WO2007016250A1 - Methode pour purifier une proteine - Google Patents

Methode pour purifier une proteine Download PDF

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Publication number
WO2007016250A1
WO2007016250A1 PCT/US2006/029206 US2006029206W WO2007016250A1 WO 2007016250 A1 WO2007016250 A1 WO 2007016250A1 US 2006029206 W US2006029206 W US 2006029206W WO 2007016250 A1 WO2007016250 A1 WO 2007016250A1
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Prior art keywords
protein
cells
interest
cell
supernatant
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PCT/US2006/029206
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English (en)
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Miguel E. CARRIÓN
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Genvec, Inc.
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Publication of WO2007016250A1 publication Critical patent/WO2007016250A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention provides a method for purifying a protein of interest, comprising (a) providing a culture comprising a population of virus packaging cells, (b) infecting the cells with a population of vector particles comprising a nucleic acid sequence that encodes the protein of interest, (c) maintaining the infected cells in culture to express the nucleic acid and produce the protein of interest in a supernatant or in the infected cells (d) collecting the supernatant or cells comprising the protein of interest at about 28 to about 68 hours after infection of the cells, wherein if the cells are collected, the cells are lysed to obtain a cell lysate (e) subjecting the supernatant or the cell lysate to filtration to obtain a filtered composition comprising the protein of interest, (1) subjecting the filtered composition to ion exchange chromatography to obtain a first eluted composition comprising the protein of interest, and (g) subjecting the first eluted composition to size-exclusion chromatography to obtain a second
  • the ratio of the amount of viral vector particle encapsidated DNA in the second eluted composition as compared to the supernatant or cell lysate of step (d) is no more than about 15:100
  • the ratio of the amount of host cell DNA in the second eluted composition as compared to the supernatant or cell lysate of step (d) is no more than about 15:100
  • the ratio of the amount of viral particle component protein in the second eluted composition as compared to the supernatant or cell lysate of step (d) is no more than about 15:100
  • the ratio of the amount of host cell protein in the second eluted composition as compared to the supernatant or cell lysate of step (d) is no more than about 15:100, such that the protein of interest is purified.
  • the invention also provides a method for purifying a protein of interest, comprising (a) providing a culture comprising a population of cells, (b) transfecting the cells with a nucleic acid sequence that encodes the protein of interest, (c) maintaining the transfected cells in culture to express the nucleic acid and produce the protein of interest in a supernatant or in the transfected cells, (d) collecting the supernatant or the cells comprising the protein of interest at about 28 to about 68 hours after infection, wherein if the cells are collected, the cells are lysed to obtain a cell lysate, (e) subjecting the supernatant or the cell lysate to filtration to obtain a filtered composition comprising the protein of interest, (f) subjecting the filtered composition to ion exchange chromatography to obtain a first eluted composition comprising the protein of interest, and (g) subjecting the first eluted composition to size-exclusion chromatography to obtain a second eluted composition comprising
  • the ratio of the amount of host cell DNA in the second eluted composition as compared to the supernatant or cell lysate of step (d) is no more than about 15:100 or the ratio of the amount of host cell protein in the second eluted composition as compared to the supernatant or cell lysate of step (d) is no more than about 15:100, such that the protein of interest is purified.
  • the invention provides a method for purifying a protein of interest.
  • the method comprises (a) providing a culture comprising a population of virus packaging cells, (b) infecting the cells with a population of vector particles comprising a nucleic acid sequence that encodes the protein of interest, (c) maintaining the infected cells in culture to express the nucleic acid and produce the protein of interest in a supernatant or in the infected cells (d) collecting the supernatant or cells comprising the protein of interest at about 28 to about 68 hours after infection of the cells, wherein if the cells are collected, the cells are lysed to obtain a cell lysate, (e) subjecting the supernatant or the cells to filtration to obtain a filtered composition comprising the protein of interest, (f) subjecting the filtered composition to ion exchange chromatography to obtain a first eluted composition comprising the protein of interest, and (g) subjecting the first eluted composition to size-exclusion chromatography to obtain a second
  • the method comprises (a) providing a culture comprising a population of cells, (b) transfecting the cells with a nucleic acid sequence that encodes the protein of interest, (c) maintaining the transfected cells in culture to express the nucleic acid and produce the protein of interest in a supernatant or in the transfected cells, (d) collecting the supernatant or the cells comprising the protein of interest at about 28 to about 68 hours after infection, wherein if the cells are collected, the cells are lysed to obtain a cell lysate, (e) subjecting the supernatant or the cell lysate to filtration to obtain a filtered composition comprising the protein of interest, (f) subjecting the filtered composition to ion exchange chromatography to obtain a first eluted composition comprising the protein of interest, and (g) subjecting the first eluted composition to size-exclusion chromatography to obtain a second eluted composition comprising the protein of interest.
  • the ratio of the amount of host cell DNA in the second eluted composition as compared to the supernatant or cell lysate of step (d) is no more than about 15:100 or the ratio of the amount of host cell protein in the second eluted composition as compared to the supernatant or cell lysate of step (d) is no more than about 15:100, such that the protein of interest is purified.
  • the ratio of the amount of viral vector particle encapsidated DNA in the second eluted composition as compared to the supernatant or cell lysate of step (d) is no more than about 15:100 or the ratio of the amount of viral particle component protein in the second eluted composition as compared to the supernatant or cell lysate of step (d) is no more than about 15:100.
  • compositions of the invention can include, and methods of the invention can be practiced, with any suitable type of viral vector particle.
  • a "viral vector particle” is any particle comprising a collection of viral proteins that form a particle with an interior volume, which transfers to a host cell and/or expresses in a host cell genetic information contained in the interior volume.
  • a viral vector particle can be based upon, derived from, or originate from any suitable virus.
  • the viral vector particle can be an unmodified naturally occurring (i.e., "wild-type") virus particle. More typically, the viral vector particle will be a modified viral particle, such as a viral gene transfer vector and/or a synthetic viral vector particle.
  • the viral vector particle contains, or is associated with, a nucleotide genome, which preferably is a DNA genome, and most preferably is a double-stranded DNA genome, as such viral genomes are typically easier to manipulate when generating a viral gene transfer vector. Due to the limitations of their genomes, viral vectors with single- stranded RNA genomes are least preferred (although such viral vector particles often still are suitable).
  • the viral vector particle desirably comprises a genome that is transcribed and replicated in the nucleus of the host cell, and the mRNAs transcribed therefrom are preferably processed posttranscriptionally and moved to the cytoplasm for translation (thus, mimicking the translation of host genes).
  • the viral vector particle's nucleic acid does not integrate into the host cell genome.
  • the viral vector particle is derived from, is based on, comprises, or consists of, a virus that normally infects animals, preferably vertebrates, such as mammals and, especially, humans.
  • Suitable viral vector particles include, for example, adenoviral vector particles (including any virus of or derived from a virus of the adenoviridae), adeno-associated viral vector particles (AAV vector particles) or other parvoviruses and parvoviral vector particles, papillomaviral vector particles, reovirus particles, and viruses of, or viral vector ' particles derived from, the arenaviridae, bunyaviridae, circoviridae, coronaviridae, filoviridae, fiaviviridae, hepadnaviridae, herpesviridae, paramyxoviridae, rhabdoviridae, orthomyxoviridae, poxviridae, retroviridae, togaviridae, bimaviridae, astroviridae, potyviridae, picornaviridae, myoviridae, tectiviridae, nodaviridae
  • the viral vector particle is preferably a non-enveloped viral vector particle.
  • suitable non-enveloped viral vector particles include adenoviral vector particles, AAV vectors, or viruses of, or viral vector particles derived from, the papillomaviral, parvoviridae, reoviridae, bimaviridae, astroviridae, potyviridae, picornaviridae, myoviridae, tectiviridae, nodaviridae, calciviridae, iridoviridae, caulimoviridae, papovaviridae, and phycodnaviridae.
  • viruses and viral vectors examples are provided in, e.g., VIROLOGY, B.N. Fields et al., eds., Raven Press, Ltd., New York (3rd ed., 1996 and 4th ed., 2001), ENCYCLOPEDIA OF VIROLOGY, R.G. Webster et al., eds., Academic Press (2nd ed., 1999), FUNDAMENTAL VIROLOGY, Fields et al., eds., Lippincott-Raven (3rd ed., 1995), Levine, "Viruses," Scientific American Library No. 37 (1992), MEDICAL VIROLOGY, D.O. White et al., eds. Academic Press (2nd ed. 1994), INTRODUCTION TO MODERN VIROLOGY, Dimock, NJ. et al., eds., Blackwell Scientific Publications, Ltd. (1994), and other references cited herein.
  • adenoviral vectors can be constructed and/or purified using the methods set forth, for example, in Graham et al., MoI. Biotechol., 33(3): 207-220 (1995), U.S. Patents 5,922,576, 5,965,358 and 6,168,941 and International Patent Applications WO 98/22588, WO 98/56937, WO 99/15686, WO 99/54441, and WO 00/32754.
  • Adeno-associated viral vectors can be constructed and/or purified using the methods set forth, for example, in U.S.
  • the viral vector particle can be a chimeric viral vector particle.
  • chimeric viral vector particles are described in, e.g., Reynolds et al., MoI. Med. Today, 5(1): 25-31 (1999), Boursnell et al., Gene, 13: 311-317 (1991), Dobbe et al., Virology, 288(2): 283-94 (2001), Grene et al., AIDS Res. Human. Retroviruses, 13(1): 41-51 (1997), Reimann et al., J.
  • viral vector particles include adeno-associated viral vector particles and adenoviral vector particles.
  • Adenovirus from various origins, subtypes, or mixture of subtypes can be used as the source of the viral genome for the adenoviral vector.
  • non-human adenovirus e.g., simian, avian, canine, ovine, or bovine adenoviruses
  • a human adenovirus preferably is used as the source of the viral genome for the adenoviral vector.
  • an adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), an unclassified serogroup (e.g., serotypes 49 and 51), or any other adenoviral serotype.
  • subgroup A e.g., serotypes 12, 18, and 31
  • subgroup B e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50
  • subgroup C e.g., serotypes 1, 2, 5, and 6
  • subgroup D e.g., serotypes
  • Adenoviral serotypes 1 through 51 are available from the American Type Culture Collection (ATCC, Manassas, VA).
  • the adenoviral vector is of human subgroup C, especially serotype 2 or even more desirably serotype 5.
  • non-group C adenoviruses can be used to prepare adenoviral gene transfer vectors for delivery of gene products to host cells.
  • Preferred adenoviruses used in the construction of non-group C adenoviral gene transfer vectors include Ad 12 (group A), Ad7 and Ad35 (group B), Ad30 and Ad36 (group D), Ad4 (group E), and Ad41 (group F).
  • Non-group C adenoviral vectors methods of producing non-group C adenoviral vectors, and methods of using non-group C adenoviral vectors are disclosed in, for example, U.S. Patents 5,801,030, 5,837,511, and 5,849,561 and International Patent Applications WO 97/12986 and WO 98/53087.
  • the adenoviral vector can comprise a mixture of subtypes and thereby be a "chimeric" adenoviral vector.
  • a chimeric adenoviral vector can comprise an adenoviral genome that is derived from two or more (e.g., 2, 3, 4, etc.) different adenovirus serotypes.
  • a chimeric adenoviral vector can comprise approximately different or equal amounts of the genome of each of the two or more different adenovirus serotypes.
  • the chimeric adenoviral vector genome is comprised of the genomes of two different adenovirus serotypes
  • the chimeric adenoviral vector genome preferably comprises no more than about 70% (e.g., no more than about 65%, about 50%, or about 40%) of the genome of one of the adenovirus serotypes, with the remainder of the chimeric adenovirus genome being derived from the genome of the other adenovirus serotype.
  • the chimeric adenoviral vector can contain an adenoviral genome comprising a portion of a serotype 2 genome and a portion of a serotype 5 genome.
  • nucleotides 1-456 of such an adenoviral vector can be derived from a serotype 2 genome, while the remainder of the adenoviral genome can be derived from a serotype 5 genome.
  • the viral vector particle is replication-deficient in host cells.
  • replication-deficient is meant that the viral vector requires complementation of one or more regions of the viral genome that are required for replication, as a result of, for example, a deficiency in at least one replication-essential gene function (i.e., such that the viral vector does not replicate in typical host cells, especially those in a human patient that could be infected by the viral vector).
  • a deficiency in a gene, gene function, gene, or genomic region, as used herein, is defined as a mutation or deletion of sufficient genetic material of the viral genome to obliterate or impair the function of the gene (e.g., such that the function of the gene product is reduced by at least about 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, or 50-fold) whose nucleic acid sequence was mutated or deleted in whole or in part. Deletion of an entire gene region often is not required for disruption of a replication- essential gene function. However, for the purpose of providing sufficient space in the viral (e.g., adenoviral) genome for one or more transgenes, removal of a majority of a gene region may be desirable.
  • Replication-essential gene functions are those gene functions that are required for replication (e.g., propagation) and are encoded by, for example, the adenoviral early regions (e.g., the El, E2, and E4 regions), late regions (e.g., the L1-L5 regions), genes involved in viral packaging (e.g., the IVa2 gene), and virus-associated RNAs (e.g., VA-RNAl and/or VA-RNA-2).
  • the replication-deficient (i.e., replication-defective) viral vector particle is preferably a replication-deficient adenoviral vector particle.
  • the replication-deficient adenoviral vector desirably requires complementation of at least one replication-essential gene function of one or more regions of the adenoviral genome.
  • the adenoviral vector requires complementation of at least one gene function of the ElA region, the ElB region, or the E4 region of the adenoviral genome required for viral replication (denoted an El-deficient or E4-deficient adenoviral vector).
  • the recombinant adenovirus also can have a mutation in the major late promoter (MLP), as discussed in International Patent Application Publication WO 00/00628.
  • MLP major late promoter
  • the adenoviral vector is deficient in at least one replication-essential gene function (desirably all replication-essential gene functions) of the El region and at least one gene function of the nonessential E3 region (e.g., an Xba I deletion of the E3 region) (denoted an El/E3-deficient adenoviral vector).
  • the adenoviral vector can be deficient in part or all of the ElA region and/or part or all of the ElB region, e.g., in at least one replication-essential gene function of each of the ElA and ElB regions, thus requiring complementation of the ElA region and the ElB region of the adenoviral genome for replication.
  • the adenoviral vector also can require complementation of the E4 region of the adenoviral genome for replication, such as through a deficiency in one or more replication-essential gene functions of the E4 region.
  • the adenoviral vector genome can comprise a deletion beginning at any nucleotide between nucleotides 335 to 375 (e.g., nucleotide 356) and ending at any nucleotide between nucleotides 3,310 to 3,350 (e.g., nucleotide 3,329) or even ending at any nucleotide between 3,490 and 3,530 (e.g., nucleotide 3,510) (based on the adenovirus serotype 5 genome).
  • the adenoviral vector genome can comprise a deletion beginning at any nucleotide between nucleotides 22,425 to 22,465 (e.g., nucleotide 22,443) and ending at any nucleotide between nucleotides 24,010 to 24,050 (e.g., nucleotide 24,032) (based on the adenovirus serotype 5 genome).
  • the adenoviral vector genome can comprise a deletion beginning at any nucleotide between nucleotides 28,575 to 29,615 (e.g., nucleotide 28,593) and ending at any nucleotide between nucleotides 30,450 to 30,490 (e.g., nucleotide 30,470) (based on the adenovirus serotype 5 genome).
  • the adenoviral vector genome can comprise a deletion beginning at, for example, any nucleotide between nucleotides 32,805 to 32,845 (e.g., nucleotide 32,826) and ending at, for example, any nucleotide between nucleotides 35,540 to 35,580 (e.g., nucleotide 35,561) (based on the adenovirus serotype 5 genome).
  • each of the aforementioned nucleotide numbers can be +/- 1, 2, 3, 4, 5, or even 10 or 20 nucleotides.
  • the adenoviral vector When the adenoviral vector is deficient in at least one replication-essential gene function in one region of the adenoviral genome (e.g., an El- or E 1/E3 -deficient adenoviral vector), the adenoviral vector is referred to as "singly replication-deficient.”
  • a particularly preferred singly replication-deficient adenoviral vector is, for example, a replication- deficient adenoviral vector requiring, at most, complementation of the El region of the adenoviral genome, so as to propagate the adenoviral vector (e.g., to form adenoviral vector particles).
  • the adenoviral vector can be "multiply replication-deficient,” meaning that the adenoviral vector is deficient in one or more replication-essential gene functions in each of two or more regions of the adenoviral genome, and requires complementation of those functions for replication.
  • the aforementioned El -deficient or E 1/E3 -deficient adenoviral vector can be further deficient in at least one replication-essential gene function of the E4 region (denoted an E1/E4- or El/E3/E4-deficient adenoviral vector), and/or the E2 region (denoted an E1/E2- or E 1/E2/E3 -deficient adenoviral vector), preferably the E2A region (denoted an E1/E2A- or E 1/E2A/E3 -deficient adenoviral vector).
  • the deficiencies can be a combination of the nucleotide deletions discussed above with respect to each individual region.
  • the vector preferably does not comprise a complete deletion of the E2A region, which deletion preferably is less than about 230 base pairs in length.
  • the E2A region of the adenovirus codes for a DBP (DNA binding protein), a polypeptide required for DNA replication.
  • DBP is composed of 473 to 529 amino acids depending on the viral serotype. It is believed that DBP is an asymmetric protein that exists as a prolate ellipsoid consisting of a globular Ct with an extended Nt domain.
  • the Ct domain is responsible for DBP 's ability to bind to nucleic acids, bind to zinc, and function in DNA synthesis at the level of DNA chain elongation.
  • the Nt domain is believed to function in late gene expression at both transcriptional and post-transcriptional levels, is responsible for efficient nuclear localization of the protein, and also may be involved in enhancement of its own expression. Deletions in the Nt domain between amino acids 2 to 38 have indicated that this region is important for DBP function (Brough et al., Virology, 196: 269-281 (1993)).
  • any multiply replication- deficient adenoviral vector contains this portion of the E2A region of the adenoviral genome.
  • the desired portion of the E2A region to be retained is that portion of the E2A region of the adenoviral genome which is defined by the 5' end of the E2A region, specifically positions Ad5(23816) to Ad5(24032) of the E2A region of the adenoviral genome of serotype Ad5.
  • This portion of the adenoviral genome desirably is included in the adenoviral vector because it is not complemented in current E2A cell lines so as to provide the desired level of viral propagation.
  • the adenoviral vector can comprise an adenoviral genome deficient in one or more replication-essential gene functions of each of the El and E4 regions (i.e., the adenoviral vector is an El/E4-deficient adenoviral vector), preferably with the entire coding region of the E4 region having been deleted from the adenoviral genome. In other words, all the open reading frames (ORFs) of the E4 region have been removed.
  • the adenoviral vector is rendered replication-deficient by deletion of all of the El region and by deletion of a portion of the E4 region.
  • the E4 region of the adenoviral vector can retain the native E4 promoter, polyadenylation sequence, and/or the right-side inverted terminal repeat (ITR).
  • the adenoviral vector when multiply replication-deficient, especially in replication-essential gene functions of the El and E4 regions, can include a spacer sequence to provide viral growth in a complementing cell line similar to that achieved by singly replication-deficient adenoviral vectors, particularly an El -deficient adenoviral vector.
  • the spacer is desirably located between the L5 fiber region and the right-side ITR.
  • the E4 polyadenylation sequence alone or, most preferably, in combination with another sequence exists between the L5 fiber region and the right-side ITR, so as to sufficiently separate the retained L5 fiber region from the right-side ITR, such that viral production of such a vector approaches that of a singly replication-deficient adenoviral vector, particularly a singly replication-deficient El deficient adenoviral vector.
  • the spacer sequence can contain any nucleotide sequence or sequences which are of a desired length, such as sequences at least about 15 base pairs (e.g., between about 15 base pairs and about 12,000 base pairs), preferably about 100 base pairs to about 10,000 base pairs, more preferably about 500 base pairs to about 8,000 base pairs, even more preferably about 1,500 base pairs to about 6,000 base pairs, and most preferably about 2,000 to about 3,000 base pairs in length.
  • the spacer sequence can be coding or non-coding and native or non-native with respect to the adenoviral genome, but does not restore the replication-essential function to the deficient region.
  • the spacer can also contain a promoter-variable expression cassette.
  • the spacer comprises an additional polyadenylation sequence and/or a passenger gene.
  • both the E4 polyadenylation sequence and the E4 promoter of the adenoviral genome or any other (cellular or viral) promoter remain in the vector.
  • the spacer is located between the E4 polyadenylation site and the E4 promoter, or, if the E4 promoter is not present in the vector, the spacer is proximal to the right-side ITR.
  • the spacer can comprise any suitable polyadenylation sequence.
  • polyadenylation sequences include synthetic optimized sequences, BGH (Bovine Growth Hormone), polyoma virus, TK (Thymidine Kinase), EBV (Epstein Barr Virus) and the papillomaviruses, including human papillomaviruses and BPV (Bovine Papilloma Virus).
  • BGH Bovine Growth Hormone
  • polyoma virus TK (Thymidine Kinase)
  • EBV Epstein Barr Virus
  • the spacer includes an SV40 polyadenylation sequence.
  • the SV40 polyadenylation sequence allows for higher virus production levels of multiply replication deficient adenoviral vectors.
  • the spacer is composed of the glucuronidase gene.
  • the use of a spacer in an adenoviral vector is further described in, for example, U.S. Patent 5,851,806 and International Patent Application WO 97/21826.
  • the spacer is preferably transcriptionally inert.
  • the adenoviral vector can be a conditionally-replicating adenoviral vector, which is engineered to replicate under conditions pre-determined by the practitioner.
  • replication-essential gene functions e.g., gene functions encoded by the adenoviral early regions
  • an inducible, repressible, or tissue-specific transcription control sequence e.g., promoter, hi this embodiment, replication requires the presence or absence of specific factors that interact with the transcription control sequence.
  • adenoviral vector replication in, for instance, lymph nodes, to obtain continual antigen production and control immune cell production.
  • Conditionally-replicating adenoviral vectors are described further in U.S. Patent 5,998,205.
  • the adenoviral genome can contain benign or non-lethal modifications, i.e., modifications which do not render the adenovirus replication-deficient, or, desirably, do not adversely affect viral functioning and/or production of viral proteins, even if such modifications are in regions of the adenoviral genome that otherwise contain replication-essential gene functions.
  • benign or non-lethal modifications i.e., modifications which do not render the adenovirus replication-deficient, or, desirably, do not adversely affect viral functioning and/or production of viral proteins, even if such modifications are in regions of the adenoviral genome that otherwise contain replication-essential gene functions.
  • modifications commonly result from DNA manipulation or serve to facilitate expression vector construction.
  • benign mutations often have no detectable adverse effect on viral functioning.
  • the adenoviral vector can comprise a deletion of nucleotides 10,594 and 10,595 (based on the adenoviral serotype 5 genome), which are associated with VA-RNA-I transcription, but the deletion of which does not prohibit production of VA-RNA-I.
  • the coat protein of a viral vector can be manipulated to alter the binding specificity or recognition of a virus for a viral receptor on a potential host cell.
  • adenovirus such manipulations can include deletion of regions of the fiber, penton, or hexon, insertions of various native or non-native ligands into portions of the coat protein, and the like.
  • Manipulation of the coat protein can broaden the range of cells infected by a viral vector or enable targeting of a viral vector to a specific cell type.
  • the adenoviral vector comprises a chimeric coat protein (e.g., a fiber, hexon pIX, pllla, or penton protein), which differs from the wild- type (i.e., native) coat protein by the introduction of a nonnative amino acid sequence, preferably at or near the carboxyl terminus.
  • a nonnative amino acid sequence is inserted into or in place of an internal coat protein sequence.
  • the nonnative amino acid sequence can be inserted within the internal coat protein sequence or at the end of the internal coat protein sequence.
  • the resultant chimeric viral coat protein is able to direct entry into cells of the adenoviral, vector comprising the coat protein that is more efficient than entry into cells of a vector that is identical except for comprising a wild-type adenoviral coat protein rather than the chimeric adenoviral coat protein.
  • the chimeric adenovirus coat protein binds a novel endogenous binding site present on the cell surface that is not recognized, or is poorly recognized, by a vector comprising a wild-type coat protein.
  • One direct result of this increased efficiency of entry is that the adenovirus can bind to and enter numerous cell types which an adenovirus comprising wild-type coat protein typically cannot enter or can enter with only a low efficiency.
  • the adenoviral vector comprises a chimeric virus coat protein not selective for a specific type of eukaryotic cell.
  • the chimeric coat protein differs from the wild-type coat protein by an insertion of a nonnative amino acid sequence into or in place of an internal coat protein sequence.
  • the chimeric adenovirus coat protein efficiently binds to a broader range of eukaryotic cells than a wild-type adenovirus coat, such as described in International Patent Application WO 97/20051.
  • Specificity of binding of an adenovirus to a given cell also can be adjusted by use of an adenovirus comprising a short-shafted adenoviral fiber gene, as discussed in U.S. Patent 5,962,311.
  • Use of an adenovirus comprising a short-shafted adenoviral fiber gene reduces the level or efficiency of adenoviral fiber binding to its cell-surface receptor and increases adenoviral penton base binding to its cell-surface receptor, thereby increasing the specificity of binding of the adenovirus to a given cell.
  • an adenovirus comprising a short-shafted fiber enables targeting of the adenovirus to a desired cell-surface receptor by the introduction of a nonnative amino acid sequence either into the penton base or the fiber knob.
  • the ability of an adenoviral vector to recognize a potential host cell can be modulated without genetic manipulation of the coat protein. For instance, complexing an adenovirus with a bispecific molecule comprising a penton base-binding domain and a domain that selectively binds a particular cell surface binding site enables one of ordinary skill in the art to target the vector to a particular cell type.
  • Suitable modifications to an adenoviral vector are described in U.S.
  • Adenoviral vectors can be constructed and/or purified using methods known in the art (e.g., using complementing cell lines, such as the 293 cell line, Per.C6 cell line, or 293-ORF6 cell line) and methods set forth, for example, in U.S. Patents 5,965,358, 5,994,128, 6,033,908, 6,168,941, 6,329,200, 6,383,795, 6,440,728, 6,447,995, 6,475,757, and 6,908,762; U.S. Patent Application Publication No.
  • the adenoviral vector particle genome preferably contains a packaging domain, such that the adenoviral genome produced from infection of suitable host cells with such particles can be packaged into an adenoviral vector particle.
  • the packaging domain can be located at any position in the adenoviral genome, so long as the adenoviral genome is packaged into adenoviral particles.
  • the packaging domain is located downstream of the El region.
  • the packaging domain is located downstream of the E4 region.
  • the replication- deficient adenoviral vector lacks all or part of the El region and the E4 region, hi this preferred embodiment, a spacer (i.e., a transcriptionally inert nucleic acid sequence) is inserted into the El region or into the E4 region, a desired heterologous nucleic acid sequence (e.g., a nucleic acid sequence encoding a TNF-a) is located in the E4 region or the El region, respectively, and the packaging domain is located downstream of the E4 region.
  • a spacer i.e., a transcriptionally inert nucleic acid sequence
  • a desired heterologous nucleic acid sequence e.g., a nucleic acid sequence encoding a TNF-a
  • the packaging domain is located downstream of the E4 region.
  • a quantity of viral vector particles sufficient for infection can be obtained using known techniques. Examples of such techniques are described in, e.g., Benton et al., In Vitro, 14(2): 192-9 (1978), Schilz et al., J. Gene Med, 3(5): 427-36 (2001), Pan et al., J. Gene Med., 1(6): 2133-40 (1999), Reiser, Gene Ther., 7(11): 910-3 (2000), Andreadis et al., Biotechnol.
  • adeno-associated viral vectors can be constructed and/or purified using the methods set forth, for example, in U.S. Patent 4,797,368 and Laughlin et al., Gene, 23: 65-73 (1983).
  • adenoviral vector particles can be constructed and/or purified using the methods set forth, for example, in U.S. Patent 5,965,358, Donthine et al., Gene Ther., 7(20): 1707-14 (2000), and International Patent Applications WO 98/56937, WO 99/15686, and WO 99/54441.
  • adenoviral transfer vectors or adenoviral genome constructs
  • adenoviral genome constructs also is well known in the art, and involves using standard molecular biological techniques such as those described in, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Press 1989) and the third edition thereof (2001), Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Wiley Interscience Publishers 1995), and Watson et al., RECOMBINANT DNA, (WH Freeman, New York, 2 nd Edition 1992), and in several of the other references mentioned herein.
  • a suitable genome encoding a recombinant adenoviral vector particle is produced by in vitro homologous recombination of two or more portions of the recombinant genome or by direct ligation of such portions to form a genome coding for the expression of the adenoviral vector particle.
  • Any suitable homologous recombination technique can be used to generate the adenoviral vector-producing plasmid. Examples of such techniques are provided in, e.g., Chinnadurai et al., J Virol, 32: 623-28 (1979), Berkner et al., Biotechniques, 6: 616-28 (1998), Chartier et al., J.
  • the viral vector particle desirably includes a heterologous nucleic acid sequence that encodes a protein of interest.
  • a "heterologous nucleic acid sequence” is a nucleic acid sequence that is not native to the viral vector particle.
  • the viral vector particle can comprise any suitable number of heterologous nucleic acid sequences.
  • the heterologous nucleic acid sequence can be PvNA or DNA, and can encode a protein, e.g., a peptide or a polypeptide, with a desired activity.
  • the heterologous nucleic acid sequence preferably comprises at least one nucleic acid sequence encoding at least one protein.
  • the nucleic acid sequence encoding the protein can be obtained from any source, e.g., isolated from nature, synthetically generated, isolated from a genetically engineered organism, and the like.
  • Any type of nucleic acid sequence e.g., DNA, RNA, and cDNA
  • the heterologous nucleic acid sequence preferably encodes a protein such as a cancer therapeutic, an angiogenic factor, an anti-angiogenic factor, or a neurotrophic factor.
  • the heterologous nucleic acid sequence can encode, for example, a member of the tumor necrosis factor superfamily of peptides (e.g., tumor necrosis factor-a (TNF-a), described in U.S. Patent 4,879,226), a vascular endothelial growth factor (VEGF) (e.g., a non-heparin- binding VEGF, such as VEGF121, VEGF145, VEGF165, VEGFl 89, or VEGF206, variously described in U.S. Patents 5,332,671, 5,240,848, and 5,219,739), or homologs thereof as described in, e.g., U.S.
  • TNF-a tumor necrosis factor-a
  • VEGF vascular endothelial growth factor
  • Patent Application Publication US 20030027751 Al and references cited therein an atonal-associated factor (e.g., MATH-I or HATH-I, described, e.g., in Birmingham et al., Science, 284: 1837-1841 (1999), and Zheng and Gao, Nature Neuroscience, 3(2): 580-586 (2000)), or an inducible nitric oxide synthase (iNOS) (described, e.g., in Yancopoulos et al., Cell, 93: 661-64 (1998) and references cited therein).
  • iNOS inducible nitric oxide synthase
  • the heterologous nucleic acid sequence desirably encodes pigment epithelial growth factor (PEDF) or a therapeutic fragment thereof (described in, e.g., U.S. Patent 5,840,686 and International Patent Applications WO 93/24529 and WO 99/04806).
  • PEDF also named early population doubling factor- 1 (EPC-I)
  • EPC-I early population doubling factor- 1
  • serpins is a secreted protein having homology to a family of serine protease inhibitors.
  • PEDF is made predominantly by retinal pigment epithelial cells and is detectable in most tissues and cell types of the body. PEDF has both neurotrophic and anti-angiogenic properties and, therefore, is useful in the treatment and study of a broad array of diseases.
  • Neurotrophic factors are thought to be responsible for the maturation of developing neurons and for maintaining adult neurons. It has been postulated that neurotrophic factors can actually reverse degradation of neurons associated with, for example, vision loss. Neurotrophic factors function in both paracrine and autocrine fashions, making them ideal therapeutic agents.
  • PEDF has been observed to induce differentiation in retinoblastoma cells and enhance survival of neuronal populations (Chader, Cell Different, 20: 209-216 (1987)). PEDF further has gliastatic activity or has the ability to inhibit glial cell growth. PEDF also has anti-angiogenic activity. Anti-angiogenic derivatives of PEDF include SLED proteins, discussed in International Patent Application Publication WO 99/04806.
  • PEDF is involved with cell senescence (Pignolo et al., J Biol. Chem., 268(12): 8949-8957 (1998)).
  • PEDF is further characterized in U.S. Patents 5,840,686, 6,319,687, and 6,451,763, and International Patent Applications WO 93/24529, 95/33480, and WO 99/04806.
  • Viral vectors comprising an exogenous nucleic acid encoding PEDF are further described in International Patent Application Publication WO 01/58494.
  • the protein to be purified can be any suitable protein (e.g., an intracellular protein), the protein of interest preferably is an extracellular (i.e., secreted) protein, such as PEDF.
  • extracellular protein i.e., secreted protein
  • secreted protein is meant any peptide, polypeptide, or portion thereof, which is released by a cell into the extracellular environment.
  • the nucleic acid can encode a protein that affects splicing or 3' processing (e.g., polyadenylation), or a protein that affects the level of expression of another gene within the cell (i.e., where gene expression is broadly considered to include all steps from initiation of transcription through production of a processed protein), such as by mediating an altered rate of mRNA accumulation or transport or an alteration in post-transcriptional regulation.
  • the expression of the nucleic acid sequence encoding the protein is controlled by a suitable expression control sequence operably linked to the nucleic acid sequence.
  • An "expression control sequence” is any nucleic acid sequence that promotes, enhances, or controls expression (typically and preferably transcription) of another nucleic acid sequence.
  • Suitable expression control sequences include constitutive promoters, inducible promoters, repressible promoters, and enhancers.
  • the nucleic acid sequence encoding the protein can be regulated by its endogenous promoter or, preferably, by a non-native promoter sequence.
  • suitable non-native promoters include the cytomegalovirus (CMV) promoters, such as the CMV immediate-early promoter (described in, for example, U.S.
  • HIV human immunodeficiency virus
  • PGK phosphoglycerate kinase
  • RSV Rous sarcoma virus
  • MMTV mouse mammary tumor virus
  • HSV promoters such as the Lap2 promoter or the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad.
  • promoters derived from SV40 or Epstein Barr virus such as the p5 promoter, the sheep metallothionein promoter, the human ubiquitin C promoter, and the like.
  • expression of the nucleic acid sequence encoding the protein can be controlled by a chimeric promoter sequence.
  • the promoter sequence is "chimeric" in that it comprises at least two nucleic acid sequence portions obtained from, derived from, or based upon at least two different sources (e.g., two different regions of an organism's genome, two different organisms, or an organism combined with a synthetic sequence).
  • the promoter can be an inducible promoter, i.e., a promoter that is up- and/or down-regulated in response to an appropriate signal.
  • Suitable inducible promoters include, for example, an ecdysone-inducible promoter, a tetracycline-inducible promoter, a zinc- inducible promoter (e.g., a metallothionein promoter), a radiation-inducible promoter (e.g., an EGR promoter), an arabinose-inducible promoter, a steroid-inducible promoter (e.g., a glucocorticoid-inducible promoter), or a pH, stress, or heat-inducible promoter.
  • the nucleic acid sequence preferably is operably linked to a radiation-inducible promoter, especially when the nucleic acid sequence encodes a TNF.
  • a radiation-inducible promoter provides control over transcription of the nucleic acid sequence, for example, by the administration of radiation to a cell or host comprising the adenoviral vector. Any suitable radiation-inducible promoter can be used in conjunction with the invention.
  • the radiation-inducible promoter preferably is the early growth region- 1 (Egr-1) promoter, specifically the CArG domain of the Egr-1 promoter.
  • the Egr-1 promoter is described in detail in U.S.
  • the promoter can be introduced into the genome of the adenoviral vector by methods known in the art, for example, by the introduction of a unique restriction site at a given region of the genome.
  • the promoter can be inserted as part of the expression cassette comprising the nucleic acid sequence coding for the protein, such as a TNF.
  • the nucleic acid sequence encoding the protein further comprises a transcription-terminating region such as a polyadenylation sequence located 3' of the region encoding the protein.
  • a polyadenylation sequence located 3' of the region encoding the protein.
  • Any suitable polyadenylation sequence can be used, including a synthetic optimized sequence, as well as the polyadenylation sequence of BGH (Bovine Growth Hormone), polyoma virus, TK (Thymidine Kinase), EBV (Epstein Barr Virus), and the papillomaviruses, including human papillomaviruses and BPV (Bovine Papilloma Virus).
  • BGH Bovine Growth Hormone
  • polyoma virus TK
  • EBV Epstein Barr Virus
  • papillomaviruses including human papillomaviruses and BPV (Bovine Papilloma Virus).
  • a preferred polyadenylation sequence is
  • Adenoviral vector particles can comprise a heterologous nucleic acid sequence in any suitable region of the adenoviral genome.
  • the adenoviral vector particle can contain more than one heterologous nucleic acid sequence.
  • the heterologous nucleic acid sequences are located in separate regions of the adenoviral genome; however, the heterologous nucleic acid sequences also or alternatively can be placed next to each other, either upstream or downstream from one another, in the same region of the adenoviral genome.
  • the heterologous nucleic acid sequence or sequences are preferably in a region of the adenoviral genome corresponding to a region wherein the adenoviral genome is deficient for a gene function required for viral propagation.
  • the nucleic acid sequence encoding the protein is preferably located in the El region of the adenoviral genome.
  • the insertion of a nucleic acid sequence into the adenoviral genome can be facilitated by known methods, for example, by the introduction of a unique restriction site at a given position of the adenoviral genome.
  • the heterologous nucleic acid sequence can be inserted into, e.g., the El region, the E2 region, the E3 region, the E4 region, or any combination thereof.
  • the cells for use in the invention include any suitable cells.
  • Particularly preferred cells include cells that are capable of complementing a replication-deficient viral vector particle (e.g., a cell capable of complementing the production of an AAV viral vector particle or a replication-deficient adenoviral vector particle by inclusion of one or more nucleic acids that provide regions necessary for the propagation of such vector particles).
  • suitable cells in this context include, e.g., 293/E4, 293-ORF6, and 293/E4/E2A cells, which are described in, e.g., U.S. Patents 5,851,806 and 5,994,106.
  • Additional appropriate cell lines can be generated using standard molecular biology techniques, such as those set forth in, e.g., Sambrook et al, supra, Ausubel et al., supra, Mulligan, Science 260, 926-932 (1987 and 1993), and Watson et al., supra. Additional molecular biology techniques related to the production of recombinant cells, vectors, and other genetically modified compositions are described in, e.g., Friedman, Therapy For Genetic Diseases (Oxford University Press, 1991), Ibanez et al., EMBO J, 10: 2105-10 (1991), Ibanez et al., Cell, 69: 329-41 (1992), and U.S.
  • a preferred cell line complements for at least one and preferably all replication- essential gene regions not present in a replication-deficient adenovirus.
  • the complementing cell line can complement for a deficiency in at least one replication-essential gene region, such as the early regions, late regions, viral packaging regions, virus-associated RNA regions, or combinations thereof, including substantially all adenoviral gene functions (e.g., to enable propagation of adenoviral amplicons, which comprise minimal adenoviral sequences, such as only inverted terminal repeats (ITRs) and the packaging signal or only ITRs and an adenoviral promoter).
  • the complementing cell line complements for a deficiency in at least one replication-essential region (e.g., two or more replication-essential regions).
  • the complementing cell line preferably complements for a deficiency in the El region of the adenoviral genome, particularly a deficiency in a replication-essential region of each of the ElA and ElB regions.
  • the complementing cell line can complement for a deficiency in the E2 (particularly as concerns the adenoviral DNA polymerase and terminal protein) and/or E4 regions of an adenoviral genome.
  • a cell that complements for a deficiency in the E4 region comprises an E4-ORF6 gene sequence (or a suitable functional (typically also structural) homolog thereof) and produces an E4-ORF6 protein or a functional homolog thereof, which desirably also is a structural homolog of a wild-type E4-ORF6 protein.
  • Such a cell desirably comprises at least E4-ORF6 and no other open reading frame (ORF) of the E4 region of the adenoviral genome.
  • the complementing cell line can complement for a deficiency in the El region and/or the E2 region (particularly as concerns the adenoviral DNA polymerase and terminal protein) and/or the E4 region of the adenoviral genome.
  • a nucleic acid encoding a complementing protein in a complementing cell preferably is transcriptionally linked to an antibiotic resistance gene.
  • Antibiotic resistance genes are well-known in the art and include such genes as, e.g., hygromycin, puromycin, and neomycin resistance genes.
  • the nucleic acid and antibiotic resistance gene can be transcriptionally linked in any suitable manner.
  • the nucleic acid molecule is transcriptionally linked to an antibiotic resistance gene using methods known in the art (see, for example, International Patent Application Publication WO 99/15686).
  • Suitable antibiotic or antibiotic analogs for the method of the invention depend on the type of antibiotic resistance gene utilized. For example, linkage of a puromycin resistance gene to the nucleic acid encoding the complementing protein will necessitate the use of puromycin or a puromycin analog to effect selection of the cell.
  • the nucleic acid sequence encoding a complementing protein in a complementing cell desirably is operably linked to a transcription control element that is upregulated in the presence of an inducer.
  • the transcriptional control element can be any suitable transcriptional control element that demonstrates increased activity in the presence of an inducer.
  • Suitable transcriptional control elements include, for example, an ecdysone- inducible promoter, a tetracycline-inducible promoter, a zinc-inducible promoter (e.g., a metallothionein promoter), a radiation-inducible promoter (e.g., an EGR promoter), an arabinose-inducible promoter, a steroid-inducible promoter (e.g., a glucocorticoid-inducible promoter), or a pH, stress, or heat-inducible promoter.
  • an ecdysone- inducible promoter e.g., a tetracycline-inducible promoter, a zinc-inducible promoter (e.g., a metallothionein promoter), a radiation-inducible promoter (e.g., an EGR promoter), an arabinose-inducible promoter, a steroid-inducible promote
  • an inducible promoter to control a nucleic acid sequence that encodes a protein that complements at least one adenovirus gene function is especially beneficial when the complementing protein is toxic to the cells. Since the promoter requires the presence of the inducer for full activation, in the absence of inducer the toxic protein will not be expressed until the promoter is induced at the required time for optimal adenoviral vector particle production.
  • Adenoviral proteins typically are toxic to a cell.
  • El proteins can be powerful transcriptional activators that induce viral replication by activating the cell replication cycle in host cells. El proteins can be oncogenic, resulting in transformation of normal cells to neoplastic cells.
  • ElA proteins have been linked to cellular transformation in vitro in cell cultures and in vivo in rodents (see, e.g., Bayley et al., Int. J. Oncol., 5: 425-444 (1994)).
  • ElA proteins can be highly toxic to cells and, in some instances, instigate cell death through apoptosis, as well as enhancing cell killing by other agents, e.g., natural killer cells, macrophages, and cytokines such as human tumor necrosis factor (see, e.g., Querido et al. J.
  • the E4/ORF6 region has oncogenic potential as well (Moore et al. Proc. Nat. Acad. Sd. USA, 93: 11295-11301, 1996).
  • at least one of the ElA and E4-ORF6 gene sequences in an adenovirus complementing cell desirably are under the control of such an inducible promoter.
  • the inducible promoter is a metallothionein promoter (e.g., a sheep metallothionein promoter).
  • the inducer is preferably zinc (alternatively copper can be used, but is less desired due to its toxic effects on cells).
  • the zinc can be added to the cell culture at any time suitable for induction of the production of the complementary protein.
  • the cell line preferably is further characterized in that it contains the complementing genes in a non-overlapping fashion with the adenoviral vector, which minimizes, and practically eliminates, the possibility of the vector genome recombining with the cellular DNA.
  • RCA replication-competent adenovirus
  • the lack of RCA in the vector composition avoids the replication of the adenoviral vector in non-complementing cells.
  • complementing cell lines involves standard molecular biology and cell culture techniques, such as those described by Sambrook et al., supra, and Ausubel et al., supra.
  • Complementing cell lines for producing the adenoviral vector include, but are not limited to, 293 cells (described in, e.g., Graham et al., J Gen.
  • the cells to be infected can be any suitable cells.
  • the cell can be a primary cell, such as a primary human retinal cell or primary African green monkey cell, or, more typically, will be an immortalized cell in a continuous cell line.
  • Suitable cells include, for example, cells of primary cell lines, such as human embryonic kidney (HEK), human embryonic lung (HEL), and human embryonic retinoblasts. More particular examples of such cells include HEK-293 cells (Graham et al., Cold Spring Harbor Svmp. Quant. Biol., 39: 637-650 (1975)) and cells derived therefrom (e.g., 293-ORF6 cells, which are discussed elsewhere herein), W162 cells (Weinberg et al., Proc. Nat. Acad. ScL, 80: 5383-5386 (1983)), gMDBP cells (Klessig et al., MoI.
  • HEK-293 cells Graham et al., Cold Spring Harbor Svmp. Quant. Biol., 39: 637-650 (1975)
  • cells derived therefrom e.g., 293-ORF6 cells, which are discussed elsewhere herein
  • W162 cells Weinberg et al., Proc.
  • Suitable cells also include human embryonic retinal (HER) cells such as 911 cells (Fallaux et al., Human Gene Therapy, 7: 215-222 (1996) and PER.C6 cells (Crucell - Lieden, Netherlands (formerly Introgene, Inc.), described in, e.g., International Patent Application Publication WO 97/00326).
  • HER human embryonic retinal
  • the cell is preferably a HeLa cell (ATCC CCL-2) or an ARPE- 19/HPV- 16 cell (ATCC CRL-2502).
  • Suitable cells also include renal carcinoma cells, WI38 cells and other human fibroblast cells, CHO cells, KB cells, SW-13 cells, MCF7 cells, and African green monkey cells (e.g., Vero cells).
  • Other suitable cells include, for example, lung carcinoma cells such as NCI- H2126 cells (ATCC No. CCL-256), NCI-H23 cells (ATCC No. CRL-5800), NCI-H322 cells (ATCC No. CRL-5806), NCI-H358 cells (ATCC No. CRL-5807), NCI-H810 cells (ATCC No.
  • CRL-5816 NCI-Hl 1,55 cells (ATCC No. CRL-5818), NCI-H647 cells (ATCC No. CRL-5834), NCI-H650 cells (ATCC No. CRL-5835), NCI-H1385 cells (ATCC No. CRL-5867), NCI-H1770 cells (ATCC No. CRL-5893), NCI-H1915 cells (ATCC No. CRL- 5904), NCI-H520 cells (HTB-182), and NCI-H596 cells (ATCC No. HTB-178).
  • squamous/epidermoid carcinoma cells that include HLF-a cells (ATCC No. CCL-199), NCI-H292 cells (ATCC No.
  • NCI-H226 cells ATCC No. CRL- 5826
  • Hs 284.Pe cells ATCC No. CRL-7228
  • SK-MES-I cells ATCC No. HTB-58
  • SW-900 cells ATCC No. HTB-59
  • large cell carcinoma cells e.g., NCI-H661 cells (ATCC No. HTB-183)
  • alveolar cell carcinoma cells e.g., SW-1573 cells (ATCC No. CRL-2170). Additional examples of suitable cells are described, for example, in U.S. Patent 5,994,106 and International Patent Application Publication WO 95/34671.
  • the cells can be maintained in any suitable medium to form a culture.
  • the culture of cells can be any culture suitable for the propagation of a viral vector particle.
  • suitable types of cultures include perfusion cultures, substrate-supported cultures, microcarrier-supported cultures, fluidized bed cultures, and suspension cultures.
  • Suspension cultures (independent of microcarriers) are particularly favored, including for example, shaker flask cultures, roller bottle cultures, and suspension bioreactor cultures.
  • Such cultures and related culturing techniques are described in, e.g., ANIMAL CELL TECHNOLOGY, Rhiel et al., eds, (Kluwer Academic Publishers 1999), Chaubard et al., Genetic Eng.
  • the medium can be any medium suitable for maintaining the cells and propagating a viral vector particle or vectors therein.
  • Mediums suitable for use in the invention, along with techniques used to develop new or modified mediums suitable for use in the context of the invention, are known in the art.
  • the medium will contain a selection of secreted cellular proteins, diffusible nutrients, amino acids, organic and inorganic salts, vitamins, trace metals, sugars, and lipids.
  • the medium can also contain additional compounds such as growth promoting substances (e.g., cytokines).
  • a suitable medium preferably has the physiological characteristics and conditions (e.g., pH, salt content, vitamin and amino acid profiles) under which the cells naturally flourish.
  • the medium can be an undefined medium or a defined medium.
  • An undefined medium is a medium where the specific contents of the medium (e.g., the type and amount of proteins and nutrients) are not known or specified by a set formula.
  • suitable undefined mediums include mediums based on animal serum (e.g., fetal bovine serum (FBS) or fetal calf serum (FCS)) or which utilize an alternative nutritional source, for example, enzymatic digestions of meat, organs, or glands, as well as milk or hydrolysates of wheat gluten.
  • animal serum e.g., fetal bovine serum (FBS) or fetal calf serum (FCS)
  • FCS fetal calf serum
  • an undefined medium in the context of the invention is a serum- free medium (SFM).
  • SFM serum-free medium
  • animal-derived components e.g., albumin, fetuin, hormones, and "undefined” components such as organ extracts.
  • a defined medium is a medium with known contents or a medium that is prepared using a specific formula.
  • a simple defined medium is, for example, a basal medium.
  • a basal medium is generally composed of vitamins, amino acids, organic and inorganic salts, and buffers. Additional defined components, such as bovine serum albumin (BSA), can be added to make a basal medium more nutritionally complex and appropriate for the nutritional needs of a specific cell type. More complex suitable defined mediums include protein-free and protein-containing mediums.
  • a defined medium in the context of the invention is an animal protein-free medium.
  • An animal protein-free medium does not contain proteins of animal origin, but can contain proteins from other sources.
  • a particularly preferred medium is an animal protein-free medium, which contains recombinant proteins and growth factors (particularly, e.g., epidermal growth factor (EGF) and insulin-like growth factor (IGF), the addition of which is described further herein), as well as lipids (e.g., cod liver extracts) and cholesterol in amounts suitable for culturing 293-derived cells (e.g., 293-ORF6 cells) to desired cell densities during the viral vector production process (preferred cell densities are discussed further elsewhere herein).
  • recombinant proteins and growth factors particularly, e.g., epidermal growth factor (EGF) and insulin-like growth factor (IGF), the addition of which is described further herein
  • lipids e.g., cod liver extracts
  • cholesterol e.g., cod liver extracts
  • Examples of commercially available preferred medias are ExCeIl 525 (JRH Biosciences), CD293 medium (GIBCO), SFMII medium (GIBCO), Gene Therapy Medium I for Retinoblastoma-like Cells (GTRB) medium (SIGMA), Pro293s medium (BioWhittaker), Gene Therapy Medium II (SIGMA), and PF293 (HyClone). It is also desirable that such media are supplemented with glutamine to obtain optimal growth. For example, cells grown in SFMII medium are preferably supplemented with glutamine to reach a glutamine concentration of about 4 niM. [0056]
  • the medium contains glucose. Any suitable concentration of glucose appropriate for culturing cells to desired cell densities is appropriate.
  • the concentration of glucose in the medium is at least about 1- 5 gm/L, more preferably about 2-4 gm/L.
  • the cells comprise a nucleic acid sequence operably lined to a transcription control element that is upregulated in the presence of an inducer (e.g., a zinc-inducible promoter)
  • an inducer e.g., a zinc-inducible promoter
  • the inducer can be added at any suitable time.
  • the zinc can be added to the cell culture at any time suitable for induction of the production of the complementary protein.
  • the zinc is added about 0-48 hours before the cell culture is infected with adenoviral vector particles.
  • the zinc is added at about 10- 36 hours before infection, still more preferably, the zinc is added at about 20- 28 hours before infection, and most preferably, the zinc is added at about 23- 25 hours before infection (e.g., at about 24 hours).
  • the inducer e.g., the zinc for a metallothionein-linked complementing sequence
  • the inducer desirably is added about 0-36 hours after the cell culture is infected with viral vector particles. More preferably, the inducer is added at about 4-24 hours after infection, and even more preferably, the inducer is added at about 8-12 hours after infection.
  • the concentration of zinc administered to the cells can be any suitable concentration appropriate for induction of the production of the complementary protein by the adenoviral vector particle packaging cells.
  • the zinc concentration is about 5 ⁇ M to about 100 ⁇ M, more preferably about 10 ⁇ M to about 80 ⁇ M, still more preferably about 20 ⁇ M to about 60 ⁇ M, even more preferably about 20 ⁇ M to about 40 ⁇ M (e.g., about 25 ⁇ M), and most preferably about 30 ⁇ M to about 40 ⁇ M (e.g., about 35 ⁇ M).
  • the culture can be prepared in any suitable manner that promotes the growth and sustenance of the cells.
  • the culture is initiated by inoculation of a suitable medium with a population of cells.
  • the cells used to inoculate the medium can be cells that were previously frozen and stored.
  • the cells were frozen under conditions suitable for maintaining a high percentage of viable cells in the culture for future use.
  • Several methods of freezing cells for future use are known in the art, for example, by using liquid nitrogen. Examples of techniques for freezing and thawing such cells, without lysing the cells, are described in, e.g., U.S. Patent 6,168,941 and references cited therein.
  • the cells are then "cultured” or cultivated under conditions to permit growth of the cells.
  • Any suitable manner of culturing the cells that permits the growth of the viral vector-producing cells is suitable in the context of the invention.
  • the method of culturing such cells will depend upon the type of cell selected. Suitable culturing methods are well known in the art, and typically involve maintaining pH and temperature within ranges suitable for growth of the cells. Preferred temperatures for culturing are about 35-40° C, more preferably about 36-38° C, and optimally about 37° C.
  • the pH of the culture is maintained at about 6-8, more preferably at about 6.7-7.8, and optimally at about 6.9-7.5.
  • the first stage, or lag phase occurs at the introduction of cells or storage culture into the medium to form the culture.
  • the cells or storage culture i.e., the "inoculum”
  • the lag phase is typically followed by a log (or exponential) phase, in which cells divide at the maximum possible growth rate, thus increasing the number of total viable cells in the culture.
  • the cell growth rate is dependent on the growth medium and growth conditions (e.g., aeration, pH level etc.), which are preferably optimized to promote cell growth during the log phase.
  • the cell growth rate is limited by the maximum doubling time that is dependent upon cell type.
  • the cell growth rate during the exponential phase is constant, but because each cell divides at a slightly different moment the growth curve rises gradually.
  • the log phase is followed by a decelerating phase, where the rate of increase in viable cells in the culture decreases.
  • the decelerating phase is followed by a stationary phase where the total number of viable cells in the culture does not increase further, an effect caused either by a lack of cell division or by a balanced ratio of cell division and cell death.
  • the culture moves through a second decelerating phase, wherein the total number of viable cells declines, followed by an exponential death phase.
  • Cell density increases throughout the growth cycle of the culture. The concentration of the cells in the medium can be monitored while culturing the cells.
  • Cell growth rates can be determined by numerous techniques known in the art. Techniques focusing on total number of cells in the culture include: determining the weight of the culture, assessing culture turbidity, determining metabolic activity in the culture, electronic cell counting, microscopic cell counting of culture samples, plate counting using serial dilutions, membrane filter counting, and radioisotope assays. Mechanical systems for measuring cell density, based upon these and other principles and particularly suited for use in bioreactors, are reviewed in, for example, Junker et al., Bioprocess Engineering, 10: 195-207 (1994).
  • any technique permissive for assessing cell density is suitable.
  • Cell density of a culture can be determined spectrophotometrically or by using a counting chamber, such as a hemocytometer.
  • a hemocytometer is used.
  • hemocytometer-based techniques involve taking a sample of the culture, counting (and possibly also examining) a statistically significant number of cells in a given space in the hemocytometer, and determining the density of cells in the culture using simple mathematical formulas.
  • Perfusion through the culture means that a certain volume of medium is added to the culture and a substantially identical amount of medium is removed from the culture without removing a significant percentage of the cells in the culture. Perfusion can be carried out by any suitable technique. A bioreactor with perfusion capabilities is usually used to accomplish such perfusion in a microcarrier-free suspension culture. For continuous perfusion cultures, perfusion of fresh medium is taking place throughout culturing in contrast to "intense perfuction" which is discussed further herein. Typically, for continuous perfusion cultures, perfusion through the culture occurs at a rate of about 1-4 volumes of medium in the culture per day.
  • Continuous perfusion is a suitable means for adding fresh medium to the culture to sustain the cells during culturing, but it is not effective in removing large amounts (e.g., over about 20%, 50%, 65%, or even higher percentages) of spent medium from the culture. Such techniques are particularly preferred with HER cells.
  • the suspension culture is maintained in a batch or fed-batch mode before and after perfusion of the fresh medium through the culture. Techniques for perfusing fresh medium through a culture are further described in U.S. Patent 6,168,941. [0065]
  • the density of cells in the medium during infusion typically is about 0.8-4.2 x 10 6 cells/mL.
  • the density of cells in the medium during an intense perfusion is typically 0.8 x 10 6 -l.l x 10 6 cells/mL, more specifically about 1.0 x 10 6 -l.l x 10 6 cells/mL, desirably in 10 liter fed batch and batch bioreactors.
  • cell densities while the fresh medium is perfused through the culture can be about 0.8 x 10 6 - 1.4 x 10 6 cells/mL, more specifically about 1.1 x 10 6 -1.3 x 10 6 cells/mL.
  • a 10-liter continuous perfusion bioreactor such densities typically will be about 2.4 x 10 6 -4.2 x 10 6 cells/mL. More particular cell densities for certain aspects of the invention are described elsewhere herein.
  • the actual density of cells in the medium at stationary phase can be any suitable density.
  • the specific stationary phase density for any culture will depend upon the specific components of the culture (e.g., type of cells and medium used), and will depend significantly on the type, and size, of culture.
  • Typical stationary phase density can be about 1-9 x 10 6 cells/mL.
  • stationary phase density is typically about 1.5 x 10 6 -2.6 x 10 6 cells/mL, more typically about 1.5 x 10 6 -2 x 10 6 cells/mL.
  • the stationary phase density often is higher, such as about 5-6 x 10 6 cells/mL for A549 cells.
  • 293 cells and cells of 293-derived cell lines grown in, for example, a 10-liter continuous perfusion bioreactor typically have a stationary stage cell density of about 7-9 x 10 6 cells/mL.
  • these cell densities represent preferred stationary phase cell densities in the practice of the invention.
  • the number of cells in the medium when the culture is in the stationary phase can be determined by allowing some portion of the culture to progress to stationary phase or by assessing substantially similar cultures wherein the density of the culture at the stationary phase is determined.
  • the cells are contacted with viral vector particles under conditions permissive for infection of the cells.
  • Any appropriate cell density within about 35-75%, for example, about 40% to about 70% (e.g., about 44-63%), more preferably about 55-70% (e.g., about 60-70%), even more preferably about 62-69% (e.g., about 65%) of the density of cells that would be (or will be) obtained in the medium when the growth of the culture is in the stationary phase is preferred, particularly for the production of adenoviral vector particles.
  • densities are achieved during the mid-to-late exponential phase of the culture.
  • Preferred cell densities for a particular cell type suitable for production of an viral vector particle composition may vary somewhat within the range of 40-70% of the stationary phase density based on the particular cell type. Suitable densities allow for the production of high yields of assembled viral vector particles, particularly active/viable viral vector particles, in contrast to the mere production of proteins by the infected cells, which typically is associated with infecting cells at cell densities well above 70% of the stationary phase density.
  • the time to reach an appropriate cell density for infection will vary depending upon the vector, type of cells, and type of culture used during the cell growth cycle. For example, starting with frozen cells with a density of less than 3 x 10 5 cells/mL, a period of about 6-10 days maybe required to achieve the aforementioned cell densities.
  • the culture can be grown in a single container or in multiple containers. For example, the culture can be grown initially in multiple roller bottles or spinner flasks until a desired cell density is achieved, then the separated culture can be unified in a single container, such as a bioreactor, in different bioreactors, or in multiple bioreactors at once.
  • a time corresponding to the cell density associated with optimal composition production can be determined for a particular composition and selected as an indicator of when the culture should be contacted with the viral vector particles in practicing the invention with a substantially similar composition (e.g., same cell type and same medium).
  • Another technique that is available is the use of mathematical growth formulas, based on one or more sample points during the growth of the culture, such as the Monrod Model. Either type of technique, or other similar techniques, can be combined with mechanical monitoring techniques or other techniques for practicing the invention under such determined parameters.
  • the culture will desirably comprise at least about 50% spent medium (medium nutritionally used by the cells and/or containing the byproducts of cellular metabolism) at the time of contact with the viral vector particles.
  • the cell culture desirably comprises at least about 60% spent medium, more preferably about 70% spent medium, even more preferably about 80% spent medium, advantageously about 90% spent medium, even more advantageously about 95% spent medium, and optimally about 100% spent medium.
  • Such techniques are particularly preferred with HEK cells and/or cells comprising a portion of the E4 region of the adenovirus genome (such as, e.g., 293-ORF6 cells).
  • the culture can comprise a portion of the spent medium, in an amount corresponding to any of the above-described percentages, which results in an increased yield in the production of viral vector particles from the cells with respect to a substantially identical culture containing less than the designated amount of the spent medium portion (or, preferably, substantially no spent medium).
  • the increased yield in the production of viral vector particles from the cells is at least about a 30% increase, e.g., at least about a 50% increase, at least about a 75% increase, more preferably at least about a 90% increase, at least about a 100% increase, at least about a 150% increase, and about a 200% increase, over a medium substantially free of the spent medium portion at lysis and/or after filtration and chromatography purification (desirably at both times).
  • the portion of the spent medium will contain metabolites that induce the production of viral vector particles at an increased rate when present in one of the above-described amounts.
  • the portion can be any suitable portion.
  • the separation of the components of the spent medium to obtain the spent medium portion can be accomplished by any suitable technique, including, e.g., cell fractionation techniques (for example, differential centrifugation, velocity sedimentation, and density gradient centrifugation), chemical extraction techniques, biochemical techniques, such as chromatography separation techniques.
  • cell fractionation techniques for example, differential centrifugation, velocity sedimentation, and density gradient centrifugation
  • chemical extraction techniques for example, chemical extraction techniques, biochemical techniques, such as chromatography separation techniques.
  • the presence of the aforementioned concentration of spent medium preferably results in an increase in the number of adenoviral vector particles produced by performing a substantially identical production and purification process with a culture comprising a substantially identical population of cells and less than about 50% spent medium, less than about 40% spent medium, less than about 25% spent medium, and less than about 10% spent medium, typically with a culture comprising substantially no spent medium.
  • the cells can be infected with viral vector particles under any suitable conditions as described elsewhere herein at any suitable time after culturing and/or at any suitable cell density in the culture. Typically, the cells are infected when the culture has a cell density of at least about 1 x 10 6 cells/mL.
  • the cell density in the culture at infection is at least about 1 x 10 5 cells/mL, more preferably at least about 1 x 10 6 cells/mL, even more preferably at least about 1 x 10 7 cells/mL, still more preferably at least about 1 x 10 8 cells/mL or higher (e.g., about 1 x 10 9 cells/mL to about 1 x 10 11 cells/mL).
  • the cells can be cultured and infected in any suitable manner.
  • the culture is capable of supporting a population of adenoviral vector packaging cells at a cell density of about 1 x 10 4 cells/mL to about 1 x 10 10 cells/mL in a fed-batch mode, more preferably about 1 x 10 6 cells/mL to about 1 x 10 8 cells/mL, most preferably about 2 x 10 6 cells/mL to about 4 x 10 6 cells/mL.
  • the cells are typically infected when the density of cells in the medium is about 40-70% of the density of the cells obtained in the medium when the growth of the culture is in the stationary phase, such as described elsewhere herein.
  • the cells are harvested between 36 and 60 hours post-infection, e.g., about 48 hours post infection.
  • At least about 1 x 10 4 adenoviral vector particle units e.g., at least about at least about 2.9 x 10 4 adenoviral vector particle units
  • more preferably at least about Ix 10 6 adenoviral vector particle units, even more preferably at least about 1 x 10 8 adenoviral vector particle units, or most preferably at least about 1 x 10 10 adenoviral vector particle units/cell are obtained at lysis and/or final purification of the adenoviral vector particle composition.
  • the viral vector packaging cells are cultured under perfusion conditions (or at least in a bioreactor or other container capable of perfusion), which can be altered such that an "intense perfusion" is performed prior to contacting the cells with a viral vector particle.
  • An "intense perfusion” occurs when fresh medium is perfused through the culture for about 1-6 hours in an amount of at least about 125%, preferably at least about 150%, and more preferably at least about 200% (e.g., about 2-3 times or about 3-4 times) the volume of the medium in the culture immediately prior to such perfusion.
  • An intense perfusion provides fresh medium and removes substantial amounts of spent medium accumulated in the culture prior to the initiation of the intense perfusion.
  • An intense perfusion can occur at any suitable rate and the ordinarily skilled artisan will readily be able to determine an appropriate rate for the particular system used.
  • An intense perfusion results in about 66% or more of the spent media being removed from the culture (and replaced with fresh medium) prior to contact with the viral vector particles.
  • an intense perfusion of fresh medium in an amount equal to about three to four times the volume of the culture results in about 95% or more of the spent medium in the culture being removed (and thus replaced with fresh medium).
  • Certain cells respond better to intense perfusion culturing than spent medium culturing with respect to the amount of viral vector particles produced.
  • Intense perfusions techniques are preferably performed with, for example, HER cells and, particularly, El -complementing HEK cells.
  • medium exchange during contact of the culture with the viral vector particles has surprisingly been found to be not necessary.
  • the perfusion of the fresh medium through the culture prior to infection can be the only medium exchange used throughout the process of producing the viral vector particle composition.
  • other nutritional supplements are not added after infection (e.g., glucose) and that the cells are cultured in a batch mode.
  • Medium exchange during or immediately after contacting the culture with the viral vector particle can result in the undesired removal of viable viral vector particles from the medium after their introduction to the culture.
  • the medium addition can be performed by any suitable technique.
  • the medium addition is performed by a perfusion method, such as an intense perfusion, or other perfusion method described herein.
  • the culture is maintained under batch conditions after such medium exchange is performed.
  • the viral vector particles are permitted to infect the cells. Infection can be carried out under any suitable conditions. Conditions for viral vector particle infection can vary depending on the type of viral vector particle and cells utilized.
  • the temperature of the culture during contact of the culture with the viral vector particles is about 35-40° C, more preferably about 36-38° C, and optimally about 37° C.
  • the pH during contact of the culture with the adenoviral vector particles is preferably about 6.7-7.8, more preferably about 6.9-7.5.
  • Suitable infection conditions for other types of viral vector particles are described in, e.g., Bachrach et al., J Virol, 74(18): 8480-6 (2000), Mackay et al, J Virol, 19(2): 620-36 (1976), and Fields et al., supra.
  • the infected cells are incubated with the serum-free medium for about 12-72 hours, more preferably about 24-68 hours (e.g., about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, and ranges thereof).
  • the cells can be incubated at any suitable temperature as described herein, any suitable carbon dioxide level described further herein, and can be agitated at any suitable rotations per minute (rpms) or can be incubated without agitation.
  • the cells are preferably suspended in a serum-free medium with continuous shaking, rocking, or rolling, typically accomplished by mechanical means such as by a shaker, rocker, or roller.
  • the contact of the viral vector particles to the cells and incubation of the viral vector particle/cell composition to produce a population of viral vector infected cells through cell infection can be performed at any suitable cell density.
  • the concentration of the cells can be desirable prior to infection (such as by concentrating the medium to a density of about 3 x 10 6 cells/mL, about 5 x 10 6 cells/mL, or even higher).
  • the cells can be concentrated in such aspects using any suitable technique, including, for example, density gradient centrifugation. m most aspects, however, the method is performed without the concentration of the cells prior to infection.
  • any suitable number of viral vector particles can be used to infect the population of cells in any aspect of the invention.
  • the number of viral vector particles used to infect the cells will depend on the number of cells in the culture, cell type, and viral vector particle type.
  • the ratio of viral vector particles contacting with the culture to the cells in the culture desirably is greater than 1, and more preferably is at least about 5 (e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, and ranges therebetween).
  • adenoviral vector particles and adenoviral vector packaging cells e.g., El -complementing HER cells, 293 cells, or 293-derived cells such as 293-ORF6 cells.
  • the culture can be, and, in most aspects preferably is, contacted with the viral vector particles without concentrating the cells prior to such contact.
  • the culture is not significantly concentrated before or during the contact of the culture with the viral vector particles and, most preferably, is not concentrated at all.
  • the avoidance of concentrating the culture during the production of the viral vector particles is desirable inasmuch as the concentration process can involve the need for large and expensive equipment (e.g., a centrifuge capable of concentrating a 10 liter culture) and intensive labor.
  • the contact of the viral vector particles under conditions permissive for infection can be performed for any suitable period of time that enables a desired level of infection of the cells with the viral vector particles.
  • the time for infection will depend at least on the titer of the virus and the specific cell type employed (because some cell types may have a greater density of the receptor which the viral vector particle uses to attach to cells) and the available surface area available to the viral vector particles (which is a function of the culture type and/or the cell type employed). Additionally, the desired period of time can be affected by the type of viral vector particle utilized (e.g., the virus can have an altered coat protein through recombinant engineering or be conjugated with a chemical entity that affects its ability to bind to cells).
  • One of ordinary skill in the art can determine an appropriate period of time for contact of the culture with the viral vector particles by taking such variables into account and using routine experimentation.
  • a period of about one hour typically is sufficient under most conditions for infection, although longer periods (e.g., at least about 2, 3, 5, 10, 15, or 24 hours, or even longer) can be used.
  • the period of contact of the cells with the viral vector particles, and the period of culture of the cells after such contact are contemporaneous, as the culture is not concentrated and no medium exchange or other significant modification to the culture occurs after contacting the culture with the viral vector particles.
  • Viral vector particles alternatively, though less preferably, can be initially obtained by transfection of the cells with a viral genome (e.g., a naked polynucleotide coding for production of the viral vector particle in the host cell).
  • the cell culture can be supplemented with any suitable growth factors in any suitable concentration.
  • the cell culture is supplemented with one or more growth factors in a concentration which increases the cell density in the culture.
  • the cell culture is supplemented with one or more growth factors in a concentration such that the yield of viral vector particles from the cells after infection and lysis (and/or after purification) is greater than the yield in the presence of a lower amount of the growth factors, such as the normal physiological amount of the growth factor or growth factors present in the cells.
  • the growth factors that are added to the cell culture in this respect can be any suitable growth factors.
  • Preferred growth factors include insulin-like growth factors (IGFs), epidermal growth factors (EGFs), members of the tumor necrosis factor- ⁇ family of proteins (additional aspects of which are discussed elsewhere herein), or protein homologs thereof.
  • the IGF can be any suitable naturally occurring IGF, such as human IGF (as described in, e.g., U.S. Patents 5,158,875 and 5,340,725).
  • the EGF can be any suitable EGF, including, for example, human EGF (as described in, e.g., U.S. Patents 4,528,186, 5,096,825, and 5,290,920).
  • the cell culture can comprise or be supplemented with any amount of IGF, EGF, or homologs thereof, capable of increasing the yield of adenoviral vector particle units per cell (and more desirably the number of fluorescent focus units produced per cell) and, thus, the amount of the protein of interest as compared to a culture of such cells having a lower amount of IGF, EGF, or homolog thereof, such as a cell culture comprising normal physiological levels of IGF or EGF.
  • the amount of IGF and/or EGF in the culture can be determined using any suitable technique. Example of techniques for assaying the level of IGF are described in, e.g., U.S. Patents 5,158,875 and 5,340,725.
  • Culturing cells in medium containing an IGF and/or an EGF can be used for the production of any suitable viral vector particle (at levels lower than those where a cell-density independent yield of viral vector particles is obtained one or both of the growth factors can increase the growth rate and/or maximum density of the packaging cells.
  • IGF and/or EGF at levels which increase the yield of viral vector particles produced in cells cultured in the presence of such growth factors is particularly advantageous in the production of adenoviral vector particles, which will result in elevated levels of transgene expression.
  • the medium can comprise any suitable amount of an EGF or EGF homolog, examples of which are described above. Typically and preferably, the medium will comprise about 5-50 ng/mL of an EGF, more preferably about 5-35 ng/niL of an EGF, even more preferably about 5-15 ng/mL of an EGF, and most preferably about 10 ng/mL of an EGF.
  • the medium also or alternatively can comprise any suitable amount of an IGF or IGF homolog, examples of which are described above.
  • the medium further comprises about 5-50 ng/mL of an IGF, more preferably about 5-35 ng/mL of an IGF, even more preferably about 5-15 ng/mL of an IGF, and most preferably about 10 ng/mL of an IGF.
  • IGF insulin growth factor
  • EGF EGF-like growth factor
  • the presence of the IGF, EGF, or both, in the medium can increase the adenoviral vector particle (and, thus, protein) yield significantly.
  • the presence of the EGF, IGF, or both, in the aforementioned concentrations preferably results in an at least about a 10% increase, desirably an at least about a 30% increase, more preferably an at least about a 50% increase, even more preferably an at least about a 60% increase, more preferably still an at least about an 80% increase, still more advantageously an at least about a 100% increase, and most preferably an at least about a 150% increase in particle unit/cell yield at lysis of the cells and/or final purification of the composition over culturing the cells (and, if applicable, purifying the composition) in a substantially identical medium lacking an increased level of EGF, IGF, or both over levels normally present in the cells.
  • the culture medium also can contain r-insulin, dextran sulfate, glutamine, and/or apluronic (preferably
  • the growth factor(s) can be added at any suitable point in the culturing of the viral vector packaging cells.
  • the cells are infected with the adenoviral vector before seven doublings of the culture (e.g., before 6 doublings, before 5 doublings, before 4 doublings, before 3 doublings or before 2 doublings).
  • the infected cells are cultured to complete production of the viral vector particle composition and of the expressed transgene of interest.
  • the infected culture can be cultured under any suitable conditions permissive for the propagation of the viral vector particles within the cells.
  • the pH of the culture is maintained at about 6.5-7.5, more preferably at about 6.9-7.3.
  • pH and/or other conditions will be maintained to optimize glucose metabolism by the cells while reducing the production of lactic acid in the culture.
  • the pH of a cell culture can be controlled by any suitable method, preferably in a manner that does not substantially inhibit the production of the viral vector particle composition or the protein of interest.
  • buffers e.g., bicarbonate or tris buffers.
  • Temperature is another factor that influences the production of the viral vector particle composition after infection. Any temperature suitable for the production of the viral vector particle composition can be utilized, preferably a temperature of about 35-40° C, more preferably about 36-38° C (e.g., about 37° C). Proper mixing of the culture is another condition which can be important to cell growth and viral vector particle production.
  • the cells can be cultured by any method suitable for production of viral vector particles in infected cells under the aforementioned conditions, it is preferred that the infected cell culture is cultured in a bioreactor (also sometimes referred to as a fermentor) to produce large scale viral vector particle compositions.
  • a bioreactor also sometimes referred to as a fermentor
  • Any suitable bioreactor can be used, which ensures proper mixing and preferably optimal pH and temperature conditions for culturing the culture, and which enables the perfusion of fresh medium through the culture in an amount equal to at least about two times the volume of the culture prior to infection.
  • bioreactors examples include stirred tank bioreactors, bubble column bioreactors, air lift bioreactors, fluid bed bioreactors, packed bed bioreactors, wave bioreactors, and flocculated cell bioreactors.
  • the bioreactor is not a microprojectile-based or microcarrier-based bioreactor, a cell factory, or a cell cube bioreactor.
  • the bioreactor is a stirred tank bioreactor, which prevents cell damage by shearing and turbulence during culture.
  • the bioreactor can be either a batch, continuous, or fed-batch bioreactor, with perfusion capabilities, and the culture preferably is maintained under batch, fed-batch, or continuous culture conditions with the exception of the perfusion of fresh culture through the medium prior to infection at a volume equal to at least about two times the volume of the medium prior to infection with the viral vector particles.
  • perfusion culture-capable bioreactors are used with variable volume fed-batch procedures (also referred to in the art as repeated fed-batch process or cyclic fed-batch culture) or batch procedures during the culturing of the cells prior to, and after, the perfusion of fresh medium through the culture. After such perfusion and infection, batch conditions typically and preferably are maintained until harvest.
  • continuous-perfusion cell culture conditions can be used in place of batch conditions during the initial growth of the cells and/or after infection, particularly in aspects where the cells are cultured by the "intense perfusion technique", where perfusion of fresh medium through the culture occurs in an amount significantly lower than the perfusion of at least two times the volume of medium in the culture performed prior to contact of the culture with the viral vector particles in such aspects.
  • nonviral transfer methods can be utilized as an alternative to the introduction of the nucleic acid sequence encoding the protein of interest by viral vector particles.
  • the invention comprises transfection of the nucleic acid sequence (e.g., naked DNA) into the suspension of cells.
  • the transfected nucleic acid sequence encodes the protein of interest operably linked to a promoter.
  • the method of transferring the nucleic acid sequence into cells can be any suitable method, such as those disclosed in Baldi et al., Biotechnol. Prog., 21(1): 148-153 (2005); Meissner et al., Biotechnol. Bioeng., 75(2): 197-203 (2001); Song et al., Nucl. Acids Res., 23(17): 3609-3611 (1995); and Durocher et al., Nucl. Acids. Res., 30(2): E9 (2002)).
  • the nucleic acid sequence can be transfected into cells suspended in any suitable media, preferably, the media is serum-free.
  • a bioreactor can be any suitable size for producing an appropriate size protein composition.
  • commercial 10 liter bioreactors, or larger bioreactors are preferred.
  • cells can be transferred to the bioreactor by any appropriate techniques, such as a peristaltic pump transmission through a closed (i.e., environmentally isolated) transfer route, such as through SCD connection tubing or a sterilized steam block, as is described further herein.
  • At least about 1 x 10 6 is provided.
  • the cells are preferably expanded to at least about 1 x 10 , more preferably at least about 1 x 10 9 , even more preferably at least about 1 x 10 10 , still more preferably at least about 1 x 10 11 or more adenoviral vector packaging cells.
  • the cells are expanded in at least one bioreactor, as further described herein.
  • the cells are preferably expanded in at least two bioreactors (i.e., expanded in a first bioreactor and then subsequently transferred to a second bioreactor for further expansion).
  • the cells are preferably expanded in the bioreactors to at least about 1 x 10 9 , more preferably at least about 1 x 10 10 , even more preferably at least about 1 x 10 12 , still more preferably at least about 1 x 10 13 , advantageously at least about 1 x 10 14 , or even more preferably at least about 1 x 10 15 or more adenoviral vector packaging cells.
  • the levels of secreted protein in the supernatant can be measured using any suitable technique, such as ELISA. Additionally, levels of protein expression can be measured by SDS-PAGE and Western Blotting using standard techniques. [0099] Typically and desirably, the level of expression ranges from about 5 pg/cell to about 100 pg/cell or more, typically about 10 pg/cell to about 80 pg/cell, more typically about 10 pg/cell to about 60 pg/cell, even more typically about 10 pg/cell to about 40 pg/cell, most typically about 10 pg/cell to about 25 pg/cell, although the actual amount will depend on the particular transgene of interest, promoter, and vector configuration.
  • the adenoviral vector particles of the invention are advantageously able to achieve such levels of gene expression consistently.
  • Measurements of transgene bioactivity can be performed using any suitable method or technique for measuring bioactivity.
  • bioactivity is measured using a bioactivity assay.
  • a bioactivity assay is typically developed based on the characteristics of the protein activity being measured.
  • the bioactivity of a vascular endothelial growth factor (VEGF) can be measured by adding the VEGF protein to a culture of endothelial cells. If the VEGF possesses suitable bioactivity, the endothelial cells will migrate toward the VEGF.
  • VEGF vascular endothelial growth factor
  • VEGF-related assays are described in, e.g., U.S. Patent Application 09/832,355, which published as U.S. Patent Application Publication 20030027751 Al, and references cited therein.
  • the bioactivity of a pigment endothelial derived growth factor (PEDF) can be determined, for example, by strategies such as determining whether cells responsive to PEDF migrate towards the growth factor upon administration; measuring apoptosis; determining capillary tube formation in vitro; determining neurite outgrowth; applying microarray technology; measuring receptor- mediated activity (e.g., phosphorylation, reporter gene expression); performing a pathway activation/hybridization test; analyzing promoter activity; or testing for anti-permeability function.
  • receptor- mediated activity e.g., phosphorylation, reporter gene expression
  • the protein of interest to be purified is a secreted or extracellular protein. Isolation of such a protein can be achieved by collecting the supernatant from the cells after culturing (e.g., see description of harvesting of cells set forth herein). Preferably, the supernatant comprising the protein of interest is collected at about 28 to about 68 hours after infection, more preferably from about 38 to about 58 hours after infection, and even more preferably, about 48 hours after infection. Methods of collecting supernatant are well known in the art, including centrifuging the medium containing the cells and protein to separate the cells from the supernatant containing the protein of interest.
  • the protein of interest can be an intracellular protein.
  • the cells are typically harvested from the culture. Any method of harvesting cells which will result in the recovery of intracellular protein of interest can be used in the context of the invention. Suitable methods of harvesting include methods of removing the cells from culture conditions such that the cells are no longer in conditions conducive to cell growth.
  • harvesting can be accomplished by removal of the cells from the bioreactor (e.g., by a closed system comprising a peristaltic pump).
  • the cells can be centrifuged down into a lower volume, or the cells can be maintained in the full amount of medium used during the infection process.
  • the cells preferably are harvested in the full amount of medium used during the infection process.
  • Cells harvested in the full amount of medium can be maintained (stored) for any suitable period of time in a suitable container, e.g., in sterile plastic bags (which are preferred due to their ability to form a closed system with the container holding the harvested cells and the next device or container to be used in purifying the viral vector composition or the protein of interest, their ability to freeze and thaw effectively due to their large surface area, their disposability, and low cost).
  • the cells can be directly subjected to lysis and further purification methods.
  • the viral vector particle-infected cells can be lysed using any suitable method to obtain a lysate.
  • Suitable methods to produce a cell lysate include, but are not limited to, sonication, hypotonic solution lysis, hypertonic solution lysis, liquid shear (e.g., microfluidization), solid shear (e.g., French pressure cell lysis, Mickle shaker lysis, and Hughes pressure cell lysis), detergent lysis, or a combination thereof.
  • liquid shear e.g., microfluidization
  • solid shear e.g., French pressure cell lysis, Mickle shaker lysis, and Hughes pressure cell lysis
  • detergent lysis or a combination thereof.
  • the use of such techniques to disrupt cells, generally, is known in the art. Additional techniques and description are known in the art and can be found in U.S. Patent 6,168,941 and International Patent Application Publication WO 03/03945.
  • the supernatant collected from the cells, if the protein of interest is extracellular, or the cell lysate, if the protein of interest is intracellular, can be purified by any suitable means.
  • the supernatant or cell lysate is subjected to the following steps: (a) filtration; (b) buffer exchange or dilution; (c) a capture column, such as ion (e.g., cation) exchange chromatography; (d) buffer exchange or dilution; (e) a purification column, such as a second ion (e.g., anion) exchange chromatography; and (f) a polishing column, such as size exclusion chromatography.
  • steps can be added or deleted from the process in order to obtain a composition of purified protein of interest.
  • the supernatant or cell lysate is desirably subjected to filtration, preferably clarification, (i.e., the removal of large particulate matter, particularly cellular components, from the supernatant or cell lysate by filtration). Clarification can be accomplished by any suitable technique. Suitable techniques include, but are not limited to, microfiltration and depth filtration. Both techniques use filters to separate large particulate matter (which is retained by the filter) from protein of interest (which passes through the filters). The microfiltration filter or filters can be formed from any suitable materials.
  • the microfiltration filter is prepared from an inert (i.e., non-protein-binding), polymeric material (e.g., cellulose acetate, polyester, polypropylene, PTFE, glass fiber, and nylon 66).
  • the microfiltration filter can be formed from glass, ceramic materials, and even metal. Examples of suitable filters formed of such materials are known in the art, and are generally described in, e.g., Sinclair, TJie Philosoph, 12(19): 18 (1998), FILTRATION IN THE BIOPHARMACEUTICAL INDUSTRY, Meltzer and Jornitz, Eds., (Marcel Dekker, Inc.
  • Suitable inert polymeric filter materials include cellulose acetate, polyester, polypropylene, PTFE, glass fiber, and nylon 66.
  • a cellulose acetate filter can be combined with a polypropylene pre-filter
  • a PTFE filter can be combined with a polypropylene pre-filter
  • a glass fiber filter can be combined with a polypropylene pre-filter
  • a nylon 66 filter can be combined with a polypropylene pre-filter
  • a cellulose acetate filter can be combined with a glass fiber pre- filter.
  • the filter may also include diatomaceous earth, perlite, or precipitated silica, which are useful in the removal of surfactants (lipids and/or detergents), DNA, or both.
  • the depth filter can be any suitable depth filter.
  • Suitable depth filters are known in related arts.
  • Materials for the depth filter include polypropylene, cellulose, acrylics, and glass fibers.
  • a depth filter consists of a network of fibrous or granular materials that produce a random porous structure that traps particles in a fluid passing through the filter.
  • the pore size of the depth filter is not typically rated, unlike membranes with pores of defined and ordered structures (see, e.g., FILTRATION IN THE BIOPHARMACEUTICAL INDUSTRY, Meltzer and Jornitz, Eds. (Marcel Dekker, Inc. 1998)).
  • the depth filter removes at least about 90% of particles of a specified size.
  • Depth filters typically filter particles of about 0.5-100 ⁇ m.
  • Depth filters can be derivatized, for example by the addition of a positive or a negative charge to the filter membrane by any suitable cationic or anionic composition, or by the addition of a binding moiety that is selective for a desired biomolecule to be bound by the depth filter (e.g., a lipid-binding moiety such as tri-n-butyl phosphate (TNBP)).
  • a lipid-binding moiety such as tri-n-butyl phosphate (TNBP)
  • the clarification filtration system can comprise any suitable number of filters having any suitable pore size.
  • the clarification filter will comprise pores with a pore size (approximate diameter) of about 20 ⁇ m (e.g., a 0.22 ⁇ m filter) to about 0.45 ⁇ m.
  • the pore size is between about 10 ⁇ m and about 0.65 ⁇ m. Ideally, the average pore size of the filter is between about 8 ⁇ m and about 0.8 ⁇ m. Additional preferred combination clarification filters are described further herein.
  • the supernatant or cell lysate is clarified by an active microfiltration (e.g., filtration through a microfiltration filter at a positive flow rate generated by any suitable technique). Any suitable flow rate can be applied in performing microfiltration clarification of the protein composition.
  • the flow rate is preferably between about 700-1500 niL/min and more preferably the flow rate is about 900-1300 mL/min per filter. Most preferably, the flow rate is about 1000-1200 mL/min per filter.
  • Microfiltration also can be characterized on the basis of the specific pressure of the microfiltration process. Any suitable specific pressure can be used.
  • the specific pressure is typically and preferably about 0-10 psi. More preferably, the pressure is about 2-8 psi. Most preferably, the specific pressure is about 4-6 psi.
  • Microfiltration in the context of the invention can be performed at any suitable filtration volume.
  • the filtration volume during microfiltration is preferably at least about 10L/ft 2 per filter. More preferably, the filtration volume is at least about 20L/ft 2 per filter. Most preferably, the filtration of volume is at least about 40L/ft 2 per filter.
  • Microfiltration e.g., clarification microfiltration
  • the filtration flow during clarification is preferably at least about 2L/min/ft 2 per filter. More preferably, the filtration flow is at least about 4L/min/ft 2 per filter. A filtration flow of at least about lOL/min/ft 2 per filter typically will be optimal.
  • the supernatant or cell lysate is passed through a series of at least two, more preferably at least three, microfiltration filters having decreasing pore size in the order in which they are contacted with the supernatant or cell lysate.
  • microfiltration filter clarification filtration can be performed at any suitable point in the purification process. Desirably, such clarification filtration is performed on supernatant or cell lysate before the filtered lysate is subjected to concentration and/or benzon nuclease digestion and additional downstream processing steps (e.g., tangential flow diafiltration and chromatography purification, as described elsewhere herein).
  • Single microfiltration filter clarification can be preferred in other points in the purification process.
  • the supernatant or cell lysate also or alternatively can be subjected to ultrafiltration.
  • Ultrafiltration can be used to filter and/or purify the protein of interest in any suitable manner.
  • purify it is meant that the composition is enriched with respect to protein by increasing the protein with respect to the total composition and/or one or more undesired biomolecules therein.
  • Preferred uses of ultrafiltration systems in the inventive method include using ultrafiltration filters, preferably tangential flow filtration ultrafiltration systems, during buffer exchange (diafiltration) and/or during concentration of the protein composition. Concentration refers to the enrichment of the composition for the protein of interest with respect do the total composition, which can be determined by measuring the increase in protein concentration brought about by the removal of contaminants or extraneous composition materials (e.g., water).
  • TFF tangential flow filtration
  • Suitable TFF techniques are known in the art. Briefly, in TFF, the protein composition flows across a membrane surface that facilitates back-diffusion of solute from the membrane surface into the bulk solution.
  • Membranes are generally arranged within various types of filter apparatus including open channel plate and frame, hollow fibers, spiral wound modules, and tubules.
  • a preferred TFF filter in the method of the invention is plate and frame.
  • the ultrafiltration filter and more particularly, for example, the TFF ultrafiltration filter or filters of the inventive techniques and systems can have any suitable pore size.
  • the pore size of the ultrafiltration filter membranes corresponds with a nominal molecular weight cutoff (NMWCO) that is about 1/5-1/3 of the molecular weight of the protein to be purified (e.g. about 10-20 kiloDaltons (kDa) when the protein to be purified is about 50 kDa)
  • NMWCO nominal molecular weight cutoff
  • Diafiltration is a method of buffer exchange based on filtration.
  • the use of ultrafiltration filters during diafiltration can facilitate the removal and exchange of salts, organic solvents, sugars, non-aqueous solvents, promote separation of free material from bound species, promote removal of material of low molecular weight, and/or facilitate the rapid change of ionic and pH levels.
  • diafiltration results in the removal of (or reduction in concentration of) at least one undesired biomolecule in the composition, such as contaminating viral vector particles.
  • Microsolutes are removed most efficiently by adding solvent to the solution being ultrafiltered at a rate equal to the ultrafiltration rate. This washes microspecies from the solution at constant volume, purifying the retained species.
  • the shear rate during diafiltration can be any suitable shear rate (e.g., about 2,000-10,000 sec "1 ).
  • the flow rate in any particular microfluidization system is proportional to the shear rate. Therefore, a preferred flow rate is one which corresponds with a preferred shear rate.
  • Ultrafiltration e.g., TFF diafiltration ultrafiltration
  • TMP transmembrane pressure
  • TMP will typically and preferably be about 1-3 bar. More preferably, the TMP will be about 1.5-2.5 bar. Most preferably, the TMP will be about 2 bar.
  • the protein composition optionally is subjected to at least one nuclease digestion, such that the amount (concentration) of contaminating (i.e., undesired), viral and non-viral encapsidated polynucleic acids (e.g., extraneous host cell DNA) is reduced.
  • Any nuclease or combination of nucleases which have DNAase activity, RNAase activity, both DNAase activity and RNAase activity, or that otherwise function to reduce the amount of nucleic acid contaminants in the supernatant or cell lysate without significant loss of protein can be added to the protein composition.
  • the nuclease is preferably an endonuclease.
  • Benzon nuclease which originates from Serratia marcescens and exhibits a high level of DNAase and RNAase activity.
  • Benzon nuclease hydrolyzes nucleic acids into nucleotides, oligonucleotides, or smaller nucleic acid fragments.
  • Benzon nuclease is marketed under the trademark, Benzonase® (Merck & Co, hie, Whitehouse Station, NJ) and is described in, e.g., U.S. Patent 5,173,418. [0123] Benzon nuclease digestion can occur at any suitable stage of the purification process.
  • a preferred time for the benzon nuclease treatment is after clarification of the supernatant or cell lysate with at least one clarification filter (e.g., at least one microfiltration clarification filter, preferably after clarification filtration using a two-part or three-part microfiltration filter system, having multiple filters of decreasing pore size as described elsewhere herein), and concentration (e.g., a concentration filtration which results in a composition about 5-10 times more concentrated than the clarified protein composition with respect to the viral vector particles), which typically is accomplished by tangential flow diafiltration.
  • a suitable benzon nuclease buffer is preferably added to the concentrated protein composition using diafiltration with tangential flow filtration.
  • the buffer preferably has an ionic strength of about 50-100 mM. More preferably, the buffer has an ionic strength of about 75 mM.
  • the ionic strength is desirably obtained by the presence of a monovalent salt in the composition, such as NaCl, which is preferred.
  • the benzon nuclease is added to the composition. Any suitable amount of benzon nuclease can be used for viral and non- viral encapsidated polynucleotide digestion.
  • the combination of benzon nuclease and the protein composition can be incubated at any suitable temperature for any suitable amount of time which results in a decrease in the amount of nucleic acid contaminants. Suitable conditions for the benzon nuclease digestion include digestion at room temperature (about 18-25° C) for about 1-4 hours, or overnight at refrigerated temperatures (0-10° C).
  • the benzon nuclease digestion is performed at room temperature for 1-4 hours.
  • Benzon nuclease digestion of the protein composition can be performed any suitable number of times.
  • protein purification by the inventive method comprises only one benzon nuclease digestion.
  • multiple (e.g., 2, 3, or more) benzon nuclease or benzon nuclease/other nuclease digestions can be performed using any suitable combination of techniques described herein or otherwise known in the art.
  • the protein composition can be subjected to any number of chromatographic purification techniques (steps) to obtain a purified protein composition.
  • Any suitable type of chromatography column or combination of columns can be used in the purification of the protein of interest.
  • the protein composition is subjected to one or more ion exchange chromatography columns.
  • the ion exchange chromatography columns comprise a cation exchange chromatography resin.
  • the cation exchange chromatography resin can be any suitable resin. Examples of preferred chromatography resins in this respect are described in International Patent Application Publication WO 99/54441.
  • the process of purifying a protein composition by chromatography can involve the use of any number of chromatography steps (i.e., columns) to achieve the desired purity.
  • the chromatography purification process can involve the use of a single step technique (i.e., one column), which is capable of purifying the protein composition to a desired level.
  • the chromatography purification process will involve the use of multiple columns, such as two or more, three or more, or even four or more columns, to achieve the desired purity, with two and three column processes being most preferred.
  • the process can include the repetition of purification by a particular type of chromatography column.
  • the method can comprise subjecting the protein composition to two cation or anion exchange chromatography resins, or one of each.
  • the method comprises subjecting the PEDF composition to a cation exchange chromatography column and an anion exchange chromatography column.
  • the protein composition is applied to (e.g., loaded on) the chromatography column(s) using any suitable technique.
  • the first column is desirably subjected to an equilibration buffer.
  • this buffer will comprise a monovalent or divalent salt, or a. mixture of both, having a certain ionic strength and a desired molarity and pH.
  • a wash buffer typically is used in conjunction with running the particle composition through the column.
  • the wash buffer generally comprises the same solution as the equilibration buffer but can contain a slightly higher concentration of the salt.
  • a portion of the protein composition is eluted, such that a purified protein composition is obtained (with respect to the protein composition loaded onto the column). Elution of the portion can be accomplished by any suitable technique. Typically, elution is accomplished with an elution buffer that is applied to the first column, which causes a population of bound protein to be released from the column. This buffer generally comprises the same solution as the above-described buffers but with a higher salt concentration than either the equilibration buffer or wash buffer.
  • the chromatography columns of the invention are prepared by any suitable technique. Typically, a prepared slurry comprising the chromatography column resin is "packed" into the column using a particular packing rate.
  • the packing rate is important during a chromatography purification process.
  • the packing rate can be any suitable packing rate and will vary with the type of chromatography column at issue among other variables.
  • the protein composition is loaded onto the column, run through the column, and finally eluted from the column.
  • the rate at which the protein composition is loaded, run, eluted or (typically) the rate at which all three processes occur is referred to herein as the flow rate.
  • the portions of the eluant containing the protein of interest are collected to obtain a purified protein composition.
  • This eluted protein composition can then be further purified by, for example, loading the eluate onto a second column (e.g., an anion exchange chromatography column), which is referred to in the art as a purification column.
  • a second column e.g., an anion exchange chromatography column
  • the eluate is preferably diluted or buffer exchange performed.
  • a purification column can comprise any suitable resin, however, an ion exchange chromatography resin is preferred. More preferably, the purification column comprises an anion exchange chromatography resin.
  • the resin is a solid that has chemically bound charged groups to which ions are electrostatically bound and can exchange these ions for ions in aqueous solution.
  • Ion exchangers can be used in column chromatography to separate molecules according to charge. Charged molecules adsorb to ion exchangers reversibly so that molecules can be bound or eluted by changing the ionic environment.
  • Separation on ion exchangers is usually accomplished in two stages: first, the substances to be separated are bound to the exchanger, using conditions that give stable and tight binding; then the column is eluted by the addition of buffer(s) of different pH, ionic strength, or composition wherein the components of the buffer(s) compete with the bound protein for the binding sites on the resin.
  • An ion exchanger is usually a three-dimensional network or matrix that contains covalently linked charged groups. If a group is negatively charged, it will exchange positive ions and is a cation exchanger. A typical group used in cation exchangers is the sulfonic group, SO 3 " .
  • the exchanger is said to be in the acid form; it can, for example, exchange one H + for one Na + or two H + for one Ca 2+ .
  • the sulfonic acid group is called a strongly acidic cationic exchanger.
  • Other commonly used groups are phenolic hydroxyl and carboxyl, both weakly acidic cation exchangers.
  • the ion exchange purification resin will preferably be functionalized with an anion exchanging tertiary amine-binding moiety, comprising at least three carbon atoms, a quaternary amine binding moiety, or both.
  • binding moieties will be more selective for the protein of interest than a DEAE binding moiety.
  • Particularly preferred purification resins are described in the above- referenced '441 PCT application.
  • Dimethylaminopropyl binding moieties are particularly preferred in the anion exchange chromatography (AEC) aspects (particularly in an AEC purification column) of the invention.
  • the binding moiety of the invention can be linked to a matrix support through any suitable (and desirably flexible) linker group, as is known in the art.
  • Sulphonamide and acrylic polymer linkers are among those suitable for use in the context of the invention.
  • the support matrix can be composed of any suitable material; however, it is preferable for the matrix support to be a material based on poly(styrene divinyl benzene) due to the inherent high protein binding capacity, and the rigidity of composite materials for high flow rates and increased tolerance to compression or shrinking and swelling of the media, a common characteristic of soft gels.
  • the matrix support be a perfusive anion exchange chromatography resin such that intraparticle mass transport is optimized.
  • Typical perfusive chromatography resins which can be used in the context of the invention have large (e.g., about 6,000-8,000 A) pores that transect the particles. A network of smaller pores, thereby limiting diffusional pathlengths, enhances the surface area of the large-pore diameters. In part due to the bimodal distribution of pore sizes, the mobile phase and proteins enter and flow through the chromatography resin particles, utilizing both convective and diffusional transport.
  • Such perfusive chromatography resins are more fully described by, e.g., Afeyan et al., J. Chromatogr. 519: 1-29 (1990), and U.S. Patents 5,384,042; 5,228,989; 5,552,041; 5,605,623; and 5,019,270.
  • Particularly preferred anion exchange chromatography resins in the context of the invention are Source 30 Q (GE Healthcare), Poros 50D (Applied Biosystems) and Poros DEAE (Applied Biosystems).
  • the purification column Prior to the protein composition being loaded onto the purification column, the purification column typically is subjected to an equilibration buffer as described above. It is also contemplated to subject the protein composition to an anion exchange chromatography resin without first performing tangential flow filtration on the composition, diluting the composition, or de-salting the composition. The protein composition is then loaded onto the purification column, and is subsequently run through the column in conjunction with a wash buffer, as described above.
  • any suitable packing rate and flow rate can be used in conjunction with chromatography purification of the protein composition in the purification column.
  • the protein composition can be subjected to size- exclusion chromatography (SEC).
  • SEC size-exclusion chromatography
  • the size-exclusion chromatography purification preferably is also a buffer exchange step, which places the size-exclusion purified protein (i.e., the portion of the composition eluted from the size-exclusion chromatography column) into a different buffer.
  • any suitable SEC column can be used in the context of the invention.
  • the column will have the ability to resolve proteins having masses of about 50 kDa (e.g., PEDF).
  • Size-exclusion chromatography resins are generally rated according to the ability to separate a globular protein from a desired product.
  • the packing and flow rates used in conjunction with the size-exclusion chromatography resin can significantly impact the ability to effectively obtain a size purified protein composition from the SEC column. For example, tighter packing reduces void volume as well as dilution of the protein during purification.
  • the alteration of specific buffer components, such as salt concentration, may improve separation of the protein composition during SEC and, thus, size purification of the protein of interest from the composition.
  • Another technique that can be used in conjunction with the invention is reverse- phase chromatography. This technique separates molecules based on differences in hydrophobicity imparted by hydrophobic amino acid residues.
  • the stationary phase (the resin) is hydrophobic and non polar.
  • the initial mobile phase (the buffer), which contains the analyte (e.g., the protein composition), is an aqueous polar solvent, such as water. Elution from reversed-phase columns is typically accomplished with strong non-polar solvents in a linear gradient.
  • Reversed-phased chromatography in conjunction with MALDI-TOF MS can be used to determine the relative amount of each protein component and how each protein might change over time in a protein composition. This technique also can be use to identify a sample, to quantitate a given sample, and, in some instances, it can provide relative purity. Accordingly, reversed-phased chromatography and MALDI-TOF MS can be used in the context of the invention for many aspects, which are important in protein production.
  • the invention also encompasses the use of one or more negative (i.e., nonprotein binding) chromatography columns, typically in addition to or in place of any of the above-described capture columns, hi such an application, the protein composition is loaded onto an anion exchange chromatography resin under specific conditions which do not allow the protein of interest to bind to the column, but viral vector particles are bound by the column, such that the vector particles are purified from the composition.
  • any suitable chromatography resin can be used.
  • the resins employed in the purification column also can be used in a negative chromatography process (e.g., POROS 50D), however, different loading conditions are used such that the negative chromatography effect is achieved.
  • the resin can be designed to bind to a known impurity, such as by the incorporation of antibodies bound to the resin, which are specific for the impurity, hi this negative chromatography step, the resin can also adsorb viral vector particles and remove them from the protein composition.
  • Other loading conditions can be altered to specifically bind impurities such as utilizing the salt concentration or the pH.
  • two columns which both comprise ion exchange chromatography resins, will be connected in series, and, optionally, the column comprising the size-exclusion chromatography resin will either be connected in series to the second ion exchange column, or will be separate from these columns in which case the eluant from the second ion exchange column will first be collected and then applied to the size-exclusion chromatography column.
  • any suitable chromatography columns can be used in the series chromatography aspects of the invention.
  • the columns-in-series comprise an ion exchange chromatography resin, which is functionalized with a tertiary amine binding moiety having at least three carbon atoms, a quaternary amine binding moiety, or both, wherein the ion exchange chromatography binding moiety is more selective for the protein of interest than a DEAE binding moiety.
  • the tertiary amine binding moiety is a dimethylaminopropyl moiety, dimethylaminobutyl moiety, dimethylaminoisobutyl moiety, or dimethylaminopentyl moiety.
  • the reverse flow technique can be used in conjunction with any number of columns and is generally only employed in the first and second purification columns.
  • a reverse flow elution technique can be employed when recovering a purified portion (eluate) of the protein composition from the column of interest.
  • the protein composition is loaded and run through the column in a first direction and is eluted from the column in the direction opposite of the first direction.
  • the invention provides a method of using such a technique for preparing a purified protein composition.
  • Such a method comprises (a) obtaining a protein composition comprising a population of protein and an undesired biomolecule, (b) loading the protein composition onto a chromatography column, (c) eluting the protein composition from the column, (d) loading the protein composition onto a column in a first direction, (e) eluting the protein composition from the column in the direction opposite of the first direction, arid (f) collecting a portion of the eluted protein composition to obtain a purified protein composition.
  • the chromatography column is a high performance liquid chromatography (HPLC) column. HPLC can be characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of suitable particles and high pressure to maintain an adequate flow rate.
  • Separation typically can be accomplished in a matter of minutes, or at most an hour.
  • dilution of the protein composition is minimized such that the volume is reduced to a level below that which would be obtainable if the elution of the protein composition in step (e) was done in the same direction as the loading direction.
  • the protein composition can be loaded onto the chromatography column at any suitable rate.
  • the protein composition is loaded onto the column at a rate of about 250-550 cm/hr.
  • the additional column(s) may be removed after an eluate is eluted from the first, saturated column and be used in purification of another protein composition.
  • an eluate from the second columns can be used as a "low dose" protein composition.
  • the protein composition is eluted from the column(s) by adding a composition comprising about 200-500 mM NaCl to the column in an amount sufficient to elute a majority of the protein of interest from the column(s).
  • the volume of the purified protein composition that is eluted from the column(s) in step (e) is at least about 15% less than the volume of a purified protein composition eluted from a column when the protein composition is eluted in the same direction as it is loaded onto the column.
  • the volume of the purified protein composition that is eluted from the column(s) in step (e) is at least about 50% less (e.g., about 60% or less) than the volume of a purified protein composition eluted from a column when the protein composition is eluted in the same direction as it is loaded onto the column.
  • the protein composition can be eluted from the column using any other suitable elution technique.
  • a salt or ionic strength gradient is used to elute the protein • composition from the column.
  • various buffers, having different concentrations of a salt typically are blended together before being applied to the column.
  • the elution process involves a step elution process.
  • the buffers are independently applied to the column in sequential order according to their molarity, with the lower concentration salts being utilized first.
  • the invention provides a method for eluting at least a portion of an protein composition from a chromatography column comprising; (a) subjecting resin such that the composition comprising a protein of interest binds to the resin, and (b) eluting at least a portion of the composition from the column in a step wise fashion by sequentially higher salt concentration than the preceding buffer immediately following a composition comprising the protein of interest.
  • this method is carried out with at least two or more buffers, and, more preferably, with at least five buffers, which each comprises a monovalent salt having a concentration of about 50 mM-500 mM.
  • the first elution buffer subjected to the column comprises a monovalent salt having a concentration of about 75 mM.
  • the final elution buffer subjected to the column comprises a monovalent salt having a concentration of about 500 mM.
  • the switching from one buffer to the next is preferably under the control of an automated programmable control system.
  • an automated programmable control system is able to monitor the pH, conductivity, or both, of each elution buffer such that a pre-determined pH level, conductivity level, or both is maintained during elution of the portion of the composition.
  • the automated programmable control system also can control the collection of fractions comprising the protein that is eluted from the column.
  • a sample solution of protein can be prepared.
  • the sample solution of the protein then can be purified by utilizing one of the aforementioned chromatography techniques while determining the absorbance of the protein composition eluted from the chromatography resin at a wavelength sensitive for quantification of a particular protein.
  • the absorbance of a standard solution of the particular protein i.e., a solution of protein of known concentration, is determined.
  • the concentration of protein in a sample solution is determined.
  • the standard absorbance can be a single standard absorbance or a series or group of standard absorbance indicative of a range of concentrations of the protein of interest.
  • the sample absorbance and standard absorbance can be presented in similar or different (though preferably similar) formats, measurements, or units as long as a useful comparison can be performed.
  • a suitable standard absorbance can be an absorbance that is determined from a standard solution of a particular protein that has been treated in the same manner as a sample solution of protein purified in accordance with the present inventive methods, and subsequently subjected to amino acid analysis to obtain an accurate concentration. Such standard solution is referred to as a calibration standard.
  • Quantification of the amount of protein is accomplished by comparing the sample absorbance to the standard absorbance in any suitable manner.
  • sample absorbance and standard absorbance can be compared by calculating a standard curve of the area under the peak corresponding to the elution of the protein of interest from the chromatography resin on an absorbance versus time chromatograph.
  • the absorbance of different known concentrations of the protein of interest can be plotted on a graph, creating a standard curve.
  • linear regression analysis the sample concentration then can be determined.
  • the purity of the protein composition can be confirmed by any method or technique suitable for determining the purity of the protein composition. Suitable methods and techniques include, for example, analysis of host cell DNA and/or host cell protein by Western Blotting, analysis of particulates in the composition, observation of the appearance of the sample, and/or other analytical biochemical methods as appropriate.
  • Laser light scattering methods can be performed using any suitable technique appropriate for measuring and quantitating laser light scattering from a solution. Preferably, the laser light scattering is performed by illuminating a sample with a fine beam of highly collimated and monochromatic light produced by a laser. The scattered light is then measured as a function of the angle between the detector and the incident beam direction.
  • the measurement may be restricted to a single fixed angle, a low angle (e.g., low angle laser light scattering (LALLS)), a high angle, or any angle in between.
  • LALLS low angle laser light scattering
  • MALS multi-angle light scattering
  • the protein composition desirably is subj ected to additional testing as needed.
  • Additional testing can be any testing method or technique necessary for assuring the safety, purity, potency, and stability of the protein composition.
  • additional testing can include testing for pH, conductivity, osmolality, seal integrity, fill volume verification, or stopper extractables.
  • Characterization of the protein composition can occur at any suitable point during the protein composition production process.
  • testing and characterization assays are performed during at least one or more of the steps of upstream processing, downstream processing, and the finished product. More preferably, testing and characterization assays are performed during at least two or more of the steps, still more preferably during at least three or more of the steps, most preferably during at least four of the steps. More than one assay can be performed during each step.
  • the total number of assays performed from the start of the process through the stability tests on the finished product is preferably about 30 to about 100, more preferably about 40 to about 90, still more preferably about 50 to about 80, and most preferably about 55 to about 70.
  • suitable tests during the upstream processing include, e.g., screening for viruses and/or performing potency assays.
  • suitable tests during the downstream processing include, for example, performing potency assays, determination of biological activity, testing for sample purity, testing for sample identity, and/or testing for the presence of viruses.
  • Appropriate tests performed on the finished product include, for example, sample identity tests, sample purity tests, sample potency tests, sterility tests, pH tests, osmolality tests, conductivity tests, seal integrity tests, fill verification tests, and/or stopper extractable tests.
  • Suitable tests on the stability of the final product include any suitable test for determining stability of the product, e.g., any of the above-mentioned tests for the finished product as appropriate.
  • the purity and homogeneity of the protein composition is assessed by SDS-PAGE, mass spectrometry, and light scattering as known in the art.
  • the purified protein composition is preferably assessed for host cell protein concentration, host cell encapsidated DNA concentration, viral encapsidated DNA viral particle component protein, or a combination thereof.
  • assays can be done using any suitable techniques, including, but not limited to, mass spectroscopy, SDS-PAGE, western blot, reverse phase HPLC, quantitative RT-PCR (e.g., TaqMan®, Perkin Elmer/Applied Biosystems), wherein the failure of the results of the assessment to meet or exceed the predetermined standards of purity results in the discarding of purified protein composition, or (if acceptable) subjecting the composition to repeated and/or additional purification steps (e.g., a repeated benzon nuclease digestion).
  • the purified protein composition is assessed for biological activity of the protein of interest.
  • the invention provides protein compositions of significant purity with respect to the impurities such as viral and non- viral encapsidated polynucleotides (e.g., viral vector particle encapsidated DNA, host cell DNA), viral and non-viral vector component proteins (e.g., viral vector particle component proteins, host cell proteins), or a combination thereof, while retaining a high concentration of the protein of interest.
  • the ratio of the amount of viral vector particle encapsidated DNA in the second eluted composition as compared to the supernatant or cell lysate of step (d) is no more than about 15:100
  • the ratio of the amount of host cell DNA in the second eluted composition as compared to the supernatant or cell lysate of step (d) is no more than about 15:100
  • the ratio of the amount of viral particle component protein in the second eluted composition as compared to the supernatant or cell lysate of step (d) is no more than about 15:100
  • the ratio of the amount of host cell protein in the second eluted composition as compared to the supernatant or cell lysate of step (d) is no more than about 15:100.
  • the ratio of each of the above is no more than about 10:100. More preferably, the ratio of each of the above is no more than about 5:100. Desirably, the ratio of each of the above is no more than about 2:100
  • the protein composition e.g., second eluted composition
  • the protein composition preferably contains at least about 85% less viral vector particle encapsidated DNA, at least about 85% less host cell DNA, at least about 85% less viral particle component protein, or at least about 85% less host cell protein than the supernatant or cell lysate collected following harvesting of the cells.
  • the final product contains at least about 90% less of the above contaminants than the supernatant or cell lysate.
  • the final product contains at least about 95% less of the above contaminants than the supernatant or cell lysate.
  • the final product contains at least about 98% less of the above contaminants than the supernatant or cell lysate.
  • host cell protein makes up about 5% or less of the total protein content of the composition.
  • the host cell protein makes up less than about 4% of the total protein of the composition. More preferably, the host cell protein makes up less than 3%, and even more preferably, less than 2%. Most preferably, the host cell protein makes up less than about 1% of the total protein of the composition. Ideally, the host cell protein makes up 0.5% or less of the total protein content of the composition. Optimally, the host cell protein makes up 0.25% or less of the total protein content of the composition.
  • Protein purified in a solution or purified from cells infected with adenovirus using anion exchange chromatography resins can be obtained in solutions that can contain high concentrations of an elution agent, e.g., NaCl.
  • the buffer composition can be readily changed by any suitable technique to any desired buffer, e.g., a sterile, isotonic buffer for mammalian injection (e.g., lactated Ringer's solution) containing suitable excipients ⁇ (stabilizers and cryopreservants) for long term storage of the purified protein.
  • Suitable techniques for changing the buffer composition include, but are not limited to, dialysis, diafiltration, and size-exclusion chromatography.
  • Suitable size-exclusion chromatography matrices include Toyopearl HW-40C, Toyopearl HW40F, and Toyopearl HW-50F (TosoHaas, Montgomeryville, PA); UniflowTM, SuperflowTM, and UltraflowTM (Sterogene, Carlsbad, CA); ShodexTM (Thomson Instruments, Chantilly, VA); Bio-SilTM and Bio-GelTM (Bio-Rad, Hercules, CA); and Superdex 200 and Superdex 75 (Amersham Biosciences).
  • Each of these chromatography resins has a suitably low protein binding potential.
  • the production and purification process of the invention involves the use of one or more automated programmable system(s) during the production and/or purification process.
  • Automation is important to the protein production and purification process for several reasons. Automation allows the key parameters of the process to be continuously monitored and recorded, it allows key parameters to be set and maintained, and it allows the process of production, recovery, and purification of the protein of interest to be maintained as a closed system. Automation also ensures a relative degree of consistency in protein composition manufacturing.
  • Preferably included in the automation process is the production of the product in bioreactors, the initial recovery of the product from the production culture using tangential flow filtration (TFF), and the purification of the product using chromatography.
  • the manufacture of drug substance i.e., the final formulation of the purified protein composition
  • filling and labeling of vials are under automated monitoring and/or control for at least one parameter.
  • the PEDF cell supernatants were either (a) buffer exchanged or (b) diluted into a Hepes/MES/Acetate buffer at pH 6.0 in order to lower the salt concentration and prepare for loading onto the capture column.
  • This material was loaded directly onto a Poros® 50 HS strong cation exchanger (cross-linked poly(styrene-divinyl benzene) surface coated with polyhydroxylated polymer fuctionalized with sulfopropyl groups).
  • a 50 to 500 mM NaCl gradient in the presence of Hepes/MES/Acetate at pH 6.0, DTT, and EDTA resulted in an elution peak at approximately 300 mM NaCl, which was collected and (a) diluted or (b) buffer exchanged into a Tris buffer at pH 7.5 in order to lower salt concentration and prepare for loading onto the purification column.
  • This material was loaded directly onto a Poros® 50 D weak anion exchanger (cross-linked poly(styrene-divinyl benzene) surface coated with polyhydroxylated polymer fuctionalized with dimethyl amino alkyl groups).
  • a 50 to 500 mM NaCl gradient in the presence of Tris at pH 7.5, DTT, and EDTA resulted in an elution peak at approximately 300 mM NaCl, which was collected and then loaded directly onto a polishing column.
  • the Superdex® 75 size exclusion column (SEC) was used for polishing and buffer exchange into phosphate buffered saline at a pH of roughly 7.4. The resulting peak flowing through at about 0.5 Column Volumes was collected, 5% Trehalose added, and finally filtered through a Sartobran® P 0.2 ⁇ m filter unit.
  • MALS multi-angle light scattering
  • Mass spectrometry of purified PEDF showed the expected molecular weight for mature glycosylated human PEDF of 46,500 Da. Multiple ionization forms of the protein were also observed (15,500 Da and 23,300 Da); however, there was a notable absence of contaminant proteins.
  • Gelatin zymogram analysis was also employed to demonstrate the removal of enzymatic activity as a function of protein purification.
  • the enzymatic activity was assessed after several steps: (i) crude cell lysate at the time of harvest (48 hours post infection with AdPEDF), (ii) PEDF elution from the capture column (cation exchanger), (iii) PEDF elution from the purification and concentration column (anion exchanger), and (iv) PEDF fraction from the polishing column (size exclusion column) were compared with an enzyme (MMP-2) control. Only the crude cell lysate and control showed enzymatic activity, indicating that the purification steps effectively removed enzymatic activity.
  • PEDF protein also was analyzed for the presence of host cell proteins and viral proteins by Western Blot.
  • goat anti-293-ORF6 cells were utilized with 293-ORF6 cell lysate proteins used as control.
  • rabbit anti-Ad5 was utilized with Ad5 proteins as a control. 500 ng of PEDF was used from each of 5 samples: (i) filtered solution, (ii) PEDF elution from the capture column (cation exchanger), (iii) PEDF elution from the purification and concentration column (anion exchanger), and (iv) PEDF fraction from the polishing column (size exclusion column).
  • the biological activity of the purified PEDF also was assessed.
  • the purified PEDF retained its biological activity as illustrated by the dose dependent inhibition of endothelial cell invasion by hPEDF.
  • purified PEDF was prepared in serum-free media at various concentrations (0.05 nM, 0.1 nM, 0.5 nM, 1 nM, and 5 nM) and administered to dermal human microvascular endothelial cells. After incubation for 22 hours, invading cells were labeled with the fluorescent dye, Calcein AM, for 90 minutes. As PEDF concentration increased, the endothelial cell invasion decreased, indicating that the purified PEDF retained its biological activity.
  • the inventive method resulted in the production and purification of the secreted protein, PEDF, without significant contamination (e.g., by host cell and viral proteins).
  • This example describes an in vivo efficacy model of purified recombinant human PEDF.
  • CNV choroidal neovascularization
  • the pupils were dilated with 1% tropicamide, and three burns of diode laser photocoagulation (75 ⁇ m spot size; 0.1 s duration, 120 mV) were made in each retina using a slit lamp delivery system and a cover glass as a contact lens. Burns were performed in the 9, 12, and 3 o'clock positions at 2-3 disk diameters from the optic nerve so that each burn could be identified postmortem. Production of a vaporization bubble at the time of laser indicated rupture of the Bruch's membrane, which is an important factor in obtaining CNV (Tobe et al., Am. J. Pathol, 153(5): 1641-1646 (1998)).
  • mice were placed into 5 test groups (3-5 mice per group).
  • Group 1 received no further treatment (i.e., no injection).
  • Group 2 received one injection of vehicle (phosphate buffered saline with 5% trehalose).
  • Group 3 received four injections of vehicle every three days.
  • Group 4 received one injection of AdPEDF.
  • Group 5 received four injections of recombinant PEDF protein purified by the method of Example 1 every three days.
  • the right eye of each mouse received a single intravitreal injection of (a) vehicle control, (b) 1 x 10 9 particles of AdPEDF, or (c) 1 ⁇ g of the recombinant PEDF protein.
  • mice of Groups 4 and 5 had statistically smaller areas of CNV (P-value ⁇ 0.05; two-tailed test) as compared to controls (Groups 1-3). Accordingly, the inventive method resulted in the production and purification of a secreted protein, PEDF, which retained its function and efficacy in an in vivo mouse model of CNV.
  • PEDF secreted protein

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Abstract

L'invention concerne des méthodes pour purifier des compositions qui comprennent une protéine intéressante, de préférence pour utilisation dans un contexte clinique.
PCT/US2006/029206 2005-07-28 2006-07-27 Methode pour purifier une proteine WO2007016250A1 (fr)

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GB2522561A (en) * 2012-02-21 2015-07-29 Cytonics Corp Systems, compositions and methods for transplantation
US9481706B2 (en) 2008-01-18 2016-11-01 Hoffmann-La Roche Inc. Purification of non-glycosylated polypeptides
EP2188371B1 (fr) 2007-08-09 2017-12-20 Wyeth LLC Utilisation de la perfusion pour améliorer la production d'une culture cellulaire en mode semi-continu dans des bioréacteurs

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2188371B1 (fr) 2007-08-09 2017-12-20 Wyeth LLC Utilisation de la perfusion pour améliorer la production d'une culture cellulaire en mode semi-continu dans des bioréacteurs
US9481706B2 (en) 2008-01-18 2016-11-01 Hoffmann-La Roche Inc. Purification of non-glycosylated polypeptides
GB2522561A (en) * 2012-02-21 2015-07-29 Cytonics Corp Systems, compositions and methods for transplantation
GB2522561B (en) * 2012-02-21 2016-09-21 Cytonics Corp Apparatus for enriching a bio-fluid with respect to the concentration of alpha-2-macroglobulin
US10265388B2 (en) 2012-02-21 2019-04-23 Cytonics Corporation Systems, compositions, and methods for transplantation
US10940189B2 (en) 2012-02-21 2021-03-09 Cytonics Corporation Systems, compositions, and methods for transplantation
US11040092B2 (en) 2012-02-21 2021-06-22 Cytonics Corporation Systems, compositions, and methods for transplantation

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