WO2010079081A1

WO2010079081A1 - Methods for recovering a virus or a viral antigen produced by cell culture

Info

Publication number: WO2010079081A1
Application number: PCT/EP2009/067485
Authority: WO
Inventors: Jean-Francois C. P. Chaubard; Roland Mainil
Original assignee: Glaxosmithkline Biologicals S.A.
Priority date: 2009-01-07
Filing date: 2009-12-18
Publication date: 2010-07-15

Abstract

A method for recovering a virus or a viral antigen thereof produced by cell culture, comprising at least the steps of: (a) separating the virus contained in the cell culture medium from the contaminating cellular material including cells and/or cell debris, (b) treating said cellular material so as to extract from it the remaining associated-virus, and (c) separating the virus-containing fraction from the treated cellular material.

Description

METHODS FOR RECOVERING A VIRUS OR A VIRAL ANTIGEN PRODUCED BY CELL CULTURE

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Aspects of this invention were made with United States government support pursuant to Contract # HHSO10020060001 1 C, from the Department of Health and Human Services; the United States government may have certain rights in the invention.

TECHNICAL FIELD

The present invention relates to a method for recovering viruses, or viral antigens, produced by cell culture. In particular, the invention provides a method for improving the virus yield.

TECHNICAL BACKGROUND

Due to the vast number of diseases caused by viruses, virology has been an intensively studied field. There has always been the demand to produce viruses efficiently in order to isolate and purify viral proteins, to generate vaccines, to prepare analytical tools, or to provide viruses for laboratory studies.

Recently, cell culture-based technologies as an alternative to the traditional egg-derived production systems have been frequently developed.

Cell culture systems appear as a suitable alternative mode of vaccine preparation in particular, simpler, flexible, consistent, allowing to improve possibilities of up-scaling vaccine production capacities and thus to reach large quantities of virus, if needed, in particular, in case of a pandemic threat or a terrorist attack.

However, after production, the cell culture-produced virus requires to be recovered from the cell culture, and, when appropriate, to be purified. Various processes of cell culture-derived virus are known in the art, including methods for recovering the virus from the cell culture and for purifying it. Such processes present the major drawback of providing a low virus yield, as virus material is lost along the different steps required for these processes. Therefore, a need remains for providing alternative and, preferably, improved methods for virus recovery and purification from cell culture.

SUMMARY OF THE INVENTION

The method according to the present invention provides a solution to overcome this drawback, as it is intended for limiting the virus loss during the recovery process, and, therefore, increasing the virus yield. In a first aspect of the present invention, there is provided a method for recovering a virus, or a viral antigen thereof, produced by cell culture, comprising at least the steps of:

(i) separating the virus contained in the cell culture medium from the contaminating cellular material including cells and/or cell debris, (ii) treating said cellular material so as to extract from it the remaining associated-virus, and (iii) separating the virus-containing fraction from the treated cellular material.

In a second aspect, there is provided a method for separating a virus, or a viral antigen thereof, from the cells which have been used for producing said virus, or said viral antigen thereof, comprising at least the steps of:

(i) centrifuging the culture medium after cells were infected with the virus and said virus was replicated and released into the medium, (ii) collecting the virus-containing supernatant, (iii) treating the cellular material-containing pellet so as to extract from it the remaining associated virus,

(iv) centrifuging the treated cellular material, (v) collecting the virus-containing supernatant, and, optionally, (vi) pooling the virus solutions collected in steps (ii) and (v).

In a third aspect, there is provided a method for the preparation of a vaccine comprising at least the step of admixing the virus obtained according to the present invention with a pharmaceutically acceptable carrier.

DESCRIPTION OF DRAWINGS

Figure 1 : Virus harvests subjected to centrifugation to separate virus from cellular material. Said cellular material was treated, as indicated, for extracting virus trapped within or attached thereto, or left untreated, as a control. After treatment, cellular material was centrifuged to recover the virus extracted in the supernatant phase and collect it. Supernatants corresponding to each treatment was subjected to an analytical sucrose gradient. Results obtained are illustrated in the form of a graph.

DETAILED DESCRIPTION

The present invention relates to an improved method of recovering and purifying viruses from cell culture that can be applied to both small and large scale virus production. The method involves, in particular, an improved step of clarification relying on a two-step process consisting in (i) separating the virus produced by cell culture which is contained in the cell culture medium from the contaminating cellular material and (ii) extracting from said cellular material the virus which may have been trapped inside or attached thereto and left associated with the cellular material after the step of separation. The resulting virus preparation may be further concentrated and/or enriched by using standard techniques employed for virus purification.

The virus prepared according to the present invention can be used for any purpose, including, for instance, purification of viral proteins, analytical assays, infection of host cells, diagnostic purposes or therapeutic or prophylactic uses such as vaccination and clinical administration.

The method of the invention is amenable to a wide range of viruses, any virus which is capable of infecting cells and using them for its replication, including, but not limited to, adenoviruses, hepadnaviruses, herpes viruses, orthomyxoviruses, papovaviruses, paramyxoviruses, picornaviruses, poxviruses, reoviruses and retroviruses. In particular, the method of invention is suitable for enveloped viruses, such as myxoviruses. In one embodiment, the viruses produced by the method of the invention belong to the family of orthomyxoviruses, in particular, influenza virus.

Viruses or viral antigens may be derived from an Orthomyxovirus, such as influenza virus. Orthomyxovirus antigens may be selected from one or more of the viral proteins, including hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein (M1 ), membrane protein (M2), one or more of the transcriptase (PB1 , PB2 and PA). Particularly suitable antigens include HA and NA, the two surface glycoproteins which determine the antigenic specificity of the Influenza subtypes.

The influenza virus can be selected from the group of human influenza virus, avian influenza virus, equine influenza virus, swine influenza virus, feline influenza virus. Influenza virus is more particularly selected in strains A, B and C.

Influenza antigens may be derived from interpandemic (annual or seasonal) influenza strains. Alternatively, influenza antigens may be derived from strains with the potential to cause a pandemic outbreak (Ae., influenza strains with new hemagglutinin compared to hemagglutinin in currently circulating strains, or influenza strains which are pathogenic in avian subjects and have the potential to be transmitted horizontally in the human population, or influenza strains which are pathogenic to humans). Depending on the particular season and on the nature of the antigen included in the vaccine, the influenza antigens may be derived from one or more of the following hemagglutinin subtypes: H1 , H2, H3, H4, H5, H6, H7, H8, H9, H10, H11 , H12, H13, H14, H15 or H16.

The cells which are used in the method according to the invention can in principle be any desired cell type of cells which can be cultured in cell culture and which can support virus replication. They can be both adherently growing cells or cells growing in suspension. They can be either primary cells or continuous cell lines. Mammalian cells are particularly suitable, for example, human, hamster, cattle, monkey or dog cells.

A number of mammalian cell lines are known in the art and include PER.C6, HEK cells, human embryonic kidney cells (293 cells), HeLa cells, CHO cells, Vero cells, and MDCK cells.

Suitable monkey cells are, for example, African green monkey cells, such as kidney cells as in the Vero cell line. Suitable dog cells are, for example, kidney cells as in the MDCK cell line.

Suitable mammalian cell lines for growing influenza virus include MDCK cells, Vero cells, or PER.C6 cells. These cell lines are all widely available, for instance, from the American Type Cell Culture (ATCC) collection.

According to a specific embodiment, the method of the invention uses MDCK cells. The original MDCK cell line is available from the ATCC as CCL-34, but derivatives of this cell line may also be used, such as the MDCK cells adapted to growth in suspension (WO 1997/37000).

Alternatively, cell lines for use in the invention may be derived from avian sources, such as chicken, duck, goose, quail or pheasant. Avian cell lines may be derived from a variety of developmental stages including embryonic, chick and adult. In particular, cell lines may be derived from the embryonic cells, such as embryonic fibroblasts, germ cells, or individual organs, including neuronal, brain, retina, kidney, liver, heart, muscle, or extraembryonic tissues and membranes protecting the embryo. Chicken embryo fibroblasts (CEF) may be used. Examples of avian cell lines include avian embryonic stem cells (WO01/85938) and duck retina cells (WO05/042728). In particular, the EB66^® cell line derived from duck embryonic stem cells is contemplated in the present invention. Other suitable avian embryonic stem cells include the EBx^® cell line derived from chicken embryonic stem cells, EB45, EB14 and EB14-074 (WO2006/108846). This EBx cell line presents the advantage of being a stable cell line whose establishment has been produced naturally and did not require any genetic, chemical or viral modification. These avian cells are particularly suitable for growing influenza viruses.

Cell culture conditions (temperature, cell density, pH value, etc ...) are variable over a very wide range owing to the suitability of the cells employed and can be adapted to the requirements of particular virus growth conditions details. It is within the skilled in the art person's capabilities to determine the appropriate culture conditions, as cell culture is extensively documented in the art (see, for example, Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and R.I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9). In a specific embodiment, host cells used in the method described in the present invention are cultured in serum-free and/or protein-free media. A "serum-free medium" (SFM) means a cell culture medium ready to use that does not require serum addition allowing cell survival and cell growth. This medium is not necessarily chemically defined and may contain hydrolyzates of various origin, from plant for instance. Such serum-free medium present the advantage that contamination with viruses, mycoplasma or unknown infectious agents can be ruled out. "Protein-free" is understood to mean cultures in which multiplication of the cells occurs with exclusion of proteins, growth factors, other protein additives and non-serum proteins. Optionally trypsin or other proteases that may be necessary for viral growth. The cells growing in such culture naturally contain protein themselves.

Serum-free media are commercially available from numerous sources, for instance, VP SFM (Invitrogen Ref 11681-020), Opti-Pro (Invitrogen, Ref 12309-019), or EX-CELL (JHR Bioscience).

Cell may be grown in various ways, for instance, in suspension, or adhering to surfaces, including growth on microcarriers, or combinations thereof. Culturing can be done in dishes, flasks, roller bottles, or in bioreactors, using batch, fed-batch, or continuous systems, such as perfusion systems.

Typically, cells are scaled-up from a master or working cell bank vial through various sizes of flasks or roller bottles and finally to bioreactors. In one embodiment, the cells employed according to the method of the invention are cultured on microcarrier beads in a serum-free medium in a stirred- bioreactor and the culture medium is provided by perfusion.

Cells can be cultured at around 37°C, more suitably at 36.5°C, at a pH ranging from 6.7 to 7.8, suitably around 6.8 to 7.5, and more suitably at around 7.2

In order to produce large quantities of cell-based viruses, it may be advantageous to inoculate cells with the desired virus strain once cells have reached a high density. Usually, the inoculation is performed when the cell density is at least around 5 x 10⁶ cells/ml, preferably 6 x 10⁶ cells/ml, more preferably 7 x 10⁶ cells/ml, or even higher.

The inoculation can be carried out at an MOI (Multiplicity Of Infection) of about 10^~1 to 10^~7, suitably about 10^~2 to 10^~6, and more suitably, about 10^~5.

The temperature and pH conditions for virus infection may vary. Temperature may range from 32°C to 39°C depending on the virus type. For Influenza virus production, cell culture infection is suitably performed at a temperature ranging from about 32 to about 34°C, in particular at about 33°C. Proteases, typically trypsin, may be added to the cell culture depending on the virus strain, to allow viral replication. The protease can be added at any suitable stage during the culture, either before, during or after infection of the cells with the virus. Once infected, cells may release into the culture medium newly formed virus particles, due to spontaneous lysis of host cells, also called passive lysis. Therefore, in one embodiment, cell-based viral harvest may be provided any time after virus inoculation by collecting the cell culture medium. This mode of harvesting is particularly suitable when it is desired to harvest cell-based virus at different time points after virus inoculation, and pooling the different harvests, if needed.

Alternatively, after virus infection, cell-based virus may be harvested by employing external factor to lyse host cells, also called active lysis. However, contrary to the previous one, such a harvesting mode requires that the cell-based viral harvest be collected at a single time point, as actively lysing the cells will immediately terminate the cell culture.

Methods that can be used for active cell lysis are known to the person skilled in the art. Useful methods in this respect are for example, freeze-thaw, solid shear, hypertonic and/or hypotonic lysis, liquid shear, high pressure extrusion, detergent lysis, or any combination thereof.

According to one embodiment, cell-based viral harvest may be provided any time after virus inoculation by collecting the cell culture supernatants, lysing the inoculated cells or both.

Before harvesting, cell infection may last for 2 to 10 days. The person skilled in the art will determine the optimal time to harvest the cell-based virus based on the determination of the infection peak. For example, the skilled person can measure the CPE (CytoPathic Effect), by monitoring the morphological changes occurring in host cells after virus inoculation, including cell rounding, disorientation, swelling or shrinking, death, detachment from the surface. He can also monitor the detection of a specific viral antigen by standard techniques of protein detection, such as a Western- blot analysis and proceed to the harvest when the desired detection level is achieved. In the particular case of influenza virus, the content of HA can be monitored any time post-inoculation of the cells with the virus, by the SRD (single radial immunodiffusion assay) assay, which is a technique familiar to a person skilled in the art (J. M. Wood et al.: An improved single radial immunodiffusion technique for the assay of influenza haemagglutinin antigen: adaptation for potency determination of inactivated whole virus and subunit vaccines. J. Biol. Stand. 5 (1977) 237-247; J. M. Wood et al., International collaborative study of single radial diffusion and Immunoelectrophoresis techniques for the assay of haemagglutinin antigen of influenza virus. J. Biol. Stand. 9 (1981 ) 317-330)).

In the context of the present invention, the cell culture phase is to be understood as encompassing any step preceding the virus harvesting step, while the virus purification phase is to be understood as encompassing any step following said harvesting step. The virus purification phase includes, in particular, a clarification step aimed at eliminating any non-viral constituents that may be present in the form of aggregates or particles, in addition to the virus, in the virus harvest. Said constituents mostly represent cellular material, including intact cells or cell debris, floating in the culture medium. The method according to the present invention involves, in particular, an improved step of clarification relying on a two-step process consisting in (i) separating the virus produced by cell culture which is contained in the cell culture medium from the contaminating cellular material and (ii) extracting from said cellular material the virus which may have been trapped inside or attached thereto and left associated with the cellular material after the step of separation. The separation may be performed, for example, based on the size or density difference of the constituents to be separated, in particular, by centrifugation.

After separation of the produced virus from the contaminating cellular material, and collection of the respective fractions, when analyzing the presence of the virus in each fraction, the inventors, surprisingly, found out that a significant proportion of the virus was still present in the cellular material fraction, suggesting that while most of the produced virus is in a cell-free form in the culture medium, some of it is in a cell-associated form and, therefore, remains trapped within the cellular material fraction after separation. Thus, the cell-associated virus form, if untreated, accounts for virus loss during the purification process.

Accordingly, in the context of the invention, a virus that is "cell-associated" refers to a virus which is attached to or trapped within the contaminating cellular material present in the culture medium, i.e. cells detached from their support and floating in the culture medium, whether cells are intact or only debris. Thus, "cell-associated" virus designates the fraction of virus which is present in the sedimented cellular material, after separation of the virus-containing cell culture medium from the contaminating cellular material,

The term "harvest" or "harvesting" specifically designates the virus-containing cell culture medium which is collected after virus infection, replication and release into the medium. The harvest includes cell-free virus, cell-associated virus and the contaminating cellular material.

The term "sediment" is to be understood as the result of the separation step, wherein heavier or denser particles within a solution or a suspension, due to their weight or their density, will migrate towards the bottom of the container containing said solution or suspension. According to the present invention, in the viral harvest the constituents which will sediment during the separation step are the cellular materials composed of floating intact cells and cell debris, while "cell-free" virus will remain at the top of the container. Therefore, in the sense of the invention a "cell-free virus" means a virus which, once released in the cell culture medium is not associated to floating cells or cell debris present in said medium. When the separation step is performed by centrifugation, the resulting sediment is specifically called the "pellet". Therefore, the term "pellet" is to be understood as the cellular material which sedimented after the viral harvest was centrifuged. Another term which may be used in the rest of the application for designating the sedimented cellular material is "cell paste". Cell paste should be considered a synonym for sedimented, or pelleted, cellular material.

According to the present invention, separated fractions may be, individually, collected, so that they can be, independently, further processed, when appropriate.

In a specific embodiment, separating the virus from the contaminating cellular material within the virus harvest is performed by centrifugation, in particular, by a continuous centrifugation.

In the context of the present invention, the inventors observed that the cell-associated virus present in the sedimented cellular material could be extracted from it and recovered, and, thus, the virus loss be reduced. Therefore, in the sense of the invention, "extracting" a virus is to be understood as converting a cell-associated virus to a cell-free virus. The term "recovering" is to be understood as collecting and isolating the extracted virus.

They observed that specific treatments of sedimented cellular material after clarification of the viral harvest, aimed at causing the dissociation of the cell-associated virus, allow to extract the virus, so as to render it cell-free. After treatment of the cellular material, the newly converted cell-free virus form, extracted from said material, may be separated and collected. This new, or second, virus population may, then, be further purified according to known in the art techniques. As an alternative, if desired, this new population may be pooled with the initial virus-containing fraction obtained after clarifying the viral harvest, i.e. after the first separation step.

In one embodiment, the first virus-containing fraction and the second virus-containing fraction are collected, optionally, pooled and, optionally, further purified.

According to the present invention, the treatment of the sedimented cellular material, which may comprise some cell-associated virus form, is any treatment allowing to dissociate the interactions between the trapped virus and the cells or the cell debris, whether of a chemical nature, or of a physico-mechanical type. As non-limiting examples of a chemical treatment, may be cited (i) the use of detergents, for instance, non-ionic detergents or ionic detergents known to be used for lysing cells, such as, Tween 80 or Triton X-100, and deoxycholate, respectively, and (ii) the use of enzymatic proteins, with a proteolytic activity, such as trypsin, chymotrypsin, pronase and collagenase, or with a nucleic acid degrading activity, such as Benzonase™. With regard to mechanical treatment, by way of example, may be cited the freeze-thawing method, crushing, sonication and ultrasonication or the use of a hypotonic solution. It is also contemplated within the scope of the present invention to combine one or more of different treatments. This combination may rely on the use of one or more different detergents, or on the use of one chemical treatment associated with one mechanical treatment. It is not necessary to proceed to the treatment of the sedimented cellular material, immediately after the separation step. If desired, the sedimented cellular material may be stored at -70⁰C before any further processing. In one embodiment of the present invention, the cellular material is stored at -70⁰C after clarification of the viral harvest.

In one embodiment, the treatment implemented for extracting the virus from the sedimented cellular material and recovering it is selected from the group of: a detergent treatment, a sonication, an enzymatic protein treatment, or any combination thereof.

The nature of the treatment(s) will be selected so as to provide the best virus extraction or recovery level, depending on what cell type and what virus are to be employed. The extent of the virus extraction or recovery can be monitored by using, for instance, any known in the art method of protein detection, such as Western-blot analysis or Theshold™ assay. In the particular case of influenza virus, the content of HA by the SRD assay may be assessed as described above. The treatments will also be operated in conditions set so as to allow achieving the best recovery level. Amongst these conditions are, in particular, the incubation time and the temperature incubation, the concentration of the reagent, the occurrence of agitation, etc ... The present invention is not limited to a single cycle of separation/extraction. If the recovery is not complete and if willing to further increase it, it is contemplated within the present invention to proceed to a second extraction and use as many cycles as necessary, in order to obtain the desired and highest possible recovery level.

Depending on whether it is required, or not, to recover a virus which is intact, it is recommended to carefully choose what agent or what treatment to use, as some of them may damage the integrity of the viral particle. If considering the influenza virus, for example, a sucrose gradient analysis can be operated allowing to distinguish intact whole virus from disintegrated virus, based on a size or density difference.

In one embodiment of the present invention, the treatment implemented for extracting the virus from the sedimented cellular material and recovering it is performed by a detergent treatment of the sedimented cellular material. In a specific embodiment, this detergent is Tween 80. The concentration of Tween 80 used for the extraction step may range between 0.1 % and 1 %.

In another embodiment, at least a sonication, whether by itself or in combination with a distinct treatment, is performed as the extraction step during the method according to the present invention. In particular, a combination of sonication and a detergent, such as Tween 80, is contemplated by the present invention.

After the treatments for extracting the virus from the sedimented cellular material, separation of each fraction (virus and cellular material) and collection of the virus fraction, the inventors observed a virus recovery yield ranging from 40% to 100%, as measured by analysing the content of HA by the SRD assay, when producing influenza virus. When using the present invention, the vast majority of the virus which is lost when separating the virus contained in the culture medium from the cellular material is recovered. In one embodiment, the method according to the present invention allows to recover from the cellular material at least 40%, in particular, at least 60%, more particularly, at least 80%, and even 100% of the cell-associated virus.

In case the separation of the virus from the cellular material within the viral harvest needs to be increased, so as to further improve the clarification of the virus harvest, the method of the present invention also contemplates the possibility of including additional clarification steps. For instance, after extracting and recovering the virus from the cellular material, this virus fraction, whether or not pooled with the initial virus-containing fraction, may be further clarified. This clarification may be done by a filtration. It is also within the scope of the present invention that the virus contained in the culture medium be separated from the cellular material within the virus harvest by filtration. Alternatively, centrifugation and/or filtration may be combined together, in any order, for achieving the desired clarification level of the virus harvest. Suitable filters may utilize cellulose filters, regenerated cellulose filters, cellulose fibers combined with inorganic filter aids, cellulose filter combined with inorganic filter aids and organic resins, or any combination thereof, and polymeric filters. Although not required, a multiple filtration process may be carried out, like a two- or three- stage process consisting, for instance, in sequentially and progressively removing impurities according to their size, using filters with appropriate nominal pore size, in particular, filters with decreasing nominal pore size, allowing to start removing large precipitates and cell debris. In addition, single stage operations employing a relatively tight filter or centrifugation may also be used for clarification. More generally, any clarification approach including, but not limited to, dead-end filtration, depth filtration, microfiltration, or centrifugation, which provide a filtrate of suitable clarity to not foul the membrane and/or resins in subsequent steps, will be acceptable to use in the clarification step of the present invention. In one embodiment, the viral clarification step is performed by depth filtration, in particular, using a three- stage train filtration composed, for example, of three different depth filters with nominal porosities of 5 μm - 0.5 μm - 0.2 μm.

Viruses or viral antigens thereof produced according to the method of the present invention may be subjected to further purification, using standard techniques employed for virus purification. For instance, the virus purification phase of cell culture-derived viruses may include a number of different filtration, concentration and/or other separation steps such as ultrafiltration, ultracentrifugation (including gradient ultracentrifugation), chromatography (such as ion exchange chromatography) and adsorption steps in a variety of combinations.

In one embodiment, during the virus purification step, the method of the invention comprises at least one step selected from the group of: filtration, ultrafiltration/diafiltration, ultracentrifugation and chromatography, or any combination thereof. Depending on the purity level that is desired, the above steps may be combined in any way.

According to the present invention, the virus suspension may be subjected to ultrafiltration (sometimes referred to as diafiltration when used for buffer exchange), for concentrating the virus and/or buffer exchange. This step is particularly advantageous when the virus to be purified is diluted, as is the case when pooling viral harvest collected by perfusion over a few days post-inoculation. The process used to concentrate the virus according to the method of the present invention can include any filtration process where the concentration of virus is increased by forcing diluent to be passed through a filter in such a manner that the diluent is removed from the virus suspension whereas the virus is unable to pass through the filter and thereby remains in concentrated form in the virus preparation.

In one embodiment, the virus purification step of the method of the invention comprises at least one ultraf iltration/d iaf iltration step, suitably at least two ultrafiltration/diafiltration steps.

The virus suspension obtained according to the method of the present invention may be further purified, by methods generally known to the person skilled in the art, such as density gradient centrifugation, for instance sucrose gradient ultracentrifugation, and/or chromatography.

In a specific embodiment, the virus purification phase comprises at least one step of sucrose gradient ultracentrifugation, a technique commonly used for purifying viruses and known in the art.

According to the method of the invention, it may be possible to combine a purification step, such as sucrose gradient ultracentrifugation, with a virus splitting step. In particular, a splitting agent may be added to the sucrose gradient. This embodiment is particularly suitable, when it is desired to minimize the total number of steps of the method of the invention, as it allows, within a single operation, to both purify and split the virus. Hence, in certain embodiments, when at least one sucrose gradient ultracentrifugation is implemented, the sucrose gradient additionally comprises a splitting agent.

Alternatively, it is also possible to purify viruses by chromatography, including ion exchange, anionic or cationic, chromatography, size exclusion, such as gel filtration or gel permeation, chromatography, hydrophobic interaction chromatography, hydroxyapatite or any combination thereof. As mentioned above, the chromatography steps may be implemented in combination with other purifications steps, such as density gradient ultracentrifugation. The person skilled in the art will be aware of theses processes, and can vary the exact way of employing these additional steps to optimize the method of the invention.

At the end of the virus purification phase, the virus preparation may be suitably subjected to sterile filtration, as is common in processes for pharmaceutical grade materials, such as immunogenic compositions or vaccines, and known to the person skilled in the art. Such sterile filtration can for instance suitably be performed by filtering the preparation through a 0.22 μm filter. After sterile preparation, the virus or viral antigens are ready for clinical use, if desired.

The immunogenic compositions, in particular vaccines, may generally be formulated in a sub-virion form, e.g. in the form of a split virus, where the lipid envelope has been dissolved or disrupted, or in the form of one or more purified viral proteins (subunit vaccine). As an alternative, the immunogenic compositions may include a whole virus, e.g. a live attenuated whole virus, or an inactivated whole virus.

Methods of splitting viruses, such as influenza viruses, are well known in the art (WO02/28422). Splitting of the virus is carried out by disrupting or fragmenting whole virus whether infectious (wild- type or attenuated) or non-infectious (inactivated) with a disrupting concentration of a splitting agent. Splitting agents generally include agents capable of breaking up and dissolving lipid membranes. Traditionally, split influenza virus was produced using a solvent/detergent treatment, such as tri-n-butyl phosphate, or diethylether in combination with Tween™ (known as "Tween-ether" splitting) and this process is still used in some production facilities. Other splitting agents now employed include detergents or proteolytic enzymes or bile salts, for example sodium deoxycholate. Detergents that can be used as splitting agents include cationic detergents e.g. cetyl thrimethyl ammonium bromide (CTAB), other ionic detergents, e.g. sodium lauryl sulphate (SLS), taurodeoxycholate, or non-ionic detergents such as Tween 80 or Triton X-100, or combination of any two or more detergents.

The splitting process may be carried out as a batch, continuous or semi-continuous process. When implemented in batch, the split virus may require an additional step of purification, such as a chromatography step. It is not necessary to implement a splitting step as such, as it is possible to perform the splitting simultaneously to a purification step. For instance, a detergent may be added to the sucrose gradient aimed at purifying viral proteins by ultracentrifugation, as described above.

For the safety of vaccines, it may be necessary to reduce infectivity of the virus suspension along different steps of the purification process. The infectivity of a virus is determined by its capacity to replicate on a cell line. Therefore, the method according to the present invention, optionally, includes at least one virus inactivation step. The inactivation may be performed by using BPL (beta- propiolactone) at any suitable step of the method. In one specific embodiment, the method according to the invention further comprises at least one BPL treatment step. In a specific embodiment, the method according to the invention further comprises at least one BPL treatment step and at least one formaldehyde treatment step. Formaldehyde and BPL may be used sequentially, in any order, for instance, formaldehyde is used after the BPL. Immunogenic compositions of the present invention, including vaccines, can optionally contain the additives customary for vaccines, in particular substances which increase the immune response elicited in a patient who receives the composition, i.e. so-called adjuvants.

In one embodiment, immunogenic compositions are contemplated, which comprise a virus or viral antigen of the present invention admixed with a suitable pharmaceutical carrier. In a specific embodiment, they comprise an adjuvant.

Adjuvant compositions may comprise an oil-in-water emulsion which comprise a metabolisable oil and an emulsifying agent. In order for any oil-in-water composition to be suitable for human administration, the oil phase of the emulsion system comprises a metabolisable oil. The meaning of the term metabolisable oil is well known in the art. Metabolisable can be defined as 'being capable of being transformed by metabolism' (Dorland's Illustrated Medical Dictionary, W. B. Sanders Company,

25th edition (1974)). The oil may be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and is capable of being transformed by metabolism. Nuts, seeds, and grains are common sources of vegetable oils. Synthetic oils are also part of this invention and can include commercially available oils such as NEOBEE® and others.

A particularly suitable metabolisable oil is squalene. Squalene (2,6,10,15,19,23-Hexamethyl- 2,6,10,14,18,22-tetracosahexaene) is an unsaturated oil which is found in large quantities in shark- liver oil, and in lower quantities in olive oil, wheat germ oil, rice bran oil, and yeast, and is a particularly preferred oil for use in this invention. Squalene is a metabolisable oil by virtue of the fact that it is an intermediate in the biosynthesis of cholesterol (Merck index, 10th Edition, entry no.8619). In a further embodiment of the invention, the metabolisable oil is present in the immunogenic composition in an amount of 0.5% to 10% (v/v) of the total volume of the composition.

The oil-in-water emulsion further comprises an emulsifying agent. The emulsifying agent may suitably be polyoxyethylene sorbitan monooleate. Further, said emulsifying agent is suitably present in the vaccine or immunogenic composition 0.125 to 4% (v/v) of the total volume of the composition.

The oil-in-water emulsion of the present invention optionally comprise a tocol. Tocols are well known in the art and are described in EP0382271. Suitably may be a tocol is alpha-tocopherol or a derivative thereof such as alpha-tocopherol succinate (also known as vitamin E succinate). Said tocol is suitably present in the adjuvant composition in an amount 0.25% to 10% (v/v) of the total volume of the immunogenic composition.

The method of producing oil-in-water emulsions is well known to the person skilled in the art. Commonly, the method comprises mixing the oil phase (optionally comprising a tocol) with a surfactant such as a PBS/TWEEN80™ (or polysorbate 80) solution, followed by homogenisation using a homogenizer. A suitable method comprises passing the mixture twice through a syringe needle would be suitable for homogenising small volumes of liquid. Alternatively, the emulsification process in microfluidiser (M110S Microfluidics machine, maximum of 50 passes, for a period of 2 minutes at maximum pressure input of 6 bar (output pressure of about 850 bar)) could be adapted by the man skilled in the art to produce smaller or larger volumes of emulsion. The adaptation could be achieved by routine experimentation comprising the measurement of the resultant emulsion until a preparation was achieved with oil droplets of the required diameter.

In an oil-in-water emulsion, the oil and emulsifier should be in an aqueous carrier. The aqueous carrier may be, for example, phosphate buffered saline.

In particular, the oil-in-water emulsion systems of the present invention have a small oil droplet size in the sub-micron range. Suitably the droplet sizes will be in the range 120 to 750 nm, more particularly sizes from 120 to 600 nm in diameter. Even more particularly, the oil-in water emulsion contains oil droplets of which at least 70% by intensity are less than 500 nm in diameter, more particular at least 80% by intensity are less than 300 nm in diameter, more particular at least 90% by intensity are in the range of 120 to 200 nm in diameter.

The oil droplet size, i.e. diameter, according to the present invention is given by intensity. There are several ways of determining the diameter of the oil droplet size by intensity. Intensity is measured by use of a sizing instrument, suitably by dynamic light scattering such as the Malvern Zetasizer 4000 or suitably the Malvern Zetasizer 3000HS. A detailed procedure is given in Example II.2. A first possibility is to determine the z average diameter ZAD by dynamic light scattering (PCS-Photon correlation spectroscopy); this method additionally give the polydispersity index (PDI), and both the ZAD and PDI are calculated with the cumulants algorithm. These values do not require the knowledge of the particle refractive index. A second mean is to calculate the diameter of the oil droplet by determining the whole particle size distribution by another algorithm, either the Contin, or NNLS, or the automatic "Malvern" one (the default algorithm provided for by the sizing instrument). Most of the time, as the particle refractive index of a complex composition is unknown, only the intensity distribution is taken into consideration, and if necessary the intensity mean originating from this distribution. The adjuvant compositions may further comprise a Toll like receptor (TLR) 4 agonist. By "TLR4 agonist" it is meant a component which is capable of causing a signalling response through a TLR4 signalling pathway, either as a direct ligand or indirectly through generation of endogenous or exogenous ligand (Sabroe et al, Jl 2003 p1630-5). The TLR 4 may be a lipid A derivative, particularly monophosphoryl lipid A or more particularly 3 Deacylated monophoshoryl lipid A (3 D - MPL). 3D-MPL can be produced according to the methods disclosed in GB 2 220 211 A. Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated chains. In particular, in the adjuvant compositions of the present invention small particle 3 D- MPL is used. Small particle 3 D - MPL has a particle size such that it may be sterile-filtered through a 0.22μm filter. Such preparations are described in International Patent Application No. WO 94/21292. Synthetic derivatives of lipid A are known and thought to be TLR 4 agonists including, but not limited to:

OM174 (2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-o-phosphono-β-D- glucopyranosyll^-^RJ-S-hydroxytetradecanoylaminol-α-D-glucopyranosyldihydrogenphosphate), (WO 95/14026)

OM 294 DP (3S, 9 R) -3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)-3- hydroxytetradecanoylamino]decan-1 ,10-diol,1 ,10-bis(dihydrogenophosphate) (WO99 /64301 and WO 00/0462 )

OM 197 MP-Ac DP ( 3S-, 9R) -3-[(R) -dodecanoyloxytetradecanoylaminoH-oxo-δ-aza-θ-KR)^- hydroxytetradecanoylamino]decan-1 ,10-diol,1 -dihydrogenophosphate 10-(6-aminohexanoate) (WO 01/46127)

Other TLR4 ligands which may be used are alkyl Glucosaminide phosphates (AGPs) such as those disclosed in WO9850399 or US6303347 (processes for preparation of AGPs are also disclosed), or pharmaceutically acceptable salts of AGPs as disclosed in US6764840. Some AGPs are TLR4 agonists, and some are TLR4 antagonists. Both are thought to be useful as adjuvants. In addition, further suitable TLR-4 agonists are disclosed in US2003/0153532 and US2205/0164988.

The invention is particularly suitable for preparing influenza virus immunogenic compositions, including vaccines. Various forms of influenza virus are currently available. They are generally based either on live virus or inactivated virus. Inactivated vaccines may be based on whole virions, spilt virions or purified surface antigens (including HA). Influenza antigens can also be presented in the form of virosomes (nucleic acid-free viral-like liposomal particles).

Virus inactivation methods and splitting methods have been described above and are applicable to influenza virus.

Influenza virus strains for use in vaccines change from season to season. In the current inter- pandemic period, vaccines typically include two influenza A strains and one influenza B strain. Trivalent vaccines are typical, but higher valence, such as quadrivalence, is also contemplated in the present invention. The invention may also use HA from pandemic strains (i.e. strains to which the vaccine recipient and the general human population are immunologically naϊve), and influenza vaccines for pandemic strains may be monovalent or may be based on a normal trivalent vaccine supplemented by a pandemic strain. Compositions of the invention may include antigen(s) from one or more influenza virus strains, including influenza A virus and/or influenza B virus. In particular, a trivalent vaccine including antigens from two influenza A virus strains and one influenza B virus strain is contemplated by the present invention. Alternatively a quadrivalent vaccine including antigens from two influenza A virus strains and two influenza B virus strains is also within the scope of the present invention.

The compositions of the invention are not restricted to including only one strain type, i.e. only seasonal strains or only pandemic strains. The invention also encompasses compositions comprising a combination of seasonal strains and of pandemic strains. In particular, a quadrivalent composition, which may be adjuvanted, comprising three seasonal strains and one pandemic strain falls within the scope of the invention.

HA is the main immunogen in current inactivated influenza vaccines, and vaccine doses are standardized by reference to HA levels, typically measured by SRD. Existing vaccines typically contain about 15 μg of HA per strain, although lower doses can be used, e.g. for children, or in pandemic situations, or when using an adjuvant. Fractional doses such as a half (Ae. 7.5 μg HA per strain) or a quarter have been used, as have higher doses, in particular, 3x or 9x doses. Thus immunogenic compositions of the present invention may include between 0.1 and 150 μg of HA per influenza strain, particularly, between 0.1 and 50 μg, e.g. 0.1-20 μg, 0.1-15 μg, 0.1-10 μg, 0.1-7.5 μg, 0.5-5 μg, etc. Particular doses include about 15, about 10, about 7.5, and about 5 μg per strain.

Once an influenza virus has been purified for a particular strain, it may be combined with viruses from other strains to make a trivalent vaccine, for example, as described above. It is more suitable to treat each strain separately and to mix monovalent bulks to give a final multivalent mixture, rather than to mix viruses and degrade DNA and purify it from a multivalent mixture.

The invention will be further described by reference to the following, non-limiting, examples.

Example 1 : Influenza virus produced in MDCK cells is partly trapped within the contaminating cellular material present in the cell culture supernatant

1. Experiment EFLTAP A01 1

The MDCK adherent cells were grown on microcarriers in a perfusion culture mode in a 20 liter stirred- bioreactor scale at 36.5°C. After the growth phase, once the appropriate cell density was reached (above 7 x 10⁶ cells/ml), cells are inoculated with the New Caledonia A strain of Influenza virus (Multiplicity of Infection of 1 x 10^~5) in a perfusion mode and the temperature was switched to 33°C. The virus was harvested by perfusion at days 2, 3, 4 and 5 post-inoculation. The perfusion harvests were pooled and the complete harvest was subjected to a centrifugation performed with a tubular centrifuge (Celeros APD-75), wherein the separation force was fixed at 5000 g (12 000 rpm) and the flow rate was 0.75 ml/min. While the supernatant containing the influenza virus was collected for further characterization, the sedimented cell paste was recovered at the end of operations, a sample of which was resuspended into a pH 7.4 - PBS buffer containing 125 mM citrate. The rest of the cell paste was stored at -70⁰C before further processing.

A significant clarification effect was observed after centrifugation, as the turbidity of the supernatant fraction (measured by reading the absorbance at 650 nm in a spectrophotometer) was reduced to 0.07.

The relative distribution of the influenza virus in each fraction (supernatant and resuspended cell paste) was assessed by measuring the content of HA according to the SRD assay. The concentration of total proteins was also measured by using the classical Lowry method. Results are presented in Table 1 in the form of percentages to be compared to the control value 100% representing the total HA content present in the starting material (harvest).

Table 1 - HA yield after centrifugation

- Results-Conclusions

The results presented in Table 1 clearly indicate that, while the majority of HA was present in the supernatant fraction, a significant fraction of HA was also detected in the cell paste fraction which is likely to represent some virus trapped within the cellular material and attached to cell debris.

- SRD method used to measure HA content

Glass plates (12.4 - 10 cm) were coated with an agarose gel containing a concentration of anti- influenza HA serum that is recommended by NIBSC. After the gel had set, 72 sample wells (3 mm diameter) were punched into the agarose. 10 μl of appropriate dilutions of the reference and the samples were loaded in the wells. The plates were incubated for 24 hours at room temperature (20 to 25°C) in a moist chamber. After that, the plates were soaked overnight with NaCI solution and washed briefly in distilled water. The gel was then pressed and dried. When completely dry, the plates were stained on Coomassie Brillant Blue solution for 10 minutes and destained twice in a mixture of methanol and acetic acid until clearly defined stained zones become visible. After drying the plates, the diameter of the stained zones surrounding antigen wells was measured in two directions at right angles. Alternatively equipment to measure the surface can be used. Dose-response curves of antigen dilutions against the surface were constructed and the results were calculated according to standard slope-ratio assay methods (Finney, D.J. (1952). Statistical Methods in Biological Assay. London: Griffin, Quoted in: Wood, JM, et al (1977). J. Biol. Standard. 5, 237-247)

2. Experiment EFLTCPB001

The MDCK cells were grown and infected as described in section 1 , except that the Influenza virus strain used is a Jiangsu B type. The virus was harvested in the same conditions as the conditions described in section 1. The complete harvest was subjected to a centrifugation performed with a discstack centrifuge (Westfalia, CSA8). Centrifugation was operated at constant speed (9300 rpm) and at a 1.5 L/min flow rate. While the supernatant containing the influenza virus was collected for further characterization, the sedimented cell paste was recovered at the end of operations, a sample of which was resuspended into a pH 7.4 - PBS buffer containing 125 mM citrate. The rest of the cell paste was stored at -70⁰C before further processing (see Example 2).

A strong clarification effect was observed after centrifugation, as the turbidity of the supernatant fraction (measured by reading the absorbance at 650 nm in a spectrophotometer) was reduced from 0.27 to 0.04.

The relative distribution of the influenza virus in each fraction (supernatant and resuspended cell paste) was assessed by measuring the content of HA according to the SRD assay. Results are presented in Table 2 in the form of percentages to be compared to the control value 100% representing the total HA amount present in the starting material (harvest).

Table 2 - HA yield after centrifugation

- Results-Conclusions

The results presented in Table 2 clearly confirm the conclusion drawn from the experiment displayed in section 1. While the majority of HA was present in the supernatant fraction, a non-negligible fraction of HA was also detected in the cell paste fraction which is likely to represent some virus trapped within the cellular material and attached to cell debris. Example 2: Extraction and recovery of the influenza virus fraction trapped within the sedimented cellular material after centrifugation

- Experiment EFLTCPB001

For recovery experiments, the frozen cell paste from the Example 1 , section 2 was thawed and resuspended in a pH 7.4 - PBS buffer containing citrate 125 mM. The following treatments were applied on the resuspended cell paste in order to extract the influenza virus that was trapped within or attached thereto: i) Tween 80 0.1 % addition and agitation at room temperature for 2 hours ii) Tween 80 1 % addition and agitation at room temperature for 2 hours iii) Tween 80 0.1 % addition and sonication (Vibracell, amplitude 70%) for 1 min and agitation at room temperature for 2 hours iv) Sonication (Vibracell, amplitude 70%) for 1 min and agitation at room temperature for 2 hours v) Benzonase™ (100 U/ml) addition and sonication (Vibracell, amplitude 70%) for 1 min and agitation at room temperature for 2 hours vi) Benzonase™ (100 U/ml) and Tween 80 1 % addition and agitation at room temperature for 2 hours vii) As a control, incubation of the resuspended cell paste at room temperature for 2 hours, without any treatment

At the end of the 2 hours incubation at room temperature, all treated resuspended cell pastes

(treatments i) to vi)) and the untreated resuspended control cell paste (condition vii)) were subjected to centrifugation at 4000 rpm for 10 minutes, twice. Supernatants were collected and characterized for their HA content by SRD and the total protein content by the Lowry method. Results are presented in Table 3, in the form of percentages to be compared to the 100% control value corresponding to the content of HA, or the content of total proteins, present in the starting material (resuspended cell paste obtained in Example 1 , Section 2).

Table 3 - HA yield and total protein content

- Results and conclusions

As illustrated in the HA column of Table 3, the best recoveries were obtained with Tween 80 1 % (ii) (104%), with sonication (iv) (104%) and with a combination of Tween 80 0.1 % and sonication (iii) (93%), compared to a 38% recovery in the untreated control (vii). Every treatment with sonication gave interesting yields.

After the different treatments of cell paste and centrifugation to recover the virus fraction extracted from said cell paste in the supernatant phase, said supernatants were also analysed by analytical sucrose gradient, which allows to evaluate the status of the influenza virus, namely intact whole virus vs. disintegrated virus, as constituents of the preparation to be tested migrate into the gradient until reaching their respective density. The whole virus is expected to be present in the fractions corresponding to a high percentage of sucrose, as opposed to virus fragments or contaminants of lower density. Given the results presented on Figure 1 , the whole virus was present in fractions corresponding to a percentage of sucrose ranging from approximately 40% to 50%. A nice peak is observed at this density region for some treatments, such as treatment iii) and iv), while for other treatments, such as treatment v), the peak is shifted to lower densities. It is to be noted that the control resuspended cell paste obtained in Example 1 , Section 2 gives a peak in the density region ranging from 40% to 50%.

Several fractions were pooled and assessed for their HA content (%) by the SRD assay, as indicated in Table 4 . The results are presented in Table 4 in the form of percentages to be compared to the 100% control value corresponding to the content of HA present in the starting material, i.e. in the supernatant obtained as described above and to be loaded on the sucrose gradient. The last column Total HA recovery represents the sum of the HA % recovered in the different pools tested.

Table 4 - HA recovery after sucrose gradient ultracentrifugation

* corresponds to a 34-51 % sucrose pool; NT is for Not Tested - Results and conclusions

Most of the HA extracted from the cell paste after the different treatments, i) to vii), was recovered in the sucrose fractions (last column, 88% in average). A significant proportion of HA was present in both fractions corresponding to the whole virus and fractions of lower density than the whole virus.

Taking into account the HA total recovery yield and the proportion of HA measured in the fractions corresponding to the whole virus (39-53% sucrose), Table 4 indicates that some treatments, such as i), iii) and iv), allow to recover from the cell paste around 50% of whole virus.

Claims

1. A method for recovering a virus or a viral antigen thereof produced by cell culture, comprising at least the steps of: (a) separating the virus contained in the cell culture medium from the contaminating cellular material including cells and/or cell debris,

(b) treating said cellular material so as to extract from it the remaining associated-virus, and

(c) separating the virus-containing fraction from the treated cellular material.

2. The method according to the preceding claim, wherein the virus-containing fractions obtained in steps (a) and (c) are collected, optionally, pooled, and, optionally, further purified.

3. The method according to claim 1 or claim 2, wherein the step (a) is performed by centrifugation.

4. A method for separating a virus or a viral antigen thereof from the cells which have been used for producing said virus or said viral antigen thereof, comprising at least the steps of:

(i) centrifuging the culture medium after cells were infected with the virus and said virus was replicated and released into the medium,

(ii) collecting the virus-containing supernatant,

(iii) treating the cellular material-containing pellet so as to extract from it the remaining associated-virus,

(iv) centrifuging the treated cellular material, (v) collecting the virus-containing supernatant, and, optionally,

(vi) pooling the virus solutions collected in steps (b) and (e).

5. The method according to claim 3 or claim 4, wherein the centrifugation is continuous.

6. The method according to any of the preceding claims, wherein the treatment of step (b) or (iii) is selected from a detergent treatment, a sonication, an enzymatic protein treatment, or any combination thereof.

7. The method according to the preceding claim, wherein the treatment is a detergent treatment.

8. The method according to the preceding claim, wherein the detergent is Tween 80.

9. The method according to claim 6, wherein the treatment is a sonication.

10. The method according to claim 6, wherein a detergent treatment and a sonication are combined.

11. The method according to claim 6, wherein the enzymatic protein is a proteolytic enzyme.

12. The method according to claim 6, wherein the enzymatic protein is an endonuclease.

13. The method according to any of the preceding claims, wherein the cellular material obtained after step (a) or (i) is stored at -70⁰C before the treatment of the step (c) or (iii).

14. The method according to any of the preceding claims, wherein the virus is influenza virus.

15. The method according to any of the preceding claims, wherein the cells are mammalian cells or avian cells.

16. The method according to the preceding claim, wherein the mammalian cells are MDCK cells.

17. The method according to claim 15, wherein the avian cells are EB66® cells.

18. A method for the preparation of a vaccine comprising at least the step of admixing the virus obtained according to the method as claimed in any of claims 1 to 17 with a pharmaceutically acceptable carrier.