Analytical ultracentrifugation method for characterizing recombinant virus particles

Abstract

The present invention provides methods for characterizing preparations of recombinant viral particles using analytical ultracentrifugation. Recombinant viral particles include recombinant adeno-associated viral particles, recombinant adenoviral particles, recombinant lentiviral particles, and recombinant herpes simplex viral particles. Different (variant) recombinant viral particles can be identified and quantified, including empty capsids and recombinant viral particles with different genomes (e.g., truncated genomes, aggregates, recombinants). The methods can be used to characterize preparations of recombinant viral particles regardless of the genome sequence of the recombinant virus or the serotype of the recombinant viral capsid.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680016288.9 (filed on January 19, 2016, entitled "Analytical Ultracentrifugation Method for Characterizing Recombinant Virus Particles").

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 105714, filed January 20, 2015, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0004] This invention relates to a method for characterizing recombinant viral vectors using analytical ultracentrifugation, such as recombinant adeno-associated viral (AAV) particles, recombinant adenovirus (rAd) particles, recombinant lentivirus particles, and recombinant herpes simplex virus (rHSV) particles. Background Technology

[0005] Recombinant viruses have shown great promise and practicality as vectors for delivering therapeutic nucleic acids in gene therapy applications. Several different recombinant viruses have been used in these gene therapy applications based on a variety of factors, including the size of the nucleic acid to be delivered, the target cells or tissues for delivery, the need for short-term or long-term performance of the therapeutic nucleic acid, and the integration of the therapeutic nucleic acid into the recipient genome. Examples of viruses used in gene therapy applications include adeno-associated virus (AAV), adenovirus, lentivirus, and herpes simplex virus (HSV).

[0006] The production of recombinant viral vectors for clinical use requires analytical methods that monitor the quality of the manufactured product regarding homogeneity, purity, and consistency; however, no method has yet been established to support this characterization. Typically, the DNA content of a recombinant viral DNA vector is measured using Southern blotting analysis with sequence-specific probes. The viral capsid or envelope can be characterized by immunoassays using antibodies that specifically bind to the capsid or envelope proteins of a particular recombinant virus. For example, Steinbach, S et al. (1997) J. Gen. Virol. ,78:1453-1462 provides an immunoassay for rAAV serotypes. What is needed is a general assay to characterize recombinant viral preparations, regardless of the nucleic acid sequence of the recombinant viral genome or the serotype of the capsid.

[0007] All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety. Summary of the Invention

[0008] In some aspects, the present invention provides a method for characterizing a preparation of recombinant viral particles, comprising: a) performing analytical ultracentrifugation on the preparation under boundary sedimentation velocity conditions, wherein the sedimentation of the recombinant viral particles is monitored at time intervals; b) plotting the sedimentation coefficient in Svedberg units (S) as a differential sedimentation coefficient distribution (C(s)); and c) integrating the area under each peak in the C(s) distribution to determine the relative concentration of each peak, wherein each peak represents a recombinant viral particle.

[0009] In some aspects, the present invention provides a method for assessing the vector genome integrity of recombinant viral particles in a preparation of recombinant viral particles, comprising: a) performing analytical ultracentrifugation on the preparation under boundary sedimentation velocity conditions, wherein the sedimentation of the recombinant viral particles is monitored at time intervals; b) plotting sedimentation coefficients expressed in Svedberg units (S) using differential sedimentation coefficient distribution values ​​(C(s)); and c) identifying the species of recombinant viral particles in the preparation by peaks corresponding to S values ​​present on the plot, wherein the genome size of a specific species of recombinant viral particles is calculated by comparing the S value of the species with a standard curve generated from the S values ​​of recombinant viral particles containing a viral genome of known nucleotide size with a capsid coating. In some embodiments, the method further includes integrating the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species.

[0010] In some aspects, the present invention provides a method for determining the presence of empty capsids or capsid particles containing recombinant viral genomes of different sizes in a preparation of recombinant viral particles, comprising the steps of: a) performing analytical ultracentrifugation on the preparation under boundary sedimentation velocity conditions, wherein the sedimentation of recombinant viral particles is monitored at time intervals; and b) plotting the sedimentation coefficients in Svedberg units (S) using differential sedimentation coefficient distribution values ​​(C(s)), wherein the presence of one or more peaks other than the peak of a complete capsid particle containing a complete recombinant viral genome indicates the presence of capsid particles containing genomes of different sizes and / or empty capsids.

[0011] In some aspects, the present invention provides a method for measuring the relative amount of empty capsids in a preparation of recombinant viral particles, comprising the steps of: a) performing analytical ultracentrifugation on the preparation under boundary settling velocity conditions, wherein the settling of recombinant viral particles is monitored at time intervals; b) plotting the settling coefficient in Svedberg units (S) against the differential settling coefficient distribution (C(s)); c) integrating the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species; and d) comparing the amount of recombinant viral particles having an S value corresponding to empty capsid particles with the amount of recombinant viral particles having an S value corresponding to recombinant viral particles containing a complete viral genome or the total amount of recombinant viral particles in the preparation.

[0012] In some aspects, the present invention provides a method for measuring the relative amounts of capsid particles or empty viral capsid particles containing different recombinant viral genomes in a recombinant viral particle preparation, comprising the steps of: a) performing analytical ultracentrifugation on the preparation under boundary sedimentation velocity conditions, wherein the sedimentation of recombinant viral particles is monitored at time intervals; b) plotting the sedimentation coefficient expressed in Svedberg units (S) with differential sedimentation coefficient distribution values ​​(C(s)); c) integrating the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species; d) comparing the amount of recombinant viral particles having an S value that does not correspond to a recombinant viral particle containing a complete viral genome with the amount of recombinant viral particles having an S value that corresponds to a recombinant viral particle containing a complete viral genome or the total amount of recombinant viral particles in the preparation.

[0013] In some aspects, the present invention provides a method for measuring the relative amount of capsid particles containing different recombinant viral genomes in a recombinant viral particle preparation, comprising: a) performing analytical ultracentrifugation on the preparation under boundary sedimentation velocity conditions, wherein the sedimentation of recombinant viral particles is monitored at time intervals; b) plotting the sedimentation coefficient expressed in Svedberg units (S) with differential sedimentation coefficient distribution values ​​(C(s)); c) integrating the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species; and d) comparing the amount of recombinant viral particles having an S value that does not correspond to a recombinant viral particle containing a complete viral genome or an empty capsid with the total amount of recombinant viral particles in the preparation.

[0014] In some aspects, the present invention provides a method for measuring the relative amount of recombinant viral particles containing a complete viral genome in a recombinant viral particle preparation, comprising: a) performing analytical ultracentrifugation on the preparation under boundary settling velocity conditions, wherein the settling of recombinant viral particles is monitored at time intervals; b) plotting the settling coefficient in Svedberg units (S) against a differential settling coefficient distribution value (C(s)); c) integrating the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species; and d) comparing the amount of recombinant viral particles having an S value corresponding to recombinant viral particles containing a complete viral genome with the amount of recombinant viral particles having an S value corresponding to empty capsid particles, with capsid particles containing different recombinant viral genomes, and / or with the total amount of recombinant viral particles in the preparation.

[0015] In some aspects, the present invention provides a method for monitoring the removal of empty capsids and / or capsid particles containing different recombinant viral genomes during the purification process of a recombinant viral particle preparation. The method includes removing a sample of recombinant viral particles from the preparation after one or more steps of the purification process, and analyzing the relative amount of empty capsids and / or capsid particles containing different recombinant viral genomes in the sample according to the method of any one of claims 5-8, wherein a decrease in the relative amount of empty capsids and / or capsid particles containing different genomes compared to a complete capsid indicates the removal of empty capsids from the recombinant viral particle preparation. In some embodiments, the presence of a peak corresponding to the S value of an empty capsid indicates the presence of empty capsid particles. In some embodiments, the presence of one or more peaks other than the peaks of complete capsid particles containing a complete recombinant viral genome or empty capsid particles indicates the presence of capsid particles containing genomes of different sizes. In some embodiments, the capsid particles containing genomes of different sizes include truncated genomes, aggregates, recombinants, and / or DNA impurities.

[0016] In some aspects, the present invention provides a method for determining the heterogeneity of recombinant viral particles in a preparation of recombinant viral particles, comprising the steps of: a) performing analytical ultracentrifugation on the preparation under boundary sedimentation velocity conditions, wherein the sedimentation of recombinant viral particles is monitored at time intervals; b) plotting sedimentation coefficients expressed in Svedberg units (S) as differential sedimentation coefficient distribution values ​​(C(s)), wherein the presence of peaks other than those representing a capsid containing a complete recombinant viral genome indicates heterogeneity of recombinant particles in the preparation. In some embodiments, the presence of the additional peaks indicates the presence of empty capsid particles and / or recombinant viral particles containing different genomes. In some embodiments, the different genomes are truncated viral genomes, aggregates, recombinants, and / or DNA impurities. In some embodiments, the method further comprises integrating the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species.

[0017] In some aspects, the present invention provides a method for monitoring the homogeneity of recombinant viral particles during the purification process of a recombinant viral particle preparation, the method comprising removing a sample of recombinant viral particles from the preparation after one or more steps of the purification process, and determining the heterogeneity of the recombinant viral particles according to the above method, wherein an increase in the relative amount of recombinant viral particles containing a complete viral genome indicates an increase in the homogeneity of complete viral particles in the recombinant viral particle preparation.

[0018] In some embodiments of the above aspects, the sedimentation of recombinant viral particles is monitored by absorbance. In some embodiments, the absorbance is about 230 nm, 260 nm, or 280 nm. In some embodiments, the absorbance is about 260 nm. In some embodiments, the sedimentation of recombinant viral particles is monitored by interference. In some embodiments, the interference is Rayleigh interference.

[0019] In some embodiments of the above aspects, the preparation is an aqueous solution. In further embodiments, the aqueous solution comprises a pharmaceutical formulation. In some embodiments, the aqueous solution comprises a buffer. In some embodiments, the buffer is at a physiological pH. In some embodiments, the buffer is at a physiological osmolality concentration. In some embodiments, the pharmaceutical formulation comprises phosphate-buffered saline (PBS). In some embodiments, the PBS has a pH of about 7.2 and an osmolality concentration of about 300 mOsm / L. In some embodiments, the monitoring further comprises comparison with a reference sample, wherein the reference sample comprises an aqueous solution free of recombinant viral particles.

[0020] In some embodiments of the above aspects, the C(S) value is determined by an algorithm incorporating Lamm equation solutions. In some embodiments, the algorithm is the SEDFIT algorithm. In some embodiments, sedimentation is monitored until the lowest density recombinant viral particles settle to the bottom of the sector of the ultracentrifuge. In some embodiments, the ultracentrifugation utilizes an ultracentrifuge containing an ultracentrifuge velocity cell. In some embodiments, sedimentation is monitored until the recombinant viral particles settle to the bottom of the ultracentrifuge velocity cell. In some embodiments, sedimentation is monitored until the lowest density recombinant viral particles settle and pass through an optical window (sediment and clear the optical window).

[0021] In some embodiments, radial concentration is recorded for at least about 0.5 hours, 0.75 hours, 1.0 hour, 1.5 hours, 2.0 hours, 3.0 hours, 4.0 hours, or 5.0 hours. In some embodiments, radial concentration is recorded for about 1.2 hours. In some embodiments, radial concentration is recorded for about 0.5 hours to about 2.0 hours. In some embodiments, radial concentration is recorded for about 1.0 hour to about 2.0 hours.

[0022] In some embodiments, at least 30 scans are used to monitor the sedimentation of recombinant viral particles. In some embodiments, about 30 scans are used to monitor the sedimentation of recombinant viral particles. In some embodiments, about 30 to about 75 scans are used to monitor the sedimentation of recombinant viral particles. In some embodiments, about 30 to about 50 scans are used to monitor the sedimentation of recombinant viral particles. In some embodiments, about 50 to about 75 scans are used to monitor the sedimentation of recombinant viral particles.

[0023] In some embodiments, regularization is applied to the fitting level with a confidence level of at least about 0.68 for the F-statistic. In some embodiments, the regularization is second-derivative regularization. In some embodiments, the regularization is maximum entropy regularization. In some embodiments, regularization is applied to the fitting level with a confidence level of about 0.68 to about 0.90 for the F-statistic. In some embodiments, regularization is applied to the fitting level with a confidence level of about 0.68 to about 0.99 for the F-statistic. In some embodiments, regularization is applied to the fitting level with a confidence level of about 0.68 for the F-statistic.

[0024] In some implementations, the following C(S) parameters remain constant: resolution is approximately 200S to approximately 5000S, S 最小值 It is from 1S to approximately 100S, S 最大值 The time interval is approximately 100 s to approximately 5000 s, and the frictional ratio is approximately 1.0 or fluctuates to a value determined by the centrifugation software. In some embodiments, the resolution is approximately 200 s to approximately 1000 s. In some embodiments, the resolution is approximately 200 s. In some embodiments, S 最小值 It is approximately 1. In some implementations, S 最大值 It is approximately 100 seconds to approximately 1000 seconds. In some implementations, S 最大值 It is approximately 200S to approximately 5000S. In some implementations, S 最大值 It is approximately 200 seconds. In some embodiments, the friction ratio is floated to a value determined by centrifugation software. In some embodiments, the friction ratio is approximately 1.0. In some embodiments, radius invariant (RI) and time invariant (TI) noise reduction are applied.

[0025] In some embodiments, the settling of recombinant viral particles is monitored approximately every 10-60 seconds. In some embodiments, the settling of recombinant viral particles is monitored (e.g., scanned) approximately every 10 seconds. In some embodiments, the settling of recombinant viral particles is monitored approximately every 60 seconds. In some embodiments, the settling rate of the recombinant virus during ultracentrifugation is determined by monitoring the settling of each recombinant viral particle more than approximately every 15 seconds, 30 seconds, 45 seconds, 1 minute (60 seconds), 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, and 25 minutes.

[0026] In some embodiments of the foregoing aspects, the boundary settlement rate is from about 3,000 rpm to about 20,000 rpm. In some embodiments, the boundary settlement rate is from about 3,000 rpm to about 10,000 rpm. In some embodiments, the boundary settlement rate is from about 10,000 rpm to about 20,000 rpm. In some embodiments, the boundary settlement rate is from about 15,000 rpm to about 20,000 rpm.

[0027] In some embodiments of the above aspects, the boundary settlement rate is carried out at a temperature of about 4°C to about 20°C. In some embodiments, the boundary settlement rate is carried out at a temperature of about 4°C.

[0028] In some embodiments of the above aspects, the recombinant viral particles are recombinant adeno-associated virus (AAV) particles, recombinant adenovirus particles, recombinant lentivirus particles, or recombinant herpes simplex virus (HSV) particles. In some implementations, the recombinant viral particles comprise AAV1 capsids, AAV2 capsids, AAV3 capsids, AAV4 capsids, AAV5 capsids, AAV6 capsids, AAV7 capsids, AAV8 capsids, AAVrh8 capsids, AAV9 capsids, AAV10 capsids, AAVrh10 capsids, AAV11 capsids, AAV12 capsids, AAV2R471A capsids, AAVAAV2 / 2-7m8 capsids, AAV DJ capsids, AAV2 N587A capsids, AAV2 E548A capsids, AAV2N708A capsids, AAV V708K capsids, goat AAV capsids, AAV1 / AAV2 chimeric capsids, bovine AAV capsids, or mouse AAV capsids rAAV2 / HBoV1 (chimeric AAV / human bocavirus 1). In some embodiments, the recombinant viral particles comprise 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, and AAV12 ITR. In some embodiments, the AAV capsid contains a tyrosine mutation or a heparin-binding mutation. In other embodiments, the recombinant viral particles are recombinant adenovirus particles. In some embodiments, the recombinant adenovirus particles comprise a capsid from adenovirus serotypes 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad 3, canine Ad 2, sheep Ad, or swine Ad 3. In some embodiments, the recombinant adenovirus particles comprise a variant of the adenovirus serotype 2 capsid or a variant of the adenovirus serotype 5 capsid. In some embodiments, the recombinant virus particles are recombinant lentivirus particles. In some embodiments, the recombinant lentiviral particles are pseudovesicular stomatitis virus (VSV), lymphocytic choriomeningovirus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokala virus, rabies virus, RD114, or variants thereof. In some embodiments, the recombinant viral particles are rHSV particles.In some implementations, the HSV particles are HSV-1 particles or HSV-2 particles.

[0029] In some aspects, the present invention provides a method for evaluating a method of producing recombinant viral particles, the method comprising the method of any one of claims 1-67, wherein an increase in the relative amount of recombinant viral particles containing a complete viral genome compared to a reference preparation of recombinant viral particles, compared to the relative amount of empty capsid particles and / or recombinant viral capsid particles with different recombinant viral genomes, indicates an improvement in the yield of recombinant viral particles. In some embodiments, the recombinant viral particles are recombinant adeno-associated virus (AAV) particles, recombinant adenovirus particles, recombinant lentivirus particles, and recombinant herpes simplex virus (HSV) particles. In some embodiments, rAAV particles are generated from producer cell lines. In some embodiments, the rAAV particles are generated by triple transfection of: i) nucleic acids encoding AAVrep and cap, ii) an rAAV vector sequence, and iii) nucleic acids encoding adenovirus helper functions. In other embodiments, the recombinant viral particles are generated by AAV / HSV hybridization. In other embodiments, the recombinant viral particles are generated from baculovirus cells. In some embodiments, recombinant viral particles are generated by transiently transfecting nucleic acids encoding AAV vector sequences, AAV rep and cap coding regions, and AAV helper virus functions into suitable host cells. In some embodiments, recombinant viral particles are generated by introducing one or more nucleic acids encoding AAV vector sequences, AAV rep and cap coding regions, and AAV helper virus functions into suitable host cells, wherein the one or more nucleic acids are introduced into the cells using a recombinant helper virus. In some embodiments, the recombinant helper virus is an adenovirus or herpes simplex virus. In some embodiments, the recombinant viral particles contain a self-complementary AAV (scAAV) genome. In some embodiments, the method is used to detect the presence of recombinant viral particles containing either a monomeric or a dimer form of the scAAV genome.

[0030] In some embodiments of the above aspects, the recombinant viral particles are generated by transiently transfecting nucleic acids encoding adenovirus vector sequences and adenovirus replication and packaging sequences into suitable host cells. In other embodiments, the recombinant viral particles are generated by transiently transfecting nucleic acids encoding lentiviral vector sequences and / or lentiviral replication and packaging sequences into suitable host cells. In other embodiments, the recombinant viral particles are generated by transiently transfecting nucleic acids encoding HSV vector sequences and / or HSV replication and packaging sequences into suitable host cells.

[0031] In some aspects, the present invention provides a method for preparing recombinant viral particles having reduced empty capsids and / or recombinant viral particles containing different genomes, the method comprising: a) culturing host cells under conditions suitable for recombinant virus production, wherein the cells contain: i) nucleic acids encoding heterologous transgenes flanked by at least one AAV ITR, ii) nucleic acids containing AAV rep and cap coding regions, wherein the nucleic acids contain a mutated p5 promoter, wherein rep expression from the p5 promoter is reduced compared to the wild-type p5 promoter, and iii) nucleic acids encoding AAV helper viral functions; b) lysing the host cells to release the recombinant viral particles; c) isolating the recombinant viral particles produced by the host cells; and d) analyzing the presence of empty capsids and / or recombinant viral particles with different genomes in the recombinant viral particles by analytical ultracentrifugation, according to the method described above. In some aspects, the present invention provides a method for preparing recombinant viral particles having reduced empty capsids and / or containing recombinant viral particles with different genomes, the method comprising: a) culturing host cells under conditions suitable for recombinant virus production, wherein the cells contain: i) nucleic acids encoding a heterologous transgene flanked by at least one AAV ITR, ii) nucleic acids containing AAV rep and cap coding regions, wherein the nucleic acids contain a mutated p5 promoter, wherein rep expression from the p5 promoter is reduced compared to the wild-type p5 promoter, and iii) nucleic acids encoding AAV helper viral functions; b) lysing the host cells to release the recombinant viral particles; c) isolating the recombinant viral particles produced by the host cells; and d) analyzing the presence of empty capsids and / or recombinant viral particles with different genomes in the recombinant viral particles by analytical ultracentrifugation, according to the method described above. In some embodiments, the p5 promoter is located at the 3' of the rep and / or cap coding region. In some implementations, the AAV auxiliary virus functionality includes adenovirus E1A functionality, adenovirus E1B functionality, adenovirus E2A functionality, adenovirus VA functionality, and adenovirus E4 orf6 functionality.

[0032] In some embodiments, the recombinant viral particles in any of the foregoing embodiments have been purified using one or more purification steps. Attached Figure Description

[0033] Figure 1A and 1B Analytical ultracentrifugation (AUC) shows that it can be used to characterize recombinant viral vector particles. Figure 1A The representative boundary sedimentation velocity scan spectrum, plotted against radius (cm) of the AAV2 mixture at 1.2-hour time intervals (T), represents the absorbance (260 nm) of the mixture. The AAV2 mixture contains empty capsids (“empty Cap”) and complete genome capsids (“complete vector”). Figure 1B Concentration plots, measured in C(S), compared with sedimentation coefficients (Svedberg units, S), show that AUC can be used to measure the concentration of empty capsids and complete genome capsids from 80% / 20% mixtures. Each peak is labeled with particle species and their corresponding sedimentation coefficient (S) and relative abundance (%).

[0034] Figure 2A and 2B Displaying an empty AAV2 shell ( Figure 2A ) and the AAV2-transgenic 1 capsid containing the genome ( Figure 2B The AUC profile of the pure population is shown. Each peak is labeled with the capsid species and its sedimentation coefficient (S).

[0035] Figure 3A and 3B This shows a comparison of AUC between the interference and absorbance detection methods. Figure 3A The sedimentation coefficient distribution values ​​c(s) were compared with the sedimentation coefficient plot expressed in Svedberg units (S) to obtain the sedimentation coefficient distribution of 1:1 mixtures of empty capsids and genomic capsids, generated using interferometric detection. The sedimentation coefficient and relative abundance (%) of each species were labeled. Figure 3B The sedimentation coefficient distribution values ​​c(s) were compared with the sedimentation coefficient plot expressed in Svedberg units (S) to obtain the sedimentation coefficient distribution of 1:1 mixtures of empty capsids and genomic capsids, generated using absorbed light detection (260 nm). The sedimentation coefficient and relative abundance (%) of each species were labeled.

[0036] Figure 4 A triple transfection method for AAV vector production is described. Three vectors containing the target gene (“pVector”), the AAVRep and Cap genes (“pHLP”), and an adenovirus component (“pIAdeno”) are labeled. Note that both genomic capsids (labeled with an “ITR-Transgene-ITR” graphic) and empty capsids (blank) are produced.

[0037] Figure 5 A producer cell line method for AAV vector production is described. As indicated by the label, the HeLa S3 cell line contains integrated Rep, Cap, and puromycin resistance genes, as well as the target transgene flanked by an ITR. This cell line is infected with adenovirus (“Ad5”) to stimulate the production of recombinant virus. Note that both a genome-containing capsid (labeled “recombinant viral vector”) and an empty capsid are produced, in addition to the adenovirus particles.

[0038] Figure 6A ,6B 6C and other methods showed that different vector preparations were obtained through vector production via producer cell lines and triple transfection methods, as revealed by AUC analysis. Figure 6A A schematic diagram of the AAV2-transgenic vector and its 3.4kb genome. Figure 6B The sedimentation coefficient distribution of the carrier preparations produced via the producer cell line method was obtained by comparing the differential sedimentation coefficient distribution value c(s) with a graph of sedimentation coefficients expressed in Svedberg units (S). The sedimentation coefficient and relative abundance (%) of each species were labeled. Figure 6C The distribution of sedimentation coefficients of the carrier preparations generated by the triple transfection method was obtained by comparing the differential sedimentation coefficient distribution value c(s) with the sedimentation coefficient plot expressed in Svedberg units (S). The sedimentation coefficient and relative abundance (%) of each species were labeled.

[0039] Figure 7A , 7B As shown by 7C, the AUC method can be used to monitor the quality and potency of vector purification. Figure 7A This image shows a graph illustrating the purification of the complete genome AAV2-transgenic 1 capsid from empty capsids using anion exchange chromatography. Peak fractions corresponding to each species are labeled. Figure 7B The sedimentation coefficient distribution of the support preparation after elution from the anion exchange column was obtained by comparing the differential sedimentation coefficient distribution value c(s) with the sedimentation coefficient expressed in Svedberg units (S). The sedimentation coefficient and relative abundance (%) of each species were labeled. Figure 7C The sedimentation coefficient distribution of the carrier preparation prior to chromatography was obtained by comparing the differential sedimentation coefficient distribution value c(s) with a graph of sedimentation coefficients expressed in Svedberg units (S). The sedimentation coefficient and relative abundance (%) of each species were labeled.

[0040] Figure 8 The linear relationship between sedimentation coefficient and vector genome size is shown. A standard curve of sedimentation coefficient (S) versus genome size is plotted, along with the best-fit line, its formula, and its associated R² value.

[0041] Figure 9A , 9B The results (9C) show a correlation between the assessment of capsid genome size using AUC data and the assessment of genome size using Southern blotting. Figure 9AThe sedimentation coefficient distribution of the scAAV9 EGFP vector preparation was obtained by comparing the differential sedimentation coefficient distribution value c(s) with the sedimentation coefficient plot expressed in Svedberg units (S). The single-stranded monomers (82S) and double-stranded dimers (101S) were labeled using the corresponding sedimentation coefficients and relative abundance values ​​(%). A schematic diagram of the vector is also provided. Figure 9B Basic Southern blotting analysis of DNA from the capsids of scAAV9 EGFP (lane 1) and single-stranded AAV9 EGFP (lane 2) vectors. Corresponding bands were labeled as described in the blotting instructions. Size standards of 4.2 and 2.4 kb were provided as indicated by the labels. Figure 9C The sedimentation coefficient distribution of the single-stranded AAV9 EGFP vector was obtained by comparing the differential sedimentation coefficient distribution value c(s) with the sedimentation coefficient plot expressed in Svedberg units (S). The 82S and 99S (complete genome) peaks were labeled with the corresponding sedimentation coefficients and relative abundance values ​​(%).

[0042] Figure 10A , 10B 10C and other data show that the Rep / Cap promoter position affects genome packaging in recombinant viral vectors generated by triple transfection methods. Figure 10A A schematic diagram of the self-assembled scAAV2 EGFP vector, and the estimated sedimentation coefficients for dimer and monomeric genome types. Figure 10B The sedimentation coefficient distribution of the scAAV2 EGFP vector preparation, generated using "wild-type" helper plasmids ("WT Rep") containing an endogenous p5 promoter driving Rep 78 / 68 expression, was obtained by comparing the differential sedimentation coefficient distribution value c(s) with the sedimentation coefficient plot expressed in Svedberg units (S). Peaks for single-stranded monomers (80S) and double-stranded dimers (100S) were labeled with their respective sedimentation coefficients and relative abundance values ​​(%). Figure 10C The sedimentation coefficient distribution of the scAAV2 EGFP vector preparation, expressed in Svedberg units (S), was obtained by comparing the differential sedimentation coefficient distribution value c(s) with the sedimentation coefficient plot. This preparation was generated using a "wild-type" helper plastid ("pHLP Rep") containing an endogenous p5 promoter (shifted downstream of the cap2 sequence) driving Rep 78 / 68 expression. Peaks for single-stranded monomers (82S) and double-stranded dimers (100S) were labeled with the corresponding sedimentation coefficients and relative abundance values ​​(%).

[0043] Figure 11A , 11B 11C and 11D show that the Rep / Cap promoter position affects genome packaging in two additional AAV vectors. Figure 11Aand 11B The sedimentation coefficient distribution of the single-stranded AAV5 Factor IX vector (AAV5 hFIX16) was obtained by comparing the differential sedimentation coefficient distribution value c(s) with the sedimentation coefficient expressed in Svedberg units (S). The vector contains a cap5 sequence generated by a helper plasmid with an endogenous p5 promoter (“WT Rep,”). Figure 11B ) or the p5 promoter downstream of the cap5 sequence (“pHLP Rep,” Figure 11A ). ( Figure 11C-11D The sedimentation coefficient distribution of the AAV5hSMN vector (AAV5SMN) was obtained by comparing the differential sedimentation coefficient distribution value c(s) with the sedimentation coefficient in Svedberg units (S). The vector contains a cap5 sequence generated by a helper plasmid with an endogenous p5 promoter (“WT Rep,”). Figure 11D ) or the p5 promoter downstream of the cap5 sequence (“pHLP Rep,” Figure 11C ).

[0044] Figure 12A and 12B The study revealed a correlation between Southern blot analysis and AUC analysis, but missed some fragmented genome segments that could be detected by AUC. Figure 12A Southern blotting analysis of vector DNA from AAV5SMN preparations was performed using pHLP helper plasmids (lane 2) or WT Rep plasmids (lane 1). Size standards of 4.6 and 2.4 kb are provided, as labeled. Figure 12B Southern blotting analysis of vector DNA from AAV5FIX preparations was performed using pHLP helper plasmids (lane 2) or WT Rep plasmids (lane 1). Size standards of 4.3, 3.0, and 1.9 kb were provided, as labeled.

[0045] Figure 13 A map of the AAV5 Factor IX vector is provided, indicating the location of the hFIX transgene, ITR, Rep origin, and AmpR marker gene, as well as other features. Note that the AmpR marker gene is upstream of the ITR, enhancer, and promoter regions.

[0046] Figure 14A and 14B This demonstrates how the WT Rep vector genome (different from the pHLP Rep vector genome) packages the sequence upstream of the 5' ITR in the AAV5Factor IX vector. Figure 14ASouthern imprinting analysis of the genomes of pHLP Rep (lane 1) and WT Rep (lane 2) vectors was compared using hFIX transgene-specific probes. Figure 14B Southern imprinting analysis of the genomes of pHLP Rep (lane 1) and WT Rep (lane 2) vectors was compared using Repori / AmpR-specific probes.

[0047] Figure 15A and 15B Excessive fragmentation of the AAV vector genome was observed, as indicated by the AUC analysis. Figure 15A Concentration C(S) versus sedimentation coefficient (S) plot of AAV vectors with excessively large genomes, generated by AUC. The genome contains a full-length chicken β-actin (CBA) promoter, which drives the expression of β-phosphodiesterase (ssAAV2 / 5CBA-βPDE). Peaks of detected species were labeled by observed sedimentation coefficient (S) and relative abundance (%). Figure 15B A concentration C(S) versus sedimentation coefficient (S) plot of AAV vectors with truncated genomes, generated by AUC. This genome contains a CBA promoter with reduced-size introns, which drives the expression of β-phosphodiesterase (AAV5 minCBAPDE6B). Peaks of detected species were labeled by observed sedimentation coefficient (S) and relative abundance (%).

[0048] Figure 16 The AUC profile of a pure population of adenovirus capsids is shown. The sedimentation coefficient (S) and interference value are given for each peak. Detailed Implementation

[0049] This invention provides a method for characterizing viral particle preparations using analytical ultracentrifugation. By performing analytical ultracentrifugation (AUC) on the preparations under boundary settling velocity conditions, the sedimentation of viral particles can be monitored at time intervals (e.g., once or multiple times). The differential sedimentation coefficient distribution (C(s)) is then plotted against the sedimentation coefficient in Svedberg units (S), and the area under each peak in the C(s) distribution is integrated to determine the relative concentration of each peak. Each peak represents the type of viral particle, reflecting its molecular weight. Types that can be detected by these methods include, but are not limited to, recombinant adeno-associated virus (rAAV) particles, recombinant adenovirus (rAd) particles, recombinant lentivirus particles, and recombinant herpes simplex virus (rHSV) particles. To use rAAV particles as an illustrative example, these methods allow for the detection of rAAV species, including capsid particles containing the complete rAAV genome (e.g., complete capsids), empty viral capsids (where the rAAV genome is not encapsulated within the viral capsid), and rAAV particle variants where the variant rAAV genome is encapsulated within the viral capsid (e.g., particles containing capsulated AAV DNA impurities, truncated viral genomes, aggregates, etc.). These methods can be applied to the preparation of recombinant viruses regardless of the nucleic acid sequence of the viral genome or the serotype of the recombinant viral capsid (for recombinant viral particles). These methods can be applied to rAAV, rAd, recombinant lentiviruses, and rHSV viral particles.

[0050] In some aspects, the present invention provides a method for assessing the vector genome integrity of recombinant viral particles in a preparation of recombinant viral particles, which involves analytical ultracentrifugation of the preparation under boundary settling velocity conditions, wherein the settling of the recombinant viral particles is monitored at time intervals (e.g., once or multiple times). By plotting the settling coefficient in Svedberg units (S) as a differential settling coefficient distribution value (C(s)), the species of recombinant viral particles in the preparation can be identified by the peaks corresponding to the S values ​​present on the graph. The genome size of a specific species of recombinant viral particle can be calculated, for example, by comparing the S value of the species with a standard curve generated from the S values ​​of recombinant viral particles containing known different-sized viral genomes with capsid coatings. Vector genomes that can be evaluated using these methods include, but are not limited to: recombinant viral capsid particles containing a complete viral genome (e.g., a complete capsid), empty viral capsids (where the rAAV genome is not encapsulated within the viral capsid), and recombinant viral particle variants, wherein the variant recombinant viral genome (e.g., particles containing capsid-coated AAV DNA impurities, truncated viral genomes, aggregates, etc.) is encapsulated within the viral capsid. In some embodiments, the viral particles are rAAV, rAd, recombinant lentivirus, or rHSV viral particles.

[0051] In some embodiments, the present invention provides a method for determining the heterogeneity of recombinant viral particles (e.g., rAAV, rAd, lentivirus, or rHSV particles) in a recombinant viral particle preparation by means of AUC under boundary settling velocity conditions, wherein the presence of peaks other than the peak representing the capsid containing the complete recombinant viral genome in the C(S) vs. S plot indicates the heterogeneity of recombinant particles in the preparation. In some embodiments, the relative amount of each recombinant viral species in the preparation is calculated by integrating the area of ​​each peak in the plot.

[0052] In some embodiments of the invention, AUC is used to determine the presence of empty capsids and / or recombinant viral particle variants in recombinant viral particle preparations (e.g., rAAV, rAd, lentivirus, or rHSV particles), wherein the presence of a peak corresponding to the S-value of an empty capsid particle and / or recombinant viral particle variant in a C(S) vs. S plot indicates the presence of an empty capsid particle and / or recombinant viral particle variant. In some embodiments, the relative amount of empty capsids and / or recombinant viral particle variants in the recombinant viral particle preparation is determined by integrating the area under each peak in the C(S) vs. S plot and comparing the amount of recombinant viral particles having an S-value corresponding to an empty capsid particle and / or recombinant viral particle variant with the amount of recombinant viral particles having an S-value corresponding to a recombinant viral particle containing a complete viral genome. In some embodiments, the amount of recombinant viral particles having an S-value corresponding to an empty capsid particle and / or recombinant viral particle variant is compared to the total amount of all recombinant viral particles in the preparation, which is obtained by integrating and summing the areas under all peaks in the plot.

[0053] In some embodiments, the present invention provides a method for monitoring the removal of empty capsids and / or recombinant viral particle variants during the purification process of a recombinant viral particle preparation (e.g., rAAV, rAd, lentivirus, or rHSV particles) using AUC. A sample of recombinant viral particles from the preparation after one or more steps of the purification process is analyzed to determine the relative amounts of empty capsids and / or recombinant viral particle variants, wherein a decrease in the relative amount of empty capsids and / or recombinant viral particle variants compared to fully capsidated particles indicates the removal of empty capsids and / or recombinant viral particle variants from the recombinant viral particle preparation.

[0054] In some embodiments, the present invention provides a method for evaluating the production of recombinant viral particles (e.g., rAAV, rAd, lentivirus, or rHSV particles) by AUC. The analysis examines the presence of complete viral capsid particles, empty particles, and / or recombinant viral particle variants in the recombinant viral particle preparation. An increase in the relative amount of recombinant viral particles containing a complete viral genome compared to a reference preparation of recombinant viral particles (e.g., a standard recombinant viral preparation) indicates an improvement in recombinant viral particle yield.

[0055] I. General Technology

[0056] The techniques and procedures described herein are well known to those skilled in the art and commonly employed using conventional methods, such as: *Molecular Cloning: A Laboratory Manual* (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); *Current Protocols in Molecular Biology* (edited by F.M. Usubel et al., 2003); *Methods in Enzymology* (Academic Press, Inc.); *PCR 2: A Practical Approach* (edited by M.J. MacPherson, B.D. Hames, and G.G. Taylor et al., 1995); *Antibodies, A Laboratory Manual* (edited by Harlow and Lane, 1988); *Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications* (R.I. Freshney, 6th ed., J. Wiley and Sons, 2010); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Methods in Molecular Biology, Humana*. Press; CellBiology: A Laboratory Notebook (JECellis, Academic Press, 1998); Introduction to Cell and Tissue Culture (JPMather and PERoberts, Plenum Press, 1998); Celland Tissue Culture: Laboratory Procedures (A.Doyle, JBGriffiths and DG Newell, J.Wiley and Sons, 1993-8); Handbook of Experimental Immunology (DMWeir and CC Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., 1994); Current Protocols in Immunology (JEColigan et al., 1991); Short Protocols in Molecular Biology (Ausubel et al., J.Wiley and Sons, 2002); Immunobiology (CA Janeway et al., 2004); Antibodies (P.Finch, 1997); Antibodies: A PracticalApproach (edited by D.Catty, IRL Press, 1988-1989); Monoclonal Antibodies: A PracticalApproach (edited by P.Shepherd and C.Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E.Harlow and D.Lane, Cold Spring Harbor Laboratory Press,1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and the widely used methods described in *Cancer: Principles and Practice of Oncology* (VT DeVita et al., eds., JBLippincott Company, 2011).

[0057] II. Definition

[0058] As used in this article, “vector” refers to a recombinant plasmid or virus containing nucleic acid to be delivered to a host cell (in vitro or in vivo).

[0059] As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length (whether ribonucleotides or deoxyribonucleotides). Therefore, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine or pyrimidine bases or other natural, chemically or biologically modified, non-natural, or derived nucleotide bases. The backbone of a polynucleotide may contain sugar and phosphate groups (as commonly found in RNA or DNA) or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide may contain a polymer of synthetic subunits (such as aminophosphates) and thus may be an oligodeoxynucleotide aminophosphate (P-NH₂) or a mixture of aminophosphate-phosphodiester oligomers. Furthermore, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products by synthesizing a complementary strand and annealing the strand under suitable conditions, or by de novo synthesis of a complementary strand using a DNA polymerase with suitable primers.

[0060] The terms "peptide" and "protein" are used interchangeably to refer to polymers of amino acid residues, and there is no minimum length requirement. Such polymers of amino acid residues can contain native or non-native amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. Both full-length proteins and fragments thereof are included in this definition. The term also includes post-expression modifications of peptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Furthermore, for the purposes of this invention, "peptide" means a protein containing modifications such as deletions, additions, and substitutions of the original sequence (generally conserved in nature), provided that the protein retains the desired activity. These modifications can be intentional, such as through directed mutagenesis; or they can be accidental, such as through mutations in the host producing the protein or due to PCR amplification errors.

[0061] "Recombinant viral vector" refers to a recombinant polynucleotide vector containing one or more heterologous sequences (i.e., non-viral original nucleic acid sequences). In the case of recombinant AAV vectors, the recombinant nucleic acid flanking sequence has at least one inverted terminal repeat (ITR). In some embodiments, the recombinant nucleic acid flanking sequence has at least two inverted terminal repeat (ITRs).

[0062] "Recombinant AAV vector (recombinant adeno-associated virus vector)" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., non-AAV original nucleic acid sequences) flanked by at least one AAV inverted terminal repeat (ITR). In some embodiments, the recombinant nucleic acid flanks at least two inverted terminal repeats (ITRs). Such recombinant viral vectors, when present in host cells that have been infected with a suitable helper virus (or exhibiting a suitable helper function) and express AAV rep and cap gene products (i.e., AAV Rep and Cap proteins), can replicate and package into infectious viral particles. When a recombinant viral vector integrates into a larger polynucleotide (e.g., a chromosome or another vector used for cloning or transfection, such as a plastid), the term "recombinant viral vector" can refer to a "pro-vector" that can be "rescued" through replication and capsid coating in the presence of AAV packaging function and a suitable helper function. Recombinant viral vectors can exist in any of many forms, including but not limited to plastids, linear artificial chromosomes, lipid complexes, encapsulated within liposomes, and capsid-coated viral particles, such as AAV particles. Recombinant viral vectors can be packaged in AAV viral capsids to produce "recombinant adeno-associated virus particles (recombinant viral particles)".

[0063] "rAAV virus" or "rAAV virus particle" refers to a viral particle consisting of at least one AAV capsid protein and a capsid-coated rAAV vector genome.

[0064] "Recombinant adenoviral vector" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., non-adenoviral original nucleic acid sequences) flanked by at least one adenoviral inverted terminal repeat (ITR). In some embodiments, the recombinant nucleic acid flanked by at least two inverted terminal repeats (ITRs). Such recombinant viral vectors, when present in host cells expressing basic adenoviral genes deleted from the recombinant viral genome (e.g., E1, E2, E4, etc.), can replicate and be packaged into infectious viral particles. When a recombinant viral vector integrates into a larger polynucleotide (e.g., a chromosome or another vector used for cloning or transfection, such as a plastid), the recombinant viral vector can refer to a "pre-vector" that can be "rescued" through replication and capsid coating in the presence of adenoviral packaging function and suitable helper functions. Recombinant viral vectors can exist in any of many forms, including but not limited to plastids, linear artificial chromosomes, lipid complexes, encapsulated within liposomes, and capsid-coated viral particles, such as adenoviral particles. Recombinant viral vectors can be packaged in adenovirus capsids to produce "recombinant adenovirus particles".

[0065] "Recombinant lentiviral vector" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., non-lentiviral original nucleic acid sequences) flanked by at least one lentiviral terminal repeat (LTR). In some embodiments, the recombinant nucleic acid flanks at least two lentiviral LTRs. Such recombinant viral vectors can replicate and package into infectious viral particles in the presence of host cells that have been infected with a suitable helper virus. The recombinant viral vector can be packaged in a lentiviral capsid to produce "recombinant lentiviral particles".

[0066] "Recombinant herpes simplex virus vector (recombinant HSV vector)" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., non-HSV original nucleic acid sequences) flanked by HSV terminal repeat sequences. Such recombinant viral vectors, when present in host cells that have been infected with a suitable helper virus, can replicate and package into infectious viral particles. When a recombinant viral vector integrates into a larger polynucleotide (e.g., a chromosome or another vector used for cloning or transfection, such as a plastid), the recombinant viral vector can be referred to as a "pre-vector," which can be "rescued" through replication and capsid coating in the presence of HSV packaging function and suitable helper functions. Recombinant viral vectors can exist in any of many forms, including but not limited to plastids, linear artificial chromosomes, lipid complexes, encapsulation within liposomes, and capsid coating within viral particles, such as HSV particles. Recombinant viral vectors can be packaged within HSV capsids to produce "recombinant herpes simplex virus particles."

[0067] "Heterologous" means originating from an individual that is genotype-completely different from the compared individual or other parts of the individual into which it is introduced or integrated. For example, polynucleotides introduced into different cell types through genetic engineering are heterologous polynucleotides (and encode heterologous polypeptides when expressed). Similarly, cellular sequences (e.g., genes or portions thereof) integrated into viral vectors are heterologous nucleotide sequences relative to the vector.

[0068] The term "transgenic" refers to a polynucleotide introduced into a cell that is capable of being transcribed into RNA and optionally translated into polynucleotides under suitable conditions. In some respects, it endows the introduced cell with desired properties or achieves desired therapeutic or diagnostic outcomes. In other respects, it can be transcribed into molecules that mediate RNA interference, such as siRNA.

[0069] The terms “genomic particles (gp),” “genomic equivalents,” or “genomic copies” used in relation to viral titers refer to the number of virions (whether infectious or functional) containing a recombinant viral DNA or RNA genome. The number of genomic particles in a specific viral preparation can be measured, for example, by the methods described in the embodiments herein, or by methods described, for example, in Clark et al., (1999) Hum. Gene Ther., 10: 1031-1039; Veldwijk et al. (2002) Mol. Ther., 6: 272-278.

[0070] The terms “infectious unit (iu),” “infectious particle,” or “replication unit” used in relation to viral titers refer to the number of infectious and replicable recombinant viral vector particles, which are determined by the infection center assay, also known as the replication center assay, as described in McLaughlin et al., (1988) J.Virol., 62: 1963-1973.

[0071] The term “transduction unit (tu)” used in relation to viral titers refers to the number of infectious recombinant viral vector particles that result in the production of functional transgenic products, which is determined by a functional assay, such as those described in the examples herein, or for example, regarding AAV, see Xiao et al., (1997) Exp. Neurobiol., 144: 113-124; or Fisher et al., (1996) J. Virol., 70: 520-532 (LFU assay).

[0072] "Inverted terminal repeat" or "ITR" sequence is a well-known term in the field, referring to a relatively short sequence found at the end of a viral genome that is reversed.

[0073] The term "AAV inverted terminal repeat (ITR)" is well-known in the art; it is a sequence of approximately 145 nucleotides located at both ends of the original single-stranded AAV genome. The outermost 125 nucleotides of the ITR can take either direction, resulting in heterogeneity between different AAV genomes and between the ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter self-complementary regions (called A, A', B, B', C, C, and D regions), allowing intrastrand base pairing within this portion of the ITR.

[0074] The "terminal resolution sequence" (TRS) is a sequence in the D region of the AAV ITR that is cleaved by the AAV rep protein during viral DNA replication. Mutated terminal resolution sequences are difficult for the AAV rep protein to cleave.

[0075] "AAV helper functions" refers to the functions that allow host cells to replicate and package AAV. AAV helper functions can be provided in any of many forms, including but not limited to helper viruses or helper virus genes that assist in AAV replication and packaging. Other AAV helper functions are known in the art, such as genotoxic agents.

[0076] "Helper viruses" used for AAV refer to viruses that allow host cells to replicate and package AAV (a defective parvovirus). Helper viruses provide "accessory functions" that allow AAV replication. Several such helper viruses have been identified, including adenoviruses, herpesviruses, poxviruses such as vaccinia and baculoviruses. Adenoviruses comprise several different subgroups, although adenovirus type 5 (Ad5) of subgroup C is the most commonly used. Many human, non-human mammalian, and avian adenoviruses are known and are available from collections such as the ATCC. Herpesviruses (also available from collections such as the ATCC) include, for example, herpes simplex virus (HSV), Epstein-Barr viruses (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV). Examples of adenovirus helper functions used for AAV replication include E1A, E1B, E2A, VA, and E4orf6 functions. Baculoviruses that can be obtained from depository institutions include the alfalfa silver-striped moth nuclear polyhedrosis virus (Autographa californica nuclear polyhedrosis virus).

[0077] When the ratio of infectious AAV particles to infectious helper virus particles is at least about 10... 2 1. At least about 10 4 1. At least about 10 6 : 1 or at least about 10 8 When the concentration is 1 or higher, the rAAV preparation is claimed to be "substantially free" of helper viruses. In some embodiments, the preparation is also free of an equal amount of helper virus proteins (i.e., proteins present due to this level of helper virus if the aforementioned helper virus particle impurities are presented in a disrupted form). Viral and / or cellular protein contamination can be observed when Coomassie staining bands are present on the SDS gel (i.e., bands appearing in addition to those corresponding to AAV capsid proteins VP1, VP2, and VP3).

[0078] The “differential coefficient distribution value” or “C(S)” used in this paper is a variant of the distribution containing the solution of the Lamm equation, used to describe the distribution of settled particles, for example, during ultracentrifugation.

[0079] The term "Svedberg unit" as used in this article refers to a unit used for settling rate. The settling rate of particles of a given size and shape measures how quickly the particles settle. One Svedberg unit is equal to 10⁻¹³ seconds. For example, Svedberg units are often used to reflect the rate of molecular motion under the influence of centrifugal force.

[0080] As used herein, “sedimentation velocity conditions” or “boundary sedimentation velocity conditions” can refer to any experimental conditions under which the sample solution is used for sedimentation velocity analysis. Sedimentation velocities allow for the study of particles over a wide range of pH and ionic strength conditions and at temperatures ranging from 4 to 40 °C. The rate of change at the sedimentation boundary is a measurement of the sedimentation coefficient of the sedimentation species. The sedimentation coefficient depends on the molecular weight (larger particles settle faster) and also on the molecular shape. The minimum width of the sedimentation boundary is related to the diffusion coefficient of the molecules; the presence of multiple species with similar sedimentation coefficients can cause the boundary to be wider than expected based solely on diffusion. Sedimentation velocity conditions can be unlimited to include any conditions related to rotor speed, distance between sample and rotor center, temperature, solvent, sample, buffer, centrifugation time, detection time interval, sector and optical window characteristics, AUC instrument (including ultracentrifugation and detection devices), equilibration dialysis of the reference solvent, and data analysis algorithms.

[0081] As used herein, the term "analytical density gradient sedimentation equilibrium" refers to methods for measuring the buoyant density of particles, or for separating different types of particles based on differences in buoyant density. These methods may utilize, for example, AUC sedimentation equilibrium techniques. In these methods, a particle solution (e.g., but not limited to solutions of peptides, polynucleotides, or viral capsids) is ultracentrifuged over a gradient solvate (such as a cesium chloride or cesium sulfate gradient) until equilibrium is reached with the solvate. At equilibrium, the particle solution will concentrate or exhibit a band at a point in the gradient where the particle density equals the solvate density. The location of the band can be used to calculate particle density, or the band can be extracted to separate a single type of particle.

[0082] The "SEDFIT algorithm" used in this paper is an algorithm that allows people to analyze hydrodynamic data (such as settling velocity) (Schuck (2000) Biophys.J., 78: 1606-19). In the SEDFIT algorithm, a grid of settling coefficients covering the expected range is generated. The settling boundary is simulated using the solutions of the Lamm equations for each settling coefficient, assuming that the particle shape and solvent friction ratio are constant.

[0083] The term "F-statistic" or "F-ratio" used in this article refers to the confidence level. This parameter controls the amount of regularization used. It has different meanings for different ranges: from 0 to 0.5, no regularization is used; values ​​from 0.5 to 0.999 correspond to the probability P (confidence level). From these P values, the ideal chi-square increase, taking into account the parsimony constraint, is calculated using the F-statistic. A value of 0.51 results in very small regularization; 0.68 to 0.90 correspond to commonly used confidence levels (typically with 50 scans or more, this corresponds to a chi-square increase of approximately 0.1% for a probability of 0.7), and values ​​close to 0.99 result in very high regularization. The relationship between these values ​​and probabilities can be tested using an F-statistic calculator. If a number >1 is entered, it is directly used as the chi-square ratio (since there is no probability >1). For example, a value of 1.1 would result in a chi-square increase with 10% regularization.

[0084] "Reduction" means lowering, reducing, or preventing activity, function, and / or quantity compared to a reference. In some embodiments, "reduction" means an ability to cause a total reduction of 20% or more. In another embodiment, "reduction" means an ability to cause a total reduction of 50% or more. In yet another embodiment, "reduction" means an ability to cause a total reduction of 75%, 85%, 90%, 95%, or more.

[0085] As used herein, "reference" means any sample, standard, or level used for comparative purposes. For example, when measuring the absorbance or refractive index of AAV in an aqueous solution, the absorbance or refractive index of said solution is compared with the absorbance or refractive index of an aqueous solution without AAV (i.e., a reference solution). In other embodiments, reference may invoke standard procedures known in the art. For example, when analyzing procedures for improving the quality of AAV production (e.g., homogeneity), AAV produced by a candidate procedure is compared with procedures known in the art (i.e., a reference procedure).

[0086] "Isolated" molecules (e.g., nucleic acids or proteins) or cells refer to those that have been identified and separated and / or recovered from components of their native environment. Therefore, isolated rAAV particles, for example, can be prepared using purification techniques to enrich them from source mixtures (e.g., culture lysates or production culture supernatants). Enrichment can be measured in various ways, such as by the proportion of DNase-resistant particles (DRP) in solution, or by infectivity, or based on a second potential interfering substance present in the source mixture (e.g., contaminants, including production culture medium contaminants or in-process contaminants, including helper viruses, culture medium components, etc., as defined below).

[0087] The reference in this document to “about” a value or parameter includes (and describes) implementations that refer to the value or parameter itself. For example, a description of “about X” includes a description of “X”.

[0088] Unless otherwise stated, the singular forms of the word “a,” “an,” and “the” used herein include plural references. For example, the phrase “rAAV particle” includes one or more rAAV particles.

[0089] It should be understood that the aspects and embodiments of the invention described herein include aspects and embodiments that are “comprising,” “composed of,” and / or “essentially composed of.”

[0090] III. Analytical ultracentrifugation

[0091] Analytical ultracentrifugation is a method for evaluating the molecular weight and hydrodynamic and thermodynamic properties of proteins or other macromolecules. The heterogeneity of proteins or macromolecules is assessed by sedimentation velocities within a range of conditions, including concentration, temperature, ionic strength, and pH. For example, proteins can be analyzed in clinically relevant formulations. The use of analytical ultracentrifugation for characterizing adenovirus preparations is described in Berkowitz, SA & Philo JS, (2007) Anal. Biochem., 362: 16-37.

[0092] In some aspects, the present invention provides methods for characterizing viral particle preparations using analytical ultracentrifugation (AUC). For example, in some embodiments, the invention provides a method for assessing the vector genome integrity of recombinant adeno-associated virus (rAAV) particles in rAAV particle preparations, the method using AUC to distinguish viral particles containing a complete, intact genome, empty viral capsids, and viral particles with different (e.g., truncated, aggregated, impurities, etc.) viral genomes. In other aspects, these methods can be applied in a similar manner to analyze adenovirus, lentivirus, and herpes simplex virus (HSV) particles. AUC analysis refers to a quantitative method for characterizing the biophysical properties of particles (e.g., peptides, polynucleotides, and viral capsids) by measuring their migration through a solvent in a centrifugal force field. AUC analysis has been well-characterized and highly versatile for over decades. Because AUC analysis relies on first-principles hydrodynamic and thermodynamic information, AUC can be applied to determine the biophysical properties of many types of particles across a wide range of particle concentrations and sizes. AUC analysis typically covers two basic experimental types: sedimentation velocity and sedimentation equilibrium. Sedimentation equilibrium analysis yields the thermodynamic properties of the particles, which can be used to measure characteristics such as stoichiometry and association constants. Sedimentation velocity yields the hydrodynamic properties of the particles, which can be used to measure characteristics such as size, shape, and concentration. A characteristic of AUC analysis of viral preparations is that different viral particle preparations can be analyzed using the same assay conditions, regardless of the viral genomic nucleotide sequence or serotype of the capsid.

[0093] Some aspects of this invention disclose the use of sedimentation velocity analysis for characterizing viral capsid properties. In some embodiments, sedimentation velocity analysis utilizes an ultracentrifugation chamber in dialysis equilibrium with two sectors (one for the experimental sample and one for a reference sample in solvent-only form), each sector containing two optical windows that allow light to pass through the chamber. Ultracentrifugation applies angular velocity to the cells and causes solute particles to settle rapidly toward the bottom of the sector. As sedimentation occurs, the solute near the meniscus at the top of the chamber is depleted, forming a sedimentation boundary between the depleted solute zone and the settled solute. The rate of movement or migration of the sedimentation boundary is measured by removing the analyte and comparing the characteristics of the sample and reference sectors at specific time intervals (typically on the order of seconds for sedimentation velocities). If multiple solute species are present, this may result in the formation of multiple sedimentation boundaries, each corresponding to a soluble / resolvable species.

[0094] Several methods are known in the art for optically detecting sedimentation boundaries and measuring their movement or migration rates (see Cole et al. (2008) Methods Cell Biol., 84: 143-79). In some embodiments, reference and sample sectors can be determined using absorbance detection. In such detection methods, the absorbance of the sample and reference sectors can be measured at specific wavelengths, said measurements being performed at different radial positions within each sector. Alternatively, the time progression of absorbance can be measured at a single radial position. Beer's Law provides a mathematical relationship between absorbance and solute extinction coefficient.

[0095] In some implementations, interferometric detection (e.g., Rayleigh interferometry) can be used to determine the reference and sample sectors. In Rayleigh interferometry, the interfering light system contains two parallel slits. A single coherent beam of light is split and passed through the two windows, and then the two beams are recombined. When the two light waves are combined, they form an interference pattern of alternating bright and dark fringes. If the sample and reference sample are to have the same refractive index, the resulting interference fringes will be perfectly straight. Increasing the concentration of the solute increases the refractive index of the solution, thereby blocking the sample beam and causing a vertical fringe shift. By measuring this fringe shift, one can measure the concentration of the solute in the sample. Unlike absorbance detection, which measures the absolute values ​​of the sample and reference, interferometry measures the relative difference between the sample and reference. However, interferometry yields an integrated peak that is directly proportional to the concentration and can be used for samples of types with insignificant absorbance. For reference on using Rayleigh interferometric optics with AUC, see Furst (1997) Eur. Biophys. J. 35: 307-10.

[0096] The velocity at the settling boundary can be measured to derive many physical properties of the solute particles. The velocity at which the boundary moves determines the settling coefficient, which is based on the particle's mass and shape (coefficient of friction). The settling coefficient of a particle, s, refers to the ratio of its velocity to the acceleration exerted on it by a centrifugal force field. The settling coefficient is expressed in Svedberg units, S (one Svedberg unit equals 10⁻⁶). -13 (seconds). The settling coefficient of particles or particle solutions depends on their properties, such as molecular weight (corrected for buoyancy), and the properties of the solvent.

[0097] The change of the solute concentration boundary over time during ultracentrifugation can be determined using the Lamm equation (Schuck (2000) Biophys.J., 78: 1606-19). In short, the Lamm equation calculates the change of the solute concentration boundary over time in response to the competitiveness of sedimentation (which concentrates the solute) and diffusion (which disperses the solute), taking into account the fan-shaped chamber and the centrifugal force field generated by the rotor. The Lamm equation can be expressed as:

[0098] Equation 1: ∂c / ∂t = D [(∂^2 c / ∂r^2) + 1 / r(∂c / ∂r)] - sω^2 [r(∂c / ∂r) + 2c]

[0099] Where c is the solute concentration, D represents the solute diffusion constant, s represents the sedimentation coefficient, ω represents the rotor angular velocity, r is the radius, and t is time.

[0100] By matching raw AUC data with solutions to the Lamm equation, it is possible to determine changes in solute characteristics such as sedimentation coefficients and concentration distribution. For example, experimentally determined rates of change of sedimentation boundaries can be modeled using the Lamm equation to determine the sedimentation coefficient, molecular weight, or concentration of the solute forming the boundary. Several procedures known in the art, such as SEDFIT (Schuck (2000) Biophys. J., 78: 1606-19), can be used to model the Lamm equation to derive AUC data. These procedures can also apply the Lamm equation to solutions containing multiple solutes or multiple sedimentation boundaries.

[0101] An example of a suitable procedure for determining solute characteristics is the SEDFIT algorithm. In some implementations, the SEDFIT algorithm can be used to calculate the differential coefficient distribution, or C(S), using AUC data from solutions containing mixtures of multiple particle types (see Schuck (2000) Biophys. J., 78: 1606-19). In the SEDFIT algorithm, a grid of sedimentation coefficients covering a desired range is generated. The sedimentation boundary is simulated using solutions to the Lamm equations for each sedimentation coefficient, assuming constant particle shape and solvent friction ratio. The actual AUC data is then adjusted to fit these Lamm solutions to obtain the differential coefficient distribution, or C(S). Many other procedures useful for analyzing AUC data can be found in Cole and Hansen (1999) J. Biomol. Tech. 10: 163-76.

[0102] In some embodiments of the invention, the recombinant viral particles are highly purified, appropriately buffered, and concentrated. In some embodiments, the viral particles are concentrated to at least about 1 x 10⁻⁶. 7 vg / mL, 2 x 10 7vg / mL、3 x 10 7 vg / mL、4 x 10 7 vg / mL、5 x 10 7 vg / mL、6 x 10 7 vg / mL、7 x 10 7 vg / mL、8 x 10 7 vg / mL、9 x 10 7 vg / mL、1 x 10 8 vg / mL、2 x 10 8 vg / mL、3 x 10 8 vg / mL、4 x 10 8 vg / mL、5 x 10 8 vg / mL、6 x10 8 vg / mL、7 x 10 8 vg / mL、8 x 10 8 vg / mL、9 x 10 8 vg / mL、1 x 10 9 vg / mL、2 x 10 9 vg / mL、3 x 10 9 vg / mL、4 x 10 9 vg / mL、5 x 10 9 vg / mL、6 x 10 9 vg / mL、7 x 10 9 vg / mL、8 x10 9 vg / mL、9 x 10 9 vg / mL、1 x 10 10 vg / mL、2 x 10 10 vg / mL、3 x 10 10 vg / mL、4 x 10 10 vg / mL、5 x 10 10 vg / mL、6 x 10 10 vg / mL、7 x 10 10 vg / mL、8 x 10 10 vg / mL、9 x 10 10 vg / mL、1 x 10 11 vg / mL、2 x 10 11 vg / mL、3 x 10 11 vg / mL、4 x 10 11 vg / mL、5 x 1011 vg / mL, 6 x 10 11 vg / mL, 7 x 10 11 vg / mL, 8 x 10 11 vg / mL, 9 x 10 11 vg / mL, 1 x 10 12 vg / mL, 2 x 10 12 vg / mL, 3 x 10 12 vg / mL, 4 x 10 12 vg / mL, 5 x 10 12 vg / mL, 6 x 10 12 vg / mL, 7 x 10 12 vg / mL, 8 x 10 12 vg / mL, 9 x 10 12 vg / mL, 1 x 10 13 vg / mL, 2 x 10 13 vg / mL, 3 x 10 13 vg / mL, 4 x 10 13 vg / mL, 5 x 10 13 vg / mL, 6 x 10 13 vg / mL, 7 x 10 13 vg / mL, 8 x 10 13 vg / mL, 9 x 10 13 Any of the following: vg / mL. In some embodiments, the viral particles are concentrated to approximately 1 x 10⁻⁶. 7 vg / mL to approximately 1 x 10 13 vg / mL, approximately 1 x 10 8 vg / mL to approximately 1 x 10 13 vg / mL, approximately 1 x 10 9 vg / mL to approximately 1 x 10 13 vg / mL, approximately 1 x 10¹⁰ vg / mL to approximately 1 x 10¹⁰ vg / mL 13 vg / mL, approximately 1 x 10 11 vg / mL to approximately 1 x 10 13 vg / mL, approximately 1 x 10 12 vg / mL to approximately 1 x 10 13 vg / mL, approximately 1 x 10 7 vg / mL to approximately 1 x 10 12 vg / mL, approximately 1 x 10 8vg / mL to approximately 1 x 10 12 vg / mL, approximately 1 x 10 9 vg / mL to approximately 1 x 10 12 vg / mL, approximately 1 x 1010 vg / mL to approximately 1 x 1010 12 vg / mL, approximately 1 x 10 11 vg / mL to approximately 1 x 10 12 vg / mL, approximately 1 x 10 7 vg / mL to approximately 1 x 10 11 vg / mL, approximately 1 x 10 8 vg / mL to approximately 1 x 10 11 vg / mL, approximately 1 x 10 9 vg / mL to approximately 1 x 10 11 vg / mL, approximately 1 x 10 10 vg / mL to approximately 1 x 10 11 vg / mL, approximately 1 x 10 7 vg / mL to approximately 1 x 10 10 vg / mL, approximately 1 x 10 8 vg / mL to approximately 1 x 10 10 vg / mL, approximately 1 x 10 9 vg / mL to approximately 1 x 10 10 vg / mL, approximately 1 x 10 7 vg / mL to approximately 1 x 10 9 vg / mL, approximately 1 x 10 8 vg / mL to approximately 1 x 10 9 vg / mL or approximately 1 x 10 7 vg / mL to approximately 1 x 10 8 vg / mL.

[0103] In some embodiments, viral particles are generated and purified in suitable host cells. In some embodiments, the viral particles are purified by affinity chromatography. Methods for purifying viral particles (e.g., AAV particles, adenovirus particles, lentivirus particles, HSV particles) are known in the art. For example, by immobilizing the viral capsid protein on a chromatographic matrix using an antibody or a binding ligand of the viral capsid protein. Examples of viral capsid affinity chromatography include, but are not limited to: AVB affinity chromatography for AAV (GE Healthcare), metal affinity chromatography for adenovirus and HSV, and heparin affinity chromatography for AAV and lentivirus, etc. Methods for purifying adenovirus particles have been discovered, for example in Bo, H et al., (2014) Eur. J. Pharm. Sci. 67C: 119-125. Methods for purifying lentivirus particles have been discovered, for example in Segura MM et al., (2013) Expert Opin Biol Ther. 13(7): 987-1011. Methods for purifying HSV particles have been discovered, for example in Goins, WF et al., (2014) Herpes Simplex Virus Methods in Molecular Biology 1144:63-79.

[0104] In some embodiments, recombinant viral particles are formulated into a pharmaceutical composition. In related embodiments, the pharmaceutical composition contains a buffer solution having a physiological pH and / or physiological osmolarity concentration. A non-limiting example of the pharmaceutical composition is phosphate-buffered saline (PBS), and in some embodiments, the PBS may be at a physiological osmolarity concentration (i.e., approximately pH 7.2 and approximately 300 mOsm / L). In some embodiments, the sample is adjusted to a target concentration, from 0.1 to 1.0, by optical density measurement at 260 nm. In some examples, this concentration yields reproducible and consistent AUC data. In some examples, the concentration of viral particles is adjusted by direct dilution with PBS or by further concentration, such further concentration being, for example, using a centrifugal filtration device.

[0105] In some embodiments of the invention, sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis is performed using analytical ultracentrifugation, which characterizes the sample in its original state under biologically relevant solution conditions (e.g., ProteomeLab™ XL-I (Beckman Coulter)). When using ProteomeLab™ XL-1, the sample is loaded into the sample sector of two velocity chambers, and a vector control (e.g., PBS without recombinant virus) is loaded into the corresponding reference sector. The sample is placed in a four-well rotor and equilibrated in the instrument until a temperature of 20°C and a complete vacuum are maintained for approximately 1 hour. In one exemplary embodiment, sedimentation velocity centrifugation is performed at approximately 20,000 RPM, approximately 20°C, and approximately 0.003 cm radius step settings, without delay or repetition. Different parameters can be used for centrifugation as described below. In some embodiments, absorbance (260 nm) and / or interference optics (e.g., Rayleigh interference optics) are used to simultaneously record the radius concentration as a function of time until the smallest sedimented component passes through the optical window. In some embodiments, the radius concentration is recorded until the lowest density recombinant virus particle settles through a sector. In some embodiments, sedimentation is monitored until the lowest density recombinant virus particle settles to the bottom of the ultracentrifugation sector. In some embodiments, sedimentation is monitored until the recombinant virus particle settles to the bottom of the ultracentrifugation velocity chamber. The sector may be part of an ultracentrifuge; for example, an ultracentrifugation velocity chamber. In some embodiments, the sector may be part of the ultracentrifuge for detecting the sample. In some embodiments, ultracentrifugation utilizes an ultracentrifuge containing an ultracentrifugation velocity chamber. In some embodiments, monitoring is performed until the recombinant virus particle settles to the bottom of the ultracentrifugation velocity chamber. In some embodiments, sedimentation is detected until the lowest density recombinant virus particle settles through an optical window. In some embodiments, the radius concentration is recorded for at least about 0.5 hours, 0.75 hours, 1.0 hours, 1.5 hours, 2.0 hours, 3.0 hours, 4.0 hours, or 5.0 hours. In some embodiments, the radius concentration is recorded for at least one of the following durations: about 0.5 hours to about 0.75 hours, about 0.75 hours to about 1.0 hour, about 1.0 hour to about 1.5 hours, about 1.5 hours to about 2.0 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, or about 4 hours to about 5 hours. In some embodiments, the radius concentration is recorded for about 1.2 hours. Optimized operating conditions may include, for example, continuing operation until all sedimentation species have completely settled to the bottom of the sector, maintaining a constant temperature of 20°C and a speed of 18,000 rpm to 20,000 rpm. Other temperatures and speeds may be used as described below.

[0106] The percentage of complete capsid was determined using the SEDFIT continuous-size C(S) distribution model by analyzing multiple (e.g., 75) scans from various detection methods. The second (2) nd Derivative regularization is applied to the fit. In some implementations, the confidence level of the F-statistic is approximately 0.68. In some implementations, the confidence level of the F-statistic is greater than any one of approximately 0.68, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 0.99. In some implementations, the confidence level of the F-statistic is approximately 0.68 to approximately 0.90. In some implementations, the confidence level of the F-statistic is approximately 0.68 to approximately 0.99. In some implementations, the C(S) parameter remains constant, and the resolution is approximately 200S to approximately 5000S. 最小值 It is from 1S to approximately 100S, S 最大值 The time is approximately 100 s to approximately 5000 s, and the friction ratio is approximately 1.0 or is adjusted to a value determined by centrifugation software. In some embodiments, the resolution is any one of approximately 200 s, 300 s, 400 s, 500 s, 600 s, 700 s, 800 s, 900 s, or 1000 s. In some embodiments, the resolution is approximately 200s to approximately 1000s, 200s to approximately 900s, 200s to approximately 800s, 200s to approximately 700s, 200s to approximately 600s, 200s to approximately 500s, 200s to approximately 400s, 200s to approximately 300s, 300s to approximately 1000s, 300s to approximately 900s, 300s to approximately 800s, 300s to approximately 700s, 300s to approximately 600s, 300s to approximately 500s, 300s to approximately 400s, 400s to approximately 1000s, 400s to approximately 900s, 400s to approximately... Any of 800S, 400S to about 700S, 400S to about 600S, 400S to about 500S, 500S to about 1000S, 500S to about 900S, 500S to about 800S, 500S to about 700S, 500S to about 600S, 600S to about 1000S, 600S to about 900S, 600S to about 800S, 600S to about 700S, 700S to about 1000S, 700S to about 900S, 700S to about 800S, 800S to about 1000S, 800S to about 900S, or 900S to about 1000S. In some embodiments, the resolution is about 200S. In some embodiments, S 最大值 It is any one of approximately 100S, 200S, 300S, 400S, 500S, 600S, 700S, 800S, 900S, or 1000S. In some implementations, S 最大值It is about 100S to about 1000S, 100S to about 900S, 100S to about 800S, 100S to about 700S, 100S to about 600S, 100S to about 500S, 100S to about 400S, 100S to about 300S, 100S to about 200S, 200S to about 1000S, 200S to about 900S, 200S to about 800S, 200S to about 700S, 200S to about 600S, 200S to about 500S, 200S to about 400S, 200S to about 300S, 300S to about 1000S, 300S to about 900S, 300S to about 800S, 300S to about 700S, 300S to about 600S, 300S to about 500S. Any one of 0S, 300S to about 400S, 400S to about 1000S, 400S to about 900S, 400S to about 800S, 400S to about 700S, 400S to about 600S, 400S to about 500S, 500S to about 1000S, 500S to about 900S, 500S to about 800S, 500S to about 700S, 500S to about 600S, 600S to about 1000S, 600S to about 900S, 600S to about 800S, 600S to about 700S, 700S to about 1000S, 700S to about 900S, 700S to about 800S, 800S to about 1000S, 800S to about 900S, or 900S to about 1000S. In some embodiments, S 最大值 It is approximately 200S to approximately 5000S. In some embodiments, where S 最大值 It is approximately 200 seconds. In some embodiments, the friction ratio is allowed to float to a value determined by centrifugation software. In some embodiments, the friction ratio is approximately 1.0. In some embodiments, noise subtractions are applied using radius invariant (RI) and time invariant (TI). In some embodiments, the meniscus position is allowed to float, allowing the software to select the optimal position. In some embodiments, the friction ratio is allowed to float, allowing the software to select the optimal position. The model fits the data to the Lamm equation, and the resulting size distribution is a "distribution of sedimentation coefficients," which looks like a chromatogram, where the area under each peak is proportional to the concentration in units of stripes or OD 260. The sedimentation coefficient (in Svedberg units) and relative concentration (in OD units) of each component in the distribution are determined. In some implementations, multiple AUC runs are determined independently, and the individual analyses monitor the following attributes to ensure the quality of results: goodness of fit (rmsd), the ratio of interference signal to OD 260nm / stripes for each peak (A260 / IF ratio), consistency of sedimentation coefficients for each class between runs, and overall scan quality.

[0107] In some embodiments of the invention, the extinction coefficient is used from absorbance data to calculate the molar concentration and actual percentage of the intact carrier peak. The molar extinction coefficient of the empty capsid is... 260 / 衣壳 =3.72e6) and the molar extinction coefficient of the intact carrier ( 260 / 载体 =3.00e7) can be calculated based on the published formula (Sommer et al. (2003) Mol Ther., 7: 122-8). The extinction coefficient can be used for both empty capsid and intact carrier peaks. The C(S) value can be determined using the SEDFIT algorithm described in Schuck (2000) Biophys.J., 78: 1606-19. The molar concentrations of both intact carrier and empty capsid can be calculated using Beer's Law, and the percentage of intact capsid is calculated from these values. In some embodiments, the values ​​are reported as a percentage of intact capsid.

[0108] In some implementations, the extinction coefficient of a specific type of recombinant viral particle (e.g., a viral particle with a fragmented genome of unknown size and sequence) may be determined empirically. The relationship between the S-value and genome size can be established by analyzing a recombinant viral vector prepared with a capsid-coated viral genome of known nucleotide size, and the corresponding S-value is determined as described herein. The calculated S-values ​​can be plotted to generate a standard curve, which can be compared with recombinant viral species of unknown molecular weight or genome size to determine the molecular weight of the unknown species.

[0109] In some aspects, the present invention provides a method for characterizing preparations of recombinant viral particles (e.g., rAAV, rAd, lentivirus, or rHSV particles), comprising the steps of: a) performing analytical ultracentrifugation on the preparations under boundary settling velocity conditions, wherein the settling of the recombinant viral particles is monitored at time intervals (e.g., once or multiple times); b) plotting a differential settling coefficient distribution (C(s)) against a settling coefficient expressed in Svedberg units (S); and c) integrating the area under each peak in the C(s) distribution to determine the relative concentration of each peak, wherein each peak represents a recombinant viral particle. In some embodiments, the types of recombinant viral particles identified by the method of the present invention include, but are not limited to: fully recombinant viral particles containing a complete recombinant viral genome, empty recombinant viral capsid particles, and recombinant viral particles containing different recombinant viral genomes. In some embodiments, the different genomes are smaller than the complete recombinant viral genome (e.g., truncated genomes). In some embodiments, the different genomes are larger than the complete recombinant viral genome (e.g., aggregates, recombinants, etc.). In some embodiments, the present invention provides a method for assessing the vector genome integrity of recombinant viral particles in a preparation of recombinant viral particles, comprising: a) performing analytical ultracentrifugation on the preparation under boundary sedimentation velocity conditions, wherein sedimentation of the recombinant viral particles is monitored at time intervals (e.g., once or multiple times); b) plotting sedimentation coefficients expressed in Svedberg units (S) using differential sedimentation coefficient distribution values ​​(C(s)); c) identifying the species of recombinant viral particles in the preparation by peaks corresponding to S values ​​present on the plot, wherein the genome size of a specific species of recombinant viral particles is calculated by comparing the S values ​​of the species with a standard curve generated from the S values ​​of recombinant viral particles containing viral genomes of different known sizes with capsid coatings. In some embodiments, the method further comprises integrating the area under the peaks in the C(S) distribution to determine the relative concentration of each species of recombinant viral particles. In some embodiments, sedimentation of the recombinant viral particles is monitored at one time interval. In some embodiments, sedimentation of the recombinant viral particles is monitored at more than one time interval.

[0110] In some embodiments of the invention, sedimentation of recombinant viral particles (e.g., rAAV, rAd, lentivirus, or rHSV particles) is monitored by measuring optical density or absorbance at 260 nm. Methods for measuring absorbance are known in the art. In some embodiments, an ultracentrifuge for AUC is equipped with means for measuring absorbance. In other embodiments, sedimentation of recombinant viral particles is monitored by interference. In some embodiments, sedimentation of recombinant viral particles is monitored by Rayleigh interference. Methods for measuring interference are known in the art (Furst (1997) Eur. Biophys. J. 35: 307-10). In some embodiments, an ultracentrifuge for AUC is equipped with means for measuring interference. In some embodiments, sedimentation of recombinant viral particles is monitored by both absorbance and interference. In some embodiments, absorbance and / or interference are measured using a reference standard. In some embodiments, the reference standard is matched to a solution of the recombinant viral preparation, except where the recombinant virus is absent. For example, the recombinant viral preparation may contain the recombinant virus in a buffer solution, such as phosphate-buffered saline. In this example, the reference standard could be phosphate-buffered saline without recombinant viral particles.

[0111] In some embodiments of the invention, the preparation of the virus particles is in the form of a pharmaceutical formulation. Such formulations are well known in the art (see, for example, Remington's Pharmaceutical Sciences, 15th edition, pp. 1035-1038 and 1570-1580). Such pharmaceutical formulations can be sterile water, such as water, and oils, including petroleum, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, etc. Saline solutions and aqueous solutions of dextran, polyethylene glycol (PEG), and glycerol can also be used as liquid carriers, especially for injectable solutions. The pharmaceutical formulation may also contain additional ingredients such as preservatives, buffers, tensile agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, thickeners, etc. In some embodiments of the invention, the pharmaceutical formulation comprises phosphate-buffered saline.

[0112] In some embodiments of the invention, the settling velocity of viral particles during ultracentrifugation is determined by continuously monitoring the settling of viral particles during ultracentrifugation. Optimizing the AUC parameters for different types of viral particles is within the understanding of those skilled in the art. Without being bound by theory, the range of AUC settings that allow for the analysis of both AAV and lentiviral particles should enable the analysis of other viral particles, including lentiviruses and HSVs, since the sizes of HSV and lentiviral particles fall between those of AAV and adenovirus particles. In some embodiments, data acquisition from rAAV, rHSV, lentivirus, and / or rAd particles is performed with an AUC rate of approximately 3,000 to approximately 20,000 rpm. In some embodiments, data analysis for rAAV, rHSV, lentivirus, and / or rAd is performed with an AUC rate of approximately 1 second. 最小值 And about 1000S of S 最大值 The analysis of data for rAAV, rHSV, lentivirus, and / or rAd particles is performed at a resolution of approximately 200 s to approximately 1,000 s. In some embodiments, the resolution is any one of approximately 200 s, 300 s, 400 s, 500 s, 600 s, 700 s, 800 s, 900 s, or 1,000 s. In some implementations, the resolution is approximately 200s to approximately 1000s, 200s to approximately 900s, 200s to approximately 800s, 200s to approximately 700s, 200s to approximately 600s, 200s to approximately 500s, 200s to approximately 400s, 200s to approximately 300s, 300s to approximately 1000s, 300s to approximately 900s, 300s to approximately 800s, 300s to approximately 700s, 300s to approximately 600s, 300s to approximately 500s, 300s to approximately 400s, 400s to approximately 1000s, 400s to approximately 900s, 400s to approximately 800s. The resolution is any one of 00S, 400S to about 700S, 400S to about 600S, 400S to about 500S, 500S to about 1000S, 500S to about 900S, 500S to about 800S, 500S to about 700S, 500S to about 600S, 600S to about 1000S, 600S to about 900S, 600S to about 800S, 600S to about 700S, 700S to about 1000S, 700S to about 900S, 700S to about 800S, 800S to about 1000S, 800S to about 900S, or 900S to about 1000S. In some embodiments, the resolution is about 200S. In some implementations, data analysis for rAAV, rHSV, lentivirus, and / or rAd particles is accompanied by an S of approximately 100S, 200S, 300S, 400S, 500S, 600S, 700S, 800S, 900S, or 1000S.最大值 In some implementations, S 最大值 It is about 100S to about 1000S, 100S to about 900S, 100S to about 800S, 100S to about 700S, 100S to about 600S, 100S to about 500S, 100S to about 400S, 100S to about 300S, 100S to about 200S, 200S to about 1000S, 200S to about 900S, 200S to about 800S, 200S to about 700S, 200S to about 600S, 200S to about 500S, 200S to about 400S, 200S to about 300S, 300S to about 1000S, 300S to about 900S, 300S to about 800S, 300S to about 700S, 300S to about 600S, 300S to about 500S. Any one of 0S, 300S to about 400S, 400S to about 1000S, 400S to about 900S, 400S to about 800S, 400S to about 700S, 400S to about 600S, 400S to about 500S, 500S to about 1000S, 500S to about 900S, 500S to about 800S, 500S to about 700S, 500S to about 600S, 600S to about 1000S, 600S to about 900S, 600S to about 800S, 600S to about 700S, 700S to about 1000S, 700S to about 900S, 700S to about 800S, 800S to about 1000S, 800S to about 900S, or 900S to about 1000S. In some embodiments, S 最大值 It is approximately 200S to approximately 5000S. In some embodiments, where S 最大值 It is approximately 200 seconds. In some implementations, radius invariant (RI) and time invariant (TI) noise reduction are applied. In some implementations, the meniscus position is allowed to float, allowing the software to select the optimal position. In some implementations, the friction ratio is allowed to float, allowing the software to select the optimal position. In some implementations, the data analysis of rAAV and / or adenovirus particles is kept constant at 1. In some implementations, the data analysis of rAAV, HSV, lentivirus, and / or adenovirus particles is allowed to float by using the FIT command with values ​​optimized by nonlinear regression.

[0113] For recombinant viral particles (e.g., rAAV, rAd, lentivirus, or rHSV particles), in some embodiments, the settling rate of the recombinant virus during ultracentrifugation is determined by monitoring (e.g., scanning) the settling of the recombinant viral particles more than approximately every 15 seconds, 30 seconds, 45 seconds, 1 minute (60 seconds), 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes. Scans can be acquired continuously as quickly and without delay as the optical system allows. Interferometric scans are rapid, with a single scan completed in ~10⁻¹⁵ seconds, while absorbance scans require ~60 seconds. When using dual detection, the acquisition rate of scans for both detections depends on the absorbance system. In some embodiments of the invention, the sedimentation of recombinant viral particles is monitored during ultracentrifugation using more than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 scans. In some embodiments, at least 30 scans are required for analysis, and scans are collected until the sedimentation process is complete. In some embodiments, the sedimentation process can typically be described by 40 to 75 scans. In some embodiments, the sedimentation rate of the recombinant viral particles is determined based on about 75 scans. In some embodiments, the sedimentation rate of the recombinant viral particles is determined based on about 55 to about 75 scans. In some embodiments, the sedimentation rate of the recombinant viral particles is determined based on about 55 to about 60 scans. In some embodiments, the sedimentation rate of the recombinant viral particles is determined based on about 60 to about 75 scans. In some embodiments, the sedimentation rate of the recombinant viral particles is determined based on about 60 to about 70 scans. In some implementations, the sedimentation velocity of the recombinant viral particles is determined based on multiple ultracentrifugation runs. In some implementations, the sedimentation velocity of the recombinant viral particles is determined based on any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more ultracentrifugation runs. In some implementations, the sedimentation velocity is used to detect the C(S) value using the SEDFIT algorithm. In some implementations, second derivative regularization is applied to the fitting level, with a confidence level of approximately 0.68 for the F-statistic. In some implementations, the following C(S) parameters are kept constant: from approximately 100 S to approximately 200 S at resolution, S... 最小值 It is 1, S 最大值 The duration is approximately 200 to 300 seconds, and the friction ratio is approximately 1.0 to 1.2 seconds. In some implementations, radius invariant (RI) and time invariant (TI) noise reduction are applied.

[0114] In some embodiments of the invention, the boundary settling velocity of the recombinant viral particles (e.g., rAAV, rAd, lentivirus, or rHSV particles) in the recombinant viral particle preparation is determined by ultracentrifugation of the recombinant viral particle preparation at any of about 5,000 rpm; 10,000 rpm; 15,000 rpm; 20,000 rpm; 25,000 rpm; 30,000 rpm; 35,000 rpm; 40,000 rpm; 45,000 rpm; or 50,000 rpm.In some embodiments, the ultracentrifugation is performed at approximately 5,000 rpm to approximately 50,000 rpm; approximately 10,000 rpm to approximately 50,000 rpm; approximately 15,000 rpm to approximately 50,000 rpm; approximately 20,000 rpm to approximately 50,000 rpm; approximately 25,000 rpm to approximately 50,000 rpm; approximately 30,000 rpm to approximately 50,000 rpm; approximately 35,000 rpm to approximately 50,000 rpm; approximately 40,000 rpm to approximately 50,000 rpm; approximately 45,000 rpm to approximately 50,000 rpm; approximately 5,000 rpm to approximately 45,000 rpm; approximately 10 ... 0 rpm to approximately 45,000 rpm; approximately 15,000 rpm to approximately 45,000 rpm; approximately 20,000 rpm to approximately 45,000 rpm; approximately 25,000 rpm to approximately 45,000 rpm; approximately 30,000 rpm to approximately 45,000 rpm; approximately 40,000 rpm to approximately 45,000 rpm; approximately 5,000 rpm to approximately 40,000 rpm; approximately 10,000 rpm to approximately 40,000 rpm; approximately 15,000 rpm to approximately 40,000 rpm; approximately 20,000 rpm to approximately 40,000 rpm; approximately 25,000 rpm to approximately 40,000 rpm; approximately 30,000 rpm to approximately 45,000 rpm; approximately 30,000 rpm to approximately 45,000 rpm; approximately 15,000 rpm to approximately 45 ...5,000 rpm; approximately 25,000 rpm to approximately 40,000 rpm; approximately 30,000 rpm to approximately 45,000 rpm; approximately 25,000 rpm to approximately 40,000 rpm; approximately 30,000 rpm to approximately 00rpm to approximately 40,000rpm; approximately 35,000rpm to approximately 40,000rpm; approximately 5,000rpm to approximately 35,000rpm; approximately 10,000rpm to approximately 35,000rpm; approximately 15,000rpm to approximately 35,000rpm; approximately 20,000rpm to approximately 35,000rpm; approximately 25,000rpm to approximately 35,000rpm; approximately 30,000rpm to approximately 35,000rpm; approximately 5,000rpm to approximately 30,000rpm; approximately 10,000rpm to approximately 30,000rpm; approximately 15,000rpm to approximately 30,000rpm; approximately 20,000rpm to approximately 35 ... Operating at approximately 00 rpm to approximately 30,000 rpm; approximately 25,000 rpm to approximately 30,000 rpm; approximately 5,000 rpm to approximately 25,000 rpm; approximately 10,000 rpm to approximately 25,000 rpm; approximately 20,000 rpm to approximately 25,000 rpm; approximately 5,000 rpm to approximately 20,000 rpm; approximately 10,000 rpm to approximately 20,000 rpm; approximately 15,000 rpm to approximately 20,000 rpm; approximately 5,000 rpm to approximately 15,000 rpm; approximately 10,000 rpm to approximately 15,000 rpm; or approximately 5,000 rpm to approximately 10,000 rpm.In some embodiments of the present invention, the boundary settling velocity of the recombinant viral particles in the recombinant viral particle preparation is determined by ultracentrifugation of the recombinant viral particle preparation at approximately 20,000 rpm. In some embodiments of the present invention, the boundary settling velocity of the recombinant viral particles in the recombinant viral particle preparation is determined by ultracentrifugation of the recombinant viral particle preparation at approximately 15,000 rpm to approximately 20,000 rpm.

[0115] In some embodiments of the invention, the boundary settling velocity of recombinant viral particles in the recombinant viral particle preparation (e.g., rAAV, rAd, lentivirus, or rHSV particles) is determined by ultracentrifugation of the recombinant viral particle preparation at about or greater than 4°C, 10°C, 15°C, 20°C, 25°C, or 30°C. In some embodiments, the ultracentrifugation is performed at any one of the following: about 4°C to about 30°C, about 4°C to about 25°C, about 4°C to about 20°C, about 4°C to about 15°C, about 4°C to about 10°C, about 10°C to about 30°C, about 10°C to about 25°C, about 10°C to about 20°C, about 10°C to about 15°C, about 15°C to about 30°C, about 15°C to about 25°C, about 15°C to about 20°C, about 20°C to about 30°C, or about 20°C to about 25°C. In some embodiments, the boundary settling velocity of recombinant viral particles in the recombinant viral particle preparation is determined by ultracentrifugation of the recombinant viral particle preparation at about 20°C. In some implementations, the boundary settling velocity of the recombinant viral particles in the recombinant viral particle preparation is determined by ultracentrifugation of the recombinant viral particle preparation at approximately 15°C to approximately 20°C.

[0116] As disclosed herein, the methods of this invention can be used to analyze many types of recombinant viral particles (e.g., AAV, adenovirus, lentivirus, and / or HSV particles). Suitable ultracentrifugation conditions, analytical algorithms, and other parameters can be determined empirically using methods known in the art. Exemplary parameters for AAV, adenovirus, lentivirus, and HSV particles, as well as guidance for selecting specific parameter options, are provided in Table 1 below and are not intended to be limiting.

[0117] Table 1. Exemplary parameters of AAV, adenovirus, lentivirus, and HSV particles

[0118]

[0119]

[0120] In some aspects, the present invention provides a method for determining the presence of empty capsids in a preparation of recombinant viral particles (e.g., rAAV, rAd, lentivirus, or rHSV particles), comprising the steps of: a) performing analytical ultracentrifugation on the preparation under boundary settling velocity conditions, wherein the settling of the recombinant viral particles is monitored at time intervals (e.g., once or multiple times), and b) plotting the settling coefficient in Svedberg units (S) as differential settling coefficient distribution values ​​(C(s)), wherein the presence of a peak corresponding to the S value of empty capsid particles indicates the presence of empty capsid particles. In some embodiments, the present invention provides a method for measuring the relative amount of empty capsids in a preparation of recombinant viral particles, comprising the steps of: a) performing analytical ultracentrifugation on the preparation under boundary settling velocity conditions, wherein the settling of recombinant viral particles is monitored at time intervals (e.g., once or multiple times); b) plotting the sedimentation coefficient in Svedberg units (S) against a differential sedimentation coefficient distribution (C(s)); c) integrating the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species; and d) comparing the amount of recombinant viral particles having an S value corresponding to empty capsid particles with an S value corresponding to recombinant viral particles containing a complete viral genome. In some embodiments, the amount of recombinant viral particles having an S value corresponding to empty capsid particles is compared with the total amount of all recombinant viral particles in the preparation, the total amount being obtained by integrating over all peaks on the C(S) versus S plot.

[0121] In some aspects, the present invention provides a method for determining the presence of recombinant viral particle variants in a preparation of recombinant viral particles (e.g., rAAV, rAd, lentivirus, or rHSV particles), comprising the steps of: a) performing analytical ultracentrifugation on the preparation under boundary sedimentation velocity conditions, wherein the sedimentation of the recombinant viral particles is monitored at time intervals (e.g., once or multiple times); b) plotting the sedimentation coefficient in Svedberg units (S) as differential sedimentation coefficient distribution values ​​(C(s)), wherein the presence of peaks corresponding to S values ​​that differ from the S values ​​of recombinant viral capsid particles containing a fully complete recombinant viral genome indicates the presence of a recombinant viral particle variant. In some embodiments, the present invention provides a method for measuring the relative amount of recombinant viral particle variants in a preparation of recombinant viral particles, comprising the steps of: a) performing analytical ultracentrifugation on the preparation under boundary settling velocity conditions, wherein the settling of recombinant viral particles is monitored at time intervals (e.g., once or multiple times); b) plotting the sedimentation coefficient in Svedberg units (S) against a differential sedimentation coefficient distribution (C(s)); c) integrating the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species; d) comparing the amount of recombinant viral particles having an S value corresponding to empty capsid particles with an S value corresponding to recombinant viral particles containing a complete viral genome. In some embodiments, the amount of recombinant viral particles whose S values ​​differ from those of recombinant viral capsid particles containing a completely complete recombinant viral genome is compared with the total amount of all recombinant viral particles in the preparation, the total amount being obtained by integrating the C(S) versus S plot over all peaks. In some implementations, the recombinant viral particle variants contain a recombinant viral genome that is smaller (e.g., truncated) or larger than the full-length complete viral genome. Other viral capsid-coated DNA impurities can also be detected.

[0122] In some embodiments, the present invention provides a method for monitoring the removal of empty capsids and / or capsid particles containing different recombinant viral genomes during the purification process of a preparation of recombinant viral particles (e.g., rAAV, rAd, lentivirus, or rHSV particles). The method includes removing a sample of the recombinant viral particles from the preparation after one or more steps of the purification process, and analyzing the relative amount of empty capsids in the sample using the AUC described herein. A decrease in the relative amount of empty capsids and / or capsid particles containing different genomes compared to a complete capsid indicates the removal of empty capsids from the recombinant viral particle preparation.

[0123] In some embodiments, the present invention provides a method for determining the heterogeneity of recombinant viral particles in a preparation of recombinant viral particles (e.g., rAAV, rAd, lentivirus, or rHSV particles), comprising the steps of: a) performing analytical ultracentrifugation on the preparation under boundary settling velocity conditions, wherein the settling of the recombinant viral particles is monitored at time intervals (e.g., once or multiple times); b) plotting the settling coefficients, expressed in Svedberg units (S), as differential settling coefficient distribution values ​​(C(s)), wherein the presence of peaks other than those representing a capsid containing a complete viral genome indicates heterogeneity of the recombinant viral particles in the preparation. In some embodiments, the types of recombinant viral particles identified by the method of the present invention include, but are not limited to: fully recombinant viral particles containing a complete recombinant viral genome, empty recombinant viral capsid particles, and recombinant viral particles containing different recombinant viral genomes. In some embodiments, the different genomes are smaller than the complete recombinant viral genome (e.g., truncated genomes). In some embodiments, the different genomes are larger than the complete recombinant viral genome (e.g., aggregates, recombinants, etc.). In some embodiments, the different genomes include genomes smaller or larger than the complete recombinant viral genome.

[0124] This invention provides a method for monitoring the heterogeneity of recombinant viral particles during the purification process of a preparation of recombinant viral particles (e.g., rAAV, rAd, lentivirus, or rHSV particles). The method includes removing a sample of recombinant viral particles from the preparation after one or more steps of the purification process, and determining the relative amounts of complete capsids, empty capsids, and / or recombinant viral particles with different genomes containing the complete recombinant viral genome using the AUC described herein. An increase in the relative amount of recombinant viral particles containing the complete viral genome indicates an increase in the heterogeneity of complete viral particles in the recombinant viral particle preparation.

[0125] In the above-described embodiments, the recombinant viral particles have been purified using one or more purification steps. Examples of purification steps include, but are not limited to, equilibration centrifugation, anion exchange filtration, tangential flow filtration (TFF), apatite chromatography, thermal inactivation of the assist virus, hydrophobic interaction chromatography, immunoaffinity chromatography, size exclusion chromatography (SEC), nanofiltration, cation exchange chromatography, and anion exchange chromatography.

[0126] In embodiments described above, the recombinant viral particle comprises a self-assembled AAV (scAAV) genome. In some embodiments, the recombinant AAV genome comprises a first heteropolynucleotide sequence (e.g., a therapeutic transgene coding strand) and a second heteropolynucleotide sequence (e.g., a therapeutic transgene non-coding or antisense strand), wherein the first heteropolynucleotide sequence is capable of forming intrastrand base pairs with the second polynucleotide sequence for most or all of its length. In some embodiments, the first and second heteropolynucleotide sequences are linked by a sequence that promotes intrastrand base pairing; for example, a hairpin DNA structure. Hairpin structures are known in the art, for example, in siRNA molecules. In some embodiments, the first and second heteropolynucleotide sequences are linked by a mutated ITR. In some embodiments, the scAAV viral particle comprises a monomeric form of the scAAV genome. In some embodiments, the scAAV viral particle comprises a dimer form of the scAAV genome. In some embodiments, the AUC described herein is used to detect the presence of rAAV particles comprising a monomeric form of the scAAV genome. In some embodiments, the AUC described herein is used to detect the presence of rAAV particles containing the scAAV genome in a dimer form. In some embodiments, the AUC described herein is used to monitor the packaging of the scAAV genome into a capsid.

[0127] In the above-described embodiments, the rAAV particles comprise AAV1 capsids, AAV2 capsids, AAV3 capsids, AAV4 capsids, AAV5 capsids, AAV6 capsids (e.g., wild-type AAV6 capsids or variant AAV6 capsids such as ShH10, as described in USPG Pub. 2012 / 0164106), AAV7 capsids, AAV8 capsids, AAVrh8 capsids, AAVrh8R, AAV9 capsids (e.g., wild-type AAV9 capsids or modified AAV9 capsids, as described in USPG Pub. 2013 / 0323226), AAV10 capsids, AAVrh10 capsids, AAV11 capsids, AAV12 capsids, tyrosine capsid mutants, heparin-binding capsid mutants, AAV2R471A capsids, AAVAAV2 / 2-7m8 capsids, and AAV... DJ costumes (such as AAV-DJ / 8 costume, AAV-DJ / 9 costume, or any other costume described in USPGPub.2012 / 0066783), AAV2 N587A costume, AAV2 E548A costume, AAV2N708A costume, AAV V708K costume, goat AAV costume, AAV1 / AAV2 chimeric costume, bovine AAV costume, mouse AAV costume, or AAV costumes described in U.S. Patent No. 8,283,151 or International Publication No. WO / 2003 / 042397. In the embodiments described above, 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 DJ ITR, goat AAV ITR, bovine AAV ITR, or mouse AAV ITR. In some embodiments, the rAAV particles comprise an ITR from one AAV serotype and an AAV capsid from another serotype. For example, the rAAV capsid may comprise a therapeutic transgene flanked by at least one AAV2 ITR, encapsulated within an AAV9 capsid. Such combinations may be referred to as pseudotyped rAAV particles.

[0128] IV. Viral particles

[0129] The methods disclosed in this invention can be used in particular to characterize species of interest in various viral particles (e.g., viral particles with complete genomes compared to viral particles with truncated genomes and / or viral particles containing DNA impurities).

[0130] In some embodiments, the viral particle is a recombinant AAV particle containing nucleic acid containing a transgene with one or two ITRs flanking it. The nucleic acid is encapsulated within the AAV particle. The AAV particle also contains a capsid protein. In some embodiments, the nucleic acid contains a protein-coding sequence of interest (e.g., a therapeutic transgene), components operatively linked in the direction of transcription, including control sequences for transcription initiation and termination sequences, thereby forming an expression cassette. The expression cassette has at least one functional AAV ITR sequence flanking it at the 5' and 3'. “Functional AAV ITR sequence” means the ITR sequence functioning for rescuing, replicating, and packaging AAV virions. See Davidson et al., PNAS, 2000, 97(7): 3428-32; Passini et al., J.Virol., 2003, 77(12): 7034-40; and Pechan et al., Gene Ther., 2009, 16: 10-16, all of which are incorporated herein by reference in their entirety. To implement some aspects of the invention, the recombinant vector contains at least all the sequences of AAV necessary for capsid coating and the physical structure for infection by rAAV. The AAV ITR used in the vector for the purposes of this invention does not need to have a wild-type nucleotide sequence (e.g., as described in Kotin, Hum. Gene Ther., 1994, 5: 793-801) and can be altered by nucleotide insertion, deletion, or substitution, or the AAV ITR can 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 are being 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. Virol., 2003, 77(12): 6799-810. Any use of any AAV serotype is understood to be within the scope of this invention. In some implementations, the rAAV vector is a vector derived from AAV serotypes, including but not limited to: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAV11, AAV12, tyrosine capsid mutants, heparin-binding capsid mutants, AAV2R471A capsid, AAVAAV2 / 2-7m8 capsid, AAV DJ capsid, AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, or mouse AAV capsid, etc.In some embodiments, the nucleic acids in AAVs contain ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAV11, AAV12, etc. In further embodiments, rAAV particles contain capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAV11, AAV12, etc. In further embodiments, rAAV particles contain capsid proteins from AAV serotypes of clade (Clades) AF (Gao et al. J. Virol. 2004, 78(12): 6381).

[0131] Different AAV serotypes can be used to optimize the transduction of specific target cells or to target specific cell types within specific target tissues (e.g., diseased tissues). rAAV particles may contain viral proteins and viral nucleic acids of the same or mixed serotypes. For example, an rAAV particle may contain an AAV9 capsid protein and at least one AAV2 ITR, or it may contain an AAV2 capsid protein and at least one AAV9 ITR. In yet another embodiment, the rAAV particle may contain capsid proteins from both AAV9 and AAV2, and further contain at least one AAV2 ITR. This document provides any combination of AAV serotypes for generating rAAV particles, as each combination has been explicitly described herein.

[0132] In some embodiments, the AAV comprises at least one AAV1 ITR and a capsid protein from any one of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, and / or AAV12. In some embodiments, the AAV comprises at least one AAV2 ITR and a capsid protein from any one of AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, and / or AAV12. In some embodiments, the AAV comprises at least one AAV3 ITR and a capsid protein from any one of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, and / or AAV12. In some embodiments, the AAV comprises at least one AAV4 ITR and a capsid protein from any one of AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, and / or AAV12. In some embodiments, the AAV comprises at least one AAV5 ITR and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, and / or AAV12. In some embodiments, the AAV comprises at least one AAV6 ITR and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, and / or AAV12. In some embodiments, the AAV comprises at least one AAV7 ITR and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh.8, AAVrh10, AAV11, and / or AAV12. In some embodiments, the AAV comprises at least one AAV8 ITR and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAVrh.8, AAVrh10, AAV11, and / or AAV12. In some embodiments, the AAV comprises at least one AAV9 ITR and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh.8, AAVrh10, AAV11, and / or AAV12.In some embodiments, the AAV comprises at least one AAVrh8 ITR and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV11, and / or AAV12. In some embodiments, the AAV comprises at least one AAVrh10 ITR and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, and / or AAV12. In some embodiments, the AAV comprises at least one AAV11 ITR and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAVrh10, and / or AAV12. In some implementations, AAV comprises at least one AAV12 ITR and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh8, AAV9, AAVrh10 and / or AAV11.

[0133] Self-complementary AAV viral genome

[0134] In some aspects, the present invention provides viral particles comprising a recombinant, self-complementary genome. AAV viral particles containing a self-complementary genome and methods for using a self-complementary AAV genome 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 is incorporated herein by reference in its entirety. rAAV containing a self-complementary genome rapidly forms a double-stranded DNA molecule by virtue of its partially complementary sequences (e.g., the complementary coding and non-coding strands of a transgene). In some embodiments, the present invention provides AAV viral particles comprising an AAV genome, wherein the rAAV genome comprises a first heteropolynucleotide sequence (e.g., a therapeutic transgene coding strand) and a second heteropolynucleotide sequence (e.g., a therapeutic transgene noncoding or antisense strand), wherein the first heteropolynucleotide sequence is capable of forming intrastrand base pairs with the second polynucleotide sequence for most or all of its length. In some embodiments, the first and second heteropolynucleotide sequences are linked by a sequence that promotes intrastrand base pairing; for example, a hairpin DNA structure. Hairpin structures are known in the art, for example, in siRNA molecules. In some embodiments, the first and second heteropolynucleotide sequences are linked by a mutated ITR (e.g., a right ITR). The mutated ITR comprises a deletion of a D region, which contains an end-resolved sequence. As a result, during the replication of the AAV viral genome, the rep protein does not cleave the viral genome at the mutated ITR. Similarly, a recombinant viral genome containing the following in a 5' to 3' sequence is packaged in the viral capsid: AAV ITR, a first heteropolynucleotide sequence including the regulatory sequence, a mutated AAV ITR, a second heteropolynucleotide in the opposite direction to the first heteropolynucleotide, and a third AAV ITR.

[0135] In some embodiments, the viral particle is an adenovirus particle. In some embodiments, the adenovirus particle is a recombinant adenovirus particle, such as a polynucleotide vector containing one or more heterologous sequences (i.e., non-adenovirus-native nucleic acid sequences) between two ITRs. In some embodiments, the adenovirus particle lacks or contains defective copies of one or more E1 genes, resulting in adenovirus replication defects. Adenoviruses comprise a linear, double-stranded DNA genome within a large (~950 Å), non-enveloped icosahedral capsid. Adenoviruses have large genomes capable of integrating heterologous sequences larger than 30 kb (e.g., replacing E1 and / or E3 regions), making them uniquely suited for use with larger heterologous genes. They are also known to infect dividing and non-dividing cells and not naturally integrate into the host genome (although hybrid variants may have this ability). In some embodiments, the adenovirus vector may be a first-generation adenovirus vector in which the E1 region is replaced by a heterologous sequence. In some embodiments, the adenovirus vector may be a second-generation adenovirus vector with deletions or additional mutations in E2A, E2B, and / or E4. In some implementations, the adenovirus vector may be a third-generation or gutted adenovirus vector, which lacks all viral coding genes, retains only the ITR and packaging signals, and requires in-trans helper adenovirus for replication and packaging. The use of adenovirus particles as vectors for transient transfection of mammalian cells and as gene therapy vectors has been investigated. For further description, see Danthinne, X. and Imperiale, MJ (2000) Gene Ther. 7:1707-14, and Tatsis, N. and Ertl, HC (2004) Mol. Ther. 10:616-29.

[0136] In some embodiments, the viral particle is a recombinant adenovirus particle containing nucleic acid, said nucleic acid containing transgenes. Use of any adenovirus serotype is considered within the scope of this invention. In some embodiments, the recombinant adenovirus vector is a vector derived from an adenovirus serotype, including but not limited to: AdHu2, AdHu3, AdHu4, AdHu5, AdHu7, AdHu11, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad 3, canine Ad 2, sheep Ad, and porcine Ad 3. The adenovirus particle also contains a capsid protein. In some embodiments, the recombinant viral particle comprises an adenovirus particle combined with one or more foreign viral capsid proteins. Such combinations may be referred to as pseudotyped recombinant adenovirus particles. The foreign viral capsid proteins used in the pseudotyped recombinant adenovirus particles are derived from a foreign virus or from another adenovirus serotype. In some implementations, the foreign viral capsid protein is derived from, but is not limited to, reovirus type 3. Examples of vector and capsid protein combinations used in pseudotyped adenovirus particles can be found in the following references (Tatsis, N. et al. (2004) Mol. Ther. 10(4): 616-629 and Ahi, Y. et al. (2011) Curr. Gene Ther11(4): 307-320). Different adenovirus serotypes can be used to optimize transduction to specific target cells or to target specific cell types within specific target tissues (e.g., diseased tissues). Tissues or cells targeted by specific adenovirus serotypes include, but are not limited to, lung (e.g., HuAd3), spleen and liver (e.g., HuAd37), smooth muscle, synovial cells, dendritic cells, cardiovascular cells, tumor cell lines (e.g., HuAd11), and dendritic cells (e.g., HuAd5 pseudotyped reovirus type 3, HuAd30, or HuAd35). For further description, please see Ahi, Y. et al. (2011) Curr. Gene Ther. 11(4): 307-320, Kay, M. et al. (2001) Nat. Med. 7(1): 33-40, and Tatsis, N. et al. (2004) Mol. Ther. 10(4): 616-629.

[0137] In some embodiments, the viral particles are lentiviral particles. In some embodiments, the lentiviral particles are recombinant lentiviral particles, such as polynucleotide vectors containing one or more heterologous sequences (i.e., non-lentiviral original nucleic acid sequences) between two ITRs. The lentivirus is a positive-sense ssRNA retrovirus with a genome of approximately 10 kb. Lentivirals are known to integrate into dividing and non-dividing cells. Lentiviral particles can be generated, for example, by transfecting multiple plastids (typically separating the lentiviral genome from the replication and / or desired genes to avoid viral replication) into packaging cell lines that package the modified lentiviral genome into lentiviral particles. In some embodiments, lentiviral particles may refer to first-generation vectors lacking envelope proteins. In some embodiments, lentiviral particles may refer to second-generation vectors lacking all genes (except the gag / pol and tat / rev regions). In some implementations, lentiviral particles can refer to third-generation vectors containing only endogenous rev, gag, and pol genes and possessing chimeric LTRs for transduction without the tat gene (see Dull, T. et al. (1998) J. Virol. 72: 8463-71). For further description, see Durand, S. and Cimarelli, A. (2011) Viruses 3: 132-59.

[0138] In some embodiments, the viral particle is a recombinant lentiviral particle containing nucleic acid, said nucleic acid containing transgenes. The use of any adenovirus serotype is considered to be within the scope of this invention. In some embodiments, the lentiviral vector is derived from lentiviruses including, but not limited to: human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), sheep visna virus (VV), and caprine arthritis encephalitis virus (CAEV). The lentiviral particle also contains a capsid protein. In some embodiments, the recombinant viral particle comprises a lentiviral vector combined with one or more foreign viral capsid proteins. Such combinations may be referred to as pseudotyped recombinant lentiviral particles. In some embodiments, the foreign viral capsid protein used in the pseudotyped recombinant lentiviral particle is derived from a foreign virus. In some implementations, the foreign viral capsid protein used in the pseudotyped recombinant lentiviral particles is the vesicular stomatitis virus glycoprotein (VSV-GP). VSV-GP interacts with ubiquitous cellular receptors, generating broad tissue tropism against the pseudotyped recombinant lentiviral particles. Furthermore, VSV-GP is believed to provide high stability to the pseudotyped recombinant lentiviral particles.In other embodiments, the foreign viral capsid protein is derived from, but is not limited to: Chandipura virus, rabies virus, Mokola virus, lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Sindbis virus, Semliki Forest virus (SFV), Venezuelan equine encephalitis virus, Ebola virus Reston, Ebola virus Zaire, Marburg virus, Lassa virus, avian leukosis virus (ALV), sheep pulmonary adenocarcinoma retrovirus (JSRV), Moloney mouse leukosis virus (MLV), Gibbon ape leukemia virus (GALV), feline endogenous retrovirus (RD114), human T-lymphotropic virus 1 (HTLV-1), human foamy virus, and Maedi-visna virus. The virus can be MVV, SARS-CoV, Sendai virus, respiratory syncytial virus (RSV), human parainfluenza virus type 3, hepatitis C virus (HCV), influenza virus, avian disease virus (FPV), or Autographa californica multiplenucleopolyhedro virus (AcMNPV). Examples of vector and capsid protein combinations used in pseudotyped lentiviral particles can be found, for example, in Cronin, J. et al. (2005). Curr. Gene Ther. 5(4): 387-398. Different pseudotyped recombinant lentiviral particles can be used to optimize transduction of specific target cells or to target specific cell types in specific target tissues (e.g., diseased tissues). For example, tissues targeted by specific pseudotyped recombinant lentiviral particles include, but are not limited to, the liver (e.g., pseudotyped with VSV-G, LCMV, RRV, or SeV F proteins), the lungs (e.g., pseudotyped with Ebola, Marburg, SeV F, and HN or JSRV proteins), pancreatic islet cells (e.g., pseudotyped with LCMV protein), the central nervous system (e.g., pseudotyped with VSV-G, LCMV, rabies, or Mokola proteins), the retina (e.g., pseudotyped with VSV-G or Mokola proteins), monocytes or muscle (e.g., pseudotyped with Mokola or Ebola proteins), the hematopoietic system (e.g., pseudotyped with RD114 or GALV proteins), or cancer cells (e.g., pseudotyped with GALV or LCMV proteins).For further description, see J. et al. (2005). Curr. Gene Ther. 5(4): 387-398 and Kay, M. et al. (2001) Nat. Med. 7(1): 33-40.

[0139] In some embodiments, the viral particles are herpes simplex virus (HSV) particles. In some embodiments, the HSV particles are rHSV particles, such as polynucleotide vectors containing one or more heterologous sequences (i.e., non-HSV native nucleic acid sequences) between two TRs. HSV is an enveloped double-stranded DNA virus with a genome of approximately 152 kb. Advantageously, about half of its genes are non-essential and can be deleted to accommodate heterologous sequences. HSV particles infect non-dividing cells. Furthermore, they spontaneously form delays in neurons, move via retrograde transport, and can be transferred across synapses, making them advantageous for neuronal transfection and / or gene therapy pathways involving the neuronal system. In some embodiments, HSV particles may be replication-defective or replicable (e.g., capable of a single replication cycle through the inactivation of one or more late genes). For further description, see Manservigi, R. et al. (2010) OpenVirol. J. 4: 123-56.

[0140] In some embodiments, the viral particles are rHSV particles containing nucleic acids, said nucleic acids containing transgenes. Any use of an HSV vector is considered to be within the scope of this invention. In some embodiments, the HSV vector is derived from HSV serotypes including, but not limited to, HSV-1 and HSV-2. The HSV particles also contain capsid proteins. In some embodiments, the recombinant viral particles comprise an HSV vector combined with one or more foreign viral capsid proteins. Such combinations may be referred to as pseudotyped rHSV particles. In some embodiments, the foreign viral capsid protein used in the pseudotyped rHSV particles is derived from a foreign virus or from another HSV serotype. In some embodiments, the foreign viral capsid protein used in the pseudotyped rHSV particles is vesicular stomatitis virus glycoprotein (VSV-GP). VSV-GP interacts with ubiquitous cellular receptors, producing broad tissue tropism for pseudotyped rHSV particles. Furthermore, VSV-GP is believed to provide high stability to pseudotyped rHSV particles. In other embodiments, the foreign viral capsid protein may be derived from different HSV serotypes. For example, an HSV-1 vector may contain one or more HSV-2 capsid proteins. Different HSV serotypes can be used to optimize transduction of specific target cells or to target specific cell types within specific target tissues (e.g., diseased tissues). Tissues or cells targeted by specific adenoviruses include, but are not limited to, the central nervous system and neurons (e.g., HSV-1). For further description, see Manservigi, R. et al. (2010) OpenVirol J 4:123-156, Kay, M. et al. (2001) Nat. Med. 7(1):33-40, and Meignier, B. et al. (1987) J. Infect. Dis. 155(5):921-930.

[0141] V. Production of viral vectors

[0142] Many methods are known in the art for producing rAAV vectors, including transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybridization, herpesvirus-AAV hybridization (Conway, JE et al., (1997) J. Virology 71(11): 8780-8789) and baculovirus-AAV hybridization. All rAAV production cultures used to produce rAAV viral particles require: 1) suitable host cells, such as human-derived cell lines like HeLa, A549, or 293 cells, or, for baculovirus production systems, insect-derived cell lines like SF-9; 2) suitable helper virus function, provided by hydrocarbon-assisted wild-type or mutant adenoviruses (e.g., temperature-sensitive adenoviruses), herpesviruses, baculoviruses, or plastid constructs; 3) AAV rep and cap genes and gene products; 4) transgenes flanked by at least one AAV ITR sequence (e.g., therapeutic transgenes); and 5) suitable culture media and media components to support rAAV production. In some embodiments, the AAV rep and cap gene products can be derived from any AAV serotype. Typically, but not necessarily, the AAV rep gene product is of the same serotype as the ITR of the rAAV vector genome, provided the rep gene product can function to replicate and package the rAAV genome. The rAAV vector can be produced using suitable culture media known in the art. These media include, but are not limited to: Hyclone Laboratories and JRH, including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), commonly used formulations such as those described in U.S. Patent No. 6,566,118, and Sf-900 IISFM medium described in U.S. Patent No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly for commonly used culture medium formulations for the production of recombinant AAV vectors. In some embodiments, the AAV helper function is provided by adenovirus or HSV. In some implementations, the AAV helper function is provided by a baculovirus and the host cell is an insect cell (e.g., Spodopterafrugiperda (Sf9) cell).

[0143] Suitable rAAV production media of the present invention can be supplemented with serum or serum-derived recombinant protein at a level of 0.5%-20% (v / v or w / v). Alternatively, as is known in the art, rAAV vectors can be produced under serum-free conditions, also known as animal-derived product-free media. It will be understood by one of ordinary skill in the art that commercial or custom media intended to support the production of rAAV vectors can be supplemented with one or more cell culture components known in the art, including but not limited to glucose, vitamins, amino acids, and / or growth factors, thereby increasing the titer of rAAV in the production culture.

[0144] In some aspects, the present invention provides a method for preparing rAAV particles with reduced empty capsids, comprising: a) culturing host cells under conditions suitable for rAAV production, wherein the cells comprise: i) nucleic acids encoding a heterologous transgene flanked by at least one AAV ITR; ii) nucleic acids comprising AAV rep and cap coding regions, wherein the nucleic acids comprise a mutant p5 promoter, wherein expression of the p5 promoter is reduced compared to a wild-type p5 promoter; and iii) nucleic acids encoding AAV helper viral functions; b) lysing the host cells to release rAAV particles; c) isolating the rAAV particles produced by the host cells; and d) analyzing the presence of empty capsids and / or rAAV particles with different genomes in the rAAV particles by analytical ultracentrifugation as described above. In some embodiments, the p5 promoter encoding the nucleic acid of the AAV rep and cap regions is located at the 3' of the rep and / or cap coding regions. In some embodiments, the nucleic acids encoding the AAV rep and cap regions are plasmid pHLP, pHLP19, or pHLP09 (see U.S. Patent Nos. 5,622,856; 6,001,650; 6,027,931; 6,365,403; 6,376,237; and 7,037,713; the contents of which are incorporated herein by reference in their entirety). In some embodiments, the AAV helper viral functions include adenovirus E1A function, adenovirus E1B function, adenovirus E2A function, adenovirus VA function, and adenovirus E4 orf6 function.

[0145] rAAV production media can be grown under a variety of conditions suitable for the specific host cells used (spanning a wide range of temperatures, culture times, etc.). As is known in the art, rAAV production cultures include attachment-dependent cultures that can be cultured in suitable attachment-dependent containers, such as, for example, roller flasks, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. rAAV carrier production cultures can also include host cells suitable for suspension, 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 wave bag systems.

[0146] The rAAV carrier particles of the present invention can be harvested from rAAV production cultures by lysing the host cells of the production culture or by harvesting the used culture medium from the production culture, provided that the cells are cultured under conditions known in the art that cause the release of rAAV particles from intact cells into the culture medium, as described in more detail in U.S. Patent No. 6,566,118. Suitable methods for lysing cells are also known in the art and include, for example, multiple freeze / thaw cycles, microfluidization, and treatment with chemicals such as detergents and / or proteases.

[0147] Many methods for producing adenovirus vector particles are known in the art. For example, for a hollowed-out adenovirus vector, the adenovirus vector genome and the helper adenovirus genome can be transfected into a packaging cell line (e.g., the 293 cell line). In some embodiments, the helper adenovirus genome may contain recombination sites located flanking its packaging signal, and both genomes can be transfected into packaging cell lines expressing recombinases (e.g., using the Cre / loxP system), thereby packaging the adenovirus vector of interest more efficiently than the helper adenovirus (see, for example, Alba, R. et al. (2005) GeneTher. 12 Suppl 1: S18-27). Adenovirus vectors can be harvested and purified using standard methods, such as those described herein.

[0148] Many methods for producing lentiviral vector particles are known in the art. For example, for third-generation lentiviral vectors, a vector containing the lentiviral genome of interest (which contains the gag and pol genes) can be co-transfected with a vector containing the rev gene into a packaging cell line (e.g., the 293 cell line). The lentiviral genome of interest also contains a chimeric LTR that promotes transcription in the absence of Tat (see Dull, T. et al. (1998) J.Virol. 72: 8463-71). Lentiviral vectors can be harvested and purified using methods described herein (e.g., Segura MM, et al., (2013) Expert Opin Biol Ther. 13(7): 987-1011).

[0149] Many methods for producing HSV particles are known in the art. HSV vectors can be harvested and purified using standard methods, such as those described herein. For example, for replication-deficient HSV vectors, the HSV genome of interest lacking all early intermediate (IE) genes can be transfected into a supplementary cell line that provides the genes required for virus production, such as ICP4, ICP27, and ICP0 (see, for example, Samaniego, LA et al. (1998) J. Virol. 72:3307-20). HSV vectors can be harvested and purified using methods described (e.g., Goins, WF et al., (2014) Herpes Simplex Virus Methods in Molecular Biology 1144:63-79).

[0150] VI. Production of rAAV vectors

[0151] At harvest, the rAAV production culture of the present invention may contain one or more of the following: (1) host cell proteins; (2) host cell DNA; (3) plastid DNA; (4) helper virus; (5) helper virus proteins; (6) helper virus DNA; and (7) culture medium components, including, for example, serum proteins, amino acids, transferrin, and other low molecular weight proteins. Furthermore, the rAAV production culture also includes 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, or AAV12. In some embodiments, the rAAV production culture also contains empty AAV capsids (e.g., rAAV particles containing capsid proteins but not the rAAV genome). In some embodiments, the rAAV production culture further comprises rAAV particles containing a distinct rAAV genome (e.g., rAAV particles containing an rAAV genome different from the full-length rAAV genome). In some embodiments, the rAAV production culture further comprises rAAV particles containing a truncated rAAV genome. In some embodiments, the rAAV production culture further comprises rAAV particles containing AAV-capsid-coated DNA impurities.

[0152] In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters, such as Class DOHC Millipore Millistak+HC Pod filters, Class A1HC Millipore Millistak+HC Pod filters, and 0.2μm Filter Opticap XL1O Millipore Express SHC hydrophilic membrane filters. Clarification can be achieved using a variety of standard techniques known in the art, such as centrifugation or filtration through any cellulose acetate membrane with a pore size of 0.2μm or larger.

[0153] In some embodiments, the rAAV production culture harvest is further treated with Benzonase® to digest any high molecular weight DNA present in the production culture. In some embodiments, 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®, a temperature range of room temperature to 37°C, and a time range of 30 minutes to several hours.

[0154] rAAV particles can be separated or purified using one or more of the following purification steps: equilibration centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) for concentrating rAAV particles; rAAV capture by apatite chromatography; thermal inactivation by assisting virus; rAAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and rAAV capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps can be used alone, in various combinations, or in different sequences. In some embodiments, the method includes all steps in the order described below. Methods for purifying rAAV particles are found, for example, in Xiao et al., (1998) Journal of Virology 72: 2224-2232; U.S. Patent Nos. 6,989,264 and 8,137,948 and WO2010 / 148143. Methods for purifying adenovirus particles can be found, for example, in Bo, H et al., (2014) Eur. J. Pharm. Sci. 67C: 119-125. Methods for purifying lentivirus particles can be found, for example, in Segura MM et al., (2013) Expert Opin Biol Ther. 13(7): 987-1011. Methods for purifying HSV particles can be found, for example, in Goins, WF et al., (2014) Herpes Simplex Virus Methods in Molecular Biology 1144: 63-79.

[0155] Example

[0156] The invention will be more fully understood by referring to the following embodiments. However, they should not be considered as limiting the scope of the invention. It is understood that the embodiments and implementations described herein are for illustrative purposes only, and various modifications or variations thereunder will be indicated to those skilled in the art and are covered within the spirit and scope of this application and the appended claims.

[0157] Example 1: Characterization of recombinant adeno-associated virus vector preparation by analytical ultracentrifugation

[0158] Adeno-associated virus (AAV) possesses characteristics that make it a compelling vector for gene therapy. Wild-type AAV consists of two open reading frames (rep and cap), encoding all the structural and regulatory elements required for assembly, replication, and infection. The rep ORF encodes Rep 78 and 68 proteins, which are involved in genome replication, and Rep 52 and 40 proteins, which are involved in single-strand replication and packaging. The cap ORF encodes three structural capsid proteins: VP1, VP2, and VP3. Recombinant AAV vectors are typically generated using a triple transfection method with a "gutless" vector approach (Xiao, X, et al., 1998, J. Virol. 3: 2224-2232). The rep and cap are replaced with a therapeutic gene and its regulatory elements sandwiched between 5' and 3' inverted terminal repeats (ITRs). The rep and cap genes are presented in trans form on isolated plastids, and a third plastid provides the required adenoviral helper genes. Assuming the viral capsid is fully assembled and the ITR located flanking the vector genome is inserted into the capsid through capsid pores (Myers, MW & Carter, BJ, 1980, Virology, 102: 71-82), the resulting capsid population contains both empty capsids (without genome) and capsids containing genome. Furthermore, the capsids may contain incomplete portions of the recombinant viral genome. The vector preparation can then be purified by separating the capsids from cell debris using affinity chromatography, and further processed by anion exchange chromatography to enrich the complete vector.

[0159] Based on their recent approval for use in gene therapy, adeno-associated virus (AAV) vectors have emerged as an important new class of biopharmaceutical products. The production of AAV vector products requires analytical methods to monitor product quality related to heterogeneity, purity, and manufacturing consistency; however, no method has yet been available to support AAV vector characterization. To address this requirement, the potential use of analytical ultracentrifugation (AUC) as a technique for characterizing the homogeneity of AAV vectors has been investigated.

[0160] method

[0161] Sample preparation

[0162] To support accurate AUC assessment, the vector product (AAV2-transgenic 2) was highly purified, appropriately buffered, and concentrated to a concentration greater than 5 x 10⁻⁶. 11vg / mL. To achieve this target, cell supernatant was purified using AVB affinity chromatography (GE Healthcare) and exchanged for PBS pH 7.2 with 10K MWCO Slide-a-Lyzer (Thermo Scientific) buffer. Product concentration was determined spectrophotometrically at 260 nm (OD 260) by densitometric measurement. To generate reproducible and consistent AUC data, samples were adjusted to target concentrations from 0.1 to 1.0 at 260 nm by densitometric measurement, either by direct dilution with PBE or further concentration using an Amicon Ultra-0.5 / 30K MWCO centrifuge filter.

[0163] Settlement velocity AUC data acquisition

[0164] Sedimentation velocity analytical ultracentrifugation (SV-AUC) was performed using a ProteomeLab™ XL-I (Beckman Coulter). 400 μL of sample was loaded into the sample sector of each of the two velocity chambers, and 400 μL of PBS was loaded into the corresponding reference sector. The sample was placed in a four-well rotor and allowed to equilibrate in the instrument until maintained at 20 °C and full vacuum for 1 hour. Sedimentation velocity centrifugation was performed at 20,000 RPM, 20 °C, and a radius of 0.003 cm, with no delay and no repetition. Radial concentration as a function of time was synchronously recorded using absorbance (260 nm) and Raleigh interferometry optics until the smallest sedimented component passed through the optical window (1.2 hours). The assay throughput was limited to a single sample per run, based on absorbance scan collection times greater than 1 minute, and the large size and rapid sedimentation of the AAV.

[0165] AUC Data Analysis

[0166] The percentage of complete capsid was determined by analyzing approximately 75 scans from each detection method using the SEDFIT (NIH / see webpage analyticalultracentrifugation.com) continuous-size C(S) distribution model. The second-order (2) nd Derivative regularization is applied to the fit, with a confidence level of 0.68 for the F-statistic. The following C(S) parameters are kept constant: resolution = 200S, S... 最小值 =1,S 最大值 =200, and friction ratio =1.0. RI and TI noise reduction are applied, and the meniscus position is allowed to fluctuate, letting the software select the optimal position. The model fits the data from the Lamm equation, and the resulting size distribution is a "distribution of sedimentation coefficients," which looks like a chromatogram, with the area under each peak expressed in units of fringes or OD. 260Concentrations expressed in units are proportional. Determine the sedimentation coefficient (in Svedberg units) and relative concentration (in OD units) for each component in the distribution. Each AUC run is an independent determination, and the following properties are monitored for each analysis to ensure the quality of the results: goodness of fit (rmsd), OD for each peak, etc. 260nm The ratio of the interferometric signal (represented by fringes) (A260 / IF ratio), the consistency of the sedimentation coefficient between runs, and the overall quality of the scan.

[0167] Absorbance optics (260nm)

[0168] The molar concentration and actual percentage of the intact carrier peak were calculated from absorbance data using the extinction coefficient. Empty capsid ( 260 / 衣壳 =3.72e6) and complete vector ( 260 / 载体 The molar extinction coefficient of the absorbance (=3.00e7) was calculated based on a publicly available formula (Sommer et al. (2003) Mol Ther., 7: 122-8). The extinction coefficient can be used for both empty capsid and intact support peaks. The C(S) value was determined using the SEDFIT algorithm described in Schuck (2000) Biophys.J., 78: 1606-19. The molar concentrations of both intact support and empty capsid were calculated using Beer's Law, and the percentage of intact capsid was calculated from these values. Values ​​were reported based on the percentage of intact capsid.

[0169] Generate AUC standard curve

[0170] Since it is impossible to determine the extinction coefficient of fragmented genomes of unknown size and sequence based on experience, a relationship between S value and genome size was established. To achieve this, rAAV vector preparations with viral genomes of known nucleotide size and capsid coating were analyzed by AUC, and the corresponding S values ​​were determined as described above.

[0171] rAAV is generated through transient transfection.

[0172] Recombinant AAV vectors were generated using a triple transfection method with an "empty" vector (Xiao et al. (1998) J. Virol., 3: 2224-32). In this method, the rep and cap genes were replaced with therapeutic genes and their regulatory elements (sandwiched between 5' and 3' inverted terminal repeats (ITRs)). The rep and cap genes were provided trans-present on the isolated vector, and the third plastid provided the required adenoviral helper genes. Without being bound by theory, it was assumed that the viral capsid was fully assembled and that the ITRs located on the flanking side of the vector genome were inserted into the capsid through capsid pores (Myers, MW & Carter, BJ, 1980, Virology, 102: 71-82). The resulting capsid population contained both capsids without genomes (empty capsids) and capsids with genomes.

[0173] rAAV produced through the producer cell platform

[0174] AAV producer cell lines are an alternative production platform for generating clinical rAAV vectors. Using this method, HeLa S3 cells adapted for suspension growth are designed to have integrated copies of the AAV rep and cap genes, which are required for vector replication and packaging, along with the vector sequence and selection markers (see, for example, Puro: Thorne et al. (2009) Hum. Gene Ther., 20: 707-14). Once transfected with WT adenovirus (which provides the necessary auxiliary functions for replication), the cells produce recombinant AAV vectors along with the adenovirus, which are subsequently removed by ion-exchange chromatography during the purification process.

[0175] Other methods

[0176] The synthesized transgene was cloned into plastids containing a selected promoter and bovine growth hormone polyadenylation signal (polyA). The entire transgene expression cassette was then inserted into the previral plastid vector pAAVDC64 containing AAV2 inverted terminal repeats. The total size of the AAV genome obtained in each expression plastid (including regions with flanking ITRs) was 4–4.6 kb. Recombinant vectors were produced by triple transfection of 293 cells with pAdHelper (Stratagene, La Jolla, CA USA) using helper plastids expressing rep2 / cap sequences and adenovirus helper functions. The rep / cap helper was expressed from AAV serotype 2, while the cap sequence encoded one of the following sequences: AAV cap 1, 2, 5, 9, or rh8R. The vectors were purified by affinity chromatography and, in some cases, further purified to remove empty particles (see, for example, Qu et al. (2007) J.Virol.Methods. 140:183–92).

[0177] result

[0178] Analytical ultracentrifugation (AUC) using classical boundary settling velocities was employed to reveal particle heterogeneity in recombinant adeno-associated virus (rAAV) vector preparations. A mixture containing 20% ​​rAAV2 particles with a complete genome and 80% empty capsids was produced by mixing purified empty capsids and purified genomic capsids in a defined ratio. The mixture of empty and complete capsids was purified via a CsCl2 gradient followed by triple transfection to produce both empty and complete capsids. To monitor the movement of rAAV2 particles in response to centrifugal force, the rAAV2 capsid mixture was scanned at defined time intervals along the centrifugal force field at an absorbance of 260 nm. Figure 1A This displays representative scan patterns of the AAV2 mixture after centrifugation at 20,000 rpm for 1.2 hours (until the smallest sedimentation species passed through the optical window). Scans represent the concentration data obtained as a function of radius r at time t, producing a series of concentration scans that reveal the complete migration pattern of the component carrier particles in the rAAV2 vector preparation. In these S-shaped curves or boundaries, the leading edge of the curve represents the faster sedimentation species (i.e., rAAV2 capsids containing the genome), while the trailing edge represents the slower sedimentation species (i.e., “empty” rAAV2 capsids). Figure 1A ).

[0179] Plotting the sedimentation coefficient (in Svedberg units, S) against the differential sedimentation coefficient distribution value C(S) yielded different peaks. Both empty capsids and capsids containing genomes exhibited unique sedimentation coefficients. Figure 1B The C(S) value was determined using the SEDFIT algorithm described in Schuck (2000) Biophys.J., 78: 1606-19. To calculate the molar concentration and percentage values ​​for each capsid species from the absorbance data, extinction coefficients were used according to Table 2.

[0180] Table 2. Extinction coefficients and molar concentrations for various capsid types

[0181]

[0182] Empty shell (ε) 260 / 衣壳 =3.72e6) and genome-containing capsids (ε 260 / 衣壳The molar extinction coefficients of the two capsids (=3e7) were calculated using genome size and a publicly available formula (Sommer et al., 2003). Then, Beer's law was used to calculate the molar concentrations of both genome-containing and empty capsids. The relative abundance of each species was calculated using its molar concentration, expressed as a percentage of total capsids (Figure 1). These results demonstrate that AUC can be used to accurately distinguish and quantify empty and genome-containing capsids from heterogeneous vector preparations.

[0183] For the rAAV2 vector preparations shown in Figure 1, the capsid containing the complete genome is represented by a peak settling at 94, accounting for 21% of the vector preparations. Empty capsids precipitate with an S value of 64S, accounting for 79% of the vector preparations. These sedimentation coefficient values ​​are derived from the empty capsid (…). Figure 2A ) or containing genome particles ( Figure 2B AUC analysis of the pure preparations of rAAV2 was used for confirmation. The AUC plot of the pure rAAV2 empty capsid revealed a single peak with a sedimentation coefficient of 64S, while the AUC plot of the pure rAAV2 capsid containing the genome revealed a single peak with an even higher sedimentation coefficient of 94S. These results are consistent with the values ​​generated from heterogeneous preparations and further confirm that the AUC method can be used to quantify both genome-containing and empty AAV capsids from heterogeneous preparations containing two species.

[0184] The reproducibility of the AUC method was further evaluated by performing five independent AUC runs on the same vector sample (scAAV2 / 9 LP2), as shown in Table 3. The sedimentation coefficients for both genomic and empty AAV2 capsids were highly reproducible, yielding coefficients of variation of 0.5–0.6%. Similarly, the relative abundance (expressed as a percentage of the total abundance) of genomic capsids was determined with a coefficient of variation of approximately 2%. These results indicate that the AUC method used for quantifying genomic and empty AAV2 capsids produces highly reproducible and consistent values.

[0185] Table 3. Five independent determinations of scAAV2 / 9 LP2 samples

[0186]

[0187] Example 2: Comparison of Interference and Absorbance Detection Methods for AUC

[0188] We also evaluated an alternative optical detection method for AUC, Rayleigh interferometry. This method measures sample concentration based on the refractive index difference between a reference solution and an AAV-containing sample. Like absorbance detection, interferometry can be applied to any rAAV, regardless of its genomic sequence. Unlike absorbance detection, which requires an extinction coefficient, interferometry yields an integrated peak that is directly proportional to the concentration.

[0189] The pure preparations of empty and genome-containing AAV2 capsids were mixed in a 1:1 ratio and subjected to AUC interference ( Figure 3A ) and absorbance detection ( Figure 3B The two methods were analyzed. Interference detection revealed two populations of AAV capsids at the expected ratio of approximately 43% empty capsids and 57% genomic capsids. Figure 3A Both detection methods produced similar abundance ratios. However, comparing the peak sizes produced by the two methods using absorbance detection indicated a disconnect between peak height and concentration (compare). Figure 3A and 3B (The size of the "empty cap" peak). The data generated by the two methods are compared in Table 4. The ratio of absorbance signal to interference signal (A 260nm / IF) can be used in a similar manner to the 260 / 280 ratio of absorbance data, and it helps to identify peaks in the C(S) distribution.

[0190] Table 4. S-values ​​and relative abundances generated by absorbance and interferometry.

[0191]

[0192] While interferometric optics offers precision and resolution, it may require high-concentration samples. Furthermore, interferometric optics can be affected by mismatches between the reference and AAV sample buffers. However, AAV samples typically contain low protein concentrations, and may be necessary for a perfect match between the AAV sample and the reference solution.

[0193] Example 3: The effect of production method on the heterogeneity of AAV carriers

[0194] Previous examples have demonstrated that the AUC method is a highly accurate and reproducible method for distinguishing and quantifying empty and genomically contained AAV capsids from heterogeneous mixtures. This capability can be advantageous for a variety of applications evaluating the quality of AAV vector preparations. For example, a major problem in producing pure AAV vector preparations is the presence of capsids containing partial or fragmented genomes. To illustrate the practicality of the AUC method for distinguishing these types, AAV vectors produced by two different methods (referred to as the "triple transfection" and "producer cell line" methods) were analyzed by AUC.

[0195] Used for triple transfection method ( Figure 4 ) or producer cell line method ( Figure 5 The AAV2 vector carrying transgene 2 was produced. These methods are described in Example 1. After chromatographic purification, both vector preparations were analyzed by AUC. Figure 6A A schematic diagram of the AAV2 vector genome is shown.

[0196] The AUC profiles of vector preparations produced by these methods are significantly different. Using the producer cell line method, 74% of the capsids contain the complete genome, represented by 92S species ( Figure 6B 19% were empty capsids, with the remainder containing fragmented genomes (7% of 75S strains). In contrast, 82% of capsids produced via triple transfection were empty, and 64S strains (…). Figure 6C ), while capsids containing fragmented genomes (76S and 84S species) accounted for 11%, while capsids with complete genomes (94S) accounted for only 8%.

[0197] These results demonstrate that vector preparations produced using producer cell line technology can be of high quality, primarily containing capsids with complete genomes. The vast majority of capsids produced via triple transfection were empty, while a larger proportion contained fragmented genomes. These results also highlight the ability of the AUC method to distinguish capsids with fragmented genomes, in addition to those containing complete genomes and empty capsids. Furthermore, they indicate the effectiveness of the AUC method in assessing the quality and homogeneity of vector preparations.

[0198] Example 4: Use of AUC to evaluate the removal of empty capsids from carrier preparations

[0199] The AUC method has been evaluated as a tool for monitoring the removal of empty capsids using chromatographic methods (see Qu et al. (2007) J. Virol. Methods, 140: 183-92). Separation of empty and genome-containing capsids was performed using anion exchange chromatography. Figure 7A AUC was performed at the resolved peak to demonstrate that genome-containing rAAV2 particles were enriched in fractions subsequently eluted from resin. Figure 7A (The "complete genome capsid" in the text).

[0200] like Figure 7B As shown, AUC analysis revealed that the genome-containing capsid represented 94% of the vector preparation upon elution from the column. This subsequent fractionation produced a single peak with a sedimentation coefficient of 92S. In contrast, the rAAV2 vector preparation prior to the chromatographic step ( Figure 7CThe AUC analysis revealed two peaks with S values ​​of 63 and 93, with the 63S peak (empty capsid) representing 52% of the total capsid population. These results demonstrate that the chromatographic method is highly efficient for removing empty capsids from AAV carrier preparations. Importantly, they demonstrate the practicality of applying the AUC method to assess carrier quality during purification. The AUC method is a useful tool for evaluating different carrier purification schemes or techniques.

[0201] Example 5: Assessing viral genome integration using the AUC method

[0202] As described in Example 3, AAV vector preparations may contain a capsid packaged with fragmented genomes, in addition to complete genomes and empty species. A major problem in producing homogeneous AAV preparations for therapeutic or research applications is the presence of capsids containing fragmented genomes, which can lead to aberrant or absent expression of transgenes of interest. Indeed, heterogeneity associated with AAV vector preparations has been reported to result in the packaging of fragmented genomes or DNA impurities coated with AAV capsids (Kapranov et al. (2012) Hum. Gene Ther., 23: 46-55). Therefore, AUC is considered a tool for quantifying aberrant packaging of fragmented genomes in rAAV vector preparations.

[0203] Since it is impossible to determine the extinction coefficient of fragmented genomes of unknown size and sequence empirically, a relationship between S-value and genome size was established. To achieve this, rAAV vector preparations with known nucleotide sizes of capsid-coated viral genomes were analyzed by AUC, and their corresponding S-values ​​were determined, as shown in Table 5. A standard curve relating genome size and S-value was then generated. Figure 8 This demonstrates a highly linear relationship between sedimentation coefficient and genome size (R0). 2 =0.9978).

[0204] Table 5. S-values ​​of rAAV vectors with known genome size

[0205]

[0206] To demonstrate the practicality of AUC detection of genomic fragments, a vector containing self-complementary AAV2 ITRS, a minimal CBA promoter, and an EGFP transgene were packaged into an AAV9 capsid (AAV2 / 9minCBAEGFP; see [link to documentation]). Figure 9A(See schematic diagram). Vector particles were purified to remove empty capsids and analyzed by AUC. Genome size was then assigned to each resolved genome-containing capsid using a standard curve. Approximately 25% of the vector preparations settled to 10¹S species, representing a ~4.3 kb genome encapsulated in the capsid. Figure 9A The 101S peak represents the double-stranded dimer vector genome, predicted to be ~4.3kb in size. However, the majority (75%) of the vector preparations precipitated with an S value of 82, corresponding to a vector genome size of ~2kb. Figure 9A This is consistent with the packaging of single-stranded monomers. The packaging of monomeric genomes with their complementary vectors is well documented and is often due to accidental end-distinguishing at pseudo-"trs-like" sequences, despite the presence of ITRs with mutated D sequences (McCarty et al. (2001) Gene Ther., 8: 1248-54).

[0207] Figure 9B Basic Southern blotting of the same vector (scAAV9 EGFP) revealed two vector populations with genome sizes of ~4.3kb and ~2kb, confirming that... Figure 9A The AUC values ​​were also observed. Southern blotting also confirmed that the monoclonal viral genome was preferentially packaged over the dimeric genome. Interestingly, AUC analysis of the single-stranded AAV9 EGFP vector (~4kb) revealed a single main peak with a measured S value of 99S, corresponding to approximately 4.1kb, through the standard curve and 84% capsid abundance. Figure 9C These results indicate that single-stranded AAV vectors can be packaged in a more homogeneous manner than double-stranded vectors. Similarly, consistent with the AUC method, Southern blot analysis of this vector preparation revealed homogeneous capsidation of the predicted ~4kb viral genome (lane 2, ...). Figure 9B These results demonstrate that the AUC method can be used to measure the genome size of AAV vectors, yielding genome size data conforming to standard Southern blotting techniques. Using the AUC method, single-stranded AAV vectors were found to produce more homogeneous vector preparations than double-stranded ones. These results show that the AUC method is a powerful tool for identifying and quantifying capsid species containing incomplete genomes from vector preparations.

[0208] Example 6: Use of AUC to assess factors influencing vector genome packaging

[0209] The AUC method was then used as a tool to identify factors that affect the packaging of the complete AAV vector genome.

[0210] As described in Example 3, the production of rAAV vectors via transient transfection requires the use of three types of plasmids, including rep / cap helper plasmids, ITR vector plasmids, and pAd helper plasmids. Figure 4 The effect of rep / cap helper plasmids on vector genome packaging (for both single-stranded and self-complementary AAV vectors) was evaluated using AUC. First, self-complementary AAV vectors carrying EGFP transgenes were generated using one of two methods. Figure 10A In the first method ( Figure 10B ), using an auxiliary plastid, where rep 78 / 68 expression is driven by an endogenous p5 promoter (“WT Rep” construct). In the second approach ( Figure 10C The helper plasmid was modified by moving the p5 promoter downstream of the cap2 sequence and mutating the TATA box, thereby reducing 78 / 68 expression (“pHLP Rep” construct). The complete scAAV2 EFGP capsid was predicted to have sedimentation coefficients of 100S (in the dimer genome form) and 80S (in the monomer genome form). Figure 10A ).

[0211] AUC analysis of these scAAV2EGFP vector preparations revealed significant differences in vector genome packaging. In the presence of reduced rep78 / 68 (pHLP), over half (55%) of the vector preparations contained a dimer genome, represented by 100S species (…). Figure 10C This is the expected sedimentation coefficient for a capsid containing a 4.4 kb dimeric genome. In contrast, the scAAV2EGFP preparation produced with full complement of rep78 / 68 had significantly less packaged dimeric genome (26%), with most capsids containing monomeric genomes and sedimenting at 80S. Figure 10B These results reveal significant differences in genome packaging induced by the P5 promoter via the mobile helper plasmid, resulting in reduced rep78 / 68 protein levels.

[0212] A single-stranded AAV5 Factor IX vector (AAV5FIX) was generated using rep / cap helpers that differ from those mentioned above in rep expression. Figure 11A -B) and single-chain AAV5hSMN vector ( Figure 11C-D), but the cap sequence of AAV2 is replaced by the cap sequence of AAV5. Based on the nucleotide size of the FIX expression cassette (4.3 kb), the predicted sedimentation coefficient for the AAV5 FIX vector capsid is approximately 10¹S. AUC analysis of AAV2 / 5FIX in the presence of reduced rep78 / 68 (“pHLP19 Rep”) revealed a homogeneity profile, with most vectors (90%) sedimenting at the expected S value (~10¹S). Figure 11A In contrast, the AAV5 FIX vector (“WT Rep”) generated using the rep / cap5 helper expressing wild-type levels of the 78 / 68 protein produced significantly different AUC profiles. Figure 11B This map reveals further capsid heterogeneity, with most AAV5 FIX (80%) settling at a lower S value of 86S (instead of the main peak at 101S), possibly indicating the packaging of fragmented genomes. Furthermore, in this vector sample, only 15% of the AAV5 FIX vector capsids settled at the correct S value of ~104S. Figure 11B ).

[0213] AAV5SMN vectors derived from these same wild-type and mutant p5 rep / cap replicons also exhibited significantly different AUC profiles. As observed by AUC analysis in single-stranded AAV5FIX vectors, AAV5 SMN vectors generated in the presence of reduced rep78 / 68 showed lower heterogeneity, with individual capsid species settling at an S value of 101S, consistent with the predicted genome size of ~4.4kb. Figure 11C In contrast, AUC profiles of the same vector packaged with “wild-type” levels of rep78 / 68 protein revealed three distinct AAV vector species with sedimentation coefficients of 100S (the predicted S value for a 4400 nt complete vector genome), 92S (representing approximately 3300 nt of fragmented genome), and 80S (representing approximately 2000 nucleotides of fragmented genome). Figure 11D These results confirm the significant differences in genome packaging induced by moving the p5 promoter of the helper plasmid with two additional AAV vectors.

[0214] Further analysis of the AAV5SMN and AAV5FIX vector preparations was performed using Southern blotting analysis of the vector DNA. Following the AUC method, Southern blotting analysis of AAV5SMN generated from wild-type rep78 / 68 protein revealed the packaging of both full-length (4.4 kb) and fragmented (less than 4.4 kb) SMN genomes. Figure 12ALane 1). In contrast, the AAV5SMN vector generated in the presence of reduced rep78 / 68 protein mainly contains a capsid with a full-length SMN genome ( Figure 12A (lane 2). Interestingly, a comparison of the two AAV5FIX vector preparations by Southern analysis revealed the presence of the full-length FIX genome, even when the vectors were generated with wild-type level rep 78 / 68 ( Figure 12B Lane 2). However, the AUC analysis of this AAV5FIX vector ( Figure 11B The results showed that 80% of the capsid contained fragmented genomes (~3000 nucleotides) that could not be detected by the FIX probe.

[0215] By generating probes targeting discrete regions (including the scaffold region) of the carrier plasmid, the FIX carrier preparations generated under two experimental conditions were further analyzed. Figure 13 The image of this vector is provided. Figure 14 shows Southern blot analysis using these probes to compare these FIX vector preparations generated under different conditions. Figure 14A As shown, both vector preparations (pHLP rep, lane 1; WT rep, lane 2) contain the hFIX gene. However, Figure 14B Lane 2 confirmed that in the WT Rep preparation ( Figure 11B The study observed approximately 3kb fragments of the vector genome deposited at 86S. Furthermore, this fragment was associated with an Amp R-specific probe (…). Figure 14B The reaction in lane 2 indicates that upstream packaging of the 5' ITR occurs in a rep-dependent manner. In contrast, there was no evidence of Amp R-containing fragments in the rAAV5FIX vector preparations produced in the presence of reduced rep 68 / 78 levels. Figure 14B Lane 1).

[0216] Also by using specific targeting of Amp R Q-PCR of primers and probes was used to assess DNA impurities in AAV FIX preparations. Approximately 35% Amp was detected in the AAV5FIX vector preparations generated in the presence of “wt” rep by Q-PCR. R The titer, in contrast, was less than 1% when the same vector was used to generate the AAV5FIX vector in the presence of reduced rep68 / 78 (data not shown). These results highlight the utility of AUC in revealing the presence of genes packaged in ways that are undetectable by gene-specific Southern blot analysis.

[0217] The packaging capability of AAV vectors has been extensively studied, and while many reports have demonstrated successful transduction with vectors containing oversized AAV genomes, the latter have been fragmented into subgenomic-length DNA. To further explore the applicability of the AUC method, the heterogeneity of AAV vectors generated with oversized genomes was evaluated. Expression cassettes carrying full-length CBA promoters driving β-phosphodiesterase expression were packaged into 5.4 kb oversized genomes (…). Figure 15A ) or packaged as a wild-type size genome of 4.6kb ( Figure 15B To generate a 4.6kb genome, the CBA promoter was truncated by reducing the size of the introns, as previously reported (Gray, SJ et al., (2011) Hum. Gene Ther. 22(9): 1143-1153).

[0218] like Figure 15A As shown, the AUC profiles of AAV vector preparations generated with excessively large vector genomes demonstrate that nearly half of the preparations settled at 93S, consistent with the packaging of a fragmented vector genome of approximately 3.5 kb. 30% of the preparations were represented by another subgenome vector species, approximately 4.9 kb, settling at 105S. There was no evidence of packaging a full-length 5.4 kb genome, predicted to settle at 108–109S. In contrast, AUC analysis revealed that the same transgenes under the control of the abbreviated CBA promoter settled primarily as 102S vector species, consistent with the predicted packaging of a full-length 4.6 kb vector genome. Figure 15B These results demonstrate the practicality of AUC analysis for profiling AAV vectors with excessively large genomes, and that this profiling is crucial given the observed rate of gene fragmentation.

[0219] This example demonstrates that the AUC method is highly efficient for analyzing the genome size of AAV vector capsids in heterogeneous preparations. By distinguishing genome-containing capsids (e.g., dimer and monomeric genomes, or fragments thereof) based on size, the AUC method represents a powerful tool for determining the quality of AAV vector preparations produced under different conditions. Furthermore, results from three different vector systems demonstrate the broad usefulness of the AUC method for quality control and condition optimization to produce improved AAV vector preparations. Importantly, the AUC method can detect fragmented genomes that are undetectable by Southern blotting analysis. Given that Southern blotting depends on the presence of a DNA probe sequence for detection, the AUC method is sequence-independent. The AUC method also demonstrates its effectiveness as a tool for analyzing excessively large AAV genomes. Overall, these results demonstrate that the AUC method is highly advantageous and efficient for analyzing a wide variety of AAV vector preparations, and its highly variable effects on genome packaging have been found.

[0220] Example 7: Characterization of recombinant adenovirus vector preparation by analytical ultracentrifugation

[0221] Adenovirus (Ad) vectors possess characteristics that make them compelling candidates for gene therapy. The production of Ad vector products requires analytical methods to monitor product quality, which relates to homogeneity, purity, and manufacturing consistency. To meet these requirements, the potential use of analytical ultracentrifugation (AUC) as a technique for characterizing the homogeneity of Ad vectors was investigated.

[0222] method

[0223] Sample preparation

[0224] To support accurate AUC assessment, a recombinant adenovirus serotype 2 vector (Ad2) was prepared and purified by CsCl gradient ultracentrifugation to enrich the particulate-containing genome. The AUC was then analyzed spectrophotometrically at 260 nm (OD200). 260 The product concentration was determined by optical density measurement at 260 nm. To produce reproducible and consistent AUC data, the sample was adjusted to the target concentration by optical density measurement from 0.1 to 1.0 at 260 nm, either by direct dilution with PBS or by further concentration using an Amicon Ultra-0.5 / 30K MWCO centrifugal filter device.

[0225] Settlement velocity AUC data acquisition

[0226] Sedimentation velocity analytical ultracentrifugation (SV-AUC) was performed using a ProteomeLab™ XL-I (Beckman Coulter). 400 μL of sample was loaded into the sample sector of each of the two velocity chambers, and 400 μL of PBS was loaded into the corresponding reference sector. The sample was placed in a four-well rotor and allowed to equilibrate in the instrument until maintained at 20 °C and full vacuum for 1 hour. Sedimentation velocity centrifugation was performed at 6,000 RPM, 20 °C, and a 0.003 cm radius step setting, with no delay and no repetition. Radieigh interferometric optics were used to synchronously record the radius concentration as a function of time until the smallest sedimented component passed through the optical window (1.2 hours). The assay throughput was limited to a single sample / run, based on absorbance scan collection times greater than 1 minute, and the large size and rapid sedimentation of Ad2.

[0227] AUC Data Analysis

[0228] The percentage of complete capsids was determined from approximately 75 scans using an interferometric detection method by employing a SEDFIT (NIH / see analyticalultracentrifugation.com) continuous-size C(S) distribution model. Second-order (2nd) derivative regularization was applied to the fit, with a confidence level / ratio of 0.68 for the F-statistic. The following C(S) parameters were kept constant: resolution = 250S, S... 最小值 =10, S 最大值 =1500, and friction ratio =1.86935. RI and TI noise reduction are applied, and the meniscus position is allowed to fluctuate, letting the software select the optimal position. The model fits the data from the Lamm equation, and the resulting size distribution is a "distribution of sedimentation coefficients," which looks like a chromatogram, with the area under each peak and the value expressed in units of stripes or OD. 260 Concentrations expressed in units are proportional. Determine the sedimentation coefficient (in Svedberg units) and relative concentration (in OD units) for each component in the distribution. Each AUC run is an independent determination, and the following properties are monitored for each analysis to ensure the quality of the results: goodness of fit (rmsd), OD for each peak, etc. 260nm The ratio of the interferometric signal (represented by fringes) (A260 / IF ratio), the consistency of the sedimentation coefficient between runs, and the overall quality of the scan. The rmsd value for this representative example is 0.006584.

[0229] result

[0230] Analytical ultracentrifugation (AUC) using classical boundary sedimentation velocities was employed to reveal particle heterogeneity in recombinant adenovirus serotype 2 (rAd2) vector preparations. To monitor the motion of rAd2 particles in response to centrifugal force, the mixture of rAd2 capsids was scanned along the centrifugal force field at defined time intervals using interferometric optics. Scans represent the concentration data obtained as a function of radius *r* at time *t*, producing a series of concentration scans that revealed the complete migration patterns of the component vector particles in the rAd2 vector preparation. Plotting the sedimentation coefficients (in Svedberg units, *S*) against the differential sedimentation coefficient distribution C(S) yielded distinct peaks for different rAd2 species with unique sedimentation coefficients. Figure 16 The C(S) value was determined using the SEDFIT algorithm described in Schuck (2000) Biophys.J., 78: 1606-19.

[0231] for Figure 16 The rAd2 vector preparation shown exhibits a sedimentation rate of 87.8% with an S value of 731, consistent with vector preparations primarily composed of a genome-containing capsid. These data confirm that adenovirus particles can be resolved via AUC.

[0232] This disclosure relates to the following implementation plan:

[0233] 1. A method for characterizing recombinant virus particle preparations, comprising the following steps:

[0234] a) The preparation was subjected to analytical ultracentrifugation under boundary settling velocity conditions, wherein the settling of recombinant virus particles was monitored at time intervals.

[0235] b) Plot the settlement coefficient in Svedberg units (S) against the differential settlement coefficient distribution values ​​(C(s)).

[0236] c) Integrate the area under each peak in the C(s) distribution to determine the relative concentration of each peak, where each peak represents a recombinant viral particle.

[0237] 2. A method for assessing the vector genome integrity of recombinant viral particles in a recombinant viral particle preparation, comprising:

[0238] a) The preparation was subjected to analytical ultracentrifugation under boundary settling velocity conditions, wherein the settling of recombinant virus particles was monitored at time intervals.

[0239] b) Plot the settlement coefficient in Svedberg units (S) against the differential settlement coefficient distribution values ​​(C(s)).

[0240] c) The species of recombinant viral particles in the preparation are identified by the peaks corresponding to the S values ​​present in the figure, wherein the genome size of the specific species of recombinant viral particles is calculated by comparing the S value of the species with a standard curve generated by the S value of recombinant viral particles containing a known nucleotide-sized viral genome with a capsid coating.

[0241] 3. The method according to Implementation Scheme 2 further includes integrating the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species.

[0242] 4. A method for determining the presence of empty capsids or capsid particles containing recombinant viral genomes of different sizes in a recombinant viral particle preparation, comprising the following steps:

[0243] a) The preparation was subjected to analytical ultracentrifugation under boundary sedimentation velocity conditions, wherein the sedimentation of recombinant virus particles was monitored at time intervals, and

[0244] b) Plot the sedimentation coefficient in Svedberg units (S) with differential sedimentation coefficient distribution values ​​(C(s)), where the presence of one or more peaks other than the peak of a fully capsid particle containing a complete recombinant viral genome indicates the presence of capsid particles containing genomes of different sizes and / or empty capsids.

[0245] 5. A method for measuring the relative amount of empty capsid in a recombinant virus particle preparation, comprising the following steps:

[0246] a) The preparation was subjected to analytical ultracentrifugation under boundary settling velocity conditions, wherein the settling of recombinant virus particles was monitored at time intervals.

[0247] b) Plot the settlement coefficient in Svedberg units (S) against the differential settlement coefficient distribution values ​​(C(s)).

[0248] c) Integrate the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species, and

[0249] d) Compare the amount of recombinant viral particles with an S value corresponding to empty capsid particles with the amount of recombinant viral particles with an S value corresponding to recombinant viral particles containing a complete viral genome or the total amount of recombinant viral particles in the preparation.

[0250] 6. A method for measuring the relative amount of capsid particles or empty viral capsid particles containing different recombinant viral genomes in a recombinant viral particle preparation, comprising the following steps:

[0251] a) The preparation was subjected to analytical ultracentrifugation under boundary settling velocity conditions, wherein the settling of recombinant virus particles was monitored at time intervals.

[0252] b) Plot the settlement coefficient in Svedberg units (S) against the differential settlement coefficient distribution values ​​(C(s)).

[0253] c) Integrate the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species.

[0254] d) Compare the amount of recombinant viral particles with an S value that does not correspond to a recombinant viral particle containing a complete viral genome with the amount of recombinant viral particles with an S value that corresponds to a recombinant viral particle containing a complete viral genome or the total amount of recombinant viral particles in the preparation.

[0255] 7. A method for measuring the relative amount of capsid particles containing multiple recombinant viral genomes in a recombinant viral particle preparation, comprising:

[0256] a) The preparation was subjected to analytical ultracentrifugation under boundary settling velocity conditions, wherein the settling of recombinant virus particles was monitored at time intervals.

[0257] b) Plot the settlement coefficient in Svedberg units (S) against the differential settlement coefficient distribution values ​​(C(s)).

[0258] c) Integrate the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species.

[0259] d) Compare the amount of recombinant viral particles with an S value that does not correspond to a recombinant viral particle containing a complete viral genome or an empty capsid with the total amount of recombinant viral particles in the preparation.

[0260] 8. A method for measuring the relative amount of recombinant viral particles containing a complete viral genome in a recombinant viral particle preparation, comprising:

[0261] a) The preparation was subjected to analytical ultracentrifugation under boundary settling velocity conditions, wherein the settling of recombinant virus particles was monitored at time intervals.

[0262] b) Plot the settlement coefficient in Svedberg units (S) against the differential settlement coefficient distribution values ​​(C(s)).

[0263] c) Integrate the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species.

[0264] d) Compare the amount of recombinant viral particles having an S value corresponding to a recombinant viral particle containing a complete viral genome with the amount of recombinant viral particles having an S value corresponding to an empty capsid particle, with capsid particles containing different recombinant viral genomes, and / or with the total amount of recombinant viral particles in the preparation.

[0265] 9. A method for monitoring the removal of empty capsids and / or capsid particles containing different recombinant viral genomes during the purification process of a recombinant viral particle preparation, the method comprising removing a sample of recombinant viral particles from the preparation after one or more steps of the purification process, and analyzing the relative amount of empty capsids and / or capsid particles containing different recombinant viral genomes in the sample according to any one of embodiments 5-8, wherein a decrease in the relative amount of empty capsids and / or capsid particles containing different genomes compared to a complete capsid indicates the removal of empty capsids from the recombinant viral particle preparation.

[0266] 10. The method according to any one of embodiments 4-9, wherein the presence of the peak corresponding to the S value of the empty capsid particle indicates the presence of the empty capsid particle.

[0267] 11. The method according to any one of embodiments 4-9, wherein the presence of one or more peaks other than the peaks of fully capsid particles containing complete recombinant viral genomes or empty capsid particles indicates the presence of capsid particles containing genomes of different sizes.

[0268] 12. The method according to embodiment 11, wherein the capsid particles containing genomes of different sizes contain truncated genomes, aggregates, recombinants and / or DNA impurities.

[0269] 13. A method for determining the heterogeneity of recombinant viral particles in a recombinant viral particle preparation, comprising the following steps:

[0270] a) The preparation was subjected to analytical ultracentrifugation under boundary settling velocity conditions, wherein the settling of recombinant virus particles was monitored at time intervals.

[0271] b) Plot the sedimentation coefficient in Svedberg units (S) against the differential sedimentation coefficient distribution (C(s)), where the presence of peaks other than the peak representing the capsid containing the complete recombinant viral genome indicates the heterogeneity of the recombinant particles in the preparation.

[0272] 14. The method according to embodiment 13, wherein the presence of the additional peak indicates the presence of empty capsid particles and / or recombinant viral particles containing different genomes.

[0273] 15. The method according to embodiment 14, wherein the different genomes are truncated viral genomes, aggregates, recombinants and / or DNA impurities.

[0274] 16. The method according to any one of embodiments 13-15, further comprising integrating the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species.

[0275] 17. A method for monitoring the homogeneity of recombinant viral particles during the purification process of a recombinant viral particle preparation, the method comprising removing a sample of recombinant viral particles from the preparation after one or more steps of the purification process, and determining the heterogeneity of the recombinant viral particles according to the method of embodiment 16, wherein an increase in the relative amount of recombinant viral particles containing a complete viral genome indicates an increase in the homogeneity of complete viral particles in the recombinant viral particle preparation.

[0276] 18. The method according to any one of the implementation schemes 1-17, wherein the sedimentation of recombinant virus particles is monitored by absorbance.

[0277] 19. The method according to embodiment 18, wherein the absorbance is about 230 nm, 260 nm or 280 nm.

[0278] 20. The method according to embodiment 18 or 19, wherein the absorbance is about 260 nm.

[0279] 21. The method according to any one of implementation schemes 1-20, wherein the sedimentation of recombinant viral particles is monitored by interference.

[0280] 22. The method according to embodiment 21, wherein the interference is Rayleigh interference.

[0281] 23. The method according to any one of embodiments 1-22, wherein the preparation is an aqueous solution.

[0282] 24. The method according to embodiment 23, wherein the aqueous solution contains a pharmaceutical preparation.

[0283] 25. The method according to embodiment 23 or 24, wherein the aqueous solution contains a buffer.

[0284] 26. The method according to embodiment 25, wherein the buffer is at a physiological pH.

[0285] 27. The method according to embodiment 25 or 26, wherein the buffer is at a physiological osmotic concentration.

[0286] 28. The method according to any one of embodiments 24-27, wherein the pharmaceutical preparation comprises phosphate-buffered saline (PBS).

[0287] 29. The method according to embodiment 28, wherein the pH of the PBS is about 7.2 and the osmotic concentration is about 300 mOsm / L.

[0288] 30. The method according to any one of embodiments 24-30, wherein the monitoring further includes comparison with a reference sample, wherein the reference sample comprises an aqueous solution free of recombinant virus particles.

[0289] 31. The method according to any one of embodiments 1-30, wherein the C(S) value is determined by an algorithm incorporating solutions to the Lamm equation.

[0290] 32. The method according to implementation scheme 31, wherein the algorithm is the SEDFIT algorithm.

[0291] 33. The method according to any one of implementation schemes 1-32, wherein sedimentation is monitored until the lowest density recombinant virus particles settle to the bottom of the sector of the ultracentrifuge.

[0292] 34. The method according to any one of embodiments 1-32, wherein the ultracentrifugation utilizes an ultracentrifuge comprising an ultracentrifugation velocity chamber.

[0293] 35. The method according to any one of the implementation schemes 1-33, wherein sedimentation is monitored until the recombinant virus particles settle to the bottom of the ultracentrifugation chamber.

[0294] 36. The method according to implementation scheme 34, wherein sedimentation is monitored until the recombinant virus particles with the lowest density settle through an optical window.

[0295] 37. The method according to any one of embodiments 33-36, wherein at least 30 scans are used to monitor the sedimentation of recombinant viral particles.

[0296] 38. The method according to implementation plan 37, wherein approximately 30 scans are used to monitor the sedimentation of recombinant viral particles.

[0297] 39. The method according to implementation scheme 37, wherein approximately 30 to approximately 75 scans are used to monitor the sedimentation of recombinant viral particles.

[0298] 40. The method according to implementation scheme 37, wherein approximately 30 to approximately 50 scans are used to monitor the sedimentation of recombinant viral particles.

[0299] 41. The method according to implementation scheme 37, wherein approximately 50 to approximately 75 scans are used to monitor the sedimentation of recombinant viral particles.

[0300] 42. The method according to any one of implementation schemes 31-41, wherein regularization is applied to the fit level with a confidence level of at least about 0.68 for the F-statistic.

[0301] 43. The method according to implementation scheme 42, wherein the regularization is second derivative regularization.

[0302] 44. The method according to implementation scheme 42, wherein the regularization is maximum entropy regularization.

[0303] 45. The method according to any one of embodiments 42-44, wherein regularization is applied to the fit level with a confidence level of about 0.68 to about 0.90 for the F-statistic.

[0304] 46. ​​The method according to any one of embodiments 42-44, wherein regularization is applied to the fit level with a confidence level of about 0.68 to about 0.99 for the F-statistic.

[0305] 47. The method according to any one of implementation schemes 42-44, wherein regularization is applied to the fit level with a confidence level of approximately 0.68 for the F-statistic.

[0306] 48. The method according to any one of embodiments 31-47, wherein the following C(S) parameter remains constant: the resolution is from approximately 200S to approximately 5000S, S 最小值 It is from 1S to approximately 100S, S 最大值 It is approximately 100S to approximately 5000S, and the friction ratio is approximately 1.0 or is allowed to fluctuate to a value determined by centrifugation software.

[0307] 49. The method according to embodiment 48, wherein the resolution is about 200S to about 1000S.

[0308] 50. The method according to implementation scheme 48 or 49, wherein the resolution is approximately 200 s.

[0309] 51. According to the method of any one of implementation schemes 48-50, wherein S 最小值 It is approximately 1.

[0310] 52. According to the method of any one of implementation schemes 48-51, where S 最大值 It is approximately 100S to approximately 1000S.

[0311] 53. According to the method of any one of implementation schemes 48-51, wherein S 最大值 It is approximately 200S to approximately 5000S.

[0312] 54. According to the method of any one of implementation schemes 48-51, where S 最大值 It is approximately 200S.

[0313] 55. The method according to any one of embodiments 48-54, wherein the friction ratio is floated to a value determined by centrifugation software.

[0314] 56. The method according to any one of embodiments 48-54, wherein the friction ratio is about 1.0.

[0315] 57. The method according to any one of implementation schemes 31-56, wherein radius invariant (RI) and time invariant (TI) noise reduction are applied.

[0316] 58. The method according to any one of the implementation schemes 1-57, wherein the settling of each recombinant viral particle is monitored at approximately every 10-60 seconds.

[0317] 59. The method according to implementation plan 58, wherein the settling of recombinant viral particles is monitored at approximately every 10 seconds.

[0318] 60. The method according to implementation plan 58, wherein the settling of recombinant viral particles is monitored at approximately every 60 seconds.

[0319] 61. The method according to any one of embodiments 1-60, wherein the boundary settlement rate is from about 3,000 rpm to about 20,000 rpm.

[0320] 62. The method according to embodiment 61, wherein the boundary settlement rate is from about 3,000 rpm to about 10,000 rpm.

[0321] 63. The method according to embodiment 61, wherein the boundary settlement rate is from about 10,000 rpm to about 20,000 rpm.

[0322] 64. The method according to embodiment 61, wherein the boundary settlement rate is from about 15,000 rpm to about 20,000 rpm.

[0323] 65. The method according to any one of embodiments 1-64, wherein the boundary settlement rate is carried out at about 4°C to about 20°C.

[0324] 66. The method according to embodiment 65, wherein the boundary settlement velocity is carried out at approximately 4°C.

[0325] 67. The method according to any one of embodiments 1-66, wherein the recombinant viral particle is a recombinant adeno-associated virus (AAV) particle, a recombinant adenovirus particle, a recombinant lentivirus particle, or a recombinant herpes simplex virus (HSV) particle.

[0326] 68. A method for evaluating the production of recombinant viral particles, comprising the method according to any one of embodiments 1-67, wherein an increase in the relative amount of recombinant viral particles containing a complete viral genome compared to a reference preparation of recombinant viral particles indicates an improvement in the yield of recombinant viral particles.

[0327] 69. The method of implementation scheme 68, wherein the viral particles are rAAV particles.

[0328] 70. The method of embodiment 69, wherein the rAAV particles are generated from a producer cell line.

[0329] 71. The method according to embodiment 69, wherein the rAAV particles are generated by triple transfection of: i) nucleic acids encoding AAV rep and cap, ii) rAAV vector sequence, and iii) nucleic acids encoding adenovirus helper functions.

[0330] 72. The method according to embodiment 69, wherein the recombinant viral particles are generated by AAV / HSV hybridization.

[0331] 73. The method according to embodiment 69, wherein the recombinant virus particles are generated from insect cells.

[0332] 74. The method according to embodiment 69, wherein the recombinant viral particles are generated by transiently transfecting a nucleic acid encoding an AAV vector sequence, AAVrep and cap coding regions, and AAV helper viral functions into a suitable host cell.

[0333] 75. The method according to embodiment 69, wherein the recombinant viral particles are generated by introducing one or more nucleic acids encoding an AAV vector sequence, an AAV rep and cap coding region, and an AAV helper virus function into a suitable host cell, wherein the one or more nucleic acids are introduced into the cell using a recombinant helper virus.

[0334] 76. The method according to embodiment 75, wherein the recombinant helper virus is an adenovirus, herpes simplex virus, or baculovirus.

[0335] 77. The method according to embodiment 68, wherein the recombinant viral particles are generated by transiently transfecting nucleic acids encoding an adenovirus vector sequence and an adenovirus replication and packaging sequence into a suitable host cell.

[0336] 78. The method according to embodiment 68, wherein the recombinant viral particles are generated by transiently transfecting nucleic acids encoding lentiviral vector sequences and / or lentiviral replication and packaging sequences into suitable host cells.

[0337] 79. The method according to embodiment 68, wherein the recombinant viral particles are generated by transiently transfecting nucleic acids encoding HSV vector sequences and / or HSV replication and packaging sequences into suitable host cells.

[0338] 80. A method for preparing recombinant viral particles having reduced empty capsids and / or recombinant viral particles containing different genomes, the method comprising:

[0339] a) Culture of host cells under conditions suitable for recombinant virus production, wherein the cells comprise:

[0340] i) A nucleic acid encoding a heterologous transgene, flanked by at least one AAV ITR.

[0341] ii) Nucleic acids containing AAV rep and cap coding regions, wherein said nucleic acids contain a mutated p5 promoter, wherein rep expression from said p5 promoter is reduced compared to the wild-type p5 promoter, and

[0342] iii) Nucleic acids encoding AAV helper viral functions;

[0343] b) Lysing host cells to release recombinant viral particles;

[0344] c) Isolate recombinant viral particles produced by host cells; and

[0345] d) Analyze the presence of empty capsids and / or recombinant viral particles with different genomes in recombinant viral particles by analytical ultracentrifugation according to any one of the methods in Implementation Scheme 1-79.

[0346] 81. The method according to embodiment 80, wherein the p5 promoter is located at 3' of the rep and / or cap coding region.

[0347] 82. The method according to embodiment 80 or 81, wherein the AAV auxiliary virus function includes adenovirus E1A function, adenovirus E1B function, adenovirus E2A function, adenovirus VA function and adenovirus E4 orf6 function.

[0348] 83. The method according to any of the foregoing embodiments, wherein the recombinant viral particles have been purified by one or more purification steps.

[0349] 84. The method according to embodiments 67, 69-76, 80-83, wherein the recombinant viral particle contains a self-complementary AAV (scAAV) genome.

[0350] 85. The method according to embodiment 84, wherein the method is used to detect the presence of recombinant viral particles comprising either a monomeric scAAV genome or a dimerized scAAV genome.

[0351] 86. The method according to any one of embodiments 67, 69-76, 80-85, wherein the recombinant viral 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 AAVV708K capsid, a goat AAV capsid, an AAV1 / AAV2 chimeric capsid, a bovine AAV capsid, or a mouse AAV capsid rAAV2 / HBoV1 (a chimeric AAV / human bocavirus 1).

[0352] 87. The method according to any one of embodiments 67, 69-76, 80-86, wherein the recombinant viral particles comprise 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 DJ ITR, goat AAV ITR, bovine AAV ITR, or mouse AAV ITR.

[0353] 88. The method according to embodiment 86 or 87, wherein the AAV capsid contains a tyrosine mutation or a heparin-binding mutation.

[0354] 89. The method according to embodiment 67 or 68, wherein the recombinant viral particle is a recombinant adenovirus particle.

[0355] 90. The method according to embodiment 89, wherein the recombinant adenovirus particle comprises a capsid from adenovirus serotypes 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad 3, canine Ad 2, sheep Ad, or swine Ad 3.

[0356] 91. The method according to embodiment 90, wherein the recombinant adenovirus particle comprises a variant of adenovirus serotype 2 capsid or a variant of adenovirus serotype 5 capsid.

[0357] 92. The method according to embodiment 67 or 68, wherein the recombinant viral particle is a recombinant lentiviral particle.

[0358] 93. The method according to embodiment 92, wherein the recombinant lentiviral particle is pseudovesicular stomatitis virus (VSV), lymphocytic choriomeningovirus (LCMV), Ross river virus (RRV), Ebola virus, Marburg virus, Mokala virus, rabies virus, RD114 or a variant thereof.

[0359] 94. The method according to embodiment 67 or 68, wherein the recombinant viral particles are rHSV particles.

[0360] 95. The method according to embodiment 94, wherein the HSV particles are HSV-1 particles or HSV-2 particles.

Claims

1. A method for characterizing recombinant virus particle preparations, comprising the following steps: a) The preparation was subjected to analytical ultracentrifugation under boundary settling velocity conditions, wherein the settling of recombinant virus particles was monitored at time intervals. b) Plot the settlement coefficient in Svedberg units (S) against the differential settlement coefficient distribution values ​​(C(s)). c) Integrate the area under each peak in the C(s) distribution to determine the relative concentration of each peak, where each peak represents a recombinant viral particle.

2. A method for assessing the vector genome integrity of recombinant viral particles in a recombinant viral particle preparation, comprising: a) The preparation was subjected to analytical ultracentrifugation under boundary settling velocity conditions, wherein the settling of recombinant virus particles was monitored at time intervals. b) Plot the settlement coefficient in Svedberg units (S) against the differential settlement coefficient distribution values ​​(C(s)). c) The species of recombinant viral particles in the preparation are identified by the peaks corresponding to the S values ​​present in the figure, wherein the genome size of the specific species of recombinant viral particles is calculated by comparing the S value of the species with a standard curve generated by the S value of recombinant viral particles containing a known nucleotide-sized viral genome with a capsid coating.

3. The method of claim 2, further comprising integrating the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species.

4. A method for determining the presence of empty capsids or capsid particles containing recombinant viral genomes of different sizes in a recombinant viral particle preparation, comprising the following steps: a) The preparation was subjected to analytical ultracentrifugation under boundary sedimentation velocity conditions, wherein the sedimentation of recombinant virus particles was monitored at time intervals, and b) Plot the sedimentation coefficient in Svedberg units (S) with differential sedimentation coefficient distribution values ​​(C(s)), where the presence of one or more peaks other than the peak of a fully capsid particle containing a complete recombinant viral genome indicates the presence of capsid particles containing genomes of different sizes and / or empty capsids.

5. A method for measuring the relative amount of empty capsid in a recombinant virus particle preparation, comprising the following steps: a) The preparation was subjected to analytical ultracentrifugation under boundary settling velocity conditions, wherein the settling of recombinant virus particles was monitored at time intervals. b) Plot the settlement coefficient in Svedberg units (S) against the differential settlement coefficient distribution values ​​(C(s)). c) Integrate the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species, and d) Compare the amount of recombinant viral particles with an S value corresponding to empty capsid particles with the amount of recombinant viral particles with an S value corresponding to recombinant viral particles containing a complete viral genome or the total amount of recombinant viral particles in the preparation.

6. A method for measuring the relative amount of capsid particles or empty viral capsid particles containing different recombinant viral genomes in a recombinant viral particle preparation, comprising the following steps: a) The preparation was subjected to analytical ultracentrifugation under boundary settling velocity conditions, wherein the settling of recombinant virus particles was monitored at time intervals. b) Plot the settlement coefficient in Svedberg units (S) against the differential settlement coefficient distribution values ​​(C(s)). c) Integrate the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species. d) Compare the amount of recombinant viral particles with an S value that does not correspond to a recombinant viral particle containing a complete viral genome with the amount of recombinant viral particles with an S value that corresponds to a recombinant viral particle containing a complete viral genome or the total amount of recombinant viral particles in the preparation.

7. A method for measuring the relative amount of capsid particles containing multiple recombinant viral genomes in a recombinant viral particle preparation, comprising: a) The preparation was subjected to analytical ultracentrifugation under boundary settling velocity conditions, wherein the settling of recombinant virus particles was monitored at time intervals. b) Plot the settlement coefficient in Svedberg units (S) against the differential settlement coefficient distribution values ​​(C(s)). c) Integrate the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species. d) Compare the amount of recombinant viral particles with an S value that does not correspond to a recombinant viral particle containing a complete viral genome or an empty capsid with the total amount of recombinant viral particles in the preparation.

8. A method for measuring the relative amount of recombinant viral particles containing a complete viral genome in a recombinant viral particle preparation, comprising: a) The preparation was subjected to analytical ultracentrifugation under boundary settling velocity conditions, wherein the settling of recombinant virus particles was monitored at time intervals. b) Plot the settlement coefficient in Svedberg units (S) against the differential settlement coefficient distribution values ​​(C(s)). c) Integrate the area under each peak in the C(s) distribution to determine the relative concentration of each recombinant viral particle species. d) Compare the amount of recombinant viral particles having an S value corresponding to a recombinant viral particle containing a complete viral genome with the amount of recombinant viral particles having an S value corresponding to an empty capsid particle, with capsid particles containing different recombinant viral genomes, and / or with the total amount of recombinant viral particles in the preparation.

9. A method for monitoring the removal of empty capsids and / or capsid particles containing different recombinant viral genomes during the purification process of a recombinant viral particle preparation, the method comprising removing a sample of recombinant viral particles from the preparation after one or more steps of the purification process, and analyzing the relative amount of empty capsids and / or capsid particles containing different recombinant viral genomes in the sample according to the method of any one of claims 5-8, wherein a decrease in the relative amount of empty capsids and / or capsid particles containing different genomes compared to a complete capsid indicates the removal of empty capsids from the recombinant viral particle preparation.

10. The method according to any one of claims 4-9, wherein the presence of the peak corresponding to the S value of the empty capsid particle indicates the presence of the empty capsid particle.

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