WO2003076462A1

WO2003076462A1 - Use of recombinant trypsin for vaccine production

Info

Publication number: WO2003076462A1
Application number: PCT/NL2002/000157
Authority: WO
Inventors: Maria Grazia Pau; Alphonsus Gerardus Cornelis Maria Uytdehaag
Original assignee: Crucell Holland B.V.
Priority date: 2002-03-08
Filing date: 2002-03-08
Publication date: 2003-09-18
Also published as: AU2002239151A1

Abstract

The present invention provides the use of recombinantly produced serine proteases, such as bovine trypsin produced in corn, for the production and propagation of viruses in vitro cell cultures and discloses methods for the production of viral batches in serum-free tissue culture, without using animal-derived trypsin.

Description

Title : Use of recombinant trypsin for vaccine production .

FIELD OF THE INVENTION

The invention relates to the field of medicine. More particularly, the invention relates to a cell-based production process of viruses to be used for the manufacturing of vaccines, such as influenza vaccines.

BACKGROUND OF THE INVENTION

Influenza viruses are pleiomorphic spherical-shaped particles of approximately 100-120 ran. The genome consists of eight segments that are encapsidated by a virally encoded nucleoprotein to form the nucleocapsid. Of these eight segments, six encode for single gene products: the viral polymerases PB1, PB2 and PA, the trans embrane glycoproteins hemagglutinin (HA) , neuraminidase (NA) and the nucleoprotein NP. By the use of alternative splicing, the two remaining segments encode for several proteins, namely the matrix Ml protein and the trans-membrane ion-channel M2 protein, the non- structural protein NS1 and the nuclear export protein NEP or NS2. The nucleocapsid is surrounded by a lipid envelope membrane containing embedded the HA and NA surface antigens. The HA protein is the major antigenic component of the virus, eliciting neutralizing antibodies. The protein is responsible for the binding of the virion to the sialic acid-containing cell surface receptors and, after internalization and endocytosis, for mediating fusion of the viral and cellular membranes. This process results in the release of the viral segments into the nucleus where viral replication takes place. The protein is synthesized as a HAO precursor of about 75 kD that non-covalently associates as a homotrimer. This polypeptide is post- • translationally cleaved by cellular proteases in two subunits, HA1 and HA2, which remain linked by a disulfide bridge (Skehel and Wiley 2000) . This cleavage exposes the fusion domain of the HA2 subunit favoring a pH-dependent conformational change essential for the infectivity of the virus. Cleavage of HA is therefore a fundamental prerequisite for spread of the virus in the infected host and viral pathogenicity (Steinhauer 1999) . Highly pathogenic strains of avian influenza viruses, responsible for lethal systemic infections, are activated by ubiquitous membrane-bound proteases. The HA cleavage site of these viruses consists of multibasic amino acids which is recognized by the subtilisin family of proteases such as furin. However, the a-pathogenic avian strains, as well as the mammalian influenza viruses, are activated by proteases secreted from a limited number of cell types. The HA molecule of these viruses displays a monobasic cleavage signal recognized by serine family proteases, such as trypsin. Other examples of serine- proteases are proteinase-K, plasmin (Lazarowitz and Choppin 1975) , the blood-clotting factor X in the allantoic fluid of chicken eggs (Gotoh et al . 1990) and bacterial proteases (Scheiblauer et al . 1992). Most cells that have been cultured in vitro for prolonged periods of time are no longer able to produce sufficient amounts of serine-proteases for this process. Therefore, the addition of trypsin is required to promote infectivity and subsequent replication of mammalian influenza viruses in continuous tissue cultures. Interestingly, besides influenza viruses, other viruses need processing through serine-proteases to be infectious. Examples of other viruses that require such proteases for infectiousness are, but are not limited to, Para-influenza Virus (PIV) rotavirus (Kido H et al . 1999; Henrickson et al . 1994; Crawford et al. 2001) and metapneumovirus (MPV) , such as human MPV (Van den Hoogen et al . 2001) . The PER.C6™ cell line (ECACC deposited under nr. 96022940), which originates from an adenovirus transformed human embryonal retina cell, is an important, platform on which numerous viruses (including influenza virus and rotavirus) can be grown for the production of safer vaccines (this feature of the cell line has been described in great detail in WO 01/38362, which is included as a reference herein in its entirety). PER.C6 cells can be used for the production of several viruses in serum-free media and in sterile settings. Moreover, PER.C6 cells can be grown to high densities and in large volumes. The viruses that are produced in this well- controlled manner are processed further for the production of vaccines.

Clearly, the cleavage of the influenza hemagglutinin HA precursor to HAl and HA2 subunits is a step of great importance in the viral life cycle and, consequently, it has an enormous impact in any study aimed at the optimization of influenza virus and -vaccine production. Among human influenza viruses, cleavage of HA in continuous tissue cultures, including PER.C6 cells, is promoted by the addition of trypsin. The trypsin enzyme that is commonly applied in tissue culture is generally animal-derived, with bovine and porcine pancreas being the primary source of the enzyme. Numerous disadvantages are associated with obtaining trypsin from these sources. For instance, there is always a considerable contamination with other proteins such as non-serine proteases that might hamper a correct cleaving. Recombinant proteases can be produced in different systems, ranging from bacteria to plants. Several groups have described a number of different production systems for obtaining recombinant trypsin in commercially interesting quantities (WO 00/05384; WO 01/55429; Hood and Jilka 1999) . The use of serum and/or products derived from animal/human sources in mammalian cell culture is widely applied, but is generally recognized as being unsafe, for instance in view of the possible presence of prions and unwanted adventitious viruses. It is appreciated in the art that there is a need for methods that employ clean, safe and animal component-free production systems and set-ups.

SUMMARY OF THE INVENTION

The invention relates to methods for producing a virus and/or a viral vector encoded protein, comprising the steps of: a) providing a cell capable of supporting the growth of said virus and/or the production of said viral vector encoded protein with a nucleic acid encoding said virus and/or said viral vector encoded protein; b) culturing said cell in a suitable medium comprising a serine protease; c) allowing for expression of said virus and/or said viral vector encoded protein; and d) harvesting said virus and/or viral vector encoded protein from said medium and/or said cell, wherein said serine protease is substantially free from animal and/or human components. The invention further relates to the use of a serine protease, such as recombinant bovine trypsin, for the production of virus particles in tissue culture cells, wherein said serine protease is substantially free from animal and/or human components. The invention also relates to virus particles or viral vector encoded proteins obtainable by methods according to the invention, or by a use according to the invention, optionally followed by one or more purification steps, and to the use of such virus particles or a viral vector encoded proteins for the prophylactic or therapeutic treatment of a viral disease, such disease, for instance, being caused by a virus selected from the group of: Influenza virus, Para- influenza Virus, rotavirus and metapneumovirus . DETAILED DESCRIPTION

The present invention provides the use of recombinantly produced serine proteases, such as bovine trypsin produced in corn, for the production and propagation of viruses in in vi tro cell cultures (see for details of the generation of the transgenic corn: WO 00/05384) . Other suitable platforms for the recombinant production of trypsin are Escherichia coli and yeast (WO 01/55429) . Preferred according to the invention is the use of recombinant trypsin produced in transgenic corn.

The present invention discloses methods for the production of viral batches in serum-free tissue culture, without using animal-derived trypsin. This is beneficial for the manufacturing of vaccines, since it significantly lowers the risk of transfer of unwanted components such as animal prions, viruses or other proteases that might have contaminated batches that are derived from animal tissue. Importantly, the present invention also shows that recombinantly produced trypsin is more efficient than animal-derived trypsin in supporting propagation of influenza viruses in cell culture.

Clearly, as mentioned above, other viruses than influenza virus need similar processes (such as the cleavage of HAO into HAl and HA2 in the case of influenza viruses) to become infectious and to enter host cells in a proper and efficient way. Viruses that can be propagated in vi tro using serine proteases such as trypsin include but are not limited to: influenza virus, Para-influenza Virus (PIV) , metapneumovirus (MPV) and rotavirus. Many of such viruses have also been found to infect PER.C6 cells. It is therefore also part of the invention to use recombinant trypsin for viruses other than influenza to propagate on cells, such as PER.C6™ cells (ECACC deposit no. 96022940).

The invention discloses a method for producing a virus and/or a viral vector encoded protein, comprising the steps of: a) providing a cell capable of supporting the growth of said virus and/or the production of said viral vector encoded protein with a nucleic acid encoding said virus and/or said viral vector encoded protein; b) culturing said cell in a suitable medium comprising a serine protease; c) allowing for expression of said virus and/or said viral vector encoded protein; and d) harvesting said virus and/or viral vector encoded protein from said medium and/or said cell, wherein said serine protease is substantially free from animal and/or human components. The nucleic acid may be provided to said cell with the means of a wild type virus, a recombinant virus particle, through naked nucleic acid transfections, electroporations or through 'other means that are useful and generally known to be used in the transfer of nucleic acid into a cell. Said nucleic acid may be DNA and/or RNA. The nucleic acid may be present in the form of a circular or linear plasmid, a cosmid, in genomic structures, etc. The viral vector encoded proteins may be viral proteins, but may also be non-viral proteins that may be used in therapeutics or for the preparation of medicaments according to the present invention. Non- limiting examples of viruses or virus particles that can be produced according to the methods of the invention are influenza virus, rotavirus, parainfluenza virus and metapneumovirus. Substantially free from animal and/or human components is defined as such that the protease is present in a form that was produced in an environment in which in essence no animal and/or human components are present. Such production environments are for instance bacterial cell cultures, plants, plant cell cultures or yeast production platforms. It can however not be excluded that such production systems make use of media that harbor components that are of animal and/or human origin. The protease is produced in non-animal (and non- human) systems, leading to a product that is substantially free from animal and/or human components and/or contaminants. It cannot be excluded that cells that are used according to the methods provided by the invention are cultured in the presence of components, present in the culture media, that were originally not free from animal and/or human derived components, or even that such components are animal and/or human derived. It is to be understood that at least the trypsin formulation that is used in these methods is being produced in animal and/or human component free conditions . Before the present invention, mammalian cell cultures for the production of viruses and viral encoded proteins made use of proteases, such as trypsin, that were derived from, e.g. porcine or bovine pancreas. Therefore, in an important embodiment, the invention provides a method, wherein said serine protease is produced recombinantly, for example in a production system such as a bacterial cell, a yeast cell, a plant cell or a plant. Many different serine proteases are known in the art. However, for the production of vaccines against viruses such as influenza, the protease of choice is generally trypsin. Therefore, one embodiment of the invention makes use of trypsin as the serine protease, wherein said trypsin is preferably bovine trypsin. In a preferred embodiment, the invention provides a method, wherein said trypsin is present in a concentration of less than 3 μg/ml, preferably less than 1 μg/ml, more preferably in a range between 0.1 to 0.5 μg/ml. In another embodiment, the invention provides methods according to the invention, wherein said cell capable of supporting the growth of said virus and/or the production of said viral vector encoded protein, is immortalized by the expression of a viral sequence, or a functional derivative thereof, of an adenovirus. Several immortalizing viral sequences are known in the art. The immortalizing sequences within adenovirus are the genes present in the so-called early region-1 (El) of the viral genome. This region encodes mainly two sets of proteins: the E1A and the E1B proteins that together can transform and immortalize a cell. Such cell is preferably not derived from tumor material, but instead preferably derived from healthy donor material. In one embodiment of the invention, the methods of the present invention are performed on cells that are immortalized by viral sequences that are based on, or derived from, the El region of an adenovirus. It is possible not to use the exact wild-type viral sequences from the El region to obtain a similar level of transformation and/or immortalisation. Therefore such sequences can be based on (which might mean synthetically generated) or derived from such sequences (for instance via sub-cloning with or without introducing heterologous nucleic acid sequences, mutations, swaps, deletions, etc.), as long as it is functionally equivalent to the wild-type sequences. In a highly preferred embodiment of the present invention the methods are performed with a human non-tumor derived transformed embryonic retina cell, wherein said cell is a PER.C6™ cell (ECACC deposit no. 96022940) or a derivative thereof. PER.C6 cells are known to support the growth of numerous viruses and support the production of several types of proteins (see WO 01/38362 and WO 00/63403) . The invention further provides the use of a serine protease for the production of whole viruses and/or virus particles in tissue culture cells, wherein said serine protease is substantially free from animal and/or human components . Preferably, such serine protease is produced recombinantly. More preferably, said recombinant serine protease is produced by a bacterial cell, a yeast cell, a plant cell or a plant. More preferably, said serine protease is trypsin, while it is even more preferred that said trypsin is (recombinantly produced) bovine trypsin. The present invention provides also virus particles and/or viral vector encoded proteins that are obtainable by a method according to the invention, or by a use according to the invention, optionally followed by one or more purification steps. The purification methods for such viruses, viral particles and/or proteins from the tissue culture medium and/or from the used cells, are generally known to the person skilled in the art. The invention also provides virus particles or viral vector encoded proteins according to the invention for the prophylactic or therapeutic treatment of a viral disease. Moreover, the invention provides the use of a virus particle or a viral vector encoded protein according to the invention for the preparation of a medicament for the prevention and/or treatment of a disease caused by a virus selected from the group of: Influenza virus, Para- influenza Virus, rotavirus and metapneumovirus. EXAMPLES

Example 1. Use of recombinant bovine trypsin from corn for the growth of influenza virus in PER.C6™ cells. Suspension cultures of PER.C6 were cultured in

ExCell-525 medium (JRH Biosciences) supplemented with 4 mM L-Glutamin (Gibco) , at 37°C and 10% C0₂ in 490 cm² tissue culture roller bottles during continuous rotation at 1 rp . On the day of infection, PER.C6 suspension cells were seeded in 6-well plates at a density of lxlO⁶ cell/ml (2 ml final volume) , in ExCell-525 medium supplemented with L-Glutamin and increasing amounts of recombinant bovine trypsin (ranging from 0.1 to 3 μg/ml) that was produced in transgenic corn (Prodigene cat. #9002-07-7). For the generation of the transgenic plants, the cloning of the cDNA and the expression of the bovine trypsin protein, see WO 00/05384. Also, separate cells were seeded in the presence of 3 μg/ml of non-recombinant porcine-derived trypsin-EDTA (Gibco) to serve as a control. Subsequently, infection was performed at a multiplicity of infection (moi) of 10^~4 pfu/cell with the PER-C6-grown influenza virus X-127 (egg-reassortant for A/Beijing/262/95) . Furthermore, mock-infected cells were included in the experiment for macroscopic observations of cells. Infected cells were incubated as static cultures, at 35°C and 10% C0₂ for six days. Samples were retrieved throughout the experiment and processed as follows. One ml of cell suspension was centrifuged for 4 min at 5000 rpm in eppendorf microfuge at room temperature. Clarified supernatants were transferred to a new eppendorf tube, and stored at -80°C until use in plaque assay (see below) . 900 μl of the remaining cell suspension was mixed with 100 μl of 10% zwittergent, which is a detergent commonly used in single radial im unodiffusion assay (Schild et al. 1975), for 30 min at room temperature, rapidly frozen in liquid N₂ and stored at -80°C until use in SRID.

Virus infectious titers were studied by scoring for plaque formation in Madin Darbin Canine Kidney (MDCK) cells inoculated with virus supernatants, using methods generally known to persons skilled in the art. MDCK cells are generally useful for such plaque assay experiments. Briefly, a total of 1 ml of 10-fold diluted viral supernatants were inoculated on MDCK cells which were grown until 95% confluence in 6-well plates in DMEM (Gibco) supplemented with 2 mM L-glutamin (Gibco) and 4 μg/ml Trypsin-EDTA (Gibco) . After approximately 1 h at 35°C the cells were washed twice with PBS (Gibco) and overloaded with 3 ml of agarose mix (1.2 ml 2.5% agarose, 1.5 ml 2x MEM, 30 μl 200 mM L-Glutamin, 24 μl Trypsin- EDTA, 250 μl PBS) . The cells were then incubated in a humid, 10% C0₂ atmosphere at 35°C for approximately 48 h and viral plaques were visually scored and counted. As shown in figure 1, recombinantly produced bovine trypsin from transgenic corn supported production of infectious virions at any of the concentrations tested. Already a concentration of 0.3 μg/ml of recombinantly produced trypsin yielded higher infectivity titers when compared with the animal-derived trypsin-EDTA control.

Next to the issue that recombinantly produced trypsin is safer than animal-derived trypsin, these results clearly show that recombinant trypsin is more efficient in influenza propagation processes. Next, influenza HA yields were determined by Single Radial Immunodiffusion (SRID) assay as described elsewhere (Wood et al . 1977). Briefly, the SRID method involves incorporating a suitably diluted antiserum into agarose, through which antigen diffuses forming a precipitation ring. Antigen at higher concentration diffuses further from the well before it falls to the level giving precipitation with antibody near optimal proportions. By incorporating standards (Reference Preparations) of known antigen concentration, a curve can be obtained and used to determine the amount of antigen in the test sample.

The assay was performed using glass plates onto which the agarose (immunodiffusion grade; 1% w/v) was poured using a Perspex mould. The agarose contained hyperimmune serum provided by NIBSC (National Institute for Biological Standards and Control) prepared against purified haemagglutinin of the influenza X-127 strain. Samples were pre-treated with the ionic detergent Zwittergent 3-14 (Calbiochem, cat. #693017) to disrupt the virions and release the HA. Undiluted and diluted samples were then inoculated into circular 4 mm diameter wells that are cut in the agarose (20 μl per well) . Once the samples were absorbed into the agarose, the plates were left to incubate for 3 days at room temperature in humidified containers. After this, dried gels were stained with a Coomassie stain. The diameters of the precipitation zone were measured and a standard curve was constructed using the diameters obtained for the dilutions of the standard preparation. The HA concentration of test samples was calculated from the standard curve. As shown in figure 2, the HA yield derived from the culture treated with 0.1 μg/ml of recombinantly produced trypsin was below detection level whereas recombinantly produced trypsin concentrations ranging from 0.3 to 3 μg/ml yielded quantities comparable with the non-recombinant animal-derived trypsin-EDTA control. These data show that recombinant trypsin produced in transgenic plants can efficiently support growth of influenza viruses and therefore replace the commonly used animal-derived non-recombinant trypsin.

Example 2. E fect of recombinant trypsin on influenza virus growth in PER.C6 cells cultured in two different serum-free media .

The performance of the recombinant trypsin produced in plants (corn) in PER.C6 cells cultured in two different serum-free media was investigated. On the day of infection, suspension grown PER.C6 cells were seeded in 6-well plates at a density of lxlO⁶ cell/ml (2 ml final volume), in ExCell-525 or AEM medium (Invitrogen) supplemented with L-Glutamine in the presence of 0.3 and 3 μg/ml recombinant bovine trypsin (Prodigene, see also example 1) . As a control, cells were seeded in the presence of the same concentrations of non-recombinant porcine-derived trypsin-EDTA (Gibco) . Influenza propagation was monitored by direct immunofluorescence (I.F.) assay. As a negative control, cells were cultured in the absence of trypsin. Cells were either mock infected or inoculated with the PER.C6-grown virus strain X-127 with a moi of 10^~4 pfu/cell. Cultures were incubated at 35°C, 10% C0₂. Samples were retrieved throughout the experiment for direct I.F. assay.

The direct I.F. assay for the detection of influenza virus kinetic of infection was carried out in infected PER.C6 cells using IMAGEN Influenza A and B kit (Dako) according to the protocol provided by the supplier. Briefly, infected cells were centrifuged for 5 min. The supernatant was removed and the pellet resuspended in PBS. 20 μl of cell suspension was added to each of two wells of an I.F. slide. This was allowed to dry at room temperature. The cells were fixed by adding 20 μl of acetone to each well and air-dried. To each well, 20 μl of the appropriate IMAGEN influenza reagent was added. The slide was then incubated for 15 min at 37 °C on a damp tissue. Excess reagent was washed away with PBS and then rinsed for 5 min in PBS. The slide was air-dried at room temperature. One drop of IMAGEN mounting fluid was added to each well and a cover slip placed over the slide. Samples were viewed microscopically using epifluorescence illumination. Infected cells were characterized by apple- green fluorescence. The approximate percentage of cells that showed positive (fluorescent green) compared with negative (red) cells was recorded. As shown in figures 3 and 4, influenza virus propagation was equally supported at 0.3 and 3 μg/ml of recombinant trypsin in both ExCell- 525 and AEM media, whereas only the higher concentration of 3 μg/ml of trypsin-EDTA allowed replication of the influenza virus. No major differences were observed between the two media. These data show that recombinant trypsin from transgenic corn plants is more efficient in supporting growth of influenza viruses on suspension cells and can therefore advantageously replace non- recombinant trypsin in vaccine production processes.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph showing the plaque-forming-units (pfu) per ml calculated from influenza virus batches grown in PER.C6 cells using different concentrations of recombinant and porcine-derived trypsin. Pfu' s were determined using MDCK cells and agarose overlay.

Figure 2 shows the yield of HA protein from influenza infected PER.C6 cells grown in suspension determined by SRID assay. Cells were treated with different concentrations of recombinant trypsin from transgenic plants and porcine-derived trypsin.

Figure 3 is a graph showing the rate of immunofluorescence of PER.C6 cells infected with influenza virus in serum-free ExCell-525 medium in the presence of different concentrations of recombinant and porcine-derived trypsin.

Figure 4 is a graph showing the rate of immunofluorescence of PER.C6 cells infected with influenza virus in serum-free AEM medium in the presence of different concentrations of recombinant and porcine- derived trypsin.

REFERENCES

Crawford SE, Mukherjee SK, Estes MK, Lawton JA, Shaw AL, Ramig RF and Prasad BV. (2001) Trypsin cleavage stabilizes the rotavirus VP4 spike. J Virol 75:6052-6061

Gotoh B, Ogasawara T, Toyoda T, Inocencio NM, Hamaguchi M and Nagai Y. (1990) An endoprotease homologous to the blood clotting factor X as a determinant of viral tropism in chick embryo. EMBO J 9:4189-4195

Henrickson KJ, Kuhn SM, Savatski LL and Sedmak J. (1994) Recovery of human parainfluenza virus types one and two. J Virol Methods 46:189-205

Hood and Jilka (1999) Plant-based production of xenogenic proteins. Curr Opin Biotechnol 10:382-386

Kido H, Murakami M, Oba K, Chen Y and Towatari T. (1999) Cellular proteinases trigger the infectivity of the influenza A and Sendai viruses. Mol Cells 9:235-244

Lazarowitz SG and Choppin PW. (1975) Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. Virology 68:440-454

Scheiblauer H, Reinacher M, Tashiro M and Rott R. (1992) Interactions between bacteria and influenza A virus in the development of influenza pneumonia. J Infect Dis 166:783-791

Schild GC, Wood JM and Newman RW. (1975) A single-radial- immunodiffusion technique for the assay of influenza haemagglutinin antigen. Proposals for an assay method for the haemagglutinin content of influenza vaccines. Bull World Health Organ 52:223-231

Skehel JJ and Wiley DC. (2000) Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69:531-569

Steinhauer DA. (1999) Role of hemagglutinin cleavage for the pathogenicity of influenza virus. Virology 258:1-20

Van den Hoogen BG, De Jong JC, Groen J, Kuiken T, De Groot R, Fouchier RAM and Osterhaus ADME . (2001) A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nature Med 7:719-724

Wood JM, Schild GC, Newman RW and Seagroatt V. (1977) An improved single-radial-immunodiffusion technique for the assay of influenza haemagglutinin antigen: application for potency determinations of inactivated whole virus and subunit vaccines. J Biol Stand 5:237-247

Claims

1. A method for producing a virus and/or a viral vector encoded protein, comprising the steps of: a) providing a cell capable of supporting the growth of said virus and/or the production of said viral vector encoded protein with a nucleic acid encoding said virus and/or said viral vector encoded protein; b) culturing said cell in a suitable medium comprising a serine protease; c) allowing for expression of said virus and/or said viral vector encoded protein; and d) harvesting said virus and/or viral vector encoded protein from said medium and/or said cell, wherein said serine protease is substantially free from animal and/or human components.

2. Method according to claim 1, wherein said serine protease is produced recombinantly.

3. Method according to claim 1 or 2, wherein said (recombinant) serine protease is produced by a bacterial cell, a yeast cell, a plant cell or a plant.

4. Method according to any one of claims 1-3, wherein said serine protease is trypsin.

5. Method according to claim 4, wherein said trypsin is bovine trypsin.

6. Method according to claim 4 or 5, wherein said trypsin is present in a concentration of less than 3 μg/ml, preferably less than 1 μg/ml, more preferably in a range between 0.1 to 0.5 μg/ml.

7. Method according to any one of claims 1-6, wherein said cell is immortalized by the expression of a viral sequence, or a functional derivative thereof, of an adenovirus .

8. Method according to claim 7, wherein said viral sequence is based on, or derived from, the El region of an adenovirus.

9. Method according to claim 8, wherein said cell is a PER.C6™ cell (ECACC deposit no. 96022940) or a derivative thereof.

10. Method according to any one of claims 1-9, wherein said virus is selected from the group of: Influenza virus, Para-influenza Virus, rotavirus and metapneumovirus .

11. Method according to any one of claims 1-9, wherein said viral vector encoded protein is derived from a virus selected from the group of: Influenza virus, Para- influenza Virus, rotavirus and metapneumovirus.

12. Use of a serine protease for the production of virus particles in tissue culture cells, wherein said serine protease is substantially free from animal and/or human components.

13. Use according to claim 12, wherein said serine protease is produced recombinantly.

14. Use according to claim 13, wherein said recombinant serine protease is produced by a bacterial cell, a yeast cell, a plant cell or a plant.

15. Use according to any one of claims 12-14, wherein said serine protease is trypsin.

16. Use according to claim 15, wherein said trypsin is bovine trypsin.

17. A virus particle or a viral vector encoded protein obtainable by a method according to any one of claims ,1-

11, or by a use according to any one of claims 12-16, optionally followed by one or more purification steps.

18. A virus particle or a viral vector encoded protein according to claim 17 for use as a prophylactic or therapeutic treatment of a viral disease.

19. Use of a virus particle or a viral vector encoded protein according to claim 17 for the preparation of a medicament for the prevention and/or treatment of a disease caused by a virus selected from the group of: Influenza virus, Para-influenza Virus, rotavirus and metapneumovirus .