U.S. patent application number 11/242018 was filed with the patent office on 2006-05-25 for refrigerator-temperature stable influenza vaccine compositions.
This patent application is currently assigned to MEDIMMUNE VACCINES, INC.. Invention is credited to George Kemble, Richard Schwartz, George Trager.
Application Number | 20060110406 11/242018 |
Document ID | / |
Family ID | 46322831 |
Filed Date | 2006-05-25 |
United States Patent
Application |
20060110406 |
Kind Code |
A1 |
Kemble; George ; et
al. |
May 25, 2006 |
Refrigerator-temperature stable influenza vaccine compositions
Abstract
Methods and compositions for the optimization and production of
refrigerator-temperature stable virus, e.g., influenza,
compositions are provided. Formulations and immunogenic
compositions comprising refrigerator-temperature stable virus
compositions are provided.
Inventors: |
Kemble; George; (Saratoga,
CA) ; Trager; George; (Emerald Hills, CA) ;
Schwartz; Richard; (San Mateo, CA) |
Correspondence
Address: |
JOHNATHAN KLEIN-EVANS
ONE MEDIMMUNE WAY
GAITHERSBURG
MD
20878
US
|
Assignee: |
MEDIMMUNE VACCINES, INC.
Gaithersburg
MD
|
Family ID: |
46322831 |
Appl. No.: |
11/242018 |
Filed: |
October 4, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10788236 |
Feb 25, 2004 |
|
|
|
11242018 |
Oct 4, 2005 |
|
|
|
60616711 |
Oct 6, 2004 |
|
|
|
60450181 |
Feb 25, 2003 |
|
|
|
Current U.S.
Class: |
424/209.1 ;
435/235.1 |
Current CPC
Class: |
A61K 39/12 20130101;
C12N 7/00 20130101; A61K 39/145 20130101; A61P 31/16 20180101; C12N
2760/16151 20130101; A61P 37/04 20180101; C12N 2760/16134 20130101;
C12N 2760/16251 20130101; A61K 2039/70 20130101; C12N 2760/16234
20130101 |
Class at
Publication: |
424/209.1 ;
435/235.1 |
International
Class: |
A61K 39/145 20060101
A61K039/145; C12N 7/00 20060101 C12N007/00 |
Claims
1. A method of making a refrigerator stable influenza virus
composition, the method comprising: infection of a population of
host eggs or into a population of host cells with influenza
viruses; culturing the population of host eggs or population of
host cells at an appropriate temperature; recovering influenza
viruses in a viral harvest; clarifying the viral harvest by
filtration, thereby producing a clarified viral harvest; subjecting
the clarified viral harvest to continuous flow centrifugation,
thereby producing a further clarified viral harvest; and
sterilizing said further clarified viral harvest by filtration.
2. (canceled)
3. (canceled)
4. A refrigerator stable influenza virus composition produced by
the method of claim 1.
5. A refrigerator stable influenza virus composition which
comprises at least one strain of influenza virus and one or more
members selected from the group consisting of: a) 6-8% sucrose b)
1-2% arginine monohydrochloride; c) 0.05-0.1% glutamic acid,
monosodium monohydrate; and d) 0.5-2% gelatin hydrolysate.
6. The refrigerator stable influenza virus composition of claim 5,
which comprises 6.84% sucrose, 1.21% arginine monohydrochloride,
0.094% glutamic acid, monosodium monohydrate; and 1% gelatin
hydrolysate.
7. The refrigerator stable influenza virus composition of claim 4,
wherein said composition has a potency loss of less than 1.0 logs
when stored at 4.degree. C. for at least 3 months.
8. The refrigerator stable influenza virus composition of claim 5,
6, or 7, wherein said composition has a potency loss of less than
1.0 logs when stored at 4.degree. C. for at least 3 months.
9. The refrigerator stable influenza virus composition of claim 8,
wherein said composition further comprises a cold adapted
temperature-sensitive attenuated influenza virus.
10. An immunogenic composition comprising the refrigerator stable
influenza virus composition of claim 7.
11. An immunogenic composition comprising the refrigerator stable
influenza virus composition of claim 9.
12. (canceled)
13. A vaccine comprising the immunogenic composition of claim
11.
14-17. (canceled)
18. A refrigerator stable immunogenic composition comprising at
least one live influenza virus, wherein the composition has a
potency loss of less than 1.0 logs when stored for a period of 3
months at 4-8.degree. C.
19. (canceled)
20. The refrigerator stable immunogenic composition of claim 18
comprising three influenza virus strains.
21. A vaccine comprising the refrigerator stable immunogenic
composition of claim 18 or 20.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C .sctn.
119(e) of U.S. Provisional Application No. 60/616,711, filed on
Oct. 6, 2004; and is a continuation-in-part of and claims the
benefit under 35 U.S.C. .sctn. 120 of U.S. patent application Ser.
No. 10/788,236, filed Feb. 25, 2004, which claims the benefit under
35 U.S.C .sctn. 119(e) of U.S. Provisional Application No.
60/450,181, filed Feb. 25, 2003. All of the foregoing applications
are incorporated herein by reference in their entirety.
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
immunocompromised 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 are 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 capable of being quickly and economically produced and that
is capable of easy storage/transport is thus quite desirable. Even
more desirable would be such a vaccine that would be capable of
storage/transport at refrigerator temperatures (e.g., approximately
2-8.degree. C.).
[0004] To date, all influenza vaccines commercially available in
the U.S. 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, methods to
increase stability (e.g., at refrigerator temperatures) of the
produced vaccine, are greatly desirable as they can prevent
deterioration of vaccine stock, which would otherwise necessitate
new production, etc.
[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);
therefore, any methods to increase vaccine composition stability
(e.g., storage/transport at refrigerator temperature) in these
systems as well are 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., U.S. patent application Nos. 60/375,675
filed Apr. 26, 2002, PCT/US03/12728 filed Apr. 25, 2003, Ser. No.
10/423,828 filed Apr. 25, 2003, and PCT/US05/017734 filed May 20,
2005.
[0007] The present invention provides vaccine compositions that
have stability at, for example, refrigerator temperatures (e.g.,
4.degree. C.) and methods of producing the same. Aspects of the
current invention are applicable to traditional hen egg and new
cell culture vaccine production methods (and also combined systems)
and comprise numerous other benefits that will become apparent upon
review of the following.
SUMMARY OF THE INVENTION
[0008] The current invention provides liquid vaccine formulations
that are substantially stable at temperatures ranging from
4.degree. C. to 8.degree. C. These and other liquid formulations,
which are specific embodiments of the invention are referred to
herein, for example, as "vaccine formulations of the invention,"
"refrigerator stable vaccine formulations," "liquid formulations of
the invention," "formulations of the invention,"
"refrigerator-temperature stable (RTS) formulations of the
invention," or simply "compositions of the invention" or "virus
compositions of the invention."
[0009] The present invention provides liquid vaccine formulations
that are substantially stable at temperatures ranging from
4.degree. C. to 8.degree. C. In one specific embodiment of the
invention, liquid vaccine formulations of the invention are
substantially stable at temperatures ranging from 2.degree. C. to
8.degree. C. or at 4.degree. C. for a period of 3 months, or 4
months, or 5 months, or 6 months, or 9 months, or 12 months, or 18
months, or 24 months, or 36 months, or 48 months, in that there is
an acceptable loss of potency (e.g., influenza virus potency loss),
for example, a potency loss of between 0.5-1.0 logs, or less than
0.5 logs, or less than 1.0 logs of potency, at the end of such
time.
[0010] In one embodiment, refrigerator stable vaccine formulations
of the invention are provided that comprise live influenza viruses.
For instance, formulations of the invention may comprise one or
more of the following: an attenuated influenza virus, a
cold-adapted influenza virus, a temperature-sensitive influenza
virus, an attenuated cold-adapted temperature sensitive influenza
virus, an influenza A virus, and an influenza B virus. In one
embodiment, liquid vaccine formulations of the invention comprise
two influenza A virus strains and one influenza B virus
strains.
[0011] Alternatively, formulations of the invention may comprise
other live viruses such as paramyxoviruses (e.g., RSV, measles
virus, mumps virus, Sendai, New Castle Disease viruses) and
parainfluenza virus.
[0012] The present invention further provides immunogenic
compositions comprising formulations of the invention. The present
invention further provides vaccines (e.g., influenza vaccines)
comprising formulations and immunogenic compositions of the
invention.
[0013] The present invention further includes methods of producing
such liquid vaccine formulations. For instance, in one specific
embodiment, methods of producing liquid formulations comprising one
or more influenza viruses are provided herein. In one specific
embodiment, methods of producing a liquid formulation of the
invention includes one or more of the following steps: 1)
introducing a plurality of vectors [one or more of which
incorporates (or encodes) a portion of an influenza virus genome]
into a population of host eggs or into a population of host cells,
which population of host eggs or host cells is capable of
supporting replication of influenza virus; 2) culturing the
population of host eggs or population of host cells at an
appropriate temperature; 3) recovering influenza viruses in a viral
harvest; 4) addition of a stabilizer (e.g., sucrose and
glutamate-containing solutions as described herein); 5) clarifying
the viral harvest (e.g., by depth or membrane filtration), thereby
producing a clarified viral harvest; 6) subjecting the viral
harvest to a centrifugation step (e.g., continuous zonal
centrifugation, continuous flow centrifugation); 7) a sterile
filtration step (e.g., use of 0.2, or 0.2-0.5 micron filter (with
or without heating during filtration); and 8) storage at -60
degrees C.
[0014] In another specific embodiment, methods of producing a
liquid formulation of the invention includes one or more of the
following steps: 1) infection of a population of host eggs or into
a population of host cells with influenza viruses; 2) culturing the
population of host eggs or population of host cells at an
appropriate temperature; 3) recovering influenza viruses in a viral
harvest; 4) addition of a stabilizer; 5) clarifying the viral
harvest (e.g., by depth filtration and/or passing through one or
more filters ranging from 0.2-0.8 microns; or 0.8 or 1.5 micron
followed by 0.2 micron), thereby producing a clarified viral
harvest; 6) subjecting the viral harvest to a centrifugation step
(e.g., continuous zonal centrifugation, continuous flow
centrifugation); 7) a sterile filtration step (e.g., use of 0.2, or
0.2-0.5 micron filter (with or without heating during filtration);
and 8) storage at -60 degrees C.
[0015] In other specific embodiment, methods of producing an
influenza virus composition of the invention comprises one or more
of the following steps: 1) infection of a population of host eggs
or into a population of host cells with influenza viruses; 2)
culturing the population of host eggs or population of host cells
at an appropriate temperature; 3) recovering influenza viruses in a
viral harvest; 4) clarifying the viral harvest by filtration,
thereby producing a clarified viral harvest; 5) subjecting the
clarified viral harvest to centrifugation (e.g., continuous flow
centrifugation), thereby producing a further clarified viral
harvest; 6) addition of stabilizers (e.g., one or more of the
following: 6-8% sucrose; 1-2% arginine monohydrochloride; 0.05-0.1%
glutamic acid, monosodium monohydrate; and 0.5-2% gelatin
hydrolysate); and 6) sterilizing said further clarified viral
harvest by filtration.
[0016] In other specific embodiment, methods of producing an
influenza virus composition of the invention comprises all of the
following steps: 1) infection of a population of host eggs or into
a population of host cells with influenza viruses; 2) culturing the
population of host eggs or population of host cells at an
appropriate temperature; 3) recovering influenza viruses in a viral
harvest; 4) clarifying the viral harvest by filtration, thereby
producing a clarified viral harvest; 5) subjecting the clarified
viral harvest to centrifugation (e.g., continuous flow
centrifugation), thereby producing a further clarified viral
harvest; and 6) sterilizing said further clarified viral harvest by
filtration.
[0017] In other specific embodiment, methods of producing an
influenza virus composition of the invention comprises one or more
of the following steps: 1) infection of a population of host eggs
or into a population of host cells with influenza viruses; 2)
culturing the population of host eggs or population of host cells
at an appropriate temperature; 3) recovering influenza viruses in a
viral harvest; 4) clarifying the viral harvest; 5) subjecting the
clarified viral harvest to centrifugation (e.g., continuous flow
centrifugation), thereby producing a further clarified viral
harvest; and 6) sterilizing said further clarified viral harvest by
filtration.
[0018] In other specific embodiment, methods of producing an
influenza virus composition of the invention comprises one or more
of the following steps: 1) infection of a population of host eggs
or into a population of host cells with influenza viruses; 2)
culturing the population of host eggs or population of host cells
at an appropriate temperature; 3) recovering influenza viruses in a
viral harvest; 4) clarifying the viral harvest; and 5) subjecting
the clarified viral harvest to diafiltration.
[0019] In other specific embodiment, methods of producing an
influenza virus composition of the invention comprise one or more
of the following steps: 1) infection of a population of host eggs
or into a population of host cells with influenza viruses; 2)
culturing the population of host eggs or population of host cells
at an appropriate temperature; 3) recovering influenza viruses in a
viral harvest; 4) clarifying the viral harvest; 5) subjecting the
clarified viral harvest to diafiltration; and 6) addition of
stabilizers (e.g., one or more of the following: 6-8% sucrose; 1-2%
arginine monohydrochloride; 0.05-0.1% glutamic acid, monosodium
monohydrate; and 0.5-2% gelatin hydrolysate).
[0020] 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
[0021] FIG. 1: Displays a flow chart illustrating a CTM Process
Flow.
[0022] FIG. 2: Displays a flow chart illustrating a CTM Process
Flow.
[0023] FIG. 3: Displays a table illustrating Centrifuge Loading and
Temperature Studies.
[0024] FIG. 4: Displays a table illustrating a Summary of QC test
data.
[0025] FIG. 5: Displays a table illustrating average yields for a
number of influenza strains.
[0026] FIG. 6: Displays a table with a summary of QC test data for
monovalent bulk release assay results.
DETAILED DESCRIPTION
[0027] The present invention provides liquid vaccine formulations
that are substantially stable at temperatures ranging from
4.degree. C. and 8.degree. C. In one specific embodiment of the
invention, liquid vaccine formulations of the invention are
substantially stable at temperature ranging from 2-8.degree. C. or
4.degree. C. for a period of at least 1 month, or at least 2
months, or at least 3 months, or at least 4 months, or at least 5
months, or at least 6 months, or at least 9 months, or at least 12
months, or at least 18 months, or at least 24 months, or at least
36 months, or at least 48 months, in that there is an acceptable
loss of potency (e.g., influenza virus potency loss) at the end of
such time, for example, a potency loss of between 0.5-1.0 logs (as
measured by, e.g., TCID.sub.50 or Fluorescent Focus Assay
(FFA).
[0028] The present invention provides liquid vaccine formulations
that are substantially stable at temperatures ranging from
4.degree. C. and 8.degree. C. In one specific embodiment of the
invention, liquid vaccine formulations of the invention are
substantially stable at temperature ranging from 2-8.degree. C. or
4.degree. C. for a period of at least 1 month, or at least 2
months, or at least 3 months, or at least 4 months, or at least 5
months, or at least 6 months, or at least 9 months, or at least 12
months, or at least 18 months, or at least 24 months, or at least
36 months, or at least 48 months, in that there is an acceptable
loss of potency (e.g., influenza virus potency loss) at the end of
such time, for example, a potency loss of less than 10%, or less
than 20%, or less than 30%, or less than 40%, or less than 50%, or
less than 60%, or less than 70%, or less than 80%, or less than
90%.
[0029] The present invention further provides immunogenic
compositions comprising formulations of the invention. The present
invention further provides vaccines (e.g., influenza vaccines)
comprising formulations and/or immunogenic compositions of the
invention.
[0030] In one embodiment, liquid vaccine formulations of the
invention are provided that comprise live influenza viruses. For
instance, formulations of the invention may comprise one or more of
the following: an attenuated influenza virus, a cold-adapted
influenza virus, a temperature-sensitive influenza virus, an
attenuated cold-adapted temperature sensitive influenza virus, an
influenza A virus, and an influenza B virus. In one embodiment,
liquid vaccine formulations of the invention comprise two influenza
A virus strains and one influenza B virus strains.
[0031] Alternatively, formulations of the invention may comprise
other live viruses such as paramyxoviruses (e.g., RSV,
parainfluenza virus, measles virus, mumps virus, Sendai, New Castle
Disease viruses).
[0032] The present invention further includes methods of producing
such liquid vaccine formulations. For instance, in one specific
embodiment, methods of producing liquid formulations comprising one
or more influenza viruses are provided herein. In one specific
embodiment, methods of producing a liquid formulation of the
invention includes one or more of the following steps: 1)
introducing a plurality of vectors [one or more of which
incorporates (or encodes) a portion of an influenza virus genome]
into a population of host eggs or into a population of host cells,
which population of host eggs or host cells is capable of
supporting replication of influenza virus; 2) culturing the
population of host eggs or population of host cells at an
appropriate temperature; 3) recovering influenza viruses in a viral
harvest; 4) addition of a stabilizer (e.g., sucrose and
glutamate-containing solutions as described herein); 5) clarifying
the viral harvest (e.g., by depth or membrane filtration), thereby
producing a clarified viral harvest; 6) subjecting the viral
harvest to a centrifugation step (e.g., continuous zonal
centrifugation, continuous flow centrifugation); 7) a sterile
filtration step (e.g., use of 0.2, or 0.2-0.5 micron filter (with
or without heating during filtration); and 8) storage at -60
degrees C.
[0033] In another specific embodiment, methods of producing a
liquid formulation of the invention includes one or more of the
following steps: 1) infection of a population of host eggs or into
a population of host cells with influenza viruses; 2) culturing the
population of host eggs or population of host cells at an
appropriate temperature; 3) recovering influenza viruses in a viral
harvest; 4) addition of a stabilizer; 5) clarifying the viral
harvest (e.g., by depth filtration and/or passing through one or
more filters ranging from 0.2-0.8 microns; or 0.8 or 1.5 micron
followed by 0.2 micron), thereby producing a clarified viral
harvest; 6) subjecting the viral harvest to a centrifugation step
(e.g., continuous zonal centrifugation, continuous flow
centrifugation); 7) a sterile filtration step (e.g., use of 0.2, or
0.2-0.5 micron filter (with or without heating during filtration);
and 8) storage at -60 degrees C.
[0034] In other specific embodiment, methods of producing an
influenza virus composition of the invention comprises one or more
of the following steps: 1) infection of a population of host eggs
or into a population of host cells with influenza viruses; 2)
culturing the population of host eggs or population of host cells
at an appropriate temperature; 3) recovering influenza viruses in a
viral harvest; 4) clarifying the viral harvest by filtration,
thereby producing a clarified viral harvest; 5) subjecting the
clarified viral harvest to centrifugation (e.g., continuous flow
centrifugation), thereby producing a further clarified viral
harvest; 6) addition of stabilizers (e.g., one or more of the
following: 6-8% sucrose; 1-2% arginine monohydrochloride; 0.05-0.1%
glutamic acid, monosodium monohydrate; and 0.5-2% gelatin
hydrolysate); and 6) sterilizing said further clarified viral
harvest by filtration.
[0035] In other specific embodiment, methods of producing an
influenza virus composition of the invention comprises all of the
following steps: 1) infection of a population of host eggs or into
a population of host cells with influenza viruses; 2) culturing the
population of host eggs or population of host cells at an
appropriate temperature; 3) recovering influenza viruses in a viral
harvest; 4) clarifying the viral harvest by filtration, thereby
producing a clarified viral harvest; 5) subjecting the clarified
viral harvest to centrifugation (e.g., continuous flow
centrifugation), thereby producing a further clarified viral
harvest; and 6) sterilizing said further clarified viral harvest by
filtration.
[0036] In other specific embodiment, methods of producing an
influenza virus composition of the invention comprises one or more
of the following steps: 1) infection of a population of host eggs
or into a population of host cells with influenza viruses; 2)
culturing the population of host eggs or population of host cells
at an appropriate temperature; 3) recovering influenza viruses in a
viral harvest; 4) clarifying the viral harvest; 5) subjecting the
clarified viral harvest to centrifugation (e.g., continuous flow
centrifugation), thereby producing a further clarified viral
harvest; and 6) sterilizing said further clarified viral harvest by
filtration.
[0037] In other specific embodiment, methods of producing an
influenza virus composition of the invention comprises one or more
of the following steps: 1) infection of a population of host eggs
or into a population of host cells with influenza viruses; 2)
culturing the population of host eggs or population of host cells
at an appropriate temperature; 3) recovering influenza viruses in a
viral harvest; 4) clarifying the viral harvest; and 5) subjecting
the clarified viral harvest to diafiltration.
[0038] In other specific embodiment, methods of producing an
influenza virus composition of the invention comprise one or more
of the following steps: 1) infection of a population of host eggs
or into a population of host cells with influenza viruses; 2)
culturing the population of host eggs or population of host cells
at an appropriate temperature; 3) recovering influenza viruses in a
viral harvest; 4) clarifying the viral harvest; 5) subjecting the
clarified viral harvest to diafiltration; and 6) addition of
stabilizers (e.g., one or more of the following: 6-8% sucrose; 1-2%
arginine monohydrochloride; 0.05-0.1% glutamic acid, monosodium
monohydrate; and 0.5-2% gelatin hydrolysate).
[0039] In one embodiment, methods of producing a liquid formulation
of the invention may include the step of freezing such
formulations. The freezing step may be done, for example, prior to
final stability testing and distribution and/or prior to storage at
refrigerator temperatures (e.g., 4-8 degrees Celsius). Freezing the
vaccine formulations prior to storage under refrigerator
temperatures may increase stability of the vaccine formulations of
the invention by at least 10%, or at least 20%, or at least 30%, or
at least 40%, or at least 50%, or at least 80%.
[0040] The invention further provides methods of producing one or
more influenza virus compositions by filtering an influenza virus
harvest, whereby the virus harvest is heated during the filtering.
Included in these specific 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).
Stabilizers and Buffers
[0041] Stabilizers of the invention include, for example, one or
more of the following: arginine (e.g., 0.5-1%, 1-2%; 1%; 1.2%;
1.5%, 0.75-2%); poloxamer; sucrose (e.g., 2-8%; 2%; 6-8%; 3%; 4%;
5%; 6%; 7%, or 8%); hydrolyzed gelatin (e.g., 1%; 0.5-2%; 1.5%;
0.5%; 0.75%); and glutamate (e.g., 0.05-0.1%, 0.02-0.15%, 0.03%,
0.04%, 0.06%, 0.02-0.3%, or 0.094%)
[0042] Buffers of the invention include, for example, one or more
of the following: phosphate buffer (mono or dibasic or both) (e.g.,
10-200 mM, pH 7-7.5; 100 mM, pH 7.2; 100 mM, pH 7-7.3); and
histidine buffers (e.g., 25-50 mM histidine, pH 7-7.5, 50-100 mM
Histidine, pH 7-7.5).
Process Yield
[0043] In one embodiment, methods of producing a liquid formulation
of the invention results in an actual or average process yield
(from Viral Harvest (VH) to final formulation) of less than 10%, of
less than 16%, less than 20%, less than 30%, less than 40%, less
than 50%, less than 60%, less than 70%, less than 80%, less than
90%, or less than 93%, or less than 95%.
[0044] It will be appreciated by those skilled in the art that the
various steps of the methods described herein are not required to
be performed or required to exist in the same production series.
Thus, while in some preferred embodiments, all steps and/or
compositions herein are performed or exist, in other embodiments,
one or more steps are optionally, e.g., omitted, changed (in scope,
order, placement, etc.) or the like.
[0045] 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.
essentially stable at 2-8.degree. C. The individual steps are
described in greater detail below.
[0046] Further, the present invention provides methods of using
such liquid vaccine formulations. For example, vaccine formulations
may be administered to a human in order to prevent or reduce the
effects of a viral infection, e.g., influenza infection. In one
embodiment, formulations of the invention are administered as an
immunogenic composition to prevent or reduce the effects of an
influenza virus infection.
Refrigerator-Stable CAIV Formulations
[0047] Prior work by the inventor and co-workers has resulted in
the development of a trivalent, live, cold-adapted influenza
vaccine (CAIV-T, FluMist.RTM., which is referenced throughout as
FluMist, but should be assumed to be FluMist.RTM.) administered by
nasal spray. The current invention involves the development of a
formulation of CAIV-T, which has improved stability profile at
refrigerated temperatures. Methods of producing such improved
formulations are provided herein and may include one or more of the
following steps: a sterile filtration step to reduce contamination
risk, ultracentrifugation (e.g., rate zonal centrifugation), and
diafiltration. In addition, included herein are a number of methods
of producing liquid FluMist and other refrigerator-temperature
stable (RTS) formulations of the invention.
[0048] The development of CAIV strains was assisted by Dr. John
Maassab of the University of Michigan in the 1960s who serially
passaged influenza A and B strains in PCK cells at decreasing
temperatures until the resulting strains reproducibly showed the
phenotypic properties of cold-adaptation (virus grows well at
reduced temperatures compared to wild type virus), temperature
sensitivity (virus does not grow well in elevated temperatures in
vitro), and attenuation (virus replication is restricted in
ferrets). Through development by, e.g., the inventor and coworkers,
these properties were used as the basis for development of an
annual trivalent vaccine reflecting the CDC-designated vaccines
strains for a particular year, through the process of 6:2 genetic
reassortment. For example, a 6:2 CAIV strain is produced by in
vitro co-infection of the relevant A or B strain Master Donor Virus
(MDV) with the circulating flu strain of interest, and
antibody-mediated selection of the proper reassortant. The target
6:2 reassortant contains HA and NA genes from the circulating
strain, and the remaining genes from the cold adapted master donor
virus (MDV). The reassortant retains the cold adapted phenotypic
properties described above. Further development of CAIV has been
conducted by the inventors and coworkers. FluMist has demonstrated
a safe profile and shown efficacy against viral challenge and is
approved for commercial pharmaceutical use in many situations.
[0049] The original formulation of FluMist contained virus harvest
(VH) produced by infecting specific pathogen-free chicken eggs with
Manufacturer's Working Virus Seed (MWVS) of the selected strain,
followed by incubation for two to three days, and harvesting
infected allantoic fluid. VH was stabilized by the addition at
1/10.sup.th volume of a 10.times. sucrose phosphate glutamate (SPG)
solution. Trivalent FluMist was produced by combining VH from each
of the three strains in the vaccines for a given year with
stabilized normal allantoic fluid (NAF) to a target concentration
of 7.3 log.sub.10TCID.sub.50/mL of each strain. The resulting blend
was then filled into sprayers fitted with a spray tip allowing
intranasal delivery of FluMist vaccine. This product format was
used as "frozen FluMist", which was stored in a frozen form. It
will be appreciated that the MWVS virus could also optionally be
manufactured by, e.g. plasmid reassortment. See, e.g., U.S. patent
application Nos. 60/375,675 filed Apr. 26, 2002, PCT/US03/12728
filed Apr. 25, 2003, Ser. No. 10/423,828 filed Apr. 25, 2003,
PCT/US05/017734 filed May 20, 2005, and US20050186563.
[0050] While frozen FluMist can serve as a marked vaccine when
frozen after manufacture and held frozen until time of use, a form
of FluMist that is stable for transport/storage at refrigerator
temperatures is quite desirable. Such "refrigerator-temperature
stable" (or RTS) forms are characterized by one or more (but not
necessarily all in each embodiment) of the following: retention of
stability when distributed as a refrigerated liquid; are passed
through sterilizing filters (e.g., 0.2 micron) to provide assurance
of a sterile product; have reduced content of egg protein (e.g.,
substantially free of NAF); have eliminated the need for
manufacture of NAF diluent; have a reduced volume of a dose; and
comprises either or both arginine and gelatin as excipients (e.g.,
as stabilizers). In certain aspects herein, formulations having one
or more such characteristics are referred to as "liquid FluMist" or
RTS or various similar terms, to distinguish from other versions of
CAIV-T vaccine, such as frozen. The current invention presents
these and other aspects.
[0051] In producing/testing a liquid RTS virus composition of the
invention, numerous development batches were conducted. Development
batches ranged from 2000 to 20,000 eggs per lot, while GMP batches
were approximately 10,000 eggs each. It will be appreciated that
while various examples and protocols are given herein for
production of MWVS viruses (e.g., reassortants), the viruses are
optionally produced through different means in different
embodiments. For example, in certain embodiments, the viruses
herein are optionally made through the protocols shown herein,
while in other embodiments, the MWVS viruses are optionally made
through, e.g., plasmid reassortment or "plasmid rescue"
technologies. See, e.g., U.S. patent application Nos. 60/375,675
filed Apr. 26, 2002, PCT/US03/12728 filed Apr. 25, 2003, U.S. Ser.
No. 10/423,828 filed Apr. 25, 2003, Ser. No. 10/788,236 filed Feb.
25, 2004, PCT/US05/017734 filed May 20, 2005, and US20050186563,
which are each incorporated by reference herein. Accordingly, as
used herein "infection of a population of host cells" encompasses
host cells infected by virus created by or during plasmid
reassortment.
[0052] In various embodiments, the invention comprises virus and
vaccine compositions that are substantially stable, e.g., do not
show unacceptable losses in potency, e.g., potency loss of between
0.5-1.0 logs, or less than 0.5 logs, or less than 1.0 logs, over
selected time periods (typically for at least 1 month, for at least
2 months, for at least 3 months, for at least 4 months, for at
least 5 months, for at least 6 months, for at least 7 months, for
at least 8 months, for at least 9 months, for at least 10 months,
for at least 11 months, for at least 12 months, for at least 13
months, for at least 14 months, for at least 15 months, for at
least 16 months, for at least 17 months, for at least 18 months,
for at least 19 months, for at least 20 months, for at least 21
months, for at least 22 months, for at least 23 months, or for at
least 24 months, or for greater than 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., or between the ranges of 2.degree. C. to 4.degree.
C., or between the ranges of 2.degree. C. to 8.degree. C.).
[0053] While a number of aspects of the invention herein are
exemplified or illustrated with FluMist, the principles embodied by
the invention are applicable to other virus/vaccine compositions as
well and should not necessarily be limited to particular
strains/viruses herein. Thus other live attenuated influenza virus
and vaccines and compositions are also within the purview of the
invention, e.g., ones created through rational means, by human
intervention, etc. Also, other viruses of other influenza strains,
etc., such as influenza A strains, influenza B strains, attenuated
and non-attenuated influenza strains, cold adapted and non-cold
adapted influenza strains, temperature sensitive and
non-temperature sensitive influenza strains, etc. are all
optionally within the embodiments of the current invention. Such
other virus and vaccine can be used, e.g., as new vaccine and/or as
controls for testing other vaccine either in humans or animals,
etc. In addition, other live viral vaccine compositions
particularly those comprising live viruses grown in chicken cells
or eggs (e.g., measles virus) are embodiments of the invention.
Furthermore, the principles embodied by the invention are also
largely applicable to virus and vaccine compositions comprising
live viruses grown in mammalian cells. See, e.g., U.S. Pat. Nos.
6,244,354; 6,146,873; and 6,656,720.
Bulk Virus Harvest Production
[0054] Purification of the cold-adapted influenza virus (or other
similar viruses) and the actual formulation of the compositions are
features of RTS, or liquid, virus compositions. Separation of
influenza virus from allantoic fluid had been practiced as a part
of commercial processes for manufacture of inactivated vaccines.
The method of choice for such inactivated vaccine has been
ultra-centrifugation. Commercial scale continuous flow
ultracentrifuge became available in 1969 and was quickly applied to
the preparation of inactivated influenza vaccines. While
chromatographic purification of live influenza virus would be an
attractive alternative, robust large-scale processes that retain
viral activity are not yet available. This is thought to be due to
the membrane coat and pleiomorphic nature of the influenza virus
particle.
[0055] Recovery of live virus (as opposed to inactivated virus)
purified by ultra-centrifugation was achieved by the inventors and
coworkers and is an embodiment of the current invention. Further
work demonstrated the ability of depth filtration as a commercially
viable alternative to swinging-bucket centrifugation prior to
ultra-centrifugation, and acceptable recoveries of live virus
following filtration through a 0.2 micron filter, and again such is
an embodiment of the current invention.
[0056] In one specific embodiment, the median process yield for the
VH clarification step of the methods of the invention is at least
30%, or at least 40%, or at least 50%, or at least 60%, or at least
70%, or at least 80%, or at least 90%.
[0057] In another specific embodiment, the median process yield for
the ultracentrifugation (e.g., zonal centrifugation) step of the
methods of the invention is at least 30%, or at least 40%, or at
least 50%, or at least 60%, or at least 70%, or at least 80%, or at
least 90%.
[0058] In another specific embodiment, the median process yield for
the peak dilution and sterile filtration step of the methods of the
invention is at least 30%, or at least 40%, or at least 50%, or at
least 60%, or at least 70%, or at least 80%, or at least 90%.
[0059] The ultra-centrifugation step provides the benefit of
concentrating CAIV. This supports the use of a smaller delivered
volume (0.1 mL per nostril rather than 0.25 mL per nostril, e.g.,
as might be used for frozen FluMist). Such reduced volume is more
typical of nasally administered products and can increase consumer
acceptance and reduce product losses due to swallowing or vaccine
dripping out of the nose. Infected allantoic fluid harvest titers
for CAIV have typically been between 8.3 and 9.5
log.sub.10TCID.sub.50/mL, which is more than an order of magnitude
above the target product concentration for frozen FluMist (7.3
log.sub.10 TCID.sub.50/mL, or 7.0 log.sub.10 TCID.sub.50 per dose).
In the event that a very low titer strain is included in the annual
vaccine recommendation, a liquid or RTS FluMist improves the
chances of producing a full strength trivalent vaccine despite the
increased virus concentration in the final product compared to
frozen FluMist. Liquid or RTS FluMist is optionally formulated to a
final concentration of 7.7 log.sub.10 TCID.sub.50/mL, which
delivers the same amount of live virus per dose as frozen
FluMist.
[0060] In one specific embodiment, a low titer influenza vaccine
composition is provided whereby the viral titer is less than 7.3
log.sub.10 TCID.sub.50/mL, or less than 7.0 log.sub.10
TCID.sub.50/mL, or less than 6.0 log.sub.10 TCID.sub.50/mL, or less
than 5.0 log.sub.10 TCID.sub.50/mL, or less than 4.0 log.sub.10
TCID.sub.50/mL, or less than 3.0 log.sub.10 TCID.sub.50/mL, or less
than 2.0 log.sub.10 TCID.sub.50/mL. Such low titer influenza
vaccine compositions may further comprise a pharmaceutically
acceptable adjuvant, e.g., E. coli heat-labile toxin (or fragments
thereof), pertussis toxin, aluminum. Other adjuvants include, but
are not limited to aluminum phosphate, aluminum hydroxide,
MPL..TM.. (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem
Research, Inc., Hamilton, Mont., now Corixa), synthetic lipid A
analogs such as 529 (Corixa), Stimulon..TM.. QS-21 (Aquila
Biopharmaceuticals, Framingham, Mass.), IL-12 (Genetics Institute,
Cambridge, Mass.), synthetic polynucleotides such as
oligonucleotides containing a CpG motif (U.S. Pat. No. 6,207,646
(28)), and cholera toxin (either in a wild-type or mutant form, for
example, where the glutamic acid at amino acid position 29 is
replaced by another amino acid, preferably a histidine, in
accordance with published International Patent Application Number
WO 00/18434).
[0061] In one specific embodiment, diafiltration may be used in the
preparation of virus compositions of the invention. For instance,
diafiltration may be used to concentrate the virus prior to
formulation. Diafiltration may be used in addition to or instead of
ultracentrifugation.
[0062] After an initial phase described later herein, cGMP
production was initiated at the scale of 10,000 eggs per lot.
A/Beijig/262/95 (H1N1), A/Sydney/05/97 (H3N2) and B/Ann Arbor/1/94
(B/harbin/7/94-like) purified monovalent CAIV bulks were
manufactured to support subsequent clinical trials. The B/Ann Arbor
strain is hereafter referred to as B/Harbin/7/94-like. For
convenience, strain designations are often abbreviated (e.g.,
A/Beijing). The current application describes most heavily the
process steps unique to the liquid FluMist, however, steps involved
in both or common to both liquid and frozen FluMist are also
described herein.
[0063] Methods for egg management and preparation of fresh virus
harvest were essentially the same in various embodiments of the
current invention (with the exception of automated inoculation and
harvesting) for frozen FluMist as for liquid FluMist and those of
skill in the art will be aware of similar or equivalent steps which
are capable of use with the current invention. In the current
invention, ultra-centrifugation was performed using a Hitachi CP40Y
zonal centrifuge and an RP40CT Type D continuous-flow rotor (rotor
volume=3.2 L). Virus harvest was first pooled, stabilized with
sucrose phosphate glutamate (SPG) to facilitate filtration, then
passed through a 5 micron polypropylene filter. The filtrate was
then loaded onto a 10% to 60% sucrose gradient, banded for one hour
at 40,000 rpm, and eluted into 100-ml fractions.
[0064] Fractions containing high hemagglutinin (HA) levels were
pooled, diluted to 0.2M sucrose concentration, and then sterile
filtered using a polyvinylidene fluoride (PVDF) 0.2 micron filter.
The resulting bulk purified monovalent CAIV was frozen in 1 L
bottles below -60 degrees C. and held for further processing.
Formulation and Filing
[0065] Initial formulation screening determined that the liquid
phase stability of purified CAIV at refrigerated temperatures was
suitable for an annual vaccine. Hydrolyzed animal gelatin added to
the frozen FluMist stabilizer SPG provided the best stability
results, with a least stable strain and lot from the CTM-1 campaign
showing a loss of one log over 6.9 months. Further formulation
development studies showed that the stability of liquid FluMist
could be further improved by storing frozen just after manufacture,
and thawing before final distribution. The stability of the
worst-case strain was also improved by addition of arginine. While
animal gelatins can raise concerns with regard to transmissible
spongiform encephalopathies (TSE), available formulations lacking
gelatin did not achieve the required stability in all embodiments.
Thus, porcine gelatin was chosen for the some embodiments of liquid
FluMist formulation due to its stabilizing properties and the fact
that there are no reported occurrences of TSE in pigs. The harsh
chemical processing steps used in collagen hydrolysis are also
thought to cause inactivation of prion-sized proteins.
[0066] Frozen, purified monovalent bulk CAIV was shipped and
trivalent vaccine was produced under cGMP to support clinical
testing of liquid FluMist. A total of six fills were performed.
Blending was performed at small scale using a 4-liter glass
aspirator flask, and 0.5 mL Becton Dickinson (BD) Hypack SCF
(sterile, clean, ready to fill) glass sprayers were filled using an
INOVA automated filler/stopperer and associated equipment. The
manufacture of filled trivalent liquid FluMist is described in more
detail below.
SPECIFIC FORMULATION EMBODIMENTS OF THE INVENTION
[0067] In one embodiment, the vaccine formulations of the invention
comprise one or more of the following in the final formulations:
sucrose: 6-8% weight/volume (w/v); arginine monohydrochloride 1-2%
w/v; glutamic acid, monosodium monohydrate 0.05-0.1% w/v; gelatin
hydrolysate, porcine Type A (or other sources) 0.5-2% w/v;
potassium phosphate dibasic 1-2%; and potassium phosphate monobasic
0.25-1% w/v.
[0068] In one specific embodiment, vaccine formulations comprise
one or more of the following: sucrose: 6.84% weight/volume (w/v);
arginine monohydrochloride 1.21% w/v; glutamic acid, monosodium
monohydrate 0.094 w/v; gelatin hydrolysate, porcine Type A (or
other sources) 1% w/v; potassium phosphate dibasic 1.13%; and
potassium phosphate monobasic 0.48% w/v. In another specific
embodiment, vaccine formulations comprise all of the following:
sucrose: 6.84% weight/volume (w/v); arginine monohydrochloride
1.21% w/v; glutamic acid, monosodium monohydrate 0.094% w/v;
gelatin hydrolysate, porcine Type A (or other sources) 1% w/v;
potassium phosphate dibasic 1.13%; and potassium phosphate
monobasic 0.48% w/v.
[0069] In another specific embodiment, vaccine formulations
comprise all of the following (within 10% variation of one or more
component): sucrose: 6.84% weight/volume (w/v); arginine
monohydrochloride 1.21% w/v; glutamic acid, monosodium monohydrate
0.094% w/v; gelatin hydrolysate, porcine Type A (or other sources)
1% w/v; potassium phosphate dibasic 1.13%; and potassium phosphate
monobasic 0.48% w/v.
[0070] In another specific embodiment, vaccine formulations
comprise all of the following (within 10% variation of one or more
component): sucrose: 6.84% weight/volume (w/v); arginine
monohydrochloride 1.21% w/v; gelatin hydrolysate, porcine Type A
(or other sources) 1% w/v. In such embodiments, formulation are in
buffer (e.g., a potassium phosphate buffer (pH 7.0-7.2)).
[0071] In another specific embodiment, vaccine formulations
comprise all of the following (within 20% variation of one or more
component): sucrose: 6.84% weight/volume (w/v); arginine
monohydrochloride 1.21% w/v; glutamic acid, monosodium monohydrate
0.094% w/v; gelatin hydrolysate, porcine Type A (or other sources)
1% w/v; potassium phosphate dibasic 1.13%; and potassium phosphate
monobasic 0.48% w/v.
[0072] In another specific embodiment, vaccine formulations
comprise all of the following (within 30% variation of one or more
component): sucrose: 6.84% weight/volume (w/v); arginine
monohydrochloride 1.21% w/v; glutamic acid, monosodium monohydrate
0.094% w/v; gelatin hydrolysate, porcine Type A (or other sources)
1% w/v; potassium phosphate dibasic 1.13%; and potassium phosphate
monobasic 0.48% w/v.
[0073] In another specific embodiment, vaccine formulations
comprise all of the following (within 40% variation of one or more
component): sucrose: 6.84% weight/volume (w/v); arginine
monohydrochloride 1.21% w/v; glutamic acid, monosodium monohydrate
0.094% w/v; gelatin hydrolysate, porcine Type A (or other sources)
1% w/v; potassium phosphate dibasic 1.13%; and potassium phosphate
monobasic 0.48% w/v.
[0074] In another specific embodiment, vaccine formulations
comprise all of the following (within 1% variation of one or more
component): sucrose: 6.84% weight/volume (w/v); arginine
monohydrochloride 1.21% w/v; glutamic acid, monosodium monohydrate
0.094% w/v; gelatin hydrolysate, porcine Type A (or other sources)
1% w/v; potassium phosphate dibasic 1.13%; and potassium phosphate
monobasic 0.48% w/v.
[0075] In another specific embodiment, vaccine formulations
comprise all of the following (within 3% variation of one or more
component): sucrose: 6.84% weight/volume (w/v); arginine
monohydrochloride 1.21% w/v; glutamic acid, monosodium monohydrate
0.094% w/v; gelatin hydrolysate, porcine Type A (or other sources)
1% w/v; potassium phosphate dibasic 1.13%; and potassium phosphate
monobasic 0.48% w/v.
[0076] In a specific embodiment, formulations of the invention may
contain, e.g., potassium phosphate (e.g., at least 50 mM, or at
least 100 mM, or at least 200 mM, or at least 250 mM) as a buffer
or alternatively, histidine (e.g., at least 50 mM, or at least 100
mM, or at least 200 mM, or at least 250 mM).
OTHER SPECIFIC EMBODIMENTS OF THE INVENTION, E.G., DOSING,
POTENCY
[0077] In another specific embodiment, methods of administering the
vaccine formulations of the invention intranasally are included.
For instance, vaccine formulations of the invention may be
administered intranasally in final doses of 0.5 mL/dose; 0.75
mL/does; 0.1 mL/does; 0.15 mL/does; 0.2 mL/dose; 0.25 mL/dose;
0.5-0.1 mL/dose; 0.5 mL-2 mL/dose; or 0.5-2.5 mL/dose.
[0078] In one embodiment, vaccine formulations of the invention
comprise approximately 10.sup.7 fluorescent focus units (FFU) or
10.sup.7 TCID.sub.50 of each of three different reassortant strains
of influenza. In another specific embodiment, vaccine formulations
of the invention comprise a potency of 7.0 (+/-0.5) log10 FFU/dose
(or TCID.sub.50/dose) for each strain of influenza virus. In
another specific embodiment, vaccine formulations of the invention
comprise a potency of 7.0 (+/-0.5) log10 FFU/dose (or
TCID.sub.50/dose) for at least one strain of influenza virus. In
another specific embodiment, vaccine formulations of the invention
have a potency of at least (or equal to) 6.0 log10 FFU/dose, 6.5
log10 FFU/dose, 6.7 log10 FFU/dose 6.7 log10 FFU/dose, 6.8 log10
FFU/dose, 6.9 log10 FFU/dose, 7.0 log10 FFU/dose, 7.1 log10
FFU/dose, 7.2 log10 FFU/dose, 7.3 log10 FFU/dose, 7.4 log10
FFU/dose, 7.5 log10 FFU/dose, 7.6 log10 FFU/dose, 7.7 log10
FFU/dose, 7.8 log10 FFU/dose, 7.9 log10 FFU/dose, 8.1 log10
FFU/dose, 8.5 log10 FFU/dose or 8.8 log10 FFU/dose.
[0079] Potency may be measured by TCID.sub.50 instead of FFU in
each embodiment of the invention. A Fluorescent Focus Assay (FFA)
is a direct measure of the infectivity in the vaccine while
TCID.sub.50 is an indirect measure which measures the ability of
productive replication in MDCK or other cells.
[0080] In another specific embodiment, vaccine formulations of the
invention have a potency of between 6.5-7.5 log 10 FFU per dose
(e.g., per 0.2 mL dose).
[0081] In another specific embodiment, vaccine formulations of the
invention have a potency of at least (or equal to) 7.0 log.sub.10
TCID.sub.50 per dose, or at least 6-8 TCID.sub.50 per dose, or at
least 6.4 TCID.sub.50 per dose, or at least 6.6 log.sub.10
TCID.sub.50 per dose.
[0082] In one embodiment, vaccine formulations of the invention
have pH of 6.7-7.7, or 6.6, or 6.7, or 6.8, or 6.9, or 7.0, or 7.1,
or 7.2, or 7.3, or 7.4, or 7.5, or 7.6, or 7.7.
[0083] In another specific embodiment, the vaccine formulations of
the invention have no greater than 60 EU/mL of endotoxin, or have
no greater than 200 EU/mL of endotoxin. For instance, the vaccine
formulations of the invention have less than or equal to 5 EU/mL of
endotoxin. In another specific embodiment, the vaccine formulations
of the invention have less than or equal to 1.0 EU/7.0 log10
FFU.
[0084] In another specific embodiment, the vaccine formulations of
the invention are free of one or more (or all) of the following:
mycoplasma, retrovirus, avian leucosis virus, and mycobacterium
(e.g., M. tuberculosis). Methods of the producing such formulations
of the invention may comprise the step(s) of testing for such
impurities.
[0085] In another specific embodiment, the vaccine formulations of
the invention may contain influenza viruses having HA and NA genes
from three different viruses as in FluMist.
[0086] In one specific embodiment, formulations of the invention
comprise one or more (or all) of the following per dose (e.g., per
0.2 mL dose): sucrose (13.68 mg), dibasic potassium phosphate (2.26
mg), monobasic potassium phosphate (0.96 mg), gelatin hydrolysate
(2.0 mg), arginine hydrochloride (2.42 mg), and monosodium
glutamate (0.188 mg).
[0087] In one specific embodiment, formulations of the invention
comprise one or more (or all) of the following per dose (e.g., per
0.2 mL dose): sucrose (13.68 mg), potassium phosphate (100 mM),
gelatin hydrolysate (2.0 mg), arginine hydrochloride (2.42 mg), and
monosodium glutamate (0.188 mg).
[0088] In another specific embodiment, formulations of the
invention comprise one or more (or all) of the following (plus or
minus 10%, or 20% or 30% or 40%) per dose: sucrose (13.68 mg),
dibasic potassium phosphate (2.26 mg), monobasic potassium
phosphate (0.96 mg), gelatin hydrolysate (2.0 mg), arginine
hydrochloride (2.42 mg), and monosodium glutamate (0.188 mg).
[0089] In another specific embodiment, formulations of the
invention comprise one or more (or all) of the following (plus or
minus 10%, or 20% or 30% or 40%) per dose: sucrose (13.68 mg),
potassium phosphate (100 mM), gelatin hydrolysate (2.0 mg),
arginine hydrochloride (2.42 mg), and monosodium glutamate (0.188
mg).
[0090] In another specific embodiment, formulations of the
invention comprise an influenza virus comprising the genetic
backbone of one or more of the following influenza viruses: A/Ann
Arbor/6/60 (A/AA/6/60) B/Ann Arbor/1/66 virus, the FluMist MDV-A
(ca A/Ann Arbor/6/60), the FluMist MDV-B (ca B/Ann Arbor/1/66),
A/Leningrad/17 donor strain backbone, and PR8.
[0091] In another specific embodiment, the vaccine formulations of
the invention comprise an influenza virus comprising an HA and an
NA polypeptide sequence (or at least 90% identical or at least 95%
identical to such sequences) from one or more of the following:
B/Yamanashi; A/New Caledonia; A/Sydney; A/Panama; B/Johannesburg;
B/Victoria; B/Hong Kong; A/Shandong/9/93; A/Johannesburg/33/94;
A/Wuhan/395/95; A/Sydney/05/97; A/Panama/2007/99;
A/Wyoming/03/2003; A/Texas/36/91; A/Shenzhen/227/95;
A/Beijing/262/95; A/New Caledonia/20/99; B/Ann Arbor/1/94;
B/Yamanashi/166/98; B_Johannesburg.sub.--5.sub.--99;
B/Victoria/504/2000; B/Hong Kong/330/01;
B_Brisbane.sub.--32.sub.--2002; B/Jilin/20/03; an H1N1 influenza A
virus, an H3N2 influenza A virus, H9N2 influenza A virus, an H5N1
influenza A virus; an influenza B virus; and a pandemic influenza
strain (either designated by WHO or not circulating in the human
population). See, e.g., US 20050042229.
[0092] In another specific embodiment, the vaccine formulations of
the invention are sterile.
Description of Representative Steps in Vaccine Production
[0093] For ease in discussion and description, the various steps of
vaccine composition production in general, can be thought of as
comprising or falling into four broad groups (roughly similar to
the presentation outlined previously above). The first group
comprises such aspects as co-infection, reassortment, selection of
reassortants, and cloning of reassortants. The second group
comprises such aspects as purification and expansion of
reassortants. The third group comprises further expansion of
reassortants in eggs, along with harvesting and purification of
such harvested virus solutions. The fourth group comprises
stabilization of harvested virus solutions and potency/sterility
assays of the virus solutions. 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. Again, it will be appreciated that other steps (both
similar and different) are optionally used with the methods and
compositions of the invention (e.g., the methods and compositions
for RTS vaccine compositions).
[0094] As mentioned above, for ease in discussion and description,
the various steps of vaccine production 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.
[0095] Group 1
[0096] The aspects of vaccine composition production 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 reasserted 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, e.g., U.S. patent
application Nos. 60/375,675 filed Apr. 26, 2002, PCT/US03/12728
filed Apr. 25, 2003 and U.S. application Ser. Nos. 10/423,828,
filed Apr. 25, 2003, PCT/US05/017734, filed May 20, 2005; and
US20050186563.
[0097] 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, sometimes large
numbers of reassortments need to be done. After being reasserted,
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).
[0098] 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.
[0099] Optimization of Reassortment
[0100] Various embodiments utilizing the steps in Group 1 can
optimize the reassortment process in order to reduce the number of
reassortments needed (and thus increase the throughput/stability of
the vaccine production process), etc. 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. See, e.g., U.S. patent application Ser. Nos. 10/788,236
and PCT/US04/05697 both filed Feb. 25, 2004, which are incorporated
by reference in their entirety for all purposes, both within this
section and throughout the specification.
[0101] Other methods of reassortment of influenza virus can
optionally 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. 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.
[0102] Selection and Cloning of Reassortments
[0103] The steps in Group 1 also comprise selection of reassorted
influenza viruses. Reassorted influenza A strains are capable of
selection in either cell culture (e.g., CEK cells) or in eggs.
However, reasserted 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. Various
methods of vaccine composition production, see, e.g., See, e.g.,
U.S. patent application Ser. Nos. 10/788,236 and PCT/US04/05697
both filed Feb. 25, 2004, utilize different steps for virus
reassortment, e.g., selection, such steps can optionally be used to
create virus for the vaccine compositions.
[0104] Characterization of Reassortments
[0105] Yet other methods of virus/vaccine production 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. 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 methods can 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) Clin. Micro.
J38(2):839-845 which is incorporated herein by reference for all
purposes.
[0106] Prevention of Bacterial Contamination
[0107] Some methods of virus/vaccine production can comprise steps
to detect and/or prevent/detect microbial contamination of eggs in
which influenza virus is produced. The microbial detection
strategies of the invention are useful for rapid/high throughput
microbial detection and, thus, as with many other steps, are useful
for increasing throughput and optionally stability in virus/vaccine
production.
[0108] Many current influenza vaccine production strategies, which
can optionally be used with 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. 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.
[0109] After expansion of viruses for 2 to 3 days in the eggs, the
top of the eggshell is typically removed for manual harvesting of
the allantoic fluid containing virus within the egg. See, above.
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 prior to use in the blending and
filling to ensure microbial-free product.
[0110] After incubation, the "traditional" method of candling is
used to identify infertile and dead eggs that 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.
[0111] 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. 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). Again, see, e.g., U.S. patent application Ser. Nos.
10/788,236 and PCT/US04/05697 both filed Feb. 25, 2004, for
descriptions of various techniques capable of use with the current
invention.
[0112] Group 2
[0113] Aspects of virus/vaccine production that fall into Group 2
include further purification and virus expansion, etc. 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. Purification protocols
can involve, e.g., ultra-centrifugation 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.
[0114] Group 3
[0115] Aspects of virus/vaccine production that fall under the
heading of Group 3 include, e.g., 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.
[0116] For example, conditioning, washing, candling, and incubating
eggs which contain the reasserted 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 or Viral
Harvest (VH)) from the eggs; and clarification of the virus
solution can all fall within such category. 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 virus/vaccine production that 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. See, e.g., U.S. patent
application Ser. Nos. 10/788,236 and PCT/US04/05697 both filed Feb.
25, 2004, give further steps such as rocking, etc. that can also be
used with the methods and compositions of the current invention.
Other similar steps can include specific filtering and warming of
compositions, again, see, the same.
[0117] Filtering and Warming
[0118] The current invention involves aspects of
ultra-centrifugation, see above, which can fall into the current
grouping. In addition, U.S. patent application Ser. Nos. 10/788,236
and PCT/US04/05697 both filed Feb. 25, 2004 also give other
filtering and warming steps that can optionally be used with the
methods and compositions of the current invention. As described,
the FluMist.TM. manufacturing process can use embryonated chicken
eggs to generate master virus seeds (MVS), manufacturer's working
virus seeds (MWVS) and virus harvests (VH). 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. 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.
[0119] Group 4
[0120] Group 4 of the aspects of vaccine formulation/composition
production comprises, e.g., steps primarily concerned with
stabilization (e.g., through addition of components, alterations in
buffer/NAF ratios, etc.) and assays of potency/sterility of virus
containing solutions. The description of the current invention
above, gives various aspects which can optionally be grouped within
the current category. See, above.
[0121] In some embodiments, the final viral solutions/vaccines
comprising live viruses are stable in liquid form for a period of
time sufficient to allow storage "in the field" (e.g., on sale and
commercialization when refrigerated at 2-8.degree. C., 4.degree.
C., 5.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. In other embodiments, such
solutions/vaccines are stable in liquid form at from about
2.degree. C. to about 8.degree. C., e.g., refrigerator temperature.
For example, if a 0.3 log potency loss was acceptable and the
storage period were 9 months, then an 0.05 log/month decrease in
potency would be acceptable. As another 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 when stored at from about 2.degree. C. to about
8.degree. C. The stability of the composition can comprise between
about 1 day and 2 years, between about 10 days and about 2 years,
between about 20 days and about 2 years, between about 1 month and
about 2 years, between about 2 months and about 2 years, between
about 3 months and about 2 years, between about 4 months and about
2 years, between about 5 months and about 2 years, between about 6
months and about 2 years, between about 7 months and about 2 years,
between about 8 months and about 2 years, between about 9 months
and about 2 years, between about 10 months and about 2 years,
between about 11 months and about 2 years, between about 12 months
and about 2 years, between about 13 months and about 2 years,
between about 14 months and about 2 year, between about 15 months
and about 2 years, between about 16 months and about 2 years,
between about 17 months and about 2 years, between about 18 months
and about 2 years, between about 19 months and about 2 years,
between about 20 months and about 2 years, between about 21 months
and about 2 years, between about 22 months and about 2 years,
between about 23 months and about 2 years, or greater than about 2
years.
[0122] One may test the stability of a formulation of the invention
by a number of methods. For instance, one may first incubate a
formulation at freezing temperatures (e.g., -25 or -70.degree. C.
(+/-10, 20, 30, or 40.degree. C.) and then store (or "incubate")
the formulation at 4-8.degree. C. for a length of time.
Alternatively, one may test the stability of a formulation of the
invention by simply storing (or "incubating") the formulation at
4-8.degree. C. for a length of time. Potency could be measured by a
number of methods as described herein or otherwise known.
[0123] Concentration/Diafiltration of Virus Harvests
[0124] In some methods of vaccine composition production, virus
harvests are optionally concentrated using an appropriate column.
See, U.S. patent application Ser. Nos. 10/788,236 and
PCT/US04/05697 both filed Feb. 25, 2004.
[0125] Stabilizers/Buffers
[0126] Vaccine composition production can also optionally include
various dilutions of NAF (typically unfractionated NAF) comprising
the virus of interest and combinations of, e.g., sucrose, arginine,
gelatin, EDTA, etc. See, e.g., U.S. patent application Ser. Nos.
10/788,236 and PCT/US04/05697, for examples of various combinations
possible in different vaccine formulations. Such methods and
compositions are preferably stable, e.g., do not show unacceptable
losses in potency, e.g., a potency loss of between 0.5-1.0 logs, or
less than 0.5 logs, or less than 1.0 logs, 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.).
[0127] In some formulations, compositions can comprise a 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 U.S. patent
application Ser. Nos. 10/788,236 and PCT/US04/05697. Also, in many
virus solutions/vaccine solutions a base solution of SPG (sucrose,
potassium phosphate and monosodium glutamate) is optionally
utilized.
[0128] U.S. patent application Ser. Nos. 10/788,236 and
PCT/US04/05697 both filed Feb. 25, 2004 give other/additional
methods of virus/vaccine composition stabilization, e.g., NAF level
manipulation, etc.
[0129] Definitions
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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 that
regulates transcription in a specific tissue type or cell type, or
types.
[0134] 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, but not necessarily
exclusively, the vectors of herein refer to plasmids.
[0135] 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.
[0136] 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.
[0137] In the context herein, 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.
[0138] 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.
[0139] 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 neuramimidase, 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 neuramimidase, from a different parental
virus.
[0140] 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.
[0141] 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, COS7 cells).
[0142] Influenza Virus
[0143] 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 neuramimidase (NA) envelope glycoprotein.
Segment 7 encodes two matrix proteins, designated M1 and M2, which
are translated from differentially spliced mRNAs. Segment 8 encodes
NS1 and NS2, two nonstructural proteins, which are translated from
alternatively spliced mRNA variants.
[0144] The eight genome segments of influenza B encode 11 proteins.
The three largest genes code for components of the RNA polymerase,
PB1, PB2 and PA. Segment 4 encodes the HA protein. Segment 5
encodes NP. Segment 6 encodes the NA protein and the NB protein.
Both proteins, NB and NA, are translated from overlapping reading
frames of a biscistronic mRNA. Segment 7 of influenza B also
encodes two proteins: M1 and M2. The smallest segment encodes two
products, NS1 which is translated from the full length RNA, and NS2
which is translated from a spliced mRNA variant.
[0145] Influenza Virus Vaccine
[0146] 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 neuramimidase 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).
[0147] 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, another method 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 stability in storage time and/or of storage at a
more convenient temperature (e.g., refrigerator temperature of
about 2-8.degree. C., e.g., as through use of the methods and
compositions of the current invention), are thus quite
desirable.
[0148] 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 inventor
and his 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, e.g., U.S. patent
application Nos. 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, PCT/US05/017734 filed May 20, 2005. 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 through use of the current
invention in whole or part.
[0149] 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.
[0150] FluMist.RTM.
[0151] As mentioned previously, numerous examples and types of
influenza vaccine exist. An exemplary influenza vaccine is FluMist
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 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 and their compositions.
[0152] 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).
[0153] 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 reasserted virus using B strains of influenza is more
difficult, however.
[0154] Recent work, see, see, e.g., U.S. patent application Nos.
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, PCT/US05/017734
filed May 20, 2005 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.
[0155] 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., reasserted influenza viruses for vaccine
production to produce viruses for vaccines in a more stable,
consistent and productive manner.
[0156] Cell Culture
[0157] 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.
[0158] 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.
[0159] In a specific embodiment of the invention, host cells of the
invention are cultured and/or infected under serum-free conditions,
in the presence or absence of trypsin, and are cultured and/or
infected at temperatures ranging from 30 degrees Celsius to 39
degrees Celsius; or 30 degrees Celsius, or 31 degrees Celsius, 32
degrees Celsius, 33 degrees Celsius, 34 degrees Celsius, 35 degrees
Celsius, 36 degrees Celsius, 37 degrees Celsius, 38 degrees
Celsius, or 39 degrees Celsius.
[0160] Cells for production of influenza virus can be cultured in
serum-containing or serum free medium. In some cases, 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).
[0161] 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.).
[0162] In some methods (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.
[0163] 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. See, e.g., US20050158342, which is
incorporated by reference herein.
[0164] Kits
[0165] To facilitate use of the methods and compositions of the
invention, any of the vaccine components and/or compositions, e.g.,
virus in 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.
EXAMPLE 1
Development of Liquid FluMist
[0166] A total of 19 monovalent bulk lots of virus were initiated
under protocol, including four development lots (CB0006H, CB0008H,
CG0017H, CB0018H and CB0019H). Lots CB0018H and CB0019H were
conducted to supply material for various studies and are not
discussed further in this application. Two lots (CB00180H and
CB0012H) were combined after the ultra-centrifugation step to
produce A/Sydney monovalent bulk CB0020H. Each lot was initiated
with about 2000 eggs.
[0167] Egg handling and incubation conditions were set up similar
to the process used for production of frozen FluMist, however,
manual inoculation and harvesting were used due to the smaller
scale, etc. The ultracentrifuge process was scaled down to smaller
equipment made by the same manufacturer (Discovery 90, made by
Hitachi and marketed by Sorvall/heraus), with a Model P32CT rotor
having approximately 470 mL of total capacity. Clarified,
stabilized virus harvest was loaded onto a 20% to 60% sucrose
gradient, banded for one hour at 32,000 rpm, and eluted into 20 mL
fractions. Fractions containing peak HA levels were pooled, diluted
to 0.2M sucrose concentration, filtered through a 0.2 micron
filter, and then transferred into 250 mL flexible polymer bags
(Stedim) which were frozen and held below -60.degree. C. for
further manufacture.
[0168] After a short series of test runs, cGMP manufacturing was
initiated to support further clinical testing of liquid FluMist.
A/Beijing/262/95 (H1N1) and A/Sydney/05/97 (H3N2) were again
manufactured, and the B strain was changed to B/Yamanashi/166/98.
Monovalent bulks produced were frozen and shipped for blending,
filing, and packaging. Of course, it will be appreciated here and
throughout, that use of particular strains (e.g., Beijing, etc.)
should not necessarily be taken as limiting unless specifically
stated to be so. Thus for example, the methods and formulations
herein can optionally use different strains each influenza season
to produce different RTS compositions, etc.
[0169] Development and Clinical Trials
[0170] The process for liquid compositions was developed and
included monovalent bulk lots CAZ001-024 and CAZ035-043. The
clinical trial material (CTM) included monovalent bulk lots
CAZ025-CAZ034.
[0171] The liquid FluMist manufacturing process developed and used
for CTM-1 is divided into six discrete process stages below. The
process stages 1 through 5 were defined to match the major process
steps as conducted using separate manufacturing instruction
documents. Stage 6 includes the entire blend and fill process. It
will be appreciated that such stages can be roughly compared to the
generalized steps outlined below for manufacturing/production of
vaccine compositions in general.
[0172] Stages
[0173] Stage 1: receipt and sanitization of SPF eggs; Stage 2:
production of virus harvest; Stage 3: concentration of virus by
zonal centrifugation; Stage 4: pooling and dilution of peak
fractions; Stage 5: sterile filtration and monovalent bulk storage;
and, Stage 6: blend and fill
[0174] A process flow diagram for the CTM-1 monovalent bulk
production is shown in FIG. 1. The blend and fill process flow is
shown in FIG. 2.
[0175] Receipt and Sanitization of SPF Eggs
[0176] Specific pathogen-free (SPF) embryonated eggs were purchased
from SPAFAS, Inc. which utilizes flock management and surveillance
programs designed to provide suitable assurance of disease-free
eggs. Eggs were laid in the United States and sanitized using
Clorwash and Quat 800 sprays. The eggs were then transported by air
and road using packaging sufficient to maintain cleanliness and
avoid freezing or overheating. Upon receipt, the cold fertile eggs
were inspected and stored in an SPF incubation unit at 14.degree.
C.+/-2.degree. C. with 60-80% relative humidity (RH) for a maximum
of 7 days. The eggs were transferred to trays from the cartons
supplied by the vendor and a batch number assigned. Eggs were
placed on 36-egg Jamesway trays and the eggshell surfaces sanitized
with Chlorwash and Quat 800 in an automated egg sanitization system
to minimize bioburden prior to placing them in the incubator.
Chlorwash was prepared at a range of 43-44.degree. C. and
48-49.degree. C. and Quat 800 was prepared at a range of
48-49.degree. C. Following sanitization, the eggs were placed on
trolleys and air dried at ambient temperature for not less than 2
hours. The eggs were then placed in a Buckeye Incubator and
incubated at 37.5.degree. C.+/-1.degree. C. with 60-80% RH for 264
hours +/-12 hours. After incubation, the SPF eggs were transferred
from the SPF egg unit to a candling area. Using a fiber optic lamp,
the position of the air sac was located in each egg. Dead or
infertile eggs were discarded, and the fertile eggs marked for
inoculation.
[0177] Production of Virus Harvests
[0178] The fertile eggs were surface sanitized with 70% industrial
methylated spirits (IMS) prior to inoculation and then transferred
for mass inoculation. In preparation of the diluted manufacturer's
working virus seed (MWVS), the master virus seeds used to produce
the MWVS for phase 1 clinical trials were produced using the
classical reassortment method. The MWVS, was transferred into the
AVU and serial dilutions of the thawed MWVS were prepared in
sterile 0.01M phosphate buffered saline, pH 7.7 within a
microbiological safety cabinet using a sterile disposable pipette
for each transfer. The inoculation occurred under laminar flow in a
class 10,000 room using a semi-automatic Bibby inoculation machine,
which penetrates and inoculates 36 eggs per tray. The target titer
of the inoculum was log.sub.102.1 TCID.sub.50 per 0.1 mL and the
dilution was calculated from the predetermined titer of the MWVS.
Preparation of each virus inoculum was completed within 2 hours of
the removal of the vial from the freezer. Aliquots of the inoculum
were stored at 5 +/3.degree. C. until use. Inoculum was used within
8 hours of preparation.
[0179] Inoculation of SPF Eggs
[0180] Each tray of eggs was hand-fed into the Bibby automated
inoculation machine. A punch was used to pierce the eggshell and
penetrate the egg to a preset adjustable depth. The inoculation
needles extended into the egg and the dosing pump delivered 0.1 mL
of the MWVS inoculum into each egg after which the punch was
withdrawn. Following inoculation, the egg tray was removed by an
operator and the process repeated with a new tray of eggs. The CAIV
inoculated eggs were incubated at 33+/-1.degree. C. for a time
determined by the growth curve of the virus strain. After
incubation, the SPF embryonated eggs were chilled to 28.degree. C.
for 18+/-6 hours. At the end of chilling, the eggs were transferred
for the harvesting step.
[0181] Concentration of Virus by Zonal Centrifugation
[0182] Virus Harvest (VH)
[0183] Eggs were harvested with an automatic harvest machine
(Bibby). The trays were hand fed to a decapitation station where
the upper section of the eggshell was removed to create an access
hole for harvest needle entry. The eggs were visually inspected
prior to harvest and unsuitable eggs (e.g., discolored) rejected.
The acceptable eggs proceeded to a harvest station, where the
allantoic fluid was withdrawn using needles and suction provided by
a vacuum system. The harvest was collected mixed in a 100 L
jacketed stainless vessel with a jacket temperature of 2-8.degree.
C. Samples of the virus harvest were collected from the VH pool and
tested for: potency (TCID.sub.50), safety, avian leucosis, M.
tuberculosis, mycoplasma, extraneous virus, identity, reverse
transcriptase assay, virus genotype, virus phenotype, and
attenuation.
[0184] Stabilization and Clarification
[0185] The pooled virus harvest (VH) was immediately stabilized at
2-8.degree. C. to a final concentration of 0.2 M sucrose, 0.01M
phosphate, 0.005M glutamate (SPG), with 10.times.SPG (9 parts VH to
1 part 10.times.SPG). Samples were collected for potency and
bioburden prior to clarification. The stabilized virus harvest was
clarified by 5 .mu.m depth filtration to remove particulates prior
to purification by high-speed continuous flow centrifugation.
Clarified virus harvest was sampled for potency.
[0186] Zonal Centrifugation, Collection, and HA Assay of Peak
Fractions
[0187] Prior to use, the centrifuge rotor was sanitized with a 1:20
dilution of concentrated formaldehyde solution. The clarified VH
was loaded onto a sucrose gradient (10-60% sucrose in phosphate
buffer, pH 7.2) for continuous high-speed centrifugation using a
Hitachi CP40Y zonal centrifuge. The gradient was formed by addition
to the centrifuge of a 60% sucrose solution followed by a 10%
sucrose solution in phosphate buffer. The centrifuge speed was set
to 4,000 rpm for 20 minutes to allow the sucrose solutions to form
a density gradient. Centrifuge temperature was set at 2-8.degree.
C. during gradient formation. Following gradient formation, buffer
flow was initiated and the rotor speed was increased to 40,000 rpm,
before the clarified VH was loaded onto the gradient at a rate of
20 L per hour at 40,000 rpm. For a typical CTM-1 batch with 10,000
eggs harvested and 6 mL/egg, the duration of the loading step was
about three hours. Following loading of the clarified VH,
centrifugation was continued at 40,000 rpm for an additional hour
to allow the virus to band. The virus particles migrated to the
38-45% sucrose portion of the gradient and concentrate into a
"band." At the end of this step, the centrifuge was gradually
slowed and then stopped to allow collection of the virus peaks. 100
mL fractions were collected under laminar flow into 125 mL sterile
polycarbonate bottles. Fractions were held at 2-8.degree. C. for
approximately 1 hour while fractions were assayed for HA activity.
Peak fractions were typically found between 38-45% of the sucrose
gradient.
[0188] Pooling and Dilution of Peak Fractions
[0189] The centrifuge peak fractions, as identified by the HA
assay, were pooled under a laminar flow hood by aseptically pouring
the fractions into a 5 L sterile glass bottle, and mixed by
swirling. The refractive index (RI) was read to determine sucrose
concentration and the pool sampled for potency. The centrifuge peak
fraction pool (CP) was diluted by aseptically adding a calculated
volume of sterile cold (2-8.degree. C.) phosphate-glutamate buffer,
pH 7.2 (PBG buffer) to a final concentration of 0.2M sucrose, 0.1M
phosphate, and 0.005 M glutamate. This was typically a 1:6
dilution. The diluted centrifuge peak fraction pool (DCP) was
sampled for potency and bioburden.
[0190] Sterile filtration and Monovalent Bulk Storage
[0191] The DCP was pumped through sterile tubing to a Class 100
filling room for sterile filtration. The DCP was sterile filtered
with a 0.22 .mu.m filter into a sterile 5 L glass bottle under a
Laminair Air Flow hood. The filtered monovalent bulk was mixed and
sampled for potency, identity, and sterility testing. The 0.22
.mu.m filter was integrity tested before and after the filtration
process. The MB was then aliquoted into sterile one liter
polycarbonate bottles. The MB was collected in 500 mL aliquots with
a total volume of three to four liters. The sterile one-liter PC
bottles were stored at -60.degree. C. or below. The titer of the
bulk virus was determined by TCID.sub.50 assay. Monovalent bulk was
then shipped at .ltoreq.-60.degree. C. for blending and
testing.
[0192] Blend and Fill
[0193] The major steps for the blend and fill process of the
preparation of the bulk trivalent blend were: Thawing, Preparation
of Diluent, Blending, Filling, and Packaging.
[0194] Thawing and Blending
[0195] The appropriate 1 bottles of monovalent bulk were removed
from frozen storage (.ltoreq.-60.degree. C. for MB) and transferred
to a thaw room. The required quantities of the three individual
monovalent bulks and SPG diluent were calculated based on the bulk
infectivity titer and the required formulation strength. The
bottles of MB were loaded into a 33.+-.3.degree. C. water bath and
manually agitated every five minutes. Thawing was monitored
visually to ensure that all bottles were thawed before leaving the
water bath, and that all thawed bottles were removed every 15
minutes. Once thawed, the bottles of MB were moved to a
5.+-.3.degree. C. refrigerator and held until all of the required
bottles had been thawed.
[0196] Sterile sucrose phosphate glutamate (SPG) diluent was
manufactured by BioWhittaker (Walkersville, Md.). Just prior to
manufacture of the liquid FluMist blend, about one-third of the
amount of SPG diluent required for the blend was added to a sterile
2-L bottle. The bottle was warmed to room temperature, then
hydrolyzed porcine gelatin (powder) was added in the amount need to
reach a level of 10 mg/ml in the final blend. The SPG diluent was
then passed through the filter (washing through any residual
gelatin) to reach the target volume specified in the blend
calculation and stored at 5.+-.3.degree. C. until use.
[0197] Thawed bottles were moved to a blend room and virus
aseptically transferred to a 5 L glass process vessel. The vessel
was kept at 5.+-.3.degree. C. throughout the blending process and
the subsequent filling process by using refrigerated cold packs.
The SPG-gelatin diluent was added after the three virus strains had
been added, and, if necessary, the pH was adjusted with HCl to
7.2.+-.0.3. The three virus strains and diluent were blended by
continuous mixing with a magnetic stir bar and stir plate.
[0198] Filling and Packaging
[0199] Following pH adjustment, the bulk trivalent blend vessel was
moved to a filling room and connected to an INOVA filler. The INOVA
filling machine filled a preset volume of product into a row of
eight BD HYPAK disposable sprayers and then stoppered the sprayers.
Sprayer filling and stoppering operations and weight check
verifications continued as directed. A new tub of sprayers was
manually placed at the in-feed stations upon completion of each
fill cycle and the tub of filled sprayers removed to the discharge
station. The blend vessel was maintained at 5.+-.3.degree. C. with
cold packs during the filling process.
[0200] Following filling of the nasal sprayers, the filled
trivalent vaccines were loaded into tubs and immediately
transported via cart to a packaging area for final packaging and
labeling. Packaged and labeled sprayers were stored at
5.+-.3.degree. C.
[0201] Optimization of Upstream Operating Parameters
[0202] Clarification of Harvest by Filtration (5 .mu.m)
[0203] Filtration vs. Low Speed Centrifugation: Clarification
potency losses using the standard low-speed centrifugation for
frozen FluMist are typically estimated at 0.2 to 0.3 log.sub.10
TCID.sub.50/mL. An estimated centrifugation loss for two strains
was: titer loss was negligible for one strain and 0.3 logs (59%
step yield) for a second strain using the standard conditions of
3400 g for 20 minutes. When a 5 micron depth filter was used as an
alternative to centrifugation during pre-CTM development runs, the
average clarification step yield was estimated at 41%. When
TCID.sub.50 assay variability was taken into consideration, it was
concluded that either method of clarification provided better
results than adding after clarification. Filtration was chosen as
the clarification method based on ease of operation and
scalability.
[0204] Comparison of Depth Filter Pore Sizes: Development lots
CAZ015-CAZ017 plus the ten CTM-1 lots CAZ025 through CAZ034 were
used to estimate depth filtration losses using the CTM-1 process.
A/Beijing, A/Sydney, and B/Harbin have clarification step yields of
147%, 78%, and 73% respectively for the 5 .mu.m clarification
step.
[0205] The 5 .mu.m (Pall Profile II) filter was compared to a 20
.mu.m depth filter (Pall Profile Star), which was used for lots
CAZ035 (A/Sydney), CAZ036 (A/Beijing) and CAZ037 (B/Harbin).
A/Beijing, A/Sydney, and B/Harbin had clarification step yields of
65%, 35%, and 209% respectively for the 20 .mu.m clarification
step. These yields were comparable to 5 .mu.m clarification
results, however a process change to the 20 .mu.m filter was not
recommended for all embodiments due to significant contamination of
clarified harvests by red blood cells when larger pore size was
used.
[0206] Optimization of Downstream Operating Parameters:
[0207] Centrifuge Scale
[0208] Centrifuge loading and temperature studies were performed
using the large-scale CP40Y centrifuge and RP40CT Type D
continuous-flow rotor; these compared various egg batch sizes and
centrifuge temperature set points (see FIG. 3). CA0Z15 (B/Harbin),
CAZ018 (A/Sydney), CAZ020 (A/Beijing) and CAZ022 (A/Sydney) had
batch sizes of 10K eggs and a centrifuge set temperature of
4.degree. C. CAZ 016 (B/Harbin), CAZ019 (A/Sydney), and CAZ021
(A/Beijing) had batch sizes of 20K eggs and a centrifuge set
temperature of 14.degree. C. A loading flow rate of 20 L/hr was
used for all of these studies. Percent recovery in the diluted
peak-fraction pool (step yield) was based on the clarified harvest
titer in the figure. As can be seen, lots with 10K egg batches had
a recovery range from 40-184% whereas the 20K egg batches ranged
from 40-55%. The average recovery for the 10K batch size was 113%
and the 20K egg batch recoveries averaged 46%. A batch size of
10,000 eggs per run was selected for CTM manufacturing as a
conservative limit in this step of the development process.
[0209] Temperature
[0210] Development runs CAZ015 through CAZ022 were performed using
ultracentrifuge rotor set point temperature either at 4.degree. C.
or 14.degree. C. With one exception, the development runs at
14.degree. C. were also run at larger scale than typical (20,000
eggs per batch). In general these batches had lower recoveries of
live virus, however this could be due to centrifuge loading rather
that temperature effects. In view of the better results at
4.degree. C. and the possibility of increased microbial growth
rates at higher temperatures, a temperature of 4.degree. C. was
selected as the ultracentrifuge temperature set point at this point
in the development of the process.
[0211] Clinical Trial 1 (CTM-1)
[0212] The monovalent virus lots for CTM-1 were produced and
included monovalent bulk lots CAZ025 through CAZ034. In-process QC
assay results for the CTM-1 monovalent bulk lots are presented in
FIG. 3 and herein. Average step yields per strain are presented in
FIG. 3 and herein. Results are summarized below.
[0213] Potency: The potencies (log.sub.10TCID.sub.50 mL) of
B/Harbin and A/Beijing virus harvest lots were equal or greater
than 8.1. A/Sydney ranged from 7.5 to 8.8 except for CAZ030, which
showed very low titer (6.0) because of yolk contamination in the
harvest. The titer of the monovalent bulk for A/Beijing was 9.3 or
above, for B/Harbin the titer ranged from 8.4 to 9.85 and for
A/Sydney ranged from 7.9 to 8.6 (excluding CAZ030). For CAZ030 the
titer was below the acceptable level for blending as discussed
below.
[0214] Process Yield: Process yields for each lot are referenced in
the Figures and discussed further herein. Process yields were not
corrected for sampling losses. A/Beijing had an average overall
process yield of 16% (163 doses/egg), A/Sydney had an average yield
of 13% (6 doses/egg), and B/Harbin had an average yield of 93% (86
doses/egg). See figures. The erratic yield estimates reflect assay
variability as well as the actual process performance. In
particular, the B/Harbin results were skewed upward by a very high
estimate of Lot CAZ028 monovalent bulk titer (resulting in
estimated 252% process yield and 246 doses/egg). The diluted peak
pool titer in this case was determined at 0.95 logs higher than the
centrifuge peak pool, though a decline would be expected due to
dilution. Likewise the filtered monovalent bulk titer was higher
than the prefiltered material, which can be accounted for by assay
variability. The other two B/Harbin lots, (CAZ026 and CAZ027) had
process yields of 20% and 8% respectively, and an egg yield of 7
doses/egg for both lots. The trivalent blends that included bulk
lot CAZ028 (CBF1004 and CBF1007) resulted in B/Harbin potencies in
final product sprayers that were 0.6 and 0.4 log.sub.10 TCID.sub.50
per dose. This further suggests that the CAZ028 monovalent bulk
titer was overestimated.
[0215] Bioburden and Endotoxin: Virus harvest, stabilized harvest
and diluted centrifuge peak fractions were tested for bioburden.
Bioburden test results do not give a clear picture of organism
loads through the process: test results on virus harvest and
diluted centrifuge peak fraction pool samples consisted primarily
of "none detected" (eight readings); or ">100 cfu/mL" (ten
readings) with only two intermediate values. Endotoxin values for
the monovalent bulks varied from <5 EU/mL to 587 EU/mL.
[0216] Sucrose Concentration: The centrifuge peak fraction pools
had sucrose concentrations ranging from 39 to 43.5% (CAZ030 was not
included in the range). The monovalent bulks had an average sucrose
concentration of 7.6%.
[0217] The monovalent bulk release assay results are presented in
the figures. All of the CTM-1 lots except for CAZ030 (which was not
tested) passed the bulk release assays.
[0218] SDS-PAGE Analysis: Monovalent bulks were further
characterized by 10% SDS-PAGE gels stained with Gel Code Blue.
Based on the calculated protein molecular weights and number of
copies per particle for influenza A and B strains, gel bands in the
region of 50-60 kDa (HA1protein), 56 kDa (NP protein) and 27 kDa
(M1protein) would be expected to be prominent in gels of purified
influenza virus. Ha2 protein (23 to 30 kD) may also be present.
[0219] SDS-PAGE gels for A/Beijing/262/95 CAZ031, and 33 and
A/Sydney/05/97 CAZ029, 30, and 34 were compared. The range of
resolution for the 10% gels was nominally 200-31 kDa. Because of
this limitation, interpretation of HA1 protein (23-30 kDa) and M
protein bands (21-27 kDA) should be made with caution.
[0220] For A/Beijing and A/Sydney monovalent bulk (MB) lots, a dark
band was observed in the expected region of NP protein (just above
the 55 kDa marker) for the A/Beijing samples, and a band was seen
just above 116 kDa for both strains. Both strains contained a band
near the gel front (below 31 kD) consistent with M protein. The HA1
band expected at or just above the 66 kD marker was less strong,
possibly due to heterogeneity in glycoprotein molecular weight
resulting in a more dispersed banding pattern. Staining of the
putative NP1 protein also seems to be diminished. For the A/Sydney
samples a wider variety of bands were observed, consistent with a
greater proportion of egg protein in the monovalent bulk.
[0221] Doses per Lot and Egg: The dose calculation for all three
strains was based on a 0.24 mL average fill volume containing
1.2E+07 TCID.sub.50 particles. Based on the dose calculation above,
the A/Beijing monovalent lots had an average of 1,576,022 total
doses per lot. Average total doses per lot for B/Harbin and
A/Sydney were 601,446 and 50,825 doses per lot respectively, or 38%
and 3.2% of the average A/Beijing total doses per lot respectively.
These results translate to the following values for monovalent
doses per egg: 163 doses/egg for A/Beijing, 86 doses/egg for
B/Harbin-like and 6 doses/egg for A/Sydney. The calculation for
doses/egg was based on the total number of eggs harvested to
manufacture the lot.
[0222] Summary of Egg Yields: A/Sydney had the highest average
harvest volume per egg yield, followed by A/Beijing and B/Harbin.
The yield per egg averaged 5.6 mL/egg for B/Harbin, 6.5 mL/egg for
A/Beijing, and 6.8 mL/egg for A/Sydney. The overall yield of viable
eggs for production averaged 82%. Egg yields were based on the
total number of eggs harvested compared to total number of eggs for
production. Percent eggs rejected pre-inoculation averaged 10%.
Percent rejection post-incubation averaged 5%, with the exception
of CAZ028, which had a 29% rejection rate.
[0223] Purification Yield Summary (TCID.sub.50): Clarified harvest
potency values were higher than the starting material for some of
the lots, resulting in step yields greater than 100%. Step yield
estimates are affected by the variability of the TCID.sub.50 assay,
which had a standard deviation above 0.3 log.sub.10 TCID.sub.50/mL
as performed at the time of the production runs. In addition, the
step yield at the Diluted Centrifuge Pak Fraction Pool step varies
widely for all three strains and is greater than 100% for all ten
CTM lots. This appears to be due to a systematic downward bias of
titers for Centrifuge Peak Fraction Pools (as suggested by the
>100 yield of the next step), thought to be related to the high
sucrose levels in these samples.
[0224] Comments on CTM-1 Lots
[0225] In the various embodiments and example herein it is
preferable that no yolk contamination take place of the harvest
fluids which could occur, e.g., with improper harvest machine
settings.
[0226] 0.2 micron filtration step: The batch record in effect
specified a Pall Kleenpak disposable filter, however this was not
used for any CTM lots. Lots CAZ025-030 used a cartridge filter
(AB1DFL7PH4--Pall hydrophilic PVDF Fluorodyne II filter) with
housing instead of the Kleenpak. The filters had similar materials
of construction (hydrophilic PVDF), however the filter areas were
5100 cm.sup.2 for the Fluorodyne II cartridge filter vs. 1500
cm.sup.2 for the Kleenpak Fluorodyne II filter. For lots
CAZ031/032/033/034 a disposable Pall NovaSip C3DFLP1 filter
assembly was used instead of the Kleenpak. The membrane surface
area for the C3DFLP1 filter was the same as the Kleenpak filter
(1500 cm.sup.2).
[0227] Temperature excursions: During the second incubation step
for CAZ031, the temperature rose to 34.4.degree. C. for 13 hours
and for CAZ032 the temperature dropped to 31.5.degree. C. for 4
hours. These lots reached normal harvest titers above 9.0 log10
TCID50/mL, so the excursions were not regarded as having impacted
the product.
[0228] Summary
[0229] Based on two Clinical Trial Manufacturing Campaigns (CTM-1
and CTM-2), the liquid FluMist process development and
manufacturing work indicates that the process is suitable to
produce clinical supplies that pass release specifications.
Aggregate data from both CTM runs and various development studies
leads to the following estimates of median process yield:
[0230] Clarification: 50% yield
[0231] Ultracentrifuge purification: 50% yield
[0232] Peak dilution and sterile filtration: 20 to 50% yield
EXAMPLE 2
Stability Testing
[0233] A trivalent vaccine formulation was prepared comprising
three different reassortant influenza viruses (7.0+/-0.5 log.sub.10
FFU/dose [approximately 7.0+/-0.5 log.sub.10TCID.sub.50/dose]) and
comprising 200 mM sucrose; 1% (w/v) porcine gelatin hydrolysate;
1.21% (w/v) arginine monohydrocloride [equivalent to 1% (w/v)
arginine base]; and 5 mM monosodium glutamate in 100 mM potassium
phosphate buffer (pH 7.2). The stability of the formulation was
stored at -25.0 degrees C+/-5.0 degrees for greater than or equal
to 24 hours, but less than or equal to two weeks and then stored at
2-8 degrees C. for various time periods. This formulation (e.g.,
lot 0141500003) was determined to be stable for at least 12 weeks
at 4-8 degree C. In particular, the potency for each strain of
virus remained within 0.5 log.sub.10 of the beginning potency prior
to 4-8 degree C. storage.
[0234] Other studies have shown that equivalent formulations
(comprising three different reassortant influenza viruses
(7.0+/-0.5 log.sub.10 FFU/dose [approximately 7.0+/-0.5 log.sub.10
TCID.sub.50/dose]) and comprising 200 mM sucrose; 1% (w/v) porcine
gelatin hydrolysate; 1.21% (w/v) arginine monohydrocloride
[equivalent to 1% (w/v) arginine base]; and 5 mM monosodium
glutamate in 100 mM potassium phosphate buffer, pH 7.2) of
different clinical lots (e.g., "Campaign 3") are stable for about
12-15 months at 5.0 (+/-3.0) degrees C.
[0235] Subsequent studies have shown that formulations comprising
only 200 mM sucrose; 1% (w/v) gelatin hydrolysate; 1.21% (w/v)
arginine monohydrocloride [equivalent to 1% (w/v) arginine base] in
100 mM potassium phosphate buffer, pH 7.2, but without glutamate,
had equivalent stability as the above formulations with
glutamate.
[0236] 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.
* * * * *