DESC:FIELD OF INVENTION
The present invention relates to method culturing of mammalian cells
expressing recombinant proteins. In particular the cell culture method provided
5 ensures consistency in product quality during scale up operations.
BACKGROUND OF INVENTION
Recombinant biotherapeutics like monoclonal antibodies have evolved into a
major therapeutic category in the last quarter century. They are invariably
produced using genetically modified cells, amongst which mammalian cells
10 are predominantly favoured. Like any other pharmaceutical drugs,
development of bioprocess methods are undertaken at smaller scales in early
stages which helps save time and resources. However scaling up of those
early stage bioprocess methods to manufacturing scales poses additional
challenges due to the inherent complexities of the recombinant
15 biotherapeutics and the cell culture systems used for their production.
Recombinant biotherapeutics are heterogeneous molecules and the ones
produced in mammalian cell cultures undergo post-translational modifications.
These modifications are more often critical to the quality and efficacy of those
molecules. Therefore the aim of bioprocess methods development is to ensure
20 favourable productivity as well as product characteristics.
It is well recognised that various aspects of mammalian cell culture parameters
provide levers to control the product quality and productivity. It is imperative
that biotherapeutics production process is well characterised so as to identify
key process parameters that would affect the product quality and quantity.
25 The present application provides experimentally characterised cell culture
parameters which ensures consistent product quality and productivity upon
scale-up of early scale methods to manufacturing scales.
3
SUMMARY OF THE INVENTION
Mammalian cell culture methods for production of biotherapeutics invaraibly
involves early scale development in smaller bioreactors which provides
eficiency in terms of resources and time. Several process parameters can be
5 screened in parallel to arrive at a process that provides favourable productivity
and product profile. However scaling up of those early stage process to
manufacturing scale poses is a challenge as it is known that various cell
culture parameters can affect the product quality as well and the quantity.
Herein a scale up strategy based on volumetric mass transfer coefficients of
10 O2 and CO2, which ensures productivity as well as product quality.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Contour plots of agitation vs aeration for kLaO2 values at (a) 600L,
(b) 800L and (c) 1000L scales with 20µm sparger.
Figure 2: Variation of kLaO2 with incremental air flow rate and agitation with
15 20µm sparger at 1000L scale.
Figure 3: kLaCO2 at 95, 110 and 125 RPM with ? change in pH units over air
flow rates through DHS sparger at 1000L scale.
Figure 4: Viable cell densities cell cultures (a) N-1 seed bioreactor stage (b)
production stage
20 Figure 5: Viability of cell culture (a) N-1 seed bioreactor stage, (b) production
stage
Figure 6: Air flow rate utilized for day wise stripping of excessive pCO2 in the
production batches
4
DETAILED DESCRIPTION
Definitions
The term “about” refers to a range of values that are similar to the stated
reference value to a range of values that fall within 25, 20, 19, 18, 17, 16, 15,
5 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 percent or less of the stated reference
value.
The term “afucosylated glycans” refers to glycans wherein fucose is not linked
to the non-reducing end of N-acetlyglucosamine and includes M3NAG, G0,
G1A, G1B and G2. Further, the term “total afucosylated glycans” refers to
10 glycans wherein fucose is not linked to the non-reducing end of Nacetlyglucosamine and includes mannose glycans. Without limitation,
examples of total afucosylated glycans include G0, G1A, G1B, G2, M3-
M9NAG, M3-M9 (see Table 1).
As used herein, the term biotherapeutics and recombinant biotherapeutics has
15 been used interchangeably. It refers to biologic products that can be produced
in various expression systems in-vitro using recombinant DNA technology.
The exemplary biotherapeutics include but not limited to cytokines, growth
factors, hormones, interferons and antibodies.
The terms “cell culture medium”, “culture medium”, "media", "medium", as
20 used herein refer to a solution containing nutrients which are required to
support the growth of the cells in cell culture. A "basal medium" refers to a cell
culture medium that contains all of the essential ingredients useful for cell
metabolism. This includes for instance amino acids, lipids, carbon source,
vitamins and mineral salts. DMEM (Dulbeccos' Modified Eagles Medium),
25 RPMI (Roswell Park Memorial Institute Medium) or medium F12 (Ham's F12
medium) are examples of commercially available basal media. Alternatively,
said basal medium can be a proprietary medium fully developed in-house, in
which all of the components can be described in terms of the chemical
formulas and are present in known concentrations.
5
The term “cell culture process” as used herein refers to a process of culturing
a population of cells that are capable of producing recombinant protein of
interest or antibody.
It is well known that proteins expressed in eukaryotic cells undergo post5 translation modifications and most notable of them is glycosylation which
involves covalent addition of sugar residues to the polypeptide chain forming
the protein. The term "glycan" refers to the sugar residue which can be a
monosaccharide or polysaccharide moiety. The term "glycoprotein" refers to
protein or polypeptide having at least one glycan moiety. Thus, any
10 polypeptide attached to a saccharide moiety is termed as glycoprotein.
Recombinant biotherapeutics including monoclonal antibodies are examples
of glycoproteins. The term "glycoform" or "glycovariant" used interchangeably
herein refers to various oligosaccharide entities or moieties linked to the
glycoprotein. Examples of such glycans and their structures are listed in Table
15 1. However, Table 1 may not be considered as limitations of this invention.
The term “G0F glycans” refers to glycan moieties with fucose linked to the nonreducing end of N-acetlyglucosamine, and does not contain any terminal
galactose residues (see Table 1).
The term “galactosylated glycans” refer to glycan moieties containing terminal
20 galactose residues such as G1A, G1B, G1AF, G1BF, G2, G2F and G2SF (see
Table 1).
The term “high mannose glycans” refers to glycan moieties containing
unsubstituted terminal mannose sugars (see Table 1). High mannose glycans
contain more than 4 mannose residues attached to the GlcNAc2) core.
25
6
Table 1: Representative table of various glycans.
The terms "perfusion" or “perfusion process” refers to a cell culture process
5 wherein high cell densities and viabilities are achieved and maintained for
Glycan Structure Code Glycan Structure Code
M3 G1AF
M3NAG G1BF
M3NAGF M6
G0 G2F
G0F M7
M5 G2SF
G1A M8
G1B G2S2F
Mannose
N-Acetyl Glucosamine
Galactose
2-AB label
Fucose
Sialic Acid
7
extended period of time by use of cell retention device/system together with
continuous media exchange. Fresh growth medium (“n+1” culture medium) is
added or one or more times during cell culture and the spent culture medium
(“n” culture medium, which was already present in the bioreactor), which may
5 contain the recombinant product expressed by the cells, is harvested from the
bioreactor continually or one or more times during cell culture. The term
“concentrated fed batch” (CFB) refers to a perfusion cell culture wherein
ultrafiltration membranes are used to retain cells as well as the recombinant
product being expressed by the cell inside the bioreactor while removing the
10 waste product in the spent media. The fresh growth medium (“n+1” culture
medium) used in the CFB may be different than the spent culture medium (“n”
culture medium). Further, the fresh growth medium (“n+1” culture medium)
may be a concentrated form than the spent culture medium (“n” culture
medium). In some examples, the fresh growth medium (“n+1” culture medium)
15 may be added as a dry powder.
The “product quality” as used herein refers to desirable attributes in the
biotherapeutic molecule. This may include, but are not limited to the specific
glycosylation pattern or glycovariant composition of the biotherapeutic
molecule.
20 The term "reference product" refers to a currently or previously marketed
recombinant biotherapeutic, also described as the "originator product" or
"branded product" serving as a comparator in the biosimilarity studies. The
term “biosimilar” interchangeably used with biosimilar drug refers to a
biotherapeutic drug which is highly similar to the reference product in terms of
25 purity, structure, and bioactivity and further have been determined to have no
meaningful difference, in terms of safety, purity or potency (safety and
effectiveness), from the reference product.
The term “target/predetermined glycosylation profile” refers to the
glycosylation profile of a recombinant biotherapeutic that would form an
30 acceptance criteria for biosimilarity of a biosimilar to the reference product.
8
As used herein, the term “turndown ratio” refers to the ratio of the maximum
working volume to the minimum working volume of a bioreactor. For example,
a bioreactor with a maximum operating volume of 200 liters and a minimum
operating volume of 40 liters has a turndown ratio of 5:1.
5 The term “temperature shift” refers to the change in temperature during the
cell culture process in order to produce the therapeutic antibody. The term
“normal temperature shift” refers to a temperature shift wherein the cell is
cultured at a first temperature for a period of time and change the cell culture
temperature to a second temperature, wherein the second temperature is
10 lower than the first temperature. The term “reverse temperature shift” refers to
a temperature shift wherein the cell is cultured at a first temperature for a
period of time and change the cell culture temperature to a second
temperature, wherein the second temperature is higher than the first
temperature.
15 The term “viability” refers to a measure of the metabolic state of a cell
population which is indicative of the potential for growth. The viability of cells
may be measured by methods well known in art such as classical trypan blue
exclusion assay, propidium iodide (PI) exclusion assay, fluorescein di-acetate
(FDA) inclusion assay. The term “viable cell density (VCD)” is defined as
20 number of live cells per unit volume.
Unless defined otherwise, the technical and scientific terms used herein are to
be accorded the same meaning as would be commonly understood by one of
skill in art to which the subject matter herein belongs.
Description of the Embodiments
25 Stainless steel bioreactors (SSBs) and fed-batch methods have been the
mainstays of biotherapeutic industry. But of late single-use bioreactors (SUBs)
and concetrated fed-batch (CFB) based methods are fast becoming the tools
of choice in biopharmaceutical industry. The risks associated in operation and
time associated with traditional stainless steel bioreactors has been a major
9
driver for adoption of SUBs while CFB based methods are being favoured for
reduction in cost of goods provided. The challenges of scaling up of a
bioprocess methods is shared commonly by SSBs and SUBs – it is important
to ensure that the product profile and productivity arrived at in early, smaller
5 scales are maintained upon scaling up the process.
In a typical cell culture bioprocess the influence of the process parameters like
temperature, pH, pCO2, pO2 on the cell physiology, metabolism, product
formation kinetics and the product critical quality attributes (CQAs) cannot be
ignored. Amongst the above enumerated factors, pCO2 is a critical process
10 parameter owing to the multiple roles CO2 plays in the production of
biotherapeutics using mammalian cell culture. pCO2 impacts the cell culture
performance by altering the internal organelle pH of the mammalian cells
affecting the growth and metabolism. The pCO2 also affects post translational
modifications present on biotherapeutics thus influencing their quality
15 attributes. Hence it is imperative that pCO2 in the cell culture medium be
maintained at optimal levels and ensure that it does not exceeds its threshold.
CO2 accumulation is the most commonly faced challenge during bench scale
process scale-up to a manufacturing scale, due to longer residence time of the
gas in large scale bioreactors. The CO2 levels in cell culture is dependent on
20 parameters like bioreactor shape, volume, agitation rate, aeration etc. The
volumetric mass transfer coefficient of CO2 (kLaCO2) is an operational process
parameter which can factors in these variables. Hence, an understanding of
the mass transfer capacity of CO2 is imperative to control the gas not to exceed
its threshold value. Furthermore, a CO2 stripping model can also be developed
25 and implemented in large scale manufacturing process with the kLa data
generated for CO2 gas.
In an embodiment, the cell culture process of the present invention is
applicable to various mammalian cell lines that can be engineered to express
a biotherapeutics, including but not limited to Chinese hamster cells (CHO),
10
baby hamster kidney (BHK) cells, human embryo kidney (HEK) cells, mouse
myeloma (NS0) cells, and human retinal cells.
The cell culture methods of the present invention is not limited to any specific
class of recombinant biotherapeutics. In exemplary aspects the cell culture
5 methods of the present invention is applicable to monoclonal antibodies.
In an embodiment the cell culture process of the present invention is applicable
to stainless steel bioreactors. In another embodiment the cell culture process
of the present invention is applicable to single use bioreactors.
In an embodiment the cell culture process of the present invention is applicable
10 to fed batch cell cultures. In an embodiment, the cell culture of the present
invention is applicable to perfusion cell cultures. In yet another embodiment
the, cell culture method of the present invention is applicable to concentrated
fed batch cell cultures.
In an embodiment, the present application provides for a cell culture method
15 for production of a recombinant biotherapeutic composition having a target
glycosylation profile, the said process comprising,
(a) providing/culturing mammalian cells expressing the said recombinant
biotherapeutic,
(b) performing the N-1 seed stage and production stage in the same
20 bioreactor, wherein a 5:2 turndown ratio is used for seed stage to
production stage expansion along with calculated O2 volumetric mass
transfer coefficient (kLaO2) from Van’t Riet equation
(c) performing the production phase in a perfusion mode
(d) maintaining the CO2 volumetric mass transfer coefficient (kLaCO2) of
about 0.10 h-1 to about 0.27 h-1 25 , and the O2 volumetric mass transfer
coefficient (kLaO2) of about 8.0 h-1 to about 10.5 h-1
(e) recovering the said recombinant biotherapeutic composition from the
culture,
11
wherein, the target glycosylation profile is characterised is terms of glycan
variants including G0F glycans, total afucosylated glycans, galactosylated
glycans.
In an embodiment, the cell culture process of present invention would
5 comprise use of a temperature shift in the production phase so as to obtain
recombinant biotherapeutic composition with the target glycoprofile. In an
embodiment, the cell culture process of the present invention would comprise
more than one temperature shift, wherein the individual temperature shift
might be result in subsequent lower temperature or higher temperature. For
10 example in a cell culture process having two temperature shift, the following
combinations are encompassed: 1st high temperature ? 2nd low temperature
? 3rd lower temperature, 1st high temperature ? 2nd low temperature ? 3rd
high temperature, 1st low temperature ? 2nd high temperature ? 3rd low
temperature. In a preferred embodiment, the cell culture method of present
15 invention includes a single temperature shift, wherein the second temperature
is lower than the first temperature. In yet another embodiment, the cell culture
method of present invention includes a single temperature shift which marks
the start of production phase in the production stage of cell culture. As an
exemplification of the temperature shift strategy, the production stage of the
20 cell culture is initiated at about 37°C and is lowered to about 35°C on about
day 6 of the production stage and the culture is continued at this temperature
till harvest.
In an embodiment, the cell culture of the present invention uses alternating
tangential flow for perfusion.
25 The cell culture methods of the present invention is not limited to any specific
class of recombinant biotherapeutics. In exemplary aspects the cell culture
methods of the present invention is applicable to cytokines, growth factors,
hormones, interferons and antibodies. In an embodiment, the cell culture of
the present invention is applicable in production of recombinant
12
biotherapeutics like darbepoetin, rituximab, trastuzumab, pertuzumab,
bevacizumab, etanercept, aflibercept, abatacept, denosumab etc.
Those skilled in the art will recognize that several embodiments are possible
within the scope and spirit of this invention. The invention will now be
5 described in greater detail by reference to the following non-limiting examples.
The following examples further illustrate the invention but, of course, should
not be construed as in any way limiting its scope.
EXAMPLES:
Estimation of kLaO2 and kLaCO2:
10 Single use bioreactor setup:
A sparger customized Single Use Bioreactor (SUB) of 1000L scale (Xcellerex
XDR1000, GE Healthcare) was utilized for the study. The sparger
configuration is a dual sparger system with 20µm X 8 sparger discs for air, O2
and CO2; and a Drilled Hole Sparger (DHS) with 20 holes of 2mm hole
15 diameter for sparging air to strip off CO2 were used. The bioreactor
specifications being 1.5 aspect ratio of 1000L maximum working volume
capacity, 30 inch tank internal diameter, single use bottom mounted 0.33 inch
M40E impeller located at 15o off centre to the tank.
Experimental Design:
20 A full factorial DOE was used to create design space with the three key factors:
volume, agitation and aeration. Experiments were carried out in static gassing
out method with volume factor at three levels 600L, 800L and 1000L, agitation
rates of 95-125 RMP and aeration rates of 2-8 LPM. 1XPBS was used as test
solution with a pH of 7.39 and 281 mOSM/Kg osmolality, mimicking the
25 properties of in-house cell culture media in use. The head space air exchange
is performed for 3 cycles to reduce the residual nitrogen concentration
between each set of study in the gassing phase resulting in a more precise
and real time measurement of oxygen transfer.
13
For the kLaO2 measurements, air was sparged through 20µm x 8 sparger discs
at 37°C process temperature and the %DO change was captured in every 20
second interval. The kLaCO2 experiment was carried out by sparging CO2 gas
through T-sparger with 20 drilled holes of 2mm diameter at 37°C and
5 correspondingly the ?pH, pCO2 values were recorded. The trials were carried
out with the highest volume first, followed by gradual removal of the required
test solution from the bioreactor for kLaO2. However kLaCO2 study was limited
to 1000L capacity.
kLa measurement:
10 The volumetric mass transfer coefficient of oxygen has been calculated from
general mass balance equation .
???????? ?
(????*-????????1)
(????*-????????2)
? = ????????????.???? Equation 1
Where, C* - Saturation concentration of oxygen in liquid phase, CLConcentration of oxygen in liquid phase, t – time required
15 A graph is being plotted and the slope is determined, considering a straight
line equation. The O2 concentration in liquid (mmol/L) is calculated by gas
solubility from Henry’s Law:
H
Py = C o2
L
Equation 2
P = Gas phase partial pressure of oxygen; H = Henry’s coefficient at
20 temperature T; yo2 = mole fraction of Oxygen
Compensating Henry’s constant for temperature by the below relationship:
H-1 = 1.385 - 0.02635(T - 20) + 0.0004288 (T - 20)2 Equation 3
Where T is Temperature in Centigrade (°C). The unit of Henry’s constant is
atm m3/mol
25 The mass transfer coefficient of dissolved CO2 has been calculated from the
overall mass balance equation
14
????[????????2]
????????
= - ????????????????????2 . ????????2 Equation 4
Solving the equation with time limits ‘0’ to ‘t’ the equation becomes,
???????? [????????2]2
[????????2]1
= - ????????????????????2???? Equation 5
The kLa is determined from the slope of the straight line equation. The liquid
5 solubility of CO2 (mmol/L) is calculated by Henry’s law.
[CO2](aq) = ???????????? 2
????????????2
Equation 6
where HCO2 is Henry’s law constant for CO2, and [CO2]aq is in mm Hg
Compensating Henry’s constant for temperature by the below relationship:
????????????2 = 101.33(11.549-2440.4
???? )
Where T is absolute Temperature in Kelvin (°K).
10 The unit of Henry’s constant is in mm Hg L/M
The most commonly used correlation for kLaO2 is expressed in terms of
power input per unit volume and superficial gas velocity and is known as Van’t
Riet equation.
kLa = A (P/V)a(Vs)ß Equation 7
15 where A, a and ß are constants.
Cell expansion and ATF setup:
A CHO cell line producing IgG1 monoclonal antibodies and an in-house
process utilizing proprietary defined basal media and feed media were
operated. The seed was expanded from vial thaw in shake flasks till 700mL
20 for 3 passages every 3-4 days in 1Xbasal media with incubator settings 37°C
temperature maintenance, 5% CO2 and 75% humidity. The culture from shake
flask was inoculated to rocking wave bioreactors (WAVE Bioreactor, GE
Healthcare). The culture was further scaled-up into 50L SUB (Xcellrex XDR50,
GE Healthcare) and 5:2 turndown ratio seed expansion in 1000L SUB
15
(Xcellrex XDR1000, GE Healthcare). The 5:2 turndown ratio is atypical from
the regular practical applications, however in the current process design
compensating the N-1 seed stage and the production bioreactor stage the half
maximal working volume was developed. During the production bioreactor
5 stage the seed bioreactor is topped up to the maximum capacity in
concentrated fed-batch mode.
For perfusion in concentrated fed-batch (CFB) mode, an alternating tangential
flow (ATF) system with a 50 kDa filter was used (ATF10, Repligen). The ATF
was operated at a pressure and exhaust flowrates (ATF exchange rate) of 54
10 LPM. The initial seeding density was 1.5±0.2 million cells/mL with =90%
viability. A temperature shift was applied on the 6th day of the production
bioreactor stage whereby the culture temperature was reduced from 37°C to
35°C. The process was maintained at 40±10% DO, agitation rate of 110 RPM,
and 7.05 ± 0.1 pH.
15 Online process monitoring was done every 4 hours and daily offline sampling
was performed by collecting cell culture samples from the bioreactor. Viable
cell density (VCD) and viability were measured using the manual counting
trypan blue exclusion method and on Vi-CELL XR cell counter, Beckman
Coulter. Offline pH, pO2, and pCO2 were measured using Rapid lab 348-
20 Siemens blood gas analyzer.
Results:
The experimentally determined KLaO2 are extrapolated as contour plots in
Figure 1. The KLaO2 values increases linearly with the flow rate and the
agitation speed for all the for all the bioreactor volumes determined. Figure 2
25 depicts the KLaO2 values determined for commonly used manufacture scales
and conditions, viz. 1000L with agitation rates ranges between 95-125 RPM
and 2 LPM flow rate variation; the highest KLaO2 value resulted with the largest
flow rate and agitation speed. The Van’t Riet constants (A - 4230, a - 0.28, ß
– 0.72) were calculated based on the generated data sets.
16
The current study aims at banking the KLaCO2 data using 2mm X 20 DHS
sparger, which can also be a measure of estimating the stripping capacity of
the sparger during an on-going cell culture process at 1000L scale. Figure 3
shows the KLaCO2 measurements with change in pH units in relation to the
5 aeration supplied through DHS at the same agitation rates used for measuring
KLaO2. In practice, this data has the decisive advantage while choosing the
precise air flow rate through DHS sparger for stripping out a determined
amount of pCO2 while a cell culture process batch is in operation. Efficient
pCO2 removal mechanism can be strategized employing Figure 3, with
10 calculated stripping capacity of each flow rate with regards to linear pH unit
variability at each of the agitation rates.
The cell culture profile results for the N-1 seed bioreactor stage mentioned in
Figure 4 and Figure 5 were plausible and consistent with all the five production
batches executed for the study, which is indicative of metabolic similarity
15 between the two stages.
In continuation to the seed bioreactor, the production bioreactor was operated
in CFB mode, wherein adequate aeration and agitation strategy was
implemented at full scale. An efficient pCO2 control mechanism was employed
to maintain optimal pCO2 through-out the cultivation for cellular metabolism.
20 The viable cell count cited in Figure 4 was consistent for all the five batches
executed, importantly because the pO2 was tightly maintained with the desired
consumption rate. Figure 6 presents the air flow rate that was used for
stripping the excessive pCO2 in the five 1000L batches. The measurements
from Figure 6 quantitatively describes the proportionate air flowrate employed
25 from DHS sparger for step-to-step stripping of the excessive pCO2 in the
running batch, which if not degassed in appropriate can retard the cell growth
and pose toxicity for the culture. The glycosylation profile and the titer of the
product in the 1000L production batches (Table 1) was consistent, which
shows that KLaCO2 based aeration/agitation strategy enables achievement of
30 a target product profile without any loss in productivity.
17
Table 1. The product quality and titer in the various 1000L production
batches
Batch No G0-F (%) TAF (%) Gal (%) Titer (g/L)
P1 52 3.7 46.7 3.0
P2 52 3.7 46.3 3.0
P3 53 3.4 46 2.9
P4 53 3.3 46 2.8
P5 51 3 47.9 3.2 ,CLAIMS:We claim:
1. A cell culture method for production of a recombinant biotherapeutic
composition having a target glycosylation profile, the said process comprising,
(a) providing/culturing mammalian cells expressing the said recombinant
5 biotherapeutic,
(b) performing the N-1 seed stage and production stage in the same
bioreactor,
(c) a 5:2 turndown ratio is used for seed stage to production stage
expansion along with calculated O2 volumetric mass transfer coefficient
10 (kLaO2) from Van’t Riet equation
(d) maintaining the CO2 volumetric mass transfer coefficient (kLaCO2) of
about 0.10 h-1 to about 0.27 h-1, and the O2 volumetric mass transfer
coefficient (kLaO2) of about 8.0 h-1 to about 10.5 h-1
(e) recovering the said recombinant biotherapeutic composition from the
15 culture,
wherein, the target glycosylation profile is characterised in terms of glycan
variants including G0F glycans, total afucosylated glycans, galactosylated
glycans.
2. A cell culture method of claim 1, wherein the production stage includes a
20 temperature shift.
3. A cell culture method of claim 1 or 2, wherein the production phase is
operated in perfusion mode.
4. A cell culture method of claims 2-3, wherein the temperature difference of
the cell culture before and after the temperature shift from about 7°C to about
25 2°C.
5. A cell culture method of claims 2-4, wherein the cell culture temperature
before the temperature shift is about 37°C and the cell culture temperature
after the temperature shift is selected from about 30°C, about 31°C, about
32°C, about 33°C, about 34°C, about 35°C.
19
6. A cell culture method of claims 2-5, wherein the recombinant biotherapeutic
composition so produced comprises of about 51% - about 53% G0-F, about
3.0% – about 3.7 %TAF, about 46% - about 48% galactosylated glycans.
7. A cell culture method of claims 2-6, wherein the recombinant biotherapeutic
5 is an IgG1 monoclonal antibody.