US20070207461A1 - Virus Purification Methods - Google Patents

Virus Purification Methods Download PDF

Info

Publication number
US20070207461A1
US20070207461A1 US10/590,427 US59042705A US2007207461A1 US 20070207461 A1 US20070207461 A1 US 20070207461A1 US 59042705 A US59042705 A US 59042705A US 2007207461 A1 US2007207461 A1 US 2007207461A1
Authority
US
United States
Prior art keywords
virus
nacl
host cells
anion exchange
adenovirus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/590,427
Other languages
English (en)
Inventor
Miranda Weggeman
Emile Joannes Josephus Van Corven
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Janssen Vaccines and Prevention BV
Original Assignee
Crucell Holand BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=34878416&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20070207461(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Crucell Holand BV filed Critical Crucell Holand BV
Assigned to CRUCELL HOLLAND B.V. reassignment CRUCELL HOLLAND B.V. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: VAN CORVEN, EMILE J. J. M., WEGGEMAN, MIRANDA
Publication of US20070207461A1 publication Critical patent/US20070207461A1/en
Priority to US12/220,828 priority Critical patent/US8124106B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • 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
    • C12N2710/10051—Methods of production or purification of viral material

Definitions

  • the invention belongs to the field of purification of virus, more in particular recombinant adenovirus, from host cells.
  • Viruses are used for vaccination and in the field of gene therapy. It is possible for many viruses or virus-like particles to safely and efficiently propagate these in host cells (see for instance WO 01/38362, which describes the propagation of various viruses in host cells being E1-immortalized retina cells).
  • Recombinant adenoviruses are a preferred class of viral vectors for use in gene therapy and for vaccination purposes. Such recombinant adenoviruses are usually deficient in at least the E1 region, and are propagated in complementing cells providing the E1-region, such as 293 cells, or E1-immortalized retina cells such as PER.C6TM cells (see for instance U.S. Pat. No. 5,994,128).
  • the methods comprise growing host cells, infecting the host cells with adenovirus, harvesting and lysing the host cells, concentrating the crude lysate, exchanging the buffer of the crude lysate, treating the lysate with nuclease, and further purifying the virus using chromatography.
  • FIG. 1 Scheme of the known method of harvesting the cells (T/B) versus the method according to the invention (B/T), see example 1.
  • T Triton
  • B Benzonase. p.i.: post infection.
  • FIG. 2 Host cell protein removal at clarification after T/B vs. B/T process (see FIG. 1 for scheme).
  • a silver-stained SDS-PAGE (4-12% bis-tris NuPAGE, Invitrogen) analysis of in process samples of 5 separate purifications is shown (see example 1 and Table 1 for samples).
  • Panel 2 is from a T/B harvest, wherein lysis preceded nuclease addition;
  • panels 3-7 are from a B/T harvest, wherein nuclease was added before lysis.
  • the harvest (lanes 1) was clarified by a 0.5 ⁇ m Clarigard filter (lanes 2), followed by a 0.8/0.45 ⁇ m Sartopore 2 filter (lanes 3).
  • M marker, M w in kD is shown alongside.
  • FIG. 3 Diafiltration with high salt removes histones during process (see example 2). A silver-stained SDS-PAGE is shown.
  • FIG. 4 Scheme of a preferred process according to the invention (see example 1).
  • FIG. 5 Removal of Ebola nucleoprotein (NP) from recombinant virus preparations (see example 3, experiment 3.1 for details). A silver-stained SDS-PAGE (4-12% bis-tris NuPAGE, Invitrogen) is shown. A: starting material. B: incubation with 1% Tween 20. C: incubation with 2.5 M NaCl. The arrow denotes NP.
  • NP Ebola nucleoprotein
  • FIG. 6 Experiment for removal of Ebola nucleoprotein from recombinant virus preparations (see example 3, experiment 3.3 for details).
  • FIG. 7 Non-reduced SDS-PAGE (panel 1) and Western blot (panel 2) analysis of removal of Ebola nucleoprotein (NP) from recombinant virus preparations (see example 3, experiment 3.3 for details). Lanes A, B, C contain product A, B and C, respectively (see FIG. 6 and experiment 3.3). For the Western blot analysis, an antibody recognizing NP was used. The arrows denote NP.
  • FIG. 8 RP-HPLC analysis of removal of Ebola nucleoprotein (NP) from recombinant virus. Products A, B and C were analysed. For details see example 3, experiment 3.3.
  • the vertical axis are in AU ( ⁇ 10 ⁇ 3 ). Under the horizontal axis (elution time), arrow 1 indicates the peak of hexon protein, arrow 2 indicates peak of NP.
  • FIG. 9 SDS-PAGE (panel A) and Western blot (panel B) showing the removal of Ebola nucleoprotein (NP) from recombinant virus preparations using high salt and filtration.
  • NP Ebola nucleoprotein
  • FIG. 10 Chromatogram of Ad35 TFF retentate (example 6) loaded on a Q-XL column (panel A) and on a charged filter (panel B).
  • the circle in panel B indicates the extra peak, which is only separated from the virus peak using the charged filter.
  • FIG. 11 Disc centrifugation analysis of two fractions of the charged filter chromatogram.
  • Panel A shows the sedimentation profile of the Ad35 virus peak
  • panel B shows the sedimentation profile of the extra peak (circled in FIG. 10 ).
  • FIG. 12 SDS-PAGE analysis of chromatography fractions Ad35 (see example 6). 4-12% bis-tris gel, stained with silver.
  • Gel A shows the fractions of the charged filter run: 1. marker; 2. start material; 3. flowthrough; 4. peak 1 (circled in FIG. 10 ); 5. Ad35 peak.
  • Gel B shows the fractions of the Q-XL run: 1. start material; 2. flowthrough; 3. Ad35 peak.
  • the present invention provides a method for the purification of a virus from a host cell, said method comprising the steps of: a) culturing host cells that are infected with a virus, b) adding nuclease to the cell culture, and c) lysing said host cells to provide a lysate comprising the virus.
  • the method further comprises: d) clarification of the lysate.
  • the method further comprises: e) further purifying the adenovirus, preferably with at least one chromatography step.
  • a nuclease is added before lysing the cells.
  • the purified virus batch resulting from this process contains less host cell DNA than with the method wherein the lysing of cells precedes the nuclease addition.
  • the virus is a recombinant adenovirus.
  • the nuclease used in step b) is benzonase®.
  • the step of lysing the host cells (step c) is performed with a detergent, which in one embodiment thereof is Triton-X100.
  • the clarification of the lysate (step d) comprises depth filtration and membrane filtration.
  • said membrane filtration is performed using a combination of filters having a pore size of 0.8 ⁇ m and 0.45 ⁇ m, such as a combination filter comprising two asymmetric polyethersulfone membranes with pore sizes of 0.8 and 0.45 ⁇ m, such as a SartoporeTM-2 combination filter.
  • the clarified lysate (resulting from step d) is subjected to ultrafiltration and/or diafiltration.
  • the diafiltration results in buffer exchange against a solution comprising 0.8-2.0 M NaCl, or another salt providing an equivalent ionic strength.
  • further purification of the virus (step e) comprises anion exchange chromatography.
  • said further purification of the virus (step e) comprises a size exclusion chromatography step, preferably in group separation mode.
  • step e) comprises both anion exchange chromatography and size exclusion chromatography.
  • the clarified lysate and further purified virus are in buffers that are free of detergent, magnesiumchloride and sucrose.
  • the invention provides a batch of recombinant adenovirus comprising a transgene chosen from the group consisting of: an Ebolavirus nucleoprotein, an Ebolavirus glycoprotein, a Plasmodium falciparum circumsporozoite gene, and measles virus hemagglutinin, said batch characterized in that it contains less than 0.1 ng host cell DNA per 1E11 viral particles.
  • the invention further provides a method for the production of a virus comprising a nucleic acid sequence coding for a nucleic acid binding protein, comprising the steps of: a) culturing host cells that have been infected with virus, b) subjecting said culture of host cells and said virus therein produced to lysis of the host cells to provide a lysate comprising said virus, c) subjecting the virus to anion exchange chromatography, characterized in that after anion exchange chromatography the virus containing mixture is buffer exchanged with a solution comprising at least 1 M NaCl, or another salt providing an equivalent ionic strength.
  • said solution comprises at least 1.5 M NaCl, more preferably at least 2 M NaCl, still more preferably at least 3 M NaCl, still more preferably about 5 M NaCl, or another salt providing an equivalent ionic strength.
  • said virus is further purified using filtration through a hydrophilic filter, preferably with a pore size not larger than 1.2 ⁇ m, and/or by size exclusion chromatography.
  • the virus preferably is a recombinant virus, more preferably a recombinant adenovirus.
  • the nucleic acid binding protein may be a nuclear protein, such as a nucleoprotein of a haemorrhagic fever virus, such as Ebola, Marburg or Lassa virus, preferably Ebola virus.
  • a host cell according to the present invention can be any host cell wherein a desired virus can be propagated.
  • the propagation of recombinant adenovirus vectors is done in host cells that complement deficiencies in the adenovirus.
  • host cells preferably have in their genome at least an adenovirus E1 sequence, and thereby are capable of complementing recombinant adenoviruses with a deletion in the E1 region.
  • the adenovirus may have a deletion in the E3 region, which is dispensable from the Ad genome, and hence such a deletion does not have to be complemented.
  • Any E1-complementing host cell can be used, such as human retina cells immortalized by E1, e.g. 911 (see U.S. Pat. No.
  • E1-transformed amniocytes See EP patent 1230354
  • E1-transformed A549 cells see e.g. WO 98/39411, U.S. Pat. No. 5,891,690
  • GH329:HeLa Gao et al, 2000, Human Gene Therapy 11: 213-219
  • PER.C6TM cells U.S. Pat. No. 5,994,128, or cells derived therefrom are used as host cells, as they are suitable for the propagation of various different viruses (see e.g. WO 01/38362), including but not limited to recombinant adenoviruses.
  • Examples of other useful mammalian cell lines that may be used directly as host cells for propagating viruses or converted into complementing host cells for replication deficient virus are Vero and HeLa cells and cell lines of Chinese hamster ovary, W138, BHK, COS-7, HepG2, 3T3, RIN and MDCK cells, as known to the person skilled in the art.
  • Host cells according to the invention are cultured to increase cell and virus numbers and/or virus titers. Culturing a cell is done to enable it to metabolize, and/or grow and/or divide and/or produce virus of interest according to the invention. This can be accomplished by methods as such well known to persons skilled in the art, and includes but is not limited to providing nutrients for the cell, for instance in the appropriate culture media. The methods may comprise growth adhering to surfaces, growth in suspension, or combinations thereof. Culturing can be done for instance in dishes, roller bottles or in bioreactors, using batch, fed-batch, continuous systems, hollow fiber, and the like.
  • the present invention comprises subjecting cultured host cells that are infected with virus to lysis.
  • Culturing host cells and infecting them with a virus is well known to the person skilled in the art. Infecting of host cells can for instance simply be accomplished by exposing the virus to the appropriate host cell under physiological conditions, permitting uptake of the virus. For certain viruses it is not even necessary to start with virus per se, as nucleic acid sequences may be used to reconstitute the virus in the cultured cells.
  • viruses including but not limited to adenoviruses, pox viruses, iridoviruses, herpes viruses, papovaviruses, paramyxoviruses, orthomyxoviruses (such as influenza), retroviruses, adeno-associated virus, vaccinia virus, rotaviruses, etc.; adenoviruses being particularly preferred.
  • the viruses are preferably recombinant viruses, but can include clinical isolates, attenuated vaccine strains, and so on.
  • the present invention is used for concentrating recombinant viruses, preferably adenoviruses, carrying a heterologous transgene for use in gene therapy or for vaccination purposes.
  • viruses preferably adenoviruses
  • a heterologous transgene for use in gene therapy or for vaccination purposes.
  • the invention will be described in more detail for recombinant adenovirus, but is in no way limited thereto.
  • the adenoviral vector is deficient in at least one essential gene function of the E1 region, e.g., the E1a region and/or the E1b region, of the adenoviral genome that is required for viral replication.
  • the vector is deficient in at least one essential gene function of the E1 region and at least part of the nonessential E3 region (e.g., an Xba I deletion of the E3 region).
  • the adenoviral vector can be “multiply deficient,” meaning that the adenoviral vector is deficient in one or more essential gene functions in each of two or more regions of the adenoviral genome.
  • the aforementioned E1-deficient or E1-, E3-deficient adenoviral vectors can be further deficient in at least one essential gene of the E4 region and/or at least one essential gene of the E2 region (e.g., the E2A region and/or E2B region).
  • Adenoviral vectors deleted of the entire E4 region can elicit lower host immune responses.
  • adenoviral vectors include adenoviral vectors that lack (a) all or part of the E1 region and all or part of the E2 region, (b) all or part of the E1 region, all or part of the E2 region, and all or part of the E3 region, (c) all or part of the E1 region, all or part of the E2 region, all or part of the E3 region, and all or part of the E4 region, (d) at least part of the E1a region, at least part of the E1b region, at least part of the E2a region, and at least part of the E3 region, (e) at least part of the E1 region, at least part of the E3 region, and at least part of the E4 region, and (f) all essential adenoviral gene products (e.g., adenoviral amplicons comprising ITRs and the packaging signal only).
  • all essential adenoviral gene products e.g., adenoviral amplicons comprising ITRs and the packaging
  • the functions encoded by these regions have to be provided in trans, preferably by the host cell, i.e. when parts or whole of E1, E2 and/or E4 regions are deleted from the adenovirus, these have to be present in the host cell, for instance integrated in the genome, or in the form of so-called helper adenovirus or helper plasmids.
  • the replication-deficient adenoviral vector can be generated by using any species, strain, subtype, or mixture of species, strains, or subtypes, of an adenovirus or a chimeric adenovirus as the source of vector DNA (see for instance WO 96/26281, WO 00/03029), which for instance may provide the adenoviral vector with the capability of infecting certain desired cell types.
  • the adenoviral vector can be any adenoviral vector capable of growth in a cell, which is in some significant part (although not necessarily substantially) derived from or based upon the genome of an adenovirus.
  • the adenoviral vector may comprise an adenoviral genome of a wild-type adenovirus of group C, especially of serotype 5 (i.e., Ad5) or Ad2.
  • the adenoviral vector may also comprise an adenoviral genome or at least a fiber protein derived from an adenovirus of group B, for instance Ad11, Ad35, Ad51, etc. (see e.g. WO 00/70071), which embodiments have the advantage that less neutralizing antibodies against these serotypes are encountered in the population, and confer the possibility of targeting other cell types, since the tropism of these adenoviral vectors differs from those derived from Ad5.
  • Ad5 serotype 5
  • Adenoviral vectors, methods for construction thereof and methods for propagating thereof are well known in the art and are described in, for example, U.S. Pat. Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806, 5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191, and 6,113,913, and Thomas Shenk, “Adenoviridae and their Replication”, M. S.
  • adenoviral vectors are well understood in the art and involves the use of standard molecular biological techniques, such as those described in, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
  • the virus according to the invention is a wild type virus, or a mutant or part thereof that is still infectious in host cells according to the invention.
  • the virus is a recombinant virus comprising heterologous information, which may be used in a therapeutic setting for gene therapy purposes, or as an antigen for vaccination purposes.
  • heterologous information is referred to as ‘transgene’.
  • the methods according to the present invention are applicable with a virus, preferably adenovirus, comprising any transgene, and hence the nature of the transgene is in itself not material to the present invention.
  • Transgenes that may be present in a virus according to the invention may for instance be therapeutic genes, such as tumor suppressor genes, including but not limited to p53, p16, APC, DCC, NF-1, WT-1, p21, BRCA1, BRCA2, and the like; enzymes, such as cytosine deaminase, HGPRT, glucocerebrosidase, HSV thymidine kinase or human thymidine kinase, etc; hormones, such as growth hormone, prolactin, erythropoietin, chorionic gonadotropin, thyroid-stimulating hormone, leptin, ACTH, angiotensin, insulin, glucagon, somatostatin, calcitonin, vasopressin, and the like; interleukins and cytokines, such as IL-1,
  • HIV-1 derived gag, env, nef, or modifications thereof including codon optimized versions see for instance WO 02/22080), Ebola, Marburg, Lassa virus; or bacterial antigens, fungal antigens, parasitic (including trypanosomes, tapeworms, roundworms, helminths, malaria, etc) antigens, and the like.
  • the person skilled in the art will choose the gene of interest that is useful in the envisaged therapeutic setting, be it in gene therapy and/or in vaccination, and is not confined to the list above.
  • control regions for the transgene are preferably present in recombinant viral vectors aimed at expression of the transgene, for instance including a promoter and a polyadenylation signal. These are all aspects well known to the person skilled in the art, and need not further be elaborated here. Several control regions are discussed in WO 98/22588, p. 49-55.
  • adenoviruses used in the present invention are further discussed in the examples.
  • the lytic characteristics of adenovirus therefore permits two different modes of virus production.
  • the first mode is harvesting virus prior to cell lysis, employing external factors to lyse the cells.
  • the second mode is harvesting virus supernatant after (almost) complete cell lysis by the produced virus (see e.g. U.S. Pat. No. 6,485,958, describing the harvesting of adenovirus without lysis of the host cells by an external factor).
  • longer incubation times are required in order to achieve complete cell lysis, and hence high yields of virus.
  • the gradual spill of the host cell contents into the medium may be detrimental to the integrity and yield of the obtained viruses.
  • the cells are lysed using at least one detergent.
  • a detergent for lysis has the advantage that it is an easy method, and that it is easily scalable.
  • the cells are lysed by shear using hollow fiber ultrafiltration, such as described in WO 03/084479.
  • Detergents that can be used according to the present invention, and the way they are employed, are generally known to the person skilled in the art. Several examples are for instance discussed in WO 98/22588, p. 29-33.
  • Detergents can include anionic, cationic, zwitterionic, and nonionic detergents.
  • Exemplary detergents include but are not limited to taurocholate, deoxycholate, taurodeoxycholate, cetylpyridium, benzalkonium chloride, ZWITTERGENT-3-14®, CHAPS (3-[3-Cholamidopropyl)dimethylammoniol]-1-propanesulfonate hydrate, Aldrich), Big CHAP, Deoxy Big CHAP, Triton X-100®, Triton X-114®, C12E8, Octyl-B-D-Glucopyranoside, PLURONIC-F68®, TWEEN-20®, TWEEN-80® (CALBIOCHEM® Biochemicals), Thesit®, NP-40®, Brij-58®, octyl glucoside, and the like.
  • the concentration of the detergent may be varied, for instance within the range of about 0.1%-5% (w/w).
  • the detergent is present in the lysis solution at a concentration of about 1% (w/w).
  • use of Triton resulted in less viscous solutions than some other detergents tested (Tween 20, Tween 80, deoxycholate).
  • the detergent used is Triton X-100.
  • the present invention employs nuclease to remove contaminating, i.e. mostly host cell, nucleic acids.
  • nucleases suitable for use in the present invention include Benzonase®, Pulmozyme®, or any other DNase and/or RNase commonly used withing the art.
  • the nuclease is Benzonase®, which rapidly hydrolyzes nucleic acids by hydrolyzing internal phosphodiester bonds between specific nucleotides, thereby reducing the viscosity of the cell lysate.
  • Benzonase® can be commercially obtained from Merck KGaA (code W214950).
  • the concentration in which the nuclease is employed is preferably within the range of 1-100 units/ml.
  • the nuclease is employed before the cells are lysed. It may be added just seconds prior to (or virtually concomitant with) the lysis step, but preferably the nuclease is added to the culture at least one minute before the lysis step.
  • the cell culture with the added nuclease can then be incubated above process temperature, e.g. around 40° C., or at the culturing temperature (e.g. between about 35° C. to about 37° C.), or at room temperature (around 20° C.) or lower (e.g. around 0° C.), wherein in general longer incubation times are required at lower temperature to achieve the same result (see Benzonase® brochure Merck KGaA code W 214950).
  • the incubation can for instance be performed at about 37° C., for about 10 minutes, after which the cells are lysed.
  • the nuclease can and preferably will still actively degrade nucleic acid after the lysis step, and in certain embodiments according to the present invention the incubation of the cells with endonuclease after lysis is prolonged for about 50 minutes (resulting in a total time of the nuclease treatment of about 1 hour, although this time may effectively be still longer, because it is anticipated that the nuclease will still be functional until it is removed in subsequent purification steps). This is considerably shorter than the overnight incubation disclosed in WO 98/22588.
  • the ‘lysis step’ i.e. subjecting the cells containing the virus produced therein to lysis
  • the ‘lysis step’ is meant to be a lysis step employing external factors (see under ‘lysing host cells’ above), such as a detergent.
  • such lysis in the absence of external factors has occurred in less than 50%, preferably less than 40%, more preferably less than 30%, still more preferably less than 20% of the host cells, when nuclease treatment is started, i.e. preferably nuclease is added when the cells have a viability of at least 50%, 60%, 70%, 80%, respectively.
  • nuclease during the later stages of the culture, i.e. preferably when the host cells wherein the virus is propagated still have a viability of at least 5%, more preferably at least 10%, still more preferably at least 20% (i.e. when less than 95%, 90%, 80% of the cells are lysed, respectively).
  • a method for the purification of a virus that is capable of lysing host cells from host cells comprising the steps of: a) culturing host cells comprising a virus capable of lysing said host cells, b) harvesting virus following their release into culture fluid without lysis of the host cells by an external factor, characterized in that a nuclease is added to the culture before 95% of the host cells has been lysed.
  • the nuclease is added to the culture before 90%, preferably 80% of the host cells has been lysed.
  • nuclease corresponding to the optimal percentage of cells that has been lysed
  • the nuclease will depend on the amount of nuclease added and the decrease in specific activity of the nuclease during incubation, and can be empirically found by the person skilled in the art, now the advantage of the addition of nuclease to the culture per se has been disclosed by the present inventors.
  • the obtained lysate according to this aspect of the invention can be further purified employing methods and steps as discussed herein, such as filtration and chromatography.
  • the host cell lysate comprising the virus is clarified. Clarification may be done by a filtration step, removing cell debris and other impurities.
  • Suitable filters may utilize cellulose filters, regenerated cellulose fibers, cellulose fibers combined with inorganic filter aids (e.g. diatomaceous earth, perlite, fumed silica), cellulose filters combined with inorganic filter aids and organic resins, or any combination thereof, and polymeric filters (examples include but are not limited to nylon, polypropylene, polyethersulfone) to achieve effective removal and acceptable recoveries. In general, a multiple stage process is preferable but not required.
  • An exemplary two or three-stage process would consist of a course filter(s) to remove large precipitate and cell debris followed by polishing second stage filter(s) with nominal pore sizes greater than 0.2 micron but less than 1 micron.
  • the optimal combination may be a function of the precipitate size distribution as well as other variables.
  • single stage operations employing a relatively tight filter or centrifugation may also be used for clarification. More generally, any clarification approach including dead-end filtration, microfiltration, centrifugation, or body feed of filter aids (e.g. diatomaceous earth) in combination with dead-end or depth filtration, which provides a filtrate of suitable clarity to not foul the membrane and/or resins in the subsequent steps, will be acceptable to use in the clarification step of the present invention.
  • filter aids e.g. diatomaceous earth
  • depth filtration and membrane filtration is used.
  • Commercially available products useful in this regard are for instance mentioned in WO 03/097797, p. 20-21.
  • Membranes that can be used may be composed of different materials, may differ in pore size, and may be used in combinations. They can be commercially obtained from several vendors.
  • the virus suspension is subjected to ultrafiltration/diafiltration at least once during the process, e.g. for concentrating the virus and/or buffer exchange, and/or for concentration and diafiltration of the clarified harvest.
  • the process used to concentrate the virus according to the method of the present invention can include any filtration process (e.g., ultrafiltration (UF)) where the concentration of virus is increased by forcing diluent to be passed through a filter in such a manner that the diluent is removed from the virus preparation whereas the virus is unable to pass through the filter and thereby remains, in concentrated form, in the virus preparation.
  • UF is described in detail in, e.g., Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A.
  • TFF Tangential Flow Filtration
  • the particular ultrafiltration membrane selected will have a pore size sufficiently small to retain virus but large enough to effectively clear impurities.
  • adenovirus nominal molecular weight cutoffs between 100 and 1000 kDa may be appropriate, for instance membranes with 300 kDa or 500 kDa NMWC.
  • the membrane composition may be, but is not limited to, regenerated cellulose, polyethersulfone, polysulfone, or derivatives thereof.
  • the membranes can be flat sheets or hollow fibers.
  • UF is generally referred to filtration using filters with a pore size of smaller than 0.1 ⁇ m. Products are generally retained, while volume is reduced through permeation.
  • hollow fiber modules for ultrafiltration and microfiltration were developed by Amicon and Ramicon in the early 1970s (Cheryan, M. Ultrafiltration Handbook), even though now there are multiple vendors including Spectrum and A/G Technology.
  • the hollow fiber modules consist of an array of self-supporting fibers with a dense skin layer that give the membranes its permselectivity. Fiber diameters range from 0.5 mm-3 mm.
  • An advantage of hollow fiber modules is the availability of filters from small membrane areas (ca. 16 cm 2 ) to very large membrane areas (ca. 28 m 2 ) allowing linear and simple scale-up.
  • hollow fibers are used for TFF. These are reported to give less shear and a better viral particle/infectious unit (VP/IU) ratio than flat screen membranes.
  • hollow fibers of 0.05 ⁇ m are used according to the invention.
  • Diafiltration (DF), or buffer exchange, using ultrafilters is an ideal way for removal and exchange of salts, sugars, non-aqueous solvents separation of free from bound species, removal of material of low molecular weight, or rapid change of ionic and/or pH environments.
  • Microsolutes are removed most efficiently by adding solvent to the solution being ultrafiltered at a rate equal to the UF rate. This washes microspecies from the solution at a constant volume, purifying the retained virus.
  • the present invention utilizes a DF step to exchange the buffer of the lysate prior to further chromatography or other purification steps.
  • DF by TFF is performed for buffer exchange, wherein the addition of buffer equals the removal of permeate.
  • UF/DF can be used to concentrate and/or buffer exchange the virus suspensions according to the present invention in different stadia of the purification process, e.g. the lysate and/or further purified virus suspensions such as those that have undergone chromatography.
  • the lysate is concentrated by UF/DF 5-fold, and the resulting concentrated virus suspension is buffer exchanged with 6 diafiltration volumes (DFV) of a buffer comprising 1 M NaCl, using a constant volume diafiltration method. It was found that this high salt concentration significantly improves the quality of the resulting virus, as many undesired proteins were lost during this step (see example 2). It is therefore a preferred embodiment according to the invention that the clarified lysate is exchanged against a solution comprising 0.8-2.0 M NaCl, e.g. around 1 M NaCl, or another salt providing an equivalent ionic strength. It will be clear to the person skilled in the art that both the anion and the cation of the salt can be changed.
  • DFV diafiltration volumes
  • the virus suspension Before the virus suspension is subjected to anion exchange chromatography, it may be buffer exchanged with a buffer comprising 0.4 M NaCl, or another salt providing an equivalent ionic strength. In one embodiment, this is accomplished by constant volume diafiltration, using 4 DFVs of the desired buffer.
  • the virus suspension that has been obtained by the method according to the present invention preferably after clarification of the lysate, is further purified, e.g. by methods generally known to the person skilled in the art. This may for instance be achieved by density gradient centrifugation, as for instance discussed in WO 98/22588, p. 59-61.
  • further purification employs at least one chromatography step, as for instance discussed in WO 98/22588, p. 61-70.
  • chromatography steps are included in the process. The person skilled in the art will be aware of these processes, and can vary the exact way of employing chromatographic steps to optimize the process of the invention.
  • a reversed-phase adsorption step might also be used, as for instance described in WO 03/097797, p. 26.
  • the virus may be sufficiently pure.
  • a size exclusion chromatography step is further performed to increase the robustness of the process. This step may be prior to or after the anion exchange chromatography step.
  • other purification steps may also be suitably combined with an anion exchange chromatography step.
  • anion exchange chromatography for adenovirus purification has been extensively described, and this aspect is therefore well within the reach of the person skilled in the art.
  • Many different chromatography matrices have been employed for purification of adenovirus and are suitable, and the person skilled in the art can easily find the optimal anion exchange material for purifying the virus, for instance guided by the following art.
  • U.S. Pat. No. 5,837,520 (see also Huyghe et al., 1995, Human Gene Therapy 6: 1403-1416) describes a method of purifying adenovirus wherein the host cell lysate is treated with a nuclease, followed by anion exchange and metal ion affinity chromatography.
  • U.S. Pat. No. 6,485,958 describes the use of strong anion exchange chromatography for purification of recombinant adenovirus.
  • Anion exchange chromatography has been employed with fluidized bed columns for the purification of adenovirus particles, see WO 00/50573.
  • anion exchange membrane chromatography products such as those produced by Pall (e.g. MustangTM series) and Sartorius (e.g. Sartobind series) are suitable.
  • Pall e.g. MustangTM series
  • Sartorius e.g. Sartobind series
  • filters for use of these filters and their advantages in adenovirus purification see for instance WO 03/078592.
  • employment of such filters also falls within the scope of the term ‘anion exchange chromatography’ as used herein.
  • U.S. Pat. No. 6,537,793 describes the purification of adenoviral particles from host cells using ion-exchange chromatography, in particular teaching a preference for Q Sepharose XL types of chromatographic support for this purpose.
  • an adenovirus is further purified using a Q Sepharose XL column.
  • the process may further suitably employ a size exclusion chromatography step.
  • U.S. Pat. No. 6,261,823 describes a method for purifying adenovirus wherein the adenovirus preparation is subjected to anion exchange chromatography followed by size exclusion chromatography.
  • size exclusion step a group separation of viral particles from impurities of low molecular weight is achieved.
  • about 15-30%, preferably about 20% of the column volume is loaded on the size exclusion column (group separation mode of size exclusion chromatography).
  • an adenovirus suspension that has been prepared according to the method of the invention is further purified using an anion exchange chromatography step and a size exclusion chromatography step.
  • WO 03/078592 describes the use of high throughput anion exchange filters (i.e. a charged filter that contains anion exchange groups) for adenovirus (Ad5) purification.
  • anion exchange filters i.e. a charged filter that contains anion exchange groups
  • Ad5 adenovirus
  • the following advantages are described for such charged filters compared to anion exchange columns: (i) faster flow rates, (ii) higher binding capacity, (iii) higher virus recovery, (iv) no packing or cleaning validation required for clinical use, and (v) no lifetime issues or storage issues when disposable filter cartridges are used.
  • anion exchange filters is an embodiment of the present invention, and is an embodiment considered included within the scope of ‘anion exchange chromatography’ in the present invention.
  • adenovirus serotype 35 Ad35
  • an anion exchange filter certain adenovirus proteins that were not incorporated into adenovirus particles are separated from the adenovirus particles by use of an anion exchange filter, not by an anion exchange column.
  • Such free adenovirus proteins were not previously found in preparations of recombinant adenovirus particles and would normally go undetected, but now can be removed using the step of subjecting a recombinant adenovirus preparation comprising free adenovirus proteins to a charged filter that contains anion exchange groups.
  • WO 03/078592 does not disclose the employment of anion exchange filters for the purification of Ad35, or other adenovirus particles of subgroup B.
  • the invention therefore provides a method for removing free adenovirus proteins from a recombinant adenovirus preparation, comprising the step of: subjecting a recombinant adenovirus preparation comprising free adenovirus proteins to a charged filter that contains anion exchange groups.
  • WO 2004/001032 gives rise to the hitherto undetected free adenovirus proteins that appear not incorporated into adenovirus particles.
  • this particular method according to the invention may be particularly beneficial for purification of recombinant adenovirus of subgroup B, such as Ad35, Ad11, etc.
  • the method improves purification of the more stable Ad5 or Ad2 based adenovirus.
  • the invention provides the use of an anion exchange filter for the removal of free (i.e. not incorporated into viral particles) adenovirus proteins from a recombinant adenovirus preparation.
  • said recombinant adenovirus preparation comprises recombinant subgroup B adenovirus, such as recombinant Ad35.
  • the invention also provides a method for purification of recombinant subgroup B adenovirus particles, such as Ad35 particles, the method comprising a step of subjecting the recombinant subgroup B, such as Ad35, particles to an anion exchange filter purification step.
  • Anion exchange filters suitable for use in these methods of the invention are known in the art and commercially available (see WO 03/078592, paragraphs [40]-[41]), e.g. from Pall (e.g. MustangTM series) and from Sartorius (e.g. Sartobind series).
  • buffers used during purification of the virus according to the present invention can be used during purification of the virus according to the present invention.
  • buffers used for UF/DF and anion exchange chromatography in general contained 0.4-1.0 M NaCl/50 mM TRIS pH 7.5, wherein the concentrations of NaCl were dependent on the process step.
  • the buffers used after clarification are free of detergent, magnesium chloride and sucrose. The absence of these additives distinguishes these buffers from those used in known established protocols. Nevertheless, when the methods according to the present invention are employed, a purified and substantially non-aggregated adenovirus is obtained.
  • An advantage of the use of buffers without these additives is that they are easier to prepare, cheaper, and that there is no need to test for removal of the additives.
  • the adenovirus is buffer exchanged during group separation to—and finally stored in—the buffer that is also used for the Adenovirus World Standard (Hoganson et al, Development of a stable adenoviral vector formulation, Bioprocessing March 2002, p. 43-48): 20 mM Tris pH 8, 25 mM NaCl, 2.5% glycerol.
  • the transgene itself is generally regarded as irrelevant for the purification process.
  • the transgene may in specific cases by its expression in the host cell or in the virus influence properties of the virus or may have an influence on the process of purifying the virus.
  • the transgene is the Ebolavirus nucleoprotein.
  • Purifying an adenoviral vector containing the Ebolavirus nucleoprotein gene with the standard purification procedure results in co-purifying the expressed Ebolavirus nucleoprotein.
  • No co-purification of several other transgene expressed proteins was observed (for instance not with Ebola glycoprotein dTM (Sudan), Ebola Glycoprotein dTM (Zaire), measles hemagglutinin protein (MV-H)). This suggests a specific interaction between the Ebola nucleoprotein and Adenovirus, which seems to depend on the characteristics of the Ebola nucleoprotein.
  • nucleic acid binding proteins are expected to have similar characteristics and are expected to have an interaction with Adenovirus resulting in co-purification as well.
  • nucleic acid binding proteins such as nucleoproteins, such as Ebolavirus nucleoprotein
  • Buffer exchange may suitably be performed by TFF.
  • the buffer exchange step preferably takes place after the elution of the virus from anion exchange chromatography, and preferably before a further purification step.
  • a further purification step may for instance be a size exclusion step in group separation mode.
  • the further purification step may comprise a filtration of the virus suspension comprising the high salt concentration through a hydrophilic filter, such as a Durapore PVDF filter (e.g. Millipac from Millipore) or a Sartopore 2 filter.
  • a hydrophilic filter such as a Durapore PVDF filter (e.g. Millipac from Millipore) or a Sartopore 2 filter.
  • the filter preferably has a pore size of 1.2 ⁇ m, more preferably smaller, e.g. 1.0 ⁇ m, still more preferably smaller, e.g. 0.8 ⁇ m, 0.45 ⁇ m or 0.22 ⁇ m.
  • the nucleoprotein (NP) of Ebolavirus was found to be separated from a recombinant adenovirus under these conditions by being retained by the filter, while NP—having a molecular weight of about 100 kD—was expected to pass through the filter pores together with the adenovirus.
  • Use of these filters provides a fast solution for separating the nucleoprotein from the virus, as no prolonged incubation in high salt is required for this procedure, while it allowed complete removal of the nucleoprotein from the virus ( FIG. 9 ).
  • a size exclusion chromatography step may still be employed after such a filtration step, to remove other minor contaminants and/or for buffer exchange.
  • the invention further provides a method for the production of a virus comprising a nucleic acid sequence coding for a nucleic acid binding protein, comprising the steps of: a) culturing host cells that have been infected with virus, b) subjecting said culture of host cells and said virus therein produced to lysis of the host cells to provide a lysate comprising said virus, c) subjecting the virus to anion exchange chromatography, characterized in that after anion exchange chromatography the virus containing mixture is buffer exchanged with a solution comprising at least 1 M NaCl, or another salt providing an equivalent ionic strength.
  • the virus is further purified using at least one step comprising either filtration though a hydrophilic filter, and/or using at least one step comprising size exclusion chromatography.
  • a solution comprising at least 1 M NaCl or another salt providing equivalent ionic strength is referred to as a ‘high salt’ solution.
  • both the anion and the cation can be varied as is known to the person skilled in the art, as long as sufficient ionic strength is provided without precipitation or other undesired side-effects such as inactivation of the virus, as the method likely depends on the breaking of ionic interactions between the DNA binding protein and the purified virus.
  • NaCl may be in part or wholly substituted for other salts, such as for instance KCl, sodium phosphate, CsCl, LiCl, (NH 4 ) 2 SO 4 , NH 4 Cl, NaBr, NaI, KBr, KI, KNO 3 , NaHCO 3 , KHSO 4 , etc.
  • a 5 ⁇ dilution of the buffer used in the example of the invention (comprising 5 M NaCl) had a conductivity of 78-79 mS/cm. Buffers containing other salts, and having a similar or higher conductivity can for instance now easily be tested for suitability in removing DNA binding proteins from partially purified virus, according to the invention.
  • the solution comprises at least 1.5 M NaCl, or another salt providing an equivalent ionic strength. More preferably the solution comprises at least 2 M NaCl, or another salt providing an equivalent ionic strength. More preferably, the solution comprises at least 3 M NaCl, or another salt providing an equivalent ionic strength. More preferably, the solution comprises at least 4 M NaCl, or another salt providing an equivalent ionic strength. Even more preferably, the solution comprises around 5 M NaCl, or another salt providing an equivalent ionic strength.
  • the high salt solution comprising the virus may be incubated for a certain time, preferably at least one hour, more preferably at least two hours.
  • the examples show an increased purification of the DNA binding protein from the virus when incubation is longer, at least up to overnight. Further, a higher ionic strength appears to improve the purification.
  • the virus is a recombinant adenovirus.
  • said nucleic acid binding protein is a nucleoprotein of a virus.
  • the nucleic acid binding protein is the Ebolavirus nucleoprotein.
  • the buffer exchange step takes place after anion exchange chromatography and before a filtration and/or size exclusion chromatography step.
  • nuclease treatment of the lysate whereby preferably the nuclease is added to the cell culture before lysis is complete, in accordance with other aspects of the invention.
  • detergent may be added to purify the virus from contaminating DNA binding protein.
  • the inventors have shown that addition of 1% Tween 20 also significantly reduced the contaminating nucleoprotein from recombinant adenovirus expressing Ebola nucleoprotein.
  • other detergents can suitably be tested, and the concentration may be varied, e.g. between bout 0.2% and 5%, to find optimal conditions for removal of DNA binding proteins from recombinant virus preparations according to the invention.
  • at least 1% detergent is added.
  • the first experiments of the inventors however have indicated a higher reproducibility of high salt incubation for this purpose, and therefore this is preferred.
  • the invention provides a batch of recombinant adenovirus comprising a transgene chosen from the group consisting of: an Ebolavirus nucleoprotein, an Ebolavirus glycoprotein, a Plasmodium falciparum circumsporozoite gene, measles virus hemagglutinin, said batch characterized in that it contains less than 0.1 ng host cell DNA per 1E11 viral particles.
  • transgenes optionally may contain deletions, additions, and/or mutations compared to the wild-type sequences found in nature, including all isolates or subtypes, without deviating from the scope of this aspect of the invention.
  • the batch is characterized in that it contains less than 0.08 ng, more preferably less than 0.06 ng, still more preferably less than 0.04 ng host cell DNA per 10 11 viral particles.
  • the samples were then further purified. Clarification was performed by depth filtration (0.5 ⁇ m Clarigard filter, Millipore) followed by further clarification over a 0.8/0.45 ⁇ m Sartopore 2 (Sartorius) filter.
  • the clarified material was concentrated 5 times over a 0.05 ⁇ m hollow fiber (Spectrum), followed by diafiltration with subsequently 6 volumes of 1.0 M NaCl/50 mM TRIS pH 7.5 and 4 volumes of 0.4 M NaCl/50 mM Tris pH 7.5.
  • the diafiltered retentate was loaded onto a Sepharose Q-XL (Amersham) column and the virus fraction was eluted with 0.55 M NaCl/50 mM TRIS pH 7.5.
  • This fraction was further purified and buffer exchanged with a Sepharose 4 FF (Amersham) column.
  • the generated purified bulk was concentrated to the desired concentration with a hollow fiber (0.05 ⁇ m poresize, Spectrum), 0.22 ⁇ m filtered and aliquotted. Purified bulk samples were analysed for residual host cell DNA by Q-PCR.
  • T/B treatment resulted in a reduction of DNA that after further downstream processing could just meet the required specification in the filled and finished material.
  • Regulatory requirements for residual host cell DNA for life virus formulations are ⁇ 10 ng per dose (assumed that a dose contains 1E11 viral particles).
  • Benzonase® Upon addition of Benzonase® the DNA released from cells lysed due to virus production can already be digested. As soon as DNA is released from cells lysed by Triton, the Benzonase® is present to immediately digest the DNA, thereby preventing the formation of large DNA aggregates. Digestion of non-aggregated DNA is probably more effective than digestion of major DNA aggregates.
  • histon-DNA complexes are formed when the DNA is digested immediately upon release and these larger particles are retained by the clarification filters. Retainment of histon-DNA complexes during clarification might also have contributed to reduction of residual host cell DNA.
  • anion exchange resins e.g. QAE 550C and Super Q 650M (purchased from Tosoh), Q Sepharose HP, ANX Sepharose 4FF, DEAE Sepharose, Q Sepharose XL, Q Sepharose Big Bead and Q Sepharose FF (purchased from Amersham). Although all these resins were suitable for the purification of the recombinant adenoviruses, we found that Q Sepharose XL was best suitable for our purpose based on separation of virus from host cell proteins and host cell DNA, and flow characteristics.
  • Sepharose 4 FF size exclusion resins
  • Amersham Several size exclusion resins were tested e.g. Sephacryl S300, Sephacryl S500 Sepharose 4FF and Sepharose 6 FF (all purchased from Amersham). Although all these resins were suitable for the purification of the recombinant adenoviruses, we found Sepharose 4 FF best suitable for our purpose based on ability to separate virus from host cell proteins and DNA.
  • FIG. 4 a preferred process according to the invention is shown schematically in FIG. 4 .
  • PER.C6 cells were grown in a 10 L bioreactor and infected with Ad5.Adapt.MV-H (with measles virus hemagglutinin as transgene, described in WO 2004/037294). 2.5 days after infection the cells were lysed with 1% Triton® X-100, after 30 minutes Benzonase® (50 units/ml) and MgCl 2 were added and incubated for another 30 minutes. The harvest was clarified over a 0.5 ⁇ m Clarigard filter and subsequently by a Millistak DE 30/60 filter (Millipore).
  • the clarified harvest was diluted with an equal volume of 0.6 M NaCl/50 mM HEPES pH 7.5, resulting in a final concentration of 0.3 M NaCl.
  • the diluted clarified harvest was concentrated 4 times with a 500 kD flatscreen cassette (Biomax 500, Pellicon 2 module Millipore) and subsequently diafiltered with 2 diafiltration volumes (DFV) of 0.3 M NaCl/50 mM HEPES pH 7.5; 2 DFV of 0.6 M NaCl/50 mM HEPES pH 7.5; 2 DFV of 1.0 M NaCl/50 mM HEPES pH 7.5; and 3 DFV of 0.3 M NaCl/50 mM HEPES pH 7.5.
  • DFV diafiltration volumes
  • the conductivity of the generated permeates was measured and the samples were analysed by SDS-PAGE ( FIG. 3 ).
  • the data showed that histones (M w around 10-20 kD on gels, identity confirmed by mass spectrometry) are passing the membrane pores when the salt concentration of the permeate (and therefore of the retentate) is in the range of 0.55 and 0.85 M NaCl, or higher.
  • the cells were lysed first and subsequently treated with nuclease (T/B), it is anticipated that the diafiltration against buffer with high salt strength (higher than 0.55 M NaCl, for instance 1 M NaCl) is also beneficial in the process according to the invention wherein the nuclease is added to the cells before they are lysed (B/T, see example 1), even though in the B/T process there is already less histon contamination (see FIG. 2 ).
  • buffer with high salt strength higher than 0.55 M NaCl, for instance 1 M NaCl
  • the clarified lysate is exchanged against a solution comprising 0.8-2.0 M NaCl, preferably about 1 M NaCl, or another salt providing an equivalent ionic strength (see example 1 and FIG. 4 ).
  • Example 5 Generation of recombinant adenovirus with Ebola nucleoprotein as a transgene is described in example 5. In this example, the purification of such virus is described.
  • Ad5dE3x.Adapt.Ebo.NP was purified with the described protocol (see example 1, FIG. 4 ). This method resulted in co-purification of the expressed Ebola nucleoprotein (NP) transgene with the virus. Filled and finished product was diluted 1:2 with a buffer containing either 5 M NaCl (final conc 2.5 M), or 2% Tween 20 (final conc 1%) and incubated for 1 hr at room temperature before loading onto a Sepharose 4 FF column. The void and retarded fractions were analysed by SDS-PAGE. The results ( FIG. 5 ) show that the void fraction contained Adenovirus type 5 without contaminating intact NP. Thus far, the results with the high salt appeared reproducible, whereas those with the detergent were not, and hence high salt is preferred. Optimal conditions for detergent however can be tested by varying the detergent used and its concentration.
  • the Ad5dE3x.Adapt.Ebo.NP vector can be purified from the Ebola nucleoprotein by incubation in a buffer containing either 2.5 M NaCl or 1% Tween, preferably 2.5 M NaCl, followed by separation on 4 FF sepharose.
  • Ad5dE3x.Adapt.Ebo.NP was purified with the described protocol (see example 1, FIG. 4 ). Filled and finished product was dialysed with a 10 kD membrane against a 50 mM TRIS buffer pH 7.5 containing 1, 2, 3 or 5 M NaCl. The Ad5.Ebo.NP was incubated in these buffers for 2 hours or overnight before loading onto a Sepharose 4 FF column. The void and retarded fractions were analysed by SDS-PAGE. The results show that the void fraction contained Adenovirus type 5 with significantly less NP. As shown in Table 2, the amount of removal of NP relates to the salt concentration and incubation time.
  • the Ad5dE3x.Adapt.Ebo.NP vector can be purified from the Ebola nucleoprotein by incubation in a buffer containing either 2-5 M NaCl followed by separation on 4 FF sepharose. A longer incubation time and a higher salt concentration before separation on the 4 FF column results in higher purity of the Ad5.Ebo.NP vector (more removal of nucleoprotein).
  • Concentrations of 1 M and 1.5 M NaCl are tested with longer incubation times (e.g. 2 days, 1 week) according to this same method to find out whether a longer incubation time may suffice for purification at these salt strengths.
  • PERC.6 cells were grown in a 10 L bioreactor and infected with Ad5.dE3x.Adapt.Ebo.NP. 2.5 days after infection Benzonase® (50 units/ml) and MgCl 2 were added to the cell culture, after 10 minutes the cells were lysed with 1% Triton® X-100, and incubated for another 50 minutes. The harvest was clarified over a 0.5 ⁇ m Clarigard filter and subsequently by a Sartopore 2 filter (0.8/0.45 ⁇ m, Sartorius).
  • the clarified harvest was split in two portions. One portion was concentrated 5 times and diafiltered against a buffer containing 5 M NaCl/50 mM Tris pH 7.5 by use of a 0.5 ⁇ m hollow fiber (Spectrum). This resulted in an increase of trans membrane pressure (TMP) and a reduction in permeate flux while the visual appearance of the retentate turned to white and less transparent, indicating precipitation of proteins.
  • TMP trans membrane pressure
  • the second portion of clarified harvest was concentrated 5 times and diafiltrated with 6 DFV of 1.0M NaCl/50 mM TRIS pH 7.5 followed by 4 DFV of 0.4 M NaCl/50 mM TRIS pH 7.5 by use of a 0.5 ⁇ m hollow fiber (Spectrum).
  • the final retentate was purified over a Sepharose Q-XL column (Amersham).
  • the Q-XL eluate was also divided into two portions. One portion was further purified and buffer exchanged to 25 mM NaCl/20 mM TRIS/2.5% glycerol (formulation buffer) over a size exclusion column (Sepharose 4 FF) in group separation mode (loading of 20% of column volume); this is product A in FIG. 6 . The other portion was diafiltered against 6 DFV of 5 M NaCl/50 mM TRIS pH 7.5 by use of a 0.05 ⁇ m hollow fiber (Spectrum): this is further called the high salt virus fraction.
  • the poresizes of the hollow fiber (0.05 ⁇ m, about 800 kD) are large enough to allow passage of a 100 kD nucleoprotein, no nucleoprotein could be detected in the permeate and no reduction of the amount of nucleoprotein was seen in the retentate.
  • the adaptation of one or more TFF parameters e.g. increase in shear
  • the high salt virus fraction was again split into two portions: one portion was directly purified and buffer exchanged to formulation buffer over a size exclusion (group separation) column (product B in FIG. 6 ), while the second fraction was stored overnight at room temperature before further purifying and buffer exchanging over a size exclusion (group separation) column (product C in FIG. 6 ).
  • the three purified bulk lots were analysed to determine purity, infectivity, yield, aggregation and transgene expression.
  • FIG. 7 SDS-PAGE and Western analysis is shown in FIG. 7 , and shows that the intact nucleoprotein, as well as NP degradation products (confirmed by mass spectrometry to be NP degradation products), are increasingly removed from product A, B and C respectively.
  • FIG. 8 Reverse phase analysis (RP-HPLC)( FIG. 8 ) shows that the amount of intact nucleoprotein, as well as NP degradation product (eluting at 39 minutes), was reduced by introducing the high salt diafiltration step from about 50% (product A) to ⁇ 5% (product B) and after overnight storage in 5 M NaCl at room temp even to below the detection limit of 1% (product C). Using both analysis methods, no effect on viral proteins was observed.
  • a virus suspension that was buffer exchanged with 5M NaCl was directly filtered using a 0.45 ⁇ m hydrophilic filter (Millipac 20).
  • This experiment is repeated with filters of different pore sizes (e.g. 1.2, 1.0, 0.8, 0.22 ⁇ m) to determine the range of possible pore sizes.
  • a 0.8/0.45 ⁇ m Sartopore-2 combination is also tested.
  • This filtration step may suitably be combined with a subsequent size exclusion chromatography step, and may require shorter incubation times of the virus in the high salt solution, resulting in a possible savings in process time.
  • nucleic acid binding proteins such as nucleoproteins, e.g. nucleoprotein of Ebolavirus
  • nucleoproteins e.g. nucleoprotein of Ebolavirus
  • PER.C6 cells were grown in a 10 L bioreactor and infected in separate experiments with different recombinant adenoviruses. 2.5 days after infection the cells were lysed with 1% Triton® X-100, after 30 minutes Benzonase® (50 units/ml) and MgCl 2 were added and incubated for another 30 minutes. The harvest was used for clarification experiments.
  • Depth filters e.g. Clarigard and Polygard had high recovery (>90%) and good removal of cell debris (microscopic analysis), and were found suitable as an initial clarification filter. However the filtrate still looked opalescent.
  • Millistak DE 30/60 and CE50 were found to be less suitable for filtering T/B harvest due to loss of virus (20-45%). In later fractions the yield increased but the retention of opalescence decreased, indicating that the filter capacity was reached.
  • the clarified harvest was concentrated and diafiltrated with flatscreen or hollow fiber modules.
  • filters were tested to filter the final retentate, preferably with a 0.45 ⁇ m poresize, in order to make the final retentate suitable for chromatography, e.g.: Millipack 20, Lifegard 1.0 ⁇ m, Polygard 0.6 ⁇ m, Intercept Q, Milligard 1.2/0.5 ⁇ m.
  • the Sartopore 2 filter was the only filter among those tested that had a good retention of the opalescence, a high capacity as well as a high virus yield (>95%).
  • a combination of 0.8 ⁇ m and 0.45 ⁇ m filters preferably a Sartopore 2 filter, is used.
  • viruses can for instance be generated by homologous recombination in the packaging cells of a left-end part (sometimes referred to as ‘adapter-plasmid’, useful for easy cloning of the transgene) and a right-end part of the genome according to methods known from the art, such as for instance described in EP 0955373, WO 03/104467 and WO 2004/001032.
  • the viruses can be propagated in packaging cells known from the art, such as for instance 293 cells, PER.C6TM cells (exemplified by cells deposited at the ECACC under no. 96022940, see U.S. Pat. No.
  • the gene encoding the Ebola subtype Zaire nucleoprotein was amplified by polymerase chain reaction using primers; forward 6401 5′ GCA CCG GTG CCG CCA TGG ATT CTC GTC CTC A 3′ (SEQ. ID. NO. 1) and reverse 6401 5′ GCG CTA GCT CAC TGA TGA TGT TGC AG 3, (SEQ. ID. NO. 2) in order to introduce restriction endonuclease recognition sites and a consensus sequence for optimal translation initiation (Kozak M, 1987, At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol.
  • the PCR product of the correct size was digested with PinA I (Isoschizomer of Age I) and ligated into the pAdAptTM vector digested with PinA I and Hpa I. After ligating the fragment for 2 hours at room temperature, 50% of the mixture was transformed to E. coli DH5 ⁇ T1R cells by heatshock transformation and plated onto LB agar plates supplemented with 50 ug/ml ampicillin. Twenty colonies were picked and grown overnight at 37° C. in LB supplemented with ampicillin. Miniprep DNA was extracted using the Qiagen miniprep Spin kit as described by the manufacturer. After restriction enzyme analysis with Hind III and Xba I a correct clones was selected and further checked by DNA sequence analysis.
  • PinA I Isoschizomer of Age I
  • the gene encoding the Ebola subtype Zaire full-length glycoprotein was amplified by PCR using primers Forward 6001 (5′ CCC AAG CTT GCC GCC ATG GGC GTT ACA GG 3′) (SEQ. ID. NO. 3) and Reverse 6001 (5′ GGC TCT AGA TTA CTA AAA GAC AAA TTT GC 3′) (SEQ. ID. NO. 4).
  • PCR reactions were performed in a Biometra T1 or T3 thermal cycler using 10 uM of each primer, 100 ng and 25 ng DNA of VRC6001 (see-WO 03/028632), 1.5 units Pwo DNA polymerase, 5 ul 10 ⁇ PCR buffer, 0.5 ul 20 mM dNTPs using the following conditions: 1 cycle 5′ 94° C., 1′ 55° C., 4′ 72° C., 5 cycles 1′ 94° C., 1′ 55° C., 4′ 72° C., 20 cycles 1′ 94° C., 1′ 64° C., 4′ 72° C., 1 cycle 1′ 94° C., 1′ 64° C., 10′ 72° C.
  • the PCR product of the correct size was digested with Hind III and Xba I and ligated into the likewise digested pAdAptTM vector. After ligating the fragment for 2 hours at room temperature, 50% of the mixture was transformed to E. coli DH5 ⁇ T1R cells by heatshock transformation and plated onto LB agar plates supplemented with 50 ug/ml ampicillin. Colonies were picked and grown overnight at 37° C. in LB supplemented with ampicillin. Miniprep DNA was extracted using the Qiagen miniprep Spin kit as described by the manufacturer. After restriction enzyme analysis with Hind III and Xba I correct clones were selected and further checked by DNA sequence analysis.
  • codon optimized sequences encoding one of the Ebola subtypes Zaire and Sudan/Gulu glycoprotein with a deletion of the C-terminal 29 amino acids long transmembrane domain (GPdTM(Z), and GPdTM(S), respectively, see also WO 03/028632), were cloned into pAdapt.
  • the pAdapt plasmids with the different inserts were used to form recombinant adenoviruses by homologous recombination with plasmids comprising the remainder of the adenovirus type 5 genome (plasmid pWE/Ad.AflII-rITRsp ⁇ E3, which is pWE/Ad.AflII-rITRsp (see EP 0955373) with a deletion of 1878 bp in the E3 region (XbaI region) was used for the right end of the adenovirus genome), according to well known methods such as for instance described in EP 0955373, resulting in viruses named Ad5dE3x.Adapt.Ebo.NP, Ad5dE3x.Adapt.Ebo.GP(Z), Ad5d
  • transgenes can similarly be cloned in adenovirus vectors of different serotypes, such as Ad35, to generate recombinant adenovirus derived from those serotypes (see e.g. WO 00/70071).
  • CS circumsporozoite
  • Plasmodium falciparum A codon optimized circumsporozoite (CS) gene of Plasmodium falciparum was synthesized and cloned into pCR-script (Stratagene), giving clone 02-659, as described in WO 2004/055187.
  • the CS gene was cloned into pAdapt and pAdapt535 (see WO 2004/001032) for generation of respectively recombinant Ad5 and recombinant Ad35 vectors.
  • Clone 02-659 and both pAdapt vectors were digested with Hind III and BamH I and joined by ligation. After ligating the fragment for 2 hours at room temperature, 50% of the mixture was transformed to E.
  • coli DH5 ⁇ T1R cells by heatshock transformation and plated onto LB agar plates supplemented with 50 ⁇ g/ml ampicillin. Colonies were picked and grown overnight at 37° C. in LB supplemented with ampicillin. Miniprep DNA was extracted using the Qiagen miniprep Spin kit. After restriction enzyme analysis with Hind III and Xba I correct clones were selected and further checked by DNA sequence analysis.
  • Recombinant adenovirus serotype 5 with the P. falciparum CS gene was generated as follows (see for instance EP 0955373; also described in WO 2004/055187).
  • pAdapt.CS.Pfalc was digested by PacI restriction enzyme to release the left-end portion of the Ad genome.
  • Plasmid pWE/Ad.AflII-rITRsp ⁇ E3 containing the right-end part of the Ad5 genome has a deletion of 1878 bp in the E3 region (XbaI deletion), and was also digested with PacI.
  • the digested constructs were co-transfected into PER.C6 cells, such as deposited at the ECACC under number 96022940. Upon homologous recombination of the overlapping sequences, recombinant virus named Ad5 ⁇ E3.CS.Pfalc was formed.
  • Recombinant adenovirus serotype 35 with the P. falciparum CS gene was generated similarly, but now PacI-digested pAdapt535.CS.Pfalc was used for the left-end of the virus genome, and NotI-digested pWE.Ad35.pIX-rITR ⁇ E3 (see WO 2004/001032) was used for the right-end of the virus genome, and both were transfected into PER-E1B55K producer cells (having E1B-55K sequences derived from Ad35; cells have been described in U.S. Pat. No. 6,492,169).
  • Ad35 ⁇ E3.CS.Pfalc recombinant virus named Ad35 ⁇ E3.CS.Pfalc was formed.
  • E4-orf6 protein in the backbone of the Ad35 virus into E4-orf6 of Ad5, to render it possible to propagate such viruses on packaging cells that express the E1B protein of Ad5, such as PER.C6 or 293 cells (see WO 03/104467).
  • Ad5 ⁇ E3.CS.Pfalc and Ad35 ⁇ E3.CS.Pfalc are purified according to the methods of the present invention.
  • Ad35 vector with the CS gene based on pAdapt535.CS.Pfalc with an Ad35 backbone was constructed, having a deletion in E3 and further comprising E4-orf6 of Ad5: this vector is further referred to as Ad35.CS.
  • the filled and finished (F&F) products were analysed for purity by reverse phase and SDS-PAGE and found to be purified near homogeneity (except for the presence of the Ebola nucleoprotein in the preparations of the vectors having Ebola nucleoprotein as a transgene).
  • the amount of residual host cell DNA was measured by Q-PCR and was below 100 pg DNA/1E11 VP (as shown in Table 1)
  • Aggregation was measured by optical density measurements at 320 and 260 nm, and also by disc centrifugation. None of the batches showed aggregation. Potency was shown in all batches by a VP/IU ratio below 10, and transgene expression was shown in A549 cells.
  • PER.C6 cells were grown in a stirred tank to cell density of about 1 million cells/ml.
  • the cells were infected with the Ad35.CS vector with a MOI of 40.
  • After 4 days of virus production the infected cell culture was treated with Benzonase and Triton X-100 (B/T method) as described in example 1.
  • B/T harvest was clarified as described in example 1.
  • the clarified harvest was concentrated 5 times by TFF (using a 0.05 ⁇ m hollow fiber), and subsequently diafiltered against 10 diafiltration volumes of 0.1 M NaCl, 0.05% PS80, 50 mM Tris pH 7.5.
  • the concentrated and diafiltered retentate was filtered over a 0.45 um filter, and loaded onto the capturing column or filter.
  • a Q-XL column (3 ml column, 15 cm bedheight) or a Sartobind 75 filter (charged filter containing anionic groups, Sartorius) were tested.
  • the bound components were eluted with a gradient from 0 to 1 M NaCl in a TRIS-based buffer.
  • the elution profile of the charged filter shows an extra peak at the beginning of the gradient, which is separated from the Ad35 peak.
  • the Ad35 virus peak elutes from the charged filter in a sharper peak at a higher salt concentration, 0.44 M NaCl (start 0.41, end 0.49 M NaCl) compared to the Q-XL resin, 0.39 M NaCl (start 0.19, end 0.53 M NaCl).
  • the eluted fractions were analysed by SDS-PAGE, HPLC-AEX, disc centrifugation and TCID50.
  • the extra peak does not behave as intact Ad35 virus particles, when analysed by HPLC-AEX chromatography and disc centrifugation ( FIG. 11 ). SDS-PAGE analysis of the chromatography fractions shows the following results ( FIG. 12 ): In the flowthrough of both runs no or very low amounts of proteins are visible. The extra peak from the charged filter chromatogram shows some but not all Ad35 proteins. In the extra peak viral proteins IIIa, V, VI and VII appear to be missing, while viral proteins II, III, IV and 52.55 k are present.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
US10/590,427 2004-02-23 2005-02-21 Virus Purification Methods Abandoned US20070207461A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US12/220,828 US8124106B2 (en) 2004-02-23 2008-07-28 Virus purification methods

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EPEP04/050190 2004-02-23
EP2004050190 2004-02-23
PCT/EP2005/050739 WO2005080556A2 (en) 2004-02-23 2005-02-21 Virus purification methods

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2005/050739 A-371-Of-International WO2005080556A2 (en) 2004-02-23 2005-02-21 Virus purification methods

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/220,828 Division US8124106B2 (en) 2004-02-23 2008-07-28 Virus purification methods

Publications (1)

Publication Number Publication Date
US20070207461A1 true US20070207461A1 (en) 2007-09-06

Family

ID=34878416

Family Applications (2)

Application Number Title Priority Date Filing Date
US10/590,427 Abandoned US20070207461A1 (en) 2004-02-23 2005-02-21 Virus Purification Methods
US12/220,828 Expired - Fee Related US8124106B2 (en) 2004-02-23 2008-07-28 Virus purification methods

Family Applications After (1)

Application Number Title Priority Date Filing Date
US12/220,828 Expired - Fee Related US8124106B2 (en) 2004-02-23 2008-07-28 Virus purification methods

Country Status (13)

Country Link
US (2) US20070207461A1 (https=)
EP (2) EP1780269B1 (https=)
JP (1) JP2007522814A (https=)
KR (1) KR101211329B1 (https=)
CN (2) CN101343625A (https=)
AT (1) ATE435906T1 (https=)
AU (1) AU2005214090B2 (https=)
CA (1) CA2555412C (https=)
DE (1) DE602005015332D1 (https=)
DK (1) DK1780269T3 (https=)
ES (1) ES2329607T3 (https=)
NZ (1) NZ548495A (https=)
WO (1) WO2005080556A2 (https=)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090110695A1 (en) * 2006-03-27 2009-04-30 Menzo Jans Emko Havenga Compositions Comprising a Recombinant Adenovirus and an Adjuvant
US20090123989A1 (en) * 2005-04-11 2009-05-14 Crucell Holland B.V. Virus purification using ultrafiltration
WO2009100521A1 (en) * 2008-02-12 2009-08-20 Sanofi Pasteur Limited Methods using ion exchange and gel filtration chromatography for poxvirus purification
US20090253207A1 (en) * 1998-11-20 2009-10-08 Crucell Holland B.V. Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells
US20090325284A1 (en) * 2008-06-18 2009-12-31 Richard Truran Virus purification
US20110293660A1 (en) * 2009-02-06 2011-12-01 Bruno Rene Andre Novel method
US20110309038A1 (en) * 2009-02-16 2011-12-22 Kuraray Co., Ltd. Filtering device and method of manufacturing same
US8236293B2 (en) 1995-06-15 2012-08-07 Crucell Holland B.V. Means and methods for nucleic acid delivery vehicle design and nucleic acid transfer
US20130164821A1 (en) * 2010-09-28 2013-06-27 Sartorius Stedim Biotech Gmbh Method for separating viruses from a contaminant-containing liquid
US20130203152A1 (en) * 2010-08-12 2013-08-08 Yisheng Biopharma Holdings Ltd. Method for Reducing DNA Impurities in Viral Compositions
US9644187B2 (en) 2010-04-14 2017-05-09 Emd Millipore Corporation Methods of producing high titer, high purity virus stocks and methods of use thereof
US10041049B2 (en) 2008-11-03 2018-08-07 Janssen Vaccines & Prevention B.V. Method for the production of adenoviral vectors
CN111394390A (zh) * 2020-03-26 2020-07-10 赛诺（深圳）生物医药研究有限公司 用于新型冠状病毒基因疫苗的批量生产重组腺病毒的方法
CN114080453A (zh) * 2019-06-25 2022-02-22 普西奥克瑟斯医疗有限公司 纯化包含b组腺病毒的组合物的方法
WO2023017411A1 (en) * 2021-08-10 2023-02-16 Biocon Limited Purification of liraglutide
WO2023187691A1 (en) * 2022-03-30 2023-10-05 Csl Behring Llc Methods of purifying an enveloped virus
WO2024130163A1 (en) * 2022-12-16 2024-06-20 Avirmax Biopharma Inc. Methods for manufacturing viruses and viral particles

Families Citing this family (75)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070207461A1 (en) 2004-02-23 2007-09-06 Crucell Holland B.V. Virus Purification Methods
ES2525518T3 (es) 2005-11-01 2014-12-26 Novartis Vaccines And Diagnostics Gmbh Vacunas virales derivadas de células con niveles reducidos de ADN celular residual
US7875446B2 (en) * 2006-04-20 2011-01-25 Wyeth Llc Purification processes for isolating purified vesicular stomatitis virus from cell culture
EP2195418B1 (en) 2007-10-02 2012-12-12 PTC Phage Technology Center GmbH Process and system for the industrial scale purification of bacteriophages intended for bacteriophage therapy
US8785173B2 (en) 2008-09-24 2014-07-22 Medimmune, Llc Methods for purification of viruses
JP2010193720A (ja) * 2009-02-23 2010-09-09 Asahi Kasei Chemicals Corp アニオン交換基が固定された多孔膜を用いたウイルスの分離方法
US8609392B2 (en) * 2009-05-12 2013-12-17 Transgene S.A. Method for orthopoxvirus production and purification
ES2565335T3 (es) * 2009-08-17 2016-04-04 Merck Patent Gmbh Método para aislar virus
JP5393896B2 (ja) 2009-10-15 2014-01-22 クルセル ホランド ベー ヴェー アデノウイルス粒子の精製方法
CA2777116C (en) 2009-10-15 2020-09-01 Crucell Holland B.V. Process for adenovirus purification from high cell density cultures
EP2531592A1 (en) 2010-02-04 2012-12-12 Vivalis Fed-batch process using concentrated cell culture medium for the efficient production of biologics in eb66 cells
MX2012007936A (es) 2010-02-15 2012-11-22 Crucell Holland Bv Metodo para la produccion de vectores adenovirales del serotipo 26 del adenovirus.
JP5945270B2 (ja) * 2010-07-20 2016-07-05 バヴァリアン・ノルディック・アクティーゼルスカブ 発現産物を採取するための方法
US8771709B2 (en) 2010-09-20 2014-07-08 Crucell Holland B.V. Therapeutic vaccination against active Tuberculosis
BR112013004582A2 (pt) 2010-09-27 2016-09-06 Crucell Holland Bv método para induzir uma resposta imune em um sujeito contra um antígeno de um parasita que causa a malária
US9273289B2 (en) * 2010-11-26 2016-03-01 Probiogen Ag Depletion of host cell components from live vector vaccines
KR20140038517A (ko) * 2011-06-13 2014-03-28 머크 샤프 앤드 돔 코포레이션 천연 또는 돌연변이 형태의 디프테리아 독소의 정제 방법
ES2726975T3 (es) * 2011-07-27 2019-10-11 Flash Therapeutics Método para la producción de partículas retrovíricas útiles para transducir células eucariotas
US20130122038A1 (en) 2011-11-14 2013-05-16 The United States Of America As Represented By The Secretary Of The Department Heterologous prime-boost immunization using measles virus-based vaccines
US8932607B2 (en) 2012-03-12 2015-01-13 Crucell Holland B.V. Batches of recombinant adenovirus with altered terminal ends
SI2825640T1 (sl) 2012-03-12 2016-08-31 Crucell Holland B.V. Šarže rekombinantnega adenovirusa s spremenjenimi konci
JP5805908B2 (ja) 2012-03-22 2015-11-10 クルセル ホランド ベー ヴェー Ｒｓｖに対するワクチン
US9119813B2 (en) 2012-03-22 2015-09-01 Crucell Holland B.V. Vaccine against RSV
CN103571800B (zh) * 2012-07-27 2018-04-24 江苏康润生物科技有限公司 一种去除疫苗中宿主dna的方法
RU2493872C1 (ru) * 2012-08-08 2013-09-27 Федеральное государственное унитарное предприятие "Научно-производственное объединение по медицинским иммунобиологическим препаратам "Микроген" Министерства здравоохранения Российской Федерации ( ФГУП "НПО "Микроген" Минздрава России) Способ очистки вируса гриппа
CN103768589B (zh) * 2012-10-18 2016-12-21 辽宁成大生物股份有限公司 利用中空纤维膜去除人用乙型脑炎疫苗制品中残留dna的方法
BR112015021297A2 (pt) * 2013-02-28 2017-10-10 Psioxus Therapeutics Ltd um processo para a produção de adenovírus.
KR102236497B1 (ko) 2013-04-25 2021-04-06 얀센 백신스 앤드 프리벤션 비.브이. 안정화된 가용성 예비융합 ｒｓｖ ｆ 폴리펩타이드
CA2914790C (en) 2013-06-14 2024-02-27 Psioxus Therapeutics Limited A dosing regime and formulations for type b adenoviruses
CN105408348B (zh) 2013-06-17 2021-07-06 扬森疫苗与预防公司 稳定化的可溶性融合前rsv f多肽
EP3172317B1 (en) * 2014-07-24 2019-05-01 Janssen Vaccines & Prevention B.V. Process for the purification of poliovirus from cell cultures
GB201417042D0 (en) * 2014-09-29 2014-11-12 Fkd Therapies Oy Method
RS58080B1 (sr) 2014-11-04 2019-02-28 Janssen Vaccines & Prevention Bv Terapeutske vakcine protiv hpv16
JP6735288B2 (ja) 2015-04-14 2020-08-05 ヤンセン ファッシンズ アンド プリベンション ベーフェーＪａｎｓｓｅｎ Ｖａｃｃｉｎｅｓ ＆ Ｐｒｅｖｅｎｔｉｏｎ Ｂ．Ｖ． 双方向性プロモーターを有する２つの導入遺伝子を発現する組換えアデノウイルス
US10954492B2 (en) 2015-06-10 2021-03-23 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Processes for production and purification of nucleic acid-containing compositions
US10457708B2 (en) 2015-07-07 2019-10-29 Janssen Vaccines & Prevention B.V. Stabilized soluble pre-fusion RSV F polypeptides
EA039065B1 (ru) 2015-07-07 2021-11-29 Янссен Вэксинс Энд Превеншн Б.В. Вакцина против rsv
JP6470872B2 (ja) 2015-08-20 2019-02-13 ヤンセン ファッシンズ アンド プリベンション ベーフェーＪａｎｓｓｅｎ Ｖａｃｃｉｎｅｓ ＆ Ｐｒｅｖｅｎｔｉｏｎ Ｂ．Ｖ． 治療用ｈｐｖ１８ワクチン
JP7233928B2 (ja) 2016-04-05 2023-03-07 ヤンセン ファッシンズ アンド プリベンション ベーフェー Ｒｓｖに対するワクチン
IL262108B2 (en) 2016-04-05 2023-04-01 Janssen Vaccines Prevention B V Soluble pre-fusion rsv f proteins are stabilized
JP7053491B2 (ja) 2016-05-02 2022-04-12 ヤンセン ファッシンズ アンド プリベンション ベーフェー 治療用ｈｐｖワクチン組み合わせ
CN109154000B (zh) 2016-05-12 2022-07-05 扬森疫苗与预防公司 有效和平衡的双向启动子
CN109311946A (zh) 2016-05-30 2019-02-05 扬森疫苗与预防公司 稳定化的融合前rsv f蛋白
RU2745500C2 (ru) 2016-06-20 2021-03-25 Янссен Вэксинс Энд Превеншн Б.В. Эффективный и сбалансированный двунаправленный промотор
WO2018011196A1 (en) 2016-07-14 2018-01-18 Janssen Vaccines & Prevention B.V. Hpv vaccines
CN106434571A (zh) * 2016-10-19 2017-02-22 深圳源兴基因技术有限公司 一种wave无血清培养悬浮细胞制备重组腺病毒的方法
CN108085301B (zh) * 2016-11-22 2021-10-26 贺道耀 从宿主细胞提取和纯化腺相关病毒和腺病毒的方法及其组分和试剂盒
US10294452B2 (en) 2016-11-22 2019-05-21 Dao-Yao He Lysis, extraction and purification of adeno-associated virus and adenovirus from host cells
US11034978B2 (en) 2017-02-09 2021-06-15 Janssen Vaccines & Prevention B.V. Potent and short promoter for expression of heterologous genes
CN106868025B (zh) * 2017-03-13 2020-01-21 中国人民解放军军事医学科学院生物工程研究所 用酵母制备三聚体埃博拉病毒糖蛋白突变体的方法
AU2018267971A1 (en) 2017-05-17 2019-11-07 Janssen Vaccines & Prevention B.V. Methods and compositions for inducing protective immunity against RSV infection
CN107603959A (zh) * 2017-08-30 2018-01-19 四川大学 提高缓冲液盐离子浓度纯化病毒的方法
SG11202001458SA (en) 2017-09-15 2020-03-30 Janssen Vaccines & Prevention Bv Method for the safe induction of immunity against rsv
ES2991988T3 (es) 2017-10-30 2024-12-05 Takeda Pharmaceuticals Co Detergentes compatibles con el medio ambiente para la inactivación de virus con envoltura lipídica
US11603527B2 (en) * 2017-12-27 2023-03-14 Global Life Sciences Solutions Usa Llc Method and kit for viral vector isolation
GB201814590D0 (en) * 2018-09-07 2018-10-24 Oxford Biomedica Ltd Viral vector production system
CN110317832B (zh) * 2018-03-28 2022-07-05 西比曼生物科技（香港）有限公司 Gmp级规模化制备重组慢病毒载体的纯化制剂的方法
WO2019209632A1 (en) 2018-04-24 2019-10-31 Merck Sharp & Dohme Corp. Scalable chromatography process for purification of human cytomegalovirus
US11690907B2 (en) 2018-06-12 2023-07-04 Kbio Holdings Limited Vaccines formed by virus and antigen conjugation
US11529413B2 (en) 2018-06-12 2022-12-20 Kbio Holdings Limited Virus and antigen purification and conjugation
IL279295B2 (en) 2018-06-12 2026-03-01 Kentucky Bioprocessing Inc Purification and cross-linking of virus and antigen
US11696948B2 (en) 2018-06-12 2023-07-11 Kbio Holdings Limited Vaccines formed by virus and antigen conjugation
MX2021005607A (es) 2018-11-13 2021-06-30 Janssen Vaccines & Prevention Bv Proteinas f de prefusion del vrs estabilizadas.
CA3120129A1 (en) * 2018-11-21 2020-05-28 Western Oncolytics Ltd. Manufacture of virus
MX2021013950A (es) * 2019-05-14 2022-01-04 Janssen Biotech Inc Eliminacion eficaz de impurezas usando un proceso de diafiltracion.
CN114173827B (zh) * 2019-06-28 2025-01-28 武田药品工业株式会社 腺相关病毒纯化方法
IL295154A (en) * 2020-01-28 2022-09-01 Lonza Walkersville Inc Methods for determining viral titer
CN111249456A (zh) * 2020-03-13 2020-06-09 武汉生物制品研究所有限责任公司 一种狂犬病病毒灭活疫苗的纯化方法
US20230302381A1 (en) * 2020-08-18 2023-09-28 Sartorius Bia Separations D.O.O. Metal-affinity extraction of host cell dna
US12577540B2 (en) 2020-12-10 2026-03-17 Sarepta Therapeutics, Inc. Suspension mode seed train development for adherent cells
AU2022279062A1 (en) 2021-05-17 2024-01-04 Sarepta Therapeutics, Inc. Production of recombinant aav vectors for treating muscular dystrophy
CN113817689B (zh) * 2021-11-22 2023-10-03 北京艺妙神州医药科技有限公司 慢病毒纯化工艺
CN115807064A (zh) * 2022-07-20 2023-03-17 上海泰昶生物技术有限公司 重组腺相关病毒载体制品中游离和错误包装dna的检测方法
CN115141813A (zh) * 2022-07-29 2022-10-04 深圳源兴基因技术有限公司 一种高效去除宿主细胞残留蛋白的腺病毒纯化方法
EP4590838A1 (en) 2022-09-23 2025-07-30 Sarepta Therapeutics, Inc. Recombinant aav vectors for treating muscular dystrophy

Citations (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5173418A (en) * 1985-05-10 1992-12-22 Benzon Pharma, A/S Production in Escherichia coli of extracellular Serratia spp. hydrolases
US5559099A (en) * 1994-09-08 1996-09-24 Genvec, Inc. Penton base protein and methods of using same
US5789390A (en) * 1994-01-28 1998-08-04 Rhone-Poulenc Rorer S.A. Method for preparing recombinant adeno-associated viruses (AAV), and uses thereof
US5837511A (en) * 1995-10-02 1998-11-17 Cornell Research Foundation, Inc. Non-group C adenoviral vectors
US5837520A (en) * 1995-03-07 1998-11-17 Canji, Inc. Method of purification of viral vectors
US5840565A (en) * 1995-08-22 1998-11-24 The Regents Of The University Of California Methods for enhancing the production of viral vaccines in PKR-deficient cell culture
US5846782A (en) * 1995-11-28 1998-12-08 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US5851806A (en) * 1994-06-10 1998-12-22 Genvec, Inc. Complementary adenoviral systems and cell lines
US5891690A (en) * 1996-04-26 1999-04-06 Massie; Bernard Adenovirus E1-complementing cell lines
US5948410A (en) * 1997-04-09 1999-09-07 Duphar International Research B.V. Influenza vaccine
US5965541A (en) * 1995-11-28 1999-10-12 Genvec, Inc. Vectors and methods for gene transfer to cells
US5981225A (en) * 1998-04-16 1999-11-09 Baylor College Of Medicine Gene transfer vector, recombinant adenovirus particles containing the same, method for producing the same and method of use of the same
US5994134A (en) * 1998-05-04 1999-11-30 Canji, Inc. Viral production process
US5994106A (en) * 1994-06-10 1999-11-30 Genvec, Inc. Stocks of recombinant, replication-deficient adenovirus free of replication-competent adenovirus
US5994128A (en) * 1995-06-15 1999-11-30 Introgene B.V. Packaging systems for human recombinant adenovirus to be used in gene therapy
US6008036A (en) * 1995-08-10 1999-12-28 Pasteur Merieux Serums Et Vaccins Method for purifying viruses by chromatography
US6020191A (en) * 1997-04-14 2000-02-01 Genzyme Corporation Adenoviral vectors capable of facilitating increased persistence of transgene expression
US6040174A (en) * 1993-05-28 2000-03-21 Transgene S.A. Defective adenoviruses and corresponding complementation lines
US6113913A (en) * 1998-06-26 2000-09-05 Genvec, Inc. Recombinant adenovirus
US6146873A (en) * 1994-11-10 2000-11-14 Baxter Aktiengesellschaft Production of orthomyxoviruses in monkey kidney cells using protein-free media
US6168941B1 (en) * 2000-04-07 2001-01-02 Genvec, Inc. Method of producing adenoviral vector stocks
US6168944B1 (en) * 1997-01-31 2001-01-02 Schering Corporation Methods for cultivating cells and propagating viruses
US6261823B1 (en) * 1996-12-13 2001-07-17 Schering Corporation Methods for purifying viruses
US6309650B1 (en) * 1997-08-28 2001-10-30 Cheil Jedang Corporation Attenuated Japanese encephalitis virus adapted to Vero cell and a Japanese encephalitis vaccine
US6342384B1 (en) * 1998-08-21 2002-01-29 The University Of Va Patent Foundation Production of adenoviral vectors using serum-free suspension cell culture in a hollow fiber system
US6485958B2 (en) * 1996-07-01 2002-11-26 Gencell Sa Method for producing recombinant adenovirus
US20020177215A1 (en) * 1996-11-20 2002-11-28 Introgen Therapeutics, Inc. Methods for producing purified adenoviral vectors
US20020182723A1 (en) * 1998-12-01 2002-12-05 Introgen Therapeutics, Inc. An improved method for the production and purification of adenoviral vectors
US6492169B1 (en) * 1999-05-18 2002-12-10 Crucell Holland, B.V. Complementing cell lines
US6537793B2 (en) * 1998-12-31 2003-03-25 Aventis Pharma S.A. Method of separating viral particles
US6586226B2 (en) * 1998-04-22 2003-07-01 Genvec, Inc. Efficient purification of adenovirus
US20060078628A1 (en) * 2004-10-09 2006-04-13 Karl Koman Wound treating agent

Family Cites Families (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE434914B (sv) * 1975-07-07 1984-08-27 Jonsson U R S Forfarande for att ur en suspension av bakterier och ett vetskeformigt medium genom filtrering avskilja mediet under samtidig anrikning av bakterierna
US4435289A (en) * 1981-12-23 1984-03-06 Romicon, Inc. Series ultrafiltration with pressurized permeate
US4808315A (en) * 1986-04-28 1989-02-28 Asahi Kasei Kogyo Kabushiki Kaisha Porous hollow fiber membrane and a method for the removal of a virus by using the same
SE9500724D0 (sv) 1994-06-23 1995-02-24 Pharmacia Ab Filtrering
US5770442A (en) 1995-02-21 1998-06-23 Cornell Research Foundation, Inc. Chimeric adenoviral fiber protein and methods of using same
WO1997008298A1 (en) * 1995-08-30 1997-03-06 Genzyme Corporation Chromatographic purification of adenovirus and aav
FR2751343B1 (fr) * 1996-07-16 1998-12-18 Transgene Sa Procede de conservation de virus recombinants infectieux, suspension aqueuse virale et utilisation comme medicament
EP0981601B1 (en) 1997-01-31 2006-09-06 Schering Corporation Methods for cultivating cells and propagating viruses
CA2283253A1 (en) 1997-03-04 1998-09-11 Baxter International Inc. Adenovirus e1-complementing cell lines
US5947689A (en) * 1997-05-07 1999-09-07 Scilog, Inc. Automated, quantitative, system for filtration of liquids having a pump controller
US6210683B1 (en) 1997-09-05 2001-04-03 Merck & Co., Inc. Stabilizers containing recombinant human serum albumin for live virus vaccines
IL137510A0 (en) 1998-02-17 2001-07-24 Schering Corp Compositions comprising viruses and methods for concentrating virus preparations
US20030017138A1 (en) 1998-07-08 2003-01-23 Menzo Havenga Chimeric adenoviruses
DK1133316T3 (da) 1998-11-16 2009-05-25 Introgen Therapeutics Inc Adenovirus-formulering til genterapi
JP4802366B2 (ja) 1999-02-22 2011-10-26 トランスジェン・ソシエテ・アノニム 精製ウイルス調製物の取得方法
US6120820A (en) * 1999-02-22 2000-09-19 Land O'lakes, Inc. Method of modifying the color of a dairy material
ES2372823T3 (es) 1999-05-17 2012-01-26 Crucell Holland B.V. Adenovirus recombinante del serotipo ad11.
WO2003104467A1 (en) 2002-04-25 2003-12-18 Crucell Holland B.V. Means and methods for the production of adenovirus vectors
DE19955558C2 (de) 1999-11-18 2003-03-20 Stefan Kochanek Permanente Amniozyten-Zelllinie, ihre Herstellung und Verwendung zur Herstellung von Gentransfervektoren
EP1103610A1 (en) 1999-11-26 2001-05-30 Introgene B.V. Production of vaccines from immortalised mammalian cell lines
JP5118798B2 (ja) 2000-03-07 2013-01-16 メルク・シャープ・エンド・ドーム・コーポレイション アデノウイルス製剤
DE10022258A1 (de) * 2000-05-08 2001-11-15 Bayer Ag Reinigung von Proteineinschlusskörpern durch Querstrom-Mikrofiltration
JP2004508064A (ja) 2000-09-15 2004-03-18 メルク　エンド　カムパニー　インコーポレーテッド コドン最適化ｈｉｖ１−ｇａｇ、ｐｏｌ、ｎｅｆおよび修飾体を発現する増強された第１世代アデノウイルスワクチン
US6365395B1 (en) * 2000-11-03 2002-04-02 Millipore Corporation Process for removing protein aggregates and virus from a protein solution
US20020064860A1 (en) 2000-11-29 2002-05-30 Schering Corporation Method for purifying adenoviruses
IL152420A0 (en) 2001-02-23 2003-05-29 Novartis Ag Novel oncolytic adenoviral vectors
ATE496120T1 (de) 2001-03-01 2011-02-15 Bayer Schering Pharma Ag Konzentration und lyse von adenovirus-infizierten zellen in derselben prozesseinheit
CA2776391C (en) 2001-10-01 2015-03-31 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Development of a preventive vaccine for filovirus infection in primates
US6951752B2 (en) 2001-12-10 2005-10-04 Bexter Healthcare S.A. Method for large scale production of virus antigen
CA2469623C (en) 2001-12-12 2012-05-29 F H Faulding & Co Limited Composition for the preservation of viruses
US20030180936A1 (en) 2002-03-15 2003-09-25 Memarzadeh Bahram Eric Method for the purification, production and formulation of oncolytic adenoviruses
EP1492890A4 (en) 2002-03-29 2006-10-18 Merck & Co Inc EXPLOSIVE PROCEDURE FOR THE PRODUCTION OF ADENOVIRUS AND ADENOVIRUS STEM MIXTURES
EP1497440B1 (en) 2002-04-25 2008-08-20 Crucell Holland B.V. Stable adenoviral vectors and methods for propagation thereof
ATE360683T1 (de) * 2002-05-14 2007-05-15 Merck & Co Inc Verfahren zur reinigung von adenovirus
US20050095705A1 (en) 2003-04-15 2005-05-05 Michael Kadan Method for production of oncolytic adenoviruses
EP1633321A4 (en) * 2003-06-18 2006-11-02 Onyx Pharma Inc METHOD FOR CLEANING VIRUS
US20070207461A1 (en) 2004-02-23 2007-09-06 Crucell Holland B.V. Virus Purification Methods

Patent Citations (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5173418A (en) * 1985-05-10 1992-12-22 Benzon Pharma, A/S Production in Escherichia coli of extracellular Serratia spp. hydrolases
US6040174A (en) * 1993-05-28 2000-03-21 Transgene S.A. Defective adenoviruses and corresponding complementation lines
US5789390A (en) * 1994-01-28 1998-08-04 Rhone-Poulenc Rorer S.A. Method for preparing recombinant adeno-associated viruses (AAV), and uses thereof
US5994106A (en) * 1994-06-10 1999-11-30 Genvec, Inc. Stocks of recombinant, replication-deficient adenovirus free of replication-competent adenovirus
US5851806A (en) * 1994-06-10 1998-12-22 Genvec, Inc. Complementary adenoviral systems and cell lines
US5559099A (en) * 1994-09-08 1996-09-24 Genvec, Inc. Penton base protein and methods of using same
US6146873A (en) * 1994-11-10 2000-11-14 Baxter Aktiengesellschaft Production of orthomyxoviruses in monkey kidney cells using protein-free media
US5837520A (en) * 1995-03-07 1998-11-17 Canji, Inc. Method of purification of viral vectors
US5994128A (en) * 1995-06-15 1999-11-30 Introgene B.V. Packaging systems for human recombinant adenovirus to be used in gene therapy
US6008036A (en) * 1995-08-10 1999-12-28 Pasteur Merieux Serums Et Vaccins Method for purifying viruses by chromatography
US5840565A (en) * 1995-08-22 1998-11-24 The Regents Of The University Of California Methods for enhancing the production of viral vaccines in PKR-deficient cell culture
US5837511A (en) * 1995-10-02 1998-11-17 Cornell Research Foundation, Inc. Non-group C adenoviral vectors
US5965541A (en) * 1995-11-28 1999-10-12 Genvec, Inc. Vectors and methods for gene transfer to cells
US5846782A (en) * 1995-11-28 1998-12-08 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US5891690A (en) * 1996-04-26 1999-04-06 Massie; Bernard Adenovirus E1-complementing cell lines
US6485958B2 (en) * 1996-07-01 2002-11-26 Gencell Sa Method for producing recombinant adenovirus
US20020177215A1 (en) * 1996-11-20 2002-11-28 Introgen Therapeutics, Inc. Methods for producing purified adenoviral vectors
US6261823B1 (en) * 1996-12-13 2001-07-17 Schering Corporation Methods for purifying viruses
US6168944B1 (en) * 1997-01-31 2001-01-02 Schering Corporation Methods for cultivating cells and propagating viruses
US5948410A (en) * 1997-04-09 1999-09-07 Duphar International Research B.V. Influenza vaccine
US6020191A (en) * 1997-04-14 2000-02-01 Genzyme Corporation Adenoviral vectors capable of facilitating increased persistence of transgene expression
US6309650B1 (en) * 1997-08-28 2001-10-30 Cheil Jedang Corporation Attenuated Japanese encephalitis virus adapted to Vero cell and a Japanese encephalitis vaccine
US5981225A (en) * 1998-04-16 1999-11-09 Baylor College Of Medicine Gene transfer vector, recombinant adenovirus particles containing the same, method for producing the same and method of use of the same
US6586226B2 (en) * 1998-04-22 2003-07-01 Genvec, Inc. Efficient purification of adenovirus
US5994134A (en) * 1998-05-04 1999-11-30 Canji, Inc. Viral production process
US6113913A (en) * 1998-06-26 2000-09-05 Genvec, Inc. Recombinant adenovirus
US6342384B1 (en) * 1998-08-21 2002-01-29 The University Of Va Patent Foundation Production of adenoviral vectors using serum-free suspension cell culture in a hollow fiber system
US20020182723A1 (en) * 1998-12-01 2002-12-05 Introgen Therapeutics, Inc. An improved method for the production and purification of adenoviral vectors
US6537793B2 (en) * 1998-12-31 2003-03-25 Aventis Pharma S.A. Method of separating viral particles
US6492169B1 (en) * 1999-05-18 2002-12-10 Crucell Holland, B.V. Complementing cell lines
US6168941B1 (en) * 2000-04-07 2001-01-02 Genvec, Inc. Method of producing adenoviral vector stocks
US20060078628A1 (en) * 2004-10-09 2006-04-13 Karl Koman Wound treating agent

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8236293B2 (en) 1995-06-15 2012-08-07 Crucell Holland B.V. Means and methods for nucleic acid delivery vehicle design and nucleic acid transfer
US20090253207A1 (en) * 1998-11-20 2009-10-08 Crucell Holland B.V. Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells
US7968087B2 (en) 1998-11-20 2011-06-28 Crucell Holland B.V. Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells
US20090123989A1 (en) * 2005-04-11 2009-05-14 Crucell Holland B.V. Virus purification using ultrafiltration
US8574595B2 (en) 2005-04-11 2013-11-05 Crucell Holland B.V. Virus purification using ultrafiltration
US20090110695A1 (en) * 2006-03-27 2009-04-30 Menzo Jans Emko Havenga Compositions Comprising a Recombinant Adenovirus and an Adjuvant
US20110165645A1 (en) * 2008-02-12 2011-07-07 Yelin Xiong Methods Using Ion Exchange and Gel Filtration Chromatography for Poxvirus Purification
WO2009100521A1 (en) * 2008-02-12 2009-08-20 Sanofi Pasteur Limited Methods using ion exchange and gel filtration chromatography for poxvirus purification
CN102257134B (zh) * 2008-02-12 2014-03-05 赛诺菲巴斯德有限公司 使用离子交换和凝胶过滤色谱进行痘病毒纯化的方法
US9169491B2 (en) * 2008-06-18 2015-10-27 Oxford Biomedica (Uk) Limited Virus purification
US20090325284A1 (en) * 2008-06-18 2009-12-31 Richard Truran Virus purification
US10041049B2 (en) 2008-11-03 2018-08-07 Janssen Vaccines & Prevention B.V. Method for the production of adenoviral vectors
US20110293660A1 (en) * 2009-02-06 2011-12-01 Bruno Rene Andre Novel method
US20110309038A1 (en) * 2009-02-16 2011-12-22 Kuraray Co., Ltd. Filtering device and method of manufacturing same
US9050563B2 (en) * 2009-02-16 2015-06-09 Kuraray Co., Ltd. Filtering device and method of manufacturing same
US9644187B2 (en) 2010-04-14 2017-05-09 Emd Millipore Corporation Methods of producing high titer, high purity virus stocks and methods of use thereof
US20130203152A1 (en) * 2010-08-12 2013-08-08 Yisheng Biopharma Holdings Ltd. Method for Reducing DNA Impurities in Viral Compositions
US8778653B2 (en) * 2010-08-12 2014-07-15 Yisheng Biopharma Holdings Ltd. Method for reducing DNA impurities in viral compositions
US20130164821A1 (en) * 2010-09-28 2013-06-27 Sartorius Stedim Biotech Gmbh Method for separating viruses from a contaminant-containing liquid
US10315133B2 (en) * 2010-09-28 2019-06-11 Sartorius Stedim Biotech Gmbh Method for separating viruses from a contaminant-containing liquid
CN114080453A (zh) * 2019-06-25 2022-02-22 普西奥克瑟斯医疗有限公司 纯化包含b组腺病毒的组合物的方法
CN111394390A (zh) * 2020-03-26 2020-07-10 赛诺（深圳）生物医药研究有限公司 用于新型冠状病毒基因疫苗的批量生产重组腺病毒的方法
WO2023017411A1 (en) * 2021-08-10 2023-02-16 Biocon Limited Purification of liraglutide
WO2023187691A1 (en) * 2022-03-30 2023-10-05 Csl Behring Llc Methods of purifying an enveloped virus
WO2024130163A1 (en) * 2022-12-16 2024-06-20 Avirmax Biopharma Inc. Methods for manufacturing viruses and viral particles

Also Published As

Publication number Publication date
WO2005080556A9 (en) 2006-10-26
DK1780269T3 (da) 2009-10-12
WO2005080556A3 (en) 2006-02-23
CA2555412C (en) 2013-06-25
DE602005015332D1 (de) 2009-08-20
JP2007522814A (ja) 2007-08-16
AU2005214090B2 (en) 2008-09-11
WO2005080556A2 (en) 2005-09-01
KR101211329B1 (ko) 2012-12-12
EP1780269A2 (en) 2007-05-02
ES2329607T3 (es) 2009-11-27
EP1780269A3 (en) 2007-07-18
CN101343625A (zh) 2009-01-14
EP1718738A2 (en) 2006-11-08
ATE435906T1 (de) 2009-07-15
CN1922308A (zh) 2007-02-28
AU2005214090A1 (en) 2005-09-01
US20090017523A1 (en) 2009-01-15
EP1780269B1 (en) 2009-07-08
US8124106B2 (en) 2012-02-28
NZ548495A (en) 2009-05-31
CA2555412A1 (en) 2005-09-01
KR20070001163A (ko) 2007-01-03

Similar Documents

Publication Publication Date Title
US8124106B2 (en) Virus purification methods
US8574595B2 (en) Virus purification using ultrafiltration
US8460920B2 (en) Method for the purification of adenovirus particles
CN102203242B (zh) 产生腺病毒载体的方法
US20120202268A1 (en) Process for adenovirus purification from high cell density cultures
Carina Silva et al. Adenovirus vector production and purification
HK1174948B (en) Method for the purification of adenovirus particles from high cell density cultures

Legal Events

Date Code Title Description
AS Assignment

Owner name: CRUCELL HOLLAND B.V., NETHERLANDS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WEGGEMAN, MIRANDA;VAN CORVEN, EMILE J. J. M.;REEL/FRAME:018249/0242

Effective date: 20050221

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION