U.S. patent application number 11/855769 was filed with the patent office on 2008-11-20 for mdck cell lines supporting viral growth to high titers and bioreactor process using the same.
This patent application is currently assigned to MEDIMMUNE VACCINES, INC.. Invention is credited to Mridul Ghosh, Simon Sheng-Tsiung Hsu, Jonathan Liu, Luis Jorge Camilo Maranga, Richard Schwartz, Ajit Subramanian, Mark Thompson.
Application Number | 20080286850 11/855769 |
Document ID | / |
Family ID | 39721761 |
Filed Date | 2008-11-20 |
United States Patent
Application |
20080286850 |
Kind Code |
A1 |
Liu; Jonathan ; et
al. |
November 20, 2008 |
MDCK CELL LINES SUPPORTING VIRAL GROWTH TO HIGH TITERS AND
BIOREACTOR PROCESS USING THE SAME
Abstract
The present invention relates to novel MDCK cells which can be
to grow viruses, e.g., influenza viruses, in cell culture to higher
titer than previously possible. The MDCK cells can be adapted to
serum-free culture medium. The present invention further relates to
cell culture compositions comprising the MDCK cells and cultivation
methods for growing the MDCK cells. The present invention further
relates to methods for producing influenza viruses in cell culture
using the MDCK cells of the invention.
Inventors: |
Liu; Jonathan; (Milpitas,
CA) ; Schwartz; Richard; (San Mateo, CA) ;
Thompson; Mark; (Morgan Hill, CA) ; Maranga; Luis
Jorge Camilo; (Santa Clara, CA) ; Hsu; Simon
Sheng-Tsiung; (Palo Alto, CA) ; Ghosh; Mridul;
(San Jose, CA) ; Subramanian; Ajit; (Berkeley,
CA) |
Correspondence
Address: |
MEDIMMUNE, LLC;Jonathan Klein-Evans
ONE MEDIMMUNE WAY
GAITHERSBURG
MD
20878
US
|
Assignee: |
MEDIMMUNE VACCINES, INC.
Gaithersburg
MD
|
Family ID: |
39721761 |
Appl. No.: |
11/855769 |
Filed: |
September 14, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60845121 |
Sep 15, 2006 |
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60871721 |
Dec 22, 2006 |
|
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60917008 |
May 9, 2007 |
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60951813 |
Jul 25, 2007 |
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Current U.S.
Class: |
435/239 ;
435/350; 435/394 |
Current CPC
Class: |
C12N 2760/16152
20130101; C12N 2760/16151 20130101; C12N 7/00 20130101; C12N
2760/16251 20130101; C12N 2760/16252 20130101 |
Class at
Publication: |
435/239 ;
435/350; 435/394 |
International
Class: |
C12N 7/00 20060101
C12N007/00; C12N 5/06 20060101 C12N005/06 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] One or more inventions described herein were made with
Government support under Contract No. HHS0100200600010C awarded by
Health and Human Services. Accordingly, the Government may have
certain rights in such inventions.
Claims
1. A Madin-Darby Canine Kidney (MCDK) cell, wherein a cell culture
composition comprising a plurality of the MDCK cells supports
replication of a cold-adapted influenza virus to a base 10
logarithm of the median tissue culture infection dose per
milliliter (log.sub.10 TCID.sub.50/mL) of at least about 7.8 or to
a base 10 logarithm of fluorescent focus units per milliliter
(log.sub.10 FFU/mL) of at least about 7.8.
2. The MDCK cell of claim 1, wherein the cell culture composition
is serum free.
3. The MDCK cell of claim 1, wherein the MDCK cell is adherent.
4. The MDCK cell of claim 1, wherein the MDCK cell is
non-tumorigenic and/or non-oncogenic.
5. The MDCK cell of claim 1, wherein the MDCK cell is identified by
ATCC Accession No. PTA-7909 or PTA-7910
6. The MDCK cell of claim 1, wherein the influenza virus is also
attenuated and temperature sensitive.
7. The MDCK cell of claim 1, wherein the influenza virus comprises
one or more gene segments of influenza strain A/Ann Arbor/6/60 or
B/Ann Arbor/1/66.
8. A method for proliferating the MDCK cell of claim 1 to a cell
density of at least about 1.times.10.sup.6 cells/ml in a Single Use
Bioreactor (SUB) system comprising inoculating a serum free cell
culture medium with the MDCK cell of claim 1 at a seeding density
of between about 1.times.10.sup.4 to about 12.times.10.sup.4
cells/mL and culturing the cells while maintaining one or more
culture conditions selected from the group consisting of: a. an
agitation rate of between about 50 to 150 rpm; b. a pH of between
about 6.0 to about 7.5; c. dissolved oxygen (DO) between about 35%
to about 100%; and d. a temperature of between about 33.degree. C.
to about 42.degree. C.
9. The method of claim 8, wherein the cell culture medium is
selected from the group consisting of MediV-105, MediV-105
supplemented with glucose, M-32, M-32 supplemented with glucose,
MediV-107, MediV-107 supplemented with glucose.
10. The method of claim 8, wherein the MDCK cell is an adherent
MDCK cell and wherein a microcarrier is used for culturing the
cells.
11. A method for producing cold adapted influenza viruses to a
log.sub.10 TCID.sub.50/mL of at least about 7.8 in cell culture,
comprising: a. proliferating the MDCK cell of claim 1 to a cell
density of at least about 1.times.10.sup.6 cells/ml in a Single Use
Bioreactor (SUB) system comprising inoculating an animal protein
free cell culture medium with the MDCK cell of claim 1 at a seeding
density of between about 1.times.10.sup.4 to about
12.times.10.sup.4 cells/mL and culturing the cells while
maintaining one or more culture conditions selected from the group
consisting of an agitation rate of between about 50 to 150 rpm, a
pH of between about 6.0 to about 7.5, and dissolved oxygen (DO)
between about 35% to about 100%; b. infecting the proliferated MDCK
cells with an influenza virus; c. incubating the infected
proliferated MDCK cells under conditions that permit replication of
the influenza virus; and d. isolating influenza viruses from the
cell culture composition.
12. The method of claim 11, wherein fresh medium or additional
medium components are added to the cell culture during step
(a).
13. The method of claim 11, wherein none or some of the cell
culture medium is removed and replaced with fresh medium prior to
or during step (b)
14. The method of claim 11, wherein the cell culture medium is
selected from the group consisting of MediV-105, MediV-105
supplemented with glucose, M-32, M-32 supplemented with glucose,
MediV-107, MediV-107 supplemented with glucose.
15. The method of claim 11, wherein the MDCK cell is an adherent
MDCK cell and wherein a microcarrier is used for culturing the
cells.
16. The method of claim 11, wherein step (b) is carried out at a
Multiplicity Of Infection (MOI) of between about 0.00001 to about
0.003 FFU/cell.
17. The method of claim 11, wherein the conditions of step (c) are
selected from the group consisting of an agitation rate of between
about 50 to 150 rpm, a pH of between about 6.0 to about 7.5,
dissolved oxygen (DO) between about 35% to about 100%, and a
temperature of between about 30.degree. C. to about 35.degree.
C.
18. The method of claim 11, wherein the MDCK cell is identified by
ATCC Accession No. PTA-7909 or PTA-7910.
19. The method of claim 11, wherein the influenza virus is also
attenuated and temperature sensitive.
20. The method of claim 11, wherein the influenza virus comprises
one or more gene segments of influenza strain A/Ann Arbor/6/60 or
B/Ann Arbor/1/66.
21. A method of eliminating DNA contaminants from a viral
preparation comprising: a. passing the viral preparation over an
affinity chromatography media under conditions wherein the DNA
contaminants are not retained on the affinity chromatography media
and the virus present in the viral preparation are retained; b.
washing the affinity chromatography media to remove the DNA
contaminants; and c. eluting the virus present in the viral
preparation from the affinity chromatography media.
22. The method of claim 21, wherein between steps (a) and (b) a
non-specific endonuclease preparation is passed over the affinity
chromatography media.
23. The method of claim 21, wherein the viral preparation is an
influenza virus preparation.
24. The method of claim 23, wherein the influenza virus preparation
was prepared from mammalian cells.
25. The method of claim 21, wherein the affinity chromatography
media is Cellufine.TM. Sulfate resin, and the conditions used in
step (a) are 1.times.SP buffer at about pH 7.2, and the virus are
eluted in step (c) in 1.times.SP buffer containing about 1 M NaCl
at about pH 7.2.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn.
19(e) of the following U.S. Provisional Application Nos. 60/845,121
filed Sep. 15, 2006; 60/871,721 filed Dec. 22, 2006; 60/917,008
filed May 9, 2007; and 60/951,813 filed Jul. 25, 2007. The priority
applications are hereby incorporated by reference herein in their
entirety for all purposes.
FIELD OF THE INVENTION
[0003] The present invention relates to novel MDCK cells which can
be used to grow viruses, e.g., influenza viruses, particularly
cold-adapted, and/or temperature sensitive, and/or attenuated
influenza viruses, in cell culture to high titer. The MDCK cells
can be adapted to or genetically modified to grow in serum-free
culture medium. The present invention further relates to cell
culture compositions comprising the MDCK cells, cultivation methods
for growing the MDCK cells, and methods for identifying such cells.
The present invention further relates to methods for producing
influenza viruses in cell culture using the MDCK cells of the
invention. In particular the present invention relates to novel
bioreactor processes for growing adherent cells (e.g., MDCK cells)
which can be used to grow viruses, (e.g., influenza viruses,
particularly cold-adapted, and/or temperature sensitive, and/or
attenuated influenza viruses), in cell culture to high titer. The
bioreactor processes may utilize serum-free culture medium. The
present invention further relates to vaccine compositions generated
using the bioreactor processes of the invention.
BACKGROUND OF THE INVENTION
[0004] Vaccination is the most important public health measure for
preventing disease caused by annual epidemics of influenza. The
effective use of vaccines is dependent on being able to quickly
produce large quantities of vaccine material (e.g., virus) from a
stable and easy to cultivate source. The rapid development of
vaccines and their abundant availability is critical in combating
many human and animal diseases. Delays in producing vaccines and
shortfalls in their quantity can cause problems in addressing
outbreaks of disease. For example, recent studies suggest that
there is cause for concern regarding the long lead times required
to produce vaccines against pandemic influenza. See, for example,
Wood, J. M., 2001, Philos. Trans. R. Soc. Lond. B. Biol. Sci.,
356:1953. Accordingly, recent efforts to produce vaccines have
focused on growth of viruses for vaccines in cell culture.
[0005] Madin Darby Canine Kidney (MDCK) cells have been
traditionally used for the titration of influenza viruses (Zambon
M., in Textbook of Influenza, ed Nicholson, Webster and Hay, ch 22,
pg 291-313, Blackwell Science (1998)). These cells were established
in 1958 from the kidney of a normal male cocker spaniel. The ATCC
list the MDCK (CCL 34) line as having been deposited by S. Madin
and N. B. Darby. However, existing MDCK cell lines suffer from
several defects, including possible tumorigenicity, the requirement
for animal serum in cell culture, and low yields of influenza
viruses suitable for use in vaccines. Accordingly, there remains an
unmet need for MDCK cell lines, preferably non-tumorigenic MDCK
cell lines that can grow such influenza strains to high titer,
preferably, in serum free media. These and other unmet needs are
provided by the present invention.
SUMMARY OF THE INVENTION
[0006] The present invention provides MDCK cells which can support
the growth of influenza viruses, e.g., cold-adapted, and/or
temperature sensitive, and/or attenuated influenza viruses, to high
titer. The MDCK cells can grow in either serum containing or
serum-free media formulations including animal protein-free (APF)
formulations, but preferably grow in serum-free and/or APF media
formulations. Accordingly, in a first aspect, the invention
provides a Madin-Darby Canine Kidney (MCDK) cell, wherein a cell
culture composition comprising a plurality of the MDCK cells
supports replication of a cold-adapted, and/or temperature
sensitive, and/or attenuated influenza virus to a base 10 logarithm
of the median tissue culture infection dose per milliliter
(log.sub.10 TCID.sub.50/mL) of at least about 7.0. In some
embodiments, the MDCK cells of the invention are adherent. In other
embodiments, the MDCK cells of the invention are non-adherent
(e.g., capable of growth under non-adherent conditions). In some
embodiments, the MDCK cells of the invention are non-tumorigenic.
In some embodiments, the MDCK cells of the invention have an
epithelial morphology. In some embodiments, the MDCK cells of the
invention are adherent and have an epithelial morphology. In some
embodiments, the MDCK cells of the invention are adapted or
selected to grow under non-adherent conditions. In some
embodiments, the MDCK cells of the invention are adherent and
non-tumorigenic.
[0007] Viruses that can be grown in the MDCK cells of the invention
include but are not limited to negative strand RNA viruses,
including but not limited to influenza, RSV, parainfluenza viruses
1, 2 and 3, and human metapneumovirus, as well as other viruses,
including DNA viruses, retroviruses, positive strand RNA viruses,
negative strand RNA viruses, double-stranded RNA viruses,
including, but not limited to, papovavirus, vesicular stomatitis
virus, vaccinia virus, Coxsackie virus, reovirus, parvovirus,
adenovirus, poliomyeltitis virus, measles virus, rabies virus, and
herpes virus.
[0008] The present invention further provides methods and media
formulations useful for the derivation, propagation and maintenance
of MDCK cells that can support the growth of influenza viruses,
e.g., cold-adapted, and/or temperature sensitive, and/or attenuated
influenza viruses, to high titer. The MDCK cells of the invention
are particularly useful for the production of vaccine material such
as, for example, viruses. Accordingly, in another aspect, the
invention provides a cell culture composition comprising MDCK cells
and a cell culture medium, wherein the cell culture composition
supports replication of a cold-adapted, and/or temperature
sensitive, and/or attenuated influenza virus to a log.sub.10
TCID.sub.50/mL of at least about 7.0.
[0009] Other aspects of the invention include methods of producing
vaccine material (e.g., virus) by culturing any MDCK cell of the
invention, in a suitable culture medium under conditions permitting
production of vaccine material and, isolating the material from one
or more of the cell or the medium in which it is grown. Thus, in
some embodiments, the invention provides a method for producing
influenza viruses in cell culture, comprising infecting a cell
culture composition of the invention with an influenza virus,
incubating the cell culture composition under conditions that
permit replication of the influenza virus; and isolating influenza
viruses from the cell culture composition.
[0010] In another aspect, the invention provides immunogenic
compositions. For example, in some embodiments, the invention
provides immunogenic compositions comprising the vaccine material
produced as described above and, optionally, an excipient such as a
pharmaceutically acceptable excipient or one or more
pharmaceutically acceptable administration component.
[0011] Methods of producing immunogenic responses in a subject
through administration of an effective amount of one or more above
described immunogenic compositions to a subject are also within the
current invention. Additionally, methods of prophylactic or
therapeutic treatment of a viral infection (e.g., viral influenza)
in a subject through administration of one or more above described
immunogenic compositions in an amount effective to produce an
immunogenic response against the viral infection are also part of
the current invention. Subjects for such treatment can include
mammals (e.g., humans), avian species (e.g., poultry).
Additionally, such methods can also comprise administration of a
composition of one or more viruses produced in the MDCK cells of
the invention and a pharmaceutically acceptable excipient that is
administered to the subject in an amount effect to prophylactically
or therapeutically treat the viral infection.
[0012] These and other objects and features of the invention will
become more fully apparent when the following detailed description
is read in conjunction with the accompanying figures appendix.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 presents a graphical representation of reassortant
influenza virus strains comprising HA and NA gene segments from
wild type influenza virus strains A/Panama, A/New Calcdonia, or
B/Jilin yields in MDCK clones 1, 5, 36, 39, 40, and 55.
[0014] FIG. 2 presents a graphical representation of cell growth of
MDCK subclones 1-A, 1-B, and 1-C in MediV 105 serum free
medium.
[0015] FIG. 3 presents a graphical representation of yields of
reassortant influenza virus strains comprising HA and NA gene
segments from wild type influenza virus strains A/New
Calcdonia/20/99, A/Hiroshima/52/05, B/Malaysia/2506/04, or
A/Vietnam/1203/2004 and the remaining gene segments from a
cold-adapted, temperature sensitive, attenuated virus in MDCK
subclones 1-A, 1-B, and 1-C 3 and 4 days post infection (DPI).
[0016] FIG. 4 presents a table showing yields of reassortant
influenza virus strains comprising HA and NA gene segments from
wild type influenza virus strains A/New Calcdonia/20/99,
A/Hiroshima/52/05, B/Malaysia/2506/04, or A/Vietnam/1203/2004 and
the remaining gene segments from a cold-adapted, temperature
sensitive, attenuated virus in MDCK subclones 1-A, 1-B, 1-C, and
1-D 3 and 4 days post infection (DPI) in OptiPro.TM. media and in
MediV 105.
[0017] FIG. 5 presents the flow chart of MDCK Subclone 1-B serum
free cell bank preparation. Panel A presents the selection steps
performed in serum containing media. Panel B presents the steps for
adaptation to serum free media.
[0018] FIG. 6 presents the growth of subclone 1-A in MediV 105 and
M18M media.
[0019] FIG. 7 presents the doubling time of subclone 1-A in MediV
105 and M18M media.
[0020] FIG. 8 presents a comparison of the cell density of subclone
1-A in M18M media comprising four different microcarriers 30 and 60
minutes post-inoculation.
[0021] FIG. 9 presents a comparison of the cell yield of subclone
1-A in M18M media comprising four different microcarriers.
[0022] FIG. 10 outlines one cell culture scale up process which can
be utilized for commercial scale production of vaccine
material.
[0023] FIG. 11 outlines two purification processes which can be
utilized for commercial scale purification of vaccine material from
cell-culture.
[0024] FIG. 12 presents the results of Cellufine Sulfate (CS)
chromatography after or in combination with Benzonase treatment.
Panel A) The OD profile of column chromatography using Cellufine
Sulfate is shown in the left panel, arrows indicate the time the
load, wash and elution were started. Agarose gel electrophoresis
(right panel) show that the DNA contaminant is present in the
starting material (lane 2) and the flow through (lane 3) but is
absent in the material eluted from the column (lane 4), lane 1 is
molecular weight marker. Panel B) Depicts the scheme for MDCK dsDNA
Degradation Using Benzonase On-Column Treatment.
[0025] FIG. 13 presents several curves of the 30 L SUB process for
the production of B/Malaysia/2506/04 in MDCK subclone 1-B. Top
panel is the growth curve of the cells during the growth phase. The
metabolite profiles for glucose (middle panel, solid line), lactate
(middle panel, dotted line), glutamine (bottom panel, solid line)
and ammonium ion (bottom panel, dotted line) for this run were
measured by Bioprofile.
[0026] FIG. 14 presents the results of pilot studies without media
exchange for the SUB process. A) Plots of the viral titers obtained
for medium exchange ratios between 0% and 100% at 2 and 3 dpi (top
and bottom, respectively). B) Plots of the peak viral titer at 2
and 3 dpi for effective TrypLE concentrations of between 0.04 and
1. C) Plots of the viral titers over time for B/Malaysia/2506/04
(to) and A/Vietnam/1203/2004 (bottom) after infection with
(triangles) or without (squares) media exchange.
[0027] FIG. 15 plots the A/Solomon Islands/3/06 viral titer over
time (hours post infection) for different MOIs used. The viral
yields from 20 to 96 hours post infection are boxed and this area
of the plot is expanded to the right. The peak viral harvest of the
culture infected at 2000 FFU/mL is circled.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention is based in part on the discovery that
cloned MDCK cell lines can be obtained that support the replication
of influenza viruses, particularly cold-adapted, and/or temperature
sensitive, and/or attenuated influenza viruses, to high titer.
Thus, the present invention provides, in one aspect, MDCK cell
lines which have been adapted to a variety of cell culture
conditions, including serum-free media formulations, that can
support the replication of influenza viruses, e.g., cold-adapted,
and/or temperature sensitive, and/or attenuated influenza viruses,
to high titer and are referred to herein as "cells of the
invention".
[0029] In addition, the present invention provides cell culture
compositions comprising cells of the invention and other
components, which can include, but are not limited to, media (e.g.,
a media disclosed herein), media components, buffers, chemical
compounds, additional cell types, viral material (e.g., viral
genomes, viral particles) and heterologous proteins.
[0030] The present invention also provides methods and media
formulations useful for the cultivation of MDCK cells, with one or
more specific characteristics including but not limited to, being
non-tumorigenic (e.g., not forming nodules in a nude mouse) and/or
being non-oncogenic and/or growth as adherent cells and/or growth
as non-adherent cells and/or having an epithelial-like morphology
and/or supporting the replication of various viruses including but
not limited to orthomyxoviruses, paramyxoviruses, rhabdoviruses and
flavoviruses and/or supporting the growth of influenza viruses,
including cold-adapted, and/or temperature sensitive, and/or
attenuated influenza viruses, to high titer. The culture conditions
of the present invention include serum containing and serum-free
media formulations, as well as animal protein-free (APF)
formulations.
[0031] In addition, the present invention also provides methods of
producing vaccine material (e.g., influenza virus) in MDCK cells,
preparing vaccine material from MDCK cells, and methods of
preventing influenza infection utilizing vaccine materials produced
in MDCK cells. The cells of the invention are particularly useful
for the production of cold adapted/temperature sensitive/attenuated
(ca/ts/att) influenza strains (e.g., those in FluMist.RTM.) which
do not replicate as efficiently in other mammalian cell lines
(e.g., Vero, PerC6.RTM., HEK-293, MRC-5 and WI-38 cells).
Definitions
[0032] Tumorigenicity, as used herein, has the ordinary meaning
attributed to this term by one skilled in the art. Tumorigenicity
is, in one embodiment, determined by the adult nude mouse model
(e.g., Stiles et al., 1976, Cancer Res, 36:1353, and Example 5
below). Tumorigenicity may also be tested by other assays, for
example, by injection into a chick embryo and/or topical
application to the chorioallantois (Leighton et al., 1970, Cancer,
26:1024).
[0033] The term "recombinant" indicates that the material (e.g., a
nucleic acid or protein) has been artificially or synthetically
(non-naturally) altered by human intervention. 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, the virus is recombinant when it is
produced by the expression of a recombinant nucleic acid.
[0034] The term "reassortant," when referring to a virus, indicates
that the virus includes genetic and/or polypeptide components
derived from more than one parental viral strain or source. For
example, a 7:1 reassortant includes 7 viral genomic segments (or
gene segments) derived from a first parental virus, and a single
complementary viral genomic segment, e.g., encoding hemagglutinin
or neuraminidase, from a second parental virus. A 6:2 reassortant
includes 6 genomic segments, most commonly the 6 internal genes
from a first parental virus, and two complementary segments, e.g.,
hemagglutinin and neuraminidase, from a different parental
virus.
[0035] The term "about," as used herein, unless otherwise
indicated, refers to a value that is no more than 10% above or
below the value being modified by the term. For example, the term
"about 5 .mu.g/kg" means a range of from 4.5 .mu.g/kg to 5.5
.mu.g/kg. As another example, "about 1 hour" means a range of from
54 minutes to 66 minutes.
[0036] The terms "temperature sensitive," "cold adapted" and
"attenuated" are well known in the art. For example, the term
"temperature sensitive" ("ts") indicates that the virus exhibits a
100 fold or greater reduction in titer at a higher temperature,
e.g., 39.degree. C. relative to a lower temperature, e.g.,
33.degree. C. for influenza A strains, and that the virus exhibits
a 100 fold or greater reduction in titer at a higher temperature,
e.g., 37.degree. C. relative to a lower temperature, e.g.,
33.degree. C. for influenza B strains. For example, the term "cold
adapted" ("ca") indicates that the virus exhibits a higher growth
rate at a lower temperature, e.g., 25.degree. C. within 100 fold of
its growth at a higher temperature, e.g., 33.degree. C. For
example, the term "attenuated" ("att") indicates that the virus
replicates in the upper airways of ferrets but is not detectable in
lung tissues, and does not cause influenza-like illness in the
animal. It will be understood that viruses with intermediate
phenotypes, i.e., viruses exhibiting titer reductions less than 100
fold at 39.degree. C. (for A strain viruses) or 37.degree. C. (for
B strain viruses), exhibiting growth at 25.degree. C. that is more
than 100 fold than its growth at 33.degree. C. (e.g., within 200
fold, 500 fold, 1000 fold, 10,000 fold less), and/or exhibit
reduced growth in the lungs relative to growth in the upper airways
of ferrets (i.e., partially attenuated) and/or reduced influenza
like illness in the animal, are also useful viruses encompassed by
the invention. Growth indicates viral quantity as indicated by
titer, plaque size or morphology, particle density or other
measures known to those of skill in the art.
Cell Characteristics
[0037] The cells according to the invention are in one embodiment,
vertebrate cells. In another embodiment, the cells of the invention
are mammalian cells, e.g., from hamsters, cattle, monkeys or dogs,
in particular kidney cells or cell lines derived from these. In
still another embodiment, the cells of the invention are MDCK cells
(e.g., lineally related to ATCC CCL-34 MDCK) and are specifically
referred to herein as "MDCK cells of the invention" and are
encompassed by the term "cells of the invention". In a specific
embodiment, the cells of the invention are derived from ATCC CCL-34
MDCK. Cells of the invention may be derived from CCL-34 MDCK cells
by methods well known in the art. For example, the CCL-34 MDCK
cells may be first passaged a limited number of times in a serum
containing media (e.g., Dulbecco's Modified Eagle Medium (DMEM)+10%
Fetal Bovine Serum (FBS)+4 mM glutamine+4.5 g/L glucose, or other
media described herein) followed by cloning of individual cells and
characterization of the clones. Clones with superior biological and
physiological properties including, but not limited to, doubling
times, tumorigenicity profile and viral production, can be selected
for the generation of a master cell bank (MCB).
[0038] In a first aspect, the invention provides a Madin-Darby
Canine Kidney (MCDK) cell, wherein a cell culture composition
comprising a plurality of the MDCK cells supports replication of an
influenza virus. In a specific aspect, the MDCK cells support the
replication of an influenza virus having one or more of the
following characteristics: cold adapted, attenuated, and
temperature sensitive. In certain embodiments the ability of the
MDCK cells to support viral replication is determined by measuring
the yield of virus obtained from an infected cell culture (e.g.,
using a median tissue culture infectious dose (TCID.sub.50) assay
or fluorescent focus assay (FFA)). In certain embodiments, the MDCK
cells support replication of the influenza virus to a base 10
logarithm of the median tissue culture infection dose per
milliliter (log.sub.10 TCID.sub.50/mL) of at least about 7.0. In
certain embodiments, the MDCK cells support replication of the
influenza virus to a log.sub.10 TCID.sub.50/mL of at least about
7.2. In certain embodiments, the MDCK cells support replication of
the influenza virus to a log.sub.10 TCID.sub.50/mL of at least
about 7.4. In certain embodiments, the MDCK cells support
replication of the influenza virus to a log.sub.10 TCID.sub.50/mL
of at least about 7.6. In certain embodiments, the MDCK cells
support replication of the influenza virus to a log.sub.10
TCID.sub.50/mL of at least about 7.8. In certain embodiments, the
MDCK cells support replication of the influenza virus to a
log.sub.10 TCID.sub.50/mL of at least about 8.0. In certain
embodiments, the MDCK cells support replication of the influenza
virus to a log.sub.10 TCID.sub.50/mL of at least about 8.2. In
certain embodiments, the MDCK cells support replication of the
influenza virus to a log.sub.10 TCID.sub.50/mL of at least about
8.4. In certain embodiments, the MDCK cells support replication of
the influenza virus to a log.sub.10 TCID.sub.50/mL of at least
about 8.6. In certain embodiments, the MDCK cells support
replication of the influenza virus to a log.sub.10 TCID.sub.50/mL
of at least about 8.8. In certain embodiments, the MDCK cells
support replication of the influenza virus to a log.sub.10
TCID.sub.50/mL of at least about 9.0. Alternatively, or optionally,
viral yield can be quantified by determining the concentration of
virus present in a sample according to a fluorescent focus assay
(described as Example 6, and known in the art, see e.g., Stokes et
al., 1988, J Clin Microbiol. 26:1263-6 and U.S. Patent Publication
20040265987). The FFA values are often reported as log.sub.10
FFU/mL (fluorescent focus units/mL). Accordingly, in certain
embodiments the MDCK cells support replication of the influenza
virus to a base 10 logarithm of fluorescent focus units per
milliliter (log.sub.10 FFU/mL) of at least about 7.0, or to a
log.sub.10 FFU/mL of at least about 7.2, or to a log.sub.10 FFU/mL
of at least about 7.4, or to a log.sub.10 FFU/mL of at least about
7.6, or to a log.sub.10 FFU/mL of at least about 7.8, or to a
log.sub.10 FFU/mL of at least about 8.0, or to a log.sub.10 FFU/mL
of at least about 8.2, or to a log.sub.10 FFU/mL of at least about
8.4, or to a log.sub.10 FFU/mL of at least about 8.6, or to a
log.sub.10 FFU/mL of at least about 8.8, or to a log.sub.10 FFU/mL
of at least about 9.0.
[0039] In certain embodiments, the cells of the invention are
propagated in culture to generate a cell culture composition (also
referred to herein as "a cell culture composition of the
invention"). In one embodiment, a cell culture composition of the
invention comprises as the only host cell type MDCK cells of the
invention, wherein the cell culture composition supports
replication of an influenza virus having one or more of the
following characteristics: cold-adapted, attenuated, and
temperature sensitive to a log.sub.10 TCID.sub.50/mL and/or a
log.sub.10 FFU/mL of at least about 7.0, at least about 7.2, at
least about 7.4, at least about 7.6, at least about 7.8, at least
about 8.0, at least about 8.2, at least about 8.4, at least about
8.6, at least about 8.8, at least about 9.0, at least about 9.2, at
least about 9.4, at least about 9.6, at least about 9.8, at least
about 10.0, at least about 10.2, at least about 10.4, at least
about 10.6, at least about 10.8 or at least about 11.0.
[0040] In one aspect, the cells of the invention are adapted to
growth in a media of choice (e.g., a serum-free or APF media, such
as those described herein). Such adaptation may occur prior to,
concurrently with, or subsequent to the cloning of individual
cells. In certain embodiments, cells of the invention are adapted
to grow in MediV 101, MediV 102, MediV 103, MediV 104, MediV 105,
M-32, MediV 107, M18M or growth optimized derivatives thereof, as
described hereinafter. Accordingly, the cells of the invention can
be propagated in a media as disclosed herein to generate a cell
culture composition of the invention. In one embodiment, a cell
culture composition of the invention comprises as the only host
cell type MDCK cells of the invention, wherein the growth media is
a serum-free medium.
[0041] In a specific embodiment of the invention the cells are of
the cell lines including, but not limited to, those which have been
deposited with the American Type Culture Collection (10801
University Boulevard, Manassas, Va. 20110-2209) on Jan. 5, 2005 and
assigned ATCC Deposit Nos. PTA-6500, PTA-6501, PTA-6502, PTA-6503
and those subclones 1-A and 1-B, deposited on Oct. 5, 2006 and
assigned ATCC Deposit Nos. PTA-7909 and PTA-7910, respectively.
These deposits will be maintained under the terms of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure. In one
embodiment, the MDCK cells of the invention are used to generate a
cell bank useful for the preparation of vaccine material suitable
for approval by the U.S. Food and Drug Administration for human
use. In one embodiment, a cell culture composition of the invention
comprises as the only host cell type MDCK cells deposited as ATCC
Accession number PTA-6500, PTA-6501, PTA-6502, PTA-6503, PTA-7909,
or PTA-7910. In a specific embodiment, a cell culture composition
of the invention comprises as the only host cell type MDCK cells
deposited as ATCC Accession number PTA-7909. In another specific
embodiment, a cell culture composition of the invention comprises
as the only host cell type MDCK cells deposited as ATCC Accession
number PTA-7910.
[0042] In some embodiments, the invention provides MDCK cell lines
derived from the cell line MDCK (CCL 34) by passaging and selection
with respect to one or more specific characteristics including but
not limited to, growing as adherent cells either in serum
containing, or serum-free media or animal protein-free media,
growing as non-adherent cells either in serum containing, or
serum-free media or animal protein-free media, having an
epithelial-like morphology, being non-tumorigenic (e.g., not
forming nodules in a nude mouse), and/or being non-oncogenic,
and/or supporting the replication of various viruses including but
not limited to orthomyxoviruses, paramyxoviruses, rhabdoviruses and
flavoviruses.
[0043] In one embodiment, the MDCK cells of the invention are
non-tumorigenic. In another embodiment, a cell culture composition
of the invention comprises as the only host cell type MDCK cells of
the invention, wherein the MDCK cells of the invention are
non-tumorigenic. Methods for determining if cells are tumorigenic
are well known in the art (see, for example, Leighton et al., 1970,
Cancer, 26:1024 and Stiles et al., 1976, Cancer Res, 36:1353), the
method currently preferred by the U.S. Food and Drug Administration
uses the nude mouse model detailed in Section 9.7 below. In a
specific embodiment, the MDCK cells of the invention are
non-tumorigenic in the adult nude mouse model (see, Stiles et al.,
Id and Section 9.7 below). In another specific embodiment, the MDCK
cells of the invention are non-tumorigenic when injected into a
chick embryo and/or topically applied to the chorioallantois (see,
Leighton et al., Id). In still another embodiment, the MDCK cells
of the invention are non-tumorigenic in the adult nude mouse model
but not when injected into a chick embryo and/or topically applied
to the chorioallantois. In yet another embodiment, the MDCK cells
of the invention are non-tumorigenic in the adult nude mouse model
and when injected into a chick embryo and/or topically applied to
the chorioallantois. In still another embodiment, the MDCK cells of
the invention are non-tumorigenic after at least 20 passages, or
after at least 30 passages, or after at least 40 passages, or after
at least 50 passages, or after at least 60 passages, or after at
least 70 passages, or after at least 80 passages, or after at least
90 passages, or after at least 100 passages in a medium. In yet
another specific embodiment the medium is a media described herein
(e.g., Medi 105).
[0044] Tumorigenicity may be quantified in numerous ways known to
one of skill in the art. One method commonly utilized is to
determine the "TD.sub.50" value which is defined as the number of
cells required to induce tumors in 50% of the animals tested (see,
e.g., Hill R. The TD.sub.50 assay for tumor cells. In: Potten C,
Hendry J, editors. Cell clones. London: Churchill Livingstone;
1985. p. 223). In one embodiment, the MDCK cells of the invention
have a TD.sub.50 value of between about 10.sup.10 to about
10.sup.1, or between about 10.sup.8 to about 10.sup.3, or between
about 10.sup.7 to about 10.sup.4. In a specific embodiment, the
MDCK cells of the invention have a TD.sub.50 value of more than
about 10.sup.10, or of more than about 10.sup.9, or of more than
about 10.sup.8, or of more than about 10.sup.7, or of more than
about 10.sup.6, or of more than about 10.sup.5, or of more than
about 10.sup.4, or of more than about 10.sup.3, or of more than
about 10.sup.2, or of more than about 10.sup.1.
[0045] In one embodiment, the MDCK cells of the invention are
non-oncogenic. In another embodiment, a cell culture composition of
the invention comprises as the only host cell type MDCK cells of
the invention, wherein the MDCK cells are non-oncogenic. Methods
for determining if cells are oncogenic are well known in the art
and generally involve the inoculation of cell lysates and/or DNA
into newborn rodent species and evaluation of any tumor formation
over time (see, for example, Nowinski and Hays, 1978, J. Virol.,
27: 13-8; Peeper, et al., 2002, Nat Cell Biol., 4:148-53; Code of
Federal Regulation (CFR), "Oncogenicity", Title 40, Vol. 8, Chapter
1, section 798.330, pp. 160-164). For example, cell lysates and/or
DNA from at least 10.sup.7 cell equivalents are injected into
newborn rodents (e.g., hamster, nude mice, rats) typically less
then 4 days old which are then monitored for up to five months or
more. Oncogenicity assays are routinely performed by commercial
testing companies (e.g., BioReliance, see Protocols #001031 and
#001030). In one embodiment, cell lysates and/or DNA from at least
10.sup.5, or at least 10.sup.6, or at least 10.sup.7 MDCK cells of
the invention do not induce tumor formation in 2 months, or in 3
months, or in 4 month, or in 5 months, or in 6 months, or longer,
when injected into a newborn rodent species. In another embodiment,
0.01 mg, or 0.02 mg, or 0.03 mg, or 0.04 mg, or 0.05 mg, or 0.06
mg, or 0.07 mg, or 0.08 mg, or 0.09 mg, or 0.10 mg, or more, DNA
from an MDCK cell of the invention does not induce tumor formation
in 2 months, or in 3 months, or in 4 month, or in 5 months, or in 6
months, or longer, when injected into a newborn rodent species.
[0046] In another embodiment, the cells of the invention grow as
adherent cells either in serum-containing or serum-free media or
animal protein-free media. In yet another embodiment, the cells of
the invention grow as non-adherent cells (e.g., capable of growth
under non-adherent conditions) either in serum containing or
serum-free media or animal protein-free media. In still another
embodiments, the cells of the invention have an epithelial-like
morphology. In yet another embodiment, the MDCK cells of the
invention support the replication of various viruses including but
not limited to orthomyxoviruses, paramyxoviruses, rhabdoviruses and
flavoviruses. It is contemplated that the MDCK cells of the
invention may have any combination of one or more specific
characteristics including but not limited to, being
non-tumorigenic, being non-oncogenic, growing as adherent cells,
growing as non-adherent cells, having an epithelial-like
morphology, supporting the replication of various viruses, and
supporting the growth of influenza viruses to high titer, e.g., a
log.sub.10 TCID.sub.50/mL of at least about 7.0, at least about
7.2, at least about 7.4, at least about 7.6, at least about 7.8, at
least about 8.0, at least about 8.2, at least about 8.4, at least
about 8.6, at least about 8.8, at least about 9.0, at least about
9.2, at least about 9.4, at least about 9.6, at least about 9.8, at
least about 10.0, at least about 10.2, at least about 10.4, at
least about 10.6, at least about 10.8 or at least about 11.0 and/or
a log.sub.10 FFU/mL of at least about 7.0, at least about 7.2, at
least about 7.4, at least about 7.6, at least about 7.8, at least
about 8.0, at least about 8.2, at least about 8.4, at least about
8.6, at least about 8.8, at least about 9.0, at least about 9.2, at
least about 9.4, at least about 9.6, at least about 9.8, at least
about 10.0, at least about 10.2, at least about 10.4, at least
about 10.6, at least about 10.8 or at least about 11.0. In certain
embodiments, a cell culture composition of the invention comprises
as the only host cell type MDCK cells of the invention, wherein the
MDCK cells of the invention have any combination of one or more
specific characteristics including but not limited to, being
non-tumorigenic, being non-oncogenic, growing as adherent cells,
growing as non-adherent cells, having an epithelial-like
morphology, supporting the replication of various viruses, and
supporting the growth of influenza viruses to high titer (e.g.,
log.sub.10 TCID.sub.50/mL and/or a log.sub.10 FFU/mL of at least
about 7.8).
[0047] It is contemplated that each and every passage of the MDCK
cells of the invention can be documented in sufficient detail such
that the complete lineage of each cell line is available. The
documentation of each and every passage may facilitate approval by
the U.S. Food and Drug Administration and other regulatory bodies
around the world for the use of the MDCK cells of the invention for
the preparation of vaccine material.
[0048] In another embodiment, the MDCK cells of the invention are
free of microbial contaminants (e.g., bacterial, viral and fungal
contaminants). Methods for testing for the presence of bacterial
and fungal contaminants are well known in the art and routinely
performed by commercial contractors (e.g., BioReliance.RTM.,
Rockville, Md.). Accepted microbial sterility and mycoplasma tests
are detailed in Section 9.7 below. Specific examples of microbial
agents which may be tested for are listed in Table 4.
[0049] In yet another embodiment, the MDCK cells of the invention
support the replication of viruses including but not limited to
orthomyxoviruses (including influenza A and/or B strains),
paramyxoviruses (including RSV A and/or B, human metapneumovirus
and parainfluenza 1, 2 and/or 3), rhabdoviruses and
flavoviruses.
[0050] In a specific embodiment, the MDCK cells of the invention
support the replication of cold adapted/temperature sensitive
(ca/ts) influenza viruses such as those found, for example, in
FluMist.RTM. (Belshe et al, 1998, N Engl J Med 338:1405; Nichol et
al., 1999, JAMA 282:137; Jackson et al., 1999, Vaccine, 17:1905)
and/or reassortant viruses comprising the backbone (e.g., the
remaining gene segments) of these viruses or comprising the
backbone (or one or more vRNA segment(s)) of influenza viruses
having one or more of the following characteristics: cold adapted,
attenuated, and temperature sensitive. One indication of the
ability of a cell to support viral replication is the yield of
virus obtained from an infected cell culture. Viral yield can be
determined by numerous methods known to one skilled in the art. For
example, viral yield can be quantified by determining the
concentration of virus present in a sample according to a median
tissue culture infectious dose (TCID.sub.50) assay that measures
infectious virions or fluorescent focus assay (FFA). The
TCID.sub.50 values are often reported as the log.sub.10
TCID.sub.50/mL and the FFA values are often reported as log.sub.10
FFU/mL (fluorescent focus units/mL).
[0051] In one embodiment, the MDCK cells of the invention support
the replication of influenza viruses (e.g., ca/ts strains) to a
log.sub.10 TCID.sub.50/mL of at least 6.0, or at least 6.2, or at
least 6.4, or at least 6.6, or at least 6.8, or at least 7.0, or at
least 7.2, or at least 7.4, or at least 7.6, or at least 7.8, or at
least 8.0, or at least 8.2, or at least 8.4, or at least 8.6, or at
least 8.8, or at least 9.0, or at least 9.2, or at least 9.4, or at
least 9.6, or at least 9.8. In another embodiment, the MDCK cells
of the invention support the replication of influenza viruses
(e.g., ca/ts strains) to a log.sub.10 TCID.sub.50/mL of at least
about 6.0, or at least about 6.2, or at least about 6.4, or at
least about 6.6, or at least about 6.8, or at least about 7.0, or
at least about 7.2, or at least about 7.4, or at least about 7.6,
or at least about 7.8, or at least about 8.0, or at least about
8.2, or at least about 8.4, or at least about 8.6, or at least
about 8.8, or at least about 9.0, or at least about 9.2, or at
least about 9.4, or at least about 9.6, or at least about 9.8. In
still another embodiment, the MDCK cells of the invention support
the replication of influenza viruses (e.g., ca/ts strains) to a
log.sub.10 FFU/mL of at least 6.0, or at least 6.2, or at least
6.4, or at least 6.6, or at least 6.8, or at least 7.0, or at least
7.2, or at least 7.4, or at least 7.6, or at least 7.8, or at least
8.0, or at least 8.2, or at least 8.4, or at least 8.6, or at least
8.8, or at least 9.0, or at least 9.2, or at least 9.4, or at least
9.6, or at least 9.8. In yet another embodiment, the MDCK cells of
the invention support the replication of influenza viruses (e.g.,
ca/ts strains) to a log.sub.10 FFU/mL of at least about 6.0, or at
least about 6.2, or at least about 6.4, or at least about 6.6, or
at least about 6.8, or at least about 7.0, or at least about 7.2,
or at least about 7.4, or at least about 7.6, or at least about
7.8, or at least about 8.0, or at least about 8.2, or at least
about 8.4, or at least about 8.6, or at least about 8.8, or at
least about 9.0, or at least about 9.2, or at least about 9.4, or
at least about 9.6, or at least about 9.8.
[0052] It is well known in the art that the wild-type viruses used
in preparation of the vaccine strains for annual vaccination
against epidemic influenza are recommended annually by the Vaccines
and Related Biological Products Advisory Committee to the Centers
for Biologics Evaluation and Research (CBER) or the World Health
Organization (WHO) and the European Medicines Evaluation Agency
(EMEA), and are provided to manufacturers by the FDA or the Centers
for Disease Control and Prevention (CDC). These strains may then
used for the production of reassortant vaccine strains which
generally combine the NA and/or HA genes of the wild-type viruses
with the remaining gene segments derived from a donor virus (often
referred to as a master donor virus or MDV) which will have certain
desirable characteristics. For example, an MDV strain may be
cold-adapted, and/or temperature sensitive, and/or attenuated,
and/or have a high growth rate. The embodiments that follow
immediately below relate to cold-adapted, and/or temperature
sensitive, and/or attenuated versions of different influenza
strains (e.g., wild type strains recommended by one or more health
organization). As one skilled in the art is aware, such
cold-adapted, and/or temperature sensitive, and/or attenuated
influenza viruses can be readily made by obtaining recombinant
and/or reassortant influenza viruses that comprise the HA and NA
gene segments from the strain of interest and the remaining gene
segments from a suitable cold-adapted, and/or temperature
sensitive, and/or attenuated influenza strain (also referred to
herein as a "cold-adapted, temperature sensitive, attenuated
backbone") such as, for example, the cold-adapted, temperature
sensitive, attenuated influenza viruses found in FluMist.RTM., as
well as strain A/Ann Arbor/6/60 or B/Ann Arbor/1/66. As used herein
a recombinant and/or reassortant virus that comprises HA and NA
gene segments from a wild type influenza virus strain and the
remaining gene segments from cold-adapted, temperature sensitive,
attenuated influenza virus are also referred to by the wild type
strain designation preceded by the identifier "ca", for example a
recombinant and/or reassortant virus that comprises HA and NA gene
segments from A/New Calcdonia/20/99 and the remaining segments from
a cold-adapted, temperature sensitive, attenuated influenza virus
may be designated "ca A/New Calcdonia/20/99." In some embodiments,
the reassortant influenza virus comprises at least one gene segment
from A/Ann Arbor/6/60, B/Ann Arbor/1/66, A/Leningrad/134/47/57,
B/Leningrad/14/17/55 or A/Puerto Rico/8/34.
[0053] In certain embodiments, the MDCK cells of the invention
support the replication of a cold-adapted, and/or temperature
sensitive, and/or attenuated version (e.g., reassortant) of at
least one influenza strain (e.g., an influenza A strain, an
influenza B strain) recommended and/or provided annually by one or
more health organization including, but not limited to, the CBER,
the WHO, the EMEA, the FDA and the CDC, to a log.sub.10
TCID.sub.50/mL and/or a logo FFU/mL of at least 6.0, or at least
6.2, or at least 6.4, or at least 6.6, or at least 6.8, or at least
7.0, or at least 7.2, or at least 7.4, or at least 7.6, or at least
7.8, or at least 8.0, or at least 8.2, or at least 8.4, or at least
8.6, or at least 8.8, or at least 9.0, or at least 9.2, or at least
9.4, or at least 9.6, or at least 9.8. In one embodiment, a cell
culture composition of the invention comprises as the only host
cell type MDCK cells of the invention, wherein the cell culture
composition supports replication of a cold-adapted, and/or
temperature sensitive, and/or attenuated version (e.g.,
reassortant) of at least one influenza strain (e.g., an influenza A
strain, an influenza B strain) recommended and/or provided annually
by one or more health organization including, but not limited to,
the CBER, the WHO, the EMEA, the FDA and the CDC, to a log.sub.10
TCID.sub.50/mL and/or a log.sub.10 FFU/mL of at least 6.0, or at
least 6.2, or at least 6.4, or at least 6.6, or at least 6.8, or at
least 7.0, or at least 7.2, or at least 7.4, or at least 7.6, or at
least 7.8, or at least 8.0, or at least 8.2, or at least 8.4, or at
least 8.6, or at least 8.8, or at least 9.0, or at least 9.2, or at
least 9.4, or at least 9.6, or at least 9.8
[0054] In certain other embodiments, the MDCK cells of the
invention support the replication of a cold-adapted, and/or
temperature sensitive, and/or attenuated version of at least one
influenza A strain to a log.sub.10 TCID.sub.50/mL and/or a
log.sub.10 FFU/mL of at least 6.0, or at least 6.2, or at least
6.4, or at least 6.6, or at least 6.8, or at least 7.0, or at least
7.2, or at least 7.4, or at least 7.6, or at least 7.8, or at least
8.0, or at least 8.2, or at least 8.4, or at least 8.6, or at least
8.8, or at least 9.0, or at least 9.2, or at least 9.4, or at least
9.6, or at least 9.8. In one embodiment, a cell culture composition
of the invention comprises as the only host cell type MDCK cells of
the invention, wherein the cell culture composition supports
replication of a cold-adapted, and/or temperature sensitive, and/or
attenuated version of at least one influenza A strain to a
log.sub.10 TCID.sub.50/mL and/or a log.sub.10 FFU/mL of at least
6.0, or at least 6.2, or at least 6.4, or at least 6.6, or at least
6.8, or at least 7.0, or at least 7.2, or at least 7.4, or at least
7.6, or at least 7.8, or at least 8.0, or at least 8.2, or at least
8.4, or at least 8.6, or at least 8.8, or at least 9.0, or at least
9.2, or at least 9.4, or at least 9.6, or at least 9.8. It is
contemplated that the influenza A strain may be of any subtype
(e.g., H.sub.1N.sub.1, H.sub.3N.sub.2, H.sub.7N.sub.7,
H.sub.5N.sub.1, H.sub.9N.sub.2, H.sub.1N.sub.2, H.sub.2N.sub.2).
Presently at least 16 different HA and 9 different NA subtypes have
been identified in influenza A viruses. Accordingly, the influenza
A strain may comprise any combination of HA and NA subtypes
currently known or identified in the future.
[0055] In certain other embodiments, the MDCK cells of the
invention support the replication of a cold-adapted, and/or
temperature sensitive, and/or attenuated version of at least one
influenza B strain to a log.sub.10 TCID.sub.50/mL and/or a
log.sub.10 FFU/mL of at least 6.0, or at least 6.2, or at least
6.4, or at least 6.6, or at least 6.8, or at least 7.0, or at least
7.2, or at least 7.4, or at least 7.6, or at least 7.8, or at least
8.0, or at least 8.2, or at least 8.4, or at least 8.6, or at least
8.8, or at least 9.0, or at least 9.2, or at least 9.4, or at least
9.6, or at least 9.8. In one embodiment, a cell culture composition
of the invention comprises as the only host cell type MDCK cells of
the invention, wherein the cell culture composition supports
replication of a cold-adapted, and/or temperature sensitive, and/or
attenuated version of at least one influenza B strain to a
log.sub.10 TCID.sub.50/mL and/or a log.sub.10 FFU/mL of at least
6.0, or at least 6.2, or at least 6.4, or at least 6.6, or at least
6.8, or at least 7.0, or at least 7.2, or at least 7.4, or at least
7.6, or at least 7.8, or at least 8.0, or at least 8.2, or at least
8.4, or at least 8.6, or at least 8.8, or at least 9.0, or at least
9.2, or at least 9.4, or at least 9.6, or at least 9.8. Influenza B
viruses are not currently divided into subtypes based upon their
hemagglutinin and neuraminidase proteins, rather they are
classified by lineage. Presently, influenza B virus strains are
divided into two lineages, the B/Yamagata and the B/Victoria
lineages of which there are numerous sublineages. Accordingly, the
influenza B strain may be derived from any lineage and/or
sublineage currently known or identified in the future.
[0056] In certain embodiments, the MDCK cells of the invention
support the replication of a cold-adapted, and/or temperature
sensitive, and/or attenuated version of influenza strain A/New
Calcdonia (i.e., ca A/New Calcdonia) to a log.sub.10 TCID.sub.50/mL
and/or a logo FFU/mL of at least 6.0, or at least 6.2, or at least
6.4, or at least 6.6, or at least 6.8, or at least 7.0, or at least
7.2, or at least 7.4, or at least 7.6, or at least 7.8, or at least
8.0, or at least 8.2, or at least 8.4, or at least 8.6, or at least
8.8, or at least 9.0, or at least 9.2, or at least 9.4, or at least
9.6, or at least 9.8. In certain embodiments, the MDCK cells of the
invention support the replication of a cold-adapted, and/or
temperature sensitive, and/or attenuated version of influenza
strain A/Hiroshima (i.e., ca A/Hiroshima) to a log.sub.10
TCID.sub.50/mL and/or a logo FFU/mL of at least 6.0, or at least
6.2, or at least 6.4, or at least 6.6, or at least 6.8, or at least
7.0, or at least 7.2, or at least 7.4, or at least 7.6, or at least
7.8, or at least 8.0, or at least 8.2, or at least 8.4, or at least
8.6, or at least 8.8, or at least 9.0, or at least 9.2, or at least
9.4, or at least 9.6, or at least 9.8. In certain embodiments, the
MDCK cells of the invention support the replication of a
cold-adapted, and/or temperature sensitive, and/or attenuated
version of influenza strain B/Malaysia (i.e., ca B/Malaysia) to a
log.sub.10 TCID.sub.50/mL and/or a logo FFU/mL of at least 6.0, or
at least 6.2, or at least 6.4, or at least 6.6, or at least 6.8, or
at least 7.0, or at least 7.2, or at least 7.4, or at least 7.6, or
at least 7.8, or at least 8.0, or at least 8.2, or at least 8.4, or
at least 8.6, or at least 8.8, or at least 9.0, or at least 9.2, or
at least 9.4, or at least 9.6, or at least 9.8. In certain
embodiments, the MDCK cells of the invention support the
replication of a cold-adapted, and/or temperature sensitive, and/or
attenuated version of influenza strain A/Vietnam (i.e., ca
A/Vietnam) to a log.sub.10 TCID.sub.50/mL and/or a logo FFU/mL of
at least 6.0, or at least 6.2, or at least 6.4, or at least 6.6, or
at least 6.8, or at least 7.0, or at least 7.2, or at least 7.4, or
at least 7.6, or at least 7.8, or at least 8.0, or at least 8.2, or
at least 8.4, or at least 8.6, or at least 8.8, or at least 9.0, or
at least 9.2, or at least 9.4, or at least 9.6, or at least 9.8. In
certain embodiments, the MDCK cells of the invention support the
replication of a cold-adapted, and/or temperature sensitive, and/or
attenuated version of influenza strain A/Wisconsin (i.e., ca
A/Wisconsin) to a log.sub.10 TCID.sub.50/mL and/or a logo FFU/mL of
at least 6.0, or at least 6.2, or at least 6.4, or at least 6.6, or
at least 6.8, or at least 7.0, or at least 7.2, or at least 7.4, or
at least 7.6, or at least 7.8, or at least 8.0, or at least 8.2, or
at least 8.4, or at least 8.6, or at least 8.8, or at least 9.0, or
at least 9.2, or at least 9.4, or at least 9.6, or at least
9.8.
[0057] In certain embodiments, the MDCK cells of the invention
support the replication of a cold-adapted, and/or temperature
sensitive, and/or attenuated version of each of influenza strains
A/New Calcdonia and A/Hiroshima to a log.sub.10 TCID.sub.50/mL
and/or a log.sub.10 FFU/mL of at least 6.0, or at least 6.2, or at
least 6.4, or at least 6.6, or at least 6.8, or at least 7.0, or at
least 7.2, or at least 7.4, or at least 7.6, or at least 7.8, or at
least 8.0, or at least 8.2, or at least 8.4, or at least 8.6, or at
least 8.8, or at least 9.0, or at least 9.2, or at least 9.4, or at
least 9.6, or at least 9.8. In certain embodiments, the MDCK cells
of the invention support the replication of a cold-adapted, and/or
temperature sensitive, and/or attenuated version of each of
influenza strains A/New Calcdonia and B/Malaysia to a log.sub.10
TCID.sub.50/mL and/or a logo FFU/mL of at least 6.0, or at least
6.2, or at least 6.4, or at least 6.6, or at least 6.8, or at least
7.0, or at least 7.2, or at least 7.4, or at least 7.6, or at least
7.8, or at least 8.0, or at least 8.2, or at least 8.4, or at least
8.6, or at least 8.8, or at least 9.0, or at least 9.2, or at least
9.4, or at least 9.6, or at least 9.8. In certain embodiments, the
MDCK cells of the invention support the replication of a
cold-adapted, and/or temperature sensitive, and/or attenuated
version of each of influenza strains A/New Calcdonia and A/Vietnam
to a log.sub.10 TCID.sub.50/mL and/or a logo FFU/mL of at least
6.0, or at least 6.2, or at least 6.4, or at least 6.6, or at least
6.8, or at least 7.0, or at least 7.2, or at least 7.4, or at least
7.6, or at least 7.8, or at least 8.0, or at least 8.2, or at least
8.4, or at least 8.6, or at least 8.8, or at least 9.0, or at least
9.2, or at least 9.4, or at least 9.6, or at least 9.8.
[0058] In certain embodiments, the MDCK cells of the invention
support the replication of a cold-adapted, and/or temperature
sensitive, and/or attenuated version of each of influenza strains
A/Hiroshima and B/Malaysia to a log.sub.10 TCID.sub.50/mL and/or a
log.sub.10 FFU/mL of at least 6.0, or at least 6.2, or at least
6.4, or at least 6.6, or at least 6.8, or at least 7.0, or at least
7.2, or at least 7.4, or at least 7.6, or at least 7.8, or at least
8.0, or at least 8.2, or at least 8.4, or at least 8.6, or at least
8.8, or at least 9.0, or at least 9.2, or at least 9.4, or at least
9.6, or at least 9.8. In certain embodiments, the MDCK cells of the
invention support the replication of a cold-adapted, and/or
temperature sensitive, and/or attenuated version of each of
influenza strains A/Hiroshima and A/Vietnam to a log.sub.10
TCID.sub.50/mL and/or a log.sub.10 FFU/mL of at least 6.0, or at
least 6.2, or at least 6.4, or at least 6.6, or at least 6.8, or at
least 7.0, or at least 7.2, or at least 7.4, or at least 7.6, or at
least 7.8, or at least 8.0, or at least 8.2, or at least 8.4, or at
least 8.6, or at least 8.8, or at least 9.0, or at least 9.2, or at
least 9.4, or at least 9.6, or at least 9.8. In certain
embodiments, the MDCK cells of the invention support the
replication of a cold-adapted, and/or temperature sensitive, and/or
attenuated version of each of influenza strains B/Malaysia and
A/Vietnam to a log.sub.10 TCID.sub.50/mL and/or a log.sub.10 FFU/mL
of at least 6.0, or at least 6.2, or at least 6.4, or at least 6.6,
or at least 6.8, or at least 7.0, or at least 7.2, or at least 7.4,
or at least 7.6, or at least 7.8, or at least 8.0, or at least 8.2,
or at least 8.4, or at least 8.6, or at least 8.8, or at least 9.0,
or at least 9.2, or at least 9.4, or at least 9.6, or at least
9.8.
[0059] In certain embodiments, the MDCK cells of the invention
support the replication of a cold-adapted, and/or temperature
sensitive, and/or attenuated version of each of influenza strains
A/New Calcdonia, A/Hiroshima and B/Malaysia to a log.sub.10
TCID.sub.50/mL and/or a logo FFU/mL of at least 6.0, or at least
6.2, or at least 6.4, or at least 6.6, or at least 6.8, or at least
7.0, or at least 7.2, or at least 7.4, or at least 7.6, or at least
7.8, or at least 8.0, or at least 8.2, or at least 8.4, or at least
8.6, or at least 8.8, or at least 9.0, or at least 9.2, or at least
9.4, or at least 9.6, or at least 9.8. In certain embodiments, the
MDCK cells of the invention support the replication of a
cold-adapted, and/or temperature sensitive, and/or attenuated
version of each of influenza strains A/New Calcdonia, A/Hiroshima
and A/Vietnam to a log.sub.10 TCID.sub.50/mL and/or a log.sub.10
FFU/mL of at least 6.0, or at least 6.2, or at least 6.4, or at
least 6.6, or at least 6.8, or at least 7.0, or at least 7.2, or at
least 7.4, or at least 7.6, or at least 7.8, or at least 8.0, or at
least 8.2, or at least 8.4, or at least 8.6, or at least 8.8, or at
least 9.0, or at least 9.2, or at least 9.4, or at least 9.6, or at
least 9.8. In certain embodiments, the MDCK cells of the invention
support the replication of a cold-adapted, and/or temperature
sensitive, and/or attenuated version of each of influenza strains
A/New Calcdonia, B/Malaysia and A/Vietnam to a log.sub.10
TCID.sub.50/mL and/or a log.sub.10 FFU/mL of at least 6.0, or at
least 6.2, or at least 6.4, or at least 6.6, or at least 6.8, or at
least 7.0, or at least 7.2, or at least 7.4, or at least 7.6, or at
least 7.8, or at least 8.0, or at least 8.2, or at least 8.4, or at
least 8.6, or at least 8.8, or at least 9.0, or at least 9.2, or at
least 9.4, or at least 9.6, or at least 9.8. In certain
embodiments, the MDCK cells of the invention support the
replication of a cold-adapted, and/or temperature sensitive, and/or
attenuated version of each of influenza strains B/Malaysia,
A/Hiroshima and A/Vietnam to a log.sub.10 TCID.sub.50/mL and/or a
log.sub.10 FFU/mL of at least 6.0, or at least 6.2, or at least
6.4, or at least 6.6, or at least 6.8, or at least 7.0, or at least
7.2, or at least 7.4, or at least 7.6, or at least 7.8, or at least
8.0, or at least 8.2, or at least 8.4, or at least 8.6, or at least
8.8, or at least 9.0, or at least 9.2, or at least 9.4, or at least
9.6, or at least 9.8. In certain embodiments, the MDCK cells of the
invention support the replication of a cold-adapted, and/or
temperature sensitive, and/or attenuated version of each of
influenza strains A/New Calcdonia, A/Hiroshima, B/Malaysia and
A/Vietnam to a log.sub.10 TCID.sub.50/mL and/or a log.sub.10 FFU/mL
of at least 6.0, or at least 6.2, or at least 6.4, or at least 6.6,
or at least 6.8, or at least 7.0, or at least 7.2, or at least 7.4,
or at least 7.6, or at least 7.8, or at least 8.0, or at least 8.2,
or at least 8.4, or at least 8.6, or at least 8.8, or at least 9.0,
or at least 9.2, or at least 9.4, or at least 9.6, or at least
9.8.
[0060] In yet another aspect, the invention provides a method for
growing cold-adapted, and/or temperature sensitive, and/or
attenuated influenza viruses to a log.sub.10 TCID.sub.50/mL and/or
a log.sub.10 FFU/mL of at least 8.2, or at least 8.4, or at least
8.6, or at least 8.8, or at least 9.0, or at least 9.2, or at least
9.4, or at least 9.6, or at least 9.8 or at least 10.0, comprising
growing the cells in MediV105, M-32, MediV 107 or M18M or a growth
optimized derivative thereof, prior to infection with the influenza
viruses, then adding fresh media or media components (e.g.,
glucose, amino acids, lipids) during or post infection. In yet
another aspect, the invention provides a method for growing
cold-adapted, and/or temperature sensitive, and/or attenuated
influenza viruses to a log.sub.10 TCID.sub.50/mL and/or a
log.sub.10 FFU/mL of at least 8.0, or at least 8.2, or at least
8.4, or at least 8.6, or at least 8.8, or at least 9.0, or at least
9.2, or at least 9.4, or at least 9.6, or at least 9.8 or at least
10.0, comprising growing the cells in a serum free medium,
preferably an animal protein free medium and adding a protease
e.g., TrypLE (1:10-1:100) prior to, during or after infecting the
cells with influenza viruses. In certain embodiments, the
cold-adapted, and/or temperature sensitive, and/or attenuated
influenza viruses grow to a log.sub.10 TCID.sub.50/mL and/or a
log.sub.10 FFU/mL of at least 6.0, or at least 6.2, or at least
6.4, or at least 6.6, or at least 6.8, or at least 7.0, or at least
7.2, or at least 7.4, or at least 7.6, or at least 7.8, or at least
8.0, or at least 8.2, or at least 8.4, or at least 8.6, or at least
8.8, or at least 9.0, or at least 9.2, or at least 9.4, or at least
9.6, or at least 9.8 or at least 10.0. In certain embodiments, the
fresh media is MediV 105 supplemented with a protease, e.g., TrypLE
(1:10-1:100). In certain embodiments, the fresh media is M-32
supplemented with a protease, e.g., TrypLE (1:10-1:100). In certain
embodiments, the fresh media is MediV 107 supplemented with a
protease, e.g., TrypLE (1:10-1:100). Any protease known by one
skilled in the art to be useful in cleaving influenza proteins can
be used in these methods. In certain embodiments, the fresh media
is M18M supplemented with a protease, e.g., TrypLE (1:10-1:100). In
certain embodiments, the fresh media is DMEM/F12 supplemented with
4.5 g/L glucose, 4 mM glutamine, and a protease, e.g., TrypLE
(1:10-1:100).
[0061] It will be understood by one of skill in the art that the
cells of the invention can frequently be used as part of a cell
culture composition. The components of a cell culture composition
can vary according to the cells and intended use. For example, for
cultivation purposes a cell culture composition may comprise cells
of the invention and a suitable media for growth of the cells.
Accordingly, the present invention provides cell culture
compositions comprising cells of the invention and other components
which can include, but are not limited to, media (e.g., a media
disclosed herein), media components, buffers, chemical compounds,
additional cell types, viral material (e.g., viral genomes, viral
particles) and heterologous proteins. In one embodiment, a cell
culture composition comprises cells of the invention and a media or
components thereof. Media which may be present in a cell culture
composition include serum-free media, serum containing media, and
animal protein-free media. In one embodiment, a cell composition
comprises a serum-free media, e.g., MediV 101, MediV 102, MediV
103, MediV 104, MediV 105, M-32, MediV 107 or M18M, or components
or a growth optimized derivative thereof.
Methods and Media Formulations
[0062] The present invention further provides methods and media
formulations for the cultivation of MDCK cells that support the
replication of influenza viruses to high titer in serum containing
media. The present invention further provides methods for the
adaptation to and subsequent cultivation of the MDCK cells in
serum-free media, including animal protein free media formulations.
In certain aspects of the invention, the media are formulated such
that the MDCK cells retain one or more of the following
characteristics including but limited to, being non-tumorigenic,
being non-oncogenic, growing as adherent cells, growing as
non-adherent cells, having an epithelial-like morphology,
supporting the replication of various viruses when cultured, and
supporting the replication of influenza virus to high titer as
described herein. It is contemplated that the media formulations
disclosed herein or components thereof, may be present in a cell
culture composition.
[0063] Serum containing media formulations are well known in the
art. Serum containing media formulations include but are not
limited to, Dulbecco's Modified Eagle Medium (DMEM)+Fetal Bovine
Serum (FBS)+glutamine+glucose. In one embodiment, FBS is present in
a serum containing media at a concentration between about 1% and
about 20%, or between about 5% and about 15%, or between about 5%
and about 10%. In a specific embodiment, FBS is present in a serum
containing media at a concentration of 10%. In another embodiment,
glutamine is present in a serum containing media at a concentration
of between about 0.5 mM and about 10 mM, or between about 1 mM and
10 mM, or between about 2 mM and 5 mM. In a specific embodiment,
glutamine is present in a serum containing media at a concentration
of 4 mM. In still another embodiment, glucose is present in a serum
containing media at a concentration of between about 1 g/L and
about 10 g/L, or between about 2 g/L and about 5 g/L. In a specific
embodiment, glucose is present in a serum containing media at a
concentration of 4.5 g/L. In yet another embodiment, a serum
containing media formulation comprises, FBS at a concentration
between about 1% and about 20%, glutamine at a concentration of
between about 0.5 mM and about 10 mM, and glucose a concentration
of between about 1 g/L and about 10 g/L. In a specific embodiment,
a serum containing media formulation comprises, Dulbecco's Modified
Eagle Medium (DMEM)+10% Fetal Bovine Serum (FBS)+4 mM glutamine+4.5
g/L glucose. DMEM is readily available from numerous commercial
sources including, for example, Gibco/BRL (Cat. No. 11965-084). FBS
is readily available from numerous commercial sources including,
for example, JRH Biosciences (Cat. No. 12107-500M). While FBS is
the most commonly applied supplement in animal cell culture media,
other serum sources are also routinely used and encompassed by the
present invention, including newborn calf, horse and human.
[0064] In one embodiment, serum adapted MDCK cells of the invention
are derived from Madin Darby Canine Kidney Cells (MDCK) cells
obtained from the American type Culture Collection (ATCC CCL34) by
culturing them in a chemically defined media supplemented with
serum. In a specific embodiment, MDCK cells (ATCC CCL34) are
expanded in a chemically defined media supplemented with serum to
generate a serum adapted MDCK cell line as follows: the MDCK (ATCC
CCL34) cells are passaged in Dulbecco's Modified Eagle Medium
(DMEM) supplemented with fetal bovine serum (10% v/v), 4 mM
glutamine and 4.5 g/L glucose to obtain sufficient cells to prepare
a frozen pre Master Cell Bank (PreMCB). In another specific
embodiment, the cells are cultured using the methods detailed in
Examples 1 and 2, below. It is specifically contemplated that the
MDCK serum-adapted cells are passaged for another 20 passages or
more, from a vial of PreMCB and tested for tumorigenicity in an in
vivo adult nude mice model and karyology in a karyotype assay. In
certain embodiments, the expanded MDCK cells will not produce
tumors when injected subcutaneously into adult nude mice and will
have a modal chromosome number of 78 with a range of chromosome
numbers of no more then about 60-88, or of no more then about
65-85, or of no more than about 65-80, or of no more then about
70-85. In one embodiment, the MDCK-S cells are non-tumorigenic
after at least 20 passages, or after at least 30 passages, or after
at least 40 passages, or after at least 50 passages, or after at
least 60 passages, or after at least 70 passages, or after at least
80 passages, or after at least 90 passages, or after at least 100
passages in a medium (e.g., a media described herein).
[0065] It will be appreciated by one of skill in the art that the
use of serum or animal extracts in tissue culture applications may
have drawbacks (Lambert, K. J. et al., In: Animal Cell
Biotechnology, Vol 1, Spier, R. E. et al., Eds., Academic Pres New
York, pp. 85-122 (1985)). For example, the chemical composition of
these supplements may vary between lots, even from a single
manufacturer. In addition, supplements of animal or human origin
may also be contaminated with adventitious agents (e.g.,
mycoplasma, viruses, and prions). These agents can seriously
undermine the health of the cultured cells when these contaminated
supplements are used in cell culture media formulations. Further,
these agents may pose a health risk when substances produced in
cultures contaminated with adventitious agents are used in cell
therapy and other clinical applications. A major fear is the
presence of prions which cause spongiform encephalopathies in
animals and Creutzfeld-Jakob disease in humans. Accordingly, the
present invention further provides serum-free media formulations
comprising an MDCK cell of the invention.
[0066] Serum-free media formulations of the invention include, but
are not limited to, MediV 101 (Taub's+Plant Hydrolysate), MediV 102
(Taub's+Lipids), MediV 103 (Taub's+Lipds+Plant Hydrolysate), MediV
104 (Taub's+Lipds+Plant Hydrolysate+growth factor), MediV 105 (same
as MediV 104 except transferrin is replaced with Ferric ammonium
citrate/Tropolone or Ferric ammonium sulfate/Tropolone)(see, for
example, U.S. Patent Publication No. 2006/0188977), M-32 (same as
MediV 105 supplemented with trace elements A, B and C (see Table
9), MediV 107 (see Table 10) and M18M (see Table 11). It is
specifically contemplated that Taub's SF medium (Taub and
Livingston, 1981, Ann NY Acad. Sci., 372:406) is a 50:50 mixture of
DMEM and Ham's F12 supplemented with hormones, 5 .mu.g/mL insulin,
5 .mu.g/mL transferrin, 25 ng/mL prostaglandin E1, 50 nM
hydrocortisone, 5 pM triidothyronine and 10 nM Na.sub.2SeO.sub.3,
4.5 g/L glucose, 2.2 g/L NaHCO.sub.3 and 4 mM L-glutamine. Taub's
SF medium is also referred to herein as Taub's medium or simply
"Taub's". Specific media formulations and methods of preparing them
are provide infra (see, e.g., Section 9.10).
[0067] Plant hydrolysates include but are not limited to,
hydrolysates from one or more of the following: corn, cottonseed,
pea, soy, malt, potato and wheat. Plant hydrolysates may be
produced by enzymatic hydrolysis and generally contain a mix of
peptides, free amino acids and growth factors. Plant hydrolysates
are readily obtained from a number of commercial sources including,
for example, Marcor Development, HyClone and Organo Technie. It is
also contemplated that yeast hydrolysates my also be utilized
instead of, or in combination with plant hydrolysates. Yeast
hydrolysates are readily obtained from a number of commercial
sources including, for example, Sigma-Aldrich, USB Corp, Gibco/BRL
and others. In certain embodiments, synthetic hydrolysates can be
used in addition or in place of plant or yeast hydrolysates.
[0068] Lipids that may be used to supplement culture media include
but are not limited to chemically defined animal and plant derived
lipid supplements as well as synthetically derived lipids. Lipids
which may be present in a lipid supplement includes but is not
limited to, cholesterol, saturated and/or unsaturated fatty acids
(e.g., arachidonic, linoleic, linolenic, myristic, oleic, palmitic
and stearic acids). Cholesterol may be present at concentrations
between 0.10 mg/ml and 0.40 mg/ml in a 100.times. stock of lipid
supplement. Fatty acids may be present in concentrations between 1
.mu.g/ml and 20 .mu.g/ml in a 100.times. stock of lipid supplement.
Lipids suitable for media formulations are readily obtained from a
number of commercial sources including, for example HyClone,
Gibco/BRL and Sigma-Aldrich.
[0069] In one embodiment, Taub's media is supplemented with a plant
hydrolysate and a final concentration of at least 0.5 g/L, or at
least 1.0 g/L, or at least 1.5 g/L, or at least 2.0 g/L, or at
least 2.5 g/L, or at least 3.0 g/L, or at least 5.0 g/L, or at
least 10 g/L, or at least 20 g/L. In a specific embodiment, Taub's
media is supplemented with a wheat hydrolysate. In another specific
embodiment, Taub's media is supplemented with a wheat hydrolysate
at a final concentration of 2.5 g/L. The present invention provides
a serum-free media referred to herein as MediV 101 comprising
Taub's media supplemented with a wheat hydrolysate at a final
concentration of 2.5 g/L (see, e.g., Section 9.10).
[0070] In another embodiment, Taub's media is supplemented with a
lipid mixture at a final concentration of at least 50%, or at least
60%, or at least 70%, or at least 80%, or at least 90%, or at least
100%, or at least 125%, or at least 150%, or at least 200%, or at
least 300% of the manufacturers recommended final concentration. In
a specific embodiment, Taub's media is supplemented with a
chemically defined lipid mixture. In another specific embodiment,
Taub's media is supplemented with a chemically defined lipid
mixture at a final concentration of 100% of the manufacturers'
recommended final concentration (e.g., a 100.times. stock obtained
from a manufacture would be added to the media to a final
concentration of 1.times.). The present invention provides a
serum-free media referred to herein as MediV 102 comprising Taub's
media supplemented with a chemically defined lipid mixture at a
final concentration of 100% of the manufacturers recommended final
concentration (see, e.g., Section 9.10).
[0071] In still another embodiment, Taub's media is supplemented
with a plant hydrolysate at a final concentration of at least 0.5
g/L, or at least 1.0 g/L, or at least 1.5 g/L, or at least 2.0 g/L,
or at least 2.5 g/L, or at least 3.0 g/L, or at least 5.0 g/L, or
at least 10 g/L, or at least 20 g/L and with a lipid mixture at a
final concentration of at least 50%, or at least 60%, or at least
70%, or at least 80%, or at least 90%, or at least 100%, or at
least 125%, or at least 150%, or at least 175%, or at least 200% of
the manufacturers recommended concentration. In a specific
embodiment, Taub's media is supplemented with wheat hydrolysate and
a chemically defined lipid mixture. In another specific embodiment,
Taub's media is supplemented with a wheat hydrolysate at a final
concentration of 2.5 g/L and a chemically defined lipid mixture at
a final concentration of 100% of the manufacturers recommended
final concentration. The present invention provides a serum-free
media referred to herein as MediV 103 comprising Taub's media
supplemented with a wheat hydrolysate at a final concentration of
2.5 g/L and a chemically defined lipid mixture at a final
concentration of 100% of the manufacturers recommended final
concentration (see, e.g., Section 9.10).
[0072] In yet another embodiment, Taub's media is supplemented with
a growth hormone. Growth hormones which may be used include but are
not limited to, Epidermal Growth Factor (EGF), Insulin Growth
Factor (IGF), Transforming Growth Factor (TGF) and Fibroblast
Growth Factor (FGF). In a particular embodiment, the growth hormone
is Epidermal Growth Factor (EGF). In one embodiment, Taub's media
is supplemented with a growth factor at a final concentration of
between about 0.1 to about 50.0 ng/ml, or between about 0.5 to
about 25.0 ng/ml, or between about 1.0 to about 20 ng/ml, or
between about 5.0 to about 15.0 ng/ml, or between about 8 ng/ml to
about 12 ng/ml. In a specific embodiment, Taub's media is
supplemented with a EGF at a final concentration of about 10 ng/ml.
In still other embodiments, Taub's media is supplemented with a
growth factor at a final concentration of between about 0.1 to
about 50.0 ng/ml, or between about 0.5 to about 25.0 ng/ml, or
between about 1.0 to about 20 ng/ml, or between about 5.0 to about
15.0 ng/ml, or between about 8 ng/ml to about 12 ng/ml and with a
plant hydrolysate at a final concentration of at least 0.5 g/L, or
at least 1.0 g/L, or at least 1.5 g/L, or at least 2.0 g/L, or at
least 2.5 g/L, or at least 3.0 g/L, or at least 5.0 g/L, or at
least 10 g/L, or at least 20 g/L and with a lipid mixture at a
final concentration of at least 50%, or at least 60%, or at least
70%, or at least 80%, or at least 90%, or at least 100%, or at
least 125%, or at least 150%, or at least 175%, or at least 200% of
the manufacturers recommended concentration. In another specific
embodiment, Taub's media is supplemented with a wheat hydrolysate
at a final concentration of 2.5 g/L and a chemically defined lipid
mixture at a final concentration of 100% of the manufacturers
recommended final concentration and EGF at a final concentration of
about 10 ng/ml. The present invention provides a serum-free media
referred to herein as MediV 104 comprising Taub's media
supplemented with a wheat hydrolysate at a final concentration of
2.5 g/L and a chemically defined lipid mixture at a final
concentration of 100% of the manufacturers recommended final
concentration and EGF at a final concentration of about 10 ng/ml
(see, e.g., Section 9.10).
[0073] It will also be appreciated by one skilled in the art that
animal protein-free media formulations may be desirable for the
production of virus used in the manufacture of vaccines.
Accordingly, in certain embodiments one or more or all of the
animal derived components of the serum-free media disclosed herein
(e.g., MediV 101, MediV 102, MediV 103, MediV 104, MediV 105, M-32,
MediV 107, M18M) can be replaced by an animal-free derivative. For
example, commercially available recombinant insulin derived from
non-animal sources (e.g., Biological Industries Cat. No. 01-818-1)
may utilized instead of insulin derived from an animal source.
Likewise, iron binding agents (see, e.g., U.S. Pat. Nos. 5,045,454;
5,118,513; 6,593,140; and PCT publication number WO 01/16294) may
be utilized instead of transferrin derived from an animal source.
In one embodiment, serum-free media formulations of the invention
comprise tropolone (2-hydroxy-2,4,6-cyclohepatrien-1) and a source
of iron (e.g., ferric ammonium citrate, ferric ammonium sulfate)
instead of transferrin. For example, tropolone or a tropolone
derivative will be present in an excess molar concentration to the
iron present in the medium for at a molar ratio of about 2 to 1 to
about 70 to 1, or of about 10 to 1 to about 70 to 1 In a specific
embodiment, a serum-free media of the present invention comprises
Ferric ammonium citrate at a final concentration of 200 .mu.g/L and
Tropolone at a final concentration of 250 .mu.g/L (see, e.g.,
Section 9.10). Accordingly, where the iron concentration in the
medium is around 0.3 .mu.M, the tropolone or derivative thereof may
be employed at a concentration of about 1.5 .mu.M to about 20
.mu.M, e.g. about 3 .mu.M to about 20 .mu.M. The iron may be
present as ferrous or ferric ions, for example resulting from the
use of simple or complex iron salts in the medium such as ferrous
sulfate, ferric chloride, ferric nitrate or in particular ferric
ammonium citrate. The present invention provides a serum-free media
referred to herein as MediV 105 comprising Taub's media without
transferrin supplemented with a wheat hydrolysate at a final
concentration of 2.5 g/L and a chemically defined lipid mixture at
a final concentration of 100% of the manufacturers recommended
final concentration and EGF at a final concentration of about 10
ng/ml and Ferric ammonium citrate:Tropolone or Ferric ammonium
sulfate:Tropolone at a ratio of between 2 to 1 and 70 to 1. In a
specific embodiment, a serum-free media of the present invention
comprises Ferric ammonium citrate at a final concentration of 200
.mu.g/L and Tropolone at a final concentration of 250 .mu.g/L (see,
e.g., Section 9.10).
[0074] In certain embodiments, one or more of the media disclosed
herein are supplemented with trace elements (e.g., Trace Element
Solutions A, B and C, Table 9). Trace elements which may be used
include but are not limited to, CuSO.sub.4.5H.sub.2O,
ZnSO.sub.4.7H.sub.2O, Selenite.2Na, Ferric citrate,
MnSO.sub.4.H.sub.2O, Na.sub.2SiO.sub.3.9H.sub.2O, Molybdic
acid-Ammonium salt, NH.sub.4VO.sub.3, NiSO.sub.4.6H.sub.2O,
SnCl.sub.2 (anhydrous), AlCl.sub.3.6H.sub.2O, AgNO.sub.3,
Ba(C.sub.2H.sub.3O.sub.2).sub.2, KBr, CdCl.sub.2,
CoCl.sub.2.6H.sub.2O, CrCl.sub.3 (anhydrous), NaF, GeO.sub.2, KI,
RbCl, ZrOCl.sub.2.8H.sub.2O. Concentrated stock solutions of trace
elements are readily obtained from a number of commercial sources
including, for example Cell Grow (see Catalog Nos. 99-182, 99-175
and 99-176). The present invention provides a serum-free media
referred to herein as M-32 comprising Taub's media without
transferrin supplemented with a wheat hydrolysate at a final
concentration of 2.5 g/L and a chemically defined lipid mixture at
a final concentration of 100% of the manufacturers recommended
final concentration and EGF at a final concentration of about 10
ng/ml and Trace Element Solutions A, B and C (Table 9), and Ferric
ammonium citrate:Tropolone or Ferric ammonium sulfate:Tropolone at
a ratio of between 2 to 1 and 70 to 1. In a specific embodiment, a
serum-free media of the present invention comprises Ferric ammonium
citrate at a final concentration of 200 .mu.g/L and Tropolone at a
final concentration of 250 .mu.g/L) (see, e.g., Section 9.10). It
is also contemplated that one or more of the media disclosed herein
are supplemented with additional glucose. In one embodiment, a
serum free media of the present invention comprises an additional
1-5 g/L of glucose for a final glucose concentration of between
about 4.5 to about 10 g/L.
[0075] In one embodiment, MDCK cells adapted for growth in MediV
101, MediV 102, MediV 103, MediV 104, MediV 105, M-32, MediV 107 or
M18M serum-free media are derived from Madin Darby Canine Kidney
Cells (MDCK) cells obtained from the American type Culture
Collection (ATCC CCL34) by culturing in a chemically defined media
supplemented with serum for at least one passage and then passaging
them in a serum-free media such as, for example, the serum-free
medias described supra. In a specific embodiment, MDCK cells (ATCC
CCL34) are adapted to serum-free media as follows: The MDCK (ATCC
CCL34) cells are passaged in Dulbecco's Modified Eagle Medium
(DMEM) supplemented with fetal bovine serum (10% v/v), 4 mM
glutamine and 4.5 g/L glucose at least once and then passaged in
serum-free media. The MDCK cells are then passaged as needed in
serum-free media to obtain enough serum-free media-adapted cells to
prepare a frozen pre Master Cell Bank (PreMCB). In certain
embodiments, the cells are passaged in a serum containing media
(e.g., Dulbecco's Modified Eagle Medium (DMEM) supplemented with
fetal bovine serum (10% v/v), 4 mM glutamine and 4.5 g/L glucose)
between 1 and 5 times, or between 4 and 10 time, or between 9 and
20 times, or more than 20 times, and then passaged in serum-free
media (e.g., MediV 101, MediV 102, MediV 103, MediV 104, MediV 105,
M-32, MediV 107 and M18M, see, e.g., Section 9.10).
[0076] It is specifically contemplated that the serum-free
media-adapted MDCK cells are passaged for another 20 passages or
more, from a vial of PreMCB and tested for tumorigenicity in an
vivo adult nude mice model and karyology in a karyotype assay. In
certain embodiments, the expanded serum-free media-adapted MDCK
cells will not produce nodules when injected subcutaneously into
adult nude mice and/or will have a modal chromosome number of 78.
In another embodiment, the expanded serum-free media-adapted MDCK
cells will have a modal chromosome number of 78 with a range of
chromosome numbers of no more then about 60 to about 88, or of no
more then about 65 to about 85, or of no more then about 65-80, or
of no more then about 70 to about 85. In one embodiment, the
MDCK-SF cells are non-tumorigenic after at least 20 passages, or
after at least 30 passages, or after at least 40 passages, or after
at least 50 passages, or after at least 60 passages, or after at
least 70 passages, or after at least 80 passages, or after at least
90 passages, or after at least 100 passages in a medium (e.g., a
media described herein).
[0077] In one embodiment, the serum-free media used for the
derivation of serum-free media-adapted MDCK cells is MediV 101. In
another embodiment, the serum-free media used for the derivation of
serum-free media-adapted MDCK cells is MediV 102. In yet another
embodiment, the serum-free media used for the derivation of
serum-free media-adapted MDCK cells is MediV 103. In still another
embodiment, the serum-free media used for the derivation of
serum-free media-adapted MDCK cells is MediV-104. In another
embodiment, the serum-free media used for the derivation of
serum-free media-adapted MDCK cells is MediV 105. In other
embodiments, the serum-free media used for the derivation of
serum-free media-adapted MDCK cells is M-32. In other embodiments,
the serum-free media used for the derivation of serum-free
media-adapted MDCK cells is MediV 107. In another embodiment, the
serum-free media used for the derivation of serum-free
media-adapted MDCK cells is M18M. In yet another embodiment, the
serum-free media used for the derivation of serum-free
media-adapted MDCK cells is an APF media. It is contemplated that
the media described herein may be formulated to eliminate animal
proteins. For example bovine transferrin may be replaced with a
recombinant transferrin derived from a non animal source. Specific
media formulations and methods of preparing them are provided infra
(see, e.g., Section 9.10).
[0078] In another embodiment, the cells of the invention are not
adapted for growth in a serum-free media, but rather are simply
grown in serum free medium without adaptation. Thus, in one
embodiment, the cells are grown in MediV 101. In another
embodiment, the cells are grown in MediV 102. In yet another
embodiment, the cells are grown in MediV 103. In still another
embodiment, the cells are grown in MediV-104. In another
embodiment, the cells are grown in MediV 105. In another
embodiment, the cells are grown in M-32. In another embodiment, the
cells are grown in MediV 107. In another embodiment, the cells are
grown in M18M. In yet another embodiment, the cells are grown in an
APF media. It is contemplated that the media described herein may
be formulated to eliminate animal proteins. For example bovine
transferrin may be replaced with a recombinant transferrin derived
from a non animal source
Culture Conditions
[0079] The present invention provides methods for the cultivation
of MDCK cells of the invention and other animal cells in serum
containing and serum-free media formulations as set forth above. It
is specifically contemplated that additional culture conditions may
play a role in the maintenance of the properties of the MDCK cells
of the invention, including being non-tumorigenic, being
non-oncogenic, growing as adherent cells, growing as non-adherent
cells, having an epithelial-like morphology, supporting the
replication of various viruses, and supporting the growth of
influenza viruses (e.g., cold-adapted, and/or temperature
sensitive, and/or attenuated) to high titer, e.g., a log.sub.10
TCID.sub.50/mL and/or a log.sub.10 FFU/mL of at least about 7.4, or
at least about 7.6, or at least about 7.8, or at least about 8.0,
or at least about 9.0. These culture conditions include, but are
not limited to, the choice of adherent surface, cell density,
temperature, CO.sub.2 concentration, method of cultivation,
dissolved oxygen content and pH.
[0080] It is specifically contemplated that one skilled in the art
may adapt the culture conditions in a number of ways to optimize
the growth of the MDCK cells of the invention. Such adaptations may
also result in a increase in the production of viral material
(e.g., virus), as described, for example, in US Patent Application
Publication No. 2005/0118698. Alternatively, one skilled in the art
may adapt the culture conditions to optimize the production of
vaccine material from the MDCK cells of the invention without
regard for the growth of the cells. These culture conditions
include but are not limited to adherent surface, cell density,
temperature, CO.sub.2 concentration, method of cultivation,
dissolved oxygen content and pH.
[0081] In one embodiment, the MDCK cells of the invention are
cultivated as adherent cells on a surface to which they attach.
Adherent surfaces on which tissue culture cells can be grown on are
well known in the art. Adherent surfaces include but are not
limited to, surface modified polystyrene plastics, protein coated
surfaces (e.g., fibronectin and/or collagen coated glass/plastic)
as well as a large variety of commercially available microcarriers
(e.g., DEAE-Dextran microcarrier beads, such as Dormacell, Pfeifer
& Langen; Superbead, Flow Laboratories; styrene
copolymer-tri-methylamine beads, such as Hillex, SoloHill, Ann
Arbor; Cytodex 1 and Cytodex 3, GE Healthcare Life Science).
Microcarrier beads are small spheres (in the range of 100-200
microns in diameter) that provide a large surface area for adherent
cell growth per volume of cell culture. For example a single liter
of medium can include more than 20 million microcarrier beads
providing greater than 8000 square centimeters of growth surface.
The choice of adherent surface is determined by the methods
utilized for the cultivation of the MDCK cells of the invention and
can be determined by one skilled in the art. It will be understood
by one of skill in the art that during the process of subculturing
adherent cells (i.e., proliferating the cells, expanding the cell
culture) the cells must be transferred from a confluent support
surface (e.g., flask surface, microcarrier, etc) onto a new support
surface. A number of methods can be utilized to effect such cell
transfer. For example, proteases, including trypsin, TrypLE and
collagenase, may be used to remove cells from flasks or
microcarriers the cells are then washed and diluted into a larger
flask or into a larger volume of microcarrier containing media for
expansion. It is preferable to use a non-animal derived protease
for such applications such as, TrypLE (Invitrogen, Carlsbad,
Calif.). Alternatively, in microcarrier cultures direct bead to
bead transfer methods may be utilized, wherein fresh beads and
media are mixed with the confluent beads and the culture is
incubated under conditions which facilitate the transfer of cells
to the new beads. In certain embodiments, a combination of protease
treatment and bead to bead transfer is utilized. In a specific
embodiment, a cell culture of MDCK cells of the invention growing
as adherent cells on microcarriers are treated with a protease
(e.g., TrypLE), the protease is then inactivated (e.g., by the
addition of a protease inhibitor such as lima bean trypsin
inhibitor), fresh media and microcarrier beads may then be added to
the culture. In one embodiment, a portion or all of the growth
medium is removed prior to protease treatment. In another
embodiment, a portion or all of the growth medium is replaced with
a buffer prior to protease treatment. In still another embodiment,
a chelating agent is added prior to or during protease treatment.
In some embodiments, the protease treated culture is transferred to
a larger culture vessel before, during or after the addition of
fresh media and microcarriers.
[0082] In one embodiment, the MDCK cells of the invention are
cultivated as non-adherent cells (e.g., capable of growth under
non-adherent conditions) in suspension. Suitable culture vessels
which can be employed in the course of the process according to the
invention are all vessels known to the person skilled in the art,
such as, for example, spinner bottles, roller bottles, fermenters
or bioreactors. For commercial production of viruses, e.g., for
vaccine production, it is often desirable to culture the cells in a
bioreactor or fermenter. Bioreactors are available in volumes from
under 1 liter to in excess of 10,000 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).
[0083] In one embodiment, the MDCK cells of the invention are
cultivated as adherent cells in a batch culture system. In a
specific embodiment, the MDCK cells of the invention are cultivated
as adherent cells in a fed batch culture system wherein additional
nutrients (e.g., carbon source, amino acids, etc) are added as they
are depleted from the starting media to facilitate growth to high
cell densities. In still another embodiment, the MDCK cells of the
invention are cultivated as adherent cells in a perfusion culture
system. It is specifically contemplated that the MDCK cells of the
invention will be cultured in a perfusion system, (e.g., in a
stirred vessel fermenter, using cell retention systems known to the
person skilled in the art, such as, for example, centrifugation,
filtration, spin filters and the like) for the production of
vaccine material (e.g., virus). Additional guidance regarding
culture of MDCK cells as adherent cells may be found, for example,
in US Patent Application Publication Nos. 2003/0108860 and
2005/0118140. In another embodiment, the MDCK cells of the
invention are cultivated as non-adherent cells in a batch or fed
batch culture system. In still another embodiment, the MDCK cells
of the invention are cultivated as non-adherent cells in a
perfusion culture system.
[0084] In certain embodiments, a reactor system comprising
disposable elements such as a flexible plastic bag for culturing
cells is utilized. Such reactor systems are known in the art and
are available commercially. See for example International Patent
Publications WO 05/108546; WO 05/104706; and WO 05/10849 and
Section 9.12 infra. Reactor systems comprising disposable elements
(also referred herein as "single use bioreactor(s)" or by the
abbreviation "SUB(s)") may be pre-sterilized and do not require a
steam-in-place (SIP) or clean-in-place (CIP) environment for
changing from batch to batch or product to product in a culture or
production system. As such, SUBs require less regulatory control by
assuring zero batch-to-batch I contamination and can, thus, be
operated at a considerable cost-advantage and with minimal or no
preparation prior to use. Additionally, since SUBs do not require
cleaning or sterilizing they can be rapidly deployed to facilitate
production of large quantities of vaccine material (e.g., virus)
from cell culture. In particular embodiments, a disposable reactor
system is a stirred-tank reactor system which allows for a
hydrodynamic environment for mixing the cell culture which allows
for more efficient nutrient, O.sub.2 and pH control.
[0085] In one embodiment, the MDCK cells of the invention are
cultivated at a CO.sub.2 concentration of at least 1%, or of at
least 2%, or of at least 3%, or of at least 4%, or of at least 5%,
or of at least 6%, or of at least 7%, or of at least 8%, or of at
least 9%, or of at least 10%, or of at least 20%.
[0086] In one embodiment the dissolved oxygen (DO) concentration
(pO.sub.2 value) is advantageously regulated during the cultivation
of the MDCK cells of the invention and is in the range from 5% and
95% (based on the air saturation), or between 10% and 60%. In a
specific embodiment the dissolved oxygen (DO) concentration
(pO.sub.2 value) is at least 10%, or at least 20%, or at least 30%,
or at least 50%, or at least 60%.
[0087] In another embodiment, the pH of the culture medium used for
the cultivation of the MDCK cells of the invention is regulated
during culturing and is in the range from pH 6.4 to pH 8.0, or in
the range from pH 6.8 to pH 7.4. In a specific embodiment, the pH
of the culture medium is at about 6.4, or at about 6.6, or at about
6.8, or at about 7.0, or at about 7.2, or at about 7.4, or at about
7.6, or at about 7.8, or at least 8.0.
[0088] In a further embodiment, the MDCK cells of the invention are
cultured at a temperature of 25.degree. C. to 39.degree. C. It is
specifically contemplated that the culture temperature may be
varied depending on the process desired. For example, the MDCK
cells of the invention may be grown at 37.degree. C. for
proliferation of the cells and at a lower temperature (e.g.,
25.degree. C. to 35.degree. C.) of for the production of vaccine
material (e.g., virus). In another embodiment, the cells are
cultured at a temperature of less than 30.degree. C., or of less
than 31.degree. C., or of less than 32.degree. C., or of less than
33.degree. C., or of less than 34.degree. C. for the production of
vaccine material. In another embodiment, the cells are cultured at
a temperature of 30.degree. C., or 31.degree. C., or 32.degree. C.,
or 33.degree. C., or 34.degree. C. for the production of vaccine
material.
[0089] In order to generate vaccine material (e.g., virus) it is
specifically contemplated that the MDCK cells of the invention are
cultured such that the medium can be readily exchanged (e.g., a
perfusion system). The cells may be cultured to a very high cell
density, for example to between 1.times.10.sup.6 and
25.times.10.sup.6 cells/mL. The content of glucose, glutamine,
lactate, as well as the pH and pO.sub.2 value in the medium and
other parameters, such as agitation, known to the person skilled in
the art can be readily manipulated during culture of the MDCK cells
of the invention such that the cell density and/or virus production
can be optimized.
[0090] The present invention provides methods for proliferating
cells (e.g., MDCK cells of the present invention) in culture to
high cell density by culturing said cells in a SUB. In certain
embodiments, MDCK cells are cultured in a SUB system to a cell
density of at least 5.times.10.sup.5 cells/mL, a least
7.5.times.10.sup.5 cells/mL, at least 1.times.10.sup.6 cells/mL, at
least 2.5.times.10.sup.6 cells/mL, at least 5.times.10.sup.6
cells/mL, at least 7.5.times.10.sup.6 cells/mL, at least
10.times.10.sup.6, at least 15.times.10.sup.6 cells/mL, at least
20.times.10.sup.6 cells/mL, or at least 25.times.10.sup.6 cells/mL.
In a specific embodiment, MDCK cells are cultured in a SUB a
serum-free medium such as those described infra (see, for e.g.,
Section 9.10) that has be supplemented with additional glucose. For
example, MediV-105 supplemented with an additional 4.5 g/L of
glucose (9.0 g/L total glucose concentration) can be utilized. In
yet another specific embodiment, MDCK cells are cultured in a SUB
as adherent cells on a microcarrier. In one embodiment, the
microcarrier is used at a concentration of between about 1 to about
4 g/L. In another embodiment, the microcarrier is used at a
concentration of between about 2 to about 3 g/L. In certain
embodiments the SUB is seeded with the MDCK cells to be cultured at
a seeding density of about 5 to about 15.times.10.sup.4 cells/mL.
In a specific embodiment, the seeding density is between about 6 to
about 14.times.10.sup.4 cells/mL, or between about 7 to about
13.times.10.sup.4 cells/mL, or between about 8 to about
12.times.10.sup.4 cells/mL, or between about 9 to about
11.times.10.sup.4 cells/mL. It will be apparent to one of skill in
the art that the seeding density can also be calculated on a per
microcarrier basis. Accordingly, in certain embodiments the SUB is
seeded with the MDCK cells to be cultured at a seeding density of
about 2 to about 30 cells/microcarrier, or of about 2 to about 25
cells/microcarrier, cells/microcarrier, or of about 2 to about 20
cells/microcarrier, or of about 2 to about 15 cells/microcarrier,
or of about 2 to about 10 cells/microcarrier, or of about 5 to
about 30 cells/microcarrier, or of about 10 to about 30
cells/microcarrier, or of about 15 to about 30 cells/microcarrier,
or of about 20 to about 30, cells/microcarrier, or of about 5 to
about 30 cells/microcarrier, or of about 10 to about 25
cells/microcarrier, or of about 15 to about 20
cells/microcarrier.
[0091] In certain embodiments, MDCK cells are cultured in a
stirred-tank SUB in one or more parameters selected from the group
consisting of temperature, agitation rate, pH, dissolved oxygen
(DO), O.sub.2 and CO.sub.2 flow rate, are monitored and/or
controlled. In one embodiment, the temperature is maintained at
between about 30.degree. C. to about 42.degree. C., or between
about 33.degree. C. to about 39.degree. C., or between about
35.degree. C. to about 38.degree. C. In a specific embodiment, the
temperature is maintained at about between about 36.degree. C. to
about 37.degree. C. In one embodiment, the agitation rate is
maintained at between about 0 to 150 rpm. In a specific embodiment
the rate of agitation is maintained at between about 80 to about
120 rpm, or between about 90 to about 100 rpm. Agitation rates are
controlled by means well known in the art. In another embodiment,
the pH of the culture is maintained at between about 6.0 to about
7.5. In a specific embodiment the pH of the starting culture is
between about 6.0 to about 7.5 and the pH of the culture is
maintained at about 7.0 to about 7.5 during the culture process. It
will be understood by one of skill in the art that the initial pH
may be lower or higher then the desired range and that the pH may
be allowed to increase or decrease to the desired level (e.g., 7.4)
where it is maintained. The pH is maintained by any method known in
the art. For example the pH may be controlled by sparging CO.sub.2
and/or by adding acid (e.g., HCL) or base (e.g., NaOH) as needed.
In still another embodiment the acceptable range for the DO is
between about 100 to about 35%. In a specific embodiment, the DO is
maintained at between about 35% to about 50%, or at about 50%. In
another specific embodiment, the DO should not drop below about
35%. It will be understood by one of skill in the art that the
initial DO may be 100% and that the DO may be allowed to drop down
to a predetermined level (e.g., 50%) where it is maintained. The DO
is maintained used any method known in the art, such as, for
example, by sparging O.sub.2. In certain embodiments, the O.sub.2
flow rate is maintained at less then about 2.0 L/min. In certain
embodiments, the CO.sub.2 flow rate is maintained at less then
about 0.4 L/min.
Production of Vaccine Material (e.g., Virus)
[0092] The present invention provides a method for the production
of viruses in cell culture in which MDCK cells are used to produce
viruses. In certain embodiments of the method, the MDCK cells of
the invention are used to produce viruses. In one embodiment the
process comprises the following steps:
[0093] a. infecting a cell culture composition comprising an MDCK
cell of the invention with a virus,
[0094] b. incubating the cell culture composition under conditions
that permit replication of the virus; and
[0095] c. isolating viruses from the cell culture composition.
[0096] In one embodiment the MDCK cells of the invention are
proliferated prior to step (a) as adherent cells. In another
embodiment, the MDCK cells of the invention are proliferated prior
to step (a) as non-adherent cells. The MDCK cells of the invention
can be cultured in the course of the process in any media
including, but not limited to, those described supra. In certain
embodiments, the MDCK cells of the invention are cultured in the
course of the process in a serum-free medium such as, for example,
MediV-101, MediV-102, MediV-103, MediV-104, MediV-105, MediV-107,
M18M and APF formulations thereof. In a specific embodiment, the
MDCK cells of the invention are cultured in a serum-free medium
supplemented with glucose. Optionally, the MDCK cells of the
invention can be cultured in the course of the process in a serum
containing media (e.g., DMEM+10% FBS+4 mM glutamine+4.5 g/L
glucose). Additional culture conditions such as, for example,
temperature, pH, pO.sub.2, CO.sub.2 concentration, and cell density
are described in detail supra. One skilled in the art can establish
a combination of culture conditions for the proliferation of the
MDCK cells of the invention for the production of virus.
[0097] The temperature for the proliferation of the cells before
infection with viruses is in one embodiment between 22.degree. C.
and 40.degree. C. In certain embodiments, the temperature for the
proliferation of the cells before infection with viruses is less
then 39.degree. C., or less than 38.degree. C., or less than
37.degree. C., or less than 36.degree. C., or less than 35.degree.
C., or less than 34.degree. C., or less than 33.degree. C., or less
than 32.degree. C., or less than 30.degree. C., or less than
28.degree. C., or less than 26.degree. C., or less than 24.degree.
C. In a specific embodiment, the temperature for the proliferation
of the cells before infection with viruses is between about
33.degree. C. to about 39.degree. C. Culturing for proliferation of
the cells can be carried out in one embodiment of the method in a
perfusion system, e.g. in a stirred vessel fermenter, using cell
retention systems known to the person skilled in the art, such as,
for example, centrifugation, filtration, spin filters,
microcarriers, and the like. In a specific embodiment, culturing
for proliferation of the cells is carried out in a SUB system.
[0098] In such embodiments, the cells can, for example, be in this
case proliferated for 1 to 20 days, or for 3 to 11 days. Exchange
of the medium is carried out in the course of this, increasing from
0 to approximately 1 to 5 fermenter volumes per day. Alternatively,
the growth medium is supplemented with and/or comprises additional
components (e.g., glucose, trace mineral, amino acids, etc) such
that media exchange is not required. The cells can be proliferated
up to high cell densities in this manner, for example up to at
least 1.times.10.sup.6-25.times.10.sup.6 cells/mL. The perfusion
rates during culture in the perfusion system can be regulated via
the cell count, the content of glucose, glutamine or lactate in the
medium and via other parameters known to the person skilled in the
art. Alternatively, the cells can be cultured in a batch process or
fed batch process.
[0099] In one embodiment of the process according to the invention,
the pH, pO.sub.2 value, glucose concentration and other parameters
of the culture medium to culture the cells are regulated during
culturing as described above using methods known to the person
skilled in the art.
[0100] In certain embodiments, a portion of the medium is exchanged
prior to step (a). In one embodiment, the portion of the medium to
be exchanged is between about 20% to about 100%, or between about
30% to about 80%, or between about 30% to about 60%, or between
about 66% to about 80%. In one embodiment, the medium is exchange
with an equal volume of medium. In another embodiment, the medium
is exchange with a reduced volume of medium, effectively
concentrating the cells. The medium may be exchanged for a medium
having the same or different composition. In one embodiment, a
growth medium used for proliferation of the MDCK cells is exchange
for an infection medium (i.e., a medium used during infection and
viral replication). In a specific embodiment, the MDCK cells are
proliferated in MediV-105, MediV-107 or M18M and prior to infection
a portion of the medium is exchanged for an infection medium.
Alternatively, the growth medium is supplemented with and/or
comprises additional components (e.g., glucose, trace mineral,
amino acids, etc) such that media exchange is not required. In
another specific embodiment, the infection medium comprises a
serine protease (e.g., trypsin, TrypLE, etc). In other embodiments
where the media is not exchanged, a serine protease (e.g., trypsin,
TrypLE, etc) is added shortly before, during or shortly after
infection.
[0101] In certain embodiments, a protease is added prior to or at
the same time as the cells are infected with virus.
[0102] In some embodiments, the infection of the cells with virus
is carried out at an m.o.i. (multiplicity of infection, also
abbreviated herein as "MOI") of about 0.00001 to about 10, or about
0.00001 to about 1, or about 0.00001 to about 0.0003, or about
0.00001 to about 0.0001, or about 0.0001 to about 10, or about
0.0005 to about 5, or about 0.002 to about 0.5, or about 0.001 to
about 0.003. In still another embodiment, the infection of the
cells with virus is carried out at an m.o.i. (multiplicity of
infection) of 0.0001 to 10, or 0.0005 to 5, or 0.002 to 0.5 or
0.001 to 0.003. Alternatively, to the infection of cells with virus
is determined by the final concentration of virus in the culture.
For example, virus may be added at a final concentration of about
0.001.times.10.sup.3/mL to about 0.2.times.10.sup.3/mL, or about
0.01.times.10.sup.3/mL to about 2.times.10.sup.3/mL, or about
0.1.times.10.sup.3/mL to about 0.2.times.10.sup.3/mL, or about
1.times.10.sup.3/mL to about 4.times.10.sup.3/mL. After infection,
the infected cell culture is cultured further to replicate the
viruses, in particular until a maximum cytopathic effect or a
maximum amount of virus antigen can be detected. In one embodiment,
after infection the cells are cultured at a temperature of between
22.degree. C. and 40.degree. C. In certain embodiments, after
infection with viruses the cells are cultured at a temperature of
less then 39.degree. C., or less than 38.degree. C., or less than
37.degree. C., or less than 36.degree. C., or less than 35.degree.
C., or less than 34.degree. C., or less than 33.degree. C., or less
than 32.degree. C., or less than 30.degree. C., or less than
28.degree. C., or less than 26.degree. C., or less than 24.degree.
C. In certain embodiments, after infection with viruses the cells
are cultured at a temperature of 33.degree. C. In another
embodiment, after infection the cells are cultured at a temperature
of less than 33.degree. C. In still another embodiment, after
infection the cells are cultured at a temperature of 31.degree. C.
In certain embodiments, the culturing of the cells is carried out
for 2 to 10 days. The culturing can be carried out in the perfusion
system or optionally in the batch process or fed batch process.
[0103] In such embodiments, the cells can, for example, be cultured
after infection with viruses (step (b)) such that the pH and
pO.sub.2 value are maintained as described above. During the
culturing of the cells prior to step (a) and/or virus replication
according to step (b) of the process, a substitution of the cell
culture medium with freshly prepared medium, medium concentrate or
with defined constituents such as amino acids, vitamins, lipid
fractions, phosphates etc. for optimizing the antigen yield is also
possible. The cells can either be slowly diluted by further
addition of medium or medium concentrate over several days or can
be incubated during further perfusion with medium or medium
concentrate. The perfusion rates can in this case in turn be
regulated by means of the cell count, the content of glucose,
glutamine, lactate or lactate dehydrogenase in the medium or other
parameters known to the person skilled in the art. A combination of
the perfusion system with a fed-batch process is further
possible.
[0104] In one embodiment of the process, the harvesting and
isolation of the produced viruses (step (c)) is carried out after a
sufficient period to produce suitable yields of virus, such as 2 to
10 days, or optionally 3 to 7 days, after infection. In one
embodiment of the process, the harvesting and isolation of the
produced viruses (step (c)) is carried out 2 days, or 3 days, or 4
days, or 5 days, or after 6 days, or 7 days, or 8 days, or 9 days,
or 10 days, after infection.
[0105] Viruses which may be produced in the MDCK cells of the
present invention include but are not limited to, animal viruses,
including families of Orthomyxoviridae, Paramyxoviridae,
Togaviridae, Herpesviridae, Rhabdoviridae, Retroviridae,
Reoviridae, Flaviviridae, Adenoviridae, Picornaviridae,
Arenaviridae and Poxyiridae.
[0106] Systems for producing influenza viruses in cell culture have
also been developed in recent years (See, e.g., Furminger. in
Textbook of Influenza, ed Nicholson, Webster and Hay, pp. 324-332,
Blackwell Science (1998); Merten et al. in Novel Strategies in The
Design and Production of Vaccines, ed Cohen & Shafferman, pp.
141-151, Kluwer Academic (1996)). Typically, these methods involve
the infection of suitable host cells with a selected strain of
virus. While eliminating many of the difficulties related to
vaccine production in hen's eggs, not all pathogenic strains of
influenza grow well and can be produced according to established
tissue culture methods. In addition, many strains with desirable
characteristics, e.g., attenuation, temperature sensitivity and
cold adaptation, suitable for production of live attenuated
vaccines, have not been successfully grown, especially at
commercial scale, in tissue culture using established methods.
[0107] The present invention provides MDCK cell lines which have
been adapted to grow in either serum containing or serum-free
medias and which are capable of supporting the replication of
viruses, including, but not limited to, influenza, when cultured.
These cells lines are suitable for the economical replication of
viruses in cell culture for use as vaccine material. The MDCK cells
of the present invention are particularly useful for the production
of cold adapted, temperature sensitive (ca/ts) strains of influenza
(e.g., the influenza strains found in FluMist.RTM.) which do not
grow well using other established cell lines. Further, the MDCK
cells of the present invention are useful for the production of
strains of influenza which may not grow in embryonated eggs such as
avian influenza viruses which can also cause disease in humans
(e.g., a "pandemic" strain).
[0108] Influenza viruses which may be produced by the process of
the invention in the MDCK cells of the invention include but are
not limited to, reassortant viruses that incorporate selected
hemagglutinin and/or neuraminidase antigens in the context of an
attenuated, temperature sensitive, cold adapted (ca/ts/att) master
strain. For example, viruses can comprise the backbones (or one or
more vRNA segment) of master strains that are one or more of, e.g.,
temperature-sensitive (ts), cold-adapted (ca), or an attenuated
(att) (e.g., A/Ann Arbor/6/60, B/Ann Arbor/1/66, PR8,
B/Leningrad/14/17/55, B/14/5/1, B/USSR/60/69, B/Leningrad/179/86,
B/Leningrad/14/55, B/England/2608/76 etc.). Methods for the
production of reassortant influenza vaccine strains in either eggs
or cell lines are known in the art and include, for example,
Kilbourne, E. D. in Vaccines (2.sup.nd Edition), ed. Plotkin and
Mortimer, WB Saunders Co. (1988) and those disclosed in PCT
Application PCT Patent Publication Nos. WO 05/062820 and WO
03/091401, and in U.S. Pat. Nos. 6,951,754, 6,887,699, 6,649,372,
6,544,785, 6,001,634, 5,854,037, 5,824,536, 5,840,520, 5,820,871,
5,786,199, and 5,166,057 and U.S. Patent Application Publication
Nos. 20060019350, 20050158342, 20050037487, 20050266026,
20050186563, 20050221489, 20050032043, 20040142003, 20030035814,
and 20020164770. Other influenza viruses which may be produced by
the process of the invention in the MDCK cells of the invention
include recombinant influenza viruses which may express a
heterologous gene product, see for example, U.S. Patent Publication
Nos. 2004/0241139 and 2004/0253273.
[0109] In one embodiment, the cells are proliferated, and the cells
are then infected with influenza viruses. In certain embodiments,
the infection is carried out at an m.o.i. (multiplicity of
infection) of 0.0001 to 10, or of 0.0005 to 5, or of 0.002 to 0.5
or of 0.0001 to 0.002 or of 0.00001 to 0.002. In other embodiments,
the infection is carried out at an m.o.i. (multiplicity of
infection) of about 0.0001 to about 10, or of about 0.0005 to about
5, or of about 0.002 to about 0.5, or of about or of 0.0001 to
about 0.002 or of about 0.00001 to about 0.002. Optionally a
protease can be added which brings about the cleavage of the
precursor protein of hemagglutinin [HA.sub.0] and thus the
adsorption of the viruses on the cells. The addition of a protease
can be carried out according to the invention shortly before,
simultaneously to or shortly after the infection of the cells with
influenza viruses. If the addition is carried out simultaneously to
the infection, the protease can either be added directly to the
cell culture to be infected or, for example, as a concentrate
together with the virus inoculate. The protease is, in certain
aspects of the invention, a serine protease, or a cysteine
protease, or an asparagine protease. In one embodiment, trypsin is
used. In a specific embodiment, TPCK-treated trypsin is used. In
another embodiment, the protease from Streptomyces griseus
described in U.S. application Ser. No. 11/455,818 is used. The
trypsin can be from an animal source, or, more preferably, is from
a recombinant source.
[0110] In one embodiment, trypsin is added to the cell culture up
to a final concentration of 1 to 5000 mU/ml, or 5 to 1000 mU/ml, or
100 to 500 mU/ml. In an alternative embodiment, trypsin is added to
the cell culture up to a final concentration of 1 to 200 .mu.g/ml,
or 5 to 50 .mu.g/ml, or 5 to 30 .mu.g/ml in the culture medium.
During the further culturing of the infected cells according to
step (iii) of the process according to the invention, trypsin
reactivation can be carried out by fresh addition of trypsin in the
case of the batch or fed batch process or in the case of the
perfusion system by continuous addition of a trypsin solution or by
intermittent addition.
[0111] After infection, the infected cell culture is cultured
further to replicate the viruses, in particular until a maximum
cytopathic effect or a maximum amount of virus and/or virus antigen
can be detected. In certain embodiments, the culturing of the cells
is carried out for 2 to 10 days. The culturing can in turn be
carried out in the perfusion system or optionally in the batch or
fed batch process. In a further embodiment, the cells are cultured
at a temperature of 25.degree. C. to 36.degree. C., or of
29.degree. C. to 34.degree. C., after infection with influenza
viruses. The culturing of the infected cells at temperatures below
33.degree. C., in particular in the temperature ranges indicated
above, leads to the production of higher yields of certain
influenza viruses, such as, for example B strains (see, e.g., U.S.
Patent Publication 2006/0153872). Furthermore, the culturing of the
infected cells at temperatures below 35.degree. C. is contemplated
for the production of temperature sensitive, cold adapted (ts/ca)
influenza virus. It is contemplated that ts/ca viruses may also be
attenuated (att). In another embodiment, the cells are cultured at
a temperature of less than 30.degree. C., or of less than
31.degree. C., or of less than 32.degree. C., or of less than
33.degree. C., or of less than 34.degree. C. for the production of
ts/ca influenza strains. In a specific embodiment, the cells are
cultured at a temperature of 31.degree. C., for the production of
influenza virus B strains.
[0112] The culturing of the cells after infection with influenza
viruses (step (iii)) is in turn carried out, for example, as
described supra
[0113] In one embodiment of the process, the harvesting and
isolation of the produced influenza viruses (step (iii)) is carried
out after a sufficient period to produce suitable yields of virus,
such as 2 to 10 days, or 3 to 7 days, after infection. Viruses are
typically recovered from the culture medium, in which infected
cells have been grown. Typically crude medium is clarified prior to
concentration of influenza viruses. Common methods include
filtration, ultrafiltration, adsorption on barium sulfate and
elution, and centrifugation. For example, crude medium from
infected cultures can first be clarified by centrifugation at,
e.g., 1000-2000.times.g for a time sufficient to remove cell debris
and other large particulate matter, e.g., between 10 and 30
minutes. Alternatively, the medium is filtered through a 0.8 .mu.m
cellulose acetate filter to remove intact cells and other large
particulate matter. Optionally, the clarified medium supernatant is
then centrifuged to pellet the influenza viruses, e.g., at
15,000.times.g, for approximately 3-5 hours. Following resuspension
of the virus pellet in an appropriate buffer, such as STE (0.01 M
Tris-HCl; 0.15 M NaCl; 0.0001 M EDTA) or phosphate buffered saline
(PBS) at pH 7.4, the virus may be concentrated by density gradient
centrifugation on sucrose (60%-12%) or potassium tartrate
(50%-10%). Either continuous or step gradients, e.g., a sucrose
gradient between 12% and 60% in four 12% steps, are suitable. The
gradients are centrifuged at a speed, and for a time, sufficient
for the viruses to concentrate into a visible band for recovery.
Alternatively, and for most large scale commercial applications,
virus is elutriated from density gradients using a zonal-centrifuge
rotor operating in continuous mode. Additional details sufficient
to guide one of skill through the preparation of influenza viruses
from tissue culture are provided, e.g., in Furminger, in Textbook
of Influenza pp. 324-332 Nicholson et al. (ed); Merten et al., in
Novel Strategies in Design and Production of Vaccines pp. 141-151
Cohen & Shafferman (ed), and U.S. Pat. No. 5,690,937. If
desired, the recovered viruses can be stored at -80.degree. C. in
the presence of a stabilizer, such as sucrose-phosphate-glutamate
(SPG).
[0114] In certain embodiments of the process, the virus is treated
with Benzonase.RTM. or other a non-specific endonuclease.
Optionally, the Benzonase.RTM. treatment occurs early in the
harvesting and isolation of the produced influenza viruses. In
other embodiments of the process, following Benzonase.RTM.
treatment, the material is clarified. Methods useful for
clarification include but are not limited to, direct flow
filtration (DFF). Additional steps which may be utilized for the
harvesting and isolation of the produced influenza virus
(step(iii)) include but are not limited to, tangential flow
filtration (TFF), affinity chromatography as well as ion-exchange
chromatography and/or hydroxyapatite chromatography. In certain
embodiments, affinity chromatography is used in process. It will be
understood by one of skill in the art that a variety of affinity
chromatography media are available with similar separation
properties, for example numerous affinity chromatography media are
available for the concentration and purification of a number of
viruses and viral proteins. In a specific embodiment, Cellufine.TM.
Sulfate (Chisso Corp.) affinity media is utilized for affinity
chromatography. In another embodiment, FluSelect (GE Healthcare) is
utilized for affinity chromatography. In one embodiment, the virus
is treated with Benzonase.RTM. at the same time as an affinity
chromatography process. In certain embodiments, membrane
chromatography is used in the process. In one embodiment, ion
exchange chromatography is used in the process. In a specific
embodiment, cation exchange chromatography is used in the process.
In certain embodiments, cation exchange chromatography is performed
at high pH. In a specific embodiment, anion exchange chromatography
is used in the process. In certain embodiments, anion exchange
chromatography is performed at low pH. Anion membranes useful for
ion exchange chromatography include, but are not limited to, anion
membrane adsorbers (e.g., Sartobind.RTM. Q15, D15) and cation
membrane adsorbers (e.g., Sartobind.RTM. S15 and C15). Other steps
are exemplified in the Examples section below.
Vaccine Compositions and Methods of Use
[0115] The invention further relates to viruses (e.g., influenza)
which are obtainable by a process of the invention. These viruses
can be formulated by known methods to provide a vaccine for
administration to humans or animals. The viruses can be present as
intact virus particles (e.g., live attenuated viruses) or as
inactive/disintegrated virus (e.g., treated with detergents of
formaldehyde). Optionally, a defined viral component (e.g.,
protein) may be isolated from the viruses by methods know to the
person skilled in the art, and used in the preparation of a
vaccine. Methods for the generation and formulation of
inactive/disintegrated virus particles for vaccine compositions are
well known in the art and have been utilized for over 40 years.
[0116] The formulation of intact virus particles (e.g., live
attenuated viruses) may include additional steps including, but not
limited to, a buffer exchange by filtration into a final
formulation followed by a sterilization step. Buffers useful for
such a formulation may contain 200 mM sucrose and a phosphate or
histidine buffer of pH 7.0-7.2 with the addition of other amino
acid excipients such as arginine. In certain embodiments,
stabilization protein hydrolysates such as collagen or gelatin
(e.g., porcine, piscine, avian gelatin) are added. In some
embodiments, the final viral solutions/vaccines of the invention
can comprise live viruses that are stable in liquid form for a
period of time sufficient to allow storage "in the field" (e.g., on
sale and commercialization when refrigerated at 2-8.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, methods and compositions for formulating
a refrigerator stable attenuated influenza vaccine are described in
PCT Patent Publication No. WO/2006/041819; also see PCT Publication
WO/2005/014862.
[0117] Thus, in certain embodiments, the invention provides a
refrigerator stable vaccine formulation comprising one or more of
the following (within 10% variation of one or more component) in
the final formulations: 1-5% arginine; 1-4% gelatin; 5-10% sucrose
(optionally in a phosphate buffer); 0.01-0.1% glutamic acid
(monosodium, monohydrate); 10-150 mM potassium phosphate and 80-150
mM histidine.
[0118] In one specific embodiment, the vaccine formulation
comprises one or more of the following (within 10% variation of one
or more component): 1-2% arginine; 2% gelatin; 7-10% sucrose
(optionally in a phosphate buffer); and 100 mM histidine. In
another specific embodiment, the vaccine formulation comprises one
or more of the following (within 10% variation of one or more
component): 1-2% arginine; 1% gelatin; and 7-10% sucrose in a
phosphate buffer.
[0119] In certain other embodiments, the invention provides a
refrigerator stable vaccine formulation comprising 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 such as piscine or avian) 0.5-2%
w/v; potassium phosphate dibasic 1-2%; and potassium phosphate
monobasic 0.25-1% w/v.
[0120] In one specific embodiment, the vaccine formulation
comprises 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, the vaccine formulation comprises 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.
[0121] In another specific embodiment, the vaccine formulation
comprises 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. In another specific embodiment, the vaccine
formulation comprises 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, formulations
are in buffer {e.g., a potassium phosphate buffer (pH 7.0-7.2)). 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.
[0122] In yet another specific embodiment, the vaccine formulation
comprises 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. In still another specific embodiment, the
vaccine formulation comprises 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.
[0123] In another specific embodiment, the vaccine formulation
comprises 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. In another specific embodiment, the vaccine
formulation comprises 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. In a specific embodiment,
the vaccine formulation 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).
[0124] Optionally, spray drying, a rapid drying process whereby the
formulation liquid feed is spray atomized into fine droplets under
a stream of dry heated gas, may be utilized to extend storage time
of a vaccine formulation. The evaporation of the fine droplets
results in dry powders composed of the dissolved solutes (see,
e.g., US Patent Publication No. 2004/0042972).
[0125] Generally, virus or viral components can be administered
prophylactically in an appropriate carrier or excipient to
stimulate an immune response specific for one or more strains of
virus. Typically, the carrier or excipient is a pharmaceutically
acceptable carrier or excipient, such as sterile water, aqueous
saline solution, aqueous buffered saline solutions, aqueous
dextrose solutions, aqueous glycerol solutions, ethanol or
combinations thereof. The preparation of such solutions insuring
sterility, pH, isotonicity, and stability is effected according to
protocols established in the art. Generally, a carrier or excipient
is selected to minimize allergic and other undesirable effects, and
to suit the particular route of administration, e.g., subcutaneous,
intramuscular, intranasal, etc.
[0126] Optionally, the formulation for prophylactic administration
of the viruses, or components thereof, also contains one or more
adjuvants for enhancing the immune response to the influenza
antigens. Suitable adjuvants include: saponin, mineral gels such as
aluminum hydroxide, surface active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil or hydrocarbon
emulsions, Bacille Calmette-Guerin (BCG), Corynebacterium parvum,
and the synthetic adjuvants QS-21 and MF59.
[0127] Generally, vaccine formulations are administered in a
quantity sufficient to stimulate an immune response specific for
one or more strains of influenza virus. Preferably, administration
of the viruses elicits a protective immune response. Dosages and
methods for eliciting a protective immune response against one or
more viral strain are known to those of skill in the art. For
example, inactivated influenza viruses are provided in the range of
about 1-1000 HID.sub.50 (human infectious dose), i.e., about
10.sup.5-10.sup.8 pfu (plaque forming units) per dose administered.
Alternatively, about 10-50 .mu.g. e.g., about 15 .mu.g HA is
administered without an adjuvant, with smaller doses being
administered with an adjuvant. Typically, the dose will be adjusted
within this range based on, e.g., age, physical condition, body
weight, sex, diet, time of administration, and other clinical
factors. The prophylactic vaccine formulation is systemically
administered, e.g., by subcutaneous or intramuscular injection
using a needle and syringe, or a needleless injection device.
Alternatively, the vaccine formulation is administered
intranasally, either by drops, large particle aerosol (greater than
about 10 microns), or spray into the upper respiratory tract. While
any of the above routes of delivery results in a protective
systemic immune response, intranasal administration confers the
added benefit of eliciting mucosal immunity at the site of entry of
the influenza virus. For intranasal administration, attenuated live
virus vaccines are often preferred, e.g., an attenuated, cold
adapted and/or temperature sensitive recombinant or reassortant
influenza virus. While stimulation of a protective immune response
with a single dose is preferred, additional dosages can be
administered, by the same or different route, to achieve the
desired prophylactic effect. These methods can be adapted for any
virus including but not limited to, orthomyxoviruses (including
influenza A and B strains), paramyxoviruses (including RSV, human
metapneumovirus and parainfluenza), rhabdoviruses and
flavoviruses.
[0128] Influenza Virus
[0129] The methods, processes and compositions herein primarily
concerned with production of influenza viruses for vaccines.
Influenza viruses are made up of an internal ribonucleoprotein core
containing a segmented single-stranded RNA genome and an outer
lipoprotein envelope lined by a matrix protein. Influenza A and
influenza B viruses each contain eight segments of single stranded
negative sense RNA. The influenza A genome encodes eleven
polypeptides. Segments 1-3 encode three polypeptides, making up a
RNA-dependent RNA polymerase. Segment 1 encodes the polymerase
complex protein PB2. The remaining polymerase proteins PB1 and PA
are encoded by segment 2 and segment 3, respectively. In addition,
segment 1 of some influenza strains encodes a small protein,
PB1-F2, produced from an alternative reading frame within the PB1
coding region. Segment 4 encodes the hemagglutinin (HA) surface
glycoprotein involved in cell attachment and entry during
infection. Segment 5 encodes the nucleocapsid nucleoprotein (NP)
polypeptide, the major structural component associated with viral
RNA. Segment 6 encodes a neuraminidase (NA) envelope glycoprotein.
Segment 7 encodes two matrix proteins, designated M1 and M2, which
are translated from differentially spliced mRNAs. Segment 8 encodes
NS1 and NS2, two nonstructural proteins, which are translated from
alternatively spliced mRNA variants.
[0130] 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.
[0131] Reassortant viruses are produced to incorporate selected
hemagglutinin and neuraminidase antigens in the context of an
approved master strain also called a master donor virus (MDV).
FluMist.RTM. makes use of approved cold adapted, attenuated,
temperature sensitive MDV strains (e.g., A/AnnArbor/6/60 and B/Ann
Arbor/1/66).
[0132] A number of methods are useful for the generation of
reassortant viruses including egg-based methods and more recently
cell culture methods See, e.g., PCT Publications WO 03/091401; WO
05/062820 and U.S. Pat. Nos. 6,544,785; 6,649,372; 6,951,75, and
U.S. patent application Ser. Nos. 11/455,818, 11/455,734, and
11/501,067. It is contemplated that the MDCK cells, media and
methods of the invention are useful for the production of influenza
viruses including, but not limited to, the influenza strains
disclosed herein (e.g., A/AnnArbor/6/60 and B/AnnArbor/1/66) and
reassortant viruses comprising genes of the A/AnnArbor/6/60,
B/AnnArbor/1/66, PR8. It is further contemplated that that the MDCK
cells, media and methods of the invention are useful for the
production of influenza viruses, including reassortant viruses,
having one or more of the following phenotypes, temperature
sensitive, cold adapted, attenuated. Reassortants may be generated
by classical reassortant techniques, for example by co-infection
methods or optionally by plasmid rescue techniques (see, e.g., PCT
Publications WO 03/091401 and WO 05/062820; U.S. Pat. Nos.
6,544,785, 6,649,372, 6,951,754, 6,887,699, 6,001,634, 5,854,037,
5,824,536, 5,840,520, 5,820,871, 5,786,199, and 5,166,057; U.S.
Patent Application Publication Nos. 20060019350, 20050158342,
20050037487, 20050266026, 20050186563, 20050221489, 20050032043,
20040142003, 20030035814, and 20020164770; and Neumann et al.
(1999) Generation of influenza A virus entirely from cloned cDNAs.
Proc Natl Acad Sci USA 96:9345-9350; Fodor et al. (1999) Rescue of
influenza A virus from recombinant DNA. J. Virol 73:9679-9682;
Hoffmann et al. (2000) A DNA transfection system for generation of
influenza A virus from eight plasmids Proc Natl Acad Sci USA
97:6108-6113; WO 01/83794; Hoffmann and Webster (2000),
Unidirectional RNA polymerase I-polymerase II transcription system
for the generation of influenza A virus from eight plasmids,
81:2843-2847; and Hoffmann et al (2002), Rescue of influenza B
viruses from 8 plasmids, 99(17): 11411-11416.
[0133] Accordingly, the invention in another aspect provides an
MDCK cell of the invention that comprises one or more genomic
segments of an influenza virus. In certain embodiments, the cell
comprises all eight genomic segments of an influenza virus. In
certain embodiments, the eight genomic segments are each from the
same influenza virus. In certain embodiments, the eight genomic
segments are from one, two, or more different influenza viruses. In
certain embodiments, the eight genomic segments comprise two
segments encoding HA and NA, respectively, from any influenza
strain known to one skilled in the art without limitation and the
remaining genomic segments are from a cold-adapted, and/or
temperature, sensitive, and/or attenuated influenza virus. In
certain embodiments, the cell comprises any influenza genomic
segment described in any of the publications described above.
SPECIFIC EMBODIMENTS
[0134] 1. A Madin-Darby Canine Kidney (MCDK) cell, wherein a cell
culture composition comprising a plurality of the MDCK cells
supports replication of an attenuated, cold-adapted, temperature
sensitive influenza virus to a base 10 logarithm of the median
tissue culture infection dose per milliliter (log.sub.10
TCID.sub.50/mL) of at least about 7.0 or to a base 10 logarithm of
fluorescent focus units per milliliter (log.sub.10 FFU/mL) of at
least about 7.0.
[0135] 2. The MDCK cell of embodiment 1, wherein the MDCK cells
support replication of the influenza virus to a log.sub.10
TCID.sub.50/mL of at least about 7.2 and/or to a log.sub.10 FFU/mL
of at least about 7.2.
[0136] 3. The MDCK cell of embodiment 1, wherein the MDCK cells
support replication of the influenza virus to a log.sub.10
TCID.sub.50/mL of at least about 7.4 and/or to a log.sub.10 FFU/mL
of at least about 7.4.
[0137] 4. The MDCK cell of embodiment 1, wherein the MDCK cells
support replication of the influenza virus to a log.sub.10
TCID.sub.50/mL of at least about 7.6 and/or to a log.sub.10 FFU/mL
of at least about 7.6.
[0138] 5. The MDCK cell of embodiment 1, wherein the MDCK cells
support replication of the influenza virus to a log.sub.10
TCID.sub.50/mL of at least about 7.8 and/or to a log.sub.10 FFU/mL
of at least about 7.8.
[0139] 6. The MDCK cell of embodiment 1, wherein the MDCK cells
support replication of the influenza virus to a log.sub.10
TCID.sub.50/mL of at least 8.0 and/or to a log.sub.10 FFU/mL of at
least about 8.0.
[0140] 7. The MDCK cell of embodiment 1, wherein the MDCK cells
support replication of the influenza virus to a log.sub.10
TCID.sub.50/mL of at least 8.2 and/or to a log.sub.10 FFU/mL of at
least about 8.2.
[0141] 8. The MDCK cell of embodiment 1, wherein the MDCK cells
support replication of the influenza virus to a log.sub.10
TCID.sub.50/mL of at least 8.4 and/or to a log.sub.10 FFU/mL of at
least about 8.4.
[0142] 9. The MDCK cell of embodiment 1, wherein the MDCK cells
support replication of the influenza virus to a log.sub.10
TCID.sub.50/mL of at least 8.6 and/or to a log.sub.10 FFU/mL of at
least about 8.6.
[0143] 10. The MDCK cell of embodiment 1, wherein the MDCK cells
support replication of the influenza virus to a log.sub.10
TCID.sub.50/mL of at least 8.8 and/or to a log.sub.10 FFU/mL of at
least about 8.8.
[0144] 11. The MDCK cell of embodiment 1, wherein the MDCK cells
support replication of the influenza virus to a log.sub.10
TCID.sub.50/mL of at least 9.0 and/or to a log.sub.10 FFU/mL of at
least about 9.0.
[0145] 12. The MDCK cell of embodiment 1, wherein the MDCK cell
grows in serum-free medium.
[0146] 13. The MDCK cell of embodiment 1, wherein the serum free
media is an animal protein free media.
[0147] 14. The MDCK cell of embodiment 1, wherein the MDCK cell is
adherent.
[0148] 15. The MDCK cell of embodiment 1, wherein the MDCK cell is
non-adherent.
[0149] 16. The MDCK cell of embodiment 1, wherein the MDCK cell is
non-tumorigenic.
[0150] 17. The MDCK cell of embodiment 1, wherein the MDCK cell is
non-oncogenic.
[0151] 18. The MDCK cell of embodiment 1, wherein the MDCK cell is
derived from an MDCK cell line identified by American Type Culture
Collection (ATCC) Accession No. CCL34.
[0152] 19. The MDCK cell of embodiment 1, wherein the MDCK cell is
derived from an MDCK cell line identified by ATCC Accession No.
PTA-6500, PTA-6501, PTA-6502 or PTA-6503.
[0153] 20. The MDCK cell of embodiment 1, wherein the MDCK cell is
identified by ATCC Accession No. PTA-7909 or PTA-7910.
[0154] 21. The MDCK cell of embodiment 1, wherein the influenza
virus is an influenza A virus.
[0155] 22. The MDCK cell of embodiment 1, wherein the influenza
virus is an influenza B virus.
[0156] 23. The MDCK cell of embodiment 1, wherein the influenza
virus is a cold adapted virus.
[0157] 24. The MDCK cell of embodiment 1, wherein the influenza
virus is a temperature sensitive virus.
[0158] 25. The MDCK cell of embodiment 1, wherein the influenza
virus is an attenuated virus.
[0159] 26. The MDCK cell of embodiment 1, wherein the influenza
virus is an attenuated, cold adapted, and temperature sensitive
virus.
[0160] 27. The MDCK cell of embodiment 1, wherein the influenza
virus comprises one or more gene segments of a temperature
sensitive, attenuated and cold adapted influenza virus.
[0161] 28. The MDCK cell of embodiment 1, wherein the influenza
virus comprises one or more gene segments of influenza strain A/Ann
Arbor/6/60.
[0162] 29. The MDCK cell of embodiment 1, wherein the influenza
virus comprises one or more gene segments of B/Ann Arbor/1/66.
[0163] 30. A method for proliferating the MDCK cell of any of the
preceding embodiments to a cell density of at least about
1.times.10.sup.6 cells/ml in a SUB system comprising inoculating a
cell culture medium with the MDCK cell of any of the preceding
embodiments at a seeding density of between about 8.times.04 to
about 12.times.10.sup.4 cells/mL and culturing the cells while
maintaining one or more culture conditions selected from the group
consisting of: [0164] a. an agitation rate of between about 50 to
150 rpm; [0165] b. a pH of between about 6.0 to about 7.5; [0166]
c. dissolved oxygen (DO) between about 35% to about 100%; and
[0167] d. a temperature of between about 33.degree. C. to about
42.degree. C.
[0168] 31. The method of embodiment 30, wherein the cell culture
medium is a serum free medium.
[0169] 32. The method of embodiment 30, wherein the cell culture
medium is an animal protein free medium.
[0170] 33. The method of embodiment 30, wherein the cell culture
medium is MediV-105 supplemented with glucose, or M-32 or
MediV-107.
[0171] 34. The method of embodiment 30, wherein the agitation rate
is between about 90 to about 100 rpm.
[0172] 35. The method of embodiment 30, wherein the DO is between
about 35% to about 100%.
[0173] 36. The method of embodiment 30, wherein the temperature is
between about 36.degree. C. and about 38.degree. C.
[0174] 37. The method of embodiment 30, wherein a microcarrier is
used for culturing an adherent MDCK cell.
[0175] 38. The method of embodiment 37, wherein the microcarrier
concentration is between about 1 to about 4 g/L.
[0176] 39. A cell culture composition produced by the method of any
one of embodiments 30 to embodiment 38.
[0177] 40. A cell culture composition comprising MCDK cells and a
cell culture medium, wherein the cell culture composition supports
replication of an influenza virus to a log.sub.10 TCID.sub.50/mL of
at least about 7.0 and/or to a log.sub.10 FFU/mL of at least about
7.0.
[0178] 41. The cell culture composition of embodiment 40, wherein
the MDCK cell culture composition supports replication of the
influenza virus to a log.sub.10 TCID.sub.50/mL of at least about
7.2 and/or to a log.sub.10 FFU/mL of at least about 7.2.
[0179] 42. The cell culture composition of embodiment 40, wherein
the MDCK cell culture composition supports replication of the
influenza virus to a log.sub.10 TCID.sub.50/mL of at least about
7.4 and/or to a log.sub.10 FFU/mL of at least about 7.4.
[0180] 43. The cell culture composition of embodiment 40, wherein
the MDCK cell culture composition supports replication of the
influenza virus to a log.sub.10 TCID.sub.50/mL of at least about
7.6 and/or to a log.sub.10 FFU/mL of at least about 7.6.
[0181] 44. The cell culture composition of embodiment 40, wherein
the MDCK cell culture composition supports replication of the
influenza virus to a log.sub.10 TCID.sub.50/mL of at least about
7.8 and/or to a log.sub.10 FFU/mL of at least about 7.8.
[0182] 45. The cell culture composition of embodiment 40, wherein
the MDCK cell culture composition supports replication of the
influenza virus to a log.sub.10 TCID.sub.50/mL of at least about
8.0 and/or to a log.sub.10 FFU/mL of at least about 8.0.
[0183] 46. The cell culture composition of embodiment 40, wherein
the MDCK cell culture composition supports replication of the
influenza virus to a log.sub.10 TCID.sub.50/mL of at least about
8.2 and/or to a log.sub.10 FFU/mL of at least about 8.2.
[0184] 47. The cell culture composition of embodiment 40, wherein
the MDCK cell culture composition supports replication of the
influenza virus to a log.sub.10 TCID.sub.50/mL of at least about
8.4 and/or to a log.sub.10 FFU/mL of at least about 8.4.
[0185] 48. The cell culture composition of embodiment 40, wherein
the MDCK cell culture composition supports replication of the
influenza virus to a log.sub.10 TCID.sub.50/mL of at least about
8.6 and/or to a log.sub.10 FFU/mL of at least about 8.6.
[0186] 49. The cell culture composition of embodiment 40, wherein
the MDCK cell culture composition supports replication of the
influenza virus to a log.sub.10 TCID.sub.50/mL of at least about
8.8 and/or to a log.sub.10 FFU/mL of at least about 8.8.
[0187] 50. The cell culture composition of embodiment 40, wherein
the MDCK cell culture composition supports replication of the
influenza virus to a log.sub.10 TCID.sub.50/mL of at least about
9.0 and/or to a log.sub.10 FFU/mL of at least about 9.0.
[0188] 51. The cell culture composition of embodiment 40, wherein
the cell culture composition does not comprise animal serum.
[0189] 52. The cell culture composition of embodiment 40, wherein
the cell culture composition does not comprise a protein purified
from an animal.
[0190] 53. The cell culture composition of embodiment 40, wherein
the cell culture composition comprises a recombinantly-expressed
protein.
[0191] 54. The cell culture composition of embodiment 53, wherein
the protein is expressed by at least one of the MDCK cells.
[0192] 55. The cell culture composition of embodiment 53, wherein
the protein is expressed in a recombinant expression system and
then added to the cell culture composition.
[0193] 56. The cell culture composition of embodiment 53, wherein
the recombinantly-expressed protein is insulin or trypsin.
[0194] 57. The cell culture composition of embodiment 40, wherein
at least some of the MDCK cells are adherent.
[0195] 58. The cell culture composition of embodiment 40, wherein
the MDCK cells are adherent.
[0196] 59. The cell culture composition of embodiment 40, wherein
at least some of the MDCK cells are non-adherent.
[0197] 60. The cell culture composition of embodiment 40, wherein
the MDCK cells are non-adherent.
[0198] 61. The cell culture composition of embodiment 40, wherein
the MDCK cells are non-tumorigenic.
[0199] 62. The cell culture composition of embodiment 40, wherein
the MDCK cells are derived from the MDCK cell line identified by
American Type Culture Collection (ATCC) Accession No. CCL34.
[0200] 63. The cell culture composition of embodiment 40, wherein
the MDCK cells are derived from an MDCK cell line identified by
ATCC Accession No. PTA-6500, PTA-6501, PTA-6502 or PTA-6503.
[0201] 64. The cell culture composition of embodiment 40, wherein
the MDCK cells are identified by ATCC Accession No. PTA-7909 or
PTA-7910.
[0202] 65. The cell culture composition of embodiment 40, wherein
the influenza virus is an influenza A virus.
[0203] 66. The cell culture composition of embodiment 40, wherein
the influenza virus is an influenza B virus.
[0204] 67. The cell culture composition of embodiment 40, wherein
the influenza virus is a cold adapted virus.
[0205] 68. The cell culture composition of embodiment 40, wherein
the influenza virus is an attenuated virus.
[0206] 69. The cell culture composition of embodiment 40, wherein
the influenza virus comprises one or more gene segments of a
temperature sensitive, attenuated and cold adapted influenza
virus.
[0207] 70. The cell culture composition of embodiment 40, wherein
the influenza virus comprises one or more gene segments of
influenza strain A/Ann Arbor/6/60.
[0208] 71. The cell culture composition of embodiment 40, wherein
the influenza virus comprises one or more gene segments of B/Ann
Arbor/1/66.
[0209] 72. The cell culture composition of embodiment 40, wherein
the MDCK cells are cultured at between about 25.degree. C. and
about 33.degree. C. during the replication of the influenza
virus.
[0210] 73. The cell culture composition of embodiment 40, wherein
the MDCK cells do not comprise detectable oncogenic DNA.
[0211] 74. The cell culture composition of embodiment 40, wherein
the cell culture composition does not comprise detectable
mycoplasma.
[0212] 75. The cell culture composition of embodiment 40, wherein
the cell culture composition does not comprise detectable
bacteria.
[0213] 76. The cell culture composition of embodiment 40, wherein
the cell culture composition does not comprise a detectable virus
other than an influenza virus.
[0214] 77. The cell culture composition of embodiment 40 wherein
the detectable virus is a virus that infects canine or human
cells.
[0215] 78. The cell culture composition of embodiment 40, wherein
the MDCK cells do not comprise a latent virus.
[0216] 79. The cell culture composition of embodiment 40, wherein
the MDCK cells do not comprise a retrovirus.
[0217] 80. The cell culture composition of embodiment 40, wherein
the MDCK cells are grown to a cell density of at least about
1.times.10.sup.5 cells/ml.
[0218] 81. The cell culture composition of embodiment 40, wherein
the MDCK cells are grown to a cell density of at least about
5.times.10.sup.5 cells/ml.
[0219] 82. The cell culture composition of embodiment 40, wherein
the MDCK cells are grown to a cell density of at least about
1.times.10.sup.6 cells/ml.
[0220] 83. The cell culture composition of embodiment 40, wherein
the MDCK cells are grown to a cell density of at least about
2.5.times.10.sup.6 cells/ml.
[0221] 84. The cell culture composition of embodiment 40, wherein
the MDCK cells are grown to a cell density of at least about
5.times.10.sup.6 cells/ml
[0222] 85. A method for producing influenza viruses in cell
culture, comprising: [0223] a. infecting the cell culture
composition of any of embodiments 40-84 with an influenza virus,
[0224] b. incubating the cell culture composition under conditions
that permit replication of the influenza virus; and [0225] c.
isolating influenza viruses from the cell culture composition.
[0226] 86. The method of embodiment 85, wherein fresh medium or
additional medium components are added to the cell culture prior to
or during step (a).
[0227] 87. The method of embodiment 85, wherein none or some of the
cell culture medium is removed and replaced with fresh medium prior
to or during step (a).
[0228] 88. The method of embodiment 85, wherein step (a) is carried
out at a Multiplicity Of Infection (MOI) of between about 0.00001
to about 0.00003 FFU/cell.
[0229] 89. The method of embodiment 85, wherein step (a) is carried
out at an MOI of between about 0.0001 to about 0.0003 FFU/cell.
[0230] 90. The method of embodiment 85, wherein step (a) is carried
out at an MOI of between about 0.001 to about 0.003 FFU/cell.
[0231] 91. The method of embodiment 85, wherein the conditions of
step (b) are selected from the group consisting of: [0232] a. an
agitation rate of between about 50 to 150 rpm; [0233] b. a pH of
between about 6.0 to about 7.5; [0234] c. dissolved oxygen (DO)
between about 35% to about 100%; and [0235] d. a temperature of
between about 30.degree. C. to about 35.degree. C.
[0236] 92. The method of embodiment 85, wherein the influenza virus
replicates to a log.sub.10 TCID.sub.50/mL of at least about 7.0
and/or to a log.sub.10 FFU/mL of at least about 7.0.
[0237] 93. The method of embodiment 85, wherein the influenza virus
replicates to a log.sub.10 TCID.sub.50/mL of at least about 7.2
and/or to a log.sub.10 FFU/mL of at least about 7.2.
[0238] 94. The method of embodiment 85, wherein the influenza virus
replicates to a log.sub.10 TCID.sub.50/mL of at least about 7.4
and/or to a log.sub.10 FFU/mL of at least about 7.4.
[0239] 95. The method of embodiment 85, wherein the influenza virus
replicates to a log.sub.10 TCID.sub.50/mL of at least about 7.6
and/or to a log.sub.10 FFU/mL of at least about 7.6.
[0240] 96. The method of embodiment 85, wherein the influenza virus
replicates to a log.sub.10 TCID.sub.50/mL of at least about 7.8
and/or to a log.sub.10 FFU/mL of at least about 7.8.
[0241] 97. The method of embodiment 85, wherein the influenza virus
replicates to a log.sub.10 TCID.sub.50/mL of at least about 8.0
and/or to a log.sub.10 FFU/mL of at least about 8.0.
[0242] 98. The method of embodiment 85, wherein the influenza virus
replicates to a log.sub.10 TCID.sub.50/mL of at least about 8.2
and/or to a log.sub.10 FFU/mL of at least about 8.2.
[0243] 99. The method of embodiment 85, wherein the influenza virus
replicates to a log.sub.10 TCID.sub.50/mL of at least about 8.4
and/or to a log.sub.10 FFU/mL of at least about 8.4.
[0244] 100. The method of embodiment 85, wherein the influenza
virus replicates to a log.sub.10 TCID.sub.50/mL of at least about
8.6 and/or to a log.sub.10 FFU/mL of at least about 8.6.
[0245] 101. The method of embodiment 85, wherein the influenza
virus replicates to a log.sub.10 TCID.sub.50/mL of at least about
8.8 and/or to a log.sub.10 FFU/mL of at least about 8.8.
[0246] 102. The method of embodiment 85, wherein the influenza
virus replicates to a log.sub.10 TCID.sub.50/mL of at least about
9.0 and/or to a log.sub.10 FFU/mL of at least about 9.0.
[0247] 103. An influenza virus produced according to the method of
embodiment 85.
[0248] 104. An immunogenic composition comprising polypeptides of
the influenza virus of embodiment 103 in a pharmaceutically
acceptable carrier or diluent.
[0249] 105. An immunogenic composition comprising the influenza
virus of embodiment 103 in a pharmaceutically acceptable carrier or
diluent.
[0250] 106. The immunogenic composition of embodiment 105, wherein
the immunogenic composition is refrigerator stable.
[0251] 107. A method of eliminating DNA contaminants from a viral
preparation comprising: [0252] (a) passing the viral preparation
over affinity chromatography media under conditions wherein the DNA
contaminants are not retained on the affinity chromatography media
and the virus present in the viral preparation are retained; [0253]
(b) washing the affinity chromatography media to remove the DNA
contaminants; and [0254] (c) eluting the virus present in the viral
preparation from the affinity chromatography media.
[0255] 108. The method of 107, wherein the affinity chromatography
media is Cellufine Sulfate resin.
[0256] 109. The method of embodiment 107, wherein between steps (a)
and (b) a non-specific endonuclease preparation is passed over the
affinity chromatography media.
[0257] 110. The method of embodiment 108, wherein the non-specific
endonuclease is a Benzonase preparation comprises Benzonase in
1.times.SP buffer at about pH 7.2.
[0258] 111. The method of embodiment 107, wherein the viral
preparation is an influenza virus preparation.
[0259] 112. The method of embodiment 108, wherein the influenza
virus preparation was prepared from mammalian cells.
[0260] 113. The method of embodiment 112, wherein the mammalian
cells are MDCK cells or Vero cells, or PerC6 cells.
[0261] 114. The method of embodiment 107, wherein the conditions
used in step (a) are 1.times.SP buffer at about pH 7.2.
[0262] 115. The method of embodiment 107, wherein the virus present
in the viral preparation are eluted in 1.times.SP buffer containing
about 1 M NaCl at about pH 7.2.
EXAMPLES
[0263] The invention is now described with reference to the
following examples. These examples are provided for the purpose of
illustration only and the invention should in no way be construed
as being limited to these examples but rather should be construed
to encompass any and all variations which become evident as a
result of the teachings provided herein.
Example 1
Identification of a MDCK Cell Line that Supports High Viral
Replication in Serum Containing Media
[0264] This example describes identification and selection of an
MDCK cell line that supports replication of influenza viruses to
high titers when the MDCK cell line was cultured in Dulbecco's
Modified Eagle's Medium (DMEM) media comprising 10% Fetal Bovine
Serum (FBS). The process is outlined in FIG. 5A.
[0265] One vial of MDCK cells (ATCC Accession No. CCL-34; Lot
1805449; passage 54) obtained from the ATCC was thawed and
inoculated into a T-25 flask (Corning) containing 10 ml of
Dulbecco's Modified Eagle's Medium with L-glutamine (DMEM) and 10%
fetal bovine serum (FBS, Defined). Cells (passage 55) were
incubated at 37.+-.1.degree. C. with 5.+-.1% CO.sub.2 for 3 days.
On day 3, the cells were passaged to a T-225 flask (passage 56).
Three days after seeding, cells were passaged to 4.times.T-225
flasks (Passage 57). For each of the passages in DMEM with
L-glutamine and 10% FBS in a T-25 or T-225 flask, the procedure was
as follows.
[0266] Cells were washed twice with Dulbecco's Phosphate Buffered
Saline without Ca.sup.++ and Mg.sup.++ (DPBS), and 1.5 ml (for
T-25) or 7.5 ml (for T-225) of trypsin 0.25% were added to the
cells. The cell monolayer was incubated and allowed to release for
15 to 20 minutes, at which time 1.5 ml (for the T-75 flask) or 7.5
ml (for the T-225 flask) of DMEM with L-glutamine and 10% FBS were
added to neutralize the trypsin. The cells were then counted using
a hemacytometer, and the amount necessary to inoculate
5.times.10.sup.4 cells per ml was transferred to a T-225 flask
containing 100 ml of DMEM with L-glutamine and 10% FBS, and
incubated as above for 3 days. Cells from 4 T-225 flasks were
trypsinized, pooled, and serum containing growth medium was added
as described above. The cells were then mixed and counted. The cell
suspension was centrifuged and the cell pellet was resuspended with
10 ml of DMEM with L-glutamine and 10% FBS. This suspension was
counted again. Ten ml of 2.times. freezing medium (10% FBS DMEM
with L-glutamine and 15% v/v dimethyl sulfoxide) was added, the
cells were mixed thoroughly, and 1 ml was aliquoted into each of 20
cryovials. The cells were frozen at -80.degree. C. in Nalgene
freezer containers, and then transferred to storage in vapor phase
of liquid nitrogen. The frozen cells represented the MDCK cells at
passage 57 and are referred to herein as MDCK Pre-MCB lot 1.
[0267] Next, one vial of the MDCK Pre-MCB lot 1 was thawed and
inoculated into a T75 flask containing 35 ml of DMEM and 10% FBS.
Cells (passage 58) were incubated at 37.degree. C. with 5% CO.sub.2
for 3 days. On day 3 the cells were passaged to 2.times.T225 flasks
(passage 59). Three days after seeding, cells were passaged to
4.times.T225 flasks (Passage 60). On day 3 post seeding, a complete
medium exchange was performed. Four days after seeding, cells were
passaged to 25.times.T225 flasks (Passage 61). For each of the
passages in DMEM with L-glutamine and 10% FBS in a T75 or T225
flask, the procedure was as follows.
[0268] Cells were washed two times with Dulbecco's Phosphate
Buffered Saline without Ca.sup.++ and Mg.sup.++ (DPBS), and 3 ml
(for T75) or 7.5 ml (for T225) of trypsin 0.25% was added to the
cells. The cell monolayer was incubated and allowed to release for
15 to 20 minutes, at which time 3 ml (for T75) or 7.5 ml (for T225
flask) of DMEM with L-glutamine and 10% FBS was added to neutralize
the trypsin. The cells were then counted in a hemacytometer, and
the amount necessary to inoculate 5.times.10.sup.4 cells per ml was
transferred to a T225 flask containing 100 ml of DMEM with
L-glutamine and 10% FBS, and incubated as above for 3 days. Cells
from 24 of 25 T225 flasks were trypsinized, pooled, and serum
containing growth medium was added. The cell suspension were
centrifuged, and resuspended cell pellet with 50 ml of DMEM with
L-glutamine and 10% FBS.
[0269] This suspension was then counted. To make 60 ml of
1.times.10.sup.7 cells/ml cell suspension, 39.5 ml cell suspension
was combined with 20.5 ml of 10% FBS DMEM medium. Then 60 ml of
2.times. freezing medium (10% FBS DMEM with L-glutamine and 15% v/v
dimethyl sulfoxide) was added to 60 ml of 1.times.10.sup.7 cells/ml
cell suspension, the cells were mixed thoroughly, and 1 ml were
aliquoted into each of 100 cryovials. The cells were frozen at
-60.degree. C. in Nalgene freezer containers, and then transferred
to storage in liquid nitrogen. The frozen cells represented the
MDCK cells at passage 61. These vials were designated MDCK Pre-MCB
lot 2. This bank was deposited with the ATCC and is identified by
ATCC Accession Number PTA-6500.
[0270] Next, one vial of the MDCK Pre-MCB lot 2 (Passage 61) was
thawed and inoculated into a T75 flask containing 35 ml DMEM and
10% FBS. Cells (passage 62) were incubated at 37.degree. C. with 5%
CO.sub.2 for 3 days. On day 3 the cells were passaged to
2.times.T75 flasks (passage 63). An additional 3 passages to a new
T75 flask were performed followed by a passage to a T225 flask
(Passage 67).
[0271] Next, the cells were trypsinized and cloned by a dilution
method. In particular, the cells were seeded at 0.5 cells per 100
.mu.L per well in 96 well plates (1:1 ratio of fresh to conditioned
media). The next day, cells were visualized under the microscope
and wells which contained one cell were identified, then the plates
returned to incubate. After 7 days incubation, the plates were
checked to assess cell growth and another 100 .mu.L fresh growth
medium was added to each well. Three days later, a complete medium
exchange (200 .mu.L per well) was performed. Two weeks after
initial cloning seeding, cells were trypsinized and passaged to 2
sets of 24 well plates if they reached 100% confluence. If cells
had not reached 100% confluence, they were refed with fresh growth
medium.
[0272] The clones were expanded sequentially (24 well
plate.fwdarw.T25 flasks or 6 well plates.fwdarw.T75 or T225 flask)
and a total of 54 clones were selected as shown in Table 1, below,
and frozen at either passage 4 or 5 post cloning in 10% FBS DMEM
with 7.5% DMSO and stored in liquid nitrogen. In addition, clones
56, 57 and 58 were isolated from a second round of screening
performed essentially as described above.
TABLE-US-00001 TABLE 1 List of 54 of Serum MDCK Clones (in freezing
order) Clone ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
21 22 23 24 25 26 27 28 29 30 31 55 33 34 35 36 37 38 39 40 41 42
43 44 45 46 47 48 49 50 51 52 53 54
[0273] Initial screening of virus productivity of clones was
performed using one of the sets of 24 well plates produced above.
To do so, the cells, cultured for 3 days in DMEM with 4 mM
glutamine, were infected with influenza strain A/New Calcdonia
reassortant at an MOI of 0.001. 500 mU/ml TPCK trypsin was added
once at time of infection. Virus titer was determined using the
semi automated TCID.sub.50 assay (n=12 per sample) as described in
Example 5, below. The virus titer obtained from each clone varied
from 7.0 to 8.5, with a distribution as shown in Table 2.
TABLE-US-00002 TABLE 2 Distribution of Viral Titers from 54 clones
grown in DMEM containing 10% FBS Titer Range Number of Clones
(Log.sub.10 TCID.sub.50/mL) (total of 54) 7.0-7.5 6 7.6-8.0 35
8.1-8.5 12 >8.5 1
[0274] Based on the virus productivity data, six clones were
selected for further analysis: clones 1, 5, 36, 39, 40, and 55. The
clones were expanded in T-flasks and a set T-25 (8 flasks per
clone) were used for infection with A/New Calcdonia, A/Panama and
B/Jilin reassortants at MOI 0.001 using DMEM+4 mM glutamine as the
post infection media (2 flasks per virus strain). These flasks were
harvested 4 days post infection and the samples from each flask
were analyzed for potency using the semi automated TCID.sub.50
assay (n=12 per flask, n=24 per virus strain) of Example 5. Results
from these experiments are presented as FIG. 1, which shows that
the clones 1 and 55, which are the highest producers for
reassortant A/New Calcdonia, are also the highest producers for
A/Panama and B/Jilin reassortants. Accordingly, clone 1 was
selected for further subcloning and adaptation to serum free
medium.
[0275] In additional rounds of screening performed as described
above, more than 1000 clones were screened for ability to produce
high titers of A/New Calcdonia. Sixty three of these clones
screened for ability to produce high titers of A/Panama and B/Jilin
reassortants, none produced more virus than clone 1. Accordingly,
none of these clones was selected for further study and no data
relating to these clones is presented herein.
[0276] Next, clone 1 (P4/P71, 4 passages since isolation from a
single clone, 71 total passages) was thawed and inoculated into a
T75 flask containing 35 ml of Dulbecco's Modified Eagle's
Medium/Ham F12 with L-glutamine (DMEM/F12) and 10% FBS. Cells were
incubated at 37.degree. C. with 5% CO.sub.2 for 3 days. On day 3,
the cells were passaged to a T225 flask. Then cells were passaged 8
times in either a T75 or T225 flask every 3 or 4 days after
seeding. After these passages, the cells (P13/P80) were trypsinized
and sub-cloned by dilution as follows.
[0277] The cells were seeded at 0.5 cells per 100 .mu.L per well in
10.times.96 well plates (1:1 ratio of fresh to conditioned media).
The next day, cells (P1/P81, P1 since subclone, P81 total passage)
were visualized under the microscope and wells were marked that
contained one cell per well. The cells were allowed to grow for 7
days and the plates were checked to see if the marked wells
contained growing cells. The cells were fed with 100 .mu.L fresh
growth medium at this time point, and then a complete medium
exchange (200 .mu.L per well) was performed 3 days later. Two weeks
after initial cell seeding, single cell clones were trypsinized and
passaged to a 96 well plate if they reached>50% confluence.
Cells less than 50% confluent were refed with fresh growth medium
and allowed to continue to grow. The clones which reached>50%
confluence were expanded sequentially (24 well plate.fwdarw.6 well
plates.fwdarw.T75 flask) and a total of 63 subclones were frozen at
either passage 5 or 6 since the beginning of this round of
subcloning in 10% FBS DMEM/F12 with 7.5% DMSO and stored in liquid
nitrogen.
[0278] During clone expansion, clones were also set up in
3.times.96 well plates for virus infection (A/Panama and B/Jilin
reassortants) at a MOI of 0.001. Cells were grown in DMEM/F12 with
4 mM glutamine, cells were infected at 3 days post seeding using
DMEM/F12 with 4 mM glutamine as the post-infection media, and
viruses were harvested 4 days post infection and stabilized with
sucrose phosphate. The A/Panama virus titer was determined using a
FFA assay as described in Example 4, below. A/Panama virus titer
produced by each subclone varied from 7.0 to 8.5, with a
distribution as shown in Table 3, below.
TABLE-US-00003 TABLE 3 Distribution of Viral Titers from 63
subclones of clone 1 grown in DMEM + 10% FBS Number of Subclones
Titer Range (Log.sub.10 FFU/ml) (total of 63) <6.1 28 6.2-6.9 18
7.0-7.5 13 .gtoreq.7.6 (less than 8.0) 4
[0279] Of the 63 clones, MDCK subclone 1-A (P6/P86), subclone 1-B
(P5/P85) and subclone 1-C (P6/P86) produced virus titer of 7.6
log.sub.10 FFU/ml, while subclone 1-D produced a virus titer of 7.8
log.sub.10 FFU/ml.
Example 2
Adaptation of an MDCK Cell Clones to Growth in Serum-Free Media
[0280] This example describes adaptation of MDCK Clones 1, 55, 56,
57, and 58 and Subclones 1-A, 1-B (P5/P85), 1-C and 1-D to growth
in MediV 105 serum-free media. Clones 56, 57, and 58 were derived
from MDCK cells (ATCC Accession No. CCL-34) and adapted to growth
in media containing serum in a manner similar to that described in
Example 1. The process is outlined in FIG. 5B.
[0281] First, one vial of the MDCK Clone Subclone 1-D (frozen at
passage 5 since subclone, P85 in total) was thawed and inoculated
into a T75 flask containing 35 ml of Dulbecco's Modified Eagle's
Medium/Ham F12 (DMEM/F12) with L-glutamine and 10% fetal bovine
serum (FBS, Defined), and incubated at 37.degree. C. with 5%
CO.sub.2 for 3 days. On day 3 the cells were passaged to a T225
flask (Passage 7/P87). Next, the MDCK Subclone D cells were adapted
in serum-free medium MediV 105 for 5 passages.
[0282] At passage 5 in MediV 105, cells were frozen as an accession
bank. In addition, one flask of cells (clone 1-D) was set up to
check cell stability in MediV 105 serum free medium. SF MDCK
Subclone D cells started dying after 8 passages in MediV 105 serum
free medium.
[0283] In addition, serum MDCK clones 1, 55, 56, 57, and 58 and
subclones 1-A, 1-B (P5/P85), and C were adapted to MediV 105 serum
free medium. First, one vial of each serum MDCK clones 1, 55, 56,
57, and 58 and subclones 1-A, 1-B (P5/P85), and C were thawed into
a T75 flask containing 35 ml of 10% FBS DMEDM/F12 medium and
incubated at 37.degree. C. with 5% CO.sub.2 for 3 days. The cells
were trypsinized and seeded into a new T225 flask at
5.times.10.sup.4 cells/ml seeding density. On day 3 after seeding,
cells were passaged to a T75 flask in serum containing growth
medium.
[0284] For each of the passages in DMEM/F12 with L-glutamine and
10% FBS in a T75 or T225 flask, the procedure was as follows. Cells
were washed two times with Dulbecco's Phosphate Buffered Saline
without Ca.sup.++ and Mg.sup.++ (DPBS), and 3 ml (for the T75
flask) or 7.5 ml (for the T225) of TrypLE were added to the cells.
The cell monolayer was incubated and allowed to release for 15-20
minutes, at which time 3 ml (for T75) or 7.5 ml (for T225 flask) of
10% FBS DMEM/F12 with L-glutamine was added to neutralize the
TrypLE activity. The cells were then counted by Cedex cell count,
and the amount necessary to inoculate 5.times.10.sup.4 cells per ml
was transferred to a T75 flask or T225 flask containing sufficient
media to bring the volume to 35 ml (T75) or 100 ml (T225) of 10%
FBS DMEM with L-glutamine, and incubated as above for 3 or 4
days.
[0285] Next, each of the clones in T75 flasks (3 passages in serum
medium after thawing) was adapted for growth in MediV 105 serum
free medium. Cells from the T75 flasks were passaged for 3 passages
in T75 flasks containing 35 ml of MediV 105. At the fourth passage,
the cells were passaged to a T225 flask containing 100 ml MediV
105. Cells from the T225 flask were seeded to 2 or 3 T225 flasks
based on cell count for the fifth passage. On day 3 or day 4 post
seeding, clones 1, 56, and 57, and subclones 1-A, 1-B, 1-C, and 1-D
were frozen as an accession bank. Clones 55 and 58 were each
passaged an additional time in serum free medium before the cells
were banked.
[0286] For each of the passages in MediV 105 serum free media in a
T75 or T225 flask, the procedure was as follows, for the first
passage to MediV 105, spent medium from the T75 flask with cells at
passage 3 after vial thawing was removed, the cells were washed
with DPBS, 3 ml of TrypLE was added, and the cells were incubated
and allowed to release for 15-20 minutes. Then 3 ml of lima bean
trypsin inhibitor solution (Worthington) were added to neutralize
the TrypLE, and the cells were counted by Cedex cell counter. The
amount of cells necessary to inoculate 5.times.10.sup.4 cells per
ml of media was transferred to a T75 flask containing 35 ml of
MediV 105. All flasks were incubated at 37.degree. C. with 5%
CO.sub.2 for 3-4 days at which time the cells were again
enzymatically detached as described above except using 2.5 ml (for
T75) or 5 ml (for T225) of TrypLE, then 2.5 ml (for T75) or 5 ml
(for T225) of lima bean trypsin inhibitor solution used to stop
TrypLE activity, and cell suspensions were transferred to flasks
with fresh serum free medium. All seedings were calculated to
inoculate 5.times.10.sup.4 cells per ml of media.
[0287] For the banking of each clone/subclone the procedure was as
follows: Cells from the multiple flasks were trypsinized, pooled,
and trypsin neutralizing solution was added. The cells were then
mixed and counted. The cells were centrifuged, and resuspended in
saved spent medium to make 1.times.10.sup.7 cells/ml cell
suspension. Next, 2.times. freezing medium (MediV 105 with 15% v/v
dimethyl sulfoxide, and an equal volume of spent medium) was added,
the cells were mixed thoroughly, and 1 ml aliquots were placed into
2 ml size cryovials. These vials were designated as SF MDCK
accession banks. The cells were frozen at -60.degree. C. in Nalgene
freezer containers, and then transferred to storage in liquid
nitrogen. FIG. 5 is a flow chart of the entire selection and
adaptation process for clone 1 and subclone 1-B.
[0288] Along with banking, a T75 flask of each serum free-adapted
clone was set up for cell growth stability in serum free medium and
virus infectivity study. In the study, cells were seeded at
5.times.10.sup.4 cells per ml of media and passaged every 3 or 4
days after seeding. Clones 56 and 57 started dying at sixth passage
in MediV 105.
[0289] Each of the other clones and subclones were continued to be
cultured in MediV 105. At Passage 9 or 10, 5 T75 flasks for each
clone were set up for virus infection. The clones were infected
with reassortants of A/New Calcdonia, A/Hiroshima, B/Malaysia and
A/Vietnam at MOI 0.001, using DMEM/F12 with 4 mM glutamine+500
mU/ml TPCK trypsin as the post infection media. Viruses were
harvested at 3 and 4 days post infection and stabilized with
10.times. sucrose phosphate buffer. Virus titers were determined by
FFA assay as described below in Example 4. The results of these
experiments (data not shown) showed that the subclones of clone 1
produced more virus than the other tested MDCK cell clones.
Accordingly, subclones 1-A, 1-B, and 1-C were selected for further
experiments to assess cell growth in MediV 105. Results of this
experiment are shown as FIG. 2. As shown in FIG. 2, each of
subclones 1-A, 1-B, and 1-C exhibited essentially similar growth
characteristics.
[0290] In addition, subclones 1-A, 1-B, and 1-C were retested for
virus infectivity at passage 12. 9.times.T75 flasks for each
subclone were infected with influenza viruses under the same
conditions as at passage 9, described immediately above, in
duplicate (2 T75 flasks per clone per virus strain. Results from
this experiment are presented as FIG. 3. As shown in FIG. 3, each
of the subclones supported growth of the tested viruses to
relatively high titer, and none of the different subclones
supported the highest titer of each tested virus strain.
[0291] Finally, to assess the effects of the MediV 105 medium on
virus growth, virus infectivity was assessed for subclones 1-A,
1-B, and 1-C in both MediV 105 and OptiPro.TM. medium (GIBCO),
while subclone 1-D was tested in OptiPro.TM. media alone, as
described above. Tabular results from each of these virus
infectivity experiments are presented as FIG. 4. As shown in FIG.
4, no significant difference in virus productivity was observed
between the two media.
Example 3
Comparison of MDCK Cell Growth in MediV 105 and M18M
[0292] This example describes the results of an experiment to
assess the relative growth of MDCK cells in MediV 105 and M18M
media. The formulations of MediV 105 and M18M are described in
Example 10, below.
[0293] In these experiments, 1 vial of serum free-adapted subclone
1-A was thawed and inoculated into a T-75 flask containing either
MediV 105 or M18M, respectively. The T-75 flasks were then placed
in a 37 C incubator supplied with 5% CO.sub.2 and the cells were
allowed to grow under these conditions for 3 to 4 days. Cell growth
rate and viability were monitored the end of incubation by
trypsinizing the cells from the T-75 flasks followed by counting
the total and viable cells using a Cedex cell counter or over the
next 88 hours by Cedex and/or NucleoCounters.
[0294] Results from this experiment are presented as FIG. 6. As
shown in FIG. 6, subclone 1-A was able to replicate in both MediV
105 and M18M. However, cells decreased in viability over time in
M18M, while cell viability in MediV 105 remained relatively
constant. In addition, the doubling times of the MDCK cells were
calculated and are presented as FIG. 7. FIG. 7 indicates that the
doubling time of MDCK cell subclone 1-A was 39 hours in MediV 105
and 36 hours in M18M.
Example 4
Comparison of MDCK Cell Growth on Different Microcarriers
[0295] This Example describes the results of experiments designed
to assess the growth of MDCK cells in M118 media using different
microcarriers. In particular, growth of MDCK cells was compared for
the microcarriers cytodex 1, cytodex 3, cytopore 1, and cytopore 2
(GE Healthcare).
[0296] In the experiments, MDCK cell subclone 1-A was inoculated
into a 125 ml flask containing M18 media. Next, 2 g/L cytodex 1,
cytodex 3, cytopore 1, or cytopore 2, respectively, was added to
each flask. The density of unattached MDCK cells was determined at
30 and 60 minutes post-inoculation as shown in FIG. 8. As seen in
FIG. 8, the MDCK cells attached quickly to each of the different
microcarriers, and ultimately attached better to the cytopore
microcarriers than the cytodex microcarriers.
[0297] In addition, the cells were grown for approximately 5 days
in M18 in the presence of the different microcarriers (30 ml
microcarrier w/v in 125 ml media shaken at 120 RPM), and the total
cell density determined daily by trypsinization followed by Cedex
counting. Results from this experiment are presented as FIG. 9. As
shown in FIG. 9, the cytodex microcarriers yielded greater cell
densities relative to the cytopore microcarriers. Further, cytodex
3 yielded a greater cell density than cytodex 1.
Example 5
Replication of Influenza Viruses in MDCK Cells
[0298] T-75 flasks were seeded at 5.times.10.sup.4 cells/mL (35 mL
of DMEM+10% FBS+4 mM glutamine) and grown in an incubator
maintained at 37.degree. C. and 5% CO.sub.2 for 3 days. Cells in
one of these T-flasks were trypsinized with trypsin EDTA and
counted using the Trypan-Blue Exclusion method. The remaining
T-flasks were then infected as follows. The growth media was
aspirated off and cells washed twice with 10 mL DPBS (no
Ca.sup.2+/Mg.sup.2+) per flask. The amount of virus to infect each
T-flask at the desired multiplicity of infection (MOI) of (e.g.,
0.01 to 0.001) was determined as per the equation below:
Amount of virus ( mL ) = Total Cells per flask * M O I 10 ^ ( log T
C I D 50 / mL ) ##EQU00001##
[0299] MOI being defined as the virus particles per cell added
[0300] The required amount of virus was then added to 35 mL of post
infection medium in each T-flask. (DMEM+4 mM glutamine+500 mU/mL
TPCK trypsin). The T-flasks were then incubated at 33.degree. C.,
5% CO.sub.2 and samples taken each day for 6 days. One tenth volume
of sample volume of 10.times.SP was added to each sample as a
stabilizer and the samples were stored at <-70.degree. C. prior
to testing for infectivity.
[0301] The concentration of virus present in each sample was
determined according to a median tissue culture infectious dose
(TCID.sub.50) assay that measures infectious virions. Briefly, MDCK
cells were grown to confluent monolayers in 96-well microtiter
plates and a serial dilutions of ca/ts influenza virus sample was
added. The samples in the MDCK cell assay plate were typically
diluted to final dilutions of 10.sup.-4 to 10.sup.-10. The wells in
columns 1-5 and 8-12 contained virus-diluted sample and wells in
columns 6-7 received only virus diluent and served as cell
controls. This format produced two data points (n=2) for each
sample dilution per plate. Replication of virus in the MDCK cells
resulted in cell death and cytopathic effect (CPE). It also
released progeny viruses into the culture supernatant. The progeny
virus infected other cells, repeating the infection and resulting
in the eventual destruction of the monolayer. Infection of
monolayer cells lasted for a period of six days at 33.+-.1.degree.
C. in a CO.sub.2 environment. The plates were then removed from the
incubator, the media in the wells discarded, and 100 .mu.l of
MEM/EBSS+1.times. non-essential amino acids+2 mM
glutamine+penicillin/streptomycin+MTT was added to each well. The
plates were incubated for 3-4 hrs at 37.degree. C. 5% CO2 and the
number of wells showing CPE was determined by visual inspection of
the color formed in each well (yellow/orange signifies CPE wells
and solid purple signifying no CPE). The number of wells showing
CPE in each half plate was used to calculate the titer (log.sub.10
TCID.sub.50/mL) based on the Karber modification of the Reed-Muench
method.
Example 6
Fluorescent Focus Assay for Viral Growth
[0302] MDCK cells were grown in 96 well black plates over 4 days in
DMEM/EBSS+1.times. non-essential amino acids+2 mM
glutamine+PEN/Strep. Each well was then infected with the serially
diluted viral samples (e.g., ca/ts influenza B-strains (B/Hong
Kong/330/01 and B/Yamanashi/166/98)) and incubated for
approximately 20 hrs at 33.+-.1.degree. C. in a CO.sub.2
environment. The virus infected plates were fixed and
immuno-stained as follows to determine the virus titer of the
samples. The medium containing virus was removed from each plate
and the plates washed once with 200 .mu.l/well with DPBS (no
Ca2+/Mg2+) followed by fixation in 200 .mu.l/well of cold 4% (v/v)
formalin in PBS for 15 minutes. The plates were washed twice with
200 .mu.l/well of DPBS (no Ca.sup.2+/Mg.sup.2+) followed by
incubation of the cells with primary antibody specific for either A
strains or B strains. The primary antibodies were diluted to the
desired dilution in 0.1% saponin, 1% BSA in PBS. After incubation
for an hour, the primary antibody was removed, cells were washed
thrice with 0.1% Tween 20 in PBS, and the wells were incubated with
fluorescent dye conjugated secondary antibody (e.g., rabbit anti
sheep labeled with FITC) prepared to the desired dilution in 0.1%
saponin, 1% BSA in PBS. After washing twice as described above and
blot drying with paper towels the wells with fluorescent staining
were visualized daily using a fluorescence microscope and the
images were taken daily using SPOT program.
Example 7
Assays for Testing MDCK Cells for Karyology, Tumorigenicity, and
Adventitious Agents
[0303] This example describes representative assays suitable for
testing MDCK cells for karyology, tumorigenicity, and the presence
of adventitious agents.
[0304] Karyology Testing:
[0305] Briefly, MDCK cells for testing are grown in T-225 flasks,
maintained and subcultured as described above. When the cells are
thought to have enough mitotic cells, the cells are harvested for
mitotic analysis. The cells are then treated with colcemid (0.02
.mu.g/mL) for 150 minutes at 37.degree. C. The cells are then
harvested by trypsinization, and centrifuged for 5 minutes at
200.times.g. The supernatant is aspirated off and the cells
resuspended in prewarmed hypotonic solution and incubated at
37.degree. C. for 10 minutes. The swollen cells are pelleted by
centrifugation and then fixed by incubation in Carnoy's solution
(3:1 methanol:glacial acetic acid) at room temperature for 40
minutes. The cells are again centrifuged and washed at least twice
with Carnoy's fixative. After the last centrifugation, the cells
are resuspended in 1 to 3 ml of fresh fixative to produce an
opalescent cell suspension. Drops of the final cell suspension are
placed on clean slides and air dried.
[0306] Cells are stained by addition of Wright's stain solution in
phosphate buffer to the slides and incubating for 7-10 minutes. The
slides are then washed with tap water after 7-10 minutes and then
air dried. The cells are scanned with low power objectives
(10.times.) to find cells in the metaphase stage of cell division
and the chromosomes of cells in metaphase are analyzed via a high
power oil immersion lens (100.times.). About 100 cells in metaphase
are analyzed for cytogenic abnormalities and chromosome count.
About 1000 cells are scanned to determine polyploid frequency and
mitotic index (percent of cells under going mitosis).
[0307] Sterility Testing: Bacteriostatic, Fungistatic and Four
Media Sterility
[0308] Bacteriostatic and Fungistatic test determine whether there
is any inhibitory effects to the growth of control organisms (e.g.,
Bacillus subtilis, Candida albicans, Clostridium sporogenes,
Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus Niger)
in a test sample. Briefly, the test article is inoculated into
three tubes of TSB (soybean-casein digest medium), four tubes of
THIO (fluid thioglycollate medium), two tubes of SAB (Sabourand
Dextrose Agar) and one tube of PYG (peptone yeast extract). Each
control organism inoculum containing less that 100 cfu of control
organism is then inoculated into the appropriate media type.
Positive controls may consist of Bacillus subtilis in TSB and THIO,
Candida albicans in TSB and SAB (at 20-25.degree. C. and
30-35.degree. C.), Clostridium sporogenes in THIO and PYG,
Pseudomonas aeruginosa, Staphylococcus aureus and Aspergillus niger
in THIO and/or TSB. The negative control is sterile PBS. The media
are incubated for 3-5 days and checked for growth of organisms.
[0309] To test whether a test culture meets sterility requirements
defined in USP 26, EP and 21CFR610.12, the test culture is
inoculated in two tubes of TSB (soybean-casein digest medium), two
tubes of THIO (fluid thioglycollate medium), three tubes of SAB
(Sabourand Dextrose Agar) and two tubes of PYG (peptone yeast
extract). The media are incubated at appropriate temperatures (SAB
slants are incubated at two temperatures) and all tubes observed
over a 14 day period with the tubes checked on third/fourth or
fifth day, seventh or eight day and fourteenth day of testing. Any
test article inoculated tubes which appear turbid are plated out
and gram stains are performed on the plate to determine the gram
stain type of the organism(s) contained in the test sample.
Negative controls are sterile PBS.
[0310] Mycoplasma/Mycoplasmastasis Assay
[0311] The cells are expanded and cultured in T-flasks as explained
above. Cell lysates at a concentration of 5.times.10.sup.5 cells/mL
are prepared and frozen at -70.degree. C. The test article is then
tested for ability to inhibit growth of Mycoplasmapneumoniae,
Mycoplasma orale and Mycoplasma hyorhinis either in agar
broth/plates and/or in VERO cells.
[0312] For the agar isolation assay, the test article is tested
either spiked or unspiked on agar plates or broth bottles. The test
article is spiked with Mycoplasmapneumoniae and Mycoplasma orale to
achieve a dilution of 10 to 100 cfu/0.2 mL (for the Agar test) and
10 to 100 cfu/10 mL (for the semi broth assay). A portion of the
test sample is not spiked. 4 semi solid broth bottles are
inoculated with 10 ml each of spiked (2 bottles) or unspiked (2
bottles). One bottle each of spiked/unspiked is incubated either
aerobically or anaerobically at appropriate temperatures. 10 type A
agar plates and 10 type B agar plates are inoculated with each
spiked sample or unspiked sample. Half the type A agar plates and
type B agar plates are incubated either aerobically or
anaerobically at appropriate temperatures. Uninoculated mycoplasma
semi-solid broth serves as the uninoculated negative control. All
broth bottles are observed for 21 days. Each broth bottle (with
exception of uninoculated negative control) is subcultured on days
3, 7 and 14 onto Type A agar plates or Type B agar plates (10
plates each, 0.2 mL/plate) and incubated under the same conditions
as the appropriate bottle. They are examined once a day for 21
days.
[0313] For the enhanced VERO cell culture assay, the test article
is tested spiked or unspiked. The test article is spiked with M.
orale and M. hyorhinis at a concentration of 10-100 cfu/0.2 mL. The
spiked test articles, unspiked test articles, positive controls and
negative controls are each inoculated onto T-75 flasks of VERO cell
cultures. After 3-5 days of incubation, the cells from each flask
are scraped and snap frozen. Two tenths of one mL of cell lysate
from each flask is inoculated into each of well of a six well plate
containing VERO cells. In addition, positive and negative controls
are inoculated into appropriate wells of six well plates containing
VERO cells. After 3-5 days, the cells are fixed and stained with
DNA binding HOECHT dye and evaluated for presence of
mycoplasma.
[0314] Tumorigenicity Test in Nude Mice:
[0315] Evaluation of tumor formation in nude (nu/nu) athymic mice
is performed as follows. Briefly, about two hundred thirty athymic
mice (4 weeks old) are each injected subcutaneously with 0.2 mL
(1.times.10.sup.7 cells/mice) of either positive control (HeLa
cells), negative control (Phosphate buffered Saline (PBS)) or the
test cells (MDCK cells). The animals are randomized before
injection and all mice are injected using a 22 gauge needle on the
same day. All animals are observed every working day and the
injection site is palpated twice a week for lesion development for
a period of eighty four days. Each lesion is measured and the
animals are held as long as there is no visible increase in size of
the lesion, for a maximum of 6 months. Animals that appear moribund
will be euthanized. These animals and all mice surviving to the end
of 6 month observation period are sacrificed and necropsied. The
injection site, lungs, scapular lymph nodes and gross lesions are
analyzed by histopathological methods.
[0316] Additional Assays
[0317] Other exemplary PCR and/or antibody-specific tests for
available viral agents are conducted, as shown in Table 4,
below.
TABLE-US-00004 TABLE 4 Additional Testing Procedures General tests
PCR*/Ab specific Sterility AAV Types 1&2 Mycoplasma HCMV
Adventitious agents in vitro EBV (multiple cell lines) Adventitious
agents in vivo HSV PERT Hepatitis A, B & C Co-cultivation HHV
6, 7 & 8 Karyology HIV 1&2 Electron microscopy HPV
Tumorigenicity using intact cells HTLV I & II Oncogenicity
using cellular DNA Polyomavirus (BK and JC viruses) Oncogenicity
using cellular lysate Circovirus Bovine viruses per 9CFR Canine
Parvovirus Porcine viruses per 9CFR Canine distemper Adenovirus
SV40
Example 8
Process and Formulation of Vaccine Material
[0318] Use of a highly scalable microcarrier technology, similar to
that used for the production of the currently licensed Polio
vaccine, can be applied to the production of influenza in MDCK
cells, as discussed in Example 4, above. Spherical beads made of
dextran support excellent growth of MDCK cells and in 2 to 10 L
bioreactors. Parental MDCK cells grown in MediV 105 or OptiPro.TM.
medium were found to be capable of growing on Cytodex 3
microcarriers to a density of 2.times.10.sup.6 nuclei per mL in
batch mode in both spinner flasks and MDCK cells have been grown to
>2.5.times.10.sup.6 cell/mL in bioreactors up to 10 L scale.
[0319] These MDCK cells (or similar non-adherent MDCK cells) are
tested for production of vaccine influenza strains to high titer in
a serum-free process and compared to the productivity obtained
using serum grown cells in T-flasks. For clinical manufacturing,
influenza virus is produced in MDCK cells at the 20 L or 150 L
scale, while commercial scale production utilizes bioreactors up to
about 2,500 L. FIG. 10 outlines one process that may be used for
cell culture scale up to commercial production levels. The working
cell bank is first expanded sequentially from a T-75 flask to T-225
flasks to 1 liter spinner flasks to a 20 liter then 300 liter
bioreactors which are finally expanded to a 2500 liter bioreactor.
When the optimal cell density is obtained the culture is inoculated
with the vaccine strain. The virus is then bulk harvested from the
culture supernatant. Example 12 details the implementation of
single use bioreactors (SUBs) for the production of high titer
viral material, which may be used for the production of vaccine
material.
[0320] The purification process for cell culture based influenza
vaccines is modeled on purification of egg-based influenza vaccines
(see, e.g., PCT Publication WO 05/014862 and PCT Patent Application
PCT/US05/035614 filed Oct. 4, 2005). The purification of viral
vaccine materials from cells may include any or all of the
following processes, homogenation, clarification centrifugation,
ultrafiltration, adsorption on barium sulfate and elution,
tangential flow filtration, density gradient ultracentrifugation,
chromatography, and sterilization filtration. Other purification
steps may also be included. For example, crude medium from infected
cultures or virus harvest can first be clarified by centrifugation
at, e.g., 1000-2000.times.g for a time sufficient to remove cell
debris and other large particulate matter, e.g., between 10 and 30
minutes. Alternatively, the medium is filtered through a 0.8 .mu.m
cellulose acetate filter to remove intact cells and other large
particulate matter. Optionally, the clarified medium supernatant is
then centrifuged to pellet the influenza viruses, e.g., at
15,000.times.g, for approximately 3-5 hours. Following resuspension
of the virus pellet in an appropriate buffer, such as STE (0.01 M
Tris-HCl; 0.15 M NaCl; 0.0001 M EDTA) or phosphate buffered saline
(PBS) at pH 7.4, the virus may be concentrated by density gradient
centrifugation on sucrose (60%-12%) or potassium tartrate
(50%-10%). Either continuous or step gradients, e.g., a sucrose
gradient between 12% and 60% in four 12% steps, are suitable. The
gradients are centrifuged at a speed, and for a time, sufficient
for the viruses to concentrate into a visible band for recovery.
Alternatively, and for most large scale commercial applications,
virus is elutriated from density gradients using a zonal-centrifuge
rotor operating in continuous mode.
[0321] A feature which may be included in the purification of viral
vaccine materials from cells is the use of Benzonase.RTM., a
non-specific endonuclease, early in the process. While MDCK
cellular DNA does not pose an oncogenic risk based on studies
evaluating oncogenicity of cellular DNA, Benzonase.RTM. treatment
would virtually eliminate any potential or hypothetical risk. In
one purification process, following Benzonase.RTM. treatment, the
material is clarified by direct flow filtration (DFF) which will
also remove any residual intact mammalian cells in the bulk
material. The filtered bulk is then concentrated by tangential flow
filtration (TFF) prior to further purification steps. Purification
methods including affinity chromatography as well as ion-exchange
chromatography and/or hydroxyapatite which, have worked well for
other viral systems are useful for cell culture based influenza
vaccine production. The highly purified viral material obtained by
the process developed is then utilized in the production of vaccine
material. For example, for use in a live attenuated vaccine
production (e.g., FluMist.RTM.) the viral material may be subjected
to a buffer exchange by filtration into a final formulation
followed by a sterilization step. Buffers useful for such a
formulation may contain 200 mM sucrose and a phosphate or histidine
buffer of pH 7.0-7.2 with the addition of other amino acid
excipients such as arginine. If necessary for stabilization,
protein hydrolysates such as gelatin (e.g., porcine, avian, piscine
gelatin) may also be added. Ideally the vaccine material is
formulated to be stable for an extended storage time. One method
which may be utilized to extend storage time is spray drying, a
rapid drying process whereby the formulation liquid feed is spray
atomized into fine droplets under a stream of dry heated gas. The
evaporation of the fine droplets results in dry powders composed of
the dissolved solutes (see, e.g., US Patent Publication
2004/0042972). Spray drying offers the advantages of ease of
scalability and manufacturing cost as compared to conventional
freeze-drying processes. Alternatively, the vaccine material is
formulated to be stable as a refrigerator stable liquid formulation
using methods known in the art. For example, methods and
compositions for formulating a refrigerator stable attenuated
influenza vaccine are described in PCT Patent Application
PCT/US2005/035614 filed Oct. 4, 2005.
[0322] In-process characterization steps are incorporated into the
purification scheme to monitor the production. Characterization
steps which may be utilized include but are not limited to the
Fluorescent Focus Assay (described as Example 6, and known in the
art, see e.g., Stokes et al., 1988, J Clin Microbiol. 26:1263-6)
which uses a simple antibody binding and fluorescent staining
method to determine virus infectivity. Total protein and DNA
determination which may be performed using numerous methods known
to one of skill in the art are used to determine the percent of the
initial impurities remaining. The specific activity of the
preparation may be determined by calculating the viral infectivity
per quantity of vaccine (e.g., infectivity/mg).
[0323] Outlined in FIG. 11A is one purification process that may be
used. Briefly, the monovalent influenza viral harvest is stabilized
with a suitable buffer (e.g., sucrose-phosphate buffer). Benzonase,
a non-specific endonuclease, is then added to the stabilized viral
harvest to break down DNA to less than 300-basepair fragments.
After Benzonase treatment, the virus harvest is subjected to
filtration to remove any remaining intact MDCK cells and most
cellular debris. In particular, Direct Flow Filtration (DFF) can be
utilized. Various filter membranes with different pore sizes,
membrane compositions, and configurations (such as multimedia or
single filter) and process parameters, including maximum flow rate
and scale-up factor, are readily determined. The clarified virus
harvest is then concentrated by Tangential Flow Filtration (TFF)
using ultrafiltration membranes and the concentrated virus is then
diafiltered against a suitable buffer (e.g., sucrose-phosphate
buffer). The concentrated, diafiltered harvest is then subjected to
column chromatography or membrane chromatography. Affinity
chromatography and ion-exchange chromatography may be used to
further remove MDCK cell protein and DNA. The chromatographically
purified virus harvest then concentrated and diafiltered into a
formulation buffer and, finally, subjected to sterile filtration.
Outlined in FIG. 11B is an alternative purification process that
may be used which combines the Benzonase.RTM. step with affinity
chromatography. The use of such a process can reduce downstream
processing steps. Briefly, the monovalent influenza viral harvest
is stabilized with a suitable buffer (e.g., sucrose-phosphate
buffer). The stabilized virus is clarified by filtration, for
example by Direct Flow Filtration (DFF) using 1.2- and 0.45-.mu.m
filters. The clarified virus is then conditioned/concentrated by
TFF using ultrafiltration membranes and the concentrated virus is
then diafiltered against a suitable buffer (e.g., sucrose-phosphate
buffer) using, for example 500 KD TFF (5.times.UF/5.times.DF). The
conditioned virus is then subjected to on column Benzonase.RTM.
treatment and the purified virus eluate is then concentrated and
diafiltered into a formulation buffer using for example, 500 KD TFF
and 8.times.DF processes. The formulated virus bulk is then sterile
filtered, for example through 0.45- and 0.2-.mu.m filters.
[0324] Cellufine Sulfate Chromatography
[0325] It was determined that MDCK DNA contains a Benzonase.RTM.
resistant fragment of .about.12 kB and that was not removed by TFF
or ultracentrifugation using a sucrose density gradient (data not
shown). As described above, chromatography is utilized to ensure
removal of all contaminants. Cellufine Sulfate chromatography resin
consists of sulfate ester covalently bonded on the 6-position of
cellobiose and attached to a cellulose bead. The resin mimics the
affinity of heparin or dextran sulfate. A column chromatography
using Cellufine Sulfate (CS) was tested and demonstrated to
efficiently remove the contaminating DNA band. Briefly, a
2.6.times.2 cm (10 mL) column, was equilibrated in buffer A
(1.times.SP (218 mM sucrose, 11 mM potassium phosphate), pH 7.2)
and the TFF-purified virus (A/New Calcdonia reassortant) was
loaded. The column was washed with 5 column volumes of buffer A and
eluted with a gradient of 0-100% buffer B (1.times.SP+1 M NaCl, pH
7.2). The flow rate was maintained at 3 mL/min. The OD profile is
shown in the left panel of FIG. 12A. Shown in Table 5 are the DNA
content, total HAU and the FFA infectivity of the starting
material, the flow through and the elution fractions from the CS
column.
TABLE-US-00005 TABLE 5 Cellufine Sulfate Chromatography Total DNA
Total HAU FFA Infectivity Sample (.mu.g) (Log.sub.10/mL) (per mL)
TFF/UF material 26.7 5.8 1.5 .times. 10.sup.10 Flow Thru 12.7 4.0
6.5 .times. 10.sup.7 (47%) (1.6%) (0.4%) Elute 8.5 5.75 1.1 .times.
10.sup.10 (32%) (88%) (70%)
[0326] The starting material, the flow through and the elution
fractions from the CS column were analyzed by agarose gel
electrophoresis (FIG. 12A, right panel). The DNA contaminate is
present in both the starting material (lane 2) and the flow through
(lane 3) but is absent in the material eluted from the column (lane
4). These data indicate that the use of this affinity
chromatography resin is more effective than ultracentrifugation
alone at removing contaminants from culture media and host
cells.
[0327] On Column Benzonase.RTM. Treatment
[0328] To reduce handling steps and enhance purity the
Benzonase.RTM. treatment may be combined with Cellufine Sulfate
chromatography. The scheme for degradation of the MDCK dsDNA using
Benzonase.RTM. On-Column Treatment is shown in FIG. 12B.
[0329] The details of the process are as follows: The entire
process is carried out at 22.degree. C. (room temperature).
TFF-purified virus is warmed up to 22-24.degree. C. before
performing chromatography as needed. Loading on the column is based
on total virus infectivity unit per FFA assay. Target loading on
the column is 9-9.5 log.sub.10 FFU per mL of column volume. The
flow rates for equilibration, loading, washings and elution remain
same (155 cm/hr) except the flow rate is reduced as shown in Table
6 while washing with 1.times.SP buffer containing Benzonase.RTM..
The column (1.times.15 cm) is equilibrated with 1.times.SP (218 mM
sucrose -11 mM potassium phosphate, pH 7.0.+-.0.2) until the
conductivity and pH reach 2-3 mS/cm and 7.0.+-.0.2, respectively.
The virus is loaded on the column and the flow through is
collected. After completion of loading, the column is washed (wash
#1) with 1 column volume (CV) of 1.times.SP and the wash is
collected together with the flow through fraction. The column is
then washed (wash #2) at different flow rates (ranging from 0.33,
0.46, 0.65, 0.98, and 1.3 mL/min for each experiment performed by
repeating steps 1-3 with the same virus load material) with 2.5 CV
of 1.times.SP containing 2 mM MgCl.sub.2 and 50 units of
Benzonase.RTM. per mL of 1.times.SP. After wash #2, the column is
washed with another 2 CV of 1.times.SP (wash #3). The virus is
eluted from the column using 1.times.SP containing 1 M NaCl. The
eluted material is collected as soon as the A.sub.280 nm value
reads 5 mAU and the collection is continued until A.sub.280 nm
absorbance value returns to 5 mAU. The column is cleaned with 5 CV
of 0.1 N NaOH and left in base until it is used again. The data for
the chromatography run is captured in the data sheet at the end of
this protocol. Multiple copies of the data sheet may be made for
each chromatography run performed.
TABLE-US-00006 TABLE 6 Benzonase .RTM. Contact Time with Virus and
Flow Velocity for Runs 1-5 Contact Time of Wash Volume Flow Rate
Benzonase with Flow Velocity Run # (wash #2) (mL/min) Virus (min)
(cm/hr) 1 2.5 CV 0.33 102 25 2 2.5 CV 0.46 73 35 3 2.5 CV 0.65 51
50 4 2.5 CV 0.98 34 75 5 2.5 CV 1.3 26 100
[0330] Residual MDCK dsDNA in the eluted chromatography fraction is
quantitated using the PicoGreen quantitation assay kit as described
by Invitrogen. Fluorescence is measured using the Molecular Devices
Gemini EM fluorescence plate reader and the amount of dsDNA
degradation is calculated using SoftMax Pro version 4.8
software.
[0331] Table 7 summarizes the purification yields for several
developmental runs using both the Benzonase.RTM. treatment in bag
and the Benzonase.RTM. treatment on column.
Example 9
Preclinical Animal Models
[0332] The ferret is a robust animal model used to evaluate the
attenuation and immunogenicity of attenuated influenza vaccines and
component vaccine strains. The performance of cell derived
influenza strains produced from the MDCK cell culture are compared
to the same strains produced in eggs. Head to head comparison of
these materials in controlled studies enables a high level of
assurance of the comparability of these viral products.
[0333] In order to evaluate the ability of the two vaccines to
infect or achieve a "take" in the ferret, animals are lightly
anesthetized and inoculated intranasally with either the cell or
egg produced viral preparations. Nasal wash material is collected
at several time points following inoculation and the quantity of
virus is evaluated by one of several available methods in order to
evaluate the kinetics and extent of viral replication in the
animals' upper respiratory tract. Experiments are performed with a
range of doses and include multiple strains and different trivalent
mixtures to generalize the relative infectivity of cell culture
grown strains to egg produced strains. These same studies are also
used to evaluate the immunogenicity of the influenza strains, a
property that is inherently linked to the ability of the virus to
initiate infection. Animals are bled and nasal washes are harvested
at various points (weeks) post inoculation; these specimens are
used to assess the serum antibody and nasal IgA responses to
infection. The culmination of these data, infectivity, serum
antibody and mucosal antibody responses, will be used to compare
and evaluate the relative infectivity of the cell-produced vaccine
to the egg produced vaccine. The most likely outcome is predicted
to be that the cell and egg produced vaccine strains have similar
infectivity and immunogenicity. If the cell derived vaccine
appeared to be more infective or more immunogenic than the
egg-derived product, further studies evaluating the possibility of
lower dosage are performed.
TABLE-US-00007 TABLE 7 Summary of TVCC-1 Downstream Process Dev Run
Dev 2 Dev 3 Dev 4 Dev 5 Dev 6 Dev 7 Dev 8 Dev 9 CTM #2 Strain A/Wis
A/Wis A/NC B/Mal B/Mal A/NC A/Wis B/Mal B/Mal Harvest (hr) 60-65
60-65 60-65 60-65 60-65 60-65 48 48 48 Column BPG200 BPG200 BPG200
BPG200 BPG100 BPG100 BPG100 BPG100 BPG100 Loading (log10/mL) 8.32
8.73 8.76 8.40 9.18 9.44 9.33 8.97 9.56 Process* TVCC-1a TVCC-1a
TVCC-1a TVCC-1b TVCC-1b TVCC-1b TVCC-1b TVCC-1b TVCC-1b VH Titer
(CCD) 8.4 8.4 8.4 8.3 8.6 8.4 8.6 8.0 8.3 VH Titer (PD)
7.7{circumflex over ( )} 8.4 8.6 8.1 8.3 8.3 8.4 7.9 8.3 Final
Titer 8.3 8.5 8.5 8.5 8.9 8.6 9.2 9.0 9.7 Overall Yield .sctn.
60.9% 27.0% 6.3% 25.4% 22.0% 21.2% 30.4% 56.3% 57.5% VH DNA (ng/mL)
n/a 7200 5480 n/a 5720 10800 3440 2070 2050 Bulk DNA (ng/dose) 76.7
n/a 36.4 31.9 n/a n/a 0.4 0.3 0.06 PicoGreen Bulk DNA (ng/dose) n/a
n/a 0.92 n/a 0.23 0.18 0.032 0.164 n/a PCR VH HCP (.mu.g/mL) 278
392 269 231 250 233 135 174 74 Bulk HCP (.mu.g/dose) 1.10 n/a n/a
n/a n/a n/a 0.13 0.29 0.14 Benzonase .RTM. (ng/mL) 0.9 n/a 0.69 2.1
6.6 3.6 0.27 0.52 LOD Note: *TVCC-1a: Benzonase .RTM. treatment in
bag; TVCC-1a: Benzonase .RTM. treatment on column {circumflex over
( )}FFA assay based on Anti-NA instead of Anti-HA .sctn. Based on
VH-PD titer
[0334] A number of immunogenicity and replication studies are
performed in the ferret model to evaluate the cell culture-derived
vaccines with a single unit human dose. Infection with ca/ts/att
strains generally elicits strong and rapid antibody responses in
ferrets. In addition, individual ca/ts/att strains are routinely
tested and shown to express the attenuated (att) phenotype by
replicating to relatively high titers in the nasopharynx but to
undetectable levels in the lung of these animals. The impact of
cell culture growth on these biological traits is also assessed.
However, it is unlikely that any differences will be seen, since
the att phenotype is an integral part of the genetic composition of
these strains. The growth kinetics and crossreactivity of these
strains is evaluated following administration of a single human
dose in these animals. Live attenuated vaccines generated from egg
derived material elicit serum antibodies that cross-react with
multiple strains within a genetic lineage; and it is expected that
a cell-derived vaccine will have the same capability.
[0335] These comparability evaluations should provide significant
insight into potential biochemical and/or biophysical differences
of the primary virus product and demonstrate the impact of these
epigenetic differences on the performance of the ca/ts/att strains
measured by first passaging the virus in human cells or animal
studies. Based on the sequence information to date, there is no
expected impact on the ca/ts/att strains immunogenic performance
resulting from production on MDCK cells.
[0336] Ferrets are a well documented animal model for influenza and
are used routinely to evaluate the attenuation phenotype and
immunogenicity of ca/ts/att strains. In general, 8-10 week old
animals are used to assess attenuation; typically study designs
evaluate n=3-5 animals per test or control group. Immunogenicity
studies are evaluated in animals from 8 weeks to 6 months of age
and generally require n=3-5 animals per test article or control
group. These numbers provide sufficient information to obtain
statistically valid or observationally important comparisons
between groups. During most studies Influenza-like signs may be
noticed, but are not likely. Ferrets do not display signs of
decrease in appetite or weight, nasal or ocular discharge;
observing signs of influenza-like illness is a necessary part of
the study and interventions such as analgesics are not warranted.
Other signs of discomfort, such as open sores or significant weight
loss, would result in appropriate disposition of the animal
following discussion with the attending veterinarian.
Example 10
Formulation of Serum Free Media for Cell Culture
[0337] This Example describes several serum free media formulations
suitable for the culture of cells of the invention. While certain
of such media have been described above, for completeness and ease
of use, each is described in full below.
[0338] Formulation of Taub's Serum-free Media: Taub's media (Taub
and Livingston, 1981, Ann NY Acad. Sci., 372:406) is a serum-free
media formulation that consists of DMEM/HAM F 12 (1:1) containing
4.5 g/L glucose and 4 mM glutamine as the basal media formulation,
to which the hormones/factors are added as indicated in Table
8.
TABLE-US-00008 TABLE 8 Hormones and growth factors added to
serum-free media formulations Name of Component Final Concentration
Insulin 5 .mu.g/mL Transferrin 5 .mu.g/mL Triiodothyronine
(T.sub.3) 5 .times. 10.sup.-12 M Hydrocortisone 5 .times. 10.sup.-8
M Prostaglandin E.sub.1 25 ng/mL Sodium Selenite 10.sup.-8 M
[0339] Taub's SFM is made fresh at the time of passaging or refeed
by the addition of stock solutions of hormone supplements to SF
DMEM/Ham F12 medium+4 mM glutamine+4.5 g/L glucose+10.sup.-8 M
sodium selenite. 100 mL of Taub's Media is made by addition of 100
.mu.L of insulin stock (5 mg/mL) solution, 100 .mu.L transferrin
stock solution (5 mg/mL), 100 .mu.L triiodothyronine (T3) stock
solution (5.times.10.sup.-9 M), 5 .mu.L of hydrocortisone stock
solution (10.sup.-3 M) and 500 .mu.L of prostaglandin E1 stock
solution (50 .mu.g/mL) to basal DMEM/Ham F12 medium+4 mM
glutamine+4.5 g/L glucose+10.sup.-8 M sodium selenite. All stocks
solutions are prepared as follows:
[0340] Insulin Stock Solution--A 5 mg/mL stock solution is made by
dissolving the appropriate amount of insulin in 0.01 N HCl. The
solution is passed through a 0.2 micron sterilizing grade filter
and aliquoted into Nalgene cryovial and stored at 4-20.degree.
C.
[0341] Transferrin Stock Solution--A 5 mg/ml stock solution is made
by dissolving the appropriate amount of transferrin in MilliQ
water. The solution is passed through a sterilizing grade filter
and then aliquoted into Nalgene cryovial and store <-20.degree.
C.
[0342] Triiodothyronine (T.sub.3) Stock Solution--A stock solution
is made by dissolving the appropriate amount of T3 in 0.02 N NaOH
to obtain a 10.sup.-4 M solution. This is stock solution is further
diluted to a concentration of 5.times.10.sup.-9 M stock solution
with 0.02 N NaOH, passed through a sterilizing grade filter,
aliquoted into Nalgene cryovial and stored at <-20.degree.
C.
[0343] Hydrocortisone Stock Solution--A 10.sup.-3 M stock solution
is made by dissolving the appropriate amount of hydrocortisone in
100% ethyl alcohol and aliquoted into Nalgene cryovials. The vials
are stored at 4.degree. C. for 3-4 months.
[0344] Prostaglandin E.sub.1 Stock Solution--A 50 .mu.g/mL stock
solution made by dissolving the appropriate amount of PGE1 in 100%
sterile Ethyl alcohol and aliquoted into Nalgene cryovial and
stored at <-20.degree. C.
[0345] Na.sub.2SeO.sub.3 Stock Solution--A 10.sup.-2 M stock
solution is made by dissolving the appropriate amount of sodium
selenide in WFI water or MilliQ water. This is further diluted in
water to a final concentration of 10.sup.-5 M passed through a
sterilizing grade filter and stored at 4.degree. C.
[0346] Ferric ammonium citrate (FAC) Stock Solution--A 200 mg/L
stock solution is made by dissolving the appropriate amount of
ferric ammonium citrate in WFI water or MilliQ water passed through
a sterilizing grade filter and stored at 4.degree. C.
[0347] Tropolone Stock Solution--A 250 mg/L stock solution is made
by dissolving the appropriate amount of tropolone in WFI water or
MilliQ water passed through a sterilizing grade filter and stored
at 4.degree. C.
[0348] Formulation of MediV Serum-free Medias (MediV 101, 102, 103,
104, and 105): Each MediV serum-free media formulation uses Taub's
media as a basal media and adds supplements as follows:
[0349] MediV 101: Taub's+2.5 g/L Wheat Peptone E from Organo
Technie (cat no 19559). Wheat Peptone E1 is stored in water as a
sterile 250 g/L stock solution.
[0350] MediV 102: Taub's+100.times. chemically defined lipid
concentrate from GIBCO BRL (cat no. 11905) added to a final
concentration of 1.times..
[0351] MediV 103: Taub's+1.times. final concentration lipid
concentrate from GIBCO+2.5 g/L Wheat Peptone E1 from Organo
Technie.
[0352] MediV 104: Taub's+1.times. final concentration lipid
concentrate from GIBCO+2.5 g/L Wheat Peptone E1 from Organo
Technie+5 .mu.g/L EGF (multiple sources).
[0353] MediV 105: Taub's without Transferrin, +1.times. final
concentration lipid concentrate from GIBCO+2.5 g/L Wheat Peptone E1
from Organo Technie+5 .mu.g/L EGF+0.2 mg/L Ferric ammonium
citrate+0.25 mg/L Tropolone.
[0354] M-32: MediV 105 having a glucose concentration of between 4
g/L and 4.5 g/L+Trace Element Solutions A, B and C (Table 9) at a
final concentration of 1.times.. Optionally, M-32 is supplemented
with an additional 4 g/L to 4.5 g/L of glucose (M-32+G)
[0355] MediV 107: another serum-free medium based on MediV 105
including certain trace elements. The final formulation of MediV
107 in shown in Table 10.
[0356] Formulation of M18M Media: in addition, M18M is another
serum-free medium that can be used to culture cells of the
invention. M18M is a serum free medium based on DMNSO-7 powder that
contains supplements as set forth in Table 11, below.
TABLE-US-00009 TABLE 9 1000X Trace Element Solutions A, B and C
Components mg/L Trace Elements Soln. A CuSO.sub.4.cndot.5H.sub.2O
1.60 ZnSO.sub.4.cndot.7H.sub.2O 863.00 Selenite.cndot.2Na 17.30
Ferric citrate 1155.10 Trace Elements Soln. B
MnSO.sub.4.cndot.H.sub.2O 0.17 Na.sub.2SiO3.cndot.9H2O 140.00
NH.sub.4VO.sub.3 0.65 NiSO.sub.4.cndot.6H.sub.2O 0.13 SnCl.sub.2
(anhydrous) 0.12 Molybdic acid, 1.24 Ammonium salt Trace Elements
Soln. C AlCl.sub.3.cndot.6H.sub.2O 1.20 AgNO.sub.3 0.17
Ba(C.sub.2H.sub.3O.sub.2).sub.2 2.55 KBr 0.12 CdCl.sub.2 2.28
CoCl.sub.2.cndot.6H.sub.2O 2.38 CrCl.sub.3 (anhydrous) 0.32 NaF
4.20 GeO.sub.2 0.53 KI 0.17 RbCl 1.21 ZrOCl.sub.2.cndot.8H.sub.2O
3.22
TABLE-US-00010 TABLE 10 MediV 107 Formulation Component g/L Salts
Calcium Chloride, Anhydrous 0.1166 Magnesium Chloride 0.0286
Magnesium Sulfate, Anhydrous 0.0488 Potassium Chloride 0.3118
Sodium Chloride 6.8600 Sodium Phosphate, Monobasic, Monohydrate
0.0625 Sodium Phosphate, Dibasic, Anhydrous 0.0710 Carbohydrates
MOPS 3.1389 Putrescine, 2HCl 0.0001 Sodium Pyruvate 0.0550
Nucleosides Adenosine 0.0175 Guanosine 0.0175 Hypoxanthine, Na salt
0.0103 D-Ribose 0.0175 Thymidine 0.0004 Uridine 0.0175 Amino Acids
L-Alanine 0.0223 L-Arginine HCl 0.2739 L-Asparagine H.sub.2O 0.0339
L-Aspartic Acid 0.0333 L-Cysteine HCl H.sub.2O 0.0686 L-Glutamic
Acid 0.0368 Glycine 0.0338 L-Histidine HCl H.sub.2O 0.0735
L-Isoleucine 0.1069 L-Leucine 0.1115 L-Lysine HCl 0.1638
L-Methionine 0.0323 L-Phenylalanine 0.0685 L-Proline 0.0403
L-Serine 0.0473 L-Threonine 0.1011 L-Tryptophan 0.0192 L-Tyrosine
2Na, Dihydrate 0.0918 L-Valine 0.0997 Vitamins d-Biotin (vit B7 and
vit H) 0.0000035 D-Calcium Pantothenate 0.00224 Choline Chloride
0.00898 Cyanocobalamin (vit B12) 0.00068 Folic Acid 0.00265
myo-Inositol 0.0126 Niacinamide 0.00202 Pyridoxine HCl (vit B6)
0.002031 Riboflavin 0.000219 Thiamine HCl (vit B1) 0.00217
.sup..alpha. Linoleic Acid, sodium salt 0.000045 .sup..alpha.
DL-Lipoic Acid 0.000105 Tropolone 0.00025 Trace Metals
NH.sub.4VO.sub.3 6.5E-07 AgNO.sub.3 1.7E-07 Aluminum Chloride
6H.sub.2O 0.0000012 Ba (C.sub.2H.sub.3O.sub.2).sub.2 2.55E-06
Cadmium Chloride (CdCl.sub.2) 2.28E-06 Chromium Chloride
(CrCl.sub.3, anhydrous) 3.2E-07 Cobalt Chloride 6H.sub.20 2.38E-06
Cupric Sulfate, Pentahydrate 0.0000029 Ferric Nitrate, Nonahydrate
0.00005 Ferric Ammonium Citrate 0.0014 Ferrous Sulfate,
Heptahydrate 0.000417 GeO.sub.2 5.3E-07 MnSO.sub.4 H.sub.20 1.7E-07
Molybdic Acid ammonium Salt 1.24E-06 Nikelous Sulfate (NiSO.sub.4
6H.sub.20) 1.3E-07 Potassium Bromide 1.2E-07 Potassium Iodide
1.7E-07 Rubidium Chloride 1.21E-06 Sodium Selenite 0.000019 Sodium
Fluoride 0.0000042 Sodium Meta-Silicate.cndot.9H.sub.2O 0.00014
Stannous Chloride 1.2E-07 Zinc Sulfate, Heptahydrate 0.001295
ZrOCl.sub.2 8H.sub.20 3.22E-06 Other Components CDLC 3X Glucose
(45%) 4.5 g/L L-Glutamine (200 mM) 4 mM CD Lipids (100X) 3X Wheat
Peptone (25%) 2.5 g/L Insulin (5 mg/mL) 5 ug/mL T.sub.3 (5 .times.
10.sup.-9 M) 5 .times. 10.sup.-12 M Hydrocortisone (10.sup.-3 M) 5
.times. 10.sup.-8 M PGE1 (50 .mu.g/mL) 25 ng/mL EGF (1 .mu.g/.mu.L)
5 ug/mL Osmolality 360 pH 7.2~7.4
TABLE-US-00011 TABLE 11 Formulation of M18M Final Amount or
Component Concentration DMNSO-7 powder concentrate 21.22 g/L Ferric
ammonium citrate (FAC) Stock Soln. 1 mL/L (1000X) Polyethylene
Glycol 2 g/L .beta.-mercaptoethanol 55 .mu.m Ethanolamine 2.44
mg/mL Tropolone 5 .mu.M Wheat Peptone 2.5 g/L
2-Hydroxypropyl-b-Cyclodextrin 125 mg/L L-Proline 183.4 mg/L Copper
Sulfate 1.6 .mu.g/L CS5-20 (cholesterol source) 25 mg/L Chemically
Defined Lipid Concentrate (CDLC) 10 mL/L Triiodo-L-Thyronine Sodium
Salt (T3) 5 pM Sodium Bicarbonate 3.024 g/L Glutamine 4 mM Choline
Chloride 50 mg/L L-Serine 60.9 mg/L Insulin 20 mg/L PGE.sub.1 250
ng/L Hydrocortisone 5-.sup.11 M EGF 5 .mu.g/L
Example 11
Growth of Influenza Viruses to Very High Titers
[0357] This example describes the results of experiments showing
growth of temperature sensitive, cold-adapted and attenuated
influenza viruses to very high titer. In particular, these
experiments resulted in virus titers of log.sub.10 TCID.sub.50/ml
of 9 for four such viruses.
[0358] MDCK subclone 1-A or 1-B are grown in either MediV 105 or
M18M for three days post-seeding, then immediately prior to
infection the growth media is removed and fresh media, such as
MediV 105; M18M or DMEM/F12 medium supplemented with 4.5 g/L
glucose, 4 mM glutamine, and TrypLE (1:100) (Invitrogen) is added.
Cells are then infected with reassortant temperature sensitive,
cold-adapted, attenuated influenza viruses comprising the
FluMist.TM. backbone (e.g., all the gene segments except those
encoding the HA and NA proteins) and the HA and NA proteins from
A/New Calcdonia, A/Wisconsin, A/Vietnam, or B/Malaysia.
[0359] Results from one experiment are presented in Table 12. Table
12 demonstrates that these procedures can result in viral titers of
at least log.sub.10 TCID.sub.50/ml of 8.2 and as high as a
log.sub.10 TCID.sub.50/ml of 9.1 at 2, 3, 4, and 5 days post
infection. These data indicate that a media change or a
supplementation of depleted nutrients prior to or during infection
will result in increased in increased viral yields.
TABLE-US-00012 TABLE 12 Growth To Titers of >log.sub.10
TCID.sub.50/ml 8.0 Strains 2 DPI 3 DPI 4 DPI 5 DPI Control ca A/New
Caledonia #1 9.0 .+-. 0.06 9.0 .+-. 0.12 8.7 .+-. 0.00 8.7 .+-.
0.06 7.8 .+-. 0.06 ca A/New Caledonia #2 8.9 .+-. 0.06 9.0 .+-.
0.06 8.9 .+-. 0.10 8.8 .+-. 0.00 ca A/Wisconsin #1 8.5 .+-. 0.06
8.6 .+-. 0.06 8.6 .+-. 0.00 8.5 .+-. 0.06 8.3 .+-. 0.00 ca
A/Wisconsin #2 8.4 .+-. 0.06 8.7 .+-. 0.06 8.9 .+-. 0.12 8.8 .+-.
0.10 ca A/Vietnam #1 8.8 .+-. 0.00 9.1 .+-. 0.06 9.0 .+-. 0.10 9.0
.+-. 0.00 8.2 .+-. 0.06 ca A/Vietnam #2 8.8 .+-. 0.06 9.0 .+-. 0.06
9.1 .+-. 0.06 9.0 .+-. 0.10 ca B/Malaysia #1 8.5 .+-. 0.00 8.5 .+-.
0.00 8.3 .+-. 0.00 8.2 .+-. 0.06 7.9 .+-. 0.15 ca B/Malaysia #2 8.5
.+-. 0.00 8.4 .+-. 0.00 8.3 .+-. 0.00 8.2 .+-. 0.00
Example 12
Single Use Bioreactor Process
[0360] The standard bioreactors or fermenters (i.e., stainless
steel or glass reactors) typically used for the production of
vaccine material require cleaning, sterilization and validation
before each use. To mitigate the need for cleaning and validation a
disposable cell culture process has been developed using disposable
bioreactor technology. This process allows for a shortened
processing time, provides a significant cost savings and reduces
the infrastructure required for production of vaccine material. The
process makes use of a Single Use Bioreactor (SUB). Numerous SUB
systems are commercially available and may be utilized in the
process. Briefly, the SUB process involves growth of SF MDCK cells
on microcarriers in growth medium for .about.4 days, followed by
infection of cells with the influenza virus after performing a
medium exchange of replacing the growth medium with the infection
medium. Alternatively, infection of the cells with the influenza
virus may proceed directly, with no media exchange. The cells for
seeding the SUB may be adherent and may be obtained from roller
bottles or other readily scalable culture method used for growth of
adherent cells.
[0361] Pilot studies demonstrated that while agitation rates of
50-100 rpm supported cell growth cells grown at 90-100 rpm lead to
improved cell growth. Higher agitation rates were not tested in
these studies. Pilot studies also demonstrated that a microcarrier
concentration of about 2-3 g/L and a cell seeding density of
.about.9.0.times.10.sup.4 cells/mL (corresponds to .about.10-15
cells/MC) lead to improved cell growth and viral yields. In
addition, the use of a glucose supplemented media also resulted in
improved cell growth and viral yields. Based on these and other
pilot studies SUB methods with and without a media exchange prior
to infection were developed.
[0362] Materials
[0363] The A SUB from Hyclone (Hyclone, Part Nos. SH30715.01,
SH30720.01 and SH3B1744.01) was used for this set of experiments.
The SUB consists of the three primary components: 1. Outer support
container with a mixer drive complete with control unit and an
electrical heater jacket, 2. Single-Use Bioreactor BioProcess
Container (BPC)-- complete with mixer, sparger, vent filter inlet
and outlet ports, plus ports for integration of sensor probes, and
3. Mixer Shaft Rod which is inserted into the bioreactor BPC
through the mixing drive motor and locks into the disposable
agitator assembly. Numerous custom alterations can be made to one
or more components of the SUB apparatus, for example the outlet
port can be enlarged to facilitate harvest and media exchange,
similarly and in-line microcarrier filter can also facilitate
harvest and media exchange.
[0364] MedIV 105 (see section 9.10) or MedIV 105 plus an additional
4.5 g/L glucose (final concentration 9.0 g/L, referred to as "MedIV
105+G") is utilized as the growth medium. When MedIV 105 is
utilized the culture may be supplemented with 20 mM of Glucose on
day 2 to 3 post-inoculation to prevent glucose depletion. The
higher initial glucose concentration of MedIV+G can eliminate the
need for glucose supplementation.
[0365] The infection medium consists of DMEM/F12, Glucose,
Glutamine and TrypLE select. Table 13 shows the components and
concentration of each in the infection medium.
TABLE-US-00013 TABLE 13 Infection Medium Amount Added per liter
Component Final Concentration of DMEM/F12 DMEM/F12 1 L/L 1000 mL
Glucose 4.5 g/L 10 mL L-Glutamine 4 mM 20 mL TrypLE Select 1:33 to
1:100 20 mL
[0366] Method with Media Exchange
[0367] A microcarrier stock solution is prepared by swelling the
microcarrier in buffer followed by a buffer wash and sterilization.
Prior to use the buffer is removed and the appropriate media is
added. For example 60 g of Cytodex 3 microcarrier (2 g/L of total
working volume in SUB) is soaked in 3.0 L of Ca.sup.2+ and
Mg.sup.2+ free PBS of pH 7.4 (50 mL/g Cytodex3) in a 5 L glass
feeding bottle for at least 3 hours at room temperature. The
supernatant is then aspirated out and replaced with 1.5 L of fresh
Ca.sup.2+ and Mg.sup.2+ free PBS of pH 7.4. The microcarriers are
then sterilized by autoclaving this feed bottle at 121.degree. C.
for 30 minutes. Just prior to inoculation the PBS solution is
aspirated off and 4.0 L of DMEM/F12 medium is added and the sterile
microcarriers are added to the SUB under sterile conditions.
Alternatively, the Cytodex 3 microcarriers can be sterilized
in-situ (i.e., inside SUB bags) using .gamma.-irradiation.
[0368] Clone 1-B cells for seeding the SUB are obtained by scaling
up from 1 frozen vial. Cells are grown in MedIV 105 or MedIV 105+G
and may be scaled up as follows: on day 1 thaw vial into a T-75
flask; on day 3 split cells into T-225 flasks (seeding
density.apprxeq.5.times.10.sup.4 cells/mL); on day 7 split cells
into roller bottles (seeding density.apprxeq.6.7.times.10.sup.4
cells/mL); on day 10 split cells into additional roller bottles
(seeding density.apprxeq.6.7.times.10.sup.4 cells/mL); on day 14
the cells from .about.30-36 roller bottles are trypsinized and used
to inoculate SUB bioreactor. The inoculation parameters are
indicated in Table 14. Pooled trypsinized cells collected from
roller bottles are transferred to the SUB containing Cytodex 3
microcarriers in 30 L of SFMV105 medium through the inoculum
addition line of the BPC using a Peristaltic pump. The cultures may
be supplemented with 20 mM Glucose on day 3 post-inoculation to
prevent glucose depletion.
[0369] The cells are grown for 4 days under the growth parameter
conditions detailed in Table 15. The pH is controlled using the
Applikon controller, initially by sparging CO.sub.2 and at later
cultures stages by adding base (NaOH, 1M). DO is controlled at
.gtoreq.50% using the Applikon controller by sparging O.sub.2.
During cell growth is acceptable for DO to be as high as 100% and
drop as low as 35%. Temperature is controlled at the appropriate
values with the Hyclone controller. Agitation is controlled with
the Hyclone controller at 100 rpm.
TABLE-US-00014 TABLE 14 Inoculation Parameters Working Volume 30
.+-. 1 L Microcarrier (MC) concentration 2 to 3 .+-. 0.2 g/L Amount
of microcarrier 60 to 90 .+-. 1 g Cells/MC (calculated) 15 .+-. 5
Seeding density (cells/mL) 9.0 .+-. 1.5 .times. 10.sup.4
TABLE-US-00015 TABLE 15 Growth Parameters Agitation 100 .+-. 10 rpm
Temperature 37 (.+-.0.5) .degree. C. pH 7.4 (.+-.0.1) Dissolved
Oxygen (DO) [Air saturation] .gtoreq.35% [Controlled at 50%]
O.sub.2 Flow rate [maximum] (L/min) 1.0 .+-. 0.2 CO.sub.2 Flow rate
[maximum] (L/min) 0.20 .+-. 0.04
[0370] Infection is done at 4.+-.0.5 days post seeding. Prior to
infection, a nuclei count may be performed. Cells should reach
between 0.5-2.0.times.10.sup.6 cells/mL at this time and are
generally expected to reach a cell density of at least
.about.1.times.10.sup.6 cells/mL. After the nuclei count if
desired, all control loops are disabled and the micro carrier beads
are allowed to settle for .about.45 minutes. A medium exchange is
then performed where the growth medium is pumped out through the
medium exchange port of the SUB and infection medium is added
through the medium addition port to a final volume of 30 L.
Approximately 20-24 L are removed and the same amount of fresh
infection medium is added. This corresponds to approximately 66-80%
medium exchange. The parameters for infection are given in Table
16.
TABLE-US-00016 TABLE 16 Parameters for Infection Working Volume 30
L Agitation 100 .+-. 10 rpm Temperature 33 (.+-.0.5) .degree. C. pH
7.4 (.+-.0.1) Dissolved Oxygen (DO) [Air saturation] .gtoreq.35%
[Controlled at 50%] O2 Flow rate [maximum] (L/min) 1.0 .+-. 0.2 CO2
Flow rate [maximum] (L/min) 0.20 .+-. 0.04
[0371] The infection may be done at an MOI (Multiplicity of
Infection) of .about.0.001-0.003 FFU/cell (refer to the formula
below).
[0372] Amount of viruses in .mu.L added to S.U.B
=Total cells in SUB.times.MOI(FFU/cell)/10.sup.Virus
FFATiter(FFU/mL).times.1000.
Alternatively, to minimize process steps 2.times.10.sup.3 FFU/mL of
virus may be added. This will correspond to an MOI of
.about.0.001-0.003 FFU/cell. Under these conditions the amount of
virus in .mu.L added to S.U.B
=Volume in reactor(mL).times.2.times.10.sup.3FFU/mL/10.sup.Virus
FFATiter(FFU/mL).times.1000.
[0373] In-process sampling procedures may be utilized at several
steps for monitoring. Pre-infection 2.times.10 mL of cell
suspension is collected daily from day 0 to day 4 post seeding for
nuclei count, pictures and pH and metabolite (glucose, glutamine,
lactate, NH.sub.4.sup.+) analysis. Post-infection 2.times.5 mL
samples are drawn on day 2 and day 3 post infection. The samples
are stabilized with Sucrose Phosphate (ratio of Sucrose phosphate
to Virus sup=1:9). These samples will be frozen immediately and
stored at -80.degree. C. and may be used to determine viral
titers.
[0374] Virus harvest is obtained on day 3 post infection (+/-12 h).
The controllers on the SUB and Applikon are turned-off and the
microcarriers are allowed to settle for at least 45 min. Then the
supernatant is transferred to a sterile disposable bag and
stabilized with sucrose phosphate at a 1:9 ratio (V/V) (Sucrose
phosphate:Virus Harvest=1:9). The Sucrose phosphate should be added
by volume and not by weight.
[0375] Results with Media Exchange
[0376] Summarized here are the results of multiple SUB production
runs testing the different medium, inoculation and infection
parameters described in section 9.12.2. As shown in Table 18, all
the variation tested resulted in peak viral titers of at least 8.0
log.sub.10 FFU/mL demonstrating that the SUB process with media
exchange is robust.
[0377] For one B/Malaysia production run (SUB run A) the
microcarrier (MC) concentration was 3 g/L of working volume (30 L)
and the cell seeding density was 10 cells/MC or
.about.9.0.times.10.sup.4 cells/mL. The culture was supplemented
with 20 mM of Glucose on day 3 post-inoculation to prevent glucose
depletion. MDCK subclone 1-B was used and the cell density reached
.about.1.3.times.10.sup.6 cells/mL by day 4 post inoculation. The
remaining growth parameters shown in Table 15 were maintained as
described throughout the growth phase. Table 17 shows the cell
growth data and the doubling time for the B/Malaysia production
run. The cell growth curve is plotted in FIG. 13, as well as, the
metabolite analysis of glucose, lactate, glutamine and ammonium ion
concentration measured by Bioprofile for the B/Malaysia production
run.
TABLE-US-00017 TABLE 17 Cell Growth Total Cell Density .times. Time
(h) 10.sup.6 cells/mL Doubling Time (h) 0.15 0.09 21.67 0.10 141.55
46.00 0.30 15.35 65.33 0.60 19.57 70.00 0.69 21.84 88.83 1.30
20.61
[0378] Doubling time is about 20 h during the exponential phase. On
day 4 post seeding .about.67% of the medium was exchanged for
infection medium (see above) containing TrypLE select at a final
concentration of 1:100. The cells were then infected with
B/Malaysia/2506/04 at an MOI of 0.001 FFU/cell. The infection
parameters shown in Table 16 were maintained throughout the
infection phase. Samples taken at 2 and 3 days post infection (dpi)
were analyzed using the Focal Fluorescent Assay (FFA) to determine
the virus infectivity. Virus titer was seen to peak at .about.2 dpi
at around 8.0 log.sub.10 FFU/mL. While the peak viral titers
obtained using TrypLE at a final concentration of 1:100 from this
and several other runs were at least 8.0 log.sub.10 FFU/mL, lower
titers were occasionally seen (data not shown), and so higher
TrypLE concentrations (1:33 to 1:50) were generally used.
[0379] Two SUB runs were performed using a microcarrier
concentration of 2 g/L and MedIV 105+G as the growth medium without
any additional glucose supplementation. MDCK subclone 1-B was used
at a seeding density of .about.9.0.times.10.sup.4 cells/mL
(corresponds to 15 cells/MC). Prior to infection .about.80% of the
medium was exchanged and TrypLE select was added at a final
concentration of 1:100 (SUB run B) or 1:50 (SUB run C). The cells
were infected at a virus concentration of 2.times.10.sup.3 FFU/mL.
The peak viral titers for these runs were 8.4 log.sub.10 FFU/mL
A/Wisconsin (SUB run B) and 8.7 log.sub.10 FFU/mL A/New Calcdonia
(SUB run C).
[0380] Six additional production runs (SUB runs D-I) were performed
using a microcarrier concentration of 2 g/L and MedIV 105+G as the
growth medium without any additional glucose supplementation. As
before the MDCK subclone 1-B was used at a seeding density of
.about.9.0.times.10.sup.4 cells/mL, which here corresponds to
.about.15 cells/MC. The remaining growth parameters were maintained
as detailed in Table 15. On day 4.+-.0.5 post seeding .about.66% of
growth media (MedIV 105+G) were removed and the same amount of
infection medium (see Table 13) was added containing TrypLE select
at a final concentration of 1:33. The cells were then infected with
A/New Calcdonia/20/99; A/Wisconsin/67/05; or B/Malaysia/2506/04 at
a virus concentration of 2.times.10.sup.3 FFU/mL and the infection
parameters shown in Table 16 were maintained throughout the
infection phase. The peak viral titers for the SUB runs are shown
in Table 18 and range from 8.55-8.75 log.sub.10 FFU/mL. The growth,
glucose, lactate, glutamine and ammonium ion profiles were
comparable to that seen for SUB run A (see FIG. 13 and data not
shown)
TABLE-US-00018 TABLE 18 Peak Viral Titers for SUB runs Peak Titer
SUB (log.sub.10 run FFU/mL) Virus A 8.0 B/Malaysia B 8.4
A/Wisconsin C 8.7 A/New Caledonia D 8.6 B/Malaysia E 8.7 B/Malaysia
F 8.7 A/New Caledonia G 8.8 A/New Caledonia H 8.6 A/Wisconsin I 8.6
A/Wisconsin
[0381] Results without Media Exchange
[0382] Elimination of the media exchange step will reduce costs and
improve process efficiency. Initial testing at a TrypLE dilution of
1:100 (.about.0.01.times.) suggested that conditioned growth media
may comprise one or more components which inhibit the action of the
TrypLE and thus inhibit the growth of virus (data not shown). Pilot
experiments were performed in which the concentration of TrypLE was
varied. Briefly, MDCK cells grown in a 2 L bioreactor for 4 days
under standard conditions (mother culture). The mother culture was
then used to inoculate shake flasks with different levels of medium
exchange and TrypLE concentrations, just prior to infection with
A/New Calcdonia. Four different dilutions/concentrations of TrypLE
were used 1:100 (.about.0.01.times.); 1:50 (.about.0.02.times.);
1:33 (.about.0.03.times.); and 1:25 (.about.0.04.times.). Flasks
were sampled at 2 and 3 dpi for virus titer. The viral titers
obtained for each medium exchange ratio at 2 and 3 dpi are plotted
in FIG. 14A. These data show that even without any media exchange,
adding TrypLE at 1:25-1:33, yields a titer close to 8 log.sub.10
FFU/mL. Based on these data a 1:16 dilution of TrypLE should yield
an high titer without any medium exchange. A similar experiment was
performed at higher TrypLE concentrations. Briefly, a mother
culture was prepared as described above and used to inoculate shake
flasks with no media exchange at 1:1-1:25 (corresponding to
0.5.times.-0.04.times. TrypLE concentrations) just prior to
infection with A/New Caledonia. The peak viral titer was determined
at 2 and 3 dpi and plotted in FIG. 14B. Here, viral titers of
greater then 8 logs were obtained for the first time without media
exchange. These data indicate that the optimal TrypLE concentration
is between 1:25-1:12.5 dilution and that higher concentrations of
TrypLE do not improve viral yield. Based on these results the
production of two additional viral strains, B/Malaysia/2506/04 and
A/Vietnam/1203/2004, were examined with and without media exchange
(using 1:33 and 1:12.5 dilution of TrypLE, respectively). The viral
titers over time are plotted in FIG. 14C. The peak viral titers for
B/Malaysia/2506/04 were 8.9 and 8.7 log.sub.10 FFU/mL (with and
without media exchange, respectively). Similarly, the peak viral
titers for A/Vietnam/1203/2004 were 8.6 and 8.0 log.sub.10 FFU/mL
(with and without media exchange, respectively). Thus, increasing
the amount of TrypLE up to 1:12.5 dilution (corresponding to
0.08.times.) can compensate for the effects of the conditioned
media resulting in peak viral titers without media exchange of at
least 8 log 10 FFU/mL.
Example 13
Optimization of MOI
[0383] Because of the continual emergence (or re-emergence) of
different influenza strains, new influenza vaccines are generated
each season based on the circulating influenza strains.
Unfortunately, some influenza vaccine strains (e.g., cold adapted
temperature reassortant vaccine strains) are more difficult to grow
to high titers. The titer of the bioreactor not only defines
production capacity but also impacts the cost of manufacturing
product thus improving viral titer (i.e., peak viral titer) is
desirable. As mentioned above a number of parameters has been
examined to optimize productivity of vaccine strains. Summarized
here are the results of the studies for increasing productivity
(i.e., viral titer) for several strains. These studies identified
the MOI (virus particles used for infection per MDCK cell) as a
parameter which can be readily tested and adjusted to optimize
yield and allow for the rapid scale up and production of seasonal
and pandemic vaccine strains.
[0384] These studies were carried out by growing the MDCK subclone
1-B cells in a bioreactor and infecting the cells in shake flasks
with different amounts of virus. The details of the study are as
follows: M-32+G containing Cytodex 3 micro carrier beads at 2 g/L
was inoculated with MDCK subclone 1-B cells at .about.15
cells/microcarrier in a 3 L bioreactor vessel. The cells were grown
at 37.degree. C., 90 rpm, pH 7.4 and, 50% DO (controlled using
O.sub.2 and CO.sub.2 sparge). At .about.4 days post seeding (dps),
66% of the growth medium in the bioreactor was exchanged with
infection medium (DMEM/F12+4.5 g/L D-glucose+4 mM
L-glutamine+1.times. TrypLE select at 1:33 final dilution). Equal
amounts of culture (30 ml) were transferred to different 125 ml
shake flasks. These shake flasks were infected with different
amounts of a specific virus strain (i.e., 2, 20, 200, 2000 and
20000 FFU/ml, corresponding to approximately 1.times.10.sup.-6,
1.times.10.sup.-5, 1.times.10.sup.-4, 1.times.10.sup.-3 and
1.times.10.sup.-2 FFU/cell, respectively). Post infection the
flasks were incubated at 33.degree. C. and, 100 rpm. A number of
parameters were monitored including the viable cell density,
metabolite concentration (both before and after infection) as well
as the viral titer at various times post infection (e.g., 1, 2, 3
and, 4 days post infection (dpi)). The peak viral titer results for
four strains tested in these studies are shown in Table 19. For
each strain tested the peak viral titer was seen to increase when
the MOI was reduced from .about.1.times.10-3 FFU/cell (the MOI used
in the SUB process described in Section 9.12 above) to
.about.1.times.10.sup.-4 FFU/cell. The observed increase in viral
peak titer ranged from 0.3 log.sub.10FFU/ml to 1.3
log.sub.10FFU/ml. It should be noted that in some instances the
peak viral titers were obtained on different days post infection
(i.e., 2 dpi or 3 dpi). This may be due to differences in viral
amplification kinetics at a lower MOI of 1.times.10.sup.-4 FFU/cell
compared to a MOI of 1.times.10.sup.-3 FFU/cell, should this trend
be seen in production bioreactors the viral harvest times should be
adjusted accordingly.
[0385] A bioreactor study was performed to confirm the shake flask
results. For this study five parallel master cell cultures were
prepared in 3 L bioreactors as described above. The viable cell
density and cell metabolism profiles of glutamine, NH4+, glucose
and lactate were comparable in all the bioreactors (data not
shown). At .about.4 days post seeding (dps), 66% of the growth
medium in the bioreactors was exchanged for infection medium
DMEM/F12+4.5 g/L D-glucose+4 mM L-glutamine+10.times. TrypLE select
at 1:330 final dilution). The five cultures were infected with
A/Solomon Islands/3/06 at different amounts 2, 20, 200, 2000 or
20000 FFU/ml (corresponding to MOIs of approximately
1.times.10.sup.-6, 1.times.10.sup.-5, 1.times.10.sup.-4,
1.times.10.sup.-3 and 1.times.10.sup.-2 FFU/cell, respectively) and
incubated at 33.degree. C. All other growth parameters
post-infection were the same as for the growth of the master cell
cultures pre-infections. FIG. 15 plots the viral titer over time
(hours post infection) obtained using different MOIs. The boxed
area (expanded to the right) shows that at three days post
infection the culture infected at 2000 FFU/mL had a peak viral
titer of 8.3 log.sub.10FFU/mL while the culture infected at 20
FFU/mL had a peak viral titer of 8.5 log.sub.10FFU/mL (a 0.2
log.sub.10FFU/mL improvement). Similarly, at four days post
infection the culture infected at 2 FFU/mL also reached a peak
titer of 8.5 log.sub.10FFU/mL. Together, these studies indicate
that decreasing the MOI can result in increased viral titers and
such a method may prove useful for increasing the production yield
of certain vaccine strains. These studies further indicate that the
optimum harvest time may have to be determined based on the MOI
used.
TABLE-US-00019 TABLE 19 Optimization of MOI in Shaking Flasks Peak
Virus Titer at MOI (in FFU/cell) of Virus Strain 0.001-0.003*
0.0001** Improvement in titer A/Wisconsin/67/05 8.7 9 0.3
Log.sub.10FFU/mL A/Solomon Islands/3/06 8.3 9.2 0.9
Log.sub.10FFU/mL A/California/07/2004 7.1 7.9 0.8 Log.sub.10FFU/mL
A/Hong Kong/491 H5 + 7.9 9.2*** 1.3 Log.sub.10FFU/mL 486 N1/1997
Note: *MOI for SUB-like process = 2000 FFU/mL **MOI corresponds to
200 FFU/mL ***Peak viral titer observed at MOI of 1.0E-06
FFU/cell
Example 14
Bead to Bead Transfer
[0386] Large scale cultivation of cells requires a scale up of the
number of cell in the culture. When adherent cells are used the
scale up process generally involves sequential dissociation of
cells from flasks or microcarriers, for example by protease
treatment, dilution of the dissociated cells into a larger flask or
into a larger number of microcarriers. Minimizing the number of
washing and/or medium exchange steps during the scale up process
can enhance efficiency and reduce the likelihood of contamination.
The SUB method described above requires the use of cells harvested
from 30 to 36 separate roller bottles each of which must be
trypsinized and harvested separately. Described below is one method
that can be utilized to reduce the number of handling steps used to
scale up from a 3 L vessel to a 20 L vessel. Similar strategies can
be implemented for use in larger bioreactor process such as the 30
L SUB process described above.
[0387] 3 L Bioreactor Preparation: 1. Add 4 g of Cytodex3 to a 3 L
bioreactor. Add 500 mL of DPBS (PBS w/o Ca, Mg) to hydrate the
microcarriers for 4-6 hours. 2. Use the dip tube to remove 300 mL
of DPBS without disturbing microcarriers from the bottom. Add 300
mL of fresh DPBS and autoclave vessel for 30 minutes at 121 C. 3.
After reactor has cooled, remove 300 mL of DPBS and add 300 mL of
medium (M-32) to the vessel. Stir vessel contents for 10 minutes at
200 rpm to completely mix reactor contents and to get all
microcarriers off the bottom of the vessel. 4. Stop agitation and
remove 300 mL of medium after all microcarriers have settled. 5.
Add 1.6 L of fresh basal medium into the reactor and allow
parameters to stabilize overnight. The process parameters are: pH
7.2, Temperature 37 C, Agitation 120 rpm, Air sparge rate 50
mL/min.
[0388] 20 L Bioreactor Preparation: Add 28 g of Cytodex3 to a 5 L
bottle. Add 3 L of DPBS (w/o Ca, Mg) to hydrate the microcarriers
for 4-6 hours. Remove 2 L of DPBS without disturbing microcarriers
from the bottom. Add 2 L of fresh DPBS and autoclave vessel for 30
minutes at 121 C. 2. Remove 2 L of DPBS and add 2 L of medium to
the bottle. Shake bottle vigorously to ensure microcarriers are in
suspension. Allow microcarriers to settle before removing 2 L of
medium. Add fresh medium to the microcarriers to bring microcarrier
solution to a total volume of 3 L. 3. Add fresh medium and
microcarrier solution to ensure total volume in bioreactor is 14 L
and allow process parameters to stabilize overnight. The process
parameters are: pH 7.2, Temperature 37 C, Agitation 120 rpm, Air
sparge rate 400 mL/min.
[0389] 3 L Bioreactor Growth Phase Operation: Calibrate pH and
Dissolved Oxygen readings after sampling and analysis through NOVA
Bioprofile. 2. Add culture harvested from cell factories to
inoculate bioreactor at a target cell density of 9E4 cells/mL (15
cells per microcarrier bead). Add medium to reach a total working
volume of 2 L. 3. Start D.O. control with a set-point of 50%. 4.
Sample everyday for analysis with NOVA, Nucleocounter and for
microscope imaging.
[0390] Bead to Bead Transfer Protocol at Scale: After 96 hours of
cell growth in the 3 L vessel, switch off the agitator, gas flow
and DO and temperature controls. Allow microcarriers to settle. 2.
Remove medium (>80%) through dip tube but ensure that
microcarriers are not disturbed from the bottom of the vessel. 3.
Add DPBS (PBS w/o Ca, Mg) to bring the volume up to the original
working volume. 4. Increase agitation set point to 180 rpm. Switch
on the agitator for a period of 10 minutes to wash microcarriers of
any remaining medium. 5. Switch off the agitator and allow
microcarriers to settle to the bottom. 6. Remove 50% of the liquid
in the bioreactor through the dip tube. Ensure that the temperature
probe and agitator are still completely immersed after the removal.
(Volume remaining is approximately 1 L). 7. Switch on the agitator
and temperature control. Wait till the temperature in the reactor
is 37 C. 8. Add 5.times. TrypLE (5-7% of remaining volume) to the
bioreactor. 9. Add 1M Sodium Carbonate to adjust the pH of the
reactor contents to 7.9+/-0.1. 10. Allow trypsinization for 50+/-10
minutes with intermittent sampling and observation under the
microscope to ensure cells have detached. 11. Add 5.times. lima
bean trypsin inhibitor (LBTI) in exactly the same volume as the
TrypLE. 12. Add fresh medium to bring up the volume to original
working volume (2 L). 13. Transfer all reactor contents to the 20 L
bioreactor (1:8 split).
[0391] Infection Parameters: Under the bead to bead transfer
conditions utilized here, the cells exhibited a slightly slower
growth post bead to bead transfer which lead to infection being
delayed by one day (infection on day .about.5) as compared to
transfer from roller bottles (infection on day .about.4). Infection
was performed when cell density reached .about.1.times.10.sup.6
cells/mL essentially as described for the SUB process (see, Section
9.12 above). Although infection was delayed by one day, the peak
viral titers using bead to bead transfer were comparable to those
obtained using transfer conditions similar to those described in
Section 9.12 above (see Table 20). Accordingly, the use of bead to
bead transfer methods can reduce the number of manipulations
without sacrificing viral yield.
TABLE-US-00020 TABLE 20 Peak Virus Titers Bead to Bead Transfer
from Virus Strain Transfer Roller Bottles B/Malaysia/2506/04 8.8
8.5 A/Wisconsin/67/05 8.5 8.5 A/Solomon Islands/3/06 8.1 8.2
[0392] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations. 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. Citation or discussion
of a reference herein shall not be construed as an admission that
such is prior art to the present invention, and citation of a
patent shall not be construed as an admission of its validity.
* * * * *