deamidation-depleted adeno-associated viral products

Abstract

An adeno-associated virus (AAV) product is provided that contains less nonfunctional capsids, such as deamidated empty AAV capsids, deamidated AAV capsids containing partial transgenes, and deamidated full capsids, compared to currently used AAV products. The AAV product contains a higher ratio of functional capsids to nonfunctional capsids (deamidated hydrophobic capsids) compared to a control AAV product purified by ultracentrifugation alone, by anion exchange chromatography (AEX) without the use of a fatty acid mobile phase. Methods of making and using the AAV product are also provided, including separating functional capsids from nonfunctional capsids using anion exchange chromatography (using a fatty acid mobile phase), HIC, or chromatography using a mixed mode resin. Such methods also include maintaining transfected cells in a bioreactor for about two days (optionally at about 30-38°C) post-transfection, followed by separating functional capsids from nonfunctional capsids.

Description

Background Technology

[0001] Adeno-associated virus (AAV) vectors are commonly used to deliver nucleic acids into cells. The success of AAV-mediated gene substitution, gene silencing, and gene editing has made AAV an ideal therapeutic vector, and AAV-based therapies have received regulatory approval in Europe and the United States. However, existing AAV purification systems are often unable to remove impurities from AAV products and frequently fail to produce sufficient quantities of vector genomes for robust gene therapy applications. Therefore, there is a need for improved AAV products and AAV product purification systems. Summary of the Invention

[0002] This disclosure relates to an AAV product containing fewer non-functional capsids, such as deamided empty capsids, deamided capsids containing only partial transgenes, and deamided intact capsids, compared to AAV products used concurrently. The AAV product of the present invention contains fewer deamided hydrophobic capsids, which represent non-functional capsids. Because the improved AAV product has fewer non-functional capsids, it results in better transgene expression at a given capsid concentration. Furthermore, the improved product leads to lower immunoreactivity in subjects administered the AAV product because the amount of capsid protein in a single effective dose is reduced by decreasing the number of empty capsids or capsids containing only partial transgenes.

[0003] Therefore, this document provides an AAV product comprising a functional capsid containing a genetically modified organism (GMO) and a less deamidated AAV capsid compared to a control AAV product. For example, in some cases, the AAV product comprises: (a) a functional capsid containing a GMO; and (b) a less deamidated capsid compared to a control AAV product purified solely by ultracentrifugation or by AEX without a fatty acid mobile phase. In some embodiments, the AAV product comprises less than 10%, less than 5%, or less than 1% of a capsid having deamidated N57 on VP1. In some embodiments, the AAV product comprises a less N57 deamidated capsid compared to a control AAV product purified solely by AEX without a fatty acid mobile phase.

[0004] Also provided is an adeno-associated virus (AAV) product comprising a functional capsid containing functional transgenes, and wherein the product is less hydrophobic compared to a control AAV product purified solely by ultracentrifugation or by AEX without a fatty acid mobile phase. This adeno-associated virus (AAV) product contains more functional capsid than the control AAV product purified solely by ultracentrifugation or by AEX without a fatty acid mobile phase.

[0005] In some embodiments, the AAV product contains less than 10% nonfunctional capsid. Given the reduction in nonfunctional capsid, the AAV product has a higher ratio of functional to nonfunctional capsid, and a given concentration of capsid exhibits higher transgenic expression in host cells than a control AAV product of the same concentration purified by AEX using only ultracentrifugation or without a fatty acid mobile phase. Therefore, an AAV product comprising a transgenic AAV capsid is also provided, wherein a certain concentration of AAV capsid exhibits higher transgenic expression in host cells than a control AAV capsid of the same concentration purified by AEX using only ultracentrifugation or without a fatty acid mobile phase.

[0006] In some embodiments, the AAV product has a lower VP1 to VP2 ratio than a control AAV product purified by AEX alone or without a fatty acid mobile phase. Therefore, this document provides an AAV product comprising a transgenic AAV capsid, wherein the AAV product has a lower VP1 to VP2 ratio than a control AAV product purified by AEX alone or without a fatty acid mobile phase.

[0007] An AAV product is also provided, which essentially consists of a functional capsid containing a functional transgene and a buffer solution. In some embodiments, the functional capsid is separated from an AAV capsid group containing both the functional capsid and a deamide capsid to produce the AAV product. In some embodiments, the AAV product is relatively free of capsids in which VP1 has a deamide N57.

[0008] A method for removing at least one nonfunctional capsid subpopulation from an adeno-associated virus (AAV) product is also provided, comprising separating the transgenic functional capsid from the nonfunctional capsid subpopulation based on hydrophobicity. An AAV product produced by any of the methods described herein is also provided.

[0009] In some methods, the separation of the functional capping from the non-functional capping is further based on charge. In some methods, the removed non-functional capping comprises a deamide capping. In some methods, the removed deamide capping comprises VP1 having deamide N57.

[0010] Optionally, the separation of functional capsids containing functional genetically modified organisms from non-functional capsid subgroups includes a hydrophobic interaction chromatography (HIC) step, an AEX step using a fatty acid mobile phase, or a mixed-mode AEX step. When using a HIC step, an AEX step can also be performed (e.g., without using a fatty acid mobile phase). In this case, the HIC step is optionally performed prior to the AEX step. When the separation step includes an AEX step using a fatty acid mobile phase, the fatty acid mobile phase optionally contains octanoic acid (also known as octanoate, caprylate, or caprylic acid) or heptanoic acid (also known as heptanoate and enanthic acid). When the separation includes a mixed-mode resin chromatography step, the mixed-mode resin is optionally a hydrophobic anion exchange resin.

[0011] A method for reducing the number of nonfunctional capsids is also provided, which involves maintaining the cells in a bioreactor for approximately two days after transfection, optionally while maintaining the temperature in the reactor at approximately 30-38°C, and using AEX (with or without a fatty acid mobile phase and with or without a mixed-mode resin) or HIC to separate functional capsids containing functional transgenes from nonfunctional capsid subpopulations.

[0012] A method for treating an object that requires a protein produced by a genetically modified organism is also provided, comprising administering any of the AAV products described herein to the object. Attached Figure Description

[0013] The accompanying drawings described below are intended to illustrate certain embodiments and / or features of the compositions and methods, and to supplement any description of the compositions and methods. These drawings do not limit the scope of the compositions and methods unless the written description expressly indicates otherwise.

[0014] Figure 1A This is a schematic diagram showing an empty capsid, a partial capsid, and a complete capsid containing intact target nucleic acids within it. Theoretically, the charge on the capsid increases with the presence and quantity of nucleic acids within it. Figure 1B This is an example of an AEX linear gradient, showing a leading empty capsid, followed by a product containing a functional transgene, and a tailed non-product peak consisting of a non-functional capsid (i.e., a deamidated capsid).

[0015] Figure 2A The deamidation of VP1 showed that it conferred a negative charge, and the N-terminus of VP1 was hydrophobic in AAV serotypes. N57 deamidation of VP1 led to loss of titer and decreased transgene expression, presumably caused by VP1 extrusion from the capsid surface. Figure 2B The percentage of deamidation of residues N329, N57, Q456, and N452 of VP1 is shown for 0–4 weeks at 25°C (top) and 0–10 days at 40°C (bottom). The relative potency of the AAV product is also shown in each figure, which decreases over time with increasing deamidation.

[0016] Figure 3 The chromatograms of anion exchange (AEX) chromatography are shown, comparing the chromatographic performance of the control Poros HQ50 resin (ThermoScientific, Waltham, MA) and Nuvia aPrime 4A resin in enriching DNA-containing capsids. AEX separated the capsid into two distinct peaks (separated by a vertical black dashed line): a product peak from Library 1, primarily containing intact capsids (left), and a non-product peak from Library 2, primarily containing empty capsids (right). Compared to the control Poros HQ50 resin, Nuvia aPrime 4A resin provided better chromatographic separation between Library 1 and Library 2.

[0017] Figure 4 This study demonstrates the effect on chromatographic performance when the mobile phase of the POROS HQ50 AEX chromatogram was modified to incorporate fatty acid heptate esters. The mobile phases tested included: AEX chromatography without a fatty acid mobile phase; AEX chromatographic mobile phase containing sodium heptate and ammonium acetate; and AEX chromatographic mobile phase containing sodium heptate and glycine. Compared to the control, the AEX chromatographic mobile phase containing heptate esters increased the chromatographic resolution between Library 1 and Library 2.

[0018] Figure 5 This is a representative chromatogram from a hydrophobic interaction chromatography (HIC) run, as described in the examples.

[0019] Figure 6 This is a representative chromatogram, showing... Figure 5 The HIC is run with gradient elution.

[0020] Figure 7 This is a graph showing vector genome (VG) and capsid yield data normalized to mass equilibrium based on affinity (AF) product titers. VG and capsid titers in the flow-through and wash (FTW) fractions are below the limits of quantification and are not shown here. HIC library 1 contained the majority of the loaded material, with approximately 76% capsid and 76% VG yields. The VG to capsid titer ratio for HIC library 1 was 19%, and for HIC library 2 it was 22%, indicating that this step did not enrich DNA-containing capsids.

[0021] Figure 8 The ultracentrifugation spectra of HIC Library 1 (top) and HIC Library 2 (bottom) are shown.

[0022] Figure 9 The chromatogram is obtained by AEX purification of HIC library 1 using buffer exchange.

[0023] Figure 10 The chromatogram is obtained by AEX purification of HIC library 2 using buffer exchange.

[0024] Figure 11 It is two runs of AEX ( Figure 9 and Figure 10 The chromatograms are superimposed.

[0025] Figure 12 A representative AEX chromatogram of gradient elution using affinity (AF) product material is shown.

[0026] Figure 13 VG and capsid yield data for the AEX chromatographic step using BE HIC Library 1 are shown. Yields were calculated based on titers from the BE HIC library (due to the variability of AEX loading titers) and normalized to mass equilibrium.

[0027] Figure 14 VG and capsid yield data for the AEX chromatographic step using BE HIC Library 2 are shown. Yields were calculated based on titers from the BE HIC library (due to the variability of AEX loading titers) and normalized to mass equilibrium.

[0028] Figure 15 The AUC spectra of fractions from AEX Library 1 (top) and AEX Library 2 (bottom) using the AEX chromatographic steps of BE HIC Library 1 are shown.

[0029] Figure 16 This is a bar graph showing the percentage of amino acid deamidation abundance at various residues in VP1 for each chromatographic peak corresponding to HIC chromatography (S1: product peak of library 1 and S2: non-product peak of library 2), AEX chromatography of BE HIC library 1 (S3: product peak 1 and S4: non-product peak 2), and AEX chromatography of BE HIC library 2 (S5: non-product peak 2). Compared to other examined amino acid residues, peaks S4 and S5 (HIC library 1-AEX library 2 and HIC library 2-AEX peak 2) showed the highest deamidation abundance at asparagine 57 (N57). Peaks S4 and S5 showed the latest elution times, indicating that the species containing these peaks (mainly empty capsids) have a high net negative charge. These data are derived from the above analysis of... Figure 5-15 The HIC and AEX operations are described.

[0030] Figure 17A and Figure 17B It is a bar graph showing VG ( from AEX chromatography) Figure 17A ) and capsid ( Figure 17BThe adjusted yield was calculated as a percentage of the total yield, with yield data normalized to quality balance. The adjusted yield was measured at AAV harvest on days 2 and 3 (i.e., days post-transfection), with transfection conditions of 37°C or 39°C. Figure 17A In both temperatures, on day 3 compared to day 2, VG ​​titers showed a slight increase in loss to strip, indicating a stronger negative charge on the feed stream. Figure 17B In the study, the capillary titration showed that the FTW loss decreased on day 3 compared to day 2, while the capillary peeling loss increased on day 3 compared to day 2, indicating that the feed stream was more negatively charged.

[0031] Figure 18 The AUC percentage bar charts after AEX chromatography on days 2 and 3 following AAV harvest at 37°C or 39°C are shown. From left to right, the percentage of empty product capsids is shown in medium blue, partially filled product capsids in light blue, and complete product capsids in dark blue. Empty capsids from stripped non-product peaks are shown in orange, partially filled capsids from stripped non-product peaks are shown in green, and complete capsids from stripped non-product peaks are shown in red. On day 3, a higher percentage of empty capsids was observed in the product peaks compared to day 2, and a higher percentage of complete capsids was observed in the non-product peaks from stripping, further supporting a stronger electronegativity in the feed stream. Optimal AAV packaging was observed on day 2 at 39°C.

[0032] Figure 19A The bar graph shows the percentage of relative potency in the product peaks after AEX chromatography. A decrease in potency was observed from day 2 to day 3 at both temperatures, indicating that the capsids harvested at 37°C on day 2 had the highest potency. Figure 19B A bar graph showing the percentage of deamidation at N57 is also presented. For both the affinity (AF) and AEX product peaks, an increase in N57 deamidation was observed from day 2 to day 3 at both temperatures. Notably, the lowest relative potency percentage and the highest N57 deamidation percentage were observed at 39°C on day 3.

[0033] Figure 20A and Figure 20B The bar graph shows the VG and capsid productivity harvested on day 2 or day 3 at different temperatures. For both VG and capsid, an increase in upstream productivity was observed from day 2 to day 3 at both 37°C and 39°C. However, it is also noted that the total VG and capsid recovered throughout the process subsequently decreased from day 2 to day 3 at both temperatures. In summary, these data suggest that 37°C on day 2 is the optimal condition for VG and capsid productivity throughout the process. Detailed Implementation

[0034] The following description illustrates various aspects and embodiments of the compositions and methods disclosed herein. No particular embodiment is intended to limit the scope of the compositions and methods. Rather, these embodiments merely provide non-limiting examples of various compositions and methods that are at least within the scope of the compositions and methods disclosed herein. This description is intended to be read by one of ordinary skill in the art; therefore, information well known to those skilled in the art is not necessarily included.

[0035] AAV products

[0036] Typically, AAV products are generated in cell cultures, such as in bioreactors, and then purified from the cell cultures by eliminating at least a portion of impurities (e.g., removal of host cell proteins, culture medium components, and / or free nucleic acids from the cell cultures) and empty capsids (i.e., capsids lacking transgenes, including empty capsids). As disclosed herein, the generation of nonfunctional capsids can be reduced by limiting the time cells remain in the bioreactor post-transfection before separating and purifying the AAV product. For example, fewer nonfunctional capsids are present in the unseparated capsid population when cells remain in the bioreactor for two days or less.

[0037] Purification further provides the opportunity to eliminate nonfunctional capsids. Purification typically includes purification steps (e.g., using differential centrifugation, density centrifugation, and / or filtration) for removing cells and cell debris, and one or more downstream chromatographic steps to separate AAV products from various impurities from a clarified cell culture feed. However, until this invention, it was possible to consistently remove nonfunctional capsids (i.e., deamidated empty capsids, deamidated partially capsids, or deamidated intact capsids) to less than about 10% of the total capsids simply by using ultracentrifugation or AEX (without a fatty acid mobile phase) methods. This problem hindered the production of commercial quantities of AAV products with sufficient purity and potency using high-throughput systems. Solving this problem provides AAV products that exhibit higher transgenic expression capacity (i.e., potency) due to a higher ratio of functional to nonfunctional capsids, and lower immunoreactivity at effective doses due to a reduced number of nonfunctional capsids. To obtain AAV products of this purity, the larger negative charge of intact capsids and the smaller negative charge of nonfunctional capsids are utilized. See also Figure 1A and 1BIt is noteworthy that negative charge is correlated with hydrophobicity. The greater the negative charge, the stronger the hydrophobicity. Therefore, AEX conditions can be manipulated to remove empty capsids before the functional capsid main peak; however, unless the elution conditions are modified (e.g., to increase hydrophobicity) to allow the non-product peak to adhere to the resin, a tailed "non-product" peak will be produced. The tailed "non-product" peak mainly consists of deamidated empty capsids, followed by deamidated capsids containing partial transgenes and intact deamidated capsids. Compared to the product peak containing functional transgenes, the non-product peak also contains more than 50% deamidated VP1. Deamidation imparts a negative charge to the N-terminus of VP1, which is highly hydrophobic, leading to loss of titer and decreased transgene expression. See also Figure 2A and 2B Baseline separation between product and non-product peaks cannot be achieved using conventional AEX conditions. Conditions that promote retention of non-functional capsids result in the elution of AAV products with less than 10% non-functional capsids.

[0038] Therefore, this document provides an AAV product comprising a functional capsid containing a transgene and a reduced amount of deamided AAV capsid compared to a control AAV product. The AAV product comprises: a functional capsid containing a transgene that can be expressed in host cells; and a reduced amount of deamided AAV capsid compared to a control AAV product purified solely by ultracentrifugation or by AEX without a fatty acid mobile phase. In some embodiments, the amount of deamided AAV capsid is reduced by at least 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% compared to the AAV product control. As used throughout, the control AAV product comprises an AAV capsid of the same serotype and containing the same transgene as the AAV product described herein, but purified by a different method (e.g., ultracentrifugation or AEX without a fatty acid mobile phase).

[0039] This article also provides an AAV product containing a functional capsid containing a transgene that can be expressed in host cells, and wherein the product is less hydrophobic compared to a control AAV product purified solely by ultracentrifugation or by AEX without using a fatty acid mobile phase.

[0040] As used herein, a functional capsid is a complete or nearly complete AAV capsid containing a transgene, wherein the complete or nearly complete AAV capsid expresses the functional gene product encoded by the transgene in a host cell. A functional capsid is also called a functional recombinant AAV (rAAV) virion or a functional rAAV particle. As used herein, a complete capsid contains the full length of the desired genetic material (i.e., the transgene); a nearly complete capsid contains genetic material sufficient to express the functional protein encoded by the transgene once introduced into a host cell; a partially complete capsid contains a portion of the transgene, but that portion is insufficient to express the transgene in a host cell; an empty capsid contains no or relatively no functional genetic material, i.e., the transgene. As used herein, the term nonfunctional capsid refers to a deamidated capsid that cannot express the transgene in a host cell. As used herein, complete and nearly complete capsids that are amidated at the N57 position of VP1 and capable of expressing the transgene in a host cell are collectively referred to as “functional” capsids.

[0041] As used throughout, an AAV product is a composition comprising a plurality of AAV caps. In some embodiments, the AAV product comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% functional caps. In some embodiments, the AAV product comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% intact or nearly intact caps. In some embodiments, the AAV product comprises less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% non-functional caps.

[0042] In the AAV products and methods described herein, the nonfunctional capsid is more hydrophobic than the functional capsid. This hydrophobicity in the nonfunctional capsid is at least partially attributable to deamidation. For example, this deamidation occurs at the N57 position of VP1. Without intending to be theoretically limited, misconfigured capsids may have more N57 of VP1 extruded or exposed from the surface of the nonfunctional capsid, and this exposure may allow for deamidation. In some embodiments, the AAV products provided herein are relatively free of deamidated N57. For example, the AAV product may contain less than 10% deamidated N57 on the VP1 of the capsid, a level lower than that achievable solely by ultracentrifugation or AEX (without using a fatty acid mobile phase). An exemplary VP1 capsid sequence is shown under GenBank accession number QEU45513.1. See also Figure 2A The VP1 sequences of various AAV serotypes are shown.

[0043] In some embodiments, the AAV product has a lower VP1:VP2 ratio than a control AAV product purified solely by ultracentrifugation or AEX (without using a fatty acid mobile phase). For example, when the hydrophobic capsid (e.g., those with deamidated VP1) is removed, the 1:1 VP1:VP2 ratio in the AAV product prepared according to methods known in the art will become smaller (i.e., <1:1 VP1:VP2).

[0044] The amount of deamidation in any product described herein can be detected by methods known in the art, such as liquid chromatography or mass spectrometry.

[0045] Alternatively, the AAV product may consist primarily of a functional capsid containing genetic material and a buffer solution. As used herein, "consistent with or substantially composed of a functional capsid" means that the AAV product contains less than 10% empty capsids.

[0046] In some embodiments, the AAV product exhibits higher transgene expression in host cells than a control AAV product purified solely by ultracentrifugation or by AEX without a fatty acid mobile phase. Any AAV product disclosed herein can be introduced into cells (e.g., using any techniques known in the art) to express proteins encoded by the transgene in the AAV product. Therefore, this document provides a recombinant cell and a recombinant cell population comprising the AAV product disclosed herein.

[0047] In some AAV products, the transgene can be a DNA or RNA sequence encoding the therapeutic product. Upon introduction into a host cell, the DNA sequence can be transcribed into a target RNA molecule. Alternatively, the transgene can be a target RNA molecule. Target RNA molecules include, but are not limited to, miRNAs, shRNAs, siRNAs, antisense RNAs, gRNAs, antagomirs, miRNA sponges, RNA aptamers, RNA aptamers, mRNAs, lncRNAs, ribozymes, and synthetic RNAs known in the art.

[0048] In some embodiments, the transgene encodes one or more polypeptides or functional fragments thereof. Such transgenes may contain the complete coding sequence of the polypeptide or only a fragment of the coding sequence of the polypeptide. In some embodiments, the transgene encodes a polypeptide that can be used to treat a disease or condition of the subject. Suitable polypeptides include, but are not limited to: β-globin; hemoglobin; tissue plasminogen activator; coagulation factors; colony-stimulating factor (CSF); interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, etc.; growth factors, such as keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), bone morphogenetic protein (BMP), epidermal growth factor (EGF), growth differentiation factor- GDF-9, hepatocellular carcinoma-derived growth factor (HDGF), myostatin-8, nerve growth factor (NGF), neurotrophic factor, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor α (TGF-a), transforming growth factor β (TGF-β), etc.; soluble receptors, such as soluble TNF-α receptors, soluble interleukin receptors (e.g., soluble IL-1 receptors and soluble type II IL-1 receptors), soluble γ / ΔT cell receptors, soluble receptor ligand-binding fragments, etc.; enzymes, such as α-glucosidase, imiglucerase. Enzymes include: β-glucocerebrosidase and alglucerase; enzyme activators, such as tissue plasminogen activator; chemokines, such as IP-10, interferon-γ (Mig)-induced mononuclear factor, Groα / IL-8, RANTES, MIP-1α, MIP-1β, MCP-1, PF-4, etc.; angiogenic agents, such as vascular endothelial growth factor (VEGF, such as VEGF121, VEGF165, VEGF-C, VEGF-2), glioma-derived growth factors, angiopoietin, angiopoietin-2, etc.; and anti-angiogenic agents, such as soluble VEGF receptors. Body; protein vaccines; neuroactive peptides, such as nerve growth factor (NGF), bradykinin, cholecystokinin, gastrin, secretin, oxytocin, gonadotropin-releasing hormone, β-endorphin, enkephalin, substance P, somatostatin, prolactin, lycopene, somatostatin-releasing hormone, bufotoxin, dynorphin, warfarin, neurotensin, motilin, thyroid-stimulating hormone, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, sleep peptide, etc.; thrombolytic agents; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle-stimulating hormone (FSH);Human α-1 antitrypsin; Leukemia inhibitory factor (LIF); Tissue factor; Macrophage activating factor; Tumor necrosis factor (TNF); Neutrophil chemotactic factor; Tissue metalloproteinase inhibitor; Vasoactive intestinal peptide; Angiopoietin; Angiotropin; Fibrin; Hirudin; IL-1 receptor antagonist; Ciliary neurotrophic factor (CNTF); Brain-derived neurotrophic factor (BDNF); Neurotrophic factors 3 and 4 / 5 (NT-3 and 4 / 5); Glial cell-derived neurotrophic factor (GDNF); Aromatic amino acid decarboxylation Enzymes (AADC); Factor VIII, Factor IX, Factor X; dystrophin or mini dystrophin; lysosomal acid lipase; phenylalanine hydroxylase (PAH); glycogen storage disease-associated enzymes, such as glucose-6-phosphatase, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase, glucose transporter, aldolase A, β-enolase, glycogen synthase; lysosomal enzymes, such as iduronate-2-sulfatase (I2S) and arylsulfatase A; and mitochondrial proteins, such as coagulants.

[0049] In some embodiments, the transgene encodes a protein that is defective in subjects suffering from one or more lysosomal storage diseases. Suitable proteins include, but are not limited to, α-sialidase, cathepsin A, α-mannosidase, β-mannosidase, glycosylasparaginase, α-fucosidase, α-N-acetylglucosamine glycosidase, β-galactosidase, β-hexosamine α-subunit, β-hexosamine β-subunit, GM2 activator protein, glucocerebrosidase, sphingolipid activator protein C, arylsulfatase A, sphingolipid activator protein B, formicylglycine synthase, β-galactosylceramide enzyme, α-galactosidase A, iduronate sulfatase, α-iduronate, heparin N-sulfatase, and acetyl-CoA transaminase. Phosphotransferase, N-acetylglucosidase, β-glucuronidase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, galactose 6-sulfatase, hyaluronidase, α-glucosidase, acid sphingomyelinase, acid ceramide enzyme, acid lipase, cathepsin K, tripeptidyl peptidase, palmitoyl protein thioesterase, cystinosin, sialin, UDP-N-acetylglucosamine, phosphotransferase γ-subunit, mucoprotein-1, LAMP-2, NPC1, CLN3, CLN6, CLN8, LYST, MYOV, RAB27A, melanophilin, and AP3 β-subunit.

[0050] In some embodiments, the transgene encodes an antibody or a fragment thereof (e.g., Fab, scFv, or a full-length antibody). Suitable antibodies include, but are not limited to, muromonab-cd3, efalizumab, tositumomab, daclizumab, nebacumab, catumaxomab, edrecolomab, abciximab, rituximab, basiliximab, palivizumab, infliximab, and trastuzumab. Trastuzumab, adalimumab, ibritumomabtiuxetan, omalizumab, cetuximab, bevacizumab, natalizumab, panitumumab, ranibizumab, eculizumab, certolizumab, ustekinumab, canakinumab mab), golimumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, brentuximab vedotin, pertuzumab, raxibacumab, obinutuzumab, alemtuzumab, siltuximab, ramucirumab The following are listed: ucirumab, vedolizumab, blinatumomab, nivolumab, pembrolizumab, idarucizumab, necitumumab, dinutuximab, secukinumab, mepolizumab, alirocumab, evolocumab, and daratumumab.Elotuzumab, ixekizumab, reslizumab, olaratumab, bezlotoxumab, atezolizumab, obiltoxaximab, inotuzumabozogamicin, brodalumab, guselkumab, dupilumab, sarilumab, avelumab, and others. Cleurotus ostreatus (ocrelizumab), emicizumab, benazezumab, gemtuzumabozogamicin, durvalumab, burosumab, erenumab, galcanezumab, lanadelumab, mogamulizumab, tildrakizumab, cimiplimab, remannetumab (fr The following are listed: emanezumab, ravulizumab, emapalumab, ibalizumab, moxetumomab, caplacizumab, romosozumab, risankizumab, polotuzumab, eptinezumab, leronlimab, sacituzumab, brolucizumab, isatuximab, and teprotumumab.

[0051] In some implementations, the transgene encodes a nuclease. Suitable nucleases include, but are not limited to, zinc finger nucleases (ZFNs) (see, for example, Porteus and Baltimore (2003) Science 300: 763; Miller et al. (2007) Nat. Biotechnol. 25:778-785; Sander et al. (2011) Nature Methods 8:67-69; and Wood et al. (2011) Science 333:307, each of which is incorporated herein by reference in its entirety), transcription activator-like effector nucleases (TALENs) (see, for example, Wood et al. (2011) Science 333:307; Boch et al. (2009) Science 326:1509-1512; Moscou and Bogdanove (2009) Science326:1501; Christian et al. (2010) Genetics 186:757-761; Miller et al. (2011) Nat. Biotechnol. 29:143-148; Zhang et al. (2011) Nat. Biotechnol. 29:149-153; and Reyon et al. (2012) Nat. Biotechnol. 30(5): 460-465, each of which is incorporated herein by reference in its entirety), homing endonuclease, meganuclease (see, for example, U.S. Patent Publication No. US 2014 / 0121115, which is incorporated herein by reference in its entirety) and RNA-guided nuclease (see, for example, Makarova et al. (2018) The CRISPR Journal 1(5): 325-336; and Adli (2018) Nat. Communications 9:1911, each of which is incorporated herein by reference in its entirety).

[0052] In some implementations, the transgene encodes an RNA-guided nuclease. Suitable RNA-guided nucleases include, but are not limited to, class I and class II clustered regularly spaced short palindromic repeat (CRISPR)-related nucleases. Class I is divided into types I, III, and IV, including, but not limited to, type I (Cas3), type IA (Cas8a, Cas5), type IB (Cas8b), type IC (Cas8c), type ID (Cas10d), type IE (Cse1, Cse2), type IF (Csy1, Csy2, Csy3), type IU (GSU0054), type III (Cas10), type III-A (Csm2), type III-B (Cmr5), type III-C (Csx10 or Csx11), type III-D (Csx10), and type IV (Csf1). Class II is divided into types II, V, and VI, including but not limited to type II (Cas9), type II-A (Csn2), type II-B (Cas4), type V (Cpf1, C2c1, C2c3), and type VI (Cas13a, Cas13b, Cas13c). RNA-guided nucleases also include naturally occurring class II CRISPR nucleases, such as Cas9 (type II) or Cas12a / Cpf1 (type V), as well as other nucleases derived therefrom or obtained therefrom. Exemplary Cas9 nucleases that can be used in this invention include, but are not limited to: Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria meningitidis Cas9 (NmCas9), Campylobacter jejuni Cas9 (CjCas9), and Bacillus geyserum Cas9 (GeoCas9).

[0053] In some embodiments, the transgene encodes one or more reporter sequences that produce a detectable signal upon expression. Such reporter sequences include, but are not limited to, DNA sequences encoding: β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), red fluorescent protein (RFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane-bound proteins including, for example, CD2, CD4, CD8, influenza hemagglutinin, and fusion proteins including membrane-bound proteins comprising an antigen tag domain appropriately fused to hemagglutinin or Myc, etc.

[0054] Methods to reduce non-functional capping in AAV products

[0055] This article provides a method for reducing the amount of nonfunctional capsids in AAV products, achieved by limiting the duration of target cells in a bioreactor post-transfection, before separation and purification of unseparated capsids, and / or by removing at least one nonfunctional capsid subset from the adeno-associated virus (AAV) product. This method involves separating transgenic functional capsids from nonfunctional capsid subsets based on hydrophobicity. For example, cells may optionally be retained in the bioreactor for approximately two days post-transfection and purified using AEX (with or without a fatty acid mobile phase and with or without a mixed-mode resin) or HIC. In some embodiments, purification is performed using AEX or a mixed resin with a fatty acid mobile phase or with HIC without limiting the duration of cell presence in the bioreactor.

[0056] This article also provides methods for reducing the amount of nonfunctional capsid in AAV products by varying the post-transfection duration and / or the temperature at which cells are maintained in the bioreactor. The transfected cells are retained in the bioreactor for approximately 2 days (i.e., approximately 48 hours post-transfection). For example, approximately 2 days can include approximately 1.5 days to approximately 2.5 days, including, for example, 1.5 days, 1.6 days, 1.7 days, 1.8 days, 1.9 days, 2 days, 2.1 days, 2.2 days, 2.3 days, 2.4 days, and 2.5 days. Optionally, the post-transfection temperature is set to approximately 30–38°C (e.g., approximately 30°C, approximately 31°C, approximately 32°C, approximately 33°C, approximately 34°C, approximately 35°C, approximately 36°C, approximately 37°C, or approximately 38°C). As used herein, approximately 37°C refers to ±0.5°C. For example, approximately 37°C may include 36.5°C, 36.6°C, 36.7°C, 36.8°C, 36.9°C, 37.0°C, 37.1°C, 37.2°C, 37.3°C, 37.4°C, or 37.5°C. For example, transfected cells may be held in the bioreactor for approximately 2 days, and the post-transfection temperature may be set to approximately 37°C. The post-transfection temperature change (e.g., to approximately 37°C) is performed before the separation and purification of the unseparated capsid. For example, the post-transfection temperature may be from approximately 30°C to approximately 38°C. In some embodiments, the cells are maintained at approximately 37°C in the bioreactor post-transfection temperature. After approximately 2 days of maintenance in the bioreactor, the transfected cells are lysed and purified. Purification can then be performed using AEX (with or without a fatty acid mobile phase and with or without a mixed-mode resin) or HIC. In some embodiments, purification is performed using AEX or mixed resins with a fatty acid mobile phase or with HIC, without altering the post-transfection temperature of the bioreactor.

[0057] Existing AAV purification methods, such as ultracentrifugation, are associated with AAV product yield loss, are not scalable, and are unsuitable for high-throughput processing, often resulting in AAV products containing unacceptable levels of non-functional capsids. Furthermore, existing methods are typically limited by their inability to effectively separate functional AAV capsids from AAV product-associated impurities (i.e., non-functional capsids). However, product-associated impurities can lead to an immune response to capsid proteins in the host. This method optimizes the concentration of functional capsids by removing non-functional capsids. Moreover, this method reduces the immunogenicity of effective doses of AAV products by removing capsid proteins associated with non-functional capsids.

[0058] This document provides a method for removing at least one nonfunctional capsid subpopulation from adeno-associated virus (AAV) product, comprising separating transgenic functional capsids from nonfunctional capsid subpopulations based on hydrophobicity. The purification method targets hydrophobic capsids, such as those with deamide VP1, to remove them, thereby forming a final AAV product relatively free of deamide capsids. Specifically, capsids deamided at the N57 position of VP1 are nonfunctional capsids. As used herein, removal of nonfunctional capsid subpopulations includes removing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nonfunctional capsids. For example, the removed capsids can result in the removal of more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the capsids with deamide N57 at VP1.

[0059] As described herein, nonfunctional capsids can be separated from functional capsids (i.e., capsids containing functional transgenes and lacking deamidation N57 on VP1) based on the hydrophobicity of the nonfunctional capsid. The hydrophobicity of the nonfunctional capsid is at least partially related to the deamidation of VP1 (e.g., at the N57 position of VP1), while in the functional capsid, VP1 is amidated.

[0060] As used throughout, deamidation is a chemical reaction in which the amide functional group in asparagine or glutamine in a polypeptide (e.g., VP1) is converted to another functional group, such as aspartic acid or glutamic acid, thereby introducing a negative charge into the protein. Deamidation of VP1 also results in increased hydrophobicity of the non-functional capsid compared to the functional capsid. By utilizing the difference in deamidation (and hydrophobicity) between functional and non-functional capsids, the deamidated capsid can be separated from the AAV capsid population containing the functional capsid to produce AAV products with less non-functional capsid (e.g., less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% non-functional capsid) compared to AAV products purified solely by ultracentrifugation or by AEX without a fatty acid mobile phase.

[0061] Hydrophobicity-based separation can be optionally combined with charge-based separation. For example, HIC can be performed followed by AEX (with or without a fatty acid mobile phase).

[0062] By separating and removing non-functional capsids based on hydrophobicity or hydrophobicity and charge, the resulting AAV product exhibits a higher potency at a given capsid concentration compared to comparable AAV products in which non-functional capsids were not removed based on hydrophobicity or hydrophobicity and charge. Increased capsid potency signifies increased transgene expression following introduction of a given concentration of AAV product into host cells. The increase in capsid potency is surprising given the decreased VP1:VP2 ratio, as VP1 is typically associated with higher capsid infectivity. However, deamidation of VP1 (e.g., at the N57 position) is associated with decreased capsid infectivity, making the deamidated capsid a suitable target for eliminating impurities associated with AAV products. The purification method described herein provides increased yields of functional capsids and increased potency of the product at a given capsid concentration due to the presence of more functional AAV capsids compared to AAV products prepared using existing AAV purification methods. Furthermore, by removing the deamide cap, the VP1:VP2:VP3 ratio can be reduced from the typical cap formation ratio of about 1:1:8 to 1:1:10 to <1:1:8 to <1:1:10.

[0063] The method described herein includes one or more chromatographic steps in which an AAV product source is loaded onto, passed through, or applied to a chromatographic substrate to achieve differential separation of various components. The AAV product source may be cell culture medium containing AAV product or material containing AAV product separated from pre-chromatographic steps. It should be understood that the material eluted in the chromatographic step may be applied to a subsequent chromatographic substrate to obtain further purified AAV product.

[0064] Separating functional capsids containing functional genetically modified organisms from non-functional capsid subpopulations includes HIC steps, AEX steps using fatty acid mobile phases, or mixed-mode AEX steps. These techniques separate them based on differences in the surface hydrophobicity of biomolecules. Alternative methods based on hydrophobicity separation can be used instead of these methods.

[0065] The HIC step may include one or more HIC steps. HIC methods are known in the art, and exemplary methods are described in the examples. The HIC chromatographic medium contains hydrophobic ligands, such as straight-chain hydrocarbons (e.g., propyl, butyl, hexyl, or octyl). A salt (e.g., ammonium sulfate) is used to enhance the hydrophobic interaction, and the addition of the salt drives protein capture onto the HIC medium. Therefore, HIC resins are typically loaded at high salt concentrations and eluted at lower salt concentrations. Those skilled in the art will understand that the concentration of the salt (e.g., ammonium sulfate) can be manipulated to achieve optimal binding and elution concentrations. HIC may be used in conjunction with an AEX step (i.e., an AEX step with or without a fatty acid mobile phase). Optionally, HIC is performed prior to AEX.

[0066] In some embodiments, the separation of functional capsids containing functional genetic material from non-functional capsid subpopulations includes one or more AEX steps, wherein at least one AEX step is performed using a fatty acid mobile phase. In some embodiments, the fatty acid mobile phase comprises caprylic acid or heptanoic acid. Fatty acids impart a negative charge to capsids with exposed VP1, which causes non-functional capsids to bind more tightly to the chromatographic resin and exhibit better separation from functional capsids. Anion exchange chromatography methods are described in the examples and are known to those skilled in the art. See, for example, U.S. Patent Nos. 6,008,036, 6,586,226, 5,837,520, 6,261,823, 6,537,793, and International Patent Application Publications Nos. WO 00 / 50573, WO 02 / 44348, and WO 03 / 078592, the contents of which are incorporated herein by reference in their entirety.

[0067] In some embodiments, the separation of functional capsids containing functional genetically modified organisms from non-functional capsid subgroups includes a mixed-mode chromatography step. Such a step may optionally utilize a mixed-mode resin comprising a hydrophobic resin. As used herein, the term “mixed-mode” or “multi-mode” refers to a chromatographic substrate having the capability of more than one separation mode. For example, mixed-mode chromatography may include separating molecules based on hydrophobicity and charge. Mixed-mode chromatography methods are described in the examples. Other methods are known to those skilled in the art. See, for example, Lesellier et al., "Mixed-Mode Chromatography-A Review," LCGC Supplements 30(6): 22-33 (2107); and Zhang and Liu, "Mixed-mode chromatography in pharmaceutical and biopharmaceutical applications," Journal of Pharmaceuticals and Biomedical Analysis 28(5): 73-88 (2016). Both references are incorporated herein by reference in their entirety. Mixed-mode resins are commercially available, including, for example, Nuvia aPrime 4A. The resin and form used for the mixing mode are determined within the scope of the art.

[0068] The methods presented in this paper enable better separation of functional and non-functional AAV capsids. By eliminating the non-functional capsid, the heterogeneity of the final AAV product is reduced. These methods provide AAV products with less than 10% non-functional capsid and are more effective. By removing the non-functional capsid, the effective dose of this AAV product has reduced immunogenicity compared to the effective dose of a product containing a non-functional capsid with immunogenic capsid proteins.

[0069] Treatment

[0070] This article also provides a method for treating a subject that requires RNA or polypeptides provided or produced by a genetically modified organism, which is achieved by administering to the subject an effective amount of any AAV product described herein (including any AAV product produced by any of the methods described herein for producing AAV products).

[0071] Transgenic sequences contained in the functional capsid of an AAV product can be heterologous to the cells in that object. As used herein, the phrase “heterologous” refers to a situation that is not normally present in nature. Thus, a heterologous nucleotide sequence can be (a) foreign to its host cell (i.e., exogenous to the cell); (b) naturally present in the host cell (i.e., endogenous) but present in the cell in a non-natural amount (i.e., greater than or less than the amount naturally present in the host cell); or (c) naturally present in the host cell but located outside its natural locus.

[0072] As used throughout, the term "effective dose" is defined as any amount necessary to produce a desired physiological response (e.g., reduction or delay of one or more effects or symptoms of a disease or condition). The effective dose and regimen for administering any AAV product described herein can be determined empirically, and such determination is within the scope of the art. The range of doses administered is an amount sufficiently large to produce the desired effect of affecting (e.g., alleviating or delaying) one or more symptoms of a disease or condition. The dose should not be so large as to cause substantial adverse side effects, such as undesirable cross-reactions, undesirable cell death, etc. Typically, the dose will vary depending on the species, age, weight, general health condition, sex, and diet of the subject, the method and timing of administration, and the severity of the specific condition, and can be determined by those skilled in the art. In the event of any contraindications, the dose may be adjusted by an individual physician. The dose may be varied and may be administered in one or more doses.

[0073] The effective dose of any AAV product described herein will vary and can be determined by those skilled in the art through experiments and / or clinical trials. For example, the effective dose may be approximately 10 6 To about 10 18 One recombinant rAAV virus, or any value within that range, such as approximately 10. 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 10 14 10 15 10 16 10 17 10 18 The number of recombinant AAV particles. Therefore, the number of rAAV particles applied to the subject can be approximately 10. 6 Up to 10 18 The order of magnitude is on the order of vector genomes (vg) / ml, for example, about 10 6 10 7 10 8 10 9 1010 10 11 10 12 10 13 10 14 10 15 10 16 10 17 10 18 vg / ml. In some implementations, the number of rAAV particles applied to the subject can be approximately 10. 6 Up to 10 15 vg / kg, or any value between these amounts, such as about 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 10 14 Or 10 15 vg / kg. Other effective dosages can be readily determined by those skilled in the art through routine experiments to establish dose-response curves. Using the AAV products described herein requires lower doses of AAV compared to those prepared using conventional purification methods.

[0074] The compositions described herein can be administered in a variety of ways, depending on whether local or systemic treatment is desired. The compositions can be administered via any of several routes of administration, including intraparenchymal injection, intravenous injection, intrathecal injection, intramuscular injection, intracisional injection, coronary artery injection, intramyocardial injection, intradermal injection, subendocardial injection, or combinations thereof. The effective dose for any of the administration methods described herein can be extrapolated from dose-response curves derived from in vitro or animal model testing systems.

[0075] As used throughout, “object” refers to an individual. An object can be an adult object or a pediatric object. Pediatric objects include those from birth to eighteen years of age. Preferably, the object is an animal, such as a mammal like a primate, and more preferably a human. Non-human primates are also objects. The term “object” includes domesticated animals, such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.). Therefore, this document considers veterinary uses and pharmaceutical preparations.

[0076] As used throughout, “treat, treating, and treatment” refers to a method of reducing or delaying one or more effects or symptoms of a disease or condition. The subject may be a patient diagnosed with the disease or condition. Treatment may also refer to a method of reducing an underlying pathology rather than merely symptoms. The effect applied to the subject may have, but is not limited to, the following effects: reducing one or more symptoms of the disease, alleviating the severity of the disease, completely eliminating the disease, or delaying the onset or worsening of one or more symptoms. For example, if one or more symptoms of the disease are reduced by approximately 10% compared to the subject before treatment or compared to a control subject or control value, the disclosed method is considered treatment. Therefore, the reduction can be any amount of reduction, ranging from approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or between.

[0077] Unless otherwise stated herein, descriptions of ranges herein are intended merely as a simplified way of referring to each individual value falling within that range, and each individual value is incorporated into the specification as if it were described individually herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values ​​such as 2% to 40%, 10% to 30%, or 1% to 3% be explicitly listed in this specification. These are merely examples of specific intent, and all possible combinations of values ​​between the listed minimum and maximum values, and including them, are considered to be explicitly stated in this disclosure.

[0078] This document discloses materials, compositions, and components that can be used in, in conjunction with, or as products of the disclosed methods and compositions, and that can be used to prepare or as products of the disclosed methods and compositions. These and other materials are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups, etc., of these materials are disclosed, each is specifically considered and described herein, although specific references to every various individual and collective combination and arrangement of these compounds may not be explicitly disclosed. For example, if a method is disclosed and discussed, and many modifications that can be made to the many molecules included in the method are discussed, then each and every combination and arrangement of the method, as well as possible modifications, are specifically considered unless specifically indicated otherwise. Similarly, any subsets or combinations of these are also specifically considered and disclosed. This concept applies to all aspects of this disclosure, including but not limited to steps in methods using the disclosed compositions. Therefore, if multiple additional steps are possible, it should be understood that each of these additional steps can be performed in conjunction with or in combination with any particular method step or method step of the disclosed method, and each such combination or sub-combination is specifically considered and should be considered disclosed.

[0079] The publications cited in this article, and the materials they cite, are explicitly incorporated herein by reference in their entirety.

[0080] Additional Definitions

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0082] The articles “a” and “one” are used in this text to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “nucleic acid sequence” means at least one nucleic acid sequence, but may include more than one nucleic acid sequence.

[0083] Any and all examples or exemplary language (e.g., "such as") provided herein are intended only to better illustrate the invention and, unless otherwise required, do not constitute a limitation on the scope of the invention.

[0084] The terms “may,” “possibly,” and related terms are intended to indicate that the subject matter in question is optional (i.e., the subject matter exists in some examples but not in others), rather than to indicate the subject matter’s capability or probability, unless the context clearly indicates otherwise.

[0085] The term “about” is used to provide flexibility for the endpoints of a numerical range by allowing a given value to be slightly above or slightly below the endpoint without affecting the expected result.

[0086] The terms “optional” and “optionally” mean that the event, situation, or material described below may or may not occur or not exist, and the description includes instances in which the event, situation, or material occurs or exists as well as instances in which it does not occur or not exist.

[0087] The use of the terms “comprising,” “including,” or “having,” and variations thereof in this document is intended to cover the elements listed thereafter and their equivalents, as well as any additional elements. Embodiments described as including, containing, or having specific elements are also considered to consist substantially of and comprise of those specific elements. As used herein, “and / or” refers to and covers any and all possible combinations of one or more of the associated listed items, and non-combinations are interpreted in the alternative (“or”) context.

[0088] Implementation

[0089] 1. An adeno-associated virus (AAV) product comprising:

[0090] (a) A functional capsid containing genetically modified organisms; and

[0091] (b) Less deamidated coating compared to control AAV products purified only by ultracentrifugation or by anion exchange chromatography (AEX) without the use of a fatty acid mobile phase.

[0092] 2. The AAV product according to Embodiment 1, wherein the AAV product contains less N57 deamidated coating compared to a control AAV product purified solely by AEX without using a fatty acid mobile phase.

[0093] 3. The AAV product according to embodiment 1 or 2, wherein the AAV product contains less than 10% of a cap having deamide N57 on VP1.

[0094] 4. The AAV product according to embodiment 3, wherein the AAV product contains less than 5% of a cap having deamide N57 on VP1.

[0095] 5. The AAV product according to embodiment 4, wherein the AAV product contains less than 1% of a cap having deamide N57 on VP1.

[0096] 6. The AAV product according to any one of embodiments 1-5, wherein the AAV product contains less than 10% non-functional coating.

[0097] 7. The AAV product according to any one of embodiments 1-6, wherein the AAV product has higher transgenic expression in host cells than the control AAV product purified by ultracentrifugation or AEX without using a fatty acid mobile phase.

[0098] 8. The AAV product according to any one of embodiments 1-7, wherein the AAV product has a lower VP1 to VP2 ratio than the control AAV product purified by ultracentrifugation or by AEX without using a fatty acid mobile phase.

[0099] 9. The AAV product according to any one of embodiments 1-8, wherein the amount of deamide is detected by liquid chromatography-mass spectrometry.

[0100] 10. An adeno-associated virus (AAV) product, which is essentially composed of a functional capsid containing genetically modified organisms.

[0101] 11. The AAV product according to embodiment 10, wherein the functional capping is separated from an AAV capping group comprising the functional capping and the deamide capping to produce the AAV product.

[0102] 12. The AAV product according to embodiment 10 or 11, wherein the AAV product is relatively free of a cap in which VP1 has deamide N57.

[0103] 13. An adeno-associated virus (AAV) product comprising a functional capsid containing a genetically modified organism, wherein the product is less hydrophobic than a control AAV product purified solely by ultracentrifugation or by anion exchange chromatography (AEX) without the use of a fatty acid mobile phase.

[0104] 14. The AAV product according to embodiment 13, wherein the AAV product contains less than 10% non-functional coating.

[0105] 15. The AAV product according to embodiment 13 or 14, wherein the AAV product has higher transgenic expression in host cells than the control AAV product purified by ultracentrifugation or AEX without the use of a fatty acid mobile phase.

[0106] 16. The AAV product according to any one of embodiments

[0007] -15, wherein the AAV product has a lower VP1 to VP2 ratio than the control AAV product purified by ultracentrifugation or by AEX without using a fatty acid mobile phase.

[0107] 17. An adeno-associated virus (AAV) product comprising a capsid, wherein the AAV product comprises less than 10% of a capsid having deamidated N57 on VP1.

[0108] 18. The AAV product according to embodiment 17, wherein the AAV product contains less than 5% of a cap having deamide N57 on VP1.

[0109] 19. The AAV product according to embodiment 18, wherein the AAV product contains less than 1% of a cap having deamide N57 on VP1.

[0110] 20. An adeno-associated virus (AAV) product comprising a transgenic AAV capsid, wherein the AAV product exhibits higher transgenic expression in host cells than a control AAV product purified by ultracentrifugation or by AEX without the use of a fatty acid mobile phase.

[0111] 21. An adeno-associated virus (AAV) product comprising a genetically modified AAV capsid, wherein the AAV product has a lower VP1 to VP2 ratio than a control AAV product purified by ultracentrifugation or by AEX without the use of a fatty acid mobile phase.

[0112] 22. A method for removing at least one nonfunctional capsid subgroup from an adeno-associated virus (AAV) product, comprising separating the transgenic functional capsid from the nonfunctional capsid subgroup based on hydrophobicity.

[0113] 23. The method according to embodiment 22, wherein the separation of the functional cap and the non-functional cap is further based on charge.

[0114] 24. The method according to embodiment 22 or 23, wherein the removed nonfunctional coating comprises a deamide coating.

[0115] 25. The method according to embodiment 24, wherein the removed deamide capping comprises VP1 having deamide N57.

[0116] 26. The method according to any one of embodiments 22-25, wherein separating the functional capsid containing the functional transgene from the non-functional capsid subgroup comprises:

[0117] (a) Hydrophobic interaction chromatography (HIC) procedure,

[0118] (b) Anion exchange chromatography using a fatty acid mobile phase, or

[0119] (c) Mixed-mode anion exchange chromatography procedure.

[0120] 27. The method according to embodiment 26, wherein the separation includes the HIC step.

[0121] 28. The method according to embodiment 27, wherein the separation further includes an anion exchange chromatography (AEX) step.

[0122] 29. The method according to embodiment 28, wherein the AEX step is performed without using a fatty acid mobile phase.

[0123] 30. The method according to embodiments 27-29, wherein the HIC step is performed before the AEX step.

[0124] 31. The method according to embodiment 26, wherein the separation includes an anion exchange chromatography step using a fatty acid mobile phase.

[0125] 32. The method according to embodiment 31, wherein the fatty acid mobile phase comprises octanoic acid or heptanoic acid.

[0126] 33. The method according to embodiment 26, wherein the separation includes a mixed-mode resin chromatography step.

[0127] 34. The method according to embodiment 33, wherein the mixed-mode resin is a hydrophobic anion exchange resin.

[0128] 35. An AAV product produced by any one of embodiments 22-34.

[0129] 36. An adeno-associated virus (AAV) product comprising:

[0130] (a) A functional capsid containing genetically modified organisms; and

[0131] (b) A less hydrophobic capsid compared to a control AAV product purified solely by ultracentrifugation or by anion exchange chromatography (AEX) without a fatty acid mobile phase.

[0132] 37. The AAV product according to embodiment 36, wherein the AAV product contains less N57 deamidated coating compared to a control AAV product purified solely by AEX without using a fatty acid mobile phase.

[0133] 38. The AAV product according to embodiment 36 or 37, wherein the AAV product contains less than 10% of a cap having deamide N57 on VP1.

[0134] 39. The AAV product according to embodiment 38, wherein the AAV product contains less than 5% of a cap having deamide N57 on VP1.

[0135] 40. The AAV product according to embodiment 39, wherein the AAV product contains less than 1% of a cap having deamide N57 on VP1.

[0136] 41. The AAV product according to any one of embodiments 36-40, wherein the AAV product contains less than 10% non-functional coating.

[0137] 42. The AAV product according to any one of embodiments 36-41, wherein the AAV product has higher transgenic expression in host cells than the control AAV product purified by ultracentrifugation or AEX without the use of a fatty acid mobile phase.

[0138] 43. The AAV product according to any one of embodiments 36-42, wherein the AAV product has a lower VP1 to VP2 ratio than the control AAV product purified by ultracentrifugation or by AEX without using a fatty acid mobile phase.

[0139] 44. The AAV product according to any one of embodiments 36-43, wherein the amount of deamide is detected by liquid chromatography-mass spectrometry.

[0140] 45. An adeno-associated virus (AAV) product comprising essentially a functional capsid containing a genetically modified organism, wherein the functional capsid is separated from a hydrophobic AAV capsid group to produce the AAV product.

[0141] 46. ​​The AAV product according to embodiment 45, wherein the AAV product is relatively free of a cap in which VP1 has deamide N57.

[0142] 47. The AAV product according to embodiment 45 or 46, wherein the AAV product contains less than 10% non-functional coating.

[0143] 48. The AAV product according to embodiment 45 or 46, wherein the AAV product has higher transgenic expression in host cells than the control AAV product purified by AEX without using a fatty acid mobile phase.

[0144] 49. The AAV product according to any one of embodiments 45-48, wherein the AAV product has a lower VP1 to VP2 ratio than the control AAV product purified by ultracentrifugation or by AEX without using a fatty acid mobile phase.

[0145] 50. A method of treating a subject who requires a protein produced by a genetic modification, comprising administering to the subject the AAV product described in any one of embodiments 1-21 and 35-49.

[0146] 51. An adeno-associated virus (AAV) product that is relatively free of non-functional capsids.

[0147] 52. An adeno-associated virus (AAV) product consisting essentially of a functional capsid containing genetic material and a buffer solution.

[0148] 53. The method according to any one of embodiments 22-34, wherein the AAV capsid is harvested approximately 2 days after transfection.

[0149] 54. The method according to embodiment 53, wherein the post-transfection temperature is about 30°C to about 38°C.

[0150] 55. The method according to embodiment 54, wherein the post-transfection temperature is about 37°C, and wherein the AAV product is harvested about 2 days after transfection.

[0151] 56. The method according to embodiment 22, wherein separating the functional capsid from the non-functional capsid includes harvesting the AAV capsid approximately 2 days (e.g., 1.5-2.5 days) after transfection.

[0152] 57. The method according to embodiment 56, wherein the post-transfection temperature is about 36°C, 37°C or 38°C.

[0153] 58. The method according to embodiment 57, wherein the post-transfection temperature is about 37°C, and wherein the AAV product is harvested about 2 days after transfection.

[0154] Example

[0155] Example 1: Comparison of chromatographic resolution between Nuvia aPrime 4A anion exchange column and Poros HQ50 anion exchange column for purifying DNA-containing capsids.

[0156] Capsule preparation

[0157] AAV capsids were generated using transient transfection with three plasmids in a 50 L bioreactor. Cells were lysed after 72 hours to expose the AAV capsids generated in the upstream process. The lysed material was then clarified using a constant flux deep filter. The product of this process is referred to as the clarified lysate. The clarified lysate was then captured on an affinity chromatography resin under binding and elution conditions. Briefly, the clarified lysate was loaded onto the resin, washed to remove host cell-related impurities, and then eluted under acidic conditions. The affinity eluent was neutralized to pH 8.0 using Tris buffer. This is referred to as the affinity product. The affinity product was aliquoted and frozen at -80 °C. Appropriate volumes of aliquoted samples were thawed at room temperature for use in the experiments performed and described in this application.

[0158] Sample preparation

[0159] AAV cap (2x10) 14 The sample concentration (capsids per mL) was diluted with loading dilution buffer (28 mM ammonium acetate, 2 mM MgCl2, 0.01% (w / v) poloxamer 188) to bring the final loading concentration to below 2 x 10⁻⁶. 13 Capsules / mL. The sample pH was adjusted to 9.3 using 1N NaOH or 1N acetic acid, and the conductivity was adjusted to 3 mS / cm using 0.01% (w / v) poloxamer 188 and 2 mM MgCl2 solution.

[0160] Resin balance

[0161] Prepare 1 mL of Nuvia aPrime 4A anion exchange column (Bio-rad, Hercules, Cat# 12007392) and 1 mL of Poros HQ50 anion exchange column (ThermoFisher, Waltham, MA, Cat# PN4481315). First, wash away the stored ethanol with 10 column volumes (CV) of wash solution (28 mM ammonium acetate, 2 mM magnesium chloride, 0.01% (w / v) poloxamer 188, pH 9.3, conductivity: 3 mS / cm), then sterilize with 5 CV (0.5 N sodium hydroxide) for 30 minutes. Equilibrate by first pre-equilibrating the column in 1 M ammonium acetate (pH approximately 6.7) to remove chloride ions, then pump in 5 CV of equilibration buffer A (28 mM ammonium acetate, 2 mM magnesium chloride, 0.01% (w / v) poloxamer 188, pH 9.3). All equilibration steps were pumped into the system at a rate of 1 mL / min for 1 minute.

[0162] Anion exchange (AEX) chromatography

[0163] Diluted samples were applied to two equilibrated columns at a rate of 0.33 mL / min, with a residence time of 3 minutes. After sample loading, the columns were washed with 5 CV buffer A, followed by 5 CV wash buffer (10 mM ammonium acetate, 0.01% (w / v) poloxamer 188, pH 9.3). Column elution was performed using a 60 CV linear gradient from 0-100%, stepwise increasing by 0.01% in buffer B (1 M ammonium acetate, pH 9.3), followed by 20 CV elution with 100% buffer B. 1 mL fraction was collected in pre-neutralization buffer. After sample collection, the column was stripped with 5 CV stripping buffer (50 mM Tris, 2 M sodium chloride, 0.01% (w / v) poloxamer 188, pH 8), followed by 5 CV wash buffer (0.5 N NaOH). The pH was then rebalanced with 1 M ammonium acetate (pH approximately 6.7), and finally stored in 5 CV of 100 mM sodium phosphate, 18% ethanol (v / v%), pH 7.

[0164] Chromatographic results

[0165] For comparison, 1 mL pre-filled Nuvia aPrime 4A columns and Poros HQ50 (control) columns were run. The results are shown in Table 1.

[0166] Table 1: Chromatographic column operation results

[0167]

[0168] Chromatograms of the Nuvia aPrime 4A run and the control (POROS HQ 50) run were generated. Analysis of these chromatograms showed that Nuvia aPrime 4A provided sufficient resolution between Library 1 (mainly intact capsids) and Library 2 (mainly empty capsids). To better understand the chromatographic performance of this new technique, the two chromatograms were overlaid and presented below. Figure 3 .

[0169] Data Analysis

[0170] The areas under the absorbance curves at 260 nm and 280 nm and the A260 / A280 ratio for Nuvia aPrime 4A and Poros HQ50 columns were calculated, indicating the presence of both intact and empty capsids. This data is provided in Table 2. Analysis of Table 2 shows that for library 1 (predominantly intact capsids), Nuvia aPrime 4A and Poros HQ50 have similar area ratios and performance. It was found that Nuvia aPrime 4A library 2 (predominantly empty capsids) and the stripped A260 / 280 ratio were higher than those of Poros HQ50 library 2 and the stripped library.

[0171] Table 2: Comparison of Area Under the Curve for 260nm and 280nm

[0172]

[0173] Vector genome and capsid quality balance results

[0174] The vector genome (VG) quality balance of the two columns is shown in Table 3, and the capsid quality balance is shown in Table 4.

[0175] Table 3: VG Yields of Nuvia aPrime 4A and Poros HQ50 Columns

[0176]

[0177] Table 4: Capsule Yields of Nuvia aPrime 4A and Poros HQ50 Columns

[0178]

[0179] Analysis of Tables 3 and 4 shows that the VG and capping recovery rates of Library 1 (product peak) for Nuvia aPrime 4A and Poros HQ50 columns are comparable. Library 2 (mainly empty capping peak) for Nuvia aPrime 4A was found to have lower VG and capping yields compared to the Poros HQ50 column. The flow-through / washing libraries for Nuvia aPrime 4A were found to have higher VG and capping recovery rates compared to Poros HQ50. The VG and capping recovery rates of the stripping libraries for both resins were found to be comparable.

[0180] Analytical ultracentrifugation (AUC) was performed on samples from Nuvia aPrime 4A and Poros HQ50 containing the product peak (C1) and empty capsid peak (2). The AUC sample data for Nuvia aPrime 4A C1 and C2 are shown in Tables 5 and 6, respectively.

[0181] Table 5: AUC Sample Data for Nuvia aPrime 4A Library 1

[0182]

[0183] Table 6: AUC Sample Data for Nuvia aPrime 4A Library 2

[0184]

[0185] The AUC spectra and sample data for PorosHQ50 libraries 1 and 2 are shown in Tables 7 and 8, respectively.

[0186] Table 7: AUC Sample Data of PorosHQ50 Library 1

[0187]

[0188] Table 8: AUC Sample Data of PorosHQ50 Library 2

[0189]

[0190] Analysis of the AUC spectra of library 1 of Nuvia aPrime 4A and Poros HQ50 showed that Poros HQ50 had a higher percentage of intact capsids compared to Nuvia aPrime 4A. The AUC spectra of library 2 of Nuvia aPrime 4A and Poros HQ50 were also compared. It was found that the library 2 spectra of the two columns were comparable and consisted mainly of empty capsids.

[0191] The reduction in intact caps recovered using Nuvia aPrime 4A and PorosHQ50 was slight. This is not intended to be theoretically limited, and is likely because even some intact caps are deamided and therefore nonfunctional. The removal of deamided intact caps was measured as a reduction in intact caps, but reflects the elimination of nonfunctional intact caps.

[0192] Example 2: Introducing fatty acids into the AEX mobile phase to increase chromatographic resolution

[0193] As described above, AEX chromatography was performed using a POROS HQ50 column, the difference being the incorporation of fatty acid heptanoates into the mobile phase. For example... Figure 4 As shown, the chromatographic conditions using heptanoic acid esters in the mobile phase resulted in increased chromatographic separation between the product peak of Ku1 and the non-product peak of Ku2.

[0194] Example 3: Two-step chromatographic method using hydrophobic interaction chromatography (HIC) coupled with AEX chromatography

[0195] Sample preparation

[0196] The AAV capsid (2.6 x 10) 14 The sample was diluted 7-fold with sample dilution buffer (1.5 M ammonium sulfate, 100 mM sodium phosphate, 0.01% (w / v) poloxamer-188, pH 7.0, 187 mS / cm). The salt concentration was adjusted to a final concentration of 1.5 M ammonium sulfate by adding 60% (v / v) salt spiking buffer (4 M ammonium sulfate, 100 mM sodium phosphate, 0.01% (w / v) poloxamer-188) per volume of capsid sample. pH and conductivity were measured after sample dilution.

[0197] The steps for HIC chromatography and subsequent procedures are described below.

[0198]

[0199]

[0200]

[0201] HIC chromatography

[0202] Using a Bio-Rad pre-filled 44mL Nuvia aPrime 4A column, a challenge of 2.6 x 10⁻⁶ was achieved. 14 One capsid per mL of resin. The AF product was prepared by diluting it 7-fold in 1.5 M ammonium sulfate loading dilution buffer, followed by adding 60% (v / v%) 4 M ammonium sulfate spiked buffer per volume of AF product used. The chromatograms and corresponding gradient elutions for this HIC mode run are shown below. Figure 5 and Figure 6 .

[0203] The final conductivity of the loaded material was 189 mS / cm, close to that of the 1.5 M ammonium sulfate equilibration buffer (188 mS / cm). Using this loading preparation method for HIC operations, conductivity up to 2.6 x 10⁻⁶ mS / cm was achieved. 14 No penetration was observed at a rate of 1 capsid / mL resin. Figure 5 See also Figure 6 ).

[0204] The materials were separated into two distinct peaks using gradient elution with decreasing conductivity, and collected into two libraries. At higher conductivity, the material in HIC library 1 eluted first and was less hydrophobic. Elution of HIC library 1, measured by ATKA, started at 188 mS / cm and peaked at 172 mS / cm. The material in HIC library 2 was more tightly bound to the resin and was more hydrophobic than the material in HIC library 1. Elution of HIC library 2 started at 124 mS / cm and peaked at 84 mS / cm. Although baseline separation between the two peaks was not obtained in this experiment, this is one of the best separations observed to date in a linear gradient.

[0205] Table 9 shows the 260 nm and 280 nm areas, normalized 260 nm area (%A260), and A260 / A280 ratio for each process fraction. The 260 nm area of ​​HIC library 1 is larger than that of HIC library 2, corresponding to 59% of the normalized 260 nm area. The A260 / A280 ratio is 1.07 in both elution libraries. This indicates that the separation in HIC chromatography does not enrich DNA-containing capsids and is therefore independent of the presence of DNA in the capsid.

[0206] Table 9: HIC chromatogram areas at 260 nm and 280 nm (mAU*mL)

[0207]

[0208] HIC chromatography VG and capsid mass balance

[0209] The combined volumes of HIC library 1 and HIC library 2 were 700 mL and 795 mL, respectively. Figure 7 The VG and capsid yield data are shown after normalization to mass equilibrium based on AF product titers. VG and capsid titers in the FTW fraction were below the limits of quantitation and are not shown here. HIC library 1 contained the majority of the loaded material, approximately 76% of the capsid yield and 76% of the VG yield. This result confirms the higher 260 nm area of ​​peak 1 observed in the chromatogram. The VG to capsid titer ratio for HIC library 1 was 19%, and for HIC library 2 it was 22%, indicating that this step did not enrich DNA-containing capsids.

[0210] HIC analysis using ultracentrifugation (AUC)

[0211] The AUC results for the loading solution, HIC library 1, HIC library 2, and low pH stripping are shown in the table. Figure 8 The AUC spectra of HIC Library 1 and HIC Library 2 are shown. The percentages of intact and empty cells were similar across all fractions, and the spectra of HIC Library 1 and HIC Library 2 were also similar. These results indicate that HIC chromatography does not enrich DNA-containing capsids, but rather separates capsids based on surface differences.

[0212] Table 10: Summary of AUC results for HIC Library 1 and HIC Library 2

[0213]

[0214] Tangential flow filtration

[0215] To concentrate and buffer exchange (BE) materials for HIC Library 1 and HIC Library 2, a MicroKross 40 cm⁻¹ was used. 2 100K mPES membrane. The initial volume of HIC library 1 was 700 mL, and the coating titer was 9.4 x 10⁻⁶. 12 One capsid per mL resulted in a membrane loading challenge of 1.8 x 10⁻⁶. 18 One capsid / m 2 The initial volume of HIC library 2 was 795 mL, and the capsid titer was 2.2 x 10⁻⁶. 12 One capsid per mL resulted in a membrane loading challenge of 5.5 x 10⁻⁶. 17 One capsid / m 2 These sample-loading challenges are targeted as 5x10 in TFF2. 17 Up to 2x10 18 One capsid / m 2 Within the scope. Each HIC library is condensed to achieve 1x10. 14 The target is a capsid titer per unit of capsid.

[0216] Table 11 shows the initial volume and titer, as well as a summary of the titers obtained after TFF-2 and percolation.

[0217] Table 11: Titers of HIC Library 1 and Library 2 before and after TFF-2 and percolation

[0218]

[0219] During concentration, the permeate flow rate of HIC library 1 was approximately 1 mL / min, and concentration of this library took two days (560 minutes). The membrane was flushed after the first day, and a new membrane was used on the second day. The permeate flow rate of HIC library 2 was approximately 3 mL / min, and concentration was completed in one day (200 minutes).

[0220] After concentration, the solutions were buffer-exchanged using a neutralized affinity buffer (100 mM glycine, 100 mM NaCl, pH 3.3, containing 2% (v / v%) 1 M Tris, 100 mM NaCl, pH 10.9). Seven perfiltration volumes were planned for each buffer-exchange step; however, the solutions became turbid after reaching 193 mL and 234 mL for HIC library 1 and HIC library 2, respectively. At this point, both buffer-exchange processes were stopped. The buffer-exchanged and concentrated libraries were then filtered through a 25 mm EDF syringe filter. These samples were subsequently named BE HIC library 1 and BE HIC library 2.

[0221] AEX chromatography

[0222] Using pre-filled 10 mL Poros HQ50 resin, buffer-exchanged BE HIC library 1 and library 2 samples were further processed in an AEX procedure (as described above). The estimated loading challenge for BE HIC library 1 was 1.7 x 10⁻⁶. 14 One capsid per mL of resin, BEHIC library 2 is 1.1 x 10⁻⁶. 14 One capsid per mL of resin.

[0223] Chromatograms obtained from the AEX purification step using BE HIC library 1 and BE HIC library 2 are shown below. Figure 9 and Figure 10 . Figure 11 The image shows a superimposed chromatogram of two AEX runs. Figure 12 The typical AEX chromatogram for gradient elution using AF product material is shown.

[0224] Chromatograms using the AEX step of BE HIC library 1 ( Figure 7 The chromatogram exhibits typical flow patterns and gradient elution with peak 1. However, gradient elution with peak 2 is almost nonexistent. On the other hand, the chromatogram obtained using the AEX step of the BE HIC library 2 ( Figure 8 There was no flow; there was a small peak (gradient 1) and a high peak (gradient 2) elution. AEX chromatograms of BE HIC library 1 and BE HIC library 2 were overlaid. Figure 11 Similar to a typical AEX chromatogram. Figure 12 ).

[0225] The 260 nm and 280 nm areas of each AEX fraction of the BE HIC library 1 and BE HIC library 2 loading materials are shown in Tables 12 and 13, respectively. For BE HIC library 1 loading, AEX library 1 accounted for 73% of the normalized 260 nm area, while FTW accounted for 16%, and AEX library 2 accounted for only 10% (Table 12). The A260 / A280 ratio of HIC library 1 was 1.38, indicating that the library was enriched with DNA-containing capsids.

[0226] Table 12: Chromatogram areas at 260 nm and 280 nm (mAU*mL) of BE HIC library 1-AEX

[0227]

[0228] For BE HIC library 2, the FTW UV absorbance was below the detection limit, and the normalized 260 nm area of ​​AEX library 1 was <5%, indicating that the capsid concentration in these libraries was low. AEX library 2 had most of the normalized 260 nm area (92%), but the A260 / A280 ratio was low (1.08), indicating that the DNA capsid concentration in this library was low (Table 13).

[0229] Table 13: Chromatogram areas at 260 nm and 280 nm (mAU*mL) of BE HIC library 2-AEX

[0230]

[0231] AEX chromatography VG and capsid mass balance

[0232] VG and capsid yield data for AEX chromatographic steps using materials loaded from BE HIC Library 1 and BE HIC Library 2 are shown below. Figure 13 and Figure 14 Yields were calculated based on titers from the BE HIC library (due to the variability in titers from AEX loading) and normalized to mass balance.

[0233] Based on the yield data, the capsid of BE HIC library 1 was distributed across the AEX FTW, AEX library 1, and AEX library 2 fractions. However, most of the VG in HIC library 1 was recovered in AEX library 1 (the product library). The VG yields in FTW and library 2 were <5%. These results are consistent with the 260 nm normalized area of ​​these libraries and the high A260 / A280 ratio of AEX library 1 (Table 12).

[0234] from Figure 14 The BE HIC library 2 material mainly consists of capsids and VG recovered from the AEX non-product library 2 fraction (>94%). VG and capsid yields in AEX library 1 and the FTW fraction are <6%. These results are consistent with the 260 nm normalized area of ​​these libraries (Table 13).

[0235] AEX analytical ultracentrifugation (AUC)

[0236] The AUC results for each fraction using the AEX chromatographic steps of materials from BE HIC Library 1 and BE HIC Library 2 are shown in Tables 14 and 15, respectively.

[0237] Table 14: Summary of AUC results for each level in the BE HIC library 1 - AEX step

[0238]

[0239] Table 15: Summary of AUC results for each level in the BE HIC library 2 - AEX step

[0240]

[0241] AUC results from the AEX chromatographic step using BE HIC library 1 showed that AEX FTW and AEX library 2 consisted mainly of empty capsids (Table 14). The product peak (AEX library 1) consisted of 87.7% intact capsids, similar to the AUC results of the HQ1 product obtained in DEV-000169 (82.4% intact capsids).

[0242] For the BE HIC library 2 material (Table 15), the AUC results for AEX library 1 (product peak) showed a higher percentage of empty capsid (18.8%) and a lower percentage of intact capsid (72.9%), indicating that this enrichment step was less successful than BE HIC library 1. The AEX library 2 fraction had a higher percentage of intact capsid, comparable to the loaded sample. The AEX FTW fraction of this process was not submitted for AUC analysis due to its low titer.

[0243] Figure 15 The AUC spectra of AEX Library 1 and AEX Library 2 fractions using the AEX chromatographic steps of BE HIC Library 1 are shown.

[0244] Residues, CE and SEC results

[0245] Table 16 shows the capillary electrophoresis (CE) results of three runs (HIC chromatography, AEX chromatography of BE HIC library 1, and AEX chromatography of BE HIC library 2) for libraries 1 and 2. All libraries were concentrated prior to testing. The concentration of HIC library 2—AEX library 1 was too low to be submitted for CE.

[0246] Table 16: Results of CE execution

[0247]

[0248] All samples had a purity percentage above 99%. The electrophoresis patterns of BE HIC library 1 and HIC library 1-AEX library 1 showed three peaks at a retention time of approximately 24 minutes, a typical characteristic of DNA. These are the libraries of interest (the product libraries of each process). HIC library 1-AEX library 1 had the highest DNA peak and the highest percentage of intact AUC. Other electrophoresis patterns showed very small to non-existent DNA peaks.

[0249] The area percentages of VP1, VP2, and VP3 varied among samples. For HIC chromatographic samples, the more hydrophobic BE HIC library 2 had a lower VP2 area and a higher VP3 area than the less hydrophobic BE HIC library 1 (Table 16). It is unclear whether this variation is related to increased capsid hydrophobicity. The maximum VP2 peak value in HIC library 2—AEX library 2 was also lower than the maximum VP1 peak value, but the VP2 area of ​​this sample was similar to other AEX elution libraries (area % VP2 ~ 10.4%).

[0250] Size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) results are shown in Table 17. Except for BE HIC library 1 and BE HIC library 2, which were concentrated, buffer-exchanged samples, the SEC samples were not concentrated before analysis.

[0251] Table 17: Results of the SEC Execution

[0252]

[0253] Typically, the capsid titer is below 4 x 10⁻⁶. 12 Samples with 1 capsid / mL had high aggregate percentages. These samples had low absorbance signals and high noise in the monomer peaks, which increased the aggregate percentage. HIC Library 1 and HIC Library 2 were the original fractions from the HIC step, while BE HIC Library 1 and BE HIC Library 2 were samples from the HIC step after concentration, buffer exchange, and EDF filtration. For both libraries, the aggregate percentage decreased after TFF-2. The lowest aggregate percentage (0.40%) was obtained for the product peak HIC Library 1 – AEX Library 1. Table 18 shows the hcDNA and HCP remnants of the two major product peaks of this process. Table 19 shows the plasmid remnants of the major product peaks of this process. For BE HIC Library 1 and HIC Library 1 – AEX Library 1, the concentrations of all process remnants were low.

[0254] Table 18: Residual hcDNA and HCP in the main product library

[0255]

[0256] Table 19: Plasmid Residues in Major Product Libraries

[0257]

[0258] According to the experiment described in this embodiment, HIC was first performed to separate HIC library 1 (the material with the lowest hydrophobicity), and then AEX was performed to separate AEX library 1 (the material with the lowest charge). Figure 16 As shown, the target product resulted in 0% deamidation of N57.

[0259] Example 4: Identifying and controlling AAV charge heterogeneity through optimized bioreactor operation

[0260] The minute charge differences within the capsid of recombinant adeno-associated virus (rAAV) have traditionally been utilized by anion exchange chromatography (AEX) to separate empty and intact capsids. During this step with rAAV9, non-uniform charge distribution of the capsid was observed and was found in various AEX fractions eluted at different salt concentrations. The observed broad distribution of the capsid indicates the presence of heterogeneous charge distribution. To understand the origin of this heterogeneity, production time in the bioreactor was manipulated to determine the location of maximum heterogeneity in the system (intracellular or extracellular).

[0261] HEK293 cells were transfected using a transient transfection platform and incubated for 3 or 6 days before harvesting. In one group, detergent lysis was performed on day 3 post-transfection, and the complete lysate was collected. For the other group, cells and supernatant were separated by centrifugation on days 3 and 6 post-transfection. The cell pellet was resuspended and lysed to extract the inner capsid. After harvesting, all feed streams were purified using affinity chromatography, followed by an AEX purification step to enrich the intact capsid and examine the observed charge distribution.

[0262] Empty capsids exhibited a more heterogeneous charge distribution than DNA-containing capsids, and surprisingly, inner capsids carried more negative charge than outer capsids. Further investigation revealed that outer capsids consisted of fewer empty capsids, which helps explain their more uniform charge distribution. LC-MS / MS of various feed streams unexpectedly showed that inner capsids fresh from the bioreactor contained more asparagine N57 deamidation than outer capsids, with levels increasing from day 3 to day 6 post-transfection. A linear correlation was found between N57 deamidation and capsid detachment loss to AEX, demonstrating that N57 deamidation was the cause of the increased charge heterogeneity observed via AEX mass balance. For all tested feed streams, N57 deamidation in the product was reduced to approximately half by AEX; however, this step was insufficient to completely remove the deamidated species. For all feed streams, titer (measured by GFP expression and fluorescence) decreased significantly from day 3 to day 6 post-transfection.

[0263] Therefore, deamidation at N57 occurs within the bioreactor, leading to the charge heterogeneity observed in the AEX step. While AEX is capable of removing some deamidated species generated via the bioreactor process, harvest time is a more critical parameter that can be controlled to reduce N57 deamidation and increase the titer of the rAAV product. Finally, scaled-up data show that by shortening the bioreactor duration to two days post-transfection, N57 deamidation can be further reduced and titer increased without significantly sacrificing titer.

[0264] Example 5: Comparison of upstream processes on day 2 and day 3 at different post-transfection temperatures

[0265] To determine the impact of upstream processes on functional capsid production, cells were transfected with AAV9 virus, and the post-transfection temperature was maintained at 37°C or transitioned to 39°C prior to harvest. At each temperature, cells were harvested from the bioreactor on days 2 and 3 post-transfection. The transfected cells were then lysed and purified. Purification included affinity chromatography followed by an AEX purification step to further enrich intact capsids.

[0266] After AEX purification, the VG and capsid yields in the FTW fraction, product fraction, and stripping fraction were evaluated. See Figure 17A and 17B From day 2 to day 3, a slight decrease in product yield was observed at both 37°C and 39°C. Close examination suggested this was likely due to an increase in VG found in the stripping fraction. Interestingly, for both temperatures, the VG titer on day 3 showed a slight increase in stripping loss compared to day 2, indicating a stronger negative charge in the feed stream. Regarding capping yield, a decrease in FTW loss was observed on day 3 compared to day 2, and an increase in capping stripping loss was observed on day 3 compared to day 2. These findings also support the feed stream having a greater negative charge. AUC studies of the day 2 and day 3 harvests showed a higher percentage of empty caps in the product peaks and a higher percentage of intact caps in the non-product peaks from stripping on day 3 compared to day 2, further supporting the increased negative charge in the feed stream. See [link to relevant documentation]. Figure 18 .

[0267] Given the high correlation between potency and deamidation percentage, both parameters were examined after AEX purification. Capsids harvested on day 2 post-transfection exhibited higher potency compared to day 3; furthermore, these capsids showed the highest overall relative potency when the post-transfection temperature was set at 37°C. Figure 19A Notably, the detected percentage of N57 deamidation was correlated with titer. As deamidation increased, titer decreased. This was particularly evident in cells harvested on day 3 at 39°C, as these cells exhibited significantly lower titers and the highest percentage of N57 deamidation. See [link to relevant documentation]. Figure 19B .

[0268] Studies of VG and capsid productivity in the overall upstream process showed that both VG and capsid productivity increased from day 2 to day 3; however, the overall recovery decreased throughout the upstream process. This was observed at both post-transfection temperatures. See Figure 20A and 20B .

Claims

1. An adeno-associated virus (AAV) product comprising: (a) A functional capsid, wherein the functional capsid contains a genetically modified organism; and (b) Less deamidated coating compared to control AAV products purified only by ultracentrifugation or by anion exchange chromatography (AEX) without the use of a fatty acid mobile phase.

2. The AAV product of claim 1, wherein the AAV product contains less N57 deamidation coating than a control AAV product purified solely by AEX without using a fatty acid mobile phase.

3. The AAV product according to claim 1 or 2, wherein the AAV product comprises less than 10% of a cap having deamide N57 on VP1.

4. The AAV product of claim 3, wherein the AAV product comprises less than 5% of a cap having deamide N57 on VP1.

5. The AAV product of claim 4, wherein the AAV product comprises less than 1% of a cap having deamide N57 on VP1.

6. The AAV product of claim 1, wherein the AAV product comprises less than 10% non-functional capping.

7. The AAV product of claim 1, wherein the AAV product exhibits higher transgenic expression in host cells than a control AAV product purified by AEX using only ultracentrifugation or without the use of a fatty acid mobile phase.

8. The AAV product of claim 1, wherein the AAV product has a lower VP1 to VP2 ratio than the control AAV product purified by AEX alone or without the use of a fatty acid mobile phase.

9. The AAV product according to claim 1, wherein deamidation is detected by liquid chromatography-mass spectrometry.

10. An adeno-associated virus (AAV) product, which is essentially composed of a functional capsid containing genetically modified organisms.

11. The AAV product of claim 10, wherein the functional capping is separated from the AAV capping group comprising the deamide capping to produce the AAV product.

12. The AAV product according to claim 10 or 11, wherein the AAV product is relatively free of a cap in which VP1 has deamide N57.

13. An adeno-associated virus (AAV) product comprising a functional capsid, wherein the functional capsid contains a genetically modified organism, and wherein the product is less hydrophobic than a control AAV product purified solely by ultracentrifugation or by anion exchange chromatography (AEX) without the use of a fatty acid mobile phase.

14. The AAV product of claim 13, wherein the AAV product comprises less than 10% non-functional coating.

15. The AAV product according to claim 13 or 14, wherein the AAV product exhibits higher transgenic expression in host cells than a control AAV product purified solely by ultracentrifugation or by AEX without the use of a fatty acid mobile phase.

16. The AAV product of claim 13, wherein the AAV product has a lower VP1 to VP2 ratio than a control AAV product purified by ultracentrifugation or by AEX without using a fatty acid mobile phase.

17. An adeno-associated virus (AAV) product comprising a capsid, wherein the AAV product comprises less than 10% of a capsid having deamidide N57 on VP1.

18. The AAV product of claim 17, wherein the AAV product comprises less than 5% of a cap having deamide N57 on VP1.

19. The AAV product of claim 18, wherein the AAV product comprises less than 1% of a cap having deamide N57 on VP1.

20. A method for removing at least one nonfunctional capsid subgroup from an adeno-associated virus (AAV) product, comprising separating the transgenic functional capsid from the nonfunctional capsid subgroup based on hydrophobicity.

21. The method of claim 20, wherein the separation of the functional capping shell from the non-functional capping shell is further based on charge.

22. The method of claim 20 or 21, wherein the removed nonfunctional capping comprises a deamide capping.

23. The method of claim 22, wherein the removed deamidated cap comprises VP1 having deamidated N57.

24. The method of claim 20, wherein separating the functional capsid containing the functional transgene from the non-functional capsid subgroup comprises: (a) Hydrophobic interaction chromatography (HIC) procedure, (b) Anion exchange chromatography using a fatty acid mobile phase, or (c) Mixed-mode anion exchange chromatography procedure.

25. The method of claim 24, wherein the separation includes the HIC step.

26. The method of claim 25, wherein the separation further comprises anion exchange chromatography (AEX) step.

27. The method of claim 26, wherein the AEX step is performed without using a fatty acid mobile phase.

28. The method of claim 26 or 27, wherein the HIC step is performed prior to the AEX step.

29. The method of claim 24, wherein the separation comprises an anion exchange chromatography step using a fatty acid mobile phase.

30. The method of claim 29, wherein the fatty acid mobile phase comprises octanoic acid or heptanoic acid.

31. The method of claim 24, wherein the separation comprises a mixed-mode resin chromatography step.

32. The method according to claim 31, wherein the mixed-mode resin is a hydrophobic anion exchange resin.

33. The method of claim 24, further comprising transducing target cells in a bioreactor to produce unseparated capsids, wherein the unseparated capsids are maintained in the bioreactor for up to two days after target cell transfection.

34. An AAV product produced by the method of claim 20.

35. A method of treating a subject requiring a protein produced by a genetic modification, comprising administering the AAV product of claim 1 to said subject.

36. An adeno-associated virus (AAV) product comprising a transgenic AAV capsid, wherein the AAV product exhibits higher transgenic expression in host cells than a control AAV product purified solely by ultracentrifugation or by AEX without the use of a fatty acid mobile phase.

37. An adeno-associated virus (AAV) product comprising a genetically modified AAV capsid, wherein the AAV product has a lower VP1 to VP2 ratio than a control AAV product purified by ultracentrifugation or by AEX without the use of a fatty acid mobile phase.

38. The method of claim 20, wherein the AAV capsid is harvested approximately 2 days after transfection.

39. The method according to claim 20, wherein the post-transfection temperature is 37°C.

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