Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Methods for Adapting Mammalian Cells
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is copending with, shares at least one common inventor
with,
and claims priority to United States provisional patent application number
60/732,818,
filed November 2, 2005, the contents of which are hereby incorporated by
reference in
their entirety.
BACKGROUND OF THE INVENTION
[0002] This disclosure relates generally to methods of adapting mammalian
cells,
e.g. untransfected mammalian cells, for production of a therapeutic protein of
interest.
Particularly this disclosure relates to adapting untransfected mammalian cells
for
superior performance in bioreactors.
[0003] Proteins can be produced by using well known recombinant techniques.
Transformed cells are commonly cultured in a controlled environment, such as a
bioreactor. Most large-scale commercial manufacturing strategies employ
suspension
cell cultures grown in large stirred-tank reactors. Most cell lines, however,
do not readily
perform well in high cell density protein production processes (e.g., fed-
batch
processes) and/or cannot reach the desired high cell densities. Many
inhibitors are
present or accumulate in the cell culture medium during a production run;
these
inhibitors may be by-products generated from metabolic processes such as
lactate or
ammonia, among others. The cell lines are typically grown and maintained
initially in
conditions that are designed to aid in the propagation and/or survival of
these cell lines.
Conditions used during protein production (e.g., conditions encountered in
production
bioreactors), however, can be quite different, with e.g., secondary
metabolites and/or
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other components present and/or accumulated as protein production progresses,
which
can have potential deleterious effects on the cell line. As non-limiting
examples, such
deleterious effects may comprise a decrease in the viable cell density and/or
a decrease
in the final titer, as well as a decrease in the amount and/or quality of the
produced
protein.
[0004] Practicing ordinary methods, numerous experiments are performed to
determine whether a cell line has adapted to growth and/or production in a
protein
production process such as a fed-batch process in a bioreactor. Such
experimentation
often requires multiple clonal cell lines and/or transfection pools, typically
followed by
adaptation of the cells to growth in media that will be used in a production
bioreactor
(e.g., a serum free suspension or a "defined medium"). Once a cell line is
adapted,
additional rounds of screening and optimization in bench-scale bioreactors are
typically
performed. Individual clones that exhibit good expression phenotypes in the
preliminary
research are then typically subjected to additional experimentation to
determine which
clones also exhibit acceptable growth and/or viability characteristics in a
bioreactor, for
example a fed-batch bioreactor. Such extensive screening processes are costly
and
particularly arduous for the practitioner.
[0005] Therefore, what is needed are methods for adapting cells to protein
production conditions including, but not limited to, bioreactor conditions.
What is further
needed is a method of adapting untransfected host cells prior to being
transformed in
such a manner that there are no or minimal detrimental effects on one or more
cell
culture characteristics (e.g., titer, viability, protein quality, etc.) after
transfection and
transition into protein production conditions.
SUMMARY
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_,_ ., . _.., ..... -.. ._.. _
[0006] The present disclosure relates to methods for adapting mammalian cells
to a
high density protein production process such as, for example, a fed-batch
process.
Inventive processes can involve both untransfected mammalian cells and
genetically
manipulated host cells. In certain embodiments, untransfected mammalian cells
are
adapted to production-matched conditions such as those used during protein
production. For example, untransfected mammalian cells may be adapted to a
production medium used in a bioreactor during a production run.
[0007] Different methods may be employed to adapt the untransfected mammalian
cells to production conditions. In certain embodiments, cells are cultured in
a production
and/or adaptation medium. In certain embodiments, cells are cultured in an
adaptation
medium with standard iterative splitting cycles performed. For example,
subpopulations
of the cells may be passaged one or more times, e.g. every three or four days.
In
certain embodiments, a production and/or adaptation medium generally has
higher
levels of nutrients, vitamins, and/or trace elements compared to a standard
growth
medium. In certain embodiments, the cells are then allowed a recovery period
between
passaging, where the passaged cells are grown in a standard growth medium,
e.g.
during one of the cycles, before returning the cells to a production and/or
adaptation
medium.
[0008] In certain embodiments, cells are adapted by passaging them in batch-
refeed
mode, where subpopulations of the cells are split or passaged every seven or
eight
days. In certain embodiments, passaging the cells after such a longer duration
allows
an accumulation of secondary metabolites in the cell culture medium. In
certain
embodiments, adapted cells gain tolerance to and/or begin to take up such
secondary
metabolites. In certain embodiments, such secondary metabolites comprise
inhibitors
and/or metabolites that typically accumulate in a bioreactor during a
production run,
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such as, for example, lactate and/or ammonia. In certain embodiments, cells
are
adapted by growth in a medium that includes one or more inhibitors including,
but not
limited to, lactate, ammonia, alanine, glutamine, and/or acetolactate. In
certain
embodiments, such inhibitors and/or metabolites are consistent with inhibitors
and/or
metabolites typically found in a bioreactor during a production run.
[0009] In certain embodiments, untransfected mammalian cells may be adapted by
being cultured (and optionally split every three or four days) in a standard
growth
medium which is supplemented with inhibitors such as alanine, glutamine,
acetolactate,
ammonia, and lactate. In certain embodiments, cells adapted in such a manner
are
passaged every three or four days. In certain embodiments, the concentrations
of such
inhibitors correspond to concentrations typically found in a bioreactor during
conditions
used for protein production. In certain embodiments, concentrations of some
inhibitors
include about 2 to about 10 g/L of lactate or about 0.1 to about 0.5 g/L of
ammonia,
mimicking typical bioreactor conditions. In certain embodiments, cells adapted
to
bioreactor conditions by one or more methods of the present invention exhibit
certain
characteristics or phenotypes and/or can develop such phenotypes in which the
cells
begin to uptake lactate and/or other secondary metabolites, such that the
levels of
lactate and/or other secondary metabolites actually decrease during one or
more time
periods during the production run. Such phenotypes may be screened-for because
they
exhibit qualities desirable in a high-density fed-batch production bioreactor.
[0010] In certain embodiments, cells adapted by one or more methods of the
present
invention are host cells that have not been transfected to produce a protein
of interest.
In certain embodiments, such adapted host cells are then screened for one or
more
desirable characteristics. In certain embodiments, once a subpopulation is
screened for
one or more desirable characteristics, after which the cells may be
genetically
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manipulated (e.g. transfected) to create a cell line that produces a protein
of interest. In
certain embodiments, such an adapted cell. line is placed in a bioreactor
where the cell
line readily adapts to a high cell density protein production process such as,
for
example, a fed batch process. In certain embodiments, as a result of the prior
adaptation of the untransfected host cells, e.g., mammalian host cells, the
genetically
manipulated cell line does not have to transition from a standard growth
medium to a
production medium and/or transitions with fewer and/or less severe deleterious
effects.
In certain embodiments, prior adaptation minimizes the potential deleterious
effects on
the cell line, and helps ensure cell line performance and accelerates
development
timelines. In certain embodiments, such an adapted cell line may uptake
secondary
metabolites during the protein production run. A person of ordinary skill in
the cell
culture art can readily determine what components make-up a standard growth
medium
and a standard production medium. In certain embodiments, growth and protein
production conditions differ in the composition of the cell culture media
used. During
growth and/or protein production phases, conditions of a bioreactor may be
altered
and/or supplements may be added in order to increase the productivity and/or
maintain
viability of the cell line. Supplements may include a feed medium and/or one
or more
additives. Those of ordinary skill in the art will be able to select
appropriate media
supplements. Further supplements to the mediums will depend on the desired
protein
product, the parameters (e.g., components, pH, etc.) of the cell culture
medium, the
methods employed throughout the growth and/or production process, and or any
of a
variety of other factors known to those of ordinary skill in the art.
[0011] In certain embodiments, untransfected mammalian cells are adapted by a
method comprising culturing untransfected mammalian cells in an adaptation
medium,
performing one or more iterative splitting cycles (e.g. every 3 or 4 days)
comprising
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splitting the untransfected mammalian cells in the adaptation medium, allowing
a
recovery period where the cells are cultured in a standard growth medium, and
screening the cells and selecting a subpopulation that exhibits an improved
growth
and/or viability phenotype compared to an unadapted version of the cells when
cultured
under conditions of a production bioreactor. In certain embodiments, the cells
are
transfected with a gene encoding a protein of interest and cultured in a
production
bioreactor to express the protein of interest. In certain embodiments, an
adaptation
medium contains increased levels of nutrients, vitamins, and/or trace elements
compared to said standard growth medium. In certain embodiments, an adaptation
medium contains an increased amount of inhibitory metabolites as compared to a
standard production medium prior to cell culture.
[0012] In certain embodiments, untransfected mammalian cells are adapted by a
method comprising culturing untransfected mammalian cells in an adaptation
medium
that has been supplemented with side products of primary carbon metabolism,
performing one or more iterative splitting cycles comprising splitting the
untransfected
mammalian cells about every 3 or 4 days in the adaptation medium, allowing a
recovery
period where the cells are cultured in a standard growth medium, accumulating
levels of
the side products, and screening the untransfected mammalian cells and
selecting a
subpopulation that exhibits an improved phenotype compared to an unadapted
version
of the untransfected mammalian cells when cultured in said the adaptation
medium. In
certain embodiments, such side products are one or more of lactate, ammonia,
alanine,
glutamine, and/or acetolactate. In certain embodiments, lactate is initially
present at a
concentration of about 2 to about 10 g/L. In certain embodiments, ammonia is
initially
present in a concentration of about 0.1 to about 0.5 g/L. In certain
embodiments, the
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untransfected mammalian cells are adapted to take up said side products of
primary
carbon sources.
[0013] In certain embodiments, untransfected mammalian cells are adapted by a
method comprising culturing untransfected mammalian cells for a duration of
about 7 or
8 days in a previously-conditioned medium having an accumulation of at least
one
inhibitory metabolite before splitting the untransfected mammalian cells, and
screening
the untransfected mammalian cells and selecting a subpopulation that exhibits
an
improved phenotype in a production bioreactor. In certain embodiments, cells
are
allowed a recovery period by culturing the cells in a standard growth medium
for a
duration of about 3 to about 4 days before splitting the cells. In certain
embodiments, a
conditioned medium contains an accumulation of the inhibitory metabolites
through at
least one metabolic process of the untransfected mammalian cells. In certain
embodiments, such an inhibitory metabolite is one or more of ammonia, alanine,
glutamine, acetolactate, and/or lactate. In certain embodiments, such
inhibitory
metabolites are consistent with those found in a production bioreactor at the
end of a
typical commercial-scale batch re-feed process.
[0014] Any of a variety of suitable culture procedures and/or media (e.g.,
inoculum
media, feed media, etc.) may be used to culture the cells in the process of
protein
production. Both serum-containing and serum-free media may be used. For
example,
in certain embodiments, cells are grown in a defined medium. In certain
embodiments,
cells are grown in a complex medium. In addition, one or more specific
culturing
methods may be used, altered and/or optimized to culture the cells as
appropriate for
the specific cell type and protein product. Such procedures are well known and
understood by workers and those of ordinary skill within the cell culture art.
Other
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features and advantages of the disclosure will be apparent from the following
description
of certain embodiments, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 a shows Seven Day Batch-Refeed Viable Cell Density
[0016] Figure 1 b shows Standard Splitting Viable Cell Density
[0017] Figure 1 c shows Viable Cell Density
[0018] Figure 2a shows Growth Rate of Monoclonal Antibody Cell Line
[0019] Figure 2b shows Accumulated Integral Viable Cell Density During Fed-
Batch
[0020] Figure 3 shows Cell Densities of Cell Cultures Adapted to Lack of
Insulin and
Control Cell Cultures.
[0021] Figure 4 shows Viability of Cell Cultures Adapted to Lack of Insulin
and
Control Cell Cultures.
[0022] Figure 5 shows Accumulated IVCD of Cell Cultures Adapted to Lack of
Insulin
and Control Cell Cultures.
[0023] Figure 6 shows Cell Densities of Cell Cultures Adapted to Lack of
Insulin and
Control Cell Cultures.
[0024] Figure 7 shows Viability of Cell Cultures Adapted to Lack of Insulin
and
Control Cell Cultures.
[0025] Figure 8 shows Accumulated IVCD of Cell Cultures Adapted to Lack of
Insulin
and Control Cell Cultures.
[0026] Figure 9 shows Specific Lactate Consumption Rate of Cell Cultures
Adapted
to Lack of Insulin and Control Cell Cultures.
DEFINITIONS
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[0027] Following long-standing convention, the terms "a" and "an" mean "one or
more" when used in this application, including the claims. Even though the
invention
has been described with a certain degree of particularity, it is evident that
many
alternatives, modifications, and variations will be apparent to those skilled
in the art in
light of the disclosure. Accordingly, it is intended that all such
alternatives,
modifications, and variations, which fall within the spirit and scope of the
invention, be
embraced by the claims.
[0028] The phrase "host cell" refers to cells which are capable of being
genetically
manipulated and/or are capable of growth and survival in a cell culture
medium.
Typically, the cells can express a large quantity of an endogenous or
heterologous
protein of interest and can either retain the protein or secrete it into the
cell culture
medium.
[0029] Host cells are typically "mammalian cells," which comprise the
nonlimiting
examples of vertebrate cells, including include baby hamster kidney (BHK),
Chinese
hamster ovary (CHO), human kidney (293), normal fetal rhesus diploid (FRhL-2),
and
murine myeloma (e.g., SP2/0 and NSO) cells. One of ordinary skill in the art
will be
aware of other host cells that may be used in accordance with methods and
compositions of the present invention.
[0030] The term "celi culture medium" refers to cells in a solution containing
nutrients
to support cell survival under conditions in which the cells can grow and/or
produce a
desired protein. The phrase "inoculation medium" or "inoculum medium" refers
to a
solution or substance containing nutrients in which a culture of cells is
initiated. In
certain embodiments, a cell culture is supplemented at one or more times with
a "feed
medium", with which the cells are fed after initiation of the culture. In
certain
embodiments, a "Feed medium" contains similar nutrients as the inoculation
medium,
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but is a solution or substance but is a solution or substance with which the
cells are fed
subsequent to initiation of the culture. In certain embodiments, a feed medium
contains
one or more components not present in an inoculation medium. In certain
embodiments, a feed medium lacks one or more components present in an
inoculation
medium. A person of ordinary skill in the cell culture art will know without
undue
experimentation what components make-up such inoculation and feed mediums.
Typically, these solutions provide essential and non-essential amino acids,
vitamins,
energy sources, lipids, and/or trace elements required by a cell for growth
and survival.
[0031] The term "cell culture characteristic" as used herein refers to an
observable
and/or measurable characteristic of a cell culture. Methods and compositions
of the
present invention are advantageously used to improve one or more cell culture
characteristics. In certain embodiments, improvement of a cell culture
characteristic
comprises increasing the magnitude of a cell culture characteristic. In
certain
embodiments, improvement of a cell culture characteristic comprises decreasing
the
magnitude of a cell culture characteristic. As non-limiting examples, a cell
culture
characteristic may be improved growth, increased viability, increased
integrated viable
cell density, increased titer, and/or increased cell specific productivity.
One of ordinary
skill in the art will be aware of other cell culture characteristics that may
be improved
using methods and compositions of the present invention.
[0032] The phrase "growth medium" or "standard growth medium" refers to a
medium
that contains nutrients and supplements that allows cells or a cell line to
divide and
grow. In certain embodiments, the phrase "production medium" refers to an
enriched
growth medium that permits high levels of a protein of interest to be
expressed. In
certain embodiments, a production medium, generally, contains higher levels of
nutrients, vitamins, trace elements, and/or other medium components when
compared
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to a standard growth medium. The phrase "adaptation medium" refers to a medium
that
subjects the cells to protein production conditions that exist in bioreactors
(e.g., late-
stage production bioreactors), prepares the cells for protein production
conditions,
and/or minimizes the potentially deleterious effects of transitioning the
cells from growth
conditions to protein production conditions. In certain embodiments, an
adaptation
medium includes the presence of one or more secondary metabolites, e.g. those
generated from metabolic processes including but not limited to lactate and/or
ammonia.
In certain embodiments, an adaptation medium includes one or more inhibitors
including, but not limited to, lactate, ammonia, alanine, glutamine, and/or
acetolactate.
In certain embodiments, an adaptation medium comprises a production medium. In
certain embodiments, an adaptation medium mimics one or more characteristics
of a
production medium. In certain embodiments, adapting cells in an adaptation
medium
results in cells that exhibit decreased or less severe deleterious effects
when such
adapted cells are switched from a growth or adaptation medium to a production
medium. Such adaptation media can be production matched, for example, by the
supplementation of production media with such metabolites and/or inhibitors
and/or by
the accumulation of such metabolites and/or inhibitors in an extended duration
cell
culture.
[0033] The term "defined medium" as used herein refers to a medium in which
the
composition of the medium is both known and controlled.
[0034] The term "complex medium" as used herein refers to a medium contains at
least one component whose identity or quantity is either unknown or
uncontrolled.
[0035] The phrase "cell line" refers to, generally, primary host cells that
have been
transfected with exogenous DNA, e.g. DNA coding for the desired protein of
interest. In
certain embodiments, cells derived from the genetically modified cells form
the cell line
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and are placed in a cell culture medium to grow and produce the protein
product of
interest. In some embodiments, primary host cells are transfected with
exogenous DNA
coding for a desired protein and/or containing control sequences that activate
expression of linked sequences, whether endogenous or heterologous. In certain
embodiments, a cell line comprises primary host cells that have been
transfected with
exogenous DNA and express an heterologous protein of interest. In certain
embodiments, a cell line comprises primary host cells that have not been
transfected
with exogenous DNA and express an endogenous protein of interest.
[0036] The "growth phase" of a cell culture medium refers to the period when
the
cells are undergoing rapid division and growing exponentially, or close to
exponentially.
Growth phase conditions may include a temperature at about 35 C to 42 C,
generally
about 37 C. The length of the growth phase and the culture conditions in the
growth
phase can vary but are generally known to a person of ordinary skill in the
cell culture
art. Typically, during the growth phase, cells are grown in a "growth medium"
or
"standard growth medium". In certain embodiments, a cell culture medium in a
growth
phase is supplemented with a feed medium.
[0037] [0038] The "transition phase" occurs during the period when the cell
culture medium is being shifted from conditions consistent with the growth
phase to
conditions consistent with the production phase. During the transition phase,
factors like
temperature, among others, are often changed. In certain embodiments, a cell
culture
medium in a transition phase is supplemented with a feed medium. Methods of
the
present invention are useful in minimizing the potentially deleterious effect
of switching a
cell culture from growth phase to production phase conditions.
[0038] The "production phase" occurs after both the growth phase and the
transition
phase. The exponential growth of the cells has ended and protein production is
the
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principal objective. Typically, during the production phase, cells are grown
in a
production medium. A production medium can be supplemented to initiate
production.
In certain embodiments, a cell culture medium in a production phase is
supplemented
with a feed medium. In certain embodiments, the temperature of the cell
culture
medium during the production phase is lower, generally, than during the growth
phase.
As is known in the art, in many instances such a decreased temperature
facilitates
protein production. The production phase continues until a desired endpoint is
achieved.
[0039] The phrases "splitting", "passaging", and "subculturing" refer to the
process of
dividing a population of cells into two or more subpopulations. For example, a
population of cells growing in a cell culture medium may be passaged or
subcultured by
removing a subpopulation of cells from the cell culture medium and diluting
that
subpopulation to a lower viable cell density. In certain embodiments, a
subpopulation of
cells may be diluted in a similar volume with fresh medium. In certain
embodiments, the
subpopulation of cells is diluted with a cell culture medium that is similar
or identical to
the cell culture medium in which the original population of cells was growing.
For
example, if the original population of cells was growing in a production
medium, the
subpopulation may be diluted with the same or similar production medium. In
certain
embodiments, the subpopulation of cells is diluted with a cell culture medium
that differs
from the cell culture medium in which the original population of cells was
growing. For
example, if the original population of cells was growing in a growth medium a
subpopulation of cells may be diluted with a production or adaptation medium.
In
certain embodiments, the original population of cells has stopped growing
(e.g.,
increasing in cell number) prior to passaging or subculturing a subpopulation.
In certain
embodiments, a population is passaged two or more times during the adaptation
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process. For example, a population may be passaged, 2, 3, 4, 5, 6, 7,8 ,9 10,
11,12, 13,
14, 15, 16, 17, 18, 19, 20 times or more during the adaptation process.
Standard
practice maintains passaging or "splitting" a cell culture medium every three
or four days
using a standard growth medium. In certain embodiments, cells are adapted to
protein
production conditions by growing a population of cells for a longer period of
time before
passaging. For example, cells may be grown 5, 6, 7, 8, 9, 10, 11, 12, 13, 14
days or
more before being passaged.
[0040] The phrase "viable cell density" refers to the total number of cells
that are
surviving in the cell culture medium in a certain volume. The phrase "cell
viability" refers
to number of cells that are alive compared to the total number of cells, both
dead and
alive, expressed as a percentage.
[0041] "Integrated Viable Cell Density", "IVCD": The terms "integrated viable
cell
density" or "IVCD" as used herein refer to the average density of viable cells
over the
course of the culture multiplied by the amount of time the culture has run.
When the
amount of protein produced is proportional to the number of viable cells
present over the
course of the culture, integrated viable cell density is a useful tool for
estimating the
amount of protein produced over the course of the culture.
[0042] As used herein, the term "antibody" includes a protein comprising at
least one,
and typically two, VH domains or portions thereof, and/or at least one, and
typically two,
VL domains or portions thereof. In certain embodiments, the antibody is a
tetramer of
two heavy immunoglobulin chains and two light immunoglobulin chains, wherein
the
heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide
bonds.
The antibodies, or a portion thereof, can be obtained from any origin,
including, but not
limited to, rodent, primate (e.g., human and non-human primate), camelid, as
well as
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recombinantly produced, e.g., chimeric, humanized, and/or in vitro generated,
as
described in more detail herein.
[0043] Examples of binding fragments encompassed within the term "antigen-
binding
fragment" of an antibody include, but are not limited to, (i) a Fab fragment,
a monovalent
fragment consisting of the VL, VH, CL and CH I domains; (ii) a F(ab')2
fragment, a
bivalent fragment comprising two Fab fragments linked by a disulfide bridge at
the hinge
region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv
fragment
consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb
fragment,
which consists of a VH domain; (vi) a camelid or camelized heavy chain
variable domain
(VHH); (vii) a single chain Fv (scFv); (viii) a bispecific antibody; and (ix)
one or more
fragments of an immunoglobulin molecule fused to an Fc region. Furthermore,
although
the two domains of the Fv fragment, VL and VH, are coded for by separate
genes, they
can be joined, using recombinant methods, by a synthetic linker that enables
them to be
made as a single protein chain in which the VL and VH regions pair to form
monovalent
molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988)
Science
242:423-26; Huston et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5879-83).
Such single
chain antibodies are also intended to be encompassed within the term "antigen-
binding
fragment" of an antibody. These fragments may be obtained using conventional
techniques known to those skilled in the art, and the fragments are evaluated
for
function in the same manner as are intact antibodies.
[0044] The "antigen-binding fragment" can, optionally, further include a
moiety that
enhances one or more of, e.g., stability, effector cell function or complement
fixation.
For example, the antigen binding fragment can further include a pegylated
moiety,
albumin, or a heavy and/or a light chain constant region.
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[0045] Other than "bispecific" or "bifunctional" antibodies, an antibody is
understood
to have each of its binding sites identical. A "bispecific" or "bifunctional
antibody" is an
artificial hybrid antibody having two different heavy/light chain pairs and
two different
binding sites. Bispecific antibodies can be produced by a variety of methods
including
fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai &
Lachmann,
Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-
1553
(1992).
[0046] The phrases "screen" or "screening" refer to a method of selecting a
subpopulation of cells with a certain phenotype that exhibits one or more
advantageous
characteristics. In certain embodiments, a cell line is screened for one or
more cell
culture characteristics that are advantageous in a protein production process
including,
but not limited to improved growth, increased viability, increased integrated
viable cell
density, increased titer, and/or increased cell specific productivity. In
certain
embodiments, in a cell line for is screened for one or more cell culture
characteristics
that are advantageous in a fed-batch bioreactor process, e.g., strong growth
and/or
viability.
[0047] The phrase "bioreactor" refers to a vessel in which a cell culture
medium can
be contained and internal conditions of which can be controlled during the
culturing
period, e.g., pH and temperature. One of ordinary skill in the art will be
aware of useful
and/or appropriate bioreactor conditions that may be controlled, as well as
methods of
controlling such bioreactor conditions. A "production bioreactor" refers to a
bioreactor
that is utilized during a protein production process. For example, a
production
bioreactor may comprise a large commercial-scale vessel from which a large
amount of
protein may be produced, although a production bioreactor is not limited to
such large
commercial-scale vessels. In certain embodiments, the volume of a bioreactor
is at
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least 1 liter and may be 10, 100, 250, 500, 1,000, 2,500, 5,000, 8,000,
10,000, 12,000
liters or more, or any volume in between. In addition, bioreactors that may be
used
include, but are not limited to, a stirred tank bioreactor, fluidized bed
reactor, hollow fiber
bioreactor, or roller bottle.
[0048] The phrase "secondary side-products" and "secondary metabolites" refer
to
small molecules typically generated as a result of cellular metabolic
activity. As is
understood by those of ordinary skill in the art, such secondary side-products
are often
detrimental to cell growth and/or viability. Thus, their minimization or
elimination from a
cell culture is desirable. Non-limiting examples of secondary side-products
include
lactate and ammonium ions. In certain embodiments, cells are adapted to
protein
production conditions by growing the cell in the presence of such secondary
side-
products. In certain embodiments, cells adapted to protein production
conditions exhibit
the ability to take up such secondary side-products, such that the levels of
secondary
side-products decrease over time. One of ordinary skill in the cell culture
art will be
aware of other metabolic side-products that may be used to adapt cells in
accordance
with the present invention.
[0049] A "fed batch culture" refers to a method of culturing cells in which
cells are
first inoculated in a bioreactor with an inoculum medium. The cell culture
medium is
then supplemented at one or more points throughout the production run with a
feed
medium containing nutritional components and/or other supplements.
[0050] A "batch culture" refers to a method of culturing cells in which cells
are
inoculated in a bioreactor with all the necessary nutrients and supplements
for the
entirety of the production run. No nutrients, media, etc. are added to the
cell culture
medium after the cell culture is initiated.
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[0051] A "perfusion culture" refers to a method of culturing cells that is
different from
a batch or fed-batch culture method, in which the culture is not terminated,
or is not
necessarily terminated, prior to isolating and/or purifying an expressed
protein of
interest, and in which new nutrients and other components are periodically or
continuously added to the culture, during which the expressed protein is
periodically or
continuously harvested. The composition of the added nutrients may be changed
during
the course of the cell culture, depending on the needs of the cells, the
requirements for
optimal protein production, and/or any of a variety of other factors known to
those of
ordinary skill in the art.
[0052] The phrase "batch-refeed" refers to a mode of operating a bioreactor or
a
method of passaging cells. In certain embodiments, a batch-refeed process
comprises
passaging cells every 7 or 8 days compared to other modes in bioreactors where
cells
are not passaged or passaged less frequently. On a smaller scale employing
standard
splitting methods, cells are generally passaged sooner, e.g. every 3 or 4
days,
compared to batch-refeed. In certain embodiments, batch-refeed methods are
used to
adapt a cell culture such that it is able to achieve an improved cell culture
characteristic
including, but not limited to, an improved growth rate, increased viability,
increased
integrated viable cell density, increased titer, and/or increased cell
specific productivity
In certain embodiments, a batch-refeed process utilizes a more enriched
culture and/or
a medium that contains secondary metabolites and/or other inhibitors in order
to adapt
cells to protein production conditions. The phrase "secondary metabolites"
refers to by-
products of metabolic processes of cell functions.
[0053] The phrase "expression" refers to the transcription and the translation
that
occurs within a host cell. The level of expression relates, generally, to the
amount of
protein being produced by the host cell.
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[0054] The phrase "protein" or "protein product" refers to one or more chains
of
amino acids. As used herein, the term "protein" is synonymous with
"polypeptide" and,
as is_generally understood in the art, refers to at least one chain of amino
acids liked via
sequential peptide bonds. In certain embodiments, a "protein of interest" is a
protein
encoded by an exogenous nucleic acid molecule that has been transformed into a
host
cell. In certain embodiments, where the "protein of interest" is encoded by an
exogenous DNA with which the host cell has been transformed, the nucleic acid
sequence of the exogenous DNA determines the sequence of amino acids. In
certain
embodiments, a "protein of interest" is a protein encoded by a nucleic acid
molecule that
is endogenous to the host cell. In certain embodiments, expression of such an
endogenous protein of interest is altered by transfecting a host cell with an
exogenous
nucleic acid molecule that may, for example, contain one or more regulatory
sequences
and/or encode a protein that enhances expression of the protein of interest.
Methods
and compositions of the present invention may be used to produce any protein
of
interest, including, but not limited to proteins having pharmaceutical,
diagnostic,
agricultural, and/or any of a variety of other properties that are useful in
commercial,
experimental and/or other applications. In addition, a protein of interest can
be a protein
therapeutic. Namely, a protein therapeutic is a protein that has a biological
effect on a
region in the body on which it acts or on a region of the body on which it
remotely acts
via intermediates. Examples of protein therapeutics are discussed in more
detail below.
In certain embodiments, proteins produced using methods and/or compositions of
the
present invention may be processed and/or modified. For example, a protein to
be
produced in accordance with the present invention may be glycosylated.
[0055] The term "titer" as used herein refers to the total amount of
recombinantly
expressed protein produced by a cell culture in a given amount of medium
volume. Titer
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is typically expressed in units of milligrams or micrograms of protein per
milliliter of
medium.
[0056] One of skill in the art will recognize that the methods disclosed
herein may be
used to culture many of the well-known mammalian cells routinely used and
cultured in
the art, i.e., the methods disclosed herein are not limited to use with only
the instant
disclosure.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
[0057] It has been discovered that mammalian cells, e.g. untransfected
mammalian
cells, including, e.g., Chinese hamster ovary cells, can be adapted to
environments
consistent with production bioreactor conditions to improve cell growth during
subsequent production of a protein of interest. Such methods are also
applicable to
both untransfected mammalian cells and to transfected mammalian cells (e.g.,
mammalian cells transfected with a cDNA or genomic construct causing the
expression,
as from an expression vector, of a desired recombinant protein). The present
invention
may be used to adapt cells for the advantageous production of any therapeutic
protein,
such as, for example, pharmaceutically or commercially relevant enzymes,
receptors,
antibodies (e.g., monoclonal and/or polyclonal antibodies), Fc fusion
proteins, cytokines,
hormones, regulatory factors, growth factors, coagulation/clotting factors,
antigen
binding agents, etc. One of ordinary skill in the art will be aware of other
proteins that
can be produced in accordance with the present invention, and will be able to
use
methods disclosed herein to produce such proteins.
[0058] Methods of the present invention for adapting untransfected mammalian
cells
have the potential for identifying cell lines that yield superior
productivities and/or exhibit
superior protein production characteristics. Furthermore, methods of the
present
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invention may help in the identification of suitable candidate cell lines more
quickly and
with less effort as compared to standard cell line development procedures. For
example, standard processes for identifying cell line candidates typically
require
numerous experiments on different scales and additional testing for
robustness.
[0059] Host cells, prior to being transformed are considered untransfected.
Traditionally, when creating a new cell line for development, untransfected
host cells
were initially transfected, or transformed, with exogenous DNA to express of a
protein of
interest. Using such prior methods, the host cells were not generally
experimented with
or altered prior to transfection; experimentation and research began on cell
line
development after the host cells were genetically manipulated to produce a
protein
product.
[0060] In certain embodiments, untransfected mammalian cells can be adapted to
protein production conditions with improved performance in batch, fed-batch,
and/or
perfusion bioreactor processes according to one or more methods of the present
invention. In certain embodiments, such adapted cells are capable of
maintaining an
improved phenotype, for example exhibiting stronger growth, increased
viability,
increased integrated viable cell density, increased titer, increased cell
specific
productivity and/or higher cell densities through such development. Certain
embodiments of inventive methods described herein may be employed to adapt
untransfected mammalian cells to a batch, fed-batch, and/or perfusion protein
production process having a superior performance.
[0061] Any of a variety of commercially available media such as, for example,
Minimal Essential Medium (MEM, Sigma), Ham's F I O (Sigma), or Dulbecco's
Modified
Eagle's Medium (DMEM, Sigma) may be used as the base medium. Such base media
may then be supplemented with amino acids, vitamins, inorganic salts, trace
elements,
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and/or other components to produce growth and/or production mediums. In
certain
embodiments, cells may be adapted by culturing transfected or untransfected
mammalian cells in a production medium similar or identical to a production
medium. In
certain embodiments, cells are adapted by growing the cells under conditions
similar or
identical to conditions typically encountered under production conditions in a
bioreactor
(e.g., in a medium consistent with the medium typically found in a bioreactor)
operated
in batch mode, fed-batch mode, and/or perfusion mode. In certain embodiments,
cells
to be adapted are untransfected. In certain embodiments, cells to be adapted
have
been transfected with an exogenous nucleic acid molecule, for example a
nucleic acid
molecule that expresses a protein of interest. In certain embodiments, one or
more cell
culture characteristics is improved during a protein production phase by
adapting cells to
protein production conditions prior to transfection with an exogenous nucleic
acid
molecule. In certain embodiments, such an improved cell culture characteristic
includes, without limitation, improved growth, improved the overall cell
viability,
increased integrated viable cell density, increased titer, and/or increased
cell specific
productivity. A production medium is typically more enriched than a growth
medium and
is, for example, supplemented with higher levels of nutrients, vitamins, trace
elements,
and/or other media components compared to a growth medium.
[0062] It is typical practice in the art for cells to be continuously cultured
in growth
medium during cell line development and not encounter production medium until
the
start of a fed-batch production assay. In certain embodiments, adaptation of
untransfected mammalian cells in a protein production medium and/or under
protein
production conditions prior to transfection, rather than growth medium and/or
growth
conditions, minimizes the potentially deleterious effects of transitioning the
cells from
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one environment to another, e.g., from growth conditions to protein production
conditions post-transfection.
[0063] In certain_embodiments, methods of the present invention are useful for
generating a host cells line that is adapted to protein production conditions.
Such an
adapted host cell line is capable of being transfected with any of a variety
of proteins of
interest. In certain embodiments, such an adapted, transfected cell line is
placed
directly into protein production conditions. In certain embodiments, such an
adapted,
transfected cell line is grown to a desired cell density and/or a desired cell
number
during a growth phase, after which the cell line is transitioned into a
protein production
phase. According to the present invention, such an adapted, transfected cell
line
exhibits fewer and less severe deleterious effects during the transition phase
compared
to an non-adapted, transfected cell line.
[0064] In certain embodiments, in order to produce a protein of interest,
adapted host
cells are transfected with an exogenous nucleic acid molecule. In certain
embodiments,
a nucleic acid molecule introduced into the cell encodes the protein desired
to be
expressed according to the present invention. In certain embodiments, a
nucleic acid
molecule contains a regulatory sequence or encodes a gene product that induces
or
enhances the expression of the desired protein by the cell. As a non-limiting
example,
such a gene product may be a transcription factor that increases expression of
the
protein of interest.
[0065] In certain embodiments, a nucleic acid that directs expression of a
protein is
stably introduced into the host cell. In certain embodiments, a nucleic acid
that directs
expression of a protein is transiently introduced into the host cell. One of
ordinary skill
in the art will be able to choose whether to stably or transiently introduce
the nucleic
acid into the cell based on experimental, commercial or other needs.
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[0066] A gene encoding a protein of interest may optionally be linked to one
or more
regulatory genetic control elements. In some embodiments, a genetic control
element
directs constitutive expression of the protein. In some embodiments, a genetic
control
element that provides inducible expression of a gene encoding the protein of
interest
can be used. Use of an inducible genetic control element (e.g., an inducible
promoter)
allows for modulation of the production of the protein in the cell. Non-
limiting examples
of potentially useful inducible genetic control elements for use in eukaryotic
cells include
hormone- regulated elements (see e.g., Mader, S. and White, J.H., Proc. Natl.
Acad.
Sci. USA 90:5603-5607, 1993), synthetic ligand-regulated elements (see, e.g.
Spencer,
D.M. et al., Science 262:1019-1024, 1993) and ionizing radiation-regulated
elements
(see e.g., Manome, Y. et al., Biochemistry 32:10607-10613, 1993; Datta, R. et
al., Proc.
Natl. Acad. Sci. USA 89:10149-10153, 1992). Additional cell-specific or other
regulatory
systems known in the art may be used in accordance with methods and
compositions
described herein.
[0067] Any protein that is expressible in a host cell may be produced in
accordance
with methods and compositions of the present invention. The protein may be
expressed
from a gene that is endogenous to the host cell, or from a heterologous gene
that is
introduced into the host cell. The protein may be one that occurs in nature,
or may
alternatively have a sequence that was engineered or selected by the hand of
man. A
protein to be produced may be assembled from protein fragments that
individually occur
in nature. Additionally or alternatively, the engineered protein may include
one or more
fragments that are not naturally occurring.
[0068] Any host cell susceptible to cell culture, and to expression of
proteins, may be
utilized in accordance with the present invention. In certain embodiments,
host cells are
mammalian cells, such as, for example, Chinese hamster ovary (CHO) cells.
Other
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non-limiting examples of mammalian cells that may be used in accordance with
the
present invention include BALB/c mouse myeloma line (NSO/l, ECACC No:
85110503);
human retinoblasts (PER.C6 (CruCell, Leiden, The Netherlands)); monkey kidney
CV1
line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line
(293
or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen
Virol.,
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster
ovary
cells +/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216
(1980));
mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey
kidney
cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-
1
587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells
(MDCK,
ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung
cells
(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor
(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.,
383:44-
68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[0069] In certain embodiments, cells (e.g. untransfected mammalian cells) are
adapted by culturing cells in growth medium supplemented with inhibitors. In
certain
embodiments, such inhibitors include inhibitors that are typically found when
cells are
grown under protein production conditions. Non-limiting examples of such
inhibitors
include lactate, ammonia, alanine, glutamine, and/or acetolactate. One of
ordinary skill
in the art will be aware of other inhibitors that may be used in accordance
with the
present invention to adapt cells to protein production conditions.
[0070] In certain embodiments, cells (e.g. untransfected mammalian cells) are
adapted by culturing cells in an adaptation medium that lacks or comprises a
reduced
concentration of one or more components traditionally found in production
media. For
example, traditional production media typically contain insulin at a
concentration of
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about 10mg/L or greater. Thus, in certain embodiments, cells are adapted by
culturing
cells in an adaptation medium that lacks insulin. In certain embodiments,
cells are
adapted by culturing cells in an adaptation medium that contains insulin at a
concentration lower than an insulin concentration traditionally found in
production media.
One of ordinary skill in the art will be aware of other components that are
typically
present in production media, and will be able to use methods of the present
invention to
adapt cells to media lacking or comprising a reduced concentration such
components.
[0071] In certain embodiments, cells are adapted by culturing (e.g.,
continuous
culturing) of untransfected mammalian cells in growth medium supplemented with
one
or more inhibitors. Non-limiting examples of such inhibitors include secondary
side-
products of primary carbon sources. For example, some secondary side-products
include, but are not limited to, lactate, alanine, glutamine, acetolactate,
and/or ammonia.
In certain embodiments, the concentrations of such secondary side-products
present in
and/or added to an adaptation medium mimic those typically encountered in
bioreactor
conditions, such as, for example, about 2.0 to about 10.0 g/L of lactate
and/or about 0.1
to about 0.5 g/L of ammonia. In certain embodiments, cells are adapted under
conditions in which the concentration of lactate in the adaptation medium is
about 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 g/L or higher. Additionally or
alternatively, in
certain embodiments, cells are adapted under conditions in which the
concentration of
ammonia in the adaptation medium is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0,
1.1, 1.2, 1.3, 1.4, 1.5 g/L or higher.
[0072] In certain embodiments, individual untransfected mammalian cells and/or
subpopulations of untransfected mammalian cells that have been adapted to
protein
production conditions according to one or more methods of the present
invention may
exhibit phenotypes consistent with the uptake of such secondary side-products,
such
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that levels of secondary side products actually decrease over time. n certain
embodiments, such adapted untransfected mammalian cells retain the ability to
take up
secondary side-products after they are transfected with an exogenous nucleic
acid
molecule to produce a protein of interest. In certain embodiments, such
adapted,
transfected mammalian cells generally perform better in a subsequent protein
production process (e.g. a fed-batch bioreactor process) relative to
untransfected
mammalian cells that do not uptake the side-products and/or that have not been
adapted to uptake such secondary side-products. For example, such adapted,
transfected mammalian cells perform better in a subsequent batch, fed-batch
and/or
perfusion protein production processes. In a production reactor (e.g., a fed-
batch
production reactor), such secondary side-products may often accumulate to
levels that
are generally inhibitory to further cell growth and/or to viability. In
certain embodiments,
subpopulations of untransfected mammalian cells that have been adapted
according to
methods of the present invention grow in a supplemented medium can grow well
under
the inhibitory and stressful conditions typically encountered in a production
bioreactor.
[0073] As is known in the cell culture art, in many instances the temperature
of a cell
culture is decreased the cell culture is switched from growth conditions to
protein
production conditions. In certain embodiments, cells are adapted by culturing
the cells
at a temperature conducive to the production of a protein product. In certain
embodiments, cells are adapted by culturing them at a temperature of about 31
C,
although methods of the present invention are not limited to such a
temperature. For
example, cells may be adapted by culturing them at a temperature of about 20,
21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, or '
45 C. One of ordinary skill in the art will be aware of temperature(s)
suitable for protein
production, which temperature may depend, at least in part, on the cell line,
the protein
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to be produced, other culture conditions, and/or any other factor deemed to be
important
by those of ordinary skill in the art.
[0074] In certain embodiments, cells are adapted by continuous culturing of
untransfected mammalian cells for a longer duration in a "batch-refeed" mode.
In
certain embodiments, untransfected cells are adapted by such "batch-refeed"
adaptation
methods. In certain embodiments, transfected cells are adapted by such "batch-
refeed"
adaptation methods. Typical cell culture operations may employ a cell culture
management regimen of splitting, passaging, or sub-culturing a cell culture
every three
or four days. In certain embodiments of "batch-refeed" adaptation methods,
untransfected mammalian cells are cultured for longer durations, such as seven
or eight
days, before being sub-cultured. In certain embodiments, mammalian cells are
cultured
for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more before being sub-cultured.
Such longer
duration cultures have the dual effect of adapting cells for protein
production conditions
by increasing cell density and/or by accumulating higher levels of secondary
metabolites
in the conditioned medium to which the cells can adapt.
[0075] In certain embodiments, untransfected mammalian cells may be
continuously
cultured under "production-matched" conditions. In certain embodiments,
untransfected
cells may be cultured with iterative cycles of cell culture under production-
matched
conditions followed by passaging subpopulations of the cells after 3 or 4 days
under
"recovery" or standard growth conditions with the option of using a growth
medium. In
certain embodiments, a population is passaged multiple times during the
adaptation
process. For example, a population may be passaged, 2, 3, 4, 5, 6, 7,8, 9, 10,
11, 12,
13, 14, 15, 16, 17, 18, 19, 20 times or more during the adaptation process. In
certain
embodiments, cells are adapted to protein production conditions by growing a
population of cells for a longer period of time before passaging. For example,
cells may
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be grown 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more before being passaged.
Such
passaged subpopulations may be screened for one or more desirable
characteristics. In
certain embodiments, after several cycles of iterative or continuous
adaptation,
subpopulations of cells will emerge that exhibit superior characteristics
including, for
example, improved growth and/or viability relative to the unadapted starting
population,
in the production-matched conditions. In addition, in certain embodiments,
certain
subpopulations will begin to take up side products, e.g. lactate, such that
the level of
that side product decreases over time, enhancing the performance of that cell
line in a
production bioreactor. In certain embodiments, one or more screened
subpopulations of
cells that exhibit one or more desirable characteristics (e.g., strong
growth)are then
transformed such that they produce a protein of interest, after which the
transfected
subpopulations are placed into protein production conditions (e.g., conditions
consistent
with those to which the cells have been adapted).
[0076] In certain embodiments, cells are grown in accordance with any of the
cell
culture methods described in United States Patent Application Serial Nos.
11/213,308,
11/213,317 and 11/213,633 each of which was filed August 25, 2005, and each of
which
is herein incorporated by reference in its entirety. For example, in certain
embodiments,
the cells may be grown in a culture medium in which the cumulative amino acid
concentration is greater than about 70 mM. In certain embodiments, the cells
may be
grown in a culture medium in which the molar cumulative glutamine to
cumulative
asparagine ratio is less than about 2. In certain embodiments, the cells may
be grown
in a culture medium in which the molar cumulative glutamine to cumulative
total amino
acid ratio is less than about 0.2. In certain embodiments, the cells may be
grown in a
culture medium in which the molar cumulative inorganic ion to cumulative total
amino
acid ratio is between about 0.4 to 1. In certain embodiments, the cells may be
grown in
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a culture medium in which the combined cumulative glutamine and cumulative
asparagine concentration is between about 16 and 36 mM. In certain
embodiments, the
cells may be grown in a culture medium that contains two, three, four or all
five of the
preceding medium conditions. Use of such media allows high levels of protein
production and lessens accumulation of certain undesirable factors such as
ammonium
and/or lactate.
[0077] In some embodiments, the cells are grown under one or more of the
conditions described in United States Provisional Patent Application Serial
No.
60/830,658, filed July 13, 2006 and incorporated herein by reference in its
entirety. For
example, in some embodiments, cells are grown in a culture medium that
contains
manganese at a concentration between approximately 10 and 600 nM. In some
embodiments, cells are grown in a culture medium that contains manganese at a
concentration between approximately 20 and 100 nM. In some embodiments, cells
are
grown in a culture medium that contains manganese at a concentration of
approximately
40 nM. Use of such media in growing glycoproteins results in production of a
glycoprotein with an improved glycosylation pattern (e.g. a greater number of
covalently
linked sugar residues in one or more oligosaccharide chains).
[0078] Components and/or supplements of the production medium and growth
medium can be readily determined by one skilled in the cell culture art. As is
known in
the art, components and/or supplements may vary depending on the host cell
used and
the desired protein of interest. In addition, the conditions and amount of
side-products
produced may or will vary with different bioreactor conditions and with each
different cell
line. The present disclosure is further illustrated by the following, non-
limiting examples.
Any modifications that might become necessary in the course of the adaptation
of
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mammalian cells to cell culture medium for production of different proteins
are well
within the art of cell culture.
EXAMPLES
Example 1: Adaptation of Untransfected Chinese Hamster Ovary Cells to
Production
Matched Conditions
[0079] Untransfected CHOK1 cells were cultured either in standard 3 day/4 day
batch-refeed conditions in growth medium, components listed in Table 1 below,
or
cultured in a 7-day batch-refeed mode in enriched production medium,
components
listed in Table 2 below, for multiple cycles. The cells were cultured at 37 C
in a working
volume from 10 to about 30ml. The cell numbers from the experiment are shown
in
Figures 1 a and 1 b. At around day 58 of the batch-refeed method of Figure 1
a, the cells
were passaged for 9 days, rather than 7, and the subsequent passaged did not
grow
well. After the poor 7-day passage, however, the cells were able to recover.
TABLE 1
Component mg/L
alanine 17.80
arginine 347.97
asparagine=H20 75.00
aspartic acid 26.20
cysteine=HCI=H20 70.19
cystine=2HCI 62.25
glutamic acid 29.40
monosodium glutamate 0.00
glutamine 1163.95
glycine 30.00
histidine=HCI=H2O 46.00
isoleucine 104.99
leucine 104.99
lysine=HCI 145.99
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methionine 29.80
phenylalanine 65.99
proline 68.99
serine 126.00
threonine 94.99
tryptophan 16.00
tyrosine=2Na=2 H20 103.79
valine 93.99
biotin 0.20
calcium pantothenate 2.24
choline chloride 8.98
folic acid 2.65
inositol 12.60
nicotinamide 2.02
pyridoxal=HCI 2.00
pyridoxine=HCI 0.03
riboflavin 0.22
thiamine=HCI 2.17
vitamin B12 0.78
CaCl2 116.09
KCI 311.77
Na2HP04 70.99
NaCI 5539.00
NaH2PO4=H20 62.49
MgSO4 48.83
MgCl2 28.61
NaHCO3 2440.00
Sodium Selenite 0.005
Fe(N03)3=9H20 0.050
CuSO4 0.001
FeSO4=7H20 0.840
ZnSO4=7H20 0.430
Hydrocortisone 0.036
Putrescine=2HCI 1.080
linoleic acid 0.040
thioctic acid 0.100
D-glucose (Dextrose) 6150.7
PVA 2400.000
Nucellin 10.000
Sodium Pyruvate 54.995
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TABLE 2
Components mg/L
alanine 24.87
arginine 423.43
asparagine=H20
173.90
aspartic acid 52.72
cysteine=HCI=H20
70.01
cystine=2HCI 62.09
glutamic acid 41.08
monosodium glutamate 0.00
glutamine 1162.40
glycine 35.92
histidine=HCI=H20
75.27
isoleucine 151.90
leucine 172.69
lysine=HCI 218.38
methionine 53.55
phenylalanine 98.81
proline 96.40
serine 273.07
threonine 132.81
tryptophan 28.99
tyrosine=2Na=2H20 145.10
valine 131.17
biotin 0.36
calcium pantothenate 4.03
choline chloride 16.11
folic acid 4.76
inositol 22.64
nicotinamide 3.61
pyridoxal=HCI 1.99
pyridoxine=HCI 1.67
riboflavin 0.40
thiamine=HCI 3.92
vitamin B12 1.34
CaC12
115.8
KCI 310.9
Na2HPO4
70.8
NaCI 3705.0
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NaH2P04=H20
114.3
MgSO4
48.7
MgSO4=7H20
8.6
MgCl2
28.5
NaHCO3
2440.0
Sodium Selenite 0.007
Fe(N03)3=9H20 0.050
CuSO4
0.001
CuSO4=5H2O
0.007
FeSO4=7H20
1.543
ZnSO4=7H20
1.384
MnSO4*H20 0.0002
Na2SiO3*9H20 0.140
(NH4)6Mo7O24*4H20 0.001
NH4V03 0.001
Hydrocortisone 0.086
Putrescine=2HCI 2.480
linoleic acid 0.057
thioctic acid 0.142
D-glucose (Dextrose) 11042.2
PVA 2520.0
Nucellin 14.000
Sodium Pyruvate 54.849
[0080] Cells were then evaluated in a fed-batch production assay, where the
working
volume was between 10 and 30m1. The cells were cultured at 37 C for four days
and
then the temperature was shifted to 31 dC until day 12, when the assay
completed. The
cell density and viability were compared and the results are illustrated in
Figure 1 c.
Cells cultured in 7-day batch-refeed mode exhibited higher cell densities than
cells
cultured in standard 3-day/4 day batch refeed mode. Similarly, two different
groups of
cells that were adapted for growth in production medium, components listed in
Table 3
below, achieved higher cell densities than the unadapted control starting
population.
TABLE 3
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Component mg/L
alanine 17.86
arginine 698.24
asparagine=H20
3000.00
aspartic acid 220.16
cysteine=HCI=H20
70.63
cystine=2HCI 468.75
monosodium glutamate 33.91
glutamine 584.00
glycine 115.87
histidine=HCI=H20 476.13
isoleucine
572.57
leucine 1034.01
lysine=HCI 1405.91
methionine 388.65
phenylalanine 508.63
proline 541.23
serine 1055.38
threonine 566.62
tryptophan 275.04
tyrosine=2Na=2H20 746.00
valine
751.41
biotin 2.69
calcium pantothenate 22.00
choline chloride 158.97
folic acid 26.01
inositol 164.51
nicotinamide 26.31
pyridoxal=HCI 2.04
pyridoxine=HCI 36.24
riboflavin 2.42
thiamine=HCI 39.56
vitamin B12 21.24
CaC12 116.92
KCI
313.91
KH2P04 0.00
Na2HPO4
56.78
NaH2PO4=H20 647.92
MgSO4
138.44
MgC12
28.59
NaHCO3
2000.00
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Sodium Selenite
0.069
Fe(N03)3=9H20 0.050
CuSO4 0.010
CuSO4=5H20
0.100
FeSO4=7H20
4.170
ZnSO4=7H20
2.649
MnSO4*H20
0.034
Na2SiO3*9H20
0.284
(NH4)6Mo7O24*4H20 0.247
NH4VO3 0.002
NiSO4*6H20 0.005
SnC12*2H20 0.001
IC13*6H20 0.001
CrCI3 0.016
KI 0.033
H3B03 0.012
Hydrocortisone 0.540
Putrescine=2HCI 15.000
linoleic acid 0.291
thioctic acid 0.718
D-glucose (Dextrose) 15016.08
PVA 2560.00
Nucellin 50.00
Sodium Pyruvate 55.18
Example 2: Adaptation of Transfected Product Cell Line to Production-matched
Conditions
[0081] A cell line expressing the heavy and light chain genes of a monoclonal
antibody was cultured under standard 3 day/4 day batch refeed conditions,
using either
standard growth medium, components listed in Table 1, or two versions of
production
medium, "A" and "B". Production medium A, components listed in Table 2, was
moderately enriched relative to growth medium, and production medium B,
components
listed in Table 4 below, was significantly enriched relative to growth medium.
The
effects of methotrexate (MTX) were also tested in production medium "B", 1.5
pM of
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MTX was added into one culture with production medium "B" and not into
another. The
cells were cultured at 37 C in a working volume from 10 to about 30m1. Growth
rates
during adaptation in these media are shown in Figure 2a.
TABLE 4
Components mg/L
alanine 5.95
arginine 232.75
asparagine=HZO 750.00
aspartic acid 73.39
cysteine=HCI=H20 23.54
cystine=2HCI 250.00
glutamic acid 0.00
monosodium glutamate 44.30
glutamine 1160.00
glycine 38.62
histidine=HCI=H20 158.71
isoleucine 190.86
leucine 344.67
lysine=HCI 468.64
methionine 129.55
phenylalanine 169.54
proline 180.41
serine 351.79
threonine 188.87
tryptophan 91.68
tyrosine=2Na=2H20 250.00
valine 250.47
biotin 0.90
calcium pantothenate 7,33
choline chloride 52.99
folic acid 8.67
inositol 54.84
nicotinamide 8.77
pyridoxal=HCI 0.68
pyridoxine=HCI 12.08
riboflavin 0.81
thiamine=HCI 13.19
vitamin B12 7.08
CaC12 109.0
KCI 604.6
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NaZHPO4 18.9
NaCI 2000.0
NaH2PO4=H2O 216.0
MgSO4 46.1
MgSO4=7H20 80.0
MgCIZ 9.5
NaHCO3 3300.0
Sodium Selenite 0.035
Fe(N03)3=9H20 0.017
CuSO4 0.003
CuSO4=5H20 0.050
FeSO4=7H20 2.500
ZnSO4=7H20 0.883
MnSO4*H20 0.0169
Na2SiO3*9H20 0.142
(NH4)6Mo7O24*4H20 0.124
NH4VO3 0.001
NiSO4*6H20 0.003
CrCI3 0.008
KI 0.017
H3B03 0.006
Hydrocortisone 0.180
Putrescine=2HCI 5.000
linoleic acid 0.097
thioctic acid 0.239
D-glucose (Dextrose) 10072.0
PVA 2560.0
Nucellin 20.000
Sodium Pyruvate 18.392
[0082] After continuous culture in these mediums, the cell lines were
evaluated in a
fed-batch production assay, in which cells were evaluated in production medium
B,
illustrated in Figure 2b. The cells were cultured at 37 C for four days and
then the
temperature was shifted to 31 C until day 12, when the assay was complete.
The
results indicate that cell lines that over express recombinant protein may
also be
adapted under production-matched conditions to achieve superior growth
characteristics
in a fed-batch production assay.
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Example 3: Adaptation of Untransfected Chinese Hamster Ovary Cells to Low
Insulin
Conditions
[0083] Insulin directly impacts the metabolism of glucose by mammalian cells.
The
rapid consumption of glucose in cell culture is frequently coupled with the
excretion of
lactic acid as a metabolic waste product. Lactic acid can inhibit cell growth
and have
negative effects on cell viability. Insulin is also reported to be growth
factor for
mammalian cells.
[0084] Cells from the CHOK1 host cell line were taken from normal growth media
(containing 10 mg/L nucellin) and put into media completely lacking insulin.
After an
initial lag phase in which the cells demonstrated diminished growth, the
growth rate
eventually climbed to a rate comparable to that of the CHOK1 control culture
in insulin
containing media, suggesting that cells cultured in the absence of insulin
have adapted
to these conditions. The viability of the cells was not affected and remained
in the mid-
90s throughout adaptation. This adaptation was continued for approximately 100
population doublings. When the adapted CHOK1-insulin cells were banked and
then
thawed, they maintained their ability to grow in insulin-free media. CHOK1
control cells
thawed into insulin-free media showed a reduction in growth rate suggesting
that the
K1-insulin adaptation indeed altered the cells, allowing them to grow
independent of
exogenous insulin. Continuing investigations include combining the insulin-
free
phenotype with the 7-day passage adaptation phenotype.
[0085] The first fed-batch production assay was set up in a non-pH adjusted
format.
The experiment included non-adapted CHOK1 cells in standard production media
(20
mg/mL nucellin) as a positive control, and non-adapted CHOK1 cells cultured in
insulin-
free production media as a negative control. The insulin-free adapted CHOK1
cells
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were cultured in insulin-free production media. Insulin was included in the
feed media
on days 3 and 7 (at a final concentration of 0.006 mg/mL). Examination of the
data
suggests the CHOK1 cells adapted to grow in insulin-free media have very
similar
growth, viability and IVCD characteristics when compared to the CHOK1 positive
control
sample (see Figures 3-5). The negative control CHOK1 sample showed a reduction
in
growth rate (see Figure 3). These results confirm that CHOK1 cells adapted to
insulin-
free growth are inherently different from the non-adapted CHOK1 cells, given
their ability
to grow well in media that does not contain insulin.
[0086] The insulin-free adapted CHOKI cells were then evaluated in a second
fed
batch experiment, under conditions with pH control. In this experiment the non-
adapted
CHOK1 cells and insulin-free adapted CHOK1 cells were set up in three separate
conditions. The first condition contained insulin in both the base media and
the feed (50
mg/L and 0.006 mg/mL respectively), symbolized in Figures 6-9 as (+/+). The
second
condition contained insulin-free base media with an insulin containing feed
(symbolized
by (-/+) in Figures 6-9) and the third condition contained insulin-free base
media and
insulin-free feed media (symbolized by (-/-) in Figures 6-9). The insulin-free
adapted
CHOK1 cells demonstrate better growth, viability and IVCD when cultured in
insulin-free
media as compared to the non-adapted CHOK1 cells (see Figures 6-9). The CHOK1-
insulin adapted cell lines seem to produce less lactate and almost completely
consume
whatever lactate they do produce (see Figure 9). This elimination of a
detrimental
byproduct leads to a healthier cell culture and is seen as a very promising
phenotype.
This promising phenotype provides better growth conditions and allows the
cells to
reach higher densities then the associated control. It should be noted that
this
phenotype is directly related to the elimination of insulin from the media, as
the CHOK1-
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insulin adapted cell lines has similar lactate production rates to the control
sample when
cultured in media containing insulin.
[0087] Example 3 demonstrates that adaptation of CHOK1 cells to an insulin-
free
conditions leads to desirable phenotypes when cells are cultured in
industrially relevant
production modes. The improved metabolic phenotypes lead to increased cell
growth
and viability, which are expected to have a significant positive impact on the
volumetric
productivity of a recombinant CHO cell culture.
[0088] Although certain embodiments of the disclosure have been described
herein,
the above description is merely illustrative. Further modification of the
embodiments
herein disclosed will occur to those skilled in the cell culture art and all
such
modifications are deemed to be within the scope of the embodiments as defined
by the
appended claims.
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