APPLICATION NOTE No.

410 I April 2019

Small Scale Perfusion Using an ATF Cell Retention

Device with DASGIP® Parallel Bioreactor System
Amanda Suttle, Stacey Willard, and Ma Sha
Eppendorf, Inc., Enfield, CT, USA
Contact: [email protected]

Abstract

Experimentation at small scale is crucial for the cost- of 60 x 106 cells/mL. Cell growth, peak density, and
efficient development of bioprocesses, which then antibody production at the 1 L scale were comparable to
can be transferred to larger production volumes. To those in a previously performed perfusion process with
facilitate perfusion cell culture process development at a working volume of 3.75 L. Our results demonstrate the
small scale, we tested the feasibility of connecting an feasibility of ATF-based perfusion at small scale using
alternating tangential flow (ATF) filtration device with the DASGIP Parallel Bioreactor System.
a DASGIP Parallel Bioreactor System. In a working
volume of 1 L we reached a peak viable density

Introduction

Upstream bioprocessing in perfusion mode holds great Apart from being used for the production of secreted
promise for the industrial production of cells and biologics. proteins or viral vectors, perfusion is used in the earlier
In perfusion, fresh medium is constantly added to the sub-steps of a bioprocess, as in the production of high-
bioreactor and used medium is harvested, while the cells density seed trains, or to reach high cell densities before
are retained in the bioreactor. As a result, the composition transfection/infection of cells for viral vector production. In
of the cell culture medium stays quite constant during the all cases, perfusion bioprocessing requires a cell retention
process. This has several advantages. In perfusion, higher device within or attached to the bioreactor.
cell densities can be reached than in batch and fed-batch Cell retention devices based on ATF are filters that hold
processes and therefore the volumetric productivity will be back the cells and let the liquid and small molecules pass. In
enhanced. As the medium composition may influence the tangential flow filtration (TFF) the liquid flows past the pores
cell metabolism and therefore product characteristics, more of hollow fiber filters tangentially, rather than being forced
constant process conditions can lead to a more consistent through them orthogonally, thus reducing the likelihood of
product quality. Products which pass the cell retention clogging. ATF devices use the same principle of tangential
device are constantly harvested. The time they reside in the flow, but reverse the direction of flow regularly to minimize
culture medium is reduced, which is advantageous for the fouling and reduce shear forces on the cells. ATF perfusion is
production of less stable products. suitable for perfusion processes with suspension cells.
APPLICATION NOTE I No. 410 I Page 2

ATF cell retention devices support a broad range of feasibility of utilizing ATF for small scale (1 L) perfusion of
bioreactor scales from small to pilot/production. The CHO cells in the DASGIP Parallel Bioreactor System.
objective of this proof of concept study was to demonstrate

Material and Methods

Connection of DASGIP Parallel Bioreactor System with barb fitting).

ATF cell retention device We installed the dip tube opposite of the gas sparger as
We used a DASGIP Parallel Bioreactor System with Bioblock. recommended by the manufacturer, ensuring no bubbles
The system was equipped with a DASGIP MP8 Multi Pump from gassing would enter the ATF filter. The filter had a
Module, a DASGIP TC4SC4 temperature and agitation pore size of 0.2 µm. The device was connected to an ATF
control module, a DASGIP PH4PO4 module for pH and controller (C24 Controller, Repligen). The ATF circulation
DO monitoring, and a DASGIP MX4/4 gas mixing module, rate was set to 0.5 L/min.
supporting gas flow rates of up to 250 sL/h. High maximum Medium was added and used medium was harvested
flow rates of air and oxygen are beneficial for cell culture using the system’s integrated pumps. Depending on the
perfusion bioprocesses, because high cell densities can be pump head tube diameter, the pumps support feed rates of
reached and the oxygen demand of the culture may become 0.3 to 9.5 mL/h or 13 to 420 mL/h. The broad feed rate range
high. allows the adjustment of the perfusion rate during the run.
We used a DASGIP Bioblock Stirrer Vessel with a working In the experiments here we used pump head tubing with an
volume of 200 mL – 1 L equipped with a pitched-blade inner diameter of 0.5 mm, supporting feed rates of 1.3 to
impeller. 42 mL/h. Feeding and harvest volumes can also be matched
The connection of a DASGIP Parallel Bioreactor System using two integrated scales, one for feeding and one for
with ATF cell retention device is shown in Figure 1. For harvesting. This allows for tight control over liquid additions
medium harvest the bioreactor was equipped with a dip tube and the removal of wastes from the vessel, maintaining exact
(length 250 mm, diameter 12 mm, Repligen®, USA). The vessel volume.
dip tube was connected to a XCell™ ATF 2 (Repligen) via
silicone tubing and a CPC™ AseptiQuik® fitting (3/8” hose

Fig. 1:
Connection of
DASGIP Parallel
Bioreactor
ATF controller
C24 System with ATF
cell retention
device.
A: Schematic
overview
B: Photo of the
setup
Bioreactor

ATF filter
APPLICATION NOTE I No. 410 I Page 3

Cell line and medium a polarographic sensor (Mettler Toledo®, Switzerland) and
We used a suspension CHO cell line from TPG Biologics, controlled it at 50 % by sparging air and/or O2. The pH was
Inc., expressing an hMAb. We cultivated the cells in controlled at 7.0 (deadband = 0.2) via a cascade to CO2 (acid)
Dynamis™ AGT™ Medium (Thermo Fisher Scientific®, USA). and 0.45 M sodium bicarbonate (base). We inoculated the
The medium was supplemented with 8 mM L-glutamine and culture with a final cell density of 0.9 x 106 cells/mL. We
1 % Gibco® Anti-Clumping Agent (Thermo Fisher Scientific). cultivated the cells at 37 °C.
Cultures were perfused with Dynamis AGT Medium
supplemented with 2 mM L-glutamine and 1 % Anti- Sampling and analytics
Clumping Agent. Glutamine feeding concentration was We took two 3-mL samples from the bioreactor daily, one in
decreased to reduce ammonia production during the run. the morning and one in the evening, to analyze cell density,
viability, and the concentrations of glucose, ammonia,
Inoculum preparation lactate, and IgG. The samples were taken through a dip tube
We prepared the bioreactor inoculum by cultivating the connected to a swabable valve. To collect the highest quality
cells in single-use baffled polycarbonate shake flasks in a sample from the growing culture, we connected a sterile
New Brunswick™ S41i CO2 incubator shaker set at 125 rpm 5 mL syringe to the sample port Luer Lock and removed
and 8 % CO2 with passive humidification. Cells from a and discarded a dead volume of 3 mL. We then collected
cryopreserved stock vial were inoculated at a density of a second 3-mL sample in a new syringe to provide a fresh,
0.3 x 106 cells/mL in a 125 mL flask with a 20 % fill volume. viable sample for analytics. The total bioreactor volume was
After one week of passaging every other day, we scaled-up readjusted to compensate volume lost through sampling
the culture volume by increasing the flask size from 125 mL after every sample.
to 250 mL, while keeping the inoculation density, percentage We measured cell density and viability (via the trypan blue
fill, and all other parameters constant. A flow chart exclusion method) using a Vi-Cell® XR Viability Analyzer
representing the cell expansion process from initial thaw to (Beckman Coulter®, USA), and pH using an Orion Star™
inoculation of the bioreactor is shown in Figure 2. 8211 pH meter (Thermo Fisher Scientific). Using the offline
pH value, we restandardized the controller pH calibration
Bioreactor control and process parameters daily, if necessary, to prevent any discrepancy between
The glass vessel was equipped with a pitched-blade impeller. online and offline measurements. Glucose, ammonia, lactate,
The culture was agitated at 309 rpm. We measured DO using and hMAb concentrations were measured using a Cedex®
Bio Analyzer (Roche Diagnostics®, Germany).
Cryopreserved cells (1 x 107 cells)
Feeding and perfusion control
1 x 125 mL shake flask
30 mL working volume
Our perfusion target was to keep the ammonia concentration
inoculated at 0.3 x 106 cells/mL < 4 mM. We also aimed to keep the glucose concentration
> 3 g/L. We adjusted the perfusion rate and fed the culture
2 x 250 mL shake flask with glucose based on the metabolite concentrations
60 mL working volume
grown to 1.8 x 106 cells/mL determined offline.
We turned on the ATF device a few hours prior to
4 x 250 mL shake flask inoculation and began media circulation through the filter at
60 mL working volume our setpoint for the run (0.5 L/min), as recommended by the
grown to 4 x 106 cells/mL
manufacturer. The filter could then be properly wetted by
media circulation and allow for any air bubbles to gradually
240 mL inoculum work themselves out of the filter prior to adding cells to our
vessel. As per manufacturer recommendations, we started
Bioreactor (1 L working volume) the ATF circulation of media and cells 5 hours before it was
Inoculated with 0.9 x 106 cells/mL
needed to initiate perfusion so that our cells could adjust to
the alternating tangential flow-related stress.

Fig. 2: Cell expansion from initial thaw to bioreactor inoculation

APPLICATION NOTE I No. 410 I Page 4

Results

Metabolite concentrations
We aimed to keep the ammonium concentration < 4 mM and The IgG concentrations in the bioreactor and harvest
the glucose concentration > 3 g/L in the course of the run. increased up to approximately 500 mg/L. As expected, IgG
On day 2 the ammonia concentration exceeded 3 mM and production steadily increased following the cell growth
we started perfusion at 0.2 vessel volumes per day (VVD). profile (Figure 5).
Based on the ammonia concentrations determined offline,
we gradually increased the perfusion rate up to 1.4 VVD. The 80 100

ammonia concentration stayed below 4 mM until day 15. The 90

70
lactate concentration increased up to 1.9 g/L on day 2. With 80
60
the start of perfusion on day 2 it decreased, and was kept 70

Cell viability (%)

Cell density (x 106)
50
below 1 g/L from day 6. When the glucose concentration 60

dropped below 3 g/L, we fed the culture by pumping in 40 50

40
the appropriate amount of 200 g/L sterile glucose into the 30
30
culture twice daily, in addition to replenishing the glucose 20
20
throughout the perfusion process (Figure 3). 10
10

0 0
0 2 4 6 8 10 12 14 16
Cell growth and antibody production Cell Density Cell Viability Time (days)

We had inoculated the culture at a density of 0.9 x 106 cells/

mL. On day 13, the culture reached a peak cell density of
60.88 x 106 cells/mL. At that time point, 90 % of the cells
were viable (Figure 4). Fig. 4: Cell growth and viability. Samples were taken twice daily
and cell number and viability were analyzed offline.

8
600
Glucose conc. (g/L), lactate conc. (g/L)

7
IgG concentration in bioreactor
500
IgG concentration (mg/L)

6
IgG concentration in harvest
NH3 conc. (mmol/L)

5 400

4
300

3
200
2

100
1

0 0
0 2 4 6 8 10 12 14 16 18
0 2 4 6 8 10 12 14 16 18
Glucose Lactate Time (days) Time (days)
NH3

Fig. 3: Metabolic profile. Samples were taken twice daily and Fig. 5: IgG production. Samples were taken twice daily from
the concentrations of glucose, lactate, and ammonia were the culture medium in the bioreactor and the harvest. The IgG
determined offline. production in culture medium and harvest were determined
offline.
APPLICATION NOTE I No. 410 I Page 5

Conclusion
The results described above demonstrated the feasibility of ATF-2 device from Repligen and controlled with a BioFlo 320
cultivating CHO cells using perfusion in a DASGIP Parallel bioprocess control station [1]. Likewise, an ammonium
Bioreactor System equipped with an ATF cell retention concentration of less than 4 mM and a glucose concentration
device. The system allowed cell culture perfusion at small higher than 3 g/L were targeted. The peak viable cell density
scale, which is well suited for process development. The was 74 x 106 cells/mL at day 15. The fact that we achieved
system’s pumps support a broad range of perfusion rates. similar cell densities in CHO cell perfusion processes with
The system’s broad gas flow ranges help meeting the oxygen working volumes of 1 L and 3.75 L indicates the scalability of
demand of high-density cultures. the system. Larger ATF filtration devices are available, which
In a previous study we had cultivated the same cell line in principle support perfusion cultures with working volumes
in perfusion using a custom BioBLU c Single-Use Vessel of more than 1,000 L.
(maximum working volume of 3.75 L) equipped with an

Literature

[1] Willard S, et al. Comparing Culture Methods in Monoclonal Antibody Production. Bioprocess International.
15 (3): 38–46. 2017
APPLICATION NOTE I No. 410 I Page 6

Ordering information
Description Order no.
DASGIP® Parallel Bioreactor System, for microbial applications, max. 250 sL/h gassing, 4-fold system with 76DG04MBBB
DASGIP® Bioblock
DASGIP® Vessel, SR0700ODLS, 200 mL – 1.0 L 76SR0700ODLS
New Brunswick™ S41i, 170 L, CO2 incubator shaker with inner shelf and touch screen control, 1 (2. optional) S41I120010
shelves, orbit diameter 2.5 cm (1 in)

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Repligen® is a registered trademark of Repligen Corp., USA. XCell™ is a trademark fo Repligen Corp., USA. Thermo Fisher Scientific® is a registered trademark of Thermo Fisher Scientific Inc., USA. Dynamis™
AGT™ and Orion Star™ are trademarks of Thermo Fisher Scientific Inc., USA. Gibco® is a registered trademark of Life Technologies Corporation, USA. Aseptiquick® is a registered trademark of Colder Products
Company, USA. Beckman Coulter® and VI-CELL® are registered trademarks of Beckman Coulter, Inc., USA. Roche Diagnostics® and CEDEX® are registered trademarks of Roche Diagnostics GmbH, Germany.
Mettler Toledo® is a registered trademark of Mettler-Toledo GmbH, Switzerland. Eppendorf®, the Eppendorf Brand Design, and BioBLU® are registered trademarks of Eppendorf AG, Germany. New Brunswick™
is a trademark of Eppendorf AG, Germany. DASGIP® is a registered trademarks of DASGIP Information and Process Technology GmbH, Germany. BioFlo® is a registered trademark of Eppendorf, Inc., USA.
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