Electrophoresis-mediated characterization of DNA content of adeno-associated virus capsid

Abstract

Aspects of a method of characterizing a population of particles presumed to comprise recombinant nucleic acids in a fluid sample according to the present disclosure include extracting nucleic acids from particles, labeling the extracted recombinant nucleic acids, separating the labeled nucleic acids by size, and comparing the separated labeled nucleic acids to a standard, and determining one or more of the following: 1) the ratio of intact virus particles to empty virus particles in the fluid sample, 2) the ratio of intact virus particles or empty virus particles to partial intact virus particles, and 3) the ratio of virus particles comprising intact recombinant genomes to virus particles comprising incomplete recombinant genomes, thereby characterizing a population of particles in the fluid sample. Optionally, the method includes extracting a protein from the particle, labeling the extracted protein, separating the labeled protein according to size, and comparing the separated labeled protein to a standard.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 348,166, filed on June 2, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure generally relates to evaluating the DNA content of a population of particles presumed to contain a recombinant nucleic acid. According to specific aspects, the present disclosure relates to evaluating a population of particles presumed to contain a recombinant nucleic acid to determine: 1) the proportion of particles in a sample that correctly contain an intact recombinant nucleic acid ("intact"), 2) the proportion of particles in a sample that contain an incorrectly sized recombinant nucleic acid ("partially intact"), 3) the proportion of viral capsids in a sample that do not contain a detectable recombinant nucleic acid ("empty"), or 4) any combination of 1), 2), and 3). Background Art

[0004] Particles containing recombinant nucleic acids, such as viral vectors, have various uses, including in recombinant technology and gene therapy. In current gene therapy, viral vectors, including adeno-associated virus (AAV), have been shown to provide successful, long-term gene transfer in vivo.

[0005] AAV is a single-stranded DNA (ssDNA), non-enveloped virus that belongs to the Parvoviridae family, dependent Parvovirus genus. AAV particles consist of an icosahedral capsid that includes three types of structural proteins, VP1, VP2, and VP3, and a single-stranded DNA genome. There are multiple naturally occurring and recombinant serotypes of AAV that specifically target specific tissues and cells, and AAV is able to infect both dividing and non-dividing cells.

[0006] However, the AAV production process results in the production of a mixture of viral particles, some of which correctly contain the desired recombinant DNA, also known as "complete" capsids, some of which are capsids that contain only a portion of the desired recombinant DNA, also known as "partially complete", and some of which are capsids that incorrectly contain the DNA, also known as "empty" capsids.

[0007] Similarly, the production process of non-viral particles that serve as carriers of recombinant nucleic acids (i.e., non-viral vector particles) results in the production of a mixture of non-viral vector particles, some of which correctly contain the desired recombinant DNA, also referred to as "complete" non-viral vector particles, some of which contain only a portion of the desired recombinant DNA, also referred to as "partially complete", and some of which are non-viral vector particles that incorrectly contain DNA, also referred to as "empty" non-viral vector particles.

[0008] There continues to be a need for methods to characterize a population of particles putatively containing recombinant nucleic acids in a sample to determine the proportion of particles in the sample that correctly contain complete recombinant nucleic acids (i.e., "intact") versus particles in the sample that incorrectly contain complete recombinant nucleic acids (i.e., "empty" and / or "partially intact"). Summary of the invention

[0009] A method for characterizing a population of particles presumed to contain recombinant nucleic acids in a fluid sample to determine one or more of: 1) the ratio of intact particles to empty particles in the fluid sample, and 2) the ratio of intact particles or empty particles to partially intact particles, comprising: extracting recombinant nucleic acids from a first aliquot of the fluid sample containing the particles to produce extracted recombinant nucleic acids; labeling the extracted recombinant nucleic acids to produce labeled extracted recombinant nucleic acids; passing the labeled extracted recombinant nucleic acids through a polymeric separation medium in a microchannel into a detection zone in fluid communication with the microchannel, the detection zone being in signal communication with a sensor capable of detecting a signal of a detectable nucleic acid label from the labeled extracted recombinant nucleic acids, thereby separating the labeled extracted recombinant nucleic acids according to size by passing the labeled extracted recombinant nucleic acids through the polymeric separation medium in the microchannel; detecting the detectable label of the labeled extracted recombinant nucleic acids in the detection zone to determine: a) the amount of time it takes the labeled extracted recombinant nucleic acids to flow through the polymeric separation medium in the microchannel into the detection zone, which is indicative of the ratio of the number of recombinant nucleic acids in the fluid sample to the number of empty particles; and b) the amount of time it takes the labeled extracted recombinant nucleic acids to flow through the polymeric separation medium in the microchannel into the detection zone, which is indicative of the ratio of the number of recombinant nucleic acids in the fluid sample to the number of empty particles. The size of the nucleic acid, and / or b) the signal intensity of the detectable label in the detection zone, which represents the amount of the labeled extracted recombinant nucleic acid present and indicates the concentration of the labeled extracted recombinant nucleic acid in the fluid sample; and comparing a) with a reference standard representing intact particles, and based on the comparison, determining the ratio of intact particles to partially intact particles to produce a first measurement result, and / or comparing b) with a reference standard representing intact particles, and based on the comparison, determining the ratio of intact particles to empty particles in the fluid sample to produce a first measurement result; thereby characterizing the particle population in the fluid sample.

[0010] A method for characterizing a population of viral particles presumed to contain recombinant DNA in a fluid sample to determine one or more of: 1) the ratio of intact viral particles to empty viral particles in the fluid sample, and 2) the ratio of intact viral particles or empty viral particles to partially intact viral particles, comprising: extracting recombinant DNA from a first aliquot of the fluid sample containing the viral particles to produce extracted recombinant DNA (if present); labeling the extracted recombinant DNA to produce labeled extracted recombinant DNA; allowing the labeled extracted recombinant DNA to flow through a polymer separation medium in a microchannel into a detection zone connected to the microchannel fluid, the detection zone being connected to a sensor signal, the sensor being capable of detecting a signal from a detectable label of the labeled extracted recombinant DNA, whereby Separating the labeled extracted recombinant DNA according to size by passing the labeled extracted recombinant DNA through a polymer separation medium in a microchannel; detecting the detectable label of the labeled extracted recombinant DNA in the detection zone to determine: a) the amount of time it takes for the labeled extracted recombinant DNA to flow through the polymer separation medium in the microchannel into the detection zone, which is indicative of the size of the recombinant DNA in the fluid sample, and / or b) the signal intensity of the detectable label in the detection zone, which is representative of the amount of labeled extracted recombinant DNA present and indicative of the concentration of the labeled extracted recombinant DNA in the fluid sample; and comparing a) with a reference standard representing intact viral particles and, based on the comparison, determining the ratio of intact viral particles to partially intact viral particles to generate a first assay result (R DNA ), and / or b) is compared with a reference standard representing complete virus particles, and based on the comparison, the ratio of complete virus particles to empty virus particles in the fluid sample is determined to produce a first measurement result; thereby characterizing the virus particle population in the fluid sample.

[0011] According to aspects of the method of characterizing a population of particles presumed to contain recombinant nucleic acids in a fluid sample of the present disclosure, labeling the extracted recombinant nucleic acids comprises introducing the extracted recombinant nucleic acids into wells and / or microchannels of a microfluidic device, wherein the wells and / or microchannels comprise a polymer separation medium and a detectable nucleic acid label, whereby the detectable nucleic acid label binds to the extracted recombinant nucleic acids to produce labeled extracted recombinant nucleic acids in the wells and / or microchannels.

[0012] According to aspects of the present disclosure, a method of characterizing a population of particles putatively comprising a recombinant nucleic acid in a fluid sample further comprises determining an amount of particle protein present in the fluid sample.

[0013] According to aspects of the method of characterizing a population of particles putatively containing a recombinant nucleic acid in a fluid sample of the present disclosure, the amount of particle protein in the fluid sample is determined using information about the total number of particles in the fluid sample without assaying the particle protein in the sample.

[0014] According to aspects of the present disclosure, a method of characterizing a population of particles putatively comprising a recombinant nucleic acid in a fluid sample further comprises assaying a second aliquot of the fluid sample for particle protein to determine an amount of particle protein present in the fluid sample.

[0015] According to aspects of the method for characterizing a population of particles presumed to contain a recombinant nucleic acid in a fluid sample of the present disclosure, determining the amount of particle protein in a second aliquot of the fluid sample to determine the amount of particle protein present in the fluid sample comprises: extracting protein from the second aliquot of the fluid sample containing the particles to produce extracted protein of the particles; labeling the extracted protein of the particles to produce labeled extracted protein of the particles; allowing the labeled extracted protein of the particles to flow through a polymer separation medium in a microchannel to enter a detection zone connected to the microchannel fluid, the detection zone being connected to a sensor signal, the sensor being capable of detecting a signal from the particles; The method comprises the steps of: detecting a detectable label of a labeled extracted protein, whereby the labeled extracted protein is separated according to size by passing the extracted protein through a polymer separation medium in a microchannel; detecting the detectable label of the labeled extracted protein to determine: c) the amount of time it takes for the labeled extracted protein to flow through the polymer separation medium in the microchannel into a detection zone, and d) the intensity of the signal of the detectable label, which is representative of the amount of labeled extracted protein present; comparing c) and d) to a reference standard, the reference standard representing a known amount of the protein, thereby determining the relative amount of the protein in the sample, and thereby generating a second assay result (R p ); and comparing the first assay result and the second assay result to determine one or more of the following: 1) the ratio of complete virus particles to empty virus particles in the fluid sample, 2) the ratio of complete virus particles or empty virus particles to partially complete virus particles, and 3) the ratio of virus particles containing a complete recombinant genome to virus particles containing an incomplete recombinant genome, thereby characterizing the recombinant virus particle population in the fluid sample.

[0016] According to aspects of the method of characterizing a population of particles putatively containing recombinant nucleic acids in a fluid sample of the present disclosure, labeling the extracted proteins of the particles comprises introducing the extracted proteins of the particles into wells and / or microchannels of a microfluidic device, the wells and / or microchannels comprising a polymer separation medium and a detectable protein label, whereby the detectable protein label binds to the extracted proteins of the particles, producing labeled extracted proteins of the particles in the wells and / or microchannels.

[0017] According to aspects of the method of characterizing a population of particles putatively containing recombinant nucleic acids in a fluid sample of the present disclosure, the particles are recombinant viral particles having a single-stranded DNA or RNA genome ranging in length from 500-7000 nucleotides.

[0018] According to aspects of the method of characterizing a population of particles putatively comprising a recombinant nucleic acid in a fluid sample of the present disclosure, the particles are selected from the group consisting of: recombinant adeno-associated virus (AAV) particles, recombinant retroviral particles, recombinant lentiviral particles, and recombinant adenoviral particles.

[0019] According to aspects of the method of characterizing a population of particles putatively containing a recombinant nucleic acid in a fluid sample of the present disclosure, the particles are present in an amount of about 1×10 10 Recombinant virus particles / ml is about 1×10 14 The amount of recombinant virus particles present in the fluid sample is 100 recombinant virus particles / ml.

[0020] According to aspects of the method for characterizing a population of particles presumed to contain recombinant nucleic acids in a fluid sample of the present disclosure, extracting nucleic acids from a first aliquot of the fluid sample containing particles includes contacting the first aliquot of the fluid sample containing particles with a protease and a denaturant. According to aspects of the method for characterizing a population of particles presumed to contain recombinant nucleic acids in a fluid sample of the present disclosure, the denaturant is heat, a chaotropic agent, a detergent, or a mixture thereof.

[0021] According to aspects of the method of characterizing a population of particles presumed to contain recombinant nucleic acids in a fluid sample of the present disclosure, extracting nucleic acids from a first aliquot of the fluid sample containing particles comprises contacting the first aliquot of the fluid sample containing particles with a serine protease and a denaturant. According to aspects of the method of characterizing a population of particles presumed to contain recombinant nucleic acids in a fluid sample of the present disclosure, the denaturant is a chaotropic agent, a detergent, or a mixture thereof.

[0022] According to aspects of the method for characterizing a population of particles presumed to contain recombinant nucleic acids in a fluid sample of the present disclosure, extracting nucleic acids from a first aliquot of the fluid sample containing particles comprises contacting the first aliquot of the fluid sample containing particles with a protease and a denaturant, wherein the protease is selected from the group consisting of proteinase K, subtilisin, trypsin, chymotrypsin, thrombin, plasmin, elastase, pronase, and lactoferrin. According to aspects of the method for characterizing a population of particles presumed to contain recombinant nucleic acids in a fluid sample of the present disclosure, the denaturant is heating, a chaotropic agent, a detergent, or a mixture thereof.

[0023] According to aspects of the present disclosure, a method for characterizing a population of viral particles putatively containing a recombinant nucleic acid in a fluid sample to determine one or more of the following: 1) intact viral particles (N) in the fluid sample;f ) and empty virus particles (N e ), 2) the ratio of complete virus particles or empty virus particles to partially complete virus particles, comprising: extracting viral nucleic acid from a first aliquot of the fluid sample; determining the extracted viral nucleic acid by microfluidic electrophoresis; calculating the ratio of the relative nucleic acid concentration in the fluid sample to the nucleic acid concentration in a reference standard, calculating the ratio of the relative protein concentration in the fluid sample to the protein concentration in the reference standard, and calculating the fractional amount β of complete capsids in the sample according to the following formula:

[0024]

[0025] where N is the total number of capsids, f and e refer to complete capsids and empty capsids, respectively, and β s is the percentage of intact capsids in the reference standard, R DNA is the ratio of the concentration of viral nucleic acid in the sample to the concentration of viral nucleic acid in the reference standard, R P It is the ratio of the concentration of viral protein in the sample to the concentration of viral protein in the reference standard.

[0026] According to aspects of the present disclosure, a method for characterizing a population of viral particles putatively containing a recombinant nucleic acid in a fluid sample to determine one or more of the following: 1) intact viral particles (N) in the fluid sample; f ) and empty virus particles (N e ), 2) the ratio of complete virus particles or empty virus particles to partially complete virus particles, comprising: extracting viral nucleic acid from a first aliquot of the fluid sample; determining the extracted viral nucleic acid by microfluidic electrophoresis; extracting viral protein from a second aliquot of the fluid sample; determining at least one of the extracted viral proteins by microfluidic electrophoresis; calculating the ratio of the relative nucleic acid concentration in the fluid sample to the nucleic acid concentration in a reference standard, calculating the ratio of the protein concentration in the fluid sample to the protein concentration in the reference standard, and calculating the fraction β of complete capsids in the sample according to the following formula:

[0027]

[0028] where N is the total number of capsids, f and e refer to complete capsids and empty capsids, respectively, and β s is the percentage of intact capsids in the reference standard, R DNA is the ratio of the concentration of viral nucleic acid in the sample to the concentration of viral nucleic acid in the reference standard, R P It is the ratio of the concentration of viral protein in the sample to the concentration of viral protein in the reference standard.

[0029] According to aspects of the present disclosure, a method for characterizing a population of viral particles putatively containing a recombinant nucleic acid in a fluid sample to determine one or more of the following: 1) intact viral particles (N) in the fluid sample; f ) and empty virus particles (N e ), 2) the ratio of complete virus particles or empty virus particles to partially complete virus particles, comprising: extracting viral nucleic acid from a first aliquot of the fluid sample; determining the extracted viral nucleic acid by microfluidic electrophoresis; calculating the ratio of the nucleic acid concentration in the fluid sample to the nucleic acid concentration in a reference standard, calculating the ratio of the protein concentration in the fluid sample to the protein concentration in the reference standard, wherein the protein concentration in the fluid sample is calculated by reference to the standard and no protein assay is performed, and calculating the fraction β of complete capsids in the sample according to the following formula:

[0030]

[0031] where N is the total number of capsids, f and e refer to intact capsids and empty capsids, respectively, βs is the percentage of intact capsids in the reference standard, and R DNA is the ratio of the concentration of viral nucleic acid in the sample to the concentration of viral nucleic acid in the reference standard, R P It is the ratio of the concentration of viral protein in the sample to the concentration of viral protein in the reference standard.

[0032] According to aspects of the present disclosure, a kit for characterizing the nucleic acid content of a particle population presumed to contain recombinant nucleic acids in a fluid sample is provided, comprising: a nucleic acid marker, a gel, a nucleic acid gradient standard, a nucleic acid storage buffer, a nucleic acid sample buffer, a protease, and a denaturant. According to aspects of the present disclosure, the nucleic acid marker is a fluorescent nucleic acid intercalator. According to aspects of the present disclosure, the protease is a serine protease.

[0033] According to aspects of the present disclosure, a kit for characterizing the nucleic acid content of a particle population presumed to contain recombinant nucleic acids in a fluid sample is provided, comprising: a nucleic acid marker, a gel, a nucleic acid gradient standard, a nucleic acid storage buffer, a nucleic acid sample buffer, a protease, a denaturant, a protein dye, a protein standard, a protein storage buffer, a protein sample buffer, and a wash buffer. According to aspects of the present disclosure, the nucleic acid marker is a fluorescent nucleic acid intercalator. According to aspects of the present disclosure, the protease is a serine protease.

[0034] According to aspects of the present disclosure, a kit for characterizing the nucleic acid content of a population of viral particles presumed to contain recombinant nucleic acids in a fluid sample is provided, comprising: a nucleic acid marker, a gel, a nucleic acid gradient standard, a nucleic acid storage buffer, a nucleic acid sample buffer, a protease, and a denaturant. According to aspects of the present disclosure, the nucleic acid marker is a fluorescent nucleic acid intercalator. According to aspects of the present disclosure, the protease is a serine protease. According to aspects of the present disclosure, the viral particles are AAV viral particles, and an AAV standard containing a known number of AAV viral particles is included in the kit for characterizing the nucleic acid content of a population of AAV particles presumed to contain recombinant nucleic acids in a fluid sample.

[0035] According to aspects of the present disclosure, a kit for characterizing the nucleic acid content of a population of viral particles presumed to contain recombinant nucleic acids in a fluid sample is provided, comprising: a nucleic acid marker, a gel, a nucleic acid gradient standard, a nucleic acid storage buffer, a nucleic acid sample buffer, a protease, a denaturant, a protein dye, a protein standard, a protein storage buffer, a protein sample buffer, and a wash buffer. According to aspects of the present disclosure, the nucleic acid marker is a fluorescent nucleic acid intercalator. According to aspects of the present disclosure, the protease is a serine protease. According to aspects of the present disclosure, the protein standard is or includes a protein gradient standard. According to aspects of the present disclosure, the viral particles are AAV viral particles, and an AAV standard containing a known number of AAV viral particles is included in a kit for characterizing the content of a population of AAV particles presumed to contain recombinant nucleic acids in a fluid sample.

[0036] According to aspects of the present disclosure, a kit is provided for characterizing the nucleic acid content of a population of particles putatively containing a recombinant nucleic acid in a fluid sample, wherein the protease is selected from the group consisting of proteinase K, subtilisin, trypsin, chymotrypsin, thrombin, plasmin, elastase, pronase and lactoferrin.

[0037] According to aspects of the present disclosure, a kit for characterizing the nucleic acid content of a population of particles putatively containing a recombinant nucleic acid in a fluid sample is provided, wherein the denaturing agent is a chaotrope, a detergent, or a mixture thereof.

[0038] According to aspects of the present disclosure, a kit for characterizing the nucleic acid content of a population of particles putatively containing a recombinant nucleic acid in a fluid sample is provided, comprising at least one protease and at least one chaotropic agent, wherein the at least one protease is proteinase K and the at least one chaotropic agent is urea.

[0039] According to aspects of the present disclosure, a kit for characterizing the nucleic acid content of a particle population presumed to contain a recombinant nucleic acid in a fluid sample is provided, comprising one or more standards. According to aspects of the present disclosure, the standards included may be samples comprising a known ratio of complete particles: empty particles, a known ratio of complete particles: partially complete particles, or a known ratio of empty particles: partially complete particles, two or more thereof, or all three thereof. According to aspects of the present disclosure, the standards included may be samples comprising a known ratio of complete AAV particles: empty AAV particles, a known ratio of complete AAV particles: partially complete AAV particles, or a known ratio of empty AAV particles: partially complete AAV particles, two or more thereof, or all three thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a schematic diagram illustrating aspects of a method for characterizing a population of particles putatively containing a recombinant nucleic acid in a fluid sample to determine one or more of the following according to the present disclosure: 1) the ratio of intact particles to empty particles in the fluid sample, 2) the ratio of intact particles or empty particles to partially intact particles; in the illustrated embodiment, the method enables viral capsid nucleic acid and protein content analysis;

[0041] Figure 2 is a schematic diagram illustrating aspects of a method for characterizing a population of particles putatively containing a recombinant nucleic acid in a fluid sample to determine one or more of the following according to the present disclosure: 1) the ratio of intact particles to empty particles in the fluid sample, 2) the ratio of intact particles or empty particles to partially intact particles; in the illustrated embodiment, the method enables viral capsid nucleic acid and protein content analysis;

[0042] Figure 3A is a typical protein electropherogram showing peaks representing VP3, VP2, and VP1 AAV capsid proteins from samples containing either complete or empty AAV capsids, from smallest to largest mass (left to right);

[0043] Figure 3B yes Figure 3A The graph of the results shown in , which shows the total area under the curve ("area") for each of the three major protein peaks for VP1, VP2, and VP3, respectively, demonstrates that there is no statistical difference between the VP peaks produced by intact and empty viral capsids;

[0044] Figure 4A is a typical nucleic acid electropherogram showing the results obtained using samples containing complete or empty AAV capsids; note that the results using samples containing complete AAV capsids produce a peak, while the results using samples containing empty AAV capsids do not produce a peak.

[0045] Figure 4Byes Figure 4A , which shows the total area under the curve ("Area") for each sample containing either complete or empty AAV capsids; note that for all three runs, the empty AAV sample did not produce a peak, showing a significant statistical difference between the ssDNA profiles of the empty and complete samples;

[0046] Figure 5A is a typical electropherogram showing capsid protein results obtained using samples containing different percentages of intact capsids, including 75%, 50%, 25%, and 4% intact capsids; the electropherogram in this figure shows peaks for VP3, VP2, and VP1 capsid proteins representing all samples (from smallest to largest mass (left to right));

[0047] Figure 5B yes Figure 5A A graph of the results shown in , which shows the total area under the curve ("Area") of the VP protein peak for four samples containing different percentages of intact capsids as shown;

[0048] Figure 5C is a typical nucleic acid electropherogram showing nucleic acid results obtained using samples containing different percentages of intact capsids (including 75%, 50%, 25% and 4% intact capsids); note that the DNA peak is observed to decrease as the percentage of intact capsids decreases;

[0049] Figure 5D yes Figure 5C , which shows the total area under the curve ("Area") of the viral nucleic acid peaks for four samples containing different percentages of intact capsids as shown; note that the empty AAV sample did not produce a peak for all three runs;

[0050] Figure 6 is a graph showing the prediction accuracy between the protein area prediction method based on equation (6) and the concentration prediction method based on equation (8) compared to the actual or reported intact capsid percentage for each sample; the sample numbers correspond to those reported in Tables 6-8; in addition, all samples include an error interval of 10% of the total value (in each direction) to account for inaccuracies in the reported standards used to prepare the standards and samples used in this study;

[0051] Figure 7 is a protein electropherogram showing the integration of the VP3 peak from an empty / full analysis of AAV proteins from samples containing empty or full AAV capsids;

[0052] Figure 8 is an electropherogram showing ssDNA peak integration of empty / intact analysis of AAV nucleic acid from samples containing empty or intact AAV capsids;

[0053] Fig. 9 is a nucleic acid electropherogram showing ssDNA peak integration from a partially intact / intact analysis of AAV nucleic acid from a sample containing either partially intact capsids or intact AAV capsids;

[0054] Fig.10 is an electropherogram showing RNA peak integration of RNA ladder without (top line) or with (bottom line) benzonase treatment;

[0055] Fig.11 is an overlay electropherogram of three sips of AAV8 ssDNA recovered after the cleanup procedure using an ssDNA purification kit (QIAquick PCR);

[0056] Fig. 12A is an overlay electropherogram showing the separation of two nucleic acid samples of size 2.4 kilonucleotide (knt) ssDNA and 3.3 knt ssDNA in a separate capillary electrophoresis run;

[0057] Fig. 12B is an overlay electropherogram showing the separation of two nucleic acid samples of size 2.4 kilonucleotide (knt) ssDNA and 3.3 knt ssDNA combined in a single capillary electrophoresis run;

[0058] Fig.13A is a set of electropherograms showing the results of assays of extracted viral proteins for each indicated serotype (AAV2, AAV9, or AAV8, shown from top to bottom). The viral protein resolution and full width at half maximum (FWHM) of AAV2, AAV8, or AAV9 viral particles are similar;

[0059] Fig. 13B 70% intact AAV8 virus particles (F-70), 50% intact AAV8 virus particles (F-50), 25% intact AAV8 virus particles (F-25) and empty AAV8 virus particles (F-0) are shown in the table. For each pair of bars on the graph, the left bar represents the calculated theoretical percentage of intactness, and the right bar represents the average percentage of intactness measured and calculated using equation (6);

[0060] Fig. 13Cis a graph showing the results of the extracted nucleic acid assay of 82% intact AAV9 viral particles (F-82), 50% intact AAV9 viral particles (F-50), 25% intact AAV9 viral particles (F-25), and empty AAV9 viral particles (F-0). For each pair of bars on the graph, the left bar represents the calculated theoretical percentage of intactness, and the right bar represents the average percentage of intactness measured and calculated using equation (6); and

[0061] Fig.13D 62% intact AAV2 virus particles (F-62), 50% intact AAV2 virus particles (F-50), 25% intact AAV2 virus particles (F-25) and empty AAV2 virus particles (F-0) are shown in the figure. For each pair of bars on the graph, the left bar represents the calculated theoretical percentage of intact, and the right bar represents the average percentage of intact measured and calculated using equation (6). DETAILED DESCRIPTION

[0062] Scientific and technical terms used herein are intended to have the meanings that are commonly understood by one of ordinary skill in the art. Such terms are defined and used in various standard references, which exemplarily include J. Sambrook and D. W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; FM Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002; DL Nelson and MM Cox, Lehninger Principles of Biochemistry, 4th Ed., WH Freeman & Company, 2004; Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st Ed., 2005; LV Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th Ed., Philadelphia, PA: Lippincott, Williams & Wilkins, 2004; and L. Brunton et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill Professional, 12th Ed., 2011.

[0063] The singular terms "a," "an," and "the" are not intended to be limiting and include plural referents unless expressly stated otherwise or the context clearly dictates otherwise.

[0064] When used in this specification, the terms "includes, including", "comprises, comprising", "has, having" and grammatical variations thereof are not intended to be limiting and specify the presence of stated features, elements and / or components, but do not preclude the presence or addition of one or more other features, elements, components and / or groups thereof.

[0065] As used herein, the term "about" when referring to a number is used herein to include numbers that are 1%, 5%, 10% or 20% greater or less than the stated or implied value.

[0066] Specific combinations of features are recorded in the claims and / or disclosed in the specification, and these combinations of features are not intended to limit the disclosure of the various aspects. Such combinations of features are not specifically recorded in the claims and / or disclosed in the specification. Although each dependent claim listed below may be directly dependent on only one claim, the disclosure of the various aspects includes the combination of each dependent claim with each other claim in the claim set. As used herein, a phrase referring to "at least one" in a list of items refers to any combination of those items, including single members. As an example, "at least one of a, b or c" is intended to cover a alone; b alone; c alone; a and b; a, b and c; b and c; a and c and any combination with multiple identical elements, such as a and a; a, a and a; a, a and b; a, a and c; a, b and b; a, c and c; and any other combination or ordering of a, b and c.

[0067] The terms "first", "second", etc. are used herein to describe various features or elements, but these features or elements are not intended to be limited by these terms, but are only used to distinguish one feature or element from another feature or element. Therefore, without departing from the teachings of the present disclosure, a first feature or element may be referred to as a second feature or element, and vice versa.

[0068] According to aspects of the present disclosure, methods are provided for characterizing a population of particles putatively containing a recombinant nucleic acid in a fluid sample to determine one or more of: 1) the ratio of intact particles to empty particles in the fluid sample, and 2) the ratio of intact or empty particles to partially intact particles.

[0069] As used herein, the term "particle" and its grammatical equivalents refer to: 1) a virus, ie, a viral particle, or 2) a non-viral particle that is a carrier of a recombinant nucleic acid, ie, a non-viral vector particle.

[0070] As used herein, the term "viral particle" refers to a "complete" viral particle including a capsid and a viral genome coated with the capsid. For example, when referring to a recombinant adeno-associated virus (AAV) viral particle, the viral particle includes an adeno-associated virus capsid and a recombinant genome coated with the capsid.

[0071] As used herein, the term "recombinant viral particle" refers to a viral capsid comprising a heterologous nucleic acid, wherein the recombinant viral particle is produced by using recombinant techniques and / or recombinant technology. As used herein, the term "heterologous nucleic acid" refers to a nucleic acid encoding a corresponding nucleic acid and / or polypeptide that is not encoded by a naturally occurring virus from which the recombinant viral particle is derived.

[0072] The recombinant nucleic acid presumed to be present in the particle population to be analyzed can be any type of nucleic acid, and the term "nucleic acid" as used herein refers to any form of RNA or DNA molecule having more than one nucleotide, including single-stranded, double-stranded, oligonucleotide or polynucleotide. According to aspects of the present disclosure, the particle is a viral particle and the nucleic acid is a single-stranded RNA or DNA. According to aspects of the present disclosure, the particle is a viral particle and the nucleic acid is a single-stranded DNA.

[0073] The embodiments herein describe methods according to the present disclosure with respect to adeno-associated viruses (AAV), and such methods can be used with other viruses, including but not limited to lentiviruses, adenoviruses, MMLV retroviruses, MSCV retroviruses, baculoviruses, vesicular stomatitis virus, and herpes simplex virus.

[0074] AAV subtypes were initially identified serologically, with 12 classical serotypes identified: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. With the advent of molecular analysis techniques, many naturally occurring genetic variants have been identified and many synthetic genetic variants have been generated.

[0075] Methods according to aspects of the present disclosure are not limited to analyzing specific serotypes or genetic variations of viruses. In specific aspects, methods according to aspects of the present disclosure are not limited to analyzing specific serotypes or genetic variations of AAV.

[0076] Non-viral particles that are carriers of recombinant nucleic acids (ie, non-viral vector particles) can contain or be associated with recombinant nucleic acids and can be used to transport the nucleic acid to a desired location, for example into an organism, a cell or a cell compartment.

[0077] The non-viral vector particles can be or include liposomes, micelles, unilamellar or multilamellar vesicles, polymer particles (eg, hydrogel particles, polyglycolic acid particles, or polylactic acid particles), inorganic particles, and inorganic / organic particles.

[0078] The non-viral vector particles can be selected from lipid particles, polymer particles, inorganic particles, inorganic / organic particles, or any mixture of two or more thereof. Mixtures of particle types can also be included as particulate pharmaceutically acceptable carriers.

[0079] The non-viral vector particles can be nanoparticles and / or microparticles. The non-viral vector particles are generally formulated so that the particles have an average particle size in the range of about 1 nm to 10 microns. In specific aspects of the present disclosure, the non-viral vector particles are formulated so that the particles have an average particle size in the range of about 1 nm to 100 nm.

[0080] Non-viral vector particles include, for example, nanoparticle polymers, dendrimers, liposomes, viruses, carbon nanotubes, and metals (e.g., iron oxide and gold). Exemplary polymers for preparing non-viral vector particles include natural polymers such as heparin, dextran, albumin, gelatin, alginate, collagen, and chitosan; or synthetic polymers including polyethylene glycol (PEG), polyglutamic acid (PGA), polylactic acid (PLA), polycarprolactone (PCL), and N-(2-hydroxypropyl)-methacrylamide copolymer (HPMA).

[0081] The non-viral vector particle is a lipid-based vector particle according to aspects of the present disclosure.The term "lipid-based vector particle" refers to a macromolecular structure having lipids and / or lipid derivatives as main components.

[0082] The lipids contained in the lipid-based carrier particles may be naturally occurring lipids, synthetic lipids, or a combination thereof.

[0083] The lipid-based carrier particle is a liposome according to aspects of the present disclosure.

[0084] The term "liposome" refers to a bilayer particle of amphiphilic lipid molecules surrounding an aqueous interior space. Liposomes are typically produced in the form of small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), or multilamellar vesicles (MLVs). Recombinant nucleic acids can be associated with liposomes by being encapsulated in the aqueous interior space of the liposomes, being placed in the lipid bilayer of the liposomes, and / or being associated with liposomes by binding (e.g., ionic binding) or by association with van der Waals forces. The diameter of the liposomes according to aspects of the present disclosure is typically in the range of about 1 nm-1 micron, but they are not limited in size.

[0085] The lipid-based carrier particles include one or more types of neutral, cationic and / or anionic lipids such that the liposome formulation has a surface charge or a net neutral surface charge at physiological pH. Optionally, one or more PEG-modified lipids are included.

[0086] The term cationic lipid refers to any lipid having a net positive charge at physiological pH. Examples of cationic lipids include, but are not limited to, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), dioctadecylaminoglycyl spermine (DOGS), 1,2-dipalmitoylphosphatidylethanolaminospermine (DPPES), 2,3-dioleoyloxy-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl-1-propylammonium trifluoroacetate (DOSPA), dimyristoyltrimethylammonium propane (DMTAP), (3-dimyristyloxypropyl)(dimethyl)(hydroxyethyl)ammonium (DMRIE), dioctadecyldimethylammonium chloride (DODAC), dimethyldidodecyl bromide ammonium iodide (DDAB), 3β[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), 1-[2-(9(Z)-octadecenoyloxy)-ethyl]-2-(8(Z)-heptadecenyl)-3-(2-hydroxyethyl)-imidazoline (DOTIM), biguanidinium-spermidine-cholesterol (BGTC), biguanidinium-triaminoethylamine-cholesterol (bis-guanidinium-tren-cholesterol, BGTC), 1,3-dioleoyloxy-2-(6-carboxy-sperminyl)-propionamide (DOSPER), N-[3-[2-(1,3-dioleoyloxy)propoxy-carbonyl]propyl]-N,N,N-trimethylammonium iodide (YKS-220), and pharmaceutically acceptable salts and mixtures thereof. Additional examples of cationic lipids are described in Lasic and Papahadjopoulos, Medical Applications of Liposomes, Elsevier, 1998; U.S. Pat. Nos. 4,897,355; 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,334,761; 5,459,127; 5,736,392; 5,753,613; 5,785,992; 6,376,248; 6,586,410; 6,733,777; and 7,145,039.

[0087] The term neutral lipid refers to any lipid that does not have a net charge (either uncharged or in the form of a neutrally charged zwitterion) at physiological pH. Examples of neutral lipids include, but are not limited to, L-α-phosphatidylcholine (ePC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphoethanolamine (DPPE), cephalin, ceramide, cerebroside, cholesterol, diglyceride, and sphingomyelin.

[0088] The term anionic lipid refers to any lipid with a net negative charge at physiological pH. Examples of anionic lipids include, but are not limited to, dihexadecyl phosphate (DhP), phosphatidylinositol, phosphatidylserine (e.g., dimyristoylphosphatidylserine and dipalmitoylphosphatidylserine), phosphatidylglycerol (e.g., dimyristoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol), phosphatidic acid (e.g., dimyristoyl phosphate and dipalmitoyl phosphate and diphosphatidylglycerol).

[0089] The term "modified lipid" refers to a lipid that has been modified to help, for example, inhibit aggregation and / or precipitation, inhibit immune response and / or improve in vivo circulation half-life. Modified lipids include, but are not limited to, PEGylated lipids, such as polyethylene glycol 2000 distearoylphosphatidylethanolamine

[0090] (PEG(2000)DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG-2000), and polyethylene glycol 750 octadecylsphingosine (PEG(750)C8).

[0091] In addition to lipids, polymers and inorganic materials, non-viral vector particles can contain any of a variety of molecules and substances that can contribute to the structure of the particles and / or the intended function of the particles and can be integrated into and / or complexed with non-viral vector particles, including but not limited to proteins, peptides, carbohydrates, oligosaccharides and drugs. Such molecules and substances can be biologically active molecules and substances. The term "biologically active molecules and substances" refers to molecules or substances that exert biological effects in vitro and / or in vivo, such as but not limited to nucleic acids, inhibitory RNA, siRNA, shRNA, ribozymes, antisense nucleic acids, antibodies, hormones, small molecules, aptamers, decoy molecules and toxins.

[0092] Non-viral vector particles are produced using well-known standard methods, including but not limited to those described in detail in the following references: Rangelov, S. et al. Polymer and Polymer-Hybrid Nanoparticles: From Synthesis to Biomedical Applications, CRC Press, 2013; Liposomes: A Practical Approach (The Practical Approach Series, 264), VP Torchilin and V. Weissig (Eds.), Oxford University Press; 2nd ed., 2003; N. Duzgunes, Liposomes, Part A, Volume 367 (Methods in Enzymology) Academic Press; 1st ed., 2003; LV Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th Ed., Philadelphia, PA: Lippincott, Williams & Wilkins, 2005, pp. 663-666; and ARGennaro, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed., 2005, pp. 766-767.

[0093] The term "complete" when used herein in reference to a recombinant viral particle is intended to refer to a viral capsid that properly contains a complete recombinant viral genome. The term "empty" as used herein in reference to a recombinant viral particle is intended to refer to a viral capsid that does not contain detectable nucleic acid. The term "partially complete" as used herein in reference to a recombinant viral particle is intended to refer to a viral capsid that contains an incomplete recombinant viral genome (i.e., incomplete).

[0094] The term "intact" when used herein in reference to a non-viral vector particle is intended to refer to a non-viral vector particle that correctly contains the desired recombinant nucleic acid intact. The term "empty" as used herein in reference to a non-viral vector particle is intended to refer to a non-viral vector particle that does not contain a detectable nucleic acid. The term "partially intact" as used herein in reference to a non-viral vector particle is intended to refer to a non-viral vector particle that contains an incomplete recombinant nucleic acid (i.e., incomplete).

[0095] The term "incomplete recombinant viral genome" refers to a fragment of a recombinant genome, such as a recombinant genome with one or more deleted ends, or with one or more internal deletions. The length of the incomplete recombinant genome is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more or less than the reference complete recombinant viral genome.

[0096] The term "incomplete recombinant nucleic acid" refers to a fragment of a recombinant nucleic acid, such as a recombinant nucleic acid having one or more missing ends, or having one or more internal deletions. The length of the incomplete recombinant nucleic acid is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more or less than the reference complete recombinant nucleic acid.

[0097] According to particular aspects of the present disclosure, methods are provided for characterizing a population of particles in a sample to determine the ratio or fractional percentage of "full" particles to "empty" particles and / or "empty" particles to "full" particles in the sample.

[0098] According to specific aspects, the present disclosure relates to evaluating a population of recombinant viral capsids to determine: 1) the proportion of recombinant viral capsids in a sample that correctly contain a complete recombinant viral genome ("complete"), 2) the proportion of recombinant viral capsids in a sample that contain a recombinant viral genome of the wrong size ("partially complete"), 3) the proportion of recombinant viral capsids in a sample that do not contain a detectable recombinant viral genome ("empty"), or 4) any combination of 1), 2), and 3).

[0099] According to specific aspects of the present disclosure, methods are provided for characterizing a population of viral particles in a sample to determine the ratio or fractional percentage of the following in the sample: 1) "partially intact" viral capsids to "complete" viral capsids, 2) "complete" viral capsids to "partially intact" viral capsids, 3) "partially intact" viral capsids to "empty" viral capsids, 4) "empty" viral capsids to "partially intact" viral capsids, or any combination of 1), 2), 3) and 4).

[0100] According to specific aspects, the present disclosure relates to evaluating a population of non-viral vector particles to determine: 1) the proportion of non-viral vector particles in a sample that correctly contain an intact recombinant nucleic acid ("intact"), 2) the proportion of non-viral vector particles in a sample that contain a recombinant nucleic acid of the wrong size ("partially intact"), 3) the proportion of non-viral vector particles in a sample that do not contain a detectable recombinant nucleic acid ("empty"), or 4) any combination of 1), 2), and 3).

[0101] According to specific aspects of the present disclosure, methods are provided for characterizing a population of non-viral vector particles in a sample to determine the ratio or fractional percentage of the following in the sample: 1) "partially intact" non-viral vector particles to "intact" non-viral vector particles, 2) "intact" non-viral vector particles to "partially intact" non-viral vector particles, 3) "partially intact" non-viral vector particles to "empty" non-viral vector particles, 4) "empty" non-viral vector particles to "partially intact" non-viral vector particles, or any combination of 1), 2), 3) and 4).

[0102] According to aspects of the present disclosure, a method for characterizing a population of particles presumed to contain recombinant nucleic acids in a fluid sample is provided to determine one or more of the following: 1) the ratio of intact particles to empty particles in the fluid sample, and 2) the ratio of intact particles or empty particles to partially intact particles, the method comprising extracting recombinant nucleic acids from a first aliquot of the fluid sample containing particles, producing extracted recombinant nucleic acids. In addition, and optionally, the method of characterizing a population of particles presumed to contain recombinant nucleic acids in a fluid sample of the present disclosure comprises extracting proteins from a second aliquot of the fluid sample containing particles, producing extracted proteins of the particles.

[0103] Extraction of nucleic acids or proteins from a population of particles is accomplished by any method that isolates nucleic acids or proteins from a sample containing the particle population of interest.

[0104] Extracting nucleic acids from a sample may include using a protein denaturing agent that effectively denatures proteins and releases recombinant nucleic acids from proteins in the sample. The protein denaturing agent may be or include one or more proteases, heating, one or more detergents, osmotic shock, one or more chaotropic agents, one or more organic solvents, or any combination of two or more thereof.

[0105] The denaturing agent can be or include a chaotropic agent, such as a thiocyanate (e.g., guanidinium thiocyanate, sodium thiocyanate, potassium thiocyanate, or any combination of two or more thereof), n-butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, lithium dodecyl sulfate, thiourea, formamide, urea, or any combination of two or more thereof.

[0106] The denaturant may be or include a detergent. The detergent may be an anionic, cationic, zwitterionic or nonionic detergent, such as Triton X-100, Triton X-114, NP-40, Tween-20, Tween-80, octyl-β-glucoside, octylthioglucoside, ethyltrimethylammonium bromide, sodium dodecyl sulfate (SDS), Brij-35, Brij-58, CHAPS, CHAPSO, or any combination of two or more thereof.

[0107] According to aspects of the present disclosure, the extraction of nucleic acids comprises treating the particles with a protease. According to aspects of the present disclosure, treating the particles with a protease comprises treating the particles with a serine protease. According to aspects of the present disclosure, treating the particles with a protease comprises treating the particles with a protease selected from proteinase K, subtilisin, trypsin, chymotrypsin, thrombin, plasmin, elastase, pronase, and lactoferrin.

[0108] According to aspects of the present disclosure, the extraction of nucleic acids comprises treating the particles with a protease and a second denaturant. According to aspects of the present disclosure, the second denaturant for extracting nucleic acids from the particles is a chaotropic agent, a detergent, or a mixture thereof. According to aspects of the present disclosure, the second denaturant for extracting nucleic acids from the particles is or comprises urea.

[0109] Extracting proteins from a sample can include using a nucleic acid denaturing agent that effectively denatures nucleic acids and releases proteins in the sample. The nucleic acid denaturing agent can be or include heating at a temperature of at least about 75° C.-100° C. and an optional reducing agent. According to aspects of the present disclosure, the optional reducing agent is or includes dithiothreitol.

[0110]

[0013] Methods of characterizing a population of particles according to aspects of the present disclosure include labeling an extracted recombinant nucleic acid with a detectable label, producing a labeled extracted recombinant nucleic acid.

[0111]

[0013] Methods of characterizing a population of particles according to aspects of the present disclosure include labeling extracted particle protein with a detectable label, producing labeled extracted particle protein.

[0112] The term "detectable label" refers to a material that is capable of generating a signal indicating the presence of a labeled extracted recombinant nucleic acid or a labeled extracted particle protein and that is detectable by any suitable method, including illustratively spectrally, optically, photochemically, biochemically, enzymatically, electrically, and / or immunochemically. A detectable label allows for detection based on a detectable property of the label, such as, but not limited to, a chemical property, an electrical property, a magnetic property, an optical property, a physical property, or any two or more thereof. A detectable label may include one or more of a fluorescent label, a bioluminescent label, a chemiluminescent label, a chromophore, a magnetic label, an antibody, an antigen, an enzyme, a substrate, a radioisotope, or any two or more thereof.

[0113] According to aspects of the present disclosure, the detectable label is a fluorescent label.The fluorescent label is selected based on fluorophore characteristics including, but not limited to, excitation maximum wavelength and emission maximum wavelength.

[0114] The fluorophore used as a fluorescent label can be any of a number of fluorophores, including, but not limited to, those described in Haughland, RP, The Handbook, A Guide to Fluorescent Probes and Labeling Technologies, 10th Ed., 2005; Lakowicz, JR, Principles of Fluorescence Spectroscopy, Springer, 3rd ed., 2006; 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine and its derivatives, such as acridine and acridine isothiocyanate; 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5disulfonate, Lucifer Yellow VS; N-(4-anilino-1-naphthyl)maleimide; anthranilamide, Brilliant Yellow VS; Yellow; BIODIPY fluorophores (4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes); coumarins and their derivatives, such as coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151); cyanosine; DAPOXYL sulfonyl chloride; 4',6-diaminidino-2-phenylindole (DAPI); 5',5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol red); Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylaminonaphthalene-1-sulfonyl chloride]

[0115] 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); EDANS (5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid) acid), eosin and derivatives, such as eosin isothiocyanate; erythrosine and derivatives, such as erythrosine B and erythrosine isothiocyanate; ethidium, such as ethidium bromide; fluorescein and derivatives, such as 5-carboxyfluorescein (FAM), hexachlorofluorescein, 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE) and fluorescein isothiocyanate (FITC); fluorescamine; green fluorescent protein and derivatives, such as EBFP, EBFP2, ECFP and YFP; IAEDANS (5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid) (5-({2-

[0116] [(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid), Malachite Green isothiocyanate; 4-methylumbelliferone; o-cresolphthalein; nitrotyrosine; pararosaniline; phenolsulfonphthalein (Phenol Red); B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene butyrate, 1-pyrenesulfonyl chloride and succinimidyl 1-pyrenebutyrate; QSY 7; QSY 9; Reactive Red 4 4, Cibacron.RTM. Brilliant Red 3B-A); rhodamine and derivatives, such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (rhodamine 6G), rhodamine isothiocyanate, lissamine rhodamine B sulfonyl chloride, rhodamine B, rhodamine 123, sulforhodamine B, sulforhodamine 101 and the sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N-tetramethylcarboxyrhodamine (TAMRA); tetramethylrhodamine; tetramethylrhodamine isothiocyanate (TRITC); riboflavin, rhodamine and terbium chelate derivatives, or any combination of two or more thereof.

[0117] According to aspects of the present disclosure, the detectable label is a fluorescent nucleic acid stain. Fluorescent nucleic acid stains include, but are not limited to, acridine dyes, acridine orange (AO, N, N, N', N'-tetramethylacridine-3,6-diamine), cyanine dimer dyes, cyanine monomer dyes, DAPI (4', 6-diamidino-2-phenylindole), DRAQ dyes, ethidium compounds, ethidium bromide, GelRed TM 、GelGreen TM, Hoechst dyes, iodine compounds, 7-aminoactinomycin D, oxazole dyes, PicoGreen, propidium iodide (2,7-Diamino-9-phenyl-10(diethylaminopropyl)-phenanthridiumiodide methiodide), SYBR dyes, SYTO dyes, TOTOTM dyes, thiazole dyes, thiazole orange homodimer (TOTOTM-1), thiazole red (TO- -3), Thiazole red homodimer ( -3), oxazole yellow (oxazole yellow, YO- -1), oxazole yellow homodimer (YOYOTM-1), oxazole red (YO- -3), oxazole red homodimer (YOYOTM-3), oxazole blue (PO-PROTM-1), oxazole blue homodimer (POPOTM-1), TO iodide (TO Iodide, TO-PROTM-1), BOBO, JOJO, LOLO, At BO-PRO, JO-PRO, LO-PRO, or any combination of two or more thereof.

[0118] According to aspects of the present disclosure, a method of characterizing a population of particles includes flowing a labeled extracted recombinant nucleic acid through a polymer separation medium in a microchannel of a microfluidic device.

[0119] The microfluidic devices used according to aspects of the methods of the present disclosure allow for separation of labeled extracted recombinant nucleic acids and / or extracted labeled particle proteins, and detection of the separated labeled extracted recombinant nucleic acids and / or extracted labeled particle proteins.

[0120] According to aspects of the present disclosure, the microfluidic device is a capillary electrophoresis system that allows separation of labeled extracted recombinant nucleic acids and / or extracted labeled particle proteins, and detection of separated labeled extracted recombinant nucleic acids and / or extracted labeled particle proteins. According to aspects of the present disclosure, the microfluidic device is or includes a microfluidic chip.

[0121] The microfluidic device used according to aspects of the method of the present disclosure includes at least one microchannel, and may include one or more containers (receptacle) that are communicated with one or more microchannel fluids. Microchannel and / or container are included in the microfluidic device, such as by etching, bonding, soft engraving (soft lithography) or molded into a material that is substantially inert (insert) relative to a screening matrix, dye, nucleic acid and / or protein, ceramic and semiconductor (such as glass or silicon) or polymer (such as polydimethylsiloxane (PDMS)). Part or all of the microchannel and / or container can be connected in a network as needed, and the microchannel and / or container can be communicated with one or more input and / or output fluids to allow input to the microfluidic device and / or output from the microfluidic device.

[0122] The microchannels of the microfluidic device are made of any material suitable for containing separation media and sample aliquots while remaining inert to the separation media and sample aliquots, such as but not limited to glass, silicon, plastic, quartz, or a mixture of any two or more thereof.

[0123] In some aspects, the microchannel has a length in the range of about 25 mm to about 250 mm, for example, 25 mm to about 35 mm, 35 mm to about 45 mm, 45 mm to about 55 mm, 55 mm to about 65 mm, 65 mm to about 75 mm, 75 mm to about 85 mm, 85 mm to about 95 mm, 95 mm to about 100 mm, 100 mm to about 110 mm, 110 mm to about 120 mm, 120 mm to about 130 mm, 130 mm to about 140 mm, 140 mm to about 150 mm, 150 mm to about 160 mm, 160 mm to about 170 mm, 170 mm to about 180 mm, 180 mm to about 190 mm, 190 mm to about 200 mm, 200 mm to about 210 mm, 210 mm to about 220 mm, 220 mm to about 230 mm, 230 mm to about 240 mm, or 240 mm to 250 mm.

[0124] In some aspects, the microchannel has an inner diameter between about 1 micron and about 10 millimeters, e.g., about 1 micron to about 10 microns, about 10 microns to about 20 microns, about 20 microns to about 30 microns, about 30 microns to about 40 microns, about 40 microns to about 50 microns, about 50 microns to about 60 microns, about 60 microns to about 70 microns, about 70 microns to about 80 microns, about 80 microns to about 90 microns, about 90 microns to about 100 microns, about 100 microns to about 200 microns, about 200 microns to about 300 microns, about 300 microns to about 400 microns, about 400 microns to about 500 microns, about 500 microns to about 600 microns, about 600 microns to about 700 microns, about 700 microns to about 800 microns, about 800 microns to about 900 microns, about 900 microns to about 1 millimeter, or about 1 millimeter to about 10 millimeters.

[0125] According to aspects of the present disclosure, polymer separation media (also referred to as screening matrix) is or includes polymer, such as but not limited to one or more polyacrylamide, polyvinyl pyrrolidone and agarose, or any two or more mixtures thereof. The polyacrylamide that can be included in the screening matrix includes but is not limited to linear polyacrylamide, polydimethylacrylamide, polydiethylacrylamide, hydroxyethyl cellulose, or any two or more mixtures thereof. According to aspects of the present disclosure, polymer separation media is or includes polymer gel, such as but not limited to acrylamide gel and agarose gel. The polymer contained in the screening matrix is ​​optionally cross-linked. Optionally, denaturant is included in the screening matrix. The denaturant contained in the screening matrix can be a chaotropic agent, a detergent or its mixture.

[0126] According to aspects of the present disclosure, the included sieving matrix is ​​configured to separate labeled viral nucleic acids from other materials that may be present in an aliquot of the sample, and obtain a signal from the labeled viral nucleic acids representing the amount of labeled viral nucleic acids present in the aliquot of the sample. According to aspects of the present disclosure, the included sieving matrix is ​​configured to separate labeled viral proteins from other materials that may be present in an aliquot of the sample, and obtain a signal from the labeled viral proteins representing the amount of labeled viral proteins present in the aliquot of the sample. Configuring the sieving matrix to separate different types of viral particles can include, but is not limited to, selecting one or more of: pore size, polymer type, polymer concentration, cross-linking agent, and degree of cross-linking.

[0127] According to aspects of the present disclosure, the sieving matrix included is configured to separate one or more of the labeled extracted AAV viral proteins VP1, VP2 and VP3 from other materials that may be present in an aliquot of the sample, and to obtain a signal from one or more of the labeled extracted AAV viral proteins VP1, VP2 and VP3 representing the amount of one or more of the labeled extracted AAV viral proteins VP1, VP2 and VP3 present in the aliquot of the sample.

[0128] According to aspects of the present disclosure, the included sieving matrix is ​​configured to separate labeled extracted AAV viral protein VP3 from other materials that may be present in an aliquot of the sample and to obtain a signal from the labeled extracted AAV viral protein VP3 representing the amount of labeled extracted AAV viral protein VP3 present in the aliquot of the sample.

[0129] The pore size of the sieving matrix may be uniform along the length of the microchannel (wherein the sieving matrix is ​​placed). Alternatively, the sieving matrix may be placed in the microchannel in a non-uniform manner (e.g., with a smooth gradient of pore size; or with two or more blocks of uniform pore size) to achieve a "step" gradient.

[0130] The concentration of the sieving polymer matrix used and its viscosity are adjusted according to the size of the nucleic acid and / or protein to be detected. According to aspects of the present disclosure, the sieving polymer matrix included is used at a concentration of about 1%-10% wt / vol and a viscosity range of about 5-100 cSt. According to aspects of the present disclosure, to determine the ratio of empty:intact, empty:partially intact or intact:partially intact AAV particles, a concentration of the sieving polymer matrix of about 2% to about 6% wt / vol and a viscosity of about 40-53 cSt is used.

[0131] Typically, flowing the aliquot through the polymer separation medium in the microchannel is performed at a temperature in the range of about 4° C. to about 30° C. According to aspects of the present disclosure, flowing the aliquot through the polymer separation medium in the microchannel is performed at a temperature in the range of about 4° C. to about 10° C., 10° C. to about 15° C., 15° C. to about 20° C., 20° C. to about 25° C., 25° C. to about 30° C., or about 30° C. to about 40° C.

[0132] Typically, flowing the aliquot through the polymer separation medium in the microchannel includes flowing into a detection zone that is connected to the microchannel fluid, wherein the detection zone is connected to a sensor signal that is capable of detecting a signal from a detectable label of the labeled extracted recombinant nucleic acid and / or a signal from a detectable label of the labeled extracted particle protein.

[0133] Make aliquots of the sample flow through polymer separation media to separate the recombinant viral nucleic acid or particle protein of the extraction through labeling. Flowing through the polymer separation media from a position in the microchannel to a distant position in the microchannel is achieved by capillary action, diffusion, hydrodynamic action and / or promoted by applying a pressure gradient, voltage gradient or a combination of two or more thereof. When the microchannel is approximately columnar, flowing through the polymer separation media from one end of the microchannel to a far-end opposite end of the microchannel is achieved by capillary action, diffusion, hydrodynamic action and / or promoted by applying a pressure gradient, voltage gradient, current gradient or a combination of two or more thereof along the length direction of the microchannel. According to aspects of the present disclosure, applying a voltage gradient or current gradient to promote flowing through the polymer separation media from one end of the microchannel to a far-end opposite end of the microchannel.

[0134] An aliquot of the sample is passed through a polymer separation medium in a microchannel and into a detection zone in fluid communication with the microchannel, the detection zone being in signal communication with a sensor capable of detecting a signal from a detectable label of the labeled extracted recombinant nucleic acid and / or labeled extracted particle protein.

[0135] Non-limiting examples of sensors capable of detecting signals from the detectable labels of labeled recombinant nucleic acids and / or labeled extracted particle proteins include charge coupled devices (CCDs), electron multiplying CCDs, photomultiplier tubes, photodiodes, complementary metal oxide semiconductors (CMOS), intensified charge coupled devices (ICCDs), and avalanche photodiodes.

[0136] According to aspects of the present disclosure, the detected signal may be an absorption or a fluorescence signal emitted from the label due to contact with electromagnetic radiation that excites the label.

[0137] The detected signal can be measured to determine one or more quantitative aspects of the sample, such as one or more of the following: a) the amount of time it takes for the labeled extracted recombinant nucleic acids to flow through the polymer separation medium in the microchannel into the detection zone, which is indicative of the size of the recombinant nucleic acids in the fluid sample, and / or b) the signal intensity of the detectable label in the detection zone, which is representative of the amount of labeled extracted recombinant nucleic acids present and indicative of the concentration of the labeled extracted recombinant nucleic acids in the fluid sample.

[0138] According to aspects of the present disclosure, the detector is operably connected to a computer that stores and processes the detected signal information, for example, to calculate one or more parameters associated with particles in a fluid sample. According to aspects of the present disclosure, the detector is operably connected to a computer that stores and processes the detected signal information, for example, to compare the detected signal information a) with a reference standard representing intact particles, and based on the comparison, determine the ratio of intact particles to partially intact particles, generate a first measurement result, and / or b) with a reference standard representing intact particles, and based on the comparison, determine the ratio of intact particles to empty particles in the fluid sample.

[0139] According to aspects of the present disclosure, the detector is operably connected to a computer that stores and processes detected signal information, for example to compare the detected signal information associated with the particle nucleic acid a) with a reference standard representing intact particles, and based on the comparison, determine the ratio of intact particles to partially intact particles, generate a first measurement result, and / or b) compare with a reference standard representing intact particles.

[0140] According to aspects of the present disclosure, the detector is operably connected to a computer that stores and processes detected signal information, for example to compare detected signal information associated with particle protein, which represents: c) the amount of time it takes for the labeled extracted protein to flow through the polymer separation medium in the microchannel into the detection zone, and d) the signal intensity of the detectable label, which represents the amount of labeled extracted protein present; comparing c) and d) with a reference standard representing a known amount of the protein to determine the amount of protein in the sample and thereby generate a second measurement result; and comparing the first measurement result with the second measurement result to determine one or more of the following: 1) the ratio of complete virus particles to empty virus particles in the fluid sample, 2) the ratio of complete virus particles or empty virus particles to partially complete virus particles, and 3) the ratio of virus particles containing complete recombinant genomes to virus particles containing incomplete recombinant genomes, thereby characterizing the recombinant virus particle population in the fluid sample.

[0141] According to aspects of the present disclosure, methods are provided for characterizing a population of particles in a fluid sample that are presumed to contain a recombinant nucleic acid. The term "fluid" as used herein refers to a liquid, a gel, or a combination of a liquid and a gel that can flow through a microchannel when referring to a sample. According to aspects of the present disclosure, the fluid is an aqueous fluid. According to aspects of the present disclosure, the fluid is an aqueous buffer, such as, but not limited to, Tris buffer, Tricine buffer, citrate buffer, HEPES buffer, carbonate buffer, phosphate buffer, MOPS buffer, TAPS buffer, and acetate buffer. Typically, the pH of the aqueous buffer is in the range of about pH 5.0 to about pH 9. According to aspects of the present disclosure, the pH of the aqueous buffer is about pH 5.0 to about pH 5.5, pH 6.0 to about pH 7.

[0142] The pH range of the present invention is as follows: about pH 6.5 to about pH 7.0, about pH 7.0 to about pH 7.5, about pH 7.5 to about pH 8.0, about pH 8.0 to about pH 8.3, or about pH 8.3 to about pH 9.

[0143] As used herein, the term "sample" refers to any material that includes or may include particles of interest.

[0144] Optionally, the sample is purified before an aliquot of the fluid sample is introduced into the microchannel of the microfluidic device. As used herein, the term "purified" refers to reducing at least some contaminants, such as materials associated with particle production in the sample, including but not limited to helper viruses, helper virus proteins, host cell proteins, and host cell debris. According to aspects of the present disclosure, the sample is purified so that the contaminants in the sample are reduced by at least 10% or more, at least 20% or more, at least 30% or more, at least 40% or more, at least 50% or more, at least 60% or more, at least 70% or more, at least 80% or more, at least 90% or more. Or at least 95% or higher.

[0145] The term "aliquot of a fluid sample" refers to a portion of a fluid sample for analysis. An aliquot typically has a volume in the range of about 0.5 microliter to about 50 microliters. According to aspects of the present disclosure, an aliquot has a volume in the range of about 0.5 microliter to about 5 microliters, about 1 microliter to about 10 microliters, about 1 microliter to about 20 microliters, about 5 microliters to about 10 microliters, about 5 microliters to about 15 microliters, about 5 microliters to about 20 microliters, about 10 microliters to about 15 microliters, about 15 microliters to about 20 microliters, about 25 microliters to about 30 microliters, about 30 microliters to about 35 microliters, about 35 microliters to about 40 microliters, about 40 microliters to about 45 microliters, or about 45 microliters to about 50 microliters. According to aspects of the present disclosure, the aliquot has a volume ranging from about 0.5 microliters, about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, or about 10 microliters.

[0146] According to aspects of the present disclosure, the viral particles in the fluid sample are approximately 1×10 10 Virus particles / ml is about 1×10 14 According to aspects of the present disclosure, the viral particles in the fluid sample are present in an amount of about 1×10 10 Virus particles / ml is about 0.5×10 11 The amount of virus particles / ml is about 0.5×10 11 Virus particles / ml is about 1×10 11 Virus particles / ml, 1×10 11 Virus particles / ml is about 0.5×10 12 Virus particles / ml, about 0.5×10 12 Virus particles / ml is about 1×10 12 Virus particles / ml, about 1×10 12 Virus particles / ml is about 0.5×10 13 Virus particles / ml, about 0.5×10 13 Virus particles / ml is about 1×10 13 Virus particles / ml, about 1×10 13 Virus particles / ml is about 0.5×10 14 Virus particles / mL, or about 0.5×10 14 Virus particles / ml is about 1×10 14 Virus particles / ml.

[0147] According to aspects of the present disclosure, a method for characterizing a population of viral particles in a sample is provided, comprising a microfluidic electrophoresis assay of viral nucleic acid in a sample. The assay produces an assay result representing the amount of viral nucleic acid in the sample; and the assay result is compared to a reference standard, which represents a known amount of viral nucleic acid. The amount of nucleic acid can be expressed as the concentration of nucleic acid in the sample and the reference standard. Comparison of the assay result with the reference standard provides a ratio of sample viral nucleic acid to the reference standard, see Figure 2 .

[0148] According to aspects of the present disclosure, a ratio of the amount of viral protein in the sample to the amount of protein in the reference standard is generated. The amount of protein can be expressed as the concentration of the protein in the sample and the reference standard. Comparison of the amount of viral protein in the sample to the amount of viral protein in the reference standard provides a ratio of the sample viral protein to the reference standard protein, see Figure 2 .

[0149] According to aspects of the present disclosure, information about the number of viral particles in a sample and / or reference standard is used to calculate the amount of protein in the sample and / or reference standard without measuring the protein in the sample and / or reference standard.

[0150] Optionally, according to aspects of the present disclosure, the amount of protein in the sample and / or standard is determined by a microfluidic electrophoresis assay.

[0151] According to aspects of the present disclosure, the proportion and / or fraction of intact viral capsids in a sample is determined by calculating the ratio of viral nucleic acid and viral protein in the sample compared to nucleic acid and protein in a reference standard.

[0152] According to specific aspects of the present disclosure, a method is provided for characterizing a population of viral particles in a sample to determine the proportion or fractional percentage of complete viral capsids, partially complete viral capsids and / or empty viral capsids in the sample, which comprises extracting viral nucleic acid from the sample; and determining the extracted viral nucleic acid by microfluidic electrophoresis.

[0153] According to specific aspects of the present disclosure, a method for characterizing a population of viral particles in a sample to determine the ratio or fractional percentage of intact viral capsids to empty viral capsids in the sample is provided, comprising extracting viral nucleic acid from the sample; determining the extracted viral nucleic acid by microfluidic electrophoresis; extracting viral protein from the sample; determining the extracted viral protein by microfluidic electrophoresis; calculating the ratio of the nucleic acid concentration in the sample to the nucleic acid concentration in a reference standard, calculating the ratio of the protein concentration in the sample to the protein concentration in the reference standard, and calculating the fractional amount of intact capsids in the sample according to the following formula:

[0154]

[0155] where N is the total number of capsids, f and e refer to intact capsids and empty capsids, respectively, βs is the percentage of intact capsids in the reference standard, and R DNA is the ratio of the concentration of viral nucleic acid in the sample to the concentration of viral nucleic acid in the reference standard, R P It is the ratio of the concentration of viral protein in the sample to the concentration of viral protein in the reference standard.

[0156] According to aspects of the present disclosure, a sample comprises AAV capsids and is assayed to determine the ratio of complete AAV capsids to empty AAV capsids (or vice versa), complete AAV capsids to partially complete AAV capsids (or vice versa), partially complete AAV capsids to empty AAV capsids (or vice versa), or any combination thereof.

[0157] According to aspects of the present disclosure, a kit for characterizing the nucleic acid content of a particle population presumed to contain recombinant nucleic acids in a fluid sample is provided, comprising: a nucleic acid marker, a gel, a nucleic acid gradient standard, a nucleic acid storage buffer, a nucleic acid sample buffer, a protease, and a denaturant. According to aspects of the present disclosure, the nucleic acid marker is a fluorescent nucleic acid intercalator. According to aspects of the present disclosure, the protease is a serine protease.

[0158] According to aspects of the present disclosure, a kit for characterizing the nucleic acid content of a particle population presumed to contain recombinant nucleic acids in a fluid sample is provided, comprising: a nucleic acid marker, a gel, a nucleic acid gradient standard, a nucleic acid storage buffer, a nucleic acid sample buffer, a protease, a denaturant, a protein dye, a protein standard, a protein storage buffer, a protein sample buffer, and a wash buffer. According to aspects of the present disclosure, the nucleic acid marker is a fluorescent nucleic acid intercalator. According to aspects of the present disclosure, the protease is a serine protease.

[0159] According to aspects of the present disclosure, a kit is provided for characterizing the nucleic acid content of a population of particles putatively containing a recombinant nucleic acid in a fluid sample, wherein the protease is selected from the group consisting of proteinase K, subtilisin, trypsin, chymotrypsin, thrombin, plasmin, elastase, and lactoferrin.

[0160] According to aspects of the present disclosure, a kit for characterizing the nucleic acid content of a population of particles putatively containing a recombinant nucleic acid in a fluid sample is provided, wherein the denaturing agent is a chaotrope, a detergent, or a mixture thereof.

[0161] According to aspects of the present disclosure, a kit for characterizing the nucleic acid content of a population of particles putatively containing a recombinant nucleic acid in a fluid sample is provided, comprising at least one protease and at least one chaotrope, wherein the at least one protease is proteinase K and the at least one chaotrope is urea.

[0162] According to aspects of the present disclosure, a kit for characterizing the nucleic acid content of a particle population presumed to contain a recombinant nucleic acid in a fluid sample is provided, comprising one or more standards. According to aspects of the present disclosure, the standards included may be samples comprising a known ratio of complete particles: empty particles, a known ratio of complete particles: partially complete particles, or a known ratio of empty particles: partially complete particles, two or more thereof, or all three thereof. According to aspects of the present disclosure, the standards included may be samples comprising a known ratio of complete AAV particles: empty AAV particles, a known ratio of complete AAV particles: partially complete AAV particles, or a known ratio of empty AAV particles: partially complete AAV particles, two or more thereof, or all three thereof.

[0163] Embodiments of the compositions and methods of the present invention are illustrated in the following examples. These examples are provided for illustrative purposes and are not to be construed as limiting the scope of the compositions and methods of the present invention.

[0164] Example

[0165] Example 1

[0166] Samples and sample preparation

[0167] AAV8 complete and empty reference standards were purchased from Vigene Biosciences (Vigene Biosciences, Rockville, MD). The titer of the complete reference standard at 75% complete fraction was 7.97 × 10 11 genome copies (GC) / mL, equivalent to 1.07×10 12 The latter is estimated by taking into account empty particles, which do not contribute to the genome copies. On the other hand, the purchased empty reference standard has a titer of 1.44 × 10 at the 96% empty fraction. 12 VP / mL. Since this value includes both intact particles and empty particles, the titer of empty particles alone in this sample was estimated to be 1.38×10 12VP / mL. For complete and empty reference standards, quality control was performed by SYBR Green qPCR and ELISA combined with TEM according to the certificate of analysis issued by the supplier (provider) (Vigene Biosciences) to determine the percentage of complete capsids in the sample. Although the supplier (vendor) did not provide the standard deviation of its estimated values, a 10% deviation was assumed for statistical analysis when necessary, which may be consistent with the analytical tools used for its capsid ratio estimation. For experiments comparing complete samples with empty samples, reference samples were used. For experiments using samples with different percentages of complete viral particles, these samples were prepared taking into account the empty particles in the complete reference standard and the complete particles in the empty reference standard. In addition, the samples were stored at -80°C in single-use aliquots to ensure that sample stability and degradation did not affect the experimental results.

[0168] method

[0169] In this example, the GX Touch II LabChip system (PerkinElmer, Waltham, MA) is a microfluidic electrophoresis separation system used to characterize samples containing viral particles to determine the ratio of intact: empty viral particles in the sample. For capsid protein experiments, the standard protocol of the LabChip ProteinExpress assay in the GX Touch II LabChip system was used, which is schematically shown in Figure 1 Above. Specifically, a high sensitivity protocol was followed, which requires 5 μL of sample, using an optional reducing buffer and incubating at 100°C for 5 minutes to extract proteins. Each sample was analyzed three times, with 20 nL transferred from the well plate to the detection chip each time. For ssDNA analysis, a customized ssDNA chip assay was used. First, the AAV sample was digested with a proteinase K digestion mixture (10 μL proteinase K diluted with 90 μL 2M urea) in a 1:1 (5 μL AAV and 5 μL digestion mixture) at 50°C for 60 minutes, and then the proteinase K was inactivated at 95°C for 20 minutes (note that the sample was not diluted 1:10 in the sample buffer), which is schematically shown in Figure 1Below. Although this example focuses on the detection of ssDNA, AAV can also contain self-complementary DNA, which can also be detected by minor assay modification. The samples were then analyzed with a custom ssDNA assay script to amplify the signal and allow detection of samples with lower genomic content. Each sample was analyzed three times, with 20nL transferred from the well plate to the detection chip each time. Although 5μL of sample was used for each assay, this volume can be significantly reduced if necessary without interfering with the assay. Statistical analysis for this study was performed using GraphPadPrism 9, and plotted using GraphPad and / or BioRender.

[0170] According to aspects of the methods of the present disclosure in this example, protein and ssDNA of viral particles in a sample are quantified, but since the protein content of intact and empty capsids is equivalent, analysis of protein content is optional.

[0171] Methods for determining the percentage of intact capsids comprising viral particles in a sample according to aspects of the present disclosure are described.

[0172] According to aspects of the present disclosure, only a few microliters (eg, 3-10 μL) of a standard (S) with a known fraction of intact capsids is used.

[0173] According to aspects of the methods of the present disclosure described below, mathematical formulas are used to relate the protein and DNA concentrations in the sample and the viral particle standards.

[0174] Mathematical formula for estimating the percentage of intact particles

[0175] In this example, samples containing empty, complete and / or partially complete AAV capsids were used. Empty, complete and partial AAV capsids have icosahedral symmetry. AAV particles are assembled from viral proteins (VP1, VP2, VP3) and genomic material (ssDNA).

[0176] The total number of AAV capsids in the sample and standard are denoted by N and N, respectively. s Therefore, the number of complete capsids and empty capsids can be described using equations (1) and (2), where f represents complete capsids and e represents empty capsids:

[0177] N=N(f)+N(e) (1)

[0178] N s =N s (f)+N s (e) (2)

[0179] Since the protein subunits of the AAV capsid (VP1, VP2, VP3) are identical for both intact and empty AAV, it is reasonable to assume that each capsid consists of αug of total protein. s The ratio of protein concentration in p It can be expressed in formula (3) as:

[0180]

[0181] Similarly, assuming a single ssDNA insert per intact AAV capsid, samples (c(ssDNA)) and standards (c s The following ratio R of ssDNA concentration in (ssDNA) DNA :

[0182]

[0183] Since the standard has a known percentage of intact capsids, β s , which can be defined as:

[0184]

[0185] The fraction β of intact capsids is obtained using the following relationship:

[0186]

[0187] Therefore, according to aspects of the present disclosure, an estimate of the percentage of complete AAV is obtained by using microfluidic electrophoresis and measuring the relative concentrations of total protein and ssDNA with a sample and one or more standards. The method is independent of the total capsid concentration in the sample or standard. This is usually the most important limitation in other technologies. If there is any error in the estimate obtained using equation (6), it only comes from the concentration ratio accuracy error in the electrophoresis method, rather than from the resolution accuracy of the method. In other words, instead of obtaining the total protein concentration of the sample from the protein assay (complete and empty) and subtracting the concentration obtained from the DNA assay (complete only) (which will have a compound error rate from using two different assays), the method according to aspects of the present disclosure uses a sample with a known concentration to standardize the two assays. Another key advantage of ratio measurement is that as long as all samples within the assay are treated in the same way (diluted by the same factor, or heated for the same time), the method according to aspects of the present disclosure is independent of the concentration of the sample because the protein area will explain the difference in concentration.

[0188] Capsid protein profile characterization

[0189] In order to validate the method for estimating the percentage of intact capsids in a sample according to the present disclosure, an evaluation of the protein and DNA profiles of AAV8 intact (in a "intact sample" containing 75% intact, 25% empty) and empty (in a "empty sample" containing 96% empty, 4% intact) reference standards was performed. It was confirmed by experimental analysis that the amount of protein in the intact and empty capsid reference standards remained at a given concentration. To this end, the AAV standards were denatured using a reducing buffer containing DTT. The concentration of the empty reference standard was adjusted to the concentration of the intact reference standard (1.07×10 12 Dilutions were performed to keep virus concentration and salt composition constant, which may affect labeling efficiency and sample conductivity.

[0190] according to Figure 3A The electropherogram shown, such as the total area under the corresponding curve of VP1, VP2 and VP3 ( Figure 3B ), there is no statistical difference between the VP peaks of the intact sample and the empty sample. In addition, based on the literature and normalized to their respective molecular weights, it is expected that VP1 accounts for 11.3% of the total capsid area, VP2 accounts for 9.5%, and VP3 accounts for 79.2%. In the analysis of this example, the intact sample accounted for 6.7% VP1, 11.3% VP2, and 82.1% VP3. Similarly, the empty sample accounted for 6.3% VP1, 11.6% VP2, and 82.1% VP3. The presence of these VP ratios that are different from the expected 1:1:10 ratio highlights the random nature of the VP capsid protein ratios. Therefore, in order to mitigate the impact of different VP ratios between samples and even within a given batch, the areas under the three VP peaks are integrated to obtain a method that is independent of the VP ratio. According to aspects of the method of the present invention, one, two, or all three of these peaks (i.e., the VP1 peak, the VP2 peak, and the VP3 peak) can be used for estimation. Alternatively, the VP3 peak alone can be used, as molecules with faster electrophoretic mobility generally yield higher quantification accuracy because the molecule has less time to diffuse (wider peaks), and higher concentrations (which increase peak area and height) can help visualize the tail of the peak due to diffusion.

[0191] Furthermore, if the sample and (usually provided) standard concentrations are known, these values ​​can be entered as concentrations in a modified equation (3) rather than the protein areas obtained in the protein expression assay detailed above (see equations 7-8). However, if the concentrations are unknown, this method can be used to estimate not only the percentage of intact capsids in a sample, but also the total concentration of the sample, as shown in Tables 1A and 1B. The area under the curve of the VP protein peaks for the standards and samples and the standard concentration were used to estimate the total sample concentration with an error rate of 1-16% and an average error rate of 6%. The results show that the LOD in this example is 5×10 11 and 1×10 12 The current LOD estimate for the ssDNA assay in this example is >1×10 11 GC / mL, which will generally bring the total protein content within the desired range.

[0192] Table 1A. Total protein concentration of samples estimated based on the VP peak area under the curve of standards (known concentrations) and the area under the curve of samples. The predicted total concentration was estimated using equation (3). Each group refers to an independent run in which samples were analyzed in triplicate.

[0193]

[0194] Note that as long as the sample and (usually provided) standard concentrations are known, these values ​​can be entered as concentrations in the modified equation (3) rather than the VP3 area peak obtained in the protein expression assay detailed herein (see Equations 7-8). However, if the concentration is unknown, this method can be used to estimate not only the percentage of intact capsids in the sample, but also the total concentration of the sample, as shown in Table 1B. Using the area under the curve of the VP3 peak of the standards and samples and the concentration of the standards, the total sample concentration was estimated according to aspects of the methods of the present disclosure with an error rate of 4-19%, with an average error rate of 11%.

[0195] Table 1B. Total protein concentration of samples estimated from the area under the curve for VP3 of standards (known concentrations) and the area under the curve for samples. The predicted total concentration was estimated using equation (3). Note that each group refers to an independent run in which samples were analyzed in triplicate.

[0196]

[0197]

[0198] ssDNA profile characterization

[0199] As described above, after confirming that the amount of capsid protein remained constant for both intact and empty capsids, the ssDNA content of the capsids was assessed. In this example, the sample viral particle concentration was normalized to 1.07 × 10 12 VP / mL, and then digest the samples using the standard proteinase K digestion protocol as described above. Figure 4A and 4B As shown, the intact reference standard (75% intact) exhibits a peak at approximately 62 s, while the blank standard (96% empty) produces no peak. Although the absence of a peak in the blank sample is not unexpected, it must be noted that the blank sample should still contain approximately 4.26 × 10 10 This means that the LOD is higher than in this example. Additional experiments were performed to determine that the LOD is approximately 1 × 10 11 This limitation is partly due to the lack of a compatible assay optimized for fluorescence analysis of ssDNA, and thus the labeling efficiency of this system will be limited.

[0200] Similar to the protein estimates generated using equation (3) shown in Tables 1A and 1B, the DNA information collected using this assay can be input into equation (4) to estimate the number of intact particles (or genome copies, GC) in the sample, as shown in Table 2. Using this approach, the prediction error rate ranged from 2-20%, with an average error rate of 8%.

[0201] Table 2: Estimation of sample genome concentrations based on the area under the ssDNA curve of standards (known concentrations) and the area under the curve of samples. The predicted genome concentrations were estimated using equation (4). Each group refers to an independent run in which samples were analyzed in triplicate.

[0202]

[0203]

[0204] Capsid protein and ssDNA profiles of samples with different intact percentages

[0205] To estimate the percentage of intact capsids in an unknown AAV8 sample, information was collected from samples with varying percentages of intact capsids (75% to 4%). In this example, the total protein concentration of both samples was normalized to 1.07 × 10 12 VP / mL and mixed the intact reference standard with the blank standard in different ratios to obtain the desired percent intact.

[0206] Figure 5A , 5B, 5C and 5D show the results of protein and nucleic acid analysis of samples with different percentages of intact capsids, including 75%, 50%, 25% and 4%. Figure 5A : Representative electropherograms of VP3, VP2, and VP1 capsid proteins of intact and empty samples, from least to most mass (left to right). Figure 5B : Total integrated area under the curve of the VP protein peak of four samples with different intact capsid percentages. Figure 5C : Representative electropherograms of genetic material of four samples, where a decrease in the DNA peak is observed as the percentage of intact capsids decreases. Figure 5D : Total area under the curve of the genetic material peak for each sample, note that for all three runs, the empty AAV sample produced no peak.

[0207] As expected, while protein concentrations remained approximately the same, the mean total VP area ranged from 28.6 to 34.16 ( Figure 5A and 5B ), but the ssDNA concentration decreased as the number of intact capsids decreased, with the average area decreasing from 11.4 to 0.0 ( Figure 5C and 5D ).

[0208] Experimental data were collected in three independent sets of protein and ssDNA assays and analyzed in triplicate. Using protein and ssDNA information from the complete reference standard (75% complete) and samples, the predicted percentage of β or complete capsids was estimated using equation (6). The results for each set are shown in Table 3.

[0209] Table 3. Compiled protein and ssDNA data collected and analyzed from three different sets of experiments.

[0210]

[0211] Currently, the model has an average prediction deviation of 4% from the actual percentage, a range of 2-6%, and a standard deviation of 2%.

[0212] Alternatively, if the sample concentration is known to be within an acceptable error range, the error rate of the system and sample analysis time can be significantly reduced by omitting the protein assay according to aspects of the methods of the present disclosure. Assuming a 10% error rate for the provided concentration and the intact percentage of the sample, the inventors' predicted error rates are therefore 10% because they are based on these values ​​and are the reported error rates of the detection platform, using a modified version of the following equation (6) to estimate the percentage of intact capsids using the ssDNA area and the provided concentration:

[0213]

[0214]

[0215] Where R concentration Used to replace R p , because as shown in the data in Table 2, sample concentration is correlated with protein area.

[0216] To assess the specificity of this alternative aspect of the methods of the present disclosure, the actual (reported) percentage of intact capsids for each sample was compared to the predicted values ​​obtained using equation (6) (referred to as "protein area prediction") and equation (8) (referred to as concentration prediction), as Figure 6 Note that while this aspect of the method of the present disclosure bypasses the need for protein analysis, it is still highly dependent on ssDNA analysis, as shown in equation (8).

[0217] After analyzing the results obtained using both methods, it was observed that the average prediction bias decreased from 4% (protein area prediction method) to 3% when the concentration prediction method was used, with a range of 1-9%. In other words, both the error rate and turnaround time can be reduced when the total sample concentration is provided. Importantly, the reduction in turnaround time will be limited because the longest incubation is required for ssDNA extraction from capsids. When known concentrations are used, the standard deviation of the prediction increases slightly from 2% to 3%.

[0218] Therefore, assessing ssDNA in capsids and normalizing the results to capsid protein (either by measuring the protein or by a non-measured calculation of the amount of protein) provides the desired information about the ratio of intact to empty capsids in the sample and thus the percentage of intact capsids.

[0219] The relationship between the protein and ssDNA profiles of intact and empty standards is used in methods according to the present disclosure to estimate the percentage of intact capsids in AAV8 samples of unknown concentration and composition. First, the primary assay was validated by comparing the measurements with literature values. While the protein assay did not produce high accuracy in the ratio of VP1 and VP2 (which was not surprising based on their lower concentrations and electrophoretic mobilities), the VP3 peak was completely consistent with the literature. As expected, the protein profiles of intact and empty AAV8 did not show significant differences for a given concentration. Similarly, the ssDNA assay showed consistent results when comparing intact and empty reference standards, and when analyzing samples with different percentages of intact capsids. It must also be noted that the main benefit of running these two assays simultaneously is the ability to obtain the total sample concentration and total genome concentration of the sample, as reported in Tables 1-2.

[0220] The method according to aspects of the present disclosure provides a rapid, high-throughput alternative that can be used to rapidly iterate multiple batches of samples and does not require highly specialized equipment that requires extensive training. Within 2-3 hours (including denaturation and digestion times, depending on the number of samples analyzed), the method according to aspects of the present disclosure estimates the percentage of intact capsids with an average error rate of 16% using a total volume of less than 10 μL / sample.

[0221] The rapid analytical processing time and high throughput nature of methods according to aspects of the present disclosure ensure that each additional sample analyzed produces only a minimal increase in the overall analysis time.

[0222] Example 2

[0223] AAV empty / full analysis

[0224] Five microliters of sample containing AAV particles was mixed with proteinase K / urea solution in a microtiter plate and heated at 55°C for 1 hour and then at 95°C for 20 minutes to generate extracted AAV single-stranded DNA (ssDNA).

[0225] Another 5 μl of sample containing AAV particles was mixed with 7 μl of denaturation solution in a microtiter plate and heated at 100°C for 5 min to extract VP1, VP2, and VP3 AAV capsid proteins.

[0226] The extracted AAV nucleic acid and the extracted AAV protein are labeled and subjected to capillary electrophoresis in a microfluidic device. The detection signals of the detectable labels from the extracted AAV nucleic acid and the extracted AAV protein are normalized to an AAV reference standard, and the ratio of empty: intact AAV particles is calculated.

[0227] Size Standard DNA Ladder Preparation

[0228] Thaw the DNA size standard (gradient) vial on ice. Spin down the gradient and heat denature at 70°C for 2 minutes. Immediately cool on ice for 5 minutes.

[0229] To prepare the gradient used, 1X sample buffer was prepared by adding 200 μl of sample buffer concentrate to 1800 μl of DEPC-treated or nuclease-free water.

[0230] Mix 116 μl 1X sample buffer with 4 μl denaturing gradient. Carefully pipette up and down to mix and transfer to the gradient tube.

[0231] Proteinase K solution preparation

[0232] Dilute proteinase K powder in 1.25 ml of diethylpyrocarbonate (DEPC)-treated or nuclease-free water.

[0233] AAV digestion mix preparation for nucleic acid extraction

[0234] Prepare the AAV digestion mix by mixing 90 μl of 2 M urea per 10 μl of proteinase K.

[0235] Nucleic acid extraction

[0236] Mix 5 μL of AAV sample with AAV digestion mix (Proteinase K / Urea solution) (5 microliters) into a PCR tube or plate. Carefully pipette the mixture up and down to mix.

[0237] Mix 5 μL of AAV standard with AAV digestion mix (5 microliters) into a PCR tube or plate. Carefully pipette the mixture up and down to mix.

[0238] Place the AAV sample / digestion mix and the standard / digestion mix in a thermal cycler and heat at 55 °C for 1 h, followed by 95 °C for 20 min to generate extracted ssDNA from the samples and standards.

[0239] The extracted ssDNA from the samples and standards were centrifuged and the supernatant was then transferred to a 384-well plate. The 384-well plate was quickly centrifuged after transfer.

[0240] Nucleic acid gel-dye solution preparation

[0241] 90 microlitres of dye concentrates are transferred to a test tube comprising a gel matrix. Vortex and invert the test tube several times until the resulting dye-gel mixture is fully mixed, then centrifuged rapidly for a few seconds. The dye-gel mixture is transferred to a centrifugal filter and at room temperature centrifuged at 9300 rcf for 10 minutes.

[0242] DNA chip preparation

[0243] Before use, wash with nuclease-free deionized water (Milli- or equivalent) and completely aspirate each active well twice.

[0244] 75 μl of the gel-dye mixture was added to the appropriate chip well and 120 μl of the gel-dye mixture was added to another well.

[0245] Add 100 μl of the DNA size standard ladder to one chip well.

[0246] After loading, capillary electrophoresis is performed, where the material in the well flows through the microchannels of the microfluidic device to the detection zone. The detection signal of the detectable label from the extracted AAV nucleic acid is normalized to the AAV reference sample, and the ratio of empty: intact AAV particles is calculated.

[0247] AAV empty / intact protein gel-dye solution preparation

[0248] Before use, allow the chip and reagents to equilibrate to room temperature for at least 30 minutes.

[0249] In order to prepare the gel-dye solution, use the reverse pipetting technology, 520 microlitres of gel matrix are transferred to the top basket of the centrifugal filter provided. 20 microlitres of dye concentrate are transferred to the gel matrix in the centrifugal filter. Invert, and vortex in an inverted direction until the gel-dye is uniformly blue. For the decolorizing solution, 250 microlitres of (HT) gel matrix are transferred to a second centrifugal filter. Gel-dye and decolorizing solution were at room temperature centrifuged for 8 minutes at 9300rcf.

[0250] AAV empty / intact protein gradients, buffers and sample preparation

[0251] Prepare a protein denaturing solution by pipetting 700 microliters of sample buffer into a 2 mL centrifuge vial, adding 24.5 microliters of 1 M DTT and mixing. Add 7 microliters of protein denaturing solution to 5 microliters of AAV sample and 5 microliters of protein standard. Transfer each mixture to a separate well of a microtiter plate. Place 12 microliters of a protein standard gradient into the wells on the plate. Denature the samples, standards, and gradients by heating at 100°C for 5 minutes. Next, add 32 microliters of water to each sample well and mix, and add 120 microliters of water to the gradient and mix.

[0252] AAV empty / intact protein chip preparation

[0253] 75 microliters of gel-dye mixture were added to 3 wells of the microfluidic device, and 120 microliters of gel-dye mixture were added to a separate well. Then 75 microliters of decolorizing solution were added to each well of the two separate wells. In addition, 120 microliters of marker solution were added to another separate well.

[0254] After loading, capillary electrophoresis is performed, where the material in the well flows through the microchannels of the microfluidic device to the detection zone. The detection signal of the detectable label from the extracted AAV protein is normalized to the AAV reference sample and combined with the data derived from the analysis of the extracted AAV nucleic acid and nucleic acid standards described above to calculate the ratio of empty: intact AAV particles.

[0255] AAV empty / intact protein analysis

[0256] To normalize the protein concentration of AAV samples relative to the AAV reference standard, in this example, the VP3 correction area input was used. Under the conditions used in this example, the arrival time of the VP3 peak was approximately 28-30 s. To obtain the combined correction area:

[0257] Exclude peaks assigned to VP1 (if assigned)

[0258] Exclude peaks assigned to VP2 (if assigned)

[0259] Manually extend the VP3 baseline to include all area under the VP3 peak. The corrected area of ​​the merged peak is now available in the peak table of GXTouch Reviewer. Figure 7 is an electropherogram showing the integration of the VP3 peak used for empty:intact ratio analysis in this example.

[0260] AAV empty / complete DNA analysis

[0261] The ssDNA aligned region was used to determine the empty:full ratio of the AAV sample. Under the conditions used in this example, the arrival time of the ssDNA peak was approximately 45-47 seconds. The baseline of the ssDNA peak can be set manually, such as Figure 8 An electropherogram showing the integration of the ssDNA peak is shown. The aligned area of ​​the ssDNA peak is indicated.

[0262] AAV Null Analysis using the AAV Null / Intact Calculator

[0263] To calculate the sample % ratio of intact AAV capsids to empty AAV capsids in the sample, the following values ​​were obtained by analyzing the generated electropherogram:

[0264] 1) % completeness of the standard (obtained from the label information of commercially available AAV standards);

[0265] 2) The protein content of the AAV standard measured (measured on the plate);

[0266] 3) The DNA content of the AAV standard measured (measured on the plate); and

[0267] 4) The measured protein and measured DNA content of each unknown AAV sample.

[0268] The ssDNA concentration ratio between the sample and the standard was calculated according to equation (4).

[0269] The protein concentration ratio between samples and standards was calculated according to equation (3).

[0270] The fraction of intact AAV capsids in the sample was calculated by the ratio of ssDNA and protein between the sample and the standard, and a given percentage of intact capsids in the sample was given by equation (6).

[0271] Example 3

[0272] Analysis of partially intact capsids

[0273] In this example, samples containing intact AAV capsids or partially intact AAV capsids with a viral genome content greater than 1E12 VG / mL were used, and the above-mentioned method for preparing ssDNA and viral proteins was used, wherein the sieving polymer matrix was 2% wt / vol and the viscosity was 40-53 centistokes (cSt).

[0274] Fig. 9 is an electropherogram showing the integration of the ssDNA peak from a partially intact / intact analysis of AAV nucleic acids from samples containing either partially intact capsids or intact AAV capsids.

[0275] Example 4

[0276] Remove residual DNA / RNA before empty / intact analysis

[0277] Some samples may contain residual DNA / RNA present outside the AAV capsid but in the sample solution. According to aspects of the present disclosure, residual DNA / RNA can be removed by treatment with a universal nuclease. An exemplary process is:

[0278] Dilute 10 μl of sample to 50 μl working volume, approximately 1 × 10 12 GC / mL. PBS or PBS + 0.001% Pluronic can be used as diluent.

[0279] Add 1 μl of universal nuclease solution (≥2.5-25 units / μl) to 50 μl of diluted sample.

[0280] Incubate at 37 °C for 10 min.

[0281] Add 1 μl of 30 mM ETDA.

[0282] Heat at 75°C for 10 minutes.

[0283] Use a 100 kDa molecular weight cut-off filter (e.g. The heat treated material is filtered using a centrifugal filter (Ultrasonic® Ultra-0.5 mL centrifugal filter, 100K). The final filtered volume is expected to be about 15 μL, which can then be used as a fluid sample in the method of characterizing a population of particles putatively containing a recombinant nucleic acid in a fluid sample of the present disclosure.

[0284] Fig.10 is an electropherogram showing the results of RNA ladder before and after omni-nuclease treatment. The size of the fragment peak with a size range of 200-6000nt was reduced to less than 200nt by treatment. Treatment with omni-nuclease and filtration can be applied before running the AAV DNA assay to remove residual ssDNA or RNA in the sample that is not encapsulated by AAV particles.

[0285] Example 5

[0286] Binding and recovery of ssDNA to purification columns

[0287] Optionally, a ssDNA cleanup procedure can be used. An exemplary procedure for AAV8 is:

[0288] Add 50 μL of AAV8 (pure or treated with proteinase K) to the QIAquick PCR column.

[0289] Incubate with binding buffer for 30 min at room temperature.

[0290] Wash the sample with 750 μL of purification kit wash buffer.

[0291] Elute with elution buffer matching the AAV DNA kit sample buffer (1:1 v / v PBS and 2M urea).

[0292] Fig.11 Shown are superimposed electropherograms of 3 sips of AAV8 ssDNA obtained after recovery from binding to the QIAquick PCR purification kit. The traces show the detection of AAV-DNA from samples purified using the QIAquick kit. The three traces show the reproducibility of the peak shape and area under the curve.

[0293] Example 6

[0294] Analysis of partially intact viral capsids

[0295] In this example, samples containing intact AAV capsids or partially intact AAV capsids with a viral genome content greater than 1E12 VG / mL were used, and the above-mentioned method for preparing ssDNA and viral proteins was used, wherein the sieving polymer matrix was 2% wt / vol and the viscosity was 40-53 centistokes (cSt).

[0296] Fig. 12A is an overlay of electropherograms showing the detection of 2.4 kilonucleotide (knt) ssDNA and 3.3 knt ssDNA in a separate capillary electrophoresis run.

[0297] Fig. 12B is an electropherogram showing the detection of 2.4 kilonucleotide (knt) ssDNA and 3.3 knt ssDNA in a single capillary electrophoresis run.

[0298] Example 7

[0299] Analysis of multiple AAV serotypes

[0300] In this example, samples containing either complete AAV2, AAV8, or AAV9 capsids or empty AAV2, AAV8, or AAV9 capsids were used, and the methods described above for preparing ssDNA and viral proteins were used for each serotype sample. For each assay, the extracted protein was compared to an AAV8 standard, and the extracted nucleic acid was compared to an AAV8 standard. Using AAV8 as the reference standard, the errors for AAV9 and AAV2 were within about 12% error (found when matching the AAV8 standard to the AAV8 sample).

[0301] Fig.13A is an electropherogram showing the results of the assay of extracted viral proteins for each indicated serotype. The viral protein resolution and full width at half maximum (FWHM) of AAV2, AAV8 or AAV9 viral particles were similar.

[0302] Fig. 13B is a graph showing the results of the extracted nucleic acid assay of 70% intact AAV8 viral particles (F-70), 50% intact AAV8 viral particles (F-50), 25% intact AAV8 viral particles (F-25), and empty AAV8 viral particles (F-0). For each pair of bars on the graph, the left bar represents the calculated theoretical percentage of integrity, and the right bar represents the average percentage of integrity measured and calculated using equation (6).

[0303] Fig. 13C 82% intact AAV9 virus particles (F-82), 50% intact AAV9 virus particles (F-50), 25% intact AAV9 virus particles (F-25) and empty AAV9 virus particles (F-0) are shown in the figure. For each pair of bars on the graph, the left bar represents the calculated theoretical percentage of integrity, and the right bar represents the average percentage of integrity measured and calculated using equation (6).

[0304] Fig.13D62% intact AAV2 virus particles (F-62), 50% intact AAV2 virus particles (F-50), 25% intact AAV2 virus particles (F-25) and empty AAV2 virus particles (F-0) are shown in Figure 1. For each pair of bars on the graph, the left bar represents the calculated theoretical percentage of intact, and the right bar represents the average percentage of intact measured and calculated using equation (6).

[0305] For non-viral vector particles, 5 μl of sample containing liposome particles putatively containing recombinant DNA or RNA was mixed with proteinase K / urea solution in a microtiter plate and heated at 55°C for 1 hour and then at 95°C for 20 minutes to produce extracted single-stranded DNA (ssDNA) or extracted RNA.

[0306] Another 5 microliters of sample containing liposome particles putatively containing recombinant DNA or RNA was mixed with 7 microliters of denaturation solution in a microtiter plate and heated at 100°C for 5 minutes to extract any liposomal proteins.

[0307] The extracted liposomal nucleic acids and extracted liposomal proteins are labeled and subjected to capillary electrophoresis in a microfluidic device. The detection signals of the detectable labels from the extracted liposomal nucleic acids and extracted liposomal proteins are normalized to a reference sample and the ratio of empty: intact liposomal particles and / or partially intact: intact liposomal particles is calculated using Equations 1, 2, 3, 4, 5, 6, 7, and 8, or any subset thereof.

[0308] project

[0309] Item 1.) A method for characterizing a population of particles presumed to contain a recombinant nucleic acid in a fluid sample to determine one or more of the following: 1) the ratio of intact particles to empty particles in the fluid sample, and 2) the ratio of intact particles or empty particles to partially intact particles, comprising:

[0310] extracting recombinant nucleic acid from a first aliquot of the fluid sample containing the particles to produce extracted recombinant nucleic acid;

[0311] labeling the extracted recombinant nucleic acid to produce labeled extracted recombinant nucleic acid;

[0312] allowing the labeled extracted recombinant nucleic acids to flow through the polymer separation medium in the microchannel into a detection zone in fluid communication with the microchannel, the detection zone being in signal communication with a sensor capable of detecting a signal from a detectable nucleic acid label of the labeled extracted recombinant nucleic acids, thereby separating the labeled extracted recombinant nucleic acids according to size by allowing the labeled extracted recombinant nucleic acids to flow through the polymer separation medium of the microchannel;

[0313] detecting the detectable label of the labeled extracted recombinant nucleic acids in the detection zone to determine: a) the amount of time it takes for the labeled extracted recombinant nucleic acids to flow through the polymer separation medium in the microchannel into the detection zone, which is indicative of the size of the recombinant nucleic acids in the fluid sample, and / or b) the signal intensity of the detectable label in the detection zone, which is representative of the amount of labeled extracted recombinant nucleic acids present and indicative of the concentration of the labeled extracted recombinant nucleic acids in the fluid sample; and

[0314] a) is compared with a reference standard representing intact particles, and based on the comparison, a ratio of intact particles to partially intact particles is determined to produce a first measurement result, and / or b) is compared with a reference standard representing intact particles, and based on the comparison, a ratio of intact particles to empty particles in the fluid sample is determined to produce a first measurement result;

[0315] Thereby characterizing the particle population in the fluid sample.

[0316] Item 2.) A method according to Item 1, wherein labeling the extracted recombinant nucleic acid comprises introducing the extracted recombinant nucleic acid into the holes and / or microchannels of a microfluidic device, wherein the holes and / or microchannels comprise a polymer separation medium and a detectable nucleic acid label, whereby the detectable nucleic acid label is combined with the extracted recombinant nucleic acid to produce labeled extracted recombinant nucleic acid in the holes and / or microchannels.

[0317] Item 3.) The method according to Item 1 or 2 also includes: determining the amount of particulate protein present in the fluid sample.

[0318] Item 4.) A method according to Item 3, wherein the amount of particulate protein in the fluid sample is determined using information about the total number of particles in the fluid sample without measuring the particulate protein in the sample.

[0319] Item 5.) The method of Item 3, comprising determining the particulate protein in a second aliquot of the fluid sample.

[0320] Item 6.) The method according to any one of items 1, 2, 3 or 5, comprising:

[0321] extracting protein from a second aliquot of the fluid sample containing the particles to produce extracted protein of the particles;

[0322] labeling the extracted protein of the particle to produce labeled extracted protein of the particle;

[0323] allowing the labeled extracted proteins of the particles to flow through a polymer separation medium in the microchannel into a detection zone in fluid communication with the microchannel, the detection zone being in signal communication with a sensor capable of detecting a signal from a detectable label of the labeled extracted proteins, thereby separating the labeled extracted proteins according to size by allowing the extracted proteins to flow through the polymer separation medium of the microchannel;

[0324] detecting the detectable label of the labeled extracted protein to determine: c) the amount of time it takes the labeled extracted protein to flow through the polymer separation medium in the microchannel into the detection zone, and d) the signal intensity of the detectable label, which represents the amount of labeled extracted protein present;

[0325] comparing c) and d) to a reference standard representing a known amount of the protein to thereby determine the amount of protein in the sample and thereby generate a second assay result; and

[0326] The first assay result and the second assay result are compared to determine one or more of the following: 1) the ratio of complete virus particles to empty virus particles in the fluid sample, 2) the ratio of complete virus particles or empty virus particles to partially complete virus particles, and 3) the ratio of virus particles containing a complete recombinant genome to virus particles containing an incomplete recombinant genome, thereby characterizing the recombinant virus particle population in the fluid sample.

[0327] Item 7.) A method according to Item 6, wherein labeling the extracted proteins of the particles comprises introducing the extracted proteins of the particles into the holes and / or microchannels of a microfluidic device, wherein the holes and / or microchannels comprise a polymer separation medium and a detectable protein label, whereby the detectable protein label binds to the extracted proteins of the particles to produce labeled extracted proteins of the particles in the holes and / or microchannels.

[0328] Item 8.) A method according to any one of Items 1-7, wherein the particle is a recombinant viral particle having a single-stranded DNA or RNA genome with a length ranging from 500 to 7000 nucleotides.

[0329] Item 9.) A method according to any one of Items 1-8, wherein the particles are selected from: recombinant adeno-associated virus (AAV) particles, recombinant retrovirus particles, recombinant lentivirus particles and recombinant adenovirus particles.

[0330] Item 10.) A method according to any one of Items 1-9, wherein the particles are about 1×10 10 Recombinant virus particles / ml is about 1×10 14The amount of recombinant virus particles present in the fluid sample is 100 recombinant virus particles / ml.

[0331] Item 11.) A method according to any one of Items 1-10, wherein extracting nucleic acids from a first aliquot of a fluid sample containing particles comprises contacting the first aliquot of the fluid sample containing particles with a protease and a denaturant.

[0332] Item 12.) The method according to Item 11, wherein the protease is a serine protease.

[0333] Item 13.) The method according to Item 12, wherein the protease is selected from: proteinase K, subtilisin, trypsin, chymotrypsin, thrombin, plasmin, elastase, pronase and lactoferrin.

[0334] Item 14.) A method according to Item 11, wherein the denaturing agent is heating, a chaotropic agent, a detergent or a mixture thereof.

[0335] Item 15.) A method for characterizing a population of viral particles presumed to contain recombinant nucleic acids in a fluid sample to determine one or more of the following: 1) the ratio of intact viral particles to empty viral particles in the fluid sample, 2) the ratio of intact viral particles or empty viral particles to partially intact viral particles, comprising: extracting viral nucleic acid from a first aliquot of the fluid sample; determining the extracted viral nucleic acid by microfluidic electrophoresis; calculating the ratio of the nucleic acid concentration in the fluid sample to the nucleic acid concentration in a reference standard, calculating the ratio of the protein concentration in the fluid sample to the protein concentration in a reference standard, and calculating the fraction β of intact capsids in the sample according to the following formula:

[0336]

[0337] where N is the total number of capsids, f and e refer to complete capsids and empty capsids, respectively, and β s is the percentage of intact capsids in the reference standard, R DNA is the ratio of the concentration of viral nucleic acid in the sample to the concentration of viral nucleic acid in the reference standard, R P It is the ratio of the concentration of viral protein in the sample to the concentration of viral protein in the reference standard.

[0338] Item 16.) The method according to Item 15 comprises: extracting viral proteins from a second aliquot of the fluid sample; and determining at least one viral protein among the extracted viral proteins by microfluidic electrophoresis.

[0339] Item 17.) A method according to Item 15, wherein the protein concentration in the fluid sample is calculated by reference to a standard and no protein assay is performed.

[0340] Item 18.) A kit for characterizing the nucleic acid content of a particle population presumed to contain recombinant nucleic acid in a fluid sample, comprising: a nucleic acid marker, a gel, a nucleic acid gradient standard, a nucleic acid storage buffer, a nucleic acid sample buffer, a protease, and a denaturant.

[0341] Item 19.) The kit according to Item 18 is used to characterize the protein content of a particle population in a fluid sample, and further includes: a protein dye, a protein gradient standard, a protein storage buffer, a protein sample buffer and a washing buffer.

[0342] Item 20.) A kit according to Item 18 or 19, wherein the nucleic acid marker is a fluorescent nucleic acid intercalator.

[0343] Item 21.) A kit according to any one of Items 18-20, wherein the protease is a serine protease.

[0344] Item 22.) A kit according to any one of Items 18-21, wherein the protease is selected from: proteinase K, subtilisin, trypsin, chymotrypsin, thrombin, plasmin, elastase, pronase and lactoferrin.

[0345] Item 23.) A kit according to any one of Items 18-22, wherein the denaturing agent is a chaotropic agent, a detergent or a mixture thereof.

[0346] Item 24.) A kit according to any one of Items 18-23, wherein the protease is proteinase K and the chaotropic agent is urea.

[0347] Any patents or publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0348] The compositions and methods described herein currently represent preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Those skilled in the art will appreciate variations and other uses thereof. Such variations and other uses may be made without departing from the scope of the invention as set forth in the claims.

Claims

1. A method of characterizing a population of particles presumed to contain a recombinant nucleic acid in a fluid sample to determine one or more of: 1) the ratio of intact particles to empty particles in the fluid sample, and 2) the ratio of intact particles or empty particles to partially intact particles, comprising: extracting recombinant nucleic acid from a first aliquot of the fluid sample containing the particles to produce extracted recombinant nucleic acid; labeling the extracted recombinant nucleic acid to produce labeled extracted recombinant nucleic acid; allowing the labeled extracted recombinant nucleic acids to flow through a polymer separation medium in a microchannel into a detection zone in fluid communication with the microchannel, the detection zone being in signal communication with a sensor capable of detecting a signal from a detectable nucleic acid label of the labeled extracted recombinant nucleic acids, thereby separating the labeled extracted recombinant nucleic acids according to size by allowing the labeled extracted recombinant nucleic acids to flow through the polymer separation medium in the microchannel; detecting the detectable label of the labeled extracted recombinant nucleic acid in the detection zone to determine: a) the amount of time it takes for the labeled extracted recombinant nucleic acid to flow through the polymer separation medium in the microchannel into the detection zone, which is indicative of the size of the recombinant nucleic acid in the fluid sample, and / or b) the signal intensity of the detectable label in the detection zone, which is representative of the amount of labeled extracted recombinant nucleic acid present and indicative of the concentration of the labeled extracted recombinant nucleic acid in the fluid sample; as well as a) is compared with a reference standard representing intact particles, and based on the comparison, a ratio of intact particles to partially intact particles is determined to produce a first measurement result, and / or b) is compared with a reference standard representing intact particles, and based on the comparison, a ratio of intact particles to empty particles in the fluid sample is determined to produce a first measurement result; Thereby characterizing the particle population in the fluid sample.

2. The method of claim 1, wherein labeling the extracted recombinant nucleic acid comprises introducing the extracted recombinant nucleic acid into a hole and / or microchannel of a microfluidic device, wherein the hole and / or microchannel comprises a polymer separation medium and a detectable nucleic acid label, whereby the detectable nucleic acid label binds to the extracted recombinant nucleic acid to produce labeled extracted recombinant nucleic acid in the hole and / or microchannel.

3. The method according to claim 1 or 2, further comprising: Determine the amount of particulate protein present in the fluid sample.

4. The method of claim 3, wherein the amount of particulate protein in the fluid sample is determined using information about the total number of particles in the fluid sample without measuring the amount of particulate protein in the sample.

5. The method of claim 3, comprising measuring the amount of particulate protein in a second aliquot of the fluid sample.

6. The method according to any one of claims 1, 2, 3 or 5, comprising: extracting protein from a second aliquot of the fluid sample containing the particles to produce extracted protein of the particles; labeling the extracted protein of the particle to produce labeled extracted protein of the particle; allowing the labeled extracted proteins of the particles to flow through a polymer separation medium in the microchannel into a detection zone in fluid communication with the microchannel, the detection zone being in signal communication with a sensor capable of detecting a signal from a detectable label of the labeled extracted proteins, thereby separating the labeled extracted proteins according to size by allowing the extracted proteins to flow through the polymer separation medium of the microchannel; detecting the detectable label of the labeled extracted protein to determine: c) the amount of time it takes the labeled extracted protein to flow through the polymer separation medium in the microchannel into the detection zone, and d) the signal intensity of the detectable label, which represents the amount of labeled extracted protein present; comparing c) and d) to a reference standard representing a known amount of the protein to thereby determine the amount of protein in the sample and thereby generate a second assay result; as well as The first assay result and the second assay result are compared to determine one or more of the following: 1) the ratio of complete virus particles to empty virus particles in the fluid sample, 2) the ratio of complete virus particles or empty virus particles to partially complete virus particles, and 3) the ratio of virus particles containing a complete recombinant genome to virus particles containing an incomplete recombinant genome, thereby characterizing the recombinant virus particle population in the fluid sample.

7. The method of claim 6, wherein labeling the extracted proteins of the particles comprises introducing the extracted proteins of the particles into a well and / or microchannel of a microfluidic device, wherein the well and / or microchannel comprises a polymer separation medium and a detectable protein label, whereby the detectable protein label binds to the extracted proteins of the particles to produce labeled extracted proteins of the particles in the well and / or microchannel.

8. The method according to any one of claims 1 to 7, wherein the particle is a recombinant viral particle having a single-stranded DNA or RNA genome ranging in length from 500 to 7000 nucleotides.

9. The method according to any one of claims 1-8, wherein the particle is selected from: a recombinant adeno-associated virus (AAV) particle, a recombinant retroviral particle, a recombinant lentiviral particle and a recombinant adenoviral particle.

10. The method according to any one of claims 1 to 9, wherein the particles are about 1×10 10 Recombinant virus particles / ml is about 1×10 14 The amount of recombinant virus particles present in the fluid sample is 100 recombinant virus particles / ml.

11. The method of any one of claims 1-10, wherein extracting nucleic acids from the first aliquot of the fluid sample containing particles comprises contacting the first aliquot of the fluid sample containing particles with a protease and a denaturant.

12. The method of claim 11, wherein the protease is a serine protease.

13. The method of claim 12, wherein the protease is selected from the group consisting of proteinase K, subtilisin, trypsin, chymotrypsin, thrombin, plasmin, elastase, pronase and lactoferrin.

14. The method of claim 11, wherein the denaturing agent is heat, a chaotropic agent, a detergent, or a mixture thereof.

15. A method of characterizing a population of viral particles presumed to contain a recombinant nucleic acid in a fluid sample to determine one or more of: 1) the ratio of intact viral particles to empty viral particles in the fluid sample, 2) the ratio of intact viral particles or empty viral particles to partially intact viral particles, comprising: extracting viral nucleic acid from a first aliquot of the fluid sample; Extracted viral nucleic acid was measured by microfluidic electrophoresis assay; The ratio of the nucleic acid concentration in the fluid sample to the nucleic acid concentration in the reference standard is calculated, the ratio of the protein concentration in the fluid sample to the protein concentration in the reference standard is calculated, and the fraction β of intact capsids in the sample is calculated according to the following formula: where N is the total number of capsids, f and e refer to complete capsids and empty capsids, respectively, and β s is the percentage of intact capsids in the reference standard, R DNA is the ratio of the concentration of viral nucleic acid in the sample to the concentration of viral nucleic acid in the reference standard, R P It is the ratio of the concentration of viral protein in the sample to the concentration of viral protein in the reference standard.

16. The method according to claim 15, comprising: extracting viral proteins from a second aliquot of the fluid sample; as well as At least one of the extracted viral proteins is assayed by microfluidic electrophoresis assay.

17. The method of claim 15, wherein the protein concentration in the fluid sample is calculated by reference to a standard and no protein assay is performed.

18. A kit for characterizing the nucleic acid content of a population of particles presumed to contain a recombinant nucleic acid in a fluid sample, comprising: Nucleic acid markers, gels, nucleic acid gradient standards, nucleic acid storage buffer, nucleic acid sample buffer, proteases, and denaturants.

19. The kit of claim 18 for characterizing the protein content of a particle population in a fluid sample, further comprising: Protein dye, protein gradient standard, protein storage buffer, protein sample buffer, and wash buffer.

20. The kit according to claim 18 or 19, wherein the nucleic acid label is a fluorescent nucleic acid intercalator.

21. The kit according to any one of claims 18-20, wherein the protease is a serine protease.

22. The kit according to any one of claims 18 to 21, wherein the protease is selected from the group consisting of proteinase K, subtilisin, trypsin, chymotrypsin, thrombin, plasmin, elastase, pronase and lactoferrin.

23. The kit according to any one of claims 18 to 22, wherein the denaturing agent is a chaotropic agent, a detergent or a mixture thereof.

24. The kit according to any one of claims 18-23, wherein the protease is proteinase K and the chaotropic agent is urea.

25. The kit according to any one of claims 18-24, further comprising a nucleic acid AAV standard.

26. The kit according to any one of claims 18-25, further comprising a protein AAV standard.

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