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Cell Culture
William Kelly and Jennifer Scully
Dept. of Chemical Engineering, Villanova University, 318 White Hall, 800 Lancaster Ave, Villanova, PA 19085
Alternating tangential flow (ATF) filtration has been used with success in the Biopharmaceutical industry as a lower shear technology for cell retention with perfusion cultures. The
ATF system is different than tangential flow filtration; however, in that reverse flow is used
once per cycle as a means to minimize fouling. Few studies have been reported in the literature that evaluates ATF and how key system variables affect the rate at which ATF filters
foul. In this study, an experimental setup was devised that allowed for determination of the
time it took for fouling to occur for given mammalian (PER.C6) cell culture cell densities
and viabilities as permeate flow rate and antifoam concentration was varied. The experimental results indicate, in accordance with DArcys law, that the average resistance to permeate flow (across a cycle of operation) increases as biological material deposits on the
membrane. Scanning electron microscope images of the post-run filtration surface indicated
that both cells and antifoam micelles deposit on the membrane. A unique mathematical
model, based on the assumption that fouling was due to pore blockage from the cells and
micelles in combination, was devised that allowed for estimation of sticking factors for the
cells and the micelles on the membrane. This model was then used to accurately predict the
increase in transmembane pressure during constant flux operation for an ATF cartridge
C 2014 American Institute of Chemical Engineers Biotechused for perfusion cell culture. V
nol. Prog., 30:12911300, 2014
Keywords: fouling, tangential flow, filtration, mammalian cell, perfusion
Introduction
Perfusion is the most efficient mode of production for animal cell culture products. The high cell and product concentrations are achievable because of the feeding of fresh
nutrients and removal of metabolites.1 Alternating tangential
flow (ATF) filters have been used with success in the biopharmaceutical industry as a lower shear technology for
mammalian cell retention with perfusion cultures.2 The longer growth cycles and the higher cell densities achievable
using ATFs for perfusion cell culture can result in membrane
fouling, which is detected by an inability of the permeate
pump to maintain the desired permeate flow rate. A fouling
rate is then defined by how long the filter is used before
fouling is detected. The potential mechanisms for ATF fouling are presumably the same as those that cause fouling in
tangential flow filtration (TFF) systems: complete pore
blockage (due to cells or aggregates of molecules), pore constriction (due to molecular buildup within the pores), or cake
formation (due to the buildup of a biological matrix on the
Correspondence concerning this article should be addressed to
W. Kelly at william.j.kelly@villanova.edu.
C 2014 American Institute of Chemical Engineers
V
outer membrane surface composed of cells and/or molecules).3 Deposition of yeast cells and latex beads (312 mm)
on membrane surfaces has been observed using a noninvasive Direct Observation through Membrane (DOTM) technique.4 These results indicated that at one fixed flux rate, the
larger (6.4 mm) particles were observed rolling across the
membrane surface while the smaller (3 mm) particles were
observed forming a flowing cake layer. In another study, for
a spin filter and cross flow filtration system, it was determined that filter fouling was due to the deposition of nucleic
acids and dead cells.5,6 In other studies, membrane fouling
due to antifoam has been studied.7
Mathematical models have been developed to describe
how transmembrane pressure (DP) should increase during
constant flux dead-end filtration. This increase in pressure
can be attributed most generally to an increase in total resistance (Rtot) due to the increased amounts of biological material deposited on the membrane per
DP=Q=A5Rtot
(1)
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Initial Cell
Viability (%)
Permeate
Rate (mL/min)
Antifoam
Level (ppm)
80.6
80.1
69.3
91.1
91.3
87.2
50.0
76.6
45.2
83.4
81.4
77.0
56.9
92.5
91.9
70
120
170
170
170
120
120
70
120
120
120
170
120
120
120
20
30
30
0
0
0
0
0
0
0
0
0
6,000
0
0
3,000
3,000
4,500
0
3,000
0
46.8
42.1
27.9
37.4
28.9
39.7
24.9
30.9
45.9
0
29.6
32.6
0
40.0
0
34.6
42.7
67.1
Figure 2.
Analytical methods
Throughout the duration of each experiment, 10 mL samples
were taken from the bioreactor. The sample was analyzed using
Siemens RAPIDLab 248 Systems blood gas analyzer to obtain
values for pH, pO2, and pCO2. In addition, a Cedex HiRes
Analyzer was used to measure average cell diameter (mm), viability, viable cell concentration, dead cell concentration, total
cell concentration, and cell aggregate rate. The sample was
centrifuged at 1,500 rpm for 5 min, with the supernate of the
sample then being extracted and filtered through a 0.2 mm filter. Metabolites, such as glucose, glutamine, glutamate ammonium, pH, potassium, lactate, sodium, and ionized calcium
were measured using BioProfile FLEX Analyzer. Finally, a
Cedex BioHT Analyzer measured Lactate Dehydrogenase
(LDH) levels in the supernate.
After some of the runs, the used filter cartridge was
drained by gravity and then the two lumen ends and permeate port were sterilely wrapped. The filters were stored in the
1294
Figure 3. (a) Total cell concentration vs. time during the ATF experiments and (b) cell viability vs. time during the ATF experiments.
1295
Figure 4.
that the fouling rate increases with the cell density of the
solution, when all other variables are the same.
SEM analysis of filtration membranes
The fibrous structure of pristine or unused membranes is
evident under SEM (Figure 5a). It was evident from looking at membranes that had been exposed to cells, that cells
had been deposited on the surface. In general, most of
these membranes had fouled, and the samples indicated
that much but not all of the surface was covered with a
(cell containing) cake layer. Figure 5b shows a section of
membrane surface that has a cake layer with several
hills that correspond to the size (approximately 10 mm)
of the cells used in the experiments. Two of the filtration
experiments were run with only media and antifoam, but
no cells were present. According to the vendor, the antifoam should form micelles in the media that are 2540 mm
in diameter. Figure 5c shows a large section of the inner
lumen membrane surface from an experiment with media
and antifoam (no cells present), on which particles are
deposited. At closer magnification (Figure 5d), these particles are seen to be approximately 3040 mm and are
assumed to be micelles.
(2)
17
(3)
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Figure 5. (a) SEM image: inner surface of a new fiber, (b) SEM image: inner surface, covered with cells after a perfusion run, (c)
SEM image: inner lumen surface, covered with antifoam micelles after ATF run, and (d) SEM image: inner lumen surface,
close up of micelles on membrane surface.
(4)
As [(CAp)/(RmlQ)] is not expected to change in the pressure vs. exhaust phases, Eqs. 2, 3, and 4 can be combined to
obtain
acells
phase =2
net-cycle
5acells
(5)
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Figure 7. (a) P2 sensor (see Figure 1 for sensor location) reading (psi-gauge) and TMP (psi) within three ATF cycles (Note: data
points are every 2 s) and (b) change in 1/TMP during the pressure and exhaust phases of the ATF cycle for a typical cycle.
[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Figure 9. (a) Model vs. expt (40 3 106 cells/mL, 3,000 ppm AF,
120 mL/min perm flow, 77% viability) and (b) model
prediction for membrane coverage (same conditions as
Figure 9a). [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
Figure 10. (a) Model vs. expt (40 3 106 cells/mL, 3,000 ppm
AF, 30 mL/min perm flow, 92.5% viability) and (b)
model prediction for membrane coverage (same
conditions as Figure 10a).
experimental runs with lower and higher viability cell solutions; however, it became evident that the sticking factors
for the exhaust phase of a cycle (acells_exhaust-phase) are greater
for lower viability cell solutions, while the (anti)sticking factors for the pressure phase are greater for the higher viability
cells. This is evident from Figure 11, which shows one cycle
of data for a low (50%) cell viability solution (Figure 11a)
and a high (91%) cell viability solution (Figure 11b). The
slope during the exhaust phase (which is proportional to the
sticking factor) is steeper for the low viability cells
(20.0031) as opposed to the higher viability cells
(20.0027); whereas the slope during the pressure phase is
steeper for the high viability cells (0.0025) as opposed to the
lower viability cells (0.0016). This result suggests that there
is variation between low and high viability cells in the
degree to which they attach (during the exhaust phase) as
well as detach (during the pressure phase) from the
membrane.
It was also observed that, for most of the experiments, the
sticking factor for the exhaust cycle (acells_exhaust-phase)
increased as the cycle-averaged TMP increased during the
course of a given experiment. This corresponded to a greater
decrease in TMP during the exhaust phase near the end of
the experiment as compared to the beginning. Interestingly,
the (anti)sticking factor for the pressure phase (acells_pressurephase) increased by the same degree, resulting in the same net
sticking factor (acells_net-cycle, from Figure 8) at the beginning
of an experiment (i.e., where cycle-averaged TMP is low)
beginning) as at the end of an experiment (i.e., where cycle-
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result of existing biological deposits on the membrane surface facilitating deposition of additional deposits.
Using the model to predict fouling rate in a perfusion cell
culture
A perfusion cell culture experiment was run to test the ability of the model to predict the rise in TMP vs. runtime. As
per Figure 12, the permeate flow rate was increased exponentially over the course of the 10 day run, per a predefined program. Samples were taken once per day for cell concentration
and viability, and those results indicate an exponential rise in
cell concentration (Figure 12). For a 1 h period every day, at
the time when samples were taken, TMP vs. time was
recorded and an average TMP for that period was obtained.
Antifoam is added when needed, and for this run was added
on day one and day six. A stepwise representation of the permeate flow rate was used (Figure 12) such that the model,
based on a constant permeate flow rate assumption, could be
used across 24 h segments of time. The model accounted for
the concentrations of cells and antifoam in solution on a given
day. The model also used the appropriate values of acells_netcycle from Figure 8, as the viability changed during the experiment (ranging from 8292%). In this experiment, the experiment concluded before the ATF cartridge fouled; however,
Figure 13 shows that the model predicted the rise in TMP
between days 710 quite well, as the system began to
approach the critical fouling TMP (of approximately 14 psi).
Figure 11. (a) Experimental TMP data within an ATF cycle:
low (50% viable cells) and (b) experimental TMP
data within an ATF cycle: low (91% viable cells).
[Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
Figure 12.
Perfusion experiment: Model vs. experiment al prediction of TMP increase with time. [Color figure
can be viewed in the online issue, which is available
at wileyonlinelibrary.com.]
averaged TMP is higher). Larger pressure-phase (anti)sticking factors, seen near the end of the experiments, correspond
to an enhanced rate of removal of biological deposits from
the membrane which is consistent with Stressmans observation9 that the removal rate of deposited biological solids
from a membrane surface as a result of crossflow (during
TFF) increases when there is more material deposited on the
membrane. The increase in deposition rate during the
exhaust cycle (i.e., acells_exhaust-phase) near the end of the
experiments is more difficult to explain, and might be a
Conclusions
In this study, an experimental setup was devised that
allowed for determination of how long it took for fouling to
occur for given mammalian cell culture cell densities and viabilities as permeate flow rate and antifoam concentration was
varied. The experimental results indicate, in accordance with
DArcys law, that the average resistance to permeate flow
(across a cycle of operation) increases as biological material
deposits on the membrane. SEM images of the post-run filtration surface indicated that both cells and antifoam micelles
were depositing on the membrane. A unique mathematical
model, based on the assumption that fouling was due to pore
blockage from the cells and micelles in combination, was
devised that allowed for estimation of sticking factors for the
cells and the micelles that were used to accurately predict the
fouling rate of ATF cartridges as a function of cell and antifoam concentration, cell viability, and permeate flow rate for
a perfusion cell culture run. Future work might include evaluating the effects of changing the ATF rate and evaluating the
fluid dynamic mechanisms responsible for the removal of
some biological material from the membrane surface during
the pressure phase of the ATF cycles, along with determining
how ATF behavior (i.e., sticking factor values) varies with
different cell and membrane types.
Acknowledgment
We want to thank Dr. Ronald Balsamo, Director of the
Villanova University microscope facility, for assistance with
cutting the fibers using liquid nitrogen.
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