Analyzing ultracentrifugation methods

Abstract

Methods of characterizing biomolecular formulations are provided. In some examples, there is provided a method of characterizing a viral particle formulation, the method comprising subjecting the viral particle formulation to analytical ultracentrifugation under sedimentation velocity conditions, measuring sedimentation of the viral particle at a plurality of points in time, determining a friction ratio function c (s, ff0) and a sedimentation coefficient for the viral particle formulation, mapping the sedimentation coefficient (S) with the c (s, ff0) to obtain c (s, ff0), and characterizing the c (s, ff0). Ff0) distribution, and characterizing the presence and / or relative composition of viral particles comprising a totiviral genome, a partial viral genome, an over-packaged viral genome, and / or an empty viral particle in the viral particle formulation.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 392,261, filed on July 26, 2022. Technical Field

[0003] The present disclosure relates to methods for characterizing preparations of biomolecules, particularly viral particles, using analytical ultracentrifugation. Background Art

[0004] Analytical ultracentrifugation (AUC) is a technique that uses an optical detection system to monitor sample sedimentation in real time. AUC provides information such as shape, size, molecular weight, and conformational changes of biomolecules such as proteins, nucleic acids, viruses, and other macromolecules. One application of AUC is the characterization of biomaterial preparations for therapeutic use, such as viral preparations or antibody preparations for gene therapy. Summary of the invention

[0005] The text discloses methods for analyzing biomolecule preparations using analytical ultracentrifugation. In specific examples, these methods can be used to analyze viral particle preparations, such as characterizing the presence or relative amount of viral particles including complete genomes in viral particle preparations. In some examples, under the same circumstances of data analysis, the provided methods provide advantages over previous methods, such as determining friction ratios (indicating the shape of particles), estimating molecular weights and Stokes or hydrodynamic radii of particles (indicating their size in nanometers), as well as sedimentation rates and peak areas.

[0006] Methods for characterizing biomolecule preparations are provided. In examples, the biomolecule preparation is a viral particle preparation. In some examples, the methods include characterizing the viral particle preparation by: subjecting the viral particle preparation to analytical ultracentrifugation under sedimentation velocity conditions, measuring the sedimentation of the viral particles at multiple time points, determining the friction ratio function c(s, ff0) and the sedimentation coefficient of the viral particle preparation, and plotting c(s, ff0) against the sedimentation coefficient (S) to obtain a c(s, ff0) distribution; and characterizing the presence and / or relative composition of viral particles containing full viral genomes, partial viral genomes, over-packaged viral genomes, and / or empty viral particles in the viral particle preparation.

[0007] In other examples, the methods include assessing the genomic integrity of viral particles in a viral preparation by subjecting the viral particle preparation to analytical ultracentrifugation, measuring the sedimentation of the viral particles at multiple time points, determining the friction ratio function c(s,ff0) and the sedimentation coefficient of the viral particle preparation, plotting c(s,ff0) against the sedimentation coefficient (S) to obtain a c(s,ff0) distribution, and characterizing the presence and / or relative composition of viral particles containing full viral genomes, partial viral genomes, and / or empty viral particles in the viral particle preparation, thereby assessing the genomic integrity of viral particles in the viral preparation. In some examples, c(s,ff0) is calculated using the Lamm equation, for example, using the SEDFIT software package.

[0008] In some examples, the methods include measuring the sedimentation of viral particles by measuring absorbance, such as measuring the absorbance at about 280 nm. In some examples, the methods include measuring absorbance at multiple time points, for example, at about 75 time points. In some examples, the interval between each of the multiple time points is about 20 seconds. In one example, analytical ultracentrifugation is performed at about 15,000 rpm and at a temperature of about 20°C.

[0009] The disclosed method includes characterizing the presence of virus particles including full viral genomes, partial viral genomes, over-packaging viral genomes and / or empty viral particles in a virus particle preparation and / or evaluating the genome integrity of virus particles in a preparation. These methods include identifying the peak corresponding to the sedimentation coefficient in the c(s, ff0) distribution, which corresponds to virus particles including full viral genomes, partial viral genomes, over-packaging viral genomes and / or empty viral particles. In some examples, these methods also include integrating the area under each peak identified in the c(s, ff0) distribution. The relative composition of each virus particle in the virus particles including full viral genomes, partial viral genomes, over-packaging viral genomes and / or empty viral particles in the virus particle preparation is determined by comparing the peak area values ​​in the preparation.

[0010] In some examples, the viral particles in the preparation are capsid particles. In other examples, the viral particle preparation is an adeno-associated virus (AAV) particle (e.g., AAV2 particle, AAV3 particle, AAV6 particle, AAV8 particle or AAV9 particle) preparation.

[0011] In one example, the method is a method for determining the relative composition in an adeno-associated virus (AAV) particle preparation, wherein the AAV particles include a full viral genome, a partial viral genome, an over-packaged viral genome, and / or an empty viral particle. These methods include subjecting the AAV particle preparation to analytical ultracentrifugation at 15,000 rpm, collecting absorbance data at 280 nm at 75 time points at 20 second intervals to measure the sedimentation of the viral particles, determining the friction ratio function c(s, ff0) and the sedimentation coefficient of the viral particle preparation, plotting c(s, ff0) against the sedimentation coefficient (S) to obtain a c(s, ff0) distribution, assigning a peak in the c(s, ff0) distribution to a viral particle category selected from a viral particle comprising a full viral genome, a viral particle comprising a partial viral genome, an over-packaged viral genome, and an empty viral particle, integrating the area under each peak in the c(s, ff0) distribution; and comparing the area under each peak of the AAV particles including the full viral genome, the partial viral genome, the over-packaged viral genome, and / or the empty viral particles in the AAV particle preparation to determine the relative composition.

[0012] The foregoing and other features of the present disclosure will become more apparent from the following detailed description made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a diagram illustrating exemplary species of viral particles (such as AAV capsids) that can be characterized using the provided methods. In some examples, the capsid or viral particle contains a complete viral genome (is "full"). In other examples, the capsid or viral particle does not include any viral DNA ("empty") or contains a truncated or incomplete viral genome ("partial" genome).

[0014] Figure 2 Data from an exemplary analysis of AAV8 viral particles packaged with a green fluorescent protein genome are shown. The upper figure shows a graph of absorbance at 280nm versus the radius of the AUC cell sector in centimeters. The figure shows the overall AUC scans collected during the experiment. The scans show the sedimentation process of species present in the sample loaded into the AUC cell sample sector. The negative peak represents the reference buffer meniscus in the AUC cell reference sector. The red line identifies the sample meniscus in the AUC cell sample sector in the SEDFIT software. The left green line identifies where the SEDFIT software analysis should start modeling sedimentation in the collected scans. The right green line defines the end point of SEDFIT modeling sedimentation. The middle figure shows the residuals of the experiment, indicating the difference between the observed absorbance data contained in the scan and the data values ​​predicted in the modeled distribution curve of the species present in the sample. The figure below shows the modeled distribution curve, which indicates which species are present in the sample, wherein each peak corresponds to a specific AAV species (empty, partial, full, etc.).

[0015] Figure 3 Data from an exemplary analysis of AAV virus particles for calculation of the friction ratio (f / f0), molecular weight (sw,ffow), and Stokes radius (Rs) shown are shown. The peak shown at 87.74S corresponds to the full GFP AAV species; the peak shown at 102S corresponds to the over-packaging AAV species, corresponding to a genome size larger than the full GFP AAV species. DETAILED DESCRIPTION

[0016] I. Terminology

[0017] The following explanation is provided to better describe the present disclosure and guide those of ordinary skill in the art in the practice of the present disclosure. Unless the context clearly stipulates otherwise, the singular form "one / kind" and "the / said" refer to one / kind or more than one / kind. Unless the context clearly indicates otherwise, the term "or" refers to a single element in the stated alternative element, or a combination of two or more elements. As used herein, "comprising" means "including". Therefore, "comprising A or B" means "comprising A, B, or A and B", without excluding other elements.

[0018] Unless otherwise indicated, all numbers indicating component quantities, molecular weights, percentages, temperatures, times, etc., as used in the specification or claims are understood to be modified by the term "about". Therefore, unless otherwise indicated, implicitly or explicitly, the numerical parameters are approximate values, which may depend on the desired properties sought and / or the detection limits under standard test conditions / methods. When an example is directly and clearly distinguished from the prior art discussed, the example numbers are not approximate unless the word "about" is explicitly stated.

[0019] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0020] Adeno-associated virus (AAV): A small, non-enveloped virus that infects humans and some other primates. AAV is not known to cause disease and elicits a very mild immune response. Gene therapy vectors utilizing AAV can infect both dividing and quiescent cells and can persist in an extrachromosomal state without integrating into the host cell's genome. These characteristics make AAV an attractive viral vector for gene therapy. In some examples, AAV is a recombinant AAV and is replication-defective.

[0021] Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein or virus) has been substantially separated or purified from other biological components (e.g., other chromosomal and extrachromosomal DNA and RNA, proteins and / or organelles). Nucleic acids, proteins and / or viruses that have been "isolated" include nucleic acids, proteins and viruses purified by standard purification methods. The term also includes nucleic acids, proteins and viruses prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins.

[0022] The term "isolated" (or purified) does not require absolute purity; rather, it is intended as a relative term. Thus, for example, isolated or purified nucleic acids, proteins, viruses, or other active compounds are those that are completely or partially separated from related nucleic acids, proteins, and other contaminants. In some examples, the term "substantially purified" refers to nucleic acids, proteins, viruses, or other active compounds that have been separated from cells, cell culture media, or other crude preparations and subjected to fractionation to remove various components of the initial preparation, such as proteins, cell debris, and other components.

[0023] Recombinant: A recombinant nucleic acid molecule is a nucleic acid molecule that includes a sequence that does not occur naturally or has a sequence that is produced by the artificial combination of two otherwise isolated sequence segments. This artificial combination can be accomplished by chemical synthesis or by artificial manipulation of isolated nucleic acid molecule segments, such as by genetic engineering techniques.

[0024] Similarly, a recombinant virus is a virus having a non-naturally occurring nucleic acid sequence (such as a heterologous sequence not from the virus) or a nucleic acid sequence prepared by artificially combining sequences from at least two different sources. The term "recombinant" also includes nucleic acids, proteins and viruses that are altered only by adding, replacing or deleting a portion of a natural nucleic acid molecule, protein or virus.

[0025] As used herein, "recombinant AAV" (rAAV) refers to an AAV particle into which a heterologous nucleic acid molecule has been packaged. The heterologous nucleic acid molecule of the recombinant AAV is a combination of nucleic acid sequences present and absent in the AAV genome; for example, a therapeutic nucleotide sequence flanking the inverted terminal repeat (ITR) nucleotide sequences of AAV. Therapeutic nucleic acids are nucleic acids that slow disease progression, reduce disease severity or symptoms. The slowed progression, reduced severity or symptoms of a disease can be caused by one or more transcripts of a heterologous nucleic acid molecule, translation of one or more transcripts of a heterologous transcript, or simply by the presence of a heterologous nucleic acid.

[0026] Fully recombinant AAV is an AAV in which the AAV capsid encapsulates a heterologous nucleic acid molecule that is flanked by ITRs at each end. Empty recombinant AAV is an AAV in which the capsid does not encapsulate a heterologous nucleic acid sequence. Partially recombinant AAV is an AAV in which the AAV capsid encapsulates a heterologous nucleic acid but the encapsulated heterologous nucleic acid is not full-length; for example, lacks all or part of the therapeutic nucleic acid and / or ITRs.

[0027] Reference: A sample, standard, or value used for comparison. In some examples, the reference is a sample containing the same buffer but lacking AAV particles (e.g., when analyzing an AAV viral particle preparation). In other examples, the reference is a sample that includes the same buffer as the sample being analyzed and a different biomolecule (e.g., a sample that includes 70S ribosomes can be a reference for an AAV viral particle preparation).

[0028] SEDFIT algorithm: An algorithm for analyzing hydrodynamic data, such as sedimentation velocity (Schuck, Biophys. J. 78: 1606-1619, 2000). For each sedimentation coefficient, a solution of Lamm's equation is used to simulate the sedimentation boundary, with a constant particle shape and solvent friction ratio.

[0029] Sedimentation coefficient: A measure of how quickly a particle of a given size and shape will sediment, for example under centrifugation. The sedimentation coefficient is the ratio of the sedimentation velocity of the particle to the applied acceleration. The sedimentation coefficient is expressed in Svedberg units (S), where one Svedberg unit is equal to 10 -13 Second.

[0030] Vector: A nucleic acid molecule (such as a plasmid or virus) that allows the insertion of exogenous nucleic acid without destroying the ability of the vector to replicate and / or integrate in a host cell. The vector may include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. The vector may also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of the inserted gene or genes. In some non-limiting examples, the vector is AAV.

[0031] II. Overview

[0032] Analytical ultracentrifugation (AUC) is a technique that can be used to analyze the properties of biomolecules (such as proteins, nucleic acids, or viral particles), such as molecular weight, biomolecule shape, fluid dynamic properties, and thermodynamic properties. AUC analysis can be used to characterize the biophysical properties of many types of molecules by measuring their migration through a solvent in a centrifugal field. In some examples, AUC analysis includes determining sedimentation velocity. Sedimentation velocity provides information about the hydrodynamic properties of particles, including size, shape, and concentration.

[0033] The radial sedimentation of biomolecules during ultracentrifugation is affected by a variety of factors, including the heterogeneity of the molecules in solution, intrinsic chemical interactions, and migration through the buffer. The process of radial sedimentation is described mathematically by Lamm's equation, which shows here the sedimentation of an ideal particle in the absence of interaction as a function of time t and distance r from the axis of rotation:

[0034]

[0035] where c is the particle concentration, and the parameters D, S, and ω represent the diffusion constant, sedimentation coefficient, and rotor angular velocity, respectively.

[0036] The sedimentation coefficient S depends on the particle molecular weight M, differential specific volume and its size represented by the hydrodynamic radius or Stokes radius Rs. η 0 According to the Svedberg equation (N A is Avogadro's number), S also depends on the solvent density ρ 0 And viscosity:

[0037]

[0038] In addition, D and Rs are connected by the friction coefficient f through the following Stokes relationship:

[0039] f=6πη°Rs

[0040] Relationship with Einstein-Stokes:

[0041] D=RT / N A f

[0042] where R is the gas constant and T is the temperature in Kelvin.

[0043] The friction ratio f / f0 allows the friction coefficient f (and the Stokes radius) to be compared with the minimum friction coefficient f0 corresponding to the volume of the particle being a perfect sphere with radius R0:

[0044]

[0045] The Lamm equation is solved by using an integral function that models a specific type of molecular interaction. Two such functions are c(s) and c(s,ff0). c(s) is simply a function of the sedimentation velocity and the size distribution curve plots c(s) versus s (sedimentation velocity). c(s) imposes a set friction ratio value (f / f0=1.2) because c(s) assumes a similar friction ratio value for each sedimentation species in solution. c(s,ff0) is a function of s and friction ratio (f / f0). The size distribution curve plots the function versus s and friction ratio and is represented as a 2D graph. It calculates size and shape distributions and does not impose a set value for f / f0, but rather uses a range of values. f / f0 describes the shape of the molecule, where a ratio of 1.2-1.3 describes a spherical shape for spherical compact particles and an f / f0 of about 1.5 describes a slightly asymmetric shape. Elongated rods and coil shapes have f / f0>2.0. In addition, from this function, other properties such as molecular weight and Stokes radius can be calculated. These functions can be assessed using software packages such as SEDFIT.

[0046] In some examples, the sedimentation of biomolecules is measured by monitoring absorbance. In some examples, the absorbance is measured at about 230nm-280nm. In one example, the absorbance is measured at about 230nm. In other examples, the absorbance is measured at about 260nm. In a further example, the absorbance is measured at 280nm. In another example, the absorbance is measured at about 260nm and about 280nm. In such an example, the distribution curves at 260nm and 280nm can overlap, for example, to confirm or characterize biomolecules containing DNA with higher absorbance at 260nm. In some examples, the sedimentation of biomolecules is measured using an ultracentrifuge including a detection system (such as a UV detection system (such as a UV / VIS spectrophotometer)).

[0047] In some examples, the sedimentation of the biomolecule is measured by scanning at multiple time points, such as about 30-100 scans. In some examples, sedimentation is measured by about 30-50 scans, about 50-75 scans, or about 75-100 scans (e.g., about 30 scans, about 40 scans, about 50 scans, about 60 scans, about 70 scans, about 75 scans, about 80 scans, about 90 scans, or about 100 scans). In one example, sedimentation is measured with about 75 scans. In some examples, the time between scans is about 15 seconds to about 15 minutes, such as about 15 seconds to about 45 seconds, about 20 seconds to about 1 minute, about 30 seconds to about 2 minutes, about 1 minute to about 5 minutes, about 2.5 minutes to about 10 minutes, or about 7.5 minutes to about 15 minutes (e.g., about 15 seconds, about 20 seconds, about 30 seconds, about 45 seconds, about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 7.5 minutes, about 10 minutes, or about 15 minutes). In one example, the time interval between scans is about 20 seconds. In other examples, the time interval between scans is about 180 seconds. The appropriate interval between scans can be selected, for example, based on the biomolecule being detected.

[0048] Sedimentation is measured over a period of about 15 minutes to about 2 hours, such as about 15 minutes to 30 minutes, about 20 minutes to about 40 minutes, about 25 minutes to about 45 minutes, about 40 minutes to about 1 hour, about 1 hour to 1.5 hours, or about 1.5 hours to 2 hours (e.g., about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, or about 2 hours). In one example, sedimentation is measured over a period of about 25 minutes.

[0049] In some examples, the analytical ultracentrifugation is performed at about 3000 rpm to about 50,000 rpm, such as about 3000 rpm to about 7500 rpm, about 5000 rpm to about 10,000 rpm, about 10,000 rpm to about 15,000 rpm, about 15,000 rpm to about 20,000 rpm, about 20,000 rpm to about 25,000 rpm, about 25,000 rpm to about 30,000 rpm, about 30,000 rpm to about 35,000 rpm, about 35,000 rpm to about 40,000 rpm, about 40,000 rpm to about 45,000 rpm, or about 45,000 rpm to about 50,000 rpm (e.g., In one example, the analytical ultracentrifugation is performed at about 15,000 rpm. In another example, the analytical ultracentrifugation is performed at about 40,000 rpm. The appropriate centrifugation speed can be selected based on the biomolecule being detected, for example, higher speeds are used for smaller molecules such as monoclonal antibodies compared to viral particles.

[0050] In some examples, the analytical ultracentrifugation is performed at about 4°C to about 30°C, such as about 4°C to about 10°C, about 10°C to about 15°C, about 15°C to about 20°C, about 20°C to about 25°C, or about 25°C to about 30°C (e.g., about 4°C, about 6°C, about 8°C, about 10°C, about 12°C, about 15°C, about 20°C, about 22°C, about 25°C, or about 30°C). In one example, the analytical ultracentrifugation is performed at about 20°C.

[0051] Exemplary biomolecules that can be analyzed or characterized using the methods provided herein include, but are not limited to, virus-like particles (such as AAV particles), viral vectors (such as LV or adenovirus), ribozymes (including ribosomes, such as 70S, 80S-human ribosomes), monoclonal antibodies, large glycoprotein polymers (e.g., antibodies. Such as IgM), aggregates, multimeric molecules (such as dimers, trimers, etc., for example, rubella or influenza virus hemagglutinin rosettes), lipid nanoparticles (LNPs) or exosomes. In some examples, the disclosed methods are used to characterize or analyze viral particle preparations. In other examples, the disclosed methods are used to characterize or analyze proteins, such as antibodies (e.g., monoclonal antibodies).

[0052] III. Methods for Characterizing Viral Particle Preparations

[0053] In examples, methods of characterizing a viral particle preparation are provided. In other examples, methods of assessing the genomic integrity of viral particles in a viral preparation are provided.

[0054] In some examples, the method of characterizing a viral particle preparation includes subjecting the viral particle preparation to analytical ultracentrifugation under sedimentation velocity conditions and measuring the sedimentation of the viral particles at multiple time points. These methods also include determining the friction ratio function c(s,ff0) sedimentation coefficient of the viral particle preparation, plotting the c(s,ff0) sedimentation coefficient (S) to obtain a c(s,ff0) distribution, and characterizing the presence and / or relative composition of viral particles (such as viral particles including a full viral genome, a partial viral genome, an over-packaged viral genome, and / or an empty viral particle) in the viral particle preparation.

[0055] In other examples, methods for assessing the genomic integrity of viral particles in a viral preparation include subjecting the viral particle preparation to analytical ultracentrifugation and measuring the sedimentation of the viral particles at multiple time points. These methods also include determining the friction ratio function c(s, ff0) and the sedimentation coefficient of the viral particle preparation, plotting c(s, ff0) against the sedimentation coefficient (S) to obtain a c(s, ff0) distribution, and characterizing the presence and / or relative composition of viral particles containing full viral genomes, partial viral genomes, and / or empty viral particles in the viral particle preparation, thereby assessing the genomic integrity of viral particles in the viral preparation. The genomic integrity of the viral particles may include viral particles having a full viral genome (e.g., an intact or substantially intact genome, such as a complete genome), a partial viral genome (e.g., an incomplete viral genome), an over-packaged viral genome (e.g., containing a genome size larger than expected), or the viral particles may be empty viral particles (e.g., lacking or substantially lacking a viral genome). Exemplary viral particle species are Figure 1 In the description.

[0056] In other examples, the methods include or further include determining the friction ratio (f / f0), estimating the molecular weight, and / or the Stokes radius of the viral particles. The friction ratio allows the determination of the shape of the biomolecule, where f / f0 is 1 and is a perfect sphere. As the ratio increases, the shape deviates from a perfect sphere to a longer elliptical shape. Empty, partial genome, and full genome viral particles (such as AAV) are substantially spherical. Aggregates have a rectangular shape. Based on the known or calculated molecular weight of empty viral particles or viral particles with full genomes, estimating the molecular weight allows the determination of the type of viral particles present. Similar to f / f0, empty, partial genome, and full genome viral particles have similar diameters (e.g., about 20nm-25nm for AAV particles); however, aggregates can be identified based on the presence of larger diameter particles.

[0057] The AUC of the sample is performed as described in Section II. In some specific examples, AUC is performed at 15,000 rpm, and absorbance data at 280 nm are collected at 75 time points at 20 second intervals to measure the sedimentation of the virus particles. The friction ratio function c (s, ff0) and the sedimentation coefficient of the virus particle preparation are determined, and c (s, ff0) is plotted against the sedimentation coefficient (S) to obtain the c (s, ff0) distribution. The c (s, ff0) function is calculated using the Lamm equation, for example, using a software package. A software package for modeling AUC data as a Lamm equation is SEDFIT (Schuck, Biophys. J. 78: 1060-1619, 2000). In some examples, the parameters of the buffer density and viscosity are adjusted to correspond to the sample buffer. In one example, the sample buffer is Dulbecco's phosphate buffered saline (D-PBS), and the buffer density is set to 1.005584, and the buffer viscosity is set to 0.0102 poise. In some examples (including AAV particle analysis), the differential specific volume was 0.73, reflecting a globular protein. Additional exemplary parameters for analyzing AAC particles are shown in Example 3. However, one of ordinary skill in the art can adjust these parameters for other sample types or preparations.

[0058] Characterizing the presence of viral particles with full viral genomes, partial viral genomes, over-packaged viral genomes, and / or empty viral particles in a viral particle preparation includes identifying a peak in the c(s,ff0) distribution corresponding to a sedimentation coefficient corresponding to a viral particle including a full viral genome, a partial viral genome, and / or an empty viral particle. In some examples, the distribution of peaks corresponding to full viral genomes, partial viral genomes, over-packaged viral genomes, and / or empty viral particles provides a qualitative analysis of the preparation.

[0059] In other examples, these methods include integrating the area under each peak in the c(s, ff0) distribution. Determine the relative composition of each type of viral particles (those with full viral genome, partial viral genome, over-packaging viral genome and / or empty viral particles) in the viral particle preparation by comparing the peak area values. In some examples, relative composition is determined by comparing the area under the peak of the viral particles with the area under the peak of the viral particles with the area under the peak of the viral particles with the partial genome and the area under the peak of the empty viral particles, to determine the relative amount of the viral particles with full viral genome in the preparation. In other examples, relative composition is determined by comparing the area under the peak of the empty viral particles with the area under the peak of the viral particles with full viral genome and the area under the peak of the viral particles with partial viral genome, to determine the relative amount of the empty viral particles in the preparation. In a further example, the area under the peak of the viral particles with partial viral genome is compared with the area under the peak of the viral particles with full viral genome and the area under the peak of the empty viral particles, to determine the relative amount of the viral particles with partial viral genome in the preparation. In another example, the area under the peak of the viral particles with excessive packaging viral genome is compared with the area under the peak of the viral particles with full viral genome and the area under the peak of the empty viral particles to determine the relative amount of viral particles with excessive packaging viral genome in the preparation. This information can be used to evaluate processing efficiency (e.g., in a purification scheme) or for GMP settings. Therefore, in some examples, the composition percentage is used to develop or continue a purification scheme or determine whether a particular preparation is suitable for further use (e.g., administration to a patient).

[0060] In some examples, the viral particle preparation is an adeno-associated virus (AAV) particle preparation. AAV is a small, non-enveloped, helper-dependent parvovirus, classified as the Dependoparvovirus genus of the Parvoviridae family. AAV has a linear, single-stranded DNA genome of about 4.7 kb. The genome is flanked by inverted terminal repeats (ITRs) and two open reading frames (ORFs): rep and cap. The rep ORF encodes four replication proteins (Rep78, Rep68, Rep52, and Rep4), and the cap ORF encodes three viral capsid proteins (VP1, VP2, and VP3) and assembly activation protein (AAP). AAV requires a helper virus (such as adenovirus, herpes simplex virus, or other viruses) to complete its life cycle. AAV is currently used in many gene therapy clinical trials worldwide. Although AAV infects humans and some other primate species, it is not known to cause disease and cause a very mild immune response. Gene therapy vectors using AAV can infect dividing and quiescent cells and persist in an extrachromosomal state without integrating into the host cell genome. AAV has several desirable characteristics for gene therapy vectors, including the ability to bind and enter target cells, enter the nucleus, express for a long time in the nucleus, and low toxicity.

[0061] ITR is the only component required for the successful packaging of heterologous proteins in the AAV capsid. Therefore, in some examples, the disclosed method can be used to analyze the preparation of the AAV vector comprising a nucleic acid encoding a target gene operably connected to a promoter. In some examples, the AAV vector includes 5 and 3 ITRs, which flank the target nucleotides operably connected to the promoter. The vector may also include other elements, such as enhancer elements (e.g., nucleic acid sequences that increase the transcription rate by increasing the activity of the promoter) and / or polyadenylation signals. Any combination of ITR, enhancer, promoter, polyadenylation signal and / or other elements can be used in the AAV vector described herein.

[0062] In one example, the methods include determining the relative composition of adeno-associated virus (AAV) particles including full viral genomes, partial viral genomes, and / or empty viral particles in an AAV particle preparation by subjecting the AAV particle preparation to analytical ultracentrifugation at 15,000 rpm, collecting absorbance data at 280 nm at 20 second intervals at 75 time points to measure sedimentation of viral particles, and determining the friction ratio function c(s, ff0) and sedimentation coefficient of the viral particle preparation. Then, c(s, ff0) is plotted against the sedimentation coefficient (S) to obtain a c(s, ff0) distribution, peaks in the c(s, ff0) distribution are assigned to viral particle categories selected from viral particles having full viral genomes, viral particles having partial viral genomes, and empty viral particles, and the area under each peak in the c(s, ff0) distribution is integrated. The relative composition is determined by comparing the area under each peak, such as comparing the area under the peak of virus particles with a complete viral genome with the area under the peak of virus particles with a partial genome and the area under the peak of empty virus particles to determine the relative amount of virus particles with a complete viral genome in the preparation.

[0063] In some examples, the viral particle preparation is an adeno-associated virus (AAV) particle preparation, such as an AAV capsid particle. Exemplary AAV capsid particles include AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAV9 capsid, AAV10 capsid, AAV11 capsid, AAV12 capsid or a hybrid serotype. Use transcapsidation (ITRs from one serotype are cross-packaged into capsids of different serotypes), changes in the surface of AAV capsids (changes in the tropism of AAV capsids, by modifying the viral protein ratio or even eliminating one or two VPs), chimeric capsid serotypes (capsid subunits from different serotypes are assembled in a single virion) or adsorbing receptor ligands to the surface of AAV capsids to change tropism to produce hybrid serotypes. Examples of hybrid serotypes include rh74, rh10, hu68, and AAV-DJ. In some specific examples, the AAV particle is an AAV2, AAV3, AAV6, AAV8, or AAV9 particle, such as an AAV2 capsid particle, an AAV3 capsid particle, an AAV6 capsid particle, an AAV8 capsid particle, or an AAV9 capsid particle.

[0064] In other examples, the viral particle preparation is an adenovirus (adenovirus) preparation, a retrovirus (lentivirus, murine leukemia virus) preparation, or a herpes virus (herpes simplex virus) preparation.

[0065] III. Methods for Characterizing Biomolecule Preparations

[0066] In an example, a method for characterizing a biomolecule preparation is provided. Exemplary biomolecules are as described above. In some examples, the biomolecule is an antibody or a fragment thereof, such as a monoclonal antibody or a fragment thereof. In other examples, the biomolecule is a bacterial 70S ribosome (such as an E. coli 70S ribosome) or a human 80S ribosome.

[0067] In some examples, the method of characterizing the biomolecule preparation includes subjecting the biomolecule preparation to analytical ultracentrifugation under sedimentation velocity conditions and measuring the sedimentation of the biomolecule at multiple time points. These methods also include determining the friction ratio function c(s, ff0) and the sedimentation coefficient of the viral particle preparation, plotting the function c(s, ff0) against the sedimentation coefficient (S) and the friction ratio (ff0) to obtain a c(s, ff0) distribution, and characterizing the biomolecule preparation based on the c(s, ff0) distribution. In other examples, these methods include determining the friction ratio (e.g., molecular shape) of the biomolecule, estimating the molecular weight and / or Stokes radius (e.g., diameter).

[0068] In other examples, the methods include plotting the function c(s, ff0) versus S and M (molecular weight), plotting the function c(s, ff0) versus S and Rs, plotting the function c(s, ff0) versus S and D (diffusion), and / or plotting the function c(s, ff0) versus ff0 and M. In addition to component identification, these plots support further understanding of sample composition because they relate sedimentation velocity and friction ratio to other specific variables such as molecular weight, diffusion, and hydrodynamic radius.

[0069] Example

[0070] The following examples are provided to illustrate certain specific features and / or examples. These examples should not be construed as limiting the disclosure to the specific features or examples described.

[0071] Example 1

[0072] Analysis and characterization of AAV viral particles

[0073] Sample preparation: Thaw samples from 80°C storage to room temperature and mix thoroughly by pipetting up and down. Obtain a buffer that matches the sample matrix composition for sample dilution and background correction. To prepare samples in the correct concentration range for SV-AUC UV detection, measure the optical density (OD) of all samples at 280 nm using a UV spectrophotometer. 280 ). Measure OD 280 Three (3) times and calculate the average. Based on the average OD 280 Determine the dilution factor so that the optical density of the sample falls between 0.4 and 0.6 OD 280If the absorbance is below 0.4, the sample can be used neat. Using a Beckman-Coulter An-50 Ti analytical 8-position titanium rotor, seven (7) full assembly SV-AUC cells were constructed using a center piece, a two-sector 12 mm flow-through piece, and two quartz windows (the 8th position is reserved for a balance block). One sector is assigned to contain the sample, while the other is assigned to the corresponding buffer: the sample volume must be 400 μL, and the buffer volume is 410 μL-420 μL. One cell was assigned to the reference material (70S ribosomes), and the other six cells contained the samples.

[0074] AUC Preparation: On a Beckman-Coulter Optima AUC instrument, set up method scans with the following parameters: AN50Ti rotor, temperature 20°C; set scans to absorbance only in module 1; method stages set to Stage 1 Equilibration: 0 RPM, Delay 2 hours; Stage 2 Analysis: 15,000 RPM, Delay 0 hours; Stage 3 Hold: 0 RPM, Delay 0 hours. In Stage 2, select cells 1-7, enter 75 scans, 280 nm, scan frequency 20 seconds, data resolution 10 μm, minimum radius (cm) 5.8, maximum radius (cm) 7.2, one (1) replicate. Perform a vacuum test prior to running samples.

[0075] Data analysis using c(s,ff0) by SEDFIT: Import raw data from Optima AUC. Load scans into SEDFIT, one pool at a time. Select c(s,ff0) under Model. Parameters under Parameters are set as: Resolution: 100S, s min 2.00000, s max 200, Check Fit RI and Time Independent Noise Box. Buffer density and buffer viscosity are adjusted according to the buffer used in the sample matrix. Vbar is maintained at 0.73. On the scan curve, the red line is set to coincide with the sample meniscus and the left green line is set to coincide with the optimal lower range value for starting the analysis. The regularization model has been set to Maximum Entropy as it is the default for c(s,ff0) analysis. The confidence level (F-ratio) is set to 0.95. The default plot is set to s-ff0. After running SEDFIT, the RMSD values ​​are recorded, the peaks are integrated, and the composition percentages are calculated based on the peak areas.

[0076] Results: The distribution curve using c(s,ff0) for AAV8 preparation is shown in Figure 2 As shown in the figure below, peaks representing empty particles, GFP-containing particles, and over-packaging particles (containing a genome size larger than the GFP genome size) can be identified. Figure 3Distribution curves using c(s,ff0) are shown, which were used to calculate the friction ratio (f / f0), molecular weight (sw,ffow), and Stokes radius (Rs) of AAV2, AAV3, AAV6, AAV8, and AAV9 formulations. The results are shown in Tables 1 to 3. Based on the calculated results, all species are spherical, have molecular weights consistent with their type, and have diameters of approximately 20nm-25nm, consistent with historical and published data.

[0077] Table 1. Friction ratio (f / f0)

[0078] Viral vectors type n AVG f / f0 %CV AAV2 GFP 2 1.000000 0 AAV3 null 1 1.044685 NA AAV3 GFP 1 1.000000 NA AAV6 null 1 1.000000 NA AAV6 GFP 1 1.000000 NA AAV8 null 3 1.000000 0 AAV8 GFP 3 1.000000 0 AAV9 null 1 1.000000 NA AAV9 GFP 1 1.166479 NA

[0079] Table 2. Molecular weight (sw, ffow)

[0080] Viral vectors type n AVG MW(MDa) %CV AAV2 GFP 1 4.06 NA AAV3 null 1 2.74 NA AAV3 GFP 1 4.10 NA AAV6 null 1 2.15 NA AAV6 GFP 1 3.97 NA AAV8 null 3 2.46 0 AAV8 GFP 3 4.02 0 AAV9 null 1 2.39 NA AAV9 GFP 1 5.16 NA

[0081] Table 3. Stokes radius (Rs)

[0082]

[0083] Example 2

[0084] Characterization of 70S ribosomes

[0085] Bacterial 70S ribosomes were selected as reference materials for AUC. This molecule is a mixture of proteins and nucleic acids, and has a molecular weight of 2.5MDa, similar to empty AAV (2.7MDa-3.0MDa). 70S has a spherical shape, 50S is also spherical, and 30S is more elongated, but is considered to be spherical. The 70S ribosome was characterized using the AUC method described in Example 1. Two main peaks corresponding to 30S and 50S subcomponents and a small peak corresponding to 70S were observed. 30S and 50S components precipitated consistently at the same rate (n=8). C(s,ff0) modeling results are provided in Table 4. The average RMSD is 0.0022 (%CV=33%). All runs met the RMSD acceptance criteria of ≤0.01.

[0086] Table 4. 70S ribosome c(s,ff0) modeling

[0087]

[0088] Example 3

[0089] Example AUC Workflow

[0090] The workflow provided is an example for analyzing AAV particles. One of ordinary skill in the art will appreciate that certain aspects of the workflow may vary, for example, depending on the biomolecule to be analyzed.

[0091] 1. Experimental setup:

[0092] a. Run samples on a Beckman Coulter Optima analytical ultracentrifuge.

[0093] b. Assign one pool to a reference sample (e.g., 70S ribosomes).

[0094] c. Assign the remaining pools to samples.

[0095] 2. Perform ultracentrifugation with data collection set to 75 scans at 280 nm.

[0096] 3. Import the raw data file into the assay folder and select all 75 scans of the pool to be analyzed.

[0097] 4. Import all files into SEDFIT and select the c(s,ff0) function.

[0098] 5. Set the analysis parameters, for example:

[0099]

[0100] 1. Set the sample meniscus lower range value for scanning.

[0101] 2. Run the calculation.

[0102] 3. Record the RMSD value.

[0103] 4. Integrate the peak area and record the sedimentation coefficient.

[0104] 5. Calculate the composition % using the peak area.

[0105] 6. Repeat steps 3-10 for each sample.

[0106] In view of the many possibilities to which the principles of the present disclosure can be applied, it should be appreciated that the examples shown should not be considered to limit the scope of the present invention. Rather, the scope of the present invention is defined by the appended claims. The inventor therefore claims that the present invention fully belongs to the scope and spirit of the claims.

Claims

1. A method for characterizing a viral particle preparation, comprising: subjecting the viral particle preparation to analytical ultracentrifugation under sedimentation velocity conditions; measuring sedimentation of the viral particles at multiple time points; Determining the friction ratio function c(s,ff0) and sedimentation coefficient of the viral particle preparation; Plotting the c(s,ff0) versus the sedimentation coefficient (S) to obtain a c(s,ff0) distribution; and Characterize the presence and / or relative composition of viral particles comprising whole viral genomes, partial viral genomes, over-packaged viral genomes and / or empty viral particles in the viral particle preparation.

2. A method for assessing the genome integrity of viral particles in a viral preparation, comprising: subjecting the viral particle preparation to analytical ultracentrifugation; measuring sedimentation of the viral particles at multiple time points; Determining the friction ratio function c(s,ff0) and sedimentation coefficient of the viral particle preparation; Plotting the c(s,ff0) versus the sedimentation coefficient (S) to obtain a c(s,ff0) distribution; and Characterize the presence and / or relative composition of viral particles containing whole viral genomes, partial viral genomes, over-packaged viral genomes and / or empty viral particles in the viral particle preparation, thereby assessing the genome integrity of the viral particles in the viral preparation.

3. The method of claim 1 or claim 2, wherein measuring the sedimentation of the viral particles comprises measuring absorbance. The method of claim 3 , wherein the absorbance is measured at about 280 nm.

5. The method of claim 3 or claim 4, wherein measuring the sedimentation of the viral particles at multiple time points comprises measuring the absorbance at about 75 time points.

6. The method according to any one of claims 1 to 5, wherein the time interval between each of the plurality of time points is 20 seconds.

7. The method according to any one of claims 1 to 6, wherein the analytical ultracentrifugation is performed at about 15,000 rpm and / or at a temperature of about 20°C.

8. The method according to any one of claims 1 to 7, wherein characterizing the presence of viral particles comprising a full viral genome, a partial viral genome, an over-packaged viral genome and / or empty viral particles in the viral particle preparation comprises identifying a peak in the c(s,ff0) distribution corresponding to a sedimentation coefficient corresponding to viral particles comprising a full viral genome, a partial viral genome and / or empty viral particles.

9. The method according to any one of claims 1 to 7, wherein characterizing the relative composition of viral particles comprising full viral genomes, partial viral genomes, over-packaged viral genomes and / or empty viral particles in the viral particle preparation comprises integrating the area under each peak in the c(s,ff0) distribution.

10. The method of claim 9, wherein the relative composition of each viral particle in the viral particle preparation comprising a full viral genome, a partial viral genome, an over-packaged viral genome and / or an empty viral particle is determined by comparing the peak area values ​​within the preparation.

11. The method according to any one of claims 1 to 10, wherein the c(s,ff0) is calculated using Lamm's equation.

12. The method of claim 10, wherein the Lamm equation is calculated using the SEDFIT software package.

13. The method according to any one of claims 1 to 13, wherein the viral particle is a capsid particle.

14. The method according to any one of claims 1 to 13, wherein the viral particle preparation is a preparation of adeno-associated virus (AAV) particles or recombinant AAV particles.

15. The method of claim 14, wherein the AAV particles comprise one or more of AAV2 particles, AAV3 particles, AAV6 particles, AAV8 particles, and AAV9 particles.

16. A method for determining the relative composition of adeno-associated virus (AAV) particles comprising whole viral genomes, partial viral genomes, over-packaged viral genomes and / or empty viral particles in a preparation of AAV particles, the method comprising: subjecting the AAV particle preparation to analytical ultracentrifugation at 15,000 rpm; The absorbance data at 280 nm were collected at 75 time points at 20-second intervals to measure the sedimentation of the virus particles; Determining the friction ratio function c(s,ff0) and sedimentation coefficient of the viral particle preparation; Plotting the c(s,ff0) versus the sedimentation coefficient (S) to obtain a c(s,ff0) distribution; assigning peaks in the c(s,ff0) distribution to viral particle classes selected from viral particles comprising a full viral genome, viral particles comprising a partial viral genome, over-packaged, and empty viral particles; Integrating the area under each peak in the c(s,ff0) distribution; and The compositions of AAV particles comprising full viral genomes, partial viral genomes and / or empty viral particles in the AAV particle preparations are compared.

17. The method of claim 16, wherein c(s,ff0) is calculated using the Lamm equation.

18. The method of claim 17, wherein the Lamm equation is calculated using the SEDFIT software package.

19. The method of any one of claims 16 to 18, wherein the AAV particle is a capsid particle.

20. The method of any one of claims 16 to 18, wherein the AAV particles comprise one or more of AAV2 particles, AAV3 particles, AAV6 particles, AAV8 particles, and AAV9 particles.

Images