Method for determining the empty / full ratio of complete viral capsids

Abstract

Methods and systems are provided for efficiently, accurately, and precisely determining the empty / full ratio of particles (e.g., intact virus capsids) in purified and crude samples using biosensors, such as BLI biosensors. Initial slope / rate analysis or saturation / end-point analysis can be used, and / or reference subtraction analysis in which a reference signal is subtracted from the signal of the set of standards or test samples is used to calculate the binding rate of particles (e.g., virus capsids).

Description

Technical Field

[0001] This disclosure relates to a method for using optical sensors to determine the empty / full (E / F) ratio on an intact viral capsid. Background Technology

[0002] Viral vectors used in gene therapy, such as adeno-associated virus (AAV) vectors, can provide durable therapeutic responses for a variety of diseases for which there are currently no effective treatments. Viral vectors can be used to deliver polynucleotides (such as DNA) encoding genes intended to treat various diseases. A natural byproduct of recombinant viral synthesis is a capsid that is not yet packaged with the polynucleotide of interest (also known as an "empty" capsid) and a capsid that is already packaged with the polynucleotide of interest (also known as a "full" capsid). Empty capsids can impair the efficacy of AAV drug products and produce adverse clinical effects. Removing empty capsids can increase transgene expression in subjects. Therefore, it is important to have effective methods to distinguish between full and empty capsids. Despite being integrated into the purification steps of the recombinant virus (such as recombinant AAV) production chain, empty capsids still account for a large portion of batch production.

[0003] Several methods have been used to attempt to determine the capsid content ratio (full capsid to empty capsid), including anion exchange chromatography (AEX), optical density (OD), transmission electron microscopy (TEM), charge detection mass spectrometry (CDMS), size exclusion chromatography-multi-angle light scattering (SEC-MALS), enzyme-linked immunosorbent assay (ELISA) tandem with quantitative polymerase chain reaction (qPCR), analytical ultracentrifugation (AUC), dynamic / static light scattering (DLS / SLS), and microfluidics. However, these techniques are time-consuming, labor-intensive, error-prone, inaccurate, and expensive, and are limited by the large amounts of sample preparation or processing time they require. For example, sample analysis times for these techniques can range from 15 minutes (OD) to 6 hours (AUC and TEM) per sample, and require significant resources, which significantly limits the number of samples that can be processed per day and makes most methods low-throughput. Furthermore, these techniques are either completely incompatible with crude samples or not directly compatible (i.e., require processing) crude samples. Summary of the Invention

[0004] In light of the foregoing, there is a need for methods that can efficiently, accurately, precisely, and rapidly determine the empty / filled capsid ratio in purified and crude samples. This disclosure generally relates to systems and methods that address these shortcomings in the art and provide additional or alternative advantages. For example, the systems and methods disclosed herein are directly compatible with crude samples without any type of processing; require only a single experiment; and are high-throughput. Furthermore, the systems and methods disclosed herein can determine the empty / filled ratio on intact viral capsids without lysing any viral capsids. By analyzing intact viral capsids, the systems and methods disclosed herein avoid the extensive optimization and validation typically required for methods measuring released viral DNA.

[0005] This disclosure provides embodiments of systems and methods for determining the empty / filled ratio on an intact viral capsid using a biosensor. The signal indicating viral capsid binding to the biosensor can be measured using an interferometer based on biolayer interferometry (BLI). The empty / filled viral capsid ratio in a sample can be calculated based on the binding signal strength, binding rate, or binding kinetics of the viral capsid by the biosensor.

[0006] In certain aspects of this disclosure, a method for determining the empty / filled particle ratio in a sample is provided. The method includes: contacting a set of standards with a biosensor to bind particles from the set of standards to the biosensor; contacting a test sample with the biosensor to bind particles from the test sample to the biosensor; detecting, for each of the set of standards and the test sample, an optical signal associated with the binding of the particles to the biosensor; and determining the empty / filled particle ratio in the test sample by comparing the optical signal associated with the binding of particles in the test sample to the biosensor with the optical signal associated with the binding of particles from the set of standards to the biosensor. The set of standards includes particles at a first concentration and different empty / filled ratios in a first buffer solution. The test sample includes particles at an unknown empty / filled ratio in a second buffer solution.

[0007] In some embodiments, the particles include viral capsids, liposomes, lipid nanoparticles, or polymer nanoparticles. In some embodiments, the viral capsid includes the capsid of adeno-associated virus (AAV).

[0008] In an embodiment, detecting the optical signal associated with particles in the set of standards binding to the biosensor and the optical signal associated with particles in the test sample binding to the biosensor includes: calculating the binding rate of particles in the set of standards to the biosensor; generating a standard curve based on the binding rate of particles in the set of standards to the biosensor; calculating the binding rate of particles in the test sample to the biosensor; and comparing the calculated binding rate of particles in the test sample with the standard curve.

[0009] In some 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 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 endpoint / saturation analysis.

[0010] In an embodiment, generating a standard curve includes comparing the calculated binding rate of particles in the set of standards with the empty / filled particle ratio in the set of standards.

[0011] In one embodiment, the test sample includes a plurality of test samples, and the method includes adjusting the plurality of test samples to contain the particles at a second concentration.

[0012] In one embodiment, the method further includes: adjusting the test sample to contain the particles at the first concentration before bringing the test sample into contact with the biosensor.

[0013] In this embodiment, the first buffer and the second buffer are the same.

[0014] In an embodiment, the set of standards is prepared by: providing a sample containing empty-shell particles and a sample containing full-shell particles; adjusting the sample containing empty-shell particles and the sample containing full-shell particles to contain particles at the first concentration; and mixing the adjusted sample containing empty-shell particles and the adjusted sample containing full-shell particles in different proportions to generate the set of standards comprising different empty / full particle ratios.

[0015] In an embodiment, the method further includes: pairing each of the set of standards or the test samples with one or more reference samples containing reference particles, wherein the one or more reference samples have the same or lower full-shell particle ratio as the set of standards or the test samples.

[0016] In an embodiment, the reference particle includes the empty shell particle. In an embodiment, the one or more reference samples have the same characteristics as the paired set of standards or the test sample, wherein the characteristics include one or more of particle size, particle composition, particle concentration, buffer content, incubation time, biosensor regeneration cycle, and experimental days.

[0017] In an embodiment, the method further includes: contacting the one or more reference samples with the biosensor to bind the reference particles in the one or more reference samples to the biosensor; and calculating the binding rate of the reference particles in the one or more reference samples to the biosensor.

[0018] In an embodiment, the method further includes: calculating the attributable binding rate (residual binding rate after subtraction) of the particles in the set of standards by subtracting the binding rate of the reference particles in the one or more reference samples paired with each of the set of standards from the binding rate of the particles in the set of standards; generating a standard curve based on the attributable binding rate (residual binding rate after subtraction) of the particles in the set of standards; calculating the attributable binding rate of the particles in the test sample by subtracting the binding rate of the reference particles in the one or more reference samples paired with the test sample from the binding rate of the particles in the test sample; and comparing the attributable binding rate (residual binding rate after subtraction) of the particles in the test sample with the standard curve to determine the empty / filled particle ratio in the test sample.

[0019] In this embodiment, the test sample is a crude sample comprising a viral capsid.

[0020] In one embodiment, the optical signal comprises light reflected from two different locations associated with the biosensor. In another embodiment, the optical signal comprises optical interference generated by biolayer interferometry (BLI).

[0021] In one embodiment, the method includes providing the set of standards and the test sample in the holes of a porous plate.

[0022] In some embodiments, the method includes sequentially contacting the set of standards and the test sample. In other embodiments, the method includes simultaneously contacting the set of standards and the test sample.

[0023] In one embodiment, a method for determining the empty / filled viral capsid ratio is provided. The method includes: acquiring optical signals of a set of standards, the set of standards comprising viral capsids at a first viral titer and different empty / filled ratios in a first buffer solution; acquiring 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 / filled ratio in the first buffer solution; calculating the attributed binding rate of particles in the set of standards by subtracting the first reference optical signal from the optical signal of each of the standards; and acquiring an optical signal of a test sample comprising viral capsids at a second viral titer and an unknown empty / filled ratio in a second buffer solution. The test sample is subjected to a viral capsid. A second reference optical signal is acquired, comprising an optical signal of a second reference sample, the second reference sample comprising a viral capsid at the second viral titer and an empty / filled capsid ratio in the second buffer solution. The attributable binding rate of particles in the test sample is calculated by subtracting the optical signal of the reference particles from the binding rate of particles in the test sample. The empty / filled capsid ratio of the test sample is determined by comparing the attributable binding rate of the particles in the test sample (the remaining binding rate after deducting the reference binding rate) with the attributable binding rate of particles in the set of standards (the remaining binding rate after deducting the reference binding rate).

[0024] In this embodiment, the optical signals of the set of standards are obtained based on the binding rate of the viral capsid in the set of standards to the biosensor. The optical signal of the first reference sample is obtained based on the binding rate of the viral capsid in the first reference sample to the biosensor. The optical signal of the test sample is obtained based on the binding rate of the viral capsid in the test sample to the biosensor. The optical signal of the second reference sample is obtained based on the binding rate of the viral capsid in the second reference sample to the biosensor.

[0025] In some embodiments, the optical signal comprises optical interference generated by biolayer interferometry (BLI). In some embodiments, the binding rate of the viral capsid to the biosensor is measured using initial slope / rate analysis. In other embodiments, the binding rate of the viral capsid to the biosensor is measured using endpoint / saturation analysis.

[0026] In one embodiment, a method for determining the empty / filled viral capsid ratio is provided. The method includes: pairing a set of standards or test samples with a reference sample, the reference sample having a filled viral capsid ratio (e.g., %filled) less than or equal to the filled viral capsid ratio of the set of standards or the test sample; contacting the set of standards and their paired reference samples with a biosensor to bind viral capsids from the set of standards or their paired reference samples to the biosensor, the set of standards comprising viral capsids at the same viral titer and different empty / filled ratios; calculating the binding rate of viral capsids from the set of standards to the biosensor; calculating the binding rate of viral capsids from the reference sample to the biosensor; subtracting the calculated binding rate of the reference sample from the calculated binding rate of viral capsids from the set of standards; and subtracting particles from the set of standards. A standard curve is generated by comparing a reference binding rate with a known empty / filled particle ratio in the set of standards; a test sample and its paired test reference sample are brought into contact with the biosensor to bind the viral capsid in the test sample or its paired test reference sample to the biosensor, the test sample including viral capsids with an unknown empty / filled ratio; the binding rate of the viral capsid in the test sample to the biosensor is calculated; the binding rate of the reference viral capsid in the test reference sample to the biosensor is calculated; the calculated binding rate of the test reference sample is subtracted from the calculated binding rate of the viral capsid in the test sample; and the empty / filled viral capsid ratio in the test sample is determined by extrapolating the binding rate of the viral capsid in the test sample (subtracted from the reference) to the standard curve.

[0027] In an embodiment, the reference sample includes a reference viral capsid of the same type as the set of standards or the test sample paired with it.

[0028] In an embodiment, each of the set of standards or the test samples has the same characteristics as the reference sample containing the reference viral capsid, wherein the characteristics include one or more of the following: viral titer, buffer content, incubation time, biosensor regeneration cycle, and experimental days.

[0029] In this embodiment, the binding rate of the viral capsid in the set of standards and the binding rate of the viral capsid in the test sample are calculated using initial slope / rate analysis.

[0030] In this embodiment, the binding rate of the viral capsid in the set of standards and the binding rate of the viral capsid in the test sample are calculated using endpoint / saturation analysis.

[0031] In this embodiment, the test reference sample is the same as the reference sample.

[0032] In one aspect of this disclosure, a system for determining the empty / filled viral capsid ratio is provided. The system includes a biosensor configured to bind viral capsids from a set of standards or a test sample, detect an optical signal associated with the viral capsid bound to the biosensor, and determine the empty / filled viral capsid ratio in the test sample based on the optical signal. The set of standards includes viral capsids at the same viral titer but with different empty / filled ratios. The test sample includes viral capsids with an unknown empty / filled ratio.

[0033] In one embodiment, the optical signal comprises light reflected from two different locations associated with the biosensor.

[0034] In this embodiment, the biosensor is a biolayer interferometry (BLI) biosensor.

[0035] In one embodiment, the system further includes a container configured to hold the set of standards or the test samples.

[0036] In one embodiment, the container is a hole in a perforated plate.

[0037] In an embodiment, 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, generate a standard curve based on a signal obtained by subtracting the calculated binding rate of the reference viral capsid from the calculated binding rate of the viral capsids in the set of standards, and determine the empty / full viral capsid ratio in the test sample based on the signal obtained by subtracting the calculated binding rate of the reference viral capsid from the calculated binding rate of the viral capsids in the test sample and the standard curve.

[0038] In one embodiment, this disclosure provides a system for determining the empty / filled particle ratio in a sample. The system includes: a biosensor configured to bind particles in the sample and detect optical signals associated with the particles bound to the biosensor; and a processor configured to perform the method provided herein for determining the empty / filled ratio of particles or viral capsids. Attached Figure Description

[0039] Various aspects of this disclosure are described below with reference to the accompanying drawings, which are incorporated in and form part of this specification.

[0040] Figures 1A-1C It is a graphical representation of the binding rate of empty and full-shell AAV capsids of the same titer, indicated by a biosensor. Figure 1A The data for the AAV8 shell is displayed. Figure 1B The data for the AAV9 capsid is displayed. Figure 1C The data for the AAV2 capsid is displayed.

[0041] Figure 2 This is a graphical representation of an example method for determining the empty / filled ratio on an intact viral capsid in a sample using initial rate analysis.

[0042] Figure 3 This is a graphical representation of an example method for using saturation analysis to determine the empty / filled ratio on an intact viral capsid in a sample.

[0043] Figure 4 The graph represents the time-varying interference signal change (nm shift), showing the higher binding rate of a fully capped AAV8 capsid to the Octet AAVX biosensor compared to an empty capsid, where the viral capsid titer is the same.

[0044] Figure 5 This is a graphic representation of one-step sampling of viral capsids with low and high capsid percentages and the same titer using the BLI biosensor to achieve an empty / capsid ratio.

[0045] Figure 6 It is a graphical representation of the time-varying interference signal change (nm shift), showing that, over a wide range of titers, a fully capped AAV8 cap has a higher binding rate at saturation compared to an empty capped AAV8 cap.

[0046] Figure 7 This is a graphic representation of a one-step sampling method using the BLI biosensor to sample capsids with different viral titers in an empty / filled ratio.

[0047] Figure 8 It is a graphical representation of the time-varying interference signal change (nm shift), which indicates the empty / filled ratio of the AAVX biosensor used to determine the same titer of viral capsids.

[0048] Figure 9 It is a graphical representation of the binding rate of viral capsids with different full-shell percentages and the same titer, analyzed using the initial rate.

[0049] Figure 10 It is a graphical representation of the time-varying interference signal change (nm shift), which indicates the AAVX biosensor used to determine the empty / filled ratio of the viral capsid at variable titers.

[0050] Figure 11It is a graphical representation comparing the binding rates of viral capsids with different full-capsule percentages and titers, all using saturation analysis, with standard curves generated from viral capsids with different full-capsule percentages and the same titer.

[0051] Figure 12 This is a graphical representation of an example empty / full-scale measurement design on the Octet AAVX biosensor.

[0052] Figure 13A This is a graphical representation of the time-varying interference signal change (nm offset) after pairing reference subtraction, indicating the AAVX biosensor used to determine the empty / full ratio of the same sample at different titers.

[0053] Figure 13B This is a graphical representation of the difference in net BLI signal (nm) between a full-shell AAV sample and its paired empty-shell capsid reference at various titers.

[0054] Figure 14A This is a graphical representation of the time-varying interference signal change (nm offset) after reference subtraction, indicating the empty / full ratio of the AAVX biosensor used to determine the sample (standard) in Octet sample dilution buffer.

[0055] Figure 14B This is a graphical representation of the time-varying interference signal change (nm shift) after reference subtraction, indicating the parameters used to determine the presence of interference signals in crude cell lysates. Figure 14A The same sample with an empty / full ratio of AAVX biosensor was used.

[0056] Figure 15A This is a graphical representation of the time-varying interference signal change (nm offset) after reference subtraction, indicating the AAVX biosensor used to determine the empty / full ratio of the sample diluted and analyzed on day 1.

[0057] Figure 15B This is a graphical representation of the time-varying variation (nm shift) of the interference signal after reference subtraction, indicating the parameters used to determine the relationship between the interference signal and the reference signal. Figure 15A The same sample with an empty / full ratio of AAVX biosensor was used, but the same sample was freshly diluted and analyzed on day 3.

[0058] Figure 16A This is a graphical representation of the time-varying interference signal change (nm offset) after reference subtraction, indicating the new AAVX biosensor used to determine the empty / filled ratio of a sample.

[0059] Figure 16BThis is a graphical representation of the time-varying variation (nm shift) of the interference signal after reference subtraction, indicating the parameters used to determine the relationship between the interference signal and the reference signal. Figure 16A The same sample was used with an empty / full ratio of the AAVX biosensor that underwent three regenerations.

[0060] Figure 17 This is a graphical representation of the signals in a sample containing a full-shell viral capsid, a sample containing a mixture of full-shell and empty-shell viral capsids, and a sample containing an empty-shell viral capsid, before and after reference subtraction, according to embodiments of the present disclosure.

[0061] Figure 18 This is a graphical representation of pairing a set of standards or test samples with reference samples containing the same viral titer, buffer solution, and assay batch, according to embodiments of this disclosure.

[0062] Figure 19 It is a graphical representation of reference subtraction and the signal obtained from reference subtraction according to embodiments of the present disclosure.

[0063] Figure 20 This is a flowchart of a method for determining the empty / filled particle ratio in a sample according to embodiments of the present disclosure.

[0064] Figure 21 This is a flowchart of a method for detecting optical signals associated with particles bound to the biosensor in accordance with embodiments of the present disclosure, based on the set of standards or the test sample.

[0065] Figure 22 This is a flowchart of a method for reference subtraction of optical signals associated with the detection of particles in the set of standards or the test sample, according to embodiments of the present disclosure.

[0066] Figure 23 This is a schematic representation of a system for determining the empty / filled particle ratio in a sample according to embodiments of the present disclosure. Detailed Implementation

[0067] This disclosure will now be described more fully below with reference to exemplary embodiments thereof and the accompanying drawings. These exemplary embodiments are described so that this disclosure will be thorough and complete, and will fully convey the scope of this disclosure to those skilled in the art. Features from one embodiment or aspect may be combined with features from any other embodiment or aspect in any suitable combination. For example, any individual or combination of features from a method aspect or embodiment may be applied to an apparatus, product, or component aspect or embodiment, and vice versa. This disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will meet applicable legal requirements.

[0068] As used in this article, “vector” refers to a recombinant plasmid or virus, which includes nucleic acids to be delivered to host cells in vitro or in vivo.

[0069] Viral titers can be expressed in various ways, from which those skilled in the art can choose the appropriate way to express viral titers in the context. For example, the term "capsid particles (cp)" used when referring to viral titers refers to the number of viral capsids, regardless of infectivity or functionality. The terms "viral genome (vg)," "genome particles," "genome equivalent," or "genome copy" used when referring to viral titers refer to the number of viral particles containing a recombinant viral DNA genome or RNA genome, regardless of infectivity or functionality. The number of capsid particles or genome particles in a particular vector formulation can be measured by standard methods, such as using fluorescent dyes or electron microscopy. The terms "infectious unit (iu)," "infectious particle," or "replication unit" used when referring to viral titers refer to the number of infectious and replicative recombinant viral vector particles measured by an infection center assay (also known as a replication center assay). The term "transduction unit (tu)" used when referring to viral titers refers to the number of infectious recombinant viral vector particles that lead to the production of a functional transgenic product, measured in a functional assay.

[0070] As used herein, the term "isolated" or "separated" molecule (such as a viral capsid) refers to the identification, isolation, and / or recovery of the molecule from components of its native environment. Thus, for example, purification techniques can be used to enrich isolated (or isolated or purified) viral particles from a source mixture (such as culture lysate or production culture supernatant) to prepare these particles. Similarly, purification techniques can be used to isolate isolated (or isolated or purified) viral polynucleotides from a source mixture containing a viral capsid to prepare these viral polynucleotides.

[0071] In the context of loading biopolymers into a structure to assemble a biosensor, the term "loading" or "being loaded" refers to the process of bringing an equilibrium sample (such as a biopolymer) into contact with an equilibrium solid phase (thereby assembling a biosensor). Loading can be performed, for example, by using a chromatographic apparatus, i.e., by using an external force (or external driving force) to flow the sample through the apparatus (such as by gravity or pumping), or by immersing it in a well plate containing the sample.

[0072] As used herein, the term "BLI biosensor" refers to a sensing and / or analytical device that uses biomolecules (such as enzymes or antibodies) or living organisms suitable for BLI detection to detect the presence, physical characteristics, or quantity of a substance. 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.

[0073] As used herein, the term "proximal" refers to the portion of the device or its components that is closer to the user or machine using the device, and the term "distal" refers to the portion of the device or its components that is further away from the user or machine using the device.

[0074] "Analyte-binding" molecules refer 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.

[0075] "Antibody" refers to an immunoglobulin molecule having two heavy chains and two light chains, prepared by any method known in the art or developed hereafter, and includes polyclonal antibodies (such as those produced by inoculating mammals (such as goats, mice, rabbits, etc.) with an immunogen) and monoclonal antibodies produced using the well-known Kohler-Milton hybridoma fusion technique. The term includes antibodies produced using genetic engineering methods (such as, for example, using SCID mice reconstructed with human immunoglobulin genes) and antibodies humanized using surface remodeling techniques known in the art. "Antibody" also refers to antibody fragments. "Antibody fragment" refers to a fragment of an antibody molecule produced by chemical cleavage or genetic engineering techniques, as well as single-chain variable fragments (SCFvs) (such as those produced using combinatorial gene libraries and phage display techniques). Antibody fragments used according to this disclosure generally retain the ability to bind to their corresponding antigens and therefore include variable sequences and antigen-binding sites, both of which are within the scope of antibodies.

[0076] As used in this article, a “recombinant” virus or “recombinant” viral vector refers to a virus or polynucleotide vector that includes one or more heterologous sequences (in other words, nucleic acid sequences not of viral origin).

[0077] As used in this article, “binding rate” refers to a calculated output indicating the quality and properties of a biomolecule or its complex binding to a biosensor.

[0078] As used herein, “viral capsid” includes the protein shell of a viral protein, or a lipid bilayer / enveloping membrane that surrounds viral nucleic acid or recombinant viral vectors. As used herein, a “full-shell” viral capsid refers to a viral capsid containing polynucleotides. As used herein, a “full-shell capsid” or “full-shell viral capsid” refers to a viral capsid containing a higher polynucleotide content than a “hollow capsid” or “hollow viral capsid” as used herein. As used herein, a “hollow” viral capsid can be a viral capsid that does not contain polynucleotides, or a viral capsid that is substantially free of polynucleotides. As used herein, “substantially free of” when referring to a particular component (such as polynucleotides) means that the component (such as polynucleotides) constitutes less than about 5.0% by weight of the composition (such as viral capsid), 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. Empty capsids or empty viral capsids may or may not contain polynucleotides.

[0079] As used herein, a "full-shell" particle refers to a particle containing a payload. A "full-shell particle" (such as a full-shell virus particle or a full-shell nanoparticle) contains a higher payload content than an "empty-shell particle" (such as an empty-shell virus particle or an empty-shell nanoparticle). Empty-shell particles may or may not contain a payload. As used herein, an "empty-shell" particle may be a particle without a payload or a particle that is substantially free of polynucleotides. Any payload can be used in the methods and systems provided herein, including small molecules (e.g., small molecule drugs), nucleic acids or polynucleotides (e.g., mRNA, DNA, siRNA, shRNA, antisense RNA, miRNA, etc.), proteins or peptides (e.g., hormones, growth factors, enzymes, anticoagulants, interferons, interleukins, antibodies, antibody fragments, antibody-drug conjugates, etc.), exosomes, or liposomes. In some embodiments, the payload is a therapeutic molecule.

[0080] This disclosure provides a method for determining the empty / filled ratio of intact viral capsids in a sample using a biosensor such as the BLI biosensor. The method is based on the understanding that filled viral capsids have a higher density than empty viral capsids.

[0081] In some embodiments, the viral capsid binding rate is calculated by measuring the wavelength shift due to optical interference. In some embodiments, the first sample is a reference sample with a known percentage of fully capped viral capsids. In some embodiments, the second sample is an unknown sample, which may be a crude or purified sample. In some embodiments, the binding rate is calculated using initial slope / rate analysis or endpoint / saturation analysis. In some embodiments, the biosensor is a BLI biosensor, such as the AAVX affinity biosensor. In some embodiments, a fully capped AAV capsid results in a higher binding rate to the BLI biosensor than an empty capsid with the same viral titer. In some embodiments, a fully capped capsid produces a higher binding rate than an empty capsid upon saturation, regardless of titer. In some embodiments, the first set of samples is normalized to the same capsid titer as the second sample.

[0082] In some embodiments, the set of standards is prepared from the first set of samples by mixing the first viral capsid and the second viral capsid in multiple different proportions.

[0083] In some embodiments, the method further includes 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.

[0084] In some embodiments, the third viral capsid is diluted from the same viral stock solution as the first viral capsid in the first group of samples.

[0085] In some embodiments, the method further includes: subtracting the calculated binding rate of the first viral capsid from the calculated binding rate of the viral capsid in the set of standards; and subtracting the calculated binding rate of the third viral capsid from the calculated binding rate of the viral capsid in the second sample.

[0086] In some embodiments, the methods disclosed herein involve providing at least two reference samples, wherein at least one reference sample is fully shelled and at least one reference sample is empty. The percentage of fully shelled and the absolute titer of the reference samples are known or determined using known techniques such as analytical ultracentrifugation and ELISA.

[0087] Non-limiting examples of viral capsids that can be evaluated using the methods and systems disclosed herein include viral capsids derived from retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex viruses. In some embodiments, the one or more viral capsids comprise adeno-associated virus (AAV) capsids (including recombinant AAV).

[0088] AAV is a single-stranded DNA (ssDNA) non-enveloped virus belonging to the Parvoviridae family, with a measured diameter of 25 nm. AAV has at least eleven serotypes (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11), each with slightly different tropisms, including retinal, lung, muscle, liver, and brain cells. The virus consists only of protein and DNA and has three repeating capsid proteins, VP1, VP2, and VP3, in a pre-defined ratio of 1:1:10, which can vary between different serotypes and even within a given batch of particles. Depending on the tropism of a given serotype, it can infect both dividing and non-dividing cells. In some embodiments, the AAV described herein is AAV serotype 2 (AAV2), AAV serotype 8 (AAV8), or AAV serotype 9 (AAV9).

[0089] In an example embodiment, the BLI biosensor disclosed herein includes a core component that may include a silicon substrate activated with silane groups such as aminopropylsilane (APS) and epipropylsilane (EPS). The BLI biosensor is designed for specific viral binding, with negligible non-specific binding from interfering substances in the sample.

[0090] A non-limiting example of a BLI biosensor that can be used in the methods disclosed herein is the Octet® AAVX affinity biosensor (Sartorius Lab Instruments GmbH & Co. KG). This Octet® AAVX biosensor is coated with CaptureSelect™ Biotin Anti-AAVX conjugate (Thermo Fisher Scientific Inc.). Those skilled in the art will understand that other types of BLI biosensors can also be used with the methods disclosed herein.

[0091] In one example embodiment, a method for determining the empty / filled ratio of a viral capsid in a sample includes an initial rate analysis. The method may include the following steps: providing a plurality of first samples, each first sample comprising one or more viral capsids containing polynucleotides and one or more viral capsids not containing polynucleotides; providing one or more second samples, each second sample 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 and second samples with a biosensor; calculating the binding rate of the viral capsid from the set of standards and the binding rate of the viral capsid from the second sample; generating a standard curve from the set of standards; and determining the empty / filled ratio of the viral capsid in the second sample by comparing the binding rate to the standard curve.

[0092] In some embodiments, a known technique, such as ELISA, is used to determine the absolute viral titer of an unknown sample. The unknown sample may be crude or purified. In some embodiments, all samples may be normalized to a viral titer greater than about 1E11 cp / ml. In some embodiments, normalizing the reference sample involves mixing different proportions of reference samples (empty and full capsids) at the same capsid titer and diluting the unknown sample to the same titer as the reference sample. In other embodiments, an empty capsid reference sample is diluted to the same titer as the unknown sample using the same assay buffer for reference subtraction (described below).

[0093] In one embodiment, a BLI biosensor (e.g., an AAVX affinity biosensor) is immersed in the wells of a microplate containing a reference sample and an unknown sample, with a continuously set time, to determine the binding rate of the AAV vector to the AAVX antibody on the BLI biosensor. In a non-limiting example, the set time may be between approximately 10 and 30 minutes. For example, initial slope analysis can be used to determine the binding rate of the AAV vector.

[0094] In one embodiment, an initial slope binding rate equation can be used on a computing processor to fit the binding rate of a reference sample. The fitted model can then be used on the computing processor to generate a standard curve.

[0095] In one embodiment, BLI is used to compare signals from unknown samples with a standard curve to determine the empty / full viral capsid ratio in these samples. BLI is an optical technique that measures macromolecular interactions by analyzing the interference pattern of white light reflected from the surface of a BLI biosensor tip. The shift of the interference signal over time (“nm shift”) indicates the binding of a substance, such as a viral capsid, to the BLI biosensor.

[0096] As seen in Figure 1 and as a non-limiting example, a fully capped AAV capsid produces a higher BLI signal on the AAVX biosensor than an empty capsid. This is partly due to the fact that a fully capped capsid has a higher density than an empty capsid. The differential BLI signal from empty / fully capped AAV capsids allows for the determination of the empty / fully capped ratio on the AAV capsid. In this example, the fully capped or empty AAV8 capsids are first normalized using a known titer determination method (e.g., ELISA) before comparing the binding rates on the AAVX biosensor.

[0097] like Figure 4 As shown and as a non-limiting example, at any given time, a fully capped AAV capsid causes a greater spectral shift and exhibits a higher binding rate than an empty capsid of the same titer. In this example, the AAVX biosensor binds to both a fully capped and an empty AAV8 capsid with a titer of approximately 8.0 E11 cp / ml. Figure 5 As shown and as a non-limiting example, when samples have the same titer, initial rate analysis allows for one-step sampling of the empty / full volume ratio using a BLI biosensor.

[0098] like Figure 8 and Figure 9 As shown and as a non-limiting example, the E / F ratio was determined by initial slope analysis using the AAVX biosensor at the same viral titer. Figure 8 It is a graphical representation of the time-varying interference signal change (nm offset), in which the AAV8 capes with different full-shell percentages are plotted after subtracting the reference sample (empty shell AAV8). Figure 9 It is a graphical representation of the standard curve of the binding rate of AAV capsids with different full-shell percentages and the same titer.

[0099] Figure 2 A graphical representation of an example method for determining the empty / filled ratio on an intact viral capsid in a sample using initial rate analysis is shown. In this example, the method begins with the determination of the AAV capsid titer of an unknown sample. Next, a standard is prepared by mixing reference samples (empty and filled capsids) in different proportions at the same capsid titer. The preparation of the standard is performed once. The unknown sample is diluted to the same titer as the standard. The third step in this method involves determining the empty / filled ratio in the unknown sample. This step involves generating a standard curve using initial slope analysis and comparing the unknown sample to the standard curve to obtain the empty / filled ratio.

[0100] In some embodiments, the titers of a set of standards need not be matched to the titers of a set of unknown samples. However, each standard in the set of standards and each unknown sample in the set of unknown samples should maintain a constant titer. In these embodiments, each standard in the set of standards and each unknown sample in the set of unknown samples includes an empty shell reference for performing reference subtraction. As a result, a single standard curve can be used to calibrate multiple batches of samples with different titers, buffers, and experimental dates.

[0101] In one example embodiment, a method for determining the empty / filled ratio of a viral capsid in a sample includes saturation analysis. The method may include the steps of: normalizing a first sample to the same capsid titer; preparing a set of standards from the plurality of first samples; contacting each of the first and second samples with a biosensor; calculating the binding rate of the viral capsid from the set of standards and the binding rate of the viral capsid from the second sample; generating a standard curve from the set of standards; and determining the empty / filled ratio of the viral capsid in the second sample by comparing the binding rate with the standard curve.

[0102] In some embodiments, the normalized absolute viral titer of full-capsule and empty-capsule reference samples can be greater than about 5E11cp / ml. In some embodiments, a series of standards is prepared by mixing reference samples (empty-capsule and full-capsule) in different proportions at the same capsid titer.

[0103] In some embodiments, a coarse titer for the unknown sample can be determined using a BLI biosensor (e.g., an AAVX biosensor) before the reference sample and the unknown sample are brought into contact with the BLI biosensor. The unknown sample may be crude or purified. In other embodiments, the coarse titer can be simply estimated without using a BLI biosensor for determination.

[0104] In one embodiment, a BLI biosensor (e.g., an AAVX affinity biosensor) is immersed in the wells of a microplate containing a reference sample and an unknown sample containing a viral capsid (e.g., an AAV capsid) for a predetermined period of time to determine the binding rate of the AAV capsid to the AAVX antibody on the BLI biosensor. In a non-limiting example, the predetermined time may be approximately 60 minutes. For example, endpoint analysis may be used to determine the binding rate of the AAV capsid.

[0105] In one embodiment, a BLI is used to compare signals from unknown samples with a standard curve to determine the empty / full viral capsid ratio in those samples. The offset of the interference signal over time (“nm offset”) indicates the binding of a substance, such as a viral capsid, to the BLI biosensor.

[0106] like Figure 6 As shown and as a non-limiting example, regardless of titer, a fully capped AAV capsid causes a greater spectral shift and produces a higher BLI signal on the AAVX biosensor than an empty AAV capsid. In this non-limiting example, the AAVX biosensor binds to both fully capped and fully capped AAV8 capsids with viral titers ranging from approximately 2.5E11 to 4.0E12 cp / ml. Saturation analysis allows for one-step sampling of the empty / capped ratio from a wide range of sample titers. Figure 7 The results of the single experiment and one-step sampling method shown do not exhibit compound error.

[0107] like Figure 12 As shown and by way of a non-limiting example, the Octet BLI biosensor is immersed in an AAV sample to report a BLI signal. The BLI signal indicates the properties of the 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 across samples, and the net signal difference can be entirely attributed to the density difference between a given sample and an empty-shell reference. Therefore, the empty / filled ratio difference can be determined.

[0108] like Figure 10 and Figure 11 As seen and as a non-limiting example, the AAVX biosensor is used to determine the E / F ratio at variable viral titers. Figure 10 It is a graphical representation of the time-varying interference signal change (nm shift), which indicates the AAVX biosensor used to determine the empty / full ratio when the AAV capsid reaches saturation at a variable titer. Figure 11 It is a graphical representation comparing the binding rates of AAV capsids with different full-capsule percentages and different titers with standard curves generated from viral capsids with different full-capsule percentages and the same titer.

[0109] Figure 3 A graphical representation of another example method for determining the empty / filled ratio on an intact viral capsid in a sample using saturation analysis is shown. In this example, the method begins with a coarse estimate of the AAV capsid titer of an unknown sample. Next, a standard is prepared by mixing reference samples (empty and filled capsids) in different proportions at the same saturation titer. The standard is prepared once. A third step in the method involves determining the empty / filled ratio in the unknown sample. This step involves generating a standard curve using endpoint analysis and comparing the unknown sample to the standard curve to obtain the empty / filled ratio.

[0110] In some embodiments, the method for determining the empty / full ratio on an intact viral capsid involves performing a reference subtraction step after calculating the binding rate of viral capsids from a set of standards (including one or more empty capsid reference samples) and calculating the binding rate of viral capsids from a set of unknown samples (including one or more empty capsid reference samples). The binding rate of the empty capsid reference samples corresponding to the set of standards is subtracted from the binding rate of the set of standards. Similarly, the binding rate of the empty capsid reference samples corresponding to the unknown samples is subtracted from the binding rate of the unknown samples. The reference subtraction step can be performed manually or automatically using a computing processor.

[0111] In the reference subtraction embodiment, each of the set of standards or the test samples may be paired with a reference sample containing a reference particle. The reference sample has a lower ratio of fully capped particles (e.g., fully capped viral capsids) than each of the set of standards or the test samples, and therefore has a lower signal. The reference particle may be the first particle (e.g., an empty-capped particle) used to prepare the set of standards. Each of the set of standards or the test samples may have the same characteristics as the reference sample, such as the same viral titer, buffer content, incubation time, biosensor regeneration cycle (e.g., biosensor batch), and / or the experimental day on which the assay (e.g., obtaining the binding rate) was performed.

[0112] The reference subtraction method may include: contacting the reference sample with the biosensor to bind the reference particles in the reference sample to the biosensor; and calculating the binding rate of the reference particles in the reference sample to the biosensor. The reference subtraction method may further include: subtracting the calculated binding rate of the reference particles from the calculated binding rates of the particles in the set of standards; generating a standard curve based on the signal obtained by subtracting the calculated binding rate of the reference particles from the calculated binding rates of the particles in the set of standards; subtracting the calculated binding rate of the reference particles from the calculated binding rate of the particles in the test sample; and comparing the signal obtained by subtracting the calculated binding rate of the reference particles from the calculated binding rate of the particles in the test sample with the standard curve to determine the empty / filled particle ratio in the test sample.

[0113] Reference subtraction / paired reference subtraction can be achieved by ensuring that a sufficient number of empty reference wells are isolated to pair with each sample condition and each sample readout step. Ideally, the empty AAV references (minuends) used for subtraction are matched as closely as possible to the sample (minuend) in composition (e.g., titer, matrix, and volume) and detection conditions (e.g., microplate, sensor batch, and instrument time). The empty AAV references can be analyzed together with the sample to be subtracted in near real-time.

[0114] In an embodiment of reference subtraction, the method includes: acquiring optical signals of a set of standards; acquiring a first reference optical signal; calculating the attributed binding rate of the particles in the set of standards by subtracting the first reference optical signal from the optical signal of each of the optical signals in the set of standards; acquiring an optical signal of a test sample; acquiring a second reference optical signal; calculating the attributed binding rate of the particles in the test sample by subtracting the optical signal of the reference particles from the binding rate of the particles in the test sample; and determining the empty / full viral capsid ratio of the test sample by comparing the attributed binding rate of the particles in the test sample (the remaining optical signal after subtraction) with the attributed binding rate of the particles in the set of standards (the remaining optical signal after subtraction). The set of standards may include viral capsids at a first viral titer and different empty / full ratios in a first buffer solution. The test sample may include viral capsids at a second viral titer and an unknown empty / full ratio in a second buffer solution. The first reference optical signal may include the optical signal of a first reference sample, which includes viral capsids at the first viral titer and at an empty / full ratio in a first buffer solution. The second reference optical signal may include the optical signal of the second reference sample, which includes a viral capsid in the second buffer solution at the second viral titer and at an empty / full ratio.

[0115] In this embodiment, the optical signal of the set of standards is obtained based on the binding rate of the viral capsid in the set of standards to the biosensor. The optical signal of the first reference sample is obtained based on the binding rate of the viral capsid in the first reference sample to the biosensor. The optical signal of the test sample is obtained based on the binding rate of the viral capsid in the test sample to the biosensor. The optical signal of the second reference sample is obtained based on the binding rate of the viral capsid in the second reference sample to the biosensor.

[0116] In some embodiments, the optical signal comprises optical interference generated by biolayer interferometry (BLI). In some embodiments, the binding rate of the viral capsid to the biosensor is measured using initial slope / rate analysis. In other embodiments, the binding rate of the viral capsid to the biosensor is measured using endpoint / saturation analysis.

[0117] Some of the benefits associated with the paired reference subtraction described herein include: normalization of various variables (such as sample titer, buffer matrix, incubation time, biosensor regeneration cycle, and experimental day); improved 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 regeneration / reuse for saturation analysis; and allowing fair comparison of samples measured in different experiments.

[0118] For example, to process 56 samples in an Octet BLI system with an 8-channel processing capacity, one of the 8 channels should read an empty shell reference in each assay. The result would be 8 assays, each processing 7 samples and one empty shell reference, which would allow for fair comparisons between different sample reading steps.

[0119] In another example, if a standard is diluted to a first titer (titer A) in sample diluent, and an unknown sample is diluted to a second titer (titer B) in cell lysis buffer, then an empty shell reference sample diluted to titer A in sample diluent can be used as a reference to subtract the standard. The same empty shell reference sample diluted to titer B in cell lysis buffer can be used as a reference to subtract the unknown sample.

[0120] like Figure 13A and Figure 13B As observed and as a non-limiting example, similar BLI signals (nm shift) were produced at saturation for the same sample at different capsid titers after paired reference subtraction. In this example, the AAVX biosensor bound to an intact AAV8 capsid with viral titers ranging from approximately 2.5E11 to 4.0E12 cp / ml. As a result, samples could be successfully normalized at different titers using paired reference subtraction.

[0121] like Figure 14A and Figure 14B As observed and by way of a non-limiting example, after paired reference subtraction, the percentage of fully capped capsids is similar for the same sample analyzed in different buffer matrices. In this example, the AAVX biosensor binds to both full AAV8 empty shells and fully capped capsids. Figure 14AThis shows the predetermined percentage of fully capped sample from the Octet sample dilution buffer, which is used to construct the standard curve. Figure 14B The percentage of fully capsids, experimentally determined from the same sample of crude HEK293 cell lysate, is shown, comparable to standards. As a result, samples were successfully normalized in different buffer matrices using paired reference subtraction.

[0122] like Figure 15A and Figure 15B As observed and as a non-limiting example, the percentage of full-capsule coating after pairing reference subtraction is similar for the same sample diluted and analyzed on different days. In this example, the AAVX biosensor binds to a complete AAV5 capsule. Figure 15A The predetermined percentage of fully capped shells in the samples diluted and analyzed on day 1 is shown, which was used to construct the standard curve. Figure 15B The percentage of fully capped shells, determined experimentally, is shown in samples freshly diluted and analyzed on day 3, comparable to standards. The results demonstrate that samples analyzed in independent experiments can be successfully normalized using paired reference subtraction.

[0123] In some embodiments, using paired reference subtraction, AAVX biosensors can be regenerated and reused for empty / full-scale measurements, providing an efficient and cost-effective solution for high-throughput applications. Figure 16A and Figure 16B As observed and by way of non-limiting example, the percentage of full capsid after pairing reference subtraction is similar for the same sample analyzed using biosensors with different regeneration cycles. In this example, the AAVX biosensor binds to a complete AAV5 capsid. Figure 16A The predetermined percentage of fully capped shells in samples analyzed using the new AAVX biosensor is shown, which was used to construct a standard curve. Figure 16B The percentage of fully capillary membrane in samples analyzed using the biosensor after three regeneration cycles is shown, experimentally determined and comparable to a standard. As a result, samples analyzed using the biosensor with different regeneration cycles can be successfully normalized using paired reference subtraction, thus enabling the regeneration and reuse of the AAVX biosensor described in this application.

[0124] like Figure 17 As observed, by using the signal from a sample containing empty-shell particles (e.g., empty viral capsids) as a reference, and subtracting the reference signal from the signal obtained from the set of standards or the test sample (e.g., containing full-shell particles or a mixture of full-shell and empty-shell particles), the signal can be amplified to sensitively detect the ratio of full-shell particles to empty-shell particles. For example, in Figure 17In the original sensor images, signals of 8.7 nm, 8.0 nm, and 6.5 nm were provided for samples with full-shell particles, samples with a mixture of full-shell and empty-shell particles, and samples with empty-shell particles, respectively. After reference subtraction, the signals for samples with full-shell particles, samples with a mixture of full-shell and empty-shell particles, and samples with empty-shell particles became 2.2 nm, 1.5 nm, and 0 nm, respectively.

[0125] like Figure 18 As can be seen, the reference sample may have the same particle concentration (e.g., viral capsid titer), dilution / culture medium, and other assay conditions (e.g., biosensor batch, assay day on which the binding rate was obtained).

[0126] like Figure 19 As seen and disclosed herein, the set of standards and test samples may have different particle concentrations, although the particle concentrations are adjusted to be the same within the set of standards and between the test samples. The set of standards may be paired with a reference sample containing the same particle concentration as the set of standards. The test sample may be paired with a reference sample containing the same particle concentration as the test sample. Therefore, the particle concentration in a reference sample paired with the set of standards may differ from the particle concentration in a reference sample paired with the test sample.

[0127] Figure 20 An example method for determining the empty / filled particle ratio in a sample is provided. Process 2000 may begin at step 2008, contacting a set of standards with a biosensor to bind particles from the set of standards to the biosensor. Process 2000 continues to step 2010, contacting the test sample with the biosensor to bind particles from the test sample to the biosensor. Process 2000 continues to step 2012, detecting an optical signal associated with the binding of the particles to the biosensor. Process 2000 continues to step 2014, determining the empty / filled particle ratio in the test sample based on the optical signal. Process 2000 may also include optional steps 2002, 2004, and 2006 prior to step 2008. Process 2000 may begin at optional step 2002, providing a set of standards comprising particles with the same particle concentration and different empty and filled particle ratios. Process 2000 continues to step 2004, providing a test sample comprising particles with an unknown empty / filled particle ratio. Process 2000 continues to step 2006, adjusting the test samples to contain the same particle concentration. Step 2006 continues to step 2008.

[0128] In some embodiments, the empty / filled particle (e.g., capsid) ratio in the sample is 0-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0129] The particles may include viral capsids, liposomes, lipid nanoparticles, or polymer nanoparticles. In a particular embodiment, the particles are viral capsids. The viral capsid may include the capsid of adeno-associated virus (AAV).

[0130] In some embodiments, the viral particles in the sample have been purified using purification steps (such as those provided herein). In some embodiments, the viral particles in the sample have not been purified.

[0131] The full-shell and empty-shell viral capsids in any given sample can belong to any serotype, including native and recombinant serotypes, including chimeric (hybrid) and novel serotypes. It should be understood that capsids of different serotypes have different charge properties, including different charge differentiation levels between empty-shell and full-shell viral capsids, or between different recombinant constructs within a serotype. In some embodiments, the viral particles include AAV1 capsids, AAV2 capsids, AAV3 capsids, AAV4 capsids, AAV5 capsids, AAV6 capsids, AAV7 capsids, AAV8 capsids, AAVrh8 capsids, AAV9 capsids, AAV10 capsids, AAVrh10 capsids, AAV11 capsids, AAV12 capsids, AAV13 capsids, AAV14 capsids, AAV15 capsids, AAV16 capsids, AAVrh20 capsids, AAV.rh39 capsids, AAV.Rh74 capsids, AAV.RHM4-1 capsids, AAV.hu37 capsids, AAV.Anc80 capsids, AAV.Anc80L65 capsids, and AAV.PHP.B capsids. AAV2.5 caption, AAV2tYF caption, AAV3B caption, AAV.LK03 caption, AAV.HSC1 caption, AAV.HSC2 caption, AAV.HSC3 caption, AAV.HSC4 caption, AAV.HSC5 caption, AAV.HSC6 caption, AAV.HSC7 caption, AAV.HSC8 caption, AAV.HSC9 caption, AAV.HSC10 caption, AAV.HSC11 caption, AAV.HSC12 caption, AAV.HSC13 caption, AAV.HSC14 caption, AAV.HSC15 caption, AAV.HSC16 caption, AAV2R471A caption, AAV2 / 2-7m8 caption, AAV DJ capillary, AAV2 N587 A capillary, AAV2 E548A capillary, AAV2N708A capillary, AAV V708K capillary, goat AAV capillary, AAV1 / AAV2 chimeric capillary, bovine AAV capillary, or mouse AAV capillary. In some specific embodiments, the AAV particles are AAV2 particles.In some embodiments, the AAV genome is 2500 to 5500 bases, 3000 to 5500 bases, 3500 to 5500 bases, 4000 to 5500 bases, 4500 to 5500 bases, 5000 to 5500 bases, 2500 to 5000 bases, 3000 to 5000 bases, 3500 to 5000 bases, 4000 to 5000 bases, 4 The recombinant viral particle may contain 500 to 5000 bases, 2500 to 4500 bases, 3000 to 4500 bases, 3500 to 4500 bases, 4000 to 4500 bases, 2500 to 4000 bases, 3000 to 4000 bases, 3500 to 4000 bases, 2500 to 3500 bases, 3000 to 3500 bases, or 2500 to 3000 bases. In some embodiments, the recombinant viral particle comprises a self-complementary AAV (scAAV) genome.

[0132] The methods disclosed herein can be used to determine the empty / filled ratio on various gene therapy delivery vectors. The vectors can be viral or non-viral. For example, viral vectors can include adeno-associated virus (AAV), adenovirus, lentivirus, herpes simplex virus, and retrovirus. Non-viral vectors can be lipid-based (e.g., liposomes, solid lipid nanoparticles (LNPs)) and / or polymer-based. Non-viral vectors can have their surfaces coated with various types of polymers.

[0133] Figure 21 An example method is provided for detecting optical signals associated with particles bound to the biosensor in the aforementioned set of standards or test samples, the step of which involves... Figure 20 This is described as step 2012. Process 2100 begins at step 2012 by calculating the binding rate of the particles in the set of standards to the biosensor. Process 2100 continues to step 2104, generating 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, calculating the binding rate of the particles in the test sample to the biosensor. Process 2100 continues to step 2108, comparing the calculated binding rates of the particles in the test sample with the standard curve.

[0134] In embodiments, as exemplified in steps 2102 and 2106, an initial slope / rate analysis is used to calculate the binding rate of the particles from the set of standards and the binding rate of the particles from the test sample. In other embodiments, an endpoint / saturation analysis is used to calculate the binding rate of the particles from the set of standards and the binding rate of the particles from the test sample.

[0135] Generating a standard curve (as exemplified in step 2014) may include comparing the calculated binding rate of the first particle in the set of standards with the empty / filled particle ratio in the set of standards.

[0136] The method provided herein may further include adjusting the test sample to contain the same particle concentration as the set of standards before contacting the test sample with the biosensor. The set of standards may be prepared by: providing a sample containing a first particle and a sample containing a second particle, wherein the first particle has a lower payload 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 mixing the adjusted sample containing the first particle and the adjusted sample containing the second particle in different proportions to generate the set of standards comprising different ratios of empty and full-shell particles.

[0137] Figure 22An example method for reference subtraction is provided. Process 2200 begins at step 2202, pairing each of the set of standards or the test samples with a reference sample containing reference particles, wherein the reference sample has a lower full-shell particle ratio than each of the set of standards or the test samples. The reference particles may be the first particles used to prepare the set of standards, such as empty-shell particles. As provided in this disclosure, in order to subtract the reference (background) signal associated with each of the set of standards or the test samples, each of the set of standards or the test samples may have the same characteristics as the reference sample, such as the same viral titer, buffer concentration, incubation time, biosensor regeneration cycle, experimental day, or other assay conditions. Process 2200 continues to step 2204, contacting the reference sample with the biosensor to bind the reference particles in the reference sample to the biosensor. Process 2200 continues to step 2206, calculating the binding rate of the reference particles in the reference sample to the biosensor. Process 2200 continues to step 2208, subtracting the calculated binding rate of the reference particle from the calculated binding rate of the particle in the set of standards. Process 2200 continues to step 2210, generating a standard curve based on the signal obtained by subtracting the calculated binding rate of the reference particle from the calculated binding rate of the particle in the set of standards. Process 2200 continues to step 2212, subtracting the calculated binding rate of the reference particle from the calculated binding rate of the particle in the test sample. Process 2200 continues to step 2214, comparing the signal obtained by subtracting the calculated binding rate of the reference particle from the calculated binding rate of the particle in the test sample with the standard curve to determine the empty / filled particle ratio in the test sample.

[0138] The optical signal may include light reflected from two different locations associated with the biosensor. In one embodiment, the optical signal includes optical interference generated by biolayer interferometry (BLI).

[0139] The method may include providing the set of standards and the test samples in a container (such as holes in a multi-well plate).

[0140] The method may include sequentially contacting the set of standards and the test sample. In other embodiments, the method includes simultaneously contacting the set of standards and the test sample. Simultaneous measurement enables rapid and efficient assessment (e.g., high-throughput assessment) of the ratio of full-shell particles to empty-shell particles in the sample.

[0141] Therefore, a method for determining the empty / filled viral capsid ratio is provided. The method includes: providing a set of standards comprising viral capsids at the same viral titer and different empty and filled capsid ratios; providing a test sample comprising viral capsids at an unknown empty / filled capsid ratio; adjusting the test sample to contain the same capsid titer among itself; contacting the set of standards with a biosensor to bind the viral capsids in the set of standards to the biosensor; calculating the binding rate of the viral capsids in the set of standards to the biosensor; contacting the test sample with the biosensor to bind the viral capsids in the test sample to the biosensor; and calculating the binding rate of the viral capsids in the test sample to the biosensor. The method further includes analysis using reference subtraction: pairing each of the set of standards or the test samples with a reference sample containing a reference viral capsid, wherein the reference sample has a lower full-capsid ratio than each of the set of standards or the test samples, and wherein each of the set of standards or the test samples has the same characteristics as the reference sample containing the reference viral capsid, wherein the characteristics include one or more of viral titer, buffer concentration, incubation time, biosensor regeneration cycle, and experimental day; contacting the reference sample with the biosensor to bind the reference viral capsid in the reference sample to the biosensor; and calculating the reference viral capsid in the reference sample. The binding rate of the viral capsid to the biosensor; subtracting the calculated binding rate of the reference viral capsid from the calculated binding rate of the viral capsid in the set of standards; generating a standard curve based on the signal obtained by subtracting the calculated binding rate of the reference viral capsid from the calculated binding rate of the viral capsid in the set of standards; subtracting the calculated binding rate of the reference viral capsid from the calculated binding rate of the viral capsid in the test sample; and determining the empty / full viral capsid ratio in the test sample based on the signal obtained by subtracting the calculated binding rate of the reference viral capsid from the calculated binding rate of the viral capsid in the test sample and the standard curve.

[0142] In some embodiments, initial slope / rate analysis is used to calculate the binding rate of the first viral capsid from the set of standards and the test viral capsid, as well as the binding rate of the viral capsid from the test sample. In some embodiments, endpoint / saturation analysis is used to calculate the binding rate of the first viral capsid from the set of standards and the test viral capsid, as well as the binding rate of the viral capsid from the test sample.

[0143] In one aspect, a system is provided for determining the empty / filled viral capsid ratio. The system includes a biosensor configured to bind viral capsids from a set of standards or a test sample, detect an optical signal associated with the viral capsids bound to the biosensor, and determine the empty / filled 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 and filled viral capsid ratios, and the test sample includes viral capsids with an unknown empty / filled viral capsid ratio.

[0144] The optical signal may include light reflected from two different locations associated with the biosensor. In one embodiment, the biosensor is a biolayer interferometry (BLI) biosensor.

[0145] The system may further include a container configured to hold the set of standards or the test samples. In an embodiment, the container is a hole in a multi-well plate.

[0146] 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 a signal obtained by subtracting the calculated binding rate of the reference viral capsid from the calculated binding rate of the viral capsid in the set of standards, and to determine the empty / full viral capsid ratio in the test sample based on the signal obtained by subtracting the calculated binding rate of the reference viral capsid from the calculated binding rate of the viral capsid in the test sample and the standard curve.

[0147] In one embodiment, this disclosure provides a system for determining the empty / filled particle ratio (such as the empty / filled viral capsid ratio) in a sample. The system includes: a biosensor configured to bind particles in the sample and detect optical signals associated with the particles bound to the biosensor; and a processor configured to perform the method provided herein for determining the empty / filled particle ratio (such as the empty / filled viral capsid ratio) in a sample.

[0148] Figure 23An example system for determining the empty / full viral capsid ratio is provided. System 2300 includes a biosensor 2302 and a processor 2304. Biosensor 2303 is configured to bind viral capsids in a sample 2306 (including a set of standards and a test sample), detect an optical signal associated with the viral capsids bound to biosensor 2302, and determine the empty / full viral capsid ratio in the test sample 2306 based on the optical signal. The set of standards includes viral capsids at the same viral titer and different empty and full capsid ratios, and the test sample includes viral capsids with an unknown empty / full viral capsid ratio. Processor 2304 acquires optical signals or binding rate information from biosensor 2302 and performs the method provided herein for determining the empty / filled particle ratio (such as the empty / filled viral capsid ratio) in a sample, including: detecting and calculating the binding rate of particles in the set of standards or the test sample (e.g., using initial slope / rate analysis or endpoint / saturation analysis); generating a standard curve; comparing the calculated binding rate of particles in the test sample with the standard curve; and reference subtraction analysis (e.g., calculating the binding rate of the reference particles in the reference sample to the biosensor; calculating the binding rate of the particles in the set of standards). The binding rate of the reference particle is subtracted from the calculated binding rate of the particles in the test sample; a standard curve is generated based on the signal obtained by subtracting the calculated binding rate of the reference particle from the calculated binding rate of the particles in the set of standards; the calculated binding rate of the reference particle is subtracted from the calculated binding rate of the particles in the test sample; and the signal obtained by subtracting the calculated binding rate of the reference particle from the calculated binding rate of the particles in the test sample is compared with the standard curve to determine the empty / filled particle ratio in the test sample.

[0149] In some embodiments, the biosensor includes an optical fiber having a proximal end portion and a distal end portion, the proximal end portion being configured to receive light from a light source and to transmit reflected light to a detector. The distal end portion is configured to bind an analyte thereto such that the light reflected from the distal end portion undergoes a phase shift based on the thickness of the analyte bound to the distal end portion. In some embodiments, the biosensor further includes an optical resonator at the distal end portion of the optical fiber, the optical resonator including a first reflective surface and a second reflective surface, the first reflective surface being configured to reflect light with a first phase, and the second reflective surface being configured to reflect light with a second phase, the second phase being phase-shifted based on the thickness of the analyte bound to the optical resonator.

[0150] In some embodiments of the methods provided herein, signals (such as those generated by viral capsid binding to a biosensor) are measured by a detector. The signals can be detected based on any label-free technique used to detect changes in the properties of the sensor surface, such as BLI, surface plasmon resonance (SPR), surface acoustic wave (SAW), quartz crystal microbalance (QCM), and reflectance interferometry (RIfS). In certain embodiments, the signals are measured by an interferometer. The interferometer may include the biosensor and may constitute a BLI sensor. In some embodiments, the interferometer further includes: a first optical waveguide configured to receive light from a light source; a second optical waveguide configured to transmit reflected light to a detector; and an optical coupler spatially separating 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. In some embodiments, the interferometer further includes a light source optically communicated with and configured to provide light to the first optical waveguide. In some embodiments, the interferometer further includes a detector configured to receive light from the second optical waveguide. In some embodiments, the first and second optical waveguides are disposed in an optical fiber bundle.

[0151] For example, an interferometer may include a light source, optical components, and a detector unit. The biolayer interferometry (BLI) sensor or optical component serves as a sensing element or detector tip to detect analytes attached to its end. The detector unit detects interference signals generated by interference light waves reflected from the optical component. The light source directs light to the optical component, and the light is reflected back to the detector unit through an optical coupling assembly. The coupling assembly includes a first optical waveguide or fiber extending from the light source to the optical component, a second optical waveguide or fiber transmitting the reflected light from the optical component to the detector, and an optical coupler optically coupling the first and second optical waveguides. In some embodiments, the coupling assembly includes a lens system configured to focus a light beam on the upper surface of the optical component and to direct reflected interference light from the optical component to the detector.

[0152] The light source can be a white light source (such as a light-emitting diode (LED)) that produces light across a broad spectrum, for example, 400 nm or less to 700 nm or greater, typically within a spectral range of at least 100 nm. In some embodiments, the light source can be multiple light sources, each with a different characteristic wavelength, such as LEDs designed to emit light at different selected wavelengths within the visible light range. The same function can be achieved by a single light source (such as a white light source) in conjunction with suitable filters to direct light with different selected wavelengths to the optical components.

[0153] The detector may be a spectrometer (such as a charge-coupled device (CCD)) capable of recording the spectrum of reflected interference light from the optical component. In some embodiments, where the light source operates to direct different selected wavelengths to the optical component, the detector may be a simple photodetector for recording the light intensity at each different illumination wavelength. In some embodiments, the detector may include one or more filters that allow the detection of light intensity (e.g., the light intensity from a white light source) at each of a plurality of selected wavelengths of the interference reflected wave.

[0154] The first optical waveguide and / or the second optical waveguide may be in the form of a fiber bundle (FOB). As shown, the first optical waveguide includes a plurality of fiber elements surrounding a single fiber element of the second optical waveguide. This arrangement separates the transmission of light from the light source from the transmission of reflected light from the optical components to the detector. It should be understood that other arrangements of the first and second optical waveguides may allow spatial separation of light from the light source and reflected light from the optical components. Separation of light from the light source from the reflected light from the optical components can improve the signal-to-noise ratio (SNR) of the device. In some embodiments, the first optical waveguide is a single fiber, and the second fiber waveguide is formed of multiple fibers.

[0155] When the BLI sensor is attached to the optical coupler, the distal tip of the fiber bundle can be aligned with the proximal end portion of the fiber. The BLI sensor can be fixedly attached to the optical coupler to align and maintain the position of the proximal end portion relative to the tip.

[0156] The BLI sensor may include an 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. The BLI sensor may have an optical resonator having a first reflective surface and a second reflective surface located distal to the first reflective surface. The optical fiber is substantially transparent between its proximal end and its distal end. The optical resonator may be transparent between the first reflective surface and the second reflective surface. The distance between the first reflective surface and the second reflective surface defines the thickness of the optical resonator. The thickness of the optical resonator may be in the range of 50 nm to 5,000 nm, such as between 400 nm and 1,000 nm.

[0157] The second reflective surface is formed of a layer of analyte-binding molecules that effectively bind analyte molecules in a specific manner and with high affinity. That is, the analyte and antianalyte molecules are opposing members of a binding pair, which may include, but is not limited to, antigen-antibody pairs, complementary nucleic acid pairs, and receptor-binding agent pairs. In a particular embodiment, the analyte and antianalyte molecules carry opposite charges. For example, the analyte and antianalyte molecules carry negative and positive charges, respectively.

[0158] The refractive index of the optical fiber can be similar to that of the second reflective surface, such that light reflected from the second reflective surface primarily occurs in the layer formed by the analyte-bound molecules, rather than at the interface between the optical fiber and the analyte-bound molecules. Similarly, as analyte molecules bind to the distal end portion of the optical component, light reflected from the distal end portion of the component primarily occurs in the layer formed by the analyte-bound molecules and the bound analyte, rather than at the interface region.

[0159] The first reflective surface of the optical component is formed as a transparent material layer with a refractive index significantly different from that of the optical fiber, such that the layer reflects a portion of the light guided to the optical component.

[0160] The thickness of the analyte-binding layer disposed in the distal end portion of the optical element can be designed to optimize overall sensitivity based on specific hardware and optical components. Conventional immobilization chemical methods are used to chemically (e.g., covalently) attach the analyte-binding molecule layer to the lower surface of the optical element.

[0161] In some embodiments, the analyte binding layer is formed under conditions in which the distal end surface of the optical fiber is densely coated, thereby causing analyte molecules to bind to the layer and thus varying the thickness of the layer, rather than filling the layer. The analyte binding layer can be a single-layer or multi-layer matrix.

[0162] The presence, concentration, and / or binding rate of an analyte to the optical component are measured by the interference of reflected light beams from two reflective surfaces in the optical component. Specifically, the average thickness of the first reflective layer changes accordingly as analyte molecules attach to or detach from the surfaces. Since the thickness of all other layers remains constant, the interference wave formed by the light waves reflected from the two surfaces undergoes a phase shift according to this thickness change.

[0163] Assume there are two reflected light beams, a first beam reflected from a first reflective surface, and a second beam reflected at a second reflective surface from the analyte-bound molecules, the bound analyte, and the surrounding medium. The conversion of a phase shift into a change in the thickness of the bound analyte is well known in the art.

[0164] While several embodiments of the present disclosure have been shown in the accompanying drawings, they are not intended to be limited thereto, but rather are intended to be as broad as permitted in the art, and the description should be understood in the same manner. Any combination of the above embodiments is also contemplated and falls within the scope of the appended claims. Therefore, the above description should not be construed as restrictive, but merely as illustrative of particular embodiments. Other modifications that fall within the scope of the appended claims will be contemplated by those skilled in the art.

Claims

1. A method for determining the empty / full particle ratio, the method comprising: A set of standards is brought into contact with a biosensor to bind particles from the set of standards to the biosensor, the set of standards comprising particles at a first concentration and different empty / filled ratios in a first buffer solution; The test sample is brought into contact with the biosensor to bind particles in the test sample to the biosensor, the test sample comprising particles in a second buffer solution with an unknown empty / full ratio; For each of the set of standards and the test sample, detect the optical signal associated with the particle binding to the biosensor; as well as The empty / filled particle ratio in the test sample is determined by comparing the optical signals associated with particles bound to the biosensor in the test sample with the optical signals associated with particles bound to the biosensor in the set of standards.

2. The method according to claim 1, wherein, The particles include viral capsids, liposomes, lipid nanoparticles, or polymer nanoparticles.

3. The method according to claim 2, wherein, The viral capsid includes the capsid of adeno-associated virus (AAV).

4. The method according to claim 1, wherein, Detecting the optical signals associated with particles in the set of standards binding to the biosensor and the optical signals associated with particles in the test sample binding to the biosensor includes: Calculate the binding rate of particles from the set of standards to the biosensor; A standard curve is generated based on the binding rate of particles from the set of standards to the biosensor. Calculate the binding rate of particles in the test sample to the biosensor; and The calculated binding rate of the particles in the test sample is compared with the standard curve.

5. The method according to claim 4, wherein, The binding rates of particles from the set of standards and the binding rates of particles from the test samples were calculated using initial slope / rate analysis.

6. The method according to claim 4, wherein, The binding rates of particles from the set of standards and the binding rates of particles from the test samples were calculated using endpoint / saturation analysis.

7. The method according to claim 4, wherein, Generating a standard curve involves comparing the calculated binding rate of particles in the set of standards with the empty / filled particle ratio in the set of standards.

8. The method according to claim 1, wherein, The test sample includes multiple test samples, and the method includes adjusting the multiple test samples to contain particles at a second concentration.

9. The method of claim 1, further comprising: Before bringing the test sample into contact with the biosensor, the test sample is adjusted to contain particles at the first concentration.

10. The method according to claim 1, wherein, The first buffer and the second buffer are the same.

11. The method according to claim 1, wherein, The set of standards was prepared in the following manner: The samples containing empty-shell particles and the sample containing full-shell particles were adjusted to contain particles at the first concentration; and Samples containing empty shell particles and samples containing full shell particles were mixed and adjusted in different proportions to generate a set of standards with different empty / full particle ratios.

12. The method according to any one of claims 4 to 11, further comprising pairing each of the set of standards or the test samples with one or more reference samples containing reference particles, wherein, The one or more reference samples have the same or lower full-shell particle ratio as the set of standards or the test samples.

13. The method according to claim 12, wherein, The reference particle includes the empty shell particle.

14. The method according to claim 12, wherein, The one or more reference samples have the same characteristics as the paired set of standards or the test samples, wherein the characteristics include one or more of particle size, particle composition, particle concentration, buffer content, incubation time, biosensor regeneration cycle, and experimental days.

15. The method of claim 12, further comprising: The one or more reference samples are brought into contact with the biosensor to bind the reference particles in the one or more reference samples to the biosensor; as well as Calculate the binding rate of the reference particles in the one or more reference samples to the biosensor.

16. The method of claim 15, further comprising: The attributable binding rate of particles in the set of standards is calculated by subtracting the binding rate of the reference particles in one or more reference samples paired with each of the set of standards from the binding rate of particles in the set of standards. A standard curve is generated based on the attribution binding rate of the particles in the set of standards; The attributable binding rate of particles in the test sample is calculated by subtracting the binding rate of reference particles in one or more reference samples paired with the test sample from the binding rate of particles in the test sample. as well as The attribution binding rate of particles in the test sample is compared with the standard curve to determine the empty / filled particle ratio in the test sample.

17. The method according to claim 1, wherein, The test sample is a crude sample including a viral capsid.

18. The method according to claim 1, wherein, The optical signal comprises light reflected from two different locations associated with the biosensor.

19. The method according to claim 1, wherein, The optical signal includes optical interference generated by biological layer interferometry (BLI).

20. The method of claim 1, comprising: The set of standards and the test samples are provided in the holes of the multi-hole plate.

21. The method according to claim 1, comprising: The test sample and the set of standards are contacted in sequence.

22. The method according to claim 1, comprising: Simultaneously contact the set of standards and the test sample.

23. A method for determining the empty / full viral capsid ratio, the method comprising: Acquire the optical signal of a set of standards, the set of standards comprising viral capsids at a first viral titer and different empty / filled ratios in a first buffer solution; Acquire a first reference optical signal, the first reference optical signal including the optical signal of a first reference sample, the first reference sample including a viral capsid in a first buffer solution at the first virus titer and at an empty / full ratio; The attribution binding rate of particles in the set of standards is calculated by subtracting the first reference optical signal from the optical signal of each of the standards. Acquire the optical signal of the test sample, which comprises a viral capsid in a second buffer solution at a second viral titer and an unknown empty / filled ratio; Acquire a second reference optical signal, the second reference optical signal including the optical signal of a second reference sample, the second reference sample including a viral capsid in the second buffer solution at the second virus titer and at an empty / filled ratio; The attributable binding rate of the particles in the test sample is calculated by subtracting the optical signal of the reference particle from the binding rate of the particles in the test sample. as well as The empty / full viral capsid ratio of the test sample is determined by comparing the attributable binding rate of particles in the test sample with the attributable binding rate of particles in the set of standards.

24. The method according to claim 23, wherein, The optical signals of the set of standards are obtained based on the binding rate of the viral capsid in the set of standards to the biosensor. The optical signal of the first reference sample is obtained based on the binding rate of the viral capsid in the first reference sample to the biosensor. The optical signal of the test sample is obtained based on the binding rate of the viral capsid in the test sample to the biosensor; and The optical signal of the second reference sample is obtained based on the binding rate of the viral capsid in the second reference sample to the biosensor.

25. The method according to claim 23, wherein, The optical signal includes optical interference generated by biological layer interferometry (BLI).

26. The method according to claim 23, wherein, The binding rate of the viral capsid to the biosensor was measured using initial slope / rate analysis.

27. The method according to claim 23, wherein, The binding rate of the viral capsid to the biosensor was measured using endpoint / saturation analysis.

28. A method for determining the empty / full viral capsid ratio, the method comprising: A set of standards or test samples is paired with a reference sample, wherein the reference sample has a full-shell virus capsid ratio that is less than or equal to that of the set of standards or the test sample. A set of standards and their paired reference samples are brought into contact with a biosensor to bind viral capsids from the set of standards or their paired reference samples to the biosensor, the set of standards comprising viral capsids at the same viral titer and different empty / filled ratios. Calculate the binding rate of the viral capsid in the set of standards to the biosensor; Calculate the binding rate of the viral capsid in the reference sample to the biosensor; Subtract the calculated binding rate of the reference sample from the calculated binding rate of the viral capsid in the set of standards; A standard curve is generated by comparing the binding rate of particles in the set of standards (subtracted from the reference) with the known empty / full particle ratio in the set of standards. The test sample and its paired test reference sample are brought into contact with the biosensor to bind the viral capsid in the test sample or its paired test reference sample to the biosensor, the test sample comprising a viral capsid in an unknown empty / full ratio. Calculate the binding rate of the viral capsid in the test sample to the biosensor; Calculate the binding rate of the reference virus capsid in the test reference sample to the biosensor; Subtract the calculated binding rate of the test reference sample from the calculated binding rate of the viral capsid in the test sample; as well as The empty / full viral capsid ratio in the test sample is determined by extrapolating the reference-subtracted binding rate of the viral capsid in the test sample to the standard curve.

29. The method according to claim 28, wherein, The reference sample includes a reference viral capsid of the same type as the set of standards or the test sample paired with it.

30. The method according to claim 28, wherein, Each of the set of standards or the test samples has the same characteristics as the reference sample containing the reference viral capsid, wherein the characteristics include one or more of the following: viral titer, buffer content, incubation time, biosensor regeneration cycle, and experimental days.

31. The method according to claim 28, wherein, The binding rates of the viral capsids in the set of standards and the viral capsids in the test samples were calculated using initial slope / rate analysis.

32. The method according to claim 28, wherein, The binding rates of the viral capsid in the set of standards and the viral capsid in the test samples were calculated using endpoint / saturation analysis.

33. The method according to claim 28, wherein, The test reference sample is the same as the reference sample.

34. A system for determining the empty / full viral capsid ratio, the system comprising: A biosensor configured to bind to viral capsids in a set of standards or in a test sample, detect and associate an optical signal with the viral capsid bound to the biosensor, and determine the empty / filled viral capsid ratio in the test sample based on the optical signal. The set of standards includes viral capsids with the same viral titer and different empty / full ratios, and the test samples include viral capsids with unknown empty / full ratios.

35. The system according to claim 34, wherein, The optical signal comprises light reflected from two different locations associated with the biosensor.

36. The system according to claim 34, wherein, The biosensor is a biolayer interferometry (BLI) biosensor.

37. The system of claim 34, further comprising a container configured to contain the set of standards or the test samples.

38. The system according to claim 34, wherein, The container is a hole in a perforated plate.

39. The system according to claim 34, wherein, 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, generate a standard curve based on a signal obtained by subtracting the calculated binding rate of the reference viral capsid from the calculated binding rate of the viral capsid in the set of standards, and determine the empty / full viral capsid ratio in the test sample based on the signal obtained by subtracting the calculated binding rate of the reference viral capsid from the calculated binding rate of the viral capsid in the test sample and the standard curve.

40. A system for determining the empty / filled particle ratio in a sample, the system comprising: A biosensor configured to bind particles in the sample and detect optical signals associated with the particles bound to the biosensor; as well as A processor configured to perform the method according to any one of claims 1 to 33.

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