This poster explores key elements of bioreactor design and automation strategies that enable successful implementation of seed train intensification via perfusion: - Sparger performance characterization - Cell retention device connection - Evaluation of the Hamiltion® Incyte viable cell density (or permittivity) sensor - Cell culture case studies To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: www.merckmillipore.com/mlab

1. MerckMillipore.com
The life science business of Merck operates as
MilliporeSigma in the U.S. and Canada.
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VCD,e6cells/mL
Culture Age, days
ATF-6, 20 L
ATF-6, 40 L
Cellicon TFF,
50 L (1)
Cellicon TFF,
50 L (2)
Cellicon™
TFF Filter,
50 L (1)
Cellicon™
TFF Filter,
50 L (2)
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kLa,hr-1
Measuredin1XPBS,55W/m3
Total Gas Flow, L / min
ActualVCD(e6c/mL)
ModeledMaxVCD(e6c/mL)
model-predicted max
VCD capability with this
cell line ~150 e6 c/mL
OUR = OTR
VCD x qO2 = kLa x (C* - CL)
Introduction
With recent advancements in media development and cell
retention technologies, perfusion processes are rapidly
emerging as a viable means to successfully intensify
upstream biomanufacturing operations. The fresh supply of
key nutrients coupled with the removal of waste products
while retaining the thriving cell culture in the bioreactor can
lead to higher accumulated cell densities, improved
viabilities, and greater volumetric productivities. These
process advancements can translate to savings in time,
footprint, and/or cost in the manufacturing space. In this
poster we explore key elements of the bioreactor design and
automation strategies that enable the successful
implementation of seed train intensification via perfusion.
Sparger performance characterization, cell retention device
connection, and evaluation of the Hamilton® Incyte viable cell
density (or permittivity) sensor are specifically explored. Cell
culture case studies are also presented to demonstrate the
effectiveness of using the Mobius® single-use bioreactor for
intensified seed train applications.
Perfused N-1 CoGS
Assessment
A comprehensive cost modeling assessment was performed
to examine more closely how specific production scenarios
can be impacted by implementing a perfused N-1 process
step followed by a fed-batch N (production). Figure 1 below
highlights a subset of the data generated.
Figure 1 Assumptions:
• 1 Seed Train - 1 Production bioreactor - 1 DSP
• 200 L N-1 bioreactor
• 2000 L Production bioreactor (X0=5 x106 cells/mL)
• 14 day production for both traditional and high seed
• Industry average costs
Figure 1:
CoGS, throughput, and batches/year are compared for
various scenarios of increasing production titer. The increase
in titer results when a perfused N-1 process enables a high-
seed fed-batch production. Four of the five scenarios
assume the production is carried out for a comparable
number of days compared to the traditional fed-batch
process (14 days), and a fifth scenario (bar 2) examines the
impact of perfused N-1 enabling a high-seed production
reactor to achieve comparable titer to the traditional process
but within a shorter duration of 10 days. For more
information watch the on demand webinar: A Cost Analysis
and Evaluation of Perfused Seed Train Scenarios Through
Process Modeling
Anne Hansen, Allyson Fournier, Alison Dupont, Marisa Maher, Hiral Gami, Habib Horry, Jeffrey Barna, Arshan Nazempour, Amy Wood
Merck
Hamilton® Incyte VCD Sensor
Exploration
The capability to incorporate new sensor technology such as viable cell
density (or permittivity) into recipes and PID control loops may help to
advance perfusion processing operations. Figures 5, 6 and 7 explore
how a VCD sensor can be used in perfusion processes in the Mobius®
single-use bioreactors.
Figures 5 & 6:
Offline VCD data is plotted with InCyte sensor permittivity data for the
growth phase of 4x 3L and 2x 50 L trials (Figure 5, top). When
plotted on an x-y scatter, the slope of the linear fit line gives a cell
factor of 1.45 (Figure 6, bottom). Using the offset value from the cell
free media “zero” prior to inoculation, the 1.45 cell factor was applied
retrospectively to the data collected during the 2nd 50 L trial, and
plotted as InCyte VCD in Figures 5 and 7 below. This historical
correlation model, using a single cell factor, showed strong correlation
between offline and inline VCD.
Figure 7:
Implementing CSPR control (cell specific perfusion rate, purple) during
a 50 L N-1 perfusion process using the Hamilton® InCyte VCD Sensor
provided a steady ratio of nutrients to cells, drove towards more
consistent cell culture conditions, and enabled a better estimate of
media usage. Note, CSPR control was not implemented until the
second day of perfusion (day 4 of process), due to the limited ability
for the pump to operate in a continuous mode at low speeds.
Operating the harvest pump in on/off mode is not recommended due
to potential negative impact on CRD performance.
Cell Culture & Sparger Performance Results
• 4 N-1 perfusion trials were performed with ring sparger
prototype flexware; 2 with Xcell™ ATF devices, 2 with
prototype Cellicon™ 50 L filters (operating in TFF mode)
• The 4 processes demonstrate consistent capability to support
VCDs of at least 50-90 e6 c/mL with the ring sparger
• Using the real-time gas flow rates and corresponding VCD
data from a cell culture trial, the specific oxygen uptake rate,
qO2, can be calculated. Together with measured kLa, the
maximum VCD that the system can support can be modeled.
In the example shown in Figure 4, using pure O2 and 55
W/m3, the system is expected to be able to support
sustaining up to 150 e6 c/mL with this cell line
Exploring Intensified Seed Train
Through Advancements in Perfusion
Processing Technologies
© 2020 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. Merck, Millipore, Cellicon, Mobius and
the vibrant M are trademarks, and the vibrant M are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All
other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly
accessible resources. Lit. No. MK_PS6000EN V.1.0
Incyte VCD Sensor Exploration Results
• As shown in the 2nd 50 L trial in Figures 5 and 7, the offline
VCD had a strong correlation to the InCyte sensor VCD
during the growth phase of this process when using the
historical correlation model.
• This approach was successful during the growth phase of a
culture up to ~ 50 e6 c/mL
• Adjustments to the cell factor are likely to be needed to
maintain a strong correlation if higher densities and/or
subsequent culture phases occur in the process.
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HarvestRate,L/day
VCD,e6cells/mL
Weight,kg
CSPR,pL/cell.day
Culture Age, Days
InCyte VCD
Offline VCD
y = 1,4479x - 0,7763
R² = 0,9773
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pF/cm
e6cells/mL
Culture Age, days
Permittivity
(pF/cm)
Offline VCD
InCyte VCD
50L Trials3L Trials
4
5
NEW: entirely flexible film
construction @ bag base
NEW: 2 x ¾” AseptiQuik®
Connectors to install cell
retention technologies
2
NEW: Ability to include CO2 sensor
with control loop and use additional
spare sensor in PID control loops (ex:
Capacitance / VCD sensor for cell
bleed or CSPR control)
1
NEW: Built-in X-baffle;
promotes homogeneous mixing
and consistency across scales
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4
6 NEW: load cell control loop
3 NEW: Ring sparger option
• Implementing a perfused N-1 operation can directly
translate to an improvement in throughput and/or
reduction in CoGS
• Decreased process duration, made possible by
perfused seed train operations, allows for more
batches per year
• Development of a high-seed fed-batch production
process, enabled by the perfused N-1 operation, is a
potential way to improve overall titer, drive down
costs, and improve throughput without the need of
additional batches or volume capacity
• Perfusion seed train operations can prove to be an
effective method of process intensification, while
keeping the production bioreactor operating in fed-
batch mode
CoGS Assessment Results
Bioreactor Advancements
Supporting Perfusion
Several upgrades have been made to the 50 and 200 L Mobius®
single-use bioreactors in order to meet the needs of perfused N-1
processes; refer to Figure 2 below.
Figure 2:
Key features in Mobius® 50 and 200 L single-use bioreactors
targeted to support perfused N-1 processes
Cell Culture and Sparger
Performance Results
Four N-1 perfusion process trials were conducted in prototype
flexware bags containing the ring sparger to assess performance.
Both ATF and TFF type CRDs were used.
Figure 3:
Several trials confirm that the Mobius® bioreactor system is capable
of supporting upwards of 50 e6 c/mL, with a max of 90 e6 c/mL
realized using a 50 L prototype of the Cellicon™ TFF filter (green).
Although the ring sparger was used in all 4 cases, two different cell
lines were used and process conditions were varied by design,
therefore identical performance was not expected across the trials.
Figure 4:
A model predicted capability to support a max VCD of 150 e6 c/mL
(pink) was determined based on measured kLa (purple), real-time
gas profiles (not shown), and VCD obtained during trial (blue)