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ARTICLE

Cell-Controlled Hybrid Perfusion Fed-Batch CHO


Cell Process Provides Significant Productivity
Improvement Over Conventional Fed-Batch
Cultures
Gregory W. Hiller, Ana Maria Ovalle, Matthew P. Gagnon, Meredith L. Curran,
Wenge Wang

Pfizer, Inc., 1 Burtt Road, Andover, Massachusetts 01810; telephone: þ978-247-3173;


fax: 646-348-8248; e-mail: gregory.hiller@pfizer.com

groups continue to manipulate and improve the CHO cell expression


ABSTRACT: A simple method originally designed to control lactate systems and metabolism to overcome these limits and achieve a
accumulation in fed-batch cultures of Chinese Hamster Ovary higher producing culture (Du et al., 2015). However, determining
(CHO) cells has been modified and extended to allow cells in culture the identity and ultimately controlling the accumulation of growth
to control their own rate of perfusion to precisely deliver nutritional inhibitors, or otherwise avoiding negative culture conditions, can be
requirements. The method allows for very fast expansion of cells to
high density while using a minimal volume of concentrated extremely difficult in a culture in which every molecule generated
perfusion medium. When the short-duration cell-controlled generally remains in the culture.
perfusion is performed in the production bioreactor and is Due to the limits of fed-batch culture the biopharmaceutical
immediately followed by a conventional fed-batch culture using industry has begun to reconsider the use of continuous perfusion.
highly concentrated feeds, the overall productivity of the culture is Perfusion culture simultaneously removes inhibitory compounds,
approximately doubled when compared with a highly optimized
state-of-the-art fed-batch process. The technology was applied with provides nutrients, and restores environmental parameters to
near uniform success to five CHO cell processes producing five ensure continued cell growth and production. Continuous
different humanized monoclonal antibodies. The increases in suspension culture perfusion bioreactors often operate with
productivity were due to the increases in sustained viable cell perfusion rates between one and three reactor volumes per day
densities. (Gray et al., 1996; Mercille et al., 1999; Voisard et al., 2002). Such
Biotechnol. Bioeng. 2017;9999: 1–10. high perfusion rates allow for high cell densities and high
ß 2017 Wiley Periodicals, Inc. volumetric productivities to be achieved, but often result in low titer
KEYWORDS: CHO cell culture; mammalian cell culture; hybrid concentrations and by default large volume perfusion medium
perfusion fed-batch; glucose limitation; HIPDOG; HIPCOP
preparation, storage, and handling equipment. Additionally, most
commercial biopharmaceutical perfusion processes can be catego-
rized as “sustainable” perfusion processes, meaning that they
maintain high cell viability and a continuous non-zero cell growth
Introduction rate by means of a “cell bleed”—a continuous removal of a fraction
of the cell mass from the bioreactor (Clincke et al., 2013; Desch^enes
The fed-batch mode of operation for CHO or other mammalian cells et al., 2006; Hiller et al., 1993). For some cell lines the per cell, or cell
has been the dominant production method for biopharmaceutical specific productivity, usually expressed in picograms/cell/day, is
production for more than two decades (Rouiller et al., 2013). higher when cells are not in an active, growing state (Bi et al., 2003;
If culture parameters such as nutrient availability, osmotic Suzuki and Ollis, 1990).
strength, pH, dissolved oxygen, and carbon dioxide can be Somewhere between the two extremes of a purely fed-batch or
maintained in an appropriate range, cells likely ultimately cease purely continuous sustainable perfusion mode of operation, we
division due to accumulation of growth-inhibitory compounds have begun to investigate hybrid processes in which perfusion with
which in-turn limit the final productivity of the culture. Many complete cell retention is performed for a few days in the production
bioreactor and then the bioreactor operational mode switches to
fed-batch for the remainder of the batch. A smooth ramp up in
Correspondence to: G. W. Hiller
Received 14 July 2016; Revision received 21 November 2016; Accepted 25 January 2017 perfusion rates as the cell number increases enables a peak cell
Accepted manuscript online xx Month 2016; density far in excess of that achievable with a conventional fed-
Article first published online in Wiley Online Library batch operation. If the perfusion is then discontinued abruptly, and
(wileyonlinelibrary.com).
DOI 10.1002/bit.26259 the culture is continued as a fed-batch process supplying sufficient

ß 2017 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2017 1
nutrients for additional growth, as we will show oftentimes cell limiting cells take up lactic acid and the bulk culture pH rises, when
division will continue for another 24–48 h. Since the process ends as glucose is in excess cells make and likely secrete lactic acid which
a fed-batch with a single discrete harvest, conventional purification suppresses pH. In perfusion culture at this point a pump is activated
trains designed for legacy fed-batch upstream processes can still be that delivers perfusion medium containing a high concentration of
employed provided they are scaled sufficiently to deal with the glucose directly to the culture. Since the culture is a perfusion
increase in solids removal and overall productivity. Additionally, it reactor with a cell retention system, the volume of the bioreactor is
is likely that at least during the phase of culture in which the maintained constant through the simultaneous activation of a pump
majority of the protein is produced, such a hybrid perfusion/fed- that removes cell-free culture from the bioreactor system (level or
batch process would result in culture conditions similar in many weight-based control systems, or merely linked to the medium
aspects to a conventional fed-batch, and therefore, less likely to addition pump). As with the HIPDOG control scheme, the HIPCOP
produce protein with a product quality profile dramatically different control system can operate over the time span of minutes.
from legacy fed-batch processes (Meuwly et al., 2006). Therefore, even though the perfusion medium addition and cell-free
While undergoing rapid cell division, particularly in the early harvest pumps are turned on and off repeatedly, the culture is still
stages of batch growth in nutrient rich medium, mammalian cells effectively operating in a continuous perfusion mode. As the cell
commonly exhibit aerobic glycolysis; meaning that a large fraction densities increase, the cells smoothly increase their own perfusion
of the glucose consumed by the cells is converted to lactic acid via rate with a continuous feedback control based on pH, without the
the glycolysis pathway in spite of high availability of oxygen (Miller need for significant operator intervention, or timed finite step
et al., 1987, 1988). In environmentally controlled bioreactors, the changes in perfusion rate. Additionally, since the rate of addition of
lactic acid formed and secreted into the bulk culture fluid is perfusion medium is directly dependent upon the concentration of
neutralized with basic titrant to maintain a stable pH, and the glucose in the perfusion medium when the HIPCOP scheme is
resulting accumulation of lactate and increase in osmotic strength operating, simply decreasing the concentration of glucose in the
can negatively affect cell growth and productivity (Gagnon et al., perfusion medium will increase the volume of perfusion medium
2011; Hassell et al., 1991; Yoon et al., 2006). We have previously delivered to the culture for any particular cell density, and therefore
described a method of high-end pH-controlled delivery of glucose more effectively flush other inhibitory metabolites from the culture.
(HIPDOG) which when used in fed-batch cultures of CHO cells As with HIPDOG in fed-batch cultures during the growth phase,
completely suppresses the accumulation of lactate during the when HIPCOP is operating in a continuous perfusion culture,
growth phase (Gagnon et al., 2011). When HIPDOG is operating depending upon the metabolic characteristics of the cell line, the
and glucose concentrations drop to a low level, cells “signal” their levels of lactate in the culture can fall while the control system is
need for additional glucose by taking up lactic acid (as the acid functioning. When the HIPDOG control scheme is operating in a
form) from the bulk culture and thereby cause the pH to rise. When fed-batch culture it is possible that the drop in concentration of
the high-end pH setpoint is reached, a small amount of lactate might be due in a roundabout way to other cell-produced
concentrated glucose is added which pushes pH down as lactic acids secreted into the media which have a minor suppressive effect
acid is formed again. on pH. These additional metabolic acids serve to partially
The HIPDOG technology can be considered to be a form of “cell- circumvent the normally expected control scheme in which every
controlled” glucose addition. In the current work we have extended molecule of lactic acid that is produced when glucose is
and adapted the basic premise of this technology to continuous momentarily in excess is stoichiometrically reabsorbed when
perfusion culture to allow cells to self-regulate their own rates of glucose is momentarily limited. Since the HIPDOG control scheme
delivery of perfusion medium. The new control scheme can be operating in a fed-batch culture keeps pH constant throughout the
described as high-end pH control of perfusion (HIPCOP). Figure 1 growth phase, the downward “pressure” on pH generated by these
is a pictorial representation of the hypothesized sequence of events other cell-produced acids manifests itself as a slowly decreasing
occurring in the bulk cell culture fluid when the HIPCOP control level of lactate. When the HIPCOP technology is controlling the
scheme is operating. In perfusion culture at fast growth rates cells perfusion rate of a continuous-perfusion bioreactor the phenome-
respond in a similar manner as in fed-batch; when glucose becomes non of lactate levels falling is exacerbated by the perfusion process
itself flushing lactate out of the bioreactor system. Recognizing that
very low levels of lactate in the bulk fluid of a perfusion bioreactor
might cause the HIPCOP control system to cease functioning
properly, we found it advantageous for these initial experiments to
add a low concentration (1–3 g/L) of purified sodium-L-lactate
directly to the perfusion medium.

Materials and Methods


Cells Lines
Figure 1. Pictorial representation of the hypothesized sequence of events
occurring in the bulk cell culture fluid during the growth phase of a bioreactor utilizing All experiments used CHO-K1 cells, pre-adapted to serum-free
the HIPCOP control scheme which allows for a ‘‘cell-controlled’’ ramp up of perfusion suspension growth prior to transfection. Cells were stably
rates while limiting lactic acid accumulation.
transfected with proprietary DNA vectors encoding recombinant

2 Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2017


humanized antibodies using the glutamine-synthetase (GS) pump (Broadley-James, Irvine, CA) pulling from the shell side of
expression system (Lonza, Basel, Switzerland). the hollow-fiber filtration device.
Production reactors with 1–2-L working volumes used single
Rushton impellers (4.5 cm diameter) operating at approximately
Medium and Feeds
275 RPM. At this RPM the agitation system provided a calculated
All experiments utilized highly concentrated proprietary chemi- power per unit volume of approximately 90 W/m3. Bioreactors with
cally-defined media and feed solutions developed at Pfizer. Some a 3-L working volume used a Rushton impeller on the bottom (6 cm)
details of the various basal, perfusion, and feed media used in the and a pitch blade on the top (6 cm) operating at approximately
experiment are shown in Table I. As shown in Table I, some 195 RPM and imparting approximately the same 90 W/m3
minor modifications to the perfusion medium and the feed calculated power per unit volume. Temperature was controlled at
medium used in the production reactor were made between the 36.5 C with electric heating blankets.
experiments for cell lines A and B, and those with C, D, and E. For experiments with cell lines A, B, and D, cell culture fluid was
Because the concentrated nutrient feeds contain both glucose and circulated through the external loop using a Watson-Marlow peristaltic
other nutrients in a balanced ratio, during the fed-batch portion pump (Wilmington, MA) operating at 16.7 RPM using 6.4 mm
of the production bioreactors generally all glucose entering the ID/12.7 mm OD flexible tubing (Pharmed, Cole-Parmer, Vernon Hills,
bioreactor systems came from the concentrated feeds listed in IL) for an effective flowrate of approximately 120 mL/minute.
Table I. On rare occasions if it was felt that the residual Experiments with cell lines C and E used a magnetically levitated low
concentrations of amino acids in the bioreactor were too high shear centrifugal pump (model PuraLev1 200 MU, Levitronix,
relative to the glucose concentration, small amounts of a 500 g/L Waltham, MA) which using feedback control and flow sensors
solution of glucose were added to the bioreactor and the rate of maintained the flowrate at approximately 600 mL/min by varying the
feeding of the complete nutrient solution was decreased. During RPM of the pump between 1,400 and 1,600. The flowrate in the
the fed-batch portion of the cultures nutrient feeding was nearly recirculation loop with the Levitronix pumps was 600 mL/min because
continuous. Due to the highly concentrated nature of the feeds this was near the minimum practical flowrate for these pumps. The
and the overall quantities of feeds required, daily bolus feeding volume of the recirculation loop was reduced by 11 mLwhen using the
would have resulted in abrupt increases in culture osmotic centrifugal pump.
strength that might have been detrimental to the cultures. Calculations and prior experience suggest that for all of the
experiments the dissolved oxygen should not drop below a level
which would affect cellular metabolism before the cells re-enter the
Equipment
bioreactor from the perfusion loop.
Cells were grown in 1, 2, or 3-L working volume Applikon
bioreactors (Applikon, Inc., Schiedam, The Netherlands) utilizing
Inoculum Expansion and Maintenance
Bionet computer control systems (Broadly-James, Irvine, CA).
Perfusion was accomplished using an external recirculation loop Cells were thawed from frozen vials into CD-CHO AGT TM
flowing culture through the lumen side of a 0.2 micron cutoff medium (Invitrogen, Carlsbad, CA). Shake flasks (Corning-Life
hollow-fiber filtration device (General Electric CFP-2-E-4MA) with Sciences, Oneonta, NY) were used for expansion; 50 mL working
a surface area of 420 cm2, and a lumen ID of 1 mm. The volume in 250-mL flasks and 300 mL in 1-L flasks. Cells were
recirculation loop including the lumen of the hollow-fiber cartridge inoculated at 0.3  106 viable cells/mL for 3-day passages and
had a hold up volume of approximately 120 mL and so consisted of a 0.2  106 for 4-day passages. Flasks were placed on an orbital
maximum of about 10% of the working volume of the bioreactor shaker platform (Bellco Biotechnology, Vineland, NJ) operating
system when in operation with a 1-L working volume bioreactor, at 140 RPM with either a 2.5 or 5 cm throw inside a humidified
and less so for larger working volume bioreactors. Perfusion was incubator (Thermo Fisher, 3950 Reach-In CO2) operating at 5%
accomplished by removing cell-free permeate with a peristaltic CO2 and 36.5 C.`

Table I. Medium nutrient levels and osmotic strengths.

Total amino acids Glucose Sodium–L-Lactate Osmotic strength pH after


Medium (mM) (g/L) (g/L) (mOsm) preparation
N-1 basal medium and N-1 perfusion medium for all cell lines 48 8 0 347 7.2
Basal medium for production bioreactor inoculation for all 120 4 0 280 7.1
cell lines
Production bioreactor perfusion medium for cell lines A and B 90 8 1.4 375 7.1
Production bioreactor perfusion medium for cell lines C, D, 90 8 2.5 345 7.1
and E
Concentrated feed medium cell lines A and B 607 130 0 1630 7.2
Concentrated feed medium cell lines C, D, and E 607 90 0 1322 7.2

Hiller et al.: Hybrid Perfusion Fed-Batch CHO Culture 3


Biotechnology and Bioengineering
N-1 or Seed Bioreactors—Inoculum Preparation sparging element. It was realized that the 100 micron sparging
elements were near the practical limit of their ability to transfer
The N-1 or seed bioreactors were operated as perfusion bioreactors
oxygen into the culture. For cell lines C and E a dual sparger
using a perfusion rate that was increased as a step-change once per
approach was used in which pure oxygen was sparged through a 15
day. Inoculation density of the N-1 bioreactors was 0.75–1.0  106
micron sintered steel sparger (Applikon, Inc.) to control the
viable cells/mL and perfusion was started approximately 24 h after
dissolved oxygen level, and air was sparged through a drilled hole
inoculation. The perfusion rate was increased once per day from 0.4,
(7  1 mm holes) sparger for carbon dioxide stripping/control. Via
0.8, 1.5, 2, to 2.5 reactor volumes per day. The perfusion equipment
computer control the flow rate of gas through the drilled hole
used for the N-1 cultures was identical to that used in the production
sparger was set to a volume of gas 2.5–4 times that of pure oxygen
culture bioreactors as described above. For the N-1 cultures all cell
that was used in the 15 micron sparger. This sparging strategy
lines used the peristaltic pumps for recirculation to the perfusion
effectively controlled both dissolved oxygen and carbon dioxide
loop. For cell lines A, B, C, and E, the N-1 working volume was
levels. Carbon dioxide levels were generally maintained between 6
between 1 and 3 L, and the batch length was 6 days. While equivalent
and 12% of saturation for cell lines A, B and D, and between 5 and
results were achieved in related experiments when using cells from an
15% for cell lines C and E.
N-1 bioreactor at the 1–3 L scale, for the data presented in the current
work the N-1 culture for cell line D was performed at the 1,800-L
scale. Cells were removed from the N-1 on day 5, immediately Analytical Methods
transported from the large-scale facility and used to inoculate the
Cell counts, glucose, lactate, ammonium, and osmolality (per the
bench-top production bioreactors. Perfusion at the 1,800-L scale was
freezing point method) were determined using NovaFlex Analyzers
performed with an external tangential flow filtration device
(Nova Biomedical, Waltham, MA). As a check of the continuous on-
(ProStakTM, cat. #SK2P446E0, 0.65 micron, 1.7 m2, 8 cartridges,
line readouts of pH and dissolved oxygen, once per day samples
EMD Millipore, Billerica, MA). Cells were recirculated through the cell
were analyzed using a Rapidlab 248 blood gas analyzer (Siemens
retention device using a rotary-lobe pump at a rate of 64 L/min such
Diagnostics, Deerfield, IL) and if necessary or appropriate, minor
that the residence time in the external loop was no longer than 60 s. In
changes were made to the probe calibrations. The blood gas
all cases, the final cell densities reached in the N-1 cultures were
analyzer also provided an estimate of the carbon dioxide
between 22 and 30  106 viable cells/mL which allowed for a
accumulation in the bioreactor and minor adjustments were
minimum split ratio into the production bioreactor of 4.4.
sometimes made to the sparging volumes of gas to maintain this
critical parameter within 5–15% of saturation. Titer was measured
Bioreactor Environmental Controls via protein-A HPLC (model 1100 HPLC, Aligent technologies, Inc.
Santa Clara, CA) using a POROS1 Prepacked Protein A, Affinity
The pH in the N-1 bioreactors was controlled at 7.10  0.15 using Column (Applied Biosystems, Foster City, CA).
additions of a mixture of 1 M sodium carbonate/potassium
carbonate (molar ratio of 0.94 sodium: 0.06 potassium) on the low
end, and sparging of carbon dioxide if needed on the high end. In Imaged Capillary Isoelectric Focusing (iCE)
the case of cell line D the pH was controlled at 7.10  0.2 and the Charge heterogeneity was evaluated by imaged capillary
base titrant consisted only of 1 M sodium carbonate. For the first isoelectric focusing (iCE). iCE separates based on charge
4 days after inoculation, pH in the production bioreactors for all differences in a pH gradient generated by ampholytes under
cultures was controlled at 7.1  0.025 using additions of the same the influence of an electric field. Protein charge species are
base titrant on the low end (mixed titrant), and additions of focused within a capillary under DC voltage and detected at
perfusion medium containing 8 g/L glucose (Table I) on the high 280 nm with whole capillary imaging. The sample electrophero-
end. The pH control system was well tuned such that no gram is compared to reference material.
significant pH overshoot occurred when base titrant was being
added, and that once the pH rose to the upper end of the dead band,
N-linked Oligosaccharide Analysis
no significant overshoot occurred when perfusion media was being
added to the bioreactor to push pH down. The flowrate of perfusion N-linked glycans were enzymatically released with peptide-
medium when the pump was on (only when pH is above the N-glycosidase F. The glycans are derivatized by a fluorescent agent
setpoint) was set to 0.5 reactor volumes/day and increased once per and analyzed using hydrophilic interaction liquid chromatography
day to 2 reactor volumes/day for the final day of perfusion. After and fluorescence detection. The chromatographic profile is
day 4 (when the perfusion was ended) the pH deadband was slightly compared to reference material.
increased (7.1  0.15) and pH on the high end was then controlled
by sparging of carbon dioxide if needed. The dissolved oxygen was
controlled at 40  10% of air saturation for all bioreactors by Size Exclusion HPLC (SE-HPLC)
controlled sparging of pure oxygen. Experiments with cell lines A, B, Product purity was measured by SE-HPLC. Test samples were
and D used pure oxygen sparged through 100 micron sintered steel diluted and injected onto a size-exclusion column. The content
sparging elements (Applikon, Inc.) and flushed the headspace of high molecular mass species (HMMS) and monomer is
continuously with a mixture of 7% carbon dioxide in air. For these reported as the percent of the total area for all protein-related
three cell lines, the 100 micron sparging element was the only peaks.

4 Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2017


Capillary Gel Electrophoresis (CGE) Reducing HIPCOP technology as the pH of the bulk culture fluid hovered near
the high-end pH set point of the control system and the perfusion
Purity of the reduced protein was measured by denaturing samples
feed pump and cell-free permeate pumps cycled on and off more
with SDS and heat in the presence of a reducing agent. The resulting
and more frequently. Depending upon the cell line/project, the total
heavy and light chains are electrophoretically separated in a
volume of perfusion medium used ranged between 1.6 and 2.5
capillary containing sieving medium and detected using UV
reactor volumes based upon the working volume of the bioreactor
spectroscopy. The separation allows quantitation of the resolved
during the perfusion stage (Table II). The maximum perfusion rate
heavy and light chains as well as size related impurities. The purity
for a 24 h period (which occurred during the final day of perfusion)
is reported as the total percent of heavy and light chains.
ranged between 0.7 and 1.3 reactor volumes per day (Table II). The
rate of perfusion increase (again, controlled by the cells) was nearly
Results and Discussion proportional to the increase in cell density, such that the cell specific
perfusion rate was relatively constant for any particular cell line
Figure 2 provides a pictorial representation of the timeline of events
(data not shown, though average cell specific perfusion rate in
during the hybrid perfusion fed-batch experiments. Five different
Table II).
clonal cell lines producing five different monoclonal IgG molecules
For the current experiments, the HIPCOP stage (the perfusion
were investigated. Cells were inoculated into the production
stage) of the bioreactor was terminated for all cultures 4 days after
bioreactor at a density of approximately 5  106 viable cells/mL
inoculation at which point a highly concentrated nutrient solution
(Fig. 3A) from the N-1 or seed cultures as described in the materials
was continuously fed for the remainder of the culture (fed-batch
and methods section. As cell division occurred in the production
portion of experiment) while the working volume of the bioreactor
bioreactor, within 1–2 days the residual glucose concentration fell
was allowed to increase. The feed medium flowrates during the fed-
from 3 to 4 g/L to below 1 g/L (Fig. 4A), and the accumulated lactate
batch portion of the bioreactor experiments were adjusted manually
concentration for most cell lines rose from below 1 g/L to between 2
once per day based upon cell density, lactate concentration and
and 3 g/L (Fig. 4B). For each of the cell lines the residual glucose
osmolality measurements, and an analysis of nutrient levels such as
concentration eventually reached a sufficiently low concentration
glucose and amino acids. The feed medium flowrates for all five cell
that the cells shifted to a more oxidative metabolism and began to
lines were between 11% and 13% of the instantaneous bioreactor
take up lactic acid from the bulk culture fluid. The actual residual
volume for the first 24 h after the perfusion was terminated, and
glucose concentration at which this occurs can vary slightly from
were subsequently ramped down once per day throughout the
cell line to cell line, but was always less than 1.0 g/L glucose for the
course of the culture to approximately 4–7.5% of the bioreactor
five cell lines investigated in the current work. At the cell densities
volume on the final day of culture. Some details of the compositions
achieved in the current set of experiments, once lactic acid began to
of the perfusion media and concentrated feed media are provided in
be reabsorbed from the bulk culture fluid, within 15–60 min the pH
Table I; the total volume of feed media used for each cell line/project
of the bulk culture fluid rose to the high-end setpoint (7.12) at
is listed in Table II.
which time the perfusion medium addition and the cell-free
The decision on the length of time to continue perfusion in these
permeate pumps were activated and the perfusion portion of the
experiments was a compromise between the amount of perfusion
culture was self-initiated by the cells. The self-initiation of
medium that would be required, the amount of product that might
perfusion happened between 24 and 48 h after inoculation. The
be lost to drain during the perfusion stage, and also the maximum
control of pH on the high end through the addition of perfusion
cell density (and therefore productivity) that might be achieved. At
media was very effective and resulted in sufficiently tight control
the time of termination of the perfusion, the viable cell densities
such that once the perfusion was initiated no base titrant was again
reached between 38 and 55  106 cells/mL (Fig. 3A). Additional cell
added until the perfusion stage was complete. For the next several
division occurred for another 2 days for most cell lines, but as long
days as the cell mass increased the rate of perfusion was
as an additional 6 days for cell line E during the fed-batch portion of
continuously and smoothly ramped up through the use of the
the culture (as feeds are still being added to the culture
continuously, even an unchanging cell density indicates that cell
division is occurring). Cell viabilities were generally high during the
experiment, but fell to as low as 87% by day 12 for cell line A. Amino
acid residual concentrations were determined during and after the
experiment (data not shown), as well as the commonly available
parameters such as glucose, lactate, and ammonium (Fig. 4A–C).
Based on the aforementioned analyses, there is no evidence that any
commonly known nutrients (glucose, amino acids, most trace
metals) or vitamins were depleted as the cell growth slowed.
Additional shear protectant, most nutrients, trace metals, and
vitamins known to be consumed or degraded during CHO cell
Figure 2. Timeline of operations for the hybrid perfusion fed-batch process.
culture are contained in the feeds. Based on our accumulated
Perfusion controlled via the HIPCOP technology is ‘‘self-initiated’’ by the cells when experience with the cell lines we do not think it likely that any
glucose falls to near zero and lactic acid begins to be consumed by the culture. For commonly known inhibitor such as lactate or ammonium, or the
most cultures perfusion self-started near the 24 h time point after inoculation.
increasing osmotic strength (Fig. 4D) would have significantly

Hiller et al.: Hybrid Perfusion Fed-Batch CHO Culture 5


Biotechnology and Bioengineering
Figure 3. Timecourse of a HIPCOP hybrid perfusion fed-batch culture. (A) Viable cell density. (B) Cell viability. (C) Product titer as measured by HPLC.

Figure 4. Timecourse of various nutrients, metabolites, and cell culture conditions over the course of the experiments. A: Residual glucose. B: Lactate. C: Ammonium. D:
Osmolality.

6 Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2017


Table II. Process performance metrics—perfusion volumes, peak perfusion rates, feed volumes, final titers, day-12 cell viabilities for the hybrid
perfusion/fed-batch process.

Cell line/project

Parameter A B C D E
Total volume of perfusion medium used (reactor volumes, based on final day-12 bioreactor volume) 1.6 1.4 1.4 1.8 1.0
Total volume of perfusion medium used (reactor volumes, based on bioreactor volume during perfusion phase) 2.5 2.2 2.2 2.4 1.6
Peak perfusion rate (volume/reactor volume/day in last 24 h of perfusion based on bioreactor volume during perfusion) 1.1 1.2 1.0 1.3 0.7
Cell specific perfusion rate from days 1–4 (picoliters/cell/day) 25 33 34 24 27
Hybrid process integrated viable cell density from days 4–12 (e6 cell  day/mL) 397 400 407 426 490
Optimized fed-batch integrated viable cell density (12-day culture, e6 cell  day/mL) 216 318 223 217 181
Hybrid process total feed volume used (percent of final reactor volume) 35 36 37 34 36
Hybrid process final day-12 titer (g/L) 11.8 11.3 12.0 6.1 8.2
Hybrid process percent titer improvement over optimized fed-batch process 151 95 71 22 70
Specific productivity of hybrid process (picograms/cell/day, days 4–12) 27 26 27 14 16
Specific productivity of optimized fed-batch process (picograms/cell/day) 22 18 31 23 27
Hybrid process final day-12 cell viability (%) 86.7 97.3 94.4 92.4 96.4

Specific productivities for the hybrid process are calculated using the change in titer during the time in fed-batch and the integrated viable cell densities over the same span of
time.

affected cell growth rates near the time the cell densities peaked groups. In the presence of excess ammonium, mammalian cells will
around day 5–6. While not detailed in the current work, on-going also oftentimes transaminate pyruvate to form alanine which will
experimental investigations into CHO fed-batch culture in our accumulate in the culture (Altamirano et al., 2001). In the current
laboratory suggest that numerous low molecular weight metabolites experiment, generally when ammonium levels in the culture were
accumulating in culture serve as inhibitors of cell growth. elevated, the levels of alanine were also elevated (data not shown).
After the HIPCOP portion of the bioreactor experiment, no The timecourse of osmolality of the cultures is shown in
attempt was made to continue glucose limitation for any of the Figure 4D. Within the first 24 h for most cultures, the osmolality
bioreactors. As a result, due to cell line metabolism variability, the increases slightly as some fraction of glucose is converted to lactate
lactate level for some bioreactors rose modestly from day 4 to 5, and and then neutralized by base. This osmolality increase is
then generally fell throughout the remainder of the fed-batch compensated to some extent as the glucose and amino acids
(Fig. 4B). One cell line, known from previous conventional fed- present in the medium are consumed. After the perfusion is self-
batch experiments to be particularly prone to lactic acid production initiated by the cells between 24 and 48 h after inoculation, the
when glucose is freely available, peaked at a lactate level of osmolality of the culture stabilizes and trends slightly lower for all
4.7–4.8 g/L on days 5 and 6. cultures until the perfusion stage is terminated on day 4. From an
While no attempt was made to limit glucose availability beyond analysis of residual amino acids, we estimate that between 40 and
day 4, in some cases due to the rapidly increasing cell densities from 60% of the amino acids entering in the perfusion medium are being
days 4 to 6 and the difficulty in predicting the metabolism during consumed (data not shown). To minimize high osmolality late in
this transition state, some cell lines drove the glucose concentration
down to below 1 g/L transiently (Fig. 4A). One bioreactor was
slightly overfed with concentrated glucose (cell line D days 5–7,
Fig. 4A). After completing the experiments with cell lines A and B,
because the residual lactate levels were getting to low values just
prior to the termination of perfusion, there was some concern that
the HIPCOP control scheme might be on the edge of failure. If the
culture could no longer signal a need for additional glucose through
a rise in bulk pH, the perfusion rate might no longer continue to
ramp up. For this reason the sodium-L-lactate concentration in the
perfusion medium was increased from 1.4 to 2.5 g/L for
experiments performed with cell lines C, D, and E (Table I).
Ammonium levels in the bioreactors were generally below about
6 mM until the last several days of culture. Some accumulation of
amino acids in the culture occurred due to a slight excess of
concentrated feeds (data not shown). The excess availability of
amino acids may have contributed to the slight rise in ammonium
levels from days 10–12 for some cultures as sometimes mammalian Figure 5. Final day-12 harvest titer of HIPCOP hybrid perfusion fed-batch culture
cells will utilize excess amino acids as a carbon source for the Kreb’s compared to that of the optimized fed-batch process designed for each cell line/
product.
cycle, which can lead to an accumulation of the residual amine

Hiller et al.: Hybrid Perfusion Fed-Batch CHO Culture 7


Biotechnology and Bioengineering
Table III. Selected day-12 product quality parameters from the hybrid perfusion/fed-batch process in comparison to reference standards of original
optimized fed-batch processes.

Cell line/project

Parameter A Ref.std. A B Ref.std. B C Ref.std. C E Ref.std. E


High molecular mass species (%) 1.3 1.3 1.9 2.6 1.7 0.4 0.8 2.5
Low molecular mass species (%) 0 0.3 0.4 0.4 0 0 0.3 0
Fragment (%) – – – – 0.6 0.6 0.6 0.6
% Core fucosylated 94.7 95.0 – – – – – –
% G0-core fucosylated 75.8 75.7 – – – – – –
% G1-core fucosylated 13.7 15.4 – – – – – –
% G2-core fucosylated 1.3 1.4 – – – – – –
% Terminal galactosylation 15.9 17.6 – – – – – –
% High mannose structures 3.3 2.8 – – – – – –

All bioreactor samples underwent a simplified purification process consisting of drip column proA purification. High and low molecular species were quantitated by size
exclusion chromatography; fragment was quantitated by reduced capillary gel electrophoresis; N-linked glycan analysis was by the N-linked Oligosaccharide method as
described in materials and methods. Elevated high molecular mass species in bioreactor samples from project C may be due to the truncated purification process. The reference
standards for projects B and E were created using clonal cell lines that were abandoned in later stage development partly due to elevated high molecular mass species levels. All
reference standard material was generated at 500 or 2,500 L scale with a complete purification process. Blanks in the table indicate the assay was not performed.

culture, after the experiments with cell lines A and B it was decided exceeded 375 mOsm/kg, well within a reasonable range for CHO
to slightly decrease the overall osmotic strength of the perfusion cells in culture.
medium for the experiments with cell lines C, D, and E, so that the The final day-12 cell densities in the current experiments were
osmolality of the culture is as low as possible when the fed-batch between 31 and 48  106 viable cells/mL (Fig. 3A), but it is possible
portion of the experiment begins. that a significant number of cells were lost to shear damage during
The timecourse of product titer, the concentration of the culture and therefore calculating total cell mass using the
recombinant humanized IgG antibody molecule, is plotted in percent viability measurement might be somewhat misleading. For
Figure 3C. Samples of permeate were also tested for product titer this reason careful measurements of total solids were performed by
during the perfusion stage of the experiment and the results centrifuging culture samples. This analysis showed that the percent
showed that no significant product retention occurred across the solids were between 10% and 12% by volume on the final day of
microfiltration hollow fiber unit. For this reason, during the culture which might be close to the limit for some solids removal
perfusion stage most IgG antibody is flushed out of the bioreactor technologies typically utilized for large-scale harvest operations.
system, which explains the very low antibody concentration in the
bioreactor around days 4–5. Calculations performed utilizing the
Comparison of Product QualityHybrid Perfusion
flow rates and concentrations of product during the perfusion
Fed-Batch to Optimized Fed-Batch
phase allow us to estimate that only 9–11% of total antibody
produced during the entire 12-day culture is lost to the permeate Product quality analytics were performed on some of the samples
stream. from the experiments, the results are shown in Table III and
Although extensive optimization experiments utilizing this Figure 6. For many of the parameters, there are no significant
hybrid perfusion/fed-batch approach have not been performed, a differences between the day-12 harvest sample of the hybrid
comparison can be made to the late-stage optimized fed-batch perfusion fed-batch process when compared to the reference
processes for all of the cell lines/projects utilized in the current standard that was generated at large scale (500 or 2,500-L) using an
experiments. With the possible exception of cell line D (which optimized fed-batch process and fully purified. The acidic and basic
exhibited a higher cell specific productivity in its legacy basal species quantitation illustrated by Figure 6 shows that while there
medium and used a very high inoculation density in the optimized may be differences in earlier time-point samples, usually by the
fed-batch process), Figure 5 and Table II show that substantial day-12 harvest the profiles are quite similar. A more detailed
improvements in titer were achieved, some as high as 2.5 times the analytical investigation would be required to determine the precise
fed-batch productivity. It is likely that using the hybrid perfusion/ nature of the changes to the molecule that are causing the shifts in
fed-batch approach even higher peak cell densities would likely have acidic and basic species, and the reasons for the shifts might also
been achievable if the perfusion was extended beyond day 4. Since vary from antibody to antibody. From prior work and knowledge of
the cell density typically peaks 1–2 days after the perfusion phase, hold time stability experiments performed on proteins under
and the majority of the protein of interest is produced during the development, we know that changes in acidic and basic species
fed-batch portion of the culture, optimizing the feed composition levels are common in antibodies produced in animal cell culture
and feeding rate during this stage is particularly critical. It is and can be due to chemical or enzymatic modification of molecules
interesting to note that despite using a feed solution with an osmotic already secreted, or as a result of shifts in the properties of the
strength above 1,300 mOsm/kg and a total feed volume of 37% for molecules being secreted at different times in culture. For the
cell line C, the bulk osmotic strength of the bioreactor never majority of the antibodies studied in this work we see an increase in

8 Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2017


Figure 6. A–D. Acidic and basic species quantitation by imaged capillary electrophoresis (iCE) for cell lines/projects A, B, C, and E. Time course samples from the hybrid
perfusion/fed-batch process compared with the reference standard for each project. The reference standard was obtained from fully purified material from the original conventional
fed-batch process.

the acidic species with time in culture, which is sometimes the original process with no increase in length of culture. The
indicative of increasing levels of amino acid deamidation (Du et al., HIPCOP technology functioned effectively without significant
2012). While less pronounced, we also see a slight increase in the manual intervention for all five cell lines, allowing the cells to self-
level of basic species with time in culture for the majority of the control their perfusion rates as the cell mass increased. The total
molecules. Basic species increases are often due to increases in volumes of perfusion medium utilized were modest and because the
the levels of carboxy-terminal lysine in the heavy chain, or increases perfusion takes place only for a short duration and occurs when the
in amidation of carboxy-terminal proline. The levels of high- or low- cellular viability is high, scale-up of the cell retention system with
molecular mass species and fragments are also not significantly currently available technology and equipment is also likely to be
increased when compared to the reference standards for each achievable. Potentially of more concern regarding scale-up might be
project (Table III). the control of environmental parameters when viable cell densities
exceed 80  106 cells/mL. Design modifications of bioreactor
sparging and agitation systems may be required to supply sufficient
Conclusions
oxygen and even more importantly strip adequate carbon dioxide at
The hybrid perfusion/fed-batch process demonstrated significant large scale.
increases in productivity when compared with a more conventional This hybrid perfusion/fed-batch approach provides an opportu-
optimized fed-batch process for five different CHO cell lines/ nity to increase productivity significantly without moving to a fully
projects. In one case (cell line A) the final harvest titer was 2.5 times continuous and long-duration upstream process. The hybrid

Hiller et al.: Hybrid Perfusion Fed-Batch CHO Culture 9


Biotechnology and Bioengineering
approach also allows for a single harvest and a conventional fed- Gagnon MP, Hiller GW, Luan Y-T, Kittredge A, DeFelice J, Drapeau D. 2011. High-end
batch downstream that is appropriate for many stainless-steel pH-controlled delivery of glucose effectively suppresses lactate accumulation in
CHO fed-batch cultures. Biotechnol Bioeng 108:1328–1337.
legacy facilities.
Gray D, Chen S, Howarth W, Inlow D, Maiorella BL. 1996. CO2 in large-scale and
high-density CHO cell perfusion culture. Cytotechnology 22:65–78.
Thanks to many members of the Culture Process Development group at Hassell T, Gleave S, Butler M. 1991. Growth inhibition in animal cell culture. The
Pfizer for their suggestions to improve the HIPCOP technology and hybrid effect of lactate and ammonia. Appl Biochem Biotechnol 30:29–41.
perfusion fed-batch process, and to Inge Tamm-Daniels and Elizabeth Hiller GW, Clark DS, Blanch HW. 1993. Cell retention-chemostat studies of
Eydelman for early experiments investigating these technologies. Special hybridoma cells—analysis of hybridoma growth and metabolism in continuous
thanks to Michael O’Conner for help implementing the technology at the suspension culture in serum-free medium. Biotechnol Bioeng 42:185–195.
100-L pilot scale, and to product quality analysis and performed by Jennyfer Mercille S, Johnson M, Lanthier S, Kamen A, Massie B. 1999. Understanding factors
K. Smith, Benjamin B Gerade, Rebekah Ward, and Angela Stead. that limit the productivity of suspension-based perfusion cultures operated at
high medium renewal rates. Biotechnol Bioeng 67:435–450.
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