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WO2025166267A1 - Procédés de détermination de rapports vides/complets de capsides virales intactes - Google Patents

Procédés de détermination de rapports vides/complets de capsides virales intactes

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Publication number
WO2025166267A1
WO2025166267A1 PCT/US2025/014168 US2025014168W WO2025166267A1 WO 2025166267 A1 WO2025166267 A1 WO 2025166267A1 US 2025014168 W US2025014168 W US 2025014168W WO 2025166267 A1 WO2025166267 A1 WO 2025166267A1
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WIPO (PCT)
Prior art keywords
standards
biosensor
test sample
viral
empty
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English (en)
Inventor
Tianfang GE
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Sartorius Bioanalytical Instruments Inc
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Sartorius Bioanalytical Instruments Inc
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Publication of WO2025166267A1 publication Critical patent/WO2025166267A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0205—Investigating particle size or size distribution by optical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/0606—Investigating concentration of particle suspensions by collecting particles on a support
    • G01N15/0612—Optical scan of the deposits
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7779—Measurement method of reaction-produced change in sensor interferometric
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/015—Parvoviridae, e.g. feline panleukopenia virus, human Parvovirus

Definitions

  • the present disclosure relates to methods for determining empty/full (E/F) ratios on intact viral capsids using an optical sensor.
  • Viral vectors such as adeno-associated virus (AAV) vectors
  • AAV adeno-associated virus
  • Viral vectors can be used for delivery of polynucleotides (such as DNA) encoding genes intended to treat a variety of diseases.
  • the natural byproducts of recombinant virus synthesis are capsids that have not been packaged with polynucleotides of interest, also referred to as “empty” capsids, and capsids that have been packaged with polynucleotides of interest, also referred to as “full” capsids. Empty capsids compromise AAV drug product efficacy and pose detrimental clinical effects.
  • empty capsids can increase transgene expression in subjects. Accordingly, it is important to have effective methods to differentiate full capsids from empty capsids. Despite the purification steps that have been integrated into the recombinant virus (such as recombinant AAV) production chain, empty capsids can still account for a significant portion of a batch.
  • recombinant virus such as recombinant AAV
  • capsid content ratio full vs empty capsids
  • AEX anion-exchange chromatography
  • OD optical density
  • TEM transmission electron microscopy
  • CDMS charge detection mass spectrometry
  • SEC-MALS size-exclusion chromatography multiangle light scattering
  • ELISA enzyme-linked immunosorbent assay
  • sample analysis times for these techniques can range between 15 min (OD) and 6 h (AUC and TEM) per sample, and require extensive resources, which significantly limit the number of samples that can be processed in a day and renders most approaches as low throughput.
  • these techniques are either not compatible at all or are not directly compatible (i.e., require treatments) with crude samples.
  • the disclosure herein provides embodiments of systems and methods for determining empty/full ratio on intact viral capsids using a biosensor.
  • the signals indicative of binding viral capsids to the biosensor can be measured by an interferometer based on bio-layer interferometry (BLI).
  • the empty/full viral capsid ratio in a sample can be calculated based on the binding signal intensity, binding rate, or binding kinetics of the biosensor to the viral capsids.
  • a method for determining an empty/full particle ratio in a sample includes contacting a set of standards with a biosensor, to bind particles in the set of standards to the biosensor; contacting a test sample with the biosensor, to bind particles in the test sample to the biosensor; detecting optical signals associated with binding of the particles to the biosensor for each of the set of standards and the test sample; and determining the empty/full particle ratio in the test sample by comparing the optical signal associated with binding of the particles in the test sample to the biosensor to the optical signals associated with binding of the particles in the set of standards to the biosensor.
  • the set of standards includes particles at a first concentration and different empty/full ratios in a first buffer.
  • the test sample includes particles at an unknown empty/full ratio in a second buffer.
  • the particles include viral capsids, liposomes, lipid nanoparticles, or polymeric nanoparticles.
  • the viral capsids include capsids of adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • detecting the optical signal associated with binding of the particles in the set of standards to the biosensor and the optical signal associated with binding of the particles in the test sample to the biosensor includes calculating binding rates of the particles in the set of standards to the biosensor; generating a standard curve based on the binding rates of the particles in the set of standards to the biosensor; calculating binding rates of the particles in the test sample to the biosensor; and comparing the calculated binding rates of the particles in the test sample to the standard curve.
  • the binding rates of the particles from the set of standards and the binding rates of the particles from the test sample are calculated using initial slope / rate analysis. In other embodiments, the binding rates of the particles from the set of standards and the binding rates of the particles from the test sample are calculated using end point / saturation analysis. [0010] In embodiments, generating a standard curve includes comparing the calculated binding rates of the particles in the set of standards to the empty/full particle ratio in the set of standards.
  • the test sample includes a plurality of test samples, and wherein the method includes adjusting the plurality of test samples to contain the particles at a second concentration.
  • the method further includes, prior to contacting the test sample with the biosensor, adjusting the test sample to contain the particles at the first concentration.
  • the first buffer and the second buffer are the same.
  • the set of standards is prepared by providing a sample containing an empty particle and a sample containing a full particle; adjusting the sample containing the empty particle and the sample containing the full particle to contain the particles at the first concentration; and mixing the adjusted sample containing the empty particle and the adjusted sample containing the full particle at different ratios, to generate the set of standards comprising different empty/full particle ratio.
  • the method further includes pairing each of the set of standards or the test sample with one or more reference samples containing a reference particle, wherein the one or more reference samples have a same or lower full particle ratio than the set of standards or the test sample.
  • the reference particle includes the empty particle.
  • the one or more reference samples have the same characteristics as the set of standards or the test sample that is paired with, wherein the characteristics include one or more of particle size, particle composition, particle concentration, buffer content, incubation time, biosensor regeneration cycles, and experiment day.
  • the method further includes contacting the one or more reference samples with the biosensor to bind the reference particle in the one or more reference samples to the biosensor; and calculating binding rates of the reference particle in the one or more reference samples to the biosensor.
  • the method further includes calculating attributed binding rates of the particles in the set of standards by subtracting the binding rates of the reference particle in the one or more reference samples paired with each of the set of standards from the binding rates of the particles in the set of standards; generating a standard curve based on the attributed binding rates (remaining binding rates after subtraction) of the particles in the set of standards; calculating attributed binding rates of the particles in the test sample by subtracting the binding rates of the reference particle in the one or more reference samples paired with the test sample from the binding rates of the particles in the test sample; and comparing the attributed binding rates (remaining binding rates after subtraction) of the particles in the test sample to the standard curve, thereby determining the empty/full particle ratio in the test sample.
  • test sample are crude samples comprising viral capsids.
  • the optical signal includes light reflected from two different locations associated with the biosensor.
  • the optical signal includes light interference generated by biolayer interferometry (BLI)
  • the method includes providing the set of standards and the test sample in wells in a multi-well plate. [0022] In some embodiments, the method includes contacting the set of standards and the test sample sequentially. In other embodiments, the method includes contacting the set of standards and the test sample simultaneously.
  • a method for determining an empty/full viral capsid ratio includes obtaining optical signals of a set of standards, the set of standards comprising viral capsids at a first viral titer and different empty/full ratios in a first buffer; obtaining a first reference optical signal, the first reference optical signal comprising an optical signal of a first reference sample comprising viral capsids at the first viral titer and at an empty empty/full ratio in the first buffer; calculating attributed binding rates of the particles in the set of standards by subtracting the first reference optical signal from the optical signals of each of the set of standards; obtaining an optical signal of a test sample, the test sample comprising viral capsids at a second viral titer and an unknown empty/full ratio in a second buffer; obtaining a second reference optical signal, the second reference optical signal comprising an optical signal of a second reference sample comprising viral capsids at the second viral titer and at an empty empty/full
  • the optical signals of the set of standards is obtained based on a binding rates of the viral capsids in the set of standards to the biosensor.
  • the optical signal of the first reference sample is obtained based on a binding rates of the viral capsids in the first reference sample to the biosensor.
  • the optical signal of the test sample is obtained based on a binding rates of the viral capsids in the test sample to the biosensor.
  • the optical signal of the second reference sample is obtained based on a binding rates of the viral capsids in the second reference sample to the biosensor.
  • a method for determining an empty/full viral capsid ratio includes pairing a set of standards or a test sample with a reference sample, the reference sample having a full viral capsid ratio (e g., % full) that is less than or equal to the full viral capsid ratio of the set of standards or to the test sample; contacting a set of standards and its paired reference sample with a biosensor, to bind viral capsids in the set of standards or its paired reference sample to the biosensor, the set of standards comprising viral capsids at the same viral titer and different empty/full ratios; calculating binding rates of viral capsids in the set of standards to the biosensor; calculating binding rates of the viral capsid in the reference sample to the biosensor; subtracting the calculated binding rates of the reference sample from the calculated binding rates of the viral capsids in the set of standards; generating a standard curve by comparing the reference-subtracted binding rates of particles in the set of standards
  • each of the set of standards or the test sample has the same characteristics as the reference sample containing the reference viral capsid, wherein the characteristics include one or more of viral titer, buffer content, incubation time, biosensor regeneration cycles, and experiment day.
  • the binding rates of the viral capsids in the set of standards and the binding rates of the viral capsids in the test sample are calculated using initial slope / rate analysis. [0030] In embodiments, the binding rates of the viral capsids in the set of standards and the binding rates of the viral capsids in the test sample are calculated using end point / saturation analysis.
  • test reference sample is the same as the reference sample.
  • a system for determining an empty/full viral capsid ratio includes a biosensor, configured to bind viral capsids in a set of standards or in a test sample, to detect an optical signal associated with viral capsids bound to the biosensor, and to determine a empty/full viral capsid ratio in the test sample based on the optical signal.
  • the set of standards includes viral capsids at the same viral titer and different empty/full ratios.
  • the test sample includes viral capsids at an unknown empty/full ratio.
  • the optical signal includes light reflected from two different locations associated with the biosensor.
  • the biosensor is a biolayer interferometry (BLI) biosensor.
  • BBI biolayer interferometry
  • the system further includes a container configured to house the set of standards or the test sample.
  • the container is a well in a multi-well plate.
  • the biosensor is further configured to bind a reference viral capsid in a reference sample paired with each of the set of standards or the test sample, to generate a standard curve based on signals obtained by subtracting calculated binding rates of the reference viral capsid from calculated binding rates of the viral capsids in the set of standards, and to determine an empty/full viral capsid ratio in the test sample based on the signals obtained by subtracting calculated binding rates of the reference viral capsid from calculated binding rates of the viral capsids in the test sample and the standard curve.
  • the present disclosure provides a system for determining an empty/full particle ratio in a sample.
  • the system includes a biosensor, configured to bind particles in the sample and to detect an optical signal associated with the particles bound to the biosensor; and a processor, configured to perform the method of determining an empty/full ratio of particles or viral capsids provided herein.
  • FIG. 1 A-C are graphical representations indicative of the binding rate of empty and full AAV capsids of identical titer using a biosensor.
  • FIG. 1A displays data for AAV8 capsids.
  • FIGs. IB displays data for AAV9 capsids.
  • FIG. 1C displays data for AAV2 capsids.
  • FIG. 2 is a diagrammatic representation of an example method for determining empty/full ratios on intact viral capsids in a sample using initial rate analysis.
  • FIG. 3 is a diagrammatic representation of an example method for determining empty/full ratios on intact viral capsids in a sample using saturation analysis.
  • FIG. 4 is a graphical representation of interferometry signal change (nm shift) overtime showing higher binding rate of full AAV8 capsids to an Octet AAVX biosensor as compared with empty AAV8 capsids, wherein the viral capsid titer is identical.
  • FIG. 5 is a diagrammatic representation of one-step sampling with a BLI biosensor of empty/full ratio on samples viral capsids having low percentage full and high percentage full and identical titer.
  • FIG. 6 is a graphical representation of interferometry signal change (nm shift) overtime showing higher binding rate of full AAV8 capsids at saturation as compared with empty AAV8 capsids from a wide range of titer.
  • FIG. 7 is a diagrammatic representation of one-step sampling with a BLI biosensor of empty/full ratio on samples having capsids of varying viral titer.
  • FIG. 8 is a graphical representation of interferometry signal change (nm shift) overtime that is indicative of an AAVX biosensor used to determine empty/full ratio of viral capsids at an identical titer.
  • FIG. 9 is a graphical representation of binding rates of viral capsids having different full percentage and an identical titer, using initial rate analysis.
  • FIG. 10 is a graphical representation of interferometry signal change (nm shift) over time that is indicative of an AAVX biosensor used to determine empty/full ratio at viral capsids at variable titer.
  • FIG. 11 is a graphical representation of binding rates of viral capsids having different full percentage and varying titer as compared with a standard curve generated from viral capsids having different full percentage and an identical titer, both using saturation analysis.
  • FIG. 12 is a diagrammatic representation of an example empty/full ratio assay design on an Octet AAVX biosensor.
  • FIG. 13 A is a graphical representation of interferometry signal change (nm shift) over time after paired reference subtraction that is indicative of an AAVX biosensor used to determine empty/full ratio of the same sample at different titer.
  • FIG. 13B is a graphical representation of net BLI signal (nm) difference between a full AAV sample and its paired empty capsid reference at various titer.
  • FIG. 14A is a graphical representation of interferometry signal change (nm shift) over time after reference subtraction that is indicative of an AAVX biosensor used to determine empty/full ratio for samples in Octet Sample Diluent buffer (standards).
  • FIG. 14B is a graphical representation of interferometry signal change (nm shift) over time after reference subtraction that is indicative of an AAVX biosensor used to determine empty/full ratio for the same samples used in FIG. 14A in crude cell lysate.
  • FIG. 15A is a graphical representation of interferometry signal change (nm shift) over time after reference subtraction that is indicative of an AAVX biosensor used to determine empty/full ratio for samples diluted and analyzed on Day 1.
  • FIG. 15B is a graphical representation of interferometry signal change (nm shift) over time after reference subtraction that is indicative of an AAVX biosensor used to determine empty/full ratio for the same samples used in FIG. 15 A, but freshly diluted and analyzed on Day 3.
  • FIG. 16A is a graphical representation of interferometry signal change (nm shift) over time after reference subtraction that is indicative of a new AAVX biosensor used to determine empty/full ratio for samples.
  • FIG. 16B is a graphical representation of interferometry signal change (nm shift) over time after reference subtraction that is indicative of a three-times regenerated AAVX biosensor used to determine empty/full ratio for the same samples used in FIG. 16A.
  • FIG. 17 is a graphical representation of signals before and after reference subtraction in samples containing full viral capsids, mixed full and empty viral capsids, and empty viral capsids according to the embodiments of the present disclosure.
  • FIG. 18 is a graphical representation of pairing a set of standards or test samples with a reference sample containing the same viral titer, buffer, and assay lot according to the embodiments of the present disclosure.
  • FIG. 19 is a graphical representation of reference subtraction and signals obtained from reference subtraction according to the embodiments of the present disclosure.
  • FIG. 20 is a flowchart of the method of determining the empty/full particle ratio in samples according to the embodiments of the present disclosure.
  • FIG. 21 is a flowchart of the method of detecting an optical signal associated with binding of particles in the set of standards or the test samples to the biosensor according to the embodiments of the present disclosure.
  • FIG. 22 is a flowchart of the method of reference subtraction to detect an optical signal associated with binding of particles in the set of standards or the test samples according to the embodiments of the present disclosure.
  • FIG. 23 is a schematic representation of a system for determining the empty/full particle ratio in samples according to the embodiments of the present disclosure.
  • a “vector,” as used herein, refers to a recombinant plasmid or virus that comprises a nucleic acid to be delivered into a host cell, either in vitro or in vivo.
  • Viral titers can be expressed in a number of ways, from which one skilled in the art can select a way to express viral titers suitable in the context.
  • capsid particles (cp) as used in reference to a viral titer refers to the number of viral capsids, regardless of infectivity or functionality.
  • viral genomes (vg),” “genome particles,” “genome equivalents,” or “genome copies” as used in reference to a viral titer refer to the number of virions containing the recombinant viral DNA genome or RNA genome, regardless of infectivity or functionality.
  • the number of capsid particles or genome particles in a particular vector preparation can be measured by standard methods such as using a fluorescent dye or electron microscopy.
  • isolated or “isolating” a molecule refers to identifying and separating and/or recovering the molecule from a component of its natural environment.
  • isolated (or separated or purified) viral particles may be prepared using a purification technique to enrich them from a source mixture, such as a culture lysate or production culture supernatant.
  • isolated (or separated or purified) viral polynucleotides may be prepared using a purification technique to separate them from a source mixture that contains viral capsids.
  • load or “loaded” in the context of loading a biopolymer to a structure to assemble a biosensor refers to the process of bringing the equilibrated sample (such as biopolymer) into contact with the equilibrated solid phase (such as to assemble a biosensor). Loading can be done for example with chromatography devices by causing the sample to pass through the device by means of an external force, such as by gravity or by pumping, or by dipping into a well plate that contains the sample.
  • the term “BLI biosensor” refers to a sensing and/or analytical device that detects the presence, physical characteristics, or amount of substances using a biological molecule (such as an enzyme or an antibody) or a living organism suitable for BLI detection.
  • the biosensor has a distal end and a proximal end.
  • the proximal end has a surface coated with a thin layer of analyte-binding molecules.
  • proximal refers to the portion of the device or component thereof that is closer to the user or machine using the device and the term “distal” refers to the portion of the device or component thereof that is farther from the user or machine using the device.
  • distal refers to the portion of the device or component thereof that is farther from the user or machine using the device.
  • an “analyte-binding” molecule refers to any molecule capable of participating in a specific binding reaction with an analyte molecule. Examples include, but are not limited to, antibody-antigen binding reactions and nucleic acid hybridization reactions.
  • an “antibody” refers to an immunoglobulin molecule having two heavy chains and two light chains prepared by any method known in the art or later developed and includes polyclonal antibodies such as those produced by inoculating a mammal such as a goat, mouse, rabbit, etc. with an immunogen, as well as monoclonal antibodies produced using the well-known Kohler Milstein hybridoma fusion technique.
  • the term includes antibodies produced using genetic engineering methods such as those employing, e.g., SCID mice reconstituted with human immunoglobulin genes, as well as antibodies that have been humanized using art-known resurfacing techniques.
  • An antibody also refers to an antibody fragment.
  • an “antibody fragment” refers to a fragment of an antibody molecule produced by chemical cleavage or genetic engineering techniques, as well as to single chain variable fragments (SCFvs) such as those produced using combinatorial genetic libraries and phage display technologies. Antibody fragments used in accordance with the present disclosure usually retain the ability to bind their cognate antigen and so include variable sequences and antigen combining sites, which are within the scope of antibodies.
  • a “recombinant” virus or “recombinant” viral vector as used herein refers to a virus or polynucleotide vector comprising one or more heterologous sequences (in other words, nucleic acid sequence not of viral origin).
  • a “binding rate” as used herein refers to a computational output indicative of the quality and nature of biomolecules or complexes thereof binding to a biosensor.
  • a “viral capsid” as used herein includes either a protein shell of viral protein, or a lipid bilayer coat/ envelop, that encloses viral nucleic acid or recombinant viral vector.
  • a “full” viral capsid as used herein refers to a viral capsid that contains polynucleotides.
  • An “empty” viral capsid as used herein can be a viral capsid that does not contain polynucleotides, or that is essentially free of polynucleotides. “Essentially free” as used herein in reference to a particular component (such as polynucleotides) means that the component (such as polynucleotides) present constitutes less than about 5.0% by weight, such as less than about 4.0% by weight, less than about 3.0% by weight, less than about 2.0% by weight, less than about 1.0% by weight, less than about 0.5% by weight or less than about 0.1% by weight of the composition (such as a viral capsid). An empty capsid or empty viral capsid may or may not contain polynucleotides.
  • a “full” particle as used herein refers to a particle that contains cargo.
  • a “full particle”, such as a full viral particle or a full nanoparticle contains a higher content of cargo relative to an “empty particle”, such as empty viral particle or an empty nanoparticle.
  • An empty particle may or may not contain cargo.
  • An “empty” particle as used herein can be a particle that does not contain cargo, or that is essentially free of polynucleotides. Any cargo can be used in the methods and systems provided herein, including a small molecule (e.g., a small molecule drug), a nucleic acid or polynucleotide (e.g., mRNA, DNA, siRNA.
  • the cargo is a therapeutic molecule.
  • the present disclosure provides methods for determining empty/full ratios on intact viral capsids in a sample using a biosensor, such as a BLI biosensor.
  • the methods are based on the understanding that full viral capsids have a higher density than empty viral capsids.
  • the binding rates of the viral capsids are calculated by measuring the wavelength shift due to light interference.
  • the first samples are reference samples where the percentage full of the viral capsids is known.
  • the second samples are unknown samples that can be either crude or purified samples.
  • the binding rates are calculated using either initial slope / rate analysis or using end point/ saturation analysis.
  • the biosensor is a BLI biosensor, such as an AAVX affinity biosensor.
  • full AAV capsids result in a higher binding rate to a BLI biosensors than empty capsids of identical viral titer.
  • full capsids generate a higher binding rate than empty capsids at saturation regardless of titer.
  • the set of first samples is normalized to the same capsid titer as the second samples.
  • the set of standards from the set of first samples is prepared by mixing the first viral capsids with the second viral capsids at a plurality of different ratios.
  • the method further comprises pairing each of the one or more second samples with one or more third viral capsids, wherein each of the third viral capsids has a lower polynucleotide content than each of the second viral capsids.
  • the third viral capsids are diluted from the same viral stock as the first viral capsids in the set of first samples.
  • the method further comprises subtracting the calculated binding rates of the first viral capsids from the calculated binding rates of the viral capsids from the set of standards; and subtracting the calculated binding rates of the third viral capsids from the calculated binding rates of the viral capsids from the second samples.
  • the methods disclosed herein involve providing at least two references samples, wherein at least one reference sample is full and at least one reference sample is empty.
  • the percentage full and the absolute titer of the reference samples are either already known or are determined using known techniques, such as analytical ultracentrifugation and ELISA.
  • Non-limiting examples of viral capsids that can be evaluated using the methods and systems disclosed herein include viral capsids from retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex viruses.
  • the one or more viral capsids comprise capsids of adeno-associated virus (AAV) including recombinant AAV.
  • AAV adeno-associated virus
  • AAV is a single-stranded DNA (ssDNA) nonenveloped virus that belongs to the parvovirus family and measures 25 nm in diameter.
  • serotypes of AAV AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11
  • the virus is only composed of protein and DNA and has three repeating capsid proteins, VP1, VP2, and VP3, at an expected ratio of 1 : 1 : 10, which may vary across serotypes and even within the particles of a given batch. It is capable of infecting both dividing and nondividing cells depending on the tropism of the given serotype.
  • the AAV provided herein is AAV serotype
  • AAV2 AAV2
  • AAV8 AAV serotype 8
  • AAV9 AAV9
  • the BLI biosensors disclosed herein include core components that can comprise a silicon substrate, activated with silane group, such as aminopropylsilane (APS) and epoxypropylsilane (EPS).
  • silane group such as aminopropylsilane (APS) and epoxypropylsilane (EPS).
  • APS aminopropylsilane
  • EPS epoxypropylsilane
  • the BLI biosensors are designed for specific virus binding with negligible non-specific bindings from interfering substances in samples.
  • a non-limiting example of a BLI biosensor that can be used in the methods disclosed herein is an Octet® AAVX affinity biosensor (Sartorius Lab Instruments GmbH & Co. KG).
  • the Octet® AAVX biosensor is coated with CaptureSelectTM Biotin Anti-AAVX Conjugate (Thermo Fisher Scientific Inc.).
  • CaptureSelectTM Biotin Anti-AAVX Conjugate Thermo Fisher Scientific Inc.
  • a method for determining empty/full ratio on viral capsids in a sample comprises an initial rate analysis.
  • the method can include the steps of providing a plurality of first samples, the first samples comprising one or more viral capsids that contain polynucleotides and one or more viral capsids that do not contain polynucleotides; providing one or more second samples, the second samples comprising one or more viral capsids; normalizing both the first and second samples to the same capsid titer; preparing a set of standards from the plurality of first samples; contacting each of the first samples and the second samples with a biosensor; calculating binding rates of the viral capsids from the set of standards and the binding rates of the viral capsids from the second samples; generating a standard curve from the set of standards; and determining empty/full ratio of the viral capsids in the second samples by comparing binding rate against the standard curve.
  • determining the absolute viral titer of the unknown samples is done using known techniques, such as ELISA.
  • the unknown samples may either be crude or purified.
  • all of the samples may be normalized to a viral titer of greater than about 1E11 cp/ml.
  • normalizing the reference samples comprises mixing different ratios of reference samples (empty and full) at the same capsid titer and diluting unknown samples to the same titer as the reference samples.
  • empty reference samples are diluted to the same titer as unknown samples using the same assay buffer for reference subtraction (as described below).
  • the BLI biosensor e.g., AAVX affinity biosensor
  • the BLI biosensor is dipped into microplate wells containing reference samples and unknown samples of viral capsids (e.g., AAV capsids) for a set time to determine the binding rate of AAV vector to AAVX antibody on the BLI biosensor.
  • the set time may be between about 10 to 30 minutes.
  • the binding rate of AAV vector can be determined using initial slope analysis.
  • the binding rate for the reference samples may be fitted using an initial slope binding rate equation on a computing processor.
  • a standard curve may then be generated using a fitting model on a computing processor.
  • BLI is used to compare signals from the unknown samples to a standard curve to determine a empty/full viral capsid ratio in these samples.
  • BLI is an optical technique for measuring macromolecular interactions by analyzing interference patterns of white light reflected from the surface of a BLI biosensor tip. The shift in interferometry signal (“nm shift”) over time indicates binding of a material (such as viral capsid) to the BLI biosensor.
  • AAVX biosensor BLI signal than empty AAV capsids on an AAVX biosensor. This is partially due to the fact that full capsids have a higher density than empty capsids.
  • the differential BLI signal from empty/full AAV capsids allows for the determination of empty/full ratio on the AAV capsids.
  • full or empty AAV8 capsids were first normalized by a known titering method (e.g., ELISA), before being compared on an AAVX biosensor for binding rate.
  • full AAV capsids cause more spectral shift and show higher binding rate than empty capsids of identical titer at any given time.
  • an AAVX biosensor binds to intact AAV8 empty and full capsids having a titer of about 8.0E11 cp/ml.
  • initial rate analysis allows one- step sampling with a BLI biosensor of empty/full ratio when the samples are of identical titer.
  • FIG. 8 is a graphical representation of interferometry signal change (nm shift) overtime, wherein AAV8 capsids having different percentages full are plotted after subtracting reference sample (empty AAV8).
  • FIG. 9 is a graphical representation of a standard curve comprised of binding rates of AAV capsids having different percentages full and an identical titer.
  • FIG. 2 shows a diagrammatic representation of an example method for determining empty/full ratio on intact viral capsids in a sample using initial rate analysis.
  • the method commences with AAV capsid titer determination for unknown samples.
  • standards are prepared by mixing different ratios of reference samples (empty vs. full) at the same capsid titer. The preparation of standards is done once.
  • the unknown samples are diluted to the same titer as the standards.
  • the third step in this method involves determination of empty/full ratio in unknown samples. This step involves generating a standard curve using initial slope analysis and comparing unknown samples to the standard curve to obtain the empty/full ratio.
  • the titer of a set of standards does not need to match the titer of a set of unknown samples.
  • constant titer should be maintained by each of the standards within the set of standards and by each of the unknown samples within the set of unknown samples.
  • each of the standards and each of the unknown samples includes an empty reference for performing reference subtraction.
  • a single standard curve may be used to calibrate multiple batches of samples having different titers, buffers, and experimental dates.
  • a method for determining empty/full ratio on viral capsids in a sample comprises a saturation analysis.
  • the method can include the steps of normalizing the first samples to the same capsid titer; preparing a set of standards from the plurality of first samples; contacting each of the first samples and the second samples with a biosensor; calculating binding rates of the viral capsids from the set of standards and the binding rates of the viral capsids from the second samples; generating a standard curve from the set of standards; and determining empty/full ratio of the viral capsids in the second samples by comparing binding rate against the standard curve.
  • the normalized absolute viral titer of the full and empty reference samples may be greater than about 5E11 cp/ml.
  • a standard series is prepared by mixing different ratios of reference samples (empty vs. full) at the same capsid titer.
  • a rough titer may be determined for unknown samples using a BLI biosensor (e g., an AAVX biosensor). The unknown samples may be either crude or purified. In other embodiments, a rough titer may simply be estimated, without the need for determination using a BLI biosensor.
  • the BLI biosensor e g., AAVX affinity biosensor
  • the BLI biosensor is dipped into microplate wells containing reference samples and unknown samples of viral capsids (e.g., AAV capsids) for a set time to determine the binding rate of AAV capsids to AAVX antibody on the BLI biosensor.
  • the set time may be about 60 minutes.
  • the binding rate of AAV capsids can be determined using end point analysis.
  • BLI is used to compare signals from the unknown samples to a standard curve to determine a empty/full viral capsid ratio in these samples.
  • the shift in interferometry signal (“nm shift”) over time indicates binding of a material (such as viral capsid) to the BLI biosensor.
  • full AAV capsids cause more spectral shift and generate a higher BLI signal than empty AAV capsids on an AAVX biosensor, regardless of titer.
  • an AAVX biosensor binds to intact AAV8 empty and full capsids having a viral titer ranging between about 2.5E11 and 4.0E12 cp/ml. Saturation analysis allows one-step sampling of empty/full ratio from a wide range of sample titer. As a result of the one-experiment, one-step sampling approach shown in FIG. 7, there is no compound error.
  • an Octet BLI biosensor is dipped into an AAV sample for reporting a BLI signal.
  • the BLI signal is indicative of the nature of captured viral particles, such as size, abundance, and density.
  • AAV binding is allowed to reach saturation, at which point the abundance of bound AAV is equal between samples and the net signal difference can be solely attributed to density difference between a given sample and the empty reference.
  • the empty/full ratio difference can be determined.
  • FIG. 10 is a graphical representation of interferometry signal change (nm shift) over time that is indicative of an AAVX biosensor used to determine empty / full ratio when AAV capsids reach saturation at variable titer.
  • FIG. 11 is a graphical representation of binding rates of AAV capsids having different full percentage and varying titer as compared with a standard curve generated from viral capsids having different full percentage and an identical titer.
  • FIG. 3 shows a diagrammatic representation of another example method for determining empty/full ratio on intact viral capsids in a sample using saturation analysis.
  • the method commences with rough AAV capsid titer estimation for unknown samples.
  • standards are prepared by mixing different ratios of reference samples (empty vs. full) at the same saturating titer.
  • the standards are prepared once.
  • the third step in this method involves determination of empty/full ratio in unknown samples. This step involves generating a standard curve using end point analysis and comparing unknown samples to the standard curve to obtain the empty/full ratio.
  • methods for determining empty/full ratio on intact viral capsids involves performing a reference subtraction step after calculating binding rates of viral capsids from a set of standards (including one or more empty reference samples) and calculating binding rates of viral capsids from a set of unknown samples (including one or more empty reference samples).
  • the binding rate(s) of the empty reference sample(s) corresponding to the set of standards is subtracted from the binding rates of the set of standards.
  • the binding rate(s) of the empty reference sample(s) corresponding to the unknown samples is subtracted from the binding rates of the unknown samples.
  • the reference subtraction steps may be performed manually or automatically using a computing processor.
  • each of the set of standards or the test sample can be paired with a reference sample containing a reference particle.
  • the reference sample has a lower ratio of full particles (e.g., full viral capsids) than each of the set of standards or the test sample, and thus lower signals.
  • the reference particle can be the first particle (e.g., empty particle) that is used to prepare a set of standards.
  • Each of the set of standards or the test sample can have the same characteristics as the reference sample, for example the same viral titer, buffer content, incubation time, biosensor regeneration cycles (e.g., biosensor lot), and/or experiment day on which the assay is performed (e.g., binding rates are obtained).
  • the method of reference subtraction can include contacting the reference sample with the biosensor to bind the reference particle in the reference sample to the biosensor; and calculating binding rates of the reference particle in the reference sample to the biosensor.
  • the method of reference subtraction can further include subtracting the calculated binding rates of the reference particle from the calculated binding rates of the particles in the set of standards; generating a standard curve based on the signals obtained by subtracting the calculated binding rates of the reference particle from the calculated binding rates of the particles in the set of standards; subtracting the calculated binding rates of the reference particle from the calculated binding rates of the particles in the test sample; and comparing the signals obtained by subtracting the calculated binding rates of the reference particle from the calculated binding rates of the particles in the test sample to the standard curve, thereby determining an empty/full particle ratio in the test sample.
  • Reference subtraction / paired reference subtraction may be implemented by ensuring that enough empty reference wells are sequestered to pair with each sample condition and each sample read step. It is desirable that an empty AAV reference used for subtraction (subtrahend) matches the sample for which it is to be subtracted from (minuend) as closely as possible in both composition (e.g., titer, matrix, and volume) and detection conditions (e.g., plate, sensor lot, and instrument time). The empty AAV reference may be analyzed in near real-time alongside the samples for which it is to be subtracted from.
  • composition e.g., titer, matrix, and volume
  • detection conditions e.g., plate, sensor lot, and instrument time
  • the method includes obtaining optical signals of a set of standards; obtaining a first reference optical signal; calculating attributed binding rates of the particles in the set of standards by subtracting the first reference optical signal from the optical signals of each of the set of standards; obtaining an optical signal of a test sample; obtaining a second reference optical signal; calculating attributed binding rates of the particles in the test sample by subtracting the optical signal of the reference particle from the binding rates of the particles in the test sample; and determining the empty /full viral capsid ratio of the test sample by comparing the attributed binding rates of the particles (remaining optical signal after subtraction) in the test sample to the attributed binding rates of the particles (remaining optical signal after subtraction) in the set of standards.
  • the set of standards can include viral capsids at a first viral titer and different empty/full ratios in a first buffer.
  • the test sample can include viral capsids at a second viral titer and an unknown empty/full ratio in a second buffer.
  • the first reference optical signal can include an optical signal of a first reference sample comprising viral capsids at the first viral titer and at an empty empty/full ratio in the first buffer.
  • the second reference optical signal can include an optical signal of a second reference sample comprising viral capsids at the second viral titer and at an empty empty/full ratio in the second buffer.
  • the optical signals of the set of standards is obtained based on a binding rates of the viral capsids in the set of standards to the biosensor.
  • the optical signal of the first reference sample is obtained based on a binding rates of the viral capsids in the first reference sample to the biosensor.
  • the optical signal of the test sample is obtained based on a binding rates of the viral capsids in the test sample to the biosensor.
  • the optical signal of the second reference sample is obtained based on a binding rates of the viral capsids in the second reference sample to the biosensor.
  • the optical signal includes light interference generated by biolayer interferometry (BLI).
  • BLI biolayer interferometry
  • the binding rates of the viral capsids to the biosensor is measured using initial slope / rate analysis. In other embodiments, the binding rates of the viral capsids to the biosensor is measured using end point / saturation analysis.
  • Some of the benefits associated with paired reference subtraction, as described herein, include normalizing various variables, such as sample titer, buffer matrix, incubation time, biosensor regeneration cycles, and experiment day; enhancing assay reproducibility, design flexibility and matrix compatibility; allowing a single standard curve to be used for different types of samples on different days; allowing biosensor to be regenerated/reused for saturation analysis; and allowing for fair comparisons to be made for sampled measured in different experiments.
  • an empty reference sample diluted in sample diluent to titer A can serve as reference to subtract the standards.
  • the same empty reference sample diluted in cell lysis buffer to titer B could serve as reference to subtract the unknown samples.
  • a similar BLI signal (nm shift) is generated at saturation for the same samples at different capsid titer after paired reference subtraction.
  • an AAVX biosensor binds to intact AAV8 capsids having a viral titer ranging between about 2.5E11 and 4.0E12 cp/ml.
  • samples can be successfully normalized in different titer using paired reference subtraction.
  • FIGs. 14A and 14B show the values of the percentage of full capsids after paired reference subtraction.
  • an AAVX biosensor binds to intact AAV8 empty and full capsids.
  • FIG. 14A shows the pre-determined percentage of full capsids from samples in Octet Sample Diluent Buffer, which is used to construct the standard curve
  • FIG. 14B shows the percentage of full capsids experimentally determined from the same samples in crude HEK293 cell lysate, which is comparable to the standards.
  • samples can be successfully normalized in different buffer matrices using paired reference subtraction.
  • FIGs. 15A and 15B show the values of the percentage of full capsids after paired reference subtraction.
  • an AAVX biosensor binds to intact AAV5 capsids.
  • FIG. 15A shows the pre-determined percentage of full capsids from samples diluted and analyzed on Day 1, which is used to construct the standard curve
  • FIG. 15B shows the percentage of full capsids experimentally determined from samples freshly diluted and analyzed on Day 3, which is comparable to the standards.
  • samples analyzed in independent experiments can be successfully normalized using paired reference subtraction.
  • AAVX biosensors may be regenerated and reused for empty /full ratio measurements to provide an efficient and cost-effective solution for high-throughput applications.
  • FIGs. 16A and 16B the values of the percentage of full capsids after paired reference subtraction are similar for the same samples analyzed with biosensors of different regeneration cycles.
  • an AAVX biosensor binds to intact AAV5 capsids.
  • FIG. 16A shows the pre-determined percentage of full capsids from samples analyzed with new AAVX biosensors, which is used to construct the standard curve, while FIG.
  • 16B shows the percentage of full capsids experimentally determined from samples analyzed with three times-regenerated biosensors, which is comparable to the standards.
  • the signal in the sample containing empty particles e.g., empty viral capsids
  • the reference signal can be amplified for sensitive detection of the ratio of full and empty particles.
  • raw sensogram provides signals of 8.7 nm, 8.0 nm, and 6.5 nm for samples having full particles, mixed full and empty particles, and empty particles, respectively. After reference subtraction, the signals become 2.2 nm, 1.5 nm, and 0 nm for samples having full particles, mixed full and empty particles, and empty particles, respectively.
  • the reference sample may have the same particle concentration (e.g., viral capsid titer), diluent/medium, and other assay conditions (e.g., the lot of the biosensor, day of the assay on which the binding rates were obtained).
  • particle concentration e.g., viral capsid titer
  • diluent/medium e.g., the lot of the biosensor, day of the assay on which the binding rates were obtained.
  • the set of standards and test samples can have different particle concentration, although the particle concentration is adjusted to be the same within the set of standards and among test samples.
  • the set of standards can be paired with a reference sample containing the same particle concentration as the set of standards.
  • the test samples can be paired with a reference sample containing the same particle concentration as the test samples.
  • the particle concentration in the reference sample paired with the set of standards can be different from the particle concentration in the reference sample paired with the test samples.
  • FIG. 20 provides an example method for determining an empty/full particle ratio in a sample.
  • Process 2000 may begin from step 2008, to contact the set of standards with a biosensor, to bind particles in the set of standards to the biosensor.
  • Process 2000 continues to step 2010, to contact the test sample with the biosensor, to bind particles in the test sample to the biosensor.
  • Process 2000 continues to step 2012, to detect an optical signal associated with binding of the particles to the biosensor.
  • Process 2000 continues to step 2014, to determine an empty/full particle ratio in the test sample based on the optical signal.
  • Process 2000 may also include optional steps 2002, 2004, an 2006 prior to step 2008.
  • Process 2000 may begin with optional step 2002, to provide a set of standards that includes particles at the same particle concentration and different ratios of empty and full particles.
  • Process 2000 continues to step 2004, to provide test sample that include particles at an unknown empty/full particle ratio.
  • Process 2000 continues to step 2006, to adjust the test sample to contain the same particle concentration as one another. Step 2006 continues to step 2008.
  • the viral particles in the sample have been purified using a purification step, such as those provided herein. In some embodiments, the viral particles in the sample have not been purified.
  • the full and empty viral capsids in any given sample may belong to any serotype, including natural and recombinant serotypes, including chimeric (mixed) and novel serotypes. It will be understood that capsids of different serotypes have different charge properties, including different levels of charge distinction between empty viral capsids and full capsids, or among different recombinant constructs within a serotype.
  • the viral particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAVS capsid, an AAV6 capsid, an AAV7 capsid, an AAVS capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrhlO capsid, an AAV11 capsid, an AAV12 capsid, an AAV13 capsid, an AAV14 capsid, an AAV15 capsid, an AAV16 capsid, an AA Vrh20 capsid, an AAV.rh39 capsid, an AAV.Rh74 capsid, an AAV.RHM4-1 capsid, an AAV.hu37 capsid, an AAV.Anc80 capsid, an AAV.Anc80L65 capsid, an AAV.PHP.B capsid, an AAV
  • the AAV genome is 2500 bases to 5500 bases, 3000 bases and 5500 bases, 3500 bases and 5500 bases, 4000 bases and 5500 bases, 4500 bases and 5500 bases, 5000 bases and 5500 bases, 2500 bases and 5000 bases, 3000 bases and 5000 bases, 3500 bases and 5000 bases, 4000 bases and 5000 bases, 4500 bases and 5000 bases, 2500 bases and 4500 bases, 3000 bases and 4500 bases, 3500 bases and 4500 bases, 4000 bases and 4500, 2500 bases and 4000 bases, 3000 bases and 4000 bases, 3500 bases and 4000 bases, 2500 bases and 3500 bases, 3000 bases and 3500 bases, or 2500 bases and 3000 bases.
  • the recombinant viral particles comprise a self-complementary AAV (scAAV) genome.
  • the methods disclosed herein can be used to determine empty/full ratios on various gene therapy delivery vectors.
  • the vectors can be viral or non-viral.
  • the viral vectors may comprise Adeno-associated (AAV) virus, Adenovirus, Lentivirus, Herpes simplex virus, and Retrovirus.
  • AAV Adeno-associated virus
  • the non-viral vectors can be lipid-based (e g., liposome, solid lipid nanoparticle (LNP)) and/or polymeric-based.
  • Non-viral vectors may have their surface coated with various types of polymers.
  • FIG. 21 provides an example method for detecting the optical signal associated with binding of the particles in the set of standards or test samples to the biosensor, a step depicted in FIG. 20 as step 2012.
  • Process 2100 begins at step 2012, to calculate binding rates of the particles in the set of standards to the biosensor
  • Process 2100 continues to step 2104, to generate a standard curve based on the binding rates of the particles in the set of standards to the biosensor.
  • Process 2100 continues to step 2106, to calculate binding rates of the particles in the test sample to the biosensor.
  • Process 2100 continues to step 2108, to compare the calculated binding rates of the particles in the test sample to the standard curve.
  • the binding rates of the particles from the set of standards and the binding rates of the particles from the test sample are calculated using initial slope / rate analysis. In other embodiments, the binding rates of the particles from the set of standards and the binding rates of the particles from the test sample are calculated using end point / saturation analysis.
  • Generating a standard curve can include comparing the calculated binding rates of the first particles in the set of standards to the empty/full particle ratio in the set of standards.
  • the method provided herein can further include, prior to contacting the test sample with the biosensor, adjusting the test sample to contain the same particle concentration as the set of standards.
  • the set of standards can be prepared by providing a sample containing a first particle and a sample containing a second particle, wherein the first particle has a lower cargo content than the second particle; adjusting the sample containing the first particle and the sample containing the second particle to contain the same particle concentration as each other; and by mixing the adjusted sample containing first particle and the adjusted sample containing the second particle at different ratios, to generate the set of standards comprising different ratios of empty and full particles.
  • FIG. 22 provides an example method for reference subtraction.
  • Process 2200 begins at step 2202, to pair each of the set of standards or the test sample with a reference sample containing a reference particle, wherein the reference sample has a lower ratio of full particles than each of the set of standards or the test sample.
  • the reference particle can be the first particle that is used to prepare a set of standards, e.g., the empty particle.
  • each of the set of standards or the test sample can have the same characteristics as the reference sample, for example the same viral titer, buffer content, incubation time, biosensor regeneration cycles, experiment day, or other assay conditions.
  • Process 2200 continues to step 2204, to contact the reference sample with the biosensor to bind the reference particle in the reference sample to the biosensor.
  • Process 2200 continues to step 2206, to calculate binding rates of the reference particle in the reference sample to the biosensor.
  • Process 2200 continues to step 2208, to subtract the calculated binding rates of the reference particle from the calculated binding rates of the particles in the set of standards.
  • Process 2200 continues to step 2210, to generate a standard curve based on the signals obtained by subtracting the calculated binding rates of the reference particle from the calculated binding rates of the particles in the set of standards.
  • Process 2200 continues to step 2212, to subtract the calculated binding rates of the reference particle from the calculated binding rates of the particles in the test sample.
  • Process 2200 continues to step 2214, to compare the signals obtained by subtracting the calculated binding rates of the reference particle from the calculated binding rates of the particles in the test sample to the standard curve, thereby determining an empty/full particle ratio in the test sample.
  • the optical signal can include light reflected from two different locations associated with the biosensor.
  • the optical signal comprises light interference generated by biolayer interferometry (BLI).
  • the method can include providing the set of standards and the test sample in containers, such as wells in a multi-well plate.
  • the method can include contacting the set of standards and the test sample sequentially.
  • the method includes contacting the set of standards and the test sample simultaneously. Simultaneous measurements enables fast and efficient assessment (e.g., high- throughput assessment) of the ratio of full and empty particles in samples.
  • the method further includes analysis using reference subtraction, by pairing each of the set of standards or the test sample with a reference sample containing a reference viral capsid, wherein the reference sample has a lower ratio of full viral capsids than each of the set of standards or the test sample, and wherein each of the set of standards or the test sample has the same characteristics as the reference sample containing the reference viral capsid, wherein the characteristics comprise one or more of viral titer, buffer content, incubation time, biosensor regeneration cycles, and experiment day; contacting the reference sample with the biosensor, to bind the reference viral capsid in the reference sample to the biosensor; calculating binding rates of the reference viral capsid in the reference sample to the biosensor; subtracting the calculated binding rates of the reference viral capsid from the calculated binding rates of the viral capsids in the set of standards; generating a standard curve based on the signals obtained by subtracting the calculated binding rates of the reference viral capsid from the calculated binding rates of the viral capsids in the set of standards; subtracting the
  • the binding rates of the first viral capsid and the test viral capsid from the set of standards and the binding rates of the viral capsids from the test sample are calculated using initial slope / rate analysis. In certain embodiments, the binding rates of the first viral capsid and the test viral capsid from the set of standards and the binding rates of the viral capsids from the test sample are calculated using end point / saturation analysis.
  • a system for determining an empty/full viral capsid ratio includes a biosensor, configured to bind viral capsids in a set of standards or in test sample, to detect an optical signal associated with viral capsids bound to the biosensor, and to determine a empty/full viral capsid ratio in the test sample based on the optical signal.
  • the set of standards comprises viral capsids at the same viral titer and different ratios of empty and full viral capsids, and wherein the test sample comprise viral capsids at an unknown empty/full viral capsid ratio.
  • the optical signal can include light reflected from two different locations associated with the biosensor.
  • the biosensor is a biolayer interferometry (BLI) biosensor.
  • the system can further include a container configured to house the set of standards or the test sample.
  • the container is a well in a multi-well plate.
  • the biosensor can be further configured to bind a reference viral capsid in a reference sample paired with each of the set of standards or the test sample, to generate a standard curve based on signals obtained by subtracting calculated binding rates of the reference viral capsid from calculated binding rates of the viral capsids in the set of standards, and to determine a empty/full viral capsid ratio in the test sample based on the signals obtained by subtracting calculated binding rates of the reference viral capsid from calculated binding rates of the viral capsids in the test sample and the standard curve.
  • the present disclosure provides a system for determining an empty/full particle ratio (such as an empty /full viral capsid ratio) in a sample.
  • the system includes a biosensor, configured to bind particles in the sample and to detect an optical signal associated with the particles bound to the biosensor; and a processor, configured to perform the method of determining an empty/full particle ratio (such as an empty/full viral capsid ratio) in a sample provided herein.
  • FIG. 23 provides an example system for determining an empty/full viral capsid ratio is provided.
  • the system 2300 includes biosensor 2302 and processor 2304.
  • Biosensor 2303 is configured to bind viral capsids in samples 2306, including a set of standards and test sample, to detect an optical signal associated with viral capsids bound to biosensor 2302, and to determine a empty/full viral capsid ratio in the test sample 2306 based on the optical signal.
  • the set of standards comprises viral capsids at the same viral titer and different ratios of empty and full viral capsids, and wherein the test sample comprise viral capsids at an unknown empty/full viral capsid ratio.
  • Processor 2304 obtains optical signal or binding rates information from biosensor 2302, and conducts methods determining an empty/full particle ratio (such as an empty/full viral capsid ratio) in a sample provided herein, including detect calculating binding rates of particles in the set of standards or test samples (e.g., using initial slope / rate analysis or end point / saturation analysis), a generating standard curve, comparing calculated binding rates of particles in the test samples to the standard curve, and reference subtraction analysis (e.g., calculating binding rates of the reference particle in the reference sample to the biosensor; subtracting the calculated binding rates of the reference particle from the calculated binding rates of the particles in the set of standards; generating a standard curve based on the signals obtained by subtracting the calculated binding rates of the reference particle from the calculated binding rates of the particles in the set of standards; subtracting the calculated binding rates of the reference particle from the calculated binding rates of the particles in the test sample; and comparing the signals obtained by subtracting the calculated binding rates of the reference particle from the calculated binding rates of the particles in the test sample
  • the biosensor comprises an optical fiber having a proximal end portion and a distal end portion, the proximal end portion configured to receive light from a light source and configured to deliver reflected light to a detector.
  • the distal end portion configured to have analytes bind thereto such that light reflected from the distal end portion is phase shifted based on a thickness of analytes bound to the distal end portion.
  • the biosensor further comprises an optical resonator at a distal end portion of the optical fiber, the optical resonator including a first reflective surface and a second reflective surface, the first reflective surface configured to reflect light with a first phase and the second reflective surface configured to reflect light with a second phase which is phase shifted based on a thickness of analytes bound to the optical resonator.
  • the signals such as those generated by binding of the viral capsids to the biosensor are measured by a detector.
  • the signals can be detected based on any label-free technique for detecting a change in a property of a sensor surface, such as BLI, Surface Plasmon Resonance (SPR), Surface Acoustic Wave (SAW), Quartz Crystal Microbalance (QCM), and Reflectometric Interference Spectroscopy (RIfS).
  • the signals are measured by an interferometer.
  • the interferometer can comprise the biosensor, and can constitute a BLI sensor.
  • the interferometer further comprises: a first optical waveguide configured to receive light from a light source; a second optical waveguide configured to deliver reflected light to a detector; and an optical coupler spatially separates a distal portion of the first optical waveguide from a distal portion of the second optical waveguide, wherein the biosensor is attached to the optical coupler.
  • the interferometer further comprises a light source that is in optical communication with the first optical waveguide and configured to provide light to the first optical waveguide.
  • the interferometer further comprises a detector configured to receive light from the second optical waveguide.
  • the first optical waveguide and the second optical waveguide are disposed in a fiber optic bundle.
  • an interferometer can include a light source, an optical assembly, and a detector unit.
  • the bio-layer interferometry (BLI) sensor or optical assembly functions as a sensing element or detector tip to detect analytes attached to an end thereof.
  • the detector unit detects interference signals produced by interfering light waves reflected from the optical assembly.
  • the light source directs light into the optical assembly, which is reflected back to the detector unit through an optical coupling assembly.
  • the coupling assembly includes a first optical waveguide or fiber that extends from the light source to the optical assembly, a second optical waveguide or fiber which carry reflected light from the optical assembly to the detector, and an optical coupler which optically couples the first optical waveguide and the second optical waveguide.
  • the coupling assembly includes a lens system constructed to focus a light beam on an upper surface of the optical assembly and to direct reflected interfering light from the optical assembly to the detector.
  • the light source can be a white light source, such as a light emitting diode (LED), that produces light over a broad spectrum, e.g., 400 nm or less to 700 nm or greater, typically over a spectral range of at least 100 nm.
  • the light source can be a plurality of sources each having a different characteristic wavelength, such as LEDs designed for light emission at different selected wavelengths in the visible light range. The same function can be achieved by a single light source, such as, white light source, with suitable filters for directing light with different selected wavelengths onto the optical assembly.
  • the detector may be a spectrometer, such as charge-coupled device (CCD), capable of recording the spectrum of the reflected interfering light from the optical assembly.
  • CCD charge-coupled device
  • the detector may be a simple photodetector for recording light intensity at each of the different irradiating wavelengths.
  • the detector may include one or more filters which allows detection of light intensity, for instance from a white-light source, at each of a plurality of selected wavelengths of the interference reflectance wave.
  • the first optical waveguide and/or the second optical waveguide may be in the form of a fiber optic bundle (FOB).
  • the first optical waveguide includes several fiber optic elements surrounding a single fiber optic element of the second optical waveguide. This arrangement separates delivery of light from the light source from delivery of the reflected light from the optical assembly to the detector. It will be appreciated that other arrangements of the first optical waveguide and the second optical waveguide may allow for spatial separation of the light from the light source and the reflected light from the optical assembly. The separation of the light from the light source and the reflected light from the optical assembly may improve a signal to noise ratio (SNR) of the apparatus.
  • the first optical waveguide is a single fiber and the second fiber optical waveguide is formed of a plurality of fibers.
  • the distal tip of the fiber optic bundle can be aligned with a proximal end portion of the optical fiber when the BLI sensor is attached to the optical coupler.
  • the BLI sensor may be fixedly attached to the optical coupler to align and maintain a position of the proximal end portion with respect to the tip.
  • the BLI sensor can include the optical fiber having a proximal end and a distal end.
  • the proximal end and/or the distal end of the optical fiber may be polished ends.
  • the BLI sensor can have an optical resonator having a first reflecting surface and a second reflecting surface distal of the first reflecting surface.
  • the optical fiber is substantially transparent between the proximal end and distal end thereof.
  • the optical resonator may be transparent between the first and second reflecting surfaces.
  • the distance between the first and second reflecting surfaces defines a thickness of the optical resonator.
  • the thickness of the optical resonator may be in a range of 50 nm to 5,000 nm, such as between 400 nm and 1,000 nm.
  • the second reflecting surface is formed of a layer of analyte-binding molecules which are effective to bind analyte molecules specifically and with high affinity. That is, the analyte and anti-analyte molecules are opposite members of a binding pair which can include, without limitations, antigen-antibody pairs, complementary nucleic acids, and receptor-binding agent pairs. In specific embodiments, the analyte and anti-analyte molecules are oppositely charged. For example, the analyte and anti-analyte molecules are negatively and positively charged, respectively.
  • the index of refraction of the optical fiber may be similar to that of the second reflecting surface so that light reflected from the second reflecting surface occurs predominantly from the layer formed by the analyte-binding molecules, rather than from the interface between the optical fiber and the analyte-binding molecules.
  • analyte molecules bind to distal end portion of the optical assembly, light reflected from the distal end portion of the assembly occurs predominantly from the layer formed by the analyte-binding molecules and bound analyte, rather than from the interface region.
  • the first reflecting surface of the optical assembly is formed as a layer of transparent material having an index of refraction that is substantially different from that of the optical fiber, such that this layer functions to reflect a portion of the light directed onto the optical assembly.
  • the thickness of an analyte-binding layer disposed in the distal end portion of the optical element may be designed to optimize the overall sensitivity based on specific hardware and optical components.
  • Conventional immobilization chemistries are used in chemically, such as covalently, attaching a layer of analyte-binding molecules to the lower surface of the optical element.
  • the analyte-binding layer is formed under conditions in which a distal end surface of the optical fiber is densely coated, so that binding of analyte molecules to the layer forces a change in the thickness of the layer, rather than filling in the layer.
  • the analytebinding layer can be either a monolayer or a multi-layer matrix.
  • the measurement of the presence, concentration, and/or binding rate of analyte to the optical assembly is enabled by the interference of reflected light beams from the two reflecting surfaces in the optical assembly. Specifically, as analyte molecules attach to or detach from the surface, the average thickness of the first reflecting layer changes accordingly. Because the thickness of all other layers remains the same, the interference wave formed by the light waves reflected from the two surfaces is phase shifted in accordance with this thickness change. [0164] Assuming that there are two reflected beams, the first beam is reflected from the first reflecting surface and the second beam is reflected from the analyte-binding molecules and bound analyte and the surrounding medium at the second reflecting surface. The conversion of the phase shifting to a thickness change of the bound analytes is well known in the art.

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Abstract

L'invention concerne des procédés et des systèmes pour déterminer efficacement, avec précision et avec exactitude des rapports vides/complets de particules (par exemple, des capsides virales intactes) dans des échantillons purifiés et bruts à l'aide d'un biocapteur, tel qu'un biocapteur BLI. Le taux de liaison de particules (par exemple, des capsides virales) peut être calculé à l'aide d'une analyse de pente/vitesse initiale ou à l'aide d'une analyse de saturation/point d'extrémité, et/ou à l'aide d'une analyse de soustraction de référence dans laquelle des signaux de référence sont soustraits de signaux de l'ensemble de normes ou d'échantillons de test.
PCT/US2025/014168 2024-02-01 2025-01-31 Procédés de détermination de rapports vides/complets de capsides virales intactes Pending WO2025166267A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210009964A1 (en) * 2019-07-12 2021-01-14 Sangamo Therapeutics, Inc. Separation and Quantification of Empty and Full Viral Capsid Particles
US20210017608A1 (en) * 2018-03-23 2021-01-21 Life Technologies Corporation Methods and kits to detect viral particle heterogeneity
US20230020428A1 (en) * 2021-07-12 2023-01-19 Regeneron Pharmaceuticals, Inc. Liquid Chromatography Assay for Determining AAV Capsid Ratio
WO2023205609A1 (fr) * 2022-04-18 2023-10-26 Access Medical Systems, Ltd. Procédé de détermination de la concentration d'adn dans un virus à adn
WO2023240125A2 (fr) * 2022-06-08 2023-12-14 Access Medical Systems, Ltd. Méthode de détermination de contenu de capside pleine dans virus adénoassociés

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210017608A1 (en) * 2018-03-23 2021-01-21 Life Technologies Corporation Methods and kits to detect viral particle heterogeneity
US20210009964A1 (en) * 2019-07-12 2021-01-14 Sangamo Therapeutics, Inc. Separation and Quantification of Empty and Full Viral Capsid Particles
US20230020428A1 (en) * 2021-07-12 2023-01-19 Regeneron Pharmaceuticals, Inc. Liquid Chromatography Assay for Determining AAV Capsid Ratio
WO2023205609A1 (fr) * 2022-04-18 2023-10-26 Access Medical Systems, Ltd. Procédé de détermination de la concentration d'adn dans un virus à adn
WO2023240125A2 (fr) * 2022-06-08 2023-12-14 Access Medical Systems, Ltd. Méthode de détermination de contenu de capside pleine dans virus adénoassociés

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