CELL CULTURE PROCESS FOR PRODUCING A PROTEIN
FFiieelldd ooff TThhee IInnvveennttiioonn
TThhee iinnvveennttiioonn iiss rreellaatteedd ttoo tthhee mmoonnoopphhaassiicc mmaammmmaalliiaann cceellll ccuullttuurree ccoonnddiittiioonnss ffoorr tthhee
pprroodduuccttiioonn ooff ggllyyccoossyyllaatteedd pprrootteeiinnss.. MMoorree ppaarrttiiccuullaarrllyy,, iitt rreellaatteess ttoo tthhee pprroodduuccttiioonn ooff
5 mmoonnoocclloonnaall aannttiibbooddiieess aanndd ffuussiioonn pprrootteeiinnss iinn mmaammmmaalliiaann cceellll ccuullttuurree ccoonnddiittiioonnss hhaavviinngg
mmoonnoopphhaassiicc tteemmppeerraattuurree ccoonnddiittiioonn.. MMoorree ppaarrttiiccuullaarrllyy,, tthhee iinnvveennttiioonn iiss rreellaatteedd ttoo pprroocceessss ffoorr
ccuullttuurriinngg mmaammmmaalliiaann cceellll iinn ssuuiittaabbllee cceellll ccuullttuurree ccoonnddiittiioonnss ffoorr tthhee pprroodduuccttiioonn ooff pprrootteeiinnss
pprreeffeerraabbllyy ggllyyccoossyyllaatteedd pprrootteeiinnss.. MMoorree ppaarrttiiccuullaarrllyy,, iitt aallssoo rreellaatteess ttoo tthhee pprroocceessss ooff cceellll
ccuullttuurree ppeerrffoorrmmiinngg wwiitthh ssuuiittaabbllee cceellll ccuullttuurree ccoonnddiittiioonnss ssppeecciiffiiccaallllyy mmaaiinnttaaiinniinngg mmoonnoopphhaassiicc
1100 tteemmppeerraattuurree ffoorr tthhee pprroodduuccttiioonn ooff ggllyyccoossyyllaatteedd pprrootteeiinnss
The production o f therapeutic proteins for biopharmaceutical applications typically involves
the use o f mammalian cell cultures that are known t o produce high level o f glycosylated
proteins. Control and optimization o f mammalian cell culture conditions i s critically
important for successful commercial production o f glycosylated proteins. Conventionally the
mammalian cell culture process for many o f the glycosylated proteins i s biphasic;
distinguished b y a n initial growth phase and subsequent production phase. This dual
production phases provides high cell densities and better product titer.
0
Furthermore, controlling and optimizing cell culture conditions have always been a great
challenge because these conditions drastically affect the cell viability, desired product yield,
purity and heterogeneity. The physio-chemical and pharmacokinetics properties o f the
therapeutic protein molecules critically depend over the culture conditions.
5
US7294481 discloses a method for producing a fusion protein, i.e., TNFR:Fc. During the
production phase, the host cells were cultured a t a temperature o f 25-34 °C, which showed
reduction i n disulfide scrambling i n the TNFR:Fc produced i n comparison t o the production
phase carried out at 37 °C. Further it discloses that the production phase is carried out in the
presence of an alkanoic acid or salt thereof.
The initiation of the production phase may be achieved in numerous ways, with temperature
and pH shifts being the most common. Other methods used are addition of inducing agents,
alteration of feed substrate(s) or osmolality changes.
The invention relates to the cell culture process by maintaining suitable culture condition
during the culture. Specifically, the mammalian cells are grown at a single temperature
selected from at about 34 °C to at about 37 °C. The invention relates to the cell culture
process in fed batch mode by maintaining suitable culture condition during the culture.
Summary Of The Invention
In an embodiment, the invention is related to monophasic mammalian cell culture process to
produce glycosylated proteins.
In another embodiment, the invention is related to a monophasic temperature mammalian cell
culture process to produce glycosylated proteins.
In another embodiment, the invention is related to a monophasic temperature mammalian cell
culture process to produce glycosylated proteins, wherein the temperature is set between 34
°C to 37 °C.
In another embodiment, the invention is related to mammalian cell culture process having
monophasic temperature condition to produce fusion proteins and monoclonal antibodies.
In yet another embodiment, the invention is related to the method of producing glycosylated
proteins at a monophasic temperature for mammalian cell culture wherein the cell culture is
essentially free of alkanoic acid or salt thereof.
In another embodiment, the invention is related a process of producing glycosylated protein
in a mammalian cell culture the process comprising the steps of :
a) preparing inoculum with suitable cell concentration during seed development;
b) inoculating the inoculum with suitable cell concentration in to production
bioreactor;
c) culturing the cell in production bioreactor at suitable conditions wherein the suitable
condition is monophasic temperature condition; and
d) obtaining the glycosylated protein from the cell culture.
In another embodiment, the invention is related a process of producing glycosylated protein
in a mammalian cell culture the process comprising the steps of :
a) preparing inoculum with suitable cell concentration during seed development;
b) inoculating the inoculum with suitable cell concentration in to production
bioreactor;
c) culturing the cell in production bioreactor at suitable conditions wherein the
suitable condition is monophasic temperature condition; and
d) obtaining the glycosylated protein from the cell culture,
wherein monophasic temperature does not have temperature shift during step (c).
In another embodiment, the invention is related a process of producing glycosylated protein
in a mammalian cell culture the process comprising the steps of :
a) preparing inoculum with suitable cell concentration during seed development;
b) inoculating the inoculum with suitable cell concentration in to production
bioreactor;
c) culturing the cell in production bioreactor at suitable conditions wherein the
suitable condition is monophasic temperature condition; and
d) obtaining the glycosylated protein from the cell culture,
wherein the production bioreactor in step (c) does not have any distinctive growth phase and
production phase.
In another embodiment, the invention is related to cell culture process performing with
monophasic temperature condition to produce glycosylated proteins, wherein the temperature
is maintained at a set point between at about 32 °C to about 37 °C.
In another embodiment, the invention is related to cell culture process performing with
monophasic temperature condition to produce glycosylated proteins, wherein the temperature
is maintained at a set point between at about 34 °C to about 37 °C.
In certain embodiment, the invention is related to cell culture process performing with
monophasic temperature condition to produce glycosylated proteins, wherein the temperature
is maintained at about 33°C.
In certain embodiment, the invention is related to cell culture process performing with
monophasic temperature condition to produce glycosylated proteins, wherein the temperature
is maintained at about 34°C.
In certain embodiment, the invention is related to cell culture process performing with
monophasic temperature condition to produce glycosylated proteins, wherein the temperature
is maintained at about 35°C.
In certain embodiment, the invention is related to cell culture process performing with
monophasic temperature condition to produce glycosylated proteins, wherein the temperature
is maintained at about 36 °C.
The fusion proteins and monoclonal antibodies produced by the processes of the invention
are useful for biopharmaceutical applications.
The details of one or more embodiments of the invention set forth below are illustrative in
nature only and not intended to limit to the scope of the invention. Other features, objects and
advantages of the inventions will be apparent from the description.
Brief Description of figures;
Figure 1 depicts comparative growth trends of CHO cell observed during the Etanercept
production.
Figure 2 depicts comparative viability trends of CHO cell observed during the Etanercept
production.
Figure 3 depicts comparative growth trends of CHO cell observed during the Bevacizumab
production.
Figure 4 depicts comparative viability trends of CHO cell at various time points observed
during Bevacizumab production.
Figure 5 depicts growth trends of CHO cell in Rituximab production
Figure 6 depicts viability trends of CHO cell in Rituximab production
Figure 7depicts growth trends of CHO cell in Trastuzumab production
Figure 8 depicts viability trends of CHO cell inTrastuzumab production
Detailed Description of The Invention
Definition;
The term "antibody" as referred to herein includes whole antibodies and any antigen binding
fragments or single chains thereof. An "antibody" refers to a glycoprotein comprising at least
two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an
antigen binding fragment thereof. Each heavy chain is comprised of a heavy chain variable
region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain
constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is
comprised of a light chain variable region (abbreviated herein as VL) and a light chain
constant region. The light chain constant region is comprised of one domain, CL. The VH
and VL regions may be further subdivided into regions of hypervariability, termed
complementarity determining regions (CDR) with are hypervariable in sequence and/or
involved in antigen recognition and/or usually form structurally defined loops, interspersed
with regions that are more conserved, termed framework regions (FR or FW). Each VH and
VL is composed of three CDRs and four FWs, arranged from amino-terminus to carboxyterminus
in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The amino
acid sequences of FW1, FW2, FW3, and FW4 all together constitute the "non-CDR region"
or "non-extended CDR region" of VH or VL as referred to herein.
The 'Host cell" is genetically engineered means have recombinant DNA or RNA to
expresses a gene at elevated levels or at lowered levels, or expresses a mutant form of the
gene. In other words, the cell has been transfected, transformed or transduced with a
recombinant polynucleotide molecule, and thereby altered so as to cause the cell to alter
expression of a desired polypeptide. The conventional methods of "genetic engineering" are
known in the prior art.
"Production medium" eans a cell culture medium designed to be used to culture cells
during a production phase.
As used herein, "monophasic" refers to cell culture method which involves no change in any
of the culture conditions at which the culture is maintained. The culture conditions includes
but are not limited to temperature, pH, osmolality or chemical excipients.
As used herein, "monophasic temperature" refers to cell culture method performed with the
use of a single temperature set point in the production bioreactor (referred to as passage "N").
And the temperature is maintained at a single set point during the run of production
bioreactor to obtain monophasic growth conditions. The monophasic is restrained to shift.
The monophasic temperature is between at about 34 °C to at about 37 °C.
As used herein, "glycosylated protein" refers to one or more mammalian polypeptides that
function as a discrete unit. The "glycosylated protein" includes fusion proteins and
monoclonal antibodies used for biopharmaceutical applications. Examples of fusion protein
include but are not limited to etanercept, abatacept, alefacept, rilonacept, belatacept,
aflibercept, etc. Examples of monoclonal antibodies include but are not limited to rituximab,
trastuzumab, bevacizumab, adalimumab, denosumab, palivizumab, cetuximab, omalizumab,
natalizumab, panitumumab, ustekinumab, ofatumumab, pertuzumab, etc.
As used herein, "Cell density" refers to that number of cells present in a given volume of
medium.
As used herein, "mammalian cell culture" refers to a cell population that is suspended in a
medium under conditions suitable to survival and/or growth of the cell population. It refers to
growth and propagation of mammalian cells outside of a multicellular organism or tissue.
Suitable culture conditions for mammalian cells are known in the art. Mammalian cells may
be cultured in suspension or while attached to a solid substrate.
As used herein, "fed-batch culture" refers to a method of culturing cells in which additional
components are provided to the culture at some time subsequent to the beginning of the
culture process. The provided components typically comprise nutritional supplements for the
cells which have been depleted during the culturing process.
As used herein, mammalian cell culture process refers to the use of recombinant mammalian
cell lines such as CHO DUKX-B1 1, CHO S, CHO Kl, CHO DG44.
As used herein, the starting VCC(viable cell count) after inoculation of seed in the
monophasic Production Bioreactor, are selected fromO.lx 106 cells/ mL to 10 x 106 cells/ mL
and viability >90%, more preferably at 0.3x 106 cells/ mL to 5 x 106 cells/ mL and viability
>90% and even more preferably at lx 106 cells/ mL to 2 x 106 cells/ mL and viability >90%.
The VCC may be measured during the process at suitable time intervals.
As used herein, "about" with reference to temperature refers to deviation in temperature
value wherein it covers ± 1°C e.g. about 33°C covers temperature from 32°C to 34°C.
As used herein, "about" with reference to pH refers to deviation in pH value wherein it
covers ± 0.5 e.g. about pH 6.7 covers pH from 6.2 to 7.2.
In another embodiment, the invention is related to process for culturing mammalian cell
carried out with monophasic temperature condition to produce fusion proteins and
monoclonal antibodies.
In yet another embodiment, the invention is related to the mammalian cell culture process
performed with monophasic temperature condition to produce glycosylated proteins wherein
the cell culture is essentially free of alkanoic acid or salt thereof.
In another embodiment the invention is related to cell culture process performed with
monophasic temperature condition at about 34°C to about 37°C to produce glycosylated
proteins which improve the desired confirmation of glycosylated protein.
In another embodiment the mammalian cell culture is carried out in batch, fed-batch and
continuous mode in suitable medium in fermenter or bioreactor, preferably in fed-batch
mode. The invention is related to the process of culturing the mammalian cell for the
production of glycosylated proteins. More particularly, the invention relates to specific
culture conditions which are maintained during the mammalian cell culture process.
Although a lot of processes have been developed and reported in the literature for the
production of glycosylated protein from the mammalian cell culture, however those
processes are typically biphasic, requiring two sets of temperatures and/or dissolved oxygen
and/or pH. In the biphasic cell culture condition the recombinant host cells are first grown at
a temperature which promotes rapid cell multiplication, referred to as the growth phase. This
is typically achieved by growing cells at 34-37°C, then lowering the temperature to 22-34°C
which reduces cell growth while favoring the production of the recombinant protein, referred
to as the production phase.
The cell culture process requires various parameters to carry out the process in effective and
efficient way. However, the main aspect of the invention is the use of monophasic
temperature during the culture wherein the monophasic temperature does not have
temperature shift and produce desire quality and quantity of the glycosylated protein with
significant cell viability.
The invention studies the effect of temperature over the production of protein, its quality and
cell viability by using the techniques known in the art. Temperature is selected from 37°C or
36°C or 35°C or 34°C or 33°C.
In another embodiment monophasic temperature conditionat about 34°C to about 37°C
improve the desired confirmation of glycosylated protein.
In another embodiment the invention is related to cell culture process for the production of
glycosylated proteins in monophasic cell culture condition wherein the mammalian cells are
cultured at temperature maintained between from at about 32°C to at about 37°C.
In another embodiment the invention isrelated to cell culture process for the production of
glycosylated proteins in monophasic cell culture condition wherein the mammalian cells are
cultured at temperature maintained between from at about 34°C to at about 37°C.
In one embodiment of the invention, the monophasic temperature is set at about 33°C.
In one embodiment of the invention, the monophasic temperature is set at about 34°C.
In one embodiment of the invention, the monophasic temperature is set at about 35°C.
In one embodiment of the invention, the monophasic temperature is set at about 36°C.
In another embodiment of the invention, the monophasic temperature is set at about 37 °C.
In an embodiment, the invention is related a process of producing glycosylated protein in a
mammalian cell culture the process comprising the steps of :
a) preparing inoculum with suitable cell concentration during seed development;
b) inoculating the inoculum with suitable cell concentration in to production
bioreactor;
c) culturing the cell in production bioreactor at suitable conditions wherein the
suitable condition is monophasic temperature condition; and
d) obtaining the glycosylated protein from the cell culture.
In an embodiment, the invention is related a process of producing glycosylated protein in a
mammalian cell culture the process comprising the steps of :
a) preparing inoculum with suitable cell concentration during seed development;
b) inoculating the inoculum with suitable cell concentration in to production
bioreactor;
c) culturing the cell in production bioreactor at suitable conditions wherein the
suitable condition is monophasic temperature condition; and
d) obtaining the glycosylated protein from the cell culture,
wherein monophasic temperature does not have temperature shift during step (c).
In an embodiment, the invention is related a process of producing glycosylated protein in a
mammalian cell culture the process comprising the steps of :
a) preparing inoculum with suitable cell concentration during seed development;
b) inoculating the inoculum with suitable cell concentration in to production
bioreactor;
c) culturing the cell in production bioreactor at suitable conditions wherein the
suitable condition is monophasic temperature condition; and
d) obtaining the glycosylated protein from the cell culture,
wherein the production bioreactor in step (c) does not have any distinctive growth phase and
production phase.
In another embodiment the seed development steps carried out to develop the inoculum
having suitable cell concentration. It required at least about 72 hours to develop inoculum
with desired cell concentration thereafter the inoculum is inoculated in to the production
bioreactor for further scale up and protein production.
In another embodiment the cell culture process is carried out in production fermenter or
production bioreactor. The cells may be cultured for total period of 9 to 40 days. In another
embodiment, the protein is harvested at least before the day 15, preferably on day 13, more
preferably on day 12 and most preferably on day 11. In another embodiment the protein is
harvested before the cell viability reached below < 90%. In another embodiment the protein
is harvested before the cell viability reached below < 70%. In another embodiment the
protein is harvested before the cell viability reached below < 50%.In preferred embodiment
the suitable culture condition is monophasic temperature which does not have temperature
shift. Temperature is selected from about 330C to about 370C preferably from about 330C to
about 36°C.
In another embodiment the suitable conditions during the culture may be further selected
from pH, osmolality, dissolved oxygen concentration and cell density.
The seed preparation is initiated with suitable concentration of cells which are selected from
0.1 x 106 cells/mL to 0.5 x 106 cells/mL in suitable medium. In preferred embodiment seed
preparation is initiated with 0.3 x 106 cells/mL in suitable medium. In another preferred
embodiment seed preparation is initiated with 0.3 x 106 cells/mL in suitable medium and
further supplemented with suitable concentration of glutamine and methotrexate. The
suitable concentration of glutamine and methotrexate is selected from 3mM to 6mM,
preferably 4mM and 70nM to , preferably 80nM respectively.
In another embodiment the suitable concentration of glutamine is 6mM.
In certain embodiment the seed culture is maintained at about 3% to 8% C0 2, preferably 5%
C0 2, dissolved oxygen concentration is selected from at about 30% to about 70% preferably
at 50%, relative humidity is selected from about 70% to about 90%, preferably 85%,
agitation speed is selected from about 0.2m/s to about 0.4m/s, preferably 0.3m/s and
temperature is selected from between at about 34°C to at about 37°C, preferably 37°C. In
certain embodiment the concentration of dissolved oxygen concentration is maintained by
sparging with air at 0.00 to 0.03 vvm and oxygen at 0.00 to 0.09vvm. In certain embodiment
the seed culture is further amplified by passage by diluting to at least 0.1 x 106 cells/mL. The
dilution may be performed during intervals of seed culture. In another embodiment the
dilution is performed at least on 3 day or at 72 hours.
In preferred embodiment the seed culture is further amplified by supplementing feed till the
culture grow to suitable concentration of cell. The seed culture is further amplified by
supplementing feed till the culture grows to suitable concentration of cell for suitable time
period. The seed culture may be further amplified by supplementing feed for at least 5 days
and the suitable concentration of cell i.e. inoculum is selected from about 4 x 106 cells/mL to
about 7 x 106 cells/mL preferably 5 x 106 cells/mL.
In embodiment the feed is supplemented to the cell culturing in suitable bioreactor or
fermenter in suitable culture conditions. In certain embodiment the cells are cultured at
suitable culture conditions is selected from about 3% to 6% C0 2, preferably 5% C0 2,
dissolved oxygen concentration is selected from at about 30% to about 70% preferably at
50%, relative humidity is selected from about 70% to about 90%, preferably 85%, agitation
speed is selected from about 0.2m/s to about 0.4m/s, preferably 0.3m/s and temperature is
selected from between at about 34 °C to at about 37 °C, preferably 37°C . In certain
embodiment the concentration of dissolved oxygen concentration is maintained by sparging
with air at 0.03vvm to 0,09vvm.
In embodiment the viability is cell during the seed culture is maintained at least by >70%,
preferably >80%, more preferably >90%.
In preferred embodiment the cells were taken from a cell bank and cultured in a shake flask
containing growth medium with or without methotrexate to an initial viable cell density
(VCC) of 0.3x 10° cells /mL. The seed culture flask was then maintained at a set temperature
of 37°C at 5% C0 2 concentration at 120 rpm and approx. 85% relative humidity. The seed
volume was volumetrically amplified during subsequent passages by diluting to 0.3x 106
cells /mL after every 72 hr. The N-1 seed was additionally supplemented with 10% of feed
on day 1 and day 3 in bioreactor (or flasks) and incubated for 120 hr. Log phase cells from
the N-2 bioreactor (or flasks) at a VCC of 2 to 2.5 x 106 cells/ mL and viability >90% was
used to inoculate the N-1 bioreactor (or flasks). The N-1 bioreactor was maintained at a tip
speed of -0.3 m/s with dissolved oxygen at approx. 50% of dissolved oxygen saturation by
sparging separately air at 0.00 to 0.03 vvm and oxygen at 0.00 to 0.09vvm, respectively to
achieve a final VCC of 2 to 10 x 10 cells/ mL and viability >90%, more preferably at 4 to 8
x 106 cells/ mL and viability >90% and even more preferably at 5 to 6 x 106 cells/ mL and
viability >90%.
The suitable concentration of cells obtained from seed culture is referred as inoculum which
is transferred to bioreactor or fermenter to initiate the culture at high density. In certain
embodiment the suitable concentration of inoculum is selected from about 4 x 106 cells/mL
to about 7 x 106 cells/mL preferably 5 x 106 cells/mL.
The batch or the production bioreactor is initiated with inoculum having suitable
concentration of cells which is selected from 4 x 106 cells/mL to 7 x 106 cells/mL in suitable
medium. In preferred embodiment seed density is 1.2 x 106 cells/mL (which may be
achieved by diluting 5 x 106 cells/mL cells/mL or 6 x 106 cells/mL cells/mL obtained
through seed preparation). In another preferred embodiment seed density is 1 x 106 cells/mL.
In certain preferred embodiment the cells are culture in suitable medium and further
supplemented with suitable concentration of glutamine. The suitable concentration of
glutamine is selected from 3mM to 6mM, preferably 4mM.
In certain embodiment the production bioreactor is maintained at about pH from about 6 to
about 8, preferably about 6.7 to 7.4, more preferably 7, 3% to 6% C0 2, preferably 5% C0 2,
dissolved oxygen concentration is selected from at about 30% to about 70% preferably at
50%, agitation speed is selected from about 0.2m/s to about 0.5 m/s, preferably 0.3m/s and
temperature is selected from between at about 34 °C to at about 37 °C, preferably 34 °C. In
certain embodiment the concentration of dissolved oxygen concentration is maintained by
sparging with air at 0.03vvm to 0,09vvm.
The sodium bicarbonate and C0 2gas is used to control the pH of the culture.
In embodiment the feeding is performed during the batch/bioreactor culture based on the
residual glucose level. The glucose level is adjusted to at least 2 g/L, if the glucose level is
below 2 g/L, feed is supplemented to maintain the suitable glucose level. The glucose
concentration of the culture is monitored in every 12 hr or 18hr or 24 hr.
In embodiment the feed may contain from about 30 to about 35 g/L of glucose.
In another embodiment the feed may contain from about 180 to about 220 mM, L-glutamine,
preferably 200 mM of L-glutamine solution. In one embodiment the L-glutamine solution is
added at 1% of the initiation volume to the culture at 2 days and then subsequently in every
2daystill 1 1 days. In another embodiment the L-glutamine solution is added at 1% of the
initiation volume to the culture at 4 days and then at 7 days and at 9 days.
In another embodiment the D-glucose is supplemented as feed to the culture. The
concentration of D-glucose is selected from about 60 g to about 90 g, preferably 80g. In
certain embodiment the D-glucose is supplemented as feed to the culture at least after 9 days
or at least fromlOdays.
In another embodiment the desire protein is harvested at least after 10 days or after 1 1 days
or after 12days or after 13days.
In another embodiment the harvesting of the culture is performed when culture viability
drops below 40% to 70%, preferably below 50%.
In another embodiment the production bioreactor process was initiated by inoculating N-l
seed in to a bioreactor containing growth medium at approx. 55% of the final batch volume
at the starting VCC (after inoculation of the seed) in the monophasic Production Bioreactor
ofO.l to 10 x 106 cells/ mL and viability >90%, more preferably VCC at 0.3 - 5 x 106 cells/
mL and viability >90% and even more preferably VCC at 1 to 2 x 106 cells/ mL and viability
>90%. The culture pH was maintained at pH ranging between 6.7 to 7.4 by addition of 8%
sodium bicarbonate (NaHCOs) or C0 2 gas. The agitation speed was set as per the tip speed
ranging from 0.3 to 0.5 m/s, and dissolved oxygen concentration maintained at 50%
dissolved oxygen saturation controlled by sparging air at 0.00 to 0.03 vvm and oxygen at
0.00 to 0.09vvm. The temperature was set at a single set point between 34 °C to 37°C,
preferably 34°C throughout the Production Bioreactor process (monophasic). Feeding of the
reactor was done based on the residual glucose levels. The glucose concentration of the
culture monitored every 24 hr. and adjusted to 3 g/L if the glucose level is below 2 g/L with
addition of the feed. The feed contains 33.5 g/1 glucose. 200 mM of L-glutamine solution was
added at 1% of the initiation volume to the culture at 48 hr. and every 48 hr. thereon up to
240 hr. Harvesting of the culture was done on the 264 hr. or if the culture viability drops
below 90%, whichever is attained first.
The cell culture process was initiated in bioreactor with the viable cell concentration which is
selected from about l lO6 cells/mL to 2 x 106 cells/mL, preferably 1.2 x 106 cells/mL. In
another embodiment the viable cell concentration is 1 x 106 cells/mL. . In certain
embodiment, the suitable conditions for culturing the cell is selected from pH 6.7 to 7.4,
preferably 7.0. In another embodiment the osmolality is selected from about 250 to about 550
mOSm/Kg. In another embodiment the dissolved oxygen is selected from at about 30% to
about 70% preferably at 50% set point.
In preferred embodiment the growth and production phase is not distinctive in the production
bioreactor.
In another embodiment the production phase may batch or fed-batch.
In embodiment the harvested protein is clarified by the techniques known in art to skilled
person. In preferred embodiment the harvested protein present in broth obtained from
bioreactor or fermenter. EDTA solution is added in suitable concentration which could be 5
mM and then clarification carried out depth filtration using POD system.
The media composition is very important to improve the culture longevity and production.
Basal ceil culture medium formulations are well known in the art. To these basal culture
medium formulations the skilled artisan will add components such as amino acids, salts,
sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements and the
like, depending on the requirements of the host cells to be cultured. The culture medium may
or may not contain serum and/or protein. Various tissue culture media, including serum-free
and/or defined culture media, are commercially available for ceil culture. Tissue culture
edia is defined, for purposes of the invention as a media suitable for growth of animal
cells, and preferably mammalian cells, in in vitro cell culture. Typically, tissue culture media
contains a buffer, salts, energy source, amino acids, vitamins and trace essential elements.
Any media capable of supporting growth of the appropriate eukaryotic cell in culture is used;
the invention is broadly applicable to eukaryotic cells in culture, particularly mammalian
cells, and the choice of media is no crucial to the invention. Tissue culture media suitable for
use in the invention are commercially available from, e.g., ATCC (Manassas, Va.).
example, any one or combination of the following media is used; RPMI-164G Medium,
RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential
Medium Eagle, F-12K Medium, Ham's F12 Medium, Iscove's Modified Dulbecco's Medium,
McCoy's 5A Medium, Leibovitz's L-15 Medium, and serum-free media such as EX-CELL™
300 Series. In preferred embodiment the medium is EX-CELL™ 302 medium.
In another preferred embodiment the mix feed media comprises BalanCD CHO Feed 2, EXCELL
302 Powder Medium, NaHCOS, L-Glutamine Powder, MEM Amino Acid (50 X),
MEM Non-Essential Amino Acid (100 X), MEM Vitamins Solution (100 X).
In the methods and compositions of the invention, cells may be grown in serum-free, proteinfree
growth factor-free, and/or peptone-free media. The term '"serum-free" as applied to
media includes any mammalian cell culture medium that does not contain serum, such as
fetal bovine serum.
The skilled artisan may also choose to use one of the many individualized media
formulations that have been developed to maximize cell growth, cell viability, and/or
recombinant polypeptide production in a particular cultured host cell. The methods according
to the current invention may be used in combination with commercially available ceil culture
media or with a cell culture medium that has been individually formulated for use with a
particular cell line
In preferred embodiment the medium is serum free. In another preferred embodiment the
medium is essentially free of alkanoic acid or salt thereof. The alkanoic acid and salt thereof
are selected from butyric acid, sodium butyrate or dibutyl cAMP.
In embodiment the glycosylated proteinsare selected from Abciximab; Abatacept;
Adalimumab; Abrilumab; Afutuzumab; Aflibercept; Alemtuzumab; Alefacept; Alacizumab
pegol; Anakinra; Arcitumomab; Atacicept; Atlizumab; Atorolimumab; Basiliximab;
Baminercept; Bectumomab; Belimumab; Besilesomab; Bevacizumab; Biciromab;
Belatacept; Brentuximab vedotin; Brodalumab; Canakinumab; Capromab pendetide;
Catumaxomab; Certolizumab pegol; Cetuximab; Clivatuzumab tetraxetan; Daclizumab;
Denosumab; Eculizumab; Edrecolomab; Efalizumab; Efungumab; Eloctate; Ertumaxomab;
Etanercept; Etaracizumab; Fanolesomab; Farletuzumab; Fontolizumab; Gemtuzumab
ozogamicin; Girentuximab; Golimumab; Ibritumomab tiuxetan; Igovomab; Imciromab;
Infliximab; Ipilimumab; Labetuzumab; Mepolizumab; Motavizumab; Muromonab-CD3;
Natalizumab; Nimotuzumab; Nofetumomab merpentan; Obinutuzumab; Ofatumumab;
Omalizumab; Oregovomab; Palivizumab; Panitumumab; Pemtumomab; Pertuzumab;
Ramucirumab; Ranibizumab; Raxibacumab; Rituximab; Rilonacept; Rovelizumab;
Ruplizumab; Sulesomab; Tacatuzumab tetraxetan; Tefibazumab; Tocilizumab; Trastuzumab;
Ado-Trastuzumab Emtansine; Tositumomab; TRBS07; Ustekinumab; Vedolizumab;
Visilizumab; Votumumab; Zalutumumab; Zanolimumab..
The purification of the polypeptide may include an affinity column containing agents which
will bind to the polypeptide; one or more column steps over such affinity resins as
concanavalin A-agarose, heparin-TOYOPEARL® (Toyo Soda Manufacturing Co., Ltd.,
Japan) or Cibacrom blue 3GA SEPHAROSE® (Pharmacia Fine Chemicals, Inc., New York);
one or more steps involving eiution; and/or immunoaffinity chromatography. The
polypeptide may be expressed in a form that facilitates purification. For example, it may be
expressed as a fusion polypeptide, such as those of maltose binding polypeptide (MBP),
glutathione-S-transferase (GST), or thioredoxin (TRX). Kits for expression and purification
of such fusion polypeptides are commercially available from New England BioLab (Beverly,
Mass.), Pharmacia (Piscalaway, N.J.) and InVitrogen, respectively. The polypeptide may be
tagged with an epitope and subsequently purified by using a specific antibody directed to
suc epitope. One such epitope (FLAG®) is commercially available from Kodak (New
Haven, Conn.). t is also possible to utilize a affinity column comprising a polypeptidebinding
protein, such as a monoclonal antibody to the recombinant polypeptide, to affinitypurify
expressed polypeptides. Other types of affinity purification steps may be a Protein A
or a Protein G column, which affinity agents bind to proteins that contain Fc domains.
Polypeptides may be removed from an affinity column using conventional techniques, e.g. in
a high salt eiution buffer and then dialyzed into a lower salt buffer for use or by changing p
or other components depending on the affinity matrix utilized, or may be competitively
removed using d e naturally occurring substrate of the affinity moiety.
Although certain embodiments in detail above and the invention may be illustrated by way of
examples below, those having ordinary skill in the art wil clearly understand tha many
modifications are possible in the embodiments and examples without departing from the
teachings thereof
Examples
The CHO cell line was established by co-transfection of the dihydrofolate reductase (dhfr)
and gene of interest transfected into dhfr-deficient CHO cells (DUKX-B11, ATCC CRL-
9096) followed by subsequent dhfr/MTX-mediated gene amplification. The clones were
prepared as per the techniques well known in the art related to recombinant r-DNA
technology.
Example 1: Production of Etanercept through monophasic process
Seed Expansion process
The seed expansion was initiated post revival of a vial from the cell bank at 0.3 x 106 cells
/mL in growth medium supplemented with 4 mM L-Glutamine with MTX. The concentration
of MTX was maintained at 80 nM throughout the stage of seed development. The seed
culture was maintained at 37 °C at 5% C02 concentration at 120 rpm and approx. 80%
Relative humidity in shake flask.
The seed volume was adequately amplified at every subsequent passage by diluting to 0.3x
106 cells /mL after every 72 hr. at log phase. The N-2 seed was initiated with 0.3xl0 6
cells/mL and additionally supplemented with 10% of mixed feed (of the initiation volume)
on 24 hr and 72 hr and cultured for 120 hr.
Log phase cells from the N-2 flask at a VCC of 5.5x 106 cells/ mL and viability >95% was
used to inoculate the N-l flask with an initial seed density of 1.2 xlO6 cells/mL.
The N-l seed flask was run for 120 hr with feeding with 10 % mixed feed (of the initiation
volume) at 24 hr and 72 hr to obtain a viable cell density of 6.6x 106 cells /mL and a
viability above >95%.
Production Bioreactor process
The batch process was initiated by inoculating N-1 seed in to a bioreactor containing growth
medium at approx. 55% of the final batch volume at the starting VCC (after inoculation of
the seed) in the Monophasic Production Bioreactor of 1.2 x 106 cells/ mL and viability >98%.
The culture pH was maintained at pH ranging between 6.7 to 7.4 by addition of 8% sodium
bicarbonate (NaHC0 3) or C0 2 gas. The agitation speed was set as per the tip speed ranging
from 0.3 to 0.5 m/s, and dissolved oxygen concentration maintained at 50% dissolved
oxygen saturation by sparging air at 0.00 to 0.03 vvm and oxygen at 0.00 to 0.09vvm. The
temperature was set at a single set point between 34°C throughout the Production Bioreactor
process (monophasic). Feeding of the reactor was done based on the residual glucose levels.
The glucose concentration of the culture monitored every 24 hr. and adjusted to 3 g/L every
24 hr if the residual glucose level is below 2 g/L with addition of the feed (the feed contains
33.5 g/1 glucose). 200 mM of L-glutamine solution was added at 1% of the initiation volume
to the culture at 48 hr. and every 48 hr thereon up to 240 hr. The culture attained a peak VCC
of around 1lxlO 6 cells/mL. Harvesting of the culture was done on the 264 hr.
Example 2 : Production of Bevacizumab through monophasic process
Seed Expansion process
The seed expansion was initiated post revival of a vial from the cell bank at 0.4 x 106 cells
/mL in growth medium supplemented with 6 mM L-Glutamine. The seed culture was
maintained at 37 °C at 8% C0 2 concentration at 150 rpm and approx. 80% Relative humidity
in shake flask.
The seed volume was adequately amplified at every subsequent passage by diluting to 0.4x
106 cells /mL after every 72 hr. at log phase.
The N-1 seed flask was run for 72 hr to obtain a viable cell density of 5.5x 106 cells /mL and
a viability above >95%.
Production Bioreactor process
The batch process was initiated by inoculating N-1 seed in to a bioreactor containing growth
medium at approx. 69% of the final batch volume at the starting VCC (after inoculation of
the seed) in the Monophasic Production Bioreactor of 1 x 106 cells/ mL and viability >98%.
The culture pH was maintained at pH ranging between 6.8 to 7.4 by addition of 8% sodium
bicarbonate (NaHC03) or C02 gas. The agitation speed was set as per the tip speed ranging
from 0.3 to 0.5 m/s, and dissolved oxygen concentration maintained at 50% dissolved
oxygen saturation controlled by sparging air at 0.00 to 0.03 vvm and oxygen at 0.00 to
0.09vvm. The temperature was set at a single set point between 34°C throughout the
Production Bioreactor process (monophasic). Feeding of the reactor was done based on the
residual glucose levels. The glucose concentration of the culture monitored every 24 hr. and
adjusted to 4 g/L every 24 hr if the residual glucose level is below 2 g/L with addition of the
feed (the feed contains 33.5 g/1 glucose). 200 mM of L-glutamine solution was added at 1%
of the initiation volume to the culture on 96, 168 and 216 hr. The culture attained a peak
VCC of around 1lxlO 6 cells/mL. Harvesting of the culture was done on the 264 hr.
Example 3: Production of Rituximab through monophasic process
Seed Expansion process
A vial of rituximab from liquid nitrogen was thawed and the cells were inoculated in a 125
mL shake flask containing growth medium and was cultured in C0 2 incubator shaker at 37
C, 120 rpm. Cells were passaged every 72 h ±24 h with increase in culture volume
appropriate for inoculating the production bioreactor. In each passage seeding density was
maintained at 0.3 x 106 cells/mL and target for a final VCC of 3.0 ±1.0 xlO6 cells/mL.
Production Bioreactor process
The batch process was initiated by inoculating N-1 seed in to a bioreactor containing growth
medium at approx. 40 ± 5% of the final batch volume at the starting VCC (after inoculation
of the seed) in the monophasic Production bioreactor of 0.5±0.2 xlO6 cells/ mL and viability
>90%, more preferably VCC at 0.5xl0 6 cells/ mL and viability >95% The agitation speed
was set as per the tip speed ranging from 0.4 to 0.6 m/s. The temperature was set at a single
set point i.e. 36 °C throughout the production bioreactor process. Feeding of the production
bioreactor was done at day 2, 3, 5, 6, 7 and 8 for the cell culture maintenance, productivity
and product quality attributes. Glutamine and Glucose were maintained throughout the cell
culture duration at about 2mM and 2g/L respectively for cell culture maintenance. Harvesting
of the culture was done on the <3 12 h or if the culture viability drops below 50%, whichever
is earlier.
Example 4 : Production of Adalimumab through monophasic process
Seed Expansion process
A vial of Adalimumab from liquid nitrogen was thawed and the cells were inoculated in a
125 mL shake flask containing growth medium and was cultured in C0 2 incubator shaker at
37 C, 120 rpm. Cells were passaged every 72 h ±24 h with increase in culture volume
appropriate for seeding into production bioreactor. In each passage seeding density was
maintained at 0.3 x 106 cells/mL and target for a final VCC of about 3.0 ± 1.0 xlO6
cells/mL.
Production Bioreactor process
The batch process was initiated by inoculating N-1 seed in to a bioreactor containing growth
medium at approx. 60 ±10% of the final batch volume at the starting VCC (after inoculation
of the seed) in the monophasic Production bioreactor of 0.5 ±0.2 x 106 cells/ mL and viability
>90%, more preferably VCC at 0.5 x 106 cells/ mL and viability >95%. The agitation speed
was set as per the tip speed ranging from 0.6 ±0.2 m/s. The temperature was set at a single set
point at 36 °C throughout the Production bioreactor process. Feeding was done on day 3, 6, 9
and 11 for the cell culture maintenance, productivity and product quality attributes.
Glutamine and Glucose were maintained throughout the cell culture duration at about 2mM
and 2g/L respectively for cell culture maintenance. The harvest criteria was set at <50% cell
viability or 288 h ±12 h whichever is earlier.
Example 5: Production of Trastuzumab through monophasic process
Seed Expansion process
A vial of Trastuzumab from liquid nitrogen was thawed and the cells were inoculated in a
125 mL shake flask containing growth medium and was cultured in C0 2 incubator shaker at
37 C, 120 rpm, 5% C0 2, 85% Relative humidity. Cells were passaged every 72 h ±24 h with
increase in culture volume for inoculating production bioreactor. In each passage seeding
density was maintained at 0.3 x 106 cells/mL and target for a final VCC of about 3.0 ±1.0
xlO6 cells/mL.
Production Bioreactor process
The batch process was initiated by inoculating N-l seed in to a bioreactor containing growth
medium at approx. 40 ±5% of the final batch volume at the starting VCC (after inoculation of
the seed) in the monophasic production bioreactor of 0.5 ±0.2 xlO6 cells/ mL and viability
>90%, more preferably VCC at 0.5 xlO6 cells/ mL and viability >95%. The agitation speed
was set as per the tip speed ranging from 0.4 to 0.6 m/s. The temperature was at 34 °C
throughout the production bioreactor process. Feeding of the reactor was done at day 2, 4, 6,
8 and 10 to maintain cell culture longevity, productivity and product quality attributes.
Glutamine and Glucose were maintained throughout the cell culture duration at about 2mM
and 2g/L respectively for cell culture maintenance. Harvesting of the culture was done on the
<312 h or if the culture viability drops below 50%, whichever is earlier.
All patents, patent applications and publications cited in this application are hereby
incorporated by reference in their entirety for all purposes to the same extent as if each
individual patent, patent application or publication were so individually denoted.
CLAIMS
1. A process of producing glycosylated protein in a mammalian cell culture the process
comprising the steps of :
a) preparing inoculum with suitable cell concentration during seed development,
b) inoculating the inoculum with suitable cell concentration in to production
bioreactor,
c) culturing the cell in production bioreactor at suitable conditions wherein the
suitable condition is monophasic temperature condition; and
d) obtaining the glycosylated protein from the cell culture.
2. The process as claimed in claim 1, wherein monophasic temperature is selected from
the range of about 32 °C to about 37 °C.
3. The process as claimed in claim 1, wherein monophasic temperature is selected from
the range of about 34 °C to about 37 °C.
4. The process as claimed in claim 2 or 3, wherein monophasic temperature is about 33
°C.
5. The process as claimed in claim 2 or 3, wherein monophasic temperature is about 34
°C.
6. The process as claimed in claim 2 or 3, wherein monophasic temperature is about 35
°C.
7. The process as claimed in claim 2 or 3, wherein monophasic temperature is about 36
°C.
8. The process as claimed in claim 1, wherein monophasic temperature does not
comprise temperature shift.
9. The process as claimed in claim 1, wherein the mammalian cell is selected from CHO
cell.
10. The process as claimed in claim 9, wherein the CHO cell is a dhfr- CHO cell.
11. The process as claimed in any one of the preceding claims wherein the mammalian
cells are cultured in fed-batch mode in production bioreactor.
12. The process as claimed in any one of the preceding claims wherein the cell culture
process does not have distinctive growth phase and production phase.
13. The process as claimed in claim 1, wherein the suitable conditions further comprises
pH selected from 6.7 to 7.4.
14. The process as claimed in claim 13, wherein the suitable conditions further
comprises pH is about 7.
15. The process as claimed in claim 1, wherein the suitable conditions further comprises
osmolality from about 250 to about 550 mOSm/Kg.
16. The process as claimed in claim 15, wherein the osmolality is about 270 mOSm/Kg.
17. The process as claimed in any one of the preceding claims wherein the mammalian
cells are cultured in serum containing medium or serum free medium.
18. The process as claimed in claim 17, wherein the mammalian cells are cultured in
serum free medium.
19. The process as claimed in any one of the preceding claims wherein the mammalian
cells are cultured in medium essentially free of alkanoic acid or salt thereof.
20. The process as claimed in claim 19, wherein the alkanoic acid or salt thereof are
selected from butyric acid, sodium butyrate or dibutyl cAMP.
21. The process as claimed in claim 1, wherein the suitable conditions further comprises
dissolved oxygen concentration from about 30% to about 70%.
22. The process as claimed in claim 1, wherein the suitable concentration of inoculum is
selected from about 4 x 106 cells/mL to about 7 x 106 cells/mL.
23. The process as claimed in claim 1, wherein the suitable concentration of inoculum is
obtained at least by 72 hours.
24. The process as claimed in claim 1, wherein the protein is glycosylated protein which
are selected from fusion proteins and monoclonal antibodies and fragment thereof.
25. The process as claimed in claim 24, wherein the fusion protein and monoclonal
antibody and fragment thereof are selected from Abciximab; Abatacept;
Adalimumab; Abrilumab; Afutuzumab; Aflibercept; Alemtuzumab; Alefacept;
Alacizumab pegol; Anakinra; Arcitumomab; Atacicept; Atlizumab; Atorolimumab;
Basiliximab; Baminercept; Bectumomab; Belimumab; Besilesomab; Bevacizumab;
Biciromab; Belatacept; Brentuximab vedotin; Brodalumab; Canakinumab; Capromab
pendetide; Catumaxomab; Certolizumab pegol; Cetuximab; Clivatuzumab tetraxetan;
Daclizumab; Denosumab; Eculizumab; Edrecolomab; Efalizumab; Efungumab;
Eloctate; Ertumaxomab; Etanercept; Etaracizumab; Fanolesomab; Farletuzumab;
Fontolizumab; Gemtuzumab ozogamicin; Girentuximab; Golimumab; Ibritumomab
tiuxetan; Igovomab; Imciromab; Infliximab; Ipilimumab; Labetuzumab;
Mepolizumab; Motavizumab; Muromonab-CD3; Natalizumab; Nimotuzumab;
Nofetumomab merpentan; Obinutuzumab; Ofatumumab; Omalizumab; Oregovomab;
Palivizumab; Panitumumab; Pemtumomab; Pertuzumab; Ramucirumab;
Ranibizumab; Raxibacumab; Rituximab; Rilonacept; Rovelizumab; Ruplizumab;
Sulesomab; Tacatuzumab tetraxetan; Tefibazumab; Tocilizumab; Trastuzumab; Ado-
Trastuzumab Emtansine; Tositumomab; TRBS07; Ustekinumab; Vedolizumab;
Visilizumab; Votumumab; Zalutumumab; Zanolimumab.
26. The process as claimed in claim 24 or 25 wherein the fusion protein is Etanercept.
27. The process as claimed in claim 1, wherein the culture conditions maintains a high
viable cells count.
28. The process as claimed in claim 27, wherein the viable cell count is selected from
about 5xl0 6 to about 13xl0 6 cells/mL.
29. The process as claimed in claim 28, wherein the viable cell count is 1lxlO 6 cells/mL.
30. The process as claimed in any of the preceding claims improves the desire
confirmation of glycosylated proteins.
31. The process as claimed in any of the preceding claims improves the desire
confirmation of TNFR-Fc fusion proteins such as Etanercept.
32. The process as claimed in claim 1, wherein the mammalian cells are cultured in
production bioreactor for at least about 10 days to about 13 days.
33. The process as claimed in claim 32, wherein the mammalian cells are cultured in
production bioreactor for at least 11 days.
34. A process of producing fusion protein and monoclonal antibody and fragment thereof
in a mammalian cell culture the process comprising the steps of :
a) preparing inoculum with suitable cell concentration during seed development;
b) inoculating the inoculum with suitable cell concentration in to production
bioreactor;
c) culturing the cell in production bioreactor at suitable conditions wherein the
suitable condition is monophasic temperature condition; and
d) obtaining the glycosylated protein from the cell culture,
wherein the suitable conditions are
i) Monophasic temperature selected from about 34°C to about 37°C,
ii) pH is selected from 6.7 to 7.4,
iii) Osmolality from about 250 mOSm/Kg to about 550 mOSm/Kg.
35. A process of producing fusion protein and monoclonal antibody and fragment thereof
in a mammalian cell culture the process comprising the steps of :
a) preparing inoculum with suitable cell concentration during seed development;
b) inoculating the inoculum with suitable cell concentration in to production
bioreactor;
c) culturing the cell in production bioreactor at suitable conditions wherein the
suitable condition is monophasic temperature condition; and
d) obtaining the glycosylated protein from the cell culture,
wherein the suitable conditions are
i) monophasic temperature selected from about 34°C to about 37°C,
ii) pH is selected from 6.7 to 7.4,
iii) osmolality from about 250 mOSm/Kg to about 550 mOSm/Kg,
wherein the medium essentially free of alkanoic acid or salt thereof; and
wherein the cell culture process does not have distinctive growth phase and
production phase.
A process of producing TNFR-Fc fusion protein in a mammalian cell culture the
process comprising the steps of :
a) preparing inoculum with suitable cell concentration during seed development;
b) inoculating the inoculum with suitable cell concentration in to production
bioreactor;
c) culturing the cell in production bioreactor at suitable conditions wherein the
suitable condition is monophasic temperature condition; and
d) obtaining the glycosylated protein from the cell culture,
wherein the suitable conditions are
i) monophasic temperature selected from at about 34 °C to about 37 °C,
ii) pH is selected from 6.7 to 7.4,
iii) osmolality from about 250 mOSm/Kg to about 550 mOSm/Kg.
The process as claimed in any one of the proceeding claims, wherein the cell is
selected from CHO DUKX-B1 1, CHO S, CHO Kl or CHO DG44.
A fusion protein or monoclonal antibody and fragment thereof obtained from the
process as claimed in any of the preceding claims.

International application No
PCT/IB2015/054196
A. CLASSIFICATION OF SUBJECT MATTER
INV. C12P21/00 C12N9/00 C12N15/62
ADD.
According to International Patent Classification (IPC) or to both national classification and IPC
B. FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols
C12P C12N
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
EPO-Internal WPI Data, BIOSIS, EMBASE
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.
WO 2009/0863O9 A2 (BAXTER INT [US] ; BAXTER 1-20,22.
HEALTHCARE SA [CH] ; GIOVAGNOLI ANDRE [FR] ; 23 ,27 ,
ROY) 9 July 2009 (2009-07-09) 30,34,
35 ,37
page 8 , 1i ne 16 page 9 , l ine 10; c l aims
1-13 ,43-48
page 15, l ine 25 - page 16, 1i ne 8
page 18, l ine 6 l ine 14
W0 99/28455 Al (ROCHE DIAGNOSTICS GMBH I - 9 ,
[DE] ; FRANZE REINHARD [DE] ; EBERHARDT II- 2 1 ,
H0RST [DE) 1 June 1999 (1999-06-10) 23 ,
25-27 ,
30,31 ,36
page 22, l ine 23 - page 23, l i ne 2 ;
exampl e s 5,6; tabl e 2
/ -
X Further documents are listed in the continuation of Box C. See patent family annex.
* Special categories of cited documents :
"T" later document published after the international filing date or priority
date and not in conflict with the application but cited to understand
"A" document defining the general state of the art which is not considered the principle or theory underlying the invention
to be of particular relevance
"E" earlier application or patent but published on or after the international "X" document of particular relevance; the claimed invention cannot be
filing date considered novel or cannot be considered to involve an inventive
"L" document which may throw doubts on priority claim(s) orwhich is step when the document is taken alone
cited to establish the publication date of another citation or other " document of particular relevance; the claimed invention cannot be
special reason (as specified) considered to involve an inventive step when the document is
"O" document referring to an oral disclosure, use, exhibition or other combined with one or more other such documents, such combination
means being obvious to a person skilled in the art
"P" document published prior to the international filing date but later than
the priority date claimed "&" document member of the same patent family
Date of the actual completion of the international search Date of mailing of the international search report
3 1 August 2015 12/11/2015
Name and mailing address of the ISA/ Authorized officer
European Patent Office, P.B. 5818 Patentlaan 2
NL - 2280 HV Rijswijk
Tel. (+31-70) 340-2040,
Fax: (+31-70) 340-3016 Schonwasser, D
International application No
PCT/IB2015/054196
C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT
Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.
X US 2004/185534 Al (KNUDSEN IDA MOLGAARD 1-9, 11 ,
[DK] ) 23 September 2004 (2004-09-23) 13-28,
30,31 ,
34-37
exampl e 1
X REUVENY S ET AL: " Factors affecti ng cel l 1-8,
growth and monocl onal anti body production 11-17 ,
i n sti rred reactors" , 19-21 ,
JOURNAL OF IMMUNOLOGICAL METHODS, ELSEVI ER 23 ,24,
SCI ENCE PUBLISHERS B.V. .AMSTERDAM, NL, 30,32 ,34
vol . 86, no. 1,
22 January 1986 (1986-01-22) , pages 53-59 ,
XP023676423,
ISSN : 0Q22-1759, DOI :
10. 1016/0022-1759 (86)90264-4
[retri eved on 1986-01-22]
page 54, col umn 1 , paragraph 3 - page 55 ,
col umn 1 , paragraph 1 ; f i gure 1
X TRUMMER EVELYN ET AL: "Process parameter 1-17,
shi fti ng: Part I . Effect of DOT, pH, and 19-21 ,
temperature on the performance of Epo-Fc 23 ,24,
expressing CH0 cel l s cul t i vated i n 27 ,30,
control led batch bioreactors" , 32-35 ,37
BIOTECHNOLOGY AND BIOENGINEERING, WI LEY &
SONS, H0B0KEN , NJ , US,
vol . 94, no. 6 ,
20 August 2006 (2006-08-20) , pages
1033-1044, XP002486305,
ISSN : 0006-3592, DOI : 10. 1002/BIT. 21013
[retri eved on 2006-05-30]
page 1034, col umn 1 , paragraph 3 - col umn
2 , paragraph 2 ; table I
page 1036, col umn 2 , paragraph 2 - page
1037 , col umn 1, paragraph 2 ; figure 1
A Y00N SUNG KWAN ET AL: "Effect of low 1-37
cul ture temperature on speci f i c
productivi t y and transcri ption level of
anti -4-lBB antibody i n recombi nant Chi nese
hamster ovary cel l s " ,
BIOTECHNOLOGY PROGRESS, AMERICAN INSTITUTE
OF CHEMICAL ENGINEERS, US,
vol . 19, no. 4 , 6 Apri l 2003 (2003-04-06) ,
pages 1383-1386, XP002331693 ,
ISSN : 8756-7938, DOI : 10. 1021/BP034051M
f i gure 1
-/--
International application No
PCT/IB2015/054196
C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT
Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.
MOHAMED B AL-FAGEEH ET AL: "The 1-37
Cold-Shock Response i n Cul tured Mammal ian
Cel l s : Harnessing the Response for the
Improvement of Recombi nant Protei n
Production" ,
BIOTECHNOLOGY AND BIOENGINEERING, WI LEY &
SONS, HOBOKEN , NJ , US,
vol . 93, no. 5 , 5 Apri l 2006 (2006-04-05) ,
pages 829-835 , XP007912835,
ISSN : 0006-3592, D0I : 10. 1002/BIT. 20789
[retri eved on 2005-12-02]
International application No
Information on patent family members
PCT/IB2015/054196
Patent document Publication Patent family Publication
cited in search report date member(s) date
WO 2009086309 A2 09-07-2009 AR 069956 Al 03- 03 2010
AU 2008345231 Al 09-07 2009
BR PI0821481 A2 04- 11 2014
CA 2709890 Al 09- 07 2009
CN 101910408 A 08- 12 2010
EP 2235197 A2 06-10 2010
EP 2574676 Al 03-04 2013
EP 2574677 Al 03-04 2013
J P 5727790 B2 03-06 2015
J P 2011507551 A 10- 03 2011
J P 2014100148 A 05- 6 2014
KR 20100097752 A 03-09 2010
NZ 586250 A 30-11 2012
SG 187397 Al 28-02 2013
US 2009176269 Al 09- 07 2009
W0 2009086309 A2 09-07 2009
W0 9928455 Al 10-06-1999 AR 018023 Al 31-10 2001
AR 020735 Al 29-05 2002
AT 309350 T 15- 11 2005
AU 2051899 A 16- 06 1999
DE 59813187 Dl 15- 12 2005
DK 1036179 T3 06- 03 2006
EP 1036179 Al 20-09 2000
ES 2252876 T3 16- 05 2006
P 3394240 B2 07- 04 2003
J P 4384400 B2 16-12 2009
J P 2001525342 A 11-12 2001
J P 2003180392 A 02-07 2003
US 6673575 Bl 06-01 2004
WO 9928455 Al 10-06 1999
US 2004185534 Al 23-09-2004 US 2004185534 Al 23-09-2004
US 2009075331 Al 19-03-2009
International application No.
INTERNATIONAL SEARCH REPORT PCT/IB2015/054196
Box No. II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)
This international search report has not been established in respect of certain claims under Article (2)(a) for the following reasons:
□ Claims Nos.:
because they relate to subject matter not required to be searched by this Authority, namely:
□ Claims Nos.:
because they relate to parts of the international application that do not comply with the prescribed requirements to such
an extent that no meaningful international search can be carried out, specifically:
□ Claims Nos.:
because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).
Box No. Ill Observations where unity of invention is lacking (Continuation of item 3 of first sheet)
This International Searching Authority found multiple inventions in this international application, as follows:
see addi tional sheet
□ As all required additional search fees were timely paid by the applicant, this international search report covers all searchable
aims.
□As all searchable claims could be searched without effort justifying an additional fees, this Authority did not invite payment of
additional fees.
As only some of the required additional search fees were timely paid by the applicant, this international search report covers
' ' only those claims for which fees were paid, specifically claims Nos. :
No required additional search fees were timely paid by the applicant. Consequently, this international search report is
restricted to the invention first mentioned in the claims; it is covered by claims Nos.:
1-37
Remark on Protest The additional search fees were accompanied by the applicant's protest and, where applicable, the
' ' payment of a protest fee.
The additional search fees were accompanied by the applicant's protest but the applicable protest
' ' fee was not paid within the time limit specified in the invitation.
I INo protest accompanied the payment of additional search fees.
Form PCT/ISA/21 0 (continuation of first sheet (2)) (April 2005)
International Application No. PCTY I B2Q15/ Q54196
FURTHER INFORMATION CONTINUED FROM PCT/ISA/ 210
Thi s International Searching Authori t y found mul tiple (groups of)
i nventions i n thi s international appl i cation , as fol l ows :
1. claims: 1-37
A process of producing glycosylated protein i n a mammal ian
cel l cul ture the process compri si ng the steps of a)
preparing inocul um wi t h sui table cel l concentrati on during
seed development, b) inoculating the inocul um wi t h sui table
cel l concentration i n t o producti on bioreactor, c) cul turing
the cel l i n production bi oreactor at sui table condi t i ons
wherein the sui table condi tion i s monophasi c temperature
condi tion; and d) obtaini ng the glycosylated protein from
the cel l cul ture; processes of producing fusi on protein and
monoclonal antibody and fragment thereof or a TNFR-Fc fusion
protein i n a mammal ian cel l cul ture the process compri sing
i nter al i a the step of cul turing the cel l i n producti on
bioreactor at sui tabl e condi tions wherein the sui tabl e
condi tion i s monophasi c temperature condi tion.
2. claim: 38
A fusion protein or monoclonal antibody and fragment thereof
obtai ned from the process as claimed i n any of the preceding
claims.