Title of Invention

"A CELL CULTURING PROCESS FOR INCREASED PROTEIN PRODUCTION AND CELL VIABILITY"

Abstract A cell culturing process for increased protein production and cell viability comprising: a) culturing host cells which produce soluble cytotoxic T-lymphocyte antigen -4 (CTLA4) molecules at a first temperature at 37°C under cell culture conditions and for a time period that allows for cell growth; b) then culturing the cells at a second temperature at 34°C and c) then culturing the cells at a third temperature at 32°C and d) then culturing the cells at a fourth temperature of 30°C and wherein the cells are cultured at the second temperature starting day 5 to day 7 of the culture, and wherein there are four days between the start of the second temperature and the start of the third temperature and wherein the fourth temperature starts on or two weeks from the start of the culture until the end of the culturing process.
Full Text Mammalian Cell Culture Processes for Protein Production
This invention claims priority from provisional U.S. application Serial No.
60/436,101 filed December 23, 2002, which is incorporated herein by reference in
its entirety.
FIELD OF THE INVENTION
The present invention relates to new methods and processes for culturing
mammalian cells which produce a protein product, preferably a glycosylated
protein product. Performance of the cell culturing methods and processes result in
high cell viability and can also result in high product quality and quantity, extension
of the growth phase, delay of onset of the death phase, and arrest of the death
phase.
BACKGROUND OF THE INVENTION
Animal cell culture, notably mammalian cell culture, is preferably used for
the expression of recombinantly produced, glycosylated proteins for therapeutic
and/or prophylactic applications. Glycosylation patterns of recombinant
glycoproteins are important, because the oligosaccharide side chains of
glycoproteins affect protein function, as well as the intramolecular interactions
between different regions of a protein. Such intramolecular interactions are
involved in protein conformation and tertiary structure of the glycoprotein. (See,
e.g., A. Wittweret al., 1990, Biochemistry, 29:4175-4180; Hart, 1992, Curr. Op.
CellBioL, 4:1017-1023; Goochee et al., 1991, Bio/Technol., 9:1347-1355; and
R.B. Parekh, 1991, Curr. Op. Struct. Biol., 1:750-754). In addition,
oligosaccharides may function to target a particular polypeptide to certain
structures based on specific cellular carbohydrate receptors. (M.P. Bevilacqua et
al., 1993, J. Clin. Invest, 91:379-387; R.M. Nelson et al., 1993, J. Clin. Invest,
91:1157-1166; K.E. Norgard et a!., 1993, Proc. Natl Acad. Sci. USA, 90:1068-
1072; and Y. Imai et al., 1993, Nature, 361-555-557).
The terminal sialic acid component of a glycoprotein oligosaccharide side
chain is known to have an effect on numerous aspects and properties of a
glycoprotein, including absorption, solubility, thermal stability, serum half life,
clearance from the serum, as well as its physical and chemical structure/behavior
and its immunogenicity. (A. Varki, 1993, Glycobiology, 3:97-100; R.B. Parekh, Id.,
Goochee et al., Id., J. Paulson et al., 1989, TIBS, 14:272-276; and A. Kobata,
1992, Eur. J. Biochem., 209:483-501; E.Q. Lawson et al., 1983, Arch. Biochem.
Biophys., 220:572-575; and E. Tsuda et al., 1990, Eur. J. Biochem., 188:405-411).
In general, protein expression levels in mammalian cell culture-based
systems are considerably lower than in microbial expression systems, for example,
bacterial or yeast expression systems. However, bacterial and yeast cells are
limited in their ability to optimally express high molecular weight protein products,
to properly fold a protein having a complex steric structure, and/or to provide the
necessary post-translational modifications to mature an expressed glycoprotein,
thereby affecting the immunogenicity and clearance rate of the product.
As a consequence of the limitations of the culturing of animal or mammalian
cells, particularly animal or mammalian cells which produce recombinant products,
the manipulation of a variety of parameters has been investigated, including the
employment of large-scale culture vessels; altering basic culture conditions, such
as incubation temperature, dissolved oxygen concentration, pH, and the like; the
use of different types of media and additives to the media; and increasing the
density of the cultured cells. In addition, process development for mammalian cell
culture would benefit from advances in the ability to extend run times to increase
final product concentration while maintaining high product quality. An important
product quality parameter is the degree and completeness of the glycosylation
structure of a polypeptide product, with sialic acid content commonly used as
measure of glycoprotein quality.
Run times of cell culture processes, particularly non-continuous processes,
are usually limited by the remaining viability of the cells, which typically declines
over the course of the run. The maximum possible extension of high cell viabilities
is therefore desired. Product quality concerns also offer a motivation for
minimizing decreases in viable cell density and maintaining high cell viability,
cell death can release sialidases to the culture supernatant, which may reduce the
sialic acid content of the protein expressed. Protein purification concerns offer
another motivation for minimizing decreases in viable cell density and maintaining
high cell viability. The presence of cell debris and the contents of dead cells in the
culture can negatively impact on the ability to isolate and/or purify the protein
product at the end of the culturing run. By keeping cells viable for a longer periodof time in culture, there is thus a concomitant reduction in the contamination of the
culture medium by cellular proteins and enzymes, e.g., cellular proteases and
sialidases, that can cause degradation and ultimate reduction in quality of the
desired glycoprotein produced by the cells.
Various parameters have been investigated to achieve high cell viability in
cell cultures. One parameter involved a single lowering of the culture temperature
following initial culturing at 37°C (for example, Roessler et al., 1996, Enzyme and
Microbial Technology, 18:423-427; U.S. Patent Nos. 5,705,364 and 5,721,121 to
T. Etcheverry et al., 1998; U.S. Patent No. 5,976,833 to K. Furukawa et al., 1999;
U.S. Patent No. 5,851,800 to L. Adamson et al.; WO 99/61650 and WO 00/65070
to Genentech, Inc.; WO 00/36092 to Biogen, Inc.; and U.S. Patent No. 4,357,422
to Girard et al.).
Other parameters investigated involved the addition of components to the
culture. The growth factor inhibitor suramin was shown to prevent apoptosis during
exponential growth of CHO K1:CycE cells (Zhangi et al., Biotechnol. Prog. 2000,
16, 319-325). However, suramin did not protect against apoptosis during the death
phase. As a result, suramin was capable of maintaining high viability during the
growth phase, but did not allow for an extension of culture longevity. The same
authors report that for the CHO 111-10PF cell line, dextran sulfate and polyvinyl
sulfate could, similarly to suramin, increase day 3 viable cell density and viability
relative to the control culture. The effect of dextran sulfate or polyvinyl sulfate
during the death phase was however not reported. Suramin, dextran sulfate and
polyvinyl sulfate were also reported to be effective at preventing cell aggregation.
Heparin has been supplemented to animal cell culture media in order to
adapt anchorage-dependant cell lines to suspension conditions (e .g. U.S. Patent
No. 5,348,877to McKenna and Granados, 1994). Heparin is also known to bind to
growth factors, such as the heparin-binding EGF-like growth factor (HB-
Raab and Klagsbrun, Biochim. Biophys. Acta 1997, 1333, F179-F199). Cell
surface heparan sulfate proteoglycans (HSPG) reportedly enhance HB-EGF
binding and bioactivity for certain cell types including wild-type CHO cells (Raab
and Klagsbrun, 1997). [Heparan sulfate only differs from heparin in that it has
fewer N- and O-sulfate groups and more N-acetyl groups (McKenna and
Granados, 1994). For the purpose of this disclosure, heparin and heparan sulfate
are considered equivalent and will generically be referred to as heparin.] It has
been proposed, for the heparin-binding growth factor FGF-2, that binding to HSPG
increases the local FGF-2 concentration on the cell surface, which in turn
increases the probability of FGF-2 binding to the tyrosine kinase receptors of the
cells (Raab and Klagsbrun, 1997). It has been shown that pentosan polysulfate
can block the action of heparin-binding growth factors on cultured cells (Zugmaier
et al., . J. Nat. Cancer Inst. 1992, 84, 1716-1724.
Patent literature on the use of dextran sulfate in animal cell culture pertain
to the supplementation of dextran sulfate to >a medium in order: 1) To improve
growth rate and increase the number of population doublings before senescence
for human endothelial cells (U. S. Patents Nos. 4,994,387 and
5,132,223 to Levine et al., 1991,1992); 2) To increase recombinant protein yield in
mammalian cell lines (U. S. Patents No. 5,318,898 to Israel, 1994); 3) To induce
single cell suspension in insect cell lines (U. S. Patents No. 5,728,580 to Shuler and
Dee, 1996); 4) To increase growth-promoting activity of human hepatocyte-growth
factor and to suppress its degradation (U.S. Patent Nos. 5,545,722 and 5,736,506
to Naka, 1996 and 1998); 5) To increase viable cell density and recombinant
protein expression (WO 98/08934 to Gorfien et al., 1997).
In all reported cases referring to the presence or supplementation ofdextran sulfate in a medium, dextran sulfate was present throughout the culture
time in that given medium. In no case were the benefits of a delayed addition
reported. Moreover, it has never been reported that dextran sulfate can delay the
onset of the death phase, extend the growth phase, or arrest the death phase.
With increasing product concentration in the culture, it can be observed in
cell culture processes that the product quality decreases, as determined by the
measured sialic acid content of the oligosaccharide glycostructure. Usually, a
lower limit for an acceptable sialic acid content exists as determined by drug
clearance studies. High abundance of a protein produced by cells in culture is
optimally accompanied by high quality of the protein that is ultimately recovered for
an intended use.
Recombinantly produced protein products that are properly glycosylated are
increasingly becoming medically and clinically important for use as therapeutics,
treatments and prophylactics. Therefore, the development of reliable cell culture
processes that economically and efficiently achieve an increased final protein
product concentration, in conjunction with a high level of product quality, such as is
determined by sialic acid content, fulfills both a desired and needed goal in the art.
SUMMARY OF THE INVENTION
The present invention provides new processes for the production of
proteins, preferably recombinant protein products, more preferably glycoprotein
products, by animal or mammalian cell cultures. These new processes achieve
increased cell viability.
«
One aspect of this invention concerns the use two or more temperature
shifts. In this aspect, cell culture processes of this invention can advantageously
achieve an enhanced final titer or concentration of product, e.g., glycoprotein, as
well as an enhanced sialic acid content of the glycoprotein produced by the
cultured cells. More specifically, in accordance with this invention, two or more
temperature shifts during the cell culturing period sustain a high cell viability of the
cells in the culture and can provide a high quantity and quality of produced product
throughout an entire culture run. Also, according to one aspect of the invention,
the two or more temperature shifts comprising the culturing processes can
advantageously allow for an extension of the production phase of the culture.
During the extended production phase, the titer of the desired product is
increased; the product quality, as characterized by sialic acid content, is
maintained at a high level; and cell viability is also maintained at a high level. In
addition, the extended production phase associated with the culturing processes
of the invention allows for the production of product beyond that which is produced
during a standard production phase.
In one aspect of the present invention, multi-step temperature shifts,
preferably, timed multi-step temperature shifts comprising two or more downward
temperature shifts, are used in the culturing of mammalian cells to produce a
desired protein product, particularly, a glycoprotein product. Two or more (i.e., at
least two) temperature shifts, which may be performed after the growth phase of
the culture, comprise the processes of this invention. With the at least two
temperature shifts, preferably with approximately four day increments between the
shifts, a high protein yield with a concomitant high sialic acid content of the desired
protein product can be achieved. The multiple temperature shifts comprising the
culturing methods can achieve both high quality and quantity of protein product, as
well as sustain cell viability for the duration of a culturing period.
In accordance with another aspect of this invention, the culturing processes
«(methods) involving two or more temperature shifts can allow cells to be
maintained in culture for a period of time that advantageously extends the
culturing run to achieve high quality and quantity of protein production. Such an
extension of the protein production phase advantageously provided by this
invention refers to a production phase that can be carried out beyond the protein
production that is attained when no temperature shift, or only one temperature
shift, is used in the culture run. The extended production phase is associated with
the multiple temperature shifts that comprise the described cell culturing methods.
According to the new cell culture methods of this invention, the combination of a
second, third, fourth, or further downward shift in temperature with a first
temperature shift allows the cell cultures to sustain a high cell viability and
provides, in an embodiment of the invention, for an extended production phase
during which the titer of the protein product is increased and product quality,
characterized by sialic acid content, remains high until the end of the culture run.
A culture run as used herein refers to the culturing period, preferably, the
entire culture period. For a culture run comprising two or more temperature shifts,
the length of the entire culture run can last from as short as just after the second
temperature shift (for example, about 10-14 days) to as long as about 28 to 30
days, or more. For a culture run comprising three (or more) temperature shifts, the
length of the entire run can last from as short as just after the third (or the last)
temperature shift (for example, about 14 to 21 days) to as long as about 28 to 30
days or more. Thus, in accordance with the methods of the present invention,
cells can be cultured for a total run period of greater than 10 days, greater than 14
days, or greater than 21 days. Preferably, the culture run lasts for at least about
10 to 14 days to about 21 to 30 days, or more
The total culturing run can comprise two, three, four, or more step
temperature shifts. As a nonlimiting example, a two-step temperature shift is
carried out as follows: the culture temperature is initially maintained at 37°C, or
near 37°C, from day 0 to about day 6; from about day 6 to about day 10, the
culture temperature is maintained at 34°C, or near 34°C; and from about day 10
onward, e.g., to about day 14 to 28, to about day 14 to 18, or to the end of the
culture run, the culture temperature is maintained at 32°C, or near 32°C. A threestep
temperature shift culture procedure according to this invention comprises the
following nonlimiting, exemplifying format: the cell culture temperature is
controlled at 37°C, or near 37PC, from day 0 to about day 6; from about day 6 to
about day 10, the culture temperature is maintained at 34°C, or near34°C; from
about day 10 to about day 14, the culture temperature is maintained at 32°C, or
near 32°C; and from about day 14 onward, e.g., to about day 21 to day 30, or
longer, i.e., to the end of the run, the culture temperature is maintained at 30°C, or
near 30°C.
Thus, employment of the present cell culturing methods comprising two or
more temperature shifts in which high quantity and quality of protein production is
achieved is beneficial not only for culture runs having "shorter", e.g., standard,
durations (e.g., about 10 to about 14 days), but also for culture runs which can
endure longer than the standard production run. Such longer duration culturing
runs are achieved because the methods of this invention can provide an extension
of the initial or standard production phase of protein production by the cultured
cells (the initial or standard production phase occurs, in general, at about days 6 to
14). For example, by employing two, three, or more temperature shifts in the
culture run in accordance with this invention, high quality and quantity of protein
production and cell viability can be maintained and sustained for a total run time of
about 10-14 days to a total run time of about 21 to 28 days or more, compared
with protein production and product quality in cultures employing no temperature
shift or, at most, one temperature shift.
In another of its aspects, the present invention provides cell culture
methods comprising greater than two or three temperature shifts as described
above. In such multi-step temperature shift runs, cells are cultured essentially as
described for a three step culturing period, and additional downward temperature
shifts are performed until the end of the culture period. For example, a fourth
downward temperature shift, i.e., temperature lowering, can be carried out
following the third temperature shift culture period, in which the cell culture
temperature is further shifted from about 30°C, to about 28°C or 29°C, preferably
about 29°C, on or about days 15-19, preferably, day 18, from the start of the
culture. Additional temperature shifts can be included in the cell culturing method,
- wherein the cells are maintained at a lower temperature, e.g., extend protein production until the end of the run, preferably for longer than 28-30
days. In all cases, the protein produced by the cells at the end of the culturing
period is typically recovered, e.g., isolated and/or substantially purified, as desired,
employing techniques routinely practiced in the art as described herein. In
addition, sialic acid content is assessed by conventional methods.
In one particular aspect, the present invention provides a process (or
method) in which the final titer of product is enhanced, and the sialic acid content
of the produced glycoprotein is higher, by the use of a two- or more-step
temperature shift process. In accordance with this particular aspect, the
combination of two or more timed temperature shifts sustains a high cell viability of
the culture, thereby enabling an extended production phase during which the titer
of product, preferably recombinant product, is increased and the product quality,
as characterized by sialic acid content, is maintained at high level. Such a two- or
more-step temperature shift can minimize the prevailing trade-off between protein
titer and sialic acid content in the production of product during the cell culture
process. Thus, the temperature shifts provide a positive effect on enhancing an
important performance parameter of the culturing process, i.e., the mathematical
product of "end (i.e., final) titer" x "end (i.e., final) sialic acid" ("end titerx end sialic
acid").
Accordingly, in another particular aspect, for a two-step culturing method as
newly described herein, cells are maintained in culture from day 0 to on or about
day 6 at a temperature of 37°C, or near 37°C; on or about day 6, the temperature
of the cell culture is lowered to 34°C, or near 34°C; and on or about day 10-14, the
temperature is again lowered to 32°C, or near 32°C. In one embodiment of such a
two-step temperature shift method, the production phase is extended beyond
about day 14 and continues to the end of the culture run, e.g., until about day 21,
or to about day 28 to 30, or longer, during which time the cells are maintained in
culture at the lower temperature of 32°C, or near 32°C. Protein product can be
recovered at the end of the extended production phase as further described
herein.
It is yet another aspect of the present invention to provide a method for
increasing the viability of cells in culture by subjecting the cells to two or more
shifts in temperature during the culture run. A condition, such as two or more
shifts in temperature, causes increased cell viability if cell viability in the culture is
higher for a period of time in the presence of the condition than in the absence of'
the condition. According to this aspect, the two or more temperature shift cell
culturing methods as described allow cells to remain viable for increased tirm
periods, such as beyond the standard production period. As discussed heren, a
beneficial consequence of increased cell viability of the cultured cells can be lhat
larger quantities of product (of high quality) are produced at the end of the
culturing period, under conditions that are conducive to the maintenance of viable
cells.
Another aspect of this invention is that increased cell viability resulting Irom
the practice of the two or more temperature shift cell culturing methods correktes
with a decreased amount of cell debris and released contents of dead or dyinj
cells over time in the culture. The presence of cell debris and the contents of dead
cells in the culture can negatively impact on the ability to isolate and/or purify he
protein product at the end of the culturing run. By keeping cells viable for a lager
period of time in culture, there is thus a concomitant reduction in the contamination
10
of the culture medium by cell proteins and enzymes, e.g., cellular proteases and
sialidases, that can cause degradation and ultimate reduction in quality of the
desired glycoprotein produced by the cells.
Another aspect of this invention concerns the delayed addition of
polyanionic compound to the cell culture. Delayed addition of polyanionic
compound achieves increased cell viability. Polyanionic compound preferably is
dextran sulfate. Polyanionic compound is added to the culture at a time after
innoculation.
In one aspect of this invention, polyanionic compound is added to a culture
at a time after innoculation that is before the beginning of the initial death phase,
or is during the initial growth phase, or is during the second half of the initial
growth phase, or is on or about the end of the initial growth phase. In accordance
with this aspect of the invention, the growth phase is extended and/or the onset of
the death phase is delayed for a period of time, such as several days.
Additionally, once the death phase has begun, the death rate is greatly reduced.
In another aspect of this invention, polyanionic compound is added to a
culture during the initial death phase. In accordance with this aspect of the
invention, cell death is arrested for a period of time, such as several days.
In another preferred aspect of this invention and as further described
herein, the newly developed cell culture processes, both those involving two or
more temperature shifts and those involving the delayed addition of a polyanionic
compound, are especially suitable for the production of soluble CTLA4 molecules
and soluble CTLA4 mutant molecule, such as CTLA4lg and L104EA29Ylg, by host
cells genetically engineered to express and produce these proteins. (See
Examples 1 through 11). Preferred embodiments of the present invention
encompass the culturing of cells producing CTLA4lg and L104EA29Ylg using
multiple temperature shifts during the culturing run to achieve large amounts of
high quality CTLA4lg and L104EA29Ylg products, as determined by sialic acid
measurement of the final products. Preferred embodiments of the present
invention encompass the culturing of cells producing CTLA4lg and L104EA29Ylg,
using the delayed addition of polyanionic compound.
Further aspects, features and advantages of the present invention will be
appreciated upon a reading of the detailed description of the invention and a
consideration of the drawings/figures.
DESCRIPTION OF THE DRAWINGS/FIGURES
FIG. 1 shows the impact of different temperature shift profiles on cell •
viability for cells cultured at a 5 liter (5 L) reactor scale. These results were
obtained from the experiments described in Example 3 herein. Comparison is
made among culturing methods involving no temperature shift ("no T-shift"), a
single temperature shift ("single T-shift") and two downward temperature shifts
("double T-shift").
FIG. 2 shows the impact of different temperature shift profiles on cell
viability for cells cultured at a 50 liter (50 L) reactor scale. These results were
obtained from the experiments described in Example 3 herein. Comparison is
made between culturing methods involving three downward temperature shifts
("triple T-shift") and two downward temperature shifts ("double T-shift").
FIG. 3 depicts a nucleotide sequence (SEQ ID NO:1) and encoded amino
acid sequence (SEQ ID NO:2) of a CTLA4lg having a signal peptide, a wild type
amino acid sequence of the extracellular domain of CTLA4 starting at methionine
at position +1 to aspartic acid at position +124, or starting at alanine at position -1
to aspartic acid at position +124, and an Ig region.
FIG. 4 depicts a nucleotide sequence (SEQ ID NO:3) and encoded amino
acid sequence (SEQ ID NO:4) of a CTLA4 mutant molecule (L104EA29Ylg)
having a signal peptide, a mutated extracellular domain of CTLA4 starting at
methionine at position +1 and ending at aspartic acid at position +124, or starting
at alanine at position -1 and ending at aspartic acid at position +124, and an Ig
region.
FIG. 5 depicts the nucleic acid sequence (SEQ ID NO:5) and encoded
complete amino acid sequence (SEQ ID NO:6) of human CTLA4 receptor
(referred to as "wild type" CTLA4 herein) fused to the oncostatin M signal peptide
(position -26 to -2). (U.S. Patent Nos. 5,434,131 and 5,844,095).
FIG. 6 shows the impact of delayed addition of dextran sulfate on viable cell
density, total cell density, and viability in a culture in which dextran sulfate was
added at the end of the initial growth phase. These results were obtained from the
experiments described in Example 6 herein. Comparison is made between
cultures in which dextran sulfate was added at the end of the initial growth phase,
and cultures in which no dextran sulfate was added. Average values are plotted;
error bars represent standard deviation.
FIG. 7 shows the impact of delayed addition of dextran sulfate on death
rate. It is a logarithmic representation of the viable cell densities as a function of
time. These results were obtained from the experiments described in Example 6
herein. Comparison is made between cultures in which dextran sulfate was added
at the end of the initial growth phase, and cultures in which no dextran sulfate was
added. Average values are plotted. Error bars represent standard deviation.
FIG. 8 shows the impact of delayed addition of dextran sulfate on viable cell
density, total cell density, and viability in a culture in which dextran sulfate was
added during the initial death phase. These results were obtained from the
experiments described in Example 7 herein. Comparison is made between a
culture in which dextran sulfate was added during the initial death phase, and a
culture in which no dextran sulfate was added.
FIG. 9 shows the impact of delayed addition of dextran sulfate on viable cell
density, total cell density, and viability in a culture in which dextran sulfate was
added during the initial death phase. These results were obtained from the
experiments described in Example 8 herein. Comparison is made between a
culture in which dextran sulfate was added during the initial death phase, and
culture in which no dextran sulfate was added.
FIG. 10 shows viable cell density, total cell density, and viability in cultures
in which dextran sulfate was added on day 0 of the culture. These results were
obtained from the experiments described in Example 9 herein.
FIG. 11 shows viable cell density and viability in cultures in which dextran
sulfate was added at three different times (day 3, day 4, and day 5) of the initial
growth phase. These results were obtained from the experiments described in
Example 10 herein.
13
FIG. 12 shows the impact of different temperature shift profiles on viable
cell density. These results were obtained from the experiments described in
Example 11 herein. Comparison is made among culturing methods involving no
temperature shift ("no T-shift"), a single temperature shift ("one T-shift") and two
downward temperature shifts ("two T-shifts").
FIG. 13 shows the impact of different temperature shift profiles on viaability.
These results were obtained from the experiments described in Example 11
herein. Comparison is made among culturing methods involving no temperature
shift ("noT-shift"), a single temperature shift ("one T-shift") and two downward
temperature shifts ("two T-shifts").
FIG. 14 shows the impact of different temperature shift profiles on titer.
These results were obtained from the experiments described in Example 11
herein. Comparison is made among culturing methods involving no temperature
shift ("no T-shift"), a single temperature shift ("one T-shift") and two downward
temperature shifts ("two T-shifts"). •
DETAILED DESCRIPTION OF THE INVENTION
The present invention describes new processes for the production of
proteins, preferably recombinant protein products, more preferably glycoprotein
products, in mammalian or animal cell culture. These processes achieve
increased cell viability.
Cell culturing processes involving two or more temperature shifts
The cell culturing processes according to this invention involving two or
more temperature shifts achieve increased cell viability and can achieve an
enhanced final titer or concentration of product produced by cells in culture. In
addition, the sialic acid content of the glycoprotein produced by the cultured cells
can be high, thus indicating a high quality of the protein product that is produced
during the culturing period.
More specifically in accordance with this invention, two or more temperature
shifts during the cell culturing run maintain and sustain a high cell viability of the
cells in the culture, eg. achieve increased cell viability, and can provide a high
quantity and quality of produced product throughout the culture run. Also, in
accordance with the invention, the two or more temperature shifts can
advantageously allow for an extension of the production phase of the culture.
During the extended production phase, cell viability is maintained; the titer of the
desired product is increased; and the product quality, as characterized by sialic
acid content, is maintained at a high level.
In accordance with this invention, two or more temperature shifts, preferably
downward temperature shifts, during the cell culturing period can allow for a high
quantity and quality of protein product to be produced at the end of the culturing
period, compared with culturing methods involving no temperature shift, or only
one temperature shift. Illustratively, as shown in Example 3, a culturing process
with two or three temperature shifts in accordance with the present invention yields
an increase in the quantity of protein (e.g., end titer) compared with no
temperature shift or only one temperature shift, regardless of the total length of the
culture run. In addition, the processes and methods'of the present invention are
particularly suited to cells grown and maintained as fed-batch cultures, as further
described herein.
Because the two or more temperature shifts of the culturing methods also
maintain and sustain a high viability of the cells in culture, the culturing processes
can extend the production phase for protein. In particular, during the cell culturing
methods comprising an extended production phase, cell viability remains high, the
titer of the desired product is increased, and the product quality, as characterized
by a measurable sialic acid content, is also maintained at a high level. As newly
provided by this invention, the cell culture methods comprise a production phase
that is extended beyond the production that is yielded by culturing procedures
involving no temperature shifts or, at most, only one temperature shift. .
Cell culturina methods involving multiple temperature shifts
In embodiments of the present invention, timed multi-step .temperature -
shifts are used in the culturing of mammalian cells to produce a desired protein
product, particularly, a glycoprotein product. More preferably, the cells in culture
produce a recombinantly produced protein, polypeptide or peptide product.
However, in some cases, the cells may actively produce, or overproduce, an
endogenous or natural product which can be harvested or recovered following the
practice of the methods of the present invention. As described herein, two or
more temperature shifts, preferably controlled, downward temperature shifts,
carried out at appropriately timed intervals during the culturing period, can be used
in the processes of this invention to achieve a high protein yield with a concomitant
high sialic acid content.
In accordance with the cell culturing methods and processes of this
invention (also referred to as production or fermentation runs), cells cultured in
conjunction with two or more temperature shifts during a culturing run can produce
a high quantity and quality of product during the run, as measured by end titer and
sialic acid content at the end of the run. The high quantity and quality of protein
production associated with the methods of this invention are obtained relative to
methods in which no temperature shift, or at most, one temperature shift is used,
regardless of whether a culture run is carried out for a total run time of about 10-14
days or for more than 14 days. Moreover, as a result of the two or more
temperature shifts during the culturing process, cells can be maintained in culture
• for a period of time that essentially extends the standard or initial production
phase. A standard or initial production phase is typically about 6 to 14 days.
Increased production of high quality protein, as well as sustained cell viability, are
achieved during the extended production phase of the present culturing methods
involving two or more temperature shifts.
Also according to the present culturing methods, cells can be cultured for a
total run period of greater than about 10 days, greater than about 14 days, greater
than about 21 days, or greater than about 28 days, preferably, about 14 to 30
days, or more. For example, in a culture run of this invention that comprises two
or more temperature shifts, the length of the entire run can last from as short as
just after the second (or last) temperature shift (for example, about 14 days) to as
long as about 21 to 30 days or more, preferably about 28 days or more.In an embodiment of the present invention, the extended production phase
is associated with the multiple temperature shifts that comprise the cell culturing
methods of this invention. According to the new cell culture methods of this
invention, the combination of a second, third, or further temperature shift with
first temperature shift not only allows the cell cultures to produce high quantity and
quality of product throughout the duration of the culture run, but also allows the
culture to sustain a high cell viability throughout the run and/or throughout an
extended production phase until the end of the culture run. During the culture run,
including the extended production phase, the titer of the protein product is
increased and product quality, as characterized by sialic acid content, remains
high.
More particularly, in one of its specific embodiments, the present invention
embraces cell culture methods that extend the initial production phase of protein
production by cultured cells (i.e., the standard production phase that encompasses
about days 6-14 is extended). By employing two or more temperature shifts in the
culture run in accordance with this invention, an extended production phase at
about days 14-21 was achieved. With three (or more) temperature shifts in the
culture run, the culture run was further extended to about 21-28 or 30 days, or
more, with concomitantly higher yields of protein product of high quality (e.g., high
sialic acid content), (e.g., Example 3). .
In another particular embodiment of this invention, the cell culturing (or
fermentation) process encompasses a two step downward temperature shift in
which cells are maintained at three different temperatures during the total culturing
run. In this embodiment, the total cell culturing period lasts greater than about
days, more specifically, about 14 to 28 days or more, i.e., about two to three
weeks or more, prior to obtaining the end protein product (and measuring sialic
acid content). For example, in such a two step method, cells are maintained at a
first temperature of about 36°C to 38°C, preferably, 37°C, or near 37°C, for an
initial culturing period of from day 0 to about day 6. Thereafter, from about day
to day 7, preferably day 6, to about day 10, the culture temperature is maintained
at a second temperature of about 33°C to 35°C, preferably, 34°C, or near 34°C.
Following cell culture at or near 34°C, the temperature is shifted a second time
(secondary T-shift) to a third temperature of about 31 °C to 33°C, preferably, 32°C,
or near 32°C. The secondary T-shift occurs on or about day 6 to about day 14,
preferably from about day 10 to about day 14, more preferably, on or about day
10, which in various embodiments may be during the standard production phase,
17
during the growth phase, or during the death phase. Preferably there are
approximately four day increments between the first and the second temperature
shifts, more preferably four day increments. The cells are maintained at a
temperature of 32°C, or near 32°C until the end of the total culture run, e.g., for
longer than about day 10, more specifically, to about days 12-18, or to about days
14-18, or to about days 14-28 or 30, or more. At the end of the culturing process,
the protein product is typically isolated and/or purified, for example, from the
culture supernatant, if the product is secreted into the culture medium.
Alternatively in the multiple temperature shift culturing methods of this
invention, the temperature may be first lowered based on the phase of the culture.
The first temperature shift preferably occurs before the start of the death phase.
In one embodiment, the temperature is first lowered concurrently with the slowing
of cell growth. For example, the temperature is shifted from 37°C, or near 37°C,
to 34°C, or near 34°C, when the cells are no longer in their exponential growth
phase and the culture is in the stationary phase, for example, on or about day 6 of
culture. At this time, the viable cell concentration has reached a suitable cell
density for protein production, preferably enhanced protein production, for
example, about 2-12 x 106 cells/mL, such as 2-9 x 106 cells/mL, 3-7 x 106 cells/mL,
4-5 x 106 cells/mL, 3-4 x 106 cells/mL, 2-4 x 106 cells/mL, 4-6 x 106 cells/ml, 6-8 x
106 cells/mL, 8-10x106 cells/mL, or 10-12 x 106 cells/mL. Without wishing to be
bound by theory, it is possible that the slowing of cell growth correlates with the
depletion of nutrients and/or particular components of the cell culture medium,
e.g., a nitrogen limitation in the medium.
In another embodiment, the first shift in temperature occurs during the
growth phase, for example when the viable cell concentration is about 2-12 x 106
cells/mL, such as 2-9 x 106 cells/mL, 3-7 x 106 cells/mL, 4-5 x 106 cells/mL, 3-4 x
106 cells/mL, 2-4 x 106 cells/mL, 4-6 x 106 cells/mL, 6-8 x 106 cells/mL, 8-10 x 106
cells/mL, or 10-12 x 106 cells/ml.
In yet another embodiment embracing the two-step temperature shift
culturing process, cells are cultured for a 14 day run in which the culture
temperature is maintained at or near 37°C from day 0 to day 6. From about day 6
to about day 10, the culture temperature is maintained at or near 34°C; and from
18
about day 10 to about day 14, the culture temperature is maintained at or near
32°C. As another embodiment, cells are cultured for about a 21 day period in
which the culture temperature is maintained at or near 37°C from day 0 to about
day 6; from about day 6 to about day 10, the culture temperature is maintained at
or near 34°C; and from about day 10 to about day 21, the culture temperature is
maintained at or near 32°C. As yet another embodiment, cells are cultured for
about a 28 day period in which the culture temperature is maintained at or near
37°C from day 0 to about day 6; from about day 6 to about day 10, the culture
temperature is maintained at or near 34°C; and from about day 10 to about day
28, the culture temperature is maintained at or near 32°C.
The present invention also encompasses embodiments in which the cell
culturing methods comprise three or more temperature shifts. In one embodiment
involving a three-step temperature shift culturing process, cells are initially cultured
at a first temperature of about 36°C to 38°C, preferably, at or near 37°C for about6 days; thereafter, the culture temperature is shifted and maintained at about 33°C
to 35°C, preferably, at or near 34°C for a given time period; a second shift to a
temperature of about 31 °C to 33°C, preferably, at or near 32°C occurs thereafter.
A third temperature shift to a temperature of about 29°C to 31 °C, preferably at or
near 30°C, follows the culturing period at 32°C or near 32°C; the temperature is
then held at or near 30°C until the end of the run.
In other embodiments, further temperature shifts, preferably downward
temperature shifts, can be performed following the third temperature shift of the
culture method. For example, a fourth temperature shift can follow the third shift
on or about day 15-20, preferably at about day 18 from the start of the culture. .
The fourth downward shift maintains the culture temperature at or near 28°C to
29°C, preferably, about 29°C, and increases the culture run to greater than about
28 days, e.g., to about 28-32 days or more, at which time product is obtained.
As in the two-step temperature shift culturing run procedure according to
this invention, the first shift in temperature in the multiple temperature shift
processes of the present invention can occur when the cells have essentially
stopped growing and have become stationary or approximately so. Illustratively,
the temperature shift is performed when the viable cell concentration is about 2-12
19
x 106 cells/mL, such as 2-9 x 106 cells/mL, 3-7 x 106 cells/mL, 4-5 x 106 cells/mL,
3-4 x 106 cells/mL, 2-4 x 106 cells/mL, 4-6 x 106 cells/mL, 6-8 x 106 cells/mL, 8-10
x 106 cells/mL, or 10-12 x 106 cells/mL. Alternatively, the first shift in temperature
occurs during the growth phase, for example when the viable cell concentration is
about 2-12 x 106 cells/mL, such as 2-9 x 106 cells/mL, 3-7 x 106 cells/mL, 4-5 x 106
cells/mL, 3-4 x 106 cells/mL, 2-4 x 106 cells/mL, 4-6 x 106 cells/mL, 6-8 x 106
cells/mL, 8-10 x 106 cells/mL, or 10-12 x 106 cells/mL.ln another preferred
embodiment, the multi-step cell culturing process comprises three timed and
controlled temperature shifts during a culturing period of about three to four
weeks, e.g., 21-30 days or more, preferably 28 days or more, providing extended
production of product by the cells in culture. To illustrate, the three-step
temperature shift process comprises an initial culturing period from 0 to about 6
days, preferably 6 days, during which time cells are cultured at a temperature of
37°C, or near 37°C. From about day 6 to about day 10, the cells are cultured at
34°C, or near 34°C. From about day 10 to about day 14, the culture temperature
is maintained at 32°C, or near 32°C; and from about day 14 onward, i.e., to about
day 21 to day 30 or more, or to the end of the run, the culture temperature is
maintained at 30°C, or near 30°C. Accordingly, in the three-step temperature shift
culture process of this invention, the production phase may also be extended to
yield higher end titer of protein and higher cell viability for a time period longer than
about 14 days, in contrast to the standard production phase of about 6 to 14 days
with only one or no temperature shift(s). Advantageously, the production phase
and cell viability may be further extended by the three-step T-shift method, i.e., to
about three weeks or more, with accompanying high quality of product, as
measured by sialic acid content.
In various embodiments of the present invention, the second temperature
shift to 32°C, or near 32°C allows higher quantity and quality of protein at the end
of the culture run, and is also associated with extended protein production during a
run that can last for more than about two weeks. The two or more shifts in
temperature permit the culture to stabilize a slow decline in cell viability which can
occur during the previous two weeks in culture. Yet another temperature shift from
32°C, or near 32°C, to 30°C, or near 30°C, timed at about two weeks, or
20
thereabouts, provides a further extension of the production phase, thus prolonging
the production phase of the cell culture to the end of the culturing run, e.g., to
about day 21 to day 30 or more, while maintaining cell viability without sacrificing
the quality (as determined by measurement of sialylation) of the product produced.
(See Example 3, Tables 2 and 3). Additional temperature shifts can extend cell
production beyond that of the two and three temperature shift runs.
In other embodiments, the present invention is directed to (i) a cell culturing
process, (ii) a method of increasing protein production, preferably associated with
increased cell viability, (iii) a method of enhancing sialylation of a protein product,
(iv) a method of enhancing cell viability, or (v) a method of extending protein
production, involving two or more temperature shifts, comprising: culturing host
cells which express a protein of interest at a temperature at or near 37°C under
conditions and for a time period that allow for cell growth; lowering the temperature
of the cell culture and culturing the cells at a second temperature at or near 34°C
when the culture is in the stationary phase; again lowering the temperature of the
cell culture and culturing the cells at a third temperature at or near 32°C at a time
during the standard production phase of about day 6 to day 14, e.g., on or about
ten days from the start of the culture, until the end of culturing period. As has
been noted herein, the culturing period can comprise a total run time of greater
than 10 days, greater than 14 days, greater than 21 days, or greater than 28-30
days. Following culture of the cells at 32°C, i.e., at the end of the culture run, the
produced protein product, preferably a glycoprotein, is obtained.
In other embodiments, the present invention is directed to (i) a cell culturing
process, (ii) a method of increasing protein production, preferably associated with
increased cell viability, (iii) a method of enhancing sialylation of a protein product,
(iv) a method of enhancing cell viability, or (v) a method of extending protein
production, involving two or more temperature shifts, comprising: culturing host
cells which express a protein of interest at a temperature at or near 37°C under
conditions and for a time period that allow for cell growth; lowering the temperature
of the cell culture and culturing the cells at a second temperature at or near34°C
starting about day 5 to day 7; again lowering the temperature of the cell culture
and culturing the cells at a third temperature at or near 32°C starting about day 6
21
to day 14, e.g., on or about ten days from the start of the culture, until the end of
culturing period. As has been noted herein, the culturing period can comprise a
total run time of greater than 10 days, greater than 14 days, greater than 21 days,
or greater than 28-30 days. Following culture of the cells at 32°C, i.e., at the end
of the culture run, the produced protein product, preferably a glycoprotein, is
obtained.
In another of its embodiments, the present invention provides culture
methods further comprising another temperature downshift from at or near 32°C to
at or near 30°C on or about 14 days from the start of the culture until the end of
the culturing process, thereby extending the culture period well beyond a standard
production phase. To further extend protein production during the culturing
process, as well as cell viability, the method can comprise a fourth temperature
downshift from at or near 30°C to at or near 29°C on or about 15 to 19 days,
preferably 18 days, from the start of the culture until the end of the culturing
process.
The temperature shifts of this invention are typically on or about day 6 of
the culture period, which may be during or after the growth phase of the culture,
and thereafter at approximately 4 day increments, preferably 4 day increments. In
some embodiments, the timing of the shifts in temperature may approximate the
beginning (e.g., on or about day 6), the middle (e.g., on or about day 10) and the
end (e.g., on or about day 14) of the standard production phase. In the culturing
processes or methods according to this invention in which the final titer and sialic
acid content of a produced glycoprotein is enhanced by the use of a multi-step
(e.g., two step, three step or more) temperature shift profile, the combination of at
least two, timed temperature shifts allows a total culture run to be carried out for
greater than 10 days, greater than 14 days, greater than 21 days, or greater than
28 or more days, without sacrificing end titer and sialylation of the product In
accordance with the culturing processes of this invention, the two or more
temperature shifts sustain a high cell viability of the culture and can allow more
high titer and high quality protein to be produced in a culture run compared with a
run that occurs for the same period of time, but does not include two or more
temperature shifts. Also, the two or more temperature shifts can allow the
production phase of the culture to extend beyond that of a standard production
phase and/or beyond the production of a culture having no temperature shift, or at
most, one temperature shift. Such multi-step temperature shifts, such as the twoor
more-step temperature shift, can minimize the prevailing trade-off between titer
("end titer") and sialic acid content in the production of protein product in the cell
culture process. Thus, the temperature shifts provide a positive effect on
enhancing the mathematical product of "end titer x end sialic acid", which improves
on the protein production process.
Additional embodiments in accordance with the invention of cell culture processes
involving two or more temperature shifts
In one embodiment, the present invention encompasses a cell culturing
process comprising culturing host cells which express a protein of interest at a first
temperature at or near 37°C under conditions and for a time that allow for cell
growth. Following the cell growth period, the cells are cultured at a second
temperature at or near 34°C when cell growth has slowed and becomes
approximately stationary. Thereafter, the cells are cultured at a third temperature
at or near 32°C during the standard production phase of culture, i.e., on or about
day 6 to on or about day 14. At the end of the culturing process, the produced
protein product can be obtained.
In accordance with a preferred embodiment of this invention, the cells are
cultured in a batch-fed process comprising several phases, namely, a growth
phase, during which cells are cultured at a first temperature at or near 37°C; an
initial or standard production phase, during which cells are cultured at a second
temperature at or near 34°C and at a third temperature at or near 32°C so as to
provide an extended protein production phase, which can include a fourth
temperature at or near 30°C, and optionally thereafter, additional lower
temperatures, such as at or near 29°C. In the cases of the two or more-step
temperature shift runs of this invention, extension of protein production is related .
to the two or more downward shifts in temperature. As described herein, an
extended production phase comprises a successive lowering of the temperature of
the culture at different intervals two or more times following the first temperature
switch from at or near 37°C to at or near 34°C. Relative to no temperature
23
shifting, or only one temperature shift, protein production is increased and high
product quality (as measured by sialic acid content of the final product) is attained
by the practice of these methods involving two or more downward temperature
shifts during the culturing run.
During the growth phase of cell culture, e.g., from day 0 to about day 6, the
cell density in the culture increases as the cells are typically rapidly dividing in this
period of exponential cell growth, or log phase. In the non-growth associated cell
culturing and protein production methods present in some aspects of this
invention, no significant amounts of protein product are produced during the
growth phase in which cell growth is essentially maximized under appropriate
growth conditions. Thus, as a consequence of nutrient limitations in the culture,
the cells typically enter a stationary phase on about days 4 to 6, in which rapid
growth plateaus and/or declines. In these culturing methods, the protein
production phase begins when cell growth has essentially ended (e.g., at about
day 6 to about day 10). (Examples). •
In accordance with the culturing method of one embodiment, when the cells
reach stationary phase on about day 6, the temperature is shifted downward from
at or near 37°C to at or near 34°C. Thereafter, at a time that is near the midpoint
between the first temperature shift (about day 6) and the onset of the extended
production phase (about day 14), the temperature of the culture is again lowered
from at or near 34°C to at or near 32°C. The second temperature shift allows the
culture to stabilize cell viability, which typically slowly declines through about day
14; thereafter, an extension of the production phase begins (at about day 14 to
about day 21 to day 30 or longer, preferably to about day 21 to day 28 or longer).
As has been described above, other temperature shifts, e.g., a third, fourth, or
more, can be employed during the extended production phase of the culture run.
Cell culturing methods involving delayed addtion of a polvanionic compound
In accordance with the present invention, a cell culture process involving
the delayed addition of polyanionic compound is provided. The process
comprises adding polyanionic compound to a cell culture at a time after
innoculation. The delayed addition of polyanionic compound achieves increased
cell viability as compared to that observed in the absence of addition of
polyanionic compound, or as compared to that observed when polyanionic
compound is added at inoculation.
Thus, in one embodiment, the invention is directed to a cell culturing
process comprising: culturing host cells which express a protein of interest; and
adding polyanionic compound to the cell culture at a time after innoculation.
It has been found (see Example 6) that when carrying the present invention
the percent cell viability of the cell culture is increased. Percent cell viability,
known as cell viability, is the percent of viable cells in the total number of cells. A
condition, such as delayed addition of polyanionic compound, causes increased
cell viability if cell viability in the culture is higher for a period of time in the
presence of the condition than in the absence of the condition.
Thus, in other embodiments, the invention is directed to (1) a cell culturing
process, and (2) a method of increasing the cell viability in a culture comprising:
culturing host cells which express a protein of interest; and adding polyanionic
compound to the cell culture at a time after innoeulation; wherein the cell
of the cell culture is increased. _
Polyanionic compounds include, but are not limited to, dextran sulfate
(available from Sigma-Aldrich, St. Louis, MO), heparin (available from Sigma-
Aldrich), heparan sulfate (available from Sigma-Aldrich), mannan sulfate,
chondroitin sulfate (available from Sigma-Aldrich), dermatan sulfate (available
from Sigma-Aldrich), keratan sulfate (available from Sigma-Aldrich), hyaluronate
(available from Sigma-Aldrich), poly(vinyl sulfate) (available from Sigma-Aldrich),
kappa-carrageenan (available from Sigma-Aldrich), and suramin (available from
Sigma-Aldrich). The compounds are readily available from the listed sources, or
readily obtainable through means known to one of skill in the art. These
compounds are frequently available in the form of a salt, including but not limited
to sodium salt, but may also be used in non-salt forms. A polyanionic compound
includes all forms thereof, including but not limited to salt forms, such as sodium
salts.
Preferred polyanionic compounds are poysulfated compounds, including
but not limited to: dextran sulfate, heparin, heparan sulfate, mannan sulfate,
chondroitin sulfate, dermatan sulfate, keratan sulfate, po!y(vinyl sulfate),
carrageenan, and suramin. Most preferred is dextran sulfate. Dextran sulfate
have an average molecular weight of 5,000 to 500,000 Da. Preferred is
sulfate with a molecular weight of 5,000 Da.
In accordance with the invention, polyanionic compound is added at a time
after innoculation, i.e. it is not present in the basal medium and not present at
innoculation. Preferably, the polyanionic compound is added on day 1 of the
culture or later. Innoculation takes place on day 0.
In accordance with the invention, polyanionic compound may be added to
the cell culture one time, two times, three times, or any number of times during the
specified time period (eg. at a time after innoculation). One or more polyanionic
compounds may be used in conjunction. That is, any given single addition of
polyanionic compound may include the addition of one or more other polyanionic
compounds. Similarly, if there is more than one addition of a polyanionic
compound, different polyanionic compounds may be added at the different
additions. Additional compounds and substances, including polyanionic
compounds, may be added to the culture before, with or after the addition of
polyanionic compound - either during or not during the specified time period. In apreferred embodiment, there is a single, i.e. one time, addition of polyanionic
compound. In a preferred embodiment, one polyanionic compound is added.
In accordance with the invention, polyanionic compound may be added to
the cell culture by any means. Means of adding polyanionic compound include,
but are not limited to, dissolved in water, dissolved in culture medium, dissolved in
feed medium, dissolved in a suitable medium, and in the form in which it is
obtained. Preferably, polyanionic compound is added dissolved in water.
In accordance with the invention, polyanionic compound is added to bring
the concentration in the culture to an appropriate level. As non-limiting examples,
polyanionic compound is added to a concentration of 1-1000 mg/L, 1-200 mg/L, 1-
100 mg/L, or 25-75 mg/L. Preferably polyanionic compound is added to a
concentration of 25-200 mg/L or 25-100 mg/L, more preferably about 50-100 mg/L
or 50-100 mg/L, more preferably about 50 mg/L or about 100 mg/L, most
preferably 50mg/L or 100 mg/L.
26
In accordance with the invention, the culture may be run for any length of
time after addition of polyanionic compound. The culture run time may be
determined by one of skill in the art, based on relevant factors such as the quantity
and quality of recoverable protein, and the level of contaminating cellular species
(e.g. proteins and DMA) in the supernatant resulting from cell lysis, which will
complicate recovery of the protein of interest.
In particular embodiments of the cell culturing process and method of
increasing cell viability of the invention, polyanionic compound is added at a time
after innoculation that is before the beginning of the initial death phase.
Preferably, polyanionic compound is added at a time after innoculation that is
during the initial growth phase. More preferably, polyanionic compound is added
during the second half the initial growth phase. More preferably, polyanionic
compound is added on or about the end of the initial growth phase.
The initial growth phase refers to the growth phase that is observed in the
absence of the specified addition of polyanionic compound. The initial death
phase refers to the death phase that is observed in the absence of the specified
addition of polyanionic compound.
The initial growth phase may end when the initial death phase begins, or
there may be a stationary phase of any length between the initial growth phase
and the initial death phase.
In a specific embodiment, in a cell culture in which the initial growth phase
is from day 0 to day 6 and the initial death phase begins on day 7, in a particular
embodiment polyanionic compound is added at a time after innoculation and
before day 7, In a specific embodiment, polyanionic compound is added after
innoculation and by day 6. In a specific embodiment, polyanionic compound is
added between days 1 and 6. In another specific embodiment, polyanionic
compound is added on day 4, 5 or 6. In other specific embodiments, polyanionic
compound is added on about day 6, or on day 6.
It has been found (see Examples 6 and 10), that when polyanionic
compound, is added at a time after innoculation and before the beginning of the
initial death phase, the growth phase may be extended beyond the initial growth
phase. A growth phase that is extended beyond the initial growth phase has a
27
longer duration than the initial growth phase, i.e. longer than the growth phase
observed in the absence of addition of polyanionic compound. Preferably, during
the extended growth phase a higher peak viable cell density is achieved than
peak viable cell density achieved during the initial growth phase.
Thus, in other embodiments, the invention is directed to (1) a cell culturing
process, and (2) a process for extending the growth phase of a cell culture
comprising: culturing host cells which express a protein of interest; and adding
polyanionic compound to the cell culture at a time after innoculation that is before
the beginning of the initial death phase; wherein the growth phase is extended.
In particular embodiments, the invention is directed a (1) a cell culturing process,
and (2) process for extending the growth phase of a cell culture comprising:
culturing host cells which express a protein of interest; and adding polyanionic
compound to the cell culture at a time after innoculation that is during the initial
growth phase; wherein the growth phase is extended In more particular
embodiments the invention is directed to :(1) a cell culturing process, and (2)
process for extending the growth phase of a cell culture comprising: culturing host
cells which express a protein of interest; and adding polyanionic compound to
cell culture during the second half of the initial growth phase; wherein the growth
phase is extended. In other particular embodiments the invention is directed to (1)
a cell culturing process, and (2) a process for extending the growth phase of a cell
culture comprising: culturing host cells which express a protein of interest; and
adding polyanionic compound to the cell culture on or about the end of the initial
growth phase; wherein the growth phase is extended.
The growth phase may be extended for any period of time beyond the
duration of the initial growth phase. By way of example only, the growth phase
may be extended for 1-10 days, for 2-9 days, for 3-8 days, or for about 5 days.
Preferably, the growth phase is extended for one or more days, more preferably
for two or more days, more preferably for three or more days, most preferably for
four or more days. For example, in Example 6 the growth phase is extended to
day 11 where the initial growth phase is until day 6. Thus, in Example 6 the
growth phase has been extended for 5 days beyond the duration of the initial
growth phase. The extended growth phase may be succeeded by a death 28
or by a stationary phase. Likewise, the initial growth phase may be succeeded a death phase or by a stationary phase.
It has been found (see Examples 6 and 10), that when polyanionic
compound is added at a time after innoculation and before the beginning of
initial death phase, the onset of the death phase may be delayed beyond the
onset of the initial death phase, i.e. beyond the onset of the death phase observed
in the absence of the addition of polyanionic compound. A death phase
onset is delayed begins at a later time than the initial death phase.
Thus, in other embodiments, the invention is directed to (1) a cell culturing
process, and (2) a process for delaying the death phase of a cell culture
comprising: culturing host cells which express a protein of interest; and adding
polyanionic compound to the cell culture at a time after innoculation that is before
the beginning of the initial death phase; wherein the onset of the death phase
delayed. In more particular embodiments, the invention is directed to (1) a cell
culturing process, and (2) a process for delaying the death phase of a cell culture
comprising: culturing host cells which express a protein of interest; and
polyanionic compound to the cell culture at a time after innoculation that is during
the initial growth phase; wherein the onset of the death phase is delayed. In more
particular embodiments the invention is directed to (1) a cell culturing process, and
(2) a process for delaying the death phase of a cell culture comprising: culturing
host cells which express a protein of interest; and adding polyanionic compound to
the cell culture during the second half of the initial growth phase; wherein the
onset of the initial death phase is delayed. In other particular embodiments the
invention is directed to a process for delaying the death phase of a cell culture
comprising: culturing host cells which express a protein of interest; and
polyanionic compound to the cell culture on or about the end of the initial
phase; wherein the onset of the death phase is delayed.
The onset of the death phase may be delayed for any period of time. By
way of example only, the onset of the death phase may be delayed for 1-10
for 2-9 days, for 3-8 days, or for about 5 days. Preferably, the onset of the death
phase is delayed for one or more days, more preferably for two or more days,
more preferably for three or more days, most preferably for four or more days.
another particular embodiment of the cell culture process and method of
increasing cell viability of the invention described above, polyanionic compound is
added at a time after innoculation that is during the initial death phase.
It has been found (see Examples 7 and 8) that when polyanionic
compound is added during the initial death phase, the death phase may be
arrested. To arrest the death phase means to stop, for some period of time, the
decline in viable cell density observed in the absence of the addition of polyanionic
compound. The arrest may occur immediately following the addition of the
polyanionic compound, or may occur at a later time. When the death phase is
arrested, what follows may be either a growth phase or a stationary phase.
Eventually, of course, the culture will again enter a death phase.
Thus, in other embodiments, the invention is directed to (1) a cell culturing
process, and (2) a process for arresting the death phase of a cell culture
comprising: culturing host cells which express a protein of interest; and adding
polyanionic compound to the cell culture at a time during the initial death phase;
wherein the death phase is arrested..-.- .
The death phase may be arrested for any period of time before death
phase is re-entered. By way of example only, the death phase may be arrested for1 -20 days, for 2-18 days, for 5-15 days, or for 8-13 days. Preferably, the deathphase is arrested for one or more days, more preferably for n two or more days,more preferably for three or more days, most preferably for four or more days.
Continuity of the arrest of death is not necessarily implied, i.e. there may be "local"
decreases in the viable cell density profile between two stretches of constant or
increasing viable cell density.
Run times of cell culture processes, particularly non-continuous processes,
are usually limited by the remaining viable cell density, which decreases during the
death phase. Longer run times may allow higher product liters to be achieved.
Delaying the death phase, including extending the growth phase, as much as
possible, or arresting the death phase, is therefore desirable. Product quality
concerns also offer a motivation for delaying or arresting the death phase, as cell
death can release sialidases to the culture supernatant, which may reduce the
sialic acid content of the protein expressed. Protein purification concerns offer yet
30
another motivation for delaying or arresting the death phase. The presence of cell
debris and the contents of dead cells in the culture can negatively impact on the
ability to isolate and/or purify the protein product at the end of the culturing run.
In particular embodiments, any of the herein-described cell culture
processes involving two or more temperature shifts and any of the hereindescribed
the cell culture processes involving delayed addition of polyanionic
compound are used together in a cell culture. In particular embodiments, the
invention is directed to (i) a cell culturing process, and (ii) a process for increasing
cell viability, comprising: a) culturing host cells which produce a protein of interest
at a temperature at or near 37°C under conditions and for a time period that allow
for cell growth; b) lowering the temperature of the cell culture and culturing the
cells at a second temperature at or near 34°C starting about day 5 to day 7; (c)
again lowering the temperature of the cell culture and culturing the cells at a third
temperature at or near 32°C starting about day 6 to day 14;and (d) adding
polyanionic compound to the cell culture at a time after innoculation.; :
Techniques and procedures relating to glvcoprotein purification and analysis
In the culturing methods encompassed by the present invention (both the
cell culture methods involving two or more temperature shifts and the cell culture
methods involving delayed addition of polyanionic compound), the protein
produced by the cells is typically collected, recovered, isolated, and/or purified, or
substantially purified, as desired, at the end of the total cell culture period using
isolation and purification methods as known and practiced in the art. Preferably,
glycoprotein that is secreted from the cultured cells is isolated from the culture
medium or supernatant; however, protein can also be recovered from the host
cells, e.g., cell lysates, using methods that are known and practiced in the art, and
as further described below.
The complex carbohydrate comprising the glycoprotein produced by the
processes of this invention can be routinely analyzed, if desired, by conventional
techniques of carbohydrate analysis. For example, techniques such as lectin
blotting, well-known in the art, reveal proportions of terminal mannose, or other
sugars such as galactose. Termination of mono-, bi-, tri-, or tetra-antennary
oligosaccharide by sialic acids can be confirmed by release of sugars from
protein using anhydrous hydrazine or enzymatic methods and fractionation of
oligosaccharides by ion-exchange chromatography, size exclusion
chromatography, or other methods that are well-known in the art.
The pi of the glycoprotein can also be measured, before and after treatment
with neuraminidase, to remove sialic acids. An increase in pi following
neuraminidase treatment indicates the presence of sialic acids on the
glycoprotein. Carbohydrate structures typically occur on the expressed protein core structures. N-linked glycosylation refers to the
attachment of the carbohydrate moiety via GlcNAc to an asparagine residue in the
peptide chain. The N-linked carbohydrates all contain a common Man1-6(Man1-
3)Manp1-4GlcNAcp1-4GlcNAcp-R core structure, where R in this core structure
represents an asparagine residue. The peptide sequence of the protein produced
will contain an asparagine-X-serine, asparagine-X-threonine, and asparagine-Xcysteine,
wherein X is any amino acid except proline.
In contrast, O-linked carbohydrates are characterized by a common core
structure, which is GalNAc attached to the hydroxyl group of a threonine or serine.
Of the N-linked and O-linked carbohydrates, the most important are the complex
N- and O-linked carbohydrates. Such complex carbohydrates contain several
antennary structures. The mono-, bi-, tri,-, and tetra-, outer structures are
important for the addition of terminal sialic acids. Such outer chain structures
provide for additional sites for the specific sugars and linkages that comprise the
carbohydrates of the protein products.
The resulting carbohydrates can be analyzed by any method known in the
art. Several methods are known in the art for glycosylation analysis and are useful
in the context of the present invention. These methods provide information
regarding the identity and the composition of the oligosaccharide attached to the
produced peptide. Methods for carbohydrate analysis useful in connection with
the present invention include, but are not limited to, lectin chromatography;
HPAEC-PAD, which uses high pH anion exchange chromatography to separate
oligosaccharides based on charge; NMR; Mass spectrometry; HPLC; GPC;
monosaccharide compositional analysis; and sequential enzymatic digestion.
In addition, methods for releasing oligosaccharides are known and
practiced in the art. These methods include 1) enzymatic methods, which are
commonly performed using peptide-N-glycosidase F/endo-p-galactosidase; 2) elimination methods, using a harsh alkaline environment to release mainly Olinked
structures; and 3) chemical methods using anhydrous hydrazine to release
both N-and O-linked oligosaccharides. Analysis can be performed using the
following steps: 1. Dialysis of the sample against deionized water to remove all
buffer salts, followed by lyophilization. 2. Release of intact oligosaccharide chains
with anhydrous hydrazine. 3. Treatment of the intact oligosaccharide chains with
anhydrous methanolic HCl to liberate individual monosaccharides as O- derivatives. 4. N-acetylation of any primary amino groups. 5. Derivatization to
yield per-O-trimethylsilyl methyl glycosides. 6. Separation of these derivatives capillary gas-liquid chromatography (GLC) on a CP-SIL8 column. 7. Identification
of individual glycoside derivatives by retention time from the GLC and mass
spectroscopy, compared to known standards. 8. Quantification of individual
derivatives by FID with an internal standard (13-O-methyl-D-gIucose).
Neutral and amino sugars can be determined by high performance anionexchange
chromatography combined with pulsed amperometric detection (HPAEPAD
Carbohydrate System; Dionex Corp.). For instance, sugars can be released
by hydrolysis in 20% (v/v) trifluoroacetic acid at 100°C for 6 hours. Hydrolysates
are then dried by lyophilization or with a Speed-Vac (Savant Instruments).
Residues are then dissolved in 1% sodium acetate trihydrate solution and
analyzed on an HPLC-AS6 column (as described by Anumula et al., 1991, Biochem., 195:269-280).
Alternatively, immunoblot carbohydrate analysis can be performed. In this
procedure protein-bound carbohydrates are detected using a commercial glycan
detection system (Boehringer), which is based on the oxidative immunoblot
procedure described by Haselbeck et al. (1993, Glycoconjugate J., 7:63). The
staining protocol recommended by the manufacturer is followed except that the
protein is transferred to a polyvinylidene difluoride membrane instead of a
nitrocellulose membrane and the blocking buffers contain 5% bovine serum
albumin in 10 mM Tris buffer, pH 7.4, with 0.9% sodium chloride. Detection is
carried out with anti-digoxigenin antibodies linked with an alkaline phosphate
conjugate (Boehringer), 1:1000 dilution in Tris buffered saline using the
phosphatase substrates, 4-nitroblue tetrazolium chloride, 0.03% (w/v) and 5-
bromo-4 chloro-3-indoyl-phosphate 0.03% (w/v) in 100 mM Tris buffer, pH 9.5,
containing 100 mM sodium chloride and 50 mM magnesium chloride. The bands containing carbohydrate are usually visualized in about 10 to 15 Carbohydrate associated with protein can also be analyzed by digestion
with peptide-N-glycosidase F. According to this procedure the residue is
suspended in 14 ul of a buffer containing 0.18% SDS, 18 mM betamercaptoethanol,
90 mM phosphate, 3.6 mM EDTA, at pH 8.6, and heated at
100°C for 3 minutes. After cooling to room temperature, the sample is divided into
two equal parts. One part, which is not treated further, serves as a control. The
other part is adjusted to about 1% NP-40 detergent followed by the addition of 0.2
units of peptide-N-glycosidase F (Boehringer). Both samples are warmed at 37°C
for 2 hours and then analyzed by SDS-polyacrylamide gel electrophoresis.
In addition, the sialic acid content of the glycoprotein product is assessed
by conventional methods. For example, sialic acid can be separately determined
by a direct colorimetric method (Yao et al., 1989, Anal. Biochem., 179:332-preferably using triplicate samples. Another method of sialic acid determination
involves the use of thiobarbaturic acid (TBA), as described by Warren et al., J. Biol. Chem., 234:1971-1975). Yet another method involves high performance
chromatography, such as described by H.K. Ogawa et al., 1993, J.
Chromatography, 612:145-149.
Illustratively, for glycoprotein recovery, isolation and/or purification, the cell
culture medium or cell lysate is centrifuged to remove particulate cells and cell
debris. The desired polypeptide product is isolated or purified away from
contaminating soluble proteins and polypeptides by suitable purification
techniques. The following procedures provide exemplary, yet nonlimiting
purification methods for proteins: separation or fractionation on immunoaffinity ion-exchange columns; ethanol precipitation; reverse phase HPLC;
chromatography on a resin, such as silica, or cation exchange resin, e.g., DEAE;
chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration
using, e.g., Sephadex G-75, Sepharose; protein A sepharose chromatography for
removal of immunoglobulin contaminants; and the like. Other additives, such as
protease inhibitors (e.g., PMSF or proteinase K) can be used to inhibit proteolytic
degradation during purification. It will be understood by the skilled practitioner that
purification methods for a given polypeptide of interest may require modifications
which allow for changes in the polypeptide expressed recombinantly in cell Those purification procedures that can select for carbohydrates and enrich for
sialic acid are particularly preferred, e.g., ion-exchange soft gel chromatography,
or HPLC using cation- or anion-exchange resins, in which the more acidic
fraction(s) is/are collected.
Cells, proteins and cell cultures
In the cell culture processes or methods of this invention (both the cell
culture methods involving two or more temperature shifts and the cell culture
methods involving delayed addition of polyanionic compound), the cells can be
maintained in a variety of cell culture media, i.e., basal culture media, as
conventionally known in the art. For example, the methods are applicable for use
with large volumes of cells maintained in cell culture medium, which can be
supplemented with nutrients and the like. Typically, "cell culturing medium" called "culture medium") is a term that is understood by the practitioner in the and is known to refer to a nutrient solution in which cells, preferably animal or
mammalian cells, are grown and which generally provides at least one or more
components from the following: an energy source (usually in the form of a
carbohydrate such as glucose); all essential amino acids, and generally the twenty
basic amino acids, plus cysteine; vitamins and/or other organic compounds
typically required at low concentrations; lipids or free fatty acids, e.g., linoleic acid;
and trace elements, e.g., inorganic compounds or naturally occurring elements
that are typically required at very low concentrations, usually in the micromolar
range. Cell culture medium can also be supplemented to contain a variety of
optional components, such as hormones and other growth factors, e.g., insulin,
transferrin, epidermal growth factor, serum, and the like; salts, e.g., calcium,
magnesium and phosphate, and buffers, e.g., HEPES; nucleosides and bases,
e.g., adenosine, thymidine, hypoxanthine; and protein and tissue hydrolysates,
e.g., hydrolyzed animal protein (peptone or peptone mixtures, which can be
obtained from animal byproducts, purified gelatin or plant material); antibiotics,
e.g., gentamycin; and cell protective agents, e.g., a Pluronic polyol (Pluronic F68).
Preferred is a cell nutrition medium that is serum-free and free of products or
ingredients of animal origin.
As is appreciated by the practitioner, animal or mammalian cells are
cultured in a medium suitable for the particular cells being cultured and which can
be determined by the person of skill in the art without undue experimentation.
Commercially available media can be utilized and include, for example, Minimal
Essential Medium (MEM, Sigma, St. Louis, MO); Ham's F10 Medium (Sigma);
Dulbecco's Modified Eagles Medium (DMEM, Sigma); RPMI-1640 Medium
(Sigma); HyClone cell culture medium (HyClone, Logan, UT); and chemicallydefined
(CD) media, which are formulated for particular cell types, e.g., CD-CHO
Medium (Invitrogen, Carlsbad, CA). To the foregoing exemplary media can be
added the above-described supplementary components or ingredients, including
optional components, in appropriate concentrations or amounts, as necessary desired, and as would be known and practiced by those having in the art using
routine skill.
In addition, cell culture conditions suitable for the methods of the present
invention are those that are typically employed and known for batch, fed-batch, or
continuous culturing of cells, with attention paid to pH, e.g., about 6.5 to about dissolved oxygen (Oa), e.g., between about 5-90% of air saturation and carbon
dioxide (COa), agitation and humidity, in addition to temperature. As an illustrative,
yet nonlimiting, example, a suitable cell culturing medium for the fed-batch
processes of the present invention comprises a modified CD-CHO Medium
(Invitrogen, Carlsbad, CA), e.g., Example 1. A feeding medium can also be
employed, such as modified eRDF medium (Invitrogen, Carlsbad, CA), e.g.,
Example 1 or Example 7. Preferred is a feeding medium also containing Dgalactose.
Animal cells, mammalian cells, cultured cells, animal or mammalian host
cells, host cells, recombinant cells, recombinant host cells, and the like, are all
terms for the cells that can be cultured according to the processes of this
invention. Such cells are typically cell lines obtained or derived from mammals
and are able to grow and survive when placed in either monolayer culture or
suspension culture in medium containing appropriate nutrients and/or growth
factors. Growth factors and nutrients that are necessary for the growth and
maintenance of particular cell cultures are able to be readily determined
empirically by those having skill in the pertinent art, such as is described, for
example, by Barnes and Sato, (1980, Cell, 22:649); in Mammalian Cell
Ed. J.P. Mather, Plenum Press, NY, 1984; and in U.S. Patent No. 5,721,121.
Numerous types of cells can be cultured according to the methods of the
present invention. The cells are typically animal or mammalian cells that can
express and secrete, or that can be molecularly engineered to express and
secrete, large quantities of a particular protein, more particularly, a glycoprotein of
interest, into the culture medium. It will be understood that the glycoprotein
produced by a host cell can be endogenous or homologous to the host cell.
Alternatively, and preferably, the glycoprotein is heterologous, i.e., foreign, to host cell, for example, a human glycoprotein produced and secreted by a Chinese
hamster ovary (CHO) host cell. Also preferably, mammalian glycoproteins, i.e.,
those originally obtained or derived from a mammalian organism, are attained the methods the present invention and are preferably secreted by the cells into the
culture medium.
Examples of mammalian glycoproteins that can be advantageously
produced by the methods of this invention include, without limitation, cytokines,
cytokine receptors, growth factors (e.g., EGF, HER-2, FGF-oc, FGF-p, TGF-a,
TGF-p, PDGF. IGF-1, IGF-2, NGF, NGF-0); growth factor receptors, including
fusion or chimeric proteins. Other nonlimiting examples include growth hormones
(e.g., human growth hormone, bovine growth hormone); insulin (e.g., insulin A
chain and insulin B chain), proinsulin; erythropoietin (EPO); colony stimulating
factors (e.g., G-CSF, GM-CSF, M-CSF); interleukins (e.g., IL-1 .through IL-12);
vascular endothelial growth factor (VEGF) and its receptor (VEGF-R); interferons
(e.g., IFN-cx, p, or y); tumor necrosis factor (e.g., TNF-a and TNF-p) and their
receptors, TNFR-1 and TNFR-2; thrombopoietin (TPO); thrombin; brain natriuretic
peptide (BMP); clotting factors (e.g., Factor VIM, Factor IX, von Willebrands factor,
and the like); anti-clotting factors; tissue plasminogen activator (TPA), e.g.,
urokinase or human urine or tissue type TPA; follicle stimulating hormone (FSH);
luteinizing hormone (LH); calcitonin; CD proteins (e.g., CDS, CD4, CDS, CD28,
CD19, etc.); CTLA proteins (e.g., CTLA4); T-cell and B-cell receptor proteins; bone
morphogenic proteins (BNPs, e.g., BMP-1, BMP-2, BMP-3, etc.); neurotrophic
factors, e.g., bone derived neurotrophic factor (BDNF); neurotrophins, e.g., 3-6;
renin; rheumatoid factor; RANTES; albumin; relaxin; macrophage inhibitory protein
(e.g., MIP-1, MIP-2); viral proteins or antigens; surface membrane proteins; ion
channel proteins; enzymes; regulatory proteins; antibodies; immunomodulatory
proteins, (e.g., HLA, MHC, the B7 family); homing receptors; transport proteins;
superoxide dismutase (SOD); G-protein coupled receptor proteins (GPCRs);
neuromodulatory proteins; Alzheimer's Disease associated proteins and peptides,
(e.g., A-beta), and others as known in the art. Fusion proteins and polypeptides,
chimeric proteins and polypeptides, as well as fragments or portions, or mutants,
variants, or analogues of any of the aforementioned proteins and polypeptides among the suitable proteins, polypeptides and peptides that can produced by the methods of the present invention.
Nonlimiting examples of animal or mammalian host cells suitable for
harboring, expressing, and producing proteins for subsequent isolation and/or
purification include Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCCCCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet, 12:555-556; Kolkekaret al., 1997, Biochemistry, 36:10901-10909), CHO-K1 Tet-On cell line
(Clontech), CHO designated ECACC 85050302 (CAMR, Salisbury, Wiltshire, UK),
CHO clone 13 (GEIMG, Geneva, IT), CHO clone B (GEIMG, Geneva, IT), CHOK1/
SF designated ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK), RRCHOK1
designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK),
dihydrofolate reductase negative CHO cells (CHO/-DHFR, Urlaub and Chasin,
1980, Proc. Nail. Acad. Sci. USA, 77:4216), and dp12.CHO cells (U.S. Patent No.
5,721,121); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7,
ATCC CRL-1651); human embryonic kidney cells (e.g., 293 cells, or 293 cells
subcloned for growth in suspension culture, Graham et al., 1977, J. Gen. ViroL,
36:59); baby hamster kidney cells (BHK, ATCC CCL-10); monkey kidney cells
38
(CV1, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse sertoli cells (TM4, Mather, 1980, Biol.
Reprod., 23:243-251); human cervical carcinoma cells (HELA, ATCC CCL-2);
canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells
(MMT 060562, ATCC CCL-51); buffalo rat liver cells (BRL 3A, ATCC CRL-1442);TRI cells (Mather, 1982, Annals NYAcad. Sci., 383:44-68); MCR 5 cells; FS4cells. Preferred are CHO cells, particularly, CHO/-DHFR cells.
The cells suitable for culturing in the methods and processes of the present
invention can contain introduced, e.g., via transformation, transfection, infection, or
injection, expression vectors (constructs), such as plasmids and the like, that
harbor coding sequences, or portions thereof, encoding the proteins for expression
and production in the culturing process. Such expression vectors contain the
necessary elements for the transcription and translation of the inserted coding
sequence. Methods which are well known to and practiced by those skilled in theart can be used to construct expression vectors containing sequences encodingthe produced proteins and polypeptides, as well as the appropriate transcriptional
and translational control elements. These methods include in vitro recombinant
DNA techniques, synthetic techniques, and in vivo genetic recombination. Such
techniques are described in J. Sambrook et a!., 1989, Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and in P.M.
Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
Control elements, or regulatory sequences, are those non-translated
regions of the vector, e.g., enhancers, promoters, 5' and 3' untranslated regions,that interact with host cellular proteins to carry out transcription and translation.
Such elements can vary in their strength and specificity. Depending on the vectorsystem and host cell utilized, any number of suitable transcription and translation
elements, including constitutive and inducible promoters, can be used. In
mammalian cell systems, promoters from mammalian genes or from mammalian
viruses are preferred. The constructs for use in protein expression systems aredesigned to contain at least one promoter, an enhancer sequence (optional, for
mammalian expression systems), and other sequences as necessary or required
for proper transcription and regulation of gene expression (e.g., transcriptional
initiation and termination sequences, origin of replication sites, polyadenylation
sequences, e.g., the Bovine Growth Hormone (BGH) poly A sequence).
As will be appreciated by those skilled in the art, the selection of the
appropriate vector, e.g., plasmid, components for proper transcription, expression,
and isolation of proteins produced in eukaryotic (e.g., mammalian) expression
systems is known and routinely determined and practiced by those having skill in
the art. The expression of proteins by the cells cultured in accordance with the
methods of this invention can placed under the control of promoters such as viral
promoters, e.g., cytomegalovirus (CMV), Rous sarcoma virus (RSV),
phosphoglycerol kinase (PGK), thymidine kinase (TK), or the a-actin promoter.
Further, regulated promoters confer inducibility by particular compounds or
molecules, e.g., the glucocorticoid response element (GRE) of mouse mammary
tumor virus (MMTV) is induced by glucocorticoids (V. Chandler et a!., 1983, Cell,. 33:489-499). Also, tissue-specific promoters or regulatory elements can be used(G. Swift et al., 1984, Cell, 38:639-646), if necessary or desired.
Expression constructs can be introduced into cells by a variety of gene
transfer methods known to those skilled in the art, for example, conventional genetransfection methods, such as calcium phosphate co-precipitation, liposomaltransfection, microinjection, electroporation, and infection or viral transduction.
The choice of the method is within the competence of the skilled practitioner in theart. It will be apparent to those skilled in the art that one or more constructs
carrying DMA sequences for expression in cells can be transfected into the cellssuch that expression products are subsequently produced in and/or obtained from
the cells
In a particular aspect, mammalian expression systems containing
appropriate control and regulatory sequences are preferred for use in protein
expressing mammalian cells of the present invention. Commonly used eukaryoticcontrol sequences for use in mammalian expression vectors include promoters
and control sequences compatible with mammalian cells such as, for example, the
cytomegalovirus (CMV) promoter (CDM8 vector) and avian sarcoma virus
(TcLN). Other commonly used promoters include the early and late promoters from
Simian Virus 40 (SV40) (Fiers et al., 1973, Nature, 273:113), or other viral
promoters such as those derived from polyoma, Adenovirus 2, and bovine
papilloma virus. An inducible promoter, such as hMTII (Karin et al., 1982, Nature,299:797-802) can also be used.
Examples of expression vectors suitable for eukaryotic host cells include,
but are not limited to, vectors for mammalian host cells (e.g., BPV-1, pHyg, pRSV,pSV2, pTK2 (Maniatis); pIRES (Clontech); pRc/CMV2, pRc/RSV, pSFV1 (Life
Technologies); pVPakc Vectors, pCMV vectors, pSG5 vectors (Stratagene),
retroviral vectors (e.g., pFB vectors (Stratagene)), pcDNA-3 (Invitrogen),
adenoviral vectors; Adeno-associated virus vectors, baculovirus vectors, yeastvectors (e.g., pESC vectors (Stratagene)), or modified forms of any of the
foregoing. Vectors can also contain enhancer sequences upstream or
downstream of promoter region sequences for optimizing gene expression.
A selectable marker can also be used in a recombinant vector (e.g., a
plasmid) to confer resistance to the cells harboring (preferably, having stably
integrated) the vector to allow their selection in appropriate selection medium. Anumber of selection systems can be used, including but not limited to, the Herpes
Simplex Virus thymidine kinase (HSV TK), (Wigler et al., 1977, Cell, 11:223),
hypoxanthine-guanine phosphoribosyltransferase (HGPRT), (Szybalska and
Szybalski, 1992, Proc. Natl. Acad. Sci. USA, 48:202), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell, 22:817) genes, which can beemployed in tk-, hgprt-, or aprt- cells (APRT), respectively.
Anti-metabolite resistance can also be used as the basis of selection for the
following nonlimiting examples of marker genes: dhfr, which confers resistance tomethotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA, 77:357; and O'Hare
et al., 1981, Proc. Natl. Acad. Sci. USA, 78:1527); gpt, which confers resistance to
mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. .USA,
78:2072); neo, which confers resistance to the aminoglycoside G418 (Clinical
Pharmacy, 12:488-505; Wu and Wu, 1991, Biotherapy, 3:87-95; Tolstoshev, 1993,
Ann. Rev. Pharmacol. Toxicol., 32:573-596; Mulligan, 1993, Science, 260:926-
932; Anderson, 1993, Ann. Rev. Biochem., 62:191^21; May, 1993, TIB TECH,
41
11 (5): 155-215; and hygro, which confers resistance to hygromycin (Santerre et al.,
1984, Gene, 30:147). Methods commonly known in the art of recombinant DNA
technology can be routinely applied to select the desired recombinant cell clones,
and such methods are described, for example, in Ausubel et al. (eds.), Current
Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, 1990,
Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; in
Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics,
John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981. J. Mol. Biol., 150:1,
which are incorporated by reference herein in their entireties.
In addition, the expression levels of an expressed protein molecule can be
increased by vector amplification (for a review, see Bebbington and Hentschel,
"The use of vectors based on gene amplification for the expression of cloned
genes in mammalian cells in DNA cloning", Vol. 3, Academic Press, New York,
1987). When a marker in the vector system expressing a protein is amplifiable, an
increase in the level of inhibitor present in the host cell culture will increase the
number of copies of the marker gene. Since the amplified region is associated
with the protein-encoding gene, production of the protein will concomitantly
increase (Grouse et al., 1983, Mol. Cell. Biol., 3:257).
Vectors which harbor glutamine synthase (GS) or dihydrofolate reductase
(DHFR) encoding nucleic acid as the selectable markers can be amplified in thepresence of the drugs methionine sulphoximine or methotrexate, respectively. An
advantage of glutamine synthase based vectors is the availability of cell lines (e.g.,
the murine myeloma cell line, NSO) which are glutamine synthase negative.
Glutamine synthase expression systems can also function in glutamine synthase
expressing cells (e.g., CHO cells) by providing additional inhibitor to prevent thefuncioning of the endogenous gene.
Vectors that express DHFR as the selectable marker include, but are not
limited to, the pSV2-dhfr plasmid (Subramani et al., Mol. Cell. Biol. 1:854 (1981).
Vectors that express glutamine synthase as the selectable marker include, but are
not limited to, the pEE6 expression vector described in Stephens and Cockett,
1989, Nucl. Acids. Res., 17:7110. A glutamine synthase expression system andcomponents thereof are detailed in PCT publications: W087/04462; W086/05807;
W089/01036; W089/10404; and W091/06657 which are incorporated by reference
herein in their entireties. In addition, glutamine synthase expression vectors that
can be used in accordance with the present invention are commercially available
from suppliers, including, for example, Lonza Biologies, Inc. (Portsmouth, NH).
In a particular embodiment, a nucleic acid sequence encoding a soluble
CTLA4 molecule or a soluble CTLA4 mutant molecule can be inserted into a
vector designed for expressing foreign sequences in a eukaryotic host. The
regulatory elements of the vector can vary according to the particular eukaryotic
host. Vectors which express the soluble CTLA4 or soluble CTLA4 mutant in
eukaryotic host cells can include enhancer sequences for optimizing protein
expression.
Types of cell cultures
For the purposes of understanding, yet without limitation, it will be
appreciated by the skilled practitioner that cell cultures and culturing runs for
protein production can include three general types; namely, continuous culture,
culture medium supplement (i.e., feeding medium) is provided to the cells during
the culturing period, while old culture medium is removed daily and the product is
harvested, for example, daily or continuously. In continuous culture, feeding
medium can be added daily and can be added continuously, i.e., as a drip or
infusion. For continuous culturing, the cells can remain in culture as long as is
desired, so long as the cells remain alive and the environmental and culturing
conditions are maintained.
In batch culture, cells are initially cultured in medium and this medium is
neither removed, replaced, nor supplemented, i.e., the cells are not "fed" with new
medium, during or before the end of the culturing run. The desired product is
harvested at the end of the culturing run.
For fed-batch cultures, the culturing run time is increased by supplementing
the culture medium one or more times daily (or continuously) with fresh medium
during the run, i.e., the cells are "fed" with new medium ("feeding medium") during
the culturing period. Fed-batch cultures can include various feeding regimens and
times, for example, daily, every other day, every two days, etc., more than
per day, or less than once per day, and so on. Further, fed-batch cultures can be
fed continuously with feeding medium.
The desired product is then harvested at the end of the culturing/production
run. The present invention preferably embraces fed-batch cell cultures in which
quality protein production and can extend the protein production phase beyond
that which occurs when no temperature shift is used, or when only one
temperature shift is used. The present invention preferably embraces fed-batch
cell cultures in which polyanionic compound is added at a time after innoculation.
It is also envisioned that in the culturing methods of the present invention,
the feeding medium can be supplemented to contain D-galactose, or D-galactose
can be fed to the culture through some means other than in the feeding medium.
Feeding with galactose-containing feeding medium, or other form of feeding,
preferably occurs on a daily basis (or continuously) during and until the end of the
culturing run, although other feeding schedules can apply. ln;sueh a continuous
feeding regimen including D-galactose, the cultures receive feeding medium,
example, as a continuously-supplied "drip", or infusion, or other automated
addition to the culture, in a timed, regulated, and/or programmed fashion so as to
achieve and maintain the appropriate amount of galactose in the culture. Most
preferred is a feeding regimen comprising a one time per day bolus feed with
feeding medium containing galactose on each day of the culture run, from the
beginning of the culture run to the day of harvesting the cells. In accordance withthe methods of this invention involving feeding with galactose, the D-galactose
concentration in the feeding medium is preferably provided in an amount whichaffords a sustained or maintained level of D-galactose in the culture, or reactor,during the culturing process. An amount of D-galactose suitable for use in the
feeding medium comprises from about 1 g/L to about 50 g/L, preferably about
g/L to about 25 g/L, more preferably about 3 g/L to about 20 g/L. As a specific yet
nonlimiting example, 12.5 g/L of D-galactose in the feeding medium is suitable for
use in the culturing method of the invention, particularly for example, for 50 L
reactor scale. Further, it is preferred that the residual galactose concentration in
the feeding medium used for culturing cells (e.g., in a reactor or culturing vessel) ismaintained and sustained throughout the culturing run in an amount of about 0.1-10 g/L, preferably, about 0.1-5 g/L, more preferably, about 0.2-5 g/L, morepreferably, about 0.2-2.5 g/L, even more preferably, about 0.5-2 g/L, and mostpreferably about 0.5-1.0 g/L. (See, commonly-assigned patent applications U.S.Serial No. 60/436,050, filed December 23, 2002, and U.S. Serial No. 10/ ,filed concomitantly herewith, the contents of which are hereby incorporated byreference herein in their entirety).
In the aspects of the invention involving two or more temperature shifts, the
two or more temperature shifts comprising the cell culture processes of this
invention may result in more viable cells surviving in culture until the end of theprocess or production run. The greater the number of cells that survive, the
greater the amount of protein product that is produced in a non-growth associatedprocess of protein production, such as some of those exemplified herein. Thus, insuch cases, a greater accumulated amount of a desired product results at the end
of the process. According to the present invention, the rate of protein or
glycoprotein production by individual cells in the culture (i.e., cell specific
productivity) is not affected or increased by the temperature shift culturing
processes of the invention, (e.g., see below and Example 4).
According to the present invention, cell culture can be carried out, and
glycoproteins can be produced by cells, under conditions for the large or small
scale production of proteins, using culture vessels and/or culture apparatuses thatare conventionally employed for animal or mammalian cell culture. As is
appreciated by those having skill in the art, tissue culture dishes, T-flasks and
spinner flasks are typically used on a laboratory scale. For culturing on a largerscale, (e.g., 500 L, 5000 L, and the like, for example, as described in commonlyassignedpatent application U.S. Serial No. 60/436,050, filed December 23, 2002,and U.S. Serial No. 10/ , filed concomitantly herewith, the contents of
which are incorporated by reference herein in their entirety) procedures including,but not limited to, a fluidized bed bioreactor, a hollow fiber bioreactor, roller bottleculture, or stirred tank bioreactor systems can be used. Microcarriers may or maynot be used with the roller bottle or stirred tank bioreactor systems. The systems
can be operated in a batch, continuous, or fed-batch mode. In addition, the
culture apparatus or system may or may not be equipped with a cell separator
using filters, gravity, centrifugal force, and the like.
Phases of cell culture and associated parameters
The term "innoculation" refers to the addition of cells to starting medium to
begin the culture.The growth phase of a culture is the phase during which the viable celldensity at any time point is higher than at any previous time point.
The stationary phase of a culture is the phase during which the viable cell
density is approximately constant (i.e. within measuring error) over a time period ofany length.The death phase of a culture is the phase that comes after the growth
phase or after the growth phase and the stationary phase, and during which theviable cell density at any time point is lower than at any previous time point duringthat phase
In a growth-associated culture process,
such as cases where a polyanionic compound causes an extended growth phase,the production phase may start during the extended growth phase.
In a non-growth associated culture process, the production phase of cell
culture may be the stationary phase.
Preferably, the culture medium is supplemented ("fed") during the
production phase to support continued protein production, particularly in an
extended production phase, and to attain ample quantities of high quality
glycoprotein product (as exemplified and/or determined by a high level of end
sialic acid content upon protein recovery). Feeding can occur on a daily basis, oraccording to other schedules to support cell viability and protein production.
During an extended production phase at temperatures which are shifted to
be successively lower than the temperature(s) at the growth and standard (initial)production phases, the cells are fed and remain viable. This results in theproduction of desired protein product for an extended or longer total period of timethan occurs at the initial culturing temperature, or when the temperature is shifted
from the initial culturing temperature only one time. The culturing process
according to the present invention may result in more viable cell survival until theend of the culturing period. Accordingly, in some embodiments, the more cells
that survive, the more cells that are producing the desired product. This, in turn,results in a greater accumulated amount of the product at the end of the culturingprocess, with the rate of protein production by individual cells, i.e., cell specificproductivity, remaining the same. (See, e.g., Example 4).Cell specific productivity
or cell specific rate, as known in the art, typically refers to the specific expressionrate of product produced per cell, or per measure of cell mass or volume. Cellspecific productivity is measured in grams of protein produced per cell per day, forexample, and can be measured according to an integral method involving the
following formulae:
where qp is the cell specific productivity constant; X is the number of cells or cellvolume, or cell mass equivalents; and dP/dt is the rate of protein production. Thus,
qp can be obtained from a plot of product concentration versus time integral of
viable cells (J0 Xdt "viable cell days"). According to this formula, when the
amount of glycoprotein product produced is plotted against the viable cell days,the slope is equivalent to the cell specific rate. Viable cells can be determined byseveral measures, for example, biomass, O2 uptake rate, lactase dehydrogenase
(LDH), packed cell volume or turbidity, (e.g., U.S. Patent No. 5,705,364 to T.
Etcheverry et al.)
Production of soluble CTLA4 molecules and soluble CTLA4 mutant molecules bvthe culturinq methods of the present invention
In other embodiments encompassed by the present invention, the cell
culture methods of the invention (both those involving two or more temperature
shifts and involving delayed addition of polyanionic compound) are utilized to
produce a soluble CTLA4 molecule or a soluble CTLA4 mutant molecule, as
described below. A soluble CTLA4 molecule is preferably a CTLA4 fusion protein,
preferably a CTLA4lg. More preferred is CTLA4lg that comprises amino acids
to 357 or +1 to 357 as shown in FIG. 3. Most preferred is CTLA4lg that consists
of amino acids -1 to 357 or +1 to 357 as shown in FIG. 3. A soluble CTLA4
mutant molecule is preferably L104EA29Ylg that comprises amino acids -1 to
or +1 to 357 as shown in FIG. 4, most preferably that consists of amino acids
357 or +1 to 357 as shown in FIG. 4. The two- and three-step temperature shiftcell culture methods involving extended production phases for protein product areespecially suitable for generating high quality and large amounts of soluble CTLA4molecules and soluble CTLA4 mutant molecules, by their host cells in culture.In a preferred embodiment, CTLA4lg is produced by recombinantly
engineered host cells. The CTLA4lg fusion protein can be recombinantly
produced by CHO cells transfected with a vector containing the DMA sequenceencoding CTLA4lg. (See, U.S. Patent No. 5,844,095 to P.S. Linsley et al., and
Example 2 herein). The CTLA4lg fusion protein is produced in high quantity and isappropriately sialylated when cultured in accordance with the multi-step
temperature shift processes of this invention. The invention affords the productionof high levels of recoverable protein product, e.g., sialylated CTLA4lg protein
product. In another preferred embodiment, the soluble CTLA4 mutant moleculeL104EA29Ylg that comprises amino acids -1 to 357 or +1 to 357 as shown in FIG.
4 is produced by the cell culture methods of the present invention.
A ligand for CTLA4 is a B7 molecule. As used herein, "ligand" refers to a
molecule that specifically recognizes and binds another molecule. The interaction
of a molecule and its ligand can be regulated by the products of the culturing
processes of this invention. For example, CTLA4 interaction with its ligand B7 canbe blocked by the administration of CTLA4lg molecules. As other examples, theinteraction of Tumor Necrosis Factor (TNF) with its ligand, TNF receptor (TNFR),
can be blocked by administration of etanercept or other TNF/TNFR blocking
molecules.
Wild type CTLA4 or "non-mutated CTLA4" has the amino acid sequence of
naturally occurring, full length CTLA4 as shown in FIG. 5 (and also as described in
U.S. Patent Nos. 5,434,131, 5,844,095, and 5,851,795, incorporated herein byreference in their entirety), or any portion thereof that recognizes and binds a
molecule, or interferes with a B7 molecule, so that binding to CD28 and/or CTLA4
(e.g., endogenous CD28 and/or CTLA4) is blocked. Wild type CTLA4 comprises
particular portions, including, for example, the extracellular domain of wild typeCTLA4 beginning with methionine at position +1 and ending at aspartic acid atposition +124, or the extracellular domain of wild type CTLA4 beginning withalanine at position -1 and ending at aspartic acid at position +124 as
The naturally occurring wild type CTLA4 is a cell surface protein having an
N-terminal extracellular domain, a transmembrane domain, and a C-terminal
cytoplasmic domain. The extracellular domain binds to a target molecule, such as
a B7 molecule. In a cell, the naturally occurring, wild type CTLA4 protein is
translated as an immature polypeptide, which includes a signal peptide at the
amino, or N-terminal, end. The immature polypeptide undergoes posttranslational
processing, which includes cleavage and removal of the signal
peptide to generate a CTLA4 cleavage product having a newly generated Nterminal
end that differs from the N-terminal end in the immature form. One skilled
in the art will appreciate that additional post-translational processing may occur,which removes one or more of the amino acids from the newly generated Nterminal
end of the CTLA4 cleavage product. The mature CTLA4 protein may
start at methionine at position +1 or alanine at position -1. The mature form of the
CTLA4 molecule includes the extracellular domain or any portion thereof, which
binds to B7.
A CTLA4 mutant molecule, as used herein, refers to a molecule comprising
wild type CTLA4 as shown in FIG. 5, or any portion or derivative thereof, that has
a mutation, or multiple mutations, in the wild type CTLA4 sequence, preferably in
the extracellular domain of wild type CTLA4, and binds B7. A CTLA4 mutant
molecule has a sequence that it is similar, but not identical, to the sequence ofwild type CTLA4 molecule, but still binds B7. The mutations can include one ormore amino acid residues substituted with an amino acid having conservative (e.g.,
a leucine substituted for an isoleucine) or non-conservative (e.g., a glycine
substituted with a tryptophan) structure or chemical properties, amino acid deletions,
additions, f rameshifts, or truncations.
CTLA4 mutant molecules can include a non-CTLA4 molecule therein or
attached thereto, i.e., CTLA4 mutant fusion proteins. The mutant molecules canbe soluble (i.e., circulating) or they can be bound to a cell surface (membranebound).
CTLA4 mutant molecules include L104EA29Ylg and those described in
U.S. Patent Application Serial Numbers 09/865,321, 60/214,065 and 60/287,576;in WO 01/92337 A2; in U.S. Patent Numbers 6,090,914, 5,844,095 and
5,773,253; and as described in R.J. Peach et al., 1994, J Exp Med, 180:2049-
2058. CTLA4 mutant molecules can be synthetically or recombinantly produced.CTLA4lg is a soluble fusion protein comprising an extracellular domain of
wild type CTLA4, or a portion thereof that binds B7, joined to an immunoglobulin
(Ig) molecule, or a portion thereof. The extracellular domain of CTLA4 or portionthereof is joined to an Ig moiety comprising all or a portion of an immunoglobulin
molecule, preferably all or a portion of an immunoglobulin constant region such asall or a portion of IgCyl (IgCgammal), lgGy2 (lgCgamma2), IgCyS (IgCgammaS),
1gCy4 (lgCgamma4), IgCu. (IgCmu), IgCat (IgCalphal), lgCo2 (lgCalpha2), lgC8(IgCdelta) or IgCe (IgCepsilon), rendering the fusion molecule soluble. The Igmoiety can include the hinge, CH2 and CHS domains, or the CH1, hinge, CH2 and
CHS domains, of the aforementioned constant regions or other constant regions.
Preferably, the Ig moiety is human or monkey and comprises the hinge, CH2 andCHS domains. Most preferably the Ig moiety comprises the hinge, CH2 and CHSdomains of human IgCyl, or consists of the hinge, CH2 and CHS domains of
human IgCyl. In an Ig moiety of CTLA4lg, the Ig constant region or portion
thereof can be mutated, thus resulting in a reduction of its effector functions (see,e.g., U.S. Patent Nos. 5,637,481, 5,844,095 and 5,434,131). As used herein, theterms Ig moiety, Ig constant region, Ig C(constant) domain, IgCyl (IgCgammal),
lgCy2 (lgCgamma2), IgCyS (IgCgammaS), lgCy4 (lgCgamma4), lgC|i (IgCmu),IgCal (IgCalphal), lgCo2 (lgCalpha2), lgC5 (IgCdelta) or IgCe (IgCepsilon),
include both native sequences and sequences that have been mutated, such
for example, sequences having mutations in the constant region that reduce
effector function.A particular embodiment related to CTLA4 comprises the extracellular
domain of wild type CTLA4 starting at methionine at position +1 and ending at
aspartic acid at position +124, or starting at alanine at position -1 to aspartic acidat position +124; a junction amino acid residue glutamine at position +125; and animmunoglobulin portion encompassing glutamic acid at position +126 through
lysine at position +357, as shown in FIG. 3. DNA encoding this CTLA4lg was
deposited on May 31,1991, in the American Type Culture Collection (ATCC),
10801 University Blvd., Manassas, VA 20110-2209, under the provisions of theBudapest Treaty, and has been accorded ATCC accession number ATCC 68629;
P. Linsley et al., 1994, Immunity 1:793-80. A CHO cell line expressing CTLA4lgwas deposited on May 31, 1991 in ATCC under identification number CRL-10762.
The soluble CTLA4lg molecules produced according to the methods describedherein may or may not include a signal (leader) peptide sequence. FIGS. 3 and 4include an illustration of a signal (leader) peptide sequence. Typically, themolecules do not include a signal peptide sequence.
L104EA29Ylg is a fusion protein that is a soluble CTLA4 mutant molecule
comprising an extracellular domain of wild type CTLA4 with amino acid changesA29Y (a tyrosine amino acid residue substituting for an alanine at position 29) andL104E'(a glutamic acid amino acid residue substituting for a leucine at position+104) joined to an Ig tail. FIG. 4 illustrates L104EA29Ylg. The amino acidsequence of L104EA29Ylg comprises alanine at amino acid position -1 to lysine atamino acid position +357 as shown in FIG. 4. Alternatively, the amino acid
sequence of L104EA29Ylg comprises methionine at amino acid position +1 to
lysine at amino acid position +357 as shown in FIG. 4. L104EA29Ylg comprises ajunction amino acid residue glutamine at position +125 and an Ig portion
encompassing glutamic acid at position +126 through lysine at position +357.
DNA encoding L104EA29Ylg was deposited on June 20, 2000, in the AmericanType Culture Collection (ATCC) under the provisions of the Budapest Treaty, andhas been accorded ATCC accession number PTA-2104. 104EA29Y-lg isdescribed in co-pending U.S. Patent Application Serial Numbers 09/579,927,60/287,576 and 60/214,065, and in WO/01/923337 A2, which are incorporated by
reference herein in their entireties. The soluble L104EA29Ylg molecules
produced by the culturing methods of this invention may or may not include a
signal (leader) peptide sequence. Typically, the molecules produced according to
the invention do not include a signal peptide sequence.
As used herein, the term soluble refers to any molecule, or fragment
thereof, not bound or attached to a cell, i.e., circulating. For example, CTLA4, B7or CD28 can be made soluble by attaching an Ig moiety to the extracellular
domain of CTLA4, B7 or CD28, respectively. Alternatively, a molecule such as
CTLA4 can be rendered soluble by removing its transmembrane domain.
Typically, the soluble molecules produced according to the invention do not
include a signal (or leader) sequence.
A soluble CTLA4 molecule refers to a non-cell-surface-bound (i.e.,
circulating) molecule comprising wild type CTLA4, or any portion or derivative thatbinds B7, including, but not limited to, soluble CTLA4 fusion proteins; solubleCTLA4 fsion proteins such as CTLA4lg fusion proteins (e.g., ATCC 68629),wherein the extracellular domain of CTLA4 is fused to an Ig moiety that is all or aportion of an Ig molecule, preferably all or a portion of an Ig constant region, suchas all or a portion of IgCyl (IgCgammal), lgOy2 (lgCgamma2), IgCyS
(IgCgammaS), lgCy4 lgCgamma4), IgCji (IgCmu), IgCcd (IgCalphal), lgCo2
(lgCalpha2), lgC8 (IgCdelta) or IgCe (IgCepsilon), rendering the fusion moleculesoluble; soluble CTLA4 fusion proteins in which the extracellular domain is fusedor joined with a portion of a biologically active or chemically active protein such asthe papillomavirus E7 gene product (CTLA4-E7), melanoma-associated antigen
p97 (CTLA4-p97) or HIV env protein (CTLA4-env gp120), as described in U.S.Patent No. 5,844,095, herein incorporated by reference in its entirety; hybrid
(chimeric) fusion proteins such as CD28/CTLA4lg as described in U.S. Patent No.
5,434,131, herein incorporated by reference in its entirety; CTLA4 molecules withthe transmembrane domain removed to render the protein soluble (See, e.g., M.K.
Oaks et al., 2000, Cellular Immunology, 201:144-153, herein incorporated by
reference in its entirety); the soluble CTLA4 mutant molecule L104EA29Ylg.
A soluble CTLA4 molecule can also be a soluble CTLA4 mutant molecule.
The soluble CTLA4 molecules produced according to this invention may or maynot include a signal (leader) peptide sequence. The signal peptide can be any
sequence that will permit secretion of the molecule, including the signal peptidefrom oncostatin M (Malik et al., 1989, Molec. Cell. Biol., 9:2847-2853), or CDS(N.H. Jones et al., 1986, Nature, 323:346-349), or the signal peptide from any
extracellular protein. The soluble CTLA4 molecule produced by the culturing
processes of the invention can include the oncostatin M signal peptide linked atthe N-terminal end of the extracellular domain of CTLA4. Typically, in the inventionthe molecules do not include a signal peptide sequence.
CTLA4 fusion protein as used herein refers to a molecule comprising the
extracellular domain of wild type CTLA4, or portion thereof that binds to B7, fused toa non-CTLA4 moiety that renders the CTLA4 molecule soluble, such as an Igmoiety. For example, a CTLA4 fusion protein can include the extracellular domain
of CTLA4 fused to all or a portion of an Ig constant region. Examples of Ig constantdomains (or portions thereof) that may be fused to CTLA4 include all, but are notlimited to those listed hereinabove. A CTLA4 fusion protein can also be a CTLA4mutant molecule.
As used herein, "non-CTLA4 moiety" refers to a molecule or portion thereof
that does not bind CD80 and/or CD86 and does not interfere with the binding of
CTLA4 to its ligand. Examples include, but are not limited to, an Ig moiety that is
all or a portion of an Ig molecule, a portion of a biologically active or chemically
active protein such as the papillomavirus E7 gene product (CTLA4-E7),
melanoma-associated antigen p97 (CTLA4-p97) or HIV env protein (CTLA4-envgp120) (as described in U.S. Serial No. 5,844,095, herein incorporated by
reference in its entirety). Examples of Ig moieties include all or a portion of an
immunoglobulin constant domain, such as IgCyl (IgCgammal), lgCy2
(lgCgamma2), IgCyS (IgCgammaS), lgCy4 lgCgamma4), IgCu, (IgCmu), IgCal
(IgCalphal), lgCo2 (lgCalpha2), lgC8 (IgCdelta) or IgCe (IgCepsilon). The Ig
moiety can include the hinge, CH2 and CHS domains, or the CH1, hinge, CH2 andCHS domains of the aforementioned constant regions or other constant regions.
Preferably, the Ig moiety is human or monkey and includes the hinge, GH2 andCHS domains. Most preferably the Ig moiety includes the hinge, CH2 and CHSdomains of human IgCyl, or is the hinge, CH2 and CHS domains of human IgCyl.In an Ig moiety, the Ig constant region or portion thereof can be mutated so as to
reduce its effector functions (see, e.g., U.S. Patent Nos. 5,637,481, 5,844,095 and
5,434,131).The extracellular domain of CTLA4 refers to any portion of wild type CTLA4
that recognizes and binds B7. For example, an extracellular domain of CTLA4
comprises methionine at position +1 to aspartic acid at position +124 (FIG. 5). Forexample, an extracellular domain of CTLA4 comprises alanine at position -1 toaspartic acid at position +124 (FIG. 5).
As used herein, the term mutation refers to a change in the nucleotide or
amino acid sequence of a wild type molecule, for example, a change in the DMA
and/or amino acid sequences of the wild type CTLA4 extracellular domain. A
mutation in the DMA may change a codon leading to a change in the encoded
amino acid sequence. A DMA change may include substitutions, deletions,
insertions, alternative splicing, or truncations. An amino acid change may includesubstitutions, deletions, insertions, additions, truncations, or processing orcleavage errors of the protein. Alternatively, mutations in a nucleotide sequencemay result in a silent mutation in the amino acid sequence, as is we'll understoodin the art. As is also understood, certain nucleotide codons encode the same
amino acid. Examples include nucleotide codons CGU, CGG, CGC, and CGA
which encode the amino acid, arginine (R); or codons GAU, and GAG which
encode the amino acid, aspartic acid (D).
Thus, a protein can be encoded by one or more nucleic acid molecules that
differ in their specific nucleotide sequence, but still encode protein molecules
having identical sequences. The mutant molecule may have one, or more thanone, mutation. For guidance, the amino acid coding sequence is as follows:
As used herein, a fragment or portion is any part or segment of a molecule.
For CTLA4 or CD28, a fragment or portion is preferably the extracellular domain of
CTLA4 or CD28, or a part or segment thereof, that recognizes and binds B7 or
interferes with a B7 so that it blocks binding to CD28 and/or CTLA4. Also, as used
herein, "corresponding" means sharing sequence identity.
B7, as used herein, refers to any member of the B7 family of molecules
including, but not limited to, B7-1 (CD80) (Freeman et al., 1989, J Immunol.,
143:2714-2722, herein incorporated by reference in its entirety), B7-2 (CD86)
, (Freeman et al., 1993, Science, 262:909-911, herein incorporated by .reference in.
its entirety; Azuma et al., 1993, Nature, 366:76-79, herein incorporated by
reference in its entirety) that recognizes and binds CTLA4 and/or CD28. CD28
refers to the molecule that recognizes and binds B7 as described in U.S. Serial No.
5,580,756 and 5,521,288 (herein incorporated by reference in their entireties). As
used herein, B7-positive cells include any cells with one or more types of B7
molecules expressed on the cell surface.
As used herein, a "derivative" is a molecule that shares sequence similarity
and activity of its parent molecule. For example, a derivative of CTLA4 includes a
soluble CTLA4 molecule having an amino acid sequence at least 70% similar to the
extracellular domain of wildtype CTLA4, and which recognizes and binds B7 e.g.
CTLA4lg or soluble CTLA4 mutant molecule L104EA29Ylg. A derivative means any
change to the amino acid sequence, and/or chemical quality of the amino acid e.g..,
amino acid analogs.
As used herein, to regulate an immune response is to activate, stimulate, upregulate,
inhibit, block, reduce, attenuate, down-regulate or modify the immune
response. A variety of diseases, e.g., autoimmune diseases, may be treated
regulating an immune response, e.g., by regulating functional CTLA4- and/or
CD28- positive cell interactions with By-positive cells. For example, a method ofregulating an immune response comprises contacting B7-positive cells with a
soluble CTLA4 molecule, such as those produced according to this invention, toform soluble CTLA4/B7 complexes, wherein the soluble CTLA4 molecule
interferes with the reaction of an endogenous CTLA4 and/or CD28 molecule with
the B7 molecule. To "block" or "inhibit" a receptor, signal or molecule, as referred
to herein, means to interfere with the activation of the receptor, signal or molecule,
as detected by an art-recognized test. Blockage or inhibition can be partial or
total.As used herein, "blocking B7 interaction" refers to interfering with the
binding of B7 to its ligands, such as CD28 and/or CTLA4, thereby obstructing Tcelland B7-positive cell interactions. Examples of agents that block B7
interactions include, but are not limited to, molecules such as an antibody (or
portion thereof) that recognizes and binds to the any of CTLA4, CD28 or B7
molecules (e.g., B7-1, B7-2); a soluble form (or portion thereof) of the moleculessuch as soluble CTLA4; a peptide fragment or other small molecule designed to
interfere with the cell signal through a CTLA4/CD28/B7-mediated interaction. In apreferred embodiment, the blocking agent is a soluble CTLA4 molecule, such asCTLA4lg (ATCC 68629) or L104EA29Ylg (ATCC PTA-2104); a soluble CD28
molecule, such as CD28lg (ATCC 68628); a soluble B7 molecule, such as B7-lg(ATCC 68627); an anti-B7 monoclonal antibody (e.g., ATCC HB-253, ATCC CRL-2223, ATCC CRL-2226, ATCC HB-301, ATCC HB-11341 and monoclonal
antibodies as described in U.S. Patent No. 6,113,898 or in Yokochi et al., 1982, J.Immunol., 128(2):823-827); an anti-CTLA4 monoclonal antibody (e.g., ATCC HB-304, and monoclonal antibodies as described in references 82-83); and/or an anti-CD28 monoclonal antibody (e.g. ATCC HB 11944 and MAb 9.3, as described inHansen et al., 1980, Immunogenetics, 10: 247-260, or Martin et al., 1984, J. Clin.
Immunol., 4(1 ):18-22). Blocking B7 interactions can be detected by art-recognized
tests such as determining reduction of immune disease (e.g., rheumatic disease)
associated symptoms, by determining a reduction in T-cell/B7-cell interactions, or
by determining a reduction in the interaction of B7 with CTLA4/CD28. Blockagecan be partial or total.
As used herein, an effective amount of a molecule refers to an amount that
blocks the interaction of the molecule with its ligand. For example, an effective
amount of a molecule that blocks the interaction of B7 with CTLA4 and/or CD28 isthe amount of the molecule that, when bound to B7 molecules on 67-positivecells, inhibits B7 molecules from binding endogenous ligands such as CTLA4 andCD28. Alternatively, an effective amount of a molecule that blocks the interactionof B7 with CTLA4 and/or CD28 is the amount of the molecule that, when bound toCTLA4 and/or CD28 molecules on T cells, inhibits B7 molecules from binding
endogenous ligands such as CTLA4 and CD28. The inhibition or blockage can bepartial or complete.For clinical protocols, it is preferred that the Ig moiety of a fusion protein,
such as CTLA4lg or mutant CTLA4lg, does not elicit a detrimental immune
response in a subject. The preferred moiety is all or a portion of the Ig constant
region, including human or non-human primate Ig constant regions. Examples ofsuitable Ig regions include IgCyl (IgCgammal), lgCy2 (lgCgamma2), IgOyS
(IgCgammaS), lgCy4 lgCgamma4), IgCu. (IgCmu), IgCexl (IgCalphat), lgCo2
(lgCalpha2), lgC8 (IgCdelta) or IgCe (IgCepsilon), including the hinge, CH2 andCHS domains, or the CH1, hinge, CH2 and CHS domains, which are involved in
effector functions such as binding to Fc receptors, complement-dependent
cytotoxicity (CDC), or antibody-dependent cell-mediated cytotoxicity (ADCC). The
Ig moiety can have one or more mutations therein, (e.g., in the CH2 domain to
reduce effector functions such as CDC or ADCC) where the mutation modulates
the capability of the Ig to bind its ligand by increasing or decreasing the capability
of the Ig to bind to Fc receptors. For example, mutations in the Ig moiety can
include changes in any or all of its cysteine residues within the hinge domain. Forexample, as shown in FIG. 3, the cysteines at positions +130, +136, and +139 aresubstituted with serine. The Ig moiety can also include the prolihe at position +148substituted with a serine, as shown in FIG. 3. Further, mutations in the Ig moiety
can include having the leucine at position +144 substituted with phenylalanine;
leucine at position +145 substituted with glutamic acid; or glycine at position +147substituted with alanine.
EXAMPLESThe following Examples set forth specific aspects of the invention to
illustrate the invention and provide a description of the present methods for those
of skill in the art. The Examples should not be construed as limiting the invention,
as the Examples merely provide specific methodology and exemplification that areusefl in the understanding and practice of the invention and its various aspects.
Examples 1-4 as set forth below describe experiments relating to cell
culture processes involving temperature shifts during the culture run. Examples
11 describe experiments relating to cell culture processes involving delayed
addition of polyanionic compound to the culture.
EXAMPLE 1
This Example provides materials and reagents employed in the processes
of the present invention for the culturing of recombinant cells that produce the
exemplified CTLA4lg fusion proteins as described herein in Examples 2-4.
1. Cell Culture Medium
The basal cell culture medium used for all phases of cell inoculum
generation and for growth of cultures in bioreactors, including 5 liter (5 L) and 50
liter (50 L) production reactors, was modified CD-CHO medium containing
glutamine, sodium bicarbonate, insulin and methotrexate (Invitrogen, Carlsbad,
CA), as exemplified in Table 1. The pH of the medium was adjusted to 7.0 with 1
Table 1
Modified CD-CHO
Medium
For feeding cells in the fed-batch process, a modified feed medium, i.e.,
eRDF-1 medium (Invitrogen), containing glucose, glutamine, insulin and TC
Yeastolate (Becton-Dickinson, Franklin Lakes, NJ) was employed, as shown in
Table 2. The pH of the feeding medium was adjusted to 7.0 with 1 N NaOH after
the addition of all components.
Table 2Modified eRDF Medium2. Production Phase in Bioreactor
The production bioreactor was initially operated as a batch reactor, with
temperature, pressure, pH and dissolved oxygen concentration closely monitored
and controlled. The condition of the culture was evaluated by measuring the
viable cell density and the concentration of several key metabolites. The feeding
process was initiated one day after the inoculation. The remainder of the
fermentation was the conducted in fed-batch mode.
Bioreactors of 5 L scale (glass reactor with one marine impeller), and 50 L
scale (stainless steel reactor with two marine impellers) were used, (see Example
2). A data acquisition system (Intellution Fix 32) recorded temperature, pH, and
dissolved oxygen (DO) throughout runs. Gas flows were controlled via rotameters.
Air was sparged into the reactor via a submerged frit (5 um pore size) and through
the reactor head space for CO2 removal. Molecular oxygen was sparged through
same frit for DO control. CO2 was sparged through same frit as used for pH
control.
3. Feeding Strategy
At 24 hours post inoculation, a daily minimum of 1% of culture volume of
modified eRDF-l feed medium was added into the bioreactor if the glucose
concentration was > 3.0 g/L In cases in which the glucose concentration was
below 3 g/L, the volume of the daily bolus feed was calculated to bring the glucose
concentration back up to 3.0 g/L. The daily feed amount was recorded on batch
sheets.
4. Sampling
Samples of cells were removed from the reactor on a daily basis. A sample
used for cell counting was stained with trypan blue (Sigma, St. Louis, MO). Cell
count and cell viability determination were performed via hemocytometry using a
microscope. For analysis of metabolites, additional sample was centrifuged for 20
minutes at 2000 rpm (4°C) for cell separation. Supernatant was analyzed for the
following parameters: titer, sialic acid, glucose, lactate, glutamine, glutamate, pH,
pCOs, ammonia, and, optionally, lactate dehydrogenase (LDH). Additional
back-up samples were frozen at -20°C.EXAMPLE 2
This Example describes the production of CTLA4lg, shown as -1 to 357 or
+1 to 357 in FIG. 3, (encoding DNA deposited as ATCC 68629), from cultured
CHO cells.
This Example also describes a process of this invention for producing both
high quantity and high quality CTLA4lg protein, involving culture runs having twoor
three-step temperature shifts and total run times of 14, 21, or 28-30 days. A
temperature shift (T-shift) from 37°C to 34°C occurred on day 6 (end of logarithmic
growth phase) and a second T-shift from 34°C to 32°C occurred on day 10. The
run was ended on day 14, day 21, or day 28, and for the two-step shift, the
temperature was controlled at 32°C from the shift on day 10 until the end of the
run. For the three-step shift, the temperature was controlled at 30°C from the day
of the shift until the end of the run The processes described resulted in increased
end liter of protein product, increased end cell viability, and volumetric productivity,
compared with single temperature shift or no temperature shift runs. In
accordance with the invention, the second and third T-shifts extended the run time
of the standard fermentation (culturing) process to two to three weeks (or longer),
while maintaining high cell viabilities. A close to linear increase of the titer of
product was observed throughout the production period.
CHO cells used for CTLA4lg expression were expanded in modified CDCHO
medium containing glutamine, sodium bicarbonate, insulin, and methotrexate
(see Example 1) using T-75 flasks (Coming, Corning, NY) and 250 and 500 mL
spinners (Bellco, Vineland, NJ). T-flasks and spinners were incubated at 37°C in
6% CO2. After sufficient inoculum was generated, the culture was transferred into
a either a 5 L (Applikon, Foster City, CA) or a 50 L bioreactor (Feldmeier,
Syracuse, NY) with 3 L or 30 L working volume, respectively, of the abovedescribed
medium. The initial seeding density was about 2 x 105 viable cells/mL
The 5 L vessel was a glass reactor equipped with one marine impeller
(Applikon, Foster City, CA); the 50 L vessel was a stainless steel reactor
(Feldmeier, Syracuse, NY) equipped with two marine impellers. A data acquisition
system using Intellution Fix32 (Intellution, Foxboro, MA) recorded temperature,
pH, and dissolved oxygen (DO) throughout runs. Gas flows were controlled via
rotameters (Cole Farmer, Vernon Hills, IL). Air was sparged into the reactor via a
submerged frit (5 um pore size) and through the reactor head space for COa
removal. Molecular oxygen was sparged through same frit for DO control. COa
was sparged through the same frit for high side pH control. Low side pH control
was realized by addition of 1 N NaOH. Without limitation, acceptable ranges for
pH were 6-9, preferably 6.8-7.2, and for osmolarity were 200-500 mOsm,
preferably, 280-340 mOsm.
The culture in the bioreactor was given a daily bolus feed using modified
eRDF medium (Invitrogen) as described in Example 1, Table 2, as follows:
starting one day post inoculation, a minimum of 1% culture volume was added as
feeding medium; if the glucose level fell below 3 g/L, a calculated volume was
added to bring the glucose level back to 3 g/L
The fermentation process had a duration of 21 days at 5 L scale and 28
days at 50 L scale. The longer duration of the culture run at 50 L scale correlated
with the added temperature shift for that run. Samples were taken on a daily basis
from the reactor for analysis. For example, sample used for cell count was stained
with trypan blue (Sigma, St. Louis, MO). Cell count and cell viability determination
was performed using a hemocytometer and counting viable stained cells under a
microscope. For analysis of metabolites, an additional sample aliquot was
centrifuged for 20 minutes at 2000 rpm (4°C) to pellet the cells. The supernatant
was analyzed for protein titer, sialic acid, glucose, lactate, glutamine, glutamate,
pH, pOa, pCO2, ammonia, and LDH, using techniques and protocols
conventionally practiced in the art.
EXAMPLE 3
This Example describes and presents the results of comparative
evaluations to assess various culturing procedures, including the multi-step
culturing methods performed in accordance with the present invention. The end
titer (in g/L) of glycoprotein product was determined, as were the end titer sialic
acid content of the protein, the cell viability at the end of the runs (end cell viability)
and the cell density at the end of the runs (viable end cell density).
Experiments I-A, I-B and I-C; II-A, II-B and II-C; and lll-A, III-B and III-C
refer to the same cell culture run with the same temperature shift profile assessed
at different times, i.e., for I-A, II-A, and lll-A, the product and cell parameters were
assessed after 14 days, for I-B, II-B and III-B after 21 days, and for I-C, II-C andIII-C after 28 days. These experiments were performed in a 5 L bioreactor in
which the culture conditions were controlled as follows: pH at 7.0; dissolved
oxygen at 40%; agitation at 60 rpm; and initial temperature at 37°C. The data
were obtained from fed-batch cell culture fermentations according to the methods
of the present invention.
Experiments I, II and HI were designed as follows:
Experiment I: the cell culture temperature was controlled at 37°C from day 0 to
day 21 (no temperature shift).
Experiment II: the cell culture temperature was controlled at 37°C from day 0 to
day 6; and at 34°C from day 6 to day 21 (single temperature shift).
Experiment III: the cell culture temperature was controlled at 37°C from day 0 to
day 6; at 34°C from day 6 to day 10; and at 32°C from day 10 to day 21 (two-step
temperature shift procedure of the present invention).
Experiments IV-A and V-A show the results of product titer, end cell viability
and viable end cell density assessed after a 14-day culture run with an initial
(standard) production phase; Experiments IV-B and V-B show these results
assessed after a 21-day culture run with an extended production phase; and
Experiments IV-C and V-C show the results assessed after a 28-day culture run
with a second extended production phase. Experiments IV and V were performed
63
in a 50 L bioreactor in which the culture conditions were controlled as follows: pH
at 7.0; dissolved oxygen at 40%; agitation at 30 rpm; and initial temperature at
37°C.
Experiments IV and V were designed as follows:
Experiment IV: the cell culture temperature was controlled at 37°C from day 0 to
day 6; at 34°C from day 6 to day 10; and at 32°C from day 10 to day 28 (two-step
temperature shift procedure of the present invention).
Experiment V: the cell culture temperature was controlled at 37°C from day 0 to
day 6; at 34°C from day 6 to day 10; at 32°C from day 10 to day 14; and at 30°C
from day 14 to day 28 (three-step temperature shift procedure of the present
invention). Experiments V-A, V-B and V-C refer to the same cell culture run with
the same temperature shift profile assessed at different times, i.e., for V-A, the
product and cell parameters were assessed after 14 days, for V-B, after 21 days,
and for V-C, after 28 days.
Experiments I-V represent five different culture runs as described above.
As described, runs of 14 days are designated "A"; runs of 21 days are designated
"B"; while runs of 28 days are designated "C".
Table 3 presents the results of Experiments demonstrating the impact of
different temperature shift profiles on the production of CTLA4lg by cells in culture
at the 5 L reactor scale.
As has been demonstrated by the experiments in this Example, the multi
step temperature shift profiles were found to maintain a high cell viability
throughout the culture process. Specifically, in Table 3, Experiment III-B
that the use of the timed two-step temperature shift profile maintained a higf*
viability (see also FIG. 1) throughout the 21-day culturing process (including the
extended production phase), allowing the titer to reach 3.5 g/L at a high sialic acid
content of 6.5 [molar ratio]. The success of two-step culturing procedure can also
be evidenced in the high value of the mathematical product of 'end titer x end
sialic acid'.
By contrast, a culturing process involving no temperature shift (Table 3,
Experiment I-B) led to an early decline in cell viability. Further, the use of a onestep
temperature shift (Table 3, Experiment II-B) was found to yield a lower end
titer, end sialic acid, and ('end titer x end sialic acid') - mathematical product
compared with the two-step temperature shift profile and process according to this
invention.
In addition, the results presented in Table 4 involving cell cultures
performed at a 50 L reactor scale demonstrate the advantages of the multi-step
temperature shift culturing technique of this invention. The third temperature shift
to 30°C was timed to take place on day 14. In particular, the benefits of a triple
temperature shift on cell viability and end titer using the triple shift (Table 4,
Experiment V-C; also FIG. 2) can be seen compared to a double temperature
shift. In accordance with the present methods, the triple temperature shift further
extended cell viability and thus, protein production relative to no shift or one shift
methods. Due to a scale-up effect, which is usual in cell culture, the titer
generation at the 50 L reactor scale was found to be somewhat slower than at the
5 L scale. However, it is readily appreciated that such an effect does not detract
from the advantages of the two or more temperature shift culture runs as provided
by this invention.As evidenced by the results presented in Example 3, for those runs in whicha temperature shift was performed on day 6, i.e., at the end of the logarithmic
growth phase, far better results in end titer and cell viability were obtained,
compared with control runs in the absence of a temperature shift. As observed
from the results, the volumetric productivity was increased two-fold by the use of a
single temperature shift. A second temperature shift on day 10 yielded a higher
cell viability and further increased volumetric productivity (approximately 3-
compared with runs with no T-shift), while the product quality remained high, as
determined by the sialic acid content of the glycoprotein product.
EXAMPLE 4
This Example presents data showing that the cell culture process
comprising two downward temperature shifts according to this invention has no
statistically significant effect on the amount of protein, e.g., CTLA4lg in this
Example, that is produced per cell per unit time. In accordance with the newly
presented cell culturing (fermentation) methods of this invention, the overall
production of protein in the process is the result of more viable cells surviving until
the end of the process. Because more cells survive for an extended production
time, more cells are viably producing the desired protein at the end of the process.
This, in turn, yields a greater amount of the desired protein product at the end of
the process or culture run.
Table 5 illustrates the cell specific productivity at various times in the
process encompassed by the present invention. Cell specific productivity is
determined by the formula as presented supra. The culture process designed for
the production of CTLA4lg, other soluble CTLA4 molecules, and soluble CTLA4
mutant molecules is thus a non-growth associated process in which protein
production begins on or about day 6, i.e., approximately at the start of the
stationary phase, following exponential cell growth. The data presented in Table 5
relate to the experiments conducted in Example 3.
Cell specific productivity in Table 5 was calculated using cell density and
titer measurements, as described hereinabove. As will be appreciated by the
skilled practitioner in the art, cell density measurements usually have about a 10-
20% standard deviation (SD), i.e., a high SD, and are imprecise. Therefore, the
determination of cell specific productivity has a corresponding 10-20% standard
deviation. Thus, in view of the high SD involved in these types of calculations, the
amount of product produced per cell per day for the different run times does not
differ significantly among a process having no T-shift, one T-shift, or two T-shifts.
The high levels of high quality protein product produced by the newly provided cell
culturing processes of this invention, and the overall increase in protein
production, are attributed to the higher numbers of viable cells that survive through
the entire culturing process comprising multiple downward temperature shifts.
EXAMPLE 5
Example 5A
This Example provides materials and reagents employed in the processes
of the present invention for the culturing of recombinant cells that produce the
exemplified L104EA29Ylg as described herein in Examples 5B-16.
1. Cell Culture Medium The basal cell culture medium used for all
phases of cell inoculum generation was modified CD-CHO medium containing
glutamine, sodium bicarbonate, insulin and methotrexate (Invitrogen, Carlsbad,
CA), as exemplified in Table 6. The pH of the medium was adjusted to 7.0 with 1
N HCI. The basal cell culture medium used for growth of cultures in bioreactors,
including 5 liter (5L), 10 liter (10L) and 50 liter (SOL) production reactors, was also
the modified CD-CHO medium shown in Table 6, except without methotrexate.
The pH of the medium was adjusted to 7.0 with 1 N HCI.
In all Examples except Example 8, for feeding ceils in the fed-batch
process, a modified feed medium, i.e., eRDF-1 medium (Invitrogen), containing
glucose, glutamine, insulin and TC Yeastolate (Becton-Dickinson, Franklin Lakes,
NJ) was employed, as shown in Table 7. The pH of the feeding medium was
adjusted to 7.0 with 1 N NaOH after the addition of all components.
For Example 8, the feeding medium was that described above with one
modification: eRDF-1 was at a concentration of 25.2 g/L.
2. Production Phase in Bioreactor
The production bioreactor was initially operated as a batch reactor, with
temperature, pressure, pH and dissolved oxygen concentration closely monitored
and controlled. The condition of the culture was evaluated by measuring the
viable cell density and the concentration of several key metabolites. The feeding
process was initiated one day after the inoculation. The remainder of the
fermentation was the conducted in fed-batch mode.
Bioreactors of 5 L scale (glass reactor with one marine impeller), 10 L scale
(glass reactor with two marine impellers) and 50 L scale (stainless steel reactor
with two marine impellers) were used, (see Example 2). A data acquisition
system (Intellution Fix 32) recorded temperature, pH, and dissolved oxygen (DO)
throughout runs. Gas flows were controlled via rotameters. Air was sparged into
the reactor via a submerged frit (5 jim pore size) and through the reactor head
space for CO2 removal. Molecular oxygen was sparged through same frit for DO
control. COa was sparged through same frit as used for pH control.
3. Feeding Strategy
At 24 hours post inoculation, a daily minimum of 1% of culture volume of
modified eRDF-l feed medium was added into the bioreactor if the glucose
concentration was > 3.0 g/L. In cases in which the glucose concentration was
below 3 g/L, the volume of the daily bolus feed was calculated to bring the glucose
concentration back up to 3.0 g/L. The daily feed amount was recorded on batch
sheets.
4. Sampling
Samples of cells were removed from the reactor on a daily basis. A sample
used for cell counting was stained with trypan blue (Sigma, St. Louis, MO). Cell
count and cell viability determination were performed via hemocytometry using a
microscope, or via Cedex automatic cell counter (Innovatis AG, Bielefeld,
minutes at 2000 rpm (4°C) for cell separation. Supernatant was analyzed for thefollowing parameters: titer, sialic acid, glucose, lactate, glutamine, glutamate, pH,pO2, pCO2, ammonia, and, optionally, lactate dehydrogenase (LDH). Additionalback-up samples were frozen at -20°C.
Example 5BThis Example 5B describes the production of L104EA29Ylg, shown as -1 to357 or +1 to 357 in FIG. 4, (encoding DMA deposited with the ATCC as PTA-2104), from cultured CHO cells.
This Example 5B also describes a process of this invention involving
addition of polyanionic compound, more specifically dextran sulfate, to a cell
culture.CHO cells used for L104EA29Ylg expression were expanded in modifiedCD-CHO medium (Invitrogen, CA) containing glutamine, sodium bicarbonate,insulin, and methotrexate using T-75 flasks and shake-flasks. T-flasks and shakeflaskswere incubated at 37°C and 6% CO2. After sufficient inoculum wasgenerated, the culture was transferred into 5 or 10L bioreactors using modifiedCD-CHO medium as described above, except without methotrexate. Initial seedingdensity was 200,000 viable cells/ml or 106 cells/ml.The 5L and 10L vessels were glass reactors equipped with one and twomarine impellers respectively (Applikon, CA). Gas flows were controlled viarotameters. Air was sparged into the reactor via a submerged frit (5 \m\ pore size)and through the reactor head space for CO2 removal. Molecular oxygen wassparged through the same frit for DO control. CO2 was sparged through same fritfor high side pH control. Low side pH control was realized via addition of 1 NNaOH or Na2CO3.The culture in the bioreactor was given a daily bolus feed using modifiedeRDF medium (Invitrogen, CA) with glucose, galactose, glutamine, insulin, and TCYeastolate (Becton Dickinson) in the following manner: starting one day post
inoculation, a minimum of 1% culture volume was added, or if the glucose level
was below 3 g/L, a calculated volume to bring glucose back to 3 g/L.
In all examples except example 11, the temperature was controlled at 37°C
from day 0 to 6; at 34°C from day 6 to 10; at 32°C from day 10 on.
The fermentation process had a typical duration of 14-19 days. Samples
were taken on a daily basis from the reactor. The sample used for the cell count
was stained with trypan blue (Sigma, MO). Cell count and cell viability
determinations were performed using a Cedex automatic cell counter (Innovatis
AG, Bielefeld, Germany). Supernatant was analyzed for: LEA29Ytiter, glucose,
lactate, glutamine, glutamate, pH, pOa, pCOa, ammonia.
Dextran sulfate (sodium salt, from dextran of average molecular weight
5000 Da, Sigma, MO) was dissolved into water or into medium. The solution was
sterile-filtered and was added to the reactor to a concentration of 50 mg/L. The
volume of the addition constituted at most 2% of the working volume of the reactor
at the time the addition took place.
EXAMPLE 6
This Example describes and presents the results of a comparative study to
assess the effect of addition of polyanionic compound, more specifically dextran
sulfate, at a time after innoculation.
5L and 10L bioreactors were inoculated with 0.2 x 106 cells/mL of
L104EA29Y-producing cells.
Experiments 6-a and 6-b were designed as follows:
Example 6-a: dextran sulfate was not added to the cultures ('control' cultures: 4
cultures in 5L bioreactors, 4 cultures in 10L bioreactors).
Example 6-b: dextran sulfate was added to a concentration of 50 mg/L to the
The average viability, viable cell density, and total cell density profiles, and
their standard deviations for Examples 6-a and 6-b are reported in Figure 6.
In the control cultures, a plateau in viable cell density was observed
between days 6 and 7, corresponding to the stationary phase. A decline in viable
cell density and viability was observed after day 7 (on average) for the control
cultures, corresponding to the death phase. When dextran sulfate was added
the cultures on day 6, the growth phase was extended until day 11. Viable cell
control. Viability remained above 90% during this extended growth phase. After
day 11, viable cell density and total cell density declined in a proportional fashion,
indicating cell lysis, and, as a result, the viability index remained above 90% until
Figure 7 is a logarithmic representation of the viable cell densities as a
function of time for cultures with dextran sulfate addition and for control runs.
death rates (given by the slopes of the viable cell densities in Figure 2) differed
depending on the presence or not of dextran sulfate. In the presence of dextran sulfate, death rate was approximately constant between day 12 and day 19, at avalue of 0.0012 h~1. In contrast, death rate in the average of the controls was,between days 8 and 12 where it was at a maximum, 0.0024 h"1. Thus, dextran
sulfate added on day 6 slowed down cell death rate during the death phase by afactor of two.Despite the beneficial effects of dextran sulfate described above, product
L104EA29Ylg titer was similar with or without dextran sulfate addition (Table
Table 8: impact of day 6 dextran sulfate addition on product L104EA29Ylg
EXAMPLE 7
This example shows the effect of adding dextran sulfate to a cell culture
that is in the death phase.
A 5L bioreactor of L104EA29Ylg -producing cells was inoculated at a
density of 106 cells/mL, and the death phase started on day 5. Dextran sulfate
was added on day 6.
The viability, viable cell density, and total cell density profile is presented in
Figure 8. Addition of dextran sulfate on day 6 of such a culture could prevent the
occurrence of a major decline in viable cell density up to day 17. Thus, when
dextran sulfate is added during the death phase, cell death can be arrested for
several days.
In this run, a titer of 1.9 g/L with a NANA molar ration of 6.6 was obtained
75
EXAMPLE 8
This example shows the effect of adding dextran sulfate to a cell culture
that is in the death phase.
A 10L bioreactor was inoculated with 0.2 x 106 cells/mL of L104EA29Ylgproducing
cells. In this particular example, the daily feed was of a more
concentrated formulation, and as a result the onset of the death phase was
delayed until day 10 (Figure 9). Dextran sulfate was not added until day 14.
The viability, viable cell density, and total cell density profile is presented in
Figure 9.. Addition of dextran sulfate on day 14 allowed a stabilization of the viable
cell density for a period of 4 days, after which the culture was discontinued.
This example is another illustration of the arrest of cell death during the
death phase upon dextran sulfate addition to the culture.
EXAMPLE 9
This example shows the effect of adding dextran sulfate on day 0. The
effect of delayed addition of dextran sulfate may be seen by comparing the effect
seen in this experiment with the effects seen in other experiments in which
addition of dextran sulfate was delayed.
Two repeat 5L bioreactors were inoculated with 0.2 x 106 cells/mL of
L104EA29Ylg-producing cells, and dextran sulfate was added to a concentration
of 50 mg/L on the same day as the inoculation (day 0).
The viability, viable cell density, and total cell density profiles are presented
in Figure 10. Neither culture achieved a higher cell density than cultures devoid
of dextran sulfate (Compare Figures 6 and 10). The cells entered the death phaseon day 7 or 8, as opposed to day 11 when dextran sulfate is added on day 6(Compare Figures 6 and 10). The L104EA29Ylg titers obtained on day 14 of these
runs were 0.57 g/L (run #1) and 0.86 g/L (run #2), which are significantly less than
the titers obtained in the control process or in the process with day 6 dextran
76
sulfate addition (see Example 6).
These results demonstrate the importance of the timing of dextran sulfate
addition on the outcome of the addition.
Without being bound by theory, we propose that the observed effects of
dextran sulfate addition and their dependance on the timing of the addition can be
explained by the binding of dextran sulfate to diverse autocrine factors, in a
manner similar to the binding of pentosan polysulfate to heparin-binding growth
factors (Zugmaier et al., 1992). We propose that dextran sulfate binds to these
factors at their heparin-binding domain, which becomes unavailable for binding to
cell surface heparan sulfate proteoglycans. As a result, the dextran sufate-bound
factors fail to concentrate at the cell surface, which greatly reduces the probability
of their binding to their receptors. The net effect is that these factors do no longer
exercise their normal action on the cells. In the very first few days after inoculation,
the cells produce certain growth factors, whose function is to signal cell growth.
Dextran sulfate added on day 0 irreversibly binds to those factors as they are
produced. Binding of dextran sulfate to these factors does however not greatly
affect cell growth in an adverse manner, possibly because the cells are able to
respond by increasing growth factor production. Later during the growth phase,
production of growth factors ceases, and the cells start producing autocrine factors
of a different type, whose effect at a high concentration is to signal the end of the
growth phase and the onset of cell death. These factors accumulate from a low
concentration on day 0 to a high concentration at a later time. At that point, these
factors effectively signal the cells to stop growing and to enter the stationary andthe death phases. We propose that dextran sulfate preferentially binds to thesedeath-signaling factors, thus disabling their signaling function and allowing anextension of the growth phase. Continuous induction by these factors appears tobe needed for cell death to proceed, with the result that cells that have entered the
death phase can be reset into a stationary phase when death-signalling factors are
disabled by dextran sulfate (Examples 7 and 8), even as late as on day 14 in
culture. Conversely, dextran sulfate added on day 0, that has bound to growth
factors, is still bound to these factors at the end of the growth phase, making it
unavailable for binding to death-signaling molecules. Thus, extension of the
growth phase and delayed onset of cell death do not occur in the case of cultures
where dextran sulfate is added on day 0 (Example 9) in the same way as they do
in the case where dextran sulfate is added at the end of the growth phase.
The arrest of cell growth and the onset of cell death on day 11 in cultures
supplemented with dextran sulfate on day 6 in Example 6 is hypothesized to result
• from mechanisms different from induction by dextran sulfate-binding autocrine
factors. Possibly, the exhaustion of particular nutrients in the medium following
days of growth may be accountable for the end of the growth phase in these
cultures.
EXAMPLE 10
This Example compares the effect of addition of dextran sulfate at three
different times during the initial growth phase.
Cultures of L104EA29Ylg-producing cells were inoculated at 106 cells/mL in
5-L bioreactors. Dextran sulfate was added to the cultures to a concentration of
50 mg/L at different times. In one culture, dextran sulfate was added on day 3, in
another on day 4, and in a third on day 5.
The viability and viable cell density profiles are reported in Figure 11. High
viable cell densities (> 107 cells/mL) were achieved in all three cases of additions
(day 3, 4 or 5), but the earlier additions (day 3 or 4) did not prevent a decline in theviable cell density immediately following the growth phase. In contrast, day 5
addition stabilized the viable cell density for 4 days following the growth phase.
time points past 250 h, the viable cell density in the day 5 dextran sulfate addition
culture was always higher or equal (within measuring error) than in the day 4
dextran sulfate addition culture, and the viable cell density in the day 4 dextran
sulfate addition culture was always higher or equal (within measuring error) than inthe day 3 dextran sulfate addition culture. Thus, it appears from this example thatthe optimal time for dextran sulfate addition is at the end of the initial growth phase(referring to the growth phase that would have been observed in the absence of
any dextran sulfate addition). Earlier addition can extend the growth phase but will78not be as effective at stabilizing the viable cell density following the dextran
sulfate-induced extended growth phase, whereas later addition (during the death
phase) will stabilize the viable cell density but may fail to provide a substantial
increase in viable cell density (see Examples 7 and 8). Accordingly, on day 14 a
titer of 2.7 g/L was obtained with day 5 dextran sulfate addition, whereas day 14
titers of 2.6 g/L were obtained with day 3 or day 4 dextran sulfate addition, and a
day 14 titer of 1.9 g/L was obtained with day 6 dextran sulfate addition (in Example7). The NANA molar ratios were 6.3, 6.6, 6.0, and 6.6 respectively in the runs
mentioned above, showing that higher titers obtained with optimal timing of
dextran sulfate addition came with a consistent level of sialylation.
EXAMPLE 11This example shows the effect of one and two temperature shifts in aculture producing L104EA29Ylg. The culture is also subject to delayed addition ofdextran sulfate.5L reactors were innoculated at a density of 200,000 cells/mL.
Two, one, or no T-shifts were applied. For one temperature shift, the
temperature is shifted from 37°C to 34°C on day 6. For two temperature shifts, thetemperature is shifted from 37°C to 34°C on day 6, and from 34°C to 32°C on day10. In all xcases dextran sulfate was added on day 6. The two T-shift case is theaverage of three runs, and standard deviations are shown with bars.
Viable cell density, viability, and titer are reported in Figures 12,13, and 14
respectively.The results show the benefits of applying at least one temperature shift. Inthe case where the temperature is maintained at 37°C throughout the run, the
culture enters the death phase on day 10, and the decrease in viable cell density
and viability is steep. As a result, there is a clear decrease in L104EA29Ylg
volumetric productivity after 12 days in culture. For culture times of 14 days and
longer, it is clear that cultures that have one or two temperature shifts will
outperform the constant temperature culture in terms of titer.
In the case where only one temperature shift is implemented (on
day 6, to 34°C), a steep decrease in viable cell density and viability is observed
after day 16. The culture where a second temperature shift (on day 10, to 32°C) isimplemented in addition to the first does not show as steep a decline in viable cell
density and viability after day 16. At culture times past 18 days, volumetric
productivity in the one T-shift culture is clearly inferior to that in the two T-shifts
culture. In contrast, volumetric productivity in the culture with one T-shift is
superior to that in the two T-shifts culture at culture times between day 11 and day15.In conclusion, the first temperature shift is beneficial independently of thedesired harvest time, whereas the benefit of the second temperature shift dependsnded harvest time and the viability requirements for effective
downstream processing. The absence of a second temperature shift will allow
to reach at higher product liter until day 20, but for cultures that are run for longerthan 20 days, the run with two temperature shifts will outperform the run with onetemperature shift in terms of titer. In addition, it must be considered harvests pastn higher amount of cell lysis products in the case of one T-shiftthan in the case of two T-shifts, which can complicate downstream processing.The steep decrease in viable cell density observed after day 12 in the case of the
one T-shift profile can be a concern for product quality, as the corresponding celldeath may release into the supernatant a significant load of sialidases that candecrease product sialylation.
The contents of all issued and granted patents, patent applications,
published PCT and U.S. applications, articles, books, references, reference andinstruction manuals, and abstracts as referenced or cited herein are herebyincorporated by reference in their entireties to more fully describe the state of theart to which the invention pertains.
As various changes can be made in the above-described subject matter
without departing from the scope and spirit of the present invention, it is intendedthat all subect matter contained in the above description, or defined in theappended claims, be interpreted as descriptive and illustrative of the presentinvention. Many modifications and variations of the present Invention are possiblein light of the above teachings.














WE CLAIM:
1. A cell culturing process for increased protein production and cell viability comprising:
a) culturing host cells which produce soluble cytotoxic T-lymphocyte antigen -4 (CTLA4) molecules at a first temperature at 37°C under cell culture conditions and for a time period that allows for cell growth;
b) then culturing the cells at a second temperature at 34°C and
c) then culturing the cells at a third temperature at 32°C and
d) then culturing the cells at a fourth temperature of 30°C and
wherein the cells are cultured at the second temperature starting day 5 to day 7 of the culture, and wherein there are four days between the start of the second temperature and the start of the third temperature and wherein the fourth temperature starts on or two weeks from the start of the culture until the end of the culturing process.
2. The process as claimed in claim 1, wherein the cells are cultured at the second temperature starting day 6 of the culture, and wherein the cells are cultured at the third temperature starting day 10 of the culture.
3. The process as claimed in claim 1, wherein there are approximately four day increments between the start of the second temperature and the start of the third temperature.
4. The process as claimed in claim 1, wherein the soluble CTLA4 molecule is a CTLA4 fusion protein.
5. The process as claimed in claim 4, wherein the soluble CTLA4 fusion protein is a cytotoxic T-lymphocyte antigen-4 immunoglobulin (CTLA4Ig) molecule.
6. The process as claimed in claim 5, wherein the soluble CTLA4 fusion protein is CTLA4Ig comprising amino acids -1 to 357 or +1 to 357 as shown in SEQ ID NO:2.
7. The process as claimed in claim 1, wherein the soluble CTLA4 molecule soluble CTLA4 mutant molecule.
8. The process as claimed in claim 7, wherein the soluble CTLA4 mutant molecule is L104EA29YIg comprising amino acids -1 to 357 or +1 to 357 as shown in SEQ ID NO. 4.
9. The process as claimed in claim 1, wherein the cell culturing process is a fed-batch process or a continuous process.
10. The process as claimed in claim 1, wherein the cells are cultured at 34°C from day 6 to day 10 and are cultured at 32°C from day 10 onward.
11. The process as claimed in claim 1, wherein the temperature of the culture is shifted from 37°C to 34°C when the cell density in the culture is 2-12 x 106 cells/mL.
12. The process as claimed in claim 1, wherein the temperature of the culture is shifted from 37°C to 34°C when the culture is in the stationary phase.
13. The process as claimed in claim 1, wherein the produced protein comprises enhanced sialylation during cell culturing.
14. The process as claimed in claim 1, wherein the cell culturing process comprises:

a) culturing CHO cells which produce a soluble CTLA4 molecule at 37°C under conditions and for a time period that allow the cell growth;
b) then culturing the cells at 34°C starting on day 6 of the culture; and
c) then culturing the cells at 32°C starting on day 10 of the culture.

Documents:

2519-DELNP-2005-Abstract-(11-05-2010).pdf

2519-DELNP-2005-Abstract-(12-05-2010).pdf

2519-delnp-2005-abstract.pdf

2519-delnp-2005-assignment.pdf

2519-DELNP-2005-Claims-(11-05-2010).pdf

2519-DELNP-2005-Claims-(12-05-2010).pdf

2519-delnp-2005-claims.pdf

2519-DELNP-2005-Correspondence-Others (25-09-2009).pdf

2519-DELNP-2005-Correspondence-Others-(05-05-2010).pdf

2519-DELNP-2005-Correspondence-Others-(11-05-2010).pdf

2519-delnp-2005-correspondence-others.pdf

2519-delnp-2005-description (complete).pdf

2519-delnp-2005-drawings.pdf

2519-DELNP-2005-Form-1-(11-05-2010).pdf

2519-DELNP-2005-Form-1-(12-05-2010).pdf

2519-delnp-2005-form-1.pdf

2519-delnp-2005-form-18.pdf

2519-DELNP-2005-Form-2-(11-05-2010).pdf

2519-DELNP-2005-Form-2-(12-05-2010).pdf

2519-delnp-2005-form-2.pdf

2519-DELNP-2005-Form-3 (25-09-2009).pdf

2519-delnp-2005-form-3.pdf

2519-delnp-2005-form-5.pdf

2519-delnp-2005-gpa.pdf

2519-delnp-2005-pct-101.pdf

2519-delnp-2005-pct-304.pdf

2519-delnp-2005-pct-308.pdf

2519-delnp-2005-pct-401.pdf


Patent Number 240965
Indian Patent Application Number 2519/DELNP/2005
PG Journal Number 25/2010
Publication Date 18-Jun-2010
Grant Date 10-Jun-2010
Date of Filing 11-Jun-2005
Name of Patentee BRISTOL- MYERS SQUIBB COMPANY
Applicant Address P.O. BOX 4000, ROUTE 206 AND PROVINCE LINE ROAD, PRINCETON, NEW JERSEY 08543 -4000, U.S.A.
Inventors:
# Inventor's Name Inventor's Address
1 SIVAKESAVA SAKHAMURI 241 LAFAYETTE ROAD, APT.#123, SYRACUSE, NY 13205, U.S.A.
2 STEVEN S. LEE 9105 WHISTLING SWAN LANE, MANLIUS, NY 13104, U.S.A.
3 LINDA MATLOCK 223 CANFIELD ROAD, PARISH, NY 13131, U.S.A.
4 BERNHARD M. SCHILLING 2104 EUCLID AVENUE, SYRACUSE, NY 13224, U.S.A.
5 STEPHEN G. ZEGARELLI 108 WILSHIRI DRIVE, NORTH SYRACUSE, NY 13212, U.S.A.
6 WILLIAM V. BURNETT, JR. 49 LYNDON ROAD, FAYETTEVILLE, NY 13066, U.S.A.
7 CHRISTOPH E. JOOSTEN 4702 KEHOE LANE, MANLIUS, NY 13104, U.S.A.
8 JONATHAN O. BASCH 216 WELLINGTON ROAD, DEWITT, NY 13124, U.S.A.
PCT International Classification Number C07K
PCT International Application Number PCT/US2003/040991
PCT International Filing date 2003-12-18
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/436,101 2002-12-23 U.S.A.