Methods for removing host cell DNA from virus preparations

Abstract

The present invention relates to a method for removing host cell DNA from a sample containing infectious viral particles and host cell DNA by anion exchange chromatography in the presence of at least one non-ionic detergent, sugar, and protein, and to a method for purifying recombinant infectious viral particles from a host cell culture medium using such an anion exchange chromatography step.

Description

[Technical field]

[0001] The present invention relates to a method for removing host cell DNA from a sample containing infectious viral particles and host cell DNA by anion exchange chromatography in the presence of at least one non-ionic detergent, sugar and protein, and to a method for purifying recombinant infectious viral particles from a host cell culture medium using such an anion exchange chromatography step. [Background technology]

[0002] Vaccines as a defense against viral infections have been effectively used to reduce the occurrence of disease in humans. One of the most successful techniques of viral vaccines is the immunization of animals or humans with weakened or attenuated virus strains ("live attenuated viruses"). Attenuated virus strains do not cause disease because the replication of the virus after immunization is limited. However, the limited viral replication is sufficient to express the full repertoire of viral antigens and generate a strong and long-lasting immune response against the virus. Thus, upon subsequent exposure to a pathogenic virus strain, the immunized individual is protected from disease. These live attenuated virus vaccines are one of the most successful vaccines used in public health.

[0003] To generate the live attenuated virus contained in the vaccine, a mammalian host cell, such as Vero cells, is usually infected with the virus, which then replicates within the cell, resulting in the secretion of infectious viral particles into the cell supernatant. These infectious viral particles must then be separated from impurities, such as host cell proteins and host cell DNA, and can then be used to immunize a subject.

[0004] WO02 / 024876 A2 discloses a process for the production of a whole particle vaccine, in which infected host cells are incubated in serum-free medium in the presence of nucleases and proteases.

[0005] WO2006 / 22964 A1 describes the purification of inactivated West Nile virus by affinity chromatography using Cellufine sulfate chromatography.

[0006] WO2013 / 106337 A1 discloses a method for purifying viral particles of a recombinant infectious flavivirus by tangential flow filtration followed by anion exchange chromatography performed in bind and elute mode.

[0007] WO2015 / 092287 A1 describes a method for purifying enveloped viruses by performing an anion exchange chromatography step using an acidic buffer with a pH of less than 6.0. The specification also describes that applying a tangential flow filtration step prior to anion exchange chromatography results in a significantly improved efficiency of enveloped virus purification.

[0008] Nevertheless, there remains a need for methods that allow efficient removal of host cell DNA and proteins from viral particles used in vaccines. Summary of the Invention

[0009] The present invention provides a process for efficient removal of host cell DNA from a sample containing infectious viral particles and host cell DNA, which minimizes loss of viral titer during host cell DNA removal.

[0010] Accordingly, the present invention provides a method for removing host cell DNA from a sample containing infectious viral particles and host cell DNA, comprising the steps of: (a) mixing said sample, comprising infectious viral particles and host cell DNA, with a composition comprising one or more non-ionic detergents, sugars and proteins, thereby providing a mixture; (b) subjecting the mixture of step (a) to anion exchange chromatography.

[0011] In one embodiment, the nonionic surfactant is a nonionic triblock copolymer, such as an ethylene oxide-propylene oxide (EO-PO) block copolymer. In one embodiment, the nonionic surfactant is poloxamer 407 or poloxamer 403. In one embodiment, the concentration of the nonionic surfactant in the mixture of step (a) is 0.05% to 0.5% w / v.

[0012] In one embodiment, the sugar is selected from trehalose, glucose, sucrose, fructose and maltose, preferably trehalose. In one embodiment, the concentration of the sugar in the mixture of step (a) is 1 w / v% to 7.5 w / v%.

[0013] In one embodiment, the protein is albumin, preferably human serum albumin. In one embodiment, the concentration of the protein in the mixture of step (a) is 0.005 w / v% to 0.05 w / v%.

[0014] In one embodiment, the composition comprises poloxamer 407, trehalose dihydrate and human serum albumin. In one embodiment, the mixture of step (a) comprises 0.2 w / v% poloxamer 407, 3 w / v% trehalose dihydrate and 0.02 w / v% human serum albumin.

[0015] In one embodiment, the anion exchange chromatography is operated in a flow-through mode. In one embodiment, the anion exchange chromatography uses an anion exchange chromatography membrane. In one embodiment, the anion exchange chromatography membrane comprises quaternary ammonium groups.

[0016] In one embodiment, the infectious viral particle is an enveloped infectious viral particle. In one embodiment, the infectious viral particle is an infectious flavivirus viral particle. In one embodiment, the infectious viral particle is an infectious dengue virus viral particle. In one embodiment, the infectious dengue virus viral particle is an infectious live attenuated dengue virus viral particle or an infectious chimeric dengue virus viral particle.

[0017] In one embodiment, less than 5% of the viral titer is lost during anion exchange chromatography.

[0018] In one embodiment, anion exchange chromatography reduces the host cell DNA content by at least 10-fold.

[0019] In one embodiment, the method does not include treating the sample containing infectious viral particles and host cell DNA with an endonuclease.

[0020] The present invention also provides a method for purifying infectious viral particles from a host cell culture medium, comprising the steps of: (a) harvesting a host cell culture supernatant containing infectious viral particles from the host cell culture; (b) mixing the harvested infectious viral particles with a composition comprising one or more non-ionic detergents, sugars and proteins, thereby providing a mixture; (c) subjecting the mixture of step (b) to one or more anion exchange chromatography steps and collecting the flow-through; (d) subjecting the permeate from step (c) to tangential flow filtration and collecting the retentate; (e) recovering purified infectious dengue virus particles from the retentate of step (d).

[0021] In one embodiment, the nonionic surfactant is a nonionic triblock copolymer, such as an ethylene oxide-propylene oxide (EO-PO) block copolymer. In one embodiment, the nonionic surfactant is poloxamer 407 or poloxamer 403. In one embodiment, the concentration of the nonionic surfactant in the mixture of step (a) is 0.05% to 0.5% w / v.

[0022] In one embodiment, the sugar is selected from trehalose, glucose, sucrose, fructose and maltose, preferably trehalose. In one embodiment, the concentration of the sugar in the mixture of step (a) is 1 w / v% to 7.5 w / v%.

[0023] In one embodiment, the protein is albumin, preferably human serum albumin. In one embodiment, the concentration of the protein in the mixture of step (a) is 0.005 w / v% to 0.05 w / v%.

[0024] In one embodiment, the composition comprises poloxamer 407, trehalose dihydrate and human serum albumin. In one embodiment, the mixture comprises 0.2 w / v% poloxamer 407, 3 w / v% trehalose dihydrate and 0.02 w / v% human serum albumin.

[0025] In one embodiment, the anion exchange chromatography uses an anion exchange chromatography membrane, hi one embodiment, the anion exchange chromatography membrane comprises a quaternary ammonium group.

[0026] In one embodiment, the infectious viral particle is an enveloped infectious viral particle. In one embodiment, the infectious viral particle is an infectious flavivirus viral particle. In one embodiment, the infectious viral particle is an infectious dengue virus viral particle. In one embodiment, the infectious dengue virus viral particle is an infectious live attenuated dengue virus viral particle or an infectious chimeric dengue virus viral particle.

[0027] In one embodiment, the method does not include treating the sample containing infectious viral particles and host cell DNA with an endonuclease.

[0028] In one embodiment, the host cell culture medium is a Vero cell culture medium.

[0029] In one embodiment, the host cell culture supernatant containing the infectious viral particles is harvested and then subjected to depth filtration, in one embodiment through 0.45 μm and 0.2 μm filters.

[0030] In one embodiment, the tangential flow filtration comprises filtration through one or more TFF membrane cassettes. In one embodiment, the one or more TFF membrane cassettes comprise a cellulose filtration medium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] When the terms "comprise" or "comprising" are used in the present specification and claims, these expressions do not exclude other elements or steps. For the purposes of the present invention, the term "consisting of" is considered to be any embodiment of the term "comprising". Hereinafter, when a group is defined to include at least a certain number of embodiments, this group is also understood to disclose the group consisting of only these embodiments, if desired.

[0032] When an indefinite or definite article is used in connection with a singular noun, e.g. "a" or "an", "the", this includes the plural of that noun, unless expressly stated otherwise. Vice versa, when the plural of a noun is used, it also refers to the singular.

[0033] Moreover, in the specification and claims, the terms "first," "second," "third," or (a), (b), (c), etc. are used to distinguish between similar elements and do not necessarily describe an order of occurrence or chronological order. It is to be understood that the terms used herein are interchangeable under appropriate circumstances and that the embodiments of the invention described herein can be practiced in other orders than those described or illustrated herein.

[0034] Any numerical values ​​indicated in the context of the present invention are usually accompanied by an interval of accuracy that a person skilled in the art would understand to still ensure the technical function of the feature in question. As used herein, the deviation from the indicated numerical values ​​is in the range of ±10%, preferably ±5%. The above deviations of ±10%, preferably ±5% from the indicated numerical intervals are also expressed by the terms "about" and "approximately", as used herein in relation to numerical values.

[0035] By the method of the present invention, host cell DNA is removed from a sample containing infectious viral particles and host cell DNA. The term "removing host cell DNA" means that the content of host cell DNA after the method of the present invention is performed is less than the content of host cell DNA before the method of the present invention is performed. In one embodiment, the method of the present invention results in at least a 10-fold reduction in host cell DNA content. Preferably, the method of the present invention results in at least a 12-fold or 15-fold reduction in host cell DNA content, more preferably at least an 18-fold or 20-fold reduction, most preferably at least a 22-fold reduction in host cell DNA content. The fold reduction can be calculated by dividing the host cell DNA content in the sample before the method is performed by the host cell DNA content in the sample after the method is performed.

[0036] Preferably, the host cell DNA content after the method of the invention is performed is less than the detection limit of the assay used to determine the host cell DNA content, in one embodiment the host cell DNA content after the method of the invention is performed is less than 50 ng / ml.

[0037] The method of determining the host cell DNA content in the biological sample obtained from the host cell is known in the art, and includes quantitative PCR using primers that specifically bind to the host cell DNA, not to viral nucleic acid.Kits for determining the host cell DNA content are commercially available, for example from ThermoFisher.Preferably, Picogreen® stain is used to determine the host cell DNA content.

[0038] "Host cell DNA" is derived from the cell in which the infectious viral particles were produced. The host cell DNA can be distinguished from the viral nucleic acid due to a sequence of the nucleic acid that differs from the sequence of the viral nucleic acid. In one embodiment, Vero cells are used to produce the infectious viral particles. Thus, in this embodiment, the host cell DNA is derived from Vero cells and is also referred to as "Vero cell DNA." The Vero cell DNA can be distinguished from the viral nucleic acid due to a sequence of the nucleic acid that differs from the sequence of the viral nucleic acid.

[0039] The term "infectious viral particles" refers to viral particles that the virus is capable of infecting suitable host cells, such as Vero cells, replicating in these host cells and resulting in the production of further infectious viral particles. Purified viral particles obtained by the method of the invention are still capable of infecting suitable host cells, since the virus does not lose its ability to infect suitable host cells when the method of the invention is carried out. Thus, the viral particles obtained by the method of the invention are infectious viral particles. The method of the invention does not include, inter alia, a step of inactivating the viral particles. A step of inactivating the viral particles, such as by treatment with formalin or β-propiolactone, results in the loss of infectivity of the viral particles. The term "infectious viral particles" does not include virus-like particles that are not infectious. In one embodiment, the term "infectious viral particles" includes live attenuated viruses.

[0040] In one embodiment, the infectious viral particles are derived from an enveloped virus, examples of which include, but are not limited to, poxviruses, orthomyxoviruses, paramyxoviruses, and flaviviruses.

[0041] By "enveloped virus" is meant a virus particle that has an outer coating or envelope. The envelope is derived from the infected host cell during the budding process, whereby the newly formed virus particle becomes surrounded by an outer shell made from part of the cell's plasma membrane. The budding process, in which the virus acquires an envelope, results in the release of the virus particle from the host cell in which it was used to grow.

[0042] Examples of enveloped orthomyxoviruses include, but are not limited to, influenza A viruses, such as strains such as H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2, and H10N7.

[0043] Examples of enveloped paramyxoviruses include, but are not limited to, human respiratory syncytial virus, measles virus, and mumps virus.

[0044] In certain embodiments, the enveloped virus includes viruses of the Flavivirus genus, which includes enveloped positive strand RNA viruses such as West Nile (WN) virus, Japanese encephalitis virus (JEV), Zika virus, Dengue virus, Yellow fever virus (YF), Kyasanur Forest disease virus, Malay Valley encephalitis virus, St. Louis encephalitis virus, tick-borne encephalitis virus, West Nile encephalitis virus, Central European tick-borne encephalitis (TBE-W) virus, Far Eastern tick-borne encephalitis (TBE-FE) virus, Kunjin virus, Tyuleniy virus, Ntaya virus, Uganda S virus, Modoc virus, BVDV (e.g., NADL and 890 strains), CSFV Alfort 187, BDV BD31 virus, and / or GB virus types A, B, and / or C.

[0045] In certain embodiments, the infectious virus particles are infectious dengue virus particles. Dengue virus is a single-stranded, positive-sense RNA virus of the Flaviviridae family. A systematic classification is outlined in Table 1. The Flaviviridae family includes three genera: Flavivirus, Hepacivirus, and Pestivirus. The Flavivirus genus includes highly pathogenic and potentially hemorrhagic fever viruses, such as yellow fever virus and dengue virus, Zika virus, encephalitis viruses, such as Japanese encephalitis virus, Murray Valley encephalitis virus, and West Nile virus, as well as a number of less pathogenic viruses.

[0046] [Table 1]

[0047] The genome of a flavivirus contains, from the 5' to the 3' end: 5'-non-translated region (5'-NCR), · Capsid protein (C) coding region, · precursor membrane protein (prM) coding region, · envelope protein (E) coding region, The regions coding for the nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5), and 3'-untranslated region (3'-NCR).

[0048] The viral structural proteins are C, prM and E, and the nonstructural proteins are NS1 to NS5. The structural and nonstructural proteins are translated as a single polyprotein and processed by cellular and viral proteases.

[0049] As used herein, the term "dengue serotype" refers to a type of dengue virus, where the serotype is defined by cell surface antigens and can therefore be distinguished from other dengue serotypes by serological methods known in the art. Currently, four serotypes of dengue virus are known: dengue serotype 1 (DENV-1), dengue serotype 2 (DENV-2), dengue serotype 3 (DENV-3) and dengue serotype 4 (DENV-4).

[0050] In certain embodiments, the infectious virus particle is a live attenuated dengue virus.As used herein, the term "live attenuated dengue virus" refers to a viable and infectious dengue virus whose virulence is reduced by mutation.The live attenuated dengue virus can be a dengue virus whose all components are derived from the same dengue serotype, or it can be a chimeric dengue virus that has parts from two or more dengue serotypes.

[0051] In one embodiment, the live attenuated dengue virus is a molecularly characterized and cloned dengue serotype 2 strain derived from the live attenuated DEN-2 PDK-53 virus strain (TDV-2). This attenuated TDV-2 strain was generated by complementary DNA cloning of the attenuated laboratory-derived DEN-2 PDK-53 virus strain originally isolated at Mahidol University, Bangkok, Thailand (Kinney et al. (1997) Virology 230(2):300-308). DEN-2 PDK-53 was generated by 53 serial passages at 32° C. in primary canine kidney (PDK) cells (Bhamarapravati et al. (1987) Bull. World Health Organ. 65(2):189-195). In one embodiment, the TDV-2 has the nucleotide sequence of SEQ ID NO:3 and / or the amino acid sequence of SEQ ID NO:4.

[0052] The attenuated DEN-2 PDK-53 strain (the precursor of TDV-2) is derived from the wild-type virus strain DEN-2 16681 and differs from the wild-type by nine nucleotides as follows (Kinney et al. (1997) Virology 230(2):300-308): (i) 5'-non-translated region (NCR)-57 (nt-57 C to T): the main attenuation site (ii) prM-29 Asp to Val (nt-524 A to T) (iii) nt-2055 C to T (E gene) silent mutation (iv) NS1-53 Gly to Asp (nt-2579 G to A): Major attenuation site (v) NS2A-181 Leu to Phe (nt-4018 C to T) (vi) NS3-250 Glu to Val (nt-5270 A to T): the main attenuation site (vii) nt-5547 (NS3 gene) T to C silent mutation; (viii) NS4A-75 Gly to Ala (nt-6599 G to C) (ix) nt-8571 C to T (NS5 gene) silent mutation

[0053] Three nucleotide changes located in the 5' untranslated region (NCR) (nucleotide 57) (mutation (i)), NS1 (amino acid 828 of SEQ ID NO:4) (mutation (iv)), and NS3 genes (amino acid 1725 of SEQ ID NO:4) (mutation (vi)) form the basis of the attenuated phenotype of the DEN-2 PDK-53 strain (Kinney et al. (1997) Virology 230:300-308; Butrapet et al. (2000) J. Virol. 74(7):3111-3119).

[0054] A "virus strain", particularly a "dengue virus strain", is a genetic subtype of a virus, particularly a dengue virus, characterized by a specific nucleic acid sequence. A dengue serotype may contain different strains with different nucleic acid sequences that have the same cell surface antigens and are therefore recognized by the same antibodies. A dengue virus strain can be a dengue virus in which all components are derived from the same dengue serotype, or it can be a chimeric dengue virus with parts from more than one dengue serotype.

[0055] A "chimeric virus" or "chimeric strain" or "chimeric virus strain" comprises portions from at least two different viruses. In one embodiment, a chimeric virus comprises dengue virus prM and E proteins and other proteins from another flavivirus. In one embodiment, a chimeric virus comprises dengue virus prM and E proteins and other proteins from another flavivirus, such as yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, and tick-borne encephalitis virus. In one embodiment, a chimeric virus comprises dengue virus prM and E proteins and other proteins from yellow fever virus strain YF-17D. Such chimeric viruses are present in the commercial product Dengvaxia® and are described, for example, in WO 98 / 37911, WO 03 / 101397, WO 2007 / 021672, WO 2008 / 007021, WO 2008 / 047023 and WO 2008 / 065315.

[0056] A "chimeric dengue virus" or "chimeric dengue serotype strain" or "chimeric dengue strain" comprises parts from at least two different dengue serotypes. As used herein, a chimeric dengue virus does not comprise parts from different flaviviruses, such as yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, and tick-borne encephalitis virus. In particular, the chimeric dengue viruses described herein do not comprise parts from yellow fever virus. Such chimeric dengue viruses are described in WO01 / 060847 A2, WO2014 / 150939 A2, and WO2017 / 179017 A1.

[0057] As used herein, "chimeric dengue serotype 2 / 1 strain" or "DENV-2 / 1 chimera" or "TDV-1" refers to a dengue virus chimeric construct that includes portions from DENV-2 and DENV-1. In particular, in the chimeric dengue serotype 2 / 1 strain, the prM and E proteins from DENV-2 are replaced with the prM and E proteins from DENV-1. In one embodiment, the chimeric dengue serotype 2 / 1 strain has the nucleotide sequence of SEQ ID NO:1 and / or the amino acid sequence of SEQ ID NO:2.

[0058] As used herein, "chimeric dengue serotype 2 / 3 strain" or "DENV-2 / 3 chimera" or "TDV-3" refers to a dengue virus chimeric construct that includes portions from DENV-2 and DENV-3. In particular, in a chimeric dengue serotype 2 / 3 strain, the prM and E proteins from DENV-2 are replaced with the prM and E proteins from DENV-3. In one embodiment, the TDV-3 has the nucleotide sequence of SEQ ID NO:5 and / or the amino acid sequence of SEQ ID NO:6.

[0059] As used herein, "chimeric dengue serotype 2 / 4 strain" or "DENV-2 / 4 chimera" or "TDV-4" refers to a dengue virus chimeric construct that includes portions from DENV-2 and DENV-4. In particular, in a chimeric dengue serotype 2 / 4 strain, the prM and E proteins from DENV-2 are replaced with the prM and E proteins from DENV-4. In one embodiment, the TDV-4 has the nucleotide sequence of SEQ ID NO:7 and / or the amino acid sequence of SEQ ID NO:8.

[0060] In the method of the present invention, a sample containing infectious viral particles is mixed with a composition comprising one or more non-ionic detergents, sugars and proteins. In one embodiment of the method of the present invention, a sample containing infectious viral particles is mixed with a composition comprising a non-ionic detergent. In one embodiment of the method of the present invention, a sample containing infectious viral particles is mixed with a composition comprising a non-ionic detergent, sugars and proteins.

[0061] The term "nonionic surfactant" refers to a surfactant that does not contain positively or negatively charged (i.e. ionic) functional groups. In contrast to anionic and cationic surfactants, nonionic surfactants do not ionize in solution. The nonionic surfactant may be selected from block copolymers, sorbitan esters, ethoxylated or propoxylated sorbitan esters, alkyl polyglycosides (APGs), alkoxylated mono- or di-alkylamines, fatty acid monoethanolamides (FAMAs), fatty acid diethanolamides (FADAs), ethoxylated fatty acid monoethanolamides (EFAMs), propoxylated fatty acid monoethanolamides (PFAMs), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GAs, or fatty acid glucamides (FAGAs)), and combinations thereof.

[0062] The nonionic surfactant is preferably a high molecular weight nonionic surfactant. By "high molecular weight" it is meant a molecular weight of 1500 or greater. In one embodiment, the nonionic surfactant is a nonionic triblock copolymer. In one embodiment, the surfactant is a nonionic, hydrophilic polyoxyethylene-polyoxypropylene block copolymer (i.e., EO-PO block copolymer). The EO-PO block copolymer can include a block of polyethylene oxide (-CFI2CFI20-a given EO) and a block of polypropylene oxide (-CH2CHCH30-a given PO). The PO block can be adjacent to two EO blocks in an EOx-POy-Eox configuration. Because the PO component is hydrophilic and the EO component is hydrophobic, the overall hydrophilicity, molecular weight and surfactant properties of the copolymer can be adjusted by varying x and y in the EOx-POy-Eox block structure. In aqueous solution, the EO-PO block copolymer self-assembles into micelles with a core of PO and a corona of hydrophilic EO groups.

[0063] In one embodiment, the non-ionic surfactant is a poloxamer. Poloxamers are non-ionic triblock copolymers that are composed of a central poly(propylene oxide) hydrophobic chain flanked by two poly(ethylene oxide) hydrophilic chains. The length of the polymer block can be customized, resulting in different poloxamers with slightly different properties.

[0064] In one embodiment, the non-ionic surfactant is poloxamer 407 or poloxamer 403. Preferably, the non-ionic surfactant is poloxamer 407. Poloxamer 407 is a hydrophilic non-ionic surfactant consisting of a triblock copolymer consisting of a central propylene glycol block having about 56 repeating units and two adjacent hydrophilic polyethylene glycol blocks each containing about 101 repeating units. Poloxamer 407 is also known by the trade names Pluronic F127 and Synperonic PE / F127.

[0065] The concentration of the nonionic surfactant in the mixture with the sample containing infectious virus particles and host cell DNA is 0.05 w / v% to 0.5 w / v%. Preferably, the concentration of the nonionic surfactant in the mixture with the sample containing infectious virus particles and host cell DNA is 0.08 w / v% to 0.4 w / v%, more preferably, the concentration of the nonionic surfactant in the mixture with the sample containing infectious virus particles and host cell DNA is 0.1 w / v% to 0.3 w / v%, and most preferably, the concentration of the nonionic surfactant in the mixture with the sample containing infectious virus particles and host cell DNA is 0.2 w / v%.

[0066] The concentration of the nonionic triblock copolymer in the mixture with the infectious virus particles and the sample containing the host cell DNA is 0.05 w / v% to 0.5 w / v%. Preferably, the concentration of the nonionic triblock copolymer in the mixture with the infectious virus particles and the sample containing the host cell DNA is 0.08 w / v% to 0.4 w / v%, more preferably, the concentration of the nonionic triblock copolymer in the mixture with the infectious virus particles and the sample containing the host cell DNA is 0.1 w / v% to 0.3 w / v%, and most preferably, the concentration of the nonionic triblock copolymer in the mixture with the infectious virus particles and the sample containing the host cell DNA is 0.2 w / v%.

[0067] The concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.05 w / v% to 0.5 w / v%. Preferably, the concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.08 w / v% to 0.4 w / v%, more preferably, the concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.1 w / v% to 0.3 w / v%, and most preferably, the concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.2 w / v%.

[0068] As used herein, the term "sugar" includes monosaccharides (e.g., glucose, galactose, ribose, mannose, rhamnose, talose, xylose, allose or arabinose), disaccharides (e.g., trehalose, sucrose, maltose, isomaltose, cellobiose, gentiobiose, laminaribiose, xylobiose, mannobiose, lactose or fructose), trisaccharides (e.g., acarbose, raffinose, melezitose, panose or cellotriose) and sugar polymers (e.g., dextran, xanthan, pullulan, cyclodextrin, amylose, amylopectin, starch, cellooligosaccharides, cellulose, maltooligosaccharides, glycogen, chitosan or chitin). Furthermore, as used herein, the term "sugar" also includes sugar alcohols, such as mannitol, sorbitol, arabitol, erythritol, maltitol, xylitol, glycitol, glycol, polyglycitol, polyethylene glycol, polypropylene glycol, and glycerol.

[0069] In one embodiment, the sugar is a non-reducing sugar. A non-reducing sugar does not contain an aldehyde or ketone group that can be oxidized. Examples of non-reducing sugars include sucrose and trehalose. In one embodiment, the sugar is trehalose. In one embodiment, the sugar is trehalose dihydrate.

[0070] The sugar concentration in the mixture of the infectious virus particles and the sample containing the host cell DNA is 1 w / v% to 7.5 w / v%. Preferably, the sugar concentration in the mixture of the infectious virus particles and the sample containing the host cell DNA is 1.5 w / v% to 6 w / v%, more preferably, the sugar concentration in the mixture of the infectious virus particles and the sample containing the host cell DNA is 2 w / v% to 5 w / v%, and most preferably, the sugar concentration in the mixture of the infectious virus particles and the sample containing the host cell DNA is 3 w / v%.

[0071] The concentration of the non-reducing sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 1 w / v% to 7.5 w / v%. Preferably, the concentration of the non-reducing sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 1.5 w / v% to 6 w / v%, more preferably, the concentration of the non-reducing sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 2 w / v% to 5 w / v%, and most preferably, the concentration of the non-reducing sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 3 w / v%.

[0072] The concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 1 w / v% to 7.5 w / v%. Preferably, the concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 1.5 w / v% to 6 w / v%, more preferably, the concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 2 w / v% to 5 w / v%, and most preferably, the concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 3 w / v%.

[0073] In one embodiment, the concentration of the nonionic surfactant in the mixture with the sample containing infectious virus particles and host cell DNA is 0.05 w / v% to 0.5 w / v%, and the concentration of the sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 1 w / v% to 7.5 w / v%. Preferably, the concentration of the nonionic surfactant in the mixture with the sample containing infectious virus particles and host cell DNA is 0.08 w / v% to 0.4 w / v%, and the concentration of the sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 1.5 w / v% to 6 w / v%. More preferably, the concentration of the nonionic surfactant in the mixture with the sample containing infectious virus particles and host cell DNA is 0.1 w / v% to 0.3 w / v%, and the concentration of the sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 2 w / v% to 5 w / v%. Most preferably, the concentration of the non-ionic detergent in the mixture with the sample containing infectious viral particles and host cell DNA is 0.2 w / v% and the concentration of the sugar in the mixture with the sample containing infectious viral particles and host cell DNA is 3 w / v%.

[0074] In one embodiment, the concentration of the nonionic triblock copolymer in the mixture with the sample containing infectious virus particles and host cell DNA is 0.05 w / v% to 0.5 w / v%, and the concentration of the non-reducing sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 1 w / v% to 7.5 w / v%. Preferably, the concentration of the nonionic triblock copolymer in the mixture with the sample containing infectious virus particles and host cell DNA is 0.08 w / v% to 0.4 w / v%, and the concentration of the non-reducing sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 1.5 w / v% to 6 w / v%. More preferably, the concentration of the nonionic triblock copolymer in the mixture with the sample containing infectious virus particles and host cell DNA is 0.1 w / v% to 0.3 w / v%, and the concentration of the non-reducing sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 2 w / v% to 5 w / v%. Most preferably, the concentration of the non-ionic triblock copolymer in the mixture with the sample comprising infectious viral particles and host cell DNA is 0.2% w / v and the concentration of the non-reducing sugar in the mixture with the sample comprising infectious viral particles and host cell DNA is 3% w / v.

[0075] In one embodiment, the concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.05 w / v% to 0.5 w / v%, and the concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 1 w / v% to 7.5 w / v%. Preferably, the concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.08 w / v% to 0.4 w / v%, and the concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 1.5 w / v% to 6 w / v%. More preferably, the concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.1 w / v% to 0.3 w / v%, and the concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 2 w / v% to 5 w / v%. Most preferably, the concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.2 w / v%, and the concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 3 w / v%.

[0076] The protein that may be present in the mixture with the infectious virus particles and the sample containing the host cell DNA may be any protein that is essentially inactive and does not react with the infectious virus particles. In particular, the protein does not affect the structure or infectivity of the infectious virus particles. In one embodiment, the protein is not an enzyme or an antibody. In one embodiment, the protein is a structural protein or a serum protein. In one embodiment, the protein is selected from albumin, collagen, hydrolyzed collagen, gelatin and hydrolyzed gelatin.

[0077] In one embodiment, the protein is albumin. Albumin is a family of globular non-glycosylated proteins that are present, among others, in the blood of vertebrates. Albumin is water-soluble, moderately soluble in concentrated saline, and denatured by heating. Suitable albumins for use in the method of the present invention include mammalian serum albumins, such as human serum albumin and bovine serum albumin or lactalbumin.

[0078] In one embodiment, the protein is human serum albumin. Serum albumin is one of the most common proteins in vertebrate blood and has multiple functions. This protein is 585 amino acids with a molecular weight of 66,500D. Human serum albumin is not glycosylated and has a single free thiol group. Human serum albumin can be recombinant human serum albumin or human serum albumin purified from human serum. Preferably, human serum albumin purified from human serum is used.

[0079] The protein concentration in the mixture of the infectious virus particles and the sample containing the host cell DNA is 0.005 w / v% to 0.05 w / v%. Preferably, the protein concentration in the mixture of the infectious virus particles and the sample containing the host cell DNA is 0.01 w / v% to 0.04 w / v%, more preferably, the protein concentration in the mixture of the infectious virus particles and the sample containing the host cell DNA is 0.015 w / v% to 0.03 w / v%, and most preferably, the protein concentration in the mixture of the infectious virus particles and the sample containing the host cell DNA is 0.02 w / v%.

[0080] The albumin concentration in the mixture of the infectious virus particles and the sample containing the host cell DNA is 0.005 w / v% to 0.05 w / v%. Preferably, the albumin concentration in the mixture of the infectious virus particles and the sample containing the host cell DNA is 0.01 w / v% to 0.04 w / v%, more preferably, the albumin concentration in the mixture of the infectious virus particles and the sample containing the host cell DNA is 0.015 w / v% to 0.03 w / v%, and most preferably, the albumin concentration in the mixture of the infectious virus particles and the sample containing the host cell DNA is 0.02 w / v%.

[0081] The concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.005 w / v% to 0.05 w / v%. Preferably, the concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.01 w / v% to 0.04 w / v%, more preferably, the concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.015 w / v% to 0.03 w / v%, and most preferably, the concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.02 w / v%.

[0082] In one embodiment, the concentration of the nonionic surfactant in the mixture with the sample containing infectious virus particles and host cell DNA is 0.05 w / v% to 0.5 w / v%, and the concentration of the protein in the mixture with the sample containing infectious virus particles and host cell DNA is 0.005 w / v% to 0.05 w / v%. Preferably, the concentration of the nonionic surfactant in the mixture with the sample containing infectious virus particles and host cell DNA is 0.08 w / v% to 0.4 w / v%, and the concentration of the protein in the mixture with the sample containing infectious virus particles and host cell DNA is 0.01 w / v% to 0.04 w / v%. More preferably, the concentration of the nonionic surfactant in the mixture with the sample containing infectious virus particles and host cell DNA is 0.1 w / v% to 0.3 w / v%, and the concentration of the protein in the mixture with the sample containing infectious virus particles and host cell DNA is 0.015 w / v% to 0.03 w / v%. Most preferably, the concentration of the non-ionic detergent in the mixture with the infectious virus particles and the sample containing host cell DNA is 0.2 w / v % and the concentration of the protein in the mixture with the infectious virus particles and the sample containing host cell DNA is 0.02 w / v %.

[0083] In one embodiment, the concentration of the nonionic triblock copolymer in the mixture with the sample containing infectious virus particles and host cell DNA is 0.05 w / v% to 0.5 w / v%, and the concentration of albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.005 w / v% to 0.05 w / v%. Preferably, the concentration of the nonionic triblock copolymer in the mixture with the sample containing infectious virus particles and host cell DNA is 0.08 w / v% to 0.4 w / v%, and the concentration of albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.01 w / v% to 0.04 w / v%. More preferably, the concentration of the nonionic triblock copolymer in the mixture with the sample containing infectious virus particles and host cell DNA is 0.1 w / v% to 0.3 w / v%, and the concentration of albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.015 w / v% to 0.03 w / v%. Most preferably, the concentration of the nonionic triblock copolymer in the mixture with the sample containing infectious virus particles and host cell DNA is 0.2 w / v%, and the concentration of albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.02 w / v%.

[0084] In one embodiment, the concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.05 w / v% to 0.5 w / v%, and the concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.005 w / v% to 0.05 w / v%. Preferably, the concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.08 w / v% to 0.4 w / v%, and the concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.01 w / v% to 0.04 w / v%. More preferably, the concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.1 w / v% to 0.3 w / v%, and the concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.015 w / v% to 0.03 w / v%. Most preferably, the concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.2 w / v%, and the concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.02 w / v%.

[0085] In one embodiment, the sugar concentration in the mixture with the infectious virus particles and the sample containing the host cell DNA is 1 w / v% to 7.5 w / v%, and the protein concentration in the mixture with the infectious virus particles and the sample containing the host cell DNA is 0.005 w / v% to 0.05 w / v%. Preferably, the sugar concentration in the mixture with the infectious virus particles and the sample containing the host cell DNA is 1.5 w / v% to 6 w / v%, and the protein concentration in the mixture with the infectious virus particles and the sample containing the host cell DNA is 0.01 w / v% to 0.04 w / v%. More preferably, the sugar concentration in the mixture with the infectious virus particles and the sample containing the host cell DNA is 2 w / v% to 5 w / v%, and the protein concentration in the mixture with the infectious virus particles and the sample containing the host cell DNA is 0.015 w / v% to 0.03 w / v%. Most preferably, the concentration of sugar in the mixture with the infectious viral particles and the sample comprising host cell DNA is 3 w / v % and the concentration of protein in the mixture with the infectious viral particles and the sample comprising host cell DNA is 0.02 w / v %.

[0086] In one embodiment, the concentration of the non-reducing sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 1 w / v% to 7.5 w / v%, and the concentration of albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.005 w / v% to 0.05 w / v%. Preferably, the concentration of the non-reducing sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 1.5 w / v% to 6 w / v%, and the concentration of albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.01 w / v% to 0.04 w / v%. More preferably, the concentration of the non-reducing sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 2 w / v% to 5 w / v%, and the concentration of albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.015 w / v% to 0.03 w / v%. Most preferably, the concentration of non-reducing sugars in the mixture with the sample comprising infectious viral particles and host cell DNA is 3 w / v % and the concentration of albumin in the mixture with the sample comprising infectious viral particles and host cell DNA is 0.02 w / v %.

[0087] In one embodiment, the concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 1 w / v% to 7.5 w / v%, and the concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.005 w / v% to 0.05 w / v%. Preferably, the concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 1.5 w / v% to 6 w / v%, and the concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.01 w / v% to 0.04 w / v%. More preferably, the concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 2 w / v% to 5 w / v%, and the concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.015 w / v% to 0.03 w / v%. Most preferably, the concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 3 w / v%, and the concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.02 w / v%.

[0088] In one embodiment, the concentration of the nonionic surfactant in the mixture with the sample containing infectious virus particles and host cell DNA is 0.05 w / v% to 0.5 w / v%, the concentration of the sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 1 w / v% to 7.5 w / v%, and the concentration of the protein in the mixture with the sample containing infectious virus particles and host cell DNA is 0.005 w / v% to 0.05 w / v%. Preferably, the concentration of the nonionic surfactant in the mixture with the sample containing infectious virus particles and host cell DNA is 0.08 w / v% to 0.4 w / v%, the concentration of the sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 1.5 w / v% to 6 w / v%, and the concentration of the protein in the mixture with the sample containing infectious virus particles and host cell DNA is 0.01 w / v% to 0.04 w / v%. More preferably, the concentration of the nonionic surfactant in the mixture with the sample containing infectious virus particles and host cell DNA is 0.1 w / v% to 0.3 w / v%, the concentration of the sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 2 w / v% to 5 w / v%, and the concentration of the protein in the mixture with the sample containing infectious virus particles and host cell DNA is 0.015 w / v% to 0.03 w / v%. Most preferably, the concentration of the nonionic surfactant in the mixture with the sample containing infectious virus particles and host cell DNA is 0.2 w / v%, the concentration of the sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 3 w / v%, and the concentration of the protein in the mixture with the sample containing infectious virus particles and host cell DNA is 0.02 w / v%.

[0089] In one embodiment, the concentration of the nonionic triblock copolymer in the mixture with the sample comprising infectious virus particles and host cell DNA is 0.05 w / v% to 0.5 w / v%, the concentration of the non-reducing sugar in the mixture with the sample comprising infectious virus particles and host cell DNA is 1 w / v% to 7.5 w / v%, and the concentration of albumin in the mixture with the sample comprising infectious virus particles and host cell DNA is 0.005 w / v% to 0.05 w / v%. Preferably, the concentration of the nonionic triblock copolymer in the mixture with the sample containing infectious virus particles and host cell DNA is 0.08 w / v% to 0.4 w / v%, the concentration of the non-reducing sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 1.5 w / v% to 6 w / v%, and the concentration of albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.01 w / v% to 0.04 w / v%. More preferably, the concentration of the nonionic triblock copolymer in the mixture with the sample containing infectious virus particles and host cell DNA is 0.1 w / v% to 0.3 w / v%, the concentration of the non-reducing sugar in the mixture with the sample containing infectious virus particles and host cell DNA is 2 w / v% to 5 w / v%, and the concentration of albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.015 w / v% to 0.03 w / v%. Most preferably, the concentration of the nonionic triblock copolymer in the mixture with the sample comprising infectious viral particles and host cell DNA is 0.2 w / v%, the concentration of the non-reducing sugar in the mixture with the sample comprising infectious viral particles and host cell DNA is 3 w / v%, and the concentration of albumin in the mixture with the sample comprising infectious viral particles and host cell DNA is 0.02 w / v%.

[0090] In one embodiment, the concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.05 w / v% to 0.5 w / v%, the concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 1 w / v% to 7.5 w / v%, and the concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.005 w / v% to 0.05 w / v%. Preferably, the concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.08 w / v% to 0.4 w / v%, the concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 1.5 w / v% to 6 w / v%, and the concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.01 w / v% to 0.04 w / v%. More preferably, the concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.1 w / v% to 0.3 w / v%, the concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 2 w / v% to 5 w / v%, and the concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.015 w / v% to 0.03 w / v%. Most preferably, the concentration of poloxamer 407 in the mixture with the sample containing infectious virus particles and host cell DNA is 0.2 w / v%, the concentration of trehalose dihydrate in the mixture with the sample containing infectious virus particles and host cell DNA is 3 w / v%, and the concentration of human serum albumin in the mixture with the sample containing infectious virus particles and host cell DNA is 0.02 w / v%.

[0091] Anion exchange chromatography relies on charge-charge interactions between components in the sample and the charges of immobilized functional groups. In anion exchange chromatography, the bound ions present in the sample are negative and the immobilized functional groups are positive. Most viruses are negatively charged at physiological pH and therefore bind to the immobilized positive groups. Commonly used anion exchange functional groups are Q resin, quaternary amines and DEAE resin (diethylaminoethane). In general, however, the anion exchange chromatography step can be performed using all common commercially available anion exchange resins or anion exchange membranes.

[0092] Exemplary strong anion exchange groups that can be used for the present invention include functional groups such as, for example, quaternary aminoethyl (QAE) moieties, primary amines (PA), quaternary ammonium (Q) moieties, and trimethylammonium ethyl (TMAE) groups.

[0093] Resins having a quaternary aminoethyl (QAE) moiety include, for example, Toyopearl QAE (available from Tosoh Bioscience, Germany), Selectacel QAE (a quaternary aminoethyl derivative of cellulose, available from Polysciences Inc. Pennsylvania, USA), etc. Resins having a quaternary ammonium (Q) moiety include, for example, Sartobind® Q (available from Sartorius, Germany), Mustang® Q Acrodisc (available from Pall, Germany), Q Sepharose XL, Q Sepharose FF, Q Sepharose HP, Resource Q (available from GE Healthcare, Germany), Macro Prep High Q (Bio-Rad, California, USA), Toyopearl Super Q (available from Tosoh Bioscience, Germany), UNOsphere Q (available from Bio-Rad, California, USA). Resins with trimethylammoniumethyl (TMAE) groups include, for example, Fractogel EMD TMAE (available from Merck, Germany). Resins with primary amine groups include, for example, Sartobind STIC primary amine resin (available from Sartorius, Germany).

[0094] In the method of the present invention, the anion exchange chromatography step is preferably carried out using an anion exchange chromatography membrane having quaternary ammonium groups. The membrane substrate is selected from stabilized, reinforced cellulose and polyethersulfone. Preferably, the membrane substrate is stabilized, reinforced cellulose. Preferably, the membrane substrate is stabilized, reinforced cellulose and the functional groups are quaternary ammonium groups. Preferably, the anion exchange chromatography step does not involve the use of a monolithic support. The nominal pore size of the membrane is between 0.5 μm and 5 μm, preferably greater than 3 μm. The membrane area is between 20 and 50 cm 2 and preferably 25 to 45 cm2 , more preferably 30 to 40 cm 2 and most preferably 36 cm 2 The flow rate is between 1 and 50 ml / min, preferably between 10 and 40 ml / min, more preferably 30 ml / min. Most preferably, the anion exchange chromatography is carried out using a Sartobind® Q SingleStep membrane.

[0095] Anion exchange chromatography may be performed in a bind and elute mode or in a flow-through mode. In the bind and elute mode, the substance to be purified binds to the anion exchange group while the impurities do not. The substance to be purified can be eluted from the anion exchange group by changing one or more chromatographic conditions, such as the salt conditions in the buffer or the pH of the buffer. In the flow-through mode, the impurities bind to the anion exchange group while the substance to be purified does not bind but can be collected directly from the flow-through. In the present invention, the anion exchange chromatography is preferably performed in a flow-through mode. This means that in the present invention, the host cell DNA binds to the anion exchange group, preferably the quaternary ammonium group, and the infectious virus particles are present in the flow-through.

[0096] The method of the present invention minimizes the loss of viral titer during the purification step. In one embodiment, the viral titer lost during the anion exchange chromatography step is less than 5%. Preferably, the viral titer lost during the anion exchange chromatography step is less than 4%. More preferably, the viral titer lost during the anion exchange chromatography step is less than 3%. Most preferably, the viral titer lost during the anion exchange chromatography step is less than 2% or 1.5%.

[0097] Viral titers can be determined by any method known in the art. In one embodiment, viral titers are determined by immuno-focus assays, known in the art. In immuno-focus assays, serial dilutions of virus are applied to a monolayer of adherent cells, e.g., Vero cells. After a period of time to allow infectious virus to bind and be absorbed by the cells, an overlayer containing a viscous agent, e.g., agarose or carboxymethylcellulose, is added to prevent virus spread and allow progeny viruses to infect only cells adjacent to the first infected cell. After an incubation period to allow virus replication, the cells are fixed and stained using monoclonal antibodies directed against viral proteins and a secondary antibody, e.g., an antibody labeled with alkaline phosphatase. Foci are stained by adding a suitable substrate for the enzyme attached to the secondary antibody, e.g., 5-bromo-4-chloro-3-indolyl-phosphate / nitro blue tetrazolium phosphatase substrate. The number of plaques on the plate corresponds to the plaque-forming units of virus in the solution applied to the cells. For example, a concentration of 1.000 pfu / μl indicates that 1 μl of solution applied to cells contains enough virus to produce 1.000 plaques in the cell monolayer.

[0098] In some prior art methods, host cell DNA is removed by treatment with nucleases, such as enzymes capable of degrading DNA, e.g. endonucleases. A commonly used endonuclease for host cell DNA removal is Benzonase. Benzonase is a modified form of an extracellular endonuclease from Serratia marcescens, and is available, for example, from Sigma Aldrich or Merck Millipore. Since the method of the present invention effectively removes host cell DNA without enzyme treatment, the method of the present invention does not include a step of treating the sample containing infectious virus particles and host cell DNA with a nuclease. In particular, the method of the present invention does not include a step of treating the sample containing infectious virus particles and host cell DNA with Benzonase. In one embodiment, the method of the present invention does not include any step of endonuclease treatment.

[0099] As previously discussed, infectious viral particles are prepared in host cells that can be infected with infectious viruses, where they replicate the viruses when maintained under suitable conditions. Usually, mammalian host cells are used. Suitable mammalian host cells include, but are not limited to, Vero cells, MDCK cells, BHK cells, HEK-293 cells (293), RD cells, HT-1080 cells, A549 cells, Cos-7 cells, ARPE-19 cells, and MRC-5 cells. Preferably, Vero cells are used to prepare infectious viral particles. More preferably, the Vero cells are derived from the WHO General Cell Bank 10-87.

[0100] The host cells used for the production of infectious viral particles are cultured under suitable conditions that allow for the growth of the host cells and the replication of the virus within the host cells. In one embodiment, the host cells are cultured in DMEM with 10% fetal bovine serum (FBS) and without phenol red prior to infection. In one embodiment, the host cells are cultured at a temperature between 36° C. and 38° C. prior to infection. In one embodiment, the host cells are cultured in DMEM with 10% fetal bovine serum (FBS) and without phenol red prior to infection at a temperature between 36° C. and 38° C.

[0101] The culture medium used to infect the host cells with the virus may be different from the culture medium used to culture the host cells prior to infection, hi one embodiment, the culture medium used to infect the host cells may be DMEM with 0.1% F127.

[0102] The medium used to culture the host cells after infection with the virus may be different from the medium used to culture the host cells before infection, hi one embodiment, the medium used to culture the host cells after infection may be DMEM with 0.1% F127.

[0103] After culturing the host cells for a certain period of time, the cell supernatant containing the infectious virus particles may be harvested. In one embodiment, the host cells are cultured for 4 to 10 days, and then the cell supernatant containing the infectious virus particles is harvested. In one embodiment, the cell supernatant containing the infectious virus particles is harvested on the 5th, 6th, 7th, 8th, 9th, or 10th day of culture. In one embodiment, the cell supernatant containing the infectious virus particles is harvested on the 5th, 6th, 7th, 8th, 9th, and 10th days of culture, and after harvesting the cell supernatant containing the infectious virus particles on the 5th, 6th, 7th, 8th, and 9th days, a new culture medium is added to the host cells so that the cells can continue to produce viruses until the next harvest. The cell supernatant containing the infectious virus particles obtained on each day may be purified separately, or the cell supernatant containing the infectious virus particles obtained on each day may be mixed with the cell supernatant containing the infectious virus particles obtained on the other days and then purified.

[0104] After harvesting the host cell culture supernatant, it is subjected to one or more depth filtration steps. In depth filtration, a porous filter medium is used to separate particles and solids from a liquid. Filter media that may be used in depth filtration include, but are not limited to, cellulose acetate, polypropylene, cellulose acetate protected with glass fiber fleece, and polyethersulfone. In one embodiment, the filter medium used in depth filtration is polyethersulfone. The filter used in depth filtration may have a pore size of 0.1 μm to 1 μm, preferably 0.2 μm to 0.8 μm, more preferably 0.2 μm to 0.45 μm. In one embodiment, the filter is a heterogeneous bilayer of polyethersulfone with pore sizes of 0.2 μm and 0.45 μm. Such a filter is commercially available as Sartopore 2.

[0105] After the depth filtration step, the filtrate containing the harvested infectious viral particles is mixed with a composition comprising one or more detergents, sugars and proteins, thereby providing a mixture. In one embodiment, the filtrate is mixed with a composition comprising detergents, sugars and proteins, thereby providing a mixture. The detergents, sugars and proteins and their concentrations in the mixture are defined as above. The composition comprising detergents, sugars and proteins is diluted by mixing with the filtrate. The composition comprising detergents, sugars and proteins is diluted 2-fold to 10-fold, preferably 3-fold to 8-fold, more preferably 4-fold to 6-fold, most preferably 5-fold. The final concentrations of detergents, sugars and proteins after dilution are provided above.

[0106] After anion exchange chromatography, the sample containing infectious viral particles is subjected to one or more tangential flow filtration (TFF) steps, preferably one TFF step. The TFF step acts to concentrate the sample to ensure sufficient working strength and remove low molecular weight impurities. In TFF, the majority of the feed material moves tangentially across the surface of the filter, rather than within the filter.

[0107] Suitable filter materials for TFF include, but are not limited to, crosslinked cellulose and polyethersulfone. Preferably, crosslinked cellulose-based polymers are used, which are available from Sartorius under the trade name Hydrosart. The particle size cutoff of the filter material used in tangential flow filtration determines whether a particular compound is present in the filtrate or retentate. The particle size cutoff may be smaller than 1 / 3 to 1 / 5 of the size of the virus to be purified. In one embodiment, the particle size cutoff is 50 to 300 kDa. Preferably, the particle size cutoff is 100 kDa. In one embodiment, a crosslinked cellulose-based polymer filter with a particle size cutoff of 100 kDa is used in the TFF step. Thus, infectious virus particles are present in the retentate.

[0108] The TFF step can be characterized by the flow rate and the transmembrane pressure. The flow rate is proportional to the transmembrane pressure and inversely proportional to the resistance of the membrane and the filter cake. The flow rate is obtained as the feed flow rate / unit area of ​​the membrane, i.e. liters per square meter per hour (LMH). In the TFF step of the present invention, the flow rate is 100-500 LMH, preferably 120-400 LMH, more preferably 150-300 LMH. Most preferably, the flow rate is 300 LMH. Thus, the TFF step of the method of the present invention is preferably carried out using a crosslinked cellulose-based polymer filter with a separation limit particle size of 100 kDa and a flow rate of 300 LMH. The transmembrane pressure (TMP) is the differential pressure across the membrane. The TMP may be 0.2-0.5 bar, preferably 0.25-0.45 bar, more preferably 0.35-0.45 bar, most preferably 0.35-0.40 bar. Therefore, the TFF step of the method of the present invention is preferably carried out using a crosslinked cellulose-based polymer filter having a separation limit particle size of 100 kDa, a flow rate of 300 LMH, and a TMP of 0.35 to 0.40 bar.

[0109] Two basic filter structures are commonly used in TFF: cartridge filters and cassette filters. In cartridge filters (often called hollow fiber filters), the membrane is formed by a series of parallel hollow fibers. The feed stream passes through the flow path of the fibers and the permeate is collected on the outside of the fibers. In cassette filters, several membrane plates are held apart from each other and from the cassette housing by a support grid. The feed stream passes through the space between the two sheets and the permeate is collected from the opposite side of the sheets. Cassette filters are preferably used in the TFF step of the method of the present invention. In one embodiment of the present invention, hollow fiber filters are not used in the TFF step.

[0110] Preferably, the TFF step excludes diafiltration. Also preferably, the filter membrane is flushed with a buffer 1 to 5 times, preferably 2 times. The buffer used for flushing contains a non-ionic surfactant, a non-reducing sugar, and albumin. Preferably, the buffer used for flushing contains poloxamer 407, trehalose dihydrate, and human serum albumin. The buffer used for flushing contains 0.5 to 3% of a non-ionic surfactant, 20 to 40% of a non-reducing sugar, and 0.05 to 0.3% of albumin. The buffer used for flushing contains 0.5 to 3% of poloxamer 407, 20 to 40% of trehalose dihydrate, and 0.05 to 0.3% of human serum albumin. The buffer used for flushing contains 1% of a non-ionic surfactant, 27% of a non-reducing sugar, and 0.1% of albumin. The buffer used for flushing contains 1% poloxamer 407, 27% trehalose dihydrate, and 0.1% human serum albumin.

[0111] In the TFF step, the solution containing the infectious viral particles is concentrated 2- to 10-fold, preferably 3- to 8-fold, more preferably 4- to 6-fold, and most preferably 5-fold.

[0112] After the TFF step, the purified infectious dengue virus particles are collected from the retentate. In the retentate, the purified infectious virus particles are in solution. The solution containing the purified infectious virus particles may be filtered one or more times. The solution containing the infectious virus particles may be frozen before being used to prepare the formulation.

[0113] In one embodiment, the method for purifying infectious viral particles further comprises a step (f) of preparing a formulation. The formulation may contain more than one type of infectious viral particles. For example, when the infectious viral particles are infectious dengue viral particles, the formulation may contain more than one serotype of dengue virus. Preferably, the formulation contains infectious viral particles with all four serotypes of dengue virus. Therefore, in this case, the step (f) of preparing a formulation may comprise a mixture of infectious viral particles with four dengue serotypes. The step (f) of preparing a formulation may also comprise the preparation of a dried form of the formulation, for example by freeze-drying or spray-drying. In one embodiment, the step (f) of preparing a formulation comprises a mixture of infectious viral particles with four dengue serotypes and freeze-drying the mixture. Methods for freeze-drying a mixture containing live attenuated viruses are known in the art.

[0114] In one embodiment, the formulation contains the same components as the TFF retentate, so that after the purification step, it is not necessary to remove the compounds used in the purification step. Thus, in one embodiment, the formulation contains infectious virus particles, a non-ionic detergent, a sugar and a protein. Preferably, the formulation contains infectious virus particles, poloxamer 407, trehalose dihydrate and human serum albumin. More preferably, the formulation contains infectious dengue virus particles, poloxamer 407, trehalose dihydrate and human serum albumin. Most preferably, the formulation contains infectious dengue virus particles with all four dengue serotypes, poloxamer 407, trehalose dihydrate and human serum albumin. EXAMPLES

[0115] The following examples are included to demonstrate certain aspects and embodiments of the invention that are described in the claims. However, it should be understood by one of ordinary skill in the art that the following descriptions are for illustrative purposes only and should not be taken as limiting the invention in any way.

[0116] Example 1: Preparation of Dengue Virus Strains The methods used to generate the chimeric dengue strains TDV-1, -3 and -4 were standard molecular cloning and DNA engineering methods and are described in Fluang et al. (2003) J. Virology 77(21):11436-11447. The prM-E genes of dengue serotypes 1, 3 and 4 were constructed and introduced into the TDV-2 backbone using the following well-known methods: reverse transcriptase PCR (RT-PCR), PCR, restriction enzyme digestion, DNA fragment ligation, bacterial transformation by electroporation, plasmid DNA preparation, in vitro transcription with T7 RNA polymerase, and transfection of Vero cells by electroporation. The different dengue serotypes were grown separately as described in Fluang et al. (2013) PLOS Neglected Di, 7(5):e2243.

[0117] Example 2: Purification of Dengue virus strains Vero cell supernatants were collected on days 5, 6, 7, 8, 9, and 10 post-infection, and fresh medium was added to the Vero cells on days 5, 6, 7, 8, and 9. Each harvest was filtered through 0.45 + 0.2 μm and 0.1 μm pore sizes. 2 The solution was clarified using a Sartopore 2 filter with a membrane area of ​​1000 μl.

[0118] The clarified harvest was mixed with a solution containing 3% poloxamer 407, 45% trehalose dihydrate, and 0.3% human serum albumin ("3x FTA"). Specifically, one part of 3x FTA was mixed with 14 parts of the clarified harvest so that the final concentration of FTA was 0.2x (0.2% poloxamer 407, 3% trehalose dihydrate, and 0.02% human serum albumin). The mixture was applied to a Sartobind Q SingleSep nanomembrane with stabilized and reinforced cellulose as substrate and quaternary ammonium groups as ion exchange groups. Anion exchange chromatography was performed in flow-through mode and the flow-through was collected. A TFF step was then performed using a Flydrosart membrane with a membrane separation of 100 kDa at a feed flow rate of 300 LMH and a transmembrane pressure of 0.35-0.40 bar. The membrane was flushed twice with a buffer solution containing 1% poloxamer 407, 27% trehalose and 0.1% HSA and the retentate was filtered through a Sartopore 2 filter with a pore size of 0.45 + 0.2 μm. The sample obtained in this manner is shown in Table 2 below as "0.2xFTA stabilized harvest (DMV008-2)".

[0119] In a follow-up experiment, the clarified harvest was not mixed with FTA and anion exchange chromatography was immediately performed on the clarified harvest. The clarified harvest was applied to a Sartobind Q SingleSep nanomembrane with stabilized and reinforced cellulose as the substrate and quaternary ammonium groups as the ion exchange groups. Anion exchange chromatography was performed in flow-through mode and the flow-through was collected to determine host cell DNA content. This sample is shown below in Table 2 as "Clarified Harvest (DMV005-3)".

[0120] Host cell DNA content was determined by quantitative PCR using primers that specifically bind to Vero cell DNA but not to viral nucleic acid and Picogreen® stain. Viral titers were determined by immunofocus assay.

[0121] [Table 2]

[0122] Table 2 shows that anion exchange chromatography significantly reduces the host cell DNA concentration in the samples, as evidenced by the flow-through from the anion exchange chromatography membrane containing ≦50 ng / ml host cell DNA, below the detection limit of the assay. However, there was a large difference in viral titer after anion exchange chromatography depending on the feedstock used. When the clarified harvest was not mixed with FTA to a final concentration of 0.2× FTA, but instead the clarified harvest was immediately subjected to anion exchange chromatography, there was a large loss of viral titer, i.e., 1.33 log 10 A reduction in PFU was observed, whereas minimal loss in viral titer was observed when the clarified harvest was mixed with FTA to a final concentration of 0.2X FTA and then subjected to anion exchange chromatography.

[0123] List of clauses of this invention

[0124] 1. A method for removing host cell DNA from a sample containing infectious viral particles and host cell DNA, comprising: (c) mixing the sample containing infectious viral particles and host cell DNA with a composition comprising one or more non-ionic detergents, sugars and proteins, thereby providing a mixture; (d) subjecting the mixture of step (a) to anion exchange chromatography.

[0125] 2. The method of claim 1, wherein the nonionic surfactant is a nonionic triblock copolymer.

[0126] 3. The method according to claim 1 or 2, wherein the nonionic surfactant is an ethylene oxide-propylene oxide (EO-PO) block copolymer.

[0127] 4. The method of any one of the preceding clauses, wherein the non-ionic surfactant is poloxamer 407 or poloxamer 403.

[0128] 5. The method of any one of the preceding clauses, wherein the concentration of the nonionic surfactant in the mixture of step (a) is 0.05 w / v% to 0.5 w / v%.

[0129] 6. The method according to any one of the preceding clauses, wherein the sugar is selected from trehalose, glucose, sucrose, fructose and maltose, preferably trehalose.

[0130] 7. The method according to any one of the preceding clauses, wherein the concentration of sugar in the mixture of step (a) is between 1 w / v% and 7.5 w / v%.

[0131] 8. The method according to any one of the preceding clauses, wherein the protein is albumin, preferably human serum albumin.

[0132] 9. The method of any one of the preceding clauses, wherein the concentration of protein in the mixture of step (a) is 0.005 w / v% to 0.05 w / v%.

[0133] 10. The method of any one of the preceding clauses, wherein the composition comprises poloxamer 407, trehalose dihydrate and human serum albumin.

[0134] 11. The method according to clause 10, wherein the mixture of step (a) comprises 0.2 w / v% poloxamer 407, 3 w / v% trehalose dihydrate, and 0.02 w / v% human serum albumin.

[0135] 12. The method of any one of the preceding clauses, wherein the anion exchange chromatography is operated in flow-through mode.

[0136] 13. The method of any one of the preceding clauses, wherein the anion exchange chromatography uses an anion exchange chromatography membrane.

[0137] 14. The method of claim 13, wherein the anion exchange chromatography membrane comprises quaternary ammonium groups.

[0138] 15. The method of any one of the preceding clauses, wherein the infectious viral particles are enveloped infectious viral particles.

[0139] 16. The method of any one of the preceding clauses, wherein the infectious viral particles are infectious flavivirus viral particles.

[0140] 17. The method of any one of the preceding clauses, wherein the infectious viral particles are infectious dengue virus particles.

[0141] 18. The method according to clause 17, wherein the infectious dengue virus particles are infectious live attenuated dengue virus particles or infectious chimeric dengue virus particles.

[0142] 19. The method of any one of the preceding clauses, wherein less than 5% of the viral titer is lost during anion exchange chromatography.

[0143] 20. The method of any one of the preceding clauses, wherein the host cell DNA content is reduced by at least 10-fold by anion exchange chromatography.

[0144] 21. The method of any one of the preceding clauses, which does not include a step of treating the sample containing infectious viral particles and host cell DNA with an endonuclease.

[0145] 22. A method for removing host cell DNA from a sample containing infectious dengue virus particles and host cell DNA, comprising: (a) mixing said sample, comprising infectious dengue virus particles and host cell DNA, with a composition comprising one or more non-ionic triblock copolymers, a non-reducing sugar and albumin, thereby providing a mixture; (b) subjecting the mixture of step (a) to anion exchange chromatography.

[0146] 23. A method for removing host cell DNA from a sample containing infectious dengue virus particles and host cell DNA, comprising: (a) mixing said sample, comprising infectious dengue virus particles and host cell DNA, with a composition comprising a non-ionic triblock copolymer, a non-reducing sugar and albumin, thereby providing a mixture; (b) subjecting the mixture of step (a) to anion exchange chromatography.

[0147] 24. The method according to claim 22 or 23, wherein the non-ionic triblock copolymer is an ethylene oxide-propylene oxide (EO-PO) block copolymer.

[0148] 25. The method of any one of clauses 22 to 24, wherein the non-ionic triblock copolymer is poloxamer 407 or poloxamer 403.

[0149] 26. The method of any one of clauses 22 to 25, wherein the concentration of the non-ionic triblock copolymer in the mixture of step (a) is 0.05 w / v% to 0.5 w / v%.

[0150] 27. The method of any one of clauses 22 to 26, wherein the non-reducing sugar is trehalose.

[0151] 28. A method according to any one of clauses 22 to 27, wherein the concentration of non-reducing sugars in the mixture of step (a) is between 1 w / v% and 7.5 w / v%.

[0152] 29. The method of any one of clauses 22 to 28, wherein the albumin is human serum albumin.

[0153] 30. The method of any one of clauses 22 to 29, wherein the concentration of albumin in the mixture of step (a) is 0.005 w / v% to 0.05 w / v%.

[0154] 31. The method of any one of clauses 22 to 30, wherein the composition comprises poloxamer 407, trehalose dihydrate and human serum albumin.

[0155] 32. The method according to clause 31, wherein the mixture of step (a) comprises 0.2 w / v% poloxamer 407, 3 w / v% trehalose dihydrate, and 0.02 w / v% human serum albumin.

[0156] 33. The method of any one of clauses 22 to 32, wherein the anion exchange chromatography is operated in flow-through mode.

[0157] 34. The method of any one of clauses 22 to 33, wherein the anion exchange chromatography uses an anion exchange chromatography membrane.

[0158] 35. The method of claim 34, wherein the anion exchange chromatography membrane comprises quaternary ammonium groups.

[0159] 36. The method of any one of clauses 22 to 35, wherein the infectious dengue virus particles are infectious live attenuated dengue virus particles or infectious chimeric dengue virus particles.

[0160] 37. The method of any one of clauses 22 to 36, wherein less than 5% of the viral titer is lost during anion exchange chromatography.

[0161] 38. The method of any one of clauses 22 to 37, wherein the host cell DNA content is reduced by at least 10-fold by anion exchange chromatography.

[0162] 39. The method of any one of clauses 22 to 38, which does not include a step of treating the sample containing infectious viral particles and host cell DNA with an endonuclease.

[0163] 40. A method for removing host cell DNA from a sample containing infectious dengue virus particles and host cell DNA, comprising: (a) mixing said sample, comprising infectious dengue virus particles and host cell DNA, with a composition comprising 0.2% w / v poloxamer 407, 3% w / v trehalose dihydrate and 0.02% w / v human serum albumin, thereby providing a mixture; (b) subjecting the mixture of step (a) to anion exchange chromatography.

[0164] 41. The method according to clause 40, wherein the anion exchange chromatography is operated in flow-through mode.

[0165] 42. The method of claim 40 or 41, wherein the anion exchange chromatography uses an anion exchange chromatography membrane.

[0166] 43. The method according to clause 42, wherein the anion exchange chromatography membrane comprises quaternary ammonium groups.

[0167] 44. The method of any one of clauses 40 to 43, wherein the infectious dengue virus particles are infectious live attenuated dengue virus particles or infectious chimeric dengue virus particles.

[0168] 45. The method of any one of clauses 40 to 44, wherein less than 5% of the viral titer is lost during anion exchange chromatography.

[0169] 46. ​​The method of any one of clauses 40 to 45, wherein the host cell DNA content is reduced by at least 10-fold by anion exchange chromatography.

[0170] 47. The method of any one of clauses 40 to 46, which does not include a step of treating the sample containing infectious dengue virus particles and host cell DNA with an endonuclease.

[0171] 48. A method for removing host cell DNA from a sample containing infectious dengue virus particles and host cell DNA, comprising: (a) mixing said sample, comprising infectious dengue virus particles and host cell DNA, with a composition comprising a non-ionic triblock copolymer, a non-reducing sugar and albumin, thereby providing a mixture; (b) subjecting the mixture of step (a) to anion exchange chromatography in flow-through mode using an anion exchange membrane containing quaternary ammonium groups.

[0172] 49. The method according to claim 48, wherein the non-ionic triblock copolymer is an ethylene oxide-propylene oxide (EO-PO) block copolymer.

[0173] 50. The method according to clause 48 or 49, wherein the non-ionic triblock copolymer is poloxamer 407 or poloxamer 403.

[0174] 51. A method according to any one of clauses 48 to 50, wherein the concentration of the non-ionic triblock copolymer in the mixture of step (a) is between 0.05% w / v and 0.5% w / v.

[0175] 52. The method of any one of clauses 48 to 51, wherein the non-reducing sugar is trehalose.

[0176] 53. A method according to any one of clauses 48 to 52, wherein the concentration of non-reducing sugars in the mixture of step (a) is between 1 w / v% and 7.5 w / v%.

[0177] 54. The method of any one of clauses 48 to 53, wherein the albumin is human serum albumin.

[0178] 55. A method according to any one of clauses 48 to 54, wherein the concentration of albumin in the mixture of step (a) is between 0.005 w / v% and 0.05 w / v%.

[0179] 56. The method of any one of clauses 48 to 55, wherein the composition comprises poloxamer 407, trehalose dihydrate and human serum albumin.

[0180] 57. The method according to clause 56, wherein the mixture of step (a) comprises 0.2 w / v% poloxamer 407, 3 w / v% trehalose dihydrate, and 0.02 w / v% human serum albumin.

[0181] 58. A method for removing host cell DNA from a sample containing infectious dengue virus particles and host cell DNA, comprising: (a) mixing said sample, comprising infectious dengue virus particles and host cell DNA, with a composition comprising 0.2% w / v poloxamer 407, 3% w / v trehalose dihydrate and 0.02% w / v human serum albumin, thereby providing a mixture; (b) subjecting the mixture of step (a) to anion exchange chromatography in flow-through mode using an anion exchange membrane containing quaternary ammonium groups.

[0182] 59. A method for purifying infectious dengue virus particles from a host cell culture medium, comprising: (a) harvesting a host cell culture supernatant containing infectious dengue virus particles from the host cell culture; (b) combining the harvested infectious dengue virus particles with a composition comprising one or more non-ionic triblock copolymers, a non-reducing sugar, and albumin, thereby providing a combination; (c) subjecting the mixture of step (b) to one or more anion exchange chromatography steps and collecting the flow-through; (d) subjecting the permeate from step (c) to tangential flow filtration and collecting the retentate; (e) recovering purified infectious dengue virus particles from the retentate of step (d).

[0183] 60. A method for purifying infectious dengue virus particles from a host cell culture medium, comprising: (a) harvesting a host cell culture supernatant containing infectious dengue virus particles from the host cell culture; (b) combining the harvested infectious dengue virus particles with a composition comprising a non-ionic triblock copolymer, a non-reducing sugar, and albumin, thereby providing a combination; (c) subjecting the mixture of step (b) to one or more anion exchange chromatography steps and collecting the flow-through; (d) subjecting the permeate from step (c) to tangential flow filtration and collecting the retentate; (e) recovering purified infectious dengue virus particles from the retentate of step (d).

[0184] 61. The method of claim 59 or 60, wherein the nonionic triblock copolymer is an ethylene oxide-propylene oxide (EO-PO) block copolymer.

[0185] 62. The method of any one of clauses 59 to 61, wherein the non-ionic triblock copolymer is poloxamer 407 or poloxamer 403.

[0186] 63. A method according to any one of clauses 59 to 62, wherein the concentration of the non-ionic triblock copolymer in the mixture of step (a) is between 0.05% w / v and 0.5% w / v.

[0187] 64. The method of any one of clauses 59 to 63, wherein the non-reducing sugar is trehalose.

[0188] 65. A method according to any one of clauses 59 to 64, wherein the concentration of non-reducing sugars in the mixture of step (a) is between 1 w / v% and 7.5 w / v%.

[0189] 66. The method of any one of clauses 59 to 65, wherein the albumin is human serum albumin.

[0190] 67. A method according to any one of clauses 59 to 66, wherein the concentration of albumin in the mixture of step (a) is between 0.005 w / v% and 0.05 w / v%.

[0191] 68. The method of any one of clauses 59 to 67, wherein the composition comprises poloxamer 407, trehalose and human serum albumin.

[0192] 69. The method according to clause 68, wherein the composition comprises 0.2 w / v% poloxamer 407, 3 w / v% trehalose dihydrate, and 0.02 w / v% human serum albumin.

[0193] 70. The method of any one of clauses 59 to 69, wherein the anion exchange chromatography uses an anion exchange chromatography membrane.

[0194] 71. The method according to clause 70, wherein the anion exchange chromatography membrane contains quaternary ammonium groups.

[0195] 72. The method of any one of clauses 59 to 71, wherein the infectious dengue virus particles are infectious live attenuated dengue virus particles or infectious chimeric dengue virus particles.

[0196] 73. The method of any one of clauses 59 to 72, which does not include a step of treating the sample containing infectious dengue virus particles and host cell DNA with an endonuclease.

[0197] 74. The method of any one of clauses 59 to 73, wherein the host cell culture is a Vero cell culture.

[0198] 75. The method according to any one of clauses 59 to 74, wherein the host cell culture supernatant containing the infectious dengue virus particles is harvested followed by depth filtration.

[0199] 76. The method according to clause 75, wherein the depth filtration is through 0.45 μm and 0.2 μm filters.

[0200] 77. The method of any one of clauses 59 to 76, wherein the tangential flow filtration comprises filtration through one or more TFF membrane cassettes.

[0201] 78. The method of clause 77, wherein one or more TFF membrane cassettes comprise a cellulose filtration medium.

[0202] 79. A method for purifying infectious dengue virus particles from a host cell culture medium, comprising: (a) harvesting a host cell culture supernatant containing infectious dengue virus particles from the host cell culture; (b) mixing the harvested infectious dengue virus particles with a composition comprising 0.2 w / v% poloxamer 407, 3 w / v% trehalose dihydrate, and 0.02 w / v% human serum albumin, thereby providing a mixture; (c) subjecting the mixture of step (b) to one or more anion exchange chromatography steps and collecting the flow-through; (d) subjecting the permeate from step (c) to tangential flow filtration and collecting the retentate; (e) recovering purified infectious dengue virus particles from the retentate of step (d).

[0203] 80. The method of claim 79, wherein the anion exchange chromatography uses an anion exchange chromatography membrane.

[0204] 81. The method of claim 80, wherein the anion exchange chromatography membrane contains quaternary ammonium groups.

[0205] 82. The method of any one of clauses 79 to 81, wherein the infectious dengue virus particles are infectious live attenuated dengue virus particles or infectious chimeric dengue virus particles.

[0206] 83. The method of any one of clauses 79 to 82, which does not include a step of treating the sample containing infectious dengue virus particles and host cell DNA with an endonuclease.

[0207] 84. The method of any one of clauses 79 to 83, wherein the host cell culture is a Vero cell culture.

[0208] 85. The method according to any one of clauses 79 to 84, wherein the host cell culture supernatant containing the infectious dengue virus particles is harvested, followed by deep bed filtration.

[0209] 86. The method according to clause 85, wherein the depth filtration is through 0.45 μm and 0.2 μm filters.

[0210] 87. The method of any one of clauses 79 to 86, wherein the tangential flow filtration comprises filtration through one or more TFF membrane cassettes.

[0211] 88. The method of clause 87, wherein one or more TFF membrane cassettes comprise a cellulose filtration medium.

[0212] 89. A method for purifying infectious dengue virus particles from a host cell culture medium, comprising: (a) harvesting a host cell culture supernatant containing infectious dengue virus particles from the host cell culture; (b) combining the harvested infectious dengue virus particles with a composition comprising a non-ionic triblock copolymer, a non-reducing sugar, and albumin, thereby providing a combination; (c) subjecting the mixture of step (b) to one or more anion exchange chromatography steps in flow-through mode using an anion exchange membrane containing quaternary ammonium groups and collecting the flow-through; (d) subjecting the permeate from step (c) to tangential flow filtration and collecting the retentate; (e) recovering purified infectious dengue virus particles from the retentate of step (d).

[0213] 90. The method according to claim 89, wherein the non-ionic triblock copolymer is an ethylene oxide-propylene oxide (EO-PO) block copolymer.

[0214] 91. The method according to clause 89 or 90, wherein the non-ionic triblock copolymer is poloxamer 407 or poloxamer 403.

[0215] 92. A method according to any one of clauses 89 to 91, wherein the concentration of the non-ionic triblock copolymer in the mixture of step (a) is between 0.05% w / v and 0.5% w / v.

[0216] 93. The method of any one of clauses 89 to 92, wherein the non-reducing sugar is trehalose.

[0217] 94. A method according to any one of clauses 89 to 93, wherein the concentration of non-reducing sugars in the mixture of step (a) is between 1 w / v% and 7.5 w / v%.

[0218] 95. The method of any one of clauses 89 to 94, wherein the albumin is human serum albumin.

[0219] 96. The method of any one of clauses 89 to 95, wherein the concentration of albumin in the mixture of step (a) is 0.005 w / v% to 0.05 w / v%.

[0220] 97. The method of any one of clauses 89 to 96, wherein the composition comprises poloxamer 407, trehalose and human serum albumin.

[0221] 98. The method according to clause 97, wherein the composition comprises 0.2 w / v% poloxamer 407, 3 w / v% trehalose dihydrate, and 0.02 w / v% human serum albumin.

[0222] 99. The method of any one of clauses 89 to 98, wherein the infectious dengue virus particles are infectious live attenuated dengue virus particles or infectious chimeric dengue virus particles.

[0223] 100. The method of any one of clauses 89 to 99, which does not include a step of treating the sample containing infectious dengue virus particles and host cell DNA with an endonuclease.

[0224] 101. The method of any one of clauses 89 to 100, wherein the host cell culture is a Vero cell culture.

[0225] 102. The method according to any one of clauses 89 to 101, wherein the host cell culture supernatant containing the infectious dengue virus particles is harvested, followed by depth filtration.

[0226] 103. The method according to claim 102, wherein the depth filtration is through 0.45 μm and 0.2 μm filters.

[0227] 104. The method of any one of clauses 89 to 103, wherein the tangential flow filtration comprises filtration through one or more TFF membrane cassettes.

[0228] 105. The method of clause 104, wherein one or more TFF membrane cassettes comprise a cellulose filtration medium.

[0229] 106. A method for purifying infectious dengue virus particles from a host Vero cell culture medium, comprising: (a) harvesting a host Vero cell culture supernatant containing infectious Dengue virus particles from said host Vero cell culture; (b) combining the harvested infectious dengue virus particles with a composition comprising a non-ionic triblock copolymer, a non-reducing sugar, and albumin, thereby providing a combination; (c) subjecting the mixture of step (b) to one or more anion exchange chromatography steps in flow-through mode using an anion exchange membrane containing quaternary ammonium groups and collecting the flow-through; (d) subjecting the permeate from step (c) to tangential flow filtration through one or more TFF membrane cassettes containing cellulose filtration media and collecting the retentate; (e) recovering purified infectious dengue virus particles from the retentate of step (d).

[0230] 107. The method of claim 106, wherein the nonionic triblock copolymer is an ethylene oxide-propylene oxide (EO-PO) block copolymer.

[0231] 108. The method according to clause 106 or 107, wherein the non-ionic triblock copolymer is poloxamer 407 or poloxamer 403.

[0232] 109. A method according to any one of clauses 106 to 108, wherein the concentration of the non-ionic triblock copolymer in the mixture of step (a) is between 0.05 w / v% and 0.5 w / v%.

[0233] 110. The method of any one of clauses 106 to 109, wherein the non-reducing sugar is trehalose.

[0234] 111. A method according to any one of clauses 106 to 110, wherein the concentration of non-reducing sugars in the mixture of step (a) is between 1 w / v% and 7.5 w / v%.

[0235] 112. The method according to any one of clauses 106 to 111, wherein the albumin is human serum albumin.

[0236] 113. The method of any one of clauses 106 to 112, wherein the concentration of albumin in the mixture of step (a) is 0.005 w / v% to 0.05 w / v%.

[0237] 114. The method of any one of clauses 106 to 113, wherein the composition comprises poloxamer 407, trehalose and human serum albumin.

[0238] 115. The method according to clause 114, wherein the composition comprises 0.2 w / v% poloxamer 407, 3 w / v% trehalose dihydrate, and 0.02 w / v% human serum albumin.

[0239] 116. The method according to any one of clauses 106 to 115, wherein the infectious dengue virus particles are infectious live attenuated dengue virus particles or infectious chimeric dengue virus particles.

[0240] 117. The method of any one of clauses 106 to 116, which does not include a step of treating the sample containing infectious dengue virus particles and host cell DNA with an endonuclease.

[0241] 118. The method according to any one of clauses 106 to 117, wherein the host cell culture supernatant containing the infectious dengue virus particles is harvested followed by depth filtration.

[0242] 119. The method according to claim 118, wherein the depth filtration is through 0.45 μm and 0.2 μm filters.

[0243] 120. A method for purifying infectious dengue virus particles from a host Vero cell culture medium, comprising: (a) harvesting a host Vero cell culture supernatant containing infectious Dengue virus particles from said host Vero cell culture; (b) mixing the harvested infectious dengue virus particles with a composition comprising 0.2 w / v% poloxamer 407, 3 w / v% trehalose dihydrate, and 0.02 w / v% human serum albumin, thereby providing a mixture; (c) subjecting the mixture of step (b) to one or more anion exchange chromatography steps in flow-through mode using an anion exchange membrane containing quaternary ammonium groups and collecting the flow-through; (d) subjecting the permeate from step (c) to tangential flow filtration through one or more TFF membrane cassettes containing cellulose filtration media and collecting the retentate; (e) recovering purified infectious dengue virus particles from the retentate of step (d).

[0244] 121. The method according to clause 120, wherein the infectious dengue virus particles are infectious live attenuated dengue virus particles or infectious chimeric dengue virus particles.

[0245] 122. The method of clause 120 or 121, which does not include a step of treating the sample containing infectious dengue virus particles and host cell DNA with an endonuclease.

[0246] 123. The method according to any one of clauses 120 to 122, wherein the host cell culture supernatant containing the infectious dengue virus particles is harvested followed by depth filtration.

[0247] 124. The method according to claim 123, wherein the depth filtration is through 0.45 μm and 0.2 μm filters.

Claims

1. 1. A method for removing host cell DNA from a sample containing infectious flavivirus virions and host cell DNA, comprising: (a) mixing said sample, comprising infectious flavivirus virions and host cell DNA, with a composition comprising one or more poloxamers, sugars and proteins, thereby providing a mixture; (b) subjecting the mixture of step (a) to anion exchange chromatography.

2. 2. The method of claim 1, wherein the poloxamer is poloxamer 407 or poloxamer 403.

3. 3. The method of claim 1 or 2, wherein the sugar is selected from trehalose, glucose, sucrose, fructose and maltose.

4. The method of claim 3, wherein the sugar is trehalose.

5. The method according to any one of claims 1 to 4, wherein the protein is albumin.

6. The method of claim 5, wherein the protein is human serum albumin.

7. The method of any one of claims 1 to 6, wherein the composition comprises poloxamer 407, trehalose dihydrate and human serum albumin.

8. The method according to any one of claims 1 to 7, wherein the anion exchange chromatography is operated in flow-through mode.

9. The method according to any one of claims 1 to 8, wherein the anion exchange chromatography uses an anion exchange chromatography membrane.

10. The method of any one of claims 1 to 9, wherein the infectious flavivirus virus particles are infectious dengue virus particles.

11. 1. A method for purifying infectious flavivirus virions from a host cell culture medium, comprising: (a) harvesting the host cell culture supernatant containing infectious flavivirus virions from the host cell culture; (b) mixing the harvested infectious flavivirus virus particles with a composition comprising one or more poloxamers, sugars, and proteins, thereby providing a mixture; (c) subjecting the mixture of step (b) to one or more anion exchange chromatography steps and collecting the flow-through; (d) subjecting the permeate from step (c) to tangential flow filtration and collecting the retentate; (e) recovering purified infectious viral particles from the retentate of step (d).

12. 12. The method of claim 11, wherein the poloxamer is poloxamer 407 or poloxamer 403.

13. 13. The method according to claim 11 or 12, wherein the sugar is selected from trehalose, glucose, sucrose, fructose and maltose.

14. The method of claim 13, wherein the sugar is trehalose.

15. The method according to any one of claims 11 to 14, wherein the protein is albumin.

16. The method of claim 15, wherein the protein is human serum albumin.

17. The method of any one of claims 11 to 16, wherein the composition comprises poloxamer 407, trehalose and human serum albumin.

18. The method according to any one of claims 11 to 17, wherein the anion exchange chromatography uses an anion exchange chromatography membrane.

19. The method of any one of claims 11 to 18, wherein the infectious flavivirus virus particles are infectious dengue virus particles.

20. The method according to any one of claims 11 to 19, wherein the host cell culture medium is a Vero cell culture medium.

21. The method according to any one of claims 11 to 20, wherein the host cell culture supernatant containing the infectious flavivirus virus particles is harvested followed by depth filtration.

22. 22. The method of any one of claims 11 to 21, wherein the tangential flow filtration comprises filtration through one or more TFF membrane cassettes.

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