particle comprising the method thereof, and a composition comprising at least one molecular delivery particle produced by the method thereof.

[0003] Particles used to deliver various molecules, including therapeutic compounds, genes, and nutrients, are utilized in a wide range of fields, including drug delivery systems (DDS). A DDS is a technology for efficiently delivering drugs to target sites. Drug carriers used in DDSs include lipid nanoparticles, liposomes (lipid capsules composed of phospholipid bilayers), micelles composed of polyethylene glycol (PEG) and polylactic acid (PLA), and particles composed of polylactic acid-glycolic acid copolymers (also known as PLGA). In the field of gene therapy, viral vectors are used as gene carriers.

[0004] Adeno-associated virus vectors (AAV vectors), a type of viral vector, have recently attracted attention as a gene transport tool in gene therapy. In particular, they have shown therapeutic effects in the treatment of rare congenital diseases, even in diseases for which there were previously no treatment options. More than five types of AAV vectors have already been approved in the United States as treatments for diseases such as hereditary retinal dystrophy, spinal muscular atrophy, hemophilia, and Duchenne muscular dystrophy. An AAV vector is a virus that encapsulates single-stranded DNA encoding the target gene within an icosahedral protein shell (capsid). Typically, AAV vectors are manufactured using biotechnology techniques that utilize the transfection of plasmids with human or insect cells. The manufactured AAV vectors are then purified using a combination of chromatography and centrifugation, followed by solvent exchange and concentration, and finally filter sterilization to become the AAV vector active pharmaceutical ingredient. Alternatively, AAV vectors can be manufactured using genetically modified cells that stably produce AAV vectors. In this case, the AAV vector active pharmaceutical ingredient is obtained through processes such as purification, solvent exchange, and concentration. The AAV vectors produced through these processes usually contain not only complete particles (particles containing the DNA to be introduced), which are the target component with therapeutic effects, but also impurity particle mixtures such as empty particles (particles that do not contain the DNA to be introduced), incomplete particles (particles that contain only a portion of the DNA to be introduced), and excess particles (particles that contain DNA that is longer than the DNA to be introduced, or extra DNA). Therefore, it is necessary to remove the impurity particle mixture, but it is difficult to completely remove these impurity particles because their capsid shell is almost identical to that of the complete particles containing the target component.

[0005] Several methods have been reported for quantifying each component in the particle distribution of AAV vectors, particularly empty particles and complete particles. These quantification methods include calculating purity using the absorbance value A260 / A280, enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) based tests, ultracentrifugation analysis, ultracentrifugation sedimentation velocity method (Patent Document 1), anion exchange chromatography, charge detection mass spectrometry (Non-Patent Document 1), mass photometry (MP), a type of interference scattering microscopy (Non-Patent Document 2), and dFLISA (Non-Patent Document 3). Recently, there have been reports that changing the plasmid ratio during manufacturing alters the complete particle rate (Non-Patent Document 4). Therefore, quantifying the complete particle rate under various manufacturing conditions would be useful in finding manufacturing conditions that yield high yields. However, with the exception of the dFLISA method, these currently proposed methods are inadequate in environments with unpurified AAV vectors, i.e., environments containing many types of proteins that are not AAV vector-related particles, such as AAV vectors in disrupted biological samples like cell culture media or cell lysates. In such environments, impurities from the biological sample interfere with the detection of AAV vector particles, making accurate quantification of complete AAV vector particles difficult. On the other hand, purifying AAV vectors from disrupted biological samples usually takes 3-4 days, and a certain amount of sample is also required, so these methods are not suitable for screening during vector production. Furthermore, while the dFLISA method can quantify empty and complete AAV vector particles in cell lysates, it has the drawback of requiring more than one day for measurement. Moreover, as mentioned above, AAV vector samples often contain intermediate particles and overfilled particles in addition to complete and empty particles, so there is a need to comprehensively quantify AAV vectors from the perspective of particle size distribution.

[0006] Mass photometry (MP) is a powerful analytical technique for single-molecule detection based on the principle of interference scattering microscopy (iSCAT). While it is a particle size distribution determination method, it is simpler and more cost-effective than methods such as ultracentrifugation and charge detection mass spectrometry. MP detects scattered light from particles adsorbed on a solid surface such as glass and determines the mass of the adsorbed particles from the contrast of the signal obtained at the time of adsorption (Non-Patent Literature 5). However, like other quantitative methods, MP has the drawback that it is difficult to accurately quantify complete AAV vector particles in environments containing many cell-derived impurities that are not AAV vector-related particles, such as AAV vectors in disrupted biological samples like cell lysates.

[0007] Japanese Patent Publication No. 2018-505695

[0008] Werle AK, et al. Comparison of analytical techniques to quantitate the capsid content of adeno-associated viral vectors. Mol Ther Methods Clin Dev. 2021 Sep 1;23:254-262. doi: 10.1016 / j.omtm.2021.08.009. PMID: 34703846; PMCID: PMC8505359.Wagner C, et al. Quantification of Empty, Partially Filled and Full Adeno-Associated Virus Vectors Using Mass Photometry. Int J Mol Sci. 2023 Jul 3;24(13):11033. doi: 10.3390 / ijms241311033. PMID: 37446211; PMCID: PMC10341871.Soth, Sereirath, et al. Quantification of Full and Empty Particles of Adeno-Associated Virus Vectors via a Novel Dual Fluorescence-Linked Immunosorbent Assay." Molecular Therapy Methods & Clinical Development (2024).Park S, et al. Enhancing the production of adeno-associated virus (AAV)2 and AAV9 with high full capsid ratio in HEK293 cells through design-of-experiment optimization of triple plasmid ratio. Biotechnol J. 2024 Mar;19(3):e2300667. doi: 10.1002 / biot.202300667. PMID: 38479987.Young G, et al.Quantitative mass imaging of single biological macromolecules. Science. 2018 Apr 27;360(6387):423-427. doi: 10.1126 / science.aar5839. PMID: 29700264; PMCID: PMC6103225.

[0009] The presence of hollow and intermediate particles in AAV vector-based formulations can reduce the overall therapeutic effect of the formulation or trigger undesirable immune responses. Other components, such as AAV vector aggregates, may also be present in AAV vector-based formulations. Therefore, it is necessary to eliminate particle-related impurities from AAV vectors and to accurately and reliably analyze complete particles. In other words, a high proportion of complete particles is required during AAV vector manufacturing. Furthermore, there is still a need for methods that enable the quantitative detection of desired particles in very low concentrations in contaminated environments containing fragments or large aggregates, such as biological samples, and that possess the specificity and sensitivity suitable for clinical settings.

[0010] Accordingly, the present invention includes the following embodiments: <Method for Characterizing> [1] A method for characterizing molecular delivery particles by interference scattering microscopy, comprising the step of characterizing the at least one molecular delivery particle based on measurement data obtained by measuring interference scattered light from a sample containing at least one molecular delivery particle on a translucent substrate, wherein the characterizing step comprises determining one or both of the molecular weight and / or count number of the at least one molecular delivery particle from the interference scattered light intensity derived from the molecular delivery particle de-adsorbed from the translucent substrate. [2] The method according to [1], wherein the characterizing step comprises creating a histogram of interference scattered light intensity derived from the molecular delivery particle de-adsorbed from the translucent substrate; and determining one or both of the molecular weight and / or count number of the at least one molecular delivery particle based on the histogram. [3] The method according to [1] or [2], further comprising measuring interference scattered light from a sample containing at least one molecular delivery particle on a translucent substrate for a predetermined time to obtain measurement data. [4] The method according to any one of [1] to [3], wherein the characterization step includes determining the concentration ratio of the molecular delivery particles in the sample from the count number. [5] The method according to any one of [1] to [3], wherein the characterization step includes determining the concentration of the molecular delivery particles in the sample from the count number. [6] The method according to any one of [1] to [3], wherein the characterization step includes determining the molecular weight of at least one molecular delivery particle, and determining whether or not the target molecular delivery particle is present based on the determined molecular weight and the molecular weight of the target molecular delivery particle. [7] The method according to any one of [1] to [3], wherein the sample containing at least one molecular delivery particle includes a first molecular delivery particle and a second molecular delivery particle, and the characterization step includes determining one or both of the molecular weight and / or count number of the first molecular delivery particle and the second molecular delivery particle based on the scattered light intensity or histogram.[8] The method according to [7], wherein the characterizing step comprises determining one or both of the proportion of the first molecular delivery particles (P1) and the proportion of the second molecular delivery particles (P2) in at least one type of molecular delivery particles from the count number of the first molecular delivery particles (N1) and the count number of the second molecular delivery particles (N2). [9] The method according to any one of [1] to [8], wherein the sample comprises a lysate of a biological sample. [10-1] The method according to [9], wherein the lysate comprises a cell lysate. [10-2] The method according to [9] or [10-1], wherein the cell lysate comprises a cell lysate reagent. [10-3] The method according to any one of [9] to [10-2], wherein the interference scattering light intensity derived from molecular delivery particles de-adsorbed from the translucent substrate is enhanced compared to that in a sample where the cell lysate is not present.

[0011] <Manufacturing Methods> [11-1] A method for producing a composition comprising a first molecular delivery particle, comprising the method described in any of [1] to [10-3] or a method for characterizing the molecular delivery particle. [11-2] A method for producing a composition comprising a first molecular delivery particle, comprising a method for characterizing the molecular delivery particle in the method described in any of [1] to [10-3].

[12] A method according to [11-1] or [11-2] for producing a composition comprising first molecular delivery particles, comprising the step of characterizing the at least one molecular delivery particle based on measurement data of scattered light from a sample comprising at least one molecular delivery particle on a translucent substrate, wherein the at least one molecular delivery particle comprises the first molecular delivery particle and the second molecular delivery particle, and the characterization step comprises determining the count number (N1) of the first molecular delivery particle and determining the ratio (P1) of the first molecular delivery particle to the at least one molecular delivery particle based on the scattered light intensity derived from molecular delivery particles de-adsorbed from the translucent substrate, wherein the ratio (P1) is 50%, 60%, 70%, 80%, or 90% or more and 95% or more for the composition.

[13] The method for manufacturing according to

[12] , wherein the at least one molecular delivery particle is a virus particle, the first molecular delivery particle is a complete particle containing a capsid and the DNA of the target, and the second molecular delivery particle is an empty particle containing a capsid.

[0012] <Composition>

[14] A composition manufactured by a manufacturing method described in any of [11-1] to

[13] , wherein the composition comprises at least one molecular delivery particle, the at least one molecular delivery particle comprises a first molecular delivery particle and a second molecular delivery particle, and the ratio (P1) of the first molecular delivery particle to the at least one molecular delivery particle is 80% or more.

[0013] Figure 1 is a graph showing the signals (MP signals) obtained by mass photometry (MP) measurement. In Figure 1, (A) shows the MP signal of bovine serum albumin obtained by measuring with 1×PBS in a gasket well, (B) shows the MP signal of the AAV8 vector obtained by measuring with 1×PBS in a gasket well, and (C) shows the MP signal of the AAV8 vector obtained by measuring with 0.1% formic acid water in a gasket well. The negative contrast on the right is due to adsorbed particles, and the positive contrast on the left is due to deadsorbed particles. In the figure, Counts means count, and Contrast means contrast. This graph shows the MP signals of various mixtures containing purified AAV8 vector complete particles / empty particles in ratios of 0, 15, 25, 50, 70, and 95% F / E ratio. In Figure 2, (A) shows the MP signal on the adsorbed side, and (B) shows the MP signal on the deadsorbed side. (C) shows the regression lines of the MP signal on the adsorption side (A) and the MP signal on the deadsorption side (B). (D) shows a comparison of the MP signal on the deadsorption side (B) with the theoretical value. Figure 3 is a graph showing the MP signals of vectors in cell solution. In Figure 3, (A) shows the MP signals of cell lysate, (B) shows the MP signals of cell lysate containing the AAV2 vector, and (C) shows the MP signals of cell lysate containing the AAV8 vector. In (B), the line labeled "A" shows the line peak-fitted with a Gaussian function for the signal of empty particles, and the line labeled "B" shows the line peak-fitted with a Gaussian function for the signal of perfect particles. Similarly in (C), the line labeled "A" shows the line peak-fitted with a Gaussian function for the signal of empty particles, and the line labeled "B" shows the line peak-fitted with a Gaussian function for the signal of perfect particles. This graph shows the MP signals for various mixtures containing AAV8 vectors in cell lysates at F / E ratios of 0%, 15%, 25%, 50%, 70%, and 95%. In Figure 4, (A) shows the MP signal on the deadsorption side, and (B) shows a comparison between the expected full articles and the measured full articles.Figure 5 shows the MP signals for the purified AAV8 vector, including empty particles, complete particles, and excess encapsulated particles. In Figure 5, the lines labeled "A" are peak-fitted with a Gaussian function for the signal of empty particles, the lines labeled "B" are peak-fitted with a Gaussian function for the signal of complete particles, and the lines labeled "C" are peak-fitted with a Gaussian function for the excess encapsulated particles. Figure 6 shows the AAV sample and cell lysate prepared with an F / E ratio of 50%, with the AAV sample concentration at 2.5 × 10⁻¹⁰. １１ Figure 6 shows the MP signals of mixtures prepared by mixing cell lysates at cp / mL so that the final ratios were 0, 1, 10, and 20%. (A) shows the MP signal on the deadsorption side. (B) shows a graph of the counts of that MP signal. In Figure 6, the lines labeled "A" are peak-fitted lines with a Gaussian function for the signal of empty particles, and the lines labeled "B" are peak-fitted lines with a Gaussian function for the signal of complete particles. Figure 7 is a table summarizing the AAV8 vector concentration in solution expected from AAV-FP particles of the AAV8 vector and the counts measured by the MP signal of the AAV8 vector contained in the cell lysate, with (A) being the AAV8 vector concentration measured by digital droplet PCR and the counts on the deadsorption side measured by the MP signal of the AAV8 vector contained in the cell lysate. (B) is a graph of these values. Figure 8 is a diagram of the method for determining the titer of the AAV vector by MP measurement. (A) shows the MP signal on the deadsorption side. (B) is a table summarizing the count of AAV-FP obtained, the F / E ratio, and the concentration of AAV8-FP calculated using the calibration curve in Figure 7. Figure 9 is a flowchart of the MP measurement in the present invention.

[0014] <Method for Characterization> In one embodiment, the present invention relates to a method for characterizing molecular delivery particles by interference scattering microscopy, comprising the step of characterizing the at least one molecular delivery particle based on measurement data obtained by measuring scattered light from a sample containing at least one molecular delivery particle on a translucent substrate, wherein the characterization step comprises determining one or both of the molecular weight and / or count number of the at least one molecular delivery particle from the scattered light intensity originating from molecular delivery particles de-adsorbed from the translucent substrate.

[0015] In this invention, "interference scattering microscopy (iSCAT)" is a method that does not require the addition of labeling and can provide information on the relative distribution of particle groups of various masses in a solution, and typically refers to interference scattering mass spectrometry (iSCAMMS), also known as mass photometry (MP). Mass photometry can detect and measure the mass of single objects and the complexes they form in solution. Mass photometry generally detects single molecules by light scattering as they bind nonspecifically or specifically to a surface. Each binding event leads to a change in refractive index at the surface / solution interface, which effectively alters local light scattering and can be detected with high precision by utilizing optimized interference between scattered and reflected light. The optical interference scattering signal obtained by such optimized interference has a magnitude corresponding to the molecular weight.

[0016] In this embodiment, the inventors have succeeded in directly quantifying the ratio and number of AAV vectors to other particles in cell culture media and cell lysates by utilizing a signal corresponding to de-adsorbed particles, rather than the signal of particles adsorbing onto a translucent substrate (e.g., a glass substrate), which is typically used for analysis in interference scattering microscopy, particularly mass photometry.

[0017] Here, "deadsorbed particles" refers to particles that transition from a state of contact with a translucent substrate used to measure interference scattering light to a state of suspension in the sample, or particles that have transitioned to this state. In one embodiment of the present invention, the complete particle ratio determined by the deadsorbed signal correlates with the theoretical value at the same level for both the purified vector and the vector in the cell lysate. In a further embodiment, the number of deadsorbed particles correlates well with the number of particles in the solution. In another embodiment, the signal derived from the AAV vector is significantly stronger in a solution containing a second component, such as a cell lysate, than when the purified AAV vector is measured in phosphate buffer, and it is possible to quantify the complete particle ratio and titer with a smaller sample volume than when purified, which is empirically unexpected. In this regard, the cell lysate acts as a signal enhancer that amplifies the MP signal or count. For example, the composition of the cell lysate may be 1.0 × 10⁻⁶ -6 One example is a solution obtained by adding cells / mL of cells to a solution containing 0.14% polysorbate 20, 50 mM Tris, pH 9.0, 2 mM MgCl2, 69 mM NaCl, and 15 U / μL nuclease. Here, the cells to which the cell lysate is added are not limited to cells producing AAV, but can also be cultured separately, such as HEK293 cells or HeLa cells. Unexpectedly, when the cell lysate reagent 0.14% polysorbate 20, 50 mM Tris, pH 9.0, 69 mM NaCl, 2.1 w / v% BalanCD HEK293, used to prepare the cell lysate, is added to a phosphate buffer containing purified AAV vector, the signal intensity is higher than when the purified AAV vector is measured in phosphate buffer, and it is possible to quantify the complete particle ratio and titer with a smaller sample volume than when measuring without adding the cell lysate reagent. This, too, is unexpected from an empirical perspective.

[0018] In the present invention, the "translucent substrate" can be any material as long as it is a solid substrate that transmits light and can measure the scattered light intensity of particles. Examples include glass substrates, cycloolefin resins, or substrates coated with polymer resins, etc. The shape of the translucent substrate can be any shape that can measure the scattered light intensity of particles. For example, a plate shape can be used.

[0019] In this invention, "molecular delivery particles" refer to small objects capable of delivering various molecules, including therapeutic compounds, genes, and / or nutrients, and are used in various fields, including drug delivery systems. A drug delivery system (DDS) is a technology aimed at efficiently delivering drugs to a target site. Drug carriers used in DDSs include liposomes, which are lipid capsules composed of a phospholipid bilayer; micelles composed of polyethylene glycol (PEG) and polylactic acid (PLA); and particles composed of polylactic acid-glycolic acid copolymers (also called PLGA). In the field of gene therapy, viral vectors are used as gene carriers. In other words, in this invention, molecular delivery particles include, but are not limited to, lipid nanoparticles, viral particles, liposomes, albumin particles, micelles, or polylactic acid-glycolic acid copolymer particles.

[0020] When the molecular delivery particles are viral particles, they can be used, for example, as gene therapy or viral vector vaccines. Examples of viruses include, but are not limited to, adeno-associated viruses (AAVs), adenoviruses, herpesviruses, Sendai viruses, stealth viruses, lentiviruses, or retroviruses.

[0021] The particle size of the molecular delivery particles may be, for example, 5 nm to 10,000 nm, 10 nm to 5,000 μm, 20 nm to 1,000 nm, or 30 nm to 500 nm.

[0022] The particles for molecular delivery can be prepared according to known methods. In one example, the particles for molecular delivery may be recombinant virus particles. Recombinant virus particles can be artificially prepared using genetic engineering. The particles for molecular delivery may be virus particles produced using genetic engineering, or may also be virus particles produced in cultured cells or eggs.

[0023] The molecular delivery particles can include, for example, two or more types of particles (e.g., the first molecular delivery particles and the second molecular delivery particles). When the particles for molecular delivery are virus particles, the virus particles may include, for example, complete virus particles containing a capsid that is the coat as the first molecular delivery particles and its genomic DNA molecule, and capsid particles (also referred to herein as "hollow particles") that contain a capsid as the second molecular delivery particles but do not contain a genomic DNA molecule. Here, the "coat" means a structure that separates the inside and outside of the particle. The coat contains macromolecules such as proteins or lipids, for example. The combination of two or more types of particles included in the molecular delivery particles can be appropriately set according to the purpose. In one example, complete virus particles can be used as the first molecular delivery particles, capsid particles can be used as the second molecular delivery particles, and other virus particles (e.g., dimers of complete virus particles, dimers of capsid particles, dimers of complete virus particles and capsid particles, or aggregates of virus particles, or mixtures thereof) can be used as the third molecular delivery particles. For example, when the third molecular delivery particle is a dimer of complete virus particles and capsid particles, the third molecular delivery particle includes a third coat containing two capsids and a third delivery molecule containing one genomic DNA. In another example, capsid particles can be used as the first molecular delivery particles, complete virus particles can be used as the second molecular delivery particles, and other virus particles can be used as the third molecular delivery particles.

[0024] The above examples describe types of virus particles, but the types of particles are not limited to these. For example, if the molecular delivery particle is a liposome, the liposome may include a complete particle containing a lipid bilayer as an outer covering and encapsulated molecules as the first molecular delivery particle, and a hollow particle containing the lipid bilayer but not the molecules as the second molecular delivery particle. In other examples, the liposome may include a complete particle as the first molecular delivery particle, a hollow particle as the second molecular delivery particle, and other particles (e.g., liposome aggregates) as the third molecular delivery particle. If the molecular delivery particle is an albumin particle, the albumin particle may include a particle having a first molecular weight as the first molecular delivery particle, and a particle having a second molecular weight as the second molecular delivery particle.

[0025] Molecular delivery particles can be used, for example, to deliver molecules to a predetermined location within the body of an animal. The animal may be, for example, a reptile, a bird, or a mammal. The mammal may be, for example, a human or a non-human mammal. Non-human mammals may be, for example, rodents such as mice, non-human primates such as chimpanzees, artiodactyls such as cattle, odd-toed ungulates such as horses, or companion animals such as dogs and cats. In one embodiment, the mammal is a non-human primate or a human. The predetermined location may be, for example, an organ or cell of an animal. The predetermined organ may be, for example, the heart, lungs, liver, stomach, pancreas, or kidney. The predetermined cell may be, for example, a normal cell or an abnormal cell. A normal cell may be, for example, a cardiomyocyte, hepatocyte, or hematopoietic cell. An abnormal cell may be, for example, a cancer cell.

[0026] In the present invention, the characterizing step includes determining either or both of the molecular weight and / or the count number of the molecular delivery particles from the scattered light intensity derived from the molecular delivery particles desorbed from the translucent substrate. As described above, the detection of the molecular delivery particles can be performed using light scattering, such as interference scattering microscopy (iSCAT), interference scattering mass spectrometry or mass photometry. iSCAT includes determining the interference between the light scattered by the object in the sample and the light reflected from the sample position. The interference depends on the scattering amplitude of the object (and then its polarizability, i.e., volume, density, refractive index), and is measured as the iSCAT signal.

[0027] Mass photometry is a developed form of iSCAT (Kukura et al., Nature Methods 2009, 6: 923 - 935, Ortega - Arroyo et al., Physical Chemistry Chemical Physics 2012, 14: 15625 - 15636), measures the interference scattered light by a single molecule, and directly correlates it with the molecular weight. The light scattered by the particle is linearly proportional to the volume and refractive index of the particle. The correlation between the light interference scattering signal (also referred to as the "MP signal") and the molecular weight applies to various biomolecules (glycoproteins, nucleic acids, lipids), and mass photometry is a universal analytical tool for biomolecules in solution.

[0028] Using the optical interference scattering signal, a mass can be assigned to the detected object. Furthermore, the concentration of the object can be determined from the number of times the signal is detected within a certain period of time. Therefore, the method of the present invention includes determining the MP signal, which is an optical interference scattering signal, i.e., the "interference scattering light intensity," determining the mass of the particles using the "interference scattering light intensity," and further determining the particle concentration using the number of detections (counts) of signals originating from particles with a specific interference scattering light intensity. The "number of detections" means the number of times the molecular delivery particles come into contact with the translucent substrate within a predetermined measurement time. In this disclosure, the number of detections is also referred to as the count. From the count over a predetermined time, the number of molecular delivery particles present in the sample can be determined. Therefore, in such an embodiment, for example, assuming complete particles / partial particles / overfilled particles of an AAV vector, their ratios can be determined. That is, this embodiment is the method of the present invention, which includes determining the concentration ratio of the molecular delivery particles in the sample from the count. Then, by adding a molecular delivery particle (first molecular delivery particle) whose concentration is known and creating a calibration curve, the concentration of a second molecular delivery particle, whose concentration is unknown in the sample, can be determined by the calibration curve. This embodiment is a method of the present invention, which includes determining the concentration of the molecular delivery particle in the sample from the count. In such an embodiment, the method of the present invention may further include comparing the iSCAT contrast with the calibration curve or standard curve to determine the molecular weight or concentration of the target molecular delivery particle. When mass photometry is used in the method of the present invention, the molecular weight is indicated by the mass photometry method.

[0029] In the present invention, "sample" may be a biological, industrial, or environmental sample. Biological samples include samples taken from the body or individual of a human or animal, such as blood, serum, plasma, urine, saliva, lymph, sweat, amniotic fluid, cerebrospinal fluid, breast milk, tears, secretions, synovial fluid, semen, bile, or mucus, lung fluid, and fecal samples. Body fluids may be capillaries, venous blood, or arterial blood, or plasma or serum derived therefrom. In the present invention, a sample may be a biological sample containing cell lysates such as cell lysates. In the present invention, it has been found that even samples containing such lysates can be measured well.

[0030] The present invention relates, in another embodiment, to a method of the present invention in which the characterizing step comprises: creating a histogram of scattered light intensity derived from molecular delivery particles de-adsorbed from the translucent substrate; and determining, based on the histogram, one or both of the molecular weight and / or count number of the at least one molecular delivery particle. In the present invention, the scattered light intensity histogram means a distribution diagram in which the number of detections of MP signals, which are light scattering signals, i.e., particles with different "interference scattering light intensities", is assigned in a bar graph shape for each contrast. By fitting a group of bar-shaped data markers with peaks using a function such as a Gaussian distribution, it can be determined whether the group of data markers corresponds to a single particle.

[0031] The present invention relates, in yet another embodiment, to a method of the present invention, further comprising measuring scattered light from a sample containing at least one type of molecular delivery particles on a translucent substrate for a predetermined time to obtain measurement data. In such an embodiment, the present invention provides, in yet another embodiment, a method for characterizing molecular delivery particles by interference scattering microscopy, comprising the steps of: measuring interference scattered light from a sample containing at least one type of molecular delivery particles on a translucent substrate for a predetermined time to obtain measurement data; and characterizing the at least one type of molecular delivery particle based on the measurement data, wherein the characterization step includes determining one or both of the molecular weight and / or count number of the at least one type of molecular delivery particle from the interference scattered light intensity originating from molecular delivery particles de-adsorbed from the translucent substrate.

[0032] The present invention relates, in a further alternative embodiment, to a method of the present invention in which the sample containing at least one type of molecular delivery particle contains a first molecular delivery particle and a second molecular delivery particle, and the characterization step comprises determining one or both of the molecular weight and / or count number of the first molecular delivery particle and the second molecular delivery particle based on the scattered light intensity or histogram. In such an embodiment, preferably, the present invention relates to a method of the present invention in which the characterization step comprises determining one or both of the proportion of the first molecular delivery particle (P1) and the proportion of the second molecular delivery particle (P2) in the at least one type of molecular delivery particle from the count number of the first molecular delivery particle (N1) and the count number of the second molecular delivery particle (N2). In the above embodiment, as described above, the concentration ratio of the first molecular delivery particle and the second molecular delivery particle in the sample can be determined, and the concentration of molecular delivery particles contained in the sample can be determined by a calibration curve. "Molecular delivery particle," "sample," "scattered light intensity," "histogram," and "count number" are as described above.

[0033] <Manufacturing Method> In another embodiment, the present invention relates to a method for manufacturing a composition comprising first molecular delivery particles, the method comprising the method of the present invention or a method for characterizing the molecular delivery particles in the present invention. In the method for characterizing the molecular delivery particles in the present invention, the characterization step includes determining one or both of the molecular weight and / or count number of at least one molecular delivery particle from the scattered light intensity derived from the molecular delivery particles de-adsorbed from the translucent substrate. In this method, the ratio of empty particles to complete particles and the number of particles can be directly quantified without purifying the AAV vector in the cell lysate, which was difficult to quantify accurately in conventional observation methods using the adsorption side signal, for example, because the signal of the AAV vector was masked by the signal derived from the cell lysate. This method allows for the quantification of AAV vector particles with a sample volume of only a few μL of MP and a measurement time of a few minutes, making it possible to screen a large number of manufacturing conditions in units of several hundred μL to several mL in a short time, and contributing to the mass production of molecular delivery particles, including AAV vectors.

[0034] In such a form, a preferred method of the present invention is a method of the present invention for producing a composition comprising first molecular delivery particles, comprising the step of characterizing the at least one molecular delivery particle based on measurement data of interference scattered light measured from a sample comprising at least one molecular delivery particle on a translucent substrate, wherein the at least one molecular delivery particle comprises the first molecular delivery particle and the second molecular delivery particle, and the characterization step comprises determining the count number (N1) of the first molecular delivery particle and determining the ratio (P1) of the first molecular delivery particle to the at least one molecular delivery particle based on the interference scattered light intensity derived from molecular delivery particles de-adsorbed from the translucent substrate, wherein the ratio (P1) is 80% or more, preferably 90% or more, more preferably 95% or more, and the present invention relates to a method for producing the composition wherein the ratio (P1) is low, for example 5%, 10%, 30%, 50%, 60%, 70%, 80%, or 90% or more. In this embodiment, the "translucent substrate," "molecular delivery particles," "sample," "measurement data of scattered light," etc., are as described above.

[0035] In the manufacturing method of the present invention, specifically, the at least one molecular delivery particle is a virus particle, the first molecular delivery particle is a complete particle containing a capsid and the target DNA, and the second molecular delivery particle is an empty particle containing a capsid.

[0036] <Composition> In another embodiment, the present invention relates to a composition produced by the manufacturing method of the present invention, wherein the composition comprises at least one molecular delivery particle, the at least one molecular delivery particle comprises a first molecular delivery particle and a second molecular delivery particle, and the ratio (P1) of the first molecular delivery particle to the at least one molecular delivery particle is 80% or more, preferably 90% or more, and more preferably 95% or more.

[0037] The compositions of the present invention can be used in gene therapy, in vivo gene therapy, viral vector vaccines, or modification of therapeutic cells. In such embodiments, the present invention relates, as another embodiment, to a method for performing gene therapy, in vivo gene therapy, viral vector vaccines, or modification of therapeutic cells, comprising administering the compositions of the present invention to a subject in need of such therapy. Furthermore, as another embodiment, the present invention relates to the compositions of the present invention for performing gene therapy, in vivo gene therapy, viral vector vaccines, or modification of therapeutic cells. As yet another embodiment, the present invention relates to the use of the compositions of the present invention for producing pharmaceuticals for performing gene therapy, in vivo gene therapy, viral vector vaccines, or modification of therapeutic cells.

[0038] Furthermore, as another embodiment of the present invention, the present invention relates to a composition produced by the manufacturing method of the present invention, wherein the composition comprises at least one molecular delivery particle, the at least one molecular delivery particle comprises a first molecular delivery particle and a second molecular delivery particle, and the ratio (P1) of the first molecular delivery particle to the at least one molecular delivery particle is 5%, 10%, 30%, 50%, 60%, 70%, 80%, or 90% or more. The composition of the present invention can be used as a raw material for further purification of the molecular delivery particles contained herein.

[0039] The terms and descriptions of embodiments provided herein shall apply as appropriate between the aspects and embodiments provided herein, unless otherwise specified.

[0040] The following describes specific embodiments, which are merely preferred embodiments of the present invention and do not in any way limit the invention described in the attached claims.

[0041] [Materials and Methods] 1. Sample Preparation 1-1) Adeno-associated virus (serotype 8) (AAV8) vector The adeno-associated virus (serotype 8) (AAV8) vector was prepared by co-transfection of three plasmids. Specifically, the pAAV-Rep&Cap (serotype 8), pAd helper, and target transgene (CMV-EGFP) plasmids were co-transfected in a 1:1:1 ratio into suspension HEK293T cells cultured in a 200 mL flask. After 96 hours, the transfected cells were treated with a final concentration of 0.14% polysorbate 20, 50 mM Tris, pH 9.0, 69 mM NaCl, and 2 mM MgCl. ２ The cells were lysed by adding 15 U / μL nuclease and centrifuged at 4°C and 4000 g. A portion of the supernatant was used for direct analysis of the cell lysate. The remaining supernatant was filtered through a 0.22 μm filter. The filtered AAV vector was purified by affinity chromatography using an AAVX column (Thermo Fisher Scientific, USA). Subsequently, ultracentrifugation with a cesium chloride density gradient was performed to separate complete particles containing the full-length vector genome (hereinafter referred to as "AAV8-FP") from empty particles not containing the vector genome (hereinafter referred to as "AAV8-EP"). The empty particles and complete particles were then dialyzed in 1×PBS 200 mM NaCl 0.001 w / v% poloxamer 188 solution. The PBS stock solution (x10) was purchased from Thermo Scientific (USA), NaCl from Nacalai Tesque (Tokyo), and Poloxamer 188 (European Pharmacopoeia standard substance) from BASF (Germany).

[0042] 1-2) Adeno-associated virus (serotype 2) (AAV2) vector The adeno-associated virus (serotype 2) (AAV2) vector was prepared using co-transfection of three plasmids. In detail, the procedure was essentially the same as described in section 1-1) above, except that pAAV-Rep&Cap (serotype 2) was used.

[0043] 1-3) AAV8 vectors containing empty and excess contained particles: The AAV8 vectors containing empty and excess contained particles were purchased from Vectorbuilder (USA).

[0044] 1-4) Samples for quantitative evaluation of complete particles / empty particles in purified solution: The complete particle / empty particle ratio (complete particles / (complete particles + empty particles) × 100%, F / E ratio) was 0, 15, 25, 50, 70, and 95%. The two were mixed in a manner in which AAV8-FP was added in proportion to AAV8-EP. That is, F / E = 0 means only AAV8-EP, and F / E = 95 means a mixture of 5% AAV8-EP and 95% AAV8-FP. Each sample was 1.5 × 10 before measurement. １２ The solution was diluted with 1×PBS 200 mM NaCl 0.001 w / v% poloxamer 188 solution to a concentration of cp / mL.

[0045] 1-5) Samples for measuring complete / empty particles in cell lysates: For the AAV8 vector, the supernatant obtained after cell lysis was diluted 10-fold with 1×PBS. For the AAV2 vector, the cell lysate was diluted 50-fold with 1×PBS. In addition, a sample was prepared by adding cell lysis reagents to purified AAV8 vector to a final concentration of 0.14% polysorbate 20, 50 mM Tris, pH 9.0, and 69 mM NaCl.

[0046] 1-6) Sample suspension HEK293T cells were cultured for quantitative evaluation of complete / empty particles in cell lysates, and the cells were lysed without transfection (cell lysates). AAV samples prepared with F / E ratios of 0, 15, 25, 50, 70, and 95% were 1.0 × 10⁻¹⁶ PBS 200 mM NaCl 0.001 w / v% poloxamer 188 solution. １２ The sample was diluted to cp / mL. 20 μL of the diluted AAV sample and 20 μL of the cell lysate were mixed.

[0047] 2. Various Measurements 2-1) UV Spectrum Measurement UV measurements were performed using BIOMATE160 (Thermo Fisher Scientific, USA).

[0048] 2-2) Calculation of AAV vector concentration The absorbance at 260 nm and 280 nm was measured, and the concentrations of AAV8-EP and AAV8-FP were determined using the following formula: Molar concentration of AAV8-EP (C) ｃａｐ，ｅｍｐｔｙwas calculated using Equation 1 based on Lambert-Beer's law. On the other hand, the molar concentration (C ｃａｐ，ｆｕｌｌ ) of the capsid of AAV8-FP was calculated using Equation 2 by referring to the literature (Wu D, Hwang P, Li T, Piszczek G. Rapid characterization of adeno-associated virus (AAV) gene therapy vectors by mass photometry. Gene Ther. 2022;29(12):691-697. doi:10.1038 / s41434-021-00311-4).

[0049] (Equation 1) (Equation 2)

[0050] Here, C ｃａｐ is the molar concentration (M －１ cm －１ ) of the AAV vector, L is the optical path length (1 cm －１ ), A ２８０ and A ２６０ are the absorbances at 280 nm and 260 nm, respectively, ε ｃａｐ，２８０ and ε ｃａｐ，２６０ are the molar extinction coefficients (M －１ cm －１ ) of the capsid at 280 nm and 260 nm, respectively, ε ＤＮＡ，２８０ and ε ＤＮＡ，２６０ are the molar extinction coefficients per base (M －１ cm －１ ) at 280 nm and 260 nm, respectively. ε ｃａｐ，２８０ , ε ｃａｐ，２６０ , ε ＤＮＡ，２８０ , and ε ＤＮＡ，２６０This was determined by ultracentrifugation analysis based on the literature (Maruno T, Usami K, Ishii K, Torisu T, Uchiyama S. Comprehensive Size Distribution and Composition Analysis of Adeno-Associated Virus Vector by Multiwavelength Sedimentation Velocity Analytical Ultracentrifugation. J Pharm Sci. 2021;110(10):3375-3384. doi:10.1016 / j.xphs.2021.06.031), and is 7.34 × 10⁻¹⁴. ６ M －１ cm －１ , 3.18 x 10 ６ M －１ cm －１ , 1.315 × 10 ７ M －１ cm －１ , 2.248 × 10 ７ M －１ cm －１ I used it.

[0051] 2-3) Mass photometry (MP) measurement For all samples, MP measurement was performed using TwoMP (Refeyn Ltd, Oxford, UK). A 2x3 cut culture well gasket (Grace Bio-Labs, Bend, OR) was placed on a coverslip (1.5H thickness, 24x50 mm; THORLABS, Newton, NJ). Then, 18 or 16 μL of 1x PBS was injected into the gasket well, and the focus was automatically adjusted. As a condition to suppress AAV deadsorption, 0.1% formic acid was used instead of 1x PBS for only one sample. Next, 2 or 4 μL of the sample was added to the same well to a final volume of 20 μL and mixed by pipetting. Next, 60 seconds of movie data was recorded using AcquireMP version R1.1 (Refeyn Ltd). The movie file was then analyzed using DiscoverMP version R1 (Refeyn Ltd).

[0052] 2-4) Estimation of Contrast and Corresponding Mass MP measurement detected the signal (MP signal) that occurs when scattered light and reflected light from the glass interfere when particles adsorb or deadsorb onto the glass surface. The contrast of the obtained signal is proportional to the molecular weight of the particles adsorbed or deadsorbed onto the glass surface at the time of measurement. Therefore, in order to estimate the contrast corresponding to the mass of AAV particles (empty particles, complete particles, excess contained particles), the molecular weight of each sample, which is a sample with a known mass, was used to create a Contrast-to-Mass (CTM) calibration using apoferritin (Sigma-Aldrich, A3660) and empty AAV8 particles with only VP3 capsids (AAV8-VP3-EP), and applied to the contrast distribution obtained from the samples to obtain the mass distribution.

[0053] The contrast used in this process was a negative value. When analyzing movie data using DiscoverMP version R1 (Refeyn Ltd), a negative contrast value corresponds to the signal of adsorbed particles. Subsequently, the mass and contrast corresponding to empty and complete AAV particles were obtained from the resulting mass distribution. The theoretical mass of the empty particle was calculated by determining the molecular weight of each structural protein from the amino acid composition of VP1, VP2, and VP3, which constitute the AAV capsid, and assuming that the capsid contains VP1:VP2:VP3 in a ratio of 5:5:50. The theoretical mass of the complete particle was determined by adding the mass of the ssDNA contained within the empty particle, 777498.27Da, to the theoretical mass of the obtained empty particle.

[0054] 2-5) Determination of the perfect particle / empty particle ratio: A Python script was used to peak-fit the histogram of the MP contrast distribution with a Gaussian function for empty particles, perfect particles, and excess enclosed particles (if present). (Equation 3) Here, a1 and a2 were input as the estimated counts of empty and perfect particles, mu1 and mu2 as the estimated contrasts of empty and perfect particles, respectively, and σ1 and σ2 as the estimated full width at half maximum, respectively, and the models were fitted. Subsequently, each peak was integrated to obtain the peak area, and the ratio of perfect particles to empty particles and excess contained particles (if present) was determined from the peak area. In this process, for MP measurements of purified AAV particles, both the negative and positive (adsorption and deadsorption) MP contrast distributions were peak-fitted, and for MP measurements of AAV particles in cell lysates, the positive side (deadsorption side) was peak-fitted to determine the perfect particle / empty particle ratio.

[0055] [Results] 1. Mass photometry (MP) measurement The MP signal of the AAV8 vector prepared in 1-1) was measured according to the mass photometry (MP) measurement described in 2-3). The results obtained are shown in Figure 1. In Figure 1, (A) is the MP signal of bovine serum albumin obtained by measuring with 1×PBS in the gasket well, (B) is the MP signal of the AAV8 vector obtained by measuring with 1×PBS in the gasket well, and (C) is the MP signal of the AAV8 vector obtained by measuring with 0.1% formic acid water in the gasket well. The negative contrast (right) is due to adsorbed particles, while the positive contrast (left) is due to deadsorbed particles. In bovine serum albumin, only the adsorbed signal was observed, but in the AAV vector, a signal that repeatedly adsorbed and deadsorbed was observed. However, even in the AAV vector, under specific conditions (here, in the presence of 1% formic acid water), only the adsorption signal was observed, similar to other proteins.

[0056] 2. Evaluation of MP signals of purified AAV vectors on the unadsorption side The MP signals were measured for each mixture containing complete particles / empty particles of purified AAV8 vector prepared in 1-4) in a known specific ratio. The results are shown in Figure 2. In Figure 2, (A) shows the MP signal on the unadsorption side, and (B) shows the MP signal on the adsorption side. (C) shows the regression line of the MP signal on the unadsorption side (A) and the MP signal on the adsorption side (B) and the theoretical value. (D) is a comparison of the MP signal on the unadsorption side and the MP signal on the adsorption side. (D) shows that the same level of quantitative accuracy can be obtained using the unadsorption side as with the adsorption side.

[0057] 3. Evaluation of MP signals of AAV vectors in cell lysates The MP signals were measured for the samples prepared in 1-5). The results are shown in Figure 3. In Figure 3, (A) shows the MP signal of cell lysates, (B) shows the MP signal of cell lysates containing AAV2 vector, and (C) shows the MP signal of cell lysates containing AAV8 vector. The negative contrast (right) is due to adsorbed particles, while the positive contrast (left) is due to deadsorbed particles. Therefore, from (A), it can be seen that many particles in the cell lysates do not deadsorb after adsorption, and from (B) and (C), the peaks on the deadsorbed side are mainly derived from AAV vector particles. Viral vectors that recover and lyse only the precipitated cells, such as the AAV2 vector, have relatively few impurities, and the ratio of empty particles to perfect particles can be determined on the adsorption side (B). On the other hand, in (C), it can be seen that the negative contrast on the adsorption side does not fit well to the particle signal. In other words, for vectors that need to be solubilized including the culture medium, such as the AAV8 vector, fitting is not successful on the adsorption side (negative side), making it difficult to achieve a good ratio of empty particles to complete particles. However, it is clear that fitting is successful on the deadsorption side (positive side).

[0058] 4. Evaluation of MP signals of AAV vectors in cell lysates on the deadsorption side The MP signals were measured for each mixture containing complete particles / empty particles of AAV8 vector in a known specific ratio in the cell lysates prepared in 1-6). The results are shown in Figure 4. In Figure 4, (A) shows the MP signal on the deadsorption side, and (B) shows a comparison between the predicted complete particle ratio and the observed complete particle ratio. For AAV8 vectors in cell lysates, quantitative accuracy equivalent to that of the adsorption side was obtained by using the signal on the deadsorption side.

[0059] 5. Evaluation of MP signals of excess encapsulated particles For the purified AAV8 vector, the MP signals of excess encapsulated particles were measured in addition to empty and complete particles. The results are shown in Figure 5. Figure 5 shows that it is possible to quantify not only empty and complete particles, but also systems containing excess encapsulated particles, on both the adsorption and deadsorption sides.

[0060] 6. Searching for conditions under which the signal is enhanced To search for conditions under which the signal is enhanced, an AAV sample prepared with an F / E ratio of 50% and a cell lysate or cell lysate reagent were used, with the concentration of the AAV sample being 1.0 × 10⁻⁶. １１ The cell lysate or cell lysis reagent was mixed at cp / mL so that the final ratios were 0, 1, 10, and 20%. The deadsorption MP signal was measured for different ratios of cell lysate or cell lysis reagent contained in the sample. The results are shown in Figure 6. In Figure 6, (A) shows the deadsorption MP signal when cell lysate is added. (B) shows the count graph for (A). (C) shows the deadsorption MP signal when cell lysis reagent is added. (D) shows the count graph for (C). Figures 6(A) and (B) show that the count increases per unit of measurement time when 10-20% cell lysate is included. Figures 6(C) and (D) show that the count increases per unit of measurement time when cell lysate or cell lysis reagent is included.

[0061] 7. Estimation of AVV vector concentration in cell lysate: AAV-FP is diluted 2.0 × 10⁻¹⁶ times in a 1 × PBS 200 mM NaCl 0.001 w / v% poloxamer 188 solution. １２ Dilute to vg / mL and 2.0 × 10 １２ AAV samples were prepared in a 2-fold dilution series starting from vg / mL. 15 μL of each AAV sample concentration was mixed with 15 μL of cell lysate. In detail, the AAV8 vector concentration in the solution, estimated from the AAV-FP particles of the AAV8 vector measured by digital droplet PCR, and the deadsorbed count of the AAV8 vector contained in the cell lysate, measured by MP signaling, were determined. The obtained results are shown in Figure 7. (A) is a table summarizing the AAV8 vector concentration measured by digital droplet PCR and the deadsorbed count of the AAV8 vector contained in the cell lysate, measured by MP signaling. (B) is a graph of these values. 3.13 × 10 ９ -4.0 x 10 １１ Within the range of vg / mL, the concentration of the AAV8 vector could be estimated with a relative standard deviation of 26% or less. 8. Determination of the concentration of the AAV8 vector in a sample of unknown concentration By adding an AAV vector of known concentration to a contaminated environment such as a cell lysate and performing MP measurement, a calibration curve can be obtained. Subsequently, by performing MP measurement on a sample of unknown concentration, the concentration of the AAV vector contained in the sample can be determined. 15 μL of a sample of unknown concentration and F / E ratio dissolved in 1x PBS + 200 mM NaCl + 0.001% P188 and 15 μL of cell lysate were mixed. This was measured by MP, and the count number and F / E ratio were calculated. Furthermore, the concentration was determined from the count number using the calibration curve. The obtained results are shown in Figure 8. (A) shows the MP signal on the deadsorption side. (B) is a table summarizing the count number of AAV-FP obtained, the F / E ratio, and the concentration of AAV8-FP calculated using the constituent curve in Figure 7.

[0062] The present invention offers several advantages: it enables the quantitative determination of molecular delivery particles, such as AAV vector particles, in a noisy environment using only a few μL of sample and within a few minutes of measurement; and it allows for analysis without sample loss due to purification. This makes it possible to screen numerous manufacturing conditions in units of several hundred μL to several mL in a short time. This is expected to optimize future AAV vector manufacturing methods and contribute to expanding their demand.

Claims

1. A method for characterizing molecular delivery particles by interference scattering microscopy, comprising the step of characterizing the at least one molecular delivery particle based on measurement data obtained by measuring interference scattered light from a sample containing at least one molecular delivery particle on a translucent substrate, wherein the characterization step comprises determining one or both of the molecular weight and / or count number of the at least one molecular delivery particle from the interference scattered light intensity derived from molecular delivery particles de-adsorbed from the translucent substrate.

2. The method according to claim 1, wherein the characterization step comprises creating a histogram of interference scattering light intensities derived from molecular delivery particles de-adsorbed from the translucent substrate; and determining, based on the histogram, one or both of the molecular weight and / or count number of the at least one molecular delivery particle.

3. The method according to claim 1 or 2, further comprising measuring interference scattered light from a sample containing at least one type of molecular delivery particle on a translucent substrate for a predetermined time to obtain measurement data.

4. The method according to claim 1 or 2, wherein the characterization step includes determining the concentration ratio of the molecular delivery particles in the sample from the count.

5. The method according to claim 1 or 2, wherein the characterization step includes determining the concentration of the molecular delivery particles in the sample from the count.

6. The method according to claim 1 or 2, wherein the characterization step includes determining the molecular weight of at least one molecular delivery particle, and determining whether or not the target molecular delivery particle exists based on the determined molecular weight and the molecular weight of the target molecular delivery particle.

7. The method according to claim 1 or 2, wherein the sample comprising at least one type of molecular delivery particle comprises a first molecular delivery particle and a second molecular delivery particle, and the characterization step comprises determining one or both of the molecular weights and / or count numbers of the first molecular delivery particle and the second molecular delivery particle based on the scattered light intensity or histogram.

8. The method according to claim 7, wherein the characterization step includes determining one or both of the proportion of the first molecular delivery particles (P1) and the proportion of the second molecular delivery particles (P2) in at least one type of molecular delivery particle, based on the count number of the first molecular delivery particles (N1) and the count number of the second molecular delivery particles (N2).

9. The method according to claim 1, wherein the sample includes crushed material of a biological sample.

10. The method according to claim 9, wherein the crushed material includes a cell lysate.

11. A method for producing a composition comprising a first molecular delivery particle, comprising a method for characterizing the molecular delivery particle according to claim 1.

12. A method according to claim 11 for producing a composition comprising first molecular delivery particles, comprising the step of characterizing the at least one molecular delivery particles based on measurement data obtained by measuring scattered light from a sample comprising at least one molecular delivery particles on a translucent substrate, wherein the at least one molecular delivery particles comprises the first molecular delivery particles and the second molecular delivery particles, and the characterization step comprises determining the count number (N1) of the first molecular delivery particles and determining the ratio (P1) of the first molecular delivery particles to the at least one molecular delivery particles based on the scattered light intensity derived from molecular delivery particles de-adsorbed from the translucent substrate, wherein the ratio (P1) is 50%, 60%, 70%, 80%, or 90% or more for producing the composition.

13. The method for producing a product according to claim 12, wherein the at least one molecular delivery particle is a virus particle, the first molecular delivery particle is a complete particle containing a capsid and the target DNA, and the second molecular delivery particle is an empty particle containing a capsid.

14. A composition manufactured by a manufacturing method according to any one of claims 11 to 13, wherein the composition comprises at least one molecular delivery particle, the at least one molecular delivery particle comprises a first molecular delivery particle and a second molecular delivery particle, and the ratio (P1) of the first molecular delivery particle to the at least one molecular delivery particle is 80% or more.

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