Non-tumorigenic MDCK cell line for propagating viruses (02-Mar-2010)

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of the following U.S. Provisional Application Nos.: 60/638,166 filed Dec. 23, 2004 and 60/641,139 filed Jan. 5, 2005. The priority applications are hereby incorporated by reference herein in their entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to novel non-tumorigenic MDCK cells, which can be used for the production of vaccine material. The non-tumorigenic MDCK cells may be adapted to serum-free culture medium. The present invention further relates to media formulations and cultivation methods for the propagation of the non-tumorigenic MDCK cells as well as methods for maintaining the non-tumorigenic nature of the cell lines of the invention. The present invention further relates to processes for the production of influenza viruses in cell culture using non-tumorigenic MDCK cells. The present invention also relates to the viruses (e.g., influenza) obtainable by the process described and immunogenic compositions which contain viruses of this type and/or components thereof.

BACKGROUND OF THE INVENTION

Vaccination is the most important public health measure for preventing disease caused by annual epidemics of influenza. The effective use of vaccines is dependent on being able to quickly produce large quantities of vaccine material (e.g., virus) from a stable and easy to cultivate source. The rapid development of vaccines and their abundant availability is critical in combating many human and animal diseases. Delays in producing vaccines and shortfalls in their quantity can cause problems in addressing outbreaks of disease. For example, recent studies suggest that there is cause for concern regarding the long lead times required to produce vaccines against pandemic influenza. See, for example, Wood, J. M., 2001, Philos. Trans. R. Soc. Lond. B. Biol. Sci., 356:1953. Efficient vaccine production requires the growth of large quantities of vaccine material produced in high yields from a host system. Different vaccine materials require different growth conditions in order to obtain acceptable yields. Vaccine material may be produced in embryonated eggs, primary tissue culture cells, or in established cell lines. However, these host systems currently suffer from a number of limitations detailed below.

Embryonated eggs are typically used for influenza vaccine virus production in a time-, labor-, and cost intensive process that necessitates the management of chicken breeding and egg fertilization. In addition, influenza vaccine produced in eggs is contraindicated for persons with egg allergies due to the severe immediate hypersensitivity reaction that can occur. Thus, there has been an effort by the vaccine industry to develop alternative production platforms that do not utilize eggs such as producing influenza vaccine in a cell culture system.

The use of primary tissue culture cells is hampered by the difficulties encountered in developing and maintaining a stable primary cell population. Often established cells lines are used to circumvent the technical limitations of primary cells. However, many of these cell lines are known to be tumorigenic and as such raise safety concerns and are subject to significant regulatory constraints against their use for vaccine production. In fact, the applicable guidelines of the World Health Organization indicate that only a few cell lines are allowed for vaccine production. Additional problems arise from the use of serum and/or protein additives derived from animal or human sources in cell culture media. For example, variability in the quality and composition among lots of additives and the risk of contamination with mycoplasma, viruses, BSE-agents and other infectious agents are well known. In general, serum or serum-derived substances like albumin, transferrin or insulin may contain unwanted agents that can contaminate the culture and the biological products produced from therefrom. Therefore, many groups are working to develop efficient host systems and cultivation conditions that do not require serum or serum derived products.

Consequently, there has been a demand for establishing a non-tumorigenic cell line useful for the production of vaccine materials in a low-cost, highly safe and stable manner preferably in serum-free or in animal protein-free culture conditions. Such a cell system would be particularly useful for the production of influenza vaccine material.

Madin Darby Canine Kidney (MDCK) cells have been traditionally used for the titration of influenza viruses (Zambon M., in Textbook of Influenza, ed Nicholson, Webster and Hay, pg 291-313, Blackwell Science (1998)). These cells were established in 1958 from the kidney of a normal male cocker spaniel. The ATCC list the MDCK (CCL 34) line as having been deposited by S. Madin and N. B. Darby however, numerous other lineages of MDCK cells are available. Leighton J and his coworkers published a series of papers (Leighton et al.,1969, Science 163:472; Leighton et al., 1970, Cancer 26:1022 and Leighton et al., 1972 Europ J. Cancer 8:281) documenting the oncogenic characteristics of the MDCK cells. However, the lineage and passage number of the MDCK cells used for these studies was not described and it was already known that MDCK cells from different lineages and different passages showed changes in chromosome numbers and structure (Gaush et al., 1966, Proc. Soc. Exp. Biol. Med., 122: 931) which could result in cells with tumorigenic properties.

Since one of the major considerations for the acceptability of a cell line for vaccine production concerns the potential malignancy of those cells the use of MDCK cells for the production of vaccine material using currently described cell lines is limited. Groner et al. (U.S. Pat. No. 6,656,720) and Makizumi et al. (U.S. Pat. No. 6,825,036) both purport to disclose cell lines derived from MDCK cells which have been adapted to grow in serum-free media in suspension and which can be utilized for the production of influenza virus. However, it has been reported that there is correlation between the loss of anchorage requirement and the transformation of normal animal cells to cells which are tumorigenic (Stiles et al., 1976, Cancer Res., 36:3300). Several groups (Kessler et al., 1999, Cell Culture Dev Biol Stand, 98:13; Merten et al., 1999, Cell Culture Dev Biol Stand, 98:23 and Tree et al., 2001, Vaccine, 19:3444) purport to describe the use of MDCK cells for the large-scale production of influenza virus; however, they do not address the potential transformation of the MDCK cells used.

Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention. In addition, citation of a patent shall not be construed as an admission of its validity.

SUMMARY OF THE INVENTION

The present invention provides non-tumorigenic MDCK cells which have been adapted to grow in either serum containing or serum-free media formulations including animal protein-free (APF) formulations. In one embodiment, the non-tumorigenic MDCK cells of the invention are adherent. In another embodiment, the non-tumorigenic MDCK cells of the invention have an epithelial morphology. In yet another embodiment, the non-tumorigenic MDCK cells of the invention are adherent and have an epithelial morphology. Tumorigenicity is in one embodiment, determined by the adult nude mouse model (e.g., Stiles et al., 1976, Cancer Res, 36:1353, and Example 2 below). Tumorigenicity may also be tested by other assays, for example, by injection into a chick embryo and/or topical application to the chorioallantois (Leighton et al., 1970, Cancer, 26:1022).

Viruses that can be grown in the MDCK cells of the invention include but are not limited to negative strand RNA viruses, including but not limited to influenza, RSV, parainfluenza viruses 1, 2 and 3, and human metapneumovirus.

The present invention further provides methods and media formulations useful for the derivation and maintenance of non-tumorigenic MDCK cells. The MDCK cells of the invention are particularly useful for the production of vaccine material such as, for example, viruses.

Other aspects of the invention include methods of producing vaccine material (e.g., virus) by culturing any MDCK cell of the invention, in a suitable culture medium under conditions permitting production of vaccine material and, isolating the material from one or more of the host cell or the medium in which it is grown.

Immunogenic compositions are also features of the invention. For example, immunogenic compositions comprising the vaccine material produced as described above and, optionally, an excipient such as a pharmaceutically acceptable excipient or one or more pharmaceutically acceptable administration component.

Methods of producing immunogenic responses in a subject through administration of an effective amount of one or more above described immunogenic compositions to a subject are also within the current invention. Additionally, methods of prophylactic or therapeutic treatment of a viral infection (e.g., viral influenza) in a subject through administration of one or more above described immunogenic compositions in an amount effective to produce an immunogenic response against the viral infection are also part of the current invention. Subjects for such treatment can include mammals (e.g., humans). Additionally, such methods can also comprise administration of a composition of one or more viruses produced in the MDCK cells of the invention and a pharmaceutically acceptable excipient that is administered to the subject in an amount effect to prophylactically or therapeutically treat the viral infection.

These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures appendix.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Growth of Influenza strains in cells. Panel A is a photograph showing the results of a fluorescent focus assay comparing the spread of infection of a representative ca/ts influenza strain in MDCK cells and a Vero Cell Clone (27F9). Panel B is a growth curve of influenza strain ca A/Vietnam/1203/2004 (H5N1) in MDCK cells. Titers peaked at 48 hours post infection at ˜8 log₁₀ TCID₅₀/mL and remained stable for the next 3 to 4 days.

FIG. 2 outlines the process used for the derivation of MDCK-S PreMCB (passage No. 57). The process is described in detail in Example 2.

FIG. 3 is a photograph showing that MDCK-S cells have an epithelial-like morphology. The photo was taken 3 days after seeding.

FIG. 4 is the growth curve of MDCK-S cells in 10% FBS DMEM medium. Cells had about a 1 day lag phase followed by exponential growth entering stationary phase at day 4 post seeding achieving a maximum density of ˜29×10⁶ cells on day 5.

FIG. 5 is a graph of the glucose consumption and lactate production of MDCK-S cells in 10% FBS DMEM medium. The rates were low during lag phase increasing to 2.93 mM/day and 3.43 mM/day for glucose and lactate, respectively.

FIG. 6 is a graph of the glutamine consumption and both glutamate and ammonia production of MDCK-S cells in 10% FBS DMEM medium. The glutamine consumption rate was 0.49 mM/day up to day 4 and the ammonia production rate was 0.32 mM/day up to day 5. Glutamate did not accumulate in this study.

FIG. 7 is a plot of the distributions of chromosome number in 100 metaphase low passage (P61/4) and high passage (P81/24) MDCK-S cells. The chromosome count ranged from 70 to 84 per metaphase with a modal chromosome number of 78 for both the high and low passage cells.

FIG. 8 outlines the process used for the derivation of MDCK-T PreMCB (passage No. 64/5). The process is described in detail in Example 3.

FIG. 9 is a photograph showing that MDCK-T cells have an epithelial-like morphology. The photo was taken 3 days after seeding.

FIG. 10 is the growth curve of MDCK-T cells in Taub's media. Cells had no lag phase and were in exponential growth until entering stationary phase at day 4 post seeding.

FIG. 11 is a graph of the glucose consumption and lactate production of MDCK-T cells in Taub's media. During the exponential phase the rates were 1.78 mM/day and 2.88 mM/day for glucose and lactate, respectively.

FIG. 12 is a graph of the glutamine consumption and both glutamate ammonia production of MDCK-T cells in Taub's media. The glutamine consumption rate was 0.36 mM/day up to day 4 and the ammonia production rate increased linearly up to day 7 at a rate of 0.22 mM/day. Glutamate did not accumulate in this study.

FIG. 13 is a plot of the distributions of chromosome number in 100 metaphase low passage (P61/4) and high passage (P81/24) MDCK-T cells. The chromosome count ranged from 52 to 82 per metaphase for low passage cells and from 54 to 82 for high passage cells.

FIG. 14 is a plot of the distributions of chromosome number in 100 metaphase MDCK-T, MDCK-SF101 (passage 71/9) and MDCK-SF102 cells (passage 71/9). Both SF101 and SF102 cells had a modal chromosome number of 78, with the chromosome count ranging from 70 to 82 and 60 to 80 per metaphase for SF101 and SF102, respectively.

FIG. 15 is a photograph showing that MDCK-SF103 have an have an epithelial-like cell morphology. The photo was taken 3 days after seeding.

FIG. 16 is the growth curve of MDCK-SF103 cells in MediV SFM103. Cells had about a 1 day lag phase followed by exponential growth entering stationary phase at day 4 post seeding achieving a maximum density of ˜17×10⁶ cells on day 4.

FIG. 17 is a graph of the glucose consumption and lactate production of MDCK-SF103 cells in MediV SFM103. During the exponential phase the glucose consumption and lactate production mirrored each other with lactate increasing in concentration as the glucose concentration decreased

FIG. 18 is a graph of the glutamine consumption and both ammonia and glutamate production of MDCK-SF103 cells in MediV SFM103. The ammonia production rate increased nearly linearly up to day 7. Glutamate did not accumulate in this study.

FIG. 19 is a plot of the distributions of chromosome number in 100 metaphase MDCK-SF103 cells at passage 87. SF103 cells had a modal chromosome number of 78, with the chromosome count ranging from 66 to 80.

FIG. 20 Production Scale Growth and Purifiction. Panel A is a plot of the yield obtained for several vaccine reassortant strains, B/Victoria/504/2000 (˜8 LogTCID 50/mL), A/Sydney/05/97 (˜7.85 LogTCID 50/mL) and A/New Calcdonia/20/99 (˜8.2 LogTCID 50/mL), from 250 mL spinner flasks of MDCK-SF103 grown on Cytodex beads. Panel B outlines one cell culture scale up process which can be utilized for commercial scale production of vaccine material.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the discovery that MDCK cells can be cultivated under conditions wherein they remain non-tumorigenic. The present invention provides non-tumorigenic cell lines, including MDCK cell lines and other types of cells which have been adapted to a variety of cell culture conditions including serum-free media formulations and are referred to herein as “cells of the invention”. In addition, the present invention provides cell culture compositions comprising cells of the invention and other components including, but not limited to, media (e.g., a media disclosed herein), media components, buffers, chemical compounds, additional cell types, viral material (e.g., viral genomes, viral particles) and heterologous proteins. The present invention also provides methods and media formulations useful for the cultivation of non-tumorigenic cells, including MDCK cells, with one more specific characteristics including but not limited to, being non-tumorigenic (e.g., not forming nodules in a nude mouse) and/or growth as adherent cells and/or having an epithelial-like morphology and/or supporting the replication of various viruses including but not limited to orthomyxoviruses, paramyxoviruses, rhabdoviruses and flavoviruses. The culture conditions of the present invention include serum containing and serum-free media formulations, as well as animal protein-free (APF) formulations. In addition, the present invention also provides methods of producing vaccine material (e.g., influenza virus) in non-tumorigenic cells, including MDCK cells, preparing vaccine material from non-tumorigenic cells, and methods of preventing influenza infection utilizing vaccine materials produced in non-tumorigenic cells. The cells of the invention are particularly useful for the production of cold adapted/temperature sensitive/attenuated (ca/ts/att) influenza strains (e.g., those in FluMist®) which do not replicate as efficiently in other mammalian cell lines (e.g., Vero, PerC6, HEK-293, MRC-5 and WI-38 cells).

Cell Characteristics

The cells according to the invention are in one embodiment, vertebrate cells. In another embodiment, the cells of the invention are mammalian cells, e.g., from hamsters, cattle, monkeys or dogs, in particular kidney cells or cell lines derived from these. In still another embodiment, the cells of the invention are MDCK cells (e.g., derived from ATCC CCL-34 MDCK) and are specifically referred to herein as “MDCK cells of the invention” and are encompassed by the term “cells of the invention”. In a specific embodiment, the cells of the invention are derived from ATCC CCL-34 MDCK. Cells of the invention may be derived from CCL-34 MDCK cells by methods well known in the art. For example, the CCL-34 MDCK cells may be first passaged a limited number of times in a serum containing media (e.g., Dulbecco's Modified Eagle Medium (DMEM)+10% Fetal Bovine Serum (FBS)+4 mM glutamine+4.5 g/L glucose, or other media described herein) followed by cloning of individual cells and characterization of the clones. Clones with superior biological and physiological properties including, but not limited to, doubling times, tumorigenicity profile and viral production, are selected for the generation of a master cell bank (MCB). In one aspect, the cells of the invention are adapted to growth in a media of choice (e.g., a serum-free or APF media, such as those described herein). Such adaptation may occur prior to, concurrently with, or subsequent to the cloning of individual cells. In certain embodiments, cells of the invention are adapted to grow in MediV SF101, MediV SF102, MediV SF103, MediV SF104 or MediV SF105. Cells of the invention adapted to grow in these media are referred to herein as “MDCK-SF101, MDCK-SF102, MDCK-SF103, MDCK-SF104 and MDCK-SF105” cells, respectively and as “MDCK-SF cells” collectively. In other embodiments, cells of the invention are adapted to grow in serum containing media (e.g., Dulbecco's Modified Eagle Medium (DMEM)+10% Fetal Bovine Serum (FBS)+4 mM glutamine+4.5 g/L glucose), such cells are referred to herein as “MDCK-S” cells. MDCK-SF and MDCK-S cells are also encompassed by the terms “cells of the invention” and “MDCK cells of the invention”.

In a specific embodiment of the invention the cells are of the cell lines including, but not limited to, those which have been deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, Va. 20110-2209) and assigned ATCC Deposit Nos. PTA-6500 (Deposited on Jan. 5, 2005), PTA-6501 (Deposited on Jan. 5, 2005), PTA-6502 (Deposited on Jan. 5, 2005), and PTA-6503 (Deposited on Jan. 5, 2005), these cells are referred to herein as “MDCK-S, MDCK-SF101, MDCK-SF102 and MDCK-SF103”, respectively and as “the MDCK cells of the invention” collectively. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. In one embodiment, the MDCK cells of the invention are used to generate a cell bank useful for the preparation of vaccine material suitable for approval by the U.S. Food and Drug Administration for human use.

The cells lines MDCK-S, MDCK-SF101, MDCK-SF102, MDCK-SF103, MDCK-SF104 and MDCK-SF105 are derived from the cell line MDCK (CCL 34) by passaging and selection with respect to one or more specific characteristics including but not limited to, growing as adherent cells either in serum containing, or serum-free media or animal protein-free media, having an epithelial-like morphology, being non-tumorigenic (e.g., not forming nodules in a nude mouse) and/or supporting the replication of various viruses including but not limited to orthomyxoviruses, paramyxoviruses, rhabdoviruses and flavoviruses.

In one embodiment, the MDCK cells of the invention are non-tumorigenic. Methods for determining if cells are tumorigenic are well known in the art (see, for example, Leighton et al., 1970, Cancer, 26:1022 and Stiles et al., 1976, Cancer Res, 36:1353), the method currently preferred by the U.S. Food and Drug Administration using the nude mouse model is detailed in Example 2 below. In a specific embodiment, the MDCK cells of the invention are non-tumorigenic in the adult nude mouse model (see, Stiles et al., Id and Example 2 below). In another specific embodiment, the MDCK cells of the invention are non-tumorigenic when injected into a chick embryo and/or topically applied to the chorioallantois (see, Leighton et al., Id). In still another embodiment, the MDCK cells of the invention are non-tumorigenic in the adult nude mouse model but not when injected into a chick embryo and/or topically applied to the chorioallantois. In yet another embodiment, the MDCK cells of the invention are non-tumorigenic in the adult nude mouse model and when injected into a chick embryo and/or topically applied to the chorioallantois. In still another embodiment, the MDCK(cells of the invention are non-tumorigenic after at least 20 passages, or after at least 30 passages, or after at least 40 passages, or after at least 50 passages, or after at least 60 passages, or after at least 70 passages, or after at least 80 passages, or after at least 90 passages, or after at least 100 passages in a medium. In yet another specific embodiment the medium is a media described herein (e.g., Medi SF103).

Tumorigenicity may be quantified in numerous ways known to one of skill in the art. One method commonly utilized is to determine the “TD₅₀” value which is defined as the number of cells required to induce tumors in 50% of the animals tested (see, e.g., Hill R. The TD₅₀ assay for tumor cells. In: Potten C, Hendry J, editors. Cell clones. London: Churchill Livingstone; 1985. p. 223). In one embodiment, the MDCK cells of the invention have a TD₅₀ value of between about 10¹⁰ to about 10¹, or between about 10⁸ to about 10³, or between about 10⁷ to about 10⁴. In a specific embodiment, the MDCK cells of the invention have a TD₅₀ value of more than about 10¹⁰, or of more than about 10⁹, or of more than about 10⁸, or of more than about 10⁷, or of more than about 10⁶, or of more than about 10⁵, or of more than about 10⁴, or of more than about 10³, or of more than about 10², or of more than about 10¹.

In another embodiment, the non-tumorigenic cells of the invention grow as adherent cells either in serum containing or serum-free media or animal protein-free media. In still another embodiment, the non-tumorigenic cells of the invention have an epithelial-like morphology. In yet another embodiment, the MDCK cells of the invention support the replication of various viruses including but not limited to orthomyxoviruses, paramyxoviruses, rhabdoviruses and flavoviruses. It is contemplated that the MDCK cells of the invention may have any combination of one or more specific characteristics including but not limited to, being non-tumorigenic, growing as adherent cells, having an epithelial-like morphology and supporting the replication of various viruses.

It is contemplated that each and every passage of the MDCK cells of the invention is documented in sufficient detail such that the complete lineage of each cell line is available. The documentation of each and every passage may facilitate approval by the U.S. Food and Drug Administration and other regulatory bodies around the world for the use of the MDCK cells of the invention for the preparation of vaccine material.

In another embodiment, the MDCK cells of the invention are free of microbial contaminants (e.g., bacterial, viral and fungal contaminants). Methods for testing for the presence of bacterial and fungal contaminants are well known in the art and routinely performed by commercial contractors (e.g., BioReliance®, Rockville, Md.). Accepted microbial sterility and mycoplasm tests are detailed in Example 2 below. Specific examples of microbial agents which may be tested for are listed in Table 6.

In yet another embodiment, the MDCK cells of the invention support the replication of viruses including but not limited to orthomyxoviruses (including influenza A and/or B strains), paramyxoviruses (including RSV A and/or B, human metapneumovirus and parainfluenza 1, 2 and/or 3), rhabdoviruses and flavoviruses. In a specific embodiment, the MDCK cells of the invention support the replication of cold adapted/temperature sensitive (ca/ts) influenza viruses such as those found, for example, in FluMist® (Belshe et al., 1998, N Engl J Med 338:1405; Nichol et al., 1999, JAMA 282:137; Jackson et al., 1999, Vaccine, 17:1905) and/or reassortant viruses comprising the backbone of these viruses or comprising the backbone (or one or more vRNA segment(s)) of influenza viruses having one or more of the following characteristics: cold adapted, attenuated, and temperature sensitive. One indication of the ability of a cell to support viral replication is the yield of virus obtained from an infected cell culture. Viral yield can be determined by numerous methods known to one skilled in the art. For example, viral yield can be quantified by determining the concentration of virus present in a sample according to a median tissue culture infectious dose (TCID₅₀) assay that measures infectious virions. The TCID₅₀ values are often reported as the log₁₀ TCID₅₀/mL. In one embodiment, the MDCK cells of the invention support the replication of influenza viruses (e.g., ca/ts strains) to a log₁₀ TCID₅₀/mL of at least 6.0, or at least 6.2, or at least 6.4, or at least 6.6, or at least 6.8, or at least 7.0, or at least 7.2, or at least 7.4, or at least 7.6, or at least 7.8, or at least 8.0, or at least 8.2, or at least 8.4, or at least 8.6, or at least 8.8, or at least 9.0, or at least 9.2, or at least 9.4, or at least 9.6, or at least 9.8. In another embodiment, the MDCK cells of the invention support the replication of influenza viruses (e.g., ca/ts strains) to a log₁₀ TCID₅₀/mL of at least about 6.0, or at least about 6.2, or at least about 6.4, or at least about 6.6, or at least about 6.8, or at least about 7.0, or at least about 7.2, or at least about 7.4, or at least about 7.6, or at least about 7.8, or at least about 8.0, or at least about 8.2, or at least about 8.4, or at least about 8.6, or at least about 8.8, or at least about 9.0, or at least about 9.2, or at least about 9.4, or at least about 9.6, or at least about 9.8.

It will be understood by one of skill in the art that the cells of the invention will generally be part of a cell culture composition. The components of a cell culture composition will vary according to the cells and intended use. For example, for cultivation purposes a cell culture composition may comprise cells of the invention and a suitable media for growth of the cells. Accordingly, the present invention provides cell culture compositions comprising cells of the invention and other components including, but not limited to, media (e.g., a media disclosed herein), media components, buffers, chemical compounds, additional cell types, viral material (e.g., viral genomes, viral particles) and heterologous proteins. In one embodiment, a cell culture composition comprises cells of the invention and a media or components thereof. Media which may be present in a cell culture composition include serum-free media, serum containing media and APF media. In one embodiment, a cell composition comprises a media disclosed herein (e.g., MediV SF101, MediV SF102, MediV SF103, MediV SF104 or MediV SF105) or components thereof.

Methods and Media Formulations

The present invention provides methods and media formulations for the cultivation of non-tumorigenic MDCK cells in serum containing media. The present invention also provides methods for the adaptation to and subsequent cultivation of non-tumorigenic MDCK cells in serum-free media including APF media formulations. In certain aspects of the invention, the medias are formulated such that the MDCK cells retain one or more of the following characteristics including but limited to, being non-tumorigenic, growing as adherent cells, having an epithelial-like morphology and supporting the replication of various viruses when cultured. It is contemplated that the media formulations disclosed herein or components thereof, may be present in a cell culture compostion.

Serum containing media formulations are well known in the art. Serum containing media formulations include but are not limited to, Dulbecco's Modified Eagle Medium (DMEM)+Fetal Bovine Serum (FBS)+glutamine+glucose. In one embodiment, FBS is present in a serum containing media at a concentration between about 1% and about 20%, or between about 5% and about 15%, or between about 5% and about 10%. In a specific embodiment, FBS is present in a serum containing media at a concentration of 10%. In another embodiment, glutamine is present in a serum containing media at a concentration of between about 0.5 mM and about 10 mM, or between about 1 mM and 10 mM, or between about 2 mM and 5 mM. In a specific embodiment, glutamine is present in a serum containing media at a concentration of 4 mM. In still another embodiment, glucose is present in a serum containing media at a concentration of between about 1 g/L and about 10 g/L, or between about 2 g/L and about 5 g/L. In a specific embodiment, glucose is present in a serum containing media at a concentration of 4.5 g/L. In yet another embodiment, a serum containing media formulation comprises, FBS at a concentration between about 1% and about 20%, glutamine at a concentration of between about 0.5 mM and about 10 mM, and glucose a concentration of between about 1 g/L and about 10 g/L. In a specific embodiment, a serum containing media formulation comprises, Dulbecco's Modified Eagle Medium (DMEM)+10% Fetal Bovine Serum (FBS)+4 mM glutamine+4.5 g/L glucose. DMEM is readily available from numerous commercial sources including, for example, Gibco/BRL (Cat. No. 11965-084). FBS is readily available from numerous commercial sources including, for example, JRH Biosciences (Cat. No. 12107-500M). While FBS is the most commonly applied supplement in animal cell culture media, other serum sources are also routinely used and encompassed by the present invention, including newborn calf, horse and human.

In one embodiment, MDCK-S serum adapted non-tumorigenic cells of the invention are derived from Madin Darby Canine Kidney Cells (MDCK) cells obtained from the American type Culture Collection (ATCC CCL34) by culturing them in a chemically defined media supplemented with serum. In a specific embodiment, MDCK cells (ATCC CCL34) are expanded in a chemically defined media supplemented with serum to generate the MDCK-S cell line as follows: The MDCK (ATCC CCL34) cells are passaged as need in Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (10% v/v), 4 mM glutamine and 4.5 g/L glucose to obtain enough cell to prepare a frozen pre Master Cell Bank (PreMCB) designated MDCK-S. In another specific embodiment, the cells are cultured using the process detailed in Example 2, infra. It is specifically contemplated that the MDCK-S serum adapted cell are passaged for another 20 passages or more, from a vial of PreMCB and tested for tumorigenicity in an vivo adult nude mice model and karyology in a karyotype assay. In certain embodiments, the expanded MDCK-S cells will not produce nodules when injected subcutaneously into adult nude mice and will have a modal chromosome number of 78 with a range of chromosome numbers of no more then about 60-88, or of no more then about 65-85, or of no more than about 65-80, or of no more then about 70-85. In one embodiment, the MDCK-S cells are non-tumorigenic after at least 20 passages, or after at least 30 passages, or after at least 40 passages, or after at least 50 passages, or after at least 60 passages, or after at least 70 passages, or after at least 80 passages, or after at least 90 passages, or after at least 100 passages in a medium (e.g., a media described herein).

It will be appreciated by one of skill in the art that the use of serum or animal extracts in tissue culture applications may have drawbacks (Lambert, K. J. et al., In: Animal Cell Biotechnology, Vol 1, Spier, R. E. et al., Eds., Academic Pres New York, pp. 85-122 (1985)). For example, the chemical composition of these supplements may vary between lots, even from a single manufacturer. In addition, supplements of animal or human origin may also be contaminated with adventitious agents (e.g., mycoplasma, viruses, and prions). These agents can seriously undermine the health of the cultured cells when these contaminated supplements are used in cell culture media formulations. Further, these agents may pose a health risk when substances produced in cultures contaminated with adventitious agents are used in cell therapy and other clinical applications. A major fear is the presence of prions which cause spongiform encephalopathies in animals and Creutzfeld-Jakob disease in humans. Accordingly, the present invention further provides serum-free media formulations.

Serum-free media formulations of the invention include but are not limited to MediV SF101 (Taub's+Plant Hydrolysate), MediV SF102 (Taub's+Lipids), MediV SF103 (Taub's+Lipds+Plant Hydrolysate), MediV SF104 (Taub's+Lipds+Plant Hydrolysate+growth factor) and Medi SF105 (same as MediV SF104 except transferrin is replaced with Ferric ammonium citrate/Tropolone or Ferric ammonium sulfate/Tropolone). It is specifically contemplated that Taub's SF medium (Taub and Livingston, 1981, Ann NY Acad. Sci., 372:406) is a 50:50 mixture of DMEM and Ham's F12 supplemented with hormones, 5 μg/mL insulin, 5 μg/mL transferrin, 25 ng/mL prostaglandin E1, 50 nM hydrocortisone, 5 pM triidothyronine and 10 nM Na₂SeO₃, 4.5 g/L glucose, 2.2 g/L NaHCO₃ and 4 mM L-glutamine. Taub's SF medium is also referred to herein as Taub's medium or simply “Taub's”.

Plant hydrolysates include but are not limited to, hydrolysates from one or more of the following: corn, cottonseed, pea, soy, malt, potato and wheat. Plant hydrolysates may be produced by enzymatic hydrolysis and generally contain a mix of peptides, free amino acids and growth factors. Plant hydrolysates are readily obtained from a number of commercial sources including, for example, Marcor Development, HyClone and Organo Technie. It is also contemplated that yeast hydrolysates my also be utilized instead of, or in combination with plant hydrolysates. Yeast hydrolysates are readily obtained from a number of commercial sources including, for example, Sigma-Aldrich, USB Corp, Gibco/BRL and others.

Lipids that may be used to supplement culture media include but are not limited to chemically defined animal and plant derived lipid supplements as well as synthetically derived lipids. Lipids which may be present in a lipid supplement includes but is not limited to, cholesterol, saturated and/or unsaturated fatty acids (e.g., arachidonic, linoleic, linolenic, myristic, oleic, palmitic and stearic acids). Cholesterol may be present at concentrations between 0.10 mg/ml and 0.40 mg/ml in a 100× stock of lipid supplement. Fatty acids may be present in concentrations between 1 μg/ml and 20 μg/ml in a 100× stock of lipid supplement. Lipids suitable for media formulations are readily obtained from a number of commercial sources including, for example HyClone, Gibco/BRL and Sigma-Aldrich.

In one embodiment, Taub's media is supplemented with a plant hydrolysate and a final concentration of at least 0.5 g/L, or at least 1.0 g/L, or at least 1.5 g/L, or at least 2.0 g/L, or at least 2.5 g/L, or at least 3.0 g/L, or at least 5.0 g/L, or at least 10 g/L, or at least 20 g/L. In a specific embodiment, Taub's media is supplemented with a wheat hydrolysate. In another specific embodiment, Taub's media is supplemented with a wheat hydrolysate at a final concentration of 2.5 g/L. The present invention provides a serum-free media referred to herein as MediV SFM 101 comprising Taub's media supplemented with a wheat hydrolysate at a final concentration of 2.5 g/L.

In another embodiment, Taub's media is supplemented with a lipid mixture at a final concentration of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 200%, or at least 300% of the manufacturers recommended final concentration. In a specific embodiment, Taub's media is supplemented with a chemically defined lipid mixture. In another specific embodiment, Taub's media is supplemented with a chemically defined lipid mixture at a final concentration of 100% of the manufacturers recommended final concentration (e.g., a 100× stock obtained from a manufacture would be add to the media to a final concentration of 1×). The present invention provides a serum-free media referred to herein as MediV SFM 102 comprising Taub's media supplemented with a chemically defined lipid mixture at a final concentration of 100% of the manufacturers recommended final concentration.

In still another embodiment, Taub's media is supplemented with a plant hydrolysate at a final concentration of at least 0.5 g/L, or at least 1.0 g/L, or at least 1.5 g/L, or at least 2.0 g/L, or at least 2.5 g/L, or at least 3.0 g/L, or at least 5.0 g/L, or at least 10 g/L, or at least 20 g/L and with a lipid mixture at a final concentration of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% of the manufacturers recommended concentration. In a specific embodiment, Taub's media is supplemented with wheat hydrolysate and a chemically defined lipid mixture. In another specific embodiment, Taub's media is supplemented with a wheat hydrolysate at a final concentration of 2.5 g/L and a chemically defined lipid mixture at a final concentration of 100% of the manufacturers recommended final concentration. The present invention provides a serum-free media referred to herein as MediV SFM 103 comprising Taub's media supplemented with a wheat hydrolysate at a final concentration of 2.5 g/L and a chemically defined lipid mixture at a final concentration of 100% of the manufacturers recommended final concentration.

In yet another embodiment, Taub's media is supplemented with a growth hormone. Growth hormones which may be used include but are not limited to, Epidermal Growth Factor (EGF), Insulin Growth Factor (IGF), Transforming Growth Factor (TGF) and Fibroblast Growth Factor (FGF). In a particular embodiment, the growth hormone is Epidermal Growth Factor (EGF). In one embodiment, Taub's media is supplemented with a growth factor at a final concentration of between about 0.1 to about 50.0 ng/ml, or between about 0.5 to about 25.0 ng/ml, or between about 1.0 to about 20 ng/ml, or between about 5.0 to about 15.0 ng/ml, or between about 8 ng/ml to about 12 ng/ml. In a specific embodiment, Taub's media is supplemented with a EGF at a final concentration of about 10 ng/ml. In still other embodiments, Taub's media is supplemented with a growth factor at a final concentration of between about 0.1 to about 50.0 ng/ml, or between about 0.5 to about 25.0 ng/ml, or between about 1.0 to about 20 ng/ml, or between about 5.0 to about 15.0 ng/ml, or between about 8 ng/ml to about 12 ng/ml and with a plant hydrolysate at a final concentration of at least 0.5 g/L, or at least 1.0 g/L, or at least 1.5 g/L, or at least 2.0 g/L, or at least 2.5 g/L, or at least 3.0 g/L, or at least 5.0 g/L, or at least 10 g/L, or at least 20 g/L and with a lipid mixture at a final concentration of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% of the manufacturers recommended concentration. In another specific embodiment, Taub's media is supplemented with a wheat hydrolysate at a final concentration of 2.5 g/L and a chemically defined lipid mixture at a final concentration of 100% of the manufacturers recommended final concentration and EGF at a final concentration of about 10 ng/ml. The present invention provides a serum-free media referred to herein as MediV SFM 104 comprising Taub's media supplemented with a wheat hydrolysate at a final concentration of 2.5 g/L and a chemically defined lipid mixture at a final concentration of 100% of the manufacturers recommended final concentration and EGF at a final concentration of about 10 ng/ml.

It will also be appreciated by one skilled in the art that animal protein-free media formulations may be desirable for the production of virus used in the manufacture of vaccines. Accordingly, in certain embodiments one or more or all of the animal derived components of the serum-free media disclosed herein (e.g., MediV SF101, MediV SF102, MediV SF103, MediV SF104 and Medi SF105) is replaced by an animal-free derivative. For example, commercially available recombinant insulin derived from non-animal sources (e.g., Biological Industries Cat. No. 01-818-1) may utilized instead of insulin derived from an animal source. Likewise, iron binding agents (see, e.g., U.S. Pat. Nos. 5,045,454; 5,118,513; 6,593,140; and PCT publication number WO 01/16294) may be utilized instead of transferrin derived from an animal source. In one embodiment, serum-free media formulations of the invention comprise tropolone (2-hydroxy-2,4,6-cyclohepatrien-1) and a source of iron (e.g., ferric ammonium citrate, ferric ammonium sulphate) instead of transferrin. For example, tropolone or a tropolone derivative will be present in an excess molar concentration to the iron present in the medium for at a molar ratio of about 5 to 1 to about 70 to 1, or of about 10 to 1 to about 70 to 1. Accordingly, where the iron concentration in the medium is around 0.3 μM, the tropolone or derivative thereof may be employed at a concentration of about 1.5 μM to about 20 μM, e.g. about 3 μM to about 20 μM. The iron may be present as ferrous or ferric ions, for example resulting from the use of simple or complex iron salts in the medium such as ferrous sulphate, ferric chloride, ferric nitrate or in particular ferric ammonium citrate. The present invention provides a serum-free media referred to herein as MediV SFM 105 comprising Taub's media without transferrin supplemented with a wheat hydrolysate at a final concentration of 2.5 g/L and a chemically defined lipid mixture at a final concentration of 100% of the manufacturers recommended final concentration and EGF at a final concentration of about 10 ng/ml and Ferric ammonium citrate:Tropolone or Ferric ammonium sulfate:Tropolone at a ratio of between 10 to 1 and 70 to 1.

In one embodiment, MDCK-SF101, MDCK-SF102, MDCK-SF103, MDCK-SF104 and MDCK-SF105 serum-free adapted non-tumorigenic cells (collectively referred to herein as MDCK-SF) are derived from Madin Darby Canine Kidney Cells (MDCK) cells obtained from the American type Culture Collection (ATCC CCL34) by culturing in a chemically defined media supplemented with serum for at least one passage and then passaging them in a serum-free media such as, for example, the serum-free medias described supra. In a specific embodiment, MDCK cells (ATCC CCL34) are adapted to serum-free media to generate a MDCK-SF cell line as follows: The MDCK (ATCC CCL34) cells are passaged in Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (10% v/v), 4 mM glutamine and 4.5 g/L glucose at least once and then passaged in serum-free media. The MDCK-SF cells are then passaged as needed in serum-free media to obtain enough cell to prepare a frozen pre Master Cell Bank (PreMCB). In certain embodiments, the cells are passaged in a serum containing media (e.g., Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (10% v/v), 4 mM glutamine and 4.5 g/L glucose) between 1 and 5 times, or between 4 and 10 time, or between 9 and 20 times, or more than 20 times, and then passaged in serum-free media (e.g., MediV SF101, MediV SF102, MediV SF103, MediV SF104 and Medi SF105).

It is specifically contemplated that the MDCK-SF serum-free adapted cells are passaged for another 20 passages or more, from a vial of PreMCB and tested for tumorigenicity in an vivo adult nude mice model and karyology in a karyotype assay. In certain embodiments, the expanded MDCK-SF cells will not produce nodules when injected subcutaneously into adult nude mice and/or will have a modal chromosome number of 78. In another embodiment, the expanded MDCK-SF cells will have a modal chromosome number of 78 with a range of chromosome numbers of no more then about 60 to about 88, or of no more then about 65 to about 85, or of no more then about 65-80, or of no more then about 70 to about 85. In one embodiment, the MDCK-SF cells are non-tumorigenic after at least 20 passages, or after at least 30 passages, or after at least 40 passages, or after at least 50 passages, or after at least 60 passages, or after at least 70 passages, or after at least 80 passages, or after at least 90 passages, or after at least 100 passages in a medium (e.g., a media described herein).

In one embodiment, the serum-free media used for the derivation of MDCK-SF cells is MediV SF101. In another embodiment, the serum-free media used for the derivation of MDCK-SF cells is MediV SF102. In yet another embodiment, the serum-free media used for the derivation of MDCK-SF cells is MediV SF103. In still another embodiment, the serum-free media used for the derivation of MDCK-SF cells is MediV-SF104. In another embodiment, the serum-free media used for the derivation of MDCK-SF cells is MediV SF105. In yet another embodiment, the serum-free media used for the derivation of MDCK-SF cells is an APF media. It is contemplated that the media described herein may be formulated to eliminate animal proteins. For example bovine transferrin may be replaced with a recombinant transferrin derived from a non animal source.

Culture Conditions

The present invention provides methods for the cultivation of MDCK cells (preferably non-tumorigenic) and other animal cells (tumorigenic or not) in serum containing and serum-free media formulations (supra). It is specifically contemplated that additional culture conditions may play a role in the maintenance of the MDCK-S and MDCK-SF cells in a non-tumorigenic state. These culture conditions include but are not limited to the choice of adherent surface, cell density, temperature, CO₂ concentration, method of cultivation, dissolved oxygen content and pH.

It is specifically contemplated that one skilled in the art may adapt the culture conditions in a number of ways to optimize the growth of the MDCK cells of the invention. Such adaptations may also result in a increase in the production of viral material (e.g., virus), alternatively, one skilled in the art may adapt the culture conditions to optimize the production of vaccine material from the MDCK cells of the invention without regard for the growth of the cells. These culture conditions include but are not limited to adherent surface, cell density, temperature, CO₂ concentration, method of cultivation, dissolved oxygen content and pH.

In one embodiment, the MDCK cells of the invention are cultivated as adherent cells on a surface to which they attach. Adherent surfaces on which tissue culture cells can be grown on are well known in the art. Adherent surfaces include but are not limited to, surface modified polystyrene plastics, protein coated surfaces (e.g., fibronectin and/or collagen coated glass/plastic) as well as a large variety of commercially available microcarriers (e.g., DEAE-Dextran microcarrier beads, such as Dormacell, Pfeifer & Langen; Superbead, Flow Laboratories; styrene copolymer-tri-methylamine beads, such as Hillex, SoloHill, Ann Arbor). Microcarrier beads are small spheres (in the range of 100-200 microns in diameter) that provide a large surface area for adherent cell growth per volume of cell culture. For example a single liter of medium can include more than 20 million microcarrier beads providing greater than 8000 square centimeters of growth surface. The choice of adherent surface is determined by the methods utilized for the cultivation of the MDCK cells of the invention and can be determined by one skilled in the art. Suitable culture vessels which can be employed in the course of the process according to the invention are all vessels known to the person skilled in the art, such as, for example, spinner bottles, roller bottles, fermenters or bioreactors. For commercial production of viruses, e.g., for vaccine production, it is often desirable to culture the cells in a bioreactor or fermenter. Bioreactors are available in volumes from under 1 liter to in excess of 100 liters, e.g., Cyto3 Bioreactor (Osmonics, Minnetonka, Minn.); NBS bioreactors (New Brunswick Scientific, Edison, N.J.); laboratory and commercial scale bioreactors from B. Braun Biotech International (B. Braun Biotech, Melsungen, Germany).

In one embodiment, the MDCK cells of the invention are cultivated as adherent cells in a batch culture system. In still another embodiment, the MDCK cells of the invention are cultivated as adherent cells in a perfusion culture system. It is specifically contemplated that the MDCK cells of the invention will be cultured in a perfusion system, (e.g., in a stirred vessel fermenter, using cell retention systems known to the person skilled in the art, such as, for example, centrifugation, filtration, spin filters and the like) for the production of vaccine material (e.g., virus).

In one embodiment, the MDCK cells of the invention are cultivated at a CO₂ concentration of at least 1%, or of at least 2%, or of at least 3%, or of at least 4%, or of at least 5%, or of at least 6%, or of at least 7%, or of at least 8%, or of at least 9%, or of at least 10%, or of at least 20%.

In one embodiment the dissolved oxygen (DO) concentration (pO₂ value) is advantageously regulated during the cultivation of the MDCK cells of the invention and is in the range from 5% and 95% (based on the air saturation), or between 10% and 60%. In a specific embodiment the dissolved oxygen (DO) concentration (pO₂ value) is at least 10%, or at least 20%, or at least 30%, or at least 50%, or at least 60%.

In another embodiment, the pH of the culture medium used for the cultivation of the MDCK cells of the invention is regulated during culturing and is in the range from pH 6.4 to pH 8.0, or in the range from pH 6.8 to pH 7.4. In a specific embodiment, the pH of the culture medium is at least 6.4, or at least 6.6, or at least 6.8, or at least 7.0, or at least 7.2, or at least 7.4, or at least 7.6, or at least 7.8, or at least 8.0.

In a further embodiment, the MDCK cells of the invention are cultured at a temperature of 25° C. to 39° C. It is specifically contemplated that the culture temperature may be varied depending on the process desired. For example, the MDCK cells of the invention may be grown at 37° C. for proliferation of the cells and at a lower temperature (e.g., 25° C. to 35° C.) of for the production of vaccine material (e.g., virus). In another embodiment, the cells are cultured at a temperature of less than 30° C., or of less than 31° C., or of less than 32° C., or of less than 33° C., or of less than 34° C. for the production of vaccine material. In another embodiment, the cells are cultured at a temperature of 30° C., or 31° C., or 32° C., or 33° C., or 34° C. for the production of vaccine material.

In order to generate vaccine material (e.g., virus) it is specifically contemplated that the MDCK cells of the invention are cultured such that the medium can be readily exchanged (e.g., a perfusion system). The cells may be cultured to a very high cell density, for example to between 1×10⁶ and 25×10⁶ cells/mL. The content of glucose, glutamine, lactate, as well as the pH and pO₂ value in the medium and other parameters, such as agitation, known to the person skilled in the art can be readily manipulated during culture of the MDCK cells of the invention such that the cell density and/or virus production can be optimized.

Production of Vaccine Material (e.g., Virus)

The present invention provides a process for the production of viruses in cell culture (referred to hereinafter as “the process of the invention”), in which the MDCK cells of the invention are used. In one embodiment the process comprises the following steps:

• • i) proliferation of the MDCK cells of the present invention in culture media;
  • ii) infection of the cells with virus; and
  • iii) after a further culturing phase, isolating the viruses replicated in the non-tumorigenic cells.

In one embodiment the MDCK cells of the invention are proliferated in step (i) as adherent cells. The MDCK cells of the invention can be cultured in the course of the process in any media including, but not limited to, those described supra. In certain embodiments, the MDCK cells of the invention are cultured in the course of the process in a serum-free medium such as, for example, MediV-SF101, MediV-SF102, MediV-SF103, MediV-SF104, MediV-SF105 and APF formulations thereof. Optionally, the MDCK cells of the invention can be cultured in the course of the process in a serum containing media (e.g., DMEM+10% FBS+4 mM glutamine+4.5 g/L glucose). Additional culture conditions such as, for example, temperature, pH, pO₂, CO₂ concentration, and cell density are described in detail supra. One skilled in the art can establish a combination of culture conditions for the proliferation of the MDCK cells of the invention for the production of virus.

The temperature for the proliferation of the cells before infection with viruses is in one embodiment between 22° C. and 40° C. In certain embodiments, the temperature for the proliferation of the cells before infection with viruses is less then 39° C., or less than 38° C., or less than 37° C., or less than 36° C., or less than 35° C., or less than 34° C., or less than 33° C., or less than 32° C., or less than 30° C., or less than 28° C., or less than 26° C., or less than 24° C. Culturing for proliferation of the cells (step (i)) is carried out in one embodiment of the process in a perfusion system, e.g. in a stirred vessel fermenter, using cell retention systems known to the person skilled in the art, such as, for example, centrifugation, filtration, spin filters, microcarriers, and the like.

The cells are in this case proliferated for 1 to 20 days, or for 3 to 11 days. Exchange of the medium is carried out in the course of this, increasing from 0 to approximately 1 to 5 fermenter volumes per day. The cells are proliferated up to high cell densities in this manner, for example up to at least 1×10⁶-25×10⁶ cells/mL. The perfusion rates during culture in the perfusion system can be regulated via the cell count, the content of glucose, glutamine or lactate in the medium and via other parameters known to the person skilled in the art. Alternatively, the cells in step (i) of the process according to the invention be cultured in a batch process.

In one embodiment of the process according to the invention, the pH, pO₂ value, glucose concentration and other parameters of the culture medium used in step (i) is regulated during culturing as described above using methods known to the person skilled in the art.

In another embodiment, the infection of the cells with virus is carried out at an m.o.i. (multiplicity of infection) of about 0.0001 to about 10, or about 0.0005 to about 5, or about 0.002 to about 0.5. In still another embodiment, the infection of the cells with virus is carried out at an m.o.i. (multiplicity of infection) of 0.0001 to 10, or 0.0005 to 5, or 0.002 to 0.5. After infection, the infected cell culture is cultured further to replicate the viruses, in particular until a maximum cytopathic effect or a maximum amount of virus antigen can be detected. In one embodiment, after infection the cells are cultured at a temperature of between 22° C. and 40° C. In certain embodiments, after infection with viruses the cells are cultured at a temperature of less then 39° C., or less than 38° C., or less than 37° C., or less than 36° C., or less than 35° C., or less than 34° C., or less than 33° C., or less than 32° C., or less than 30° C., or less than 28° C., or less than 26° C., or less than 24° C. In another embodiment, after infection the cells are cultured at a temperature of less than 33° C. In still another embodiment, after infection the cells are cultured at a temperature of 31° C. In certain embodiments, the culturing of the cells is carried out for 2 to 10 days. The culturing can be carried out in the perfusion system or optionally in the batch process.

The culturing of the cells after infection with viruses (step (iii)) is in turn carried out such that the pH and pO₂ value are maintained as described above. During the culturing of the cells or virus replication according to step (iii) of the process, a substitution of the cell culture medium with freshly prepared medium, medium concentrate or with defined constituents such as amino acids, vitamins, lipid fractions, phosphates etc. for optimizing the antigen yield is also possible. The cells can either be slowly diluted by further addition of medium or medium concentrate over several days or can be incubated during further perfusion with medium or medium concentrate. The perfusion rates can in this case in turn be regulated by means of the cell count, the content of glucose, glutamine, lactate or lactate dehydrogenase in the medium or other parameters known to the person skilled in the art. A combination of the perfusion system with a fed-batch process is further possible.

In one embodiment of the process, the harvesting and isolation of the produced viruses (step (iii)) is carried out after a sufficient period to produce suitable yields of virus, such as 2 to 10 days, or optionally 3 to 7 days, after infection. In one embodiment of the process, the harvesting and isolation of the produced viruses (step (iii)) is carried out 2 days, or 3 days, or 4 days, or 5 days, or after 6 days, or 7 days, or 8 days, or 9 days, or 10 days, after infection.

Viruses which may be produced in the MDCK cells of the present invention include but are not limited to, animal viruses, including families of Orthomyxoviridae, Paramyxoviridae, Togaviridae, Herpesviridae, Rhabdoviridae, Retroviridae, Reoviridae, Flaviviridae, Adenoviridae, Picornaviridae, Arenaviridae and Poxyiridae.

Systems for producing influenza viruses in cell culture have also been developed in recent years (See, e.g., Furminger. in Textbook of Influenza, ed Nicholson, Webster and Hay, pp. 324-332, Blackwell Science (1998); Merten et al. in Novel Strategies in The Design and Production of Vaccines, ed Cohen & Shafferman, pp. 141-151, Kluwer Academic (1996)). Typically, these methods involve the infection of suitable immortalized host cells with a selected strain of virus. While eliminating many of the difficulties related to vaccine production in hen's eggs, not all pathogenic strains of influenza grow well and can be produced according to established tissue culture methods. In addition, many strains with desirable characteristics, e.g., attenuation, temperature sensitivity and cold adaptation, suitable for production of live attenuated vaccines, have not been successfully grown, especially at commercial scale, in tissue culture using established methods.

The present invention provides several non-tumorigenic MDCK cell lines, which have been adapted to grow in either serum containing or serum-free medias and which are capable of supporting the replication of viruses including but not limited to influenza when cultured. These cells lines are suitable for the economical replication of viruses in cell culture for use as vaccine material. The MDCK cells of the present invention are particularly useful for the production of cold adapted, temperature sensitive (ca/ts) strains of influenza (e.g., the influenza strains found in FluMist®) which do not grow well using other established cell lines (see, Example 1, infra). Further, the MDCK cells of the present invention are useful for the production of strains of influenza which may not grow in embryonated eggs such as avian influenza viruses which can also cause disease in humans (e.g., a “pandemic” strains)

Influenza viruses which may be produced by the process of the invention in the MDCK cells of the invention include but are not limited to, reassortant viruses that incorporate selected hemagglutinin and/or neuramimidase antigens in the context of an attenuated, temperature sensitive, cold adapted (ca/ts/at) master strain. For example, viruses can comprise the backbones (or one or more vRNA segment) of master strains that are one or more of, e.g., temperature-sensitive (ts), cold-adapted (ca), or an attenuated (att) (e.g., A/Ann Arbor/6/60, B/Ann Arbor/1/66, PR8, B/Leningrad/14/17/55, B/14/5/1, B/USSR/60/69, B/Leningrad/179/86, B/Leningrad/14/55, B/England/2608/76 etc.). Methods for the production of reassortant influenza vaccine strains in either eggs or cell lines are known in the art and include, for example, Kilbourne, E. D. in Vaccines (2^nd Edition), ed. Plotkin and Mortimer, WB Saunders Co. (1988) and those disclosed in PCT Application PCT Patent Publication Nos. WO 05/062820 and WO 03/091401. Other influenza viruses which may be produced by the process of the invention in the MDCK cells of the invention include recombinant influenza viruses which may express a heterologous gene product, see for example, U.S. Patent Publication Nos. 2004/0241139 and 2004/0253273.

In one embodiment, the cells are proliferated (step (i)) as described supra, the cells are then infected with influenza viruses (step (ii)). In certain embodiments, the infection is carried out at an m.o.i. (multiplicity of infection) of 0.0001 to 10, or of 0.0005 to 5, or of 0.002 to 0.5. In other embodiments, the infection is carried out at an m.o.i. (multiplicity of infection) of about 0.0001 to about 10, or of about 0.0005 to about 5, or of about 0.002 to about 0.5. Optionally a protease is added which brings about the cleavage of the precursor protein of hemagglutinin [HA₀] and thus the adsorption of the viruses on the cells. The addition of a protease can be carried out according to the invention shortly before, simultaneously to or shortly after the infection of the cells with influenza viruses (step (ii)). If the addition is carried out simultaneously to the infection, the protease can either be added directly to the cell culture to be infected or, for example, as a concentrate together with the virus inoculate. The protease is, in certain aspects of the invention, a serine protease, or a cysteine protease, or an asparagine protease. In one embodiment, trypsin is used. In a specific embodiment, TPCK-treated trypsin is used.

In one embodiment, trypsin is added to the cell culture up to a final concentration of 1 to 5000 mU/ml, or 5 to 1000 mU/ml, or 100 to 500 mU/ml. In an alternative embodiment, trysin is added to the cell culture up to a final concentration of 1 to 200 μg/ml, or 5 to 50 μg/ml, or 5 to 30 μg/ml in the culture medium. During the further culturing of the infected cells according to step (iii) of the process according to the invention, trypsin reactivation can be carried out by fresh addition of trypsin in the case of the batch process or in the case of the perfusion system by continuous addition of a trypsin solution or by intermittent addition.

After infection, the infected cell culture is cultured further to replicate the viruses, in particular until a maximum cytopathic effect or a maximum amount of virus and/or virus antigen can be detected. In certain embodiments, the culturing of the cells is carried out for 2 to 10 days. The culturing can in turn be carried out in the perfusion system or optionally in the batch process. In a further embodiment, the cells are cultured at a temperature of 25° C. to 36° C., or of 29° C. to 34° C., after infection with influenza viruses. The culturing of the infected cells at temperatures below 33° C., in particular in the temperature ranges indicated above, leads to the production of higher yields of certain influenza viruses, such as, for example B strains. Furthermore, the culturing of the infected cells at temperatures below 35° C. is contemplated for the production of temperature sensitive, cold adapted (ts/ca) influenza virus. It is contemplated that ts/ca viruses may also be attenuated (att). In another embodiment, the cells are cultured at a temperature of less than 30° C., or of less than 31° C., or of less than 32° C., or of less than 33° C., or of less than 34° C. for the production of ts/ca influenza strains. In a specific embodiment, the cells are cultured at a temperature of 31° C., for the production of influenza virus B strains.

The culturing of the cells after infection with influenza viruses (step (iii)) is in turn carried out, for example, as described supra

In one embodiment of the process, the harvesting and isolation of the produced influenza viruses (step (iii)) is carried out after a sufficient period to produce suitable yields of virus, such as 2 to 10 days, or 3 to 7 days, after infection. Viruses are typically recovered from the culture medium, in which infected cells have been grown. Typically crude medium is clarified prior to concentration of influenza viruses. Common methods include filtration, ultrafiltration, adsorption on barium sulfate and elution, and centrifugation. For example, crude medium from infected cultures can first be clarified by centrifugation at, e.g., 1000-2000×g for a time sufficient to remove cell debris and other large particulate matter, e.g., between 10 and 30 minutes. Alternatively, the medium is filtered through a 0.8 μm cellulose acetate filter to remove intact cells and other large particulate matter. Optionally, the clarified medium supernatant is then centrifuged to pellet the influenza viruses, e.g., at 15,000×g, for approximately 3-5 hours. Following resuspension of the virus pellet in an appropriate buffer, such as STE (0.01 M Tris-HCl; 0.15 M NaCl; 0.0001 M EDTA) or phosphate buffered saline (PBS) at pH 7.4, the virus may be concentrated by density gradient centrifugation on sucrose (60%-12%) or potassium tartrate (50%-10%). Either continuous or step gradients, e.g., a sucrose gradient between 12% and 60% in four 12% steps, are suitable. The gradients are centrifuged at a speed, and for a time, sufficient for the viruses to concentrate into a visible band for recovery. Alternatively, and for most large scale commercial applications, virus is elutriated from density gradients using a zonal-centrifuge rotor operating in continuous mode. Additional details sufficient to guide one of skill through the preparation of influenza viruses from tissue culture are provided, e.g., in Furminger, in Textbook of Influenza pp. 324-332 Nicholson et al. (ed); Merten et al., in Novel Strategies in Design and Production of Vaccines pp. 141-151 Cohen & Shafferman (ed), and U.S. Pat. No. 5,690,937. If desired, the recovered viruses can be stored at −80° C. in the presence of a stabilizer, such as sucrose-phosphate-glutamate (SPG).

In certain embodiments of the process, the virus is treated with Benzonase® or other a non-specific endonuclease. Optionally, the Benzonase® treatment occurs early in the harvesting and isolation of the produced influenza viruses (step (iii)). In other embodiments of the process, following Benzonase® treatment, the material is clarified. Methods useful for clarification include but are not limited to, direct flow filtration (DFF). Additional steps which may be utilized for the harvesting and isolation of the produced influenza virus (step(iii)) include but are not limited to, tangential flow filtration (TFF), affinity chromatography as well as ion-exchange chromatography and/or hydroxyapatite chromatography. Other steps are exemplified in the Examples section infra.

Vaccine Compositions and Methods of Use

The invention further relates to viruses (e.g., influenza) which are obtainable by a process of the invention. These viruses can be formulated by known methods to provide a vaccine for administration to humans or animals. The viruses can be present as intact virus particles (e.g., live attenuated viruses) or as inactive/disintegrated virus (e.g., treated with detergents of formaldehyde). Optionally, a defined viral component (e.g., protein) may be isolated from the viruses by methods know to the person skilled in the art, and used in the preparation of a vaccine.

The formulation of intact virus particles (e.g., live attenuated viruses) may include additional steps including, but not limited to, a buffer exchange by filtration into a final formulation followed by a sterilization step. Buffers useful for such a formulation may contain 200 mM sucrose and a phosphate or histidine buffer of pH 7.0-7.2 with the addition of other amino acid excipients such as arginine. In certain embodiments, stabilization protein hydrolysates such as porcine gelatin are added. In some embodiments, the final viral solutions/vaccines of the invention can comprise live viruses that are stable in liquid form for a period of time sufficient to allow storage “in the field” (e.g., on sale and commercialization when refrigerated at 2-8° C., 4° C., 5° C., etc.) throughout an influenza vaccination season (e.g., typically from about September through March in the northern hemisphere). Thus, the virus/vaccine compositions are desired to retain their potency or to lose their potency at an acceptable rate over the storage period. In other embodiments, such solutions/vaccines are stable in liquid form at from about 2° C. to about 8° C., e.g., refrigerator temperature. For example, methods and compositions for formulating a refrigerator stable attenuated influenza vaccine are described in PCT Patent Application PCT/US2005/035614 filed Oct. 4, 2005, also see PCT Publication WO 05/014862. Optionally, spray drying, a rapid drying process whereby the formulation liquid feed is spray atomized into fine droplets under a stream of dry heated gas, may be utilized to extend storage time of a vaccine formulation. The evaporation of the fine droplets results in dry powders composed of the dissolved solutes (see, e.g., US Patent Publication 2004/0042972). Methods for the generation and formulation of inactive/disintegrated virus particles for vaccine compositions are well known in the art and have been utilized for over 40 years.

Generally, virus or viral components can be administered prophylactically in an appropriate carrier or excipient to stimulate an immune response specific for one or more strains of virus. Typically, the carrier or excipient is a pharmaceutically acceptable carrier or excipient, such as sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, ethanol or combinations thereof. The preparation of such solutions insuring sterility, pH, isotonicity, and stability is effected according to protocols established in the art. Generally, a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, etc.

Optionally, the formulation for prophylactic administration of the viruses, or components thereof, also contains one or more adjuvants for enhancing the immune response to the influenza antigens. Suitable adjuvants include: saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, bacille Calmette-Guerin (BCG), Corynebacterium parvum, and the synthetic adjuvants QS-21 and MF59.

Generally, vaccine formulations are administered in a quantity sufficient to stimulate an immune response specific for one or more strains of influenza virus. Preferably, administration of the viruses elicits a protective immune response. Dosages and methods for eliciting a protective immune response against one or more viral strain are known to those of skill in the art. For example, inactivated influenza viruses are provided in the range of about 1-1000 HID₅₀ (human infectious dose), i.e., about 10⁵-10⁸ pfu (plaque forming units) per dose administered. Alternatively, about 10-50 μg, e.g., about 15 μg HA is administered without an adjuvant, with smaller doses being administered with an adjuvant. Typically, the dose will be adjusted within this range based on, e.g., age, physical condition, body weight, sex, diet, time of administration, and other clinical factors. The prophylactic vaccine formulation is systemically administered, e.g., by subcutaneous or intramuscular injection using a needle and syringe, or a needleless injection device. Alternatively, the vaccine formulation is administered intranasally, either by drops, large particle aerosol (greater than about 10 microns), or spray into the upper respiratory tract. While any of the above routes of delivery results in a protective systemic immune response, intranasal administration confers the added benefit of eliciting mucosal immunity at the site of entry of the influenza virus. For intranasal administration, attenuated live virus vaccines are often preferred, e.g., an attenuated, cold adapted and/or temperature sensitive recombinant or reassortant influenza virus. While stimulation of a protective immune response with a single dose is preferred, additional dosages can be administered, by the same or different route, to achieve the desired prophylactic effect. These methods can be adapted for any virus including but not limited to, orthomyxoviruses (including influenza A and B strains), paramyxoviruses (including RSV, human metapneumovirus and parainfluenza), rhabdoviruses and flavoviruses.

Influenza Virus

The methods, processes and compositions herein primarily concerned with production of influenza viruses for vaccines. Influenza viruses are made up of an internal ribonucleoprotein core containing a segmented single-stranded RNA genome and an outer lipoprotein envelope lined by a matrix protein. Influenza A and influenza B viruses each contain eight segments of single stranded negative sense RNA. The influenza A genome encodes eleven polypeptides. Segments 1-3 encode three polypeptides, making up a RNA-dependent RNA polymerase. Segment 1 encodes the polymerase complex protein PB2. The remaining polymerase proteins PB1 and PA are encoded by segment 2 and segment 3, respectively. In addition, segment 1 of some influenza strains encodes a small protein, PB1-F2, produced from an alternative reading frame within the PB1 coding region. Segment 4 encodes the hemagglutinin (HA) surface glycoprotein involved in cell attachment and entry during infection. Segment 5 encodes the nucleocapsid nucleoprotein (NP) polypeptide, the major structural component associated with viral RNA. Segment 6 encodes a neuramimidase (NA) envelope glycoprotein. Segment 7 encodes two matrix proteins, designated M1 and M2, which are translated from differentially spliced mRNAs. Segment 8 encodes NS1 and NS2, two nonstructural proteins, which are translated from alternatively spliced mRNA variants.

The eight genome segments of influenza B encode 11 proteins. The three largest genes code for components of the RNA polymerase, PB1, PB2 and PA. Segment 4 encodes the HA protein. Segment 5 encodes NP. Segment 6 encodes the NA protein and the NB protein. Both proteins, NB and NA, are translated from overlapping reading frames of a biscistronic mRNA. Segment 7 of influenza B also encodes two proteins: M1 and M2. The smallest segment encodes two products, NS1 which is translated from the full length RNA, and NS2 which is translated from a spliced mRNA variant.

Reassortant viruses are produced to incorporate selected hemagglutinin and neuraminidase antigens in the context of an approved master strain also called a master donor virus (MDV). FluMist® makes use of approved cold adapted, attenuated, temperature sensitive MDV strains (e.g., A/AnnArbor/6/60 and B/Ann Arbor/1/66). A number of methods are useful for the generation of reassortant viruses including egg-based methods and more recently cell culture methods See, e.g., PCT Publications WO 03/091401; WO 05/062820 and U.S. Pat. Nos. 6,544,785; 6,649,372; 6,951,754). It is contemplated that the MDCK cells, media and processes of the invention are useful for the production of influenza viruses including, but not limited to, the influenza strains disclosed herein (e.g., A/AnnArbor/6/60 and B/AnnArbor/1/66) and reassortant viruses comprising genes of the A/AnnArbor/6/60, B/AnnArbor/1/66, PR8. It is further contemplated that that the MDCK cells, media and processes of the invention are useful for the production of influenza viruses, including reassortant viruses, having one or more of the following phenotypes, temperature sensitive, cold adapted, attenuated. Reassortants may be generated by classical reassortant techniques, for example by co-infection methods or optionally by plasmid rescue techniques (see, e.g., PCT Publications WO 03/091401; WO 05/062820 and U.S. Pat. Nos. 6,544,785; 6,649,372, 6,951,754).

EXAMPLES

The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.

Example 1

Determination of Spread of Infection of ca/ts Influenza Strains in Cell Lines and Characterization of Influenza Produced in MDCK Cells

There has been an effort by the vaccine industry to develop alternative production platforms that do not utilize eggs and to produce influenza vaccines in a mammalian or insect cell culture system. The obvious advantages are easy scalability, increased process control and removal of egg proteins that could cause allergic reaction in some vaccines. Since cell culture based systems can be rapidly scaled up, it offers an additional advantage at the time of a influenza pandemic, when there is a potential for shortage of supply of eggs and rapid production of vaccine is required. Initial studies have been performed with a total of 7 different cell lines: 2 human diploid lung fibroblast lines (MRC-5 and WI-38) (data not shown), a human retinoblastoma and a human kidney cell line both of which were genetically constructed for production of adenoviral products (PER.C6 and 293, respectively) (data not shown), a fetal rhesus lung cell line (FRhL2) (data not shown), an African green monkey kidney cell line (Vero), and a Marin-Darby canine kidney cell line (MDCK). MDCK cells were the only cell line of those tested to be capable of propagating all four types of cold adapted, temperature sensitive attenulated (ca/ts/att) reassortant influenza virus strains, H1N1, H3N2, the potential pandemic vaccine strain H5N1, as well as B strains, to commercially reasonable titers (>10⁷ Log TCID₅₀/mL) (FIG. 1 and data not shown). The genetic and antigenic characteristics of virus grown in MDCK cells was compared to that of virus grown in eggs. No significant changes in the genomic sequence were seen (data not shown) and the antigenicity as determined by HAI titers were comparable (Table 1).

Fluorescent Focus Assay: MDCK and Vero cells were grown in 96 well black plates over 4 days (DMEM+4 mM glutamine+PEN/Strep). Each well was infected with the ca/ts influenza B-strains (B/HongKong/330/01 and B/Yamanashi/166/98) at an MOI of ˜0.01 in DMEM+4 mM glutamine+60 mU/mL TPCK trypsin. The virus infected plates were fixed and immuno-stained as follows to determine the spread of infection. The medium containing virus was removed from each plate and the plates washed once with 200 μl/well with DPBS (no Ca2+/Mg2+) and the plates were then fixed by addition of 200 μl/well of cold 4% (v/v) paraformaldehyde in PBS. The plates were washed twice with 200 μl/well of DPBS (no Ca²⁺/Mg²⁺) followed by incubation of the cells with primary antibody (sheep anti B yamanshi and sheep anti B hongkong diluted in 0.1% saponin, 1% BSA in PBS at a ratio of 1:1000). After incubation for an hour, the primary antibody was removed and cells were washed thrice with 0.1% Tween 20 in PBS and the wells were incubated with secondary antibody (rabbit anti sheep labeled with FITC in 0.1% saponin, 1% BSA in PBS at 1:100 ratio dilution). The wells were visualized daily for 4 days using a fluorescence microscope and the images were taken daily using SPOT program.

Results And Discussion

A fluorescent focus assay was use to assess whether there was spread of infection of ca/ts influenza B-strains in MDCK and Vero and also assess if there was any difference in the spread of virus infection among the 50 cell clones of Vero. Since the fluorescence in the monolayer increased over 4 days in the MDCK cells but not in the Vero cells (see, FIG. 1A), it was concluded that the Vero were not permissive for the production of ca/ts B strains while MDCK were. This data was similar to the data in earlier experiments that showed that B-strains could be produced to 7-7.5 log₁₀ TCID₅₀ in MDCK cells but only to 4-4.5 log₁₀ TCID₅₀ in Vero Cells (data not shown).

The MDCK cells were also tested for their ability to support replication of a number of ca/ts/att reassortant strains including a potential pandemic vaccine strain, ca A/Vietnam/1203/2004. MDCK cells were infected at a low multiplicity of infection with ca A/Vietnam/1203/2004 and virus in the supernatant was quantified at various times post infection. By 48 hours post infection, the titers of ca A/Vietnam/1203/2004 reached approximately 8 log₁₀ TCID₅₀/mL and remained stable for the next 3 to 4 days. See FIG. 1B and Table 2.

Ca/ts/att strains of type A/H1N1, A/H5N1, A/H3N2 and B replicated to relatively high titers in MDCK cells. In addition, passaging these ca/ts/att strains in MDCK cells did not significantly alter their genomic sequence. Three ca/ts/att strains, ca A/Sydney/05/97, ca A/Beijing/262/95, and ca B/Ann Arbor/1/94 were passaged once or twice in MDCK cells and the entire coding regions of all 6 internal genes were sequenced and compared to the starting material. No nucleotide changes were observed (data not shown), demonstrating that this passaging through this substrate did not change the genetic composition of these strains. Further sequence characterizations is performed on different vaccine strains produced in MDCK cells under conditions that are expected to mimic the production process including media composition, input dose (moi), temperature of incubation and time of harvest. Based on the preliminary data, it is expected that there will be no significant changes in the genomic sequence of MDCK-produced virus.

Because the genome was genetically stable following passage in MDCK cell, the biological traits of the vaccine produced in eggs or MDCK cells are expected to be indistinguishable. However, the primary viral product from cell culture may have some subtle differences compared to the egg based product, particularly with respect to post-translational modification of viral proteins including HA and NA, or composition of lipids in the viral membrane; both of which could potentially change the overall physical properties of the virion. Preliminary preclinical data on the antigenicity of cell culture produced and egg produced vaccine demonstrated that there were no detectable differences in this important parameter. Egg stocks of several vaccine strains were passaged through MDCK cells and the antigenicity of both products was determined by measuring the HAI titers using reference antisera. As show in Table 1, all the HAI titers were within 2-fold of one another, indicating that replication of the vaccine in cells did not change the antigenicity of the vaccine compared to egg derived material.

TABLE 1
HAI Titers of strains produced in eggs and MDCK cells

HAI Titer

	Strain	Egg derived	MDCK derived

	A/Panama/20/99	256	256
	A/Wuhan/359/95	1024	2048
	A/Wyoming/03/2003	512	1024
	B/Jilin/20/2003	64	32
	B/Hong Kong/330/01	64	64
	B/Jiangsu/10/2003	128	128

Example 2

Derivation of Non-Tumorigenic Serum MDCK Cells

MDCK cells have been traditionally used for the titration of influenza viruses (Zambon, 1988, in Textbook of Influenza, ed Nicholson, Webster and Hay, ch 24, pg 324-332, Blackwell Science) and thus could be used for the propagation of influenza for the production of vaccine materials. However, MDCK cells have traditionally been grown in basal medium formulations like Eagle's Minimal Essential Medium (EMEM) supplemented with FBS. Multiple reports indicate that MDCK cells may be tumorigenic when cultivated under these conditions and/or for extended periods of time (see for example, Gaush et al., Proc Soc Exp Biol Med, 122:931; Leighton et al., 1968, Science, 163:472 and Leighton et al., 1970, Cancer, 26:1022). Thus, there is concern about the use of MDCK cells for the production of vaccine materials and efforts have focused on the development of other cell lines (e.g., PER.C6 and VERO). Unfortunately, not all influenza strains grow well in other mammalian cell lines, in particular the cold adapted influenza viruses that comprise FluMist®, a live attenuated influenza vaccine, only grow to reasonable titers (>10⁷ TCID 50/mL) in MDCK cells (see Example 1, supra). Early reports characterizing MDCK cells indicate that early passages of MDCK cells may not be tumorigenic (Gaush et al., 1966, Proc Soc Exp Biol Med. 122:931). It was the goal of this experiment to establish a culture media and passage protocol to maintain MDCK cells in a non-tumorigenic state.

MDCK cells obtained from the ATCC(CCL 34) were expanded in T-flasks using DMEM supplemented with 10% FBS, 4 mM glutamine and 4.5 g/L glucose as the growth medium. A pre-Master MDCK cell bank was established on the serum grown MDCK cells (MDCK-S cells), which was tested for bacterial/fungal contaminants and mycoplasma contamination using routine tests performed by a commercial contractor (BioReliance, Rockville, Md.). The cells were found to be negative for the presence of bacterial/fungal contaminants. The MDCK-S cells were also found to be negative for the presence of cultivatable mycoplasma. The MDCK-S cells from the bank were also tested by a karyotype assay and found to be canine in origin and had a modal chromosome number of 78 with chromosome numbers ranging from 70 to 84. The MDCK-S cells were then passaged for another 20 passages from a vial of PreMCB and tested for karyology and tumorigenicity in an vivo adult nude mice model. The karyology test showed that late passage MDCK-S cells (p 81/24) showed the same modal chromosome number (78) and range of chromosomes (70 to 84) as the early passage MDCK-S cells, showing that the cells did not change on extended passaging. 1×10⁷ MDCK-S cells when injected subcutaneously into adult nude mice did not result in the formation of any nodules and were deemed to be non tumorigenic.

Materials and Methods

Materials: MDCK cell (ATCC, Cat. No: CCL-34); T-25, T-75, T-225 flasks (Corning, Cat No.: 430639, 430641, 431082); Dulbecco's Modified Eagle's Medium (DMEM) powder (Gibco, Grand Island N.Y., Formulation No.: 01-5052EF); Fetal Bovine Serum, Gamma-irradiated (JRH, Lenexa Kans., Cat. No.: 12107-500M); L-Glutamine (JRH, Lenexa Kans., Cat. No.: 59202-100M); D-Glucose (Amresco, Cat. No.: 0188-1KG); Dulbecco's Phosphate buffered saline (DPBS) without Ca²⁺ and Mg²⁺ powder (Gibco, Grand Island N.Y., Cat. No.: 21600-069); 0.05% Trypsin-EDTA (Gibco, Grand Island N.Y., Cat. No.: 25300) Dimethylsulphoxide, DMSO (Sigma, St. Louis Miss., Cat. No.: D2650); 0.4% w/v Trypan blue dye in PBS (Sigma, St. Louis Miss., Cat. No.: T8154); CO₂ Incubator (Form a Scientific, Model No.: 3110); YSI Bioanalyzer (YSI, Model No.: 2700 select); Vitro Chemistry System (Ortho clinic, Model: DT60 II); Improved Neubaurr hemacytometer (Hausser Scientific, Brightline 0.1 mm deep/Reichert, Brightline 0.1 mm deep).

Subculturing of Serum MDCK (MDCK-S) cells in Tissue Culture Flasks: A vial of serum MDCK cells was obtained from the ATCC. The cells were grown in DMEM medium supplemented with 10% (v/v) FBS, 4.5 g/L glucose, 2.2 g/L NaHCO₃ and 4 mM L-glutamine in T-75 flasks. The cells were passaged 3 or 4 days postseeding, with a complete medium exchange performed on day 3 after seeding if the cells were passaged on day 4. The cells were recovered from T-flasks as described below.

The spent growth medium was removed and the cell monolayer washed twice with DPBS (calcium and magnesium free). The appropriate amount of trypsin-EDTA (3 mL/T-75, 7.5 mL/T-225), prewarmed in a 37° C. water batch, was added to each flask and the T-flasks incubated in a 37° C., 5% CO₂ incubator for about 15-20 min. The flasks were checked every 5 minutes to check if cells had detached and the flasks were rapped several times to help detach the cells. When the cells had completed detached from the T-flask, the trypsin was inhibited by addition of equal volumes of complete growth medium containing 10% serum (3 mL/T-75, 7.5 mL/T-225). The cell suspension was aspirated up and down with an appropriately sized pipette to break any large cell clumps. Two 0.5 mL samples of cell suspension were counted in a hemacytometer. The cell counts were repeated if the results of the two counts were not within 15% of each other. The cells were diluted to 0.05×10⁶ viable cells/mL in fresh warm growth medium (DMEM+10% FBS+4.5 g/L glucose+4 mM glutamine) in fresh flasks and seeded in T-flasks (35 mL/T75 or 100 mL/T-225). The flasks were then incubated in a 37±1° C., 5% CO₂ environment for 3 days prior to subculturing or media exchange.

Preparation of MDCK-S cell bank: MDCK-S cells were expanded in T-flasks as described above until the total required amount of cells needed for banking could be recovered (4×10⁶ cells/vial×number of vials). The MDCK-S cells were recovered when in the exponential growth phase (3 days post seeding) by trypsinization as described. The MDCK-S cell suspensions from individual flasks were pooled and cells were recovered by centrifugation at 150-250 g for 7±1 min. The supernatant was aspirated off from each tube and the cell pellets were resuspended in fresh complete growth medium (DMEM+10% FBS+4.5 g/L glucose+4 mM glutamine). The cell suspensions from different centrifuge bottles were pooled and cell suspension was aspirated up and down with a pipette several times to break any large cells clumps. The total cell number was determined and the total number of vials that could be frozen at 4×10⁶ cells/vial was determined.

The volume of cell suspension was then adjusted to the above value using fresh growth medium. Equal volumes of freshly prepared 2× freezing medium (DMEM+10% FBS+4 mM glutamine+4.5/L glucose+15% DMSO) was added to the cell suspension. Cell suspension was mixed thoroughly and 1 mL of cell suspension was dispensed into each cryovial. All the vials were transferred into Nalgene freezing containers and were placed in a ≦−60° C. freezer. The frozen vials were transferred to a liquid nitrogen storage tank.

1. A cell culture composition comprising non-tumorigenic MDCK cells, wherein said non-tumorigenic MDCK cells are of the MDCK-SF 103 cell line deposited as ATCC Accession Number PTA-6503.

2. A non-tumorigenic MDCK cell line selected from the group consisting of: MDCK-S deposited as ATCC Accession Number PTA-6500; and MDCK-SF 103 deposited as ATCC Accession Number PTA-6503.

3. The cell culture composition of claim 1, wherein said non-tumorigenic MDCK cells are non-tumorigenic in an adult nude mouse model.

4. The cell culture composition of claim 1, wherein said non-tumorigenic MDCK cells remain non-tumorigenic after at least 20 passages.

5. The non- tumorigenic MDCK cell line of claim 2, wherein said cell line is MDCK-S deposited as ATCC Accession Number PTA-6500.

6. The non- tumorigenic MDCK cell line of claim 2, wherein said cell line is MDCK-SF 103 deposited as ATCC Accession Number PTA-6503.

7. The non- tumorigenic MDCK cell line of claim 2, wherein said cell line is non-tumorigenic in an adult nude mouse model.

8. The non- tumorigenic MDCK cell line of claim 2, wherein said cell line remains non-tumorigenic after at least 20 passages.