AU2014210661A1 - Methods of producing influenza vaccine compositions - Google Patents

Abstract

Methods and compositions for the optimization of production of influenza viruses suitable as influenza vaccines are provided.

Description

METHODS OF PRODUCING INFLUENZA VACCINE COMPOSITIONS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application is a divisional application from Australian Patent Application No. 2012201973, which in turn is a divisional application from Australian Patent 5 Application No. 2008230033, which in turn is a divisional application from Australian Patent Application No. 2004263813, the entire disclosures of which are incorporated herein by reference. Australian Patent Application No. 2004263813 claims the benefit of U.S. Provisional Application No. 60/450,181 filed February 25, 2003, entitled "METHODS OF PRODUCING INFLUENZA VACCINE COMPOSITIONS". This prior application is also 10 hereby incorporated by reference in its entirety for all purposes. BACKGROUND OF THE INVENTION [0002] Vaccines against various and evolving strains of influenza are important not only from a community health stand point, but also commercially, since each year numerous individuals are infected with different strains and types of influenza virus. Infants, the elderly, 15 those without adequate health cart and immuno-compromised persons are at special risk of death from such infections. Compounding the problem of influenza infections is that novel influenza strains evolve readily, thereby necessitating the continuous production of new vaccines. [0003] Numerous vaccines capable of producing a protective immune response specific 20 for such different influenza viruses have been produced for over 50 years and include, e.g., whole virus vaccines, split virus vaccines, surface antigen vaccines and live attenuated virus vaccines. However, while appropriate formulations of any of these vaccine types is capable of producing a systemic immune response, live attenuated virus vaccines have the advantage of being also able to stimulate local mucosal immunity in the respiratory tract. A vaccine 25 comprising a live attenuated virus that is also capable of being quickly and economically produced and that is capable of easy storage/transport is thus quite desirable. [0005] To date, all commercially available influenza vaccines have been propagated in embryonated hen eggs. Although influenza virus grows well in hen eggs, the production of vaccine is dependent on the availability of such eggs. Because the supply of eggs must be 30 organized, and strains for vaccine production selected months in advance-of the-next flu season, the flexibility of this approach can be limited, and often results in delays, and shortages in production and distribution. Therefore, any methods to increase throughput and/or increase output of vaccine production in hen eggs is greatly desirable. -1- WO 2005/014862 PCT/US2004/005697 [0006] Systems for producing influenza viruses in cell culture have also been developed in recent years (See, e.g., Furminger. Vaccine Production, in Nicholson et al. (eds.) Textbook of Influenza pp. 324-332; Merten et al. (1996) Production of influenza virus in cell cultures for vaccine preparation, in Cohen & Shafferman (eds.) Novel Strategies in 5 Design and Production of Vaccines pp. 141-151). While eliminating many of the difficulties related to vaccine production in hen eggs, not all pathogenic strains of influenza grow well in cell culture, or 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 10 vaccines, have not been successfully grown in tissue culture using established methods. Therefore, any methods to increase throughput and/or increase output of vaccine production in cell culture is also greatly desirable. [0007] Considerable work in the production of influenza virus for production of vaccines has been done by the present inventors and co-workers; see, e.g., Multi-Plasmid 15 System for the Production of Influenza Virus, USSN 60/375,675 filed April 26, 2002, PCTIUS03/12728 filed April 25, 2003 and USSN 10/423,828 filed April 25, 2003, etc. The present invention provides methods of increasing/optimizing production (in both quantity/quality and speed) of such viruses, as well as for other influenza viruses, for production of vaccine compositions. Aspects of the current invention are applicable to 20 traditional hen egg and new cell culture vaccine production styles (and also combined systems) and comprise numerous other benefits that will become apparent upon review of the following. SUMMARY OF THE INVENTION [0008] The invention provides embodiments of methods of making one or more 25 influenza virus compositions by passaging an influenza virus (e.g., an A virus strain or a B virus strain, etc.) through eggs, heating the virus and filtering the virus through a membrane. In some such embodiments, the filtering comprises passage of the composition through a microfilter of a pore size ranging from 0.2 micrometers to about 0.45 micrometers. Furthermore, in various embodiments, the temperature of heating in such embodiments 30 optionally comprises from about 28'C to about 40*C or more, whileih some embodiments, the temperature comprises 31 0 C or from about 30 0 C to about 32C. The heating in such -2- WO 2005/014862 PCT/US2004/005697 embodiments optionally occurs before or during or before and during the filtration and optionally comprises from about 50 minutes to about 100 minutes, from about 60 minutes to about 90 minutes, or about 60 minutes. The invention also provides an influenza virus composition produced by such methods (including wherein the composition is a vaccine 5 composition). [0009] In other aspects, the invention comprises a method of making one or more. influenza virus composition by passaging an influenza virus through eggs, heating the virus, and purifying the virus. Such embodiments also optionally include filtering the composition through a membrane and wherein the compositions comprises a vaccine 10 composition as well as the actual vaccine composition produced by such embodiment. [0010] In related aspects, the invention comprises a method of making one or more influenza virus composition, by passaging an influenza virus through eggs which are rocked during the passage. The rocking optionally comprises tilting the eggs at a rate of about 1 cycle per minute optionally for about 12 hours. Such embodiments optionally use influenza 15 A virus strains and/or influenza B virus strains and also optionally comprise wherein a

TCID

5 0 of such rocked eggs is 0.4 log greater than a TCID5O of the same virus passaged through non-rocked eggs. Virus compositions produced by such embodiments are also features of the invention, including wherein the compositions are vaccine compositions. [0011] The invention also comprises methods of making one or more influenza virus 20 composition (e.g., biasing the reassortment of such) by introducing a plurality of vectors comprising an influenza virus genome into a population of host eggs (which are capable of supporting replication of the virus), culturing the population of eggs at a temperature less than or equal to 35'C, and recovering a plurality of influenza viruses. Such viruses optionally comprise, e.g., an attenuated virus, a cold adapted virus, a temperature sensitive 25 virus or an attenuated cold adapted temperature sensitive virus, and can also comprise, e.g., an influenza B virus. A virus composition produced by such an embodiment is also a feature of the invention (including vaccine compositions). Such aspects also optionally include further selecting for influenza viruses containing wild-type HA and NA genes (e.g., by incubating the plurality of viruses with one or more antibodies specific for non-wild-type 30 HA and NA genes (e.g., done within the one or more egg). Virus compositions produced thusly are also features of the invention, including vaccine compositions. -3- WO 2005/014862 PCT/US2004/005697 [0012] Other aspects of the invention include making one or more influenza virus composition by introducing a plurality of vectors comprising an influenza virus genome into a population of host eggs (which is capable of supporting replication of influenza virus), culturing the population of eggs at a temperature less than or equal to 35'C, recovering a 5 plurality of viruses, incubating the plurality of viruses with one or more antibodies specific for non-wild-types HA and NA genes, passaging the virus through eggs (which are rocked) and heating the virus and filtering the virus through a membrane. Viruses produced by such methods are also features of the invention (including vaccine compositions). [0013] In the various methods embodied herein, the influenza virus composition is 10 optionally assayed through use of a fluorescence focus assay. Such virus compositions optionally comprise from about 10% to about 60% unfractionated normal allantoic fluid (and optionally from about 1% to about 5% arginine). The compositions are optionally diluted with a buffer which is optionally substantially free of normal allantoic fluid. The compositions herein are optionally substantially free of gelatin. These compositions are 15 stable from about 2'C to about 8'C, or are stable at 4'C. In some compositions and methods herein, the viruses are influenza viruses, while in yet other compositions and methods herein (e.g., those involving microfiltration and/or ultrafiltration and/or heating and/or rocking) the viruses optionally comprise, e.g., non-influenza viruses (e.g., viruses that are produced through culture in eggs, e.g., myxoviruses, paramyxovirus, RSV, mumps 20 virus, measles virus, Sendi virus, yellow fever virus, plY, etc.). Thus, the methods and compositions of the invention are also applicable to such other viruses and/or to non influenza viruses. [0014] In yet other aspects, the invention comprises an influenza virus composition, wherein the composition is made by: passaging the influenza virus through eggs, heating the 25 virus, and filtering the virus through a membrane, which composition has a first TCID 50 , which first TCIDso is greater than a second TCIDso, which second TCIDso results from an influenza virus not made by: passaging the virus through eggs, heating the virus, and filtering the virus through a membrane. [0015] Other aspects of the invention include an influenza virus composition, 30 wherein the composition is made by: passaging the influenza virus through.eggs, wherein the eggs are rocked during said passage, which composition has a first TCID 5 o, which first

TCID

5 o is greater than a second TCID 5 o, which second TCID 50 results from an influenza -4- WO 2005/014862 PCT/US2004/005697 virus not made by: passaging the influenza virus through eggs, wherein the eggs are rocked during said passage. [0016] Still other embodiments herein include an influenza virus composition, wherein the composition is made by: introducing a plurality of vectors comprising an 5 influenza virus genome into a population of host eggs, which population of host eggs is capable of supporting replication of influenza virus, culturing the population of host eggs at a temperature less than or equal to 35'C, and recovering a plurality of influenza viruses, which composition has a first TCID 50 , which first TCID 50 is greater than a second TCIDso, which second TCIID 5 results from an influenza virus not made by: introducing a plurality 10 vectors comprising an influenza virus genome into a population of host eggs, which population of host eggs is capable of supporting replication of influenza virus, culturing the population of host eggs at a temperature less than or equal to 35'C, and recovering a plurality of influenza viruses. [0017] These and other objects and features of the invention will become more fully 15 apparent when the following detailed description is read in conjunction with the accompanying figures appendix. BRIEF DESCRIPTION OF THE DRAWINGS [0019] Figure 1: Displays M Genotyping after infection at 33'C and 25'C in DEK TC-24. 20 [0020] Figure 2: Displays a plaque assay, and data, showing the different titers of 5:3 and 6:2 at 33*C. [0021] Figure 3: Displays growth curves of 6:2 vs. 5:3 reassortants. [0022] Figure 4: Displays the M1 sequences of MDV-B and wild-type B viruses. [0023] Figure 5: Displays the M2 sequences of MDV-B and wild-type B viruses. 25 [0024] Figure 6: Displays mutations on the two conservative sites in MDV B-Mi. [0025] Figure 7: Displays the growth curves of the B/HK 6:2 M1 mutations. [0026] Figure 8: Shows various CEK cell infections at different MOIs. [0027] Figure 9: Shows a flow chart of potential microbial contamination during -5- WO 2005/014862 PCT/US2004/005697 vaccine production process. [0028] Figure 10: Illustrates temperature decay rates of individual eggs via infra-red imaging. [0029] Figure 11: Illustrates thermal imaging of live, infertile, and dead eggs. 5 [0030] Figure 12: Illustrates a schematic flowchart of virus harvest concentration. [0031] Figure 13: Displays comparison of the 5t wash with NAF proteins. [0032] Figure 14: Displays an assay of an N/New Caledonia/20/99 1-X-Neat sample before concentration. [0033] Figure 15: Displays an assay of an A/New Caledonia/20/99 1oX 10 concentrated sample. [0034] Figure 16, Panels A-B: Display a comparison of IX and lOX of A/New Caledonia/20/99; and 1X-W sample after 5 washes. [0035] Figure 17: Displays a comparison of A/New Caledonia/20/99 1X and 1X-W samples. 15 [0036] Figure 18, Panels A-C: Display a comparison of 1OX and 1OX-W of A/New Caledonia/20/99; Permeate of A/New Caledonia/20/99; and 5 washes of A/New Caledonia/20/99. [0037] Figure 19: Shows an analysis by SEC comparing times washed and impurities removed. 20 [0038] Figure 20: Displays lXW and 1OX-W comparison of A/New Caledonia/20/99. [0039] Figure 21: Shows a 96-well plate assay of A/New Caledonia/20/99. [0040] Figure 22: Shows a graph neuraminidase activity / virus purification in retentate and permeate. 25 [0041] Figure 23: Displays RHPLC of Control, 1OX, 1OX-W, and 1X-W. -6- WO 2005/014862 PCT/US2004/005697 [0042] Figure 24: Shows a graph of Control, 1OX, 1X-W and 1OX-w samples. [0043] Figure 25: Displays RHIPLC of permeate and washes 1 to 6. [0044] Figure 26: Shows a graph of RHPLC ovomucoid removal (peak. area). [0045] Figure 27: Shows a graph of RHPLC of lysozyme removal (peak area). 5 [0046] Figure 28: Shows a graph of RHPLC of conalburnin removal (peak area). [0047] Figure 29: Shows a graph of RHPLC of ovalbumin removal (by peak area). [0048] Figure 30: Shows a graph of ovalbumin analysis by Agilent 2100. [0049] Figure 31: Is a Western blot SDS-PAGE gel of anti-A/New Caledonia. [0050] Figure 32: Displays assays of 1OX-W, sample after 5 washes of A/New 10 Caledonia/20/99. [0051] Figure 33: Shows a graph of RNA analysis by RTPCR. [0052] Figure 34: Shows monitoring of A/Beijing -cell culture propagation by SEC. [0053] Figure 35: Shows cell culture harvest.of A/Beijing in Vero cells. 15 [0054] Figure 36: Shows concentration of 2 liters of A/Panama cell culture. [0055] Figure 37: Shows concentration of 2 liters of B/Hong Kong cell culture down to 10 ml. [0056] Figure 38: Shows 4 graphs of stability of exemplary virus storage formulations. 20 [0057] Figure 39: Shows a graph of stability of exemplary virus storage formulations with various virus strains. [0058] Figure 40: Shows a graph of stability of exemplary virus storage formulations with various virus strains. [0059] Figure 41, Panels A-C: Show stability of exemplary virus storage 25 formulations with differing citrate concentrations. -7- WO 2005/014862 PCT/US2004/005697 [0060] Figure 42, Panels A-C: Show stability of exemplary virus storage formulations with differing EDTA concentrations. . [0061] Figure 43: Displays stability of unpurified virus harvest formulations with different virus strains over 9 months. 5 [0062] Figure 44: Illustrates initial potency loss of formulations associated with phosphate buffer. [0063] Figure 45: Gives a global picture of stability slopes of various formulations at 6 months. [0064] Figure 46: Gives a global picture of stability slopes of various formulations 10 at 6 months. [0065] Figure 47: Shows stability of various formulations with gelatin and PVP/EDTA. [0066] Figure 48: Shows stability of various formulations with histidine at different pH. 15 [0067] Figure 49: Shows stability of various formulations with different amounts of sucrose. [0068] Figure 50: Histogram derived from plotting the absorbance readings from the wells, versus the frequency of the values (number of wells read at that absorbance value). 20 [0069] Figure 51: Histogram derived from plotting absorbance readings yersus frequency of values. [0070] Figure 52: Shows a generic process for generating 6:2 influenza reassortants. DETAILED DESCRIPTION 25 WO 2005/014862 PCT/US2004/005697 [0073] The present invention includes methods and compositions to increase throughput and output of viruses and virus composition productions suitable for vaccine production/use. Included are methods and compositions for, e.g., selecting for desired reassortants in virus production, temperature conditioning/filtration, rocking, antibody 5 selection, potency assays, and many additional features as described in more detail herein. [0074] It will be appreciated by those skilled in the art that the various steps herein are not required to be all performed or exist in the same production series. Thus, while in some preferred embodiments, all steps andlor compositions herein are performed or exist, e.g., as outlined in Table 1, in other enibodiments, one or more steps are-optionally, e.g., 10 omitted, changed (in scope, order, placement, etc.) or the like. [0075] The basic overview of the methods and compositions for virus production herein are outlined in Table 1. Once again, as is to be emphasized throughout, the individual steps of the invention, such as those listed in Table 1 are not necessarily mutually dependent. For example, in some embodiments, eggs containing an appropriate reassorted 15 virus solution are rocked during incubation (see below), while in other embodiments they are not; heating and filtration in Step 10 is independent of use of universal reagents in Step 13, etc. The presence of any one step/method/composition in the invention is not dependent upon the necessary presence of any other step/method/composition in the invention. Therefore, various embodiments of the current invention can include only one of the steps, 20 all of the steps, or any and all various combinations of the steps. [00761 It will also be appreciated by those skilled in the art that typical embodiments comprise steps/methods/compositions that are known in the art, e.g., candling of virus containing eggs, inoculation of eggs with viruses, etc. Therefore, those skilled in the art are easily able to determine appropriate conditions, sub-steps, step details, etc., for such known 25 steps to produce the appropriate viruses, virus solutions, compositions, etc. The individual steps are described in greater detail below. See Table 1 for listing of major steps involved in example embodiment. [0077] For ease in discussion and description, the various steps of the current invention, e.g., the various methods and compositions, can be thought of as comprising or 30 falling into four broad groups. The first group comprises such aspects as co-infection, reassortment, selection of reassortants, and cloning of reassortants (e.g., thereby roughly -9- WO 2005/014862 PCT/US2004/005697 corresponding to Steps 1 through 3 in Table 1). The second group comprises such aspects as purification and expansion of reassortants and can be thought of as roughly corresponding to Steps 4 through 6 in Table 1. The third group comprises further expansion of reassortants in eggs, along with harvesting and purification of such harvested virus 5 solutions (e.g., roughly corresponding to Steps 7 through 11 in Table -1). The fourth group comprises stabilization of harvested virus solutions and potency/sterility assays of the virus solutions (e.g., roughly corresponding to Steps 12 through 15 in Table 1). It is to be understood, however, that division of the aspects of the invention into the above four general categories is solely for explanatory/organizational purposes and no inference of 10 interdependence of steps, etc. should be made. DETAILED DESCRIPTION OF STEPS [0078] As mentioned above, for ease in discussion and description, the various steps of the current invention can be thought of as comprising four broad groups. The first group comprises such aspects as co-infection, reassortment, selection of reassortants, and cloning 15 of reassortants (e.g., thereby roughly corresponding to Steps 1 through 3 in Table 1). The second group comprises such aspects as purification and expansion of reassortants and can be thought of as roughly corresponding to Steps 4 through 6 in Table 1. The third group comprises further expansion of reassortants in eggs, along with harvesting and purification of such harvested virus solutions (e.g., roughly corresponding to Steps 7 through 11 in 20 Table 1). The fourth group comprises stabilization of harvested virus solutions and potency/sterility assays of the virus solutions (e.g., roughly corresponding to Steps 12 through 15 in Table 1). It is to be emphasized, however, that division of the aspects of the invention into the above four general categories is solely for explanatory/organizational purposes and no inference of interdependence of steps, etc. should be made. 25 GROUP [0079] The aspects of the current invention which are broadly classified herein as belonging to Group 1, comprise methods and compositions related to optimization of co infection of cell culture lines, e.g., with a master donor virus and one or more wild-type viruses in order to produce specifically desired reassorted viruses; selection of appropriate 30 reassorted viruses; and cloning of the selected reassorted viruses. Reassortment of influenza virus strains is well known to those of skill in the art. Reassortment of both influenza A -10- WO 2005/014862 PCT/US2004/005697 virus and influenza B virus has been used both in cell culture and in eggs to produce reassorted virus strains. See, e.g., Tannock et al., Preparation and characterisation of attenuated cold-adapted influenza A reassortants derived from the AlLeningrad/34/17/57 donor strain, Vaccine (2002) 20:2082-2090. Reassortment of influenza strains has also 5 been shown with plasmid constructs. See, "Multi-Plasmid System for the Production of Influenza Virus," cited above. [0080] Reassortment, in brief, generally comprises mixing (e.g., in eggs -or cell culture) of gene segments from different viruses. For example, the typical 8 segments of influenza B virus strains can be mixed-between, e.g., a wild-type strain having an epitope of 10 interest and a "donor" strain, e.g., comprising a cold-adapted strain. Reassortment between the two virus types can produce, inter alia, viruses comprising the wild-type epitope strain for one segment, and the cold-adapted strain for the other segments. Unfortunately, to create the desired reassortants, a sometimes large number of reassortments need to be done. After being reassprted, the viruses can also be selected (e.g., to find the desired 15 reassortants). The desired reassortants can then be cloned (e.g., expanded in number). Steps to decrease the time required for construction of reassortants and to enhance creation of desired reassortants are, thus, highly desirable. [0081] Traditional optimization, selection, and cloning of desired reassortants for influenza B virus, typically occurs by co-infection of virus strains into a cell culture (e.g., 20 CEK cells) followed by selection with appropriate antibodies, e.g., against material from one of the parent virus, (usually done in eggs), and cloning or expanding of virus, etc. which is typically done in cell culture. However, such traditional reassortment presents drawbacks in that thousands of reassortments are needed to create the desired segment mix. When such reassortments are done, it is apparent that truly random reassortments are not the end 25 result. In other words, pressures that bias the process exist in the systems. For influenza A strains, however, such processes do not appear to have such bias. For A strains, co infection of strains (typically into cell culture such as CEK cells) is followed by selection and cloning at the same time, again, typically in cell culture. [0082] Thus, as detailed herein, various embodiments of the invention comprise 30 steps to reduce the reassortment bias. Namely, cloning of reassortants is done in eggs (e.g., at 33'C) rather than in cell lines, or is done in cell lines, but at, e.g., 25'C. -11- WO 2005/014862 PCT/US2004/005697 Optimization of Reassortment [0083] The current invention utilizes the steps in Group 1 to optimize the reassortment process in order to reduce the number of reassortments needed (and thus increase the throughput of the vaccine production process). The steps utilizing such 5 optimization techniques are typically embodied with reassortment of influenza B strains and are typically done in cell culture, e.g., CEK cells. [00841 Other methods of reassortment of influenza virus mix dilutions of a master donor virus (MDV) and a wild-type virus, e.g., a 1:5 dilution of each no matter the concentration of the respective solutions, which are then incubated for 24 and 48 hours at 10 25'C and 33*C. However, while such an approach is often acceptable for influenza A strains, influenza B strains do not typically give positive results with such protocol. For example, to achieve the proper 6:2 assortment (i.e., 6 genes from the MDV and 2 genes, NA and HA from the wild-type virus) thousands of reassortments must often be done. [0085] Thus, typical embodiments of the steps in Group 1 of the invention comprise 15 determination of the MOI (multiplicity of infection) of the MDV strain and the wild-type strains (especially for influenza B strains used), followed by reassortments comprising those illustrated in Table 2. The incubations of such optimized reassortment mixtures is carried out at 33'C for 24 hours in eggs. In embodiments like this, proper 6:2 reassortments are typically achieved by screening hundreds of reassortment mixes as opposed to thousands of 20 reassortment mixes necessary in non-optimized systems. Selection and Cloning of Reassortments [0086] The steps in Group 1 also comprise selection of reassorted influenza viruses. The methods and compositions of the current invention are especially useful for (and are typically embodied for) selection of properly reassorted influenza B viruses. Reassorted 25 influenza A strains are capable of selection in either cell culture (e.g., CEK cells) or in eggs. However, reassorted influenza B strains present problems when reassorted in cell culture (e.g., when selected for in CEK cells). It is believed that CEK cells interfere with the M gene in influenza B strains, thus reducing the overall production. See below. The current invention takes notice of such suppression by, in some embodiments, having selection of 30 influenza B reassortments done in eggs (which are neutral in terms of selection pressure against the M gene in influenza B strains) at 33*C or, alternatively, in CEK cells-at 25C. See Figure 52. -12- WO 2005/014862 PCT/US2004/005697 [0087] Other embodiments of the invention in Group 1 include use of anti-HA (of the MDV) and anti-NA (of the MDV) antisera in the selection process, thus, achieving a stronger selection. [0088] Yet other embodiments of the invention in Group 1 include cloning of the 5 reassortments produced. As will be apparent from the previous discussion, cloning out of influenza B reassortments in CEK cell culture has proven problematic because of negative selection pressure. Thus, in some embodiments herein, B strain reassortmeiits are cloned cut in eggs at 33'C. A strain reassortments, on the other hand, are optionally cloned out and selected at the same time in CEK cell culture. 10 [0089] Even though some embodiments herein take advantage of the non-bias or non-suppression of eggs on reassortments (see above), other embodiments herein comprise selection/cloning of reassortments in cell culture, but at 25'C. Thus, some aspects of the current invention comprise embodiments which take advantage of the different properties of MDVB (master donor virus B) M gene and wild-type B virus M gene. For example, 6:2 15 and 5:3 co-infections are optionally done to produce the desired reassortments. Thus, for example, in the B/HongKong/330/01 MVS production process, the cloning from mixed wild-type and cold-adapted M viral RNAs by limiting dilution in CEK cells at 33'C, results in the dominant growth of wild-type M viral RNA. In both eggs and CEK cells, the wild type M vRNA is dominant over MDV-derived M vRNA when 6:2 is coinfected with 5:3 20 (containing wild-type M gene) at 33'C, although the chance of getting wild-type M vRNA in eggs is higher. In contrast, both MDV-derived and wild-type M vRNAs are present in comparable amount when 6:2 and 5:3 are coinfected into CEK cells at 25'C. Therefore, in some embodiments herein 25'C is used for 6:2 cloning in CEK cells in the MVS process. See, Figures 1 through 8. From the Figures it can be seen that plaque assays show that the 25 titer of B virus 6:2 at 33'C is at least 2 log10 lower than respective 5:3 at low 1M00, while 6:2 grows to the same level as 5:3 at 25*C. The growth defect of 6:2 at 33'C may account for the selection against 6:2 in MVS CEK cloning. The different growth properties of MDVB and 6:2 suggest the involvement of HA, NA in the M gene dominance. There are only two conservative amino acid differences between MDVB and wild-type B viruses. A 30 single mutation of Valine to wild-type conservative Methionine on the 6:2 M gene is able to reverse the growth defect of 6:2 in CEK cells at 33"C. -13- WO 2005/014862 PCT/US2004/005697 Characterization of Reassortments [0090] Yet other embodiments of the current invention utilize applications of a high throughput single strand conformation polymorphism/capillary electrophoresis (SSCP/CE) assay to determine the gene constellation of influenza viruses used herein. It should be 5 appreciated that such characterization aspect can also be classified into other "Groups" herein, but is discussed here for organizational purposes. Influenza viruses contain 8 gene segments and, as described above, co-infection of a single cell with two different influenza strains can produce reassortant viruses with novel gene constellations distinct from either parent. Thus, some embodiments herein use a SSCP/CE assay to rapidly determine the 10 gene segment constellation of a large number of influenza virus samples. The influenza viral gene segments are optionally amplified by RT-PCR using fluorescent-labeled primers specific for each of the eight segments. See, also, Arvin et al. (2000) J. Clin. Micro. 38(2):839-845 which is incorporated herein by reference for all purposes. [0091] In order to reduce the number of RT-PCR reactions required to genotype all 15 eight segments of the influenza genome, a multiplex reaction is optionally created in which multiple segments are simultaneously amplified in the same reaction. The RT-PCR products corresponding to each segment are differentiated by size, migration pattern and fluorescent color. The migration of a single strand DNA fragment in a non-denaturing matrix is determined not only by its size but also by its sequence content. 20 [0092] Cells are optionally co-infected with cold-adapted B/Ann Arbor/1/66 (MDV B) or similar, and one of several wild-type influenza B strains. The progeny of the co infection are cloned by limiting dilution and the nucleic acids amplified in multiplex reactions. Primers are selected and products separated by SSCP/CE at 18'C, which enhances the resolution between MDV B and wild-type strains' eight gene segments. 25 [0093] For example, to demonstrate the accuracy of the SSCP/CE assay, 400 gene segments from approximately 50 different reassortant viruses were analyzed and the SSCP/CE results were compared to those obtained by restriction fragment length polymorphism (RFLP). It was found that there was a high concordance (-98%) between the two sets of data, thereby validating the SSCP/CE assay. Furthermoreit was shown that 30 the SSCP/CE assay was capable of detecting a single nucleotide substitution within the M gene segment of influenza B virus. -14- WO 2005/014862 PCT/US2004/005697 Prevention of Bacterial Contamination [0094] Some embodiments of the current invention comprise steps to detect and/or prevent/detect microbial contamination of eggs in which influenza virus is produced. Such steps are useful in several areas as outlined in Table I and can be included in Groups .1, 2, 5 and 3, but for organizational purposes are presented with the steps of Group 1. The microbial detection strategies of the invention are useful for rapid/high throughput microbial detection and, thus, as with many other steps herein, are useful for increasing throughput in virus/vaccine production. 10095] Many current influenza vaccine production strategies, including some 10 embodiments of the invention herein, use as a component, the traditional method for influenza virus expansion in specific-pathogen-free fertile chicken eggs. Possible microbial contamination can occur in several points in the production of virus in eggs. See, e.g., Figure 9, which outlines one possible example of a virus production flowchart and possible areas of contamination therein. Unfortunately, the chicken eggs may have some 15 microorganisms outside of their shells as part of their natural flora. It is also possible to have microorganisms enclosed within the shell of the egg during the development of the chicken embryo. Fertilized chicken eggs are incubated at 37*C in high humidity for development of the embryo, which constitutes prime incubation conditions for many types of microbial contaminants as well. Another possible time of microbial contamination 20 occurs when the shell is punctured to inoculate the egg. Even though prior to virus inoculation, the eggs are often sprayed with alcohol, there is still opportunity for microorganisms to enter into the egg. [0096] After expansion of viruses for 2 to 3 days in the eggs, the top of the egg shell is typically removed for manual harvesting of the allantoic fluid containing virus within the 25 egg. This harvesting is another point where microbial contamination may originate. Unfortunately eggs with such contaminating bioburden may escape detection, necessitating pooling into multiple bottles to minimize the rejection of the entire lot due to a failed MPA test. Since three influenza strains are typically used in vaccine production, blending of the three strains is required for the final bulk. In-process MPA (microbiological purity assay) 30 testing is performed, e.g., at virus harvest (see Figure 9) prior to use-in the blending and filling to ensure microbial-free product. -15- WO 2005/014862 PCT/US2004/005697 [00971 After incubation, the "traditional" method of candling is used to identify infertile and dead eggs which are possibly dead due to natural causes or to microbial contamination (i.e., dead eggs may occur due to infectivity of the virus and/or expansion of microorganisms, both of which require detection and removal of such eggs). Candling 5 comprises, e.g., the process of holding an egg in front of a light source in a darkened room to enable visualization of the developing embryo. Dead eggs are excluded from virus inoculation. [0098] As can be seen from the above points, detection of microbial contamination can be needed at multiple steps during the manufacture of influenza vaccine. There is a 10 need to eliminate or reduce avian and environmental microbes and a need to eliminate or reduce introduction of environmental and human microbes. Thus, a need for non-invasive and rapid methods of screening eggs to identify and remove infertile, dead, or microbially contaminated eggs exists. Such methods should preferably be non-invasive and rapid. Current methods for detection of contaminating microorganisms include, e.g., compendial 15 methods (MPA and Bioburden). Current methods can include, e.g., egg candling during egg pre/post inoculation (which is typically done manually at a rate of about 500 eggs/hour/person); MPA and BioBurden tests which are typically manual and take about 14 days for MPA and about 3 days for BioBurden (which are done during virus harvest); mycoplasma testing; which is typically done manually and takes about 28 days (done during 20 virus harvest); and mycobacterium testing which is typically manual and takes about 56 days (done during virus harvest). From such, it will be appreciated that there are opportunities for significant reduction in turn around times for the traditional methods. New methods are preferable, e.g., to reduce time to result from days to 24 hours or less (and preferably 4 hours or less for in-process testing) and from weeks to a few days for Release 25 Testing. Other preferences include, e.g., to reduce to intermediate/inventory hold-time, to potentially expedite product release/approval, and to reduce cost/labor/overhead. In general, any method chosen to detect microbial contamination should consider, e.g., scientific requirements such as intended use, time to result, sample type, instrument capabilities, etc.;'regulatory requirements such as FDA guidelines (e.g., the bioburden must 30 be a measure of total viable organisms as required by the FDA), review, expectations/acceptability; compliance requirements such as vendor auditsvendor -support (instrument IOPQ or instrumentally observed perspectival quality), software validation, and -16- WO 2005/014862 PCT/US2004/005697 documentation; and business requirements such as industry trends, costs of implementation, cost per test, etc. [0099] A few potential alternative methods for detection of microbial contamination which are present within various embodiments of the invention are listed in Table-3. Thus, 5 for example, an alternative to candling of eggs, and one embodiment of the current invention, comprises pre/post virus inoculation thermal imaging. In such embodiments, the infrared radiation emitted by incubated eggs is captured with an infrared camera. Using software, the captured images are converted into temperature readings for the eggs. The camera is able to capture differences in temperature less than or equal to 0.01'C. 10 Metabolically active developing embryos lose heat slower than an infertile egg or a dead embryo, thus, resulting in a higher temperature differential. For example, to set up, as an alternative to candling of eggs, thermal imaging of pre/post virus inoculation, a tray of eggs can be thermally imaged (e.g., an infrared camera can be set below a tray of eggs (e.g., a tray with open-bottomed cells)). Software can then be set up to measure the bottom 15 temperature of each egg (or side, top, etc.). Temperature decay rates of each individual egg can be evaluated, thus, allowing identification of the time to show maximum temperature differential in problem eggs. Through such thermal imaging, temperature differentials between live embryos and infertile and dead eggs can be identified. See Figures 10 and 11. [0100] In other embodiments herein, the current invention utilizes an alternative to 20 bioburden test on virus harvest, namely, MPN or Most Probable Number, which is based upon Bacteriological Analytical Manual Online, January 2001, Appendix 2, Most Probable Number from Serial Dilutions, FDA/CFSA-BAM. For example, an MPN Test can involve a 3 replicate 96-well test, wherein 1:10 serial dilutions (e.g., 1:10, 1:100, 1:1K, 1:10K, 1:100K, and 1:1000K dilutions) can be run in triplicate for 3 different samples on a 96 well 25 microtiter plate with negative controls. TSB can be added initially to all wells as a diluent and as an enriched media to the support the growth of microorganisms. Plates can be read visually or at 600 nm. MPN bioburden tests are quite useful in comparison to membrane filtration tests for detection of contamination. While membrane filtration tests can require (for 3 samples) 15 TSA plates, a large sample volume, intensive amounts of time and labor, 30 can be difficult to automate and only sample at 1:10 and 1:100 dilutions, a 96-well MPN. test can (for 3 samples) only require one 96-well microtiter plate (with controls), a small volume of sample, a few simple disposables and reagents, gives a dilution range from 1:10 -17- WO 2005/014862 PCT/US2004/005697 to 1:100,000, and can also be visual read or automatably read with a 96-well plate reader. The results of testing 70 samples using conventional Bioburden and 96-well plate MiPN were found in complete agreement with each other. Notably, for its intended purpose, the 96-well plate MPN provided comparable results with a higher throughput. 5 [0101] As an alternative to the traditional compendia] Mycoplasma test- for Virus Harvest, the current invention, in some embodiments, comprises use of universal commercial standardized rapid nucleic acid amplification-based kits (e.g., PCR). The current compendial method (direct and indirect) detects all strains of contamination (including avian M. synoviae and M. gallisepticumn and human M. pneumoniae, i.e., all 10 avian and human mycobacterium strains). The alternative PCR detection method comprises investigator-developed primer/probe sets for real-time PCR that specifically detect a mycoplasma panel, and possibly greater than 40 species based upon sequence homology of target gene (e.g., genus and/or species specific sequences on 16s and/or 23s rRNA) such as tubercle bacterial and non-tuberculous mycobacteria (e.g., M. abscessus and M. avium). 15 Some embodiments herein utilize standardized nucleic acid amplification-based kits that rapidly detect tubercule bacteria and non-tuberculous mycobacteria, etc. GROUP 2 [0102] Aspects of the current invention which fall into Group 2 include those corresponding to Step 4 through Step 6 in Table 1. After the process of correct reassortment 20 and cloning of reassortants (i.e., the 6:2 viruses), such reassorted virus particles are further purified in embryonated hen eggs and the correct clones are expanded in quantity (again through growth in hen eggs) to generate a master virus strain (MTVS) or master virus seed, which, in turn, is further expanded to generate a master working virus strain (MiWVS) or manufacturer's working virus seed. Many aspects of purification of virus particles from 25 eggs and use of such purified virus to inoculate more eggs in order to-expand the quantity of virus particles are well known to those skilled in the art. Many such techniques are common in the current production of virus particles and have been used for at least 40 years. See, e.g., Reimer, et al. Influenza virus purification with the zonal ultracentrifuge, Science 1966, 152:1379-81. For example, common purification protocols can involve, e=.g.; 30 ultracentrifugation in sucrose gradients (e.g., 10-40% sucrose), etc. Also, as noted herein; other procedures, etc. listed in other Groups are also optionally present within Group 2, e.g., prevention of microbial contamination, etc. -18- WO 2005/014862 PCT/US2004/005697 GROUP 3 [0103] Aspects of the current invention which fall under the heading of Group 3 include Step 7 through Step 11 in Table 1. These steps primarily deal with the conditioning of the embryonated eggs (e.g., specific handling and environmental conditions involved in 5 the incubation of virus infected eggs) and the harvesting and clarification of influenza virus from the allantoic fluid of the eggs. [0104] For example, the current invention comprises conditioning, washing, candling, and incubating eggs which contain the reassorted virus to be used in a vaccine; inoculation, sealing, etc. of such eggs; candling of such eggs; harvesting of the virus 10 solution (e.g., the allantoic fluid) from the eggs; and clarification of the virus solution. Again, it should be noted that several techniques applicable to the steps in Groups 2 are equally applicable to the steps in Group 3 (e.g., candling, etc.). Several aspects of the invention which comprise Groups 3 are well known to those skilled in the art. Various aspects of candling of eggs in virus production, as well as inoculation of eggs with viruses 15 and washing, incubating, etc. of such eggs are well known techniques in the production of virus/vaccines in eggs. Of course, it will be appreciated that such well-known techniques are used in conjunction with the unique and innovate aspects of the current invention. Rocking [0105] One drawback in culturing some types of influenza strains (e.g., especially 20 influenza B strains such as Victoria/504/2000) is that they do not produce as high a titer as other strains when grown in eggs. For example, if a first strain (e.g., an influenza A strain) produces a titer of 10 8 or 10 9 log (i.e., 108 or 109 virus particles per milliliter) and a second strain (e.g., an influenza B strain) only produces 107 virus particles per milliliter, then the second strain must be, e.g., grown in a greater quantity of eggs, or the first strain must be 25 held until the second strain is grown in a second production, etc. [0106] Thus, one aspect of the current invention is to rock or gently agitate the eggs in which the virus strains are incubated (i.e., after the eggs are inoculated with the virus). It should be noted that the exact mechanism used to achieve such rocking is not-limiting. For example, the eggs are optionally rocked on a shaking platform or rocking platform (e.g., as 30 is used to incubate bacterial culture flasks, as is used in egg incubators, etc.).In some embodiments, the eggs are rocked from about 1 cycle per minute or less to about 2 cycles, per minute or more. In this context, "cycle" should be taken to mean the traveling of the -19- WO 2005/014862 PCT/US2004/005697 eggs through a full range of motion. In yet other embodiments, the eggs are rocked from abut 0.5 cycles per minute or less to about 5 cycles per minute or more. In some embodiments, the eggs are rocked at about 1 cycle per minute. When rocking was added to the incubation steps in Group 3 (i.e., post inoculation) the titer of a B-Victoria influenza 5 strain increased by 0.4 log over a control group of eggs which was not rocked. Filtering and Wanning [0107] Yet another aspect of the invention that falls under Group 3 involves the effect of viral allantoic fluid (VAF) temperature on virus potency losses during sterile filtration (typically through 0.2 um filters). In various embodiments of the current 10 invention, virus particles are harvested frcm allantoic fluid and then put through a process involving warming of the fluid followed by filtration of the fluid. See, e.g., Steps 10 and 11 in Table 1. Such steps are desirable for several reasons. For example, as pointed out herein, presence of allantoic fluid and debris in vaccine preparations can lead to allergic reactions. Also, quite importantly, filtration removes bioburden (bacteria) from the solutions. All VH 15 (virus harvest) solutions containing bioburden must be discarded. This is also true in intranasal application of live-attenuated virus vaccines. Thus, the aspects of the current invention which allow filtration and clarification of live attenuated virus in order to remove and/or reduce the presence of such bioburden, etc. is quite desirable. [0108] The effects of viral allantoic fluid (VAF) temperature and warming time 20 necessary to filter a cold-adapted (ca) virus strain (e.g., A/Sydney/05/97, H3N2 type). with acceptable potency loss through sterilizing-grade filters is used as an example herein. Conditions to acceptably filter A/Sydney/05/97 are discussed, as well as the results of five additional cold adapted influenza strains (namely: 2 x H1N1, I x H3N2, 2 x B) being filtered under similar conditions. 25 [0109] Three independent assays (TCID 5 o, neuraminidase, and hemagglutinin) were used to characterize viral allantoic fluid throughout the filtration process. The data demonstrate that the addition of a warming step (e.g., exposure to the temperature of 31 ±3'C up to 60 minutes prior to filtration) to the filtration process reduced the potency losses to acceptable levels (0 - 0.3 logio TCID 5 o) compared to the sterilizing-grade filtration 30 performed without warming step for A/Sydney/05/97, In other embodiments;,the warming temperature is optionally over 28'C, or from 28 to 36'C for a period of time of at least 30 minutes, or, in other embodiments of from about 60 to 240 minutes. It will be appreciated -20- WO 2005/014862 PCT/US2004/005697 that the warming process can, indeed, continue for long periods of time, but that after greater lengths of time, the loss in potency due to virus stability loss at such elevated temperatures becomes measurable and detrimental. The added warming step did not contribute to additional potency losses for other tested strains for the times tested, indicating 5 the warming step is an acceptable process step for sterilizing-grade filtration of cold adapted influenza viruses (CAIV). [0110] As described herein, the current FluMistTM manufacturing process uses embryonated chicken eggs to generate master virus seeds (MVS), manufacturer's working virus seeds (MWVS) and virus harvests (VH). See Step 6 in Table 1. The seeds and viral 10 harvest may contain bioburden (typically bacterial contamination), which would cause the seed or bulk virus product lots to be rejected in the vaccine production process. Through previous studies to evaluate the use of filtration for virus containing allantoic fluids, indication had been that bioburden can be reduced by the introduction of a filtration step in the process. However, based on previous work, such filtration is problematic with 15 particular viral strains (e.g., A/Sydney/05/97). Based on such studies, design proposals have been made for filtration rigs comprised of a sterile plastic media bag connected to a pre-filter and 0.2 millimeter sterilizing-grade filter combination with various associated filling, dispensing and sampling lines (see below). Of course, it will be appreciated that specific listing or description of particular product types used, sizes, etc., is not to be 20 considered limiting on the current invention unless specifically stated to be so. [0111] As seen in such studies, the majority of tested cold-adapted (ca) viral strains can be filtered with minimal potency loss though a Sartorius Sartoclean CA pre-filter followed by a Sartorius Sartopore 2 as the sterilizing-grade filter. However, other filtration studies with A/Sydney/05/97 resulted in potency losses of between 0.7 to 1.4 logio 25 TCID 5 0/mL. Further studies revealed that this loss occurred across the Sartorius Sartopore 2 sterilizing-grade filter. Again, it should be noted that other filter brands and/or filter types are optionally used in such steps and that recitation of particular filter names/types should not be construed at limiting. [0112] The purpose of the first set of experiments shown below was to test the effect 30 of VAF temperature on virus potency loss during filtration. The second part of the study was designed to define the appropriate warming time of VAP prior to filtration. The cold adapted (ca) A/Sydney/05/97 virus strain (H3N2 type) was used as a model strain to -21- WO 2005/014862 PCT/US2004/005697 determine the warm-up conditions because, as stated previously, large potency losses have been observed during filtration of this strain. [0113] The third part of this example evaluated the effect of warming the viral allantoic fluid (VAF) on potency losses caused by filtration for several other monovalent 5 virus strains. Five CAIV strains (2 x HIN1, 1 x H3N2 and 2 x B) were used in these runs. All experiments were performed at the CAIV seed-scale (MVS and MWVS) using 1.0 - 3.0 L of sucrose phosphate glutamate (SPG) stabilized VAF and appropriately scaled filters, i.e. approximately 1:30 to 1:10 of proposed maximum VH process scale, prior to removal of testing samples. Typical process scale is up to about 33 L of stabilized VH per filtration rig. 10 Such volume typically works well with 50 L bags chosen for filtration rigs and has a reasonable safety margin for volume that can be filtered using standard 10" filter capsules. However, such volume is often too large for development/exemplary work; thus, a 1/10th scale filtration was performed (i.e., about 3 L). [0114] Virus propagation for such temperature/filtration steps can be performed 15 according to commonly known methods in the art, and/or using other aspects of the current invention (see, above and below) using cold-adapted (ca) influenza strains summarized in Table 4. [0115] Samples from all stages of the experiment were assayed for potency by measuring the Tissue Culture Infectious Dose (TCID 5 o) in a manual assay (see. below for 20 other aspects of TCID 5 o measurements). Neuraminidase activity (NA) and Hemagglutinin activity (HA) were also measured. [0116] A series of filtrations through Sartorius Sartoclean CA/Sartorius Sartopore 2 filter combinations were performed in order to evaluate the effect of VAF temperature prior to filtration on: potency (TCID 5 o/mL), neuraminidase (NA) and hemagglutinin (HA) 25 activity losses. [0117] During the virus harvest, VAF was pooled into 1 L PETG bottles: Once the required volume of unstabilized VAF was collected and pooled, the filtrations were performed. The temperature (start-up temperature) of the unstabilized VAF at this stage was 15 ±3'C. The total warming time was defined as the time the VAF was in-the 33 ±lC 30 water bath and consisted of the warm-up time (from 15 ±3'C to 28 ±3'C) and warm-hold time (time greater than 28'C, e.g., at a set point). -22- WO 2005/014862 PCT/US2004/005697 [0118] The Part 1 VAF temperature effect studies (see below) were performed with the cold-adapted (ca) A/Sydney/05/97 virus strain (H3N2 type). Part 2 of the example focused on determination of the optional warm-hold time ("time at the temperature"). In part 3, the effect of the previously determined warm-hold time on five other strains (Table 5 5) was tested. In all parts of the example, 1.0 - 3.0 L of sucrose phosphate glutamate (SPG) stabilized VAF, typical virus seed-scale, and approximately 1:30 - 1:10 of proposed mVH process scale, were filtered through the rigs. [0119] In the current typical manufacturing processes, after harvest, VH is centrifuged, stabilized and frozen for further transportation. In these examples, a sample of 10 VAF withdrawn from the un-stabilized pool was centrifuged and stabilized with SPG, similarly to current manufacturing processes and this served as a control for filtered VAF in all parts of the current example. Part 1: Temnerature effect on A/Sydney/05/97 virus titer changes during filtration 15 [0120] In order to determine the effect of temperature on potency loss, two sets of. filtration experiments at various temperatures were performed. Each set consisted of three parallel experiments performed on the same day with VAF collected from the same batch of eggs. In these experiments, after harvest, VAF was stabilized with SPG, split into three pools and exposed for 60 minutes prior to filtration to either 5 ±3C (refrigerator), 20 ±3'C 20 (bench top) or 31 ±3C (water bath). During this time, VAF in the bottle was mixed by inverting every 10 minutes. After the temperature treatment, it was filtered through Sartoclean CA and Sartopore 2 filters. In the control experiment, VAF was centrifuged and stabilized. TCID 5 O results of filtration under different conditions were compared to each other and the control. 25 [0121] To determine the effect of VAF temperature on potency loss, VAF was exposed for 60 minutes prior to filtration to 5 t3"C, 20 ± 3*C or 31 3 0 C. The potency change, neuraminidase and hemagglutinin activity difference between centrifuged stabilized and post-filtration material with different temperate tUeatment is summarized in Tables 5-10. As can be seen, filtration of cold (5 ± 3C) and room temperature (20 ± 3C) VAF resulted;> 30 in potency losses between 0.7 and 1.0 loglo TCIDs/nIL (see, Tables'5 and'8). However, there was no post filtration titer loss (compared to the centrifuged stabilized VAF) when -23- WO 2005/014862 PCT/US2004/005697 VAF was warmed up to 31 ± 3C for 60 minutes (30 minutes warm up time + 30 minutes warm-hold time at the set point temperature). See, Tables 5 and 8. Additionally, the post filtration neuraminidase activity levels were higher in the filtration performed after VAF was warmed up to 31 ± 3*C compared to the levels observed in cold and room temperature 5 filtrations. See, Tables 6 and 9. Addition of the warm-up step also reduced hemagglutinin activity losses. See, Tables 7 and 10. Part 2: Determination of the warming time required for acceptable filtration potency losses of A/Sydney/05/97 [01221 In order to determine the necessary warming time, a series of experiments 10 were conducted with VAF warmed to 31 ± 3C prior to filtration in a water bath. In a control experiment, VAF was filtered immediately after stabilization with SPG. In all experiments, warming time was defined as the total time (warm up time plus warm-hold time) VAF was in the water bath (i.e., at 31 ±3'C). VAF in the bottle was mixed by inverting every 10 minutes. After temperature treatment it was filtered through Sartoclean 15 CA and Sartopore 2 filters. In the control experiment, representing the current manufacturing process, VAF was centrifuged and stabilized. TCID 5 o results of filtration under different conditions were compared to each other and the control. [0123] To determine the warming time prior to filtration that is required to filter ca A/Sydney/05/97 a series of experiments was conducted wherein VAF was warmed to 31 ± 20 3*C prior to filtration for 30, 90 or 180 minutes in one set of experiments and 30, 60 or 90 minutes in another set of experiments. In the control experiments, VAF was filtered without warming immediately after stabilization with SPG. The virus potency, neuraminidase and hemagglutinin levels between filtered VAF and control are summarized in Tables 11 - 16. 25 [0124] The data demonstrate that the exposure of VAF to 31.± 3C reduced post filtration virus potency losses and allowed partial recovery of neuraminidase and hemagglutinin activities. See, Tables 11 - 13. The temperature of un-stabi-lized VAF at the beginning of the experiments (post harvesting and prior to warming) was 15 + 2"C. The warm up time required for 1-1.5 L of VAF to reach 31 3 3C was about 20-30-minutes-. 30 Thus, a 30-minute total VAF warming time results in 0-10 minutes VAF warm hold time at 31 3 0 C. -24- WO 2005/014862 PCT/US2004/005697 [0125] The minimum warming time required to minimize filtration potency losses was determined in a second series of experiments. See, Tables 14 - 16 (first set) and Tables 17- 19 (repeat set), The post filtration potency, HA and NA losses were observed in 0 and 30 minutes total warming time experiments. In 60 and 90 minute total warming time 5 (warm-hold of 30-40 and 60-70 minutes at 31 ± C) experiments, post filtration virus potency and HA and NA levels were similar to the control (centrifuged stabilized VAF) samples. See Tables 14-19. Part 3: Effect of warming on other strains [0126] A series of experiments was conducted with 5 strains other than 10 A/SydneyI05/97, i.e., 2 x HlN1, 1 x H3N2, and 2 x B, in order to assess the effect of the warm up step on filtration of influenza virus strains other than A/Sydney/05/97. Each strain was tested twice. VAF was warmed to 31 ±3"C for 60 minutes (30 minutes ramp up time + 30 minutes time at the temperature) prior to filtration. After temperature treatment, it was filtered through Sartoclean CA and Sartopore 2 filters. In a control experiment, VAF was 15 filtered immediately after stabilization with SPG at room temperature. TCID 5 O results of filtration under different conditions were compared to each other and control experiments. [0127] For the additional 5 cold-adapted influenza virus strains tested, a short exposure (total warming time of 60 minutes) to 31 ± 3C (warm-hold time of 30-40 minutes at set point temperature) contributed to the reduction of post filtration potency losses 20 compared to the experiments without temperature treatment for A/Sydney/05/97 and B/Victoria/504/2000 and did not impact potency for the other strains. The potency (TClIDso/mL), neuraminidase and hemagglutinin levels from these experiments are summarized in Tables 20-25, below. [0128] As can be seen from the tables, the aspect of the current invention 25 comprising warming to 31 ± 3C or optionally even up to 36'C (warm-hold time of 60 to 90 minutes for 1-1.5 L of VAF in bottles) of the stabilized viral harvest prior to filtration through Sartoclean CA pre filters and Sartopore 2 sterilizing grade filters resulted in acceptable reduction of virus potency (0-0.3 logio TCIDs/ml) for A/Sydney/05/97; In the control experiments, when A/Sydney/05/97 stabilized viral harvest was filtered without 30 warming, titer losses were up to 1.0 logo TCIDSO/ml. -25- WO 2005/014862 PCT/US2004/005697 [0129] As is also seen from such tables, for all 6 cold-adapted influenza virus strains tested, a short exposure (warm up and warm hold time of 60 minutes) at 31 ± 3C (warm hold of 30 -40 minutes at 31 ± 3C) either decreased the potency losses or did not contribute to additional potency losses during filtration. In all experiments, the post 5 filtration titer loss was not higher than 0.3 log TCIY 50 /ml. The reduced activity losses of the viral surface proteins neuraminidasee and hemagglutinin) of warmed filtered VAF compared to not warmed, support the decreased potency loss data shown by TCIDso assay. [0130] Thus, the data verifies that some embodiments of the current invention which comprise a warming time required to filter CAIV (MVS, MWVS or VH) have acceptable 10 potency losses of 60 minutes (time to warm up the VAF to 31 ± 3C and warm hold (time at the set point temperature) for at least 30 minutes). Such warming tolerance is a novel and unexpected result, especially in light of other filtration attempts. See above. Again, as will be appreciated, the embodiments of the current invention comprising heating/filtration steps are not limited by the above examples. In other words, e.g., other filters and filter types, etc 15 are optionally used, without deviating from the invention. GROUP 4 [0131] Group 4 of the aspects of the current invention comprises, e.g., Steps 12-15 of Table 1. Such steps primarily concern stabilization (e.g., through addition of components, alterations in buffer/NAF ratios, etc.) and assays of potency/sterility of virus 20 containing solutions. In some embodiments, the final viral solutions/vaccines comprising live viruses are stable in liquid form at 4*C for a period of time sufficient to allow storage "in the field" (e.g., on sale and commercialization when refrigerated at 4'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 25 potency or to lose their potency at an acceptable rate over the storage period. For example, if a 0.3 log potency loss were acceptable and the storage period were 9 months, then an 0.05 log/month decrease in potency-would be acceptable. Furthermore, use of FFA allows a greater latitude in terms of acceptable loss. For example, if a loss of up to,0.75jog were allowed, a rate of less than or equal to 0.09 log/month would be sufficient-to-allow stability 30 of materials stored continuously at refrigerator temperature (e.g., 4 0 C). In other embodiments, such solutions/vaccines are stable in liquid form at from about 2 0 C to about 8C. In yet other embodiments, the solutions/vaccines are stable at room temperature. -26- WO 2005/014862 PCT/US2004/005697 Typical embodiments herein do not exhibit a decrease (or exhibit small decreases) in immunogenicity due to the NAF dilutions (see below). Concentration/Diafiltration of virus harvests [0132] In some embodiments herein, virus harvests are optionally concentrated 5 using a appropriate column. Influenza virus solutions~can be concentrated without loosing appreciable viral potency/activity. Such concentration without loss of potency is a quite surprising result because previous literature, etc. showed a loss of virus activity with concentration. Viral concentration can be done at a number of points in the purification/production process, e.g., as illustrated in Table 1, in order to enhance the viral 10 particles and remove other proteins, RNA, etc. For example, concentration can be done prior to potency assaying, or even after potency assaying, etc., but in many embodiments is done within/amongst the steps categorized in Group 4. Concentration of virus particles can be useful for purification, vaccine preparation, and for analytical characterization. See, e.g., Methods and Techniques in Virology, Pierre Payment and Michel Trudel, Marcel Dekker, 15 Inc., (1993). Due to the low amount of virus in some VAF samples, the direct analysis of the virus particles precludes some of the analytical techniques like Analytical Ultra Centrifugation (AUC), Disc Centrifuge, Matrix Assisted Laser Desertion Ionization (MALDI), and particle counting. [0133] Prior traditional viral concentrations from egg NAF, etc. were done via 20 gradient purification centrifugation. See, e.g., Concentration and Purification of Influenza Virus from Allantoic Fluid, Arora et aL, Analytical Biochemistry, 144:189-192(1985). Embodiments herein, however, utilize size exclusion columns. Concentration can be used whether the virus is produced via egg production, cell culture production (e.g., Vero cells), plasmid rescue production, etc. Also, the concentration steps can be performed on a number 25 of different viruses and/or virus strains (e.g., both influenza A and influenza B strains are amenable to such actions) as well as between different lots of one strain, e.g., to ensure product quality. Additionally, size exclusion column concentration can often be used as a track on the amount of virus particles within a harvest, e.g., within an egg, etc. Thus, for example, a peak area (i.e., of virus eluted from the column) can be used instead of, or.in 30 addition to, TCIDc measurement of such solutions. Such tracking is especially.useful for virus produced in eggs. Additionally, concentrated and purified virid'material can optionally be a starting material for generating pure HA, NA and other viral components for -27- WO 2005/014862 PCT/US2004/005697 further studies. Furthermore, SEC purified virus can provide a better insight into the virus structure and the binding mechanism with the host cells. Because in most of the VAF (virus/viral allantoic fluid) materials, virus particles are below the detection limit of UV, the concentration of the virus particles is quite helpful for further characterization. 5 [0134] In concentration of virus harvest, a size exclusion column, e.g., MidGee or QuixStand (Amersham) with hollow fiber filter under pressure can be used to remove impurities and/or unwanted buffers/fluids. The concentrated virus is, thus, also more easily suspended or stored in specific buffers/stabilizers. See below. [0135] To illustrate the concentration of a virus harvest sample, an influenza harvest 10 of A/New Caledonia was concentrated and analyzed from VAF by cross flow filtration. Of course, again, it is to be emphasized that the techniques, etc. of this section are not be limited to particular strains/types of viruses. Such concentration concentrated the virus particles, removed a majority of impurities and retained virus infectivity. As illustrated, the virus infectivity was checked by CELISA (TCID 5 o). Hemeagglutination by HA assay, 15 neuraminidase activity, SEC analysis, NAF by RHPLC, and RNA by RTPCR were also done. [0136] The virus concentration in the example below was achieved by using Amersham's Cross Flow Filtration Unit MidGee. MidGee is capable of concentrating 100 or 200 ml to 10 ml in 2-3 hours. Similarly, QuixStand can be used for concentrating the, 20 virus particles from 2 liters to 100ml in 4 to 6 hours. Concentration of virus not only enhances the virus particle count, but also removes a majority of other impurities like egg proteins, RNA, and small molecules like uric acid. [0137] The virus used in the following example was A/New Caledonia/20/99. NAF comprised cold adapted influenza virus. Chicken blood was from Colorado Serum 25 Company (Denver, CO). The instrument used for concentration was from Amersham Biosciences (A/G Technology Corporation), and was a Midlet System with Peristaltic Pump (Watson Marlowe). The column used for concentration was from Amersham Biosciences (A/G Technology Corporation) and was a MidGee Hoop Cross Flow-Filter -with a nominal molecular weight cut-off of 750,000. Yet again, however, it is to be emphasized. 30 that use or recitation of particular models, producers, etc. of equipment aremnot to be .. -28- WO 2005/014862 PCT/US2004/005697 construed as limiting upon the current invention. The buffer used for washing in this example was 1X-SPG. [0138] For SEC, the instrument used was a Heweltt Packard HP 1100 HPLC system while the column was an Ultrahydrogel 1000 from Waters with a size of 7.8 x 300 mm. 5 The.buffeX with the SEC was Dulbecco's Phosphate Buffered Saline from Hyclone Solvent. For the SEC, the method comprised an isocratic condition with a flow rate of 0.5 ml/min, monitored at 210 and 280 nm. For the RHPLC, the instrument was from Waterg and the column was a YMC C4 (reverse phase), 2.1 x 250 mm, 5 um, 300 A. The method for the RHPLC was: Mobile Phase - A: 0.1% TFA in water, B: 95% CAN 0.09% TFA; Elution 10 Conditions - Variable gradient, 13 - 100% B ; Flow Rate: 0.2 ml/ min; Column Temp, - 45 C; Injection Volume -50 ul; and Detection - 214 nm. [0139] As shown in Figure 12, Step 1, 150 ml of A/New Caledonia/20/99 was concentrated by a MidJet instrument in a cold room. The pressure between the inlet and outlet was maintained between 5 to 10 PSI. After circulating through the cross filter for two 15 hours, 150 ml of the IX sample was reduced to 15 ml of 1OX concentrated sample (Step 2). The permeate was collected separately and stored for further analysis. For analytical characterization, 4 ml of the 1OX sample was removed (Step 3). The remaining 11 ml of the 1oX sample was diluted to 110 ml with 1X-SPG, and was further concentrated down to 11 ml by removing the lX-SPG as a permeate. The permeate carries most of the impurities 20 from the retentate. This step was repeated five times with 1X-SPG as shown in Step 4 and Step 5. The washed permeate was saved for further analysis. The first and second wash showed yellow coloration. This is thought to be due to the removal of egg proteins and other small molecule impurities. The yellow color in the permeate disappeared after the 3rd and 4th wash. Following the 5th wash, the sample was diluted with 1X-SPG to 110 ml to 25 bring the concentration back to 1X. At step 6, 10 ml of the 1X-W was reserved for the assay. The remaining 100 ml of the 1X-W was further concentrated down to 1OX-W (Step 7). This concentrated sample was aliquoted into 1 ml quantities for further analysis. [0140] All the samples were analyzed by SEC. chromatography. The Ultr.ahydrogel 100 column was used for the analysis with DPBS as a solvent. Even though the data was 30 collected at 220, 260 and 280 nm, for discussion purpose, the comparison was done with the 220 nm peak areas. The chromatogram peaks were classified into three major groups: one for virus (retention time around 10.6 min), one for impurities group-1(retention time 18 to -29- WO 2005/014862 PCT/US2004/005697 21 min), and one for impurities group-2 (retention time 21 to 27 min). Three NAF proteins Ovalbumin, Conalbunin and Ovomucoid elute around retention time 18-21 min. See Figure 13. Lysozyme elutes around 27.0 min. It is thought that Group-2 impurities consist of small molecules such as uric acid and other uncharacterized materials. All the Washes 5 were checked by analytical SEC chromatogram under identical condition as the virus analysis. The CELISA, HA assay, NA assay, and RTPCR were carried out by different groups. SEC Analysis and CELISA [0141] The neat sample, 1X showed the virus peak at 11.1 minutes with a peak area 10 1,221. See Figure 14. However, the concentrated 1OX sample showed a peak area 11,192, see, Figure 15, and the increment in the peak are was about 9.16 times compared to IX. See Table 26, 11,192 / 1221. This is based on the previous experiments showing linearity between the peak area and the amount of virus sample injected. During the concentration, without any washes, some impurities have been removed but not significantly. See Table 15 26, Figure 16a-b. The impurities group-i and group-2 showed increment in the peak size between IX and 1OX (Table 26). Correspondingly the TCID 50 was increased from log 9.1 to log 10.0 (Table 27). During this step, 95.9% of the infectivity was retained. This data indicates that concentrating the 1X sample to 1OX sample retained the infectivity quite well. [0142] After the 5th wash with 1X-SPG, the virus peak area of the sample IX-W, 20 retained as 1005 compared to 1221 before the wash (Table 26). Recovery by peak area between IX and 1X-W was about 82% (1005 /1221). By comparing the 1X and 1X-W chromatogram (see Figure 17), it shows that impurities group-i and group-2 were significantly reduced (Table 26). The 1X-W showed a small decrease TCID 5 o value (Table 27, IX: 9.1, 1X-W: log 8.9). The recovery of infectivity was about 98.99 % between 1X 25 and IX-W (log 8.9 / log 9.1). The washing step improved the quality of the virus material by removing NAF proteins and other components. [0143] Similarly, by comparing the LOX and 1OX-W, the impurities group-1 and group-2 was removed to a great extent (Table 26, Fig 18). By going through the 5 washes, the virus peak area of 1OX: 11,192 was reduced to 10X-W: 10, 282 (Table 26, 91.86% by 30 peak area). The TCIDso was changed from log 10.0 (10X) to log 9.9(10X-W)with-the recovery of 99.56% (Table 27). -30- WO 2005/014862 PCT/US2004/005697 [0144] By comparing the 1X-W and 1OX-W chromatogram, the peak area increased by 10 times. See Table 26, 1X-W: Peak Area: 1005 and 1OX-W: 10,282. The TCID 5 o value also increased one log (Table 27, 1X-W: log 8.9 and 1OX-W: log 9.9). Since, the IOX-W was concentrated from 1X-W in one step, no loss in either activity or in the peak area was 5 seen (10X-W: Peak area 10282 and 1X-W Peak Area 1005). [0145] The permeate showed virus peak at 104 min with the peak area 25. This could be due to the loss of a very small amount of virus particles or some other proteins eluting along with the virus in 1X sample. Most of the impurities were eluting in group-1 and group-2. See Table 26. The CELISA values showed the infectivity was below the 10 limitation of detection. This shows that there was not many virus particles eluting through the membrane during the concentration procedure. [0146] The five washes improved the quality of the virus by removing most of the impurities of group-1 and group-2. This is illustrated in the Table 26 and Figure 19. Group-i and group-2 impurities were significantly removed after the 2nd wash. After the 15 5th wash the curves reached a plateau. Even after the 5th wash, the samples IX-W and 1OX-W showed impurities group-1 and group-2 in a very low amount. See Figure 20. The identity of the peak at 19.208 min was confirmed as ovalbumin by isolating from the 1OX W sample. SDS-PAGE also confirmed the result. HA Assay 20 [0147] The sample 1X and 1X-W showed HAU 1024. See Figure 21. The concentrated, but not washed, 1OX showed at HAU 8192. However, 1OX-W showed a false negative at HAU 2 and 4. This may be due to the large amount of virus compared to the chicken RBC. High amounts of neuraminidase reverse the hemeagglutination process. See, Virus cultivation, Detection, and Genetics, S. J. Flint, L.W. Enquist, R.M. Krug, V.R. 25 Racaniello and A.M. Skalka, "Principles of Virology,' ASM Press, Washington, p 34, (2000). The absence of HAU in the permeate shows that there was not much virus eluting in the step 1. See Figure 12. NA Assay [0148] The neuraminidase assay illustrated that lOX diluted back tdr1X shoWs , see 30 decrease in activity in comparison with lX. See Figure 22. This was thought to be due to the loss of free NA protein from the VAF material. This was supported by a small amount -31- WO 2005/014862 PCT/US2004/005697 of NA in the permeate. Samples 1X-W and 1OX-W diluted back to 1X-W retained the activity at the same level. This was because the sample 1OX-W was concentrated directly from 1X-W. All the washes have the activity below the detection level. RHPLC 5 [0149] Egg protein analysis had been optimized previously by RHPLC, therefore, all the present materials in the example were analyzed under identical condition, e.g.,' C4 column with 0.1 % TFA/Acetonitrile gradient and monitored by 214 nm. The elution pattern of the ovomucoid, lysozyme, conalbumin and ovalbumin is shown in Figure 23. The 10-X sample, before any wash, showed all the egg proteins. This matches the retention 10 time of the control sample. Also lOX showed unidentified viral protein peaks labeled as Ul, U2 and U3. Completely washed samples 1OX-W and 1X-W retained the viral proteins U1, U2 and U3. The 1OX and 1OX-W samples contained the same amount of Ul, U2 and U3 proteins. Because the ratio of these proteins was the same, the proteins might be generated from the virus particles during the exposure to acetonitrile. However, the 15 ovomucoid, lysozyme and conalbumin have been completely removed from 1OX by washing with 1X-SPG for five times. Notably, in contrast, the most obvious protein peak is ovalbumin, which is still eluting along with 1OX-W and 1X-W samples. Even though 1OX W and 1X-W have gone 6 and 5 washes, still ovalbumin bound to the virus. This may be due to the strong interaction between HA proteins and ovalbumin. This data also presented 20 in the bar graph form as in Figure 24. [01501 The permeate and all the washes were checked by RHPLC. See Figure 25. The permeate contains all the NAF proteins and other unidentified peaks. Ovomucoid was removed by two washes (see Figure 26); lysozyme by 2 washes (see Figure 27); conalbumin by two washes (see Figure 28); and ovalbumin was depleted gradually, but about 5% 25 remained even after wash number. 6. See Figure 29. Agilert Bioanalyzer [0151] Simultaneously, ovalbumin was estimated by Agilent Bioanalyzer as shown in Figure 30. Just by the concentration, without any washing step, ovalbumin was considerably removed from 1X to 1OX. The first permeate carried most of.the:oyalbuniin. 30 RHPLC showed ovalbumin in all the washes, but in the Bioanalyzer analysis it reached below the detection limit. The 1OX-W sample was diluted ten times to reach the -32- WO 2005/014862 PCT/US2004/005697 concentration close to 1X-W sample, and it showed a small amount of ovalbumin. Based on this data, 95% of the egg proteins were removed by the concentration and washing steps. SDS- PAGE and Western Blot [0152] In comparison to IX (lane 2), 1OX (lane 9) contains more intense multiple 5 silver stain bands. See Figure 31. 1OX-W (lane 10) showed fewer number of bands compared to 1OX. This was due to the removal of NAF proteins and other impurities. Similarly 1X-W(lane 8) appears cleaner than 1X. Samples IX, lOX diluted to 1X (3rd Lane), and 1OXW diluted to 1X-W (4th lane) contain the same quantity of virus except different degrees of improvement in the removal of impurities. Obviously, 1OX-W diluted 10 to 1X-W shows clearer viral protein bands. However, this sample still contained an ovalbumin band. This is compared with NAF proteins in lane 6. The 1OX-W sample was further purified by analytical SEC column and the fractions were collected. See Figure 32. The fraction collected at 19.1 min was checked by SDS-PAGE, and this fraction contains mostly ovalbumin protein (lane 5). This lit up in the Western Blot against anti-NAF. This 15 is additional evidence to show that ovalbumin strongly binds to the virus even after 6 washes. The anti-NAF gel was stripped and probed with chicken anti-A/New Caledonia. Distinct bands were observed representing the viral proteins, HAo and HA 2 or M protein. See Figure 31. RTPCR 20 [0153] RTPCR showed that the RNA was about a log higher between 1X and 1OX. See Figure 33. Similarly, there was about a ten-fold increase in the viral RNA between 1X W and 1OX-W. This indicated that most of the virus was retained during the concentrations step. Permeate does not have any detectable viral RNA, but the 1X-SPG washes showed a very small amount of RNA. This may be due to small amount of virus undergoing shearing 25 during the circulation or some viral RNA bound to the filtrate and released latter slowly during the washing cycles. [0154] In summary, the concentration of the A/New Caledonia/20/99 was achieved by using a cross-flow-filtration device. The infectivity of the virus particles was retained during this procedure, and it was confirmed by CELISA assay. Washing the- concentrated 30 material by 1X-SPG improved the quality of the virus by removing other impurities. E n after the 5th wash a small amount of ovalbumin was strongly bound to the virus. This may -33- WO 2005/014862 PCT/US2004/005697 be due to the strong interaction between ovalbumin and the HA or NA protein. RHPLC and SDS PAGE and Western Blot support this protein-protein view. The increase in the quantity of RNA between the neat and concentrated sample indicates that the majority of the virus is recovered by this procedure. 5 [0155] Similar techniques are also applicable for use with virus samples from cell culture, e.g., influenza samples grown in cells such as Vero cells, etc. To illustrate such, three viruses were grown in Vero cell culture, namely, A/Beijing (A/H1NI) used as is; AlPanama (A/H3N2) concentrated from 2L to 100 ml or 20X; and B/Hong Kong concentrated from 2L to 10 ml or 200X. It will be appreciated that since virus yields from 10 Vero cells are typically lower, the embodiments of the current section can optionally be used to concentrate the virus samples. Similar to the above illustration an Amersham MidGee and a QuixStand Instrument were used for the virus concentration [0156] Figures 34-35 show monitoring of A/Beijing cell culture propagation by SEC (Figure 34) and A/Beijing Vero cell culture harvest (Figure 35). As can be seen SEC is an 15 efficient technique for monitoring the virus propagation in a short time. The amount required for such monitoring is also typically small (e.g., 100 ul). Figure 36 illustrates concentration of a 2 liter sample of an A/Panama cell culture sample. Two liters of virus harvest were concentrated down to 100 ml by QuixStand. See above. The TCIDso of the IX mixture was non detectable, but the TCID 5 o of the 20X mixture was 4.4. There was a 20 peak area ratio of 20X to IX. The concentration of the Panama cell culture sample illustrates the advantages of cross-flow filtration, e.g., virus particles can be efficiently enhanced, low molecular weight impurities can be removed from the solution and diafiltration can be done for further "clean-up" of the solution. Figure 37 shows concentration of 2 liters down to 10 ml of a Vero cell grown culture of B/Hong Kong. At 25 IX the loglO TCID 5 /ml was 4.7, while at 18.8X it was 5.8 (the theoretical for such being 5.95) and at 200X it was 6.95 (the theoretical of which being 7.00). [0157] From the above-figures it can be seen that SEC is a useful technique for monitoring virus growth in cell culture samples; very low titer virus can be assayed after concentration of virus samples; and low titer virus can be assayed after concentration. 30 Stabilizers/Buffers .- : [0158] The invention comprises compositions of virus solution andrnethods of creating the same. Such compositions optionally comprise various dilutions of NAF -34- WO 2005/014862 PCT/US2004/005697 (typically unfractionated NAF) comprising the virus of interest and combinations of, e.g., sucrose, arginine, gelatin, EDTA, etc. as detailed herein. As will be noted, various compositions herein comprise from 10% to 60% NAF. NAF can possibly contain various enzymes such as nucleases lysozymes, etc. which could adversely affect the stability of 5 virus compositions. Such methods and compositions are preferably stable (i.e., do not show unacceptable losses in potency) over selected time periods (typically at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, etc.) at desired temperatures (e.g., typically 4C, 5'C, 8 0 C, from about 2C to about 8'C or greater than 2'C, etc.). Preferred embodiments show no decrease in potency over . 10 the desired storage period. Other embodiments show less than 10% decrease, less than 5%, less than 4%, less than 3%, less than 2o, or less than 1% decrease. The potency of virus compositions herein was measured in FFU or fluorescent focus units (see below in description of FFA assay). A target FFU value is typically set based upon the virus concentration at a time zero (e.g., due to dilution of the NAF, etc.). Preferred embodiments, 15 thus, show little or no decrease from the starting value. In various compositions herein, the virus solutions comprise from about 5% to about 10% sucrose, from about 1% to about 4% arginine, and from about 1% to about 4% gelatin. Some preferred embodiments comprise about 7-10% sucrose, about 2% arginine, and about 2% gelatin. In some embodiments, the stability is measured after storage of the virus formulation at the desired temperature in a 20 FluMist @ applicator/accuspray device or other similar device. [0159] In some embodiments, the invention comprises compositions comprising stabilizer of, e.g., arginine (of pH from about 7.0 to about 7.2), either in combination with, or in place of gelatin or gelatin related and/or derived products (e.g., gelatin hydrosylate). See, Steps 12 and 15 in Table 1. However, current regulatory concerns regarding the 25 potential of unintentional contamination from animal and animal-derived products such as gelatin, collagen, etc. (e.g., by such problems as prions, mycoplasma, or host-derived viruses), as well as concerns regarding the potential for allergenicity of animal derived products, has lead to the need for non-animal based stabilizers. Arginine used either alone or in combination with additional excipients such as metal ion chelators (e.g. 30 ethylenediaminetetraacetic (EDTA) and/cr its salts) or other amino acids (e.g; Histidine and/or its salts) offers the potential of stabilization of cold-adapted influenza virus preparations with a non-animal derived excipient. -35- WO 2005/014862 PCT/US2004/005697 [0160] In various embodiments, the arginine optionally comprises either a salt with an inorganic acid or a salt with an organic acid. Of course, the salt typically comprises a pharmaceutically acceptable salt since it is to be used as a vaccine component. Typical preferable salts comprise, e.g., hydrochloride, citrate, and sulfate. The amount of such 5 stabilizing agent used is not limited to specific particular ranges, however, typical amounts used range from about 5 mg to about 60 mg per 1 mL of the virus solution. The amount used may preferably be from about 10 mg to about 50 mg, and more preferably, from about 10 mg to about 25 mg per 1 mL of the virus solution. In other embodiments, the amount used may range from about 1%; from about 1.5%; from about 2%, from about 3%, or from 10 about 4% to about 5% arginine solution of the virus solution. The amount used can vary in different embodiments of the invention. In yet other embodiments of the invention, the virus solution/vaccine solutions optionally comprise potassium phosphate. In some embodiments, the solutions comprise about 11 mM potassium phosphate. In other embodiments, the solutions comprise from about 10 mM to about 12 mM potassium 15 phosphate. The formulated composition can optionally contain substantial amounts of egg allantoic fluid components (e.g. proteins and metabolites) and/or a buffer diluent. Additionally, acceptable compositions of vaccine can contain a buffer salt, such as a mixture of monobasic and dibasic sodium or potassium salts of phosphate at concentrations ranging from, e.g., 5 to 200 millimolar or histidine and/or its salts at concentrations ranging 20 from, e.g., 25 to 100 millimolar. In preferred embodiments, sucrose-is present at a concentration ranging from about 100 millimolar to 350 millimolar. [0161] In many virus solutions/vaccine solutions a base solution of.SPC (sucrose, potassium phosphate and monosodium glutamate) is optionally utilized. However, in some embodiments of the current invention, MSG is not part of the virus/vaccine solution. In yet 25 other embodiments, levels of MSG are reduced. The amount of sucrose that is usable in the embodiments herein is variable over a wide range. Approximately 0.2 M sucrose. is utilized (7% W/V) in typical embodiments, however, compositions comprising up to ca. 20% sucrose can present no detrimental effect on virus activity/potency. Surfactants in various embodiments of the compositions can comprise, e.g., Poloxamer 188 (polyoxyethyleie 30 Polyoxypropylene block copoloymer, e.g. Pluronic F68) and Tween 20 (polyoxyethylene sorbitan monolaurate) at concentrations in the range of ca. 0.01 to 0.1% (W/V%). In some embodiments, the combination of Poloxamer, gelatin hydrolysate and arginine is superior to -36- WO 2005/014862 PCT/US2004/005697 any solution containing only one of the components, each solution in turn being more stable than a solution containing none of the added components. [0162] In yet other embodiments, steps in Group 4 (e-g., Step 15 of Table 1) comprise replacing all or part of the normal allantoic fluid (NAF) in which the viruses are 5 suspended with a buffer of sucrose, potassium phosphate and monosodium glutamate (SPG) or other simple solutions, e.g., those with reduced MSG, etc. The use of SPG in place of some or all of the NAF diluent results in greater stability of the viruses in solution. Such stability is also a novel and unexpected benefit of the embodiments of the current invention. Representative formulations embodying some or all of the formulation attributes described 10 above were prepared and the stability of the component cold-adapted viruses was evaluated. Compositions of representative formulations are shown in Table 28. The stability of the formulations at 5oC is shown in Table 29. [01631 Various formulations of the invention were tested for their stability over a variety of months and temperatures. For example, Table 30 illustrates 12 different 15 formulations. Formulations 10 and 11 were based upon formulations used for dried virus preparations. The formulations in such tables covered a range of various components, e.g., sucrose and gelatin. Tables 31-34 show the stability of such preparations comprising 4 different virus strains over six months (two sample points for each). Figure 38 graphs the results of 4 exemplary formulations with the B/Hong Kong strain used. Table 35 shows 20 compositions of additional formulations. The compositions in Table 35 examine addition of various compounds to the basic composition (i.e., typically 10/2/2 meaning about 10% sucrose, about 2% arginine, and about 2% gelatin) to help potentially inhibit adverse components present in the NAF such as lysozyme, etc. The stability results of the formulations in Table 35 are seen in Tables 36 through 39 and in Figures 39 and 40. Tables 25 40 and 41 and Figure 41a-c look at varying concentrations of citrate in the formulations (here a base formulation of about 10% sucrose, about 1% gelatin, and about 2% arginine). Formulations with citrate showed a precipitate at about 7-8 months of storage. Tables 42 and 43 and Figure 42a-c show a similar analysis, but with varying concentrations of EDTA. Exemplary formulations from the above examples were subjected to further testing which is 30 shown in Table 44 and 45a-d. Additional formulations with varying concentrations of. sucrose, gelatin, arginine, and EDTA, etc. are shown in Tables 46 through 48. -37- WO 2005/014862 PCT/US2004/005697 [0164] To further illustrate the stability of several monovalent formulations herein, compositions comprising 60% allantoic fluid were tested for stability. Samples were stored at 5'C and examined with FFA analysis. Biweekly sampling was done for the first two months, then monthly sampling was done to 9 months. A concentration of 60% AF will 5 allow a high probability of producing VH at the necessary potency even in years with low titer strains. Some formulations utilizing unpurified VH exhibited sufficient stability forall strains test to almost consistently meet a criterion of 0.5 log loss in 7 months at 5'C. Influenza strain B/Hong Kong/330/01 appeared to be the most problematic of the strains tested for stability. See Table 30 which gives percent composition of sucrose, arginine, 10 gelatin and other components for the 13 different formulations. Figure 43 illustrates the stability of four virus strains in such formulations after 9 months. Exemplary formulations of unpurified virus composition formulations can comprise, e.g., VH, 10% sucrose, 2% arginine, 2% gelatin; VH, 10% sucrose, 2% arginine; VH, 10% sucrose, 2% arginine, 1% dextran; VH, 10% sucrose, 2% arginine, 0.5% PVP; VH, 10% sucrose, 2% arginine, 2% 15 gelatin, 2.5 mM EDTA; VH, 10% sucrose, 2% arginine, 2% gelatin, citrate buffer; and, VH, 10% sucrose, 2% arginine, 2% gelatin, histidine buffer. [0165] Other methods of virus/vaccine solution purification (e.g., for stabilization, etc.) involve such techniques as removal of all NAF through fractionization (along with addition of stabilizers) to give stability of the solutions. Various embodiments of the 20 current invention, however, involve, e.g., dilution out of the NAF in-which the virus/vaccine exists. For example, in various embodiments herein, the concentration of NAF optionally comprises from about 10% to about 60% of the solution. In other embodiments, NAF can optionally comprise from about 20% to about 50%, or from about 30% to about 40% of the solution. Such dilution of NAF concentrations allows for greater stability of the 25 virus/vaccine solutions, especially at desired temperatures (e.g., 4*C, from about 2 0 C to about 8'C, etc.) in liquid form, Additionally, some embodiments of the invention comprise reduced NAF concentrations in conjunction with use of arginine (see above). Various formulations of the current invention were compared in stability with virus compositions that were NAF free purified formulations or that were NAF reduced (but still NAF purified) 30 formulations. Table 49 illustrates the formulation of a number of compositions of the-, invention as well a number of formulations wherein the VH was purified from the.NAF various ways. It will be appreciated that the base formulations shown in Table 49 also -38- WO 2005/014862 PCT/US2004/005697 typically comprise about 2% arginine, about 2% gelatin, about 1% PVP, about 1% dextran, about 2.7 mM EDTA, and about 100 miM histidine. The numbers in Table 49 correspond to the formulations displayed in Figures 44-46. [0166] The diluted NAF embodiments of the current invention are in comparison to 5 alternative stabilization methodologies, e.g., which end up with 10-25% fractionated NAF or even 5% fractionated NAF or less in their final formulations. However, those of skill in the art will appreciate that the NAF present in some current embodiments does not comprise such fractionated NAF, but is instead comprised of un-fractionated NAF. The formulations of the invention were compared against other current virus solutions that were made from 10 purified NAF (e.g., fractionated NAF, etc.) in terms of stability. The goal in the comparison was to reach less than or equal to 1.0 log potency loss in 12 months or less than or equal to 0.080 log/month loss in potency when stored at between 2'C and 8'C, e.g., 4C. The other current virus formulations compared against the formulations of the invention were purified through, e.g., fractionation, diafiltration, etc. The different formulations were tested with 3 15 different influenza strains: a HINI strain (A/New Caledonia/20/99 or A/NC), a H3N2 strain (A/Panama/2007/99 or A/Pan or A/PA), and a B strain (B/HongKong/330/01 or B/HK) and were filled into Accusprayers (i.e., a delivery device for FluMist@). In order to mimic a likely manufacturing process, the samples were frozen at -25'C for at least 6 days as an initial step. 20 [0167] In a first comparison, a NAF purified cold-adapted trivalent formulation was compared in stability with an unpurified NAF formulation of the invention. The formulations comprised 7% sucrose, 1% gelatin, 1% arginine (which are the standards for the comparing trivalent formula) and 60% AF (allantoic fluid) for the formulation of the invention. The formulation of the invention after six months showed -0.035 t 0.016 for 25 A/NC, -0.079 ± 0.035 for A/Pan, and -0,151 ± 0.018 for B/HK. The measurements for the purified composition was -0.020 ± 0.027 for A/NC, -0.011 0.020 for A/Pan, and -0.138 0.022 for B/HK. The units above are in log FFU/month. See Table 50. Table 51 shows a comparison between a purified formulation and a formulation of-the invention when the invention formulation uses a 10/2/2 composition, see above. The high initial potency loss 30 observed is though to be attributed to freeze-thaw and/or blending loss. Table 52 shows a similar comparison, but with histidine in the FluMist@ formulation, which gave rise to a better stability with no initial potency loss observed. -39- WO 2005/014862 PCT/US2004/005697 [0168] Figure 44 illustrates the initial potency loss (freezing and/or blending loss) seen above is exclusively associated with phosphate buffered formulations. No initial potency loss was observed with histidine buffer, which exerted a positive impact on stability. The formulations shown in Figure 44 are those listed in Table 49. Figure 45 5 illustrates a "global" picture of the stability slopes of the formulations of Table 49 after 6 months. As can be seen the histidine buffered 10/2/2 formulation exhibited the best combination of stability and meeting the target goal. See above. Figure 46 gives a different view of similar data (i.e., week rather than month). Figure 47 illustrates a second study which produced results illustrating the stability of a 10/2/2 + histidine formulation with 10 either gelatin (L106) or PVP/EDTA (L104). As can be seen from the figure, the replacement of gelatin with PVP/EDTA produced stability almost as efficiently as the inclusion of gelatin. Figure 48 examines the optimal pH of a histidine-based 10/2/2 formulation of the invention. As can be seen, pH 7.0 comprises a preferred embodiment. Ranges of pH from about 6.8 to about 7.2 for these 100 mM histidine 10/2/2 formulations 15 are also included embodiments of the invention. Figure 49 shows examination of preferred embodiments of sucrose concentration in embodiments of the invention. Some preferred embodiments comprise about 10% sucrose, while others comprise about 7%. The basic formulation in Figure 49 comprises the 10/2/2 above, with the addition of sucrose histidine. In the various embodiments illustrated herein, some embodiments comprise histidine as a 20 buffering additive and/or arginine as a stabilizer and/or dextran and/or PVP in place of gelatin. [0169] Other embodiments of the current invention are optionally stabilized through use of ultrafiltration/concentration of the virus/vaccine solution. Such ultrafiltration is typically an altemate means of achieving solution stability as opposed to 25 reductions/dilutions of NAF. For example, in some situations if the titer or potency of a particular strain/solution is low, then ultrafiltration can optionally be used in place of NAF dilution (which could act to further reduce the titer/potency of the solution). The ultrafiltration in Groups 4 steps is slightly different from the microfiltration as described above. In the earlier Group the filtration was for, e.g., sterility whereas in the current Group 30 the filtration concerns stability, etc. and the viruses are kept during the filtration.: See above. -40- WO 2005/014862 PCT/US2004/005697 Potency Assays [0170] In some embodiments herein, the potency measurement for the virus/vaccine is performed by a cell-based ELISA (i.e., Cell-based ELISA, or CELISA, for Potency Measurement of FluMist- a live, attenuated influenza-virus vaccine, or for other such 5 vaccines). Such method is a simpler and faster alternative to the more traditional Median Tissue Culture Infective Dose (TCID 5 o) assay, for potency measurement of live virus. Briefly, confluent monolayers of Madin-Darby Canine Kidney (MDCK) cells in 96-well. microtiter plates are infected with sample containing live virus, fixed with fornalin 16-18 hours post-infection and reacted with influenza virus-specific monoclonal antibody (Mab). 10 Virus antigen bound Mab is then detected using anti-mouse IgG-Peroxidase and peroxidase substrate to develop soluble colored product, the optical density (OD) of which is measured spectrophotometrically. Those of skill in the art will be familiar with epitopes/antigens shared by various subtypes of influenza strains (e.g., various HA, etc.). The potency of live virus in a sample is calculated from a standard curve generated using live influenza virus 15 calibrators with known logio TCID 5 o values obtained with a validated TCID 5 0 potency assay. CELISA is shown to be linear (r2 greater than or equal to 9.95) in the range 4.9-6.7 logio TCID5 0 . Between-day, between-analyst, between-plate, within plate (residual) variability (Standard Deviation in logioTCID 5 o) were 0.06, 0.02, 0.05 and 0.03 respectively. The potency of several vaccine and wild-type influenza A/HIN1, A/H3N2 and B strains 20 measured by CELISA are comparable (± 0.3 logioTCID 5 o) to the potency measured in parallel by the validated TCID 5 o potency assay. -CELISA is capable of measuring potency of up to 10 samples/plate in 2 days in contrast to 2 samples/plate in 6 days for the validated

TCID

5 o potency assay. CELISA is optionally used in place of, or in addition. to other methods of potency assay (e.g., FFA and TCID 5 O, see, below), 25 [01711 The Median Tissue Culture Infective (or Infectious) Dose 50% (TCID 5 o) assay (see, below for more details) is a widely used method for the potency measurement of live virus and live virus vaccines. However, in some embodiments herein, Cell-based ELISA (CELISA) is optionally used as a simpler and faster alternative to the traditional, long and labor intensive TCID assay to measure potency of influenza virus in.FluMist, a 30 live, attenuated vaccine (or in other similar vaccines). [0172] In other typical embodiments, potency assays of the virus solutions optionally comprise fluorescent focus assays (FFA) as opposed to common TCID 50 assays -41- WO 2005/014862 PCT/US2004/005697 which are used in the art. Such FFAs have the added benefit that they are more amenable to automation, thus, allowing higher throughput of vaccine production. TCIDo assays usually measure the quantity of a virus suspension or solution that will infect 50% of a particular cell culture. The measurement gives accurate results, but is slower than FFA and thus can 5 use up valuable time in the production of vaccines. FFA assays typically use type and/or subtype (or even universal antigen) specific anti-influenza antibodies (typically anti HA antibodies) to detect virus antigens in infected cells. In uses wherein the antibodies do not cross react with different types/subtypes of influenza they can be used to quantitate the separate virus types in multi-virus preparations (e.g., trivalent vaccine formulations). FFA 10 assays can also be used as identity tests for specific strains. Those of skill in the art will be quite familiar with FFAs and their use in virus/vaccine testing. [0173] Fluorescent focus assays, on the other hand, do not rely on the induction of cell death (either in the infected cells or the indicator cells). Instead, they use antibody staining methods to detect virus antigens within infected cells in a cell culture monolayer. 15 These infected cells are then visualized and quantified using a fluorescent label on the virus specific antibody. Typical FFAs of the current invention use, e.g., type and subtype specific anti-influenza HA antibodies to visualize virus antigens in infected cells. [0174] In other embodiments, the FFAs (and optionally other assays herein) optionally use a universal reagent (or universal antigen) which is not specific for specific 20 type/subtype influenza antigens, but is instead specific for a generalized influenza antigen. Therefore, the universal reagent is optionally useful for FFAs for myriad different screenings and type/subtype specific antibodies do not have to be developed and created each time a different virus is assayed. [0175] Other embodiments herein comprise viral potency determination using a cell 25 based fluorometry assay (CFA). While FFA assays are quite useful in many embodiments, CFA assays are preferentially used in other embodiments. While the image processing and readout of FFA assays can be capped at about 20 plates/person/day (or about 5 plates/hour image processing), the image processing and readout from CFA assays can be up to about 4 times faster. Also, while FFA titers can differ from TCID 5 o titers for influenza B strains, 30 CFA titers have not shown significant differences from TCID 5 o (or FFA) titers due to the use of assay standard or calibrators. In brief, the CFA assay measures infectious influenza viruses in MDCK cells grown in 96 well plates. As with FFA, CFA detects-viral protein -42- WO 2005/014862 PCT/US2004/005697 expression resulting from viral infection of MDCK cells during the first infection cycle. CFA assays utilize calibrator or assay standard for titer calculation. For CFA reagents, typical antibody reagents can comprise: primary antibodies specific to HA or A strains and B strains (for influenza) and secondary antibodies of, e.g., goat anti-mouse IgG conjugated 5 with Alexa 488. Assay standards for CFA can include virus harvest of the same strain as. the samples to be tested with a known FFA or TCID titer. Assay references for CFA can include, virus harvest with known FFA or TCID titer and known linear slope (does not have to be the same strain as the samples to be tested). Sample primary antibodies can include, e.g., those specific for A/H1N1 or A/H2N2 strains (from, e.g., Takara) at, e.g., a working 10 dilution of 1:2000; those specific for A/H3N2 strains (from, e.g., Takara) at, e.g., a working dilution of 1:1000; and, those specific for B strains (from, e.g., Chemicon) at, e.g., a working dilution of 1:1000. A typical CFA assay procedure can comprise virus inoculation followed by 18 hours incubation at 33'C followed by fixation and incubation at room temperature for 15 minutes. A primary antibody incubation for 60 minutes at 37'C is then 15 followed by a secondary antibody incubation for 60 minutes at 37*C. The plates are then read with a fluorometer and the data analyzed. In some embodiments of CFA, the infection level of a well, etc. is determined via protein expression (as opposed to typical FFA assays where the number of cells infected are measured). Those of skill in the art will be aware of typical FFA assays and fluorometry and similar concerns applicable to CFA assays. 20 Semi-Automated TCID, 0 Assays [0176] As stated above, some embodiments of the current invention comprise TCIDso assays and various modifications, etc. thereof. For example, some embodiments comprise a semi-automated version such as illustrated by the following descriptions. [0177] A comparison of Manual and Semi-Automated Median Tissue Culture 25 Infective Dose (TCID 50 ) assays for Potency Measurement of a live, attenuated influenza virus vaccine, e.g., FluMist @, or other similar vaccines, is given in this section. The TCIDo potency assay is optionally used for potency measurement of FluMist or other similar vaccines. A semi-automated TCID 5 O potency assay is described in. which two labor intensive steps of the validated manual potency assay are improved. These are (i) use of an 30 automated pipetting station for sample dilutions and infection of MDCK monolayers in. place of multiple manual repeating dilution steps, and (ii) use, 6-days post-infection, of a -43- WO 2005/014862 PCT/US2004/005697 96-well plate reader to measure spectrophotometrically the product of MTT, a vital dye (3 (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) widely used as an indicator of cell health/viability, in place of manual observation under a light microscope of each of the 96 wells of all assay plates in order to assess the presence of influenza virus induced 5 cytopathic effect (CPE) in MDCK cells. [0178] The semi-automated TCID 5 o potency assay used in some embodiinents herein was developed and validated to demonstrate precision (repeatability: <0.25 logioTCID 5 o; intermediate precision: SD(Dy) <0.3 logiOTCID 5 D; SD(Analys) and SDmInstrument) <0.4 logioTC]D 50 ; and reproducibility at the 90% Confidence Interval ± 0.3 logioTCID5o), 10 linearity, accuracy and range (Slope 1 ± 0.1). The semi-automated TCID 50 potency assay using an automated pipetting station and MTT dye was shown to provide equivalent results to the validated manual TCID 5 o potency assay (at the 90% Confidence Interval ± 0.3 logioTCIDso). In brief, the results herein provide support for the use of a pipetting station and MTT dye to measure the potency of influenza virus in, e.g., Flu~fist@ productions. 15 These improvements also increase the testing throughput. Infectivity/Potency (Monovalent) Assay Validation [0179] A semi-automated version of the current manual method for potency determination of monovalent influenza strains is optionally used for manufacture of FluMistTM vaccine and other similar vaccines. The semi-automated potencyassay 20 incorporates automation of the plate washing and serial dilution steps, and an automated dye-based detection of the virus induced cytopathic effect (CPE) to replace the manual microscopic detection. Automation of the plate washing and serial dilution steps allows greater assay throughput, and reduces the risk of repetitive motion injuries -for Quality . Control analysts performing this assay. Automated dye-based detection of the virus induced 25 CPE enhances assay consistency and throughput by elimination of the microscopic detection. [01801 Some steps/aspects of the semi-automated assay are similar to more traditional TCID 5 0 assays, while other steps/aspects are quite different. The assay steps include preparation, incubation and washing of assay plates containing MadinDarby canine 30 kidney (MDCK) monolayers, infection and post-infection incubationof theassay plates, and calculation of the potency based on the number of CPE positive wells and sample -44- WO 2005/014862 PCT/US2004/005697 testing configuration in both assays. The semi-automated potency assay has been validated and the performance of the assay demonstrated to be comparable to the current manual assay by the inventors and coworkers. This semi-automated assay optionally serves as the primary method for the infectivity/potency measurement of expanded wild-type influenza 5 (eWT), Master Virus Seed (MVS), Manufacturer's Working Virus Seed (MWVS), and Virus Harvest (VH) samples. The manual assay is optionally used as a backup in a situation where the semi-automated assay cannot be performed; i.e. in the case of prolonged equipment downtime. [0181] The traditional median tissue culture infectious dose (TCID 5 o) assay is a cell 10 based method that measures infectious cytocidal virions. MDCK cells are cultured in 96 well plates, and confluent monolayers are inoculated with serial dilutions of a virus sample. Replication of virus in the MIDCK cells results in cell death. The progeny virus infects other cells, resulting in the eventual destruction of the monolayer. The CPE resulting from infection is allowed to develop during an incubation period of six days. The individual wells 15 are read microscopically, to determine the presence of CPE in each well. Four individual determinations are performed on each of three days by this procedure, and the results of all 12 titrations are averaged in order to produce one test result. In addition to samples, each analyst analyzes one monovalent control, also with four replicate determinations on each, of three days. 20 [01821 The manual assay is labor intensive and has limited sample throughput. Each individual determination involves numerous cell washing and serial dilution steps, which are performed using a manual pipettor. Each well in the 96-well assay plate-has to be microscopically scored for the presence or absence of CPE. Multiple plate washing and dilution steps pose a repetitive motion injury risk for analysts. In addition, the microscopic 25 reading of each individual well in the 96-well plates is fatiguing, which limits the number of analyses that can be performed by each analyst to about 20 plates per day. One test result is obtained as an average of twelve determinations over a three-day test period, and each analyst performs one monovalent assay control in addition to samples. This limits the assay throughput to 9 samples per analyst per 3-day testing period (average of 3 sFmples per 30 analyst per day). Because each individual sub-lot (about forty to fifty sub-lots per lot) of monovalent influenza vaccine harvest is tested by this assay, the limited throughput may limit capacity for full-scale commercialization of vaccines (e.g., FluMistTM vaccine, etc.). -45- WO 2005/014862 PCT/US2004/005697 [0183] Automation of the traditional assay, specifically the plate-washing and pipetting steps, and MTT (3-[4,5-Dimethylthiazol-2-yl] 2,5-diphenyl-tetrazolium bromide) dye-based detection of CPE, results in development of a Semi-Automated TCID 5 0 Potency Assay for Influenza Virus Monovalent. The semi-automated assay uses the SkatronTM Cell 5 Washer for the washing steps, where the debris and spent media are removed from the cell culture plates and replaced with fresh media. The Matrix SerialMate@ multichannel pipetting station is used to perform the sequential 10-fold dilutions of the virus, and for transfer of the diluted samples onto the cell culture monolayers in 96-well assay plates. Of course, other devices which perform similar functions are optionally substituted herein and 10 specific mention of particular brands or types of devices should not be construed as limiting unless specifically indicated to be so. After the six-day incubation period, the 96-well assay plates are then incubated for six hours with MTT dye, which is a widely accepted indicator of cell metabolism and viability. During the incubation period, intact and healthy cell monolayers process the dye to form the insoluble purple formazan product, which 15 accumulates intracellularly. In wells where the cell monolayer is destroyed, no dye product is formed. A solubilizing solution of 0.01 N Hydrochloric Acid, containing 20% of the surfactant sodium dodecyl sulfate (SDS) is then added, and the plates incubated overnight to dissolve the insoluble dye product. The absorbance at 570 nm is measured to quantify the purple formazan dye product. The absorbance reading is processed using a.Microsoft 20 ExcelTM Macro program (or other similar program), to identify and count the CPE positive or negative wells and calculate the TCID 5 titer. Wells containing intact cell monolayers show a higher absorbance when compared to a pre-determined cut-off value, and are identified as CPE negative, whereas CPE positive wells show absorbance readings below the cut-off value (see Figure 50). The number of wells showing CPE at each dilution is then 25 used to calculate the titer (log10 TCID 5 ohmL) based on the Karber modification of the Reed Muench method. The automation of the cell washing, serial dilution and virus inoculation steps, and the MTT dye-based CPE detection are described in detail below. Automation of Cell Washing Steps using SkatronTM Cell Washer [0184] In a manual assay, plates containing MDCK cell monolayers in 96-well 30 plates are washed twice prior to inoculation with the diluted virus samples. Spent-medium containing waste products and fetal bovine serum (FBS) from the four-day cell incubation is removed and replaced with fresh virus growth medium (VGM) without FBS. The cells are -46- WO 2005/014862 PCT/US2004/005697 then incubated at 33t1 0 C and 5±1% CO 2 for at least 10 minutes, then the VGM is removed and replaced with fresh VGM a second time. For each washing step, individual plates are inverted onto clean paper towels and gently blotted to remove media from the wells, and then each well is refilled with 200 pvL of fresh VGM using a hand-held multichannel 5 pipettor. This process is labor-intensive and time consuming when large numbers of plates are processed. [0185] The Skatron TM Skanwasher (Series 300, Model 12010) is a microprocessor controlled 96-channel cell washer, which performs these washing steps automatically. The Skanwasher is small enough to fit in a 6-foot laminar flow biosafety hood. Automation of 10 cell plate washing steps using the SkatronTm Skanwasher, involves a wash program where the spent media are aspirated from the plates, then fresh VGM is dispensed into the empty wells. Individual plates are loaded into the Skanwasher, then removed to a 33±1'C and 5±1% CO 2 incubator at the end of the wash cycle. The plates are incubated for a minimum of 10 minutes, then loaded onto the Skanwasher for the second wash, after which they are 15 transferred back into the incubator. The performance of the SkatronTM Skanwasher in these wash steps is shown to be acceptable for use in the cell washing steps. The dispensing precision for the 200 pL volume is associated with a CV < 10%, and the dispensing accuracy is within 10%. The residual volumes for the aspiration step are less than 1%. Thus the SkatronTM Skanwasher provides acceptable performance, while improving the ease 20 of use and throughput efficiency of the cell-washing step. Again, it will be appreciated that similar devices capable of performance within the same standards are also optionally used herein. Automated Serial Dilution and Virus Inoculation with Matrix SerialMate@ Multichannel Pipetting Station 25 [0186] The serial dilution and the virus inoculation steps of the traditional manual

TCID

5 o assay are carried out by hand-held multi-channel micropipettes. The serial dilutions are carried out in two steps. The first set of five serial dilutions is carried out in a 0.5 mL dilution block, and then the appropriate dilution from the first block is transferred to a 2 mL dilution block, for the final five serial dilutions. It is crucial that these serial dilutions be 30 carefully executed, because pipetting errors at any one dilution may be propagated and magnified through the subsequent series. The subsequent virus inoculation step involves -47- WO 2005/014862 PCT/US2004/005697 repetitive pipetting of diluted virus into multiple rows or columns of an assay plate containing confluent cell monolayers. Prolonged use of the hand-held multichannel micropipettes used to provide the necessary accuracy for these tasks can lead to severe muscle fatigue and tendonitis, which limits the number of plates each analyst can perform in 5 one day and, thus, the throughput of the entire process. [0187] Use of the Matrix SerialMate® pipetting station for the serial dilution and virus inoculation steps improves the ease of use and throughput of the assay, and reduces the occurrence of operator injuries, while providing the necessary precision and accuracy for these tasks. The Matrix SerialMate® pipetting station is a bench top liquid handling 10 station equipped with a 12-channel nozzle head which can aspirate and dispense volumes in the range of 5 gL - 225 pL. The unit is small enough to fit in a standard 4- or 6-foot biosafety cabinet and is easy to use. The Matrix SerialMate@ provides precision better than 0.5 gL, and accuracy better than 1.0 pL for delivery volumes of 5 pL - 225 pL. This corresponds to a precision better than t 1.7% and accuracy better than ± 3.3% for the 30 pL 15 delivery volume used in the serial dilution steps. The comparability of assay results obtained using the automated assay and the current manual assay is confirmed as described below. Again, it will be appreciated that similar devices capable of performance within the same standards are also optionally used herein. Description of MTT dye-based detection 20 [0188] The final step in a TCID 5 assay is the detection of CPE and quantitation of the virus. With the current (manual) TCID 50 assay, the individual wells are read microscopically, to look for signs of CPE in each well. These signs include areas of foci, partial or complete collapse of the cell monolayer, and the presence of rounded and darkened cells on top of the destroyed cell monolayer. It has been observed that significant 25 eye strain sets in as the analyst counts large numbers of plates, setting the practical limit for the number of plates which may be counted by one operator to about 20 plates. This step is rate limiting to the throughput of the manual assay. [0189] Tetrazolium dyes are widely used as cell viability indicators. The most commonly used dye is yellow MTT dye. Viable cells, which possess active.mitochondria,.' 30 will reduce MTT dye to an insoluble purple formazan product, which-can be detected at 570 nm after a solubilization step. In CPE positive wells where the large majority of cells have -48- WO 2005/014862 PCT/US2004/005697 been destroyed, little or no dye product is formed, and a much lower absorbance is observed. [0190] In a semi-automated TCID 50 assay, after the infection and six-day incubation of the plates, the spent medium is removed, 100 iL of a solution of 0.5 mg/mL MTT dye in 5 fresh virus growth medium is added to each well of the 96-well plates, and the cells are incubated at 37±1'C and 5±1% CO 2 for six hours. The dye product is solubilized by overnight incubation at 37±1 0 C, following addition of 100 pL of a solubilizing reagent (20% SDS in 0.01 N HCl), then the absorbance at 570 nm due to the purple formazan dye product is measured with a plate reader. The absorbance data is transferred to a validated 10 Microsoft ExcelTM Macro (or other similar program) that converts the absorbance readings to a CPE count based a pre-established cut-off value. Wells containing intact cell monolayers yield a higher absorbance when compared to a pre-determined cut-off value, and are identified as CPE negative. CPE positive wells show absorbance readings below the cut-off value. The number of wells showing CPE at each dilution is then used to 15 calculate the titer (log1OTCIDso/mL) based on the Karber modification of the Reed-Muench method. [0191] The automated dye-based detection enhances the consistency of the CPE readout and increases the assay throughput. The comparability of the dye-based detection to the manual microscopic CPE detection is ensured by extensive studies where the assay 20 was run with different vaccine and wild type virus strains, and with plates prepared with different cell passage numbers and seeding densities. In these studies the plates were read first by manual microscopic examination, and then by dye-based absorbance detection. The results from these studies were analyzed to determine a universal absorbance cut-off, which provided comparable CPE counts by both detection methods. This universal cut-off value 25 of 0.5254 for the absorbance at 570 nm was confirmed by a detailed study (see, below), in which 9 different analysts performed assays on three different instruments,.over 6 assay days, using a total of 573 assay plates. The presence or absence of CPE in each well (80 virus inoculated wells per plate, for a total of 45,840 wells) was read first by manual microscopic examination, then by dye-based absorbance detection. 30 [0192] Figure 50 shows a histogram derived from plotting the absorbance readings from the wells, versus the frequency of the values (number of wells read at that absorbance -49- WO 2005/014862 PCT/US2004/005697 value). The frequency determination shows that the absorbance cut-off value of A570 = 0.5254 is located in the left-most tail (probability = 0.007%) of the distribution of the CPE negative wells, and the right-most tail of the distribution of CPE positive wells (probability = 0.02%). Comparison of the CPE detection of each well by both methods using this cut 5 off value, showed a one-to-one correspondence in most wells (45279 of 45840,98.78%) for identification as either CPE positive or CPE negative, both by dye-based detection, and by microscopic examination. Validation of Semi-Automated Potency Assay [0193] The Semi-Automated median tissue culture infectious dose (TCID 5 ) potency 10 assay for analysis of monovalent influenza vaccine virus is intended for the infectivity/potency measurement of expanded wild-type influenza (eWT), master virus seed (MVS), manufacturer's working virus seed (MWVS), and virus harvest (VH) samples. The assay was validated to demonstrate the precision (repeatability, intermediate precision and reproducibility), linearity, accuracy, and range of a Semi-Automated TCID 50 assay, and 15 show that it provides comparable results to a manual TCIDso assay. Validation tests were carried out with three different monovalent vaccine strains, chosen to include one Type A/H1N1, one Type A/H3N2 strain, and one Type B strain. The assay validation was carried out by two separate groups in different laboratories, to demonstrate laboratory-to-laboratory reproducibility. The precision (between-test variability), linearity, accuracy and range of a 20 semi-automated assay are compared with those observed for a manual assay in Table 53. [0194] The between-test standard deviation (SD) of the semi-automated assay, was evaluated from six tests performed on each of three vaccine strains, by the same analyst group, on the same pipetting station (each test result is obtained by averaging 12 determinations obtained over three days). The acceptance criterion for the between test 25 variability of the semi-automated assay was 0.25 loglO TCIDSO/ml, which is the half-width of the 95% confidence interval for a single test result based on the highest observed variability (0.11 log10 TCIDsounits) of the manual assay. The actual SD values obtained with the semi-automated assay, for the three strains, ranged between 0.06 - 0.09 loglOTCIDso/mL. These values are within the acceptance criterion of SD < 0.25 loglO 30 TCID 5 0 units'and are comparable to the between-test variability (0.Q7.to 0.11 1ogy10 TCIDso units) observed for the manual TCID5o assay, from nine repeat tests performed on three independent lots of each of three strains. -50- WO 2005/014862 PCT/US2004/005697 [0195] The assay was demonstrated to be linear over a 105-fold dilution range(titer range of 4.2 - 9.3 loglOTCIDso/mL), by showing that the relationship of the calculated and measured TCIDo titer passed a test for lack of fit to a linear model at the 1% significance level. The assay was accurate, with slopes of 1.00 - 1.02 for the.three strains, which were 5 all within the acceptance criterion of slope of 1±0.1. The linearity, accuracy and range of the semi-automated assay are comparable with the manual assay. See Table 53. [0196] Intermediate precision of the semi-automated assay was demonstrated by fitting a random effects model to a set of 18 tests obtained by two analyst groups over nine different assay days, on one type A and one type B vaccine virus strain. The measured 10 standard deviation ranges for the between day variability (SD(day)), between analyst group variability (SD(analyst)), and between instrument variability (SD(instrument)) were respectively 0.04 -0.08, 0.14 - 0.16, and 0.000 - 0.03, which met the acceptance criteria of SD(day) <0.3, SD(analyst) <0.4, and SD(instrument) < 0.4. [0197] The inter-laboratory reproducibility of the assay was demonstrated-by 15 carrying out assays on one Type A/H1N1, one TypeA/H3N2 and one Type B strain in two different laboratories. The acceptance criterion for laboratory-to-laboratory reproducibility required the two sided 90% confidence interval for the difference in the mean results from the two laboratories to be within ± 0.3 loglOTCID5o/mL. This acceptance criterion was met, with the lower and upper bounds of the 90% confidence intervals of greater than - 0.05 20 and less than + 0.15, respectively, for all three strains. [0198] A detailed statistical comparison was performed to demonstrate the comparability of the Manual and Semi-Automated assays. Two vaccine strains, one Type A/H1N1 (A/New Caledonia/20/99) and one Type B (B/Yamanashi/166/98), were assayed manually to obtain 18 test results on each strain. The data for all 18 test results obtained 25 manually for each strain, were pooled and compared with the pooled test results from the precision and intermediate precision studies carried out for semi-automated test (18 test results per strain). The Proc Mixed method in SAS vwas used to estimate the between method mean difference and its 90% confidence interval (CI). The acceptance criterion was that the 90% CI must be within ± 0.3 log1OTCID 5 0/miL, i.e. the lower bound (LB) of the 30 90% CI must be greater than -0.3, and the upper bound (U13) must be less than 0.3. The results are presented in the assay validation report and summarized in Table 54, below. As -51- WO 2005/014862 PCT/US2004/005697 may be seen from the results in, the two-sided 90% confidence intervals were within the acceptance criteria of ± 0.3 loglOTCIDso/mL for both strains, with actual estimates of the lower and upper bounds ranging between -0.05 and 0.10 log1OTCIDso/mL. [0199] - Thus, in summary, while Manual TCID 5 0 Potency Assay for Influenza Virus 5 Monovalent, is the traditional validated assay for the infectivity/potency measurement of monovalent influenza vaccine strains in expanded wild-type influenza (eWT) Master Virus Seed (MVS), Manufacturer's Working Virus Seed (MWVS), and Virus Harvest (VH) samples, it is a labor-intensive method involving numerous manual pipetting steps, which pose a repetitive motion injury hazard to analysts. In addition it uses a manual microscopic 10 CPE readout, which limits the assay throughput to 3 tests per test day per analyst. Automation of the plate-washing and manual pipetting steps, and substitution of MTT dye based detection of CPE for the manual microscopic readout can result in development of a Semi-Automated TCID 50 Potency Assay for Influenza Virus Monovalent. The implementation of the semi-automated assay, for testing of monovalent materials optionally 15 increases the assay throughput 2-3 fold, and allows practical commercialization of vaccines such as FluMistTM Vaccine at the anticipated level of doses for market. An additional benefit is a lowered risk of repetitive motion injuries for Quality Control analysts. [0200] The semi-automated assay has been validated to demonstrate repeatability, intermediate precision, linearity, and accuracy for assay of viral materials in the titer range 20 of 4.2- 9.3 loglOTCID 5 olmL in one group. The assay was also validated to demonstrate inter-laboratory reproducibility with another group. [0201] A detailed statistical comparison of results obtained by using both the semi automated assay and the manual assay, for repeated potency measurements of one Type A and one Type B influenza strains, also showed that the two assays yield comparable results. 25 Thus, the semi-automated assay is demonstrated to be comparable to the manual assay for use in the potency measurement of expanded wild-type influenza, FluMistTM master virus seed (MVS), manufacturer's Working virus seed (MWVS), and virus harvest (VH) samples. Universal Cutoff Value of CPE in Semi-automated TCIDig Assays [0202] In yet other embodiments herein other variations and modifications of 30 TCID 5 0 assays are employed to determine potency of vaccine/viruses.-One such modification is the confirmation of the universal cutoff value for the assessment of CPE for -52- WO 2005/014862 PCT/US2004/005697 the TCID 50 SemiAutomated Potency Assay for influenza virus monovalent. The "SemiAutomated TCID 50 Potency Assay for Influenza Virus Monovalent" (see above) uses the viable cell dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphnyltetrazolium bromide) to score the cytopathic effect (CPE) in infected monolayers of MDCK cells. To reliably 5 determine virus potency values using the MTT colorimetric endpoint to detect the number of CPE-positive wells, it is useful to establish an absorbance cutoff value that reproducibly distinguishes between CPE-positive and CPE-negative wells. As described in other work by the inventors, a "universal cutoff' absorbance (A570) value of 0.5254 has been determined. In the "SemiAutomated TCID 5 O Assay for Influenza Virus Monovalent," a 10 well is considered CPE-positive with an absorbance value of A570 <0.5254; CPE-negative wells have absorbance values of A570 > 0.5254. [0203] The data summarized in this section validates the universal cutoff value determined previously. The extensive testing of cold-adapted influenza strains, A/New Caledonia/20/99 (type A/HlN1), A/Sydney/05/97 (type A/H3N2), and 15 B/Yamanashi/166/98 (type B) not only generate reinforcing data for the universal cutoff assignment, but allow comparisons among analysts and instruments. The data presented herein substantiate the robustness, reproducibility and reliability of the Semi-Automated

TCTD

5 o assay and demonstrate comparability to the validated manual potency assay. Thus, illustrating the strength of embodiments comprising these measurements. 20 [0204] As explained above, the median tissue culture infectious dose (TCIDso) assay is a cell-based assay that measures the potency of infectious cytocidal virions. Serial dilutions of a virus sample are added to confluent monolayers of Madin-Darby canine kidney (MDCK) cells grown in 96-well plates. Replication of the virus in the MDCK cells affects cell metabolism, eventually resulting in the release of progeny virus into the culture 25 supernatant and cell death. The progeny viruses in turn infect other cells, resulting in the eventual destruction of the monolayer. The cytopathic effect (CPE) resulting from the infection is allowed to develop. during an incubation period of six days. After this period of time, MTT is used to detect the presence or absence of CPE in the cell monolayer. Vital dyes like MTT have been used extensively as indicators of cell health and viability in cell 30 based bioassays (see, e.g., Denizot et al., J. Immun. Methods (1986) 89:271--277; Gerlier-et al., (1986) J. Immuno. Methods 94:57-63, Heeg, et al., J. Immuno Methods (1985) 77:237 246, Mooseman J. Immuno. Methods (1983) 65:55-63, Tada, et al., J. Immuno. Methods -53- WO 2005/014862 PCT/US2004/005697 (1986) 93:147-165, and Vistica, Cancer Research (1991) 51: 2515-2520 ). Wells containing an intact monolayer of viable cells (CPE-negative) process the dye to a purple formazan dye product and yield a high absorbance value at 570 nm (Aso). In contrast, CPE-positive wells are marked by lower absorbance values due to the partial or complete monolayer destruction 5 caused by the virus. To reliably determine virus potency values using a colorimetric endpoint to detect the number of CPE-positive wells, it is useful to establish an absorbance cutoff value that reproducibly distinguished between CPE-positive and CPE-negative wells. Used in conjunction with the universal cutoff value, absorbance values from virus test samples are scored CPE-positive or CPE-negative. The number of CPE-positive wells is 10 used to calculate the virus titer (log10 TCID5o/mL). [0205] Work by the inventors and coworkers provides an initial recommendation of the universal cutoff value based on two studies performed over several days by multiple analysts with three influenza virus strains. As described, a well was considered CPE positive with an absorbance value of A 570 < 0.5254; CPE-negative wells had absorbance 15 values of A 5 70 > 0.5254. The current section describes additional experiments designed to validate the absorbance cutoff value previously determined. Multiple analysts from two independent assay groups determined the potency of three reference virus strains using the SemiAutomated TCID 50 assay. As described, CPE assessed by the validated manual method of microscopic examination was considered the "gold standard" and compared to 20 the CPE determined by MTT. The extensive testing of A/New Caledonia/20/99 (type A/HlNl), A/Sydney/05/97 (type A/H3N2), and B/Yamanashi/166/98 (type B) not only generated reinforcing data for the universal cutoff assignment, but allowed comparisons among analysts and instruments. The data presented herein substantiate the robustness, reproducibility and reliability of the SemiAutomated TCID 5 o assay demonstrating 25 comparability to the validated manual potency assay. [0206] The development of the SemiAutomated potency assay required the use of reference virus strains with known potency values previously determined using a validated manual potency assay. The reference cold-adapted virus strains were as follows: A/New Caledonia/20/99, a type A/H1N1 virus; A/Sydney/05/97, a type A/H3N2 virus; and 30 B/Yamanashi/166/98, a type B virus. The cold-adapted control virus tstrairr AlSydney/05/97, was used to confirm system suitability. -54- WO 2005/014862 PCT/US2004/005697 [0207] The method for the SemiAutomated TCID 5 o Potency Assay for Influenza Monovalent has been developed by the inventors and coworkers as well as the overall assay configuration for half-plate replicates, as well as the visual CPE scoring method. See above. Briefly, confluent monolayers of MDCK cells in 96-well plates are washed twice with virus 5 growth medium (VGM) using a Skatron' Cell Washer. Serial ten-fold dilutions of virus samples are prepared in VGM containing TPCK-trypsin using a MatrixTM SerialMate Pipetting Station and 96-well dilution blocks. The last five serial dilutions (10-5 to 10-9) are transferred to MDCK cell plates to achieve final virus concentrations ranging from 10-6 to 10-10 relative to the initial starting titer. This format derives two potency data points 10 from each plate. Since each sample is assayed on two plates, four replicate potency values are obtained. The 16 control wells (plate columns 6 and 7) receive virus-free VGM and serve as cell controls. After a 6-day incubation (33 ± 1VC with 5 ± 1 % CO 2 ) all wells are examined using a microscope and were scored for the presence or absence of CPE. Thus, a well is scored CPE-positive if the monolayer contained any evidence of virus destruction. 15 Conversely, the monolayer in a CPE-negative well was completely intact. [0208] After visually scoring the monolayers on the plates for CPE, the media is discarded and MTT (0.5 mg/mL), (US Biochemical Corporation, Cleveland, OH), prepared in phosphate buffered saline is dispensed to each well (100 plwell). The monolayers are incubated with MIT for 6 ± 0.5 hours at 37 ± 1C with 5 ± 1 % CO 2 . Solubilization buffer 20 (100 piL of 20% SDS in 0.01N HCl) is added to each well and the plates are incubated for 16 to 20 hours at 37 + 1*C in an environment of 5 ± I % C0 2 . The absorbance values at 570 nm are determined using a PerkinElmer-Wallac 1420 Multilabel Counter Spectrophotometer and were exported into a MicrosoftTM Excel macro; a program used to calculate virus titer (logio TCIDso/mL) from the number of CPE-positive wells. 25 [0209] Acceptance criteria are applied to the embodiment within this section. Accordingly, a plate was considered valid if not more than one of the sixteen cell control wells on each plate showed visual evidence of CPE, cell toxicity, or microbial contamination. In addition, for each half-plate to be valid, no less than 5 and no more than 36 wells had to be scored CPE-positive. Finally, both the mean and standard deviation (SD) 30 of the four replicate TCIDSo titer values obtained for the monovalent virus control sample (A/Sydney/05/97) had to be within the qualified range reported in the qualified control certificate. -55- WO 2005/014862 PCT/US2004/005697 [0210] Estimates of sensitivity and specificity were calculated based on the relationship between the "gold standard" CPE and MTT-assessed CPE shown below. TP denotes "true positive", FP is "false positive," FN is "false negative," and TN is "true negative." Therefore, "all positives" would be the sum of TP+FN, and "All negatives" 5 would be the sum of FP+TN. See Table 55. The calculations are: Sensitivity for each replicate = (TP) / (All CPE positive) and Specificity for each replicate = (TN) / (All CPE negative) [0211] In order to perform an instrument to instrument comparison, potency values were determined for three, reference virus samples by six analysts in a first group using the 10 SemiAutomated TCID 5 o assay. Two sets of lab instruments AZ-039 and AZ-040 were used over three days. A second groups of testers used one instrument system, AZ-036. Three analysts from that group performed the SemiAutomated TCID 5 0 assay on three days using the three reference virus samples. [0212] In order to perform an analyst to analyst comparison, each analyst in the 15 testing (Analyst # 1-6) in Group 1 performed a SemiAutomated TCID 50 potency assay on the three reference strains using instrument AZ-039 over three days. In the second group, each of the three analysts (Analyst # 7-9) performed the SemiAutomated TCID 5 o potency assay with the same three reference virus strains over three days using instrument AZ-036. [0213] To reliably distinguish between CPE-positive and CPE-negative wells in the 20 SemiAutomated TCIDso potency assay using MTT, a universal cutoff value was statistically determined. In an effort to validate the use of this cutoff value, further independent evaluation by the two groups generated an additional 45,840 absorbance values. The results are presented below. [0214] Sensitivity and specificity measurements were calculated using the manual 25 microscopic method as the reference standard. The combined data from the two groups (n=45,840) are shown in Table 56. Using the recommended cutoff value of 0.5254 resulted in a sensitivity of 98.45% and a specificity of 99.12%. In addition, the data from the second group for sensitivity and specificity determinations were 99.15% and 99.99%, respectively. Likewise, the data from the first groups, for sensitivity and specificity were 9.8.13% and 30 98.71%, respectively. All data above (>95% sensitivity and >95% specificity) correlates; -56- WO 2005/014862 PCT/US2004/005697 with the data determined wherein a sensitivity of 99.05% and a specificity of 99.99% were determined. [0215] Figure 51 shows a histogram derived from plotting the absorbance readings versus the frequency of the values (N=45,840). In agreement with the previous information, 5 the combined data from the two groups indicate that the universal cutoff value of 0.5254 lies near the midpoint between the distribution of CPE-positive and CPE-negative wells. The frequency distribution shows that the recommended cutoff value resides in the left-most tail of the distribution of the control wells, corresponding to a probability of 0.007% in the tail extending towards the left. The cutoff value, 0.5254, also corresponds to a tail 10 probability of 0.02%, when cutoff values were estimated using absorbance values from all CPE-positive wells. Furthermore, the distribution profiles evident in Figure 51 highlight that absorbance values for CPE-positive wells are widely separated from absorbance values for CPE-negativc wells [0216] A Comparison of the Mean Absorbance Values Obtained for CPE-Negative 15 Control Wells Generated estimated a cutoff value of 0.5254 based on absorbance values from 6720 control wells was previously done. A mean absorbance value of 1.261 from the control wells was obtained with a standard deviation of 0.15. As shown in Table 57, the present study generated an additional 9168 control wells; 2880 were obtained from the second group and 6288 from the first group. Mean absorbance values of 1.226 and 1.235 20 were obtained from the second and first groups, respectively, with an overall mean absorbance value of 1.231. The difference between the combined mean absorbance value and that previously reported was only 0.03 absorbance values (see Table 57). This is a very small difference given that the data were generated over a 6-month period. The studies described previously were conducted over two consecutive months, while the studies 25 described herein were conducted in second groups four months prior to that done by the first group. [0217] Table 58 summarizes the potency values obtained for the' three reference virus strains using the different instruments in the two groups in order to perform an' instrument to instrument comparison. Six analysts from the first group performed:the 30 SemiAutomated TCID 5 Assay using two sets of instruments (designated AZ-039 and AZ 040). For A/New Caledonia/20/99 the overall mean ranged from 9.2 to 9.3 log10TCID 5 /mL and the titer did not vary more than 0.09 log1OTCID 5 0/mL between AZ -57- WO 2005/014862 PCT/US2004/005697 039 and AZ-040 (see Table 58). For A/Sydney/05/97 the overall mean ranged from 8.5 to 8.6 log1OTCID 5 o/mL and did not vary more than 0.02 loglOTCIDso/mL between AZ-039 and AZ-040. For BiYamanashi/166/98 the overall mean ranged from 8.3 to 8.4 log1OTCID 50 /mL and did not vary more than 0.12 loglOTCIDso/mL between AZ-039 and 5 AZ-040. The second group produced results using one instrument system (AZ-036). Three analysts performed the SemiAutomated TCID 5 o assay on three days with the three reference virus samples. The results of the mean difference between instruments in the second group and Quality Control Laboratory did not vary more than 0.09 log OTCID50/mL for A/New Caledonia/20/99, 0.08 loglOTCID50/mL for A/Sydney/05/97 and 0.12 loglOTCIDD/mL for 10 B/Yamanashi/166/98. The mean difference data was calculated between the two instruments in the first group (AZ-039 and AZ-040) and between the first and second groups (AZ-036). [0218] In order to do an analyst to analyst comparison, in the first group each analyst (Analyst 1 through 6) performed a SemiAutomated TCID 5 0 potency assay for 15 A/New Caledonia/20/99, A/New Sydney/05/97, and B/Yamanashi/166/98 on Instrument AZ-039 over three days. In the second group, each of three analysts (Analyst 7 through 9) performed the SemiAutomated TCID5 0 potency assay with the same virus strains over three days on Instrument AZ-036. The potency values were calculated for each virus and are shown in Table 59. The variability among the first group's results was less than or equal to 20 0.3 log1OTCID50/mL for the three virus samples tested. Similarly, the variability among the second group's potency values was less than or equal to 0.2 log1OTCIDso/mL An overall comparison between the two groups of analysts was less than t 0.3 logOTCID 5 0/mL for the three reference virus samples. The standard deviations (SD) for the test results (four replicates tested over three days) ranged between 0.11 and 0.27. Because the SD values 25 were less than the acceptance criterion value of 0.50, all were valid. [0219] The results provided in this section validate a "universal cutoff' absorbance value (0.5254). There is a high level of confidence in the universal cutoff value because the studies produce strongly concordant data despite being generated by multiple analysts in independent assay groups over a relatively long timeframe. To summarize. control (CPE 30 negative) absorbance values generated by the two groups not only agreed with each other, but were virtually the same as the mean value reported previously (see, Table 57). The sensitivity and specificity values were very similar between the two groups; and agreed with -58- WO 2005/014862 PCT/US2004/005697 previous work (see Figure 51 and Table 57). In the two groups, the "universal cutoff" for the SemiAutomated TCID 5 o potency assay, using MTT to assess CPE, produced potency values that were comparable to each other and to those obtained by the validated manual TCID5 0 potency assay. Finally, the SemiAutomated system has several procedural 5 advantages over the manual method. The use of instrumentation to replace the labor intensive steps of manually pipetting and microscopically examining assay plates increases capacity and allows for a higher throughput. In addition, the spectrophotometric CPE readout and subsequent automated potency calculations provide a printout and/or an electronic record of the results. 10 DEFINITIONS [02201 Unless defined otherwise, all scientific and technical terms are understood to have the same meaning as commonly used in the art to which they pertain. For the purpose of the present invention the following terms are defined below. [0221] The terms "nucleic acid," "polynucleotide," "polynucleotide sequence" and 15 "nucleic acid sequence" refer to single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymers, or chimeras or analogues thereof. As used herein, the term optionally includes polymers of analogs of naturally occurring nucleotides having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). Unless 20 otherwise indicated, a particular nucleic acid sequence optionally encompasses complementary sequences, in addition to the sequence explicitly indicated. [0222] The term "gene" is used broadly to refer to any nucleic acid associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. The term "gene" applies to a specific genomic sequence, as 25 well as to a cDNA or an mRNA encoded by that genomic sequence. [0223] Genes also include non-expressed nucleic acid segments that, for example, form recognition sequences for other proteins. Non-expressed regulatory sequences include "promoters" and "enhancers," to which regulatory proteins such as transcription factors bind, resulting in transcription of adjacent or nearby sequences. A "tissue-specific" 30 promoter or enhancer is one which regulates transcription in a specific tissue type or cell type, or types. -59- WO 2005/014862 PCT/US2004/005697 [0224] The term "vector" refers to the means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophage, pro-viruses, phagemids, transposons, and artificial chromosomes, and the like, that replicate autonomously or can integrate into a chromosome 5 of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a lipososfie conjugated DNA, or the like, that are not autonomously replicating. Most commonly, the vectors of herein refer to plasmids. 10 [0225] An "expression vector" is a vector, such as a plasmid that is capable of promoting expression, as well as replication of, a nucleic acid incorporated therein. Typically, the nucleic acid to be expressed is "operably linked" to a promoter and/or enhancer, and is subject to transcription regulatory control by the promoter and/or enhancer. [0226] A "bi-directional expression vector" is characterized by two alternative 15 promoters oriented in the opposite direction relative to a nucleic acid situated between the two promoters, such that expression can be initiated in both orientations resulting in, e.g., transcription of both plus (+) or sense strand, and negative (-) or antisense strand RNAs. [0227] In the context of the invention, the term "isolated" refers to a biological material, such as a nucleic acid or a protein, which is substantially free from components 20 that normally accompany or interact with it in its naturally occurring environment. The isolated material optionally comprises material not found with the material in its natural environment, e.g., a cell. For example, if the material is in its natural environment, such as a cell, the material has been placed at a location in the cell (e.g., genome or genetic element) not native to a material found in that environment. For example, a naturally occurring 25 nucleic acid (e.g., a coding sequence, a promoter, an enhancer, etc.) becomes isolated if it is introduced by non-naturally occurring means to a locus of the genome (e.g., a vector, such as a plasmid or virus vector, or amplicon) not native to that nucleic acid. Such nucleic acids are also referred to as "heterologous" nucleic acids. [0228] The term "recombinant" indicates that the material (e.g., a nucleic acid or 30 protein) has been artificially or synthetically (non-naturally) altered. .3The alteration can be., performed on the material within, or removed from, its natural environment or state. -60- WO 2005/014862 PCT/US2004/005697 Specifically, when referring to a virus, e.g., an influenza virus, is recombinant when it is produced by the expression of a recombinant nucleic acid. [0229] The term "reassortant," when referring to a virus, indicates that the virus includes genetic and/or polypeptide components derived from more than one parental viral 5 strain or source. For example, a 7:1 reassortant includes 7 viral genomic segments (or gene segments) derived from a first parental virus, and a single complementary viral genomic segment, e.g., encoding hemagglutinin or neuraminidase, from a second parental virus. A 6:2 reassortant includes 6 genomic segments, most commonly the 6 internal genes from a first parental virus, and two complementary segments, e.g., hemagglutinin and 10 neuraminidase, from a different parental virus. [0230] The term "introduced" when referring to a heterologous or isolated nucleic acid refers to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid can be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently 15 expressed (e.g., transfected mRNA). The term includes such methods as "infection," "transfection," "transformation," and "transduction." In the context of the invention, a variety of methods can be employed to introduce nucleic acids into prokaryotic cells, including electroporation, calcium phosphate precipitation, lipid mediated transfection (lipofection), etc. 20 [02311 The term "host cell" means a cell that contains a heterologous-nucleic acid, such as a vector, and supports the replication and/or expression of the nucleic acid. Host cells can be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, avian or mammalian cells, including human cells. Exemplary host cells in the context of the invention include Vero (African green monkey kidney) cells, BHK (baby 25 hamster kidney) cells, primary chick kidney (PCK) cells, Madin-Darby Canine Kidney (MDCK) cells, Madin-Darby Bovine Kidney (MDBK) cells, 293 cells (e.g., 293T cells), and COS cells (e.g., COS1, COS7 cells). Influenza Virus [0232] The compositions and methods herein are primarily concerned with 30 production of influenza viruses for vaccines. Influenza viruses are niide dp 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 -61- WO 2005/014862 PCT/US2004/005697 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 PB 1 and PA are encoded by segment 2 and segment 3, 5 respectively. In addition, segment 1 of some influenza strains encodes a small protein, PB 1 F2, produced from an alternative reading frame within the PB 1 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 10 neuraminidase (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. [0233] . The eight genome segments of influenza B encode 11 proteins. The three 15 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 BM2. The smallest segment encodes two products, NS 1 which is translated from the full 20 length RNA, and NS2 which is translated from a spliced mRNA variant. Influenza virus vaccine [0234] Historically, influenza virus vaccines have primarily been produced in embryonated hen eggs using strains of virus selected based on empirical predictions of relevant strains. More recently, reassortant viruses have been produced that incorporate 25 selected hemagglutinin and neuraminidase antigens in the context of an approved attenuated, temperature sensitive master strain. Following culture of the virus through multiple passages in hen eggs, influenza viruses are recovered and, optionally, inactivated, e.g., using formaldehyde and/or P-propiclactone (or alternatively used in live attenuated vaccines). l 30 [0235] However, production of influenza vaccine in this manner has.several significant concerns. For example, contaminants remaining from the hen eggs can be highly antigenic and/or pyrogenic, and can frequently result in significant side effects upon -62- WO 2005/014862 PCT/US2004/005697 administration. Thus, as described herein, one aspect of the current invention involves replacement of some percentage of egg components with animal free media. More importantly, virus strains designated for vaccine production must be selected and distributed, typically months in advance of the next flu season to allow time for production 5 and inactivation of influenza vaccine. Again, any improvements in production time, e.g., as through use of the methods and compositions of the current invention, are thus quite desirable. [0236] Attempts at producing recombinant and reassortant vaccines in cell culture have been ha mpered by the inability of some of the strains approved for vaccine production 10 to grow efficiently under standard cell culture conditions. Thus, prior work by the inventors and their coworkers provided a vector system, and methods for producing recombinant and reassortant viruses in culture, thus, making it possible to rapidly produce vaccines corresponding to one or many selected antigenic strains of virus. See, Multi-Plasmid System for the production of Influenza virus, cited above. Of course, such reassortments 15 are optionally further amplified in hen eggs. Typically, the cultures are maintained in a system, such as a cell culture incubator, under controlled humidity and CO 2 , at constant temperature using a temperature regulator, such as a thermostat to insure that the temperature does not exceed 35 'C. Such pioneering work, as well as other vaccine production, can be further optimized and streamlined through use of the current invention in 20 whole or part. [0237] Reassortant influenza viruses can be readily obtained by introducing a subset of vectors corresponding to genomic segments of a master influenza virus, in combination with complementary segments derived from strains of interest (e.g., antigenic variants of interest). Typically, the master strains are selected on the basis of desirable properties 25 relevant to vaccine administration. For example, for vaccine production, e.g., for production of a live attenuated vaccine, the master donor virus strain may be selected for an attenuated phenotype, cold adaptation and/or temperature sensitivity. FluMistTM [0238] As mentioned previously, numerous examples and types of influenza vaccine 30 exist. An exemplary influenza vaccine is FluMistTM which is alive, -attenuated.vaccine that protects children and adults from influenza illness (Belshe et al. (1998) The efficacy of live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine in children N. Eni. -63- WO 2005/014862 PCT/US2004/005697 J. Med. 338:1405-12; Nichol et al. (1999) Effectiveness of live, attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized controlled trial JAMA 282:137-44). In typical embodiments, the methods and compositions of the current invention are preferably adapted to, or used with, production of FluMistTM vaccine. 5 However, it will be appreciated by those skilled in the art that the steps/cQmpositions herein are also adaptable to production of similar or even different viral vaccines. [0239] FluMistTM vaccine strains contain, e.g., HA and NA gene segments derived from the wild-type strains to which the vaccine is addressed along with six gene segments, PB1, PB2, PA, NP, M and NS, from a common master donor virus (MDV). The MDV for 10 influenza A strains of FluMist (MDV-A), was created by serial passage of the wild-type A/Ann Arbor/6/60 (A/AA/6/60) strain in primary chicken kidney tissue culture at successively lower temperatures (Maassab (1967) Adaptation and growth characteristics of influenza virus at 25 degrees C Nature 213:612-4). MDV-A replicates efficiently at 25'C (ca, cold adapted), but its growth is restricted at 38 and 39 0 C (ts, temperature sensitive). 15 Additionally, this virus does not replicate in the lungs of infected ferrets (att, attenuation). The ts phenotype is believed to contribute to the attenuation of the vaccine in humans by restricting its replication in all but the coolest regions of the respiratory tract. The stability of this property has been demonstrated in animal models and clinical studies. In contrast to the ts phenotype of influenza strains created by chemical mutagenesis, the ts property of 20 MDV-A does not revert following passage through infected hamsters or in shed isolates from children (for a recent review, see Murphy & Coelingh (2002) Principles underlying the development and use of live attenuated cold-adapted influenza A and B virus vaccines Viral Immunol. 15:295-323). [0240] Clinical studies in over 20,000 adults and children involving 12 separate 6:2 25 reassortant strains have shown that these vaccines are attenuated, safe and efficacious (Belshe et al. (1998) The efficacy of live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine in children N. Engl. J. Med. 338:1405-12; Boyce et al. (2000) Safety and immunogenicity of adjuvanted and unadjuvanted subunit influenza vaccines administered intranasally to healthy adults Vaccine 19:217-26; Edwards et alk(1994)A 30 randomized controlled trial of cold adapted and inactivated vaccines for the prevention of influenza A disease J. Infect. Dis. 169:68-76 ; Nichol et al. (1999) Effectiveness of live, attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized -64- WO 2005/014862 PCT/US2004/005697 controlled trial JAMA 282:137-44). Reassortants carrying the six internal genes of MDV A and the two HA and NA gene segments of a wild-type virus (i.e., a 6:2 reassortant) consistently maintain ca, ts and att phenotypes (Maassab et al. (1982) Evaluation of a cold recombinant influenza virus vaccine inferrets J. Infect. Dis. 146:780-900). Production of 5 such reassorted virus using B strains of influenza are is more difficult; however. [0241] Recent work, see, Multi-Plasmid System for the Production of Influenza Virus, cited above, has shown an eight plasid system for the generation of influenza B virus entirely from cloned cDNA, and methods for the production of attenuated live influenza A and B virus suitable for vaccine formulations, such as live virus vaccine 10 formulations useful for intranasal administration. [0242] The system and methods described previously are useful for the rapid production in cell culture of recombinant and reassortant influenza A and B viruses, including viruses suitable for use as vaccines, including live attenuated vaccines, such as vaccines suitable for intranasal administration such as FluMist@. The methods of the 15 current invention herein, are optionally used in conjunction with or in combination with such previous work involving, e.g., reassorted influenza viruses for vaccine production to produce viruses for vaccines in a more stable, consistent and productive manner. Cell Culture [0243] As previously stated, influenza virus optionally can be grown in cell culture. 20 Typically, propagation of the virus is accomplished in the media compositions in which the host cell is commonly cultured. Suitable host cells for the replication of influenza virus include, e.g., Vero cells, BHK cells, MDCK cells, 293 cells and COS cells, including 293T cells, COS7 cells. Commonly, co-cultures including two of the above cell lines, e.g., MDCK cells and either 293T or COS cells are employed at a ratio, e.g., of 1:1, to improve 25 replication efficiency. Typically, cells are cultured in a standard commercial culture medium, such as Dulbecco's modified Eagle's medium supplemented with serum (e.g., 10% fetal bovine serum), or in serum free medium, under controlled humidity and CO 2 concentration suitable for maintaining neutral buffered pH (e.g., at pH between 7.0 and 7.2). Optionally, the medium contains antibiotics to prevent bacterial growth, e.g., penicillin, 30 streptomycin, etc., and/or additional nutrients, such as L-glutamine, sodium pyiiiirite,,noi essential amino acids, additional supplements to promote favorable growth characteristics., e.g., trypsin, $-mercaptoethanol, and the like. -65- WO 2005/014862 PCT/US2004/005697 [0244] Procedures for maintaining mammalian cells in culture have been extensively reported, and are well known to those of skill in the art. General protocols are provided, e.g., in Freshney (1983) Culture of Animal Cells: Manual of Basic Technique, Alan R. Liss, New York; Paul (1975) Cell and Tissue Culture, 5 th ed., Livingston, 5 Edinburgh; Adams (1980) Laboratory Techniques in Biochemistry and Molecular Biology Cell Culture for Biochemists, Work and Burdon (eds.) Elsevier, Amsterdam. Additional details regarding tissue culture procedures of particular interest in the production of influenza virus in vitro include, e.g., Merten et al. (1996) Production of influenza virus in cell culturesfor vaccine preparation in Cohen and Shafferman (eds.) Novel Strategies in 10 Design and Production of Vaccines, which is incorporated herein in its entirety for all purposes. Additionally, variations in such procedures adapted to the present invention are readily determined through routine experimentation and will be familiar to those skilled in the art. [0245] Cells for production of influenza virus can be cultured in serum-containing 15 or serum free medium. In some case, e.g., for the preparation of purified viruses, it is typically desirable to grow the host cells in serum free conditions. Cells can be cultured in small scale, e.g., less than 25 ml medium, culture tubes or flasks or in large flasks with agitation, in rotator bottles, or on microcarrier beads (e.g., DEAE-Dextran microcarrier beads, such as Dormacell, Pfeifer & Langen; Superbead, Flow Laboratories; styrene 20 copolymer-tri-methylamine beads, such as Hillex, SoloHill, Ann Arbor) in flasks, bottles or reactor cultures. 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. For 25 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, MN); NBS bioreactors (New Brunswick Scientific, Edison, N.J.); laboratory and commercial scale bioreactors from B. Braun Biotech International (B. Braun Biotech, 30 Melsungen, Germany), [0246] Regardless of the culture volume, in many desired aspects of the current invention, it is important that the cultures be maintained at an appropriate temperature, to -66- WO 2005/014862 PCT/US2004/005697 insure efficient recovery of recombinant and/or reassortant influenza virus using temperature dependent multi plasmid systems (see, e.g., Multi-Plasmid System for the Production of Influenza Virus, cited above), heating of virus solutions for filtration, etc. Typically, a regulator, e.g., a thermostat, or other device for sensing and maintaining the 5 temperature of the cell culture system and/or other solution, is employed to insure that the temperature is at the correct level during the appropriate period (e.g., virus replication, etc.). [0247] In some embodiments herein (e.g., wherein reassorted viruses are to be produced from segments on vectors) vectors comprising influenza genome segments are introduced (e.g., transfected) into host cells according to methods well known in the art for 10 introducing heterologous nucleic acids into eukaryotic cells, including, e.g., calcium phosphate co-precipitation, electroporation, microinjection, lipofection, and transfection employing polyamine transfection reagents. For example, vectors, e.g., plasmids, can be transfected into host cells, such as COS cells, 293T cells or combinations of COS or 293T cells and MDCK cells, using the polyamine transfection reagent Trans1T-LT1 (Mirus) 15 according to the manufacturer's instructions in order to produce reassorted viruses, etc. Approximately 1 pg of each vector to be introduced into the population of host cells with approximately 2 pl of TransIT-LT1 diluted in 160 l medium, preferably serun-free medium, in a total volume of 200 p1. The DNA:transfection reagent mixtures are incubated at room temperature for 45 minutes followed by addition of 800 pl of medium. The 20 transfection mixture is added to the host cells, and the cells are cultured as described above or via other methods well known to those skilled in the art. Accordingly, for the production of recombinant or reassortant viruses in cell culture, vectors incorporating each of the 8 genome segments, (PB 2, PB 1, PA, NP, M, NS, HA and NA) are mixed with approximately 20 pl Trans1T-LT1 and transfected into host cells. Optionally, serum-containing medium is 25 replaced prior to transfection with serum-free medium, e.g., Opti-MEM I, and incubated for 4-6 hours. [0248] Alternatively, electroporation can be employed to introduce such vectors incorporating influenza genome segments into host cells. For example, plasmid vectors incorporating an influenza A or influenza B virus are favorably introduced'into Vero cells 30 using electroporation according to the following procedure. In brief&approximately 5 x 106 Vero cells, e.g., grown in Modified Eagle's Medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS) are resuspended in 0.4 ml OptiMEM and placed in an electroporation -67- WO 2005/014862 PCT/US2004/005697 cuvette. Twenty micrograms of DNA in a volume of up to 25 Rl is added to the cells in the cuvette, which is then mixed gently by tapping. Electroporation is performed according to the manufacturer's instructions (e.g., BioRad Gene Pulser II with Capacitance Extender Plus connected) at 300 volts, 950 microFarads with a time constant of between 28-33 msec. 5 The cells are remixed by gently tapping and, approximately 1-2 minutes following electroporation, 0.7 ml MEM with 10% FBS is added directly to the cuvette. The cells are then transferred to two wells of a standard 6 well tissue culture dish containing 2 mlMEM, 10% FBS. The cuvette is washed to recover any remaining cells and the wash suspension is divided between the two wells. Final volume is approximately 3.5 mL. The cells are then 10 incubated under conditions permissive for viral growth, e.g., at approximately 33*C for cold adapted strains. Kits [0249] To facilitate use of the methods and compositions of the invention, any of the vaccine components and/or compositions, e.g., reassorted virus in allantoic fluid, and 15 various formulations, etc., and additional components, such as, buffer, cells, culture medium, useful for packaging and infection of influenza viruses for experimental or therapeutic vaccine purposes, can be packaged in the form of a kit. Typically, the kit contains, in addition to the above components, additional materials which can include, e.g., instructions for performing the methods of the invention, packaging material, and a 20 container. [0250] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the 25 true scope of the invention. For example, all the techniques and apparatus described above may be used in various combinations. All publications, patents, patent applications, or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes. 30 -68- WO 2005/014862 PCT/US2004/005697 TABLES TABLE 1. Description of Step Detail of Step Step 1. Co-infection of master donor virus (MDV) and WT virus in CEK cells. Step 2. Selection of reassorted viruses. Depending on virus strain, can be done in eggs or in CEK cells. Select for MDV NA and/or HA. Step 3. Cloning of reassorted viruses. Step 4. Purification of reassorted viruses in eggs. Step 5. Expansion of reassorted viruses in eggs to generate a master viral strain (MVS). Step 6. Expansion of MVS to produce a master working viral strain (MWVS). Step 7. Conditioning, washing, primary Eggs containing the reassorted virus are optionally incubation of the eggs, and rocked during incubation. inoculation. Step 8. Candling, inoculation, sealing, secondary incubation, etc., of eggs. Step 9. Candling of the eggs and chilling. Step 10. Harvesting of virus solution from the Virus containing solutions are optionally warmed and eggs. sterile filtered to remove impurities/contaminants (bioburden). Step 11. Clarification of the virus solution. The solution is also optionally ultrafiltered to, e.g., remove uric acid and other animal derived impurities and to stabilize the solution. Step 12. Stabilization of the virus solution. Arginine is optionally added either in addition to or in place of gelatin or gelatin hydrolysate at PH 6.6 to 8.0 to stabilize the solution. Use of arginine exclusively avoids the introduction of additional animal products. Step 13. Potency assay of the virus solutions. Optional use of a "universal reagent" and field focus assays as opposed to, e.g., TCID50 to determine potency. Step 14. Sterility assay of the virus solutions. Step 15. NAF adjustment of the virus NAF is optionally reduced/replaced with buffer, e.g., solutions. to increase stability. 69 WO 2005/014862 PCT/US2004/005697 Table 2 Tube/Well of cell MOI of MDV MOI of wild-type Target incubation culture time in hours 1 5.0 1.0 24 2 5.0 0.2 24 3 1.0 1.0 24 4 1.0 0.2 24 5 1.0 0.04 24 6 n/a n/a 24 5 10 Table 3. Manufacture Process Detection Type/Assay Time Potential Alternatives Egg pre/post Egg Candling Manual/hours Automated candling inoculation of eggs or thermal imaging of eggs Virus harvest MPA Manual/14 days Bioluminence based Bioburden Manual/3 days detection or MPN Virus Harvest Mycoplasma growth Manual/28 days PCR Virus harvest Mycobacterium Manual/56 days PCR.or clinical growth diagnostic systems 15 70 WO 2005/014862 PCT/US2004/005697 Table 4. Virus Type Strain and Isolate Number A HIN1 ca A/Beijing/262/95 A HINI ca A/New Caledonia/20/99 A H3N2 ca A/Sydney/05/97 A H3N2 ca A/Panama/2007/99 B ca B/Victoria/504/2000 B ca B/Yamanashi/166/98 5 Table 5. A/Sydney/05/97 Virus potency [[ogio TCIDso/mL]. Process step Temperature 5 ± 3 0 C 20 ± 3 0 C 31 ±3 0 C Stabilized VAF (before treatment) 8.7 ±0.3 8.6 ±0.2 8.8 ±0.2 Stabilized VAF (after treatment) 9.0 ±0.2 8.8 ±0.2 3.8 ±0.2 Filtered VAF (pool) 7.6 ±0.2 7.7 ±0.1 8.7 ±0.1 Centrifuged Stabilized VAF (control) 8.4 ±0.3 8.6 ±0.2 8.7 ±0.2 Gain/Loss Filtered vs. Control - 0.8 - 0.7 0.0 10 NA = not assayed All filtrations in Table 5 were performed from the same clay harvest. Prior to filtration through Sartoclean CA and Sartopore2 filters VAF was exposed for 60 minutes to 5 ± 3*C, 20 3*C and 31 ± 3C. 15 71 WO 2005/014862 PCT/US2004/005697 Table 6. A/Sydney/05/97 neuraminidase activity [ U/nL]. Process step Temperature 5 ± 3 0 C 20± 30C 31±3*C Stabilized VAF (before treatment) 34.4 36.4 38.4 Stabilized VAF (after treatment) 43.7 38.7 39.1 Filtered VAF (pool) BD BD 22.1 Centrifuged Stabilized VAF (control) 28.2 27.5 27.0 Gain/Loss Filtered vs. Control -28.2 - 27.5 - - 4.9 BD = below detection (less than 5 gU/mL) All filtrations in Table 6 were performed from the same day harvest. Prior to filtration through Sartoclean CA and Sartopore2 filters VAF was exposed for 60 minutes to 5 ± 3oC, 5 20 3*C and 31 ±3C. Table 7. A/Sydney/05197 hemagglutinin activity [HA titer]. Process step Temperature 5 ± 30C 20 ± 30C 31± 30C Stabilized VAF (before treatment) 128 128 256 Stabilized VAF (after treatment) 128 256 128 Filtered VAF (pool) 4 16 64 Centrifuged Stabilized VAF (control) 128 64 256 10 All filtrations in Table 7 were performed from the same day harvest. Prior to filtration through Sartoclean CA and Sartopore2 filters VAF was exposed for 60-minutes to 5 ± 3oC, 20± 3 0 C and 31 ± 3 0 C. 15 72 WO 2005/014862 PCT/US2004/005697 Table 8. A/Sydney/05/97 Virus potency [logio TCID 5 o/mL]. Process step Temperature 5 ± 3 0 C 20 ± 3 0 C 31 ±3 0 C Stabilized VAF (before treatment) 8.7 ±0.1 8.5 ±0.2 8.8 ±0.1 Stabilized VAF (after treatment) 8.9 ±0.2 8.9 ±0.2 8.7 ±0.2 Filtered VAF (pool) 7.6 ±0.2 7.5 ±0.2 8.7 ±0:2 Centrifuged Stabilized VAF (control) 8.5 ±0.2 8.5 ±0.1 8.7 ±0.1 Gain/Loss Filtered vs. Control - 0.9 - 1.0 0.0 All filtrations in Table 8 were performed from the same day harvest. Prior to filtration through Sartoclean CA and Sartopore2 filters VAF was exposed for 60 minutes to 5 3oC, 20 3"C and 31 ± 3"C*. 5 10 Table 9. A/Sydney/05/97 neuraminidase activity [Ui/mL]. Process step Temperature 5 ± 3 0 C 20 ± 3 0 C 31 ± 3 0 C Stabilized VAF (before treatment) 29.8 26.1 27.0 Stabilized VAF (after treatment) 29.3 26.1 27.3 Filtered VAF (pool) BD BD 15.4 Centrifuged Stabilized VAF (control) 21.3 16.1 20.3 Gain/Loss Filtered vs. Control - 21.3 -16.1 - 4.9 BD = below detection (less than 5 gU/mL) All filtrations were performed from the same day harvest. Prior to filtration through Sartoclean CA and Sartopore2 filters VAF was exposed for 60 minutes to 5 ±3oC, 20 ± 3 0 C and 31 ± 3*C*. 15 73 WO 2005/014862 PCT/US2004/005697 Table 10. A/Sydney/05/97 hemagglutinin activity [HA titer]. Process step Temperature 5 ± 3 0 C 20 ± 3 0 C 31 ± 3 0 C Stabilized VAF (before treatment) 256 128 256 Stabilized VAF (after treatment) 256 256 128 Filtered VAF (pool) 16 32 128 Centrifuged Stabilized VAF controll) 128 128 128 *10% of PBS was added to all experiments to adjust volume All filtrations in Table 10 were performed from the same day harvest. Prior to filtration through Sartoclean CA and Sartopore2 filters VAF was exposed for 60 minutes to 5 ± 3oC, 5 20 3"C and 31 ± 3"C*. 10 Table 11. A/Sydney/05/97virus potency [log 10 TCID 5 o/mL]. Process step Warming time 30 min 90 min 180 min Stabilized VAF (before warming)* 8.7 ±0.2 8.7 ±0.2 8.7 ±0.2 Stabilized VAF (warmed up) 8.9 ±0.2 8.9 ±0.2 8.7 ±0.2 Filtered VAF (pool) 8.5 ±0.2 8.5 ±0.2 8.9 ±0.3 Centrifuged Stabilized VAF (control)* 8.9 ±0.3 8.9 ±0.3 8.9 ±0.3 Gain/Loss FIltered vs. Control - 0.4 - 0.4' 0.0 All filtrations were performed from the same day harvest. Prior to filtration through Sartoclean CA and Sartopore 2 filters VAF was exposed to 31 ±3'C for 30, 90 or 180 minutes. 15 74 WO 2005/014862 PCT/US2004/005697 Table 12. A/Sydney/05/97 neuraminidase activity [RxU/mL]. Process step Warming time 30 min 90 min 180 min Stabilized VAF (before warming)* 35.0 35.0 35.0 Stabilized VAF (warmed up) 38.0 35.2 36.0 Filtered VAF (pool) 17.8 22.9 23.1 Centrifuged Stabilized VAF (control)* 27.9 27.9 27.9 Gain/loss Filtered vs. Control -10.1 - 5.0 - 4.8 All filtrations were performed from the same day harvest. Prior to filtration through Sartoclean CA and Sartopore 2 filters VAF was exposed to 31 ± 3C for 30, 90 or 180 minutes. 5 Table 13. A/Sydney/05/97 hemagglutinin activity [HA titer]. Process step Warming time 30 min 90 min 180 min Stabilized VAF (before warming)_ 256 256 256 Stabilized VAF (warmed up) 128 256 256 Filtered VAF (pool) 128 128 128 Centrifuged Stabilized VAF (control)* NA NA NA 10 * Stabilized VAF and Centrifuged Stabilized VAF (control) samples were taken from the pool before VAF was divided into 4 individual experiments (0, 30, 60 or 90 minutes temperature treatment). NA = not available All filtrations were performed from the same day harvest. Prior to. filtration through 15 Sartoclean CA and Sartopore 2 filters, VAF was exposed to 31 ± 3*C for 30, 90 or 180 minutes. 75 WO 2005/014862 PCT/US2004/005697 Table 14. A/Sydney/05/97virus potency [log 1o TCID 5 o/mL]. Process step Warming time 0 min 30 min 60 min 90 min Stabilized VAF (before warming)* 8.8 ±0.3 8.8 ±0.3 8.8 ±0.3 8.8 ±0.3 Stabilized VAF (warmed up) - 8.7 ±0.2 8.6 0.1 8.6 ±0.1 Filtered VAF (pool) 7.7 ±0.1 8.3 ±0.2 8.4 ±0,2 8.6 ±Q.1 Centrifuged Stabilized VAF (control)* 8.6 ±0.1 8.6 ±0.1 8.6 ±0.1 8.6 ±0.1 Gain/loss Filtered vs. Control - 0.9 - 0.3 - 0.2 - 0.0 All filtrations were performed from the same day harvest. Prior to filtration through Sartoclean CA and Sartopore 2 filters, VAF was exposed to 31 ± 3"C for 0, 30, 60 or 90 minutes. 5 10 Table 15. A/Sydney/05/97 neuraminidase activity [JU/mLI. Process step Warming time 0 min 30 min .60 min 90 min Stabilized VAF (before warming)* 44.5 44.5 44.5 44.5 Stabilized VAF (warmed up) - 44.5 41.0 47.5 Filtered VAF (pool) 6.0 17.5 26.0- 30.0 Centrifuged Stabilized VAF (control)* 33.0 33.0 33.0 33.0 Gain/loss Filtered vs. Control - 27.0 - 15.5 - 7.0 - 3.0 All filtrations were performed from the same day harvest. Prior to filtration through Sartoclean CA and Sartopore 2 filters, VAF was exposed to 31 ±3"C for 0, 30, 60 or 90 minutes. 76 WO 2005/014862 PCT/US2004/005697 ) Table 16. A/Sydney/05/97 hemagglutinin activit [HA titer]. Process step Warming time 0 min 30 min 60 min 90 mi Stabilized VAF (before warming)* 64 64 64 64 Stabilized VAF (warmed up) - 128 128 128 Filtered VAF (pool) 16 64 128 128 Centrifuged Stabilized VAF (control)* 64 64 64 64 *Stabilized VAF and Centrifuged Stabilized VAF (control) samples were taken from the pool before VAF was divided into 4 individual experiments (0, 30, 60 or 90 minutes temperature treatment). 5 All filtrations were performed from the same day harvest. Prior to filtration through Sartoclean CA and Sartopore 2 filters, VAF was exposed to 31 ±3*C for 0, 30, 60 or 90 minutes. 10 Table 17. Virus potency [log 10 TCID5o/mL]. Process step Warming time 0 min 30 min 60 min 90 min Stabilized VAF (before warming)* 0.6 ±0.2 8.6 ±0.2 8.6 0.2 8.6 ±0.2 Stabilized VAF (warmed up) - 8.6 ±0.2 8.6 ±0.2 8.5 ±0.3 Filtered VAF (pool) 8.1 ±0.2 8.1 ±0.2 8.5 ±0.2 8.5 ±0.3 Centrifuged Stabilized VAF (control)* 8.6 ±0.2 8.6 ±0.2 8.6 ±0.2 8.6 ±0.2 Gain/Loss Filtered vs. Control - 0.5 - 0.5 - 0.1 - 0.1 All filtrations were performed from the same day harvest. Prior to filtration through Sartoclean CA and Sartopore2 filters, VAF was exposed to 31 ±34C for 0 30, 60 or 90 15 minutes. 77 WO 2005/014862 PCT/US2004/005697 Table 18. Neuraminidase activity U/imL. Process step Warming time 0 min 30 min 60 min 90 min Stabilized VAF (before warming)* 35.5 35.5 35.5 35.5 Stabilized VAF (warmed up) - 36.5 36.0 34.5 Filtered VAF (pool) 7.0 9.0 14.5 - 19.5 Centrifuged Stabilized VAF (control)* 23.0 23.0 23.0 23.0 Gain/Loss Filtered vs. Control - 16 - 14 - 8.5 -3.5 All filtrations were performed from the same day harvest. Before filtration through Sartoclean CA Sartopore2 filters, VAF was exposed to 31 ±3 0 C for 0 30, 60 or 90 minutes. 5 Table 19. Hemagglutinin activity. Process step Warming time 0 min 30 min 60 min 90 min Stabilized VAF (before warming)* 64 64 64 64 Stabilized VAF (warmed up) - 64 64 128 Filtered VAF (pool) 16 32 64 64 Centrifuged Stabilized VAF (control)* 64 64 64 64 * Stabilized VAF and Centrifuged Stabilized VAF (control) samples were taken from the 10 pool before VAF was divided into 4 individual experiments (0, 30, 60 or 90 minutes temperature treatment). All filtrations were performed from the same day harvest. Before filtration through Sartoclean CA Sartopore2 filters VAF was exposed to 31 ±3"C for 0 30, 60 or 90 minutes. 15 78 WO 2005/014862 PCT/US2004/005697 Table 20. Virus potency [log 19TCIDo/mL] of six influenza strains. Influenza strain Process step Stabilized Filtered Centrifuged Potency VAF* VAF Stabilized Gain/ Loss VAF* A/Beijing/262/95 RT** 9.7 0.1 9.6 ±0.2 9.6 ±0.3 0,0 A/Beijing/262/95 31 ± 3"C 9.7 ±0.1 9.4 ±0.3 9.6 ±0.3 - 0.2 A/New Caledonia/20/99 RT** 9.6 0.2 9.3 ±0.2 9.5 ±0.2 - 0.2 A/New Caledonia/20/99 31 ± 3 2C 9.6 +02 9.3 ±0.2 9.5 ±0.2 - 0.2 A/Sydney/05/97 RT 8.8 ±0.3 7.7 ±0.1 8.6 ±0.1 - 0.9 A/Sydney/05/97 31 ± 3*C 8.8 ±0.3 8.4 ±0.2 8.6 ±0.1 - 0.2 A/Panama/2007/99 RT** 8.5 ±0.2 8.2 ±0.1 8.5 ±0.3 - 0.3 A/Panama/2007/99 31 ± 3 2 C 8.5 ±0.2 8.6 ±0.2 8.5 ±0.3 0.1 BNictoria/504/2000 RT*k 8.3 ±0.2 7.8 ±0.2 8.4 ±0.2 - 0.6 BNictoria/504/2000 31 ± 3LC 8.3 ±0.2 8.4 ±0.2 8.4 ±0.2 0.0 BNamanashi/166/98 RT** 8.4 ±0.2 8.3 ±0.2 8.6 ±0.2 - 0.3 BNarnanash/1 66/98 31 L 3*C 8.4 ±0.2 8.4 ±0.2 8.6 ±0.2 - 0.2 *Stabilized VAF and Centrifuged Stabili2ed VAF (control) samples were taken from the pool before VAF was divided into individual experiments (RT and 31 ± 3"C). * *RT room temperature 5 Both filtrations for the same strain were performed from the same day harvest, Prior to filtration through Sartoclean CA and Sartopore2 filters, VAF was exposed to 31 13'C for 0 (RT) or 60 minutes. 79 WO 2005/014862 PCT/US2004/005697 Table 21. Neuraminidase activity [jtU/iL) of six influenza strains. Influenza train Process step Stabilized Filtered Centrifuged Activity VAF VAF Stabilized Gain/Loss VAF A/Beijing'/262/95 RT** 55.5 47.5 52.0 - 4.5 A/Beijing/262/95 31 ± 3*C 55.5 51.5 52.0 - 0.5 A/New Caledonia/20/99 RT** 49.5 47.5 48.5 - 1.0 A/New Caledonia/20/99 31 ± 3QC 49.5 48.5 48.5 0.0 A/Sydney/05/97 RT** 44.5 6.0 33.0 - 27.0 A/Sydney/05/97 31 ± 3 2 C 44.5 26.0 33.0 - 7.0 A/Panama/2007/99 RT* 51.0 16.5 48.0 - 31.5 A/Panama/2007/99 31 ± 39C 61.0 40.0 48.0 - 8.0 B/Victoria/504/2000 RT** 50.5 20.5 44.0 - 23.5 B/Victoria/504/2000 31 t 32C 5B.5 37.0 44.0 - 7.0 B/Yamanashi/l 66/98 RT** 66.5 51.0 55.5 - 4.5 B/Yamanashi/1 66/98 31 ±3C 66.5 53.0 55.5 - 2.5 * Stabilized VAF and Centrifuged Stabilized VAF (control) samples were taken from the pool before VAF was divided into individual experiments (RT and 31 ± 3C). **RT = room temperature 5 Both filtrations for the same strain were performed from the same day harvest. Prior to filtration through Sartoclean and CA Sartopore2 filters, VAF was exposed to 31 ±3 0 C for 0 (RT) or 60 minutes. 80 WO 2005/014862 PCT/US2004/005697 Table 22. Hemagglutinin activity [HA titer] of six influenza strains. Influenza train Process step Stabilized Stabilized Filtered Centrifuged VAF* Warmed up VAF Stabilized VAF VAF* A/Beijing/262/95 RT** 1024 - 128 1024 A/Beijing/262/95 31 ± 3 2 C 1024 512 512 1024 A/New Caledonia/20/99 RT** 32 - 32 64 A/New Caledonia/20/99 31 ± 32C 32 32 32 64 A/Sydney/05/97 RT** 64 - 16 64 A/Sydney/05/97 31 ±32C 64 128 128 64 A/Panama/2007/99 RT** 128 - 32 128 A/Panama/2007/99 31 ±3C 128 128 64 128 B/Victoria/504/2000 RT** 128 - 32 128 B/Victoria/504/2000 31 ±3"C 128 64 64 128 B/Yamanashi/1 66/98 RT** 512 - 16 32 B/Yamanashi/1 66/98 31 ± 3QC 512 32 | 32 32 * Stabilized VAF and Centrifuged Stabilized VAF (control) samples were taken from the pool before VAF was divided into individual experiments (RT and 31 ± 3C). **RT = room temperature 5 Both filtrations for the same strain were performed from the same day harvest. Prior to filtration through Sartoclean and CA Sartopore2 filters, VAF was exposed to 31 ±3"C for 0 (RT) or 60 minutes. 81 WO 2005/014862 PCT/US2004/005697 Table 23. Virus potency [log 1

OTCID

5 o/niL] of six influenza strains. Influenza strain Process step Stabilized Filtered Centrifuged Potency VAF* VAF Stabilized Gain/ Loss VAF* A/Beijing/262/95 RT** 9.6 ±0.1 9.4 ±0.2 9.6 ±0.1 - 0.2 ABeijing/262/95 31 ± 32C 9.6 ±0.1 9.5 ±0.2 9.6 ±0.1 0.1 A/New Caledonia/20/99 RT** 9.1 ±0.2 9.5 ±0.2 9.2 ±0.2 0.3 A/New Caledonia/20/99 31 ± 32C 9.1 ±0.2 9.2 ±0.3 9.2 ±0.2 0.0 A/Sydney/05/97 RT** 8.6 ±0.2 8.1 ±0.2 8.6 ±0.2 - 0.5 A/Sydney/05/97 31 * 3 2 C 8.6 ±0.2 8.5 ±0.2 8.6 ±0.2 - 0.1 A/Panama/2007/99 RT** 8.9 ±0.2 8.3 ±0.2 8.5 ±0.2 - 0.2 A/Panama/2007/99 31 2 39C 8.9 ±0.2 8.6 ±0.1 8.5 ±0.2 0.1 B/Victoria/504/2000 RT'* 7.6 ±0.2 7.7 ±0.2 7.9 ±0.2 - 0.2 B/Victoria/504/2000 31 ± 32C 7.6 ±0.2 7.7 ±0.1 7.9 ±0.2 - 0.2 B/Yamanashi/1 66/98 RT** 8.4 ±0.1 8.2 ±0.2 8.3 ±0.3 - 0.1 B/Yamanashi/1 66/98 31 ± 3*C 8.4 ±0.1 8.3 ±0.2 8.3 ±0.3 0.0 * Stabilized VAF and Centrifuged Stabilized VAF (control) samples were taken from the pool before VAF was divided into individual experiments (RT and 31 ± 3C). **RT = room temperature. 5 Both filtrations for the same strain were performed from the same day harvest Prior to filtration through Sartoclean CA and Sartopore2 filters, VAF was exposed to 31±3"C for 0 (RT) or 60 minutes. 82 WO 2005/014862 PCT/US2004/005697 Table 24. Neuraminidase activity [UiniL] of six influenza strains. Influenza strain Process step Stabilized Filtered Centrifuged Activity VAF VAF Stabilized Gain/Loss VAF* A/Beijing/262/95 RT** 56.5 47.5 54.5 - 7.0 A/Beijing/262/95 31 ± 32C 64.5 56.0 58.5 -1.5 A/New Caledonia/20/99 RT** 46.0 38.5 40.0 - 1.5 A/New Caledonia/20/99 31 ± 39C 46.0 43.0 40.0 3.0 A/Sydney/05/97 RT** 35.5 7.0 23.0 - 16.0 A/Sydney/05/97 31 ± 3 2 C 35.5 14.5 23.0 - 8.5 A/Panama/2007/99 RT** 55.5 15.0 34.5 - 19.5 A/Panama/2007/99 31 t 32C 60.5 42.5 39.0 3.5 BNictoria/504/2000 RT'* 35.0 21.0 28.5 -7.5 BNiotoria/504/2000 31 ± 32C 39.0 25.5 31.5 - 6.0 BNamanashi/1 66/98 RT** 29.0 26.0 28.5 - 2.5 BNamanash/1 66/98 31 ± 32C 33.5 27.5 29.5 - 2.0 * Stabilized VAF and Centrifuged Stabilized VAF (control) samples were taken from the pool before VAF was divided into individual experiments (RT and 31 ± 3C). ** RT = room temperature. 5 Both filtrations for the same strain were performed from the same day harvest. Prior to filtration through Sartoclean CA and Sartopore2 filters, VAF was exposed to 31 ±3C for 0 (RT) or 60 minutes. 83 WO 2005/014862 PCT/US2004/005697 Table 25. Hemagglutinin activit [HA titer] of six influenza strains. Influenza strain Process step Stabilized Stabilized Filtered Centrifuged VAF* Warmed up VAF Stabilized VAF VAF* A/Beijing/262/95 RT** 256 - 1024 512 _A/Beijing/262/95 31 ± 3LC 256 1024 2048 512 A/New Caledonia/20/99 RT** 512 - 512 512 A/New Caledonia/20/99 31 .1-3 C 512 512 512 512 A/Sydney/05/97 RT** | 64 - 16 64. A/Sydney/05/97 31 ±32C 64 64 64 64 A/Panama/2007/99 RT** 256 - 64 256 A/Panama/2007/99 31 ±39C 256 512 512 256 BNictoria/504/2000 RT** 64 - 128 128 B/Victoria/504/2000 31 ± 32C 64 64 64 128 B/Yamanashi/166/98 RT** 128 - 32 128 B/Yamanashi/166/98 31 ± 32C. 128 64 64 128 * Stabilized VAF and Centrifuged Stabilized VAF (control) samples were taken from the pool before VAF was divided into individual experiments (RT and 31 ± 3 0 C). **RT = room temperature. 5 Both filtrations for the same strain were performed from the same day harvest. Prior to filtration through Sartoclean CA and Sartopore2 filters, VAF was exposed to 31 ±3"C for 0 (RT) or 60 minutes. 84 WO 2005/014862 PCT/US2004/005697 Table 26. Analysis by SEC - Peak Area Comparison Sample Details Sample ID Peak Area at 220 Virus Peak Impurities Group 1 Impurities Grou -10.5 min) (18 to 21 min) (21 to 27 min Neat (VH) 1X 1221 31785 339528 10 times concentrated sample loX 11192 126849 435652 1X Washed 5 times with 1X-SPG 1X-W 1005 2131 2510 1OX washed with 1 X-SPG 5 times 1 OX-W 10282 15858 2194 Permeate or filtrate Permeate 25 33837 360812 Wash-1 W-1 6626 71260 Wash-2 W-2 2296 15773 Wash-3 W-3 1879 5765 Wash-4 W-4 1046 3110 Wash-5 W-5 876 2769 5 Table 27. A/New Caledonia -CELISA Values 10 Mean +/- SD Sample Details Sample ID Replicate (N) (CELISA) Neat (VH) 1x 4 9.1 +/- 0.02 10 times concentrated sample loX 4 10.0 +/- 0.05 1X Washed 5 times with 1X-SPG 1X-W 4 8.9 +/-0.03 1OX washed with 1X-SPG 5 times 10X-W 4 9.9+-0.04 Permeate or filtrate Permeate 4 <LOQ 1OX diluted back to 1X with 1X-SPG lox to 1X 4 9.0 +/-0.08 1 0X-W diluted back to 1X-W with 1X-SPG 1OX-W to 1X-w 4 8.9 +/- 0.02 85 WO 2005/014862 PCT/US2004/005697 Table 28. Composition of Representative Formulations Formulation .p . Number Composition 1 10% Allantoic fluid in 100 millimolar phosphate buffer,.7% Sucrose, no added recipients 2 60% Allantoic fluid in 100 millimolar phosphate buffer, :7% Sucrose, no added excipients 3 10% Allantoic fluid in 100 millimiolar phosphate buffer, 7% sucrose [2] with 1% gelatin hydrolysate and 1% arginine 4 60% Allantoic fluid in 100 millimolar phosphate buffer, 7% sucrose [31 with 1% elatin hydrolysate and 1% arginine 5 60% Allantoic fluid in 100 millimolar phosphate buffer, 10% sucrose, 2% arginine, 2% gelatin hydrolsate 6 60% Allantoic fluid in 100 millimolar phosphate buffer, 10% sucrose, 2% arginine 7 60% Allantoic fluid in 100 millimolar phosphate buffer, 10% sucrose, 2% arginine. 2% gelatin hydrolysate, 2.5 mM EDTA 8 60% Allantoic fluid in 50 millimolar histidine buffer, 10% sucrose, 2% arginine, 2% gelatin hydrolvsate 9 60% Allantoic fluid in 50 millimolar histidine buffer, 10% sucrose, 2% arginine, 2% gelatin hydrolysate, 2.5 mM EDTA 5 Table 29. Stability of Virus in Representative Formulations (loss of titer in logio/mImonth) Formulation A/New Caledonia20/99 A/Panama/2007/99 B/Hong Kong/330/01 Number 1 0.030 0.133 0 156 2 0.040 0.098 0.166 3 0.042 0.080 0.151 4 0.087 0.073 0.181 5 0.021 0.093 0.107 6 No loss observed 0.090 0.097 7 0.046 0.037 0.113 8 0.068 0.072 0;061 9 0.034 0.073 0;121 86 WO 2005/014862 PCT/US2004/005697 l - §,- . Z1 cl C,04 Ql C' cl a,0 Cl - t- n - in oD -4 ;.;,d 0 cc w~ Cf l , o CC)C -~ 0 -87 WO 2005/014862 PCT/US2004/005697 ~~- CC I' tN .6 6 ID 1. 1 0 N NNN 0 0 0 00 NR - 't (n lp m VV 10 10 C 10 Z V Cl N0 C%,~ ~ i Oi C OR q C) q N q q t- VVV0 V5Va\VVaV"V'6' C7 cr cr -r VC VVVVV VVVV NVVV VV 88 WO 2005/014862 PCT/US2004/005697 V) U) Cl) fl0 0 'f ~ 0 00 0l 0 1\6 \6 6 i6666 6 W CQ o q I q ,t 6 r-'Cn %.q q Cv \0 'C ' \D ' 10 \Q 'C %D Ifl zC 'C 'C 'C \C 'C 'C 'C 'C . q 'C W) In IR 'C nC \q 'Cq "t In mC "It In It qC' C u C 'C 1f 10,0,1 10 'C Cl' 0 In Ic z ' o 0~ c r- .0 0q 0) 0R W~( C 0 N. Cl 'C'C C'C C'C C'C' .C' ' ' 'D'C 'o \0 \CC'6 \0666c\6'o C, \0'D CC \0 \ 6 (- 00 cC (- C 'C ~ ~ ~ ~ t t-- 10 00'l( 1~t-00' ~ -~ ~( 0 It; t- 10 10 'C . C' ' C . 'Dr 'C' C ' 66 6 6 ' c r 3 89 WO 2005/014862 PCT/US2004/005697 Qt P 0 cc66 -T Z) 10 t, Ci C-i \0 t- 00 v 'I.iC 0 0 \D It 0- '-n I n 'tIqi \ 0 nvic D ' o 'o 0o 0 ' G0 'o \8 'D '0e o 0 0 ' U, a o 0q a) r- 0 r- C) -r00 N0N 0~~l %D 0o 00 ai 0 0Ci0 i 0 -N N c' C i ci Ci 0 '0 '00000 0 00 000 0C g- g ' Q g 00 0 C C7Nr c g 40 N3 0N' 0'0' 000 0V0 00- - - - - - - ~9 WO 2005/014862 PCT/US2004/005697 V) LnC 0 )I -u - - - - - NeC4C C) C) C C) C) CA C) C N C 00 0000 0 00 0 0000 0 0 0 30 .30 00 00 'C CO CO CCE- CO C C 'C ' C 't r,: N9 -1N C~ Cfl N q C N t \p CN 0 q 0 100 CO: N C00-00 -t o000l N330CC0 00 CC0 ON03 Nf 0 N00 CO 03 0 N q C q'C 0 00 N 030 0 q00030000!30 .... .. 0\c0 COCOONCOOCOOCOC00CCOCOO 110COwe. 0 10 t' O0CC00 O 0 0N0003CCN0 - C'COCCCOCC'COOC'CC'CCC3~ C9 WO 2005/014862 PCT/US2004/005697 Formulations: Second Tier Control Liq15 Ingredients Liql3a [1q14 Liq15 (degassed) Liq16 Liq17 LiqI8 Liq19 Liq; KPO4 buffer, pH 7.2 (1.1 mM from virus 1.1mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 moM 1001 included) Citrate buffer, pH 7.2 100 mM Sucrose (0.7%from 7% 10% 10% 10% 10% 10% 10% 10% 10 virus included) 2%% Gelatin 2% 2% 2% 2% 2% 2% 2% 2% Arginine 2%O 2% 2% 2% 2% 2% 2% 2 O Aprotinin (PI) 0.02% Leupeptin hemisulfate (PI) 0.02% Lysozyme Inhibitor 0.1% Protease Inhibitor (0.6% Cocktail DMSO) 0.5%* PMSF 1ml Cytidine 2' monophosphate NAF (from virus) 10% | 10% 10% 10% 10% 10% 10% 10% 109 NAF (added) 50% 50% 50% 50% 50% 50% 50% 50% 5090 IN KOH or7NI.2 pH 7.2 to pH 7.0 to pH 7.2 to pH 7.2 to pH 7.2 tc pH 7.2 to pH 7.2 to pH 7.2 to pH to pH 7.2 Purified Water d q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. Ingredients Liq21 Liq22 Liq23 Liq31 Liq24 Liq25 Liq26 Liq27 KPO4 buffer, pH 7.2 (1.1 mM from virus 100 mM 100 mM 100 mM 100 mM 100 mM 100mM .100 mM 100 mM included) Suvrsein.lude 10% 10% 10% 10 % 10% 10% 9.3% virus included) ______II Gelatin 10% 2% 2% 2% 2% 2% .2% 2% Arginine 2% 2% 2% 2% 2% 2% 2% 2% L-Ascorbic Acid 2% 0.05% Ascorbic Acid 6 Palmitate 0.005% 0.001% Arbutin 0.05% Propyll Gallate 0.05% EDTA 10 mM RNAse Inhibitor, SuperAse In - 2.0 U/sL NAF (from virus) 5 mM 10% 10% 10% 10% 10% 10% 10% NAF (added) 10% 50% 50% 50% 50% 50% 50% 50% IN KOH or IN HCI 50% titrate titrate titrate titrate titrate titrate titrate to pH 7.2 5 to pH 7.2 to pH 7.2 to pH 7.2 to pH -7-2 to pH 7.2 to PH 7.2 to pH 7.2 Purified Water to pH 7.2 g.s. q.s. q.s. q.s. q.s. q.s. q.s. Table 35: 92 WO 2005/014862 PCT/US2004/005697 H R U C) Cr C 0 0 C') z C t C)C. 0t C): ) 'n i I n in n 1d '11 1 ' 'n W) ) n In In In ni Cd C d C d Cd C d C d Cd C d C d 00 M.C. ' 00 00 k~0 n. q . 0- In in4cC %;LnV -l q Cd ll- o u' ln 41 o r N ~00 410') - Nvi o o NO') 10 CI ) r: q q OR 41 Q C r-: 0 u-C~~rn CC' 00 IQ I-! Cd C N C) , 4C6 6 vi t-- so OR C-"R O n C r: I N r R ' t cl r:kCI , o t ' zd \ cd 0 CO ~ ~ ~ ~ ~ R 's 'C C)N C C N 00 OC tn 'a PC - C 1 0 0 C 1 C) " 4 0 4 O~ ,~ ~ ' V ' O C O C ccc 3 3nN 44 C)N - (C C) ' C)0C 0 1 C) N N ~ 4 '0 00 00 00 ~ C 0 ' C 4 00 14 C 4 00 '~COC93d- WO 2005/014862 PCT/US2004/005697 -p,~~. ... .. ' --.-...- - - - - - - C- -1 0 00 0 0 - n c O V 00 * ~ ' 00 0 O 00 2q m. o ' vi vi 6 tri 06 10 0 N 1.C' N 00 kn in 'D In n 'o 10 In I n I n In I n I n In I n I n I 00 N In =~ 0 0 \0 100 Vi \D Of M Z IM 00 0 Ci ~ 0 0 r oq0 IQ '0 0 10 00 NO v tN dc00 0 0 - o N%0- \,-o \n -: 6 ~ 0 30 ~ ~ ~ ~ 7 *Cr000 o ' 0 , N~' 00 \6 \00 00 rt~0 ' I.001 '-t~~000N~t I03 30 ~ 0 n - O O 0 ~ 0 N N On 00 000 N 000 c 40 c 0 00 0 ;I ;I00 '~ 0 1 ;I fq A ;I 03000 100 00 00 00 'C 'C00 0 00 00 c 0 ra0 0) 0 N 00 00 ~ N~ 0 =C94 WO 2005/014862 PCT/US2004/005697 pt 0 .2 A CSC -C In 0 rD CC (C 6 ~66 1. ; N %c V: cl l0c0 0 0 0 0 0 = 0o lo0i o Ic o CD Co r- o %o %6 6 PC . .*

.

'I CC~~~~ '0 '.0 C5C 0 N N02 Cf C 02 0 CC CO95 WO 2005/014862 PCT/US2004/005697

-----------------------------

vi 6 z ' vi v- r- 0o Do.o 000 ) 00 L) Cll16l ' Cz; InN N ~ ( t-- 'oC) ( (N wl (N C o to N r, v) (c0 \q n n~ (I~ 0l c\0 C')) M) CI) C)) Co) O ~ j 0 0 0 0 0 0 0Ci) C)) I) I) C) C) C) 0o '0)0 ~ ~ o '0 \) 0 c1 410 0 V V 0' ' 3 3 (c0 0101 11 0) '0) .0) .0)'.0)'0 C'. ... ~ N 1 96 WO 2005/014862 PCT/US2004/005697 Formulation Ingredients No Citrate 20 mM 50 mM 100 mM 200 mM Citrate Citrate Citrate Citrate Citrate buffer 0 20 mM 50 mM 100 mM 200 mM pH 7.2 KPO4 buffer (from virus 1.1 mM 1.1 mM 1.1 mM 1.1 mM 1.1 mM material) Sucrose (0.7% 10 % 10 % 10 % 10 % 10 % from virus included) Gelatin 1% 1% 1% 1% 1% Arginine 2% 2% 2% 2% 2% NAF (from virus: to% 10% 10% 10% 10% B/Hongkong; A/Panama) NAF (added) 50% 50% 50% 50% 50% IN KOH or iN titrate titrate titrate titrate titrate HCl to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 Purified Water q.s. q.s. q.s. q.s. q.s. Table 40: 97 WO 2005/014862 PCT/US2004/005697 Results: Potency by FFA Assay A/Panama A/Panama B/Hongkong B/Hongkong Aliquot I Aliquot 2 Aliquot 1 Aliquot 2 Ave. of 9 Av'e. of 6 A/ Panama, Starting material plates plates 8.1 ± 0.2 7.0 ± 0.1 (8.5) (7.9) Pre-diluted Starting Material (1:10) 8.0 ± 0.1 none n/a n/a (8.0) 0% Citrate 6.7 ± 0.1 6.9 ± 0.0 6.7 ± 0.0 6.8 ± 0.1 (7.0) (7.0) (6.9) (6.9) 20 mM Citrate 6.7 ± 0.1 6.7 ± 0.2 6.9 ± 0.0 6.8 ± 0.1 (7.0) (7.0) (6.9) (6.9) 50 mM Citrate 6.7 ± 0.1 6.7 ± 0.1 6.9 ± 0.0 6.8 ± 0.1 (7.0) (7.0) (6.9) (6.9) 100 mM Citrate 6.8 ± 0.0 6.8 ± 0.0 6.8 ± 0.1 6.9 ± 0.1 (7.0) (7.0) (6.9) (6.9) 200 mM Citrate 6.8 ± 0.1 6.8 ± 0.0 6.7 ± 0.1 6.6 ± 0.2 (7.0) (7.0) (6.9) (6.9) Base Formulation: 60% NAF, 10% Sucrose, 1% Gelatin 2% Arginine Table 41: 98 WO 2005/014862 PCT/US2004/005697 Formulation Ingredients No EDTA 0.5 mM 1.0 mM 2.0 mM 5.0 mM 10 mM EDTA EDTA EDTA EDTA EDTA KPO4 buffer, pH 7.2(1.1 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM from virus included) Sucrose (0.7% from virus 10% 10% 10% 10% 10% 10% included) Gelatin 1% 1% 1% 1% 1% 1% Arginine 2% 2% 2% 2% 2% 2% EDTA 0% 0.0186% 0.037% 0.0744% 0.186% 0.372% (0.5 mM) (1.0 mM) (2.0 mM) (5.0 mM) (10 mM) NAF (from virus: 10% 10% 10% 10% 10% 10% BJHongkong; A/Panama) NAF (added) 50% 50% 50% 50% 50% 50% iN KOH or IN titrate titrate titrate titrate titrate titrate HCl to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 Purified Water q.s. q.s. q.s. q.s. q.s. q.s. Table 42: 99 WO 2005/014862 PCT/US2004/005697 Results: Potency by FFA Assay A/Panama A/Panama B/Hongkong B/Hongkong Aliquot 1 Aliquot 2 Aliquot 1 Aliquot 2 Ave. of 9 Ave, of 6 A/ Panama, Starting material plates plates 8.1± 0.2 7.9 i 0.1 (8.5) (7.9) Pre-diluted Starting Material (1:10) 8.0 ± 0.1 none n/a n/a (8.0) 0% EDTA 6.1± 0.2 6.1 ± 0.2 6.7 ± 0.1 6.7 ± 0.1 (7.0) (7.0) (6.9) (6.9) 0.5 mM EDTA 6.3 0.2 6.3 2 0.1 6.7 ± 0.1 6.6 ± 0.1 (7.0) (7.0) (6.9) (6.9) 1.0 mM EDTA 6.5 t 0.1 6.5 ±0.1 6.8 0.1 6.6 20.1 (7.0) (7.0) (6 9) (6.9) 2.0 mM EDTA 6.6 t 0.0 6.8 ± 0.1 6.6 t 6.6 t 0.1 (7.0) (7.0) (6.9) (6.9) 5.0 mM EDTA 6.7 ±0.0 6.8 ±0.1 6.6 0.1 6.7 ±0.2 (7.0) (7.0) (6.9) (6.9) 10 mM EDTA 6.8 ± 0.1 6.7 ± 0.1 6.7 0.1 6.6 ± 0.2 (7.0) (7.0) (6.9) (6.9) Base Formulation: 100 m.M KPO4, 60% NAF, 10% Sucrose, 1% Gelatin 2% Arginine Table 43: 100 WO 2005/014862 PCT/US2004/005697 Formulations: Third Tier Ingredients Liq28 Liq29 Lig30 KPO4 buffer, pH 7.2 100 mM 100 mM (1.1 mM from virus included) Citrate buffer, pH 7.2 n/a n/a 100 mM Sucrose (0.7% from 10 % 10% 10% virus included) Gelatin 2% n/a 2% Arginine 2% 2% 2% (5 mM) (10 mM) (10 mM) EDTA 0.186 % 0.372 % 0.372 % NAF (from virus) 10% 10% 10% NAF (added) 50% 50% 50% IN HCI or titrate titrate titrate IN KOH to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 Purified Water g.s. g.s. g.s. Table 44: 101 WO 2005/014862 PCT/US2004/005697 inn w in!!i5Iti mi IJ2 rU) a, U ) en co a,~ -'-I In n d~~ dic \0 0 \0\0V M o j'0 I - 1.0 It- z n-In4o'6 ,6 'o "n . z- -j C4 4 Al o ~ ~ ~ c L ~ , ~* . n r-' -102 WO 2005/014862 PCT/US2004/005697 ForrmulatiOns: P-B 4-level Custom screen A Ingredients Liq36 Liq37 Liq38 Liq39 Liq40 Liq41 Lig42 Liq43 Liq44 Liq45 Liq46 Liq47 KPO4 buffer, pH 7.2 (1.1 mM 50 50 50 50 50 50 50 50 50 50 50 50 from virus mM mM mM mM mM mM mM miM mM mM mM mM included) Sucrose (0.7% from virus 0.0 % 7.5% 7.5% 7.5% 7.5 % 7.5% 7.5 % 10 % 10% 10% 10 % 10% included) Gelatin 0% 0% 0% 1 % 1 % 2% 2% 0% 0% 0% 1 % 1 % Arginine 2% 2% 4% 0% 4% 0% 2% 4% 2 % 4% 2% 2% EDTA 1 mM 2.7 5 mM 1 mM 2.7 5mM 1mM 5 mM 5 mM 1 mM 1 mM 2.7 mM mM mM NAF (from virus: B/Hongkong; 10% 10 % 10% 10% 10% 10% 10% 10% 10% 10% 10% 10% A/Panama) NAF (added) 50% 50% 50% 501% 50 % 50% 50% 50% 50% 50% 50% 50% IN KOH or IN titrate titrate titrate titrate titrate titrate titrate titrate titrate titrate titrate titrate HCI to pH 7.2 to pH to pH to pH to pH to pH to pH to pH to pH to pH to pH to pH to pH 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 Purified Water q.s. q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s Ingredients Liq48 Lig49 Lig50 Liq51 Liq52 Liq53 Liq54 Liq55 Liq56 Liq57 Liq58 Liq59 KPO4 buffer, pH 7.2 (1.1 mM 50 50 50 50 50 50 50 50 50 50 50 50 from virus mM mM mM uM mM mM mM mM mM mM mM mM included) Sucrose (0.7% from virus 10% 10% 10% 10% 15 15% 15 % 15 % 15% 15 % 15% 15% included) Gelatin 1 % 2% 2% 2% 0% 0% 0% 1 % 1 % 1 % 2% 2% Arginine 4% 0% 0% 4% 2% 2% 4% 0%- 0% 4% 2% 4% EDTA 5 mM 1 mM 2.7 2.7 2.7 1 mM 2.7 2.7 5 mM 1 mIM 5 mM 1 mM mM mM mM mM mM NAF (from virus: B/Hongkong; 10% 10% 10% 10 % 10% 10% 10% 10% 10% 10 % 10-% -- 10-% A/Panama) ^ - NAF(added) 50% 50% 50% 50% 50% 50% 50% 50% 50 e 50;% 50 % 509 IN KOH or IN titrate titrate titrate titrate titrate titrate titrate titrate titrate titrate titrate titrate HCI to pH 7.2 to pH to pH to pH to pH to pH to pH to pH to pH to pH to pH to pH to pH 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 Purified Water q.s. q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s Table 46: 103 WO 2005/014862 PCT/US2004/005697 F-g DO cn n co CA o 1 0 0 0 10 0 0 0f Vl cc) . 1 00000n0000 00000 0000 _ 0 0 o0 - ~ ~ ~ ~ ~ ~ ~ c \c ---------- - -- - -- - - 010 WO 2005/014862 PCT/US2004/005697 F- ~H ~ H -1 H H C''i In C) CD C 0 D C D C ) C ,n kn kni 0 0 0 0 0 0 0 n fn 'fl V) n In) fn) 'D \ qC 0 V l lu In' ' 0 0 ) 'r0 "D 'o 0 0 '0 '0 I'D "D "D ID 10 10 10 10 10 '0 pm * ~ - o~0o ' 0 o0 4 In '0r -no 0 ' PC f)e w n cr - - - - - - - - - 3 35 10 WO 2005/014862 PCT/US2004/005697 > L V V ot U - '& 0 (0 S L CDC o 0.'-4

UL.

0)i ImJ I- IL or o o-66 IL ILD 0. 0 ) ) V 00 00 4-- .4- T CR ~ o0T( I~I.106 WO 2005/014862 PCT/US2004/005697 00 m 0- CII I I CICIO) 0 CA rCl Cl0 CIO 107 WO 2005/014862 PCT/US2004/005697 ~~ C Ci) C) o ~C T-0 l C ; 0 0 cd~ _ ) ~ ~* + 6 616 10 WO 2005/014862 PCT/US2004/005697 * *) - 00 r4n 0 I +1 0. 1091 WO 2005/014862 PCT/US2004/005697 Table 53: Comparison of Method Performance Test Parameter Method Performance Manual Semi-automated Precision/ Between Test Variability SD range from 0.07 to 0.11 log S) range from 0.06 - 0.09 (SD)) TCID 50 units' logioTC]D 5 o/mL"' Passes test for lack of fit to a Pd ses test for lack of fit to a Linearity linear model at the 1% linear model at the 1% significance level. significance level. Accuracy Slope range 0.986 - 1.007. Slopes range 1.00-1.02. Range 4.7 - 9.5 logioTCIDoo/mL. 4.2 - 9.3 logioTClD 5 /mL.' lBetween-test SD from 9 tests on the same material (each test results is an average of 12 determinations over 3 days), by the same analyst group, on the same pipetting station. The materials tested include 3 independent manufacturing lots of each of three virus strains (H1N1, H3N2 and B). 2Between-test SD from 6 tests on the same material (each test results is an average of 12 determinations over 3 days), by the same analyst group, on the same pipetting station. The materials tested include one lot of each of three virus strains (H1NI, H3N2 and B). 3Validation Report for Semi-Automated TCID50 Potency Assay for Influenza Virus Monovalent. 5 10 Table 54: Inter-Assay Comparison Mean Titer (log oTCIDso/mL) Strain Manual Assay Semi-Automated Dif erenceC Assay (LB, UB) AINewCaledonia/20/99 9.40 9.42 -0.02, B/Yamanashi/166/98 8.47 8.40 0.07 . . 0.03,.p;10) 15 110 WO 2005/014862 PCT/US2004/005697 Table 55. Manual "gold standard" readout SeniAutomated CPE-positive CPE-negative MTT readout ______ ______ CPE-positive TP FP (As 5 o cutoff) CPE-negative FN TN (As 70 > cutoff) ________ ________ All positives All negatives 5 Table 56. SemiAutomated TCID 50 Potency Assay for Influenza Virus Monovalent: Sensitivity and Specificity Estimates Based on the "Gold Standard" Validated Manual CPE Readout and the MTT Assay A570 Cutoff Value of 0.5254 10 _________ 10True False Sensitivitya True False Specificityb positive negative negative positive AN=14,400) 7,091 61 99.15% 7,247 1 99.99% QC 15,835 301 98.13% 15,106 198 98.71% (N=31,440) AB and QC 22,926 362 98.45% 22,353 199 99.12% (N=45,840) Control 17,248 167 99.05% 15,882 3 99.99% ATR.0126 ASENSITIVITY = (TRUE POSITIVE)/ALL POSITIVE SPECIFICITY = (TRUE NEGATIVE)/ALL NEGATIVE 15 111 WO 2005/014862 PCT/US2004/005697 Table 57. Control Well (CPE-negative) Absorbance Values Obtained by the two groups with previous Values Reported Shown for Comparison nd Combined (previous 2 group 1St group (two groups) values) Control Well count 2880 6288 9168 6720

A

570 Average 1.226 1.235 1.231 1.261 SD 0.17 0.20 0.19 0.15 5 10 Table 58. Instrument-to-Instrument Comparison: SemiAutomated TCID 50 Potency Values for Reference Virus Strains Reference Virus Strain (Mean logioTCID5o/mL t SD) Instrument (group) A/New Caledonia/20/99 A/Sydney/05/97 B/Yamanashi/166/98 AZ-039 ( 1 s)1,2 9.2 ± 0.15 8.6 +0.09 8.4 +0.10 AZ-040 (1s)",3 9.3 +0.08 8.6 +0.01 8.4 +0,10 AZ-036 ( 2 "d)1 9.2 + 0.08 8.5 +0.05 8.3 +0.06 'Number of tests (AZ-036, N=9; AZ-039, N=5; AZ-040, N=2); 1 4 - first group, 2 nd = second group. 2 For AZ-039, one test result rejected due to failure of intra-day SD acceptance criteria For AZ-040, four test results rejected due to failure of intra-day SD acceptance criteria or mishandling of plates 15 112 WO 2005/014862 PCT/US2004/005697 Table 59. Analyst-to-Analyst Comparison: SemiAutomated TCID 5 o Potency Values for Reference Virus Strains Using Instruments AZ-039 or AZ-036 Reference Virus Strain (Mean logioTCID 50 lmL SD)a A/New Caledonia/20/99 A/Sydney/05/97 B/Yamanashi/166/98 first group AZ-039 AZ-039 AZ-039 Analyst # 1 9.3 ± 0.19 8.5 ± 0.25 8.4 ±0.26 Analyst # 2 9.1 ±0.17 8.5 ±0.27 8.4± 0.16 Analyst # 3 9.1 ±0.16 8.5 ±0.15 8.4 ±0.19 Analyst # 4 9.2 ± 0.24 8.6 ± 0.21 8.6 ± 0.24 Analyst # 5 9.1 ±0.21 8.6 ±0.19 8.3 ±0.23 Analyst # 6 9.4 ±0.21 8.7 ±0.20 - 8.6 ± 0.21 second group AZ-036 AZ-036 AZ-036 Analyst # 7 9.4 ± 0.16 8.5 ± 0.21 8.3 ± 0.18 Analyst#8 9.2 0.21 8.5 ±0.18 8.2 0.15 Analyst # 9 9.3 ± 0.16 8.5 ± 0.20 8.3 ± 0.16 5 ' Mean potency values are derived from four replicates obtained aver three test days (n=12). 113

Claims (17)

1. A refrigerator-stable live influenza virus vaccine composition, comprising one or more purified live influenza viruses; a stabilizer consisting of arginine and gelatin; and a buffer comprising sucrose, potassium phosphate and glutamate (SPG), wherein the vaccine 5 composition when stored from 2'C to 8'C exhibits less than or equal to a 1.0 log potency loss in 6 months or less than or equal to a 0.080 log potency loss per month.

2. The refrigerator-stable live influenza virus vaccine composition of claim 1, wherein at least one influenza virus is selected from: an attenuated influenza virus, a cold-adapted influenza 10 virus, a temperature-sensitive influenza virus, an attenuated cold-adapted influenza virus, a temperature-sensitive cold-adapted influenza virus, an attenuated temperature-sensitive influenza virus, and an attenuated cold-adapted temperature-sensitive influenza virus.

3. The refrigerator-stable live influenza virus vaccine composition of claim 1 or 2, wherein 15 the vaccine composition comprises about 1% (w/v) to about 5% (w/v) arginine.

4. The refrigerator-stable live influenza virus vaccine composition of claim 3, wherein the vaccine composition comprises from about 1% (w/v) to about 2% (w/v) arginine. 20

5. The refrigerator-stable live influenza virus vaccine composition of any one of claims I to 4, wherein the vaccine composition comprises from about 1% (w/v) to about 4% (w/v) gelatin.

6. The refrigerator-stable live influenza virus vaccine composition of claim 5, wherein the vaccine composition comprises about 1% (w/v) gelatin. 25

7. The refrigerator-stable live influenza virus vaccine composition of any one of claims I to 6, wherein the vaccine composition comprises from about 5% (w/v) to about 10% (w/v) sucrose.

8. The refrigerator-stable live influenza virus vaccine composition of claim 7, wherein the 30 vaccine composition comprises from about 7% (w/v) to about 10% (w/v) sucrose.

9. The refrigerator-stable live cold-adapted influenza virus vaccine composition of any one of claims I to 8, wherein the vaccine composition has a pH of about 7.2. -114-

10. The refrigerator-stable live influenza virus vaccine composition of any one of claims I to 9, wherein the vaccine composition does not comprise EDTA.

11. The refrigerator-stable live influenza virus vaccine composition of any one of claims 1 to 5 10, wherein the vaccine composition comprises less than 5% (V/V) normal allantoic fluid.

12. The refrigerator-stable live influenza virus vaccine composition of any one of claims 1 to 10, wherein the vaccine composition is substantially free of normal allantoic fluid. 10

13. The refrigerator-stable live influenza virus vaccine composition of any one of claims 1 to 12, comprising at least two influenza virus strains.

14. The refrigerator-stable live influenza virus vaccine composition of claim 13, comprising three influenza virus strains. 15

15. The refrigerator-stable live influenza virus vaccine composition of any one of claims 1 to 14, wherein the potency is measured in fluorescent focus units.

16. An immunogenic composition comprising the refrigerator-stable live influenza virus 20 vaccine composition of any one of claims I to 15.

17. A vaccine comprising the immunogenic composition of claim 16. -115-