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

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 minima! detrimental effects on one or more cell
culture characteristics (e.g., titer, viability, protein quality, etc.) after transfection and
transition into protein production conditions.

[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,

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

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

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

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

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 1c 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

[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 NS0) 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 "cell 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,

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

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

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

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

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

reconibinantly 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 CH1 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.

[0045] Other than "bispecific" or "bifunotional" 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

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 ceils 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.

[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.

[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

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, pharmaceuticaliy 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

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 ceils
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 F10 (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,

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. !n 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

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.

[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

non-limiting examples of mammalian cells that may be used in accordance with the
present invention include BALB/c mouse myeloma line (NSO/I, 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 ai., 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

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

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

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

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

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. !n 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

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 1a and 1b. At around day 58 of the batch-refeed method of Figure 1a, 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.

[0080] Cells were then evaluated in a fed-batch production assay, where the working
volume was between 10 and 30ml. 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 completed. The
cell density and viability were compared and the results are illustrated in Figure 1c.
Cells cultured in 7-day batch-refeed mode exhibited higher ceil 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

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 µM of

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 30ml. Growth rates
during adaptation in these media are shown in Figure 2a.

[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.

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

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 CHOK1 ceils 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-

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.

What is claimed is:
1. A method for adapting cells to protein production conditions comprising:
culturing the cells in an adaptation medium;
screening the cells and selecting a subpopulation of cells that exhibits an
improved cell culture characteristic when the subpopulation is grown under protein
production conditions, which characteristic differs from a corresponding cell culture
characteristic that would be observed in cells grown in a medium that is not an
adaptation medium, wherein the improved cell culture characteristic is selected from the
group consisting of: improved growth, increased viability, increased integrated viable cell
density, increased titer, increased cell specific productivity, and combinations thereof.
2. The method of claim 1, further comprising the step of passaging the cells prior to
the screening step.
3. The method of claim 2 or 3, wherein the step of passaging comprises passaging
the cells two or more times prior to the screening step.
4. The method of any one of claims 1-3, wherein the step of passaging comprises
passaging the cells after approximately 3 or 4 days in the adaptation medium.
6. The method of any one of claims 1-3, wherein the step of passaging comprises
passaging the cells after approximately 7 or 8 days in the adaptation medium.

6. The method of any one of claims 1-5, further comprising the step culturing the
cells in a medium that is not an adaptation medium after the passaging step.
7. The method of claim 6, wherein the medium that is not an adaptation medium is a
standard growth medium.
8. The method of any one of claims 1-7, wherein the cells are not transfected.
9. The method of any one of claims 1-7, wherein the cells have been transfected to
express a protein of interest.
10. The method of claim 9, wherein the protein of interest is an antibody.
11. The method of claim 9, wherein the protein of interest is a protein therapeutic.
12. The method of any one of claims 1-11, wherein the adaptation medium
comprises a production medium.
13. The method of claim 12, wherein the production medium comprises an increased
level of one or more medium components as compared to a standard growth medium,
the medium components selected from the group consisting of: nutrients, vitamins, trace
elements, and combinations thereof.

14. The method of any one of claims 1-13, wherein the adaptation medium
comprises a secondary metabolite selected from the group consisting of: lactate,
ammonia, and combinations thereof.
15. The method of claim 14, wherein the secondary metabolite is added to the
adaptation medium at the beginning of the cell culture.
16. The method of claim 14 or 15, wherein the level of the secondary metabolite is
increased as the cell culture progresses.
17. The method of claim 16, wherein the level of the secondary metabolite increases
due to metabolic activity of the cells.
18. The method of claim 16 or 17, wherein the level of the secondary metabolite
increases through addition of the secondary metabolite to the cell culture.
19. The method of any one of claims 14-18, wherein the selected subpopulation of
cells take a secondary metabolite, such that the level of the metabolite decreases when
the adapted cells are grown in a production medium.
20. The method of any one of claims 1-19, wherein the adaptation medium
comprises one or more inhibitors selected from the group consisting of: lactate,
ammonia, alanine, glutamine, acetolactate, and combinations thereof.

21. The method of any one of claims 14-20, wherein lactate is present in a
concentration of about 2 to about 10g/L.
22. The method of claim 21, wherein lactate is present at the beginning of the cell
culture in a concentration of about 2 to about 10g/L.
23. The method of any one of claims 14-20, wherein ammonia is present in a
concentration of about 0.1 to about 0.5 g/L.
24. The method of claim 23, wherein ammonia is present at the beginning of the cell
culture in a concentration of about 0.1 to about 0.5 g/L.
25. The method of any of the preceding claims, wherein the adaptation medium lacks
insulin.
26. The method of any of the preceding claims, wherein the adaptation medium
includes insulin at a concentration lower than about 10 mg/L.
27. The method of any of the preceding claims, wherein the cells are mammalian.
28. The method of any of the preceding claims, wherein the protein production
conditions comprise conditions used in a bioreactor.
29. The method of claim 28, wherein the bioreactor is a production bioreactor.

30. The method of any of the preceding claims, wherein the protein production
conditions comprise culturing the cells in a fed-batch protein production process.

Methods of adapting cells, e.g., mammalian cells, to a cell culture process are provided. When the adapted cells are
genetically modified and used for protein production, they exhibit beneficial characteristics, such as being able to attain higher cell
densities and/or achieve a higher overall yield of the produced protein.