U.S. patent application number 14/638909 was filed with the patent office on 2016-02-25 for methods of producing influenza vaccine compositions.
The applicant listed for this patent is MEDIMMUNE, LLC. Invention is credited to John Michael Berry, Weidong Cui, Richard M. Schwartz, George Robert TRAGER, Vu Truong-Le, Luisa Yee.
Application Number | 20160051660 14/638909 |
Document ID | / |
Family ID | 34135009 |
Filed Date | 2016-02-25 |
United States Patent
Application |
20160051660 |
Kind Code |
A1 |
TRAGER; George Robert ; et
al. |
February 25, 2016 |
METHODS OF PRODUCING INFLUENZA VACCINE COMPOSITIONS
Abstract
Methods and compositions for the optimization of production of
influenza viruses suitable as influenza vaccines are provided.
Inventors: |
TRAGER; George Robert;
(Redwood City, CA) ; Schwartz; Richard M.;
(Bethesda, MD) ; Truong-Le; Vu; (Campbell, CA)
; Yee; Luisa; (Los Altos, CA) ; Berry; John
Michael; (Pacifica, CA) ; Cui; Weidong; (San
Jose, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MEDIMMUNE, LLC |
Gaithersburg |
MD |
US |
|
|
Family ID: |
34135009 |
Appl. No.: |
14/638909 |
Filed: |
March 4, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13779668 |
Feb 27, 2013 |
8986705 |
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14638909 |
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12330299 |
Dec 8, 2008 |
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13779668 |
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11668587 |
Jan 30, 2007 |
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12330299 |
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10788236 |
Feb 25, 2004 |
7262045 |
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11668587 |
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60450181 |
Feb 25, 2003 |
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Current U.S.
Class: |
424/186.1 |
Current CPC
Class: |
A61K 47/26 20130101;
A61K 47/02 20130101; C12N 2760/16251 20130101; A61K 2039/525
20130101; C12N 7/00 20130101; A61K 2039/5254 20130101; A61P 31/12
20180101; A61P 31/16 20180101; B01D 61/145 20130101; C12N
2760/16234 20130101; A61K 47/42 20130101; A61P 37/04 20180101; A61K
47/183 20130101; C12N 2760/16151 20130101; A61K 39/12 20130101;
B01D 61/147 20130101; A61K 35/76 20130101; A61K 39/145 20130101;
C12N 2760/16134 20130101 |
International
Class: |
A61K 39/145 20060101
A61K039/145; A61K 47/02 20060101 A61K047/02; A61K 47/42 20060101
A61K047/42; A61K 47/26 20060101 A61K047/26; C12N 7/00 20060101
C12N007/00; A61K 47/18 20060101 A61K047/18 |
Claims
1. (canceled)
2. A method of making a liquid refrigerator-stable live influenza
virus vaccine composition comprising: (a) passaging live influenza
virus through eggs; (b) harvesting the live influenza virus of (a),
thereby generating a viral harvest; (c) filtering the viral harvest
through a membrane; (d) adding a stabilizer consisting of arginine
and gelatin to the viral harvest; and (e) replacing some or all of
normal allantoic fluid with a buffer, which buffer comprises
sucrose, potassium phosphate and glutamate (SPG), thereby producing
a liquid refrigerator-stable live influenza virus vaccine
composition.
3. The method of claim 2, wherein the eggs are rocked during
passage in (a) by a rocking process.
4. The method of claim 3, wherein the rocking process comprises
tilting the eggs at a rate of about 1 cycle per minute or less,
about 5 cycles per minute or less, or about 10 cycles per minute or
less.
5. The method of claim 3, wherein the eggs are rocked for about 12
hours.
6. The method of claim 3, wherein the eggs are rocked for about 24
to 48 hours.
7. The method of claim 2, further comprising a secondary
incubation, wherein the eggs are rocked during the secondary
incubation.
8. The method of claim 3, wherein a TCID.sub.50 of the rocked eggs
is at least 0.4 log greater than a TCID.sub.50 of the same
influenza virus passaged through non-rocked eggs.
9. The method of claim 2, wherein at least one influenza virus is
selected from: an attenuated influenza virus, a cold-adapted
influenza 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.
10. The method of claim 2, wherein the influenza virus comprises
one or more influenza A viruses and/or one or more influenza B
viruses.
11. The method of claim 2, wherein the influenza virus comprises at
least two influenza virus strains.
12. The method of claim 11, wherein the influenza virus comprises
three influenza virus strains.
13. The method of claim 2, wherein the vaccine composition
comprises from 1% (w/v) to 5% (w/v) arginine and from 1% (w/v) to
4% (w/v) gelatin.
14. The method of claim 2, wherein the vaccine composition
comprises from 1% to 2% (w/v) arginine, 1% (w/v) gelatin and from
7% (w/v) to 10% (w/v) sucrose.
15. The method of claim 2, wherein the vaccine composition when
stored from 2.degree. C. to 8.degree. 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.
16. The method of claim 15, wherein the potency is measured in
fluorescent focus units.
17. An influenza virus vaccine composition produced by the method
of claim 2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/779,668, filed Feb. 27, 2013, entitled
"METHODS OF PRODUCING INFLUENZA VACCINE COMPOSITIONS," which is a
continuation of U.S. patent application Ser. No. 12/330,299, filed
Dec. 8, 2008, entitled "METHODS OF PRODUCING INFLUENZA VACCINE
COMPOSITIONS," now abandoned, which is a continuation of U.S.
patent application Ser. No. 11/668,587, filed Jan. 30, 2007,
entitled "METHODS OF PRODUCING INFLUENZA VACCINE COMPOSITIONS," now
abandoned, which is a continuation of U.S. patent application Ser.
No. 10/788,236, filed Feb. 25, 2004, entitled "METHODS OF PRODUCING
INFLUENZA VACCINE COMPOSITIONS," now U.S. Pat. No. 7,262,045, which
is a non-provisional and claims the benefit of U.S. Provisional
Application No. 60/450,181 filed Feb. 25, 2003, entitled "METHODS
OF PRODUCING INFLUENZA VACCINE COMPOSITIONS." These prior
applications are hereby incorporated by reference in their 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, those without adequate health care 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 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 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.
[0004] 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
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.
[0005] 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 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 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.
[0006] 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 System for the Production of
Influenza Virus, U.S. Ser. No. 60/375,675 filed Apr. 26, 2002,
PCT/US03/12728 filed Apr. 25, 2003 and U.S. Ser. No. 10/423,828
filed Apr. 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 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
[0007] The invention provides embodiments of methods of making one
or more 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
optionally comprises from about 28.degree. C. to about 40.degree.
C. or more, while in some embodiments, the temperature comprises
31.degree. C. or from about 30.degree. C. to about 32.degree. C.
The heating in such 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 composition).
[0008] 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
composition as well as the actual vaccine composition produced by
such embodiment.
[0009] 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 A virus strains and/or
influenza B virus strains and also optionally comprise wherein a
TCID.sub.50 of such rocked eggs is 0.4 log greater than a
TCID.sub.50 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.
[0010] The invention also comprises methods of making one or more
influenza virus 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.degree. C., and
recovering a plurality of influenza viruses. Such viruses
optionally comprise, e.g., an attenuated virus, a cold adapted
virus, a temperature sensitive 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 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.
[0011] 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.degree. C., recovering a 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).
[0012] In the various methods embodied herein, the influenza virus
composition is 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 stable from about 2.degree. C. to about 8.degree.
C., or are stable at 4.degree. 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 virus, measles virus,
Sendi virus, yellow fever virus, pIV, etc.). Thus, the methods and
compositions of the invention are also applicable to such other
viruses and/or to non-influenza viruses.
[0013] 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 virus, and filtering
the virus through a membrane, which composition has a first
TCID.sub.50, which first TCID.sub.50 is greater than a second
TCID.sub.50, which second TCID.sub.50 results from an influenza
virus not made by: passaging the virus through eggs, heating the
virus, and filtering the virus through a membrane.
[0014] Other aspects of the invention include an influenza virus
composition, 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.sub.50, which
first TCID.sub.50 is greater than a second TCID.sub.50, which
second TCID.sub.50 results from an influenza virus not made by:
passaging the influenza virus through eggs, wherein the eggs are
rocked during said passage.
[0015] Still other embodiments herein include an influenza virus
composition, wherein the composition is made by: introducing a
plurality of 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.degree. C., and recovering a plurality of influenza viruses,
which composition has a first TCID.sub.50, which first TCID.sub.50
is greater than a second TCID.sub.50, which second TCID.sub.50
results from an influenza virus not made by: introducing a
plurality 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.degree. C., and recovering a plurality of influenza viruses.
[0016] These and other objects and features of the invention will
become more fully apparent when the following detailed description
is read in conjunction with the accompanying figures appendix.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1: Displays M Genotyping after infection at 33.degree.
C. and 25.degree. C. in DEK TC-24.
[0018] FIG. 2: Displays a plaque assay, and data, showing the
different titers of 5:3 and 6:2 at 33.degree. C.
[0019] FIG. 3: Displays growth curves of 6:2 vs. 5:3
reassortants.
[0020] FIG. 4: Displays the M1 sequences of MDV-B and wild-type B
viruses.
[0021] FIG. 5: Displays the M2 sequences of MDV-B and wild-type B
viruses.
[0022] FIG. 6: Displays mutations on the two conservative sites in
MDV B-M1.
[0023] FIG. 7: Displays the growth curves of the B/HK 6:2 M1
mutations.
[0024] FIG. 8: Shows various CEK cell infections at different
MOIs.
[0025] FIG. 9: Shows a flow chart of potential microbial
contamination during vaccine production process.
[0026] FIG. 10: Illustrates temperature decay rates of individual
eggs via infra-red imaging.
[0027] FIG. 11: Illustrates thermal imaging of live, infertile, and
dead eggs.
[0028] FIG. 12: Illustrates a schematic flowchart of virus harvest
concentration.
[0029] FIG. 13: Displays comparison of the 5.sup.th wash with NAF
proteins.
[0030] FIG. 14: Displays an assay of an N/New Caledonia/20/99
1-.times.-Neat sample before concentration.
[0031] FIG. 15: Displays an assay of an A/New Caledonia/20/99
10.times.concentrated sample.
[0032] FIG. 16A and FIG. 16B: Display a comparison of 1.times. and
10.times. of A/New Caledonia/20/99; and 1.times.-W sample after 5
washes.
[0033] FIG. 17: Displays a comparison of A/New Caledonia/20/99
1.times. and 1.times.-W samples.
[0034] FIG. 18A, FIG. 18B and FIG. 18C: Display a comparison of
10.times. and 10.times.-W of A/New Caledonia/20/99; Permeate of
A/New Caledonia/20/99; and 5 washes of A/New Caledonia/20/99.
[0035] FIG. 19: Shows an analysis by SEC comparing times washed and
impurities removed.
[0036] FIG. 20: Displays 1.times.W and 10.times.-W comparison of
A/New Caledonia/20/99.
[0037] FIG. 21: Shows a 96-well plate assay of A/New
Caledonia/20/99.
[0038] FIG. 22: Shows a graph neuraminidase activity/virus
purification in retentate and permeate.
[0039] FIG. 23: Displays RHPLC of Control, 10.times., 10.times.-W,
and 1.times.-W.
[0040] FIG. 24: Shows a graph of Control, 10.times., 1.times.-W and
10.times.-w samples.
[0041] FIG. 25: Displays RHPLC of permeate and washes 1 to 6.
[0042] FIG. 26: Shows a graph of RHPLC ovomucoid removal (peak
area).
[0043] FIG. 27: Shows a graph of RHPLC of lysozyme removal (peak
area).
[0044] FIG. 28: Shows a graph of RHPLC of conalbumin removal (peak
area).
[0045] FIG. 29: Shows a graph of RHPLC of ovalbumin removal (by
peak area).
[0046] FIG. 30: Shows a graph of ovalbumin analysis by Agilent
2100.
[0047] FIG. 31: Is a Western blot SDS-PAGE gel of anti-A/New
Caledonia.
[0048] FIG. 32: Displays assays of 10.times.-W, sample after 5
washes of A/New Caledonia/20/99.
[0049] FIG. 33: Shows a graph of RNA analysis by RTPCR.
[0050] FIG. 34: Shows monitoring of A/Beijing-cell culture
propagation by SEC.
[0051] FIG. 35: Shows cell culture harvest of A/Beijing in Vero
cells.
[0052] FIG. 36: Shows concentration of 2 liters of A/Panama cell
culture.
[0053] FIG. 37: Shows concentration of 2 liters of B/Hong Kong cell
culture down to 10 ml.
[0054] FIG. 38: Shows 4 graphs of stability of exemplary virus
storage formulations.
[0055] FIG. 39: Shows a graph of stability of exemplary virus
storage formulations with various virus strains.
[0056] FIG. 40: Shows a graph of stability of exemplary virus
storage formulations with various virus strains.
[0057] FIG. 41A, FIG. 41B and FIG. 41C: Show stability of exemplary
virus storage formulations with differing citrate
concentrations.
[0058] FIG. 42A, FIG. 42B and FIG. 42C: Show stability of exemplary
virus storage formulations with differing EDTA concentrations.
[0059] FIG. 43: Displays stability of unpurified virus harvest
formulations with different virus strains over 9 months.
[0060] FIG. 44: Illustrates initial potency loss of formulations
associated with phosphate buffer.
[0061] FIG. 45: Gives a global picture of stability slopes of
various formulations at 6 months.
[0062] FIG. 46: Gives a global picture of stability slopes of
various formulations at 6 months.
[0063] FIG. 47: Shows stability of various formulations with
gelatin and PVP/EDTA.
[0064] FIG. 48: Shows stability of various formulations with
histidine at different pH.
[0065] FIG. 49: Shows stability of various formulations with
different amounts of sucrose.
[0066] FIG. 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).
[0067] FIG. 51: Histogram derived from plotting absorbance readings
versus frequency of values.
[0068] FIG. 52: Shows a generic process for generating 6:2
influenza reassortants.
DETAILED DESCRIPTION
[0069] 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 selection, potency
assays, and many additional features as described in more detail
herein.
[0070] 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 and/or compositions herein are performed or
exist, e.g., as outlined in Table 1, in other embodiments, one or
more steps are optionally, e.g., omitted, changed (in scope, order,
placement, etc.) or the like.
[0071] 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 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, all of the steps, or any and all various combinations of the
steps.
[0072] 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 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.
[0073] 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 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 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 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
interdependence of steps, etc. should be made.
DETAILED DESCRIPTION OF STEPS
[0074] 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 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 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.
Group 1
[0075] 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 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 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 A/Leningrad/134/17/57 donor strain,
Vaccine (2002) 20:2082-2090. Reassortment of influenza strains has
also been shown with plasmid constructs. See, "Multi-Plasmid System
for the Production of Influenza Virus," cited above.
[0076] 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
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
reassorted, the viruses can also be selected (e.g., to find the
desired 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.
[0077] 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., 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 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.
[0078] Thus, as detailed herein, various embodiments of the
invention comprise steps to reduce the reassortment bias. Namely,
cloning of reassortants is done in eggs (e.g., at 33.degree. C.)
rather than in cell lines, or is done in cell lines, but at, e.g.,
25.degree. C.
Optimization of Reassortment
[0079] 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 optimization
techniques are typically embodied with reassortment of influenza B
strains and are typically done in cell culture, e.g., CEK
cells.
[0080] 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 25.degree. C. and 33.degree. 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.
[0081] Thus, typical embodiments of the steps in Group 1 of the
invention comprise 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.degree. 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 reassortment mixes necessary in
non-optimized systems.
Selection and Cloning of Reassortments
[0082] 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 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 influenza B reassortments done in eggs (which are
neutral in terms of selection pressure against the M gene in
influenza B strains) at 33.degree. C. or, alternatively, in CEK
cells at 25.degree. C. See FIG. 52.
[0083] 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.
[0084] Yet other embodiments of the invention in Group 1 include
cloning of the 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
reassortments are cloned out in eggs at 33.degree. C. A strain
reassortments, on the other hand, are optionally cloned out and
selected at the same time in CEK cell culture.
[0085] 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.degree. 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 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.degree. 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 (containing
wild-type M gene) at 33.degree. 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.degree. C. Therefore, in
some embodiments herein 25.degree. C. is used for 6:2 cloning in
CEK cells in the MVS process. See, FIGS. 1 through 8. From the
Figures it can be seen that plaque assays show that the titer of B
virus 6:2 at 33.degree. C. is at least 2 log 10 lower than
respective 5:3 at low MOI, while 6:2 grows to the same level as 5:3
at 25.degree. C. The growth defect of 6:2 at 33.degree. 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
single mutation of Valine to wild-type conservative Methionine on
the 6:2 M1 gene is able to reverse the growth defect of 6:2 in CEK
cells at 33.degree. C.
Characterization of Reassortments
[0086] 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 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
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.
[0087] In order to reduce the number of RT-PCR reactions required
to genotype all 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.
[0088] 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.degree. C., which enhances the resolution between MDV B and
wild-type strains' eight gene segments.
[0089] 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
(.about.98%) between the two sets of data, thereby validating the
SSCP/CE assay. Furthermore, it was shown that the SSCP/CE assay was
capable of detecting a single nucleotide substitution within the M
gene segment of influenza B virus.
Prevention of Bacterial Contamination
[0090] 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 1 and can be included in Groups 1, 2,
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.
[0091] Many current influenza vaccine production strategies,
including some 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., FIG. 9, which outlines one possible
example of a virus production flowchart and possible areas of
contamination therein. Unfortunately, the chicken eggs may have
some 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.degree. 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 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.
[0092] 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 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) testing is
performed, e.g., at virus harvest (see FIG. 9) prior to use in the
blending and filling to ensure microbial-free product.
[0093] 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 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.
[0094] 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 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 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 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 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 be a
measure of total viable organisms as required by the FDA), review,
expectations/acceptability; compliance requirements such as vendor
audits, vendor support (instrument IOPQ or instrumentally observed
perspectival quality), software validation, and documentation; and
business requirements such as industry trends, costs of
implementation, cost per test, etc.
[0095] 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, 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.degree. C. 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 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 FIGS. 10 and 11.
[0096] In other embodiments herein, the current invention utilizes
an alternative to 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 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,
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 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 MPN 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.
[0097] As an alternative to the traditional compendial 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. gallisepticum and human M.
pneumoniae, i.e., all 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 16 s and/or 23 s rRNA) such as tubercle
bacterial and non-tuberculous mycobacteria (e.g., M. abscessus and
M. avium). Some embodiments herein utilize standardized nucleic
acid amplification-based kits that rapidly detect tubercule
bacteria and non-tuberculous mycobacteria, etc.
Group 2
[0098] 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 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 (MVS) or master virus
seed, which, in turn, is further expanded to generate a master
working virus strain (MWVS) or manufacturer's working virus seed.
Many aspects of purification of virus particles from 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., 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.
Group 3
[0099] 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
the incubation of virus infected eggs) and the harvesting and
clarification of influenza virus from the allantoic fluid of the
eggs.
[0100] 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 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 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
[0101] One drawback in culturing some types of influenza strains
(e.g., especially 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.sup.8 or 10.sup.9 log (i.e.,
10.sup.8 or 10.sup.9 virus particles per milliliter) and a second
strain (e.g., an influenza B strain) only produces 10.sup.7 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
held until the second strain is grown in a second production,
etc.
[0102] 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 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 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 strain increased by 0.4 log over a control group of eggs
which was not rocked.
Filtering and Warming
[0103] 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 invention,
virus particles are harvested from 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 (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.
[0104] The effects of viral allantoic fluid (VAF) temperature and
warming time 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.times.H1N1, 1.times.H3N2, 2.times.B) being
filtered under similar conditions.
[0105] Three independent assays (TCID.sub.50, 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.degree. C. up to 60 minutes prior to filtration) to the
filtration process reduced the potency losses to acceptable levels
(0-0.3 log.sub.10 TCID.sub.50) compared to the sterilizing-grade
filtration performed without warming step for A/Sydney/05/97. In
other embodiments, the warming temperature is optionally over
28.degree. C., or from 28 to 36.degree. 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 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 the
warming step is an acceptable process step for sterilizing-grade
filtration of cold-adapted influenza viruses (CAIV).
[0106] As described herein, the current FluMist.TM. 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 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 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 considered
limiting on the current invention unless specifically stated to be
so.
[0107] 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 log.sub.10 TCID.sub.50/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.
[0108] The purpose of the first set of experiments shown below was
to test the effect of VAF temperature on virus potency loss during
filtration. The second part of the study was designed to define the
appropriate warming time of VAF prior to filtration. The
cold-adapted (ca) A/Sydney/05/97 virus strain (H3N2 type) was used
as a model strain to determine the warm-up conditions because, as
stated previously, large potency losses have been observed during
filtration of this strain.
[0109] 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 virus strains. Five CAIV
strains (2.times.H1N1, 1.times.H3N2 and 2.times.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. 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 1/10.sup.th scale filtration
was performed (i.e., about 3 L).
[0110] Virus propagation for such temperature/filtration steps can
be performed 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.
[0111] Samples from all stages of the experiment were assayed for
potency by measuring the Tissue Culture Infectious Dose
(TCID.sub.50) in a manual assay (see below for other aspects of
TCID.sub.50 measurements). Neuraminidase activity (NA) and
Hemagglutinin activity (HA) were also measured.
[0112] 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.sub.50/mL), neuraminidase (NA) and hemagglutinin
(HA) activity losses.
[0113] 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.degree. C. The total warming time was defined as the time
the VAF was in the 33.+-.1.degree. C. water bath and consisted of
the warm-up time (from 15.+-.3.degree. C. to 28.+-.3.degree. C.)
and warm-hold time (time greater than 28.degree. C., e.g., at a set
point).
[0114] 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) 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.
[0115] In the current typical manufacturing processes, after
harvest, VH is centrifuged, stabilized and frozen for further
transportation. In these examples, a sample of 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.
[0116] Part 1: Temperature Effect on A/Sydney/05/97 Virus Titer
Changes During Filtration
[0117] 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.+-.3.degree. C. (refrigerator),
20.+-.3.degree. C. (bench top) or 31.+-.3.degree. C. (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.sub.50 results of
filtration under different conditions were compared to each other
and the control.
[0118] To determine the effect of VAF temperature on potency loss,
VAF was exposed for 60 minutes prior to filtration to
5.+-.3.degree. C., 20.+-.3.degree. C. or 31.+-.3.degree. C. The
potency change, neuraminidase and hemagglutinin activity difference
between centrifuged stabilized and post-filtration material with
different temperate treatment is summarized in Tables 5-10. As can
be seen, filtration of cold (5.+-.3.degree. C.) and room
temperature (20.+-.3.degree. C.) VAF resulted in potency losses
between 0.7 and 1.0 log.sub.10 TCID.sub.50/mL (see, Tables 5 and
8). However, there was no post filtration titer loss (compared to
the centrifuged stabilized VAF) when VAF was warmed up to
31.+-.3.degree. C. 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.degree. C. compared to the levels observed in cold
and room temperature filtrations. See, Tables 6 and 9. Addition of
the warm-up step also reduced hemagglutinin activity losses. See,
Tables 7 and 10.
[0119] Part 2: Determination of the Warming Time Required for
Acceptable Filtration Potency Losses of A/Sydney/05/97
[0120] In order to determine the necessary warming time, a series
of experiments were conducted with VAF warmed to 31.+-.3.degree. C.
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.degree. C.). VAF in the bottle was mixed by inverting every
10 minutes. After temperature treatment it was filtered through
Sartoclean CA and Sartopore 2 filters. In the control experiment,
representing the current manufacturing process, VAF was centrifuged
and stabilized. TCID.sub.50 results of filtration under different
conditions were compared to each other and the control.
[0121] 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.+-.3.degree. 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.
[0122] The data demonstrate that the exposure of VAF to
31.+-.3.degree. C. reduced post filtration virus potency losses and
allowed partial recovery of neuraminidase and hemagglutinin
activities. See, Tables 11-13. The temperature of un-stabilized VAF
at the beginning of the experiments (post harvesting and prior to
warming) was 15.+-.2.degree. C. The warm up time required for 1-1.5
L of VAF to reach 31.+-.3.degree. C. was about 20-30 minutes. Thus,
a 30-minute total VAF warming time results in 0-10 minutes VAF warm
hold time at 31.+-.3.degree. C.
[0123] 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 (warm-hold of 30-40 and 60-70 minutes at
31.+-.3.degree. C.) experiments, post filtration virus potency and
HA and NA levels were similar to the control (centrifuged
stabilized VAF) samples. See Tables 14-19.
[0124] Part 3: Effect of Warming on Other Strains
[0125] A series of experiments was conducted with 5 strains other
than A/Sydney/05/97, i.e., 2.times.H1N1, 1.times.H3N2, and
2.times.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.degree. 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 filtered immediately after
stabilization with SPG at room temperature. TCID.sub.50 results of
filtration under different conditions were compared to each other
and control experiments.
[0126] For the additional 5 cold-adapted influenza virus strains
tested, a short exposure (total warming time of 60 minutes) to
31.+-.3.degree. C. (warm-hold time of 30-40 minutes at set point
temperature) contributed to the reduction of post filtration
potency losses 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 (TCID.sub.50/mL),
neuraminidase and hemagglutinin levels from these experiments are
summarized in Tables 20-25, below.
[0127] As can be seen from the tables, the aspect of the current
invention comprising warming to 31.+-.3.degree. C. or optionally
even up to 36.degree. 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 log.sub.10 TCID.sub.50/ml) for A/Sydney/05/97. In
the control experiments, when A/Sydney/05/97 stabilized viral
harvest was filtered without warming, titer losses were up to 1.0
log.sub.10 TCID.sub.50/ml.
[0128] 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.+-.3.degree. C. (warm-hold of 30-40
minutes at 31.+-.3.degree. C.) either decreased the potency losses
or did not contribute to additional potency losses during
filtration. In all experiments, the post filtration titer loss was
not higher than 0.3 log TCID.sub.50/ml. The reduced activity losses
of the viral surface proteins (neuraminidase and hemagglutinin) of
warmed filtered VAF compared to not warmed, support the decreased
potency loss data shown by TCID.sub.50 assay.
[0129] 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 potency losses of 60 minutes
(time to warm up the VAF to 31.+-.3.degree. C. 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 are
optionally used, without deviating from the invention.
Group 4
[0130] 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
containing solutions. In some embodiments, the final viral
solutions/vaccines comprising live viruses are stable in liquid
form at 4.degree. C. for a period of time sufficient to allow
storage "in the field" (e.g., on sale and commercialization when
refrigerated at 4.degree. C., etc.) throughout an influenza
vaccination season (e.g., typically from about September through
March in the northern hemisphere). Thus, the virus/vaccine
compositions are desired to retain their potency or to lose their
potency at an acceptable rate over the storage period. 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 0.75 log
were allowed, a rate of less than or equal to 0.09 log/month would
be sufficient to allow stability of materials stored continuously
at refrigerator temperature (e.g., 4.degree. C.). In other
embodiments, such solutions/vaccines are stable in liquid form at
from about 2.degree. C. to about 8.degree. C. In yet other
embodiments, the solutions/vaccines are stable at room temperature.
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
[0131] In some embodiments herein, virus harvests are optionally
concentrated 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 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, 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.
[0132] Prior traditional viral concentrations from egg NAF, etc.
were done via 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 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 addition to, TCID.sub.50 measurement
of such solutions. Such tracking is especially useful for virus
produced in eggs. Additionally, concentrated and purified virus
material can optionally be a starting material for generating pure
HA, NA and other viral components for 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.
[0133] 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.
[0134] To illustrate the concentration of a virus harvest sample,
an influenza harvest 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.sub.50).
Hemeagglutination by HA assay, neuraminidase activity, SEC
analysis, NAF by RHPLC, and RNA by RTPCR were also done.
[0135] 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 virus
particles from 2 liters to 100 ml 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.
[0136] 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 Company (Denver, Colo.). The
instrument used for concentration was from Amersham Biosciences
(A/G Technology Corporation), and was a MidJet 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 that use or recitation of particular models,
producers, etc. of equipment are not to be construed as limiting
upon the current invention. The buffer used for washing in this
example was 1.times.-SPG.
[0137] 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.times.300 mm. The buffer 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 Waters and the column was a YMC C4 (reverse
phase), 2.1.times.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 Conditions--Variable gradient, 13-100% B; Flow Rate: 0.2
ml/min; Column Temp, --45 C; Injection Volume--50 ul; and
Detection--214 nm.
[0138] As shown in FIG. 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 hours,
150 ml of the 1.times. sample was reduced to 15 ml of 10.times.
concentrated sample (Step 2). The permeate was collected separately
and stored for further analysis. For analytical characterization, 4
ml of the 10.times. sample was removed (Step 3). The remaining 11
ml of the 10.times. sample was diluted to 110 ml with 1.times.-SPG,
and was further concentrated down to 11 ml by removing the
1.times.-SPG as a permeate. The permeate carries most of the
impurities from the retentate. This step was repeated five times
with 1.times.-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 1.times.-SPG to
110 ml to bring the concentration back to 1.times.. At step 6, 10
ml of the 1.times.-W was reserved for the assay. The remaining 100
ml of the 1.times.-W was further concentrated down to 10.times.-W
(Step 7). This concentrated sample was aliquoted into 1 ml
quantities for further analysis.
[0139] All the samples were analyzed by SEC chromatography. The
Ultrahydrogel 100 column was used for the analysis with DPBS as a
solvent. Even though the data was 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 21 min), and one for
impurities group-2 (retention time 21 to 27 min). Three NAF
proteins Ovalbumin, Conalbumin and Ovomucoid elute around retention
time 18-21 min. See FIG. 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 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.
[0140] SEC Analysis and CELISA
[0141] The neat sample, 1.times. showed the virus peak at 11.1
minutes with a peak area 1,221. See FIG. 14. However, the
concentrated 10.times. sample showed a peak area 11,192, see, FIG.
15, and the increment in the peak are was about 9.16 times compared
to 1.times.. 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 26, FIG. 16A and FIG. 16B. The impurities
group-1 and group-2 showed increment in the peak size between
1.times. and 10.times. (Table 26). Correspondingly the TCID.sub.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 1.times. sample to 10.times. sample retained
the infectivity quite well.
[0142] After the 5th wash with 1.times.-SPG, the virus peak area of
the sample 1.times.-W, retained as 1005 compared to 1221 before the
wash (Table 26). Recovery by peak area between 1.times. and
1.times.-W was about 82% (1005/1221). By comparing the 1.times. and
1.times.-W chromatogram (see FIG. 17), it shows that impurities
group-1 and group-2 were significantly reduced (Table 26). The
1.times.-W showed a small decrease TCID.sub.50 value (Table 27,
1.times.: 9.1, 1.times.-W: log 8.9). The recovery of infectivity
was about 98.99% between 1.times. and 1.times.-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 10.times. and 10.times.-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
10.times.: 11,192 was reduced to 10.times.-W: 10, 282 (Table 26,
91.86% by peak area). The TCID.sub.50 was changed from log 10.0
(10.times.) to log 9.9(10.times.-W) with the recovery of 99.56%
(Table 27).
[0144] By comparing the 1.times.-W and 10.times.-W chromatogram,
the peak area increased by 10 times. See Table 26, 1.times.-W: Peak
Area: 1005 and 10.times.-W: 10,282. The TCID.sub.50 value also
increased one log (Table 27, 1.times.-W: log 8.9 and 10.times.-W:
log 9.9). Since, the 10.times.-W was concentrated from 1.times.-W
in one step, no loss in either activity or in the peak area was
seen (10.times.-W: Peak area 10282 and 1.times.-W Peak Area
1005).
[0145] The permeate showed virus peak at 10.4 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 1.times. 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 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 FIG. 19. Group-1 and group-2
impurities were significantly removed after the 2nd wash. After the
5th wash the curves reached a plateau. Even after the 5th wash, the
samples 1.times.-W and 10.times.-W showed impurities group-1 and
group-2 in a very low amount. See FIG. 20. The identity of the peak
at 19.208 min was confirmed as ovalbumin by isolating from the
10.times.-W sample. SDS-PAGE also confirmed the result.
[0147] HA Assay
[0148] The sample 1.times. and 1.times.-W showed HAU 1024. See FIG.
21. The concentrated, but not washed, 10.times. showed at HAU 8192.
However, 10.times.-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. 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 FIG. 12.
[0149] NA Assay
[0150] The neuraminidase assay illustrated that 10.times. diluted
back to 1.times. shows some decrease in activity in comparison with
1.times.. See FIG. 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 of NA in the permeate. Samples 1.times.-W and
10.times.-W diluted back to 1.times.-W retained the activity at the
same level. This was because the sample 10.times.-W was
concentrated directly from 1.times.-W. All the washes have the
activity below the detection level.
[0151] RHPLC
[0152] 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 FIG. 23. The 10-.times. sample, before any wash, showed
all the egg proteins. This matches the retention time of the
control sample. Also 10.times. showed unidentified viral protein
peaks labeled as U1, U2 and U3. Completely washed samples
10.times.-W and 1.times.-W retained the viral proteins U1, U2 and
U3. The 10.times. and 10.times.-W samples contained the same amount
of U1, 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 ovomucoid,
lysozyme and conalbumin have been completely removed from 10.times.
by washing with 1.times.-SPG for five times. Notably, in contrast,
the most obvious protein peak is ovalbumin, which is still eluting
along with 10.times.-W and 1.times.-W samples. Even though
10.times.-W and 1.times.-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 in the bar graph form as in FIG. 24.
[0153] The permeate and all the washes were checked by RHPLC. See
FIG. 25. The permeate contains all the NAF proteins and other
unidentified peaks. Ovomucoid was removed by two washes (see FIG.
26); lysozyme by 2 washes (see FIG. 27); conalbumin by two washes
(see FIG. 28); and ovalbumin was depleted gradually, but about 5%
remained even after wash number. 6. See FIG. 29.
[0154] Agilent Bioanalyzer
[0155] Simultaneously, ovalbumin was estimated by Agilent
Bioanalyzer as shown in FIG. 30. Just by the concentration, without
any washing step, ovalbumin was considerably removed from 1.times.
to 10.times.. The first permeate carried most of the ovalbumin.
RHPLC showed ovalbumin in all the washes, but in the Bioanalyzer
analysis it reached below the detection limit. The 10.times.-W
sample was diluted ten times to reach the concentration close to
1.times.-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.
[0156] SDS-PAGE and Western Blot
[0157] In comparison to 1.times. (lane 2), 10.times. (lane 9)
contains more intense multiple silver stain bands. See FIG. 31.
10.times.-W (lane 10) showed fewer number of bands compared to
10.times.. This was due to the removal of NAF proteins and other
impurities. Similarly 1.times.-W (lane 8) appears cleaner than
1.times.. Samples 1.times., 10.times. diluted to 1.times. (3rd
Lane), and 10.times.W diluted to 1.times.-W (4th lane) contain the
same quantity of virus except different degrees of improvement in
the removal of impurities. Obviously, 10.times.-W diluted to
1.times.-W shows clearer viral protein bands. However, this sample
still contained an ovalbumin band. This is compared with NAF
proteins in lane 6. The 10.times.-W sample was further purified by
analytical SEC column and the fractions were collected. See FIG.
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 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, HA.sub.0 and HA.sub.2 or M
protein. See FIG. 31.
[0158] RTPCR
[0159] RTPCR showed that the RNA was about a log higher between
1.times. and 10.times.. See FIG. 33. Similarly, there was about a
ten-fold increase in the viral RNA between 1.times.-W and
10.times.-W. This indicated that most of the virus was retained
during the concentrations step. Permeate does not have any
detectable viral RNA, but the 1.times.-SPG washes showed a very
small amount of RNA. This may be due to small amount of virus
undergoing shearing during the circulation or some viral RNA bound
to the filtrate and released latter slowly during the washing
cycles.
[0160] 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 material by 1.times.-SPG improved the quality of the
virus by removing other impurities. Even after the 5th wash a small
amount of ovalbumin was strongly bound to the virus. This may 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.
[0161] 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/H1N1) used as is;
A/Panama (A/H3N2) concentrated from 2 L to 100 ml or 20.times.; and
B/Hong Kong concentrated from 2 L to 10 ml or 200.times.. It will
be appreciated that since virus yields from 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
[0162] FIGS. 34-35 show monitoring of A/Beijing cell culture
propagation by SEC (FIG. 34) and A/Beijing Vero cell culture
harvest (FIG. 35). As can be seen SEC is an 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). FIG. 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
TCID.sub.50 of the 1.times. mixture was non detectable, but the
TCID.sub.50 of the 20.times. mixture was 4.4. There was a peak area
ratio of 20.times. to 1.times.. 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.
FIG. 37 shows concentration of 2 liters down to 10 ml of a Vero
cell grown culture of B/Hong Kong. At 1.times. the log 10
TCID.sub.50/ml was 4.7, while at 18.8.times. it was 5.8 (the
theoretical for such being 5.95) and at 200.times. it was 6.95 (the
theoretical of which being 7.00).
[0163] 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.
Stabilizers/Buffers
[0164] The invention comprises compositions of virus solution and
methods of creating the same. Such compositions optionally comprise
various dilutions of NAF (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 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 4.degree. C.,
5.degree. C., 8.degree. C., from about 2.degree. C. to about
8.degree. C. or greater than 2.degree. C., etc.). Preferred
embodiments show no decrease in potency over the desired storage
period. Other embodiments show less than 10% decrease, less than
5%, less than 4%, less than 3%, less than 2%, 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, 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 FluMist.RTM. applicator/accuspray device or other
similar device.
[0165] 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 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. ethylenediaminetetraacetic (EDTA) and/or
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.
[0166] 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 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 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 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 from, e.g., 25 to 100 millimolar.
In preferred embodiments, sucrose is present at a concentration
ranging from about 100 millimolar to 350 millimolar.
[0167] In many virus solutions/vaccine solutions a base solution of
SPG (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
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 (polyoxyethylene-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 any solution
containing only one of the components, each solution in turn being
more stable than a solution containing none of the added
components.
[0168] 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 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 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 5.degree. C. is shown in Table
29.
[0169] Various formulations of the invention were tested for their
stability over a variety of months and temperatures. For example,
Table 30 illustrates 12 different 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). FIG. 38 graphs the
results of 4 exemplary formulations with the B/Hong Kong strain
used. Table 35 shows 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 FIGS. 39 and 40. Tables
40 and 41 and FIG. 41A, FIG. 41B and FIG. 41C 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 FIG. 42A, FIG. 42B and
FIG. 42C show a similar analysis, but with varying concentrations
of EDTA. Exemplary formulations from the above examples were
subjected to further testing which is 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.
[0170] To further illustrate the stability of several monovalent
formulations herein, compositions comprising 60% allantoic fluid
were tested for stability. Samples were stored at 5.degree. 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 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 for all strains test to almost consistently meet a
criterion of 0.5 log loss in 7 months at 5.degree. 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, gelatin and other components for
the 13 different formulations. FIG. 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% gelatin, 2.5 mM EDTA; VH, 10% sucrose, 2% arginine, 2% gelatin,
citrate buffer; and, VH, 10% sucrose, 2% arginine, 2% gelatin,
histidine buffer.
[0171] 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 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 virus/vaccine solutions,
especially at desired temperatures (e.g., 4.degree. C., from about
2.degree. C. to about 8.degree. 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)
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
typically comprise about 2% arginine, about 2% gelatin, about 1%
PVP, about 1% dextran, about 2.7 mM EDTA, and about 100 mM
histidine. The numbers in Table 49 correspond to the formulations
displayed in FIGS. 44-46.
[0172] The diluted NAF embodiments of the current invention are in
comparison to 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 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.degree. C.
and 8.degree. C., e.g., 4.degree. C. 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 different influenza
strains: a H1N1 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.RTM.). In order to mimic a
likely manufacturing process, the samples were frozen at
-25.degree. C. for at least 6 days as an initial step.
[0173] 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.+-.0.016 for 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 observed is though to be attributed to
freeze-thaw and/or blending loss. Table 52 shows a similar
comparison, but with histidine in the FluMistO formulation, which
gave rise to a better stability with no initial potency loss
observed.
[0174] FIG. 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 FIG. 44 are those listed in
Table 49. FIG. 45 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.
FIG. 46 gives a different view of similar data (i.e., week rather
than month). FIG. 47 illustrates a second study which produced
results illustrating the stability of a 10/2/2+histidine
formulation with 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. FIG. 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 are also
included embodiments of the invention. FIG. 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
FIG. 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 buffering additive and/or
arginine as a stabilizer and/or dextran and/or PVP in place of
gelatin.
[0175] Other embodiments of the current invention are optionally
stabilized through use of ultrafiltration/concentration of the
virus/vaccine solution. Such ultrafiltration is typically an
alternate means of achieving solution stability as opposed to
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 the filtration concerns stability, etc. and the
viruses are kept during the filtration. See above.
Potency Assays
[0176] 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 vaccines).
Such method is a simpler and faster alternative to the more
traditional Median Tissue Culture Infective Dose (TCID.sub.50)
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 formalin 16-18 hours post-infection and reacted with
influenza virus-specific monoclonal antibody (Mab). Virus antigen
bound Mab is then detected using anti-mouse IgG.about.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 calibrators
with known log.sub.10 TCID.sub.50 values obtained with a validated
TCID.sub.50 potency assay. CELISA is shown to be linear (r.sup.2
greater than or equal to 9.95) in the range 4.9-6.7 log.sub.10
TCID.sub.50. Between-day, between-analyst, between-plate, within
plate (residual) variability (Standard Deviation in
log.sub.10TCID.sub.50) were 0.06, 0.02, 0.05 and 0.03 respectively.
The potency of several vaccine and wild-type influenza A/H1N1,
A/H3N2 and B strains measured by CELISA are comparable (.+-.0.3
log.sub.10TCID.sub.50) to the potency measured in parallel by the
validated TCID.sub.50 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.sub.50 potency
assay. CELISA is optionally used in place of, or in addition to
other methods of potency assay (e.g., FFA and TCID.sub.50, see,
below).
[0177] The Median Tissue Culture Infective (or Infectious) Dose 50%
(TCID.sub.50) 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 live, attenuated vaccine
(or in other similar vaccines).
[0178] In other typical embodiments, potency assays of the virus
solutions optionally comprise fluorescent focus assays (FFA) as
opposed to common TCID.sub.50 assays 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.
TCID.sub.50 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 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 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.
[0179] 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. 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.
[0180] In other embodiments, the FFAs (and optionally other assays
herein) optionally use a universal reagent (or universal antigen)
which is not specific for specific 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.
[0181] Other embodiments herein comprise viral potency
determination using a cell-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.sub.50
titers for influenza B strains, CFA titers have not shown
significant differences from TCID.sub.50 (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 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 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 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.degree. C. followed by fixation and incubation at
room temperature for 15 minutes. A primary antibody incubation for
60 minutes at 37.degree. C. is then followed by a secondary
antibody incubation for 60 minutes at 37.degree. 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.
[0182] Semi-Automated TCID.sub.50 Assays
[0183] As stated above, some embodiments of the current invention
comprise TCID.sub.50 assays and various modifications, etc.
thereof. For example, some embodiments comprise a semi-automated
version such as illustrated by the following descriptions.
[0184] A comparison of Manual and Semi-Automated Median Tissue
Culture Infective Dose (TCID.sub.50) assays for Potency Measurement
of a live, attenuated influenza virus vaccine, e.g., FluMist.RTM.,
or other similar vaccines, is given in this section. The
TCID.sub.50 potency assay is optionally used for potency
measurement of FluMist or other similar vaccines. A semi-automated
TCID.sub.50 potency assay is described in which two labor-intensive
steps of the validated manual potency assay are improved. These are
(i) use of an 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 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 cytopathic effect (CPE) in MDCK cells.
[0185] The semi-automated TCID.sub.50 potency assay used in some
embodiments herein was developed and validated to demonstrate
precision (repeatability: <0.25 log.sub.10TCID.sub.50;
intermediate precision: S.sub.(Day)<0.3 log.sub.10TCID.sub.50;
SD.sub.(Analyst) and SD.sub.(Instrument)<0.4
log.sub.10TCID.sub.50; and reproducibility at the 90% Confidence
Interval.+-.0.3 log.sub.10TCID.sub.50), linearity, accuracy and
range (Slope 1.+-.0.1). The semi-automated TCID.sub.50 potency
assay using an automated pipetting station and MTT dye was shown to
provide equivalent results to the validated manual TCID.sub.50
potency assay (at the 90% Confidence Interval.+-.0.3
log.sub.10TCID.sub.50). 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., FluMist.RTM. productions.
These improvements also increase the testing throughput.
[0186] Infectivity/Potency (Monovalent) Assay Validation
[0187] A semi-automated version of the current manual method for
potency determination of monovalent influenza strains is optionally
used for manufacture of FluMist.TM. vaccine and other similar
vaccines. The semi-automated potency assay 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 CPE enhances assay consistency and
throughput by elimination of the microscopic detection.
[0188] Some steps/aspects of the semi-automated assay are similar
to more traditional TCID.sub.50 assays, while other steps/aspects
are quite different. The assay steps include preparation,
incubation and washing of assay plates containing Madin-Darby
canine kidney (MDCK) monolayers, infection and post-infection
incubation of the assay plates, and calculation of the potency
based on the number of CPE positive wells and sample 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 (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.
[0189] The traditional median tissue culture infectious dose
(TCID.sub.50) assay is a cell-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 MDCK 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 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.
[0190] 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 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 samples per 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., FluMist.TM.
vaccine, etc.).
[0191] 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.sub.50 Potency Assay for Influenza Virus
Monovalent. The semi-automated assay uses the Skatron.TM. Cell
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.RTM. 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 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 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 Excel.TM. Macro
program (or other similar program), to identify and count the CPE
positive or negative wells and calculate the TCID.sub.50 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 FIG. 50). The number of wells
showing CPE at each dilution is then used to calculate the titer
(log 10 TCID.sub.50/mL) 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.
[0192] Automation of Cell Washing Steps Using Skatron.TM. Cell
Washer
[0193] In a manual assay, plates containing MDCK cell monolayers in
96-well 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 then incubated at 33.+-.1.degree. C. and 5.+-.1%
CO.sub.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 2000 .mu.L of fresh VGM using a hand-held
multichannel pipettor. This process is labor-intensive and time
consuming when large numbers of plates are processed.
[0194] 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 cell
plate washing steps using the Skatron.TM. 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.degree.
C. and 5.+-.1% CO.sub.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
transferred back into the incubator. The performance of the
Skatron.TM. Skanwasher in these wash steps is shown to be
acceptable for use in the cell washing steps. The dispensing
precision for the 200 .mu.L 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 Skatron.TM.
Skanwasher provides acceptable performance, while improving the
ease 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.
[0195] Automated Serial Dilution and Virus Inoculation with Matrix
SerialMate.RTM. Multichannel Pipetting Station
[0196] The serial dilution and the virus inoculation steps of the
traditional manual TCID.sub.50 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 carefully executed, because pipetting errors at any
one dilution may be propagated and magnified through the subsequent
series. The subsequent virus inoculation step involves 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 one day and, thus, the throughput of the
entire process.
[0197] Use of the Matrix SerialMate.RTM. 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.RTM. pipetting
station is a bench top liquid handling station equipped with a
12-channel nozzle head which can aspirate and dispense volumes in
the range of 5 .mu.L-225 .mu.L. The unit is small enough to fit in
a standard 4- or 6-foot biosafety cabinet and is easy to use. The
Matrix SerialMate.RTM. provides precision better than 0.5 .mu.L,
and accuracy better than 1.0 .mu.L for delivery volumes of 5
.mu.L-225 .mu.L. This corresponds to a precision better than
.+-.1.7% and accuracy better than .+-.3.3% for the 30 .mu.L
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.
[0198] Description of MTT Dye-Based Detection
[0199] The final step in a TCID.sub.50 assay is the detection of
CPE and quantitation of the virus. With the current (manual)
TCID.sub.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 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.
[0200] Tetrazolium dyes are widely used as cell viability
indicators. The most commonly used dye is yellow MTT dye. Viable
cells, which possess active mitochondria, 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 been destroyed, little or no dye product is
formed, and a much lower absorbance is observed.
[0201] In a semi-automated TCID.sub.50 assay, after the infection
and six-day incubation of the plates, the spent medium is removed,
100 .mu.L of a solution of 0.5 mg/mL MTT dye in fresh virus growth
medium is added to each well of the 96-well plates, and the cells
are incubated at 37.+-.1.degree. C. and 5.+-.1% CO.sub.2 for six
hours. The dye product is solubilized by overnight incubation at
37.+-.1.degree. C., following addition of 100 .mu.L 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
Microsoft Excel.TM. 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 calculate the titer
(log 10TCID.sub.50/mL) based on the Karber modification of the
Reed-Muench method.
[0202] 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 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
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.
[0203] FIG. 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 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-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.
[0204] Validation of Semi-Automated Potency Assay
[0205] The Semi-Automated median tissue culture infectious dose
(TCID.sub.50) potency 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.sub.50
assay, and show that it provides comparable results to a manual
TCID.sub.50 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 semi-automated assay are
compared with those observed for a manual assay in Table 53.
[0206] 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 variability of the semi-automated
assay was 0.25 log 10 TCID.sub.50/ml, which is the half-width of
the 95% confidence interval for a single test result based on the
highest observed variability (0.11 log 10 TCID.sub.50 units) of the
manual assay. The actual SD values obtained with the semi-automated
assay, for the three strains, ranged between 0.06-0.09 log
10TCID.sub.50/mL. These values are within the acceptance criterion
of SD<0.25 log 10 TCID.sub.50 units and are comparable to the
between-test variability (0.07 to 0.11 log 10 TCID.sub.50 units)
observed for the manual TCID.sub.50 assay, from nine repeat tests
performed on three independent lots of each of three strains.
[0207] The assay was demonstrated to be linear over a 105-fold
dilution range (titer range of 4.2-9.3 log 10TCID.sub.50/mL), by
showing that the relationship of the calculated and measured
TCID.sub.50 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 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.
[0208] 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
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.
[0209] The inter-laboratory reproducibility of the assay was
demonstrated by 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
log 10TCID.sub.50/mL. This acceptance criterion was met, with the
lower and upper bounds of the 90% confidence intervals of greater
than -0.05 and less than +0.15, respectively, for all three
strains.
[0210] 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 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 was 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 log 10TCID.sub.50/mL, i.e. the lower
bound (LB) of the 90% CI must be greater than -0.3, and the upper
bound (UB) must be less than 0.3. The results are presented in the
assay validation report and summarized in Table 54, below. As may
be seen from the results in, the two-sided 90% confidence intervals
were within the acceptance criteria of .+-.0.3 log 10TCID.sub.50/mL
for both strains, with actual estimates of the lower and upper
bounds ranging between -0.05 and 0.10 log 10TCID.sub.50/mL.
[0211] Thus, in summary, while Manual TCID.sub.50 Potency Assay for
Influenza Virus 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 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.sub.50 Potency Assay for Influenza Virus
Monovalent. The implementation of the semi-automated assay, for
testing of monovalent materials optionally increases the assay
throughput 2-3 fold, and allows practical commercialization of
vaccines such as FluMist.TM. Vaccine at the anticipated level of
doses for market. An additional benefit is a lowered risk of
repetitive motion injuries for Quality Control analysts.
[0212] The semi-automated assay has been validated to demonstrate
repeatability, intermediate precision, linearity, and accuracy for
assay of viral materials in the titer range of 4.2-9.3 log
10TCID.sub.50/mL in one group. The assay was also validated to
demonstrate inter-laboratory reproducibility with another
group.
[0213] 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. 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, FluMist.TM. master virus seed
(MVS), manufacturer's working virus seed (MWVS), and virus harvest
(VH) samples.
[0214] Universal Cutoff Value of CPE in Semi-Automated TCID.sub.50
Assays
[0215] In yet other embodiments herein other variations and
modifications of TCID.sub.50 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 the TCID.sub.50 SemiAutomated Potency Assay for influenza
virus monovalent. The "SemiAutomated TCID.sub.50Potency Assay for
Influenza Virus Monovalent" (see above) uses the viable cell dye
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
to score the cytopathic effect (CPE) in infected monolayers of MDCK
cells. To reliably 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.sub.50 Assay for Influenza Virus
Monovalent," a well is considered CPE-positive with an absorbance
value of A570<0.5254; CPE-negative wells have absorbance values
of A570>0.5254.
[0216] 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/H1N1), A/Sydney/05/97 (type A/H3N2), and 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 TCID.sub.50
assay and demonstrate comparability to the validated manual potency
assay. Thus, illustrating the strength of embodiments comprising
these measurements.
[0217] As explained above, the median tissue culture infectious
dose (TCID.sub.50) 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
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-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 (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
(A.sub.570). In contrast, CPE-positive wells are marked by lower
absorbance values due to the partial or complete monolayer
destruction 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
used to calculate the virus titer (log 10 TCID.sub.50/mL).
[0218] 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.sub.570<0.5254;
CPE-negative wells had absorbance values of
A.sub.570.gtoreq.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.sub.50 assay. As described, CPE
assessed by the validated manual method of microscopic examination
was considered the "gold standard" and compared to the CPE
determined by MTT. The extensive testing of A/New Caledonia/20/99
(type A/H1N1), 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.sub.50
assay demonstrating comparability to the validated manual potency
assay.
[0219] 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 B/Yamanashi/166/98, a type B virus. The cold-adapted
control virus strain A/Sydney/05/97, was used to confirm system
suitability.
[0220] The method for the SemiAutomated TCID.sub.50 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 growth medium (VGM) using a Skatron.TM.
Cell Washer. Serial ten-fold dilutions of virus samples are
prepared in VGM containing TPCK-trypsin using a Matrix.TM.
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 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.+-.1.degree. C. with 5.+-.1% CO.sub.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. Conversely, the monolayer in a
CPE-negative well was completely intact.
[0221] After visually scoring the monolayers on the plates for CPE,
the media is discarded and MTT (0.5 mg/mL), (US Biochemical
Corporation, Cleveland, Ohio), prepared in phosphate buffered
saline is dispensed to each well (100 4/well). The monolayers are
incubated with MTT for 6.+-.0.5 hours at 37.+-.1.degree. C. with
5.+-.1% CO.sub.2. Solubilization buffer (100 .mu.L 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.degree. C. in an environment of 5.+-.1%
CO.sub.2. The absorbance values at 570 nm are determined using a
PerkinElmer-Wallac 1420 Multilabel Counter Spectrophotometer and
were exported into a Microsoft.TM. Excel macro; a program used to
calculate virus titer (log.sub.10 TCID.sub.50/mL) from the number
of CPE-positive wells.
[0222] 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) of the four replicate TCID.sub.50
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.
[0223] 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" 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)
[0224] 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 SemiAutomated TCID.sub.50
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.sub.50 assay on three days using the three reference virus
samples.
[0225] In order to perform an analyst to analyst comparison, each
analyst in the testing (Analyst #1-6) in Group 1 performed a
SemiAutomated TCID.sub.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.sub.50 potency assay with the same three
reference virus strains over three days using instrument
AZ-036.
[0226] To reliably distinguish between CPE-positive and
CPE-negative wells in the SemiAutomated TCID.sub.50 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.
[0227] Sensitivity and specificity measurements were calculated
using the manual 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
98.13% and 98.71%, respectively. All data above (>95%
sensitivity and >95% specificity) correlates with the data
determined wherein a sensitivity of 99.05% and a specificity of
99.99% were determined.
[0228] FIG. 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, 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 probability of 0.02%, when
cutoff values were estimated using absorbance values from all
CPE-positive wells. Furthermore, the distribution profiles evident
in FIG. 51 highlight that absorbance values for CPE-positive wells
are widely separated from absorbance values for CPE-negative
wells
[0229] A Comparison of the Mean Absorbance Values Obtained for
CPE-Negative 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 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 described herein were
conducted in second groups four months prior to that done by the
first group.
[0230] 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
SemiAutomated TCID.sub.50 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 log 10TCID.sub.50/mL and the
titer did not vary more than 0.09 log 10TCID.sub.50/mL between
AZ-039 and AZ-040 (see Table 58). For A/Sydney/05/97 the overall
mean ranged from 8.5 to 8.6 log 10TCID.sub.50/mL and did not vary
more than 0.02 log 10TCID.sub.50/mL between AZ-039 and AZ-040. For
B/Yamanashi/166/98 the overall mean ranged from 8.3 to 8.4 log
10TCID.sub.50/mL and did not vary more than 0.12 log
10TCID.sub.50/mL between AZ-039 and AZ-040. The second group
produced results using one instrument system (AZ-036). Three
analysts performed the SemiAutomated TCID.sub.50 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 10TCID.sub.50/mL
for A/New Caledonia/20/99, 0.08 log 10TCID.sub.50/mL for
A/Sydney/05/97 and 0.12 log 10TCID.sub.50/mL for
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).
[0231] In order to do an analyst to analyst comparison, in the
first group each analyst (Analyst 1 through 6) performed a
SemiAutomated TCID.sub.50 potency assay for 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 TCID.sub.50
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 0.3 log 10TCID.sub.50/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 log 10TCID.sub.50/mL. An overall comparison between the two
groups of analysts was less than .+-.0.3 log 10TCID.sub.50/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 were less than the
acceptance criterion value of 0..sub.50, all were valid.
[0232] 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-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 previous work (see
FIG. 51 and Table 57). In the two groups, the "universal cutoff"
for the SemiAutomated TCID.sub.50 potency assay, using MTT to
assess CPE, produced potency values that were comparable to each
other and to those obtained by the validated manual TCID.sub.50
potency assay. Finally, the SemiAutomated system has several
procedural 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.
DEFINITIONS
[0233] 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.
[0234] The terms "nucleic acid," "polynucleotide," "polynucleotide
sequence" and "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 otherwise indicated, a particular nucleic acid sequence
optionally encompasses complementary sequences, in addition to the
sequence explicitly indicated.
[0235] 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 well as to a cDNA or an mRNA encoded by that genomic
sequence.
[0236] 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" promoter or enhancer is one which
regulates transcription in a specific tissue type or cell type, or
types.
[0237] 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 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 liposome-conjugated DNA, or the like, that are not
autonomously replicating. Most commonly, the vectors of herein
refer to plasmids.
[0238] 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.
[0239] A "bi-directional expression vector" is characterized by two
alternative 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.
[0240] 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 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 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.
[0241] The term "recombinant" indicates that the material (e.g., a
nucleic acid or protein) has been artificially or synthetically
(non-naturally) altered. The alteration can be performed on the
material within, or removed from, its natural environment or state.
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.
[0242] 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 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 neuraminidase, from a different parental
virus.
[0243] 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 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.
[0244] 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 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, COST cells).
Influenza Virus
[0245] The compositions and methods herein are primarily concerned
with production of influenza viruses for vaccines. Influenza
viruses are made up of an internal ribonucleoprotein core
containing a segmented single-stranded RNA genome and an outer
lipoprotein envelope lined by a matrix protein. Influenza A and
influenza B viruses each contain eight segments of single stranded
negative sense RNA. The influenza A genome encodes eleven
polypeptides. Segments 1-3 encode three polypeptides, making up a
RNA-dependent RNA polymerase. Segment 1 encodes the polymerase
complex protein PB2. The remaining polymerase proteins PB1 and PA
are encoded by segment 2 and segment 3, respectively. In addition,
segment 1 of some influenza strains encodes a small protein,
PB1-F2, produced from an alternative reading frame within the PB1
coding region. Segment 4 encodes the hemagglutinin (HA) surface
glycoprotein involved in cell attachment and entry during
infection. Segment 5 encodes the nucleocapsid nucleoprotein (NP)
polypeptide, the major structural component associated with viral
RNA. Segment 6 encodes a 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.
[0246] The eight genome segments of influenza B encode 11 proteins.
The three largest genes code for components of the RNA polymerase,
PB1, PB2 and PA. Segment 4 encodes the HA protein. Segment 5
encodes NP. Segment 6 encodes the NA protein and the NB protein.
Both proteins, NB and NA, are translated from overlapping reading
frames of a biscistronic mRNA. Segment 7 of influenza B also
encodes two proteins: M1 and BM2. The smallest segment encodes two
products, NS1 which is translated from the full length RNA, and NS2
which is translated from a spliced mRNA variant.
Influenza Virus Vaccine
[0247] 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 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 .beta.-propiolactone (or alternatively
used in live attenuated vaccines).
[0248] 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 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 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.
[0249] Attempts at producing recombinant and reassortant vaccines
in cell culture have been hampered by the inability of some of the
strains approved for vaccine production 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 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.sub.2, at
constant temperature using a temperature regulator, such as a
thermostat to insure that the temperature does not exceed
35.degree. C. Such pioneering work, as well as other vaccine
production, can be further optimized and streamlined through use of
the current invention in whole or part.
[0250] 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 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.
FluMist.TM.
[0251] As mentioned previously, numerous examples and types of
influenza vaccine exist. An exemplary influenza vaccine is
FluMist.TM. which is a live, 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. Engl. 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 FluMist.TM.
vaccine. However, it will be appreciated by those skilled in the
art that the steps/compositions herein are also adaptable to
production of similar or even different viral vaccines.
[0252] FluMist.TM. 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 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.degree. C. (ca, cold adapted), but its growth is restricted at
38 and 39.degree. C. (ts, temperature sensitive). 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 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).
[0253] Clinical studies in over 20,000 adults and children
involving 12 separate 6:2 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 al.
(1994) A 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 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 in ferrets J. Infect. Dis. 146:780-900). Production
of such reassorted virus using B strains of influenza are is more
difficult, however.
[0254] Recent work, see, Multi-Plasmid System for the Production of
Influenza Virus, cited above, has shown an eight plasmid 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
formulations useful for intranasal administration.
[0255] 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.RTM.. The
methods of the 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
[0256] As previously stated, influenza virus optionally can be
grown in cell culture. 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 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.sub.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, streptomycin, etc., and/or additional nutrients, such
as L-glutamine, sodium pyruvate, non-essential amino acids,
additional supplements to promote favorable growth characteristics,
e.g., trypsin, .beta.-mercaptoethanol, and the like.
[0257] 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.sup.th ed.,
Livingston, 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 cultures for vaccine
preparation in Cohen and Shafferman (eds.) Novel Strategies in
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.
[0258] Cells for production of influenza virus can be cultured in
serum-containing 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 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 commercial production of viruses, e.g., for vaccine production,
it is often desirable to culture the cells in a bioreactor or
fermenter. Bioreactors are available in volumes from under 1 liter
to in excess of 100 liters, e.g., Cyto3 Bioreactor (Osmonics,
Minnetonka, Minn.); NBS bioreactors (New Brunswick Scientific,
Edison, N.J.); laboratory and commercial scale bioreactors from B.
Braun Biotech International (B. Braun Biotech, Melsungen,
Germany).
[0259] 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 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 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.).
[0260] 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
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 TransIT-LT1 (Mirus)
according to the manufacturer's instructions in order to produce
reassorted viruses, etc. Approximately 1 .mu.g of each vector to be
introduced into the population of host cells with approximately 2
.mu.l of TransIT-LT1 diluted in 160 .mu.l medium, preferably
serum-free medium, in a total volume of 200 .mu.l. The
DNA:transfection reagent mixtures are incubated at room temperature
for 45 minutes followed by addition of 800 .mu.l of medium. The
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, (PB2, PB1, PA, NP, M,
NS, HA and NA) are mixed with approximately 20 .mu.l TransIT-LT1
and transfected into host cells. Optionally, serum-containing
medium is replaced prior to transfection with serum-free medium,
e.g., Opti-MEM I, and incubated for 4-6 hours.
[0261] 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 using
electroporation according to the following procedure. In brief,
approximately 5.times.10.sup.6 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
cuvette. Twenty micrograms of DNA in a volume of up to 25 .mu.l 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. 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 ml MEM, 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 incubated under conditions
permissive for viral growth, e.g., at approximately 33.degree. C.
for cold adapted strains.
Kits
[0262] 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 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 container.
[0263] 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 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.
Tables
TABLE-US-00001 [0264] 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.
TABLE-US-00002 TABLE 2 Tube/Well of cell Target incubation culture
MOI of MDV MOI of wild-type 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
TABLE-US-00003 TABLE 3 Manufacture Potential Process Detection
Type/Assay Time 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 Manual/28
days PCR growth Virus harvest Mycobacterium Manual/56 days PCR or
clinical growth diagnostic systems
TABLE-US-00004 TABLE 4 Virus Type Strain and Isolate Number A H1N1
ca A/Beijing/262/95 A H1N1 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
TABLE-US-00005 TABLE 5 A/Sydney/05/97 Virus potency [log.sub.10
TCID.sub.50/mL]. Temperature Process step 5 .+-. 3.degree. C. 20
.+-. 3.degree. C. 31 .+-. 3.degree. 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 8.8 .+-. 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 NA = not assayed All
filtrations in Table 5 were performed from the same day harvest.
Prior to filtration through Sartoclean CA and Sartopore2 filters
VAF was exposed for 60 minutes to 5 .+-. 3.degree. C., 20 .+-.
3.degree. C. and 31 .+-. 3.degree. C.
TABLE-US-00006 TABLE 6 A/Sydney/05/97 neuraminidase activity
[.mu.U/mL]. Temperature Process step 5 .+-. 3.degree. C. 20 .+-.
3.degree. C. 31 .+-. 3.degree. 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 .mu.U/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 .+-. 3.degree. C., 20 .+-. 3.degree. C. and 31 .+-.
3.degree. C.
TABLE-US-00007 TABLE 7 A/Sydney/05/97 hemagglutinin activity [HA
titer]. Temperature Process step 5 .+-. 3.degree. C. 20 .+-.
3.degree. C. 31 .+-. 3.degree. C. 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
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 .+-. 3.degree. C., 20
.+-. 3.degree. C. and 31 .+-. 3.degree. C.
TABLE-US-00008 TABLE 8 A/Sydney/05/97 Virus potency [log.sub.10
TCID.sub.50/mL]. Temperature Process step 5 .+-. 3.degree. C. 20
.+-. 3.degree. C. 31 .+-. 3.degree. 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 .+-. 3.degree. C., 20 .+-. 3.degree. C.
and 31 .+-. 3.degree. C.*.
TABLE-US-00009 TABLE 9 A/Sydney/05/97 neuraminidase activity
[.mu.U/mL]. Temperature Process step 5 .+-. 3.degree. C. 20 .+-.
3.degree. C. 31 .+-. 3.degree. 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 .mu.U/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 .+-. 3.degree. C., 20 .+-. 3.degree.0 C. and 31 .+-. 3.degree.
C.*.
TABLE-US-00010 TABLE 10 A/Sydney/05/97 hemagglutinin activity [HA
titer]. Temperature Process step 5 .+-. 3.degree. C. 20 .+-.
3.degree. C. 31 .+-. 3.degree. C. Stabilized VAF (before treatment)
256 128 256 Stabilized VAF (after treatment) 256 256 128 Filtered
VAF (pool) 16 32 128 Centrifuged Stabilized VAF (control) 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 .+-. 3.degree. C., 20 .+-.
3.degree. C. and 31 .+-. 3.degree. C.*.
TABLE-US-00011 TABLE 11 A/Sydney/05/97virus potency [log.sub.10
TCID.sub.50/mL]. Warming time Process step 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 8.9 .+-. 0.3 8.9 .+-. 0.3 8.9 .+-. 0.3
(control)* 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.degree. C. for 30, 90 or 180 minutes.
TABLE-US-00012 TABLE 12 A/Sydney/05/97 neuraminidase activity
[.mu.U/mL]. Warming time Process step 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 .+-. 3.degree. C.
for 30, 90 or 180 minutes.
TABLE-US-00013 TABLE 13 A/Sydney/05/97 hemagglutinin activity [HA
titer]. Warming time Process step 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 *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 Sartoclean CA and
Sartopore 2 filters, VAF was exposed to 31 .+-. 3.degree. C. for
30, 90 or 180 minutes.
TABLE-US-00014 TABLE 14 A/Sydney/05/97virus potency [log.sub.10
TCID.sub.50/mL]. Warming time Process step 0 min 30 min 60 min 90
min Stabilized VAF 8.8 .+-. 0.3 8.8 .+-. 0.3 8.8 .+-. 0.3 8.8 .+-.
0.3 (before warming)* Stabilized VAF -- 8.7 .+-. 0.2 8.6 .+-. 0.1
8.6 .+-. 0.1 (warmed up) Filtered VAF (pool) 7.7 .+-. 0.1 8.3 .+-.
0.2 8.4 .+-. 0.2 8.6 .+-. 0.1 Centrifuged Stabilized 8.6 .+-. 0.1
8.6 .+-. 0.1 8.6 .+-. 0.1 8.6 .+-. 0.1 VAF (control)* Gain/loss
Filtered vs. -0.9 -0.3 -0.2 0.0 Control 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.degree. C. for 0, 30, 60 or 90 minutes.
TABLE-US-00015 TABLE 15 A/Sydney/05/97 neuraminidase activity
[.mu.U/mL]. Warming time Process step 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 33.0 33.0 33.0 33.0 (control)*
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.degree. C. for 0, 30, 60 or 90 minutes.
TABLE-US-00016 TABLE 16 A/Sydney/05/97 hemagglutinin activity [HA
titer]. Warming time Process step 0 min 30 min 60 min 90 min
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 64 64 64 64 (control)* *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). 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.degree. C. for 0, 30, 60 or 90 minutes.
TABLE-US-00017 TABLE 17 Virus potency [log.sub.10 TCID.sub.50/mL].
Warming time Process step 0 min 30 min 60 min 90 min Stabilized VAF
8.6 .+-. 0.2 8.6 .+-. 0.2 8.6 .+-. 0.2 8.6 .+-. 0.2 (before
warming)* Stabilized VAF -- 8.6 .+-. 0.2 8.6 .+-. 0.2 8.5 .+-. 0.3
(warmed up) Filtered VAF (pool) 8.1 .+-. 0.2 8.1 .+-. 0.2 8.5 .+-.
0.2 8.5 .+-. 0.3 Centrifuged Stabilized 8.6 .+-. 0.2 8.6 .+-. 0.2
8.6 .+-. 0.2 8.6 .+-. 0.2 VAF (control)* Gain/Loss Filtered vs.
-0.5 -0.5 -0.1 -0.1 Control All filtrations were performed from the
same day harvest. Prior to filtration through Sartoclean CA and
Sartopore2 filters, VAF was exposed to 31 .+-. 3.degree. C. for 0
30, 60 or 90 minutes.
TABLE-US-00018 TABLE 18 Neuraminidase activity .mu.U/mL. Warming
time Process step 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 23.0 23.0 23.0 23.0 (control)* 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.degree. C. for 0
30, 60 or 90 minutes.
TABLE-US-00019 TABLE 19 Hemagglutinin activity. Warming time
Process step 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 64 64 64
64 (control)* *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). All filtrations were performed from the same day
harvest. Before filtration through Sartoclean CA Sartopore2 filters
VAF was exposed to 31 .+-. 3.degree. C. for 0 30, 60 or 90
minutes.
TABLE-US-00020 TABLE 20 Virus potency [log.sub.10TCID.sub.50/mL] of
six influenza strains. Process step Centrifuged Stabilized Filtered
Stabilized Potency Influenza strain VAF* VAF VAF* Gain/Loss
A/Beijing/262/95 9.7 .+-. 0.1 9.6 .+-. 0.2 9.6 .+-. 0.3 0.0 RT**
A/Beijing/262/95 9.7 .+-. 0.1 9.4 .+-. 0.3 9.6 .+-. 0.3 -0.2 31
.+-. 3.degree. C. A/New Caledonia/ 9.6 .+-. 0.2 9.3 .+-. 0.2 9.5
.+-. 0.2 -0.2 20/99 RT** A/New Caledonia/20/ 9.6 .+-. 0.2 9.3 .+-.
0.2 9.5 .+-. 0.2 -0.2 99 31 .+-. 3.degree. C. A/Sydney/05/97 RT 8.8
.+-. 0.3 7.7 .+-. 0.1 8.6 .+-. 0.1 -0.9 A/Sydney/05/97 8.8 .+-. 0.3
8.4 .+-. 0.2 8.6 .+-. 0.1 -0.2 31 .+-. 3.degree. C.
A/Panama/2007/99 8.5 .+-. 0.2 8.2 .+-. 0.1 8.5 .+-. 0.3 -0.3 RT**
A/Panama/2007/99 8.5 .+-. 0.2 8.6 .+-. 0.2 8.5 .+-. 0.3 0.1 31 .+-.
3.degree. C. B/Victoria/504/2000 8.3 .+-. 0.2 7.8 .+-. 0.2 8.4 .+-.
0.2 -0.6 RT** B/Victoria/504/2000 8.3 .+-. 0.2 8.4 .+-. 0.2 8.4
.+-. 0.2 0.0 31 .+-. 3.degree. C. B/Yamanashi/166/98 8.4 .+-. 0.2
8.3 .+-. 0.2 8.6 .+-. 0.2 -0.3 RT** B/Yamanashi/166/98 8.4 .+-. 0.2
8.4 .+-. 0.2 8.6 .+-. 0.2 -0.2 31 .+-. 3.degree. C. *Stabilized VAF
and Centrifuged Stabilized VAF (control) samples were taken from
the pool before VAF was divided into individual experiments (RT and
31 .+-. 3.degree. C.). **RT room temperature 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.degree. C. for 0 (RT) or 60 minutes.
TABLE-US-00021 TABLE 21 Neuraminidase activity [.mu.U/mL] of six
influenza strains. Process step Centrifuged Activity Stabilized
Filtered Stabilized Gain/ Influenza train VAF VAF VAF Loss
A/Beijing/262/95 RT** 55.5 47.5 52.0 -4.5 A/Beijing/262/95 55.5
51.5 52.0 -0.5 31 .+-. 3.degree. C. A/New Caledonia/20/99 49.5 47.5
48.5 -1.0 RT** A/New Caledonia/20/99 49.5 48.5 48.5 0.0 31 .+-.
3.degree. C. A/Sydney/05/97 RT** 44.5 6.0 33.0 -27.0 A/Sydney/05/97
44.5 26.0 33.0 -7.0 31 .+-. 3.degree. C. A/Panama/2007/99 RT** 61.0
16.5 48.0 -31.5 A/Panama/2007/99 61.0 40.0 48.0 -8.0 31 .+-.
3.degree. C. B/Victoria/504/2000 RT** 58.5 20.5 44.0 -23.5
B/Victoria/504/2000 58.5 37.0 44.0 -7.0 31 .+-. 3.degree. C.
B/Yamanashi/166/98 RT** 66.5 51.0 55.5 -4.5 B/Yamanashi/166/98 66.5
53.0 55.5 -2.5 31 .+-. 3.degree. C. *Stabilized VAF and Centrifuged
Stabilized VAF (control) samples were taken from the pool before
VAF was divided into individual experiments (RT and 31 .+-.
3.degree. C.). **RT = room temperature 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.degree. C. for 0 (RT) or 60 minutes.
TABLE-US-00022 TABLE 22 Hemagglutinin activity [HA titer] of six
influenza strains. Process step Stabilized Centrifuged Stabilized
Warmed Filtered Stabilized Influenza train VAF* up VAF VAF VAF*
A/Beijing/262/95 RT** 1024 -- 128 1024 A/Beijing/262/95 1024 512
512 1024 31 .+-. 3.degree. C. A/New Caledonia/20/99 32 -- 32 64
RT** A/New Caledonia/20/99 32 32 32 64 31 .+-. 3.degree. C.
A/Sydney/05/97 RT** 64 -- 16 64 A/Sydney/05/97 64 128 128 64 31
.+-. 3.degree. C. A/Panama/2007/99 RT** 128 -- 32 128
A/Panama/2007/99 128 128 64 128 31 .+-. 3.degree. C.
B/Victoria/504/2000 128 -- 32 128 RT** B/Victoria/504/2000 128 64
64 128 31 .+-. 3.degree. C. B/Yamanashi/166/98 512 -- 16 32 RT**
B/Yamanashi/166/98 512 32 32 32 31 .+-. 3.degree. C. *Stabilized
VAF and Centrifuged Stabilized VAF (control) samples were taken
from the pool before VAF was divided into individual experiments
(RT and 31 .+-. 3.degree. C.). **RT = room temperature 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.degree. C. for 0 (RT) or 60
minutes.
TABLE-US-00023 TABLE 23 Virus potency [log.sub.10TCID.sub.50/mL] of
six influenza strains. Process step Centrifuged Potency Stabilized
Filtered Stabilized Gain/ Influenza strain VAF* VAF VAF* Loss
A/Beijing/262/95 RT** 9.6 .+-. 0.1 9.4 .+-. 0.2 9.6 .+-. 0.1 -0.2
A/Beijing/262/95 9.6 .+-. 0.1 9.5 .+-. 0.2 9.6 .+-. 0.1 -0.1 31
.+-. 3.degree. C. A/New Caledonia/20/99 9.1 .+-. 0.2 9.5 .+-. 0.2
9.2 .+-. 0.2 0.3 RT** A/New Caledonia/20/99 9.1 .+-. 0.2 9.2 .+-.
0.3 9.2 .+-. 0.2 0.0 31 .+-. 3.degree. C. A/Sydney/05/97 RT** 8.6
.+-. 0.2 8.1 .+-. 0.2 8.6 .+-. 0.2 -0.5 A/Sydney/05/97 8.6 .+-. 0.2
8.5 .+-. 0.2 8.6 .+-. 0.2 -0.1 31 .+-. 3.degree. C.
A/Panama/2007/99 RT** 8.9 .+-. 0.2 8.3 .+-. 0.2 8.5 .+-. 0.2 -0.2
A/Panama/2007/99 8.9 .+-. 0.2 8.6 .+-. 0.1 8.5 .+-. 0.2 0.1 31 .+-.
3.degree. C. B/Victoria/504/2000 7.6 .+-. 0.2 7.7 .+-. 0.2 7.9 .+-.
0.2 -0.2 RT** B/Victoria/504/2000 7.6 .+-. 0.2 7.7 .+-. 0.1 7.9
.+-. 0.2 -0.2 31 .+-. 3.degree. C. B/Yamanashi/166/98 8.4 .+-. 0.1
8.2 .+-. 0.2 8.3 .+-. 0.3 -0.1 RT** B/Yamanashi/166/98 8.4 .+-. 0.1
8.3 .+-. 0.2 8.3 .+-. 0.3 0.0 31 .+-. 3.degree. C. *Stabilized VAF
and Centrifuged Stabilized VAF (control) samples were taken from
the pool before VAF was divided into individual experiments (RT and
31 .+-. 3.degree. C.). **RT = room temperature. 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.degree. C. for 0 (RT) or 60 minutes.
TABLE-US-00024 TABLE 24 Neuraminidase activity [.mu.U/mL] of six
influenza strains. Process step Centrifuged Activity Stabilized
Filtered Stabilized Gain/ Influenza strain VAF VAF VAF* Loss
A/Beijing/262/95 RT** 56.5 47.5 54.5 -7.0 A/Beijing/262/95 64.5
56.0 58.5 -1.5 31 .+-. 3.degree. C. A/New Caledonia/20/99 46.0 38.5
40.0 -1.5 RT** A/New Caledonia/20/99 46.0 43.0 40.0 3.0 31 .+-.
3.degree. C. A/Sydney/05/97 RT** 35.5 7.0 23.0 -16.0 A/Sydney/05/97
31 .+-. 3.degree. 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 60.5 42.5 39.0 3.5 31 .+-.
3.degree. C. B/Victoria/504/2000 RT** 35.0 21.0 28.5 -7.5
B/Victoria/504/2000 39.0 25.5 31.5 -6.0 31 .+-. 3.degree. C.
B/Yamanashi/166/98 RT** 29.0 26.0 28.5 -2.5 B/Yamanashi/166/98 33.5
27.5 29.5 -2.0 31 .+-. 3.degree. C. *Stabilized VAF and Centrifuged
Stabilized VAF (control) samples were taken from the pool before
VAF was divided into individual experiments (RT and 31 .+-.
3.degree. C.). **RT = room temperature. 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.degree. C. for 0 (RT) or 60 minutes.
TABLE-US-00025 TABLE 25 Hemagglutinin activity [HA titer] of six
influenza strains. Process step Stabilized Centrifuged Stabilized
Warmed Filtered Stabilized Influenza strain VAF* up VAF VAF VAF*
A/Beijing/262/95 RT** 256 -- 1024 512 A/Beijing/262/95 256 1024
2048 512 31 .+-. 3.degree. C. A/New Caledonia/20/99 512 -- 512 512
RT** A/New Caledonia/20/99 512 512 512 512 31 .+-. 3.degree. C.
A/Sydney/05/97 RT** 64 -- 16 64 A/Sydney/05/97 64 64 64 64 31 .+-.
3.degree. C. A/Panama/2007/99 RT** 256 -- 64 256 A/Panama/2007/99
256 512 512 256 31 .+-. 3.degree. C. B/Victoria/504/2000 64 -- 128
128 RT** B/Victoria/504/2000 64 64 64 128 31 .+-. 3.degree. C.
B/Yamanashi/166/98 128 -- 32 128 RT** B/Yamanashi/166/98 128 64 64
128 31 .+-. 3.degree. C. *Stabilized VAF and Centrifuged Stabilized
VAF (control) samples were taken from the pool before VAF was
divided into individual experiments (RT and 31 .+-. 3.degree. C.).
**RT = room temperature. 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.degree. C. for 0 (RT) or 60 minutes.
TABLE-US-00026 TABLE 26 Analysis by SEC - Peak Area Comparison Peak
Area at 220 Impurities Impurities Group 1 Group 2 Virus Peak (18 to
21 (21 to 27 Sample Details Sample ID ~(10.5 min) min) min) Neat
(VH) 1X 1221 31785 339528 10 times concentrated 10X 11192 126849
435652 sample 1X Washed 5 times 1X-W 1005 2131 2510 with 1X-SPG 10X
washed with 10X-W 10282 15858 2194 1X-SPG 5 times 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
TABLE-US-00027 TABLE 27 A/New Caledonia - CELISA Values Replicate
Mean +/- SD Sample Details Sample ID (N) (CELISA) Neat (VH) 1X 4
9.1 +/- 0.02 10 times concentrated 10X 4 10.0 +/- 0.05 sample 1X
Washed 5 times with 1X-W 4 8.9 +/- 0.03 1X-SPG 10X washed with
10X-W 4 9.9 +/- 0.04 1X-SPG 5 times Permeate or filtrate Permeate 4
<LOQ 10X diluted back to 1X 10X to 1X 4 9.0 +/- 0.08 with 1X-SPG
10X-W diluted back to 10X-W to 1X-w 4 8.9 +/- 0.02 1X-W with
1X-SPG
TABLE-US-00028 TABLE 28 Composition of Representative Formulations
Formulation Number Composition 1 10% Allantoic fluid in 100
millimolar phosphate buffer, 7% Sucrose, no added excipients 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 [3] with 1% gelatin hydrolysate and 1% arginine 5 60%
Allantoic fluid in 100 millimolar phosphate buffer, 10% sucrose, 2%
arginine, 2% gelatin hydrolysate 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 hydrolysate 9 60% Allantoic fluid in 50 millimolar
histidine buffer, 10% sucrose, 2% arginine, 2% gelatin hydrolysate,
2.5 mM EDTA
TABLE-US-00029 TABLE 29 Stability of Virus in Representative
Formulations (loss of titer in log.sub.10/mL/month) Formulation
A/New B/Hong Number Caledonia20/99 A/Panama/2007/99 Kong/330/01 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
TABLE-US-00030 TABLE 30 INGRE- avs43 avs53 DIENTS Liq01 Liq02 Liq03
Liq04 Liq05 Liq06 Liq07 Liq08 Liq09 Liq10 Liq11 Liq12 KPO4 100 mM
100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM PH
7.2 HEPES 100 mM Sucrose 15% 15% 15% 15% 10% 15% 15% 15% 20% 20%
20% Gelatin 1% 1% 1% 2% 2% 1% 2% 2% 2% Arginine 2% 2% 2% 2% 2% 2%
2% Glycine 1% Methio- 0.15%.sup. nine PVP 1% Dextran 1% Pluronic
0.02%.sup. 0.02%.sup. CAIV 10% 10% 10% 10% 10% 10% 10% 10% 10% 10%
10% 10% Monova- lent (added) NAF 50% 50% 50% 50% 50% 50% 50% 50%
50% 50% 50% 50% (added) Purified q.s. q.s. q.s. q.s. q.s. q.s. q.s.
q.s. q.s. q.s. q.s. q.s. Water
TABLE-US-00031 TABLE 31 First Tier: Potency by FFA Assay B/Hongkong
(in SPG) Target: 6.9 4.degree. C. (Monthly) Formulation 0 0 1 1 2 2
3 3 4 4 5 5 6 6 Formulation Liq 01 7.3 7.3 6.6 6.6 6.1 6.2 6.5 6.5
6.5 6.6 6.7 6.7 6.5 6.4 15Suc/1Gel/2Arg Liq 01 (Repeat) 6.9 6.8 6.7
6.6 6.6 6.5 6.6 6.5 6.4 6.4 6.4 15Suc/1Gel/2Arg Liq 02 7.3 7.3 6.6
6.4 6.3 6.4 6.3 6.3 6.4 6.4 6.6 6.7 6.1 6.3 15Suc/1Gel/1Glyc Liq 02
(Repeat) 6.7 6.6 6.3 6.6 6.5 6.6 6.4 6.3 6.4 6.3 6.2 6.2
15Suc/1Gel/1Glyc Liq 03 7.3 7.1 6.5 6.5 6.2 6.1 6.4 6.3 6.4 6.2 6.7
6.8 6.3 15Suc/1Gel Liq 03 (Repeat) 6.8 6.9 6.7 6.7 6.5 6.6 6.4 6.4
6.3 6.2 6.3 6.2 15Suc/1Gel Liq 04 7.2 7.2 6.3 6.5 6.1 6.1 6.2 6.4
6.4 6.4 6.7 6.8 6.5 6.3 15Suc/2Gel/2Arg Liq 04 (Repeat) 6.8 6.8 6.7
6.7 6.5 6.5 6.4 6.4 6.4 6.4 6.3 6.2 15Suc/2Gel/2Arg Liq 05 7.1 7.1
6.5 6.5 6.1 6.1 6.4 6.2 6.4 6.5 6.7 6.7 6.4 6.2 10Suc/2Gel/2Arg Liq
05 (Repeat) 7.0 7.1 6.8 6.6 6.6 6.6 6.7 6.9 6.7 6.8 6.9 6.9
10Suc/2Gel/2Arg Liq 06 7.1 7.0 5.8 5.7 5.4 5.3 5.1 5.3 4.6 4.1 4.4
3.6 3.7 5.0 15Suc/1Gel/2Arg, HEPES Liq 07 7.0 7.1 6.3 6.3 5.9 5.9
6.3 6.3 6.2 6.2 6.5 6.6 6.4 6.4 15Suc/1PVP/2Arg Liq 07 (Repeat) 6.7
6.6 6.4 6.5 6.3 6.4 6.4 6.3 6.3 6.2 6.1 6.2 15Suc/1PVP/2Arg Liq 08
7.0 6.9 6.4 6.4 6.0 6.0 6.3 6.4 6.4 6.1 7.1 6.7 6.4 6.3
15Suc/1Dextran/2Arg Liq 08 (Repeat) 6.7 6.8 6.6 6.7 5.8 6.7 6.4 6.6
6.4 6.4 6.2 6.2 15Suc/1Dextran/2Arg Liq 09 6.9 7.0 6.5 6.4 6.0 6.0
6.3 6.3 6.0 6.2 6.7 6.6 6.3 6.4 20Suc/2Gel/2Arg Liq 09 (Repeat) 6.9
6.9 6.7 6.8 6.6 6.6 6.6 6.6 6.3 6.4 6.4 6.3 20Suc/2Gel/2Arg Liq 10
6.9 7.0 6 6.3 5.7 6.0 6.2 6.3 5.9 6.2 6.7 6.5 6.2 6.3 20Suc/2
Ge//0.15 Meth/0.02Plur Liq 10 (Repeat) 6.9 6.9 6.5 6.5 6.5 6.3 6.3
6 6.2 6.2 6.2 6 20Suc/2 Ge//0.15 Meth/0.02Plur Liq 11 7.0 7.0 6.3
6.3 6.0 5.9 6.2 6.4 6.0 6.0 6.4 6.6 6.1 6.1 20Suc/2Ge//0.02Plur Liq
12 6.9 7.0 6.3 6.5 6.4 6.4 6.1 6.1 5.8 5.3 5.1 5.3 5.6 5.3 NAF only
(60%)
TABLE-US-00032 TABLE 32 B/Harbin (in SPG) Target: 7.0 4.degree. C.
(Monthly) Formulation 0 0 1 1 2 2 3 3 4 4 5 5 6 6 Formulation Liq
01 7.1 7.2 6.6 6.7 6.2 6.4 6.6 6.6 6.8 6.8 6.7 6.8 6.2 6.1
15Suc/1Gel/2Arg Liq 01 (Repeat) 6.9 6.9 6.7 6.8 6.7 6.6 6.6 6.6 6.6
6.4 6.4 6.5 15Suc/1Gel/2Arg Liq 02 7.1 7.1 6.7 6.6 6.2 6.0 6.5 6.5
6.5 6.7 6.7 6.7 6.0 6.0 15Suc/1Gel/1Glyc Liq 02 (Repeat) 6.9 7 6.8
6.7 6.5 6.6 6.5 6.5 6.3 6.4 6.3 6.2 15Suc/1Gel/1Glyc Liq 03 7.1 7.0
6.5 6.7 6.1 6.0 6.5 6.5 6.7 6.5 6.7 7.1 6.2 6.3 15Suc/1Gel Liq 03
(Repeat) 6.9 6.9 6.7 6.7 6.5 6.5 6.6 6.5 6.5 6.4 6.4 6.4 15Suc/1Gel
Liq 04 7.0 7.0 6.5 6.8 6.1 6.2 6.5 6.6 7.0 6.9 7.3 7.1 6.4 6.5
15Suc/2Gel/2Arg Liq 04 (repeat) 6.8 6.8 6.7 6.7 6.7 6.6 6.5 6.5 6.4
6.5 6.4 6.5 15Suc/2Gel/2Arg Liq 05 7.1 7.2 6.8 6.7 6.2 6.1 6.6 6.7
6.9 6.7 6.9 6.9 6.4 6.3 10Suc/2Gel/2Arg Liq 05 (Repeat) 7.0 7.0 6.6
6.5 6.7 6.8 6.8 6.8 6.7 6.8 6.7 6.6 10Suc/2Gel/2Arg Liq 06 7.1 7.1
6.4 6.3 5.4 5.4 5.4 5.3 5.4 5.2 4.3 4.3 3.8 ud 15Suc/1Gel/2Arg,
HEPES Liq 07 7.0 7.1 6.7 6.8 6 6.2 6.4 6.4 6.5 6.6 6.4 6.7 6.4 6.5
15Suc/1PVP/2Arg Liq 07 (Repeat) 6.9 6.8 6.6 6.5 6.5 6.6 6.4 6.4 6.3
6.2 6.4 6.4 15Suc/1PVP/2Arg Liq 08 7.1 6.9 6.6 6.9 5.9 6.1 6.4 6.4
6.6 6.9 6.7 6.7 6.2 6.4 15Suc/1Dextran/2Arg Liq 08 (Repeat) 6.8 6.9
6.7 6.6 6.6 6.6 6.4 6.4 6.2 6.4 6.4 6.3 15Suc/1Dextran/2Arg Liq 09
6.9 7.0 6.8 6.9 6.1 6.2 6.5 6.5 6.8 6.8 6.6 6.9 6.4 6.4
20Suc/2Gel/2Arg Liq 09 (Repeat) 6.7 6.7 6.6 6.5 6.4 6.4 6.3 6.2 6.4
6.2 6.5 6.6 20Suc/2Gel/2Arg Liq 10 7.0 7.0 ud ud 6.1 6.2 6.4 6.4
6.7 6.8 6.5 6.6 6.6 6.4 20Suc/2 Ge//0.15 Meth/0.02Plur Liq 10
(Repeat) 6.8 7.0 6.7 6.7 6.6 6.5 6.5 6.3 6.4 6.4 6.3 6.3 20Suc/2
Ge//0.15 Meth/0.02Plur Liq 11 6.9 6.9 ud ud 6 6.1 6.4 6.5 6.6 6.7
6.6 6.3 6.5 6.4 20Suc/2Ge//0.02Plur Liq 12 6.9 6.9 6.5 6.5 6.3 6.1
6.0 6.1 6.1 6.1 5.8 5.7 5.9 5.9 NAF only (60%)
TABLE-US-00033 TABLE 33 First Tier: Potency by FFA Assay A/New
Caledonia (in SPG) Target: 6.8 4.degree. C. (Monthly) Formulation 0
0 1 1 2 2 3 3 4 4 5 5 6 6 Formulation Liq 01 7.1 7.0 7.0 7.0 6.6
6.8 6.5 6.6 6.7 6.8 6.9 6.8 6.5 6.6 15Suc/1Gel/2Arg Liq 01 (Repeat)
7.0 7.0 6.9 6.7 6.7 6.6 6.5 6.5 6.6 6.7 6.6 6.6 15Suc/1Gel/2Arg Liq
02 7.0 7.0 6.9 7.0 6.7 6.6 6.6 6.4 6.5 6.6 6.9 6.8 6.3 6.4
15Suc/1Gel/1Glyc Liq 02 (Repeat) 7.1 7.1 6.9 6.9 6.9 6.7 6.8 6.7
6.7 6.8 6.7 6.4 15Suc/1Gel/1Glyc Liq 03 7.0 7.2 6.8 6.9 6.6 6.4 6.5
6.5 6.5 6.5 6.9 6.9 6.4 6.4 15Suc/1Gel Liq 03 (Repeat) 6.8 6.9 6.7
6.7 6.6 6.6 6.5 6.6 6.6 6.6 6.6 6.5 15Suc/1Gel Liq 04 7.2 7.2 6.8
7.0 6.7 6.4 6.5 6.5 6.5 6.7 7.0 7.1 6.5 6.5 15Suc/2Gel/2Arg Liq 04
(Repeat) 6.9 7.0 6.8 6.9 6.8 6.9 6.8 6.8 6.7 6.6 6.5 6.4
15Suc/2Gel/2Arg Liq 05 7.2 7.1 7.0 7.0 6.7 6.6 6.5 6.6 6.6 6.7 7.0
7.1 6.7 6.6 10Suc/2Gel/2Arg Liq 05 (repeat) 6.9 6.9 6.9 6.8 6.8 6.8
6.8 6.6 6.7 6.6 6.6 10Suc/2Gel/2Arg Liq 06 6.9 6.9 6.4 6.3 5.5 5.5
5.4 5.0 UD UD 4.7 4.7 3.8 4.1 15Suc/1Gel/2Arg, HEPES Liq 07 7.1 7.1
6.5 6.6 6 6.1 6.3 6.3 6.3 6.3 6.8 6.6 6.4 6.2 15Suc/1PVP/2Arg Liq
07 (Repeat) 6.9 7.0 6.4 6.5 6.3 6.4 6.6 6.5 6.4 6.3 6.5 6.2
15Suc/1PVP/2Arg Liq 08 7.1 6.9 6.8 6.9 6.3 6.4 6.4 6.4 6.5 6.5 6.8
7.3 6.4 6.4 15Suc/1Dextran/2Arg Liq 08 (Repeat) 6.7 6.7 6.6 6.5 6.5
6.5 6.5 6.6 6.3 6.5 6.5 6.3 15Suc/1Dextran/2Arg Liq 09 6.8 6.9 7.0
6.8 6.5 5.7 6.5 6.5 6.4 6.5 7.1 7.2 6.4 6.3 20Suc/2Gel/2Arg Liq 09
(Repeat) 6.8 6.9 6.8 6.7 6.6 6.6 6.7 6.6 6.5 6.6 6.6 6.3
20Suc/2Gel/2Arg Liq 10 6.7 6.9 6.8 6.8 6.6 6.5 6.4 6.3 6.2 6.2 6.9
7.0 6.3 6.3 20Suc/2 Ge//0.15 Meth/0.02Plur Liq 10 (Repeat) 6.6 6.6
6.5 6.5 6.4 6.4 6.3 6.3 6.5 6.3 6.2 6.2 20Suc/2 Ge//0.15
Meth/0.02Plur Liq 11 6.8 6.7 6.8 6.7 6.7 6.6 6.3 6.4 6.3 6.2 6.7
6.8 6.4 6.3 20Suc/2Ge//0.02Plur Liq 12 6.9 7.0 5.7 5.6 5.5 5.3 5.1
5.3 4.7 4.6 4.5 4.5 4.4 4.5 NAF only (60%)
TABLE-US-00034 TABLE 34 A/Panama (in SPG) Target: 7.4 4.degree. C.
(Monthly) Formulation 0 0 1 1 2 2 3 3 4 4 5 5 6 6 Formulation Liq
01 7.6 7.6 7.4 7.3 6.9 7.1 6.6 6.6 6.8 7.1 7.4 7.4 6.6 6.7
15Suc/1Gel/2Arg Liq 01 (Repeat) 6.8 6.8 6.8 6.6 6.6 6.7 6.7 6.8 6.6
6.6 6.7 6.7 15Suc/1Gel/2Arg Liq 02 7.6 7.6 7.3 7.3 6.9 6.4 6.6 6.6
6.9 6.8 7.3 7.4 6.6 6.6 15Suc/1Gel/1Glyc Liq 02 (Repeat) 7.3 7.0
7.3 7.1 7.1 7.4 7.3 7.2 7.2 6.7 6.7 15Suc/1Gel/1Glyc Liq 03 7.2 7.8
7.2 7.3 6.8 6.7 6.6 6.7 6.6 7.1 7.3 7.4 6.6 6.8 15Suc/1Gel Liq 03
(Repeat) 6.8 6.8 6.9 6.8 6.7 6.8 6.8 6.8 6.7 6.8 6.8 6.6 15Suc/1Gel
Liq 04 7.8 7.8 7.4 7.4 7.1 7.1 6.5 6.6 6.9 6.9 7.5 7.4 6.6 6.7
15Suc/2Gel/2Arg Liq 04 (Repeat) 7.0 7.1 6.9 6.9 6.8 6.9 6.6 6.8 6.7
6.6 6.7 6.8 15Suc/2Gel/2Arg Liq 05 7.8 7.8 7.5 7.4 6.8 6.8 6.7 6.7
7.0 7.0 7.4 7.3 6.7 6.7 10Suc/2Gel/2Arg Liq 05 (repeat) 7.0 7.0 6.6
6.5 6.7 6.8 6.8 6.8 6.7 6.8 6.7 6.6 10Suc/2Gel/2Arg Liq 06 7.7 7.6
6.9 7.0 6.3 6.4 6.2 5.9 5.8 5.8 6.0 6.0 5.0 4.8 15Suc/1Gel/2Arg,
HEPES Liq 07 7.2 7.1 7.4 7.0 7.0 6.6 6.7 6.5 7.0 7.1 7.2 7.3 6.6
6.3 15Suc/1PVP/2Arg Liq 07 (Repeat) 7.2 7.2 7.2 6.9 7.2 6.9 6.8 6.6
6.6 6.7 6.8 6.9 15Suc/1PVP/2Arg Liq 08 7.1 7.2 7.1 7.2 6.6 6.8 6.7
6.8 6.9 6.9 7.2 7.2 6.5 6.6 15Suc/1Dextran/2Arg Liq 08 (Repeat) 7.2
6.8 7.0 6.8 6.9 6.9 6.9 6.5 6.8 6.9 6.9 6.7 15Suc/1Dextran/2Arg Liq
09 7.4 7.3 6.9 6.9 6.7 7.0 6.3 6.8 6.7 7.0 7.3 7.3 6.5 6.5
20Suc/2Gel/2Arg Liq 09 (Repeat) 7.2 7.2 7.0 6.7 6.8 7.0 6.9 6.9 6.7
7.0 6.8 6.7 20Suc/2Gel/2Arg Liq 10 7.2 7.1 7.1 7.1 6.8 6.6 6.6 6.4
6.7 6.7 7.3 7.2 6.5 6.6 20Suc/2 Ge//0.15 Meth/0.02Plur Liq 10
(Repeat) 7.0 6.7 6.7 6.7 6.7 6.7 6.6 6.6 6.6 6.6 6.6 6.5 20Suc/2
Ge//0.15 Meth/0.02Plur Liq 11 7.1 6.9 7.1 7.2 6.5 6.6 6.7 6.5 6.6
6.9 7.2 7.3 6.6 6.4 20Suc/2Ge//0.02Plur Liq 12 7.2 7.3 6 6.4 6.3
5.9 4.9 5.0 5.1 4.5 4.6 4.6 3.8 4.1 NAF only (60%)
TABLE-US-00035 TABLE 35 Formulations: Second Tier Control Liq15
Ingredients Liq13a Liq14 Liq15 (degassed) Liq16 Liq17 Liq18 Liq19
Liq20 KPO4 buffer, pH 7.2 1.1 mM 100 mM 100 mM 100 mM 100 mM 100 mM
100 mM 100 mM (1.1 mM from virus included) Citrate buffer, pH 7.2
100 mM Sucrose (0.7% from 7% 10% 10% 10% 10% 10% 10% 10% 10% virus
included) Gelatin 2% 2% 2% 2% 2% 2% 2% 2% Arginine 2% 2% 2% 2% 2%
2% 2% 2% Aprotinin (PI) 0.02%.sup. Leupeptin 0.02%.sup. hemisulfate
(PI) Lysozyme Inhibitor 0.1% Protease Inhibitor (0.6% DMSO)
Cocktail 0.5% * PMSF 1 mM Cytidine 2' monophosphate NAF (from
virus) 10% 10% 10% 10% 10% 10% 10% 10% 10% NAF (added) 50% 50% 50%
50% 50% 50% 50% 50% 50% 1N KOH or iN pH 7.2 to pH 7.0 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 HCl to pH
7.2 Purified Water None q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s.
added Ingredients Liq21 Liq22 Liq23 Liq31 Liq24 Liq25 Liq26 Liq27
KPO4 buffer, pH 7.2 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100
mM 100 mM (1.1 mM from virus included) Sucrose (0.7% from 10% 10%
10% 10% 10% 10% 9.3% virus included) Gelatin 10% 2% 2% 2% 2% 2% 2%
2% Arginine 2% 2% 2% 2% 2% 2% 2% 2% L-Ascorbic Acid 2% 0.05%.sup.
Ascorbic Acid 6 0.005% 0.001% Palmitate Arbutin 0.05%.sup. Propyll
Gallate 0.05%.sup. EDTA 10 mM RNAse Inhibitor, (0.05% Glyc)
SuperAse In 2.0 U/.mu.L NAF (from virus) 5 mM 10% 10% 10% 10% 10%
10% 10% NAF (added) 10% 50% 50% 50% 50% 50% 50% 50% 1N KOH or 1N
50% titrate 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
to pH 7.2 Purified Water to pH 7.2 q.s. q.s. q.s. q.s. q.s. q.s.
q.s. q.s.
TABLE-US-00036 TABLE 36 Second Tier: Potency by FFA Assay SP
stabilized B/Hongkong Target: 6.9 15.degree. C. (weekly) 0 0 2 2 4
4 6 6 8 8 10 10 12 12 14 14 Liq 13a 6.6 6.7 6.3 6.3 5.8 5.8 5.7 5.6
5.5 5.0 5.1 5.2 5.5 5.1 4.4 4.7 no excipient (60% NAF), equil. at
15.degree. C. Liq 13b 6.6 6.6 6.4 6.4 6.0 5.8 5.6 5.5 5.0 5.3 4.8
4.9 ud 4.6 ud 5.2 no excipient (60% NAF), equil. at 33.degree. C.
Liq 13c 6.5 6.7 6.1 6.3 5.7 5.8 5.5 5.6 5.3 5.2 5.1 4.7 4.7 4.5 4.7
4.5 no excipient (60% NAF), equil. at 45.degree. C. Liq 14 6.7 6.8
6.4 6.7 6.0 6.0 5.7 5.6 5.3 5.3 5.3 5.3 5.0 4.9 4.8 4.8 Citr.,
10Suc/2Gel/2Arg, equil. at 15.degree. C. Liq 15a 6.9 6.7 6.6 6.5
6.2 6.2 6.0 5.9 5.6 5.8 5.5 5.5 5.4 5.1 5.0 5.4 10Suc/2Gel/2Arg,
equil. at 15.degree. C. Liq 15b 6.7 6.9 6.6 6.5 6.1 6.1 6.0 6.0 5.4
5.3 4.8 5.7 5.1 5.2 5.0 4.7 10Suc/2Gel/2Arg, equil. at 33.degree.
C. Liq 15c 6.7 6.8 6.4 6.4 5.9 6.0 5.7 5.8 4.8 5.3 5.2 5.2 ud 5.0
4.5 4.7 10Suc/2Gel/2Arg, equil. at 45.degree. C. Liq 15d 7.0 7.0
6.2 6.3 5.7 5.9 5.8 5.8 disc. 10Suc/2Gel/2Arg (Degassed) Liq 16 6.8
6.9 6.2 6.2 5.8 5.8 5.8 5.5 5.6 5.6 5.6 5.2 disc.
10Suc/2Gel/2Arg/0.02Aprotinin(PI) Liq 17 6.8 6.9 6.1 6.1 5.8 5.8
5.6 5.9 5.6 5.7 5.6 5.5 disc. 10Suc/2Gel/2Arg/0.02Leup.
Hemisulfate(PI) Liq 18 7.0 7.1 6.3 6.3 6.2 6.0 5.9 5.9 disc.
10Suc/2Gel/2Arg/0.1Lysozyme Inhib. Liq 19 6.6 6.6 6.2 6.2 5.9 5.6
5.7 5.6 5.7 5.5 5.3 5.2 disc. 10Suc/2Gel/2Arg/0.5Prot. Inhib.
Cocktail Liq 20 ud 4.8 ud ud ud ud dsic. 10Suc/2Gel/2Arg/1 mM PMSF
Liq 21 6.7 6.5 6.3 6.3 5.9 5.9 5.9 5.8 6.3 6.3 5.8 5.4 disc.
10Suc/2Gel/2Arg/1 mM Cytid. 2'Monophos. Liq 22 6.8 6.7 5.8 5.6 5.6
5.7 ud ud ud ud disc. 10Suc/2Gel/2Arg/0.05L-Ascorbic Acid Liq 22a
(deg) 6.5 6.5 5.2 5.9 disc. 10Suc/2Gel/2Arg/0.05L-Ascorbic Acid Liq
23 4.5 4.9 ud ud ud ud disc 10Suc/2Gel/2Arg/
0.005AscorbAcid6Palmitate Liq 24 6.8 6.7 6.4 6.5 6.3 5.9 5.8 5.7
5.3 5.5 5.8 5.3 5.1 5.1 4.8 4.8 10Suc/2Gel/2Arg/0.05Arbutin Liq 24
(deg) 6.7 6.6 6.2 6.3 6.0 5.0 disc. 10Suc/2Gel/2Arg/0.05Arbutin Liq
25 6.6 6.7 5.1 6.2 5.1 ud ud ud ud ud disc.
10Suc/2Gel/2Arg/0.05PropyllGallate Liq 25 (deg) 6.4 6.4 4.8 4.7
disc. 10Suc/2Gel/2Arg/0.05PropyllGallate Liq 26 6.8 6.8 6.6 6.4 6.3
6.3 5.8 6.0 5.4 5.5 5.4 5.6 5.3 5.4 5.3 5.3 10Suc/2Gel/2Arg/10
mMEDTA Liq 26a (deg) 6.8 6.7 6.5 6.5 6.5 6.3 10Suc/2Gel/2Arg/10
mMEDTA Liq 27 6.6 6.7 ud ud 10Suc/2Gel/2Arg/RNAse Inhibt. 2 U/.mu.L
Liq 31 ud 5.9 ud ud ud ud disc 10Suc/2Gel/2Arg/
0.001AscorbAcid6Palmitate
TABLE-US-00037 TABLE 37 Second Tier: Potency by FFA Assay SP
stabilized A/Panama Target: 7.5 15.degree. C. (weekly) 0 0 2 2 4 4
6 6 8 8 10 Liq 13a 7.0 6.9 6.4 6.4 5.3 5.5 5.4 5.3 5.3 4.7 5.1 Liq
13b 6.9 6.9 6.4 6.3 5.4 5.6 5.4 5.6 5.2 4.8 4.9 Liq 13c 6.8 6.7 6.3
6.3 5.4 5.6 5.3 5.0 5.1 4.7 4.5 Liq 14 7.3 7.3 7.2 7.2 6.8 6.8 6.7
6.8 6.5 6.5 6.7 Liq 15a 7.2 7.2 6.9 7 6.6 6.7 6.6 6.4 6.2 6.0 6.0
Liq 15b 7.3 7.1 7.0 6.9 6.3 6.5 6.7 6.6 6.0 5.8 6.1 Liq 15c 7.1 6.9
6.8 6.8 6.3 6.0 6.3 6.5 5.8 5.8 6.1 Liq 15d 6.8 6.7 6.7 6.5 6.5 6.4
6.4 6.4 6.4 6.4 Liq 16 only for B/Hongkong Liq 17 only for
B/Hongkong Liq 18 6.9 6.9 6.7 6.5 6.2 6.5 6.2 6.4 6.5 6.4 Liq 19
6.5 6.6 6.6 6.5 5.7 6.0 5.9 ud 5.4 5.4 5.5 Liq 20 6.8 6.9 6.0 5.7
ud ud ud ud disc. Liq 21 6.9 6.7 6.2 6.3 6.5 6.6 6.9 7.1 5.8 5.9
6.1 Liq 22 7.1 7.2 6.0 6.0 5.9 5.8 5.1 ud 5.7 ud ud Liq 22a (deg)
6.7 6.7 6.2 5.9 6.0 5.9 Liq 23 5.6 5.5 ud ud ud disc. Liq 24 7.0
6.9 6.7 6.8 6.6 6.6 6.4 6.5 6.2 6.2 6 Liq 24a (deg) 6.6 6.6 6.5 6.6
Liq 25 6.9 6.5 5.1 5.5 ud ud ud ud ud ud disc Liq 25a (deg) ud ud
ud ud disc Liq 26 7.4 7.3 7.2 7.1 ud(repd) ud(repd) 6.9 6.9 6.7 6.7
6.7 Liq 26a (deg) 7.1 7.2 7 7.1 7.1 6.9 Liq 27 only for B/Hongkong
Liq 31 ud ud ud ud 3.5 3.8 disc. 10 12 12 14 14 16 16 Liq 13a 4.8
4.5 4.6 4.2 3.8 no excipient (60% NAF), equil. at 15.degree. C. Liq
13b 5.0 4.8 4.6 4.3 4.6 no excipient (60% NAF), equil. at
33.degree. C. Liq 13c 4.7 4.7 4.9 4.5 4.2 no excipient (60% NAF),
equil. at 45.degree. C. Liq 14 6.6 6.4 6.5 6.1 6.2 6.5 6.5 Citr.,
10Suc/2Gel/2Arg, equil. at 15.degree. C. Liq 15a 6.0 5.7 6.1 5.7
5.7 6.1 5.8 10Suc/2Gel/2Arg. equil. at 15.degree. C. Liq 15b 6.0
6.1 5.8 5.9 5.8 6.0 6.0 10Suc/2Gel/2Arg, equil. at 33.degree. C.
Liq 15c 6.1 5.8 5.8 5.8 5.8 5.9 5.9 10Suc/2Gel/2Arg, equil. at
45.degree. C. Liq 15d 10Suc/2Gel/2Arg (Degassed) Liq 16
10Suc/2Gel/2Arg/0.02Aprotinin(PI) Liq 17 10Suc/2Gel/2Arg/0.02Leup.
Hemisulfate(PI) Liq 18 10Suc/2Gel/2Arg/0.1Lysozyme Inhib. Liq 19
5.8 5.8 5.8 10Suc/2Gel/2Arg/0.5Prot. Inhib. Cocktail Liq 20
10Suc/2Gel/2Arg/1 mM PMSF Liq 21 6.0 5.9 5.7 6 6.1 5.8 5.6
10Suc/2Gel/2Arg/1 mM Cytid. 2'Monophos. Liq 22 5.4 ud ud disc.
10Suc/2Gel/2Arg/0.05L-Ascorbic Acid Liq 22a (deg)
10Suc/2Gel/2Arg/0.05L-Ascorbic Acid Liq 23
10Suc/2Gel/2Arg/0.005AscorbAcid6Palmitate Liq 24 6.3 6.1 5.9 5.7
5.9 5.9 6.1 10Suc/2Gel/2Arg/0.05Arbutin Liq 24a (deg)
10Suc/2Gel/2Arg/0.05Arbutin Liq 25
10Suc/2Gel/2Arg/0.05PropyllGallate Liq 25a (deg)
10Suc/2Gel/2Arg/0.05PropyllGallate Liq 26 6.9 6.5 6.4 6.4 6.4
10Suc/2Gel/2Arg/10 mMEDTA Liq 26a (deg) 10Suc/2Gel/2Arg/10 mMEDTA
Liq 27 10Suc/2Gel/2Arg/RNAse Inhibt. 2 U/.mu.L Liq 31
10Suc/2Gel/2Arg/0.001AscorbAcid6Palmitate
TABLE-US-00038 TABLE 38 Second Tier: Potency of FFA Assay SP
stabilized B/Hongkong Target: 6.9 4.degree. C. (Monthly) 0 0 1 1 2
2 3 3 4 4 Liq 13a 6.6 6.7 6.4 6.5 6.2 6.2 6.4 6.3 6.1 6.2 no
excipient (60% NAF), equil. at 15.degree. C. Liq 13b 6.6 6.6 6.5
6.4 6.3 6.2 6.3 6.4 6.1 6.2 no excipient (60% NAF), equil. at
33.degree. C. Liq 13c 6.5 6.7 6.4 6.4 6.1 6.1 6.3 6.4 6 5.9 no
excipient (60% NAF), equil. at 45.degree. C. Liq 14 6.0 6.8 6.4 6.5
6.0 6.4 6.3 6.4 6.1 6.1 Citr., 10Suc/2Gel/2Arg, equil. at
15.degree. C. Liq 15a 6.9 6.7 6.7 6.8 6.4 6.3 6.7 6.7 6.5 6.5
10Suc/2Gel/2Arg, equil. at 15.degree. C. Liq 15b 6.7 6.9 6.7 6.6
6.0 6.2 6.5 6.5 6.4 6.4 10Suc/2Gel/2Arg, equil. at 33.degree. C.
Liq 15c 6.7 6.8 6.6 6.6 6.1 6.2 6.5 6.4 6.4 6.4 10Suc/2Gel/2Arg,
equil. at 45.degree. C. Liq 15d 7.0 7.0 6.8 6.8 6.5 6.3
10Suc/2Gel/2Arg (Degassed) Liq 16 6.8 6.9 6.4 6.7 6.6 6.5 6.1 6.3
10Suc/2Gel/2Arg/0.02Aprotinin(PI) Liq 17 6.8 6.9 6.7 6.6 6.5 6.5
6.1 6.3 10Suc/2Gel/2Arg/0.02Leup. Hemisulfate(PI) Liq 18 7.0 7.1
6.9 6.8 6.5 6.5 10Suc/2Gel/2Arg/0.1Lysozyme Inhib. Liq 19 6.6 6.6
6.5 6.4 6.5 6.5 10Suc/2Gel/2Arg/0.5Prot. Inhib. Cocktail Liq 20 ud
4.8 ud ud disc. 10Suc/2Gel/2Arg/1 mM PMSF Liq 21 6.7 6.5 6.6 6.6
7.0 7.0 6.6 6.6 10Suc/2Gel/2Arg/1 mM Cytid. 2'Monophos. Liq 22 6.8
6.7 5.9 UD UD UD 5.2 5.5 10Suc/2Gel/2Arg/0.05L-Ascorbic Acid Liq
22a (deg) 6.5 6.5 6 6.2 10Suc/2Gel/2Arg/0.05L-Ascorbic Acid Liq 23
4.5 4.9 ud ud disc. 10Suc/2Gel/2Arg/0.005AscorbAcid6Palmitate Liq
24 6.8 6.7 6.7 6.6 ud (rep) ud (rep) 6.4 6.3 6.3 6.2
10Suc/2Gel/2Arg/0.05Arbutin Liq 24 (deg) 6.7 6.6 7.0 7.0
10Suc/2Gel/2Arg/0.05Arbutin Liq 25 6.6 6.7 6.2 6.3 5.2 5.2 5.4 5.3
10Suc/2Gel/2Arg/0.05PropyllGallate Liq 25 (deg) 6.4 6.4 5.8 5.9
10Suc/2Gel/2Arg/0.05PropyllGallate Liq 26 6.8 6.8 6.6 6.7 6.6 6.5
6.6 6.6 6.3 6.3 10Suc/2Gel/2Arg/10 mMEDTA Liq 26a (deg) 6.8 6.7 6.9
6.9 10Suc/2Gel/2Arg/10 mMEDTA Liq 27 6.6 6.7 6.6 6.4 6.6 6.5 6.3
6.3 10Suc/2Gel/2Arg/RNAse Inhibt. 2 U/.mu.L Liq 31 ud 5.9 5.1 4.9
disc. 10Suc/2Gel/2Arg/0.001AscorbAcid6Palmitate
TABLE-US-00039 TABLE 39 Second Tier: Potency of FFA Assay SP
stabilized A/Panama Target: 7.5 4.degree. C. (Monthly) 0 0 1 1 2 2
3 3 4 4 Liq 13a 7.0 6.9 6.2 6.2 6.0 6.1 5.8 5.9 5.8 5.6 no
excipient (60% NAF), equil. at 15.degree. C. Liq 13b 6.9 6.9 5.9
6.0 6.0 6.0 5.9 5.7 5.9 6.0 no excipient (60% NAF), equil. at
33.degree. C. Liq 13c 6.8 6.7 6 6.1 5.8 6.1 5.9 5.7 5.8 5.6 no
excipient (60% NAF), equil. at 45.degree. C. Liq 14 7.3 7.3 7.2 7.2
7.3 7.2 7.1 7.5 6.8 7 Citr., 10Suc/2Gel/2Arg, equil. at 15.degree.
C. Liq 15a 7.2 7.2 6.7 6.6 6.6 6.6 6.5 7.0 6.4 6.6 10Suc/2Gel/2Arg,
equil. at 15.degree. C. Liq 15b 7.3 7.1 6.7 6.7 6.7 6.6 6.9 6.8 6.4
6.6 10Suc/2Gel/2Arg, equil. at 33.degree. C. Liq 15c 7.1 6.9 6.5
6.4 6.7 6.6 6.6 6.6 6.4 6.4 10Suc/2Gel/2Arg, equil. at 45.degree.
C. Liq 15d 6.8 6.7 6.9 6.8 6.4 6.4 10Suc/2Gel/2Arg (Degassed) Liq
16 only for B/Hongkong 10Suc/2Gel/2Arg/0.02Aprotinin(PI) Liq 17
only for B/Hongkong 10Suc/2Gel/2Arg/0.02Leup. Hemisulfate(PI) Liq
18 6.9 6.9 6.9 6.8 6.4 6.3 10Suc/2Gel/2Arg/0.1Lysozyme Inhib. Liq
19 6.5 6.6 6.2 6.4 6.3 6.6 6.3 6.3 10Suc/2Gel/2Arg/0.5Prot. Inhib.
Cocktail Liq 20 6.8 6.9 6.9 7.0 7.1 7.2 6.9 6.7 10Suc/2Gel/2Arg/1
mM PMSF Liq 21 6.9 6.7 6.7 6.8 6.7 6.8 7.0 7.1 10Suc/2Gel/2Arg/1 mM
Cytid. 2'Monophos. Liq 22 7.1 7.2 6.2 6.0 6.3 5.8 6.0 5.9 6.1 6.1
10Suc/2Gel/2Arg/0.05L-Ascorbic Acid Liq 22a (deg) 6.7 6.7 6.7 6.6
10Suc/2Gel/2Arg/0.05L-Ascorbic Acid Liq 23 5.6 5.5 ud ud disc.
10Suc/2Gel/2Arg/0.005AscorbAcid6Palmitate Liq 24 7.0 6.9 6.5 6.7
6.4 6.3 6.8 6.7 6.4 6.5 10Suc/2Gel/2Arg/0.05Arbutin Liq 24a (deg)
6.7 6.6 6.2 6.2 10Suc/2Gel/2Arg/0.05Arbutin Liq 25 6.9 6.5 5.1 7.2
5.1 ud disc. 10Suc/2Gel/2Arg/0.05PropyllGallate Liq 25a (deg) 6 6.3
rep 6.4 10Suc/2Gel/2Arg/0.05PropyllGallate Liq 26 7.4 7.3 7.3 7.3
7.3 7.3 7.4 7.3 7.0 7.1 10Suc/2Gel/2Arg/10 mMEDTA Liq 26a (deg) 7.1
7.2 7.4 7.5 7.1 7.0 10Suc/2Gel/2Arg/10 mMEDTA Liq 27 only for
B/Hongkong 10Suc/2Gel/2Arg/RNAse Inhibt. 2 U/.mu.L Liq 31 ud ud ud
ud disc. 10Suc/2Gel/2Arg/0.001AscorbAcid6Palmitate
TABLE-US-00040 TABLE 40 Formulation No 20 mM 50 mM 100 mM 200 mM
Ingredients Citrate Citrate Citrate Citrate Citrate Citrate buffer
0 20 mM 50 mM 100 mM 200 mM pH 7.2 KPO4 buffer 1.1 mM 1.1 mM 1.1 mM
1.1 mM 1.1 mM (from virus 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 10% 10% 10% 10% 10% virus: B/Hongkong; A/Panama)
NAF (added) 50% 50% 50% 50% 50% 1N KOH or 1N titrate titrate
titrate titrate titrate HCl to pH 7.2 to to pH 7.2 to pH 7.2 to pH
7.2 to pH.7.2 pH 7.2 Purified Water q.s. q.s. q.s. q.s. q.s.
TABLE-US-00041 TABLE 41 Results: Potency by FFA Assay A/Panama
A/Panama B/Hongkong B/Hongkong Aliquot 1 Aliquot 2 Aliquot 1
Aliquot 2 A/Panama, Ave. of 9 Ave. of 6 Starting material plates
plates 8.1 .+-. 0.2 7.9 .+-. 0.1 (8.5) (7.9) Pre-diluted Starting
8.0 .+-. 0.1 none n/a n/a Material (1:10) (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-US-00042 TABLE 42 Formulation No 0.5 mM 1.0 mM 2.0 mM 5.0 mM
10 mM Ingredients EDTA EDTA EDTA EDTA EDTA EDTA KPO4 buffer, pH 7.2
100 mM 100 mM 100 mM 100 mM 100 mM 100 mM (1.1 mM from virus
included) Sucrose (0.7% from 10% 10% 10% 10% 10% 10% virus
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%
B/Hongkong; A/Panama) NAF (added) 50% 50% 50% 50% 50% 50% 1N KOH or
1N 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-US-00043 TABLE 43 Results: Potency by FFA Assay A/Panama
A/Panama B/Hongkong B/Hongkong Aliquot 1 Aliquot 2 Aliquot 1
Aliquot 2 A/Panama, Ave. of 9 Ave. of 6 Starting material plates
plates 8.1 .+-. 0.2 7.9 .+-. 0.1 (8.5) (7.9) Pre-diluted Starting
8.0 .+-. 0.1 none n/a n/a Material (1:10) (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 .+-. 0.1 6.7 .+-. 0.1 6.6 .+-. 0.1
(7.0) (7.0) (6.9) (6.9) 1.0 mM EDTA 6.5 .+-. 0.1 6.5 .+-. 0.1 6.8
.+-. 0.1 6.6 .+-. 0.1 (7.0) (7.0) (6.9) (6.9) 2.0 mM EDTA 6.6 .+-.
0.0 6.8 .+-. 0.1 6.6.+-. 6.6 .+-. 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 mM KPO4,
60% NAF, 10% Sucrose, 1% Gelatin 2% Arginine
TABLE-US-00044 TABLE 44 Formulations: Third Tier Ingredients Liq28
Liq29 Liq30 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%
EDTA (5 mM) (10 mM) (10 mM) 0.186% 0.372% 0.372% NAF (from virus)
10% 10% 10% NAF (added) 50% 50% 50% 1N HCl or titrate titrate
titrate 1N KOH to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 Purified
Water q.s. q.s. q.s.
TABLE-US-00045 TABLE 45 Third Tier: Potency of FFA Assay SP
stabilized B/Hongkong Target: 6.9 15.degree. C. (weekly) A 0 0 2 2
4 4 6 6 8 8 10 10 12 12 14 14 Formulation Liq 26 6.8 6.8 6.6 6.4
6.3 6.3 5.8 6.0 5.4 5.5 5.4 5.6 5.3 5.4 5.3 5.3 10Suc/2Gel/2Arg/10
mM EDTA Liq 26a (deg) 6.8 6.7 6.5 6.5 6.5 6.3 10Suc/2Gel/2Arg/10 mM
EDTA Liq 28 6.2 5.9 5.8 5.9 6.2 6.0 5.4 5.3 5.1 5.1 disc.
10Suc/2Gel/2Arg/5 mM EDTA Liq 28a 6.5 6.5 6.2 6.0 6.0 5.9 5.8 5.6
disc. 10Suc/2Gel/2Arg/5 mM EDTA Liq 29 4.9 4.9 4.8 4.7 5.0 4.8 4.2
4.0 ud 3.6 disc. 10Suc/2Arg/10 mM EDTA Liq 29a 6.3 6.4 6.0 6.3 6.1
5.9 5.8 5.7 disc. 10Suc/2Arg/10 mM EDTA Liq 30 6.6 6.6 6.3 6.4 6.7
6.5 5.9 5.9 5.4 5.4 disc. Citr./10Suc/2Gel/2Arg/10 mM EDTA Liq 30a
6.6 6.6 6.1 6.2 6.1 5.9 5.8 5.8 disc. Citr./10Suc/2Gel/2Arg/10 mM
EDTA SP stabilized B/Hongkong Target: 6.9 4.degree. C. (Monthly) B
0 0 1 1 2 2 3 3 4 4 Formulation Liq 26 6.8 6.8 6.6 6.7 6.6 6.5 6.6
6.6 6.3 10Suc/2Gel/2Arg/10 mM EDTA Liq 26a (deg) 6.8 6.7 6.9 6.9
10Suc/2Gel/2Arg/10 mM EDTA Liq 28 6.2 5.9 6.6 6.4 6.6 6.5 5.8 5.8
10Suc/2Gel/2Arg/5 mM EDTA Liq 28a 6.5 6.5 6.7 6.8 6.5 6.6
10Suc/2Gel/2Arg/5 mM EDTA Liq 29 4.9 4.9 5.1 5.1 4.9 4.9 disc.
10Suc/2Arg/10 mM EDTA Liq 29a 6.3 6.4 6.4 6.5 6.3 6.4 10Suc/2Arg/10
mM EDTA Liq 30 6.6 6.6 6.8 7.1 6.6 6.5 6.2 6.0
Citr./10Suc/2Gel/2Arg/10 mM EDTA Liq 30a 6.6 6.6 6.9 6.7 6.4 6.0
Citr./10Suc/2Gel/2Arg/10 mM EDTA SP stabilized A/Panama Target: 7.5
15.degree. C. (weekly) C 0 0 2 2 4 4 6 6 8 8 10 10 12 12 14 14
Formulation Liq 26 7.4 7.3 7.2 7.1 ud(repd) ud(repd) 6.9 6.9 6.7
6.7 6.7 6.9 6.5 6.4 6.4 6.4 10Suc/2Gel/2Arg/10 mM EDTA Liq 26a
(deg) 7.1 7.2 7 7.1 7.1 6.9 6.4 6.8 10Suc/2Gel/2Arg/10 mM EDTA Liq
28 7.5 7.4 7.1 7.1 6.9 7.0 6.7 6.7 6.5 6.5 6.6 6.6 6.1 6.1
10Suc/2Gel/2Arg/5 mM EDTA Liq 29 7.3 7.3 6.9 6.9 7.0 7.0 6.7 6.8
6.6 6.7 6.5 6.7 6.0 6.0 10Suc/2Arg/10 mM EDTA Liq 30 7.4 7.3 7.0
7.0 6.8 6.9 6.3 6.3 6.1 6.3 6.2 6.1 5.8 5.6
Citr./10Suc/2Gel/2Arg/10 mM EDTA SP stabilized A/Panama Target: 7.5
4.degree. C. (Monthly) D 0 0 1 1 2 2 3 3 4 4 Formulation Liq 26 7.4
7.3 7.3 7.3 7.3 7.3 7.4 7.3 7.0 7.1 10Suc/2Gel/2Arg/10 mM EDTA Liq
26a (deg) 7.1 7.2 7.4 7.5 7.1 7.0 10Suc/2Gel/2Arg/10 mM EDTA Liq 28
7.5 7.4 7.3 7.3 7.1 7.2 10Suc/2Gel/2Arg/5 mM EDTA Liq 29 7.3 7.3
7.2 7.5 7.1 7.1 10Suc/2Arg/10 mM EDTA Liq 30 7.4 7.3 7.3 7.4 7.0
7.2 Citr./10Suc/2Gel/2Arg/10 mM EDTA
TABLE-US-00046 TABLE 46 Formulations: P-B 4-level Custom Screen A
Ingredients Liq36 Liq37 Liq38 Liq39 Liq40 Liq41 Liq42 Liq43 Liq44
Liq45 Liq46 Liq47 KPO4 buffer, pH 7.2 50 mM 50 mM 50 mM 50 mM 50 mM
50 mM 50 mM 50 mM 50 mM 50 mM 50 mM 50 mM (1.1 mM from virus
included) Sucrose (0.7% from 0.0% 7.5% 7.5% 7.5% 7.5% 7.5% 7.5% 10%
10% 10% 10% 10% virus 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
mM 5 mM 1 mM 2.7 mM 5 mM 1 mM 5 mM 5 mM 1 mM 1 mM 2.7 mM NAF (from
virus: 10% 10% 10% 10% 10% 10% 10% 10% 10% 10% 10% 10% B/Hongkong;
A/Panama) NAF (added) 50% 50% 50% 50% 50% 50% 50% 50% 50% 50% 50%
50% 1N KOH or 1N titrate titrate titrate titrate titrate titrate
titrate titrate titrate titrate titrate titrate HCl 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 B Ingredients Liq48
Liq49 Liq50 Liq51 Liq52 Liq53 Liq54 Liq55 Liq56 Liq57 Liq58 Liq59
KPO4 buffer, pH 7.2 50 mM 50 mM 50 mM 50 mM 50 mM 50 mM 50 mM 50 mM
50 mM 50 mM 50 mM 50 mM (1.1 mM from virus included) Sucrose (0.7%
from 10% 10% 10% 10% 15% 15% 15% 15% 15% 15% 15% 15% virus
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 mM 2.7 mM 2.7
mM 1 mM 2.7 mM 2.7 mM 5 mM 1 mM 5 mM 1 mM NAF (from virus: 10% 10%
10% 10% 10% 10% 10% 10%'.sup. 10% 10% 10% 10% B/Hongkong; A/Panama)
NAF (added) 50% 50% 50% 50% 50% 50% 50% 50% 50% 50% 50% 50% 1N KOH
or 1N titrate titrate titrate titrate titrate titrate titrate
titrate titrate titrate titrate titrate HCl 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
TABLE-US-00047 TABLE 47 P-B Custon Screen 4-level: Potency by FFA
Assay SP stabilized B/Hongkong Target: 6.9 4.degree. C. (monthly) 0
0 0 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 Liq 36 6.8 6.8 6.7 50
mMKPO4/0Suc/0Gel/2Arg/1 mmEDTA Liq 37 6.6 6.6 6.7 50
mMKPO4/7.5Suc/0Gel/2Arg/2.7 mmEDTA Liq 38 6.6 6.5 6.6 50
mMKPO4/7.5Suc/0Gel/4Arg/5 mmEDTA Liq 39 6.9 6.8 6.7 50
mMKPO4/7.5Suc/1Gel/0Arg/1 mmEDTA Liq 40 6.7 6.7 6.7 50
mMKPO4/7.5Suc/1Gel/4Arg/2.7 mmEDTA Liq 41 7.0 7.0 7.0 50
mMKPO4/7.5Suc/2Gel/0Arg/5 mmEDTA Liq 42 6.9 6.9 7.0 50
mMKPO4/7.5Suc/2Gel/2Arg/1 mmEDTA Liq 43 6.8 6.8 6.7 50
mMKPO4/10Suc/0Gel/4Arg/5 mmEDTA Liq 44 6.8 6.9 6.9 50
mMKPO4/10Suc/0Gel/2Arg/5 mmEDTA Liq 45 6.7 6.8 6.8 50
mMKPO4/10Suc/0Gel/4Arg/1 mmEDTA Liq 46 7.0 7.0 6.8 50
mMKPO4/10Suc/1Gel/2Arg/1 mmEDTA Liq 47 7.1 7.0 7.0 50
mMKPO4/10Suc/1Gel/2Arg/2.7 mmEDTA Liq 48 7.0 6.9 6.9 50
mMKPO4/10Suc/1Gel/4Arg/5 mmEDTA Liq 49 7.0 7.0 7.0 50
mMKPO4/10Suc/2Gel/0Arg/1 mmEDTA Liq 50 7.0 6.8 7.0 50
mMKPO4/10Suc/2Gel/0Arg/2.7 mmEDTA Liq 51 6.9 6.8 6.9 50
mMKPO4/10Suc/2Gel/4Arg/2.7 mmEDTA Liq 52 6.6 6.7 6.7 50
mMKPO4/15Suc/0Gel/2Arg/2.7 mmEDTA Liq 53 6.7 6.8 6.7 50
mMKPO4/15Suc/0Gel/2Arg/1 mmEDTA Liq 54 6.7 6.8 6.7 50
mMKPO4/15Suc/0Gel/4Arg/2.7 mmEDTA Liq 55 6.9 6.8 6.9 50
mMKPO4/15Suc/1Gel/0Arg/2.7 mmEDTA Liq 56 6.8 6.8 7 50
mMKPO4/15Suc/1Gel/0Arg/5 mmEDTA Liq 57 6.9 6.8 6.8 50
mMKPO4/15Suc/1Gel/4Arg/1 mmEDTA Liq 58 6.9 6.8 6.9 50
mMKPO4/15Suc/2Gel/2Arg/5 mmEDTA Liq 59 50 mMKPO4/15Suc/2Gel/4Arg/1
mmEDTA
TABLE-US-00048 TABLE 48 P-B Custom Screen 4-level SP stabilized
A/Panama Targets: 7.0 4.degree. C. (monthly) 0 0 0 1 1 1 2 2 2 3 3
3 4 4 4 5 5 5 Liq 36 6.4 6.2 6.1 50 mMKPO4/0Suc/0Gel/2Arg/1 mmEDTA
Liq 37 6.6 6.7 6.6 50 mMKPO4/7.5Suc/0Gel/2Arg/2.7 mmEDTA Liq 38 6.7
6.7 6.6 50 mMKPO4/7.5Suc/0Gel/4Arg/5 mmEDTA Liq 39 6.4 6.4 6.4 50
mMKPO4/7.5Suc/1Gel/0Arg/1 mmEDTA Liq 40 6.7 6.7 6.6 50
mMKPO4/7.5Suc/1Gel/4Arg/2.7 mmEDTA Liq 41 6.0 6.0 6.0 50
mMKPO4/7.5Suc/2Gel/0Arg/5 mmEDTA Liq 42 5.9 5.9 6.0 50
mMKPO4/7.5Suc/2Gel/2Arg/1 mmEDTA Liq 43 6.5 6.6 6.5 50
mMKPO4/10Suc/0Gel/4Arg/5 mmEDTA Liq 44 6.5 6.4 6.4 50
mMKPO4/10Suc/0Gel/2Arg/5 mmEDTA Liq 45 6.3 6.3 6.4 50
mMKPO4/10Suc/0Gel/4Arg/1 mmEDTA Liq 46 6.3 6.3 6.3 50
mMKPO4/10Suc/1Gel/2Arg/1 mmEDTA Liq 47 6.7 6.7 6.6 50
mMKPO4/10Suc/1Gel/2Arg/2.7 mmEDTA Liq 48 6.6 6.6 6.7 50
mMKPO4/10Suc/1Gel/4Arg/5 mmEDTA Liq 49 6.1 6.1 6.1 50
mMKPO4/10Suc/2Gel/0Arg/1 mmEDTA Liq 50 6.6 6.6 6.5 50
mMKPO4/10Suc/2Gel/0Arg/2.7 mmEDTA Liq 51 6.9 7.0 6.9 50
mMKPO4/10Suc/2Gel/4Arg/2.7 mmEDTA Liq 52 6.8 6.7 6.8 50
mMKPO4/15Suc/0Gel/2Arg/2.7 mmEDTA Liq 53 6.7 6.7 6.7 50
mMKPO4/15Suc/0Gel/2Arg/1 mmEDTA Liq 54 6.9 6.9 6.8 50
mMKPO4/15Suc/0Gel/4Arg/2.7 mmEDTA Liq 55 6.7 6.9 6.7 50
mMKPO4/15Suc/1Gel/0Arg/2.7 mmEDTA Liq 56 6.8 6.6 6.8 50
mMKPO4/15Suc/1Gel/0Arg/5 mmEDTA Liq 57 6.9 6.8 6.9 50
mMKPO4/15Suc/1Gel/4Arg/1 mmEDTA Liq 58 6.8 6.8 7.0 50
mMKPO4/15Suc/2Gel/2Arg/5 mmEDTA Liq 59 6.9 6.8 6.6 50
mMKPO4/15Suc/2Gel/4Arg/1 mmEDTA
TABLE-US-00049 TABLE 49 Formulations in the Comparison Study
Unpurified VH purified VH 1 DiaFiltered VH 2 10% AF 60% AF <1%
AF 10% AF 60% AF <1% AF 7% SucrosePO.sub.4 (base stabilizers) 2
3 1 16 Wyeth Clinical Formulation 7 8 4 5 6 7% SucrosePO.sub.4 Arg
17 7% SucrosePO.sub.4 Arg, Gel 18 10% SucrosePO.sub.4, Arg, Gel 9
10% SucrosePO.sub.4, Arg 10 10% SucrosePO.sub.4, Arg, PVP 12 10%
SucrosePO.sub.4, Arg, Dextran 11 10% SucrosePO.sub.4, Arg, Gel,
EDTA 13 10% Sucrose, Arg, Gel, Histidine 14 10% Sucrose, Arg, Gel,
Hist., EDTA 15
TABLE-US-00050 TABLE 50 Stability of Purified VH vs. Unpurified VH
(FluMist): when both are stabilized by the purified formulation
Stability slope @4.degree. C. (.+-.SE), at six months Purified
formulation FluMist* (Log FFU/month) (Log FFU/month) A/NC -0.020
.+-. 0.027 -0.035 .+-. 0.016 A/Pan -0.011 .+-. 0.020 -0.079 .+-.
0.035 B/HK -0.138 .+-. 0.022 -0.151 .+-. 0.018 Formulation: 7%
Sucrose, 1% gelatin, 1% arginine [`7/1/1` formulation] *60% AF
level
TABLE-US-00051 TABLE 51 Stability of purified VH vs. FluMist: When
FluMist is stabilized by `10/2/2` formulation Stability slope
@4.degree. C. (.+-.SE), Initial Potency loss at six months [log
FFU/month] (Log FFU)** Purified VH FluMist* Purified VH FluMist*
A/NC -0.020 .+-. 0.027 -0.011 .+-. 0.019 0.4 0.5 A/Pan -0.011 .+-.
0.020 -0.093 .+-. 0.032 0.9 0.9 B/HK -0.138 .+-. .022 -0.107 .+-.
0.025 0 0.3 Purified VH formulation: 7% Sucrose, 1% gelatin, 1%
arginine [`7/1/1` formulation], no added AF *FluMist formulation:
10% Sucrose, 2% gelatin, 2% arginine [`10/2/2` formulation], 60% AF
level **Based on linear regression
TABLE-US-00052 TABLE 52 Stability of purified VH fommlaiton vs.
FluMist: When FluMist is stabilized by `10/2/2 Histidine`
formulation Stability slope @4.degree. C. (.+-.SE), Initial Potency
loss** at six months [log FFU/month] (Log FFU) Purified VH FluMist*
Purified VH FluMist* A/NC -0.020 .+-. 0.027 -0.068 .+-. 0.014 0.4 0
A/Pan -0.011 .+-. 0.020 -0.072 .+-. 0.012 0.9 0 B/HK -0.138 .+-.
.022 -0.061 .+-. 0.020 0 0 Purified VH formulation: 7% Sucrose, 1%
gelatin, 1% arginine [`7/1/1` formulation], no added AF *FluMist
formulation: Histidine, 10% Sucrose, 2% gelatin, 2% arginine
[`10/2/2 His` formulation], 60% AF level **Based on linear
regression
TABLE-US-00053 TABLE 53 Comparison of Method Performance Method
Performance Test Parameter Manual Semi-automated Precision/Between
Test SD range from 0.07 to 0.11 log SD range from 0.06-0.09
Variability (SD)) TCID.sub.50 units.sup.1
log.sub.10TCID.sub.50/mL.sup.2,3 Linearity Passes test for lack of
fit to a Passes test for lack of fit to a linear model at the 1%
linear model at the 1% significance level. significance
level..sup.3 Accuracy Slope range 0.986-1.007. Slopes range
1.00-1.02..sup.3 Range 4.7-9.5 log.sub.10TCID.sub.50/mL. 4.2-9.3
log.sub.10TCID.sub.50/mL..sup.3 .sup.1Between-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). .sup.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
(H1N1, H3N2 and B). .sup.3Validation Report for Semi-Automated
TCID50 Potency Assay for Influenza Virus Monovalent.
TABLE-US-00054 TABLE 54 Inter-Assay Comparison Mean Titer
(log.sub.10TCID.sub.50/mL) Manual Semi-Automated 90% CI Strain
Assay Assay Difference (LB, UB) A/NewCaledonia/ 9.40 9.42 -0.02
(-0.05, 20/99 0.01) B/Yamanashi/ 8.47 8.40 0.07 (0.03, 166/98
0.10)
TABLE-US-00055 TABLE 55 SemiAutomated Manual "gold standard"
readout MTT readout CPE-positive CPE-negative CPE-positive TP FP
(A.sub.570 .ltoreq. cutoff) CPE-negative FN TN (A.sub.570 >
cutoff) All positives All negatives
TABLE-US-00056 TABLE 56 SemiAutomated TCID.sub.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 True False Sensi- True False
Speci- positive negative tivity.sup.a negative positive
ficity.sup.b AB 7,091 61 99.15% 7,247 1 99.99% (N = 14,400) 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 .sup.aSENSITIVITY = (TRUE POSITIVE)/ALL
POSITIVE .sup.bSPECIFICITY = (TRUE NEGATIVE)/ALL NEGATIVE
TABLE-US-00057 TABLE 57 Control Well (CPE-negative) Absorbance
Values Obtained by the two groups with previous Values Reported
Shown for Comparison (previous Combined values) 2.sup.nd group
1.sup.st group (two groups) Control Well count 2880 6288 9168 6720
A.sub.570 Average 1.226 1.235 1.231 1.261 SD 0.17 0.20 0.19
0.15
TABLE-US-00058 TABLE 58 Instrument-to-Instrument Comparison:
SemiAutomated TCID.sub.50 Potency Values for Reference Virus
Strains Reference Virus Strain (Mean log.sub.10TCID.sub.50/mL .+-.
SD) Instrument A/New B/Yamanashi/ (group) Caledonia/20/99
A/Sydney/05/97 166/98 AZ-039 (1.sup.st).sup.1,2 9.2 .+-. 0.15 8.6
.+-. 0.09 8.4 .+-. 0.10 AZ-040 (1.sup.st).sup.1,3 9.3 .+-. 0.08 8.6
.+-. 0.01 8.4 .+-. 0.10 AZ-036 (2.sup.nd).sup.1 9.2 .+-. 0.08 8.5
.+-. 0.05 8.3 .+-. 0.06 .sup.1Number of tests (AZ-036, N = 9;
AZ-039, N = 5; AZ-040, N = 2); 1.sup.st = first group, 2.sup.nd =
second group. .sup.2For AZ-039, one test result rejected due to
failure of intra-day SD acceptance criteria .sup.3For AZ-040, four
test results rejected due to failure of intra-day SD acceptance
criteria or mishandling of plates
TABLE-US-00059 TABLE 59 Analyst-to-Analyst Comparison:
SemiAutomated TCID.sub.50 Potency Values for Reference Virus
Strains Using Instruments AZ-039 or AZ-036 Reference Virus Strain
(Mean log.sub.10TCID.sub.50/mL .+-. SD).sup.a A/New B/Yamanashi/
Caledonia/20/99 A/Sydney/05/97 166/98 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
.sup.aMean potency values are derived from four replicates obtained
over three test days (n = 12).
Sequence CWU 1
1
101163PRTInfluenza B virus 1Met Gly Thr Thr Ala Thr Lys Lys Lys Gly
Leu Thr Leu Ala Glu Arg1 5 10 15Lys Met Arg Arg Cys Val Ser Phe His
Glu Ala Phe Glu Ile Ala Glu 20 25 30Gly His Glu Ser Ser Ala Leu Leu
Tyr Cys Leu Met Val Met Tyr Leu 35 40 45Asn Pro Gly Asn Tyr Ser Met
Gln Val Lys Leu Gly Thr Leu Cys Ala 50 55 60Leu Cys Glu Lys Gln Ala
Ser His Ser His Arg Ala His Ser Arg Ala65 70 75 80Ala Arg Ser Ser
Val Pro Gly Val Arg Arg Glu Met Gln Met Val Ser 85 90 95Ala Met Asn
Thr Ala Lys Thr Met Asn Gly Met Gly Lys Gly Glu Asp 100 105 110Val
Gln Lys Leu Ala Glu Glu Leu Gln Ser Asn Ile Gly Val Leu Arg 115 120
125Ser Leu Gly Ala Ser Gln Lys Asn Gly Glu Gly Ile Ala Lys Asp Val
130 135 140Met Glu Val Leu Lys Gln Ser Ser Met Gly Asn Ser Ala Leu
Val Lys145 150 155 160Lys Tyr Leu2248PRTInfluenza B virus 2Met Ser
Leu Phe Gly Asp Thr Ile Ala Tyr Leu Leu Ser Leu Thr Glu1 5 10 15Asp
Gly Glu Gly Lys Ala Glu Leu Ala Glu Lys Leu His Cys Trp Phe 20 25
30Gly Gly Lys Glu Phe Asp Leu Asp Ser Ala Leu Glu Trp Ile Lys Asn
35 40 45Lys Arg Cys Leu Thr Asp Ile Gln Lys Ala Leu Ile Gly Ala Ser
Ile 50 55 60Cys Phe Leu Lys Pro Lys Asp Gln Glu Arg Lys Arg Arg Phe
Ile Thr65 70 75 80Glu Pro Leu Ser Gly Met Gly Thr Thr Ala Thr Lys
Lys Lys Gly Leu 85 90 95Ile Leu Ala Glu Arg Lys Met Arg Arg Cys Val
Ser Phe His Glu Ala 100 105 110Phe Glu Ile Ala Glu Gly His Glu Ser
Ser Ala Leu Leu Tyr Cys Leu 115 120 125Met Val Met Tyr Leu Asn Pro
Gly Asn Tyr Ser Met Gln Val Lys Leu 130 135 140Gly Thr Leu Cys Ala
Leu Cys Glu Lys Gln Ala Ser His Ser His Arg145 150 155 160Ala His
Ser Arg Ala Ala Arg Ser Ser Val Pro Gly Val Arg Arg Glu 165 170
175Met Gln Met Val Ser Ala Met Asn Thr Ala Lys Thr Met Asn Gly Met
180 185 190Gly Lys Gly Glu Asp Val Gln Lys Leu Ala Glu Glu Leu Gln
Ser Asn 195 200 205Ile Gly Val Leu Arg Ser Leu Gly Ala Ser Gln Lys
Asn Gly Glu Gly 210 215 220Ile Ala Lys Asp Val Met Glu Val Leu Lys
Gln Ser Ser Met Gly Asn225 230 235 240Ser Ala Leu Val Lys Lys Tyr
Leu 2453248PRTInfluenza B virus 3Met Ser Leu Phe Gly Asp Thr Ile
Ala Tyr Leu Leu Ser Leu Thr Glu1 5 10 15Asp Gly Glu Gly Lys Ala Glu
Leu Ala Glu Lys Leu His Cys Trp Phe 20 25 30Gly Gly Lys Glu Phe Asp
Leu Asp Ser Ala Leu Glu Trp Ile Lys Asn 35 40 45Lys Arg Cys Leu Thr
Asp Ile Gln Lys Ala Leu Ile Gly Ala Ser Ile 50 55 60Cys Phe Leu Lys
Pro Lys Asp Gln Glu Arg Lys Arg Arg Phe Ile Thr65 70 75 80Glu Pro
Leu Ser Gly Met Gly Thr Thr Ala Thr Lys Lys Lys Gly Leu 85 90 95Ile
Leu Ala Glu Arg Lys Met Arg Arg Cys Val Ser Phe His Glu Ala 100 105
110Phe Glu Ile Ala Glu Gly His Glu Ser Ser Ala Leu Leu Tyr Cys Leu
115 120 125Met Val Met Tyr Leu Asn Pro Gly Asn Tyr Ser Met Gln Val
Lys Leu 130 135 140Gly Thr Leu Cys Ala Leu Cys Glu Lys Gln Ala Ser
His Ser His Arg145 150 155 160Ala His Ser Arg Ala Ala Arg Ser Ser
Val Pro Gly Val Arg Arg Glu 165 170 175Met Gln Met Val Ser Ala Met
Asn Thr Ala Lys Thr Met Asn Gly Met 180 185 190Gly Lys Gly Glu Asp
Val Gln Lys Leu Ala Glu Glu Leu Gln Ser Asn 195 200 205Ile Gly Val
Leu Arg Ser Leu Gly Ala Ser Gln Lys Asn Gly Glu Gly 210 215 220Ile
Ala Lys Asp Val Met Glu Val Leu Lys Gln Ser Ser Met Gly Asn225 230
235 240Ser Ala Leu Val Lys Lys Tyr Leu 2454248PRTInfluenza B virus
4Met Ser Leu Phe Gly Asp Thr Ile Ala Tyr Leu Leu Ser Leu Thr Glu1 5
10 15Asp Gly Glu Gly Lys Ala Glu Leu Ala Glu Lys Leu His Cys Trp
Phe 20 25 30Gly Gly Lys Glu Phe Asp Leu Asp Ser Ala Leu Glu Trp Ile
Lys Asn 35 40 45Lys Arg Cys Leu Thr Asp Ile Gln Lys Ala Leu Ile Gly
Ala Ser Ile 50 55 60Cys Phe Leu Lys Pro Lys Asp Gln Glu Arg Lys Arg
Arg Phe Ile Thr65 70 75 80Glu Pro Leu Ser Gly Met Gly Thr Thr Ala
Thr Lys Lys Lys Gly Leu 85 90 95Ile Leu Ala Glu Arg Lys Met Arg Arg
Cys Val Ser Phe His Glu Ala 100 105 110Phe Glu Ile Ala Glu Gly His
Glu Ser Ser Ala Leu Leu Tyr Cys Leu 115 120 125Met Val Met Tyr Leu
Asn Pro Gly Asn Tyr Ser Met Gln Val Lys Leu 130 135 140Gly Thr Leu
Cys Ala Leu Cys Glu Lys Gln Ala Ser His Ser Gln Arg145 150 155
160Ala His Ser Arg Ala Ala Arg Ser Ser Val Pro Gly Val Arg Arg Glu
165 170 175Met Gln Met Val Ser Ala Val Asn Thr Ala Lys Thr Met Asn
Gly Met 180 185 190Gly Lys Gly Glu Asp Val Gln Lys Leu Ala Glu Glu
Leu Gln Ser Asn 195 200 205Ile Gly Val Leu Arg Ser Leu Gly Ala Ser
Gln Lys Asn Gly Glu Gly 210 215 220Ile Ala Lys Asp Val Met Glu Val
Leu Lys Gln Ser Ser Met Gly Asn225 230 235 240Ser Ala Leu Val Lys
Lys Tyr Leu 2455248PRTInfluenza B virus 5Met Ser Leu Phe Gly Asp
Thr Ile Ala Tyr Leu Leu Ser Leu Thr Glu1 5 10 15Asp Gly Glu Gly Lys
Ala Glu Leu Ala Glu Lys Leu His Cys Trp Phe 20 25 30Gly Gly Lys Glu
Phe Asp Leu Asp Ser Ala Leu Glu Trp Ile Lys Asn 35 40 45Lys Arg Cys
Leu Thr Asp Ile Gln Lys Ala Leu Ile Gly Ala Ser Ile 50 55 60Cys Phe
Leu Lys Pro Lys Asp Gln Glu Arg Lys Arg Arg Phe Ile Thr65 70 75
80Glu Pro Leu Ser Gly Met Gly Thr Thr Ala Thr Lys Lys Lys Gly Leu
85 90 95Ile Leu Ala Glu Arg Lys Met Arg Arg Cys Val Ser Phe His Glu
Ala 100 105 110Phe Glu Ile Ala Glu Gly His Glu Ser Ser Ala Leu Leu
Tyr Cys Leu 115 120 125Met Val Met Tyr Leu Asn Pro Gly Asn Tyr Ser
Met Gln Val Lys Leu 130 135 140Gly Thr Leu Cys Ala Leu Cys Glu Lys
Gln Ala Ser His Ser His Arg145 150 155 160Ala His Ser Arg Ala Ala
Arg Ser Ser Val Pro Gly Val Arg Arg Glu 165 170 175Met Gln Met Val
Ser Ala Met Asn Thr Ala Lys Thr Met Asn Gly Met 180 185 190Gly Lys
Gly Glu Asp Val Gln Lys Leu Ala Glu Glu Leu Gln Ser Asn 195 200
205Ile Gly Val Leu Arg Ser Leu Gly Ala Ser Gln Lys Asn Gly Glu Gly
210 215 220Ile Ala Lys Asp Val Met Glu Val Leu Lys Gln Ser Ser Met
Gly Asn225 230 235 240Ser Ala Leu Val Lys Lys Tyr Leu
2456109PRTInfluenza B virus 6Met Leu Glu Pro Phe Gln Ile Leu Ser
Ile Cys Ser Phe Ile Leu Ser1 5 10 15Ala Leu His Phe Met Ala Trp Thr
Ile Gly His Leu Asn Gln Ile Lys 20 25 30Arg Gly Val Asn Leu Lys Ile
Arg Ile Arg Asn Pro Asn Lys Glu Thr 35 40 45Ile Asn Arg Glu Val Ser
Ile Leu Arg His Ser Tyr Gln Lys Glu Ile 50 55 60Gln Ala Lys Glu Thr
Met Lys Glu Val Leu Ser Asp Asn Met Glu Ile65 70 75 80Leu Ser Asp
His Ile Val Ile Glu Gly Leu Ser Ala Glu Glu Ile Ile 85 90 95Lys Met
Gly Glu Thr Val Leu Glu Val Glu Glu Leu Gln 100 1057109PRTInfluenza
B virus 7Met Leu Glu Pro Phe Gln Ile Leu Ser Ile Cys Ser Phe Ile
Leu Ser1 5 10 15Ala Leu His Phe Met Ala Trp Thr Ile Gly His Leu Ser
Gln Ile Lys 20 25 30Arg Gly Val Asn Met Lys Ile Arg Ile Lys Gly Pro
Asn Lys Glu Thr 35 40 45Ile Asn Arg Glu Val Ser Ile Leu Arg His Ser
Tyr Gln Lys Glu Ile 50 55 60Gln Ala Lys Glu Thr Met Lys Glu Val Leu
Ser Asp Asn Met Glu Ala65 70 75 80Leu Ser Asp His Ile Val Ile Glu
Gly Leu Ser Ala Glu Glu Ile Ile 85 90 95Lys Met Gly Glu Thr Val Leu
Glu Val Glu Glu Leu His 100 1058109PRTInfluenza B virus 8Met Leu
Glu Pro Phe Gln Ile Leu Ser Ile Cys Ser Phe Ile Leu Ser1 5 10 15Ala
Leu His Phe Met Ala Trp Thr Ile Gly His Leu Asn Gln Ile Lys 20 25
30Arg Gly Val Asn Met Lys Ile Arg Ile Lys Gly Pro Asn Lys Glu Thr
35 40 45Ile Asn Arg Glu Val Ser Ile Leu Arg His Ser Tyr Gln Lys Glu
Ile 50 55 60Gln Ala Lys Glu Thr Met Lys Glu Val Leu Ser Asp Asn Met
Glu Val65 70 75 80Leu Ser Asp His Ile Val Ile Glu Gly Leu Ser Ala
Glu Glu Ile Ile 85 90 95Lys Met Gly Glu Thr Val Leu Glu Ile Glu Glu
Leu His 100 1059109PRTInfluenza B virus 9Met Leu Glu Pro Phe Gln
Ile Leu Ser Ile Cys Ser Phe Ile Leu Ser1 5 10 15Ala Leu His Phe Met
Ala Trp Thr Ile Gly His Leu Asn Gln Ile Lys 20 25 30Arg Gly Val Asn
Met Lys Ile Arg Ile Lys Gly Pro Asn Lys Glu Thr 35 40 45Ile Asn Arg
Glu Val Ser Ile Leu Arg His Ser Tyr Gln Lys Glu Ile 50 55 60Gln Ala
Lys Glu Thr Met Lys Glu Val Leu Ser Asp Asn Met Glu Val65 70 75
80Leu Asn Asp His Ile Val Ile Glu Gly Leu Ser Ala Glu Glu Ile Ile
85 90 95Lys Met Gly Glu Thr Val Leu Glu Ile Glu Glu Leu His 100
10510109PRTInfluenza B virus 10Met Leu Glu Pro Phe Gln Ile Leu Ser
Ile Cys Ser Phe Ile Leu Ser1 5 10 15Ala Leu His Phe Met Ala Trp Thr
Ile Gly His Leu Asn Gln Ile Lys 20 25 30Arg Gly Val Asn Met Lys Ile
Arg Ile Lys Ser Pro Asn Lys Glu Thr 35 40 45Ile Asn Arg Glu Val Ser
Ile Leu Arg His Ser Tyr Gln Lys Glu Ile 50 55 60Gln Ala Lys Glu Thr
Met Lys Glu Val Leu Ser Asp Asn Met Glu Val65 70 75 80Leu Ser Asp
His Ile Val Ile Glu Gly Leu Ser Ala Glu Glu Ile Ile 85 90 95Lys Met
Gly Glu Thr Val Leu Glu Ile Glu Glu Leu His 100 105
* * * * *