Improved methods for purification of recombinant aav vectors

0001Cross-reference of related applications This application claims the priority of US Provisional Patent Application No. 61 / 187,601 filed June 16, 2009, which is incorporated herein by reference in its entirety.

0002Field of invention The invention as a whole relates to the field of purification of recombinant adeno-associated virus (rAAV) vectors that can be used for gene transfer and specifically for gene therapy or vaccination. More specifically, the present invention relates to a method for purifying a recombinant rAAV vector that is substantially free of in-process components such as cell nucleic acids, cell proteins, helper viruses and media components.

0003Background of the invention Adeno-associated viruses (AAV) have unique characteristics that make them attractive as vectors for gene therapy and gene vaccines. Infection of cultured cells with AAV is noncytopathic, and natural infections in humans and other animals are asymptomatic, asymptomatic, and are not associated with the pathogenesis of any human disease. In addition, AAV infects a wide range of cell types, including many mammalian cells, and offers the potential to target many different tissues in vivo. AAV slowly infects dividing and non-dividing cells and can essentially survive the lifespan of these cells as transcriptionally active nuclear episomes (exchromosomal elements). Copies of the rAAV vector that integrate into organs such as the liver or muscle are very rare. Efficient long-term introgression has been reported in several cell tumors, including the eye, CNS and muscle. For example, X. Xiao et al., J. Virol. 70 (11): 8098-8108 (1996) (Non-Patent Document 1); RR Ali et al., Hum. Mol. Genet. 5 (5): 591-94 (1996) (Non-Patent Document 2). Current clinical studies have primarily focused on the use of serotype 2 rAAV vectors, but several reports have shown that other AAV serotypes, including rAAV-1, rAAV-4, rAAV-5 and rAAV-8. The types have been demonstrated to have a unique in vivo biodistribution that makes them attractive viral serotypes to be tested in clinical trials.

0004Adeno-associated virus (AAV) is a replication-defective parvovirus whose single-stranded DNA genome is approximately 4.7 kb long, including 145 nucleotides of reverse-terminal repeat (ITR). The nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J. Virol., 45: 555-564 (1983) (Non-Patent Document 3), and Ruffing et al., J. . Gen. Virol., It has been corrected by 75: 3385-3392 (1994) (Non-Patent Document 4). Sith-operated sequences that direct viral DNA replication (rep), capsid formation / packaging, and host cell chromosomal integration are included in the ITR. Three AAV promoters p5, p19 and p40 (Named after its relative map position) drives the expression of two AAV internal reading slots encoding the rep and cap genes. The two rep promoters (p5 and p19) are linked to the differential splicing of a single AAV intron at nucleotide numbers 2107 and 2227, and the four rep proteins from the rep gene (rep78, rep68, rep78, rep68, It results in the production of rep52 and rep40). The rep protein possesses multiple enzymatic properties that are ultimately responsible for the replication of the viral genome. The cap gene is expressed from the p40 promoter, which encodes the three capsid proteins VP1, VP2 and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of three related capsid proteins. A single consensus sporiadenylation site is located at map position 95 of the AAV genome. AAV Life Cycle and Genetics, Muzyczka, Current Topics in Microbiology and Immunology, It is outlined in 158: 97-129 (1992) (Non-Patent Document 5).

0005AAV particles contain a proteinaceous capsid with three capsid proteins VP1, VP2 and VP3, which enclose a linear single-stranded DNA genome of approximately 4.6 kb. Each particle packages only one DNA strand, which can be either a positive or negative strand. Particles containing either strand are infectious and are replicated by the conversion of the parent's infectious single strand to a double-stranded form, followed by amplification, which results in the single strand of the offspring. Replaced and packaged in capsid. A double- or single-stranded copy of the AAV genome (sometimes referred to as "provirus DNA" or "provirus") can be inserted into a bacterial plasmid or phagemid and transfected into adenovirus-infected cells. For a review of AAV, see Carter, HANDBOOK OF PARVOVIRUSES, Vol. I, pp. 169-228 (1989) (Non-Patent Document 6) and Berns, VIROLOGY, pp. See 1743-1764, Raven Press, (1990) (Non-Patent Document 7).

0006The production of rAAV vectors generally requires four common elements: (1) tolerant host cells for replication; (2) by or instead of a suitable helper virus such as adenovirus or herpesvirus. Helper virus function that can be provided by a plasmid construct containing minimal adenovirus helper function; (3) transpacking rep-cap construct; and (4) suitable production medium.

0007Recombinant AAV particles can be produced from packaging cell lysates. See, for example, Chirico and Trempe (1998) J. Virol. Methods 76: 31-41 (Non-Patent Document 8). However, cell lysates must be isolated from various cellular components, such as host cell DNA, host cell proteins, medium components and helper viruses or helper viruses, prior to being suitable for in vivo use. Contains plasmid DNA. Recent advances in rAAV production include the use of non-adherent cell suspension steps in agitated tank bioreactors and the use of production conditions in which the rAAV vector is released into the medium or supernatant, thereby being present in the product. Although the concentration of host cell components has been reduced, it still contains significant amounts of in-process impurities. See U.S. Pat. No. 6,566,118 (Patent Document 1) and PCT WO 99/11764 (Patent Document 2). Therefore, rAAV particles can be recovered from the medium and / or cell lysates and further purified.

0008Methods involving the density gradient centrifugation used to purify the rAAV vector and specifically rAAV-2 are not suitable for scale-up. Recent reports on the rAAV-2 vector describe purification methods using ion exchange chromatography, including opposing ion exchange chromatography (including cation and anion chromatography). For example, US Pat. No. 6,566,118 and US Pat. No. 6,566,118, which disclose a method of using a combination of counterion exchange chromatography to purify a recombinant adeno-associated virus vector from culture supernatant and / or cell lysate. Please refer to PCT WO 99/11764 (Patent Document 2). Further improvements in rAAV feedstock preparation include the use of deoxycholate treatment of cell lysates, the use of iodixanol gradient separation prior to affinity chromatography, which yield high titers rAAV2 (Clark et al.,, Hum. Mol. Genet. 10 (6): 1031-39 (1999) (Non-Patent Document 9); Zolotukhin et al., Gene Therapy 6 (6): 973-985 (1999) (Non-Patent Document 10)). O'Riordan et al. (O'Riordan et al., J. Gene Med. 2: 444-454 (2000) (Non-Patent Document 11); US Pat. No. 7,015,026 (Patent Document 3)) also provides ion exchange chromatography. For recombinant adeno-associated virus vectors using hydroxyapatite chromatography, cellfine sulfate affinity chromatography and zinc chelate chromatography, and for rAAV-2 vectors, as specifically exemplified. It reports on an expandable purification process.

0009From recent data, rAAV capsid serotypes such as rAAV-1, 4, 5 and 8 can be used as purified viral reservoirs or as host cell DNA, host cell proteins, serum albumin, medium and helper virus components. It has been suggested that it binds weakly to the anion resin in the presence of in-process impurities. Therefore, purification of their capsid serotypes typically involves anion exchange chromatography in combination with other purification methods, such as iodixanol density gradient centrifugation. For example, Zolotukhin et al., Methods 28 (2): 158-167 (2002) (Non-Patent Document 12) and Kaludov et al., Hum. Gene Therapy 13: 1235-1243 (2002) (Non-Patent Document 13); See US Patent Application Publication No. 2004/0110266 A1 (Patent Document 4). However, these methods are not easily extendable to industrial scale processes.

0010Therefore, in the development of recombinant AAV vectors, such as those for use in gene therapy and gene vaccines, helper viruses, as well as helper virus proteins, cell proteins, host cell DNA and medium components present in rAAV production reservoirs are included. There is a need for a method of purifying rAAV vectors from in-process components. Such methods should be used effectively on a scale suitable for the practical application of gene therapy techniques. In addition, there is a need to develop a process for purifying the rAAV vector that is expandable to obtain a high titer, high degree of purification commercial stock useful for rAAV gene therapy and gene vaccines. More specifically, there is a need to develop a process for purifying a chromatographic resin, and specifically an rAAV vector that weakly binds to an anion resin.

0011All publications, patent applications and patent disclosures cited herein are by reference as if each individual publication, patent application or patent is specifically and individually indicated as incorporated by reference. Incorporated herein. Specifically, all publications cited herein are expressly incorporated herein by reference for the purpose of describing and disclosing compositions and methods that may be used in connection with the present invention. Although the invention provided herein is described in detail by way of illustration and examples for the purpose of clarifying understanding, the invention is made without departing from the spirit or scope of the appended claims. It will be readily apparent to those skilled in the art in light of the teachings of the present invention that certain changes and modifications can be made to it.

<p num="0012"><patcit num="1"><text>U.S. Pat. No. 6,566,118</text></patcit><patcit num="2"><text>WO 99/11764</text></patcit><patcit num="3"><text>U.S. Pat. No. 7,015,026</text></patcit><patcit num="4"><text>U.S. Patent Application Publication No. 2004/0110266 A1</text></patcit></p>

<p num="0013"><nplcit num="1"><text>X. Xiao et al., J. Virol. 70 (11): 8098-8108 (1996)</text></nplcit><nplcit num="2"><text>RR Ali et al., Hum. Mol. Genet. 5 (5): 591-94 (1996)</text></nplcit><nplcit num="3"><text>Srivastava et al., J. Virol., 45: 555-564 (1983)</text></nplcit><nplcit num="4"><text>Ruffing et al., J. Gen. Virol., 75: 3385-3392 (1994)</text></nplcit><nplcit num="5"><text>Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992)</text></nplcit><nplcit num="6"><text>Carter, HANDBOOK OF PARVOVIRUSES, Vol. I, pp. 169-228 (1989)</text></nplcit><nplcit num="7"><text>Berns, VIROLOGY, pp. 1743-1764, Raven Press, (1990)</text></nplcit><nplcit num="8"><text>Chirico and Trempe (1998) J. Virol. Methods 76: 31-41</text></nplcit><nplcit num="9"><text>Clark et al., Hum. Mol. Genet. 10 (6): 1031-39 (1999)</text></nplcit><nplcit num="10"><text>Zolotukhin et al., Gene Therapy 6 (6): 973-985 (1999)</text></nplcit><nplcit num="11"><text>O'Riordan et al. (O'Riordan et al., J. Gene Med. 2: 444-454 (2000)</text></nplcit><nplcit num="12"><text>Zolotukhin et al., Methods 28 (2): 158-167 (2002)</text></nplcit><nplcit num="13"><text>Kaludov et al., Hum. Gene Therapy 13: 1235-1243 (2002)</text></nplcit></p>

0014The present invention captures rAAV particles on an apatite chromatography medium in the presence of polyethylene glycol (PEG) to obtain a population of recombinant adeno-associated virus (rAAV) particles of any capsid serotype from in-process impurities. A method for isolation is provided. In the methods of the invention, upstream treatments (eg, centrifugation, benzonase® treatment, anion exchange filtration and / or tangential flow filtration, etc.), and downstream treatments (eg, thermal inactivation, filtration, hydrophobicity, etc.) Sex interaction chromatography, size exclusion chromatography and / or anion exchange chromatography, etc.) are required. Upstream and downstream methods may be used alone or in various combinations.

0015The present invention is a step in which (a) a feed stream containing recombinant adeno-associated virus (rAAV) particles is brought into contact with an apatite chromatography medium in the presence of polyethylene glycol (PEG), wherein the rAAV particles are the apatite. Feeding, including the steps of binding to a chromatography medium; and (b) eluting the rAAV particles bound to the apatite chromatography medium with an elution buffer containing less than 3% (w / v) PEG. Provided is a method for isolating a population of rAAV particles from in-process impurities in the stream. In certain embodiments, the apatite chromatography medium is ceramic hydroxyapatite (CHT) or ceramic fluoroapatite (CFT). In certain embodiments, the rAAV particles bound to the apatite chromatography medium are eluted with an elution buffer containing less than 3% (w / v) of PEG. In certain embodiments, the rAAV particles bound to the apatite chromatography medium are eluted with an elution buffer in the absence of PEG.

0016In some embodiments, the specific binding of the apatite chromatography medium is 10⁶~10¹⁶DNase resistant particles (DRP) / ml. In some embodiments, the specific binding of the apatite chromatography medium is 10⁸~10¹⁶DNase resistant particles (DRP) / ml. In some embodiments, the specific binding of the apatite chromatography medium is 10¹⁰~10¹⁶DNase resistant particles (DRP) / ml. In some embodiments, the specific binding of the apatite chromatography medium is 10¹²~10¹⁶DNase resistant particles (DRP) / ml. In some embodiments, the specific binding of the apatite chromatography medium is 10¹⁴~10¹⁶DNase resistant particles (DRP) / ml.

0017In some embodiments, the method further comprises an anion exchange filtration step prior to the apatite chromatography step, where the rAAV particles are in the pass-through fraction of the anion exchange filtration. In some embodiments, the method further comprises concentrating rAAV particles from the pass-through fraction of anion exchange filtration by tangential flow filtration prior to the apatite chromatography step. In some embodiments, the method further comprises binding the rAAV particles in the feed stream eluted from the apatite chromatography medium to the anion chromatography medium. In some embodiments, the method further comprises a heat inactivation step to inactivate the helper virus. In some embodiments, the method further comprises binding the rAAV particles in the feed stream to hydrophobic interaction chromatography after apatite chromatography.

0018In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of polyethylene glycol (PEG) and basic buffer. In some embodiments, the basic buffer is pH 7.2-10, pH 7.4-10, pH 7.6-10, pH 7.8-10, pH 8.010.0, pH 8.210.0, pH 8.410.0, pH 8.610.0. , PH 8.8 ~ 10, pH 9.0 ~ 10.0, pH 9.2 ~ 10, pH 9.4 ~ 10.0, pH 9.6 ~ 10.0, or pH 9.8 ~ 10.0. In some embodiments, the basic buffer has a pH of approximately any of 7.2, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8 and 10.0. Any basic buffer known in the art can be used. In some embodiments, the basic buffer comprises borate. In some embodiments, the basic buffer is borate.

0019In some embodiments, the feed stream containing the rAAV particles is contacted with an apatite chromatography medium in the presence of polyethylene glycol (PEG). For example, about 3% (w / v) to about 10% (w / v) of PEG can be used. In some embodiments, the feed stream containing rAAV particles is about 3% (w / v), about 3.5% (w / v), about 4% (w / v), about 4.5% (w / v). , About 5% (w / v), about 5.5% (w / v), about 6% (w / v), about 6.5% (w / v), about 7% (w / v), about 7.5% ( w / v), about 8% (w / v), about 8.5% (w / v), about 9% (w / v), about 9.5% (w / v) or about 10% (w / v) Contact with apatite chromatography medium in the presence of PEG.

0020In some embodiments, PEG is, for example, about 5,000 (PEG5000) grams per mole, about 6,000 (PEG6000) grams per mole, about 7,000 (PEG7000) grams per mole, about 8,000 (PEG8000) grams per mole. , Approximately 9,000 (PEG9000) grams per mole, approximately 10,000 (PEG10000) grams per mole, approximately 11,000 (PEG11000) grams per mole, approximately 12,000 (PEG12000) grams per mole, approximately 13,000 (PEG13000) grams per mole It has an average molecular weight of about 5,000 (PEG5000) grams to about 15,000 (PEG15000) grams per mole, such as about 14,000 (PEG14000) grams per mole and about 15,000 (PEG15000) grams per mole. In certain embodiments, PEG has an average molecular weight of approximately 5,000 (PEG5000) grams per mole. In certain embodiments, PEG is about 6,000 per mole. It has an average molecular weight of (PEG6000) grams. In certain embodiments, PEG has an average molecular weight of approximately 8,000 (PEG8000) grams per mole. In certain embodiments, PEG has an average molecular weight of approximately 10,000 (PEG10000) grams per mole. In certain embodiments, PEG has an average molecular weight of approximately 15,000 (PEG15000) grams per mole.

0021In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of about 3% (w / v) to about 10% (w / v) of PEG6000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 3% (w / v) PEG6000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 4% (w / v) PEG6000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 5% (w / v) PEG6000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 6% (w / v) PEG6000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 7% (w / v) PEG6000. In some embodiments, the feed stream containing rAAV particles is approximately 8%. Contact with apatite chromatography medium in the presence of (w / v) PEG6000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 9% (w / v) PEG6000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 10% (w / v) PEG6000.

0022In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of about 3% (w / v) to about 10% (w / v) of PEG8000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 3% (w / v) PEG8000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 4% (w / v) PEG8000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 5% (w / v) PEG8000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 6% (w / v) PEG8000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 7% (w / v) PEG8000. In some embodiments, the feed stream containing rAAV particles is approximately 8%. Contact with apatite chromatography medium in the presence of (w / v) PEG8000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 9% (w / v) PEG8000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 10% (w / v) PEG8000.

0023In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of about 3% (w / v) to about 10% (w / v) of PEG10000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 3% (w / v) PEG10000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 4% (w / v) PEG10000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 5% (w / v) PEG10000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 6% (w / v) PEG10000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 7% (w / v) PEG10000. In some embodiments, the feed stream containing rAAV particles is approximately 8%. Contact with an apatite chromatography medium in the presence of (w / v) PEG10000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 9% (w / v) PEG10000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 10% (w / v) PEG10000.

0024In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of about 3% (w / v) to about 10% (w / v) of PEG15000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 3% (w / v) PEG15000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 4% (w / v) PEG15000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 5% (w / v) PEG15000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 6% (w / v) PEG15000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 7% (w / v) PEG15000. In some embodiments, the feed stream containing rAAV particles is approximately 8%. Contact with an apatite chromatography medium in the presence of (w / v) PEG15000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 9% (w / v) PEG15000. In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in the presence of approximately 10% (w / v) PEG15000.

0025In some embodiments, a feed stream containing rAAV particles is contacted with an apatite chromatography medium in a buffer containing approximately 20 mM borate pH 9.0 and approximately 5% PEG (such as PEG6000). In some embodiments, the feed stream is mixed in-line with an equal volume of buffer containing about 40 mM borate pH 9.0 and about 10% PEG to give a final concentration of about 20 mM borate pH 9.0 and about 5% PEG. obtain.

0026In some embodiments, the apatite chromatography medium to which the rAAV particles are attached is washed to remove in-process impurities before elution of the rAAV particles. In some embodiments, the apatite chromatography medium is washed once or multiple times with a wash buffer containing a diminishing concentration of PEG to remove in-process impurities. In some embodiments, the apatite chromatography medium is washed once or multiple times with wash buffer containing about 3% (w / v) to about 10% (w / v) PEG. In some embodiments, the wash buffer is 10% (w / v), 9.5% (w / v), 9% (w / v), 8.5% (w / v), 8% (w / v). , 7.5% (w / v), 7% (w / v), 6.5% (w / v), 6% (w / v), 5.5% (w / v), 5% (w / v), 4.5 Contains almost any of% (w / v), 4% (w / v), 3.5% (w / v) and 3% (w / v) PEG. In some embodiments, the apatite chromatography medium is 7.5% (w / v). Wash once or multiple times with wash buffer containing PEG6000. In some embodiments, the apatite chromatography medium is washed once or multiple times with wash buffer containing 7.5% (w / v) PEG8000. In some embodiments, the apatite chromatography medium is washed once or multiple times with wash buffer containing 7.5% (w / v) PEG10000. In some embodiments, the apatite chromatography medium is washed once or multiple times with wash buffer containing 7.5% (w / v) PEG15000. In some embodiments, the apatite chromatography medium is washed once or multiple times with wash buffer containing approximately 5% (w / v) PEG6000. In some embodiments, the apatite chromatography medium is washed once or multiple times with wash buffer containing approximately 5% (w / v) PEG8000. In some embodiments, the apatite chromatography medium is about 5% (w / v). Wash once or multiple times with wash buffer containing PEG10000. In some embodiments, the apatite chromatography medium is washed once or multiple times with wash buffer containing approximately 5% (w / v) PEG15000. In some embodiments, the apatite chromatography medium is washed once or multiple times with wash buffer containing less than about 3% (w / v) of PEG6000. In some embodiments, the apatite chromatography medium is washed once or multiple times with wash buffer containing less than about 3% (w / v) of PEG8000. In some embodiments, the apatite chromatography medium is washed once or multiple times with wash buffer containing less than about 3% (w / v) of PEG10000. In some embodiments, the apatite chromatography medium is washed once or multiple times with wash buffer containing less than about 3% (w / v) of PEG15000. In some embodiments, the apatite chromatography medium is washed once or multiple times with PEG-free wash buffer.

0027In some embodiments, the cleaning buffer contains a buffer known in the art. In some embodiments, the wash buffer comprises a buffer selected from the group consisting of borate, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and Tris-HCl. In some embodiments, the wash buffer comprises or is borate. In some embodiments, the wash buffer comprises or is HEPES. In some embodiments, the wash buffer comprises Tris-HCl or is Tris-HCl. In some embodiments, the wash buffer is at basic pH. In some embodiments, the cleaning buffer is pH 7.0-pH 10.0, pH 7.2-pH 10.0, pH 7.4-pH 10.0, pH 7.6-pH 10.0, pH 7.8-pH 10.0, pH 8.0-pH 10.0, pH 8.2-. pH 10.0, pH 8.4 ~ pH 10.0, pH 8.6 ~ pH 10.0, pH 8.8 ~ pH 10.0, pH 9.0 ~ pH 10.0, pH 9.2 ~ pH 10.0, pH 9.4 ~ pH It has a pH of 10.0, pH 9.6 to pH 10.0 or pH 9.8 to pH 10.0. In some embodiments, the wash buffer has a pH of 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8 or 10.0. In some embodiments, the wash buffer comprises or is a pH 8.0 to 10.0 bolate. In some embodiments, the wash buffer comprises a pH 8.0 borate or is a pH 8.0 borate. In some embodiments, the wash buffer comprises a pH 9.0 borate or is a pH 9.0 borate. In some embodiments, the wash buffer comprises a pH 10.0 borate or is a pH 10.0 borate. In some embodiments, the wash buffer comprises HEPES at pH 7.010.0 or is HEPES at pH 7.010.0. In some embodiments, the wash buffer contains HEPES at pH 7.0 or pH. It is HEPES of 7.0. In some embodiments, the wash buffer comprises HEPES at pH 8.0 or is HEPES at pH 8.0. In some embodiments, the wash buffer comprises HEPES at pH 9.0 or is HEPES at pH 9.0. In some embodiments, the wash buffer comprises HEPES at pH 10.0 or is HEPES at pH 10.0. In some embodiments, the wash buffer comprises Tris-HCl with a pH of 7.0 to 10.0 or is Tris-HCl with a pH of 7.0 to 10.0. In some embodiments, the wash buffer comprises Tris-HCl at pH 7.0 or is Tris-HCl at pH 7.0. In some embodiments, the wash buffer comprises Tris-HCl at pH 8.0 or is Tris-HCl at pH 8.0. In some embodiments, the wash buffer comprises Tris-HCl at pH 9.0 or is Tris-HCl at pH 9.0. In some embodiments, the wash buffer contains Tris-HCl pH 10.0 or pH 10.0. 10.0 Tris-HCl. In some embodiments, the wash buffer further comprises 100-500 mM phosphate. In some embodiments, the wash buffer further comprises 50-250 mM NaCl.

0028In some embodiments, the wash step is the first wash with a wash buffer containing about 30 mM borate at pH about 9.0 and about 7.5% PEG; about 150 potassium phosphate, about 20 mM borate at pH about 9.0 and Second wash with wash buffer containing about 5% PEG; third wash with wash buffer containing about 20 mM borate at pH about 9.0 and about 5% PEG; and about 20 mM HEPES at pH about 7.0 And includes a fourth wash with wash buffer containing 150 mM NaCl.

0029In some embodiments, the rAAV particles bound to the apatite chromatography medium are eluted with elution buffer containing low concentrations of PEG or with elution buffer in the absence of PEG. In some embodiments, the elution buffer contains less than about 3% (w / v) PEG, less than about 2% (w / v) PEG or less than about 1% (w / v) PEG. In some embodiments, the elution buffer is about 2.5% (w / v), about 2% (w / v), about 1.5% (w / v), about 1% (w / v) or about 0.5%. (w / v) Contains or does not contain PEG. In some embodiments, the elution buffer contains less than about 3% (w / v) PEG6000. In some embodiments, the elution buffer contains less than about 3% (w / v) of PEG8000. In some embodiments, the elution buffer contains less than about 3% (w / v) of PEG10000. In some embodiments, the elution buffer is approximately 3%. Contains less than (w / v) PEG15000. In some embodiments, the rAAV particles bound to the apatite chromatography medium are eluted with elution buffer in the absence of PEG. In some embodiments, the elution buffer comprises a buffer selected from the group consisting of borate, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and Tris-HCl. In some embodiments, the elution buffer comprises or is bolate. In some embodiments, the elution buffer comprises or is HEPES. In some embodiments, the elution buffer comprises Tris-HCl or is Tris-HCl. In some embodiments, the elution buffer is at neutral pH. In some embodiments, the elution buffer comprises or is HEPES at neutral pH. In some embodiments, the elution buffer comprises tris-HCl at neutral pH or is Tris-HCl at neutral pH. In some embodiments, the elution buffer is 100 It further contains less than mM phosphate. In some embodiments, the elution buffer further comprises less than 50 mM phosphate. In some embodiments, the elution buffer further comprises 50-250 mM NaCl. In some embodiments, rAAV particles bound to an apatite chromatography medium are eluted with an elution buffer containing approximately 50 mM potassium phosphate, approximately 20 mM HEPES at a pH of approximately 7.0 and approximately 150 mM NaCl.

0030In some embodiments, the method of isolating rAAV particles from in-process impurities in the feed stream is (a) about 5% of the feed stream containing the rAAV particles in a basic buffer of about 9.0 pH (a). w / v) The step of contacting with the apatite chromatography medium in the presence of PEG, the step of binding the rAAV particles to the apatite chromatography medium; (b) about 30 mM borate and about 30 mM borate at pH about 9.0. The step of washing the apatite chromatography medium with a first wash buffer containing 7.5% PEG; (c) a second containing about 150 potassium phosphate, about 20 mM borate at pH about 9.0 and about 5% PEG. The step of washing the apatite chromatography medium with wash buffer; (d) the step of washing the apatite chromatography medium with a third wash buffer containing about 20 mM borate at pH about 9.0 and about 5% PEG. (e) Washing the apatite chromatography medium with a fourth wash buffer containing about 20 mM HEPES and 150 mM NaCl at a pH of about 7.0; And (f) the step of eluting the rAAV particles bound to the apatite chromatography medium with an elution buffer containing about 50 mM potassium phosphate, about 20 mM HEPES at pH about 7.0 and about 150 mM NaCl.

0031Also provided herein are (a) contacting a feed stream containing recombinant adeno-associated virus (rAAV) particles with a hydrophobic interaction chromatography (HIC) medium in high salt buffer. The step of binding the rAAV particles and in-process impurities to the HIC medium; And (b) for isolating the population of rAAV particles from the in-process impurities in the feed stream, including the step of eluting the rAAV particles bound to the HIC medium with medium salt buffer. The method. In some embodiments, the HIC medium is selected from the group consisting of Tosoh Butyl 650M, Tosoh SuperButyl 650C, Tosoh Phenyl 650C, EMD Fractogel Phenyl and Tosoh Has (butyl) resins. In some embodiments, the high salt buffer comprises 0.5 M to 2.0 M citrate (eg, sodium citrate) or is 0.5 M to 2.0 M citrate (eg, sodium citrate). In some embodiments, the high salt buffer comprises approximately any of 0.5 M, 0.75 M, 1.0 M, 1.25 M, 1.5 M, 1.75 M and 2.0 M citrate. In some embodiments, the medium salt buffer contains less than 0.5 M of citrate (eg, sodium citrate) or 0.5. Citrate less than M (eg, sodium citrate). In some embodiments, the medium salt buffer comprises 0.5 M to about 0.3 M citrate. In some embodiments, the medium salt buffer comprises approximately any of 0.45 M, 0.4 M, 0.35 M, 0.3 M and 0.25 M citrates. In some embodiments, the high salt buffer further comprises 1-100 mM phosphate. In some embodiments, the medium salt buffer further comprises 1-100 mM phosphate. In some embodiments, the medium salt buffer does not elute rAAV particles with empty capsids, partially denatured capsids, less infectious capsids, and / or partially complete capsids. Is eluted.

0032In any of the embodiments described herein, rAAV particles are AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV. It has an AAV capsid serotype selected from the group consisting of -9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16. In some embodiments, the rAAV particles have an AAV capsid serotype selected from the group consisting of AAV-1, AAV-4, AAV-5 and AAV-8. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV-1. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV-4. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV-5. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV-8. In some embodiments, the rAAV particles are AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, Includes AAV capsid proteins from AAV serum types selected from the group consisting of AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16. In some embodiments, the rAAV particles have an AAV capsid serotype, which is a weak anion binding agent. In some embodiments, the weakly anionic AAV capsid serotype is selected from the group consisting of AAV-1, AAV-4, AAV-5 and AAV-8. In some embodiments, the composition containing the rAAV particles further comprises a production culture contaminant. In some embodiments, the production culture contaminants include damaged rAAV particles, host cell contaminants, helper virus contaminants and / or cell culture contaminants. In some embodiments, the host cell contaminant comprises host cell DNA, plasmid or host cell protein. In some ways And helper virus contaminants include adenovirus particles, adenovirus DNA or adenovirus proteins. In some embodiments, the cell culture contaminants include media components, serum albumin or other serum proteins. In some embodiments, the cell culture contaminants include media components. In some embodiments, the cell culture contaminants are free of serum albumin or other serum proteins.

0033It should be understood that one, some or all of the properties of the various aspects described herein can be combined to form other aspects of the invention.<u style="single">[Invention 1001]</u><u style="single"> (a) A step of contacting a feed stream containing recombinant adeno-associated virus (rAAV) particles with an apatite chromatography medium in the presence of polyethylene glycol (PEG), wherein the rAAV particles are the apatite chromatography medium. Stage of joining to; and</u><u style="single"> (b) The step of eluting the rAAV particles bound to the apatite chromatography medium with an elution buffer containing less than 3% (w / v) of PEG.</u><u style="single">A method for isolating a population of rAAV particles from in-process impurities in a feed stream, including.</u><u style="single">[Invention 1002]</u><u style="single"> The method of the present invention 1001 in which the apatite chromatography medium is ceramic hydroxyapatite (CHT).</u><u style="single">[Invention 1003]</u><u style="single"> The method of 1001 of the present invention, wherein the apatite chromatography medium is ceramic fluoroapatite (CFT).</u><u style="single">[Invention 1004]</u><u style="single"> The specific binding of the apatite chromatography medium to the rAAV particles is 10</u>^{<u style="single">14</u>}<u style="single">~10</u>^{<u style="single">16</u>}<u style="single">The method of 1001 of the present invention, wherein DNase resistant particles / milliliter (DRP / mL).</u><u style="single">[Invention 1005]</u><u style="single"> The method of the present invention 1001 further comprises the step of binding the rAAV particles in the feed stream eluted from the apatite chromatography medium to the anion chromatography medium.</u><u style="single">[Invention 1006]</u><u style="single"> The method of the present invention 1001 in which a feed stream containing rAAV particles in step (a) is brought into contact with an apatite chromatography medium in the presence of polyethylene glycol (PEG) and a basic buffer.</u><u style="single">[Invention 1007]</u><u style="single"> The method of the present invention 1006, wherein the basic buffer has a pH of 7.6 to 10.</u><u style="single">[Invention 1008]</u><u style="single"> The method of the present invention 1006, wherein the basic buffer has a pH of 8.0 to 10.0.</u><u style="single">[Invention 1009]</u><u style="single"> The method of the present invention 1006, wherein the basic buffer has a pH of 9.0 to 10.0.</u><u style="single">[Invention 1010]</u><u style="single"> The method of the present invention 1006, wherein the basic buffer comprises borate.</u><u style="single">[Invention 1011]</u><u style="single"> The method of 1001 of the present invention, wherein the PEG has an average molecular weight of about 5,000 (PEG5000) grams per mole to about 15,000 (PEG15000) grams per mole.</u><u style="single">[Invention 1012]</u><u style="single"> The method of 1011 of the present invention, wherein said PEG has an average molecular weight of about 6,000 (PEG6000) grams per mole.</u><u style="single">[Invention 1013]</u><u style="single"> The method of the present invention 1001 in which the feed stream containing the rAAV particles in step (a) is brought into contact with the apatite chromatography medium in the presence of about 3% (w / v) to about 10% (w / v) PEG. ..</u><u style="single">[Invention 1014]</u><u style="single"> The method of the present invention 1013, wherein the feed stream is brought into contact with the apatite chromatography medium in the presence of approximately 5% (w / v) PEG6000.</u><u style="single">[Invention 1015]</u><u style="single"> The method of the present invention 1013, wherein the feed stream is brought into contact with the apatite chromatography medium in the presence of approximately 10% (w / v) PEG6000.</u><u style="single">[Invention 1016]</u><u style="single"> After contacting the feed stream with the apatite chromatography medium, it further comprises washing the apatite chromatography medium with a wash buffer before eluting the rAAV particles from the apatite chromatography medium. , The method of the present invention 1001.</u><u style="single">[Invention 1017]</u><u style="single"> Wash the apatite chromatography medium once or multiple times with wash buffer containing about 7.5% (w / v) PEG and / or wash buffer containing about 5% (w / v) PEG. The method of the present invention 1016.</u><u style="single">[Invention 1018]</u><u style="single"> The method of the present invention 1017, wherein the apatite chromatography medium is further washed with a wash buffer containing less than about 3% (w / v) of PEG and / or a wash buffer containing no PEG.</u><u style="single">[Invention 1019]</u><u style="single"> The method of the invention 1016, wherein the wash buffer comprises a buffer selected from the group consisting of borate, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and Tris-HCl.</u><u style="single">[Invention 1020]</u><u style="single"> The method of the present invention 1016, wherein the wash buffer has a basic pH.</u><u style="single">[Invention 10 21]</u><u style="single"> The method of the present invention 1020, wherein the cleaning buffer comprises a borate having a pH of about 8.0 to about 10.0.</u><u style="single">[Invention 1022]</u><u style="single"> The method of 1021 of the present invention, wherein the cleaning buffer comprises a borate having a pH of about 8.0.</u><u style="single">[Invention 1023]</u><u style="single"> The method of 1021 of the present invention, wherein the cleaning buffer comprises a borate having a pH of about 9.0.</u><u style="single">[1024 of the present invention]</u><u style="single"> The method of 1021 of the present invention, wherein the cleaning buffer comprises a borate having a pH of about 10.0.</u><u style="single">[Invention 1025]</u><u style="single"> The method of the present invention 1020, wherein the wash buffer further comprises 100-500 mM phosphate.</u><u style="single">[Invention 1026]</u><u style="single"> The method of the present invention 1020, wherein the wash buffer further comprises 50-250 mM NaCl.</u><u style="single">[Invention 1027]</u><u style="single"> The method of the present invention 1001 in which the rAAV particles bound to the apatite chromatography medium are eluted with an elution buffer containing a low concentration of PEG or with an elution buffer in the absence of PEG.</u><u style="single">[Invention 1028]</u><u style="single"> The elution buffer comprises a neutral pH buffer selected from the group consisting of borate, N-2-hydroxyethylpiperazin-N'-2-ethanesulfonic acid (HEPES) and Tris-HCl. The method of invention 1027.</u><u style="single">[Invention 1029]</u><u style="single"> The method of the present invention 1027, wherein the elution buffer contains less than about 3% (w / v) of PEG6000.</u><u style="single">[Invention 1030]</u><u style="single"> The method of 1029 of the present invention, wherein the elution buffer further comprises a phosphate of less than 100 mM.</u><u style="single">[Invention 1031]</u><u style="single"> The method of 1030 of the present invention, wherein the elution buffer further comprises 50 mM phosphate.</u><u style="single">[Invention 1032]</u><u style="single"> The method of 1031 of the present invention, wherein the elution buffer further comprises 50-250 mM NaCl.</u><u style="single">[Invention 1033]</u><u style="single"> (a) The step of contacting a feed stream containing recombinant adeno-associated virus (rAAV) particles with a hydrophobic interaction chromatography (HIC) medium in high salt buffer, the rAAV particles and within the process. The stage at which impurities bind to the HIC medium;</u><u style="single"> (b) The step of eluting the rAAV particles bound to the HIC medium with a medium salt buffer.</u><u style="single">A method for isolating a population of the rAAV particles from the in-process impurities in the feed stream, including.</u><u style="single">[Invention 1034]</u><u style="single"> The method of 1033 of the present invention, wherein the HIC medium is selected from the group consisting of Tosoh Butyl 650M, Tosoh SuperButyl 650C, Tosoh Phenyl 650C, EMD Fractogel Phenyl and Tosoh Has (butyl) resins.</u><u style="single">[Invention 1035]</u><u style="single"> The method of 1033 of the present invention, wherein the high salt buffer comprises from about 0.5 M to about 2.0 M citrate.</u><u style="single">[Invention 1036]</u><u style="single"> The method of 1035 of the present invention, wherein the high salt buffer further comprises from about 1 to about 100 mM phosphate.</u><u style="single">[Invention 1037]</u><u style="single"> The method of 1033 of the present invention, wherein the medium salt buffer comprises less than 0.5 M citrate.</u><u style="single">[Invention 1038]</u><u style="single"> The method of 1037 of the present invention, wherein the medium salt buffer further comprises from about 1 to about 100 mM phosphate.</u><u style="single">[Invention 1039]</u><u style="single"> The method of the present invention 1037, wherein the medium salt buffer comprises 0.2 M to 0.5 M citrate.</u><u style="single">[Invention 1040]</u><u style="single"> A population of rAAV particles with empty capsids, partially denatured capsids, less infectious capsid substances and / or partially complete capsids is bound to the HIC medium after elution with the medium salt buffer. The method of the present invention 1039.</u><u style="single">[Invention 1041]</u><u style="single"> The rAAV particles are AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV. The method of the present invention 1001 or the present invention 1033 comprising an AAV capsid protein derived from an AAV capsid serum type selected from the group consisting of -12, AAV-13, AAV-14, AAV-15 and AAV-16.</u><u style="single">[Invention 1042]</u><u style="single"> The method of 1041 of the present invention, wherein the rAAV particles contain an AAV capsid protein derived from an AAV capsid serotype selected from the group consisting of AAV-1, AAV-4, AAV-5 and AAV-8.</u>

0034<figref num="1">The results of benzonase® digestion of the supernatant clarified from the rAAV production culture collection are shown. This result demonstrates the absence of high molecular weight DNA after digestion of Benzonase®.</figref><figref num="2">A typical spectroscopic trace of a typical resin screened for rAAV binding affinity as described in Example 4 is shown. Absorbance (AU) and conductivity (mS / cm) are shown.</figref><figref num="3">The breakthrough capacity analysis with or without PEG is shown. The volume of apatite resin (CFT type I) was evaluated using two model rAAV-producing cultures. Top panel: Rupture under load of serum-containing or serum-free feed stream in the presence or absence of approximately 5% (w / v) PEG6000 in the loading fluid. Load refers to the starting supply flow before 1: 1 online dilution and is standardized per 1 mL of resin volume. Bottom panel: Load (ml) at the time of 1% breakthrough, and recovery at the elution fraction. The TFF collection used in the experiment was approximately 10 relative to the rAAV vector.¹⁶It was a concentration of DRP / ml. In the presence of approximately 5% (w / v) PEG6000, 150 mL of TFF collection was loaded onto 1.2 mL CFT resin without rupture. This is 1.8x10¹⁴The presence of&gt; 1% rAAV was specified in the column pass fraction, corresponding to the total rAAV DRP load.</figref><figref num="4">A typical CHTI chromatogram is shown. Shown is in-line UV absorbance A by Amersham 3 mm Skid₂₈₀ It is a measurement result of (AU, absorbance unit) and conductivity (mS / cm). The parentheses mark the main segments of the program described in Example 7. "NaOH" marks the column purification stage.</figref><figref num="5">The relative purity of the rAAV vector eluted from the apatite resin is shown. Panel A is pass-through / track (FT), high phosphate / 5% (w / v) PEG6000 wash (PO)₄), Washing to remove phosphate and PEG6000 (WII / WIII) and distribution of vectors during elution. None of the differences between cases are significant within the accuracy of the analysis, typically a lack of mass balance. Panel B shows the corresponding lanes of cypro-orange-stained SDS PAGE using the fractions eluted from the apatite column. 2 x 10 for each sample¹¹Loaded with individual DRP / lanes; apparent movement differences between lanes are an unnatural result of salt due to the fact that evaporation must concentrate the CFT eluate to a volume that is compatible with the gel. .. The only major band is believed to be the AAV capsid protein. Panel C shows the relevant lanes of the Ad5 Western blot with the lanes rearranged for clarity, demonstrating comparable elimination of the Ad5 protein.</figref><figref num="6">The evaluation of purification over the whole process by SDS-PAGE is shown. In-process samples from typical production culture collections were run on a modified / reduced 10% polyacrylamide gel and stained with cypro orange. 1 x 10 samples after all collection¹⁰Loaded with 1 DRP / lane. The two upstream samples prior to the TFF concentration step (the first clarification step and the AEX pass sample) are 1x10 due to volume constraints on the gel.⁹It could only be loaded with one DRP / lane. Beta-galactosidase (B-Gal) was loaded at 50 ng / lane and staining susceptibility and consistency throughout the gel was evaluated. Three AAV1 capsid proteins (VP1, 2 and 3) have been shown.</figref><figref num="7">The stepwise recovery of the rAAV purification described in Examples 1-12 is shown. The total DRP present in the supernatant before collection was defined as 100%. The recovery rate for each stage is the total DRP recovered for the total DRP processed over that stage. The total recovery rate for the entire process was approximately 28%. D4 supernatant: production culture; AEX FT: anion exchange (Mustang® Q) pass through; capture: apatite chromatography; heat: heat inactivation or heat killing; HIC: hydrophobic interaction chromatography; SEC : Size exclusion chromatography; AEX: Anion exchange.</figref>

0035Detailed explanation Recombinant adeno-associated virus (rAAV) of any AAV capsid serotype from production culture contaminants such as damaged rAAV particles, helper viruses, helper virus proteins, plasmids, cellular proteins and DNA, medium components, serum proteins, etc. It is an object of the present invention to provide a method for isolating a population of particles. In addition, the methods of the invention are industrially expandable, orthogonal, that meet regulatory requirements for the isolation of populations of rAAV particles from high titer rAAV-producing culture collections or feed streams. process) is provided. Populations of rAAV particles isolated by the methods of the invention include production culture contaminants such as damaged rAAV particles, helper viruses, helper viral proteins, plasmids, cellular proteins and DNA, media components, serum proteins and glucans. Alternatively, it is substantially free of impurities, including in-process impurities. The methods of the invention are particularly suitable for rAAV vector serotypes, which are weak anionic binding agents such as rAAV-1, rAAV-4, rAAV-5 and rAAV-8. The present invention is further high in rAAV particles that are substantially free of contaminants, including produced cultured contaminants and / or in-process impurities, suitable for use in gene therapy applications that do not require density gradient centrifugation. We contemplate a method for isolating titer populations.

0036Definition As used herein, the term "isolated" or "purified" refers to at least some of the other components in which the rAAV particles are naturally present or also present where they were originally prepared. A preparation of rAAV particles lacking. Thus, for example, isolated rAAV particles can be prepared using purification techniques for concentrating them from the source mixture, such as culture lysates or production culture supernatants. Concentration may be measured in a variety of ways, for example by the proportion of DNase resistant particles (DRPs) present in the solution, or by infectivity, or as defined below, helper virus, medium. It may be measured for a second, potentially interfering substance present in the source mixture, such as a production culture contaminant or an in-process contaminant, including components.

0037The rAAV preparation has a ratio of infectious AAV particles to infectious helper virus particles of at least about 10.²1; Preferably, at least about 10⁴: 1, more preferably at least about 10⁶: 1; Even more preferably, at least about 10⁸If it is 1, it is said to be "substantially free" of helper viruses. The preparation also preferably does not contain an equal amount of helper virus protein (ie, if the above helper virus particle impurities are present in a disrupted form, the protein is comparable to such level results of helper virus. Exists in). Viral and / or cellular protein contaminants can generally be observed as the presence of Coomassie-stained bands on SDS gels (eg, the appearance of bands other than those corresponding to AAV capsid proteins VP1, VP2 and VP3).

0038As used herein, the terms "weak anion-binding material" or "low-affinity anion-binding material" are used in the presence of contaminants, including production-cultured contaminants or in-process contaminants. Compatible with rAAV particles having a capsid serum type that does not bind with sufficient affinity to allow isolation of rAAV particles from production culture contaminants. Such capsid serotypes are known in the art and include, but are not limited to, AAV-1, AAV-5, AAV-8 and AAV-4. As described in the art, such weak anion-binding materials are at least one density centrifuge, including iodixanol (sold under the trade name Optiprep®) or cesium chloride gradient centrifugation. It is generally purified by a method involving steps.

0039As used herein, the terms "helper virus" or "contamination helper virus" are used to produce copies of helper virus-dependent viral vectors, such as adeno-associated viruses, which by themselves do not have the ability to replicate. A virus that is affected. Helper viruses are used to co-infect cells with a viral vector and provide the proteins needed to replicate the genome of the viral vector. These terms include intact viral particles, empty capsids, viral DNA, and the like. Commonly used helper viruses for producing rAAV particles include adenovirus, herpes simplex virus, cytomegalovirus, Epstein bar virus and vaccinia virus.

0040As used herein, the term "production culture" refers to a container containing the components required for the production of rAAV vector particles. Production cultures include, but are not limited to, the following components: (1) suitable host cells; (2) helper virus function; (3) AAV rep and cap genes and gene products; (4) AAV ITR Therapeutic transgenes adjacent to the sequence; and (5) a suitable medium containing, but not limited to, serum, serum-derived proteins, vitamins, essential and non-essential amino acids, and glucose known to support rAAV production. Medium components and media supplements.

0041As used herein, "contaminants", "production culture contaminants", "in-process impurities", "in-process impurities", "impurities" or "contaminants" are used interchangeably herein. The term, but is not limited to, media formulations known in the art to support the production of rAAV vectors; media supplements such as salt, calf serum, amino acid supplements, vitamin supplements, growth factors, serum. Albumin and other low molecular weight proteins present in media formulations known in the art; acceptable host cell, host cell protein or host cell DNA; helper virus, helper virus protein or helper virus DNA, eg wild type. Such as adenovirus or herpesvirus protein; as well as other non-rAAV or rAAV vector-producing cultures introduced during the purification process, such as chromatography buffers used in the purification of rAAV vectors from glucans or feed streams.

0042As used herein, the term "production culture collection" is by means known in the art, including but not limited to transfection steps, stable cell line production, Ad-hybrid production systems or baculovirus production systems. Defined as a solution containing rAAV vector particles produced from an rAAV vector-producing culture. Furthermore, as used herein, the term "production culture collection" refers to a substance isolated from a production culture vessel, and substances isolated by lysis of rAAV producer cells by means known in the art are also intact. Also included are substances isolated from rAAV-producing cultures that are maintained under culture conditions known in the art to obtain rAAV particles released from cells into the medium. Production culture collections can include, but are not limited to, some or all of the following: rAAV vector particles, production culture components such as media components, host cell proteins, host cell DNA, host cells, helper viruses, Helper virus protein, helper virus DNA, plasmid DNA, carrier virus DNA, serum, serum-derived proteins and media supplements, etc.

0043As used herein, the term "feed stream" refers to a source of rAAV vector particles loaded onto a chromatographic substrate, flowing through the chromatographic substrate, or applied to the chromatographic substrate. The feed stream of the invention is present in the production culture collection and as a pass from the previous step, is bound and eluted in the previous step, is present in the void volume of the previous step, or of rAAV particles. Includes substances isolated from the prechromatographic step of the invention, whether present in any fraction obtained during purification. Such feed streams are one or more "contaminants", "production culture impurities", "in-process impurities", "in-process impurities" or "impurities" or "contamination" as defined herein. Can include "things".

0044As used interchangeably herein, the terms "capture," "bond," "bond," or "bond" refer to the binding, adhesion, or adhesion of components of the feed stream to a chromatographic medium. Ingredients may be by any force or chemical property known in the art, including, but not limited to, hydrophobicity, ionicity (including anionic and cationic), affinity, metal chelation and chelation. It may be bound to a chromatography medium. The components may be attached to the chromatography medium by two or more chemistries, such as in an apatite chromatography medium.

0045The terms "apatite resin", "apatite chromatography medium", "apatite substrate" or "apatite medium" used interchangeably herein refer to and are not limited to a chromatography medium composed of an inorganic substance of calcium phosphate. Includes ceramic hydroxyapatite (CHT) and ceramic fluoroapatite (CFT) chromatography media.

0046The term "mixed mode" or "multimode" refers to a chromatographic medium capable of having two or more binding chemistries. Mixed mode chromatography media include, but are not limited to, metal affinity bonds via the calcium moiety, hydrogen bonds via the hydroxyl groups present in the skeleton, positive and negative charge repulsion via the calcium moiety, and medium. Includes an apatite chromatography medium capable of exhibiting negative charge repulsion and positive charge attraction via the phosphate moiety present in.

0047General references to "compositions" include the compositions of the invention and are applicable to the compositions of the invention.

0048As used herein, the singular forms of article "one (a)", "one (an)" and "the" include plural references unless otherwise specified. For example, the phrase "a virus particle" includes one or more virus particles.

0049References to "about" prefixed to a value or parameter herein include (and describe) aspects directed to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

0050It is understood that the aspects and aspects of the invention described herein include consisting of aspects and aspects and / or essentially consisting of aspects and aspects.

0051Production of rAAV vector Numerous methods for the production of rAAV vectors include transfection, stable cell line production, and infectious hybrid virus production systems including adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids. Known in the art. rAAV-producing cultures for the production of rAAV virus particles include: (1) human-derived cell lines such as HeLa cells, A549 cells or 293 cells, or, in the case of baculovirus production systems, insect-derived such as SF-9. Suitable host cells, including cell lines of; (2) suitable provided by wild-type or mutant adenoviruses (such as temperature-sensitive adenoviruses), herpesviruses, baculoviruses or plasmid constructs that provide helper function. Helper virus function; (3) AAV rep and cap genes and gene products; (4) Transfer genes adjacent to AAV ITR sequences (such as therapeutic transfer genes); And (5) all suitable media and media components to support rAAV production are required. A suitable medium known in the art may be used for the production of the rAAV vector. These media include, but are not limited to, modified Eagle's Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), custom formulations such as those described in US Pat. No. 6,566,118, and US Pat. No. 6,723,551. Contains media prepared by Hyclone Laboratories and JRH, including the Sf-900 II SFM medium described in, each of which is particularly specific with respect to custom media formulations for use in the production of recombinant AAV vectors. The whole is incorporated herein by reference.

0052Suitable rAAV production media of the present invention can be supplemented with serum or serum-derived recombinant proteins at levels of 0.5% to 20% (v / v or w / v). Alternatively, as is known in the art, the rAAV vector may be produced under serum-free conditions, which can also be referred to as an animal-derived product-free medium. To enhance the titer of rAAV in production cultures, those skilled in the art may use one or more cell culture components known in the art, including, but not limited to, glucose, vitamins, amino acids and / or growth factors, in rAAV vectors. It can be understood that it may be supplemented with commercially available or custom-made media designed to support the production of.

0053rAAV-producing cultures can be grown under a variety of conditions suitable for the particular host cell used (eg, over a wide temperature range, over a variety of lengths of time, etc.). As is known in the art, rAAV-producing cultures have adherence that can be cultured in suitable adhesion-dependent vessels such as roller bottles, hollow thread filters, microcarriers, and packed or fluidized layer bioreactors. Dependent cultures are included. rAAV vector-producing cultures include, for example, in suspension of HeLa cells, 293 cells and SF-9 cells that can be cultured in a variety of ways, including disposable methods such as spinner flasks, agitator bioreactors and Wavebag methods. Matched host cells may also be included.

0054The rAAV vector particles of the present invention can cause the release of rAAV particles from intact cells into the medium either by lysis of the host cells of the production culture or as more fully described in US Pat. No. 6,566,118. If the cells are cultured under conditions known in the art, they can be collected from the rAAV production culture by collection of used medium from the production culture. Suitable methods of lysing cells are also known in the art, with multiple freeze / thaw cycles, sonication, microsolutions, and chemicals such as detergents and / or proteases. Including processing.

0055Purification of rAAV vector Upon collection, the rAAV-producing cultures of the invention may contain one or more of the following: (1) host cell protein; (2) host cell DNA; (3) plasmid DNA; (4) helper virus; (5) Helper virus proteins; (6) Helper virus DNAs; and (7) medium components containing, for example, plasmid proteins, amino acids, transferases and other low molecular weight proteins. In addition, rAAV-producing cultures include AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV. Further comprising rAAV particles having an AAV capsid serotype selected from the group consisting of -11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16. In some embodiments, the rAAV particles have an AAV capsid serotype selected from the group consisting of AAV-1, AAV-4, AAV-5 and AAV-8.

0056In some embodiments, the rAAV-producing culture collection is clarified to remove host cell debris. In some embodiments, the production culture collection is filtered through a series of depth filters, including, for example, a D0HC grade Millipore Millistak + HC Pod Filter, an A1 HC grade Millipore Millistak + HC Pod Filter, and a 0.2 μm Filter Opticap XL10 Millipore Express SHC Hydrophilic Membrane filter. Is clarified by. Clarification can also be achieved by a variety of other standard techniques known in the art, such as centrifugation or filtration through any cellulose acetate filter with a pore size of 0.2 μm or larger known in the art. it can.

0057In some embodiments, the rAAV-producing culture collection is further treated with Bensonase® to digest any high molecular weight DNA present in the production culture. In some embodiments, benzonase® digestion results in a final concentration of 1-2.5 units / ml benzonase® at temperatures ranging from room temperature to 37 ° C for 30 minutes to several hours, for example. It is carried out under standard conditions known in the art including.

0058rAAV particles can be isolated or purified using one or more of the following purification steps: pass-through fraction anion exchange filtration, tangential flow filtration (TFF) to concentrate rAAV particles, rAAV by apatite chromatography. Capture, thermal inactivation of helper virus, rAAV capture by hydrophobic interaction chromatography, buffer exchange by size exclusion chromatography (SEC), nanofiltration, and rAAV capture by anion exchange chromatography. These steps may be used alone, in various combinations, or in different orders. In some embodiments, the method comprises all steps in the following order.

0059Anion exchange filtration Optionally, in some embodiments, the clarified and benzonase® treated production culture collection has the rAAV vector present in the pass-through fraction and contaminating helper virus retained on the charge filter. It is subjected to anion exchange filtration under the conditions specified. At ionic strength of rAAV-producing cultures, rAAV particles are identified as helper viruses, such as adenovirus, by passing through an anion filters such as Mustang® Q Filter (Pall Corp., East Hills, NY). Can be done. Those skilled in the art will need to achieve optimal log reduction (LRV) of adenovirus proteins and adenoviruses present in clarified, benzonase®-treated and anion-filtered production cultures. The size and number of viruses can be determined. In some embodiments, the LRV is at least 1 log and greater than 10 logs. In a preferred embodiment, the LRV is at least 2 logs and is greater than 8 logs. In a more preferred embodiment, the LRV is at least 6 logs.

0060Tangent Filtration (TFF) Concentration In some embodiments, the clarified, benzonase®-treated feed stream anion filtration-derived pass-through fraction is tangentially filtered (TFF) before being applied to an apatite chromatography medium. Concentrate through. Large-scale concentration of virus using TFF ultrafiltration R. Written by Paul et al., HUMAN GENE THERAPY, 4: 609-615 (1993). TFF enrichment of the feed stream allows a technically controllable volume of feed stream to be subjected to the chromatography step of the present invention, allowing for more rational sizing of columns without the need for long recirculation times. Is possible. In some embodiments, the rAAV feed stream is enriched between at least 2-fold and at least 10-fold. In some embodiments, the feed stream is concentrated between at least 10-fold and at least 20-fold. In some embodiments, the feed stream is concentrated between at least 20-fold and at least 50-fold. Those skilled in the art will also recognize that TFF may be used at any stage during the purification process, such as it is desirable to change the buffer before performing the next step during the purification process.

0061Capture of rAAV by apatite chromatography in the presence of polyethylene glycol (PEG) The FDA-approved process for the purification of proteins and other biopharmaceuticals suitable for use in human clinical trials and pharmaceutical formulations depends on an industrial scale orthogonal process. A multi-step purification scheme would include orthogonal steps if different separation mechanisms were used, with each step corresponding to an axis in the Cartesian coordinate space. For example, a two-step process using anion exchange chromatography and hydrophobic interaction chromatography (HIC) is considered orthogonal. The steps for removing contaminants, such as production culture contaminants or in-process impurities, from the production culture collections or feed streams described herein are various for the final product (ie, the rAAV vector). An orthogonal process involving both capture and pass-through steps on the chromatographic medium. The rAAV vector (specifically, rAAV-2) has been demonstrated in the art to bind to anion resins. rAAV vectors such as rAAV-1, rAAV-5 and rAAV-8 are much weaker than rAAV-2 in the presence of production components such as serum albumin, helper virus components, production medium components and host cell DNA for anion exchange. It has been demonstrated to provide a less efficient and lower quality purification scheme for binding to the medium.

0062Previous purification strategies described in the art for lower affinity anion-binding substances, such as AAV-1, have been produced to achieve stronger binding of the rAAV vector to anion exchangers. And included a gradual gradient with iodixanol to reduce the relative concentration of contaminants such as in-process impurities. The stepwise gradient with iodixanol is not easily extendable to the industrial scale processes described herein.

0063The inventors of the present application have discovered that rAAV vector particles can be isolated from contaminants such as production culture contaminants or in-process impurities by capture on the apatite resin and elution from the apatite resin. Thus, in addition to trapping the product from the unpurified feed stream, the apatite column eliminates various process-related impurities, including host cells and adenoviral proteins, glucans and serum proteins (Ad5). It is an additional exclusion factor for helper viruses (such as helper viruses).

0064Apatite resin is a chromatography medium composed of an inorganic substance of calcium phosphate, including, but not limited to, ceramic hydroxyapatite (CHT) and ceramic fluoroapatite (CFT). Apatite chromatography media are also referred to as mixed-mode or multi-mode media because apatite has functional groups that provide two or more binding chemistries. Although not desired to be bound by theory, apatite media include many hydroxyl residues present on the skeleton, positively charged calcium moieties and negatively charged phosphate moieties on the resin. It provides opportunities for calcium metal affinity bonds, hydrogen bonds, positive charge repulsion, positive charge attraction, negative charge repulsion and negative charge attraction via different chemical groups. The chemistry of each bond applies only to mixed mode binding when it is useful for single mode chromatography. However, unlike single-mode chromatography, the various binding and elution chemistries are not irrelevant and can work in opposite directions. For example, increasing ionic strength can drive hydrophobic bonds (T. Kawasaki, M. Niikura, and Y. Kobayashi, J. Chrom. 515: 125-148 (1990) and PS Gagnon, P. Ng, J. Zhen, C. Aberin, and J. He, BioProcess Int'l 4: 50-60 ( 2006)). Specifically, CHT and CFT are hydroxyapatite (Ca) sintered at high temperatures to convert minerals from crystalline form to ceramic form.₅(PO₄)₃OH)₂Spherical, macroporous form. This results in a chromatographic medium with a macroporous structure that provides a large surface area, limited mass transfer resistance, high mechanical strength and base resistance. Sintering at different temperatures and times results in different physical structures, type I and type II, which are chemically identical but exhibit different abilities for different classes of molecules. .. CFT differs from CHT in that it is a complex of fluoroapatite and hydroxyapatite prepared by chemically substituting a hydroxyl group with a fluorine group to increase stability under acidic conditions. CFT and CHT resins are commercially available (eg, from Bio-Rad Laboratories, Inc.).

0065The inventors of the present invention are surprised to find that the presence of polyethylene glycol (PEG) in the loading buffer determines the volume and reproducibility of the rAAV vector particles bound to the apatite resin (of the rAAV particles in the pass-through fraction). We have found a dramatic increase (by reducing variable rupture). One of the characteristics of rAAV vectors that distinguishes rAAV vectors from most of the process-related impurities is the large physical size of the particles, although we do not want to be bound by theory. This size difference includes polyethylene glycol (PEG) in the chromatographic binding buffer and the cleaning buffer to provide a larger molecular partition coefficient based on the energetically likely hydration shell sharing. It is used in the capture and cleaning stages by selectively increasing to the state. The use of PEG in purifying viral and bacteriophage vectors has been described in the art, but unlike the present invention, it physically aggregates viral particles and removes the viral particles from solution. It is mainly used as a precipitant for viruses. Since PEG is known in the art to promote the aggregation and precipitation of viral particles, rAAV has been described in the art to form aggregates with an ionic strength of less than 200 mM (Wright et al). ., Molecular Therapy 12: 171-178 (2005)), the effect of PEG on the binding of the rAAV vector to the apatite resin was unpredictable. PEG promotes the binding of immunoglobulin molecules to ion exchange resins, as described, for example, in Gagnon, J. Chromtogr. 743A: 51-55 (1996), and charged hydrophobic mixed mode resins. In the case of, for example, Gagnon et al., 22^nd It was well known in the art as described in the International IBC Conference on Antibody Production and Development, March 4-6, 2009.

0066Based on experiments using PEG6000 over a concentration range of 3-10% (w / v) in the feed stream, the inventors of the present application prefer to use PEG6000 with a relative concentration of approximately 5% (w / v). Was judged. Those skilled in the art can use PEGs of other species and molecular weights, including but not limited to PEG8000, PEG10000 and PEG15000, and at appropriate concentrations of PEG drive the rAAV vector particles in solution to bind to the apatite resin. However, it will be appreciated that the relative concentration of PEG at the final concentration in the rAAV vector solution can be experimentally determined so as not to form aggregates or physically precipitate.

0067In some embodiments, the rAAV vector particles are isolated from the produced culture contaminants by capture on the apatite resin in the presence of PEG and elution of the bound rAAV particles from the apatite resin in phosphate buffer. In a preferred embodiment, the rAAV vector particles are isolated from the produced culture contaminants by capture on the apatite resin in the presence of PEG and elution of the bound rAAV particles from the apatite resin in PEG-free buffer. In some embodiments, rAAV particles containing the weak anionic binding material capsid are isolated from the produced culture contaminants by capture on an apatite resin in the presence of PEG, and the bound rAAV particles are PEG-free. Elute from the apatite resin in buffer. In a more preferred embodiment, rAAV particles containing a serotype 1 (rAAV-1 serotype) capsid were isolated from the produced culture contaminants by capture on an apatite resin in the presence of PEG, and rAAV bound to the resin. -1 Elute particles containing serotype capsid in PEG-free buffer. In some embodiments, from the apatite resin in the step of loading the feed stream in the loading buffer in the absence of phosphate but in the presence of PEG, and in the elution buffer containing phosphate and lacking PEG. RAAV vector particles are isolated from the production culture contaminants by a method involving the step of eluting bound rAAV.

0068Apatite chromatography in the presence of PEG provides an efficient capture or binding strategy for the purification of the rAAV vector, but at pH 7.0 the apatite resin also retained more in-process impurities. Although not desired to be bound by theory, proteins present in the feed stream at basic pH (pH above 7.0) are more likely to have a net negative charge and are present on the apatite resin. It is believed to be repelled by the negative phosphate binding site, thereby reducing the overall cation exchange binding capacity of the chromatographic resin. However, given the mixed modality of the apatite resin, binding via positive charge attraction and metal affinity can still occur.

0069Boric acid buffers are routinely used in the art as basic buffers due to their desirable manufacturing properties, including non-limiting ease of preparation, optimal solubility, excellent buffering capacity and low cost. Has been done. Therefore, a boric acid buffer as a model basic buffer system was evaluated for the capture of rAAV on apatite resin. One of skill in the art can appreciate that other basic buffers can be evaluated to determine if they reduced the level of in-process impurity binding to the apatite resin in the presence of PEG. Other basic buffers may be tested and used to capture rAAV. In some embodiments, the PEG-free apatite loading buffer comprises a borate buffer. In a preferred embodiment, the borate buffer is formulated at a pH of about 8.0 to about pH 9.9. In a preferred embodiment, the borate buffer is formulated at a pH of about 9.0. In some embodiments, the borate buffer has a concentration of about 5 mM to about 500 mM. In a more preferred embodiment, the borate buffer is formulated at about 20 mM borate, pH 9.0. In some embodiments, pH 9.0 is 20 The mM borate buffer particularly reduces the capture of small molecule in-process impurities on the apatite resin.

0070In some embodiments, the feed stream is on the apatite resin in a phosphate buffer in the presence of PEG by online mixing of the feed stream with a phosphate buffer containing PEG at twice the final concentration of PEG. Be loaded. In some embodiments, the pH of the phosphate buffer is pH 6.5-pH 7.0. In some embodiments, the PEG is PEG6000. In some embodiments, the concentration of PEG6000 in the loading buffer is from about 3% (w / v) to about 10% (w / v). In a more preferred embodiment, the concentration of PEG6000 in the loading buffer is about 5% (w / v). In some embodiments, the concentration of phosphate in the loading buffer for apatite resin is 5 mM to 500 mM.

0071In some embodiments, the binding ability of the apatite resin in the presence of PEG is enhanced relative to the binding ability of the apatite resin in the absence of PEG. In some embodiments, the binding ability of the apatite resin to the rAAV vector particles in the feed stream in the presence of PEG is about half the log to about half the binding ability of the apatite resin in the absence of PEG. Increased to 10 logs. In a preferred embodiment, the binding ability of the apatite resin to the rAAV particles present in the feed stream in the presence of PEG is enhanced by 8 logs. In some embodiments, the binding ability of the apatite resin to the rAAV vector particles in the feed stream in the presence of PEG is at least about 10.⁶Approximately 10 from particle rAAV / ml resin¹⁶Particle / ml resin (eg 10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²、10¹³、10¹⁴、10¹⁵、10¹⁶(Almost any of particles / ml resin, etc.). In some embodiments, the binding capacity of the apatite resin in the presence of PEG is about 10¹⁴Particle / ml resin.

0072Approximately 10 in the presence of PEG¹²~10¹⁴This amazing binding capacity of rAAV-1 / ml apatite resin of DRP allows for high efficiency and cost effective scaling of industrial rAAV-1 purification, but the binding capacity is per 1 ml of resin. Those skilled in the art will appreciate that it represents the maximum number of rAAV-1s that can bind to and is not intended to operationally limit the scope of the invention. In fact, we have 10¹⁴~10¹⁶It was found that rAAV-1 vector collection cultures containing less than DRP / ml of rAAV-1 could be purified by the present invention.

0073In some embodiments, the rAAV particles bound to the apatite medium are washed before the rAAV particles are eluted from the resin. In some embodiments, the apatite chromatography medium is washed once or multiple times with a wash buffer containing a diminishing concentration of PEG to remove in-process impurities. In some embodiments, the apatite chromatography medium is washed once or multiple times with wash buffer containing about 3% (w / v) to about 10% (w / v) PEG. In some embodiments, the wash buffer is 10% (w / v), 9.5% (w / v), 9% (w / v), 8.5% (w / v), 8% (w / v). , 7.5% (w / v), 7% (w / v), 6.5% (w / v), 6% (w / v), 5.5% (w / v), 5% (w / v), 4.5 % (w / v), 4% (w / v), 3.5% (w / v) and 3% (w / v) Contains almost any of PEG. In some embodiments, the apatite medium is washed with a wash buffer containing PEG at a concentration higher than the PEG concentration used to allow binding of rAAV particles to the apatite medium. In some embodiments, the apatite medium is further washed with diminishing concentrations of PEG. In some embodiments, the cleaning buffer contains a buffer known in the art. In some embodiments, the wash buffer comprises a buffer selected from the group consisting of borate, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and Tris-HCl. In some embodiments, the wash buffer is at basic pH. In some embodiments, the cleaning buffer is pH 7.0-pH 10.0, pH 7.2-pH 10.0, pH 7.4-pH 10.0, pH 7.6-pH 10.0, pH 7.8-pH 10.0, pH 8.0-pH 10.0, pH 8.2-. pH 10.0, pH 8.4 ~ pH 10.0, pH 8.6 ~ pH 10.0, pH 8.8 ~ pH 10.0, pH 9.0 ~ pH 10.0, pH 9.2 ~ pH It has a pH of 10.0, pH 9.4 to pH 10.0, pH 9.6 to pH 10.0 or pH 9.8 to pH 10.0. In some embodiments, the wash buffer has a pH of 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8 or 10.0. In some embodiments, the wash buffer further comprises 100-500 mM phosphate. In some embodiments, the wash buffer further comprises 50-250 mM NaCl.

0074In some embodiments, the rAAV vector isolated from the feed stream by capture on an apatite resin in the presence of PEG is eluted into a low concentration PEG buffer. In some embodiments, the low concentration PEG is from about 2.9% (w / v) to about 0.1% (w / v) PEG. In some embodiments, the rAAV vector isolated from the feed stream by capture on an apatite resin in the presence of PEG is eluted into PEG-free buffer. In a preferred embodiment, the rAAV vector isolated from the feed stream by capture on an apatite resin in the presence of PEG is eluted in a buffer containing PEG-free phosphate.

0075In some embodiments, the rAAV vector isolated from the feed stream by capture on an apatite resin in the presence of PEG is eluted in a buffer containing phosphate. In some embodiments, the rAAV vector isolated from the feed stream by capture on an apatite resin in the presence of PEG is about 0.1 mM to about 500 mM (eg, about 1 mM to about 250 mM, about 10 mM). Elute in buffer containing phosphate at a concentration of ~ about 100 mM, etc.). In a preferred embodiment, the rAAV vector isolated from the feed stream by capture on an apatite resin in the presence of PEG is eluted in 50 mM phosphate buffer.

0076The inventors of the present application have discovered that the rAAV vector present in the feed stream can be isolated by capture on an apatite resin in the presence of PEG. However, if helper viruses used in the production culture (such as adenovirus) are present in the feed stream applied to the apatite resin, they are captured by the apatite resin in the presence of PEG. The rAAV vector particles captured by the apatite resin in the presence of PEG can be easily isolated from adenovirus by their elution profile in phosphate buffer. The rAAV vector particles bound to the apatite resin in the presence of PEG elute in a buffer containing a low concentration of about 0 mM in the absence of PEG, whereas the adenovirus particles of the helper virus are rAAV. It is retained on the apatite resin under the concentration of phosphate used to elute the vector particles. Experimentally, the rAAV vector is only 50 in the absence of PEG Helper viruses, such as adenovirus, were retained on the resin, if present, as opposed to elution with a single sharp peak at the phosphate of mM. 8 to rAAV supply stream⁹In a spike-in test in which infectious adenoviruses of DNase-resistant particles (DRP) were spiked and chromatographed against apatite resin in the presence of PEG, the rAAV vector was captured on the apatite resin and was PEG-free. Although eluted in 50 mM phosphate buffer, approximately 4 logs of adenovirus protein were retained on the apatite resin. Thus, in some embodiments, the rAAV vector present in the feed stream is simply captured from the contaminating helper virus by capture on the apatite resin in the presence of PEG and elution in phosphate buffer in the absence of PEG. Release. In some embodiments, the phosphate buffer is formulated at a concentration that retains the contaminating helper virus bound to the apatite resin. In some embodiments, 2-8 logs of adenovirus are retained per ml of apatite resin. In some embodiments, the rAAV vector present in the feed stream is (0-400 mM, 0-300 mM, 0-200 mM, 0-100 mM) under the condition of retaining the helper virus bound to the apatite resin. 0 ~ 500 (mm, 0 ~ 50 mM, etc.) Isolate by elution from apatite resin in mM phosphate buffer.

0077Production systems known in the art for producing rAAV vectors may contain serum-containing production media in the range of 0.5% to 20% (v / v) or may be completely serum-free. There is also. In addition, the purification schemes described in the art may include one or more enrichment steps that can result in an increase in serum proteins and other serum components in the feed stream applied to the apatite resin. For example, the production culture supernatants described herein with 1% (v / v) serum are concentrated approximately 20-fold at the TFF stage, resulting in serum in the feed stream loaded onto the apatite resin. It contained as much as 20% serum protein contaminants compared to the feed stream concentrate from the unblended production culture. The inventors of the present application tested the apatite capture methods provided herein using feed stream concentrates from production cultures formulated in the presence or absence of serum. It was found that the presence of serum protein in the feed stream did not affect the performance of the apatite chromatography step.

0078Heat inactivation of helper virus (heat killing treatment) If infectious adenovirus is used as a source of helper virus in production cultures for rAAV production, incorporate any heat inactivation (heat killing) step and all residual adenovirus that may be present in the feed stream. The particles can be inactivated. The heat slaughter phase takes advantage of one of the major differences between AAV and adenovirus. That is, adenovirus particles are inactivated at temperatures around 54-56 ° C, while AAV and rAAV virus particles are stable and unaffected by those temperatures. In the present invention, the present inventors have adjusted the heat inactivation stage to adapt to optimization of a larger process such as a 250 L scale production culture performed herein. Specifically, the apatite eluate is prepared for mixing in a sterile, disposable 5 L biotreatment bag on a temperature controlled rocking table set at 53 ° C, at a rocking speed of 40 RPM. Heat inactivated using a 12 ° angle (20 L radio heating pan). The apatite eluate was incubated on a table until it reached 52 ° C, then held at that temperature for an additional 10 minutes. MgCl₂Was added to the apatite eluate at a final concentration of 2 mM to stabilize the rAAV vector during heating. One of ordinary skill in the art can experimentally test the scale, final heating set point and heating time to find the optimal conditions for inactivating adenovirus particles while maintaining the infectivity and integrity of the rAAV particles. Can be understood. The heat inactivation step may be excluded for purification of rAAV particles from production cultures that use plasmid constructs to provide helper function.

0079Hydrophobic interaction chromatography Hydrophobic interaction chromatography (HIC) is a technique for separating biomolecules based on their difference in surface hydrophobicity. Therefore, HIC is considered to be a method orthogonal to other purification steps in the AAV process. The HIC chromatography medium contains a hydrophobic ligand such as a linear hydrocarbon (eg, propyl (C3), butyl (C4), hexyl (C6) or octyl (C8)) or an aromatic compound (eg, phenyl). In pure water, the hydrophobic effect is too weak for functional interactions between the ligand and the protein, or the protein itself. However, liquid-leaving salts enhance hydrophobic interactions, and salt addition drives the capture of proteins into the HIC medium. For this reason, HIC resins are usually loaded under high salt concentrations and eluted at lower salt concentrations. Ammonium sulphate [(NH₄)₂SO₄] Is most often used to control protein capture via HIC chromatography due to the high liquid separation rank of both ammonium and sulfate in the Hofmeister series, and the high solubility of its salts. It is salt. In the present invention, the rAAV particles present in the feed stream are HIC resin by in-line mixing of 2 M ammonium sulfate + 50 mM bis tris buffer (pH 7.0): feed stream at a ratio of 75:25 (volume: volume), respectively. Loaded on. In-line mixing of feed stream and loading buffer avoids the risk of precipitation of any rAAV vector due to the high concentration of ammonium sulphate present in the buffer. As one of ordinary skill in the art can understand, the concentration of salt (ammonium sulphate) can be manipulated to achieve the optimum concentration for rAAV binding. Therefore, in some embodiments, the ammonium sulphate concentration is 1 M-3 M. In some preferred embodiments, the ammonium sulphate concentration in the loading buffer is 2 mM. As will be appreciated by those skilled in the art, in-line mixing of ammonium sulphate and feed stream is performed in a unit-operated flow for convenience, but the feed stream is readily known in the art as a load buffer of appropriate concentration. Can be mixed by any means of the above, and then the feed stream + loading buffer solution can be loaded onto the HIC chromatography medium.

0080Cosolvents can also affect hydrophobic interactions. For example, ethylene glycol or propylene glycol can be useful in reducing the interaction between the protein and the immobilized ligand and thus improving the elution profile. Therefore, the HIC column is 2 M ammonium sulphate + 50 mM bistris buffer (pH 7.0): 50 mM bistris (pH 7.0) + 10% propylene glycol (v: v) buffer (EMD BioSciences) 75:25 (v: v). ) RAAV was eluted in propylene glycol (800 mM ammonium sulfate + 50 mM Bistris buffer (pH 7.0) + 4% propylene glycol) in addition to the low salt buffer. Under their elution conditions, all residual helper viruses and proteins present in the feed stream loaded on the column are believed to remain bound to the column. In this example, propylene glycol was added to the buffer to sharpen the elution profile compared to the broader elution profile of the buffer without propylene glycol, which is optional in the process.

0081Examples of suitable hydrophobic resins include, but are not limited to, Tosoh Butyl 650M, Tosoh SuperButyl 650C, Tosoh Phenyl 650C and EMD Fractogel Phenyl (Tosoh Bioscience LLC, PA).

0082Waste from the rAAV production process requires stringent contaminant removal prior to disposal for at least two reasons: (1) The product contains a viral vector; and (2) ) Live adenovirus type 5 (Ad5) is often used as a helper virus for rAAV production in production cultures. Liquid debris from chromatographic operations is typically first decontaminated with bleach upon use, followed by further decontamination by holding at a high pH prior to neutralization and disposal. ..

0083Ammonium sulphate present in HIC buffer reacts with both bleach and sodium hydroxide, releasing harmful chlorine gas and ammonia gas, respectively. Therefore, the main consideration for process optimization in the HIC chromatography step has been the development of suitable buffer systems that can safely remove contaminants by methods known in the art.

0084As those skilled in the art can understand, the high salt concentration buffers used in hydrophobic interaction chromatography limit the flow velocity or cause mixing problems, thereby causing buffers at the temperature used for storage or manipulation. Further screening must be done for viscosity issues that can result in high back pressure that can increase the risk of product precipitation due to salt crystallization in. Citric acid buffers can be used for hydrophobic interaction chromatography at concentrations ranging from 0.5 M to 2.0 M, but based on the data in Table 6 below and consideration of the above factors, we have identified HIC. 1 M sodium citrate (pH 7.0) was selected as the optimal buffer for binding the rAAV vector to the chromatography medium.

0085Buffer exchange by size exclusion chromatography (SEC) Numerous methods are known in the art for carrying out the buffer exchanges described herein, including TFF and dialysis. The use of size exclusion chromatography provides the additional protein exclusion of proteins sized to pass through the pores in the resin, and the additional advantage of being relatively fast with respect to the time required to change the buffer. Has. Buffer exchange was performed at this stage to ensure that the previous HIC eluate was replaced with a buffer for rAAV binding suitable for the final anion exchange chromatography step during the process.

0086Alien factor (virus elimination) Optionally, additional steps may be incorporated into the process to eliminate trace contaminants, such as foreign viruses that may be present in the feed stream, thereby producing an industrially rational orthogonal process. Therefore, in some embodiments, the step further comprises a virus elimination filter. Examples of such filters are known in the art and include Millipore Viresolve NFR (50 nm), Pall Ultipore VF (50 nm) and Asahi 70 nm.

0087Anion exchange chromatography The anion exchange capture step on the rAAV vector subjected to apatite chromatography was performed as the final concentration and refinement step. Suitable anion exchange chromatography media are known in the art and are not limited to Unosphere Q (Biorad, Hercules, California), and N-charged amino or imino resins such as POROS 50 PI, or any DEAE. , TMAE, tertiary or quaternary amines, or PEI-based resins known in the art (US Pat. No. 6,989,264; N. Brument et al., Mol. Therapy 6 (5): 678-686 ( 2002); G. Gao et al., Hum. Gene Therapy 11: 2079-2091 (2000)). Those skilled in the art will appreciate that rAAV remains bound to the resin while removing other in-process impurities, including but not limited to glucan, which can be introduced by leaching from the various filters used in the purification step. It can be understood that a cleaning buffer solution having a high ionic strength can be specified. In some embodiments, the wash buffer is 60 mM NaCl and the rAAV vector is eluted from the column with 130 mM NaCl, resulting in all residual traces of serum albumin or helper virus present. In-process impurities are retained on the column.

<p num="0088"><u style="single">Example 1: Collection of rAAV-1 from medium clarification and benzoase® digestion</u> Clarify used rAAV-1 production medium (supernatant) from 250 L of rAAV-1 virus-producing cultures produced by any method known in the art that contains the rAAV-1 vector. Any cells contained in the supernatant were removed. (1) Millipore Millistak +® HC Pod Filter, D0 HC Grade (Millipore Corp., Bedford, MA) (4 times); (2) Millipore Millistak +® HC Pod Filter, A1HC Grade; and (3) Opticap A series of series of filters, including the XL10 Millipore Express SHC Hydrophilic Membrane 0.2 μm Filter, was passed through the supernatant at a rate of 5 liters / minute (LPM), stepwise down to 4 LPM. ..</p><p num="0089"> All filters were prewashed in reverse osmosis / deionized (RO / DI) water according to the manufacturer's specifications. The pass-through fraction was recovered in a biotreatment bag for benzonase® digestion. A final concentration of 2.5 units / ml was achieved by dissolving benzonase® (registered trademark) (EM Industries Catalog No. 1.01695.0002) at a final concentration of 2 units / ml in rAAV-1 production medium and adding it to the clarified virus supernatant. did. In addition to the supernatant, Benzonase® was incubated at room temperature with a constant recirculation of 4 LPM to allow DNA digestion. Data for benzonase® digestion are shown in Figure 1, demonstrating the absence of high molecular weight DNA after benzonase® digestion.</p><p num="0090"><u style="single">Example 2: Removal of produced contaminants via anion exchange</u> A series of 2 "x 22" Pall Mustang® Q ("MQ") filters in series with the clarified and benzonase® digested supernatant of rAAV-1 from Example 1 ("MQ") It was passed through Pall Corp., Catalog No. NP6MSTGQP1). Prior to loading the rAAV-1 virus supernatant, the filter was sterilized at 0.5 LPM with 15 L 0.5 M NaOH for a retention time of 15 minutes and 15 L TMEG + 2 M NaCl (TMEG: 0.05 M Tris-HCl, pH). 7.5, 1 mM 2-mercaptoethanol, 1 mM Na₂It was charged by rinsing with EDTA, 10% (v / v) glycerol) at a rate of 6 LPM and equilibrated at 6 LPM with 15 L of vector production medium. The supernatant was then pumped through the series of filters at a rate of approximately 6 LPM and collected in a biotreatment bag. At the ionic strength of the production medium, the anion-exchanged MQ filter demonstrated that the rAAV-1 supernatant clarifies helper virus and residual DNA, among other impurities, from the rAAV-1 supernatant by binding of contaminants to the charged membrane. Was done. However, at the ionic strength of the production culture, the rAAV-1 vector present in the supernatant flowed through the anion exchange membrane. During the process optimization, it was experimentally confirmed that a single MQ filter was used to break through the contaminants during the process, including the Ad5 helper virus. As a result, a second filter was added in series or vertically in the process.</p><p num="0091"><u style="single">Example 3: Concentration of rAAV-1 vector supernatant</u> The complete rAAV-1 vector production supernatant treated in Examples 1 and 2 was concentrated approximately 20-fold by tangential filtration (TFF) from an initial volume of approximately 250 L to a volume of approximately 12.5 L. Molecular weight cutoff value of 100 kD, C-type sieve and 5 m²Rinse a tangential flow polyether sulfone filter cartridge (Millipore Pellicon® 2 Biomax, catalog number P2B100C05) with 50 L of RO / DI water and 15 L of 0.5 M NaOH in a 15 minute retention step. It was sterilized and rinsed again with 100 L of WIFI (HyPure WFI purified water; HyClone, Logan, UT), rinsed with 15 L TMEG + 2 M NaCl, and finally equilibrated with 15 L of rAAV-1 production medium. .. The supernatant was passed through a TFF cartridge at a flow rate of approximately 3 LPM with a recirculation rate of 16 LPM. The TFF material retained on the filter (residual) was concentrated to approximately 10.5 L and transferred to the reservoir. The filter was rinsed with approximately 2 L of production medium. The wash and concentrate were then pooled to give a final volume of approximately 12.5 L.</p><p num="0092"> Concentration of rAAV-1 to a volume of 12.5 L by TFF also concentrated rAAV-1 producing contaminants remaining in solution up to approximately 20-fold. Therefore, a production retention step was introduced after the TFF step, during which the TFF concentrate was filtered through a 4-inch Opticap 0.22 μM filter membrane (Millipore Opticap® Catalog No. KVSC04HB3). This additional filtration step allows the TFF concentrate containing rAAV-1 to proceed to the next step in a process that does not require dialysis filtration and buffer replacement. The material after TFF is only 24 hours to 3 months or less before further treatment without losing stability as measured by vector yield or infectivity as assessed by assays known in the art. It must be stored at 2-8 ° C for any period, including these periods. Alternatively, the rAAV-1 vector can be concentrated or buffer exchanged using TFF as described herein at any step during the purification process.</p><p num="0093"><u style="single">Example 4: Resin screening for binding rAAV-1 vector vs. process impurities</u> The industrial FDA-approved process for the purification of proteins and other biopharmacy depends on an industrial scale incorporation orthogonal process. Orthogonal steps are steps that include two or more steps or steps for the removal of in-process impurities, including both capture and pass-through steps for the final product, such as the rAAV-1 vector. The rAAV vector (specifically, rAAV-2) has been demonstrated in the art to bind to anion resins. rAAV vectors such as rAAV-1, rAAV-5 and rAAV-8 are much weaker than rAAV-2 in the presence of production components such as serum albumin, helper virus components, production medium components and host cell DNA for anion exchange. It has been demonstrated to provide a less efficient and lower quality purification scheme for binding to the body.</p><p num="0094"> Previous purification strategies described in the art for lower affinity anion-binding substances such as AAV-1 have been used to achieve stronger binding of the rAAV vector to anion exchangers. A gradual gradient with iodixanol to reduce relative concentrations was included. The gradual gradient with iodixanol is not easily extendable to industrial scale processes such as those described herein. Therefore, to optimize the purification of rAAV vectors against low-affinity anionic binding agents such as rAAV-1 without the need for ultracentrifugation and stepwise gradients, host cell DNA, helper virus, serum albumin, serum. Often containing proteins (if the production medium contains serum) and other low molecular weight proteins found in the production culture to develop industrially expandable, orthogonal and efficient rAAV purification steps. Several resins were screened for the ability to bind the rAAV vector or exclude the rAAV vector in the pass-through fraction as compared to the ability to bind or pass the process impurities found.</p><p num="0095"> Resin screening was performed using a 1 ml (5 cm bed height) column with a linear flow velocity recommended by the vendor for each resin, with significantly underloading capacity compared to the manufacturer's recommendations. 280 nanometer ultraviolet absorbance (A) for binding to each resin and elution from each resin₂₈₀) Spectral photometric tracking results were collected. Peaks were analyzed by appropriate assays for both the rAAV-1 vector and typical process impurities. The data shown in Figure 2 represent typical spectrophotometric tracking results for typical resins in the list. Table 2 lists some of the screened resins, as well as the relative binding affinities of the resins for the rAAV-1 vector and various process impurities.</p><p num="0096"> (Table 2) Screening of resin for binding of rAAV-1 compared to binding of process contaminants<img id="000002" he="231" wi="160" file="JP5956331B2_D0001.tif" img-format="tif" img-content="drawing" />List of abbreviations: (-) = Present in pass-through fraction (no binding); + = Weak binding (eluting very early in the gradient); ++ ~ ++++ = Stronger binding (more eluting along the gradient) ..</p><p num="0097"><u style="single">Example 5: Development of apatite chromatography in the presence of polyethylene glycol (PEG) for capture of rAAV-1</u> Based on the results of the resin screening performed in Example 4, apatite resin or ceramic apatite resin was selected as one of the trapping resins for rAAV-1. Initial experiments were performed with CFT II resin, but the resin was changed to CHT I for later purification, as discussed in detail below. The data showed that both chromatographic resins worked equally well. Experiments were also conducted to increase the rAAV-1 binding capacity of the apatite resin and to improve the resin's ability to discriminate between rAAV-1 particles and other in-process impurities. Two key improvements to the function of the apatite resin have been further developed as described herein: (1) PEG addition; and (2) development of loading buffer conditions.</p><p num="0098"> Serum albumin, helper virus, which was competitively superior to the rAAV-1 vector in binding to the column in Example 4, based on the diverse evolution of apatite columns due to capacity issues at industrially reasonable column sizes. And PEG was mixed with the TFF concentrate prior to loading the apatite resin to increase the binding of the rAAV-1 vector compared to other in-process impurities such as protein impurities (see Example 3 above).</p><p num="0099"> Although many in-process impurities were also retained by the apatite resin at pH 7.0, apatite chromatography in the presence of PEG provides an efficient capture or binding strategy for the purification of the rAAV-1 vector.</p><p num="0100"> Experiments were conducted to determine if changing the buffer conditions could improve the partitioning of rAAV-1 from other in-process impurities. A small experiment using the AKTA explorer FPLC System (GE Healthcare, Piscataway, NJ) equipped with a 1.2 mL Tricorn 5 column (GE Healthcare) filled with CFT resin at a bed height of 6 cm at a flow rate of 150 cm / hour. Was done. For binding of rAAV-1 vector capture to bovine serum albumin (BSA), a model small molecule in-process impurity, in various buffer systems in the presence or absence of 5% PEG6000 on those columns. evaluated.</p><p num="0101"> rAAV-1 or BSA was injected into the CFT column in small doses (&lt;5% total volume) in the buffer system tested in the presence or absence of 5% (w / v) PEG6000. A small amount was used to eliminate the need for sample buffer replacement. 500 mM PO₄The product was eluted along the gradient of. The system was buffered at pH = 6.50 with 50 mM 2- (N-morpholino) ethanesulfonic acid (MES) and at pH 9.0 with 20 mM borate.</p><p num="0102"> From the data shown in Table 3, the binding of the rAAV-1 vector to the apatite resin was pH 6.5 or in the presence of 5% (w / v) PEG 6000, as shown by the spectrophotometric tracking results (data not shown). Essentially the same at pH 9.0, but with BSA, the binding of model small molecule in-process impurities, was dramatically reduced at pH 9.0 in the presence or absence of 5% (w / v) PEG6000. It is proved that it was done. From further analysis by enzyme-linked immunosorbent assay (ELISA) on the reduction in the volume of BSA bound to the apatite column under basic buffer loading conditions (ie, pH = 9.0), most BSA (approximately 78%). ) Is pH Although present in the pass-through fraction under 9.0 buffer conditions, further levels of elimination or reduction of BSA binding were achieved during the subsequent wash phase (approximately 19%), approximately 19% of BSA loaded on the column. It has been demonstrated that only 0.1% may actually remain bound to the apatite resin and not be eluted at the same time as the rAAV-1 vector. The rAAV-1 particles were stable at pH = 9.0, as indicated by the lack of infectivity loss or a decrease in the number of DNase-resistant particles (DRP) eluted.</p><p num="0103"> (Table 3) Relative strength of binding of rAAV-1 and BSA to apatite resin at pH = 6.5 or pH = 9.0 with or without 5% (w / v) PEG6000<img id="000003" he="51" wi="159" file="JP5956331B2_D0001.tif" img-format="tif" img-content="drawing" />List of abbreviations: + = weak bond; ++ = intermediate bond; ++++ = strong bond.</p><p num="0104"><u style="single">Example 6: Effect of serum on rAAV-1 collection culture on capture of rAAV-1 via apatite chromatography in the presence of PEG</u> RAAV-1 vector-producing cultures or feed streams containing rAAV-1 purified by the methods described herein will produce serum and serum proteins if the production cultures are grown in serum-containing medium. Can include. The production of rAAV-1 vectors using very low concentrations (ie, 1% or less) of serum (see, eg, US Pat. No. 6,995,006) has been described, but described in Example 3. A production culture or feed stream at a concentration that is present can produce a feed stream that effectively contains 20% serum and serum protein as a result of a 20-fold concentration of production culture collection. Experiments were performed on production cultures produced in the presence or absence of PEG6000 in the presence or absence of serum to assess the effect of serum components on the performance of apatite chromatography.</p><p num="0105"> The volume of apatite resin (CFT type I) was evaluated by traditional breakthrough analysis in a total of four column loading experiments using two model rAAV-1 production cultures. In one experiment, serum was included in the production culture. In another experiment, serum was not included in the production culture. Both feed streams were tested in the presence of 5% (w / v) PEG6000 or in the absence of PEG. The feed stream was representative of the collection process and was a clarified culture supernatant that had been passed through an anion exchange filter and concentrated 20-fold by tangential flow filtration as described herein. Supply flow to CFT type I column 1: 1 Boric acid buffer with pH = 9.0 was loaded by online mixing. The buffer contained either 0% or 10% (w / v) PEG6000 (to achieve a final concentration of 5% (w / v) PEG6000). When loaded on the column, the pass-through fractions were collected in a series of fractions and analyzed for the product by DRP-PCR. Functional capacity was defined as when the product concentration at column outflow just reached 1% of the concentration inflow into the column, taking into account online dilution. In the case of column loading containing PEG, the rest of the chromatography step was then run to evaluate the recovery of the vector in the elution fraction.</p><p num="0106"> The data presented in Figure 3 demonstrate that the addition of PEG6000 increases the volume of apatite resin towards binding of the rAAV-1 vector, regardless of whether serum was present in the production culture. Although not desired to be bound by theory, the supernatant after TFF in the presence of PEG is anion-mutual with the phosphate moiety where the presence of phosphate in the elution buffer may outweigh the competition. By action, rAAV-1 is selectively bound to the apatite resin as compared with other in-process impurities. In addition, the binding of rAAV-1 to the apatite resin is further distinguished from in-process impurities by metal interactions in which the presence of salts may outweigh the competition. The binding of rAAV-1 to the phosphate moiety is not driven primarily by hydrophobicity (ie, primarily hydrophobic), as the capture and elution buffers are formulated to be predominantly ionic. The elution buffer has 50 compared to the elution buffer containing 150 mM or more phosphate, which is often used in the elution of interactively bound compositions. Includes mM phosphate). Under these high salt, low phosphate elution conditions, residual helper virus, host cell DNA and other low molecular weight proteins contained in the supernatant of the production culture, if present, are retained on the resin. It is thought that it will be done. Surprisingly, from the data, the rAAV-1 vector produced in either serum-containing or serum-free medium in the presence of PEG6000 was at least 1.2 × 10¹²1.5 x 10 from 1 DRP / mL resin (1 mL load)¹⁴It has been demonstrated that it exhibits a binding ability to apatite resins in excess of DRP / mL resins (150 mL). In the absence of 5% (w / v) PEG6000, the binding ability of the apatite resin to the TFF collection was such that the rAAV-1 vector was not recovered in the CFT eluate, resulting in a vector produced in serum-containing medium. In the case of 2.4 × 10¹²7.2 × 10 for vectors produced in DRP / mL and serum-free medium¹²It was less than 1 DRP / ml.</p><p num="0107"><u style="single">Example 7: Purification of rAAV-1 via apatite chromatography</u> CHT type I columns were filled with 2 M NaCl and sterilized with 1 M NaOH. Prior to loading, the columns were equilibrated with a 6 column volume (CV) of 20 mM borate (pH = 9) + 5% (w / v) PEG6000. The TFF feed stream concentrate was placed on a 923 ml (14 cm diameter x 6 cm bed height) CHT column prepared as described at a flow rate of 96 cm / hour via a 3 mm BioProcess Skid (GE Healthcare). Loaded. TFF feed stream concentrate mixed in-line with an equal volume of 40 mM borate (pH = 9) + 10% (w / v) PEG6000 buffer to 20 mM borate (pH = 9) + 5% (w / v). ) The final concentration of PEG6000 was obtained.</p><p num="0108"> A series of four consecutive washes were performed to remove impurities in the process while retaining the rAAV-1 vector on the column. Wash 1 (chase) at 5 CV 50:50 (volume: volume) 20 mM borate (pH = 9.0) + 5% (w / v) PEG6000: 40 mM borate (pH = 9.0) + 10 % (w / v) Performed by inline mixing of PEG6000 and all loading lines were tracked with PEG6000. Furthermore, this step was found to selectively increase the binding affinity of the rAAV-1 vector. Wash 2 was performed with 15 CV of 150 mM potassium phosphate + 20 mM borate (pH = 9) + 5% (w / v) PEG6000 to retain serum albumin and other low molecular weights while retaining rAAV-1 on the column. Most of the in-process impurities of the protein were removed. Wash 3 (WII in Figure 5) at 15 CV 20 mM borate (pH = 9) + 5% (w / v) Performed with PEG6000, once PEG6000 was removed, all remaining phosphate was removed so that rAAV-1 remained bound to the column. Wash 4 (WIII in Figure 5) was performed with 5 CV of 20 mM HEPES (pH = 7.0) + 150 mM NaCl buffer to remove PEG6000 and adjust the salt concentration thereby with rAAV-1. Allowed discrimination from other in-process impurities, such as protein contaminants, which may remain bound to all remaining helper viruses or columns.</p><p num="0109"> The rAAV-1 vector was eluted from the column with 6 CV of 50 mM potassium phosphate + 20 mM HEPES (pH = 7.0) + 150 mM NaCl buffer. Figure 4 shows the conductivity for the CHT I chromatography procedure and the UV absorbance at 280 nm (A).₂₈₀) Is shown for typical spectrophotometric tracking. FIG. 5 shows the relative purity of the rAAV vector eluted from the apatite resin.</p><p num="0110"><u style="single">Glucan elimination by apatite chromatography</u> Glucan is a cellulose-like carbohydrate that leaches during the process from a cellulose-based depth filter used to collect rAAV-1 particles from production cultures. Concentrations above approximately 1 ng / mL can interfere with standard horseshoe crab amoebocyte lysate (LAL) tests for bacterial endotoxin contamination. As demonstrated in Table 4 below, the apatite CHT type I column excluded approximately 2.5 log of glucan from the production culture. Under the buffer conditions described herein, the majority of glucans were present in the pass-through fraction and did not bind to the column.</p><p num="0111"> (Table 4) Elimination of glucan during the process<img id="000004" he="68" wi="128" file="JP5956331B2_D0001.tif" img-format="tif" img-content="drawing" />Samples were assayed for glucans using a glucan-specific LAL-based dynamic colorimetric assay (Glucatell®, Cape Cod, MA).</p><p num="0112"><u style="single">Elimination of Ad5 helper virus by apatite chromatography</u> A preliminary spike-in test was performed with the feed stream from the final upstream process to confirm that Ad5 was eliminated from the feed stream by CHT chromatography. Ad5 spike levels were set based on data from Phase I virus elimination studies with CFT II resin and used three different loading ratio feed streams: 6.6 mL; 13.5 mL; and 33 mL. Supply flow after TFF / mL CHT resin.</p><p num="0113"> The data presented in Table 5, that is, the elimination of Ad5 by CHT was comparable to the elimination of 4 LRVs, demonstrating approximately 4 logs of Ad5 virus elimination, and loaded supply within 5 times the assessed range. It was considered to be independent of the flow capacity. The low Ad5 recovery is consistent with historical data suggesting that Ad5 binds more strongly to the apatite resin than rAAV-1 under buffer conditions used.</p><p num="0114"> (Table 5) All Ad5 infection units in the CHT column fraction<img id="000005" he="65" wi="128" file="JP5956331B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0115"> 8 x 10 in total⁹Infected particles Ad5 (ie, whole particles with approximately 10 P: I) were spiked into different volumes of post-TFF feed stream and flowed onto a 1.2 mL CHT column. The cells were run on a column at 100 cm / hour, fractions were collected and vector assayed with DRP and Ad5 by infectious titer assay. A spike-controlled Ad5 infectivity assay was used because both high concentrations and CHT-eluting samples are known to interfere in cell-based assays. Ad5 exclusion was determined as a logarithmic reduction (LRV) and calculated as the logarithm (logarithm reduction) of the total load Ad5 divided by the total amount of Ad5 recovered during the elution fraction.</p><p num="0116"><u style="single">Example 8: Thermal inactivation of residual helper virus</u> A heat inactivation step was performed to inactivate and remove all residual helper virus present in the CHT I eluate. For smaller experiments, divide the CHT I eluate into two 1 L PETG (Nalgene®) bottles and MgCl to increase the stability of the rAAV-1 vector.₂Was added to a final concentration of 2 mM. The bottles were incubated in a 53.5 ° C water bath with mixing until the temperature inside the bottle reached approximately 52 ° C. The bottles were then cooled by transfer to a room temperature water bath and mixed until the temperature inside the bottles exceeded room temperature by 5 ° C or less. The heat-killed mixture was filtered through a 4-inch Opticap® 0.22 μM filter membrane (Millipore Opticap® Catalog No. KVSC04HB3). Alternatively, for larger experiments, the CHT I eluate is placed in a sterile, disposable biotreatment bag (Custom Hyclone 5 L bag, CX5-14 film) on a temperature-controlled rocker at 53 ° C. It was heat inactivated at a temperature setting point with a swing speed of 40 RPM and a mixing angle of 12 ° (20 L radio heating pan). The CHT I eluate was incubated on a table until the temperature reached 52 ° C, followed by retention for an additional 10 minutes. MgCl to stabilize rAAV-1 during heating₂Was added to a final concentration of 2 mM. After heating, the product was filtered through a 0.2 μm filter and kept at room temperature overnight to minimize the effect of temperature that could have on subsequent hydrophobic interaction columns.</p><p num="0117"><u style="single">Example 9: Capture of rAAV-1 via hydrophobic interaction chromatography (HIC)</u> HIC is a technique for separating biomolecules based on their differences in surface hydrophobicity. Therefore, HIC is considered to be a method orthogonal to other purification steps in the rAAV-1 process. The HIC medium contains hydrophobic ligands such as linear hydrocarbons (eg, propyl (C3), butyl (C4), hexyl (C6) or octyl (C8))) or aromatic hydrocarbons (eg, phenyl). In pure water, the hydrophobic effect is too weak for functional interactions between the ligand and the protein, or the protein itself. However, liquid-leaving salts enhance hydrophobic interactions, and the addition of such salts drives the adsorption of proteins on HIC media. For this reason, HIC resins are usually loaded under high salt concentrations and eluted at lower salt concentrations.</p><p num="0118"><u style="single">HIC chromatography with ammonium sulphate buffer</u> In short, 170 ml (6 cm diameter x 6 cm bed height) HIC butyl column (Toyopearl® Butyl 650 M; Tosoh Biosciences, Montgomeryville, PA; Catalog number 14702) was sterilized with a few column volumes of 0.5 M NaOH and equilibrated with a 75:25 (volume: volume) mixture of 2 M ammonium sulfate + 50 mM bistris (pH = 7.0): 50 mM bistris (pH = 7.0). It became. Heat-killed rAAV-1 vector apatite eluate was mixed in-line at a ratio of 2 M ammonium sulfate + 50 mM bistris (pH = 7.0): rAAV-1 apatite eluate at a ratio of 75:25 (volume: volume) to 3.3 L. Loaded at the speed of / hour. In-line mixing avoids the risk of precipitation of any rAAV vector-1 by ammonium sulphate present in the buffer. One or more columns Volume 75:25 (Volume: Volume) 2 M Ammonium Sulfate + 50 mM Bistris pH = 7.0 Buffer: 50 mM Bistris pH = 7.0 + 10% Propylene Glycol (Volume: Volume) (EMD) Biosciences) Washed with buffer. In this example, propylene glycol was added to the buffer to sharpen the elution profile compared to the broader elution profile of the buffer without propylene glycol, which is optional in the process. The rAAV-1 vector was eluted from the column with 800 mM ammonium sulphate + 50 mM bis tris (pH = 7.0) buffer + 4% propylene glycol. Under the elution conditions used, all residual helper viruses and proteins present in the loading fluid are believed to remain bound to the column.</p><p num="0119"> Unwanted substances from the rAAV-1 production process are stringent prior to disposal due to both the product being a viral vector and the use of live adenovirus type 5 (Ad5) as a helper virus for production. It is necessary to remove various impurities. Liquid debris from chromatographic operations is typically first decontaminated with bleach upon use and then further decontaminated by holding at a high pH prior to neutralization and disposal. Ammonium sulphate present in HIC buffer reacts with both bleach and sodium hydroxide, releasing harmful chlorine gas and ammonia gas, respectively. Therefore, the main consideration for process optimization in the HIC chromatography step has been the development of suitable buffer systems that can safely remove contaminants by methods known in the art.</p><p num="0120"><u style="single">Screening of buffers suitable for binding rAAV-1 to HIC columns</u> Relative binding efficiency was determined by loading the rAAV-1 vector onto the column under a variety of different buffer conditions and measuring the amount of rAAV-1 vector present in the pass-through fraction (Table 6). The buffers evaluated included both the high concentration release salts traditionally used in the HIC chromatography step and some low pH buffers, in this case mixed mode interactions (HIC /). Cation exchange) could potentially occur. Both Tosoh Butyl 650 M resin and EMD Phenyl resin bound the vector with some of the alternative buffers.</p><p num="0121"> High salt concentration buffers used in hydrophobic interaction chromatography limit the flow velocity or cause mixing problems and risk product precipitation due to salt crystallization at the temperature of buffer storage or manipulation. Further screening must be done for viscosity issues that can result in high back pressure that can be caused. Based on the data in Table 6 below, and taking into account the above factors, 1 M sodium citrate, pH = 7.0 as the optimal buffer for binding the rAAV-1 vector to the HIC chromatography medium. Selected.</p><p num="0122"> (Table 6) Screening for AAV1 binding in alternative buffers<img id="000006" he="72" wi="158" file="JP5956331B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0123"> Experiments were performed on a Tricorn 5/50 column (bed height 6 cm, column volume 1.2 mL) at room temperature using the purified rAAV-1 vector. Equilibrate the column with the buffers listed, approximately 2x10¹¹Loaded DRP or rAAV-1 onto the column. rAAV-1 was eluted with a linear gradient of 20 CV from 145 mM bis tris (pH = 7.0), 10% (v / v) propylene glycol. Passage was collected and assayed by DRP analysis on the rAAV-1 fraction that was applied to the column and passed or did not bind.</p><p num="0124"> Further characterization of in-process impurity elimination for HIC columns in various buffers demonstrated good binding of rAAV-1 in previous experiments. The data in Table 7 below for model contaminant binding demonstrates that both adenovirus in the feed stream and, if present, DNA are effectively identified by the HIC chromatography step.</p><p num="0125"> (Table 7) Relative binding of rAAV-1 pair model in-process impurities in different HIC buffers<img id="000007" he="75" wi="128" file="JP5956331B2_D0001.tif" img-format="tif" img-content="drawing" />Abbreviation list: "0" = no binding substance present in the pass-through fraction; "-" = "very weak binding substance"; "+" = strong binding substance; "++" = stronger binding substance.</p><p num="0126"> Experiments were performed on a Tricorn 5/50 column (bed height 6 cm, column capacity 1.2 mL) at room temperature. The column was equilibrated with the described buffer and the indicated sample was loaded onto the column. Each sample was eluted with a linear gradient of 20 CV. Samples were collected and assayed for relevant loading substances.</p><p num="0127"><u style="single">HIC chromatography with sodium citrate buffer</u> A heat-killed rAAV-1 vector apatite eluate was then loaded onto the HIC butyl column to further reduce all residual process impurities and as a concentration and desalting step. A 373 ml (6 cm diameter x 8.9 cm bed height) HIC butyl column (Tosoh Biosciences Toyopearl® Butyl 650 M Catalog No. 14702) was sterilized with a few column volumes of 0.5 M NaOH and 1 M citrate + 20 mM phosphate. Sodium: 20 mM Sodium Phosphate 75:25 (Volume: Volume) Equilibrated at 5 CV of the mixture. Heat-killed rAAV-1 vector CHT I eluate at a rate of 106 cm / hour by in-line mixing of 1 M citrate + 20 mM sodium phosphate: CHT I eluate at a ratio of 75:25 (volume: volume). Loaded with. In-line mixing avoids the risk of precipitation of any rAAV-1 vector. Column 1 M Citrate + 20 mM Sodium Phosphate: 75:25 (Volume: Volume) Mix of 20 mM Sodium Phosphate Buffer 5 Washed with CV. The rAAV-1 vector was eluted from the column with 6 CV of 0.35 M citrate + 20 mM sodium phosphate. The column was then washed with 3.5 CV of 20 mM sodium phosphate buffer. This low salt cleaning solution (20 (Mmsodium) elutes a fraction of rAAV-1 vector particles whose elution profile is hydrophobically different from that of rAAV-1 vector particles eluted in a higher salt elution buffer. In fact, if the low salt eluted fraction was isolated and reapplied to the HIC column under the conditions described, the vector population would still elute only in the low salt fraction and this fraction would be in the column. It suggested that it was not the result of a capacity breach. Infectivity analysis suggests that this rAAV-1 fraction is likely to correspond to a population containing empty capsids, partially denatured capsids, less infectious capsid substances and partially complete capsids. It is suggested. Therefore, this finding can result in improved isolation of rAAV-1 particles that are less infectious and therefore less desirable as a product. Under the elution conditions used, all residual helper viruses and proteins present in the loading fluid are believed to remain bound to the column and should therefore be present in the low salt strip fluid.</p><p num="0128"><u style="single">Example 10: Buffer exchange by size exclusion chromatography (SEC)</u> Buffer exchange by size exclusion chromatography provides further protein exclusion of proteins sized to pass through the pores in the resin and is relatively fast with respect to the time required to exchange the buffer. The buffer exchange performed at this stage was to ensure that the HIC eluate from the previous stage was replaced with a buffer for rAAV-1 binding suitable for the final anion exchange chromatography step during the process. there were. 3.2 L (14 cm in diameter x 21 cm in bed height) Amersham Superdex® 200-minute resin (Amersham / GE Healthcare, Piscataway, NJ; It was prepared by filling with Catalog No. 17-1043-04) and sterilized with 2 M NaCl + 1 M NaOH and equilibrated with 2.8 CV of 20 mM NaCl + 20 mM Tris (pH = 8.0). The HIC eluate was subdivided for SEC in 3 consecutive cycles of approximately 400 ml each, and each cycle was loaded with less than 12.5% of the SEC column volume. Product peaks from 3 SEC cycles (included in void volume) were collected in a single biotreatment bag. The HIC eluate was loaded onto the column at a flow rate of 49 cm / hour. The column was followed and rinsed with 1.4 CV of 20 mM NaCl + 20 mM Tris (pH = 8.0), and the rAAV-1 vector present in the HIC eluate was present in the void volume of the column. After recovery of the described void volume, the second and third fractions were loaded and the first fraction was continuously recovered on the same column as described above.</p><p num="0129"><u style="single">Example 11: Alien factor (virus elimination)</u> A virus elimination filter was introduced into the process as an optional step to eliminate foreign viruses that may be present as trace contaminants, thus resulting in an industrially rational orthogonal step. Examples of such filters are known in the art and include Millipore Viresolve® NFR (50 nm), Pall Ultipore® VF (50 nm) and Asahi 70 nm. Millipore Viresolve® NFR (Millipore 4 "Virosolve NFR Filter Catalog No. KZRV 04T C3) Prepare virus elimination filters according to the manufacturer's instructions, rinse with 20 mM NaCl + 20 mM Tris (pH = 8.0), SEC The eluent was filtered through a membrane. The filter was rinsed with several volumes of 20 mM NaCl + 20 mM Tris-HCl (pH 8.0) and pooled with the filtered SEC eluate.</p><p num="0130"><u style="single">Example 12: Anion Exchange Chromatography</u> Unosphere® Q Resin (Biorad, Hercules,) with a second anion exchange capture step for the rAAV-1 vector as the final concentration and refinement step. I went to CA). A 373 ml (8.9 cm diameter x 6 cm bed height) column was sterilized with several column volumes of 0.5 M NaOH and equilibrated with 7 CV 20 mM NaCl + 20 mM Tris (pH = 8.0) buffer. The SEC void volume fraction or optionally virus-filtered eluate was loaded at a rate of 309 cm / hour. The column was washed with 10 CV of 60 mM NaCl. The ionic strength of the wash solution was chosen to retain rAAV-1 bound to the resin while removing any other in-process impurities such as glucan that could be introduced by leaching from the various filters used in the purification step. did. The rAAV-1 vector was eluted from the column with 6 CV of 130 mM NaCl. It is believed that the ionic strength of 130 mM NaCl salt elution removes rAAV-1 from the column, whereas all remaining trace in-process impurities, such as serum albumin or helper virus, remain bound.</p><p num="0131"> In Figure 6, SDS-PAGE compares the degree of purification over various process steps. In-process samples from typical production culture collections were run on a modified / reduced 10% polyacrylamide gel and stained with cypro orange. 1 x 10 samples after all collection¹⁰Loaded with 1 DRP / lane. The two upstream samples prior to the TFF enrichment step (the first clarification step and the anion exchange (AEX) pass-through sample) are 1 × 10 due to volume constraints on the gel.⁹It could only be loaded with one DRP / lane. Beta-galactosidase (B-Gal) was loaded at 50 ng / lane and staining susceptibility and consistency throughout the gel was evaluated. Three AAV1 capsid proteins (VP1, 2 and 3) have been shown.</p><p num="0132"><u style="single">Example 13: Recovery rate of rAAV during purification</u> The data presented in FIG. 7 show the recovery of infectious rAAV particles from typical production cultures of the rAAV-1 vector after each step of the purification scheme. The recovery rate was calculated based on the total DRP of the rAAV-1 vector recovered from each step divided by the total number of DRPs subjected to or loaded during that purification step. The data demonstrate that approximately 60% or more recovery was achieved at each stage of the purification process. In many experiments, the range of recovery from each step was at least 60% to 90%. Notably, the range of recovery in the capture step (ie, the apatite chromatography step) in the individual experiments ranged from 57% to over 90%. In addition, the range of recovery at the HIC stage ranged from 60% to 80%.</p><p num="0133"> Although the above invention has been described in a little more detail with examples and examples for clarity of understanding, it will be apparent to those skilled in the art that certain minor changes and modifications will be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention.</p>