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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/62—Detectors specially adapted therefor
  • G01N30/74—Optical detectors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/86—Signal analysis
  • G01N30/8624—Detection of slopes or peaks; baseline correction
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14151—Methods of production or purification of viral material
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
  • G01N2030/027—Liquid chromatography
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
  • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
  • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange

Definitions

• the present invention relates to methods to analyze relative levels of full and empty capsids in compositions of recombinant adeno-associated virus (rAAV) particles.
• the methods relate to size exclusion chromatography with dual wavelength detection.
• Adeno-associated virus is a small nonpathogenic parvovirus, in which a linear single stranded DNA genome with a size of approximately 4.7 kilobases is packaged into a nonenveloped icosahedral capsid.
• the viral genome consists of three viral genes rep (replication), cap (capsid), and aap (assembly-activating protein) flanked by two inverted terminal repeats (ITRs) (Naso, M. F. et al., BioDrugs 2017, 31 (4), 317-334).
• the capsid is assembled as a 60-mer from three viral proteins (VPs) VP1, VP2, and VP3 with an approximately 5:5:50 ratio (Daya, S. et al., Clin Microbiol Rev 2008, 21 (4), 583-93).
• VPs viral proteins
• VP2 viral proteins
• VP3 viral proteins
• rAAVs therapeutic recombinant AAVs
• the viral genome is replaced with a transgene, while the ITRs are retained for proper genome replication and packaging.
• the transgene is converted into a double stranded DNA for gene expression after entering the target cell nucleus, to exert therapeutic effects.
• AUC-SV can also resolve higher order species and fragmented capsids.
• Pierson et al. used charge detection mass spectrometry (CDMS) to determine the distribution of empty, partial and full capsids by concurrently measuring the mass charge ratio (m/z) and the charge (z) of individual ions (Pierson, E. E. et al., Anal Chem 2016, 88 (13), 6718-25).
• Li et al. has recently introduced a capillary isoelectric focusing (cIEF) method to determine the empty and full ratio of AAV vectors (Li, T.
• cIEF capillary isoelectric focusing
• the invention provides methods to determine the presence of empty and/or partial capsids in a composition comprising recombinant adeno-associated virus (rAAV) particles, the method comprising: a) subjecting the composition to chromatography under conditions to separate the rAAV particles from impurities in the composition in an eluate of the chromatography; b) measuring the UV absorbance at about 250 nm to about 270 nm (A250-270) and about 220 nm to about 240 nM (A220-240) of the eluate, c) plotting chromatograms of the A 250-270 and A 220-240 of the eluate, d) integrating the area under the peaks representing the rAAV particles for the A250-270 and A220-240 plots, wherein the peak area ratio at A250-270 and A220-240 (PA260/PA230) is indicative of the presence of empty and/or partial capsids in the composition.
• rAAV adeno-associated virus
• the invention provides methods of measuring the relative amount of empty and/or partial capsids in a composition comprising rAAV particles, the method comprising: a) subjecting the composition to chromatography under conditions to separate the rAAV particles from impurities in the composition in an eluate of the chromatography; b) measuring the UV absorbance at about 250 nm to about 270 nm (A250-270) and about 220 nm to about 240 nM (A 220-240 ) of the eluate, c) plotting chromatograms of the A 250-270 and A 220-240 of the eluate, d) integrating the area under the peaks representing the rAAV particles for the A250-270 and A220-240 plots, wherein the peak area ratio at A250-270 and A220-240 (PA260/PA230) is indicative of the relative amount of empty capsids in the composition.
• the UV absorbance at about 250 nm to about 270 nm is a UV absorbance at about 260 nm.
• the UV absorbance at about 220 nm to about 240 nm is a UV absorbance at about 230 nm.
• a PA260/PA230 ratio less than the PA260/PA230 ratio of a pure sample of full rAAV capsids is indicative of empty and/or partial capsids and in the composition.
• a PA260/PA230 ratio equal to or less than a PA260/PA230 ratio of a rAAV sample with a known percentage of empty and/or partial capsids is indicative of empty and/or partial capsids in the composition.
• the relative amount of empty capsids in the composition is determined by comparing the PA260/PA230 ratio of the eluate with a linear relationship between the PA260/PA230 ratio and the percent full capsids established with a plurality of rAAV preparations with different known percentages of full capsids.
• the linear relationship between the PA260/PA230 ratio and the percent full capsids is determined by plotting the PA260/PA230 ratio versus the percent of full capsids of the plurality of rAAV preparations.
• the plurality of rAAV preparations with different known percentages of full capsids comprises three or more rAAV preparations wherein the three or more rAAV preparations comprise known ratios of full capsids to empty capsids ranging from 1:0 to 0:1.
• the plurality of rAAV preparations comprise rAAV capsids of the same serotype as the rAAV particles in the composition.
• the full capsids of the plurality of rAAV preparations comprise viral genomes that are at least about 90% in size compared to viral genomes of the rAAV particles in the composition.
• the full capsids of the plurality of rAAV preparations comprise viral genomes that are the same as the viral genomes of the rAAV particles in the composition.
• the plurality of rAAV preparations comprise capsids of the same serotype and the full capsids comprise the same viral genomes as the rAAV particles in the composition.
• the chromatography is size exclusion chromatography, ion exchange chromatography, affinity chromatography, mixed mode chromatography, hydrophobic interaction chromatography, or apatite chromatography.
• the chromatography is a column chromatography.
• the chromatography is high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC).
• HPLC high performance liquid chromatography
• UHPLC ultra-high performance liquid chromatography
• the chromatography is size exclusion chromatography that utilizes a size exclusion chromatography column.
• the size exclusion chromatography column comprises particles about 3 ⁇ m to about 10 ⁇ m in diameter. In some embodiments, the size exclusion chromatography column comprises particles about 5 ⁇ m in diameter. In some embodiments, the size exclusion chromatography column comprises particles with pores of about 100 ⁇ to about 1000 ⁇ in size. In some embodiments, the size exclusion chromatography column comprises particles with pores of about 500 ⁇ in size. In some embodiments, the particles comprise silica.
• the column has an internal diameter of between about 4.0 to about 7.8 mM and a length of between about 50 mm to about 300 mm, and in particular about 7.8 mm by about 300 mm, about 4.6 mm by about 100 mm, or about 4.6 mm by about 150 mm.
• the size exclusion chromatography further utilizes a guard column.
• the guard column comprises the same particles as the size exclusion chromatography column.
• the guard column has an internal diameter of between about 4.0 to about 7.8 mM and a length of between about 30 mm to about 50 mm, and in particular is about 7.8 mm by about 50 mm, or about 4.6 by about 50 mm.
• a mobile phase of the size exclusion chromatography comprises phosphate buffered saline (PBS).
• the PBS comprises about 100 mM to about 200 mM NaCl, about 1 mM to about 5 mM KCl, about 5 mM to about 20 mM Na2HPO4, and about 1 mM to about 3 mM KH2PO4.
• the PBS comprises about 137 mM NaCl, about 2.7 mM KCl, about 10 mM Na 2 HPO 4 , and about 1.8 mM KH2PO4.
• the PBS has a pH of about 6.5 to about 7.5 or about 7.0.
• the chromatography is performed at a flow rate of about 0.5 mL/minute to about 1.0 mL/minute, about 0.7 mL/minute to about 0.8 mL/minute, or about 0.75 mL/minute. In some embodiments, the chromatography is performed at about 4°C to about 35°C. In some embodiments, the chromatography is performed at about 25°C, about 30°C, or about 35°C. In some embodiments, about 5 ⁇ L to about 500 ⁇ L or about 10 ⁇ L to about 75 ⁇ L are subjected to the chromatography.
• the titer of rAAV in the composition is about 1 ⁇ 10 11 to about 1 ⁇ 10 14 capsid particles/mL (cp/mL) or about 5 ⁇ 10 12 cp/mL. In some embodiments, the titer of rAAV in the composition is about 1 ⁇ 10 10 to about 1 ⁇ 10 14 viral genomes/mL (vg/mL). In some embodiments, greater than about 70% of the rAAV particles in the composition are full rAAV capsids. In some embodiments, greater than about 70% to about 95% of the rAAV particles in the composition are full rAAV capsids.
• the rAAV particles in the composition have been purified using one or more purification steps.
• the recombinant viral particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAV13 capsid, an AAV14 capsid, an AAV15 capsid, an AAV16 capsid, an AAVrh20 capsid, an AAV.rh39 capsid, an AAV.Rh74 capsid, an AAV.RHM4-l capsid, an AAV.hu37 capsid, an AAV.Anc80 capsid
• the recombinant viral particle comprises an AAV1 ITR, an AAV2 ITR, an AAV3 ITR, an AAV4 ITR, an AAV5 ITR, an AAV6 ITR, an AAV7 ITR, an AAV8 ITR, an AAVrh8 ITR, an AAV9 ITR, an AAV10 ITR, an AAVrh10 ITR, an AAV11 ITR, an AAV12 ITR, an AAV-13 ITR, an AAV-14 ITR, an AAV-15 ITR, an AAV-16 ITR, an AAV.rh20 ITR, an AAV.rh39 ITR, an AAV.rh74 ITR, an AAV.rhM4-1 ITR, an AAV.hu37 ITR, an AAV.Anc80 ITR, an AAV DJ ITR, a goat AAV ITR, a bovine AAV ITR, or a mouse AAV ITR.
• the rAAV genome is 2500 bases to 5500 bases in length.
• the recombinant viral particles comprise a self-complementary AAV (scAAV) genome.
• scAAV self-complementary AAV
• the rAAV particles are rAAV5 particles.
• the rAAV particles are rAAV1 particles.
• the invention provides methods of monitoring the removal of empty capsids during the purification of a composition of rAAV particles, the method comprising collecting a sample of the composition before and following one or more steps of the purification process and analyzing each collected sample for the relative amount of empty capsids according to any of the methods described herein, wherein a decrease in the relative amount of empty capsids between the samples subsequently collected indicates removal of empty capsids from the preparation of rAAV particles.
• the invention provides methods of monitoring the removal of empty capsids during the purification of a composition of rAAV particles, the method comprising: a) determining the relative amount of empty capsids according any of the methods described herein in a first sample collected before the purification process or before a step of the purification of the process, b) determining the relative amount of empty capsids according any one of methods described herein in a second sample collected following the purification process or a step of the purification of the process, wherein a decrease in the relative amount of empty capsids between the second and first collected samples indicates removal of empty capsids from the preparation of rAAV particles.
• kits for measuring the relative amount empty capsids in a composition of rAAV particles according the any of the methods described herein comprises kits for measuring the relative amount empty capsids in a composition of rAAV particles according the any of the methods described herein.
• the kit comprises chromatography columns and/or buffers for use in the methods described herein.
• the kit comprises three or more reference standards wherein the three or more reference standards comprise known ratios of full capsids to empty capsids ranging from 1:0 to 0:1.
• FIGS.1A-1C show the results of size-exclusion chromatography (SEC) of AAV5 samples using various columns.
• FIG.1A shows SEC chromatograms of an AAV5 drug substance (DS) sample.
• the DS sample was diluted with Dulbecco's phosphate buffered saline (DPBS) to approximately 5.0u10 12 cp/mL and 25 ⁇ L of high performance liquid chromatography (HPLC) sample was applied onto the column. The elution was monitored with a photodiode array (PDA) detector.
• HPLC conditions SEC-300 column (gray trace), SEC-500 column (black trace at 260 nm, dotted black trace at 280 nm), and SEC-1000 column (dashed gray trace). For SEC-300 and SEC-1000, only traces at 260 nm were shown for clarity.
• FIG.1B shows chromatogram of a different lot of DS sample showing aggregated AAVs and monomer at 260 nm resolved by the SEC- 500 column.
• the inset shows the high molecular weight species 1 (HMWS 1, 0.5% by peak area) and high molecular weight species 2 (HMWS 2, 1.7%).
• FIG.1C shows the peak areas detected at 260 nm and 280 nm, showing a linear relationship with the number of capsids injected.
• FIG.2 shows predicted and measured A260/A280 ratios of the AAV samples containing various amounts of full capsids vs. the expected percent full capsids in the samples.
• FIGS.3A-3E show the results of SEC with dual-wavelength detection (SEC-DW) for the AAV spiked samples.
• FIG.3A shows chromatograms of a series of AAV5 HPLC standards containing various percentages of full capsids at 260 nm.
• FIG.3B shows chromatograms of the empty, spiked samples and full capsids at 280 nm.
• FIG.3C shows chromatograms of the empty, spiked samples, and full capsids at 230 nm. Chromatograms of one of the standard samples were not shown as the loading led to different absorbance intensities.
• the AAV capsids (50 ⁇ L per injection) were applied onto a size exclusion column (Sepax SRT ® SEC- 500, 5 ⁇ m, 500 ⁇ , 7.8 u 300 mm) equipped with a guard column (SRT ® SEC-500, 5 ⁇ m, 500 ⁇ , 7.8 u 50 mm) equilibrated at 25qC.
• the capsids were eluted with Dulbecco's phosphate buffered saline, pH 7.0 at a flow rate of 0.75 mL/min.
• FIG.3D shows the peak area ratios at 260 nm and 280 nm (PA260/PA280) and the peak area ratios at 260 nm and 230 nm (PA260/PA230) by SEC for the AAV5 samples.
• the curve fitting function changed from quadratic (for PA260/PA280) to linear regression (for PA260/PA230).
• the %RSD values are 0.58%, 0.54% and 5.30% for the PA260/PA230 values of the DS, WRS and empty capsids samples.
• the %RSD values are 0.73% and 0.67% for the percent full capsids of the DS and WRS samples, respectively.
• FIG.4 shows the peak area at 260 nm (PA260) and the peak area at 230 nm (PA230) relative to the number of capsids applied onto the column.
• FIGS.5A-5D show representative sedimentation velocity analytical ultracentrifugation (AUC-SV) data shown as sedimentation coefficient distribution plots.
• FIG.5A shows empty capsids.
• FIG.5B shows spiked sample 3 (see Table 3).
• FIG.5C shows spiked sample 5 (see Table 3).
• FIG.5D shows full capsids.
• the sedimentation of AAV particles was monitored at 260 nm.
• the raw data were fitted with the Lamm equation using the c(s) model in SEDFIT.
• the x axis represents the sedimentation coefficient in Svedberg unit S, and the y axis represents the concentration as a function of sedimentation coefficient.
• FIGS.6A-6B show representative cryogenic electron microscopy (Cryo-EM) images of two spiked samples.
• FIG.6A shows an image of the spiked sample 3 containing 54% full capsids by Cryo-EM.
• FIG.6B Image of the full capsid sample containing 97% full capsids by Cryo-EM.
• An empty capsid and a full capsid are indicated in Image A.
• the cross central sections of the empty and full capsids show lack of and significant internal density, respectively.
• FIGS.7A-7B show the percentage of full capsids in spiked samples as determined by SEC-DW, AUC-SV and Cryo-EM.
• FIG.7A shows the percent full capsids in the spiked samples obtained from SEC-DW, AUC-SV, and Cryo-EM. The results were plotted against the expected percent full capsids and fitted with linear regressions.
• the R 2 values are 0.997, 0.993 and 0.993 for SEC-DW, AUC-SV, and Cryo-EM.
• the slope values are 0.9892 for SEC-DW, 1.0623 for AUC-SV, and 1.0378 for Cryo-EM.
• FIG. 7B shows the results from SEC-DW, AUC-SV, and Cryo-EM are presented as a bar graph to visualize the direct comparison.
• FIG. 8 shows absorbance spectra for an empty capsids sample and a full capsids sample.
• FIGS. 9A-9D show SEC-DW results of a second AAV DS candidate.
• FIG. 9A show SEC chromatograms of the empty capsids of a different AAV5 drug substance sample (candidate 2) at 230 nm and 260 nm.
• FIG. 9B shows SEC chromatograms of the DS sample of the AAV5 drug candidate 2 at 230 nm and 260 nm.
• FIG. 9C shows extracted absorbance spectra of the empty capsids and the DS sample (candidate 2).
• FIG.9D shows the PA260 and PA230 show excellent linearity with the number of capsids (different injection volumes from the same samples) applied onto the column.
• FIG. 10 shows the peak area ratios at 260 nm and 280 nm (PA260/PA280) and the peak area ratios at 260 nm and 230 nm (PA260/PA230) by SEC for the AAV1 samples.
• the curve fitting function changed from quadratic (for PA260/PA280) to linear regression (for PA260/PA230).
• the invention provides methods to determine the presence of empty and/or partial capsids in a composition comprising recombinant adeno-associated virus (rAAV) particles, the method comprising: a) subjecting the composition to chromatography under conditions to separate the rAAV particles from impurities in the composition in an eluate of the chromatography; b) measuring the UV absorbance at about 250 nm to about 270 nm (A250-270) and about 220 nm to about 240 nM (A220-240) of the eluate, c) plotting chromatograms of the A 250-270 and A 220-240 of the eluate, d) integrating the area under the peaks representing the rAAV particles for the A 250-270 and A 220-240 plots, wherein the peak area ratio at A250-270 and A220-240 (PA260/PA230) is indicative of the presence of empty and/or partial capsids in the composition
• the invention provides methods of measuring the relative amount of empty and/or partial capsids in a composition comprising rAAV particles, the method comprising: a) subjecting the composition to chromatography under conditions to separate the rAAV particles from impurities in the composition in an eluate of the chromatography; b) measuring the UV absorbance at about 250 nm to about 270 nm (A250-270) and about 220 nm to about 240 nM (A220-240) of the eluate, c) plotting chromatograms of the A250-270 and A220-240 of the eluate, d) integrating the area under the peaks representing the rAAV particles for the A 250-270 and A 220-240 plots, wherein the peak area ratio at A 250-270 and A 220-240 (PA260/PA230) is indicative of the relative amount of empty capsids in the composition.
• a “vector,” as used herein, refers to a recombinant plasmid or virus that comprises a nucleic acid to be delivered into a host cell, either in vitro or in vivo.
• the term “polynucleotide” or “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
• this term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
• the backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups.
• the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P-NH 2 ) or a mixed phosphoramidate- phosphodiester oligomer.
• a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.
• polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition.
• the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
• a “polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
• a “recombinant viral vector” refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of viral origin).
• the recombinant nucleic acid is flanked by at least one inverted terminal repeat sequence (ITR). In some embodiments, the recombinant nucleic acid is flanked by two inverted terminal repeat sequences (ITRs).
• a “recombinant AAV vector (recombinant adeno-associated viral vector)” refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of AAV origin) that are flanked by at least one AAV inverted terminal repeat sequences (ITR).
• the recombinant nucleic acid is flanked by two inverted terminal repeat sequences (ITRs).
• ITRs inverted terminal repeat sequences
• Such recombinant viral vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e. AAV Rep and Cap proteins).
• a recombinant viral vector When a recombinant viral vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), then the recombinant viral vector may be referred to as a “pro-vector” which can be “rescued” by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions.
• a recombinant viral vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral particle, for example, an AAV particle.
• a recombinant viral vector can be packaged into an AAV virus capsid to generate a “recombinant adeno- associated viral particle (recombinant viral particle)”.
• An “rAAV virus” or “rAAV viral particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated rAAV vector genome as defined above.
• an “empty AAV capsid” is an AAV capsid in which essentially no AAV nucleic acid has been encapsidated.
• a “partial AAV capsid” is an AAV capsid that has encapsidated an incomplete AAV genome (e.g., a truncated AAV genome, a fragment of an AAV genome, or an AAV genome with internal deletion(s)).
• an incomplete AAV genome comprises or consists of less than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 25% in length of the AAV genome of a full AAV capsid (capsid (i.e., harboring full-length AAV genome).
• an incomplete AAV genome comprises or consists of less than about 70%, 60%, 50%, 40%, 30% or 25% in length of the AAV genome of a full AAV capsid.
• Heterologous means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated.
• a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide).
• a cellular sequence e.g., a gene or portion thereof
• a viral vector is a heterologous nucleotide sequence with respect to the vector.
• transgene refers to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and optionally, translated and/or expressed under appropriate conditions. In aspects, it confers a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic or diagnostic outcome. In another aspect, it may be transcribed into a molecule that mediates RNA interference, such as siRNA.
• gp gp
• gene equivalents or “genome copies” as used in reference to a viral titer, refer to the number of virions containing the recombinant viral DNA genome or RNA genome, regardless of infectivity or functionality.
• the number of genome particles in a particular vector preparation can be measured by procedures such as described in, for example, in Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.
• the terms “infection unit (iu),” “infectious particle,” or “replication unit,” as used in reference to a viral titer, refer to the number of infectious and replication-competent recombinant viral vector particles as measured by the infectious center assay, also known as replication center assay, as described, for example with AAV, in McLaughlin et al. (1988) J. Virol., 62:1963-1973.
• transducing unit (tu) refers to the number of infectious recombinant viral vector particles that result in the production of a functional transgene product as measured in functional assays such as described in, for example, Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or in Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).
• An “AAV inverted terminal repeat (ITR)” sequence is an approximately 145-nucleotide sequence that is present at both termini of the native single-stranded AAV genome.
• the outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome.
• the outermost 125 nucleotides also contains several shorter regions of self-complementarity (designated A, A', B, B', C, C' and D regions), allowing intrastrand base-pairing to occur within this portion of the ITR.
• a “terminal resolution sequence” or “trs” is a sequence in the D region of the AAV ITR that is cleaved by AAV rep proteins during viral DNA replication. A mutant terminal resolution sequence is refractory to cleavage by AAV rep proteins.
• AAV helper functions refer to functions that allow AAV to be replicated and packaged by a host cell.
• AAV helper functions can be provided in any of a number of forms, including, but not limited to, helper virus or helper virus genes which aid in AAV replication and packaging. Other AAV helper functions are known in the art such as genotoxic agents.
• a “helper virus” for AAV refers to a virus that allows AAV (which is a defective parvovirus) to be replicated and packaged by a host cell.
• a helper virus provides “helper functions” which allow for the replication of AAV.
• helper viruses include adenoviruses, herpesviruses, poxviruses such as vaccinia and baculovirus.
• the adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C (Ad5) is most commonly used.
• Ad5 Adenovirus type 5 of subgroup C
• Numerous adenoviruses of human, non-human mammalian and avian origin are known and are available from depositories such as the ATCC.
• Viruses of the herpes family which are also available from depositories such as ATCC, include, for example, herpes simplex viruses (HSV), Epstein- Barr viruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV).
• HSV herpes simplex viruses
• EBV Epstein- Barr viruses
• CMV cytomegaloviruses
• PRV pseudorabies viruses
• a preparation or composition of rAAV is said to be “substantially free” of helper virus if the ratio of infectious AAV particles to infectious helper virus particles is at least about 10 2 :l; at least about 10 4 :l, at least about 10 6 :l; or at least about 10 8 :l or more.
• preparations are also free of equivalent amounts of helper virus proteins (i.e., proteins as would be present as a result of such a level of helper virus if the helper virus particle impurities noted above were present in disrupted form).
• helper virus proteins i.e., proteins as would be present as a result of such a level of helper virus if the helper virus particle impurities noted above were present in disrupted form.
• Viral and/or cellular protein contamination can generally be observed as the presence of Coomassie staining bands on SDS gels (e.g., the appearance of bands other than those corresponding to the AAV capsid proteins VPl, VP2 and VP3).
• To “reduce” is to decrease, reduce or arrest an activity, function, and/or amount as compared to a reference. In certain embodiments, by “reduce” is meant the ability to cause an overall decrease of 20% or greater.
• by “reduce” is meant the ability to cause an overall decrease of 30% or greater. In certain embodiments, by “reduce” is meant the ability to cause an overall decrease of 40% or greater. In another embodiment, by “reduce” is meant the ability to cause an overall decrease of 50% or greater. In yet another embodiment, by “reduce” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater. [0051] A “reference” as used herein, refers to any sample, standard, or level that is used for comparison purposes.
• the amount of empty capsids when measuring the relative amount of empty capsids in a composition of rAAV particles, the amount of empty capsids may be compared to the amount of AAV particles in a preparation comprising known ratios of full capsids to empty capsids.
• the purified AAV produced when monitoring the removal of empty capsids during the purification of a composition of rAAV particles, the purified AAV produced is compared to preparations comprising known ratios of full capsids to empty capsids.
• a reference may refer to a standard procedure known in the art.
• An “isolated” molecule e.g., nucleic acid or protein
• cell means it has been identified and separated and/or recovered from a component of its natural environment.
• isolated rAAV particles may be prepared using a purification technique to enrich it from a source mixture, such as a culture lysate or production culture supernatant.
• Enrichment can be measured in a variety of ways, such as, for example, by the proportion of DNase-resistant particles (DRPs) present in a solution, or by infectivity, or it can be measured in relation to a second, potentially interfering substance present in the source mixture, such as contaminants, including production culture contaminants or in-process contaminants, including helper virus, media components, and the like, as defined below.
• DNase-resistant particles DNase-resistant particles
• the invention provides methods to determine the presence of empty and/or partial capsids in a composition comprising recombinant adeno-associated virus (rAAV) particles, the method comprising: a) subjecting the composition to chromatography under conditions to separate the rAAV particles from impurities in the composition in an eluate of the chromatography; b) measuring the UV absorbance at about 250 nm to about 270 nm (A250-270) and about 220 nm to about 240 nM (A220-240) of the eluate, c) plotting chromatograms of the A250-270 and A220-240 of the eluate, d) integrating the area under the peaks representing the rAAV particles for the A 250-270 and A 220-240 plots, wherein the peak area ratio at A 250-270 and A 220-240 (PA260/PA230) is indicative of
• PA260/PA230 refers to the ratio of peak area of any plot of UV absorbance of the eluate at between about A 250 and A 270 (e.g., any of 250 nm, 251 nm, 252 nm, 253 nm, 254 nm, 255 nm, 256 nm, 257 nm, 258 nm, 259 nm, 260 nm, 261 nm, 262 nm, 263 nm, 264 nm, 265 nm, 266 nm, 267 nm, 268 nm, 269 nm, or 270 nm) and the peak area of any plot of UV absorbance of the eluate at between about A 220 and A 240 (e.g., any of 220 nm, 221 nm, 222 nm, 223 nm, 224 nm, 225 nm, 226 n
• the invention provides methods of measuring the relative amount empty and/or partial capsids in a composition comprising rAAV particles, the method comprising: a) subjecting the composition to chromatography under conditions to separate the rAAV particles from impurities in the composition in an eluate of the chromatography; b) measuring the UV absorbance at about 250 nm to about 270 nm (A250-270) and about 220 nm to about 240 nM (A 220-240 ) of the eluate, c) plotting chromatograms of the A 250-270 and A 220-240 of the eluate, d) integrating the area under the peaks representing the rAAV particles for the A250-270 and A220-240 plots, wherein the peak area ratio at A250-270 and A220-240 (PA260/PA230) is indicative of the relative amount of empty capsids in the composition.
• the UV absorbance at about 250 nm to about 270 nm is a UV absorbance at any wavelength between about 250 nm and 270 nm.
• the UV absorbance at about 250 nm to about 270 nm is a UV absorbance at 250 nm, 251 nm, 252 nm, 253 nm, 254 nm, 255 nm, 256 nm, 257 nm, 258 nm, 259 nm, 260 nm, 261 nm, 262 nm, 263 nm, 264 nm, 265 nm, 266 nm, 267 nm, 268 nm, 269 nm, or 270 nm.
• the UV absorbance at about 250 nm to about 270 nm is a UV absorbance at any wavelength between about 255 nm and 265 nm. In some embodiments, the UV absorbance at about 250 nm to about 270 nm is a UV absorbance at about 260 nm. [0060] In some embodiments, the UV absorbance at about 220 nm to about 240 nm is a UV absorbance at any wavelength between about 220 nm and 240 nm.
• the UV absorbance at about 220 nm to about 230 nm is a UV absorbance at 220 nm, 221 nm, 222 nm, 223 nm, 224 nm, 225 nm, 226 nm, 227 nm, 228 nm, 229 nm, 230 nm, 231 nm, 232 nm, 233 nm, 234 nm, 235 nm, 236 nm, 237 nm, 238 nm, 239 nm, or 240 nm.
• the UV absorbance at about 220 nm to about 240 nm is a UV absorbance at any wavelength between about 225 nm and 235 nm. In some embodiments, the UV absorbance at about 220 nm to about 240 nm is a UV absorbance at about 230 nm.
• a PA260/PA230 ratio less than the PA260/PA230 ratio of a pure sample of full rAAV capsids is indicative of empty and/or partial capsids and in the composition. As demonstrated in the Examples, other methods for determining the relative amounts of full and empty AAV capsids are known; for example, analytical ultracentrifugation and cryo-electron microscopy.
• the relative amount of empty capsids in the composition is determined by comparing the PA260/PA230 ratio of the eluate with a linear relationship between the PA260/PA230 ratio and the percent full capsids established with a plurality of rAAV preparations with different known percentages of full capsids.
• the linear relationship between the PA260/PA230 ratio and the percent full capsids is determined by plotting the PA260/PA230 ratio versus the percent of full capsids of the plurality of rAAV preparations.
• curve fitting is used to establish the relationship between the PA260/PA230 ratio and the percent full capsids.
• curve fitting demonstrates a linear relationship with a coefficient of determination, R 2 , greater than 0.9, 0.95, 0.96, 0.97, 0.98, 0.99, 0.991, 0.992, 0.993, 0.994, or 0.995.
• the plurality of rAAV preparations with different known percentages of full capsids comprises three or more rAAV preparations wherein the three or more rAAV preparations comprise known ratios of full capsids to empty capsids ranging from 1:0 to 0:1. In some embodiments, the three or more rAAV preparations comprise known ratios of full capsids to empty capsids of any one of 1:0, 0.9:0.1, 0.8:0.2, 0.7:0.3, 0.6:0:4, 0.5:0.5, 0.4:0.6, 0.3:0.7, 0.2:0.8, 0.1:0.9, and 0:1.
• the three or more rAAV preparations include preparations with low levels of empty capsids (e.g., >75% full capsids), approximately equal amounts of full and empty capsids (e.g., ⁇ 50% full capsids), and high levels of empty capsids (e.g., ⁇ 25% full capsids).
• the plurality of rAAV preparations comprise rAAV capsids of the same serotype as the rAAV particles in the composition.
• the full capsids of the plurality of rAAV preparations comprise viral genomes that are at least about 90% in size (e.g., in length) compared to viral genomes of the rAAV particles in the composition. In some embodiments, the full capsids of the plurality of rAAV preparations comprise viral genomes that are about any of 90%, 95%, 100%, 105% or 110% in size (e.g., in length) compared to viral genomes of the rAAV particles in the composition. The percentage is calculated based on the full alignment of the two AAV genome sequences.
• the full capsids of the plurality of rAAV preparations comprise viral genomes that are essentially the same size compared to viral genomes of the rAAV particles in the composition. In some embodiments, the full capsids of the plurality of rAAV preparations comprise viral genomes that are the same as the viral genomes of the rAAV particles in the composition. In some embodiments, the full capsids of the plurality of rAAV preparations comprise viral genomes that are the different as the viral genomes of the rAAV particles in the composition, provided that the viral genomes are at least about 90% in size (e.g., in length) compared to viral genomes of the rAAV particles in the composition (as described above).
• the full capsids of the plurality of rAAV preparations comprise the same serotype capsids and the full capsids comprise the same viral genomes as the rAAV particles in the composition.
• Chromatography [0064]
• the invention provides methods of measuring the relative amount of empty and/or partial capsids in a composition comprising rAAV particles. The methods use chromatography to separate impurities from the rAAV capsids (e.g., full, empty and partial capsids). Any chromatography methods that separates rAAV capsids from impurities are contemplated. In some embodiments, full, empty and partial capsids elute together.
• Non-limiting impurities may include high molecular weight species, host cell proteins, and helper viral proteins.
• chromatography used to measure the relative amount empty and/or partial capsids in a composition comprising rAAV particles is size exclusion chromatography, ion exchange chromatography, affinity chromatography, mixed mode chromatography, hydrophobic interaction chromatography, or apatite chromatography.
• the chromatography utilizes column chromatography.
• the chromatography is high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC).
• the chromatography is an ion exchange chromatography; for example, an anion exchange chromatography or a cation exchange chromatography.
• the anion exchange chromatography material is a solid phase that is positively charged and has free anions for exchange with anions in an aqueous solution passed over or through the solid phase.
• the anion exchange material may comprise a primary amine, a secondary amine, a tertiary amine or a quarternary ammonium ion functional group, a polyamine functional group, or a diethylaminoaethyl functional group.
• the cation exchange chromatography material is a solid phase that is positively charged and has free cations for exchange with cations in an aqueous solution passed over or through the solid phase.
• the cation exchange material may comprise a carboxylic acid functional group or a sulfonic acid functional group such as, but not limited to, sulfonate, carboxylic, carboxymethyl sulfonic acid, sulfoisobutyl, sulfoethyl, carboxyl, sulphopropyl, sulphonyl, sulphoxyethyl, or orthophosphate.
• the chromatography is an affinity chromatography in which the chromatography material comprises a ligand that specifically binds AAV (e.g., an antibody).
• chromatography is a mixed-mode chromatography.
• the mixed-mode chromatography material comprises functional groups capable of one of more of the following functionalities: anionic exchange, cation exchange, hydrogen bonding, and hydrophobic interactions.
• the mixed-mode material comprises functional groups capable of anionic exchange and hydrophobic interactions.
• the mixed-mode material comprises functional groups capable of cationic exchange and hydrophobic interactions.
• the chromatography is hydrophobic interaction chromatography (HIC) chromatography.
• Hydrophobic interaction chromatography is a liquid chromatography technique that separates biomolecules according to hydrophobicity.
• the chromatography is hydroxyapatite chromatography.
• the hydroxyapatite comprises (Ca5(PO4)3OH)2.
• the hydroxyapatite chromatography is ceramic hydroxyapatite chromatography.
• the hydroxyapatite chromatography is CHT ceramic hydroxyapatite chromatography.
• the chromatography is size exclusion chromatography.
• the invention provides methods of measuring the relative amount of empty and/or partial capsids in a composition comprising rAAV particles, the method comprising: a) subjecting the composition to size exclusion chromatography under conditions to separate the rAAV particles from impurities in the composition in an eluate of the chromatography; b) measuring the UV absorbance at about 250 nm to about 270 nm (A 250-270 ) and about 220 nm to about 240 nM (A 220-240 ) of the eluate, c) plotting chromatograms of the A250-270 and A220-240 of the eluate, d) integrating the area under the peaks representing the rAAV particles for the A250-270 and A220-240 plots, where
• the size exclusion chromatography is a liquid chromatography technique that separates biomolecules according to their size, shape, hydrodynamic radius and/or volume. Size exclusion chromatography materials come in different particle sizes and pore sizes for efficient separation of molecules of particular weight ranges. In some embodiments, the size exclusion chromatography materials comprise silica. In some embodiments, the size exclusion chromatography utilizes a size exclusion chromatography column. [0072] In some embodiments, the size exclusion chromatography column comprises particles about 1 ⁇ m to about 10 ⁇ m in diameter.
• the size exclusion chromatography comprises particles of any of about 1 ⁇ m, about 2 ⁇ m, about 3 ⁇ m, about 4 ⁇ m, about 5 ⁇ m, about 6 ⁇ m, about 7 ⁇ m, about 8 ⁇ m, about 9 ⁇ m, about 10 ⁇ m, or more than about 10 ⁇ m in diameter.
• the size exclusion chromatography comprises particles between about any of 1 ⁇ m and 10 ⁇ m, 1 ⁇ m and 9 ⁇ m, 1 ⁇ m and 8 ⁇ m, 1 ⁇ m and 7 ⁇ m, 1 ⁇ m and 6 ⁇ m, 1 ⁇ m and 5 ⁇ m, 1 ⁇ m and 4 ⁇ m, 1 ⁇ m and 3 ⁇ m, 1 ⁇ m and 2 ⁇ m, 2 ⁇ m and 10 ⁇ m, 2 ⁇ m and 9 ⁇ m, 2 ⁇ m and 8 ⁇ m, 2 ⁇ m and 7 ⁇ m, 2 ⁇ m and 6 ⁇ m, 2 ⁇ m and 5 ⁇ m, 2 ⁇ m and 4 ⁇ m, 2 ⁇ m and 3 ⁇ m, 3 ⁇ m and 10 ⁇ m, 3 ⁇ m and 9 ⁇ m, 3 ⁇ m and 8 ⁇ m, 3 ⁇ m and 7 ⁇ m, 3 ⁇ m and 6 ⁇ m, 3 ⁇ m and 5 ⁇ m, 3 ⁇ m and 4 ⁇ m, 2
• the size exclusion chromatography column comprises particles with pores of about 100 ⁇ to about 1000 ⁇ in size. In some embodiments, the size exclusion chromatography column comprises particles with pores of any of about 100 ⁇ , about 200 ⁇ , about 300 ⁇ , about 400 ⁇ , about 500 ⁇ , about 600 ⁇ , about 700 ⁇ , about 800 ⁇ , about 900 ⁇ , or about 1000 ⁇ .
• the size exclusion chromatography column comprises particles with pores of between any of about 100 ⁇ and about 1000 ⁇ , about 100 ⁇ and about 900 ⁇ , about 100 ⁇ and about 800 ⁇ , about 100 ⁇ and about 700 ⁇ , about 100 ⁇ and about 600 ⁇ , about 100 ⁇ and about 500 ⁇ , about 100 ⁇ and about 400 ⁇ , about 100 ⁇ and about 300 ⁇ , about 100 ⁇ and about 200 ⁇ , about 200 ⁇ and about 1000 ⁇ , about 200 ⁇ and about 900 ⁇ , about 200 ⁇ and about 800 ⁇ , about 200 ⁇ and about 700 ⁇ , about 200 ⁇ and about 600 ⁇ , about 200 ⁇ and about 500 ⁇ , about 200 ⁇ and about 400 ⁇ , about 200 ⁇ and about 300 ⁇ , about 300 ⁇ and about 1000 ⁇ , about 300 ⁇ and about 900 ⁇ , about 300 ⁇ and about 800 ⁇ , about 300 ⁇ and about 700 ⁇ , about 300 ⁇ and about 600 ⁇ , about 300 ⁇ and about 500 ⁇ , about 200 ⁇ and
• the size exclusion chromatography column has an internal diameter of between about 4.0 to about 7.8 mM and a length of between about 50 mm to about 300 mm. In some embodiments, the size exclusion chromatography column has an internal diameter of about 4.0 mm, 4.6 mm or 7.8 mm. In some embodiments, the size exclusion chromatography column has a length of about 50 mm, 100 mm, 150 mm or 300 mm. In some embodiment, the size exclusion chromatography column is about 7.8 mm by about 300 mm, about 4.6 mm by about 100 mm, or about 4.6 mm by about 150 mm. [0075] In some embodiments, the size exclusion chromatography further utilizes a guard column.
• the guard column comprises the same particles as the size exclusion chromatography column; e.g., particles made of the same material as the size exclusion chromatography column, with particles of the same size as the size exclusion chromatography column, and with pores of the same size as the pores in the size exclusion chromatography column.
• the guard column has an internal diameter of between about 4.0 to about 7.8 mM and a length of between about 30 mm to about 50 mm. In some embodiments, the guard column has an internal diameter of about 4.0 mm, 4.6 mm or 7.8 mm. In some embodiments, the guard column has a length of about 30 mm, 40 mm, or 50 mm.
• the guard column is about 7.8 mm by about 50 mm.
• the chromatography utilizes a mobile phase.
• the mobile phase is an aqueous mobile phase.
• the aqueous phase is a salt solution (e.g., an aqueous salt solution).
• the mobile phase of the chromatography comprises phosphate buffered saline (PBS).
• the mobile phase of the size exclusion chromatography comprises PBS.
• PBS is a common buffered salt solution known in the art.
• the PBS comprises about 100 mM to about 200 mM NaCl, about 1 mM to about 5 mM KCl, about 5 mM to about 20 mM Na 2 HPO 4 , and about 1 mM to about 3 mM KH 2 PO 4 . In some embodiments, the PBS comprises about 137 mM NaCl, about 2.7 mM KCl, about 10 mM Na2HPO4, and about 1.8 mM KH2PO4. [0078] In some embodiments, the mobile phase of the chromatography has a pH of about 6.0 to about 8.0. In some embodiments, the pH of the mobile phase is any of about 6.0, 6.5, 7.0, 7.5 or 8.0.
• the pH of the mobile phase is between about any of 6.0 and 8.0, 6.5 and 8.0, 7.0 and 8.0, 7.5 and 8.0, 6.0 and 7.5, 6.5 and 7.5, 7.0 and 7.5, 6.0 and 7.0, 6.5 and 7.0, or 6.0 and 6.5.
• the mobile phase of the chromatography is PBS at a pH of about 6.0 to about 8.0.
• the mobile phase of the size exclusion chromatography is PBS at a pH of about 6.0 to about 8.0.
• the mobile phase of the size exclusion chromatography is PBS at a pH of about 7.0.
• the chromatography (e.g., size exclusion chromatography) is performed at a flow rate of about 0.3 mL/minute to about 1.0 mL/minute. In some embodiments, the chromatography is performed at a flow rate of any of about 0.3 mL/minute, about 0.5 mL/minute, 0.6 mL/minute, 0.7 mL/minute, 0.75 mL/minute, 0.8 mL/minute, 0.9 mL/minute, or 1.0 mL/minute.
• the chromatography is performed at a flow rate between any of about 0.3 mL/minute and 1.0 mL/minute, about 0.5 mL/minute and 1.0 mL/minute, about 0.6 mL/minute and 1.0 mL/minute, about 0.7 mL/minute and 1.0 mL/minute, about 0.75 mL/minute and 1.0 mL/minute, about 0.8 mL/minute and 1.0 mL/minute, about 0.9 mL/minute and 1.0 mL/minute, about 0.3 mL/minute and 0.9 mL/minute, about 0.5 mL/minute and 0.9 mL/minute, about 0.6 mL/minute and 0.9 mL/minute, about 0.7 mL/minute and 0.9 mL/minute, about 0.75 mL/minute and 0.9 mL/minute, about 0.8 mL/minute and 0.9 mL/minute, about 0.3 mL/minute and 0.8 mL/minute, about
• the size exclusion chromatography is performed at a flow rate of any of about 0.75 mL/minute.
• the chromatography e.g., size exclusion chromatography
• the chromatography is performed at about 4°C to about 35°C.
• the chromatography is performed at about 4°C, about 20°C, about 25°C, about 30°C, or about 35°C.
• the chromatography is performed as between any of about 4°C to about 35°C, about 20°C to about 35°C, about 25°C to about 35°C, about 30°C to about 35°C, 4°C to about 30°C, about 20°C to about 30°C, about 25°C to about 30°C, 4°C to about 25°C, or about 20°C to about 25°C.
• the chromatography is performed at room temperature.
• the size exclusion chromatography is performed at about 25°C. [0081] In some embodiments, about 5 ⁇ L to about 500 ⁇ L of the composition comprising rAAV particles is subjected to chromatography (e.g., size exclusion chromatography).
• about 5 ⁇ L, about 25 ⁇ L, about 50 ⁇ L, about 75 ⁇ L, about 100 ⁇ L, about 200 ⁇ L, about 300 ⁇ L, about 400 ⁇ L, or about 500 ⁇ L of the composition comprising rAAV particles is subjected to chromatography.
• any of about 5 ⁇ L and about 100 ⁇ L, about 25 ⁇ L and about 100 ⁇ L, about 50 ⁇ L and about 100 ⁇ L, about 75 ⁇ L and about 100 ⁇ L, 5 ⁇ L and about 75 ⁇ L, about 25 ⁇ L and about 75 ⁇ L, about 50 ⁇ L and about 75 ⁇ L, 5 ⁇ L and about 50 ⁇ L, about 25 ⁇ L and about 50 ⁇ L, or about 5 ⁇ L and about 25 ⁇ L of the composition comprising rAAV particles is subjected to chromatography. In some embodiments, about 50 ⁇ L of the composition comprising rAAV particles is subjected to size exclusion chromatography.
• the titer of rAAV in the composition subjected to chromatography is about 1 ⁇ 10 11 to about 1 ⁇ 10 14 capsid particles/mL (cp/mL).
• the titer of rAAV in the composition subjected to chromatography between any of about 1 ⁇ 10 11 to about 1 ⁇ 10 14 cp/mL, 5 ⁇ 10 11 to about 1 ⁇ 10 14 cp/mL, 1 ⁇ 10 12 to about 1 ⁇ 10 14 cp/mL, 5 ⁇ 10 12 to about 1 ⁇ 10 14 cp/mL, 1 ⁇ 10 13 to about 1 ⁇ 10 14 cp/mL, 5 ⁇ 10 13 to about 1 ⁇ 10 14 cp/mL, 1 ⁇ 10 11 to about 5 ⁇ 10 13 cp/mL, 5 ⁇ 10 11 to about 5 ⁇ 10 13 cp/mL, 1 ⁇ 10 12 to about 5 ⁇ 10 13 cp/mL, 5 ⁇ 10 12 to about 5 ⁇ 10 13 cp/mL, 5 ⁇ 10 12 to about 5 ⁇ 10 13 cp/mL, 1 ⁇ 10 13 to about 5 ⁇ 10 13 cp/mL, 1
• the titer of rAAV in the composition subjected to size exclusion chromatography is about 5 ⁇ 10 12 cp/mL. [0083] In some embodiments, the titer of rAAV in the composition subjected to chromatography (e.g., size exclusion chromatography) is about 1 ⁇ 10 10 to about 1 ⁇ 10 14 vector genomes/mL (vg/mL).
• the titer of rAAV in the composition subjected to chromatography between any of about 1 ⁇ 10 10 to about 1 ⁇ 10 13 vg/mL, 5 ⁇ 10 10 to about 1 ⁇ 10 13 vg/mL, 1 ⁇ 10 11 to about 1 ⁇ 10 13 vg/mL, 5 ⁇ 10 11 to about 1 ⁇ 10 13 vg/mL, 1 ⁇ 10 12 to about 1 ⁇ 10 13 vg/mL, 5 ⁇ 10 12 to about 1 ⁇ 10 13 vg/mL, 1 ⁇ 10 10 to about 5 ⁇ 10 12 vg/mL, 5 ⁇ 10 10 to about 5 ⁇ 10 12 vg/mL, 1 ⁇ 10 11 to about 5 ⁇ 10 12 vg/mL, 5 ⁇ 10 11 to about 5 ⁇ 10 12 vg/mL, 1 ⁇ 10 11 to about 5 ⁇ 10 12 vg/mL, 1 ⁇ 10 11 to about 5 ⁇ 10 12 vg/mL, 1
• the titer of rAAV in the composition subjected to size exclusion chromatography is about 5 ⁇ 10 12 vg/mL.
• greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the rAAV particles in the composition are full rAAV capsids.
• rAAV particles in the composition are full rAAV capsids.
• the rAAV particles comprise an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid (e.g., a wild-type AAV6 capsid, or a variant AAV6 capsid such as ShH10, as described in U.S.
• an AAV7 capsid an AAV8 capsid, an AAVrh8 capsid, an AAVrh8R, an AAV9 capsid (e.g., a wild-type AAV9 capsid, or a modified AAV9 capsid as described in U.S.
• an AAV10 capsid an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAV13 capsid, an AAV14 capsid, an AAV15 capsid, an AAV16 capsid, an AAVrh20 capsid, an AAV.rh39 capsid, an AAV.Rh74 capsid, an AAV.RHM4-l capsid, an AAV.hu37 capsid, an AAV.Anc80 capsid, an AAV.Anc80L65 capsid, an AAV.PHP.B capsid, an AAV2.5 capsid, an AAV2tYF capsid, an AAV3B capsid, an AAV.LK03 capsid, an AAV.HSCl capsid, an AAV.HSC2 capsid, an AAV.HSC3 capsid, an AAV.HSC4 capsid, an AAV
• the rAAV particles comprise an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAV2R471A capsid, an AAV2/2-7m8 capsid, an AAV DJ capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1/AAV2 chimeric capsid, a bovine AAV capsid, or a mouse AAV capsid
• the rAAV particles comprise a capsid which is selected from the group consisting of an AAV1 capsid and an AAV5 capsid. In embodiments of the embodiments described above, the rAAV particles comprise an AAV5 capsid. In embodiments of the embodiments described above, the rAAV particles comprise an AAV1 capsid.
• the rAAV particles comprise at least one AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAVrh8 ITR, AAV9 ITR, AAV10 ITR, AAVrh10 ITR, AAV11 ITR, AAV12 ITR, AAV-13 ITR, AAV-14 ITR, AAV-15 ITR, AAV-16 ITR, AAV.rh20 ITR, AAV.rh39 ITR, AAV.rh74 ITR, AAV.rhM4-1 ITR, AAV.hu37 ITR, AAV.Anc80 ITR, AAV DJ ITR, goat AAV ITR, bovine AAV ITR, or mouse AAV ITR.
• the rAAV particles comprise ITRs from one AAV serotype and AAV capsid from another serotype.
• the rAAV particles may comprise a therapeutic transgene flanked by at least one AAV2 ITR encapsidated into an AAV9 capsid.
• Such combinations may be referred to as pseudotyped rAAV particles.
• Recombinant Adeno-associated Virus Particles [0086] The methods described herein may be used to determine the presence of empty and/or partial capsids, and/or measure the relative amount empty and/or partial capsids in compositions of viral particles (e.g., recombinant adeno-associated virus (rAAV) particles).
• the rAAV genome is about 2500 bases to about 5500 bases in length. In some embodiments, the rAAV genome is between any of about 2500 bases and about 5500 bases, about 3000 bases and about 5500 bases, about 3500 bases and about 5500 bases, about 4000 bases and about 5500 bases, about 4500 bases and about 5500 bases, about 5000 bases and about 5500 bases, about 2500 bases and about 5000 bases, about 3000 bases and about 5000 bases, about 3500 bases and about 5000 bases, about 4000 bases and about 5000 bases, about 4500 bases and about 500 bases, about 2500 bases and about 4500 bases, about 3000 bases and about 4500 bases, about 3500 bases and about 4500 bases, about 4000 bases and about 4500, about 2500 bases and about 4000 bases, about 3000 bases and about 4500 bases, about 3500 bases and about 4500 bases, about 4000 bases and about 4500, about 2500 bases and about 4000 bases, about 3000 bases and about 4000 bases, about 3500 bases and about 4000 bases, about 2500
• the viral particle is a recombinant AAV particle comprising a nucleic acid comprising a transgene flanked by one or two ITRs (the rAAV genome).
• the nucleic acid is encapsidated in the AAV particle.
• the AAV particle also comprises capsid proteins.
• the nucleic acid comprises the protein coding sequence(s) of interest (e.g., a therapeutic transgene) operatively linked components in the direction of transcription, control sequences including transcription initiation and termination sequences, thereby forming an expression cassette.
• the expression cassette is flanked on the 5' and 3' end by at least one functional AAV ITR sequences.
• the recombinant vectors comprise at least all of the sequences of AAV essential for encapsidation and the physical structures for infection by the rAAV.
• AAV ITRs for use in the vectors of the invention need not have a wild-type nucleotide sequence (e.g., as described in Kotin, Hum. Gene Ther., 1994, 5:793-801), and may be altered by the insertion, deletion or substitution of nucleotides or the AAV ITRs may be derived from any of several AAV serotypes. More than 40 serotypes of AAV are currently known, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS, 2002, 99(18): 11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis et al., J.
• a rAAV vector is a vector derived from an AAV serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HS
• the nucleic acid in the AAV comprises an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, AAV12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh20, AAV.rh39, AAV.rh74, AAV.rhM4-1, AAV.hu37, AAV.Anc80, AAV DJ, or the like.
• the nucleic acid in the AAV comprises a goat AAV ITR, a bovine AAV ITR, or a mouse AAV ITR.
• the rAAV particle comprises capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSClO, AAV.HSCl 1, AAV.HSC12, AAV.HSC13, AAV
• the rAAV particle comprises capsid proteins of goat AAV, AAV1/AAV2 chimera, bovine AAV, mouse AAV, or rAAV2/HBoV1 (chimeric AAV / human bocavirus virus 1).
• the rAAV particle comprises capsid proteins of an AAV serotype from Clades A-F (Gao, et al. J. Virol.2004, 78(12):6381).
• the rAAV particle comprise capsid proteins selected from the group consisting of AAV1 capsid proteins and AAV5 capsid proteins.
• the rAAV particle comprises capsid proteins of AAV5.
• the rAAV particle comprises capsid proteins of AAV1.
• the rAAV particle comprise capsid selected from the group consisting of an AAV1 capsid and an AAV5 capsid.
• the rAAV particle comprises an AAV5 capsid.
• the rAAV particle comprises an AAV1 capsid [0089] Different AAV serotypes are used to optimize transduction of particular target cells or to target specific cell types within a particular target tissue (e.g., a diseased tissue).
• a rAAV particle can comprise viral proteins and viral nucleic acids of the same serotype or a mixed serotype.
• a rAAV particle can comprise AAV9 capsid proteins and at least one AAV2 ITR or it can comprise AAV2 capsid proteins and at least one AAV9 ITR.
• a rAAV particle can comprise capsid proteins from both AAV9 and AAV2, and further comprise at least one AAV2 ITR. Any combination of AAV serotypes for production of a rAAV particle is provided herein as if each combination had been expressly stated herein.
• the rAAV particle comprises at least one AAV1 ITR and capsid protein from any of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSClO, AAV.HSCl 1, AAV.HSC
• the rAAV particle comprises at least one AAV2 ITR and capsid protein from any of AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSClO, AAV.HSCl 1, AAV.HSC12, AAV
• the rAAV particle comprises at least one AAV3 ITR and capsid protein from any of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSClO, AAV.HSCl 1, AAV.HSC12, AAV
• the rAAV particle comprises at least one AAV4 ITR and capsid protein from any of AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSClO, AAV.HSCl 1, AAV.HSC12, AAV
• the rAAV particle comprises at least one AAV5 ITR and capsid protein from any of AAV1, AAV2, AAV3, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSClO, AAV.HSCl 1, AAV.HSC12, AAV
• the rAAV particle comprises at least one AAV6 ITR and capsid protein from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSClO, AAV.HSCl 1, AAV.HSC12, AAV
• the rAAV particle comprises at least one AAV7 ITR and capsid protein from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSClO, AAV.HSCl 1, AAV.HSC12, AAV.
• the rAAV particle comprises at least one AAV8 ITR and capsid protein from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAVrh.8, AAVrh10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSClO, AAV.HSCl 1, AAV.HSC12, AAV
• the rAAV particle comprises at least one AAV9 ITR and capsid protein from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh.8, AAVrh10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSClO, AAV.HSCl 1, AAV.HSC12, AAV.
• the rAAV particle comprises at least one AAVrh8 ITR and capsid protein from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSClO, AAV.HSCl 1, AAV.HSC12, AAV.HSC13,
• the rAAV particle comprises at least one AAVrh10 ITR and capsid protein from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSClO, AAV.HSCl 1, AAV.HSC12, AAV.HSC13, AAV
• the rAAV particle comprises at least one AAV11 ITR and capsid protein from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAVrh10, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSClO, AAV.HSCl 1, AAV.HSC12, AAV.HS
• the rAAV particle comprises at least one AAV12 ITR and capsid protein from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh8, AAV9, AAVrh10, AAV11, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSClO, AAV.HSCl 1, AAV.HSC12, AAV.
• the rAAV particle comprises an AAV5 capsid protein and an ITR selected from the group consisting of AAV1 ITR, an AAV2 ITR, an AAV3 ITR, an AAV4 ITR, an AAV5 ITR, an AAV6 ITR, an AAV7 ITR, an AAV8 ITR, an AAVrh8 ITR, an AAV9 ITR, an AAV10 ITR, an AAVrh10 ITR, an AAV11 ITR, an AAV12 ITR, an AAV-13 ITR, an AAV-14 ITR, an AAV-15 ITR, an AAV-16 ITR, an AAV.rh20 ITR, an AAV.rh39 ITR, an AAV.rh74 ITR, an AAV.rhM4-1 ITR, an AAV.hu37 ITR, an AAV.Anc80 ITR, an AAV DJ ITR, a goat AAV ITR, a bovine AAV ITR, and/or
• AAV viral particles with self-complementing genomes and methods of use of self-complementing AAV genomes are described in US Patent Nos. 6,596,535; 7,125,717; 7,765,583; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457; and Wang Z., et al., (2003) Gene Ther 10:2105-2111, each of which are incorporated herein by reference in its entirety.
• a rAAV comprising a self-complementing genome will quickly form a double stranded DNA molecule by virtue of its partially complementing sequences (e.g., complementing coding and non-coding strands of a transgene).
• the invention provides an AAV viral particle comprising an AAV genome, wherein the rAAV genome comprises a first heterologous polynucleotide sequence (e.g., a therapeutic transgene coding strand) and a second heterologous polynucleotide sequence (e.g., the noncoding or antisense strand of the therapeutic transgene) wherein the first heterologous polynucleotide sequence can form intrastrand base pairs with the second polynucleotide sequence along most or all of its length.
• a first heterologous polynucleotide sequence e.g., a therapeutic transgene coding strand
• a second heterologous polynucleotide sequence e.g., the noncoding or antisense strand of the therapeutic transgene
• the first heterologous polynucleotide sequence and a second heterologous polynucleotide sequence are linked by a sequence that facilitates intrastrand basepairing; e.g., a hairpin DNA structure. Hairpin structures are known in the art, for example in siRNA molecules.
• the first heterologous polynucleotide sequence and a second heterologous polynucleotide sequence are linked by a mutated ITR (e.g., the right ITR).
• the mutated ITR comprises a deletion of the D region comprising the terminal resolution sequence.
• a recombinant viral genome comprising the following in 5' to 3' order will be packaged in a viral capsid: an AAV ITR, the first heterologous polynucleotide sequence including regulatory sequences, the mutated AAV ITR, the second heterologous polynucleotide in reverse orientation to the first heterologous polynucleotide and a third AAV ITR.
• rAAV Vectors Numerous methods are known in the art for production of rAAV vectors, including transfection, stable cell line production, and infectious hybrid virus production systems which include adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, JE et al., (1997) J. Virology 71(11):8780-8789) and baculovirus-AAV hybrids.
• rAAV production cultures for the production of rAAV virus particles all require; 1) suitable host cells, including, for example, human-derived cell lines such as HeLa, A549, or 293 cells, or insect- derived cell lines such as SF-9, in the case of baculovirus production systems; 2) suitable helper virus function, provided by wild-type or mutant adenovirus (such as temperature sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct providing helper functions; 3) AAV rep and cap genes and gene products; 4) a transgene (such as a therapeutic transgene) flanked by at least one AAV ITR sequences ; and 5) suitable media and media components to support rAAV production.
• suitable host cells including, for example, human-derived cell lines such as HeLa, A549, or 293 cells, or insect- derived cell lines such as SF-9, in the case of baculovirus production systems
• suitable helper virus function provided by wild-type or mutant
• the AAV rep and cap gene products may be from any AAV serotype.
• the AAV rep gene product is of the same serotype as the ITRs of the rAAV vector genome as long as the rep gene products may function to replicated and package the rAAV genome.
• Suitable media known in the art may be used for the production of rAAV vectors. These media include, without limitation, media produced by Hyclone Laboratories and JRH including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), custom formulations such as those described in U.S. Patent No.6,566,118, and Sf-900 II SFM media as described in U.S.
• the AAV helper functions are provided by adenovirus or HSV.
• the AAV helper functions are provided by baculovirus and the host cell is an insect cell (e.g., Spodoptera frugiperda (Sf9) cells).
• the p5 promoter of the nucleic acid encoding AAV rep and cap regions is located 3’ to the rep and/or cap coding region.
• the nucleic acid encoding AAV rep and cap coding regions is plasmid pHLP, pHLP19, or pHLP09 (see US Patent Nos.5,622,856; 6,001,650; 6,027,931; 6,365,403; 6,376,237; and 7,037,713; the content of each is incorporated herein in its entirety).
• the AAV helper virus functions comprise adenovirus E1A function, adenovirus E1B function, adenovirus E2A function, adenovirus VA function and adenovirus E4 orf6 function.
• Suitable rAAV production culture media of the present invention may be supplemented with serum or serum-derived recombinant proteins at a level of 0.5%-20% (v/v or w/v).
• rAAV vectors may be produced in serum-free conditions which may also be referred to as media with no animal-derived products.
• commercial or custom media designed to support production of rAAV vectors may also be supplemented with one or more cell culture components know in the art, including without limitation glucose, vitamins, amino acids, and or growth factors, in order to increase the titer of rAAV in production cultures.
• the invention provides methods for monitoring the removal of empty capsids during the purification of a composition of rAAV particles, the method comprising collecting a sample of the composition before and following one or more steps of the purification process and analyzing each collected sample for the relative amount of empty capsids according to the methods as described herein, wherein a decrease in the relative amount of empty capsids between the samples subsequently collected indicates removal of empty capsids from the preparation of rAAV particles.
• the invention provides methods of monitoring the removal of empty capsids during the purification of a composition of rAAV particles, the method comprising: a) determining the relative amount of empty capsids according to any one of the methods described herein in a first sample collected before the purification process or before a step of the purification of the process, b) determining the relative amount of empty capsids according to a method as described herein in a second sample collected following the purification process or a step of the purification of the process, wherein a decrease in the relative amount of empty capsids between the second and first collected samples indicates removal of empty capsids from the preparation of rAAV particles.
• rAAV production cultures can be grown under a variety of conditions (over a wide temperature range, for varying lengths of time, and the like) suitable to the particular host cell being utilized.
• rAAV production cultures include attachment- dependent cultures which can be cultured in suitable attachment-dependent vessels such as, for example, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized- bed bioreactors.
• rAAV vector production cultures may also include suspension-adapted host cells such as HeLa, 293, and SF-9 cells which can be cultured in a variety of ways including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as the Wave bag system.
• rAAV vector particles may be harvested from rAAV production cultures by lysis of the host cells of the production culture or by harvest of the spent media from the production culture, provided the cells are cultured under conditions known in the art to cause release of rAAV particles into the media from intact cells, as described more fully in U.S. Patent No.6,566,118).
• Suitable methods of lysing cells include for example multiple freeze/thaw cycles, sonication, microfluidization, and treatment with chemicals, such as detergents and/or proteases.
• rAAV production cultures of the present invention may contain one or more of the following: (1) host cell proteins; (2) host cell DNA; (3) plasmid DNA; (4) helper virus; (5) helper virus proteins; (6) helper virus DNA; and (7) media components including, for example, serum proteins, amino acids, transferrins and other low molecular weight proteins.
• rAAV production cultures further include rAAV particles having an AAV capsid serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh20, AAV.rh39, AAV.Rh74, AAV.RHM4-l, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCl, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSClO, AAV.HSC
• the rAAV production cultures further comprise empty AAV capsids (e.g., a rAAV particle comprising capsid proteins but no rAAV genome).
• the rAAV production cultures further comprise rAAV particles comprising variant rAAV genomes (e.g., a rAAV particle comprising a rAAV genome that differs from an intact full-length rAAV genome).
• the rAAV production cultures further comprise rAAV particles comprising truncated rAAV genomes.
• the rAAV production cultures further comprise rAAV particles comprising AAV-encapsidated DNA impurities.
• the rAAV production culture harvest is clarified to remove host cell debris.
• the production culture harvest is clarified by filtration through a series of depth filters including, for example, a grade DOHC Millipore Millistak+ HC Pod Filter, a grade A1HC Millipore Millistak+ HC Pod Filter, and a 0.2 ⁇ m Filter Opticap XL1O Millipore Express SHC Hydrophilic Membrane filter. Clarification can also be achieved by a variety of other standard techniques known in the art, such as, centrifugation or filtration through any cellulose acetate filter of 0.2 ⁇ m or greater pore size known in the art.
• the rAAV production culture harvest is further treated with Benzonase ® to digest any high molecular weight DNA present in the production culture.
• the Benzonase ® digestion is performed under standard conditions known in the art including, for example, a final concentration of 1-2.5 units/ml of Benzonase ® at a temperature ranging from ambient to 37°C for a period of 30 minutes to several hours.
• rAAV particles may be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anionic exchange filtration; tangential flow filtration (TFF) for concentrating the rAAV particles; rAAV capture by apatite chromatography; heat inactivation of helper virus; rAAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and rAAV capture by anionic exchange chromatography, cationic exchange chromatography, or affinity chromatography.
• TFF tangential flow filtration
• SEC size exclusion chromatography
• nanofiltration nanofiltration
• kits [0103] The methods of this invention can be provided in the form of a kit.
• the kit comprises chromatography columns and/or buffers for use in the method.
• the kit comprises three or more reference standards wherein the three or more reference standards comprise known ratios of full capsids to empty capsids ranging from 1:0 to 0:1. In some embodiments, the three or more reference standards comprise known ratios of full capsids to empty capsids of any one of 1:0, 0.9:0.1, 0.8:0.2, 0.7:0.3, 0.6:0:4, 0.5:0.5, 0.4:0.6, 0.3:0.7, 0.2:0.8, 0.1:0.9, and 0:1.
• the three or more reference standards include preparations with low levels of empty capsids (e.g., >75% full capsids), approximately equal amounts of full and empty capsids (e.g., ⁇ 50% full capsids), and high levels of empty capsids (e.g., ⁇ 25% full capsids).
• empty capsids e.g., >75% full capsids
• approximately equal amounts of full and empty capsids e.g., ⁇ 50% full capsids
• high levels of empty capsids e.g., ⁇ 25% full capsids.
• a method to determine the presence of empty and/or partial capsids in a composition comprising recombinant adeno-associated virus (rAAV) particles comprising: a) subjecting the composition to chromatography under conditions to separate the rAAV particles from impurities in the composition in an eluate of the chromatography; b) measuring the UV absorbance at about 250 nm to about 270 nm (A 250-270 ) and about 220 nm to about 240 nM (A220-240) of the eluate, c) plotting chromatograms of the A250-270 and A220-240 of the eluate, d) integrating the area under the peaks representing the rAAV particles for the A 250-270 and A 220-240 plots, wherein the peak area ratio at A 250-270 and A 220-240 (PA260/PA230) is indicative of the presence of empty and/or partial capsids in the composition.
• rAAV adeno-associated virus
• a method of measuring the relative amount of empty and/or partial capsids in a composition comprising rAAV particles comprising: a) subjecting the composition to chromatography under conditions to separate the rAAV particles from impurities in the composition in an eluate of the chromatography; b) measuring the UV absorbance at about 250 nm to about 270 nm (A250-270) and about 220 nm to about 240 nM (A 220-240 ) of the eluate, c) plotting chromatograms of the A 250-270 and A 220-240 of the eluate, d) integrating the area under the peaks representing the rAAV particles for the A250-270 and A 220-240 plots, wherein the peak area ratio at A 250-270 and A 220-240 (PA260/PA230) is indicative of the relative amount of empty capsids in the composition.
• the plurality of rAAV preparations with different known percentages of full capsids comprises three or more rAAV preparations wherein the three or more rAAV preparations comprise known ratios of full capsids to empty capsids ranging from 1:0 to 0:1.
• the plurality of rAAV preparations comprise rAAV capsids of the same serotype as the rAAV particles in the composition.
• a method of measuring the relative amount of empty and/or partial capsids in a composition comprising rAAV particles comprising: a) subjecting the composition to size exclusion chromatography under conditions to separate the rAAV particles from impurities in the composition in an eluate of the chromatography; b) measuring the UV absorbance at about 250 nm to about 270 nm (A 250-270 ) and about 220 nm to about 240 nM (A220-240) of the eluate, c) plotting chromatograms of the A250-270 and A220-240 of the eluate, d) integrating the area under the peaks representing the rAAV particles for the A 250-270 and A220-240 plots, wherein the peak area ratio at A250-270 and A220-240 (PA260/PA230) is indicative of the relative amount of empty capsids in the composition.
• any one of embodiments 24-26 wherein the plurality of rAAV preparations with different known percentages of full capsids comprises three or more rAAV preparations wherein the three or more rAAV preparations comprise known ratios of full capsids to empty capsids ranging from 1:0 to 0:1 [0132] 28.
• any one of embodiments 24-28 wherein the full capsids of the plurality of rAAV preparations comprise viral genomes that are at least about 90% in size compared to viral genomes of the rAAV particles in the composition.
• 30 The method of any one of embodiments 24-29, wherein the full capsids of the plurality of rAAV preparations comprise viral genomes that are the same as the viral genomes of the rAAV particles in the composition.
• 31 The method of any one of embodiments 24-30, wherein the plurality of rAAV preparations comprise capsids of the same serotype and the full capsids comprise the same viral genomes as the rAAV particles in the composition.
• 32 The method of any one of embodiments 24-28, wherein the full capsids of the plurality of rAAV preparations comprise viral genomes that are at least about 90% in size compared to viral genomes of the rAAV particles in the composition.
• the method of any one of embodiments 20-31, wherein the size exclusion chromatography utilizes a size exclusion chromatography column.
• the guard column has an internal diameter of between about 4.0 to about 7.8 mM and a length of between about 30 mm to about 50 mm, and in particular is about 7.8 mm by about 50 mm, or about 4.6 by about 50 mm.
• a mobile phase of the size exclusion chromatography comprises phosphate buffered saline (PBS).
• the PBS comprises about 100 mM to about 200 mM NaCl, about 1 mM to about 5 mM KCl, about 5 mM to about 20 mM Na2HPO4, and about 1 mM to about 3 mM KH 2 PO 4 .
• 44. The method of embodiment 42 or 43, wherein the PBS comprises about 137 mM NaCl, about 2.7 mM KCl, about 10 mM Na2HPO4, and about 1.8 mM KH 2 PO 4 .
• 45 The method of any one of embodiments 42-44, wherein the PBS has a pH of about 6.5 to about 7.5 or about 7.0.
• the recombinant viral particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAV13 capsid, an AAV14 capsid, an AAV15 capsid, an AAV16 capsid, an AAVrh20 capsid, an AAV.rh39 capsid, an AAV.Rh74 capsid, an AAV.RHM4-l capsid, an AAV.hu37 capsid, an AAV.Anc80 capsid, an AAV.Anc80L65 capsid, an AAV
• the recombinant viral particle comprises an AAV1 ITR, an AAV2 ITR, an AAV3 ITR, an AAV4 ITR, an AAV5 ITR, an AAV6 ITR, an AAV7 ITR, an AAV8 ITR, an AAVrh8 ITR, an AAV9 ITR, an AAV10 ITR, an AAVrh10 ITR, an AAV11 ITR, an AAV12 ITR, an AAV-13 ITR, an AAV- 14 ITR, an AAV-15 ITR, an AAV-16 ITR, an AAV.rh20 ITR, an AAV.rh39 ITR, an AAV.rh74 ITR, an AAV.rhM4-1 ITR, an AAV.hu37 ITR, an AAV.Anc80 ITR, an AAV DJ ITR, a goat AAV ITR, a bovine AAV ITR, or a mouse AAV ITR.
• 57 The method of any one of embodiments 1-56, wherein the rAAV genome is 2500 bases to 5500 bases in length.
• 58 The method of any one of embodiments 1-57, wherein the recombinant viral particles comprise a self-complementary AAV (scAAV) genome.
• 59 The method of any one of embodiments 6-58, wherein the rAAV particles is selected from the group consisting of rAAV5 particles and rAAV1 particles.
• 60 The method of any one of embodiments 6-59, wherein the rAAV particles are rAAV5 particles [0165] 61.
• a method of monitoring the removal of empty capsids during the purification of a composition of rAAV particles comprising collecting a sample of the composition before and following one or more steps of the purification process and analyzing each collected sample for the relative amount of empty capsids according to the method of any one of embodiments 1-63, wherein a decrease in the relative amount of empty capsids between the samples subsequently collected indicates removal of empty capsids from the preparation of rAAV particles.
• a method of monitoring the removal of empty capsids during the purification of a composition of rAAV particles comprising: a) determining the relative amount of empty capsids according to the method of any one of embodiments 1-63 in a first sample collected before the purification process or before a step of the purification of the process, b) determining the relative amount of empty capsids according to the method of any one of embodiments 1-37 in a second sample collected following the purification process or a step of the purification of the process, wherein a decrease in the relative amount of empty capsids between the second and first collected samples indicates removal of empty capsids from the preparation of rAAV particles.
• kits for measuring the relative amount empty capsids in a composition of rAAV particles according the methods of any one of embodiments 1-65 [0171] 67.
• EXAMPLES [0173] The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention.
• Example 1 Initial size-exclusion chromatography column screening
• SEC size-exclusion chromatography
• SEC resins with a pore size from 300 to 1,000 ⁇ (30 to 100 nm) were anticipated to be suitable for separating monomeric AAVs from aggregated species as well as from low molecular weight species and small molecules that are expected to be present in a sample.
• an analytical column with a large inner diameter packed with a stationary phase consisting of a relatively large particle size would be preferred.
• Several vendors including Waters, Sepax and Wyatt manufacture columns that may be suitable for this purpose.
• Sepax SRT ® SEC columns are packed with spherical high purity silica coated with nanometer thick hydrophilic and neutral films and these materials have shown promise for separation of virus and virus-like particles.
• the monomer peak observed in the elution profile obtained for the SEC-300 column is sharp but slightly skewed, and the trace amount of aggregates, which is expected to elute prior to the monomer peak, was not observed.
• the elution profile obtained for the SEC-1000 column shows the monomer eluting too close to the peak representing low molecular weight species (buffer peaks).
• the SRT ® SEC-500 column with a 500 ⁇ pore size was therefore selected for further development as the retention time, peak shape and resolution are satisfactory.
• the peak area representing the monomer is higher when the signal is monitored at 260 nm compared to 280 nm resulting in a peak area ratio (PA260/PA280) of approximately 1.25 which is indicative of a sample containing mostly full capsids.
• the minor peaks eluting at approximately 5.8 mins and 8.6 mins are AAV aggregates (0.5% for HMWS 1 and 1.7% for HMWS 2), indicating that the method is capable of detecting low level aggregates.
• Example 2 Size-exclusion chromatography with dual-wavelength detection for the AAV spiked samples [0177] The following example describes the use of size-exclusion chromatography with dual-wavelength detection (SEC-DW) to analyze preparations of AAV capsids.
• the samples were dialyzed against 1X PBS using 10K MWCO slide-a- lyzersTM (Thermo Scientific, MA) and then against the DS formulation buffer.
• the empty and full capsids were characterized by analytical ultracentrifugation (AUC) and droplet digital PCR (ddPCR) and qPCR (Bio-Rad, Hercules, CA) to determine the capsid distribution and vector genome concentration.
• AUC analytical ultracentrifugation
• ddPCR droplet digital PCR
• qPCR Bio-Rad, Hercules, CA
• concentration of the empty capsid preparation was determined by AAV titration ELISA (Progen, Biotechnik) or A280. The results are summarized in Table 1. Table 1.
• Capsid concentration, vector genome concentration and subpopulation distribution of empty and full capsids [0181] A series of high performance liquid chromatography (HPLC) standards and spiked samples containing various amounts of full capsid were prepared by mixing the empty and full capsids at defined ratios based on their capsid concentrations (cp/mL). These materials were used to develop the SEC-DW method for empty and full capsids. In addition, for comparison purposes, the samples were evaluated using orthogonal approaches such as UV measurements, AUC characterization and cryogenic electron microscopy (Cryo-EM) analyses (see Example 3). [0182] Extinction coefficients and UV measurements.
• the extinction coefficient of empty capsids at 280 nm was calculated based on the absorptivity of the aromatic amino acids present in the primary sequences of VP1, VP2 and VP3, using a ratio of VP1:VP2:VP3 of 5:5:50 per capsid.
• the extinction coefficient of empty capsids at 260 nm was obtained by converting the H280, capsid using a previously published conversion factor of 0.59 (Sommer, J.M. et al., Mol Ther 2003, 7 (1), 122-8).
• the molecular weight (MW) of empty capsids was calculated based on the sequence information and the aforementioned protein ratio.
• the extinction coefficient of the transgene at 260 nm (H 260, gene ) and its MW were calculated based on the gene sequence, and the extinction coefficient at 280 nm (H 280, gene ) was then derived with a conversion factor of 0.555 (Sommer, J. M. et al., Mol Ther 2003, 7 (1), 122-8).
• the extinction coefficients of empty and full capsids are summarized in Table 2.
• the predicted A260/A280 ratio of the different samples used in the study was calculated based on the relative amounts of empty and full capsids present in the samples. Table 2. Theoretical extinction coefficients of empty capsids and full capsids [0183] UV absorbance of empty, full and spiked samples was measured using a nanodrop spectrometer (Thermo Fisher, MA). A260/A280 data was plotted against the expected percent full capsids and compared with the predicted A 260 /A 280 data (see FIG.2). [0184] SEC-dual-wavelength.
• AAV capsids (generally 25 ⁇ L or 50 ⁇ L per injection) were loaded onto a size exclusion column (Sepax SRT ® SEC-500, 5 ⁇ m, 500 ⁇ , 7.8 ⁇ 300 mm, Delaware) equipped with a guard column (SRT ® SEC-500, 5 ⁇ m, 500 ⁇ , 7.8 ⁇ 50 mm) equilibrated at 25oC with Dulbecco's phosphate buffered saline without CaCl 2 , without MgCl2 (DPBS).
• DPBS Dulbecco's phosphate buffered saline without CaCl 2 , without MgCl2
• the capsids were eluted with DPBS, pH 7.0 at a flow rate of 0.75 mL/min using an Agilent 1200 series HPLC (Agilent, CA). The eluate was monitored at 260 nm, 280 nm and 230 nm, and data was acquired with the Agilent OpenLab software. [0186] The peak areas of monomeric AAV at 230 nm, 260 nm and 280 nm were used for calculating the peak area (PA) ratios (e.g., PA260/PA230 and PA260/PA280). The correlation of the peak area ratios with the percent full capsids was established using the HPLC standards and the spiked samples containing various amounts of full capsids.
• PA peak area
• Results SEC with dual-wavelength detection for the AAV spiked samples [0187] As described in Example 1, the various capsid standards and samples were initially monitored at 260 nm and 280 nm, where encapsidated nucleic acids and capsid proteins have their absorbance maxima, respectively (see FIG.1A) (Porterfield, J.Z. and Zlotnick, A., Virology 2010, 407 (2), 281-8).
• FIG.3A and FIG.3B show that the monomers were well-resolved from the small aggregate peaks and the PA260/PA280 ratios obtained for the standard samples were plotted against the expected value for the percent full capsids calculated based on the ratio of the empty and full capsid stock solutions combined to prepare the samples (FIG.3D).
• the PA260/PA280 ratio approached an asymptote when the full capsids accounted for more than 70% of total capsids.
• the method had limited capability to determine the percentage full capsids with appropriate accuracy and precision when the full capsid content is greater than 70%.
• FIG.3C shows the SEC traces of the standards at 230 nm.
• the peak area monitored at 260 nm and 230 nm was proportional to the column loading and the PA260/PA230 ratio remains constant over the range tested, confirming that the intrinsic PA260/PA230 ratio was independent of the capsid concentration (see FIG.4).
• the PA260/PA230 ratio was unchanged across the loading range tested.
• the components in the formulation buffer are not expected to have any impact on the PA260/PA230 ratio.
• the Bio-Rad s gel filtration standard containing thyroglobulin, J-globulin, ovalbumin, myoglobin, and vitamin B12 was used to estimate what size of proteins may interfere with the analysis. Bovine thyroglobulin eluted at the retention time windows of 8-13 minutes, indicating that only proteins of this size (MW 670,000 Da) and higher would potentially interfere with the quantitation of empty and full capsids. This type of protein impurities is not expected to be present at a level that is impactful in the samples post affinity and/or strong anion exchange column purification.
• the injections of the mobile phase blank after 14 injections of AAV samples showed no detectable peaks by both wavelengths near the retention time of monomeric AAV5, indicating no carryover from the previous injections.
• the SEC-DW method was used to determine the percentage of full capsids in a composition representative of the drug substance (DS) and in a working reference standard (WRS). Samples were compared against a standard curve generated using HPLC standards containing different ratios of the empty and full capsids (FIG.3D). The analysis was then performed over multiple assay occasions (different mobile phase lots) on different days.
• Example 3 Comparison of determination of percent of full capsids by SEC-DW, AUC- SV and Cryo-EM [0194] The following example describes experiments comparing the ability of size- exclusion chromatography with dual-wavelength detection (SEC-DW), sedimentation velocity analytical ultracentrifugation (AUC-SV), and cryogenic electron microscopy (Cryo- EM) to determine the percentage of full capsids in AAV preparations. Materials and Methods [0195] Sedimentation Velocity Analytical Ultracentrifugation (AUC-SV).
• the AUC sample ( ⁇ 400 ⁇ L) was loaded into the sample sector of a two sector 1.2 cm Charcoal-filled Epon centerpiece (Beckman Coulter) and PBS ( ⁇ 410 ⁇ L) was loaded into the reference sector.
• the cells were inserted in a four-hole rotor and equilibrated at full vacuum and 20qC for at least one hour in the centrifuge (OptimaTM XL-I, Beckman Coulter).
• the vectors were sedimented at 20,000 rpm and the absorbance of the vectors was scanned at 260 nm in a continuous mode.
• the absorbance data was loaded into SEDFIT and fitted with a continuous c(s) distribution model.
• the meniscus was floated, and the friction ratio was fixed at 1.0 while fitting the data to the Lamm equation, with time-invariant (TI) and radius-invariant (RI) noise correction.
• the second-derivative regularization was applied to the fitting with a confidence level of 0.68.
• a range of 1-200 for sedimentation coefficients was used with a resolution of 200.
• the published density and viscosity values of PBS were used.
• the relative abundance of each species in unit of detection was converted to relative abundance in molar concentration by correcting the absorbance according to Beer’s Law. The content of each species was reported as a percent of the total. The details of the data analysis have been described in Burnham, B.
• Cryogenic electron microscopy (Cryo-EM) Grid preparation. C-flat holey carbon grids were cleaned with 20 mA for 30 seconds in a Pelco EasiGlow plasma cleaner. Vitrified specimens were prepared by loading a grid into a manual plunger (EMS-002 Rapid Immersion Freezer), adding 4 ⁇ L of virus to the grid, immediately one-side blotting the grid for 2 seconds, and freezing the sample in liquid ethane. [0199] Cryo-EM Imaging.
• Electron microscopy was performed using a Titan Krios ® electron microscope operated with an accelerating voltage at 300 kV and equipped with a K3 direct electron detector. Data was collected using SerialEM at 105,000 ⁇ nominal magnification (pixel size of 0.83 ⁇ ) at 2.5 ⁇ m defocus and a dose of ⁇ 48.0 e ⁇ / ⁇ 2 . [0200] Cryo-EM data analysis. An in-house data analysis software program developed at the Cryo-EM Core Facility of UMass Medical School was used to recognize and count the empty and full capsids. For quantitation of the percent full capsids, many images were processed and a total of approximately 2,200-8,700 vectors were analyzed and counted as empty and full capsids for each spiked sample.
• FIG.5A shows the sedimentation coefficient distribution plot for the subpopulations of the empty capsid sample (98% empty capsids (65S) and 2% partial capsids (81S)).
• FIG.5D The results for the full capsid sample purified via cesium chloride density gradient centrifugation are shown in FIG.5D and indicated that the sample contained 91% full capsids (104S) and 9% partial capsids (88S).
• the partial capsids with a sedimentation coefficient of 88S represented capsids harboring fragmented genomes of approximately 2,700 nucleotides (Nass, S.A. et al., Mol Ther Methods Clin Dev 2018, 9, 33-46).
• FIG.5B and FIG.5C show samples which were prepared by mixing the full and empty capsid containing samples. The % full capsid for these samples was calculated as 55% and 82% full capsids, respectively, based on the assigned values for the full and empty capsid samples and the combined volumes. The full capsid contents were 52% and 88% by AUC-SV for these two samples.
• Cryo-EM Cryogenic electron microscopy
• FIG.6A One of the Cryo-EM images taken for the spiked sample containing approximately 55% full capsids is shown in FIG.6A, in which the representative full capsid (displaying significant internal density) and empty capsid (absence of internal density) are circled and both the empty and full capsids are similar in size (approximately 24 nm).
• FIG.6B was selected from the images for the full capsid sample, and almost no empty capsids can be identified.
• For each spiked sample at least 2,200 particles from multiple images were counted using in-house data analysis software (Cryo-EM core facility of UMASS Medical School). The results from all images analyzed were combined to calculate the percentage of full capsids and ensure statistical significance.
• Partial capsids were treated as full capsids by Cryo-EM analyses in this study, although they can be identified by Cryo-EM via 3D classification (Subramanian, S. et al., Hum Gene Ther 2019, 30 (12), 1449-1460).
• the percentages of full capsids for the empty, spiked samples and full capsid sample are 3, 24, 37, 54, 81, 88 and 97%.
• the number of images analyzed, the total number of full capsids, the total number of empty capsids, the number of particles counted with low confidence (artifact, background signal, etc.), and the percent full capsids in the samples by Cryo-EM are summarized in Table 3.
• SEC-DW results were similar to the percent full capsids measured by Cryo-EM, and thus SEC-DW provides a viable option for good manufacturing practice testing.
• Table 3 Percent full capsids determined by Cryo-EM for the spiked samples Comparison of percent full capsids determined by SEC-DW, AUC-SV and Cryo-EM [0205] To scientifically validate the SEC-DW method, the percentage of full capsids determined by SEC-DW was compared with the results from two state-of-the-art high- resolution methods: AUC-SV and Cryo-EM which can accurately measure the percent full capsids.
• FIGS.7A-7B show the percentage of full capsids in spiked samples as determined by SEC-DW, AUC-SV and Cryo-EM.
• FIGS.7A-7B show the percentage of full capsids in spiked samples as determined by SEC- DW, AUC-SV and Cryo-EM. ), judged by the R 2 values from the linear regressions (0.997, 0.993, and 0.993 for SEC-DW, AUC-SV and Cryo-EM, respectively).
• the slopes of the linear regressions were 0.989, 1.062, and 1.038 for SEC-DW, AUC-SV and Cryo-EM, indicating that the SEC-DW method did not have any significant bias when compared to the AUC-SV and Cryo-EM methods.
• Partial capsids were treated as full capsids by Cryo-EM in this study, thus, the reported full capsids for each sample was slightly higher than those values from AUC-SV (FIGS.7A-7B show the percentage of full capsids in spiked samples as determined by SEC-DW, AUC-SV and Cryo-EM. ). Overall, the percent full capsids by SEC-DW based on PA260/PA230 agreed very well with the results from AUC-SV and Cryo- EM (FIGS.7A-7B show the percentage of full capsids in spiked samples as determined by SEC-DW, AUC-SV and Cryo-EM.
• AAV5 DS sample with a different transgene but the same AAV serotype (AAV5) was profiled by SEC-DW together with the corresponding enriched empty capsids (FIGS.9A-9D).
• the PA260/PA230 of the second DS sample was 0.58, estimated to be approximately 82% full capsids using the linear relationship shown in FIG.3D. Again, the PA260/PA230 ratios were the same across the loading range tested for both empty capsids and DS samples. Based on these results, it is envisioned that this SEC- DW method is generalizable to other AAV5 samples, and potentially to other serotypes.
• the SEC-DW method did not need additional sample preparations such as denaturation and labeling, and the impact of matrices were effectively eliminated during the size exclusion elution.
• the presence of partial capsids is thought to be inevitable using the current manufacturing processes.
• the gene sequences packaged in the partial capsids do contribute to the absorbance at 260 nm and 230 nm, and therefore introduce some variations to the reportable results. Some of these variations are built in the linear equation initially established and therefore the impact of partial capsids on the quantitation of the percent full capsids is limited.
• the light scattered by a spherical solute is inversely proportional to O (Russell, S.
• the light scattering can be corrected for absorbance at 230 nm and 260 nm using the wavelength dependent extrapolation.
• the absorbance spectra of empty and full capsids at 220 ⁇ 400 nm were extracted from the data acquired by the photodiode array (PDA) detector and no significant light scattering was observed based on the minimal to neglectable absorbance at 320 ⁇ 360 nm (see FIG.8, FIGS.9A-9D).
• the SEC-DW method described herein was established using various AAV5 DS samples and it is anticipated to be applicable for testing in-process samples as long as no interference from any other macromolecules that may elute as monomeric AAVs.
• the in- process samples can be readily concentrated with a concentrator to ensure a proper capsid concentration.
• This SEC-DW method can be applied similarly to other AAV serotypes once the linear relationship of PA260/PA230 vs. percent full capsids is established. Considering the low volume of sample and the instrument required, the SEC-DW method is anticipated to prove useful for determining empty and full capsid contents on a readily accessible instrument platform in a high throughput manner.
• Example 4 Size-exclusion chromatography with dual-wavelength detection for the AAV1 spiked samples
• SEC-DW size-exclusion chromatography with dual-wavelength detection
• the full capsids were characterized by analytical ultracentrifugation (AUC) and droplet digital PCR (ddPCR) (Bio-Rad, Hercules, CA) to determine the capsid distribution and vector genome concentration.
• AUC analytical ultracentrifugation
• ddPCR droplet digital PCR
• the concentration of the empty capsid preparation was determined by ELISA and the percent empty capsids was determined by TEM.
• Table 4 Capsid concentration, vector genome concentration and subpopulation distribution of empty and full capsids [0212]
• HPLC high-performance liquid chromatography
• spiked samples containing various amounts of full capsid were prepared by mixing the empty and full capsids at defined ratios based on their capsid concentrations (cp/mL).
• SEC-dual-wavelength AAV capsids (generally 40 ⁇ L or 50 ⁇ L per injection) were loaded onto a size exclusion column (Sepax SRT ® SEC-500, 5 ⁇ m, 500 ⁇ , 7.8 ⁇ 300 mm, Delaware) equipped with a guard column (SRT ® SEC-500, 5 ⁇ m, 500 ⁇ , 7.8 ⁇ 50 mm) equilibrated at 25oC with Dulbecco's phosphate buffered saline without CaCl2, without MgCl2 (DPBS).
• DPBS Dulbecco's phosphate buffered saline without CaCl2, without MgCl2
• the capsids were eluted with DPBS, pH 7.2 at a flow rate of 0.75 mL/min using an Agilent 1200 series HPLC (Agilent, CA). The eluate was monitored at 260 nm, 280 nm and 230 nm, and data was acquired with the Agilent OpenLab software. [0215] The peak areas of monomeric AAV at 230 nm, 280 nm and 260 nm were used for calculating the peak area (PA) ratios (e.g., PA260/PA230, and PA260/PA280). The correlation of the peak area ratios with the percent full capsids was established using the HPLC standards and the spiked samples containing various amounts of full capsids.
• PA peak area

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