Indian Patents
Title of Invention

PRODUCTION OF ANTI-AMYLOID BETA ANTIBODIES
Abstract An improved system for large scale production of anti-amyloid beta antibodies in cell culture, particularly in media characterized by one or more of i) a cumulative amino acid concentration greater than about 70 mM; ii) a molar cumulative glutamine to cumulative asparagine ratio of less than about 2; iii) a molar cumulative glutamine to cumulative total amino acid ratio of less than about 0.2; iv) a molar cumulative inorganic ion to cumulative total amino acid ratio between about 0.4 to 1; or v) a combined cumulative glutamine and cumulative asparagine concentration between about 16 and 36 mM, is provided. The use of such a system allows high levels of protein production and lessens accumulation of certain undesirable factors such as ammonium and/or lactate. Additionally, culture methods including a temperature shift, typically including a decrease in temperature when the culture has reached about 20-80% of its maximal cell density, are provided. Alternatively or additionally, the present invention provides methods such that, after reaching a peak, lactate and/or ammonium levels in the culture decrease over time.
Full Text This invention relates to production of a-ABeta.
Background of the Invention
Proteins and polypeptides have become increasingly important as therapeutic
agents. In most cases, therapeutic proteins and polypeptides are produced in cell culture,
from cells that have been engineered and/or selected to produce unusually high levels of
the particular protein or polypeptide of interest. Control and optimization of cell culture
conditions is critically important for successful commercial production of proteins and
polypeptides.
Many proteins and polypeptides produced in cell culture are made in a batch or fedbatch
process, in which cells are cultured for a period of time, and then the culture is
terminated and the produced protein or polypeptide is isolated. The ultimate amount and
quality of protein or polypeptide produced can be dramatically affected by the conditions
of the cell culture. For example, traditional batch and fed-batch culture processes often
result in production of metabolic waste products that have detrimental effects on cell
growth, viability, and production or stability of the protein or polypeptide of interest.
While efforts have been made to improve production of proteins and polypeptides in batch
and fed-batch culture processes, there remains a need for additional improvements.
Additionally, significant effort has been invested in the development of defined
media (i.e., media assembled from known individual components and lacking serum or
other animal byproducts) for use in culturing cells, particularly mammalian cells. Cell
growth characteristics can be very different in defined media as contrasted with serumderived
media. There is a particular need for the development of improved systems for
producing proteins and polypeptides by cell culture in defined media.
Summary of the Invention
The present invention provides an improved system for large scale production of
proteins and/or polypeptides in cell culture. For example, the present invention provides
commercial scale (e.g., 500 L or more) culture methods that utilize a medium characterized
by one or more of: i) a cumulative amino acid amount per unit volume greater than about mM; ii) a molar cumulative glutamine to cumulative asparagine ratio of less than about 2;
iii) a molar cumulative glutamine to cumulative total amino acid ratio of less than about iv) a molar cumulative inorganic ion to cumulative total amino acid ratio between about to 1; or v) a combined cumulative amount of
glutamine and asparagine concentration per unit volume greater than about 16 mM.
One of ordinary skill in the art will understand that "cumulative", as used above, refers
to the total amount of a particular component or components added over the course of
the cell culture, including components added at the beginning of the culture and
subsequently added components. In certain preferred embodiments of the invention, it
is desirable to minimize "feeds" of the culture over time, so that it is desirable to
maximize amounts present initially. Of course, medium components are metabolized
during culture so that cultures with the same cumulative amounts of given components
will have different absolute levels if those components are added at different times
(e.g., all present initially vs. some added by feeds).
According to the present invention, use of such a medium allows high levels of
protein production and lessens accumulation of certain undesirable factors such as
ammonium and/or lactate.
One of ordinary skill in the art will understand that the media formulations of the
present invention encompass both defined and non-defined media. In certain preferred
embodiments of the present invention, the culture medium is a defined medium in
which the composition of the medium is known and controlled.
In certain preferred embodiments of the present invention, the culture methods
include changing the culture from a first set of culture conditions to a second set of
culture conditions so that a metabolic shift of the cells is achieved. In some
embodiments, this change is performed when the culture has reached about 20-80% of
its maximal cell density. In some embodiments, the change involves changing the
temperature (or temperature range) at which the culture is maintained. Alternatively or
additionally, the present invention provides methods adjusted so th,at, after reaching a
peak, lactate and/or ammonium levels in the culture decrease over time. In other
embodiments, the shift involves shifting the pH, osmolarlity or level of chemical
inductants, such as alkanoic acids or their salts.
Cell cultures of the present invention may optionally be supplemented with
nutrients and/or other medium components including hormones and/or other growth
factors, particular ions (such as sodium, chloride, calcium, magnesium, and phosphate),
buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds
usually present at very low final concentrations), amino acids, lipids, or glucose or
other energy source. In certain embodiments of the present invention, it may be
beneficial to supplement the media with chemical inductants such as hexamethylenebis(
acetamide) ("HMBA") and sodium butyrate ("NaB"). These optional supplements
may be added at the beginning of the culture or may be added at a later point in order
to replenish depleted nutrients or for another reason. In general, it is desirable to select
the initial medium composition to minimize supplementation in accordance with the
present invention.
Various culture conditions may be monitored in accordance with the present
invention, including pH, cell density, cell viability, lactate levels, ammonium levels,
osmolarity, or titer of the expressed polypeptide or protein.
Brief Description of the Drawing
Figure 1 shows a comparison of Medium 1 and Medium 2 in shake flasks using anti-
GDF-8 cells. • .
Figure 2 shows cell growth and viability of anti-GDF-8 cells in Medium 1.
Figure 3 shows cell growth of anti-GDF-8 cell cultures in control and no glutamine
feed culture conditions.
Figure 4 shows cell viability of anti-GDF-8 cell cultures in control and no glutamine
feed culture conditions.
Figure 5 shows ammonium levels of anti-GDF-8 cell cultures in control and no
glutamine feed culture conditions.
Figure 6 shows lactate levels of anti-GDF-8 cell cultures in control and no glutamine
feed culture conditions.
Figure 7 shows anti-GDF-8 titer in control and no glutamine feed culture conditions.
Figure 8 shows cell density of anti-GDF-8 cell cultures in control and glutaminestarved
feed culture conditions.
Figure 9 shows cell viability of anti-GDF-8 cell cultures in control and glutaminestarved
feed culture conditions. '
Figure 10 shows ammonium levels of anti-GDF-8 cell cultures in control and
glutamine-starved culture conditions.
Figure 11 shows lactate levels of anti-GDF-8 cell cultures in control and glutaminestarved
culture conditions.
Figure 12 shows anti-GDF-8 titer in control and glutamine-starved culture conditions.
Figure 13 shows iron dose response of anti-GDF-8 cells in Medium 1 and Medium 2.
Figure 14 shows cell density of Glutamate and Glutamine fed cultures.
Figure 15 shows cell viability of Glutamate and Glutamine fed cultures.
Figure 16 shows anti-Lewis Y titer in Glutamate and Glutamine fed cultures.
Figure 17 shows lactate levels in Glutamate and Glutamine fed cultures.
Figure 18 shows ammonium levels in Glutamate and Glutamine fed cultures.
Figure 19 shows osmolarity of Glutamate and Glutamine fed cultures.
Figure 20 shows cell density of anti-Lewis Y cells. Each plot is the average of two
shake flasks grown using the same conditions.
Figure 21 shows cell viability of anti-Lewis Y cells. Each plot is the average of two
shake flasks grown using the same conditions.
Figure 22 shows average titer of anti-Lewis Y culture. Each plot is the average of two
shake flasks grown using the same conditions.
Figure 23 shows ammonium levels of anti-Lewis Y cells. Each plot is the average of
two shake flasks grown using the same conditions. '
Figure 24 shows an impeller jump used in fed-batch cultures.
Figure 25 shows cell growth of anti-GDF-8 cells under various experimental
conditions.
Figure 26 shows viability of anti-GDF-8 cells under varioFigure 27 shows anti-GDF-8
titer under various experimental conditions.
Figure 28 shows lactate levels of anti-GDF-8 cultures under various experimental
conditions.
Figure 29 shows ammonium levels of anti-GDF-8 cultures under various
experimental conditions.
Figure 30 shows cell growth of anti-GDF-8 cells under various experimental
conditions.
Figure 31 shows anti-GDF-8 titer under various experimental conditions.
Figure 32 shows lactate levels of anti-GDF-8 cultures under various experimental
conditions.
Figure 33 shows ammonium levels of anti-GDF-8 cultures under various
experimental conditions.
Figure 34 shows cell growth of anti-GDF-8 cells in modified Medium 9 containing
various levels of glutamine and asparagine.
Figure 35 shows cell viability of anti-GDF-8 cells in modified Medium 9 containing
various levels of glutamine and asparagine.
Figure 36 shows lactate levels of anti-GDF-8 cultures in modified Medium 9
containing various levels of glutamine and asparagine.
Figure 37 shows ammonium levels of anti-GDF-8 cultures in modified Medium 9
containing various levels of glutamine and asparagine.
Figure 38 shows glutamine levels of anti-GDF-8 cultures in modified Medium 9
containing various levels of glutamine and asparagine.
Figure 39 shows anti-GDF-8 titer in modified Medium 9 containing various levels of
glutamine and asparagine.
Figure 40 shows osmolarity of anti-GDF-8 cultures in modified Medium 9 containing
various levels of glutamine and asparagine.
Figure 41 shows cell growth of anti-GDF-8 cells in media containing various levels of
asparagine and cysteine.
Figure 42 shows lactate levels of anti-GDF-8 cultures in media containing various
levels of asparagine and cysteine.
Figure 43 shows ammonium levels of anti-GDF-8 cultures in media containing various
levels of asparagine and cysteine.
Figure 44 shows glutamine levels of anti-GDF-8 cultures in media containing various
levels of asparagine and cysteine.
Figure 45 shows glutamate levels of anti-GDF-8 cultures in media containing various
levels of asparagine and cysteine.
Figure 46 shows anti-GDF-8 titer in media containing various levels of asparagine and
cysteine.
Figure 47 shows osmolarity of anti-GDF-8 cultures in media containing various levels
of asparagine and cysteine.
Figure 48 shows cell growth of anti-GDF-8 cells in media containing various levels of
amino acids and vitamins.
Figure 49 shows lactate levels of anti-GDF-8 cultures in media containing various
levels of amino acids and vitamins.
Figure 50 shows ammonium levels of anti-GDF-8 cultures in media containing various
levels of amino acids and vitamins.
Figure 51 shows glutamine levels of anti-GDF-8 cultures in media containing various
levels of amino acids and vitamins.
Figure 52 shows anti-GDF-8 titer in media containing various levels of amino acids
and vitamins.
Figure 53 shows cell growth of anti-GDF-8 cells in media containing various levels of
vitamins, trace elements E and iron.
Figure 54 shows lactate levels of anti-GDF-8 cultures in media containing various
levels of vitamins, trace elements E and iron.
Figure 55 shows ammonium levels of anti-GDF-8 cultures in media containing various
levels of vitamins, trace elements E and iron.
Figure 56 shows anti-GDF-8 titer in media containing various levels of vitamins, trace
elements E and iron.
Figure 57 shows cell growth of anti-GDF-8 cells in Mediums 1, 3 and 9.
Figure 58 shows anti-GDF-8 titer in Medium 1, 3 and 9.
Figure 59 shows extrapolated anti-GDF-8 titers for various levels of glutamine alone
and total combined glutamine and asparagine.
Figure 60 shows cell growth of anti-ABeta cells under various media conditions tested.
Figure 61 shows cell viability of anti-ABeta cells under various rnedia conditions
tested.
Figure 62 shows lactate levels of anti-ABeta cultures under various media conditions
tested.
Figure 63 shows ammonium levels of anti-ABeta cultures under various media
conditions tested.
Figure 64 shows anti-ABeta titer in various media conditions tested.
Figure 65 shows osmolarity of anti-ABeta cultures under various media conditions
tested.
Figure 66 shows cell growth of cells expressing TNFR-Ig under various experimental
conditions.
Figure 67 shows viability of cells expressing TNFR-Ig under various experimental
conditions.
Figure 68 shows residual glucose in cultures of cells expressing TNFR-Ig under
various experimental conditions.
Figure 69 shows glutamine levels in cultures of cells expressing TNFR-Ig under
various experimental conditions.
Figure 70 shows lactate concentration in cultures of cells expressing TNFR-Ig under
various experimental conditions.
Figure 71 shows ammonium levels in cultures of cells expressing TNFR-Ig under
various experimental conditions.
Figure 72 shows TNFR-Ig relative titer under various experimental conditions.
Figure 73 shows cell densities of anti-GDF-8 cells grown in 6000 L and 1 L
bioreactors.
Figure 74 shows titers of anti-GDF-8 cells grown in 6000 L and 1 L bioreactors.
Figure 75 shows lactate levels of anti-GDF-8 cells grown in 6000 L and 1 L
bioreactors.
Figure 76 shows ammonium levels of anti-GDF-8 cells grown in 6000 L and 1 L
bioreactors.
Definitions
"About", "Approximately": As used herein, the terms "about" and
"approximately", as applied to one or more particular cell culture conditions, refer to a
range of values that are similar to the stated reference value for that culture condition or
conditions. In certain embodiments, the term "about" refers to a range of values that fall
within 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 percent or less
of the stated reference value for that culture condition or conditions.
"Amino acid": The term "amino acid" as used herein refers to any of the twenty
naturally occurring amino acids that are normally used in the formation of polypeptides,
or analogs or derivatives of those amino acids. Amino acids of the present invention are
provided in medium to cell cultures. The amino acids provided in the medium may be
provided as salts or in hydrate form.
"Antibody": The term "antibody" as used herein refers to an immunoglobulin
molecule or an immunologically active portion of an immunoglobulin molecule, such as
a Fab or F(ab')2 fragment, that contains one or more antigen binding sites which
specifically bind (immunoreact with) an antigen. The terms "monoclonal antibodies"
and "monoclonal antibody composition", as used herein, refer to a clonal population of
antibody molecules that contain only one species of an antigen binding site capable of
immunoreacting with a particular epitope of an antigen, whereas thd terms "polyclonal
antibodies" and "polyclonal antibody composition" refer to a population of antibody
molecules that contain multiple species of antigen binding sites capable of interacting
with a particular antigen. The definition of monoclonal antibodies includes both clonal
molecules derived by traditional technologies as well as molecules of defined sequence
derived by manipulation or mutation of specific residues, for example, humanized
antibodies.
"Batch culture": The term "batch culture" as used herein refers to a method of
culturing cells in which all the components that will ultimately be used in culturing the
8
cells, including the medium (see definition of "medium" below) as well as the cells
themselves, are provided at the beginning of the culturing process. A batch culture is
typically stopped at some point and the cells and/or components in the medium are
harvested and optionally purified.
"Bioreactor": The term "bioreactor" as used herein refers to any vessel used for
the growth of a mammalian cell culture. The bioreactor can be of any size so long as it is
useful for the culturing of mammalian cells. Typically, the bioreactor will be at least 1
liter and may be 10, 100, 250, 500, 1000, 2500, 5000, 8000, 10,000, 12,0000 liters or
more, or any volume in between. The internal conditions of the bioreactor, including,
but not limited to pH and temperature, are typically controlled during the culturing
period. The bioreactor can be composed of any material that is suitable for holding
mammalian cell cultures suspended in media under the culture conditions of the present
invention, including glass, plastic or metal. The term "production bioreactor" as used
herein refers to the final bioreactor used in the production of the polypeptide or protein
of interest. The volume of the large-scale cell culture production bioreactor is typically
at least 500 liters and may be 1000, 2500, 5000, 8000, 10,000, 12,0000 liters or more, or
any volume in between. One of ordinary skill in the art will be aware of and will be able
to choose suitable bioreactors for use in practicing the present invention.
"Cell density": The term "cell density" as used herein refers to that number of
cells present in a given volume of medium.
"Cell viability": The term "cell viability" as used herein refers to the ability of
cells in culture to survive under a given set of culture conditions or experimental
variations. The term as used herein also refers to that portion of cells which are alive at a
particular time in relation to the total number of cells, living and dead, in the culture at
that time.
"Culture", "Cell culture" and "Mammalian cell culture": Thqse terms as used
herein refer to a mammalian cell population that is suspended in a medium (see
definition of "medium" below) under conditions suitable to survival and/or growth of the
cell population. As will be clear to those of ordinary skill in the art, these terms as used
herein may refer to the combination comprising the mammalian cell population and the
medium in which the population is suspended.
"Fed-batch culture": The term "fed-batch culture" as used herein refers to a
method of culturing cells in which additional components are provided to the culture at
some time subsequent to the beginning of the culture process. The provided components
typically comprise nutritional supplements for the cells which have been depleted during
the culturing process. A fed-batch culture is typically stopped at some point and the cells
and/or components in the medium are harvested and optionally purified.
"Fragment": The term "fragment" as used herein refers to polypeptides and is
defined as any discrete portion of a given polypeptide that is unique to or characteristic
of that polypeptide. The term as used herein also refers to any discrete portion of a given
polypeptide that retains at least a fraction of the activity of the full-length polypeptide.
Preferably the fraction of activity retained is at least 10% of the activity of the full-length
polypeptide. More preferably the fraction of activity retained is at least 20%, 30%, 40%,
50%, 60%, 70%, 80% or 90% of the activity of the full-length polypeptide. More
preferably still the fraction of activity retained is at least 95%, 96%, 97%, 98% or 99% of
the activity of the full-length polypeptide. Most preferably, the fraction of activity
retained is 100% of the activity of the full-length polypeptide. The term as used herein
also refers to any portion of a given polypeptide that includes at least an established
sequence element found in the full-length polypeptide. Preferably, the sequence element
spans at least 4-5, more preferably at least about 10, 15, 20, 25, 30, 35, 40, 45, 50 or
more amino acids of the full-length polypeptide.
"Gene": The term "gene" as used herein refers to any nucleotide sequence, DNA
or RNA, at least some portion of which encodes a discrete final product, typically, but
not limited to, a polypeptide, which functions in some aspect of cellular metabolism or
development. The term is not meant to refer only to the coding sequence that encodes
the polypeptide or other discrete final product, but may also encompass regions
preceding and following the coding sequence that modulate the basal level of expression
(see definition of "genetic control element" below), as well as intervening sequences
("introns") between individual coding segments ("exons").
"Genetic control element": The term "genetic control element" as used herein
refers to any sequence element that modulates the expression of a gene to which it is
operably linked. Genetic control elements may function by either increasing or
decreasing the expression levels and may be located before, within or after the coding
sequence. Genetic control elements may act at any stage of gene expression by
regulating, for example, initiation, elongation or termination of transcription, mRNA
splicing, mRNA editing, mRNA stability, mRNA localization within the cell, initiation,
elongation or termination of translation, or any other stage of gene expression. Genetic
control elements may function individually or in combination with one another.
"Hybridoma": The term "hybridoma" as used herein refers to a cell created by
fusion of an immortalized cell derived from an immunologic source and an antibodyproducing
cell. The resulting hybridoma is an immortalized cell that produces
antibodies. The individual cells used to create the hybridoma can be from any
mammalian source, including, but not limited to, rat, pig, rabbit, sheep, pig, goat, and
human. The term also encompasses trioma cell lines, which result when progeny of
heterohybrid myeloma fusions, which are the product of a fusion between human cells
and a murine myeloma cell line, are subsequently fused with a plasma cell. Furthermore,
the term is meant to include any immortalized hybrid cell line that produces antibodies
such as, for example, quadromas (See, e.g., Milstein et al., Nature, 537:3053 (1983)).
"Integrated Viable Cell Density": The term "integrated viable cell density" as
used herein refers to the average density of viable cells over the course of the culture
multiplied by the amount of time the culture has run. Assuming the amount of
polypeptide and/or 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 polypeptide and/or protein produced over the course of the culture.
"Medium", "Cell culture medium", "Culture medium": These terms as used
herein refer to a solution containing nutrients which nourish growing mammalian cells.
Typically, these solutions provide essential and non-essential amino acids, vitamins,
energy sources, lipids, and trace elements required by the cell for minimal growth and/or
survival. The solution may also contain components that enhance growth and/or survival
above the minimal rate, including hormones and growth factors. The solution is
preferably formulated to a pH and salt concentration optimal for cell survival and
proliferation. The medium may also be a "defined media" - a serum-free media that
contains no proteins, hydrolysates or components of unknown composition. Defined
media are free of animal-derived components and all components have a known
chemical structure.
"Metabolic waste product": The term "metabolic waste product" as used herein
refers to compounds produced by the cell culture as a result of normal or non-normal
metabolic processes that are in some way detrimental to the cell culture, particularly in
relation to the expression or activity of a desired recombinant polypeptide or protein.
For example, the metabolic waste products may be detrimental to the growth or viability
of the cell culture, may decrease the amount of recombinant polypeptide or protein
produced, may alter the folding, stability, glycoslyation or other post-translational
modification of the expressed polypeptide or protein, or may be detrimental to the cells
and/or expression or activity of the recombinant polypeptide or protein in any number of
other ways. Exemplary metabolic waste products include lactate, which is produced as a
result of glucose metabolism, and ammonium, which is produced as a result of glutamine
metabolism. One goal of the present invention is to slow production of, reduce or even
eliminate metabolic waste products in mammalian cell cultures.
"Osmolarity" and "Osmolality": "Osmolality" is a measure of the osmotic
pressure of dissolved solute particles in an aqueous solution. The solute particles include
both ions and non-ionized molecules. Osmolality is expressed as the concentration of
osmotically active particles (i.e., osmoles) dissolved in 1 kg of solution (1 mOsm/kg H2O
at 38°C is equivalent to an osmotic pressure of 19mm Hg). "Osmolarity," by contrast,
refers to the number of solute particles dissolved in 1 liter of solution. When used herein,
the abbreviation "mOsm" means "milliosmoles/kg solution".
"Perfusion culture": The term "perfusion culture" as used herein refers to a
method of culturing cells in which additional components are provided continuously or
semi-continuously to the culture subsequent to the beginning of the culture process. The
provided components typically comprise nutritional supplements for the cells which have
been depleted during the culturing process. A portion of the cells and/or components in
the medium are typically harvested on a continuous or semi-continuous basis and are
optionally purified.
"Polypeptide": The term "polypeptide" as used herein refers a sequential chain of
amino acids linked together via peptide bonds. The term is used to refer to an amino
acid chain of any length, but one of ordinary skill in the art will understand that the term
is not limited to lengthy chains and can refer to a minimal chain comprising two amino
acids linked together via a peptide bond.
"Protein": The term "protein" as used herein refers to one or more polypeptides
that function as a discrete unit. If a single polypeptide is the discrete functioning unit
and does require permanent physical association with other polypeptides in order to form
the discrete functioning unit, the terms "polypeptide" and "protein" as used herein are
used interchangeably. If discrete functional unit is comprised of more than one
polypeptide that physically associate with one another, the term "protein" as used herein
refers to the multiple polypeptides that are physically coupled and function together as
the discrete unit.
"Recombinantly expressed polypeptide" and "Recombinant polypeptide": These
terms as used herein refer to a polypeptide expressed from a mammalian host cell that
has been genetically engineered to express that polypeptide. The recombinantly
expressed polypeptide can be identical or similar to polypeptides that are normally
expressed in the mammalian host cell. The recombinantly expressed polypeptide can
also foreign to the host cell, i.e. heterologous to peptides normally expressed in the
mammalian host cell. Alternatively, the recombinantly expressed polypeptide can be
chimeric in that portions of the polypeptide contain amino acid sequences that are
identical or similar to polypeptides normally expressed in the mammalian host cell, while
other portions are foreign to the host cell.
"Seeding": The term "seeding" as used herein refers to the process of providing a
cell culture to a bioreactor or another vessel. The cells may have been propagated
previously in another bioreactor or vessel. Alternatively, the cells may have been frozen
and thawed immediately prior to providing them to the bioreactor or yessel. The term
refers to any number of cells, including a single cell.
"Titer": The term "titer" as used herein refers to the total amount of
recombinantly expressed polypeptide or protein produced by a mammalian cell culture
divided by a given amount of medium volume. Titer is typically expressed in units of
milligrams of polypeptide or protein per milliliter of medium.
Detailed Description of Certain Preferred Embodiments
The present invention provides improved systems for the production of proteins
and/or polypeptides by cell culture. In particular, the invention provides systems that
minimize production of one or more metabolic products detrimental to cell growth,
viability, and/or protein production or quality. In a preferred embodiment of the present
invention, the cell culture is a batch or fed-batch culture. Other certain preferred
embodiments of the invention are discussed in detail below. Those of ordinary skill in
the art will understand, however, that various modifications toi these preferred
embodiments are within the scope of the appended claims. It is the claims and
equivalents thereof that define the scope of the present invention, which is not and
should not be limited to or by this description of certain preferred embodiments.
Polvpeptides
Any polypeptide that is expressible in a host cell may be produced in accordance with
the present to the host cell, or from a gene that is introduced into the host cell through genetic
engineering. The polypeptide may be one that occurs in nature, or may alternatively
have a sequence that was engineered or selected by the hand of man. An engineered
polypeptide may be assembled from other polypeptide segments that individually occur
in nature, or may include one or more segments that are not naturally occurring.
Polypeptides that may desirably be expressed in accordance with the present
invention will often be selected on the basis of an interesting biological or chemical
activity. For example, the present invention may be employed to express any
pharmaceutically or commercially relevant enzyme, receptor, antibody, hormone,
regulatory factor, antigen, binding agent, etc.
Antibodies
Given the large number of antibodies currently in use or under investigation as
pharmaceutical or other commercial agents, production of antibodies is of particular
interest in accordance with the present invention. Antibodies are proteins that have the
ability to specifically bind a particular antigen. Any antibody that can be expressed in a
host cell may be used in accordance with the present invention. In a preferred
embodiment, the antibody to be expressed is a monoclonal antibody.
In another preferred embodiment, the monoclonal antibody is a chimeric
antibody. A chimeric antibody contains amino acid fragments that are derived from
more than one organism. Chimeric antibody molecules can include, for example, an
antigen binding domain from an antibody of a mouse, rat, or other species, with human
constant regions. A variety of approaches for making chimeric antibodies have been
described. See e.g., Morrison et al, Proc. Natl. Acad. Sci. U.S.A. 81, 6851 (1985);
Takeda et al., Nature 314, 452 (1985), Cabilly et al., U.S. Patent No. 4,816,567; Boss et
al, U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent Publication EP171496;
European Patent Publication 0173494, United Kingdom Patent GB 2177096B.
In another preferred embodiment, the monoclonal antibody is a human antibody
derived, e.g., through the use of ribosome-display or phage-display libraries (see, e.g.,
Winter et al., U.S. Patent No. 6,291,159 and Kawasaki, U.S. Patent No. 5,658,754) or the
use of xenographic species in which the native antibody genes are inactivated and
functionally replaced with human antibody genes, while leaving intact the other
components of the native immune system (see, e.g., Kucherlapati et al., U.S. Patent No.
6,657,103).
In another preferred embodiment, the monoclonal antibody is a humanized antibody.
A humanized antibody is a chimeric antibody wherein the large majority of the amino
acid residues are derived from human antibodies, thus minimizing any potential immune
reaction when delivered to a human subject. In humanized antibodies, amino acid
residues in the complementarity determining regions are replaced, at least in part, with
residues from a non-human species that confer a desired antigen specificity or affinity.
Such altered immunoglobulin molecules can be made by any of several techniques
known in the art, (e.g., Teng et al, Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983);
Kozbor et al., Immunology Today, 4, 7279 (1983); Olsson et al., Meth. EnzymoL, 92, 3-
16 (1982)), and are preferably made according to the teachings of PCT Publication
WO92/06193 or EP 0239400, all of which are incorporated herein by reference).
Humanized antibodies can be commercially produced by, for example, Scotgen Limited,
2 Holly Road, Twickenham, Middlesex, Great Britain. For further reference, see Jones
et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and
Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), all of which are incorporated herein by
reference.
In another preferred embodiment, the monoclonal, chimeric, or humanized
antibodies described above may contain amino acid residues that do not naturally occur
in any antibody in any species in nature. These foreign residues can be utilized, for
example, to confer novel or modified specificity, affinity or effector function on the
monoclonal, chimeric or humanized antibody. In another preferred embodiment, the
antibodies described above may be conjugated to drugs for systemic pharmacotherapy,
such as toxins, low-molecular-weight cytotoxic drugs, biological response modifiers, and
radionuclides (see e.g., Kunz et al., Calicheamicin derivative-carrier conjugates.
US20040082764A1).
In one embodiment, the antibody is an antibody that specifically binds to the Ap
fragment of amyloid precursor protein or to other components of an amyloid plaque, and
is useful in combating the accumulation of amyloid plaques in the brain which
characterize Alzheimer's disease. (See, e.g., US Provisional Application 60/636,684.)
In another embodiment, antibodies of the present invention are directed against
cell surface antigens expressed on target cells and/or tissues in proliferative disorders
such as cancer. In one embodiment, the antibody is an IgGl anti-Lewis Y antibody.
Lewis Y is a carbohydrate antigen with the structure FucaJ —> 2GalBl —» 4[FucaJ -*
3]GlcNac61^3R (Abe et al. (1983) J. Biol. Chem., 258 11793-11797). Lewis Y antigen
is expressed on the surface of 60% to 90% of human epithelial tumors (including those
of the breast, colon, lung, and prostate), at least 40% of which overexpress this antigen,
and has limited expression in normal tissues.
In order to target Ley and effectively target a tumor, an antibody with exclusive
specificity to the antigen is ideally required. Thus, preferably, the anti-Lewis Y
antibodies of the present invention do not cross-react with the type 1 structures (i.e., the
lacto-series of blood groups (Lea and Leb)) and, preferably, do not bind other type 2
epitopes (i.e., neolacto-structure) like Lex and H-type 2 structures. An example of a
preferred anti-Lewis Y antibody is designated hu3S193 (see U.S. Patent Nos. 6,310,185;
6,518,415; 5,874,060, incorporated herein in their entirety). The humanized antibody
hu3S193 (Attia, M.A., et al. 1787-1800) was generated by CDR-grafting from 3S193,
which is a murine monoclonal antibody raised against adenocarcinoma cell with
exceptional specificity for Ley (Kitamura, K., 12957-12961). Hu3S193 not only retains
the specificity of 3S193 for Ley but has also gained in the capability to mediate
complement dependent cytotoxicity (hereinafter referred to as CDC) and antibody
dependent cellular cytotoxicity (hereinafter referred to as ADCC) (Attia, M.A., et al.
1787-1800). This antibody targets Ley expressing xenografts in nude mice as
demonstrated by biodistribution studies with hu3S193 labeled with 1251, lllln, or 18F,
as well as other radiolabels that require a chelating agent, such as 11 lln, 99mTc, or 90Y
(Clark, etal. 4804-4811).
In another embodiment, the antibody is one of the human antl-GDF-8 antibodies
termed Myo29, Myo28, and Myo22, and antibodies and antigen- binding fragments
derived therefrom. These antibodies are capable of binding mature GDF-8 with high
affinity, inhibit GDF-8 activity in vitro and in vivo as demonstrated, for example, by
inhibition of ActRIIB binding and reporter gene assays, and may inhibit GDF-8 activity
associated with negative regulation of skeletal muscle mass and bone density. See, e.g.,
Veldman, et al, U.S. Patent Application No. 20040142382.
Receptors
Another class of polypeptides that have been shown to be effective as
pharmaceutical and/or commercial agents includes receptors. Receptors are typically
trans-membrane glycoproteins that function by recognizing an extra-cellular signaling
ligand. Receptors typically have a protein kinase domain in addition to the ligand
recognizing domain, which initiates a signaling pathway by phosphorylating target
intracellular molecules upon binding the ligand, leading to developmental or metabolic
changes within the cell. In one embodiment, the receptors of interest are modified so as
to remove the transmembrane and/or intracellular domain(s), in place of which there may
optionally be attached an Ig-domain. In a preferred embodiment, receptors to be
produced in accordance with the present invention are receptor tyrosine kinases (RTKs).
The RTK family includes receptors that are crucial for a variety of functions numerous
cell types (see, e.g., Yarden and Ullrich, Ann. Rev. Biochem. 57:433-478, 1988; Ullrich
and Schlessinger, Cell 61:243-254, 1990, incorporated herein by reference). Nonlimiting
examples of RTKs include members of the fibroblast growth factor (FGF)
receptor family, members of the epidermal growth factor receptor (EGF) family, platelet
derived growth factor (PDGF) receptor, tyrosine kinase with immunoglobulin and EGF
homology domains-1 (TIE-1) and TIE-2 receptors (Sato et al, Nature 376(6535):70-74
(1995), incorporated herein be reference) and c-Met receptor, some of which have been
suggested to promote angiogenesis, directly or indirectly (Mustonen and Alitalo, J. Cell
Biol 129:895-898, 1995). Other non-limiting examples of RTK's include fetal liver
kinase 1 (FLK-1) (sometimes referred to as kinase insert domain-containing receptor
(KDR) (Terman et al., Oncogene 6:1677-83, 1991) or vascular endothelial cell growth
factor receptor 2 (VEGFR-2)), fms-like tyrosine kinase-1 (Flt-1) (DeVries et al. Science
255;989-991, 1992; Shibuya et al., Oncogene 5:519-524, 1990), sometimes referred to as
vascular endothelial cell growth factor receptor 1 (VEGFR-1), neuropilin-1, endoglin,
endosialin, and Axl. Those of ordinary skill in the art will be aware of other receptors
that can preferably be expressed in accordance with the present invention.
In a particularly preferred embodiment, tumor necrosis factor inhibitors, in the
form of tumor necrosis factor alpha and beta receptors (TNFR-1; EP 417,563 published
Mar. 20, 1991; and TNFR-2, EP 417,014 published Mar. 20, 1991) are expressed in
accordance with the present invention (for review, see Naismith and Sprang, J Inflamm.
47(l-2):l-7 (1995-96), incorporated herein by reference). According to one embodiment,
the tumor necrosis factor inhibitor comprises a soluble TNF receptor and preferably a
TNFR-Ig. In one embodiment, the preferred TNF inhibitors of the present invention are
soluble forms of TNFRI and TNFRII, as well as soluble TNF binding proteins, in
another embodiment, the TNFR-Ig fusion is a TNFR:Fc, a term which as used herein
refers to "etanercept," which is a dimer of two molecules of the extracellular portion of
the p75 TNF-.alpha. receptor, each molecule consisting of a 235 amino acid Fc portion
of human IgG.sub.l.
Growth Factors and Other Signaling Molecules • ,
Another class of polypeptides that have been shown to be effective as
pharmaceutical and/or commercial agents includes growth factors and other signaling
molecules. Growth factors are typically glycoproteins that are secreted by cells and bind
to and activate receptors on other cells, initiating a metabolic or developmental change in
the receptor cell. In one embodiment, the protein of interest is an ActRIIB fusion
polypeptide comprising the extracellular domain of the ActRIIB receptior and the Fc
portion of an antibody (see, e.g., Wolfman, et al., ActRIIB fusion polvpeptides and uses
therefor. US2004/0223966 Al). In another embodiment, the growth factor may be a
modified GDF-8 propeptide (see., e.g., Wolfman, et al., Modifed and stabilized GDF
propeptides and uses thereof. US2003/0104406 Al). Alternatively, the protein of
interest could be a follistatin-domain-containing protein (see, e.g., Hill, et al., GASP1: a
follistatin domain containing protein. US 2003/0162714 Al, Hill, et al., GASP1: a
follistatin domain containing protein. US 2005/0106154 Al, Hill, et al., Follistatin
domain containing proteins. US 2003/0180306 Al).
Non-limiting examples of mammalian growth factors and other signaling
molecules include cytokines; epidermal growth factor (EGF); platelet-derived growth
factor (PDGF); flbroblast growth factors (FGFs) such as aFGF and bFGF; transforming
growth factors (TGFs) such as TGF-alpha and TGF-beta, including TGF-beta 1, TGFbeta
2, TGF-beta 3, TGF-beta 4, or TGF-beta 5; insulin-like growth factor-I and -II (IGFI
and IGF-II); des(l-3) -IGF-I (brain IGF-I), insulin-like growth factor binding proteins;
CD proteins such as CD-3, CD-4, CD-8, and CD-19; erythropoietin; osteoinductive
factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as
interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,
GM-CSF, and G-CSF; interleukins (TLs), e.g., IL-1 to IL-10; tumor necrosis factor
(TNF) alpha and beta; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating
hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor
VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as
Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as
urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin;
thrombin, hemopoietic growth factor; enkephalinase; RANTES (regulated on activation
normally T-cell expressed and secreted); human macrophage inflammatory protein
(MIP-1-alpha); mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain;
prorelaxin; mouse gonadotropin-associated peptide; neurotrophic factors such as bonederived
neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or
NT-6), or a nerve growth factor such as NGF-beta. One of ordinary skill in the art will
be aware of other growth factors or signaling molecules that can be expressed in
accordance with the present invention.
G-Protein Coupled Receptors
Another class of polypeptides that have been shown to be effective as
pharmaceutical and/or commercial agents includes growth factors and other signaling
molecules. G-protein coupled receptors (GPCRs) are proteins that have seven
transmembrane domains. Upon binding of a ligand to a GPCR, a signal is transduced
within the cell which results in a change in a biological or physiological property of the
cell.
GPCRs, along with G-proteins and effectors (intracellular enzymes and channels
which are modulated by G-proteins), are the components of a modular signaling system
that connects the state of intracellular second messengers to extracellular inputs. These
genes and gene-products are potential causative agents of disease.
Specific defects in the rhodopsin gene and the V2 vasopressin receptor gene have
been shown to cause various forms of autosomal dominant and autosomal recessive
retinitis pigmentosa, nephrogenic diabetes insipidus. These receptors are of critical
importance to both the central nervous system and peripheral physiological processes.
The GPCR protein superfamily now contains over 250 types of paralogues, receptors that
represent variants generated by gene duplications (or other processes), as opposed to
orthologues, the same receptor from different species. The superfamily can be broken
down into five families: Family I, receptors typified by rhodopsin and the beta2-
adrenergic receptor and currently represented by over 200 unique members; Family II,
the recently characterized parathyroid hormone/calcitonin/secretin receptor family;
Family III, the metabotropic glutamate receptor family in mammals; Family IV, the
cAMP receptor family, important in the chemotaxis and development of D. discoideum;
and Family V, the fungal mating pheromone receptors such as STE2.
GPCRs include receptors for biogenic amines, for lipid mediators of
inflammation, peptide hormones, and sensory signal mediators. The GPCR becomes
activated when the receptor binds its extracellular ligand. Conformational changes in the
GPCR, which result from the ligand-receptor interaction, affect the binding affinity of a
G protein to the GPCR intracellular domains. This enables GTP to bind with enhanced
affinity to the G protein.
Activation of the G protein by GTP leads to the interaction of the G protein a
subunit with adenylate cyclase or other second messenger molecule generators. This
interaction regulates the activity of adenylate cyclase and hence production of a second
messenger molecule, cAMP. cAMP regulates phosphorylation and activation of other
intracellular proteins. Alternatively, cellular levels of other second messenger molecules,
such as cGMP or eicosinoids, may be upregulated or downregulated by the activity of
GPCRs. The G protein a subunit is deactivated by hydrolysis of the GTP by GTPase, and
the a(3 and y subunits reassociate. The heterotrimeric G protein then dissociates from the
adenylate cyclase or other second messenger molecule generator. Activity of GPCR may
also be regulated by phosphorylation of the intra- and extracellular domains or loops.
Glutamate receptors form a group of GPCRs that are important in
neurotransmission. Glutamate is the major neurotransmitter in the CNS and is believed
to have important roles in neuronal plasticity, cognition, memory, learning and some
neurological disorders such as epilepsy, stroke, and neurodegeneration (Watson, S. and
S. Arkinstall (1994) The G- Protein Linked Receptor Facts Book, Academic Press, San
Diego CA, pp. 130-132). These effects of glutamate are mediated by two distinct classes
of receptors termed ionotropic and metabotropic. lonotropic receptors contain an
intrinsic cation channel and mediate fast excitatory actions of glutamate. Metabotropic
receptors are modulatory, increasing the membrane excitability of neurons by inhibiting
calcium dependent potassium conductances and both inhibiting and potentiating
excitatory transmission of ionotropic receptors. Metabotropic receptors are classified into
five subtypes based on agonist pharmacology and signal transduction pathways and are
widely distributed in brain tissues.
The vasoactive intestinal polypeptide (VIP) family is a group of related
polypeptides whose actions are also mediated by GPCRs. Key members of this family
are VIP itself, secretin, and growth hormone releasing factor (GRF). VIP has a wide
profile of physiological actions including relaxation of smooth muscles, stimulation or
inhibition of secretion in various tissues, modulation of various immune cell activities,
and various excitatory and inhibitory activities in the CNS. Secretin stimulates secretion
of enzymes and ions in the pancreas and intestine and is also present in small amounts in
the brain. GRF is an important neuroendocrine agent regulating synthesis and release of
growth hormone from the anterior pituitary (Watson, S. and S. Arkinstall supra, pp. 278-
283).
Following ligand binding to the GPCR, a conformational change is transmitted to
the G protein, which causes the a-subunit to exchange a bound GDP molecule for a GTP
molecule and to dissociate from the py-subunits. The GTP-bound form of the a-subunit
typically functions as an effector-modulating moiety, leading to the production of second
messengers, such as cyclic AMP (e.g., by activation of adenylate cyclase), diacylglycerol
or inositol phosphates. Greater than 20 different types of oc-subunits are known in man,
which associate with a smaller pool of pandysubunits. Examples of mammalian G
proteins include Gi, Go, Gq, Gs and Gt. G proteins are described extensively in Lodish
H. et al. Molecular Cell Biology, (Scientific American Books Inc., New York, N.Y.,
1995), the contents of which is incorporated herein by reference. ' i
GPCRs are a major target for drug action and development. In fact, receptors
have led to more than half of the currently known drugs (Drews, Nature Biotechnology,
1996, 14: 1516) and GPCRs represent the most important target for therapeutic
intervention with 30% of clinically prescribed drugs either antagonizing or agonizing a
GPCR (Milligan, G. and Rees, S., (1999) TIPS. 20: 118-124). This demonstrates that
these receptors have an established, proven history as therapeutic targets.
In general, practitioners of the present invention will selected their polypeptide of
interest, and will know its precise amino acid sequence. The techniques of the present
invention have been successfully applied to production of diverse polypeptides
including, for example, a human monoclonal antibody directed to growth and
differentiation factor 8 (Examples 1, 3, 4, 7-14), humanized anti-Lewis Y antibody
(Examples 5 and 6), anti-ABeta (Example 15) and a dimeric Fc-fusion protein of tumor
necrosis factor receptor (Example 16), indicating that the present invention will be useful
for expression of a variety of different polypeptides and proteins. Any 'given protein that
is to be expressed in accordance with the present invention will have its own
idiosyncratic characteristics and may influence the cell density or viability of the cultured
cells, and may be expressed at lower levels than another polypeptide or protein grown
under identical culture conditions. One of ordinary skill in the art will be able to
appropriately modify the steps and compositions of the present invention in order to
optimize cell growth and/or production of any given expressed polypeptide or protein.
Genetic Control Elements
As will be clear to those of ordinary skill in the art, genetic control elements may
be employed to regulate gene expression of the polypeptide or protein. Such genetic
control elements should be selected to be active in the relevant host cell. Control
elements may be constitutively active or may be inducible under defined circumstances.
Inducible control elements are particularly useful when the expressed protein is toxic or
has otherwise deleterious effects on cell growth and/or viability. In such instances,
regulating expression of the polypeptide or protein through inducible control elements
may improve cell viability, cell density, and /or total yield of the expressed polypeptide
or protein. A large number of control elements useful in the practice of the present
invention are known and available in the art.
Representative constitutive mammalian promoters that may be used in
accordance with the present invention include, but are not limited to, the hypoxanthine
phosphoribosyl transferase (HPTR) promoter, the adenosine deaminase promoter, the
pyruvate kinase promoter, the beta-actin promoter as well as other constitutive promoters
known to those of ordinary skill in the art. Additionally, viral prompters that have been
shown to drive constitutive expression of coding sequences in eukaryotic cells include,
for example, simian virus promoters, herpes simplex virus promoters, papilloma virus
promoters, adenovirus promoters, human immunodeficiency virus (HIV) promoters,
Rous sarcoma virus promoters, cytomegalovirus (CMV) promoters, the long terminal
repeats (LTRs) of Moloney murine leukemia virus and other retroviruses, the thymidine
kinase promoter of herpes simplex virus as well as other viral promoters known to those
of ordinary skill in the art.
Inducible promoters drive expression of operably linked coding sequences in the
presence of an inducing agent and may also be used in accordance with the present
invention. For example, in mammalian cells, the metallothionein promoter is induces
transcription of downstream coding sequences in the presence of certain metal ions.
Other inducible promoters will be recognized by and/or known to those of ordinary skill
in the art.
In general, the gene expression sequence will also include 5' non-transcribing and
5' non-translating sequences involved with the initiation of transcription and translation,
respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
Enhancer elements can optionally be used to increase expression levels of the
polypeptides or proteins to be expressed. Examples of enhancer elements that have been
shown to function in mammalian cells include the SV40 early gene enhancer, as
described in Dijkema et al., EMBO J. (1985) 4: 761 and the enhancer/promoter derived
from the long terminal repeat (LTR) of the Rous Sarcoma Virus (RSV), as described in
Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and human cytomegalovirus,
as described in Boshart et al., Cell (1985) 41:521. • i
Systems for linking control elements to coding sequences are well known in the
art (general molecular biological and recombinant DNA techniques are described in
Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, which is
incorporated herein by reference). Commercial vectors suitable for inserting preferred
coding sequence for expression in various mammalian cells under a variety of growth
and induction conditions are also well known in the art.
Introduction of coding sequences and related control elements into host cells
Methods suitable for introducing into mammalian host cells nucleic acids
sufficient to achieve expression of the polypeptides or proteins of interest are well known
in the art. See, for example, Gething et al., Nature, 293:620-625 (1981); Mantei et al.,
Nature, 281:40-46 (1979); Levinson et al.; EP 117,060; and EP 117,058, all incorporated
herein by reference. '
For mammalian cells, preferred methods of transformation include the calcium
phosphate precipitation method of Graham and van der Erb, Virology, 52:456-457 (1978)
or the lipofectamine™. (Gibco BRL) Method of Hawley-Nelson, Focus 15:73 (1193).
General aspects of mammalian cell host system transformations have been described by
Axel in U.S. Pat. No. 4,399,216 issued Aug. 16, 1983. For various techniques for
transforming mammalian cells, see Keown et al., Methods in Enzymology (1989), Keown
et al., Methods in Enzymology, 185:527-537 (1990), and Mansour et al., Nature,
336:348-352 (1988). Non-limiting representative examples of suitable vectors for
expression of polypeptides or proteins in mammalian cells include pCDNAl; pCD, see
Okayama, et al. (1985) Mol. Cell Biol. 5:1136-1142; pMClneo Poly-A, see Thomas, et
al. (1987) Cell 51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.
In preferred embodiments, the polypeptide or protein is stably transfected into the
host cell. However, one of ordinary skill in the art will recognize that the present
invention can be used with either transiently or stably transfected mammalian cells.
Cells
Any mammalian cell or cell type susceptible to cell culture, and to expression of
polypeptides, may be utilized in accordance with the present invention. Non-limiting
examples of mammalian cells that may be used in accordance with the present invention
include BALB/c mouse myeloma line (NSO/1, 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). In a particularly preferred embodiment, the present invention is used in the
culturing of and expression of polypeptides and proteins from CHO cell lines.
Additionally, any number of commercially and non-comrnercially available
hybridoma cell lines that express polypeptides or proteins may be utilized in accordance
with the present invention. One skilled in the art will appreciate that hybridoma cell
lines might have different nutrition requirements and/or might require different culture
conditions for optimal growth and polypeptide or protein expression, and will be able to
modify conditions as needed.
As noted above, in many instances the cells will be selected or engineered to
produce high levels of protein or polypeptide. Often, cells are genetically engineered to
produce high levels of protein, for example by introduction of a gene encoding the
protein or polypeptide of interest and/or by introduction of control elements that regulate
expression of the gene (whether endogenous or introduced) encoding the polypeptide of
interest.
Certain polypeptides may have detrimental effects on cell growth, cell viability or
some other characteristic of the cells that ultimately limits production of the polypeptide
or protein of interest in some way. Even amongst a population of cells of one particular
type engineered to express a specific polypeptide, variability within the cellular
population exists such that certain individual cells will grow better and/or produce more
polypeptide of interest. In certain preferred embodiments of the present invention, the
cell line is empirically selected by the practitioner for robust growth under the particular
conditions chosen for culturing the cells. In particularly preferred embodiments,
individual cells engineered to express a particular polypeptide are chosen for large-scale
production based on cell growth, final cell density, percent cell viability, titer of the
expressed polypeptide or any combination of these or any other conditions deemed
important by the practitioner.
Cell Culture Phase
Typical procedures for producing a polypeptide of interest include batch cultures
and fed-batch cultures. Batch culture processes traditionally comprise inoculating a
large-scale production culture with a seed culture of a particular cell density, growing the
cells under conditions conducive to cell growth and viability, harvesting the culture when
the cells reach a specified cell density, and purifying the expressed polypeptide. Fedbatch
culture procedures include an additional step or steps of supplementing the batch
culture with nutrients and other components that are consumed during the growth of the
cells. A persistent and unsolved problem with traditional batch and fed-batch cultures is
the production of metabolic waste products, which have detrimental effects on cell
growth, viability, and production of expressed polypeptides. Two metabolic waste
products that have particularly detrimental effects are lactate and ammonium, which are
produced as a result of glucose and glutamine metabolism, respectively. In addition to
the enzymatic production of ammonium as a result of glutamine metabolism, ammonium
also accumulates in cell cultures as a result of non-metabolic degradation over time. The
present invention provides an improved method of large-scale production of
polypeptides that minimizes the detrimental effects of ammonium and lactate by slowing
and even reversing the accumulation of these waste products in cell cultures. One of
ordinary skill in the art will recognize that the present invention can be employed in any
system in which cells are cultured including, but not limited to, batch, fed-batch and
perfusion systems. In certain preferred embodiments of the present invention, the cells
are grown in batch or fed-batch systems.
Media , ,
Traditional media formulations, including commercially available media such as
Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma),
and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma), have contained relatively
high levels of glucose and glutamine in comparison to other amino acids. These
components have been thought to be required in abundance since they are the primary
metabolic energy sources for the cells. However, rapid consumption of these nutrients
leads to the accumulation of lactate and ammonium as described above. Additionally,
high initial levels of glucose and glutamine and the subsequent accumulation of lactate
and ammonium result in high osmolarity, a condition that by itself is often detrimental to
cell growth, cell viability and the production of polypeptides.
The present invention provides a variety of media formulations that, when used in
accordance with other culturing steps described herein, minimize and even reverse
accumulation of lactate and ammonium. Media formulations of the present invention
that have been shown to have beneficial effects on cell growth and/or viability or on
expression of polypeptide or protein include one or more of: i) a cumulative amino acid
amount per unit volume greater than about 70 mM, ii) a molar cumulative glutamine to
cumulative asparagine ratio of less than about 2, iii) a molar cumulative glutamine to
cumulative total amino acid ratio of less than about 0.2, iv) a molar cumulative inorganic
ion to cumulative total amino acid ratio between about 0.4 to 1, and v) a combined
cumulative amount of glutamine and asparagine per unit volume greater than about 16
mM. One of ordinary skill in the art will understand that "cumulative", as used above,
refers to the total amount of a particular component or components added over the course
of the cell culture, including components added at the beginning of the culture and
subsequently added components. One of ordinary skill in the art will understand that the
media formulations of the present invention encompass both defined and non-defined
media.
Traditional media formulations begin with a relatively low level of total amino
acids in comparison with the media formulations of the present invention. For example,
the traditional cell culture medium known as DME-F12 (a 50:50 mixture of Dulbecco's
Modified Eagle's medium and Ham's F12 medium) has a total amino acid content of 7.29
mM, and the traditional cell culture medium known as RPMI-1640 has a total amino acid
content of 6.44 mM (See e.g., H.J. Morton, In Vitro, 6:89-108 (1970), R.G. Ham, Proc.
Nat. Assoc. Sci. (USA), 53:288-293 (1965), G.E. Moore et al., J. Am. Medical Assn.,
199:519-24 (1967), all incorporated herein by reference). In certain embodiments of the
present invention, the amino acid concentration in the culture media is preferably greater
than about 70 mM. More preferably still, the media formulations of the present
invention contain amino acid concentrations greater than about 70 mM in the starting
media. It has been shown that when amino acid concentrations of the starting media are
in this range, cell density and titer are increased throughout the growth period of the
culture (see Example 13).
Additionally, in certain embodiments of the present invention, the molar ratio of
glutamine to asparagine in the culture media is reduced compared to other commercially
and non-commercially available media. Preferably the molar ratio of glutamine to
asparagine in the culture media is less than about two.
Additionally, in certain embodiments of the present invention, the molar ratio of
glutamine to total amino acids in the culture media is reduced compared to other
commercially and non-commercially available media. Preferably the molar ratio of
glutamine to total amino acids in the culture media less than about 0.2.
An interesting and unexpected result of lowering the molar ratio of glutamine to
asparagine or to the total concentration of amino acids in the starting media according to
the present invention was that in addition to an observed decrease in the accumulation of
ammonium, a decrease in the accumulation of lactate was seen as well. In certain
embodiments, the accumulated levels of ammonium and lactate are r>ot only lower than
those in control cultures, but in fact actually decrease after an initial accumulation (for
example, see Examples 3 and 7).
Boraston (US Patent Number 5,871,999) has disclosed a culture medium in
which the molar ratio of total inorganic ions to total amino acids is between 1 and 10.
Boraston showed that by providing culture medium in which the molar ratio of total
inorganic ions to total amino acids is in this range, aggregation of CHO cells grown in
the medium is decreased. In another preferred embodiment of the present invention, the
molar ratio of total inorganic ions to total amino acids in the culture medium is reduced
even further, to between about 0.4 to 1. As shown in Example 13, reducing this ratio
from 1.75 to approximately 0.7 results in a marked increase in cell density and
production of expressed polypeptide or protein throughout the growth period of the
culture.
In another preferred embodiment of the present invention, the culture medium
contains a combined glutamine and asparagine concentration of between about 16 and 36
mM. As shown in Example 14, Table 22, media which contain a combined total
concentration of glutamine and asparagine within this range exhibit higher titers of
expressed polypeptide than media which contain a combined total glutamine and
asparagine outside this range. One of ordinary skill in the art will be able to choose the
exact combined glutamine and asparagine concentration within this range in order to
optimize cell growth and/or viability and to maximize the production of the expressed
polypeptide.
Furthermore, one of ordinary skill in the art will recognize that any of the
conditions listed above may be used either singly or in various combinations with one
another. By utilizing media formulation which exhibit one, some or all of the above
characteristics, one of ordinary skill in the art will be able to optimize cell growth and/or
viability and to maximize the production of the expressed polypeptide.
Any of these media formulations disclosed in the present invention may
optionally be supplemented as necessary with hormones and/or other growth factors,
particular ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers,
vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually
present at very low final concentrations), amino acids, lipids, protein hydrolysates, or
glucose or other energy source. In certain embodiments of the present invention, it may
be beneficial to supplement the media with chemical inductants such as hexamethylenebis(
acetamide) ("HMBA") and sodium butyrate ("NaB"). These optional supplements
may be added at the beginning of the culture or may be added at a later point in order to
replenish depleted nutrients or for another reason. One of ordinary skill in the art will be
aware of any desirable or necessary supplements that may be included in the disclosed
media formulations.
Providing a mammalian cell culture
Various methods of preparing mammalian cells for production of proteins or
polypeptides by batch and fed-batch culture are well known in the art. As described
above, a nucleic acid sufficient to achieve expression (typically a vector containing the
gene encoding the polypeptide or protein of interest and any operably linked genetic
control elements) may be introduced into the host cell line by any number of well-known
techniques. Typically, cells are screened to determine which of the host cells have
actually taken up the vector and express the polypeptide or protein of interest.
Traditional methods of detecting a particular polypeptide or protein of interest expressed
by mammalian cells include but are not limited to immunohistochemistry,
immunoprecipitation, flow cytometry, immunofluorescence microscopy, SDS-PAGE,
Western blots, enzyme-linked immunosorbentassay (ELISA), high performance liquid
chromatography (HPLC) techniques, biological activity assays and affinity
chromatography. One of ordinary skill in the art will be aware of other appropriate
techniques for detecting expressed polypeptides or proteins. If multiple host cells
express the polypeptide or protein of interest, some or all of the listed techniques can be
used to determine which of the cells expresses that polypeptide or protein at the highest
levels.
Once a cell that expresses the polypeptide or protein of interest has been
identified, the cell is propagated in culture by any of the variety of methods well-known
to one of ordinary skill in the art. The cell expressing the polypeptide or protein of
interest is typically propagated by growing it at a temperature and in a medium that is
conducive to the survival, growth and viability of the cell. The initial culture volume can
be of any size, but is often smaller than the culture volume of the production bioreactor
used in the final production of the polypeptide or protein of interest, and frequently cells
are passaged several times in bioreactors of increasing volume prior to seeding the
production bioreactor. The cell culture can be agitated or shaken to increase oxygenation
of the medium and dispersion of nutrients to the cells. Alternatively or additionally,
special sparging devices that are well known in the art can be used to increase and
control oxygenation of the culture. In accordance with the present invention, one of
ordinary skill in the art will understand that it can be beneficial to control or regulate
certain internal conditions of the bioreactor, including but not limited to pH, temperature,
oxygenation, etc.
The starting cell density in the production bioreactor can be chosen by one of
ordinary skill in the art. In accordance with the present invention, the starting cell
density in the production bioreactor can be as low as a single cell per culture volume. In
preferred embodiments of the present invention, starting cell densities in the production
bioreactor can range from about 2 x 102 viable cells per mL to about 2 x 103, 2 x 104, 2 x
105, 2 x 106, 5 x 106 or 10 x 106 viable cells per mL and higher.
Initial and intermediate cell cultures may be grown to any desired density before
seeding the next intermediate or final production bioreactor. It is preferred that most of
the cells remain alive prior to seeding, although total or near total viability is not
required. In one embodiment of the present invention, the cells may be removed from
the supernatant, for example, by low-speed centrifugation. It may also be desirable to
wash the removed cells with a medium before seeding the next bioreactor to remove any
unwanted metabolic waste products or medium components. The medium may be the
medium in which the cells were previously grown or it may be a different medium or a
washing solution selected by the practitioner of the present invention.
The cells may then be diluted to an appropriate density for seeding the production
bioreactor. In a preferred embodiment of the present invention, the cells are diluted into
the same medium that will be used in the production bioreactor. Alternatively, the cells
can be diluted into another medium or solution, depending on the needs and desires of
the practitioner of the present invention or to accommodate particular requirements of the
cells themselves, for example, if they are to be stored for a short period of time prior to
seeding the production bioreactor.
Initial Growth Phase
Once the production bioreactor has been seeded as described above, the cell
culture is maintained in the initial growth phase under conditions conducive to the
survival, growth and viability of the cell culture. The precise conditions will vary
depending on the cell type, the organism from which the cell was derived, and the nature
and character of the expressed polypeptide or protein.
In accordance with the present invention, the production bioreactor can be any
volume that is appropriate for large-scale production of polypeptides or proteins. In a
preferred embodiment, the volume of the production bioreactor is at least 500 liters. In
other preferred embodiments, the volume of the production bioreactor is 1000, 2500,
5000, 8000, 10,000, 12,000 liters or more, or any volume in between. One of ordinary
skill in the art will be aware of and will be able to choose a suitable bioreactor for use in
practicing the present invention. The production bioreactor may be constructed of any
material that is conducive to cell growth and viability that does not interfere with
expression or stability of the produced polypeptide or protein.
The temperature of the cell culture in the initial growth phase will be selected
based primarily on the range of temperatures at which the cell culture remains viable.
For example, during the initial growth phase, CHO cells grow well at 37°C. In general,
most mammalian cells grow well within a range of about 25°C to 42°C. Preferably,
mammalian cells grow well within the range of about 35°C to 40°C. Those of ordinary
skill in the art will be able to select appropriate temperature or temperatures in which to
grow cells, depending on the needs of the cells and the production requirements of the
practitioner.
In one embodiment of the present invention, the temperature of the initial growth
phase is maintained at a single, constant temperature. In another embodiment, the
temperature of the initial growth phase is maintained within a range of temperatures. For
30
example, the temperature may be steadily increased or decreased during the initial
growth phase. Alternatively, the temperature may be increased or decreased by discrete
amounts at various times during the initial growth phase. One of ordinary skill in the art
will be able to determine whether a single or multiple temperatures should be used, and
whether the temperature should be adjusted steadily or by discrete amounts.
The cells may be grown during the initial growth phase for a greater or lesser
amount of time, depending on the needs of the practitioner and the requirement of the
cells themselves. In one embodiment, the cells are grown for a period of time sufficient
to achieve a viable cell density that is a given percentage of the maximal viable cell
density that the cells would eventually reach if allowed to grow undisturbed. For
example, the cells may be grown for a period of time sufficient to achieve a desired
viable cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95 or 99 percent of maximal viable cell density.
In another embodiment the cells are allowed to grow for a defined period of time.
For example, depending on the starting concentration of the cell culture, the temperature
at which the cells are grown, and the intrinsic growth rate of the cells] the cells may be
grown for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more
days. In some cases, the cells may be allowed to grow for a month or more. The cells
would be grown for 0 days in the production bioreactor if their growth in a seed
bioreactor, at the initial growth phase temperature, was sufficient that the viable cell
density in the production bioreactor at the time of its inoculation is already at the desired
percentage of the maximal viable cell density. The practitioner of the present invention
will be able to choose the duration of the initial growth phase depending on polypeptide
or protein production requirements and the needs of the cells themselves.
The cell culture may be agitated or shaken during the initial culture phase in
order to increase oxygenation and dispersion of nutrients to the cells. In accordance with
the present invention, one of ordinary skill in the art will understand that it can be
beneficial to control or regulate certain internal conditions of the bioreactor during the
initial growth phase, including but not limited to pH, temperature, oxygenation, etc. For
example, pH can be controlled by supplying an appropriate amount of acid or base and
oxygenation can be controlled with sparging devices that are well known in the art.
Shifting Culture Conditions
In accordance with the teaching of the present invention, at the end of the initial
growth phase, at least one of the culture conditions may be shifted so that a second set of
culture conditions is applied and a metabolic shift occurs in the culture. The
accumulation of inhibitory metabolites, most notably lactate and ammonia, inhibits
growth. A metabolic shift, accomplished by, e.g., a change in the temperature, pH,
osmolality or chemical inductant level of the cell culture, may be characterized by a
reduction in the ratio of a specific lactate production rate to a specific glucose
consumption rate. In one non-limiting embodiment, the culture conditions are shifted by
shifting the temperature of the culture. However, as is known in the art, shifting
temperature is not the only mechanism through which an appropriate metabolic shift can
be achieved. For example, such a metabolic shift can also be achieved by shifting other
culture conditions including, but not limited to, pH, osmolality, and sodium butyrate
levels. As discussed above, the timing of the culture shift will be determined by the
practitioner of the present invention, based on polypeptide or protein production
requirements or the needs of the cells themselves.
When shifting the temperature of the culture, the temperature shift may be
relatively gradual. For example, it may take several hours or days to complete the
temperature change. Alternatively, the temperature shift may be relatively abrupt. For
example, the temperature change may be complete in less than several hours. Given the
appropriate production and control equipment, such as is standard ,in the commercial
large-scale production of polypeptides or proteins, the temperature change may even be
complete within less than an hour.
The temperature of the cell culture in the subsequent growth phase will be
selected based primarily on the range of temperatures at which the cell culture remains
viable and expresses recombinant polypeptides or proteins at commercially adequate
levels. In general, most mammalian cells remain viable and express recombinant
polypeptides or proteins at commercially adequate levels within a range of about 25°C to
42°C. Preferably, mammalian cells remain viable and express recombinant polypeptides
or proteins at commercially adequate levels within a range of about 25°C to 35°C. Those
of ordinary skill in the art will be able to select appropriate temperature or temperatures
in which to grow cells, depending on the needs of the cells and the production
requirements of the practitioner.
In one embodiment of the present invention, the temperature of the subsequent
growth phase is maintained at a single, constant temperature. In another embodiment,
the temperature of the subsequent growth phase is maintained within a range of
temperatures. For example, the temperature may be steadily increased or decreased
during the subsequent growth phase. Alternatively, the temperature may be increased or
decreased by discrete amounts at various times during the subsequent growth phase.
One of ordinary skill in the art will understand that multiple discrete temperature shifts
are encompassed in this embodiment. For example, the temperature may be shifted
once, the cells maintained at this temperature or temperature range for a certain period of
time, after which the temperature may be shifted again - either to a higher or lower
temperature. The temperature of the culture after each discrete shift may be constant or
may be maintained within a certain range of temperatures.
In Example 16, data are shown that demonstrate the efficacy of employing two
successive temperature changes, although it will be understood by those of ordinary skill
in the art that in accordance with the present invention, three or more successive
temperature changes may be used to increase cell viability or density and/or increase
expression of recombinant polypeptides or proteins. The temperature or temperature
ranges of the cell culture after each successive temperature shift may be higher or lower
than the temperature(s) or temperature range(s) preceding the shift. In a preferred
embodiment of the present invention, each successive temperature or temperature range
is lower than the preceding temperature or temperature range.
Subsequent Production Phase
In accordance with the present invention, once the conditions of the cell culture
have been shifted as discussed above, the cell culture is maintained for a subsequent
production phase under a second set of culture conditions conducive 'to the survival and
viability of the cell culture and appropriate for expression of the desired polypeptide or
protein at commercially adequate levels.
As discussed above, the culture may be shifted by shifting one or more of a
number of culture conditions including, but not limited to, temperature, pH, osmolality,
and sodium butyrate levels. In one embodiment, the temperature of the culture is shifted.
According to this embodiment, during the subsequent production phase, the culture is
maintained at a temperature or temperature range that is lower than the temperature or
temperature range of the initial growth phase. For example, during the subsequent
production phase, CHO cells express recombinant polypeptides and proteins well within
a range of 25°C to 35°C. As discussed above, multiple discrete temperature shifts may
be employed to increase cell density or viability or to increase expression of the
33
recombinant polypeptide or protein.
In accordance with the present invention, the cells may be maintained in the
subsequent production phase until a desired cell density or production titer is reached. In
one embodiment, the cells are maintained in the subsequent production phase until the
titer to the recombinant polypeptide or protein reaches a maximum. In other
embodiments, the culture may be harvested prior to this point, depending on the
production requirement of the practitioner or the needs of the cells themselves. For
example, the cells may be maintained for a period of time sufficient to achieve a viable
cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or
99 percent of maximal viable cell density. In some cases, it may be desirable to allow
the viable cell density to reach a maximum, and then allow the viable cell density to
decline to some level before harvesting the culture. In an extreme example, it may be
desirable to allow the viable cell density to approach or reach zero before harvesting the
culture.
In another embodiment of the present invention, the cells are allowed to grow for
a defined period of time during the subsequent production phase. For example,
depending on the concentration of the cell culture at the start of the subsequent growth
phase, the temperature at which the cells are grown, and the intrinsic growth rate of the
cells, the cells may be grown for 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20 or more days. In some cases, the cells may be allowed to grow for a month or
more. The practitioner of the present invention will be able to choose the duration of the
subsequent production phase depending on polypeptide or protein production
requirements and the needs of the cells themselves.
In certain cases, it may be beneficial or necessary to supplement the cell culture
during the subsequent production phase with nutrients or other medium components that
have been depleted or metabolized by the cells. For example, it might be advantageous
to supplement the cell culture with nutrients or other medium components observed to
have been depleted during monitoring of the cell culture (see 'Monitoring Culture
Conditions' section below). Alternatively or additionally, it may be beneficial or
necessary to supplement the cell culture prior to the subsequent production phase. As
non-limiting examples, it may be beneficial or necessary to supplement the cell culture
with hormones and/or other growth factors, particular ions (such as sodium, chloride,
calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace
elements (inorganic compounds usually present at very low final concentrations), amino
34
acids, lipids, or glucose or other energy source.
These supplementary components may all be added to the cell culture at one
time, or they may be provided to the cell culture in a series of additions. In one
embodiment of the present invention, the supplementary components are provided to the
cell culture at multiple times in proportional amounts. In another embodiment, it may be
desirable to provide only certain of the supplementary components initially, and provide
the remaining components at a later time. In yet another embodiment of the present
invention, the cell culture is fed continually with these supplementary components.
In accordance with the present invention, the total volume added to the cell
culture should optimally be kept to a minimal amount. For example, the total volume of
the medium or solution containing the supplementary components added to the cell
culture may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50% of the volume
of the cell culture prior to providing the supplementary components.
The cell culture may be agitated or shaken during the subsequent production
phase in order to increase oxygenation and dispersion of nutrients to the cells. In
accordance with the present invention, one of ordinary skill in the art will understand that
it can be beneficial to control or regulate certain internal conditions of the bioreactor
during the subsequent growth phase, including but not limited to pH, temperature,
oxygenation, etc. For example, pH can be controlled by supplying an appropriate
amount of acid or base and oxygenation can be controlled with sparging devices that are
well known in the art.
Monitoring culture conditions
In certain embodiments of the present invention, the practitioner may find it
beneficial or necessary to periodically monitor particular conditions of the growing cell
culture. Monitoring cell culture conditions allows the practitioner to determine whether
the cell culture is producing recombinant polypeptide or protein at suboptimal levels or
whether the culture is about to enter into a suboptimal production phase. In order to
monitor certain cell culture conditions, it will be necessary to remove small aliquots of
the culture for analysis. One of ordinary skill in the art will understand that such
removal may potentially introduce contamination into the cell culture, and will take
appropriate care to minimize the risk of such contamination.
As non-limiting example, it may be beneficial or necessary to monitor
temperature, pH, cell density, cell viability, integrated viable cell density, lactate levels,
35
ammonium levels, osmolarity, or titer of the expressed polypeptide or protein.
Numerous techniques are well known in the art that will allow one of ordinary skill in
the art to measure these conditions. For example, cell density may be measured using a
hemacytometer, a Coulter counter, or Cell density examination (CEDEX). Viable cell
density may be determined by staining a culture sample with Trypan blue. Since only
dead cells take up the Trypan blue, viable cell density can be determined by counting the
total number of cells, dividing the number of cells that take up the, dye by the total
number of cells, and taking the reciprocal. HPLC can be used to determine the levels of
lactate, ammonium or the expressed polypeptide or protein. Alternatively, the level of
the expressed polypeptide or protein can be determined by standard molecular biology
techniques such as coomassie staining of SDS-PAGE gels, Western blotting, Bradford
assays, Lowry assays, Biuret assays, and UV absorbance. It may also be beneficial or
necessary to monitor the post-translational modifications of the expressed polypeptide or
protein, including phosphprylation and glycosylation.
Isolation of Expressed Polvpeptide
In general, it will typically be desirable to isolate and/or purify proteins or
polypeptides expressed according to the present invention. In a preferred embodiment,
the expressed polypeptide or protein is secreted into the medium and thus cells and other
solids may be removed, as by centrifugation or filtering for example, as a first step in the
purification process. This embodiment is particularly useful when Used in accordance
with the present invention, since the methods and compositions described herein result in
increased cell viability. As a result, fewer cells die during the culture process, and fewer
proteolytic enzymes are released into the medium which can potentially decrease the
yield of the expressed polypeptide or protein.
Alternatively, the expressed polypeptide or protein is bound to the surface of the
host cell. In this embodiment, the media is removed and the host cells expressing the
polypeptide or protein are lysed as a first step in the purification process. Lysis of
mammalian host cells can be achieved by any number of means well known to those of
ordinary skill in the art, including physical disruption by glass beads and exposure to
high pH conditions.
The polypeptide or protein may be isolated and purified by standard methods
including, but not limited to, chromatography (e.g., ion exchange, affinity, size
exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, or
36
differential solubility, ethanol precipitation or by any other available technique for the
purification of proteins (See, e.g., Scopes, Protein Purification Principles and Practice
2nd Edition, Springer-Verlag, New York, 1987; Higgins, S.J. and Hames, B.D. (eds.),
Protein Expression : A Practical Approach, Oxford Univ Press, 1999; and Deutscher,
M.P., Simon, M.I., Abelson, J.N. (eds.), Guide to Protein Purification : Methods in
Enzymology (Methods in Enzymology Series, Vol 182), Academic, Press, 1997, all
incorporated herein by reference). For immunoaffinity chromatography in particular, the
protein may be isolated by binding it to an affinity column comprising antibodies that
were raised against that protein and were affixed to a stationary support. Alternatively,
affinity tags such as an influenza coat sequence, poly-histidine, or glutathione-Stransferase
can be attached to the protein by standard recombinant techniques to allow
for easy purification by passage over the appropriate affinity column. Protease inhibitors
such as phenyl methyl sulfonyl fluoride (PMSF), leupeptin, pepstatin or aprotinin may be
added at any or all stages in order to reduce or eliminate degradation of the polypeptide
or protein during the purification process. Protease inhibitors are particularly desired
when cells must be lysed in order to isolate and purify the expressed polypeptide or
protein. One of ordinary skill in the art will appreciate that the exact purification
technique will vary depending on the character of the polypeptide or protein to be
purified, the character of the cells from which the polypeptide or protein is expressed,
and the composition of the medium in which the cells were grown. ' :
Pharmaceutical Formulations
In certain preferred embodiments of the invention, produced polypeptides or
proteins will have pharmacologic activity and will be useful in the preparation of
Pharmaceuticals. Inventive compositions as described above may be administered to a
subject or may first be formulated for delivery by any available route including, but not
limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal,
bronchial, opthalmic, transdermal (topical), transmucosal, rectal, and vaginal routes.
Inventive pharmaceutical compositions typically include a purified polypeptide or
protein expressed from a mammalian cell line, a delivery agent (i.e., a cationic polymer,
peptide molecular transporter, surfactant, etc., as described above) in combination with a
pharmaceutically acceptable carrier. As used herein the language "pharmaceutically
acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the like1, compatible with
37
pharmaceutical administration. Supplementary active compounds can also be
incorporated into the compositions.
A pharmaceutical composition is formulated to be compatible with its intended
route of administration. Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a sterile diluent such as
water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl
parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and
agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be
adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose
vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use typically include sterile
aqueous solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline, bacteriostatic water,
Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all
cases, the composition should be sterile and should be fluid to the extent that easy
syringability exists. Preferred pharmaceutical formulations are stable under 1 i the
conditions of manufacture and storage and must be preserved against the contaminating
action of microorganisms such as bacteria and fungi. In general, the relevant carrier can
be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use
of a coating such as lecithin, by the maintenance of the required particle size in the case
of dispersion and by the use of surfactants. Prevention of the action of microorganisms
can be achieved by various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol,
sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and gelatin.
38
Sterile injectable solutions can be prepared by incorporating the purified
polypeptide or protein in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating the purified
polypeptide or protein expressed from a mammalian cell line into a sterile vehicle which
contains a basic dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying and freeze-drying
which yields a powder of the active ingredient plus any additional desired ingredient
from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. For the
purpose of oral therapeutic administration, the purified polypeptide or protein can be
incorporated with excipients and used in the form of tablets, troches, or capsules, e.g.,
gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as
a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials
can be included as part of the composition. The tablets, pills, capsules, troches and the
like can contain any of the following ingredients, or compounds of a similar nature: a
binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as
starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a
lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as
peppermint, methyl salicylate, or orange flavoring. Formulations for oral delivery may
advantageously incorporate agents to improve stability within the gastrointestinal tract
and/or to enhance absorption.
For administration by inhalation, the inventive compositions comprising a
purified polypeptide or protein expressed from a mammalian cell line and a delivery
agent are preferably delivered in the form of an aerosol spray from a pressured container
or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a
nebulizer. The present invention particularly contemplates delivery of the compositions
using a nasal spray, inhaler, or other direct delivery to the upper and/or lower airway.
Intranasal administration of DNA vaccines directed against influenza viruses has been
shown to induce CDS T cell responses, indicating that at least some cells in the
respiratory tract can take up DNA when delivered by this route, and the delivery agents
of the invention will enhance cellular uptake. According to certain embodiments of the
39
invention the compositions comprising a purified polypeptide expressed from a
mammalian cell line and a delivery agent are formulated as large porous particles for
aerosol administration.
Systemic administration can also be by transmucosal or transdermal means. For
transmucosal or transdermal administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration, detergents, bile salts, and
fusidic acid derivatives. Transmucosal administration can be accomplished through the
use of nasal sprays or suppositories. For transdermal administration, the purified
polypeptide or protein and delivery agents are formulated into ointments, salves, gels, or
creams as generally known in the art.
The compositions can also be prepared in the form of suppositories (e.g., with
conventional suppository bases such as cocoa butter and other glycerides) or retention
enemas for rectal delivery.
In one embodiment, the compositions are prepared with carriers that will protect
the polypeptide or protein against rapid elimination from the body, such as a controlled
release formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
Methods for preparation of such formulations will be apparent to those skilled in the art.
The materials can also be obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected
cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically
acceptable carriers. These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Patent No. 4,522,811.
It is advantageous to formulate oral or parenteral compositions in dosage unit
form for ease of administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary dosages for the subject to be
treated; each unit containing a predetermined quantity of active polypeptide or protein
calculated to produce the desired therapeutic effect in association with the required
pharmaceutical carrier.
The polypeptide or protein expressed according to the present invention can be
administered at various intervals and over different periods of time as required, e.g., one
time per week for between about 1 to 10 weeks, between 2 to 8 weeks, between about 3
40
to 7 weeks, about 4, 5, or 6 weeks, etc. The skilled artisan will appreciate that certain
factors can influence the dosage and timing required to effectively treat a subject,
including but not limited to the severity of the disease or disorder, previous treatments,
the general health and/or age of the subject, and other diseases present. Generally,
treatment of a subject with a polypeptide or protein as described herein can include a
single treatment or, in many cases, can include a series of treatments. It is furthermore
understood that appropriate doses may depend upon the potency of the polypeptide or
protein and may optionally be tailored to the particular recipient, for example, through
administration of increasing doses until a preselected desired response is achieved. It is
understood that the specific dose level for any particular animal subject may depend
upon a variety of factors including the activity of the specific polypeptide or protein
employed, the age, body weight, general health, gender, and diet of the subject, the time
of administration, the route of administration, the rate of excretion, any drug
combination, and the degree of expression or activity to be modulated.
The present invention includes the use of inventive compositions for treatment of
nonhuman animals. Accordingly, doses and methods of administration may be selected
in accordance with known principles of veterinary pharmacology and medicine.
Guidance may be found, for example, in Adams, R. (ed.), Veterinary Pharmacology and
Therapeutics, 8th edition, Iowa State University Press; ISBN: 0813817439; 2001.
Inventive pharmaceutical compositions can be included in a container, pack, or
dispenser together with instructions for administration.
The foregoing description is to be understood as being representative only and is not
intended to be limiting. Alternative methods and materials for implementing the
invention and also additional applications will be apparent to one of skill in the art, and
are intended to be included within the accompanying claims.
Examples
Example 1: Enhanced Medium 1 for anti-GDF-8 Fed-batch Process
Traditional fed-batch processes for cultivating cell lines have several drawbacks
including the time and effort required to administer the feeds and the need for special
equipment in large-scale bioreactors. The objective was to develop a batch media for the
production of proteins of interest in large-scale bioreactors that requires minimal feeds.
Materials and Methods
41
STRAINS AND MEDIA: Chinese Hamster Ovary ("CHO") cells were
engineered to express a monoclonal antibody against growth and differentiation factor 8
("anti-GDF-8 cells") (see Veldman et al., Neutralizing Antibodies Against GDF-8 and
Uses Therefor. US20040142382 Al). Anti-GDF-8 cells were used to test a new batch
media. Medium 1 and Medium 2 were compared for their abilities to support high cell
density and viability. The compositions of these media, as well as Medium 3 are listed
in Table 1. Media are made by adding all the components save for FeSCVTFbO. The
media is then adjusted to pH 7.25, the osmolarity is recorded and FeSO4«7H2O are then
added.
CULTURE CONDITIONS: For flask experiments, anti-GDF-8 cells were grown
in shake flasks and passaged three times. For bioreactor experiments, anti-GDF-8 cells
were grown in media for 12 days, supplemented daily with either 2% by volume of 20X
Medium 4 feed medium (Table 3) or 3% by volume of 16X Medium 4 (Table 4) after
day 5. For the first 4 days, cells were grown at 37°C. On day 5, cells were shifted to
SAMPLE ANALYSIS: Daily samples were taken from the cultures and were
analyzed for amino acid, vitamin, iron, phosphate, glucose and glutamine levels.
(Table Remove)
Figure 1 shows that growth rate of anti-GDF-8 cells was similar in both Medium
1 and Medium 2 in the flask experiments.
Figure 2 shows that in bioreactors, Medium 1 exhibited a significant increase in
final cell density and viability over Medium 3. The final titer also increased
significantly, from 551 mg/L for the platform process to 976 mg/L with Medium l(data
not shown). Temperature was shifted from 37°C to 31°C on day 5. Due to the
unexpected high cell growth, the cultures were fed daily after day 5 with either 2% by
volume of 20X Medium 4 or 3% by volume of 16X Medium 4. Thus, this is not a true
batch experiment as originally intended. Asparagine and thiamine were supplemented in
the feed media beginning on day 10.
In developing a concentrated batch media, several possible concerns need to be
considered. First, concentrated nutrients might prove toxic to the cells. In the media
developed in this Example, all nutrients and components were deterrnined to be below
the toxicity limits (data not shown).
Second, the concentrated batch media necessarily has a higher osmolarity than
non-concentrated media, which has been shown to have detrimental effects on cell
growth and viability. This problem can be circumvented by lowering the amount of
NaCl in the starting media. Furthermore, the concentrated batch media contains
insufficient levels of glucose to sustain growth for the entire culture period. Thus,
cultures were supplemented daily after day 5 with a glucose feed.
Third, insulin and glutamine are susceptible to degradation during the 12 day
culture period. Thus, the culture was supplemented with these components in addition to
glucose.
Finally, iron will precipitate out of solution containing high concentrations of
phosphate at high pH. This problem can be circumvented by adding iron at the end of
the media preparation process, after the pH has been adjusted to an appropriate level.

Example 2: Development of concentrated feed medium (Medium 5) for anti-GDF-8
cells in fed-batch process.
In Example 1, a batch process for culturing anti-GDF-8 cells using Medium 1 was
developed. Due to the high cell density that resulted during the process, it was
determined that supplementation of nutrients in addition to glucose and glutamine was
still advantageous. However, supplementing the batch with 8X Medium 4 feed media
would result in excessive dilution of the culture. A more concentrated feed media was
developed in order to circumvent this problem.
Materials and Methods and Results
Table 2 lists the compositions of Medium 4A-1, Medium 4B, Trace B and Trace D used
in the formulations of Tables 3-7.
(Table Remove)
Note: Nucellin™ is manufactured by Eli Lilly (Indianapolis, IN, USA); H/P stock =
0.036 mg/mL hydrocortisone, 1.08 mg/mL Putrescine»2HCl.
The media formulation consists of 3 parts: I, II, III. Part I is the concentrated version of
8X Medium 4 with the individual components of Medium 4B except folic acid due to the
48
concerns of the solubility of this vitamin. Part II is iron stock, Trace D and acidic
cysteine, to avoid possible precipitation of iron if added in part I. Part III is folic acid
stock. Part I is added 2% by volume daily starting on day 5 and parts II and III are added
once on day 5 together with Part I.
The final pH of the feed media was adjusted to 7.0 and osmolarity was about 1064
mOsm. A 2% feed will result in a 2g/L glucose, a 2 mM Glutamine and a 14 mOsm
osmolarity increase to the culture.
2. 16X Medium 4.
To reduce the increase in osmolarity, the feed media was changed from 20X Medium 4
(2% by volume daily) to 16X Medium 4 (3% by volume daily). The media formulation
for 16X Medium 4 is provided in Table 4.
(Table Remove)
Note: Nucellin™ is manufactured by Eli Lilly (Indianapolis, IN, USA); H/P stock =
0.036 mg/mL hydrocortisone, 1.08 mg/mL Putrescine»2HCl.
In this modified 16X Medium 4, glucose was also eliminated to further reduce the
osmolarity and give some flexibility of the glucose feed. Total osmolarity of the feed
media is now 295 mOsm. ' '
3. 16X Medium 4.
Changes were made to the 16X Medium 4 formulation. Iron stock solution was added in
the feed resulting in a 0.45 uM addition each feed. Additionally, glucose was added
back to give a 1.5 g/L addition every feed. The media formulation for this modified 16X
Medium 4 is provided in Table 5.
(Table Remove)
Note: Nucellin™ is manufactured by Eli Lilly (Indianapolis, IN, USA); H/P stock =
0.036 mg/mL hydrocortisone, 1.08 mg/mL Putrescine'2HCl.
4. 16X Medium 4.
Here, the feed media (16X Medium 4) was made in combined media instead of 3
separate feeds as in the last several batches. Tests were done to ensure that folic acid
could be dissolved at the concentration required and that neither iron nor folic acid
precipitated out of solution after storage at either 4°C or at room temperature for 6 days.
The media formulation for the combined 16X Medium 4 is provided in Table 6.
[0001] Here, several changes were made to the feed media. Medium 4B
powder was used instead of addition of each individual ingredient in Medium 4B.
Medium 4B powder was mixed with glucose and dissolved separately under basic
conditions by titrating the solution to pH 10.25. Additional asparagine and
thiamine were added since the amino acid and vitamin analysis results showed
these two components were exhausted by the end of fed-batch process. Use of
12X Medium 4 further reduced the osmolarity increase when fed to the culture.
The media formulation for 12X Medium 4 is provided in Table 7.
(Table Remove)
Nucellin™ is manufactured by Eli Lilly (Indianapolis, IN, USA); H/P stock
0.036 mg/mL hydrocortisone, 1.08 mg/mL Putrescine*2HCl.
The final osmolarity is 566 mOsm. A daily feed of 4% by volume gives an
approximate osmolarity increase of 8.6, an increase in glucose of 2g/L and an increase
in glutamine of 1.5 mM. The 12X Medium 4 media formulation is also known as
Medium 5. Medium 5 is easy to make compared to 20X Medium 4 or 16X Medium 4,
and stable over 10 days either at room temperature or at 4°C (data not'shown).
Example 3: Glutamine Starvation Fed-batch Process for anti-GDF-8 Cell Culture
CHO cells require glutamine in the starting media to survive. Traditionally, initial
glutamine levels are high and glutamine is fed daily after day 5 until the end of the fedbatch
process. Traditional fed-batch processes normally result in high lactate and
ammonium levels in the cell cultures, which are known to have inhibitory effects on
cell growth, cell density and recombinant protein expression. Fed-batch processes in
which glucose is slowly added to the culture have been shown to lower lactate
production and improve cell growth, cell density and recombinant protein expression.
However, prior art methods for manipulation of glucose addition are not practical for
large-scale manufacturing. Here, by utilizing culture media with lower starting levels
of glutamine and eliminating glutamine from the feed, it is shown that lower levels of
ammonium and lactate are produced, leading to increased cell viability. Additionally,
in glutamine-starved cultures, recombinant protein expression is increased and final
osmolarity is reduced.
terials and Methods
[0002] STRAINS AND MEDIA: anti-GDF-8 cells were cultured in a fedbatch
mode in Medium 1 in 1 L Bioreactor.
[0003] CULTURE CONDITIONS: Cells were grown for'twelve days in 1L
Bioreactors. Temperature was shifted from 37°C to 31°C on either day 4 or day
5 depending on the cell growth. Three fed-batch processes were tested: a normal
(control) process, a no glutamine feed process and a glutamine starvation process.
Pertinent details of these processes are listed in Table 8 and Table 9.
Table 8. Fed-batch process in 1L Bioreactors with no glutamine feed process.
(Table Remove)
SAMPLE ANALYSIS: Daily samples were taken from the cultures and were analyzed
for cell density, cell viability, lactate, glutamine, and ammonium levels. Titer of
expressed anti-GDF-8 antibody was also measured daily.
Results and Conclusions
Figure 3 shows the cell density of cultures grown in either no glutamine feed or
control fed-batch conditions. In both cases, cell density was similar over the course of
the experiment.
Figure 4 shows percent cell viability in cultures grown in either no glutamine
feed or control fed-batch conditions. The no glutamine feed culture showed a markedly 1 !
higher cell viability toward the end of the experiment, beginning on day 6.
Figure 5 shows ammonium levels in cultures grown in either no glutamine feed
or control fed-batch conditions. The no glutamine feed culture showed a marked
decrease in ammonium levels toward the end of the experiment, beginning on day 4.
Figure 6 shows lactate levels in cultures grown in either no glutamine feed or
control fed-batch conditions. Lactate levels were slightly lower in the no glutamine
feed culture throughout the course of the experiment.
Figure 7 shows anti-GDF-8 antibody titer in cultures grown in either no
glutamine feed or control fed-batch conditions. Final anti-GDF-8 antibody titer was
higher in the no glutamine feed culture.
Figure 8 shows the cell density of cultures grown in either glutamine-starved or
control fed-batch conditions. In both cases, cell density was similar over the course of
the experiment.
Figure 9 shows cell viability in cultures grown in either glutamine-starved or
control fed-batch conditions. In both cases, cell viability was similar over the course of
the experiment.
Figure 10 shows ammonium levels in cultures grown in either glutamine-starved
or control fed-batch conditions. The glutamine-starved culture showed a marked
decrease in ammonium levels throughout the course of the experiment.
Figure 11 shows lactate levels in cultures grown in either glutamine-starved or
control fed-batch conditions. The glutamine-starved culture showed a marked decrease
in lactate levels throughout towards the end of the experiment, beginning on day 4.
Figure 12 shows anti-GDF-8 antibody titer in cultures grown in either glutaminestarved
or control fed-batch conditions. Final anti-GDF-8 antibody titer was higher in the
glutamine-starved culture.
Collectively these results indicate that decreased glutamine levels are beneficial
to cell cultures by reducing the amount of ammonium production, increasing cell
viability and increasing titer of expressed anti-GDF-8 antibody. In addition, in the
glutamine-starved cultures, low lactate levels were observed, possibly due to the
decreased glucose consumption rate. Decreased ammonium and lactate levels also have
the effect of reducing total osmolarity. Elevated osmolarity is also known to have
inhibitory effects on cell growth and viability. Low initial glutamine levels together with
the elimination of the glutamine feed also has the positive effect of reducing ammonium
produced as a result of non-enzymatic glutamine degradation in stored media.
Elimination of glutamine in the feed also simplifies the process of culturing anti-GDF-8
cells. . ,
Example 4. Iron dose response of anti-GDF-8 cells in Medium 1 and Medium 2.
Medium 1 is much more concentrated in nutrients than Medium 2. The optimum iron
levels for cell growth in Medium 1 were determined in order to avoid problems with iron
deficiency during cell culture.
Materials and Methods
Anti-GDF-8 cells were cultured in dishes for one passage in either Medium 1 or Medium
2. Iron concentrations of these media were manipulated by addition of different
amounts of stock iron solution. Final cell densities were measured by CEDEX.
Results and Conclusions
Figure 13 shows the Fe dose response of anti-GDF-8 cells in Medium 1 and
Medium 2 containing different iron concentrations. In Medium 2, the cell density was
relatively constant for iron concentrations ranging from 3 \sM to 15 uM. In Medium 1,
cell density increases with increasing iron concentration but reaches a maximum after
approximately 5 uM. This difference could be due to the high nutrient content in
Medium 1, which might reduce iron availability to the cells as a consequence of
chelation of iron in the media. These results indicate that iron levels should be kept
above 5 uM to avoid problems with iron deficiency in Medium 1.
Example 5. Substitution of Glutamate for Glutamine in the Bioreactor Process.
56
Three experiments were performed to test the effects of substituting glutamate for
glutamine in an anti-Lewis Y cell culture process.
Materials and Methods
The experiments were performed in 10L bioreactors at pH 7.1, 30% dissolved oxygen,
and a starting temperature of 37°C with a shift to 31°C on day 5. Sparge and headspace
gasses were 88% of a 93% air/7% CC>2 mix and 12% oxygen. The starting media in all
experiments was Medium 1, which contains glutamine. Feed media and feed schedule
including supplemental glucose and glutamine feeds are shown in Table 10. Columns
labeled "Glutamate" were fed with modified Medium 5, containing no glutamine, but
containing a molar concentration of glutamate equal to the molar glutamine
concentration in standard Medium 5. Columns labeled glutamine were i fed with standard
(Table Remove)
Within each experiment, cell density is similar as shown in Figure 14. Cell densities are
low in the Glutamine 2 and Glutamate 2 experiments due to a pH deviation to about 6.7
on day 3 on the process. The drop in density between day 6 and 7 in the Glutamine 3
and Glutamate 3 experiments is due to the 29% media feed on day 6.
Figure 15 shows cell viability of the glutamate and glutamine fed cultures.
Viabilities remained higher during the second half of the process in the bioreactors
containing glutamate fed cultures.
In Experiment 1, anti-Lewis Y liter is similar between the glutamate and glutamine
fed cultures. Figure 16 shows that in Experiments 2 and 3, anti-Lewis Y titers are lower
in the glutamine fed reactors. The lower anti-Lewis Y titer observed in these reactors
could be due to the high levels of lactate produced, as shown in Figure 17.
Bioreactors run with glutamate in the feed media have a lower ammonium
concentration (Figure 18) and a lower osmolarity (Figure 19).
The binding ELISA assay was used to test activity of samples from the Glutamine
1 and Glutamate 1 experiments. The activities were similar: 110% of reference for the
Glutamine 1 sample and 122% of reference for the Glutamate 1 sample (data not shown).
The substitution of glutamate for glutamine in these experiments does not have a
significant effect on cell density. However, cell viability is lower in the Bioreactors fed
with glutamine. Ammonium, lactate and osmolarity are lower in the Bioreactors fed
with glutamate compared to those fed with glutamine. On average, anti-Lewis Y titer is
higher in the Bioreactors fed with glutamate and activity is essentially the same under
both conditions.
Example 6. Substitution of Glucose and Glutamine in the Anti-Lewis Y Cell Culture
Process.
The purpose of this experiment was to test the effects of substitution of glucose
and glutamine with the feed media listed in Table 11 below in the culturing of anti-Lewis
Y cells (see Bogheart et al., Antibody-targeted chemotherapy with the calicheamicin
conjugate hu3S193-N-acetvl gamma calicheamicin dimethyl hydrazide targets Lewisv
and eliminates Lewisv-positive human carcinoma cells and xenografts. Clin. Can. Res.
58
10:4538-49 (2004)). Cell density, cell viability, anti-Lewis Y liter and ammonium levels
were measured.
Materials and Methods
The experiment was performed in 250 ml shake flasks at a starting volume of 75
ml. All shake flasks were seeded at 0.25 x 106 cells/ml in Medium 2. The flasks were
incubated at 37°C in a 7% CO2 incubator for 14 days. On days 3 and 4, the flasks were
fed with 5% by volume of Medium 6 feed medium. The composition of Medium 6 is
listed in Table 11. On days 5-13 the flasks were fed with 5% by volume of one of the
feed solutions listed in Table 12. Each condition was performed in duplicate. Samples
were taken daily for cell counts by CEDEX and assays for ammonium, glucose, and
lactate
(Table Remove)
Results and Conclusions
The highest cell density was seen when glutamate or glycylglutamine was
substituted for glutamine in the presence of either glucose or galactose in the feed media.
Cell density was generally lower in the cultures fed with glucose/glutamine,
galactose/glutamine, or glucose only (Figure 20). Final viability was highest in the
cultures fed with glucose only, followed by the cultures fed with glucose/glutamate. The
lowest viability was seen in the cultures fed with glutamine or asparagine combined with
either glucose or galactose (Figure 21).
Day 14 titer was highest in the glucose/glycylglutamine and the
glucose/glutamate fed cultures at about 700 ug/ml. Titer was lowest in the
galactose/glycylglutamine and the galactose/asparagine fed cultures at about 500 ug/ml.
Titer in the glucose/glutamine control was about 570 ug/ml (Figure 22).
The lowest ammonium levels were seen in the flasks fed with glucose/glutamate
or glucose only. The flasks fed with galactose/glutamate, glucose/glutamine,
glucose/glycylglutamine, and glucose/asparagine showed intermediate levels of
ammonium. The flasks fed with galactose/asparagine, galactose/glycylglutamine, and
galactose/glutamine had the highest levels of ammonium (Figure 23).
Glucose levels remained above 1 g/L in all flasks fed with galactose until day 11.
From day 11 through day 14, the glucose in these cultures was never completely
depleted, remaining between 0.6 and 1 g/L, with no significant differences between the
different cultures.
Glucose levels increased in all flasks fed with glucose or glucose combined with
another substrate until day 10. From day 10 through day 14 in these cultures, glucose
levels remained fairly constant and similar to each other. On day 14 about 8.4 g/L
glucose remained in the glucose/glutamate fed cultures and about 10.8g/L glucose
remained in the cultures fed with glucose only.
Lactate levels reached a high of about 2.4 g/L on day 5, when conditions were the
same for all cells, and dropped to essentially zero in all cultures by day 14. Lactate
levels were highest from day 10 through day 14 in the glucose/glutamine control, but
were below 1 g/L during this time (data not shown). '
All conditions tested in this experiment resulted in higher cell density than the
control glucose/glutamine condition. All conditions tested except the
galactose/asparagine condition resulted in higher final viability than either the
glucose/glutamine control or the galactose/glutamine fed condition. Titer in the
61
glucose/glutamine control was about 570 ug/ml compared to a high of about 700 ug/ml
in the glucose/glycylglutamine fed condition and the glucose/glutamate fed condition.
Example 7. Evaluation of a Glutamine Starved Batch Process for the Production of
anti-GDF-8.
Typical fed-batch production methods require multiple feeds over the culture
period. These feeds are designed to replace nutrients in the medium that may have been
depleted by the cells or may have degraded during the batch. These feeds create
complications when the process is scaled up to be used in larger reactors, such as the
need for an impeller jump (see Figure 24). Furthermore, the feeds dilute the amount of
anti-GDF-8 already secreted into the culture and therefore affect the harvest titer. The
use of a batch process would allow inoculation of the bioreactor at full volume, instead
of at a partial volume so as to accommodate the feeds, which would remove the necessity
of an impeller jump and greatly reduce any dilution effect on productivity.
Glutamine is one of the most important reasons that a fed-batch approach is used
since it is not stable at 37 C and it had been thought that it needed to be replenished
during a batch culture. However, results of Examples 2, 5, and 6, in which a glutamine
starvation strategy was tested, showed a significant increase in productivity compared to
a control reactor that was fed glutamine. This result was combined with the batch
process to create a glutamine starvation batch process that was tested in this Example.
aterials and Methods
Anti-GDF-8 cells were grown in 1L Bioreactors for 12 days according to the
following four growth conditions. Bioreactor parameters for all conditions were kept the
same. Dissolved oxygen was maintained at no lower than 23% of air saturation by
sparging with air and pH was maintained at 7.00 by the addition of a solution containing
sodium bicarbonate at 0.58 M and sodium carbonate at 0.71 M. The temperature of all
cultures was maintained at 37°C for the first four days of the batch. On the fourth day of
the batch the temperature of all the bioreactors was lowered to 31°C and maintained at
this point for the duration of the batch. The control and fed-batch cultures were fed with
8%, 12%, and 8% total reactor volume of their respective feed media on days 5, 7, and
10, respectively.
1) Control.
- Inoculation medium Medium 7 (see Table 13).
62
- Feed Medium 8, fed on days 5, 7, and 10 (see Table 13).
- Feed 5 mM of glutamine on day 4.
- Lower the temperature to 31 °C on day 4.
2) Fed-batch glutamine starvation.
- Inoculation medium Medium 7 with only 4 mM of glutamine (see Table 13).
- Feed Medium 8 without glutamine, fed on days 5, 7, and 10 (see Table 13).
- No glutamine feed on day 4.
- Lower the temperature to 31 °C on day 4.
3) Batch glutamine starvation.
- Inoculation medium new batch medium with only 4 mM of glutamine
(see Table 13).
- No feed medium.
- No glutamine feed.
- Lower the temperature to 31 °C on day 4.
- Add 5g/L of glucose on day 8.
4) Batch glutamine starvation supplemented on day 8.
- Inoculation medium new batch medium with only 4 mM of glutamine
(see Table 13). ' '
- No feed medium.
- No glutamine feed.
- Lower the temperature to 31 °C on day 4.
- Add 4g of glucose, 375 mg of Asparagine, 3 mL of 1 mM FeSO4 stock,
3.33 mL of 5 g/L Nucellin™ stock, 2.57 mL of 36 mg/L
Hydrocortisone and 1.0 g/L Putrescine stock solution, 0.23 mL of 50
mg/L Sodium Selenite stock, and 13.1 mg of Thiamine on day 8.
(Table Remove)
Results and Conclusions
Cell growth for the first 4 days was similar for the control and batch processes,
while the glutamine starved fed-batch process had a slightly lower cell density and
remained a little lower for the rest of the batch. Both batch processes maintained higher
cell densities for the duration of the batch, probably due to the lack of any significant
dilution (see Figure 25). Viabilities of all the cultures were the same up to day 8.
However, it is interesting to note that on day 11, the viability of the batch process that
was not supplemented was lower than the other three bioreactors and ended up
significantly lower by the final day. This suggests that the batch medium could still be
optimized since the supplemented batch process had a viability that was the same as the
fed-batch bioreactors (see Figure 26).
Cells cultured in either glutamine starved batch process or in the glutamine
starved fed-batch process outperformed the same cells cultured in the control fed-batch
process in productivity. The control fed-batch process had a harvest day titer of 685
ug/mL, as expected, while the glutamine starved fed-batch process had a harvest titer of
1080 jig/mL, about 58% higher than the control. This is similar to results seen
previously. The glutamine starved non-supplemented batch process had a harvest day
titer of 960 ug/mL, 40% higher than the control, similar to the glutamine starved fedbatch
process, while the supplemented glutamine starved batch process had the highest
titer at 1296 ug/mL. This is an 89% increase over the control (see Figure 27).
When the inhibitor levels for the four conditions were analyzed the results
showed that the lactate and ammonia levels for all three glutamine starved processes
were significantly lower than the control. In fact, after day 4, those three conditions
either stopped producing or started consuming lactate while the control continued to
produce lactate throughout the batch (see Figure 28). As expected, the ammonia levels
were much lower in the glutamine starved processes and declined after day 4, while the
control continued to produce ammonia (see Figure 29).
In this Example, combining a batch process with a glutamine starvation strategy
resulted in a 40% improvement in productivity over the control fed-batch process for
anti-GDF-8 cells. The data also suggest that with some optimization of the batch
medium, an almost 2-fold improvement in productivity can be attained. This
improvement in productivity can be attributed to two factors. First, glutamine starvation
increases productivity either directly or by keeping ammonia and lactate levels very low.
Second, because of the absence of feeds, the titer is not diluted during the batch.
66
Increased productivity together with the ease of operation inherent in a batch process
makes this an attractive option for producing recombinant polypeptides.
Example 8. Effects of Glutamine and Asparagine concentrations in batch media on
anti-GDF-8 cell culture process.
In Examples 2, 5 and 6, it was demonstrated that glutamine starvation conferred benefits
on fed-batch cultures in two cell lines, including increased cell growth, cell viability and
titer as well as decreased production of lactate and ammonium. Asparagine also seems
to play a role in batch media.
Materials and Methods
Anti-GDF-8 cells were cultured for twelve days in 1L Bioreactors in modified
Medium 9 with differing concentrations of glutamine and asparagine. Base Medium 9
composition is listed in Table 14. Experimental variations on this base composition are
listed in Table 15. The cultures were incubated at 37°C for the first 5 days with the
exception of Reactor 4, whose temperature was 30°C for the first day due to temperature
control problems. The cultures were shifted to 31°C on day 6. On day 7, the cultures
were fed once with 5% by volume Medium 5 lacking glutamine. Cultures were
measured daily for cell density, anti-GDF-8 titer, lactate and ammonium levels.
(Table Remove)
Results and Conclusions • i
Figures 30, 31, 32 and 33 show the cell growth of anti-GDF-8 cells, anti-GDF-8
titer, lactate levels and ammonium levels, respectively, throughout the course of the
experiments under the various experimental conditions.
Under all experimental conditions, 4 mM glutamine is better than 1 mM
glutamine at all the Asparagine levels tested. At comparable glutamine levels, 12 mM
and 20 mM asparagine conditions are better than 8 mM asparagine conditions.
Decreased lactate and NH» levels were observed at the end of the culture for all
conditions tested.
Example 9. Effects of Glutamine and Asparagine concentrations in batch media on
anti-GDF-8 cell culture process.
In Example 8, it was demonstrated that Medium 9 containing an initial
concentration of 4 mM glutamine performs better than media containing 1 mM
glutamine, regardless of asparagine levels. This example demonstrates the effect of
media containing 13 mM glutamine levels and various asparagine levels.
Materials and Methods
69
Anti-GDF-8 cells were cultured for twelve days in 1L Bioreactors in modified
Medium 9 with differing concentrations of glutamine and asparagine as listed in Table
16. The cultures were incubated at 37°C for the first 3 days. The cultures were then
shifted to 31°C on day 4. On day 7, the cultures were fed once with 5% by volume
Medium 5 lacking glutamine. Cultures were measured periodically for cell density, cell
viability, lactate, ammonium levels and glutamine levels, anti-GDF-8 titer, and
osmolarity.
(Table Remove)
Figures 34, 35, 36, 37, 38, 39 and 40 show the cell growth of anti-GDF-8 cells,
percent viability of anti-GDF-8 cells, lactate levels, ammonium levels, glutamine levels,
anti-GDF-8 titer, and osmolarity, respectively, throughout the course of the experiments
under the various experimental conditions.
Among all the conditions tested, only Medium 9 containing 13 mM glutamine
and 20 mM asparagine showed significant adverse effects on cell growth and titer.
Glutamine is exhausted in all the cultures at approximately the same time, regardless of
whether the culture begins with 4 mM or 13 mM glutamine. The highest anti-GDF-8
titer is obtained in cultures that contain 13 mM glutamine and 12 mM asparagine. All
culture conditions exhibit decreased lactate and ammonium levels near the end of the
culture. Ammonium levels were highest in the culture containing 13 mM glutamine and
20 mM asparagine.
Example 10. The effect of asparagine and cysteine levels on the observed decrease
in lactate and ammonium levels in anti-GDF-8 cells cultured in Medium 9.
In Examples 2, 5 and 6, it was found that cultures grown under glutamine
starvation conditions exhibit decreased lactate and ammonium levels at the end of the
culture process. However, cultures grown in Medium 9 under non-glutamine starvation
conditions still exhibit decreased lactate and ammonium levels at the end of the culture
process. This effect was not observed in other media such as Medium 1, where
glutamine starvation appears necessary for the decreased levels of lactate and
ammonium. Medium 9 and Medium 1 differ in the levels of asparagine (20 mM in
Medium 9 versus 11 mM total in Medium 1 plus feed) and acidic cystine (1.5 mM in
Medium 9 versus 0.95 mM in Medium 1). This example tests whether these two
components were responsible for the observed decrease in the lactate and ammonium
levels at the end of the culture.
Materials and Methods
Anti-GDF-8 cells were cultured in 1L BioReactors for 12 days. Cells were
initially cultured at 37°C and were shifted to 31°C on day 4 or day 5 at 8-10xl06/ml.
Table 17 lists the various experimental conditions tested. Samples were taken daily and
saved for titer analysis by Protein A HPLC.
(Table Remove)
Anti-GDF-8 cells grown in Medium 9 exhibited decreased lactate and ammonium
levels at the end of the culture process, regardless of whether the cultures were started
with 4 mM or 13 mM glutamine (see Figures 42 and 43). In contrast, Medium 1 only
exhibited decreased lactate and ammonium levels at the end of the culture process when
the cultures were started with 4 mM glutamine (see Figures 42 and 43). Addition of
extra asparagine and cystine to Medium 1 containing 13 mM glutamine did not result in
decreased lactate and ammonium levels at the end of the culture process (see Figures 42
and 43).
Cultures that exhibited decreased lactate and ammonium levels at the end of the
culture process (Medium 1 with 4 mM glutamine, Medium 9 with 4 mM glutamine and
Medium 9 with 13 mM glutamine) were also observed to have lower total osmolarity at
the end of the culture process (see Figure 47).
Medium 9 with 4 mM glutamine exhibited the highest anti-GDF-8 titer, followed
by Medium 9 with 13 mM glutamine fed on day 4 (see Figure 46). Taking the effect of
dilution of the feed into account, Medium 9 containing 4 mM glutamine had equivalent
anti-GDF-8 titer to Medium 9 containing 13 mM glutamine.
Example 11. The effect of amino acid and vitamin levels on the observed decrease
in lactate and ammonium levels in anti-GDF-8 cells cultured in Medium 9.
Example 10 tested whether difference in the asparagine and cysteine levels
between Medium 1 and Medium 9 were responsible for the observed decrease in lactate
and ammonium levels at the end of the culture process in Medium 9 that was not starved
for glutamine. It was determined that these factors were not responsible for the observed
decrease. Medium 1 and Medium 9 also differ in their amino acid and vitamin
concentrations. This example tests whether differences in amino acids and vitamin
concentrations between these two media are responsible for the observed decrease.
Materials and Methods
Anti-GDF-8 cells were cultured in 1L BioReactors for 12 days. Cells were
initially cultured at 37°C and were shifted to 31°C on day 4 at 8-10xl06/ml. Table 18
lists the various experimental conditions tested. Amino acids, vitamins, hydrocortisone
and putrescine, trace elements E (composition listed in Table 19) and iron were added to
the various experimental Medium 1 conditions such that the levels of these components
were equal to the levels in Medium 9. Samples were taken daily and saved for titer
analysis by Protein A HPLC.
(Table Remove)
All conditions tested exhibited decreased lactate and ammonium levels at the end
of the culture process except for Medium 1 containing added amino acids, indicating that
increased amino acid levels in Medium 9 compared to Medium 1 are probably not
responsible for the decreases in lactate and ammonium levels (see Figures 49 and 50).
However, Medium 1 containing added vitamins, hydrocortisone and putrescine, trace
elements E and iron exhibited lower lactate and ammonium levels at the end of the
to Medium 1 containing added amino acids (see Figures 49
and 50). This indicates that these components may be responsible for the observed
decreases in Medium 9.
Cultures grown in Medium 1 containing added vitamins, hydrocortisone and
putrescine, trace elements E and iron exhibited the lowest levels of ammonium
throughout the experiment due to the lower total amounts of asparagine and glutamine in
the starting media (see Figure 50).
Example 12. The effect of vitamin, trace elements £ and iron levels on the observed
decrease in lactate and ammonium levels in anti-GDF-8 cells cultured in Medium 9.
In Example 11, it was determined that the increased levels of vitamins,
hydrocortisone and putrescine, trace elements E and iron in Medium 9 relative to
Medium 1 might be responsible for the decrease in lactate and ammonium levels
observed at the end of the culture process. Here, these components were tested
individually and in combination to determine which, if any, were responsible for the
observed decrease. • i
Materials and Methods
Anti-GDF-8 cells were cultured in 1L BioReactors for 12 days. Cells were
initially cultured at 37°C and were shifted to 31°C on day 4 at 8-10xl06cells/ml, with the
exception of Medium 1 containing trace E elements, which were shifted on day 4 at
about 6x106 cells/ml. Table 20 lists the various experimental conditions tested.
Hydrocortisone and putrescine were added to all Medium 1 conditions such that the
levels of these components were equal to the levels in Medium 9. Vitamins, Trace
elements E (composition listed in Table 19) and iron were added to the various
experimental Medium 1 conditions such that the levels of these components were equal
to the levels in Medium 9. Samples were taken daily and saved for titer analysis by
Protein A HPLC.
(Table Remove)
3 exhibited the lowest cell
density and anti-GDF-8 titer (see Figures 57 and 58). The fact that Medium 9 produces
superior results than Medium 1 indicates that it is better to provide the media components
in the starting media rather than supplying them through multiple feeds. Additionally,
the fact that both Medium 1 and Medium 9 perform better than Medium 3 indicates that
providing amino acids in concentrations greater than about 70 mM provide superior
results than providing amino acids in concentrations less than about 70 mM. Finally,
providing amino acids in concentrations greater than about 70 mM in the starting media
results in the highest cell densities and titers (compare Medium 9 vs. Medium 1).
Example 14. Statistical analysis of optimum total glutamine and asparagine levels
in Medium 9 for anti-GDF-8 cell culture in Bioreactors.
Materials and Methods
Anti-GDF-8 cells were grown in 1L Bioreactors and were shifted from 37°C to
31°C on the days indicated in Table 23. Final titers were subjected to a T-test in order to
determine the optimum level of glutamine alone and the optimum level of total combined
glutamine and asparagine. Table 23 summarizes some relevant experimental conditions
and end results for anti-GDF-8 cells grown in Medium 9.
(Table Remove)
Figure 59 shows extrapolated anti-GDF-8 titers for various levels of glutamine
alone and total combined glutamine and asparagine. Table 24 shows the results of a Ttest
comparing normalized liter of glutamine levels between 2 and 15 mM and glutamine
levels outside this range. Table 25 shows the results of a T-test comparing normalized
liter of combined glutamine and asparagine levels between 16 and 36 mM and combined
glutamine and asparagine levels oulside Ihis range.
Bolh T-lesl resulls indicated significanl differences in anti-GDF-8 titers between
Ihe Iwo groups lhal were compared. Cullures grown in Medium 9 conlaining between 2
and 15 mM glulamine and between 16 and 36 mM combined glulamine and asparagine
exhibited higher anli-GDF-8 liters lhan cullures grown in media with glulamine and
combined glulamine and asparagine levels lhal fell oulside Ihese ranges. In all
experimenls, asparagine levels were greater lhan 9 mM.
(Table Remove)
Example 15. Effects of Medium on Cell Culture.
This example investigated the performance of three cell culture medium
variations at intermediate scale utilizing high density seed cultures. All of the media
tested were expected to show improvements over the Phase 1 medium (Medium 10 fed
with Medium 11 feed medium), based on small scale bioreactor data.
Materials and Methods
CHO cells expressing a humanized anti-Abeta peptide IgGl monoclonal antibody
("anti-ABeta cells") were tested in various media, as shown in Table 26 (see Basi et al.,
Humanized Antibodies that Recognize Beta Amyloid Peptide, WO02/46237). The pH
low end set point was 7.0 controlled with 0.95M Na2CO3 + 0.05M.foCO3, except for
Phase 1, which was controlled with a solution containing sodium bicarbonate at 0.58 M
and sodium carbonate at 0.71 M. Dissolved oxygen was controlled at 30% by sparging
on demand with air, agitation was at 60 rpm, and the feed medium was Medium 5 (with
or without glutamine, as noted). All cultures were grown at 130L scale except for
03P49B501, which was grown at 500L scale. In brief, Medium 1 is enriched in all
nutrients, without consideration for relative uptake rates, while Medium 12 was balanced
by removing apparently unnecessary nutrients from the indiscriminately enriched
version. The compositions of Mediums 10, 11 and 12 are listed in Table 27.
(Table Remove)
Results and Conclusions
Media changes led to steady improvement through the course of these
experiments. In terms of cell growth, viability, reduced lactate levels, reduced
ammonium levels, and titer, reduced glutamine levels were better than elevated ones (see
Figures 60-64) and balanced (batch) medium was better than rich medium (Medium 1,
see Figures 60-64). Cultures started from high density inoculum exhibited higher final
titer than did cultures started from lower density inoculums (see Figure 64).
Unlike what was observed in small scale bioreactors, the first medium (Medium 1
with high Gin) resulted in lower liters than did the original process (see Figure 64).
There also was no shift to lactate uptake after the temperature change (see Figure 62).
This suggests that there may be some scale sensitivity with this medium. This
conclusion is supported by small-scale (2L) parallel runs that were done along with these
intermediate scale experiments (data not shown). The later medium formulations
containing less glutamine were not sensitive to scale, at least in these experiments (see
Figures 60-65). The duplicated processes (Batches 2 and 3 and Batches 5 and 6) show
very good run-to-run reproducibility (see Figures 60-65), increasing the confidence in all
of the data gathered in this campaign.
Example 16. Production of TNFR-Ig using Medium 9.
Materials and Methods
CHO cells expressing a dimeric fusion protein consisting of the extracellular
ligand-binding portion of the human 75 kilodalton (p75) tumor necrosis factor receptor
(TNFR) linked to the Fc portion of IgGl ("TNFR-Ig cells") were seeded at high density
from a perfusion bioreactor and diluted to 3xl06 viable cells/ml in Medium 9 for the
production bioreactor step.
Results and Conclusions
Figures 66, 67, 68, 69, 70, 71 and 72 show cell growth, cell viability, residual
glucose, glutamine levels, lactate concentration, ammonium concentration, and relative
product titer, respectively. Under the range of minor modifications to the process, all
conditions yielded good cell growth, high cellular viability, and high overall final titer.
For all the conditions of this experiment, the metabolic inhibitory byproduct
lactate was either consumed, or the concentration plateaued, suggesting that lactate
production was arrested. Similarly, for the inhibitory metabolite ammonium, levels rose
initially, but at some time after the temperature shift the ammonium started to be
consumed by the cells. In this Example, the TNFR-Ig cell cultures were subjected to the
chemical inductants sodium butyrate, and HMBA.
Example 17. Comparison of Large and Small-scale Culture Conditions
Materials and Methods
To determine whether the size of the culture affected relevant culture characteristics,
anti-GDF-8 cells were grown in either small-scale 1 liter bioreactors or large-scale 6000
liter bioreactors. Cells were grown at 37°C and shifted to 31°C on day 4.
Results and Conclusions
As can be seen in Figures 73, 74, 75 and 76 (which show cell density, liter,
lactate levels and ammonium levels, respectively), there were no relevant differences
between the 6000 liter large-scale and 1 liter small-scale cultures for these
characteristics. Both lactate and ammonium levels began to decrease after the
temperature shift on day 4. This example demonstrates that the size of the culture does
not affect cell density, cell viability, lactate levels and ammonium levels when the
cultures are subjected to the same growth conditions.


^ p We claim:
1. A method of producing an anti-ABeta fragment of amyloid precursor
protein antibody in a large-scale production cell culture comprising the steps of:
providing a cell culture comprising;
mammalian cells that contain a gene encoding an anti-ABeta
fragment of amyloid precursor protein antibody, which gene is expressed under
condition of cell culture; and
a mediiun containing glutamine and having a mediimi
characteristic selected from the group consisting of: (i) a cumulative amino acid
amoimt per unit volimie greater than 70 mM, (ii) a molar cumulative glutamine to
cumulative asparagine ratio of less than 2, (iii) a molar cimiulative glutamine to
cimiulative total amino acid ratio of less than 0.2, (iv) a molar cxunulative
inorganic ion to cumulative total amino acid ratio between 0.4 to 1, (v) a
combined cumulative amount of glutamine and asparagine per imit volume of
greater than 16 mM, and combinations thereof;
maintaining said culture in an initial growth phase xmder a first set of
culture conditions for a first period of time sufficient to allow said cells to
reproduce to a viable cell density within a range of 20%-80% of the maximal
possible viable cell density if said culture were maintained under the fu^t set of
culture conditions;
changing at least one of the culture conditions, so that a second set of
culture conditions is applied;
maintaining said culture for a second period of time \mder the second set
of conditions and for a second period of time so that the antibody accumulates in
the cell culture.
2. The method as claimed in claim 1 wherein the medium contains a molar
cumulative glutamine to cimiulative asparagine ratio of less than 2; glutamine; and
said medium has two medium characteristics selected from the
group consisting of: (i) a medium containing a cimiulative amino acid
87
^ ^ amount per unit volume greater than 70 mM, (ii) a molar cumulative
glutamine to cumulative total amino acid ratio of less than 0.2, (iii) a molar
cumulative inorganic ion to cumulative total amino acid ratio between 0.4
to 1, (iv) a combined cumulative amoimt of glutamine and asparagine per
unit volume of greater than 16 mM, and combinations thereof.
3. The method as claimed in claim 1, wherein said cell culture condition in
said changing at least one of the culture conditions step is selected from the group
consisting of: (i) temperature, (ii) pH, (iii) osmolahty, (iv) chemical inductant
level, and combinations thereof
4. The method as claimed in claim 1, wherein the initial glutamine
concentration of said medium is less than or equal to 10 mM.
5. The method as claimed in claim 1, wherein the initial glutamine
concentration of said medium is less than or equal to 4 mM.
6. The method as claimed in claim 1, wherein the total cimiulative amoimt
per unit volume of glutamine of said medium is less than or equal to 10 mM.
7. The method as claimed in claim 1, wherein the total cimiulative amoimt
per unit volume of glutamine of said medium is less than or equal to 4 mM.
8. The method as claimed in claim 1, wherein glutamine is only provided in
the initial medium at the beginning of the cell culture.
9. The method as claimed in claim 1, wherein the initial density of said
mammalian cells is at least 2x10^ cells/mL.
10. The method as claimed in claim 1, wherein the initial density of said
mammaUan cells is at least 2x10^ cells/mL.
88
11. The method as claimed in claim 1, wherein the step of providing
comprises providing at least 1000 L of a culture.
12. The method as claimed in claim 1, wherein the step of providing
comprises providing at least 10,000 L of a culture.
13. The method as claimed in claim 1, wherein said first set of conditions
comprises a first temperature range that is 30 to 42 degrees Celsius.
14. The method as claimed in claim 1, wherein said first set of conditions
comprises a first temperature range that is 37 degrees Celsius.
15. The method as claimed in claim 1, wherein said second set of conditions
comprises a second temperature range that is 25 to 41 degrees Celsius.
16. The method as claimed in claim 1, wherein said second set of conditions
comprises a second temperature range that is 29 to 35 degrees Celsius.
17. The method as claimed in claim 1, wherein said second set of conditions
comprises a second temperature range that is 31 degrees Celsius.
18. The method as claimed in claim 1, further comprising a second changing
step subsequent to first said changing at least one of the culture conditions
comprising changing at least one of the culture conditions, so that a third set of
conditions is appUed to the culture.
19. The method as claimed in claim 18, wherein the second changing step
comprises changing at least one culture condition selected fix)m the group
consisting of: (i) temperature, (ii) pH, (iii) osmolality, (iv) chemical inductant
level, and combinations thereof
89
20. The method of claim 18, wherein said third set of conditions comprises a
third temperature range that is 27 to 37 degrees Celsius.
21. The method as claimed in claim 1, wherein said first period of time is
between 1-7 days.
22. The method as claimed in claim 1, wherein said first period of time is 4
days.
23. The method as claimed in claim 1, wherein the total of said first period of
time and said second period of time is at least 5 days.
24. The method as claimed in claim 1, wherein in the step of maintaining said
culture for a second period of time, the lactate level decreases subsequent to the
lactate level in the culture reaching a maximal level.
25. The method as claimed in claim 1, wherein in the step of maintaining said
culture for a second period of time, the ammoniimi level decreases subsequent to
the ammoniimi level in the culture reaching a maximal level.
26. The method as claimed in claim 1, wherein said total amount of said
produced antibody is at least 1.5-fold higher that the amount of antibody produced
under otherwise identical conditions in otherwise identical medium that lacks said
medium characteristic.
27. The method as claimed in claim 1, wherein said total amount of said
produced antibody is at least 2-fold higher that the amount of antibody produced
under otherwise identical conditions in otherwise identical medixmi that lacks said
medium characteristic.
90
^m 28. The method as claimed in claim 1, wherein said cell culture is further
provided with supplementary components.
29. The method o as claimed in claim 28, wherein said supplementary
components are provided at multiple intervals.
30. The method as claimed in claim 28 wherein said supplementary
components are selected from a group consisting of hormones and/or other growth
factors, particular ions (such as sodiimi, chloride, calciimi, magnesiiun, and
phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements
(inorganic compounds usually present at very low final concentrations), amino
acids, lipids, or glucose or other energy source.
31. The method as claimed in claim 1 wherein the mediimi is
a defined medium containing glutamine and having at least two
mediimi characteristics selected from the group consisting of: i) a starting amino
acid concentration greater than 70 mM, ii) a molar glutamine to asparagine ratio
of less than 2, iii) a molar glutamine to total amino acid ratio of less than 0.2, iv) a
molar inorganic ion to total amino acid ratio between 0.4 to 1, and v) a combined
glutamine and asparagine concentration greater than 16 mM.
32. The method as claimed in claim 1 wherein the medium is
a defmed mediimi containing glutamine, characterized by: i) a
starting amino acid concentration greater than 70 mM, ii) a molar glutamine to
asparagine ratio of less than 2, iii) a molar glutamine to total amino acid ratio of
less than 0.2, iv) a molar inorganic ion to total amino acid ratio between 0.4 to 1,
and v) a combined glutamine and asparagine concentration greater than 16 mM;
maintaining said culture in an initial growth phase under a first set of
culture conditions for a first period of time sufficient to allow said cells to
reproduce within a range of 20%-80% of the maximal possible viable cell density
if said culture were maintained under the first set of culture conditions.
91
33. The method as claimed in claim 1, wherein said medium comprises a
medium containing glutamine and having a mediimi characteristic selected from
the group consisting of:
(i) a starting amino acid concentration greater than 70 mM, (ii) a molar
starting glutamine to starting asparagine ratio of less than 2, (iii) a molar starting
glutamine to starting total amino acid ratio of less than 0.2, (iv) a molar starting
inorganic ion to starting total amino acid ratio between 0.4 to 1, (v) a combined
starting glutamine and starting asparagine concentration greater than 16 mM, and
combinations thereof
34. The method as claimed in any preceding claims, wherein:
lactate levels are lower than those levels observed under otherwise
identical conditions in otherwise identical mediimi that lacks said mediimi
characteristic;
ammoniiun levels are lower than those levels observed under
otherwise identical conditions in otherwise identical medium that lacks said
medium characteristic; and
total amount of produced antibody is at least as high as that
observed under otherwise identical conditions in otherwise identical medium that
lacks said mediimi characteristic.
35. The method as claimed in claim 1, wherein said culture is not
supplemented with additional components over the course of producing said
antibody.
36. The method of claim 1, wherein glycylglutamine is substituted for
glutamine in said culture.
92
^m 37. The method as claimed in claim 1, wherein the cumulative total amount of
histidine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and
proline per unit volume in said medium is greater than 25 mM.
38. The method as claimed in claim 1, wherein the cumulative total amount of
histidine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and
proline per imit volimie in said medium is greater than 35 mM.
39. The method as claimed in claim 1, wherein said medium has a medium
characteristic selected fix)m the group consisting of:
(i) a cimiulative total amount of histidine per unit volume greater than 1.7
mM;
(ii) a cumulative total amount of isoleucine per unit volimie greater than
3.5 mM;
(iii) a cumulative total amount of leucine per unit volume greater than 5.5
mM;
(iv) a cxmiulative total amount of methionine per unit volume greater than
2.0 mM;
(v) a cumulative total amount of phenylalanine per unit volume greater
than 2.5 mM;
(vi) a cxmiulative total amoimt of proline per xmit volume greater than 2.5
mM;
(vii) a cumulative total amoimt of tryptophan per imit volume greater than
1.0 mM; and
(viii) a cumulative total amount of tyrosine per unit volume greater than
2.0 mM.
40. The method as claimed in claim 1, wherein the cumulative total amount of
serine per unit volume in said medium is greater than 10 mM.
93
^ P 41. The method as claimed in claim 1, wherein the cumulative total amount of
asparagine per imit volume in said mediimi is greater than 8 mM.
42. The method as claimed in claim 1, wherein the cumulative total amoimt of
asparagine per unit volimie in said medium is greater than 12 mM.
43. The method as claimed in claim 1, wherein the cumulative total amoimt of
phosphorous per unit volume in said medium is greater than 5 mM.
44. The method as claimed in claim 1, wherein the cimiulative total amount of
glutamate per unit volume in said medium is less than 1 mM.
45. The method as claimed in claim 1, wherein the cumulative total amount of
calcium pantothenate per unit volimie in said medium is greater than 20 mg/L.
46. The method as claimed in claim 1, wherein the cumulative total amount of
nicotinamide per vadt volimie in said medium is greater than 25 mg/L.
47. The method as claimed in claim 1, wherein the cumulative total amount of
pyridoxine and pyridoxal per unit volume in said medium is greater than 35 mg/L.
48. The method as claimed in claim 1, wherein the cumulative total amount of
riboflavin per unit volume in said medium is greater than 2.0 mg/L.
49. The method as claimed in claim 1, wherein the cumulative total amount of
thiamine hydrochloride per unit volume in said medium is greater than 35 mg/L.
94
^ B 50. The A method of producing an anti-ABeta fiagment of amyloid precursor
protein antibody in a large-scale production cell culture is substantially such as
herein described with reference to examples and figures.
Dated this 26* day of August 2005
Mrs. L. Balasubrahmanyam
Applicant's Authorized Agent
95

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2314-delnp-2007-form-2.pdf

2314-DELNP-2007-Form-3-(16-02-2010).pdf

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Patent Number 262550
Indian Patent Application Number 2314/DELNP/2007
PG Journal Number 35/2014
Publication Date 29-Aug-2014
Grant Date 27-Aug-2014
Date of Filing 26-Mar-2007
Name of Patentee Pfizer Ireland Pharmaceuticals
Applicant Address Ringaskiddy, County Cork, Ireland
Inventors:
# Inventor's Name Inventor's Address
1 DRAPEAU, DENIS 55 OLD FARM ROAD, SALEM, NH 03079 USA
2 LUAN,YEN-TUANG ARMAND DRIVE, CHELMSFORD,MA 01824 USA
3 MERCER, JAMES,R. 226 HAMPSTEAD ROAD, DERRY, NH 03038 USA
4 WANG, WENGE 1 HOLLOWRIDGE ROAD, NORTH CHELMSFORD, MA 01863 USA
5 LASKO, DANIEL 19 GLEASON STREET, MEDFORD, MA 02155 USA
PCT International Classification Number C12P 21/08
PCT International Application Number PCT/US2005/030364
PCT International Filing date 2005-08-26
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/604,936 2004-08-27 U.S.A.