US20100158944A1 - Method for influenza virus protection - Google Patents
Method for influenza virus protection Download PDFInfo
- Publication number
- US20100158944A1 US20100158944A1 US12/309,135 US30913507A US2010158944A1 US 20100158944 A1 US20100158944 A1 US 20100158944A1 US 30913507 A US30913507 A US 30913507A US 2010158944 A1 US2010158944 A1 US 2010158944A1
- Authority
- US
- United States
- Prior art keywords
- influenza virus
- particles
- virus
- purification
- influenza
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/145—Orthomyxoviridae, e.g. influenza virus
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
- B01D15/361—Ion-exchange
- B01D15/363—Anion-exchange
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
- B01D15/424—Elution mode
- B01D15/426—Specific type of solvent
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D36/00—Filter circuits or combinations of filters with other separating devices
- B01D36/003—Filters in combination with devices for the removal of liquids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16051—Methods of production or purification of viral material
Definitions
- the invention pertains to a process for the purification of influenza virus or derivative thereof, a process for the manufacturing of influenza virus or its derivative, a fraction of influenza virus or derivative thereof, a vaccine as well as a vector comprising the influenza virus or its derivative.
- the present invention relates to the purification of different quantities of influenza virus or derivative thereof.
- the invention provides a quick, efficient and scalable process for the purification of commercial quantities of influenza virus which can be applied for human usage e.g. prophylactic application such as vaccination.
- Influenza viruses belong to the family Orthomyxoviridae and are separated into types A, B and C according to antigenic differences. Influenza A and B viruses are important respiratory pathogens, although influenza A viruses are the main cause of epidemics with high mortality rate.
- Influenza viruses have a segmented, single-stranded RNA genome of negative polarity.
- the genome of influenza A viruses consist of eight RNA molecules encoding eleven (some influenza A strains ten) protein.
- the viral envelope is composed of a lipid bilayer on a layer of matrix protein (M1). Embedded in the lipid bilayer are glycoproteins hemagglutinin (HA) and neuraminidase (NA), which are responsible for virus attachment and penetration into the cell, and release of progeny virus from the infected cells, respectively. Both proteins also determine the subtypes of the influenza A viruses. High mutation rates and frequent genetic reassortment due to the segmented genome, contribute to great immunological variability, particularly of the HA and NA antigens of influenza A viruses.
- Type B viruses do not exhibit the same degree of antigenic variation and are primarily childhood pathogens, which can occasionally cause epidemics of the same severity as type A.
- influenza associated deaths ranges from 1 to 5 days. Clinical onset is characterised by abrupt fever, headache, malaise and myalgias. Systemic symptoms usually last for 3 days. Although precise data on influenza associated deaths are not available for all countries, in the United Sates influenza associated deaths range between 30 and 150 per 100 000 population aged over 65.
- influenza virus can be adsorbed on insoluble polyelectrolytes at low pH values under 6.0 and subsequently eluted at high pH. Since influenza virus is pH sensitive and infectivity of purified virus was not determined, applicability of this method for purification of infective influenza virus is not known. Polyethylene glycol precipitation, followed by gel permeation chromatography through controlled pore glass beads was also tested for influenza purification and compared to density gradient procedures (Heyward et al., 1977).
- U.S. Pat. No. 3,632,745 describes the purification of influenza viruses by dialysing a suspension of the virus against water containing bivalent metallic cations e.g. magnesium.
- U.S. Pat. Nos. 3,485,718 and 3,547,779 both disclose the use of barium sulphate in the purification of influenza virus.
- U.S. Pat. No. 3,478,145 discloses the use of calcium phosphate in the purification of influenza virus.
- U.S. Pat. No. 3,874,999 describes a process consisting of different centrifugation steps, precipitation (with MgSO 4 ), ultra filtration and dialysis.
- EP-A-0171086 disclose a method for purification of influenza virus with column chromatography using a sulphuric acid ester of cellulose or of a crosslinked polysaccharide.
- the patent application discloses a purification of influenza virus only from allantoic fluid and does not address virus recovery in terms of virus infectivity which remains unknown.
- the virus is eluted from the column with buffer containing high NaCl concentration (1.49 M) which influences virus infectivity and lowers recovery of infectious virus.
- the method is based on a particle support exhibiting low dynamic binding capacity for virus particles which in addition strongly depends on the flow rate. These properties influences down stream process significantly, since large column dimensions are needed and only a low flow rate can be used, all resulting in rather low productivity of the process.
- U.S. Pat. No. 6,008,036 discloses a general method for purifying viruses from a cell line culture by chromatography.
- the disclosed method comprises an ion exchange chromatography step followed by a cation exchange chromatography step and optionally a metal-binding affinity chromatography step.
- influenza purification was the development of the zonal ultracentrifuge (Reimer et al., 1966). This technology revolutionized the purification process and industrial production of influenza and also other viral vaccines. Up to date, it remains the basis for the down stream process of influenza virus vaccines.
- purification of influenza virus from allantoic fluids consists of clarification by centrifugation, concentration by ultra filtration and purification by ultra centrifugation.
- New influenza epidemics and pandemic are likely to occur in the future and current egg-based vaccine production technology seems to be unable to respond to a pandemic crisis.
- a system that can rapidly produce new influenza vaccine is needed.
- One such approach is a cell culture-based process which can be easily scaled up.
- the existing purification of influenza virus vaccine cannot follow this demand.
- Recently, scalable influenza virus purification process comprising of depth filtration, inactivation, ultra filtration and gel filtration was also described (Prasad Nayak et al, 2005). Since virus was inactivated, applicability of this process for purification of infective influenza virus remains unknown.
- the present invention is directed to a process for the purification of influenza virus or derivative thereof comprising the steps of providing a source having influenza virus or derivative thereof, optionally subjecting the source to a pre-purification step, followed by at least one chromatographic step on porous particles having mean pore sizes of at least 20 nm, perfusion particles, gel-in-a-shell particles, tentacle like particles, membrane adsorbers, and monoliths followed by collecting eluting influenza virus or derivatives thereof containing fractions with the proviso that sulfuric ester of cellulose or of cross-linked polysaccharides are excluded.
- the at least one further chromatographic step is performed on materials selected from the group consisting of porous particles having mean pore sizes of at least 20 nm, perfusion particles, gel-in-a-shell particles, tentacle like particles, membrane adsorbers, and monoliths.
- tentacle like particles is well-known to the skilled person. Typical materials falling into this category are disclosed in EP-A-0337144.
- gel-in-a-shell particles is a term used in the art.
- materials produced according to the gel-in-a-shell technology comprise a structure developed from a hyperformance hydrogel which is polymerized in large pores of a solid support such as for example a ceramic support.
- the support can also be produced from organic materials.
- Such configurations are for example disclosed in U.S. Pat. No. 5,268,097 or U.S. Pat. No. 5,234,991.
- EP-A-0337144 refers to materials comprising a primary or secondary aliphatic hydroxyl group-containing support coated with at least one covalently bounded polymer by graft polymerization.
- U.S. Pat. No. 5,268,097 refers to passivated porous solid supports, exhibiting high porosity, physical rigidity, high charge density, high flow rates and chemical stability. Passivated porous solid supports are stabilized against leaching under harsh environmental conditions by the application of a thin, protective polymeric coating atop their porous surfaces and that are characterized by a reversible high sorptive capacity substantially unaccompanied by non-specific adsorption of or interaction with bio-molecules. Commercialized as HyperD®.
- U.S. Pat. No. 5,234,991 refers to porous mineral support coated with an aminated polysaccharide polymer having a cationic character.
- perfusion particles is also well-known in the art. Materials falling into this category are disclosed in U.S. Pat. No. 5,384,042, U.S. Pat. No. 5,552,041, U.S. Pat. No. 5,605,623, and U.S. Pat. No. 5,833,861 and are commercially available under the tradename POROS®.
- U.S. Pat. No. 5,384,042, U.S. Pat. No. 5,552,041, U.S. Pat. No. 5,605,623, and U.S. Pat. No. 5,833,861 disclose matrices defined by first and second interconnected sets of pores and a high surface area for solute interactions in fluid communication with the members of the second set of pores.
- the first and second sets of pores are embodied, for example, as the interstices among particles and throughpores within the particles.
- the pores are dimensioned such that, at high fluid flow rates, convective flow occurs in both pore sets, and the convective flow rate exceeds the rate of solute diffusion in the second pore set.
- the present invention is directed to a process for the manufacturing of influenza virus comprising the steps of infecting cells with influenza virus, propagating the influenza virus in the cells, harvesting of the influenza viruses, and subjecting the harvested influenza virus or derivative thereof to a purification according to the process of the invention of purification of influenza virus or derivative thereof.
- the process of the invention is suitable for the purification of laboratory and commercially useful quantities of influenza virus, e.g. either for vaccine or as viral vector use.
- the invention advantageously avoids problems associated with existing methods of purifying influenza virus and relies on ultra filtration and chromatographic techniques which enable simple scale up of the process.
- the process of the invention accomplishes to purify influenza virus particles from cell lysate employing optionally ultra filtration, followed by a step in which virus is bound and subsequently eluted from the chromatographic support and a final step in which virus is purified out of remaining impurities and at the same time changing the buffer in which virus was prior to the step.
- the present purification process greatly reduces the level of contaminants from influenza virus sample and enables the application of purified influenza virus for human use.
- Ultra filtration devices can include flat sheet or hollow fibre design
- chromatographic supports include different basic material (natural and synthetic polymers) with different active groups and different types of the bed (porous particles, membranes, monoliths).
- the process of the invention is advantageous because due to the mild conditions the potency of the influenza virus, such as immunogenicity and/or infectivity can be substantially maintained.
- the elution buffer comprises ⁇ 1M NaCl, preferably ⁇ 0.8M NaCl, preferably ⁇ 0.5M NaCl, alternatively ⁇ 0.3M NaCl.
- the influenza virus or its derivative is substantially free of DNA so that treatment with deoxyribonucleases (DNAses) can be avoided.
- FIG. 1 Chromatogram showing the elution profile of influenza virus from three CIM® QA 8 ml tube monolithic columns connected in parallel.
- FIG. 2 Chromatogram showing the elution profile of influenza virus from Sepharose 6 Fast Flow (Index 70/90, 2000 ml).
- FIG. 3 SDS-PAGE and Western blot analysis of fractions purified on CIM® QA monolithic columns and Sepharose 6 Fast Flow.
- FIG. 4 Southern blot analysis of fractions purified on CIM® QA monolithic column.
- FIG. 5 Chromatogram showing the elution profile of influenza virus from CIM® DEAE disk monolithic column.
- FIG. 6 Chromatogram showing the elution profile of influenza virus from CIM® QA disk monolithic column.
- FIG. 7 Chromatogram showing the elution profile of influenza virus from CIM® EDA disk monolithic column.
- FIG. 8 Purification of influenza virus on Q Sepharose XL Virus.
- FIG. 9 Purification of influenza virus on CIM® QA disk monolithic columns.
- FIG. 10 Purification of influenza virus on Mustang Q membrane.
- FIG. 11 Elution profile of influenza virus from Sepharose 6 Fast Flow.
- FIG. 12 Elution profile of influenza virus from Fractogel® BioSEC.
- FIG. 13 Elution profile of influenza virus from Toyopearl HW-75.
- the present invention deals with the development of the process for the purification of useful quantities of any influenza virus or its derivative, especially for vaccine or as viral vector use, either for laboratory or industrial scale needs.
- the invention avoids problems associated with existing methods of purifying influenza virus and relies on ultra filtration and chromatographic techniques which enable simple process scale up and increase productivity.
- Existing methods for purification of influenza virus are based on ultracentrifugation and as such are long lasting, inefficient and difficult to scale up.
- influenza virus or its derivative is purified by providing a source having influenza virus or derivative thereof, optionally subjecting the source to a pre-purification step, followed by at least one chromatographic step on porous particles having pore sizes of at least 20 nm, membrane adsorbers, and monoliths, collecting eluting influenza virus or derivatives thereof containing fractions.
- the porous particles of particle sizes of about 10 ⁇ m to about 50 ⁇ M have mean pore sizes of from about 20 nm to about 500 nm or even more measured by mercury porosimetry.
- At least one further chromatographic step can be performed on materials, preferably selected from the group consisting of porous particles having mean pore sizes of at least 20 nm, perfusion particles, gel-in-a-shell particles, tentacle like particles, membrane adsorbers, and monoliths.
- elution of the influenza virus is performed at mild conditions, i.e. low ionic strength of the elution buffer.
- the at least one chromatographic step on porous particles, membrane adsorbers, and monoliths may be performed with ion exchange, affinity, hydrophobic or hydrophilic interaction, size exclusion materials or combinations thereof.
- DNA nucleases can be added before or after chromatographic step.
- Suitable sources of influenza virus or its derivatives are any eukaryotic cells which support replication of the influenza virus.
- a preferred host cell is a mammalian host cell line which supports infection and replication of influenza virus.
- derivatives of influenza virus are in particular genetically modified influenza viruses or virus-like particles or influenza virus particles (viral vectors) delivering foreign material, such as biologically or pharmaceutically active substances, e.g. biopolymers but also small molecules.
- Biopolymers are in particular proteins, such as antibodies, receptors enzymes; nucleic acids (antisense nucleic acids), lipids or polysaccharides.
- Influenza virus-like particles can be generated e.g. from influenza virus proteins expressed in eukaryotic cells or by in vitro manipulation of influenza virus and/or molecules comprising influenza virus. Genetic manipulation of influenza virus genome may include mutations, deletions, insertions or any other manipulation of influenza virus genome.
- influenza virus contains at least one modification and/or deletion of the NS1 gene.
- influenza virus or derivative thereof are selected from the group consisting of influenza virus wild type, influenza virus containing modifications including substitution mutations insertions and/or deletions, and immunogenic influenza-virus-like particles.
- the process of purification according to the invention is also employed in a process for the manufacturing of influenza virus comprising the steps of infecting cells with influenza virus, propagating the influenza virus in the cells, harvesting of the influenza viruses, and subjecting the harvested influenza virus or derivative thereof to the purification process of the invention.
- the present invention discloses and claims a process for the manufacturing or purification of influenza virus or derivative thereof comprising the steps of:
- Subject matter of the invention is also a fraction of influenza virus or derivative thereof, and a vaccine optionally comprising adjuvant, and/or pharmaceutically acceptable carriers obtainable according to the process of the invention of purification of influenza virus or derivative thereof.
- Influenza viral vector can include a heterologous nucleic acid sequence, which can be transferred from influenza viral vector to a host cell.
- a heterologous nucleic acid sequence may code for therapeutic protein, therapeutic nucleic acid or protein causing immune response in the organism.
- the pre-purification step comprises centrifugation, filtration, ultra filtration, selective precipitation, expanded bed chromatography, batch chromatography including magnetic beads or combinations thereof.
- cell debris and other contaminants are removed from cell lysate which may be additionally adjusted to conditions optimal for further purification.
- the prepurification step may comprise centrifugation, filtration, and ultra filtration. Selective precipitation using polyethylene glycol or any other compound causing influenza virus or contaminants precipitation is also possible.
- Expanded bed chromatography and batch chromatography may be used as pre-purification steps to avoid problems such as column clogging and blocking with cell debris.
- influenza virus can be purified by the present invention, whether it is a wild type, a mutant, or a recombinant virus or a virus-like-particle.
- Influenza virus can be prepared and cultivated according to methods known in the art.
- the influenza virus or derivative thereof are selected from the group consisting of influenza virus wild type, influenza virus containing mutations including insertions and deletions, and influenza virus-like particles.
- Mutations are changes in nucleic acids sequences leading to changes in amino acid sequence of protein coded by relevant gene. Deletions are mutations in which a region of the nucleic acid has been eliminated. Insertions are additional stretches of base pairs inserted into nucleic acid sequence.
- Influenza-virus-like particles may be generated from influenza virus proteins expressed in different eukaryotic cells.
- influenza virus-like particles are immunogenic, which means that they are inducing an immune response after entering certain organism.
- the methods described here permit retrieval of purified infective influenza virus particles at a high concentration in aqueous media.
- the methods are suitable for the preparation of laboratory or industrial quantities of any influenza virus particles.
- Influenza virus can be propagated and prepared by any method known in the art.
- the virus can be cultured in chicken eggs or cell cultures according to known procedures explained in the literature.
- influenza virus is harvested from virus-infected cells, for example Vero cells. Cells may be infected at high multiplicity of infection in order to optimize yield. Any method suitable for recovering virus from infected cells may be utilised.
- Cell debris can be removed by centrifugation followed by filtration. Centrifugation with or without filtration is feasible, but the combination of both methods may be the most robust method of clarification in the present invention. For smaller volumes batch centrifugation is possible but for larger volumes (over 50 L) continuous centrifugation is preferred over the batch method.
- the filtration step can be contemplated to additionally remove cell debris and to increase robustness of the process. Maintaining the balance between good clarification and overall yield requires investigation of a large variety of filter types. An exemplary filtration would use 0.65-0.45 ⁇ m cellulose acetate filter.
- Cleared lysate containing influenza virus may then be subjected to the chromatographic technique or it may be initially concentrated using ultra filtration and subsequently purified on chromatographic columns.
- Ultra filtration using tangential flow is preferred and different devices can be used (e.g. Proflux and Labscale TFF System, both Millipore).
- the particular ultra filtration membrane selected will be of a size sufficient small to retain influenza virus but large enough to efficiently clear impurities.
- nominal molecular weight cut-offs between 100 and 1000 kDa may be appropriate (e.g. UFP-750-E-5A, GE Healthcare; Biomax NMWC 1000, Millipore).
- the membrane composition may be, but it is not limited to, regenerated cellulose, polyethersulfone, polysulfone.
- Membranes can be flat sheets or hollow fibres.
- the concentration factor during ultra filtration will be a function of culture conditions, including medium, cell density and viral productivity and can be adjusted by a skilled person. Approximately, e.g. in a manner per se known, a 10 fold concentration is useful.
- Typical parameters that should be optimized are flux rate and trans-membrane pressure. In combination with nominal molecular weight cut-off these two parameters should enable efficient impurity removal and high virus yield at the same time. The skilled person knows how to deal with these parameters and can easily design experiments for optimization.
- At least 80% of host cell proteins are separated from influenza virus and can be determined in the permeate.
- membranes with cut-offs 300 kDa, 500 kDa and 750 kDa typically more than 90% of proteins are separated from the virus which is maintained in the retenate.
- the virus may be further purified based on its surface charge.
- cleared lysate or concentrate prepared by ultra filtration may be purified by ion exchange chromatography. Cleared lysate or concentrate can be loaded directly onto the column or can be adjusted to certain conditions in order to achieve maximum efficiency of the method.
- Different buffer solutions can be used for adjustment and for ion exchange chromatographic step. Examples of buffers that can be used in the ion exchange chromatographic step include phosphate, phosphate citrate, Tris-HCl, MOPS, HEPES, and the like. Buffers can also be formulated incorporating a stabilizing agent, e.g. disaccharides such as sucrose.
- Ion exchange chromatographic steps allow for the use of a wide variety of commercially available chromatographic materials known to be useful in fractionation of biological materials.
- Chromatographic supports of different basic material may be used in the invention. These basic materials may be of different shape, including particle supports, membranes and monoliths.
- Anion exchange chromatography may be performed by utilizing various functional groups, including, but not limited to, DEAE (diethyl amine), EDA (ethylene diamine) and QA (quaternary amine). These functional groups may be attached to any suitable resin useful in the present invention.
- Cation exchange chromatography also may be used for influenza virus purification, including but not limited to, SO 3 (sulfonyl) and CM (carboxymethyl) functional groups attached to any suitable resin.
- Cleared lysate or concentrate prepared by ultra filtration is adjusted to conditions under which influenza virus may be bound to positively (anion) or negatively (cation) charged functional groups on the surface of the chromatographic support. Subsequently, bound virus particles are eluted from chromatographic support using increased ionic strength of the buffer.
- a monolithic support based on poly(glycidyl methacrylate-co-ethylene dimethacrylate) matrix, with quaternary amine (QA) functional groups (e.g. CIM® QA monolithic column) is used in the ion exchange chromatography step of the process of the present invention.
- Cleared cell lysate or ultra filtration concentrate may be adjusted to conditions which enable influenza virus binding to the positively charged functional groups of the chromatographic support.
- buffer with high buffer capacity at pH value between 7 and 8 is used for this step (e.g. Tris, HEPES). Under such conditions negatively charged virus particles are bound to positively charged functional groups on the surface of monolithic support. Elution of the virus particles is achieved e.g. by the increasing ionic strength of the buffer, in particular using sodium chloride.
- This anion exchange chromatography step may be used for DNA removal as well. Since host cell DNA is negatively charged it will be bound to functional groups on the surface of chromatographic support. DNA is eluted from the anion exchange support at higher ionic strength compared to influenza virus. Material eluted from an anion exchange column at ionic strength suitable for influenza virus elution will not contain host cell DNA. As a consequence the treatment of the influenza virus containing solution with deoxiribonuclease (e.g. Benzonase, Merck) is in the preferred embodiment not needed.
- deoxiribonuclease e.g. Benzonase, Merck
- Influenza virus sample eluted from anion exchange column may be further purified on the basis of its size.
- the buffer in which virus was eluted from the anion exchange column is exchanged more or less at the same time.
- size exclusion chromatography and tangential flow filtration are preferred. Both methods enable impurity removal and buffer exchange at almost the same time.
- Tangential flow ultra filtration is a method which may be used to remove residual protein and nucleic acids as well as for exchanging working buffer into a final formulation buffer.
- Ultra filtration using tangential flow is preferred and different devices can be used (e.g. Proflux and Labscale TFF System, both Millipore).
- the particular ultra filtration membrane selected will be of a filter pore size sufficient small to retain influenza virus but large enough to allow penetration of impurities.
- nominal molecular weight cut-offs between 100 and 1000 kDa may be appropriate (e.g. UFP-750-E-5A, GE Healthcare; Biomax NMWC 1000, Millipore).
- the membrane composition may be, but it is not limited to, regenerate cellulose, polyethersulfone, polysulfone.
- Membranes can be of flat sheet or hollow fibre type. Main parameters that must be optimized are flux rate and trans-membrane pressure. In combination with nominal molecular weight cut-off these two parameters will enable efficient purification and buffer exchange and high virus yield.
- Size exclusion chromatography also enables simultaneous impurity removal and buffer exchange.
- SEC allow for the use of wide variety of commercially available chromatographic materials known to be useful in SEC of biological materials.
- a chromatographic support of different basic materials (natural and synthetic polymers) may be used in the invention.
- SEC supports may have different particle and pore size.
- SEC supports with pore sizes which results in the influenza virus particles being completely excluded out of the pores of the resin particles and therefore eluting in the void volume of the column are used in this invention.
- resin particle pore sizes should enable entrance of the impurities resulting in the longer elution volume if compared to the influenza viral particle allowing further purification of the product.
- SEC support such as Sepharose 6 FF (GE Healthcare), Fractogel® EMD BioSEC (Merck kGaA) and Toyopearl® HW-75 (Tosoh Bioscience), in a group separation mode may be used for influenza virus purification.
- Buffers that can be used in a SEC step include phosphate, phosphate citrate, and other biologically compatible buffers. Buffers can also be formulated with a stabilizing agent, e.g. disaccharides such as sucrose and/or detergent. Compounds preventing ion interactions between SEC resin and influenza virus, such as sodium chloride may be also added to the buffer.
- sterile filtration may be performed to eliminate bioburden. Therefore SEC eluate or final retentate from the ultra filtration step may be filtered through a 0.22 ⁇ m filter.
- the filter may be constructed from various materials, which may include but are not limited to polypropylene, cellulose, cellulose esters, nylon, polyethersulfone, or any other material which is consistent with low unspecific influenza virus binding.
- the filter may have a single membrane layer or more than one layer or may incorporate a prefilter of the same or different material. The filtrated influenza virus can be held frozen or kept at approximately 4° C. for subsequent manipulation.
- the present invention describes an influenza virus purification process composed of ultra filtration and chromatographic methods. Scale up of ultra filtration and chromatographic methods as such are well known in the art.
- the disclosed invention can be used on a laboratory scale or can be transferred to pilot or industrial scale using previously described methods.
- last step namely sterile filtration, or last two steps, namely sterile filtration and SEC (or ultra filtration) can be omitted when there is no strict demand for high purity of the influenza viral particle (this especially applies for the laboratory scale applications).
- This example demonstrates the preferred influenza virus purification method of the present invention using tangential flow filtration, anion exchange chromatography, size exclusion chromatography and sterile filtration followed each other in this order.
- Influenza virus reassortment of A/PR/8/34 Mt Sinai with deletion in NS1 gene and IVR-116 was cultivated on Vero cells in GibcoTM OptiPROTM SFM medium. Approximately 15 000 mL of cell lysate was clarified by centrifugation at 2400 rpm at room temperature for 10 minutes. The supernatant was collected and filtered through a Sartobran P 500 cm 2 capsule filter with a cellulose acetate membrane, 0.65-0.45 ⁇ m.
- the filtrate was concentrated by tangential flow filtration using Proflux system (Millipore) and hollow fibre TFF cartridge with 750 kDa nominal cut-off and 0.12 m 2 area (GE Healthcare, UFP-750-E-5A).
- the trans-membrane pressure was 0.2 to 0.3 bar.
- the final volume of concentrate was 1400 ml.
- the concentrate was further purified using three CIM® QA 8 ml tube monolithic columns connected in parallel (BIA Separations), having a total bed volume of 24 ml.
- the following buffers were used:
- the size exclusion chromatography was used as a final purification and buffer exchanging step.
- Index 70/950 column was filled with Sepharose 6 Fast Flow (GE Healthcare, 17-0159-05) to 2000 ml bed volume (N:4664/m).
- SPG buffer (0.218 M sucrose, 0.0038 M KH2PO4, 0.0072 M K2HPO4, 0.0049 K-glutamate pH 8.0 ⁇ 0.2) was used and flow rate was 20 ml/min.
- the column was sanitised with 5 column volumes of 1M NaOH (2 hours contact time) and equilibrated with 5 column volumes of SGP buffer. 40 ml of CIM® QA eluate (2% of the columns volume) was loaded on the column.
- An influenza virus containing fraction was eluted in void volume of the column and collected in 128 ml ( FIG. 2 ).
- the collected fractions were tested for host cell protein content ( FIG. 3 ), DNA size ( FIG. 4 ), protein content by BCA method, DNA content by the Picogreen method, and virus titer was determined by using the TCID50 assay (Table 1). The results confirm that the described process enables a fast (single day) and efficient purification of influenza virus.
- FIG. 3 shows SDS-PAGE and Western blot analysis of fractions purified on CIM® QA monolithic columns and Sepharose 6 Fast Flow.
- A PAGE Gold Tris-Glycine gel (10-20%) (Cambrex) stained with a Silver staining kit PlusOne (GE Healthcare).
- Line 1 PageRuler Protein Ladder (Fermentas, 200, 150, 120, 100, 85, 70, 60, 50, 40, 30, 25, 20, 15, 10 kDa);
- Line 2 TFF concentrate;
- Line 3 CIM® QA eluate;
- Line 4 SEC eluate;
- Line 5 Vero host cell protein.
- Line 1 PageRuler Protein Ladder (Fermentas, 200, 150, 120, 100, 85, 70, 60, 50, 40, 30, 25, 20, 15, 10 kDa);
- Line 2 TFF concentrate;
- Line 3 CIM® QA eluate;
- Line 4 SEC eluate;
- Line 5 Vero host cell protein.
- FIG. 4 depicts Southern blot analysis of fractions purified on CIM® QA monolithic column A: Agarose gel electrophoresis stained by ethidium bromide.
- Line 1 DNA Standard (1 kb Plus DNA Ladder, Invitrogen);
- Line 2 DNA molecular weight marker III, DIG labelled (Roche);
- Line 3 Vero cells DNA;
- Line 4 Vero cells DNA;
- Line 5 TFF concentrate;
- Line 6 CIM® QA eluate B: Southern blot and detection of Vero cells DNA with probe labeled with digoksigenin (DIG High Prime DNA Labelling and detection Starter Kit II).
- Line 1 DNA Standard (1 kb Plus DNA Ladder, Invitrogen); Line 2: DNA molecular weight marker III, DIG labelled (Roche); Line 3: Vero cells DNA; Line 4: Vero cells DNA; Line 5: TFF concentrate; Line 6: CIM® QA eluate.
- chromatographic support three different types are compared.
- the example illustrates that the porous particle support, membranes and monoliths can be used for the purification of influenza virus.
- An aliquot of a concentrate containing influenza virus was applied to the column Q Sepharose XL, virus lincesed (1 ml bed volume), at a flow rate of 1 ml/min.
- the virus was eluated from the column in step gradient of Buffer B. Remaining impurities including host cell DNA were eluted using Buffer C ( FIG. 8 ).
- Another aliquot of the virus concentrate was applied to a Mustang Q coin (0.35 ml bed volume) at flow rate of 3 ml/min and yet another aliquote to a CIM® QA disk monolithic column (0.34 ml bed volume) at 6 ml/min.
- SEC size exclusion chromatographic
- Sepharose 6 FF GE Healthcare
- Toyopearl HW-75 Tosoh Bioscience
- Fractogel® EMD BioSEC Merck
- SPG buffer 0.218 M sucrose, 0.0038 M KH 2 PO 4 , 0.0072 M K 2 HPO 4 , 0.0049 K-glutamate pH 8.0 ⁇ 0.2
- influenza virus containing fraction eluting from the CIM QA monolithic column was loaded on different SEC supports at 1 ml/min (30 cm/h). The collected fractions were analysed for an influenza virus content by hemagglutination assay and the virus yield was calculated (Table 4). From Sepharose 6 FF ( FIG. 11 ) and Fractogel® EMD BioSEC ( FIG. 12 ) the influenza virus was eluted in the void volume of the column, and on Toyopearl HW-75 ( FIG. 13 ) the virus interacted with the support and eluted later.
- chromatographic support four different types are compared with regard to dynamic binding capacity for influenza virus particles.
- the example illustrates the difference between membranes, monoliths, and particles on one side compared to material according to EP-A-0 171 086—represented by Celufine sulphate—on other side.
- Influenza virus reassortment of A/PR/8/34 Mt Sinai with deletion in NS1 gene and IVR-116 was cultivated on Vero cells in GibcoTM OptiPROTM SFM medium. The cell lysate was clarified by centrifugation at 2400 rpm at room temperature for 10 minutes. The supernatant was collected and concentrated by a tangential flow filtration using Labscale TFF system (Millipore) and a Pellicone Biomax 300 kDa cut-off membrane (Millipore, PXB3 00C 50).
- the concentrate was used for the comparison of four different chromatographic supports: CIM® QA disk monolithic column (BIA Separations), Q Sepharose XL virus licensed (GE Healthcare), Mustang Q coin (Pall) and Celufine sulfate (Chisso).
- Dynamic binding capacity for each support was calculated at break-through (Table 5). 10 to 100 fold difference in dynamic binding capacity exist between particle supports (Celufine sulfate and Q Sepharose XL) on one and monoliths and membranes (CIM QA and Mustang Q) on the other side. Such differences may significantly influence design of purification process.
- chromatographic support In this example four different types of chromatographic support are compared.
- the example illustrates that particle support, membranes and monoliths can be used for the purification of influenza virus but a difference significantly influencing the purification process design can be observed.
- Influenza virus reasortment of A/PR/8/34 Mt Sinai with deletion in NS1 gene and IVR-116 was cultivated on Vero cells in GibcoTM OptiPROTM SFM medium. The cell lysate was clarified by centrifugation at 2400 rpm at room temperature for 10 minutes. The supernatant was collected and concentrated by a tangential flow filtration using Labscale TFF system (Millipore) and a Pellicone Biomax 300 kDa cut-off membrane (Millipore, PXB3 00C 50).
- the concentrate was used for the comparison of four different chromatographic supports: CIM® QA disk monolithic column (BIA Separations), Q Sepharose XL virus licensed (GE Healthcare), Mustang Q coin (Pall) and Celufine sulfate (Chisso).
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/309,135 US20100158944A1 (en) | 2006-07-11 | 2007-07-06 | Method for influenza virus protection |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP06116979.3 | 2006-07-11 | ||
EP06116979A EP1878791A1 (en) | 2006-07-11 | 2006-07-11 | Method for influenza virus purification |
US83033906P | 2006-07-13 | 2006-07-13 | |
US12/309,135 US20100158944A1 (en) | 2006-07-11 | 2007-07-06 | Method for influenza virus protection |
PCT/EP2007/056890 WO2008006780A1 (en) | 2006-07-11 | 2007-07-06 | Method for influenza virus purification |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2007/056890 A-371-Of-International WO2008006780A1 (en) | 2006-07-11 | 2007-07-06 | Method for influenza virus purification |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/791,691 Continuation US10570376B2 (en) | 2006-07-11 | 2015-07-06 | Method for influenza virus purification |
Publications (1)
Publication Number | Publication Date |
---|---|
US20100158944A1 true US20100158944A1 (en) | 2010-06-24 |
Family
ID=37478911
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/309,135 Abandoned US20100158944A1 (en) | 2006-07-11 | 2007-07-06 | Method for influenza virus protection |
US14/791,691 Active US10570376B2 (en) | 2006-07-11 | 2015-07-06 | Method for influenza virus purification |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/791,691 Active US10570376B2 (en) | 2006-07-11 | 2015-07-06 | Method for influenza virus purification |
Country Status (6)
Country | Link |
---|---|
US (2) | US20100158944A1 (nl) |
EP (3) | EP1878791A1 (nl) |
CN (1) | CN101490248B (nl) |
BR (1) | BRPI0714292B8 (nl) |
MX (1) | MX2009000026A (nl) |
WO (1) | WO2008006780A1 (nl) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100279329A1 (en) * | 2007-09-04 | 2010-11-04 | University Of Brighton | Method for the stabilisation of purified p-glycoprotein |
US20110272357A1 (en) * | 2009-01-21 | 2011-11-10 | Sartorius Stedim Biotech Gmbh | Apparatus and method for material separation |
US20150030565A1 (en) * | 2012-01-09 | 2015-01-29 | Sanofi Pasteur Biologics, Llc | Purification of flaviviruses |
WO2015133972A1 (en) * | 2014-03-07 | 2015-09-11 | Agency For Science, Technology And Research | Apparatus and methods for fractionation of biological products |
CN111527202A (zh) * | 2017-11-08 | 2020-08-11 | 蓝天疫苗有限责任公司 | So3色谱在病毒纯化方法中的应用 |
Families Citing this family (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2267119A4 (en) * | 2008-03-31 | 2011-05-25 | Japan Tobacco Inc | PROCESS FOR VIRUS ENRICHMENT |
BRPI0822510B8 (pt) * | 2008-04-30 | 2021-05-25 | Shanghai Zerun Biotech Co Ltd | método de preparação e uso de proteínas de lise de células vero, e kit de ensaio para a determinação de hcps de células vero. |
CN101571549B (zh) * | 2008-04-30 | 2013-08-14 | 上海泽润生物科技有限公司 | Vero细胞HCP检测试剂盒及其应用 |
CN101570566B (zh) * | 2008-04-30 | 2013-02-20 | 上海泽润生物科技有限公司 | Vero细胞裂解蛋白、制备方法及其用途 |
EP2233152A1 (en) | 2009-03-24 | 2010-09-29 | Avir Green Hills Biotechnology Research Development Trade Ag | High growth reassortant influenza A virus |
EP2519539A4 (en) * | 2009-12-28 | 2013-11-13 | Ligocyte Pharmaceuticals Inc | METHODS FOR STABILIZING VIRUS-LIKE PARTICULATE SOLUTIONS BASED ON INFLUENZA-INFLUENZA-VIRUSED VIRUSES |
KR101847405B1 (ko) | 2010-07-30 | 2018-04-10 | 이엠디 밀리포어 코포레이션 | 크로마토그래피 매질 및 방법 |
US8778653B2 (en) | 2010-08-12 | 2014-07-15 | Yisheng Biopharma Holdings Ltd. | Method for reducing DNA impurities in viral compositions |
DE102010046817A1 (de) | 2010-09-28 | 2012-03-29 | Sartorius Stedim Biotech Gmbh | Verfahren zur Abtrennung von Viren aus einem Kontaminanten enthaltenden flüssigen Medium |
TW201233803A (en) | 2010-12-02 | 2012-08-16 | Oncolytics Biotech Inc | Lyophilized viral formulations |
US9045728B2 (en) | 2010-12-02 | 2015-06-02 | Oncolytics Biotech Inc. | Liquid viral formulations |
WO2012082723A2 (en) * | 2010-12-15 | 2012-06-21 | Emd Millipore Corporation | Purification of immunogens using a non-polysaccharide matrix |
SG194449A1 (en) * | 2011-04-29 | 2013-12-30 | Oncolytics Biotech Inc | Methods of purifying viruses using gel permeation chromatography |
CN103263900A (zh) * | 2013-06-07 | 2013-08-28 | 吉林大学 | 一种纳米氧化铝材料改性的聚合物整体柱的制备方法 |
CN105316296A (zh) * | 2014-06-13 | 2016-02-10 | 亚宝药业太原制药有限公司 | 一种纯化腺病毒颗粒的方法 |
EP2966093A1 (en) | 2014-07-07 | 2016-01-13 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Process for the preparation of magnetic sulfated cellulose particles, magnetic sulfated cellulose particles and their use |
ES2877563T3 (es) | 2014-09-02 | 2021-11-17 | Emd Millipore Corp | Medios de cromotografía que comprenden conjuntos porosos discretos de nanofibrillas |
JP6665184B2 (ja) | 2014-12-08 | 2020-03-13 | イー・エム・デイー・ミリポア・コーポレイシヨン | 混床イオン交換吸着剤 |
HRP20160086A2 (hr) | 2016-01-27 | 2017-08-11 | Sveučilište u Zagrebu | Eluens za imunoafinitetnu kromatografiju virusa i proteina |
CN106124096B (zh) * | 2016-06-12 | 2019-03-12 | 京东方科技集团股份有限公司 | 光学微腔、力测量装置及方法、模量测量方法及显示面板 |
CN112105973B (zh) * | 2018-07-02 | 2022-07-05 | 株式会社Lg化学 | 光学调制元件 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3547779A (en) * | 1968-12-18 | 1970-12-15 | Merck & Co Inc | Process for producing purified concentrated influenza virus |
US6008036A (en) * | 1995-08-10 | 1999-12-28 | Pasteur Merieux Serums Et Vaccins | Method for purifying viruses by chromatography |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3478145A (en) | 1964-09-15 | 1969-11-11 | American Home Prod | Chromatographic purification of virus with brushite modified by autoclaving |
GB1214135A (en) | 1967-02-21 | 1970-12-02 | Wellcome Found | Vaccines |
US3485718A (en) | 1967-09-27 | 1969-12-23 | Lilly Co Eli | Virus purification |
US3874999A (en) | 1973-10-31 | 1975-04-01 | American Cyanamid Co | Process for the purification of virus vaccine |
US5234991A (en) | 1975-07-29 | 1993-08-10 | Pasteur Merieux Serums And Vaccines | Porous mineral support coated with an aminated polysaccharide polymer |
JPS6147186A (ja) * | 1984-08-09 | 1986-03-07 | Chemo Sero Therapeut Res Inst | インフルエンザウイルスの精製方法 |
DE3811042A1 (de) | 1988-03-31 | 1989-10-19 | Merck Patent Gmbh | Ionenaustauscher |
US5228989A (en) | 1989-07-06 | 1993-07-20 | Perseptive Biosystems, Inc. | Perfusive chromatography |
US5268097A (en) | 1992-06-19 | 1993-12-07 | Sepracor Inc. | Passivated and stabilized porous mineral oxide supports and method for the preparation and use of same |
DE102010046817A1 (de) * | 2010-09-28 | 2012-03-29 | Sartorius Stedim Biotech Gmbh | Verfahren zur Abtrennung von Viren aus einem Kontaminanten enthaltenden flüssigen Medium |
-
2006
- 2006-07-11 EP EP06116979A patent/EP1878791A1/en not_active Withdrawn
-
2007
- 2007-07-06 EP EP11164648.5A patent/EP2361975B1/en active Active
- 2007-07-06 MX MX2009000026A patent/MX2009000026A/es active IP Right Grant
- 2007-07-06 BR BRPI0714292A patent/BRPI0714292B8/pt active IP Right Grant
- 2007-07-06 EP EP07787173A patent/EP2041273A1/en not_active Withdrawn
- 2007-07-06 CN CN2007800259885A patent/CN101490248B/zh active Active
- 2007-07-06 WO PCT/EP2007/056890 patent/WO2008006780A1/en active Application Filing
- 2007-07-06 US US12/309,135 patent/US20100158944A1/en not_active Abandoned
-
2015
- 2015-07-06 US US14/791,691 patent/US10570376B2/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3547779A (en) * | 1968-12-18 | 1970-12-15 | Merck & Co Inc | Process for producing purified concentrated influenza virus |
US6008036A (en) * | 1995-08-10 | 1999-12-28 | Pasteur Merieux Serums Et Vaccins | Method for purifying viruses by chromatography |
Non-Patent Citations (22)
Title |
---|
"CIM Trademark of BIA Separations." Trademark Details and registration information. Registration date 12/17/2002. * |
"Monoliths Seen to Revitalize Bioseparations." Genetic Engineeric and Biotechnology News. 10/01/2006. http://www.genengnews.com/gen-articles/monoliths-seen-to-revitalize-bioseparations/1906/ * |
Bakry R, Stöggl WM, Hochleitner EO, Stecher G, Huck CW, Bonn GK. Silica particles encapsulated poly(styrene-divinylbenzene) monolithic stationary phases for micro-high performance liquid chromatography. J Chromatogr A. 2006 Nov 3;1132(1-2):183-9. Epub 2006 Aug 22. * |
Barut M, Podgornik A, Brne P, Strancar A. Convective interaction media short monolithic columns: enabling chromatographic supports for the separation and purification of large biomolecules. J Sep Sci. 2005 Oct;28(15):1876-92. * |
BIA Separations Enabling New Generation Biotherapeutics" dated April 2013. http://transmedri.uniri.hr/files/AlesS_About%20BIA%20Separations%20April%202013.pdf. * |
BIA Separations. Home/Education/Posters/Viruses & VLPs. http://www.biaseparations.com/education/posters/viruses-vlps. Accessed 12/22/2014. * |
Branovic K, et. al. Purification of plasmid DNA and concentration of viruses on monoliths. International Symposium on the Separation of Proteins, Peptides, and Polynucleotides (ISPPP); Nov. 5-8, 2000, Lubljana, Slovenia. * |
Branovic K, Forcic D, Ivancic J, Strancar A, Barut M, Kosutic-Gulija T, Zgorelec R, Mazuran R. Application of short monolithic columns for improved detection of viruses. J Virol Methods. 2003 Jun 30;110(2):163-71. * |
Genzel Y, Fischer M, Reichl U. Serum-free influenza virus production avoiding washing steps and medium exchange in large-scale microcarrier culture. Vaccine. 2006 Apr 12;24(16):3261-72. Epub 2006 Jan 19. * |
Jungbauer et al. Monoliths for fast bioseparation and bioconversion and their applications in biotechnology. Journal of Separation Science 2004, Vol. 27, p. 767-778. * |
Kramberger P, et. al. Monolithic Chromatographic Columns - The Media of Choice for Purification and Concentration of Viruses. 2005. * |
Kramberger P, et. al. Purification of viruses on monolthic chromatographic supports: ToMV case study. 2005. * |
Kramberger P, Petrovic N, Strancar A, Ravnikar M. Concentration of plant viruses using monolithic chromatographic supports. J Virol Methods. 2004 Sep 1;120(1):51-7. * |
Kramberger P. et. al. CHROMATOGRAPHY USING SHORT LAYER MONOLITHIC COLUMNS -METHOD OF CHOICE FOR LARGE SCALE VIRUS PURIFICATION. 2004. * |
Nayak DP, Lehmann S, Reichl U. Downstream processing of MDCK cell-derived equine influenza virus. J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Sep 5;823(2):75-81. * |
Nayak et al. Downstream processing of MDCK cell-derived equine influenza virus. Journal of Chromatography B, 2005, Vol. 823, pp. 75-81. * |
Reimer CB, Baker RS, Newlin TE, Havens ML. Influenza virus purification with the zonal ultracentrifuge. Science. 1966 Jun 3;152(3727):1379-81. * |
Strancar A, Barut M, Podgornik A, Koselj P, Josic D, Buchacher A. 1998. Convective Interaction Media: Polymer-Based Supports for Fast Separation of Biomolecules. LC-GC Int. 11:660-669. * |
Talon et al. Influenza A and B viruses expressing altered NS1 proteins: A vaccine approach. Proceedings of the National Academy of Sciences of the United States of America 2000, Vol. 97, No. 8, p. 4309-4314. * |
Transfiguracion J, Jaalouk DE, Ghani K, Galipeau J, Kamen A. Size-exclusion chromatography purification of high-titer vesicular stomatitis virus G glycoprotein-pseudotyped retrovectors for cell and gene therapy applications. Hum Gene Ther. 2003 Aug 10;14(12):1139-53. * |
Urbas L, Kosir B, Peterka M, Pihlar B, Strancar A, Barut M. Reversed phase monolithic analytical columns for the determination of HA1 subunit of influenza virus haemagglutinin. J Chromatogr A. 2011 Apr 29;1218(17):2432-7. Epub 2010 Dec 28. * |
Wickramasinghe et al. Tangential Flow Microfiltration and Ultrafiltration for Human Influenza A Virus Concentration and Purification. Biotechnology and Bioengineering, published online 22 July 2005, Vol. 92, No. 2, p. 199-208. * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100279329A1 (en) * | 2007-09-04 | 2010-11-04 | University Of Brighton | Method for the stabilisation of purified p-glycoprotein |
US20110272357A1 (en) * | 2009-01-21 | 2011-11-10 | Sartorius Stedim Biotech Gmbh | Apparatus and method for material separation |
US20150030565A1 (en) * | 2012-01-09 | 2015-01-29 | Sanofi Pasteur Biologics, Llc | Purification of flaviviruses |
WO2015133972A1 (en) * | 2014-03-07 | 2015-09-11 | Agency For Science, Technology And Research | Apparatus and methods for fractionation of biological products |
CN111527202A (zh) * | 2017-11-08 | 2020-08-11 | 蓝天疫苗有限责任公司 | So3色谱在病毒纯化方法中的应用 |
Also Published As
Publication number | Publication date |
---|---|
BRPI0714292B8 (pt) | 2021-05-25 |
US10570376B2 (en) | 2020-02-25 |
CN101490248B (zh) | 2013-05-01 |
WO2008006780A1 (en) | 2008-01-17 |
MX2009000026A (es) | 2010-01-28 |
EP1878791A1 (en) | 2008-01-16 |
EP2361975A1 (en) | 2011-08-31 |
EP2361975B1 (en) | 2014-12-10 |
EP2041273A1 (en) | 2009-04-01 |
BRPI0714292A2 (pt) | 2013-03-05 |
CN101490248A (zh) | 2009-07-22 |
BRPI0714292B1 (pt) | 2020-06-09 |
US20160002606A1 (en) | 2016-01-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10570376B2 (en) | Method for influenza virus purification | |
US10792353B2 (en) | Virus like particle purification | |
JP5129805B2 (ja) | 細胞培養物から精製水疱性口内炎ウイルスを単離するための精製プロセス | |
AU2004249199B2 (en) | Method for purifying virus | |
Sviben et al. | Recovery of infective virus particles in ion-exchange and hydrophobic interaction monolith chromatography is influenced by particle charge and total-to-infective particle ratio | |
EP3277802B1 (en) | Aseptic purification process for viruses | |
US20210254021A1 (en) | Integrated manufacturing and chromatographic system for virus production | |
AU2013242822B2 (en) | Virus like particle purification | |
EP2603234A1 (en) | Method for reducing dna impurities in viral compositions | |
CN111527202A (zh) | So3色谱在病毒纯化方法中的应用 | |
AU2016269506A1 (en) | Virus like particle purification |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: AVIR GREEN HILLS BIOTECHNOLOGY RESEARCH DEVELOPMEN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ROTHL, ELISABETH;MUSTER, THOMAS;REEL/FRAME:024127/0129 Effective date: 20090924 Owner name: BIA SEPARATIONS D.O.O.,SLOVENIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:PETERKA, MATJAZ;STRANCAR, ALES;BANJAC, MARKO;AND OTHERS;REEL/FRAME:024127/0217 Effective date: 20090217 |
|
AS | Assignment |
Owner name: BAXTER HEALTHCARE SA, AUSTRIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:AVIR GREEN HILLS BIOTECHNOLOGY RESEARCH DEVELOPMENT TRADE AG;REEL/FRAME:030034/0893 Effective date: 20130118 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |
|
AS | Assignment |
Owner name: BAXALTA GMBH, SWITZERLAND Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BAXTER HEALTHCARE SA;REEL/FRAME:036388/0729 Effective date: 20150630 |
|
AS | Assignment |
Owner name: NANOTHERAPEUTICS, INC., FLORIDA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BAXALTA GMBH;REEL/FRAME:037155/0732 Effective date: 20150819 |
|
AS | Assignment |
Owner name: NANOTHERAPEUTICS, INC., FLORIDA Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE CORRESPONDENCE DATA PREVIOUSLY RECORDED ON REEL 037155 FRAME 0732. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT;ASSIGNOR:BAXALTA GMBH;REEL/FRAME:038592/0044 Effective date: 20150819 |