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On-Line Determination of Animal

Cell Concentration

P. Ducommun, I. Bolzonella, M. Rhiel, P. Pugeaud, U. von Stockar,


I. W. Marison

Institute of Chemical Engineering, Swiss Federal Institute of Technology


(EPFL), CH-1015 Lausanne, Switzerland; telephone: +41 21 6933194;
fax: +41 21 6933680; e-mail: Ian.Marison@epfl.ch
Received 26 March 2000; accepted 28 September 2000

Abstract: A new approach for the indirect determination that has the ability to catalyze a biochemical reaction
of cell concentration in the case of nonconstant meta- (Trypan blue staining), a volume enclosed by cytoplasmic
bolic rates has been developed. The specific glucose-
uptake rate was shown to be nonconstant in batch cul- membranes (Kell et al., 1990) (dielectric spectroscopy) or a
tures of free suspended and immobilized CHO SSF3 cell with a volume above a lower limit (electrical cell
cells. Time-independent models correlating the specific counter/channelizer). It is thus essential to understand what
rate to the limiting substrate concentration were estab- is hidden behind the term biomass when comparing differ-
lished, thus providing a continuous determination of the
specific rate through on-line measurement of the limiting ent experiments. This lack of clarity has been proposed
substrate. The method could be applied to determine on- (Sonnleitner et al., 1992) as a reason to abbreviate biomass
line cell concentration in both free suspended and immo- as X, the great unknown!
bilized cell cultures. Results were verified off-line by crys- Consequently, numerous techniques have been developed
tal violet nuclei counting. The predicted cell concentra-
to measure biomass. They can be classified in two main
tion was in very good agreement with the off-line
reference during the whole exponential-growth phase, categories: direct and indirect (Konstantinov et al., 1994).
until the specific glucose-uptake rate tended to zero. Direct techniques are those directly related to the cell mass,
© 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 515–522, number or volume. In microbial systems biomass is com-
2001.
monly referred to as dry weight, but in most animal cell
Keywords: biomass; cell concentration; on-line monitor-
ing; animal cell culture systems this parameter is related to the number of cells, total
or viable, per unit of volume, for both historical and prac-
tical reasons. This direct link is very convenient and thus
INTRODUCTION direct techniques would be preferred. However, they are
generally not applicable for on-line measurement purposes
Biomass concentration is a key parameter in animal cell or often restricted to free suspended cell culture, which
culture. A knowledge of this variable is required—by defi- seriously limits the application of direct techniques such as
nition—for the determination of any specific yield, meta- flow cytometry (Duval et al., 1990; Sonnleitner, 1998), tur-
bolic rate, or mass-balance calculation and is often used as bidity sensors (Konstantinov et al., 1992), electrical cell
a criteria to evaluate different culture processes, for ex- counter/channelizer (Frame and Hu, 1990), dye-exclusion
ample, in terms of specific growth rate, maximal or final
(Becker et al., 1994) and colorimetric (Mosmann, 1983)
cell concentration. Further, biomass can be used as process
assays. Fed-batch or continuous culture with immobilized
variable to establish control strategies for fed-batch and
cells, for example, on macroporous carriers or by microen-
continuous cultures, and its measurement provides informa-
capsulation, is the most common way to reach high-cell
tion on the physiological state of the cells, a feature essential
densities and thus high-volumetric productivities (Run-
for product quality control.
stadler and Cernek, 1988). Off-line colorimetric assays have
The definition of biomass concentration arises as an in-
been developed for microcarrier beads (Margis and Boro-
teresting, nontrivial and almost philosophic problem. Cells
jevic, 1989) and encapsulated (Rollan et al., 1996) cell cul-
can be characterized according to their concentration, either
as dry mass, number or volume, or to the physiological state tures. It is possible to detach cells adhered to the surface of
and metabolic activity. Distinction has also to be made be- a carrier by trypsinization, however, in the case of macro-
tween total and “viable” cells, however, the definition of porous carriers cells are growing partly inside the pores
viability is directly related to the technique that is used to (Nikolai and Hu, 1992) and complete cell release is not
determine it. For example a “viable” cell can be either a cell achieved, thus the determination of cell concentration is not
quantitative. Other direct techniques could be applied on-
line but are restricted to high-cell-density cultures, due to
Correspondence to: I. W. Marison poor sensitivity. This is the case for dielectric spectroscopy
Contract grant sponsor: Laboratoires Serono S. A. (minimum detection limit of 2–5 × 105 cell mL−1) (Cerckel

© 2001 John Wiley & Sons, Inc.


et al., 1993; Guan et al., 1998) and acoustic resonance den- be determined on-line by maintaining a pO2 at 80% air
sitometry (106 cell mL−1) (Kilburn et al., 1989). Thus, the saturation (Ducommun et al., 2000). Cultures were per-
application of such techniques to free-suspended-cell or formed with free-suspended cells and cells immobilized on
batch culture is often limited by the relatively low-cell con- macroporous microcarriers Cytopore 2 (Pharmacia Biotech,
centrations reached in such systems. Uppsala, Sweden).
As a result of the limitations of direct techniques, indirect
techniques are commonly used for biomass determination.
These enable a convenient on-line determination of cell Direct Biomass Assays
concentration based on either metabolic rate or concentra- Culture samples were removed at intervals during the cul-
tion of metabolite measurements. Nevertheless, such tech- tures for determination of biomass. Cells and nuclei were
niques must first be calibrated using a direct technique, counted using a Neubauer-improved haemacytometer. Cell
which consists of the determination of an apparent growth- concentration and viability were determined using the
yield coefficient or a specific rate. This calibration reduces Trypan blue exclusion method (0.4%, Sigma T-8154), and
indirect techniques to a purely descriptive role, as it has to nuclei concentration using the crystal violet nuclei-counting
be established separately for each biological (Capiaumont et method (0.1%, Fluka 61135). Cell concentration was addi-
al., 1993) and bioreactor system and should not be extrap- tionally determined by electrical cell count using a Coulter
olated outside the data range (Sonnleitner, 1992). Indirect Counter ZM (Coulter Electronics Limited, Luton, England),
techniques proposed by these authors are also based on the and by dielectric spectroscopy using a Biomass Monitor 214
assumption that specific metabolic rates are constant, which M (Aber Instruments Ltd., Aberystwyth, U.K.).
is frequently not the case. Often these rates vary continu-
ously during part of, or throughout, the culture process
(Pörtner et al., 1994; Zeng et al., 1998). These variations Indirect Assays
may affect indirect models and lead to incorrect biomass Glucose concentration was determined off-line using a stan-
prediction, even when complex algorithm-like software sen- dard enzyme assay (Mannheim-Boehringer 716 251) and
sors are used (Pelletier et al., 1994). measured on-line in situ using mid-infrared spectrometry
In this article we describe a new indirect method to de- (ReactIR 1000 system equipped with a DiComp diamond
termine on-line animal cell concentration in cases where ATR probe, ASI Applied Systems, Millersville, MD). Se-
specific metabolic rates are not constant. The method is cretory component was quantified using an ELISA tech-
based on the continuous determination of specific metabolic nique. Oxygen consumption was determined by oxygen-
rates as a function of a substrate concentration during the uptake-rate measurements (Ducommun et al., 2000) based
whole culture process. Further, this approach has been cali- on stationary liquid-phase balances (Ruffieux et al., 1998).
brated with a direct off-line technique that is able to mea-
sure cell concentration with a high sensitivity (104 cell
mL−1) in both free-suspended and immobilized-cell-culture RESULTS AND DISCUSSION
systems. Hence, the domain of application of this new
method is neither restricted to high-cell-density or free- Direct Technique As Reference
suspended-cell cultures.
A number of potential techniques for direct biomass deter-
mination were investigated with the aim to select a tech-
MATERIALS AND METHODS nique which could subsequently be used for the calibration
of indirect techniques for cell-concentration determination.
Cell Culture The criteria for selection were precision, sensitivity, and
longterm accuracy. In addition, the technique should be ap-
A CHO SSF3 cell line (Novartis, Basel, Switzerland) se- plicable for both free-suspended and immobilized-cell cul-
creting human secretory component, a part of dimeric sIgA tures. A screening of the chosen direct techniques based on
antibodies, was cultivated in the serum- and protein-free these criteria revealed the crystal violet nuclei-counting
medium ChoMaster HP-1 (Dr. F. Messi, Cell Culture Tech- method to be the most suitable. This method has a low limit
nologies, Zürich, Switzerland). Cell amplification was car- of detection (104 cell mL−1) and high precision (±5%), com-
ried out in 250-mL spinner flasks at 37°C in a 5% CO2 bined with the ability to monitor cell concentration in im-
incubator. Batch cultures were operated in a 2-L (1550-mL mobilized cell cultures. This method was consequently cho-
working volume) stirred-tank bioreactor (Biolafitte, St- sen as the reference technique in this study.
Germain-en-Laye, France) at 37.0°C with a controlled pH However, the crystal violet nuclei-counting method has
of 7.2. Bubble-free aeration was achieved using 100 cm of some drawbacks that should be mentioned. The first relates
PTFE tubing (W. L. Gore and Associates GmbH, Putz- to the definition of biomass measured by this technique,
brunn, Germany). The fixed geometry of this membrane which is based on the number of nuclei counted in a sample,
provides a constant overall mass transfer coefficient (kLa) and which assumes the presence of a single nucleus per cell.
during the cultures, which enabled the oxygen-uptake rate to As a result (Fig. 1) the extrapolated cell concentration ap-

516 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 72, NO. 5, MARCH 5, 2001


has considerable potential for use with high-density-culture
systems, such as continuous cultures of immobilized cells
(Degouys et al., 1993).
Hence, the crystal violet nuclei-counting method appears
to be the most suitable direct technique to determine cell
concentration in low-density, free-suspended and immobi-
lized-cell-batch cultures. It has therefore been used as the
off-line reference in this study to calibrate indirect tech-
niques, the latter allowing a more convenient on-line deter-
mination of the cell concentration.
Two different indirect approaches were applied to predict
cell concentration in batch cultures of CHO SSF3 cells. The
first is based on apparent growth-yield coefficients and the
second on metabolic rates. In the latter approach, two meth-
Figure 1. Variation of the total cell concentration determined by crystal
ods had to be used to determine specific rates during the
violet nuclei-counting (䊊) and by Trypan blue-exclusion methods (䊉), and
variation of the capacitance (dots) during a batch culture of free-suspended culture: the integral method, in the case of constant specific
CHO SSF3 cells. rates, and the differential method, in case of nonconstant
specific rates.

pears to be systematically higher than that determined by


the Trypan blue exclusion method. Such differences have Yield Coefficient Method
been observed elsewhere and shown to be due to the pres- The easiest approach to correlate the measurement of a me-
ence of binucleated cells in the culture (Berry et al., 1997). tabolite production or consumption to the cell concentration
However, the importance of this deviation is cell line- is to determine the apparent growth-yield coefficient (YX/S)
dependent. For the free-suspended-cell cultures of CHO between the cell (X) and the species (S) concentration,
SSF3 used in this study, the difference in specific growth which stands either for a substrate or a product:
rates determined using the crystal violet and Trypan blue-
staining methods never exceeded 15%. Another drawback ⌬X
of the crystal violet method is the lack of information about YX Ⲑ S = Ⳳ (1)
⌬S
cell viability. However, this can be estimated by the use of
additional methods such as Trypan blue staining for free- Cell concentration is then simply determined by multiplying
suspended cells and fluorescence staining for immobilized the produced or consumed species concentration by the
cells. Lactate dehydrogenase activity may also be used as a yield coefficient.
method to follow cell lysis and death kinetics (Goergen et This simple approach was applied to a batch culture of
al., 1993), even if the reproducibility of such correlations free suspended CHO SSF3 cells. Yield coefficients were
may be questionable (Legrand et al., 1992). Finally, it is first established during a “calibration” culture. It is impor-
clear that the crystal violet nuclei-counting method is tant to point out that indirect techniques are purely descrip-
strictly limited to off-line analysis. Other direct techniques, tive and should not be extrapolated outside their data range.
such as acoustic resonance densitometry and dielectric spec- Thus, the calibration culture was run with the same param-
troscopy, would allow on-line monitoring of cell concen- eters and settings as the one during which cell concentration
tration but are limited by poor sensitivity. In this study the was predicted. Figure 2 shows this validation culture. The
reference technique has to be suitable for application with off-line reference-cell concentration was determined using
batch-culture systems where the maximum cell concentra- the crystal violet nuclei-counting method. Three yield co-
tion attained around 106 cell mL−1. Dielectric spectroscopy efficients were used to predict the total cell concentration,
was applied to the batch culture presented in Figure 1. A based on glucose, oxygen (obtained by integration of the
Biomass Monitor 214 M coupled to an in situ sterilizable oxygen uptake rate), and secretory component measure-
probe was operated at an optimal frequency of 0.6 MHz. ments, and were established during the calibration culture.
The probe produces an electric field and measures on-line The biomass yield values obtained were 4.49 × 1010 cell
the charge separations, i.e., capacitance that the field in- mol−1 (YX/glucose), 7.58 × 1010 cell mol−1 (YX/oxygen) and 2.11
duces in the cell membrane. During the first 35 hours of the × 1010 cell g−1 (YX/secretory).
culture, the capacitance signal was close to the detection The simplicity of this method unfortunately has severe
limit, while the noise level was significant throughout the drawbacks. As shown in Figure 2 the yield model is, by
culture. As a result, the capacitance signal correlated poorly definition, not able to predict a death phase because when a
to the cell concentration determined either by crystal violet specific metabolic rate tends to zero, the concentration of
or Trypan blue methods in this low range of cell concen- the produced or consumed metabolite remains constant, and
trations. However, the results suggest that this direct tech- the yield model would consequently predict a steady state
nique, enabling on-line cell concentration determination, instead of a decline. In addition, the yield model requires

DUCOMMUN ET AL.: ON-LINE DETERMINATION OF ANIMAL CELL CONCENTRATION 517


direct, on-line cell concentration measuring techniques and
the ability to scale-up is somewhat limited. If RS is not
directly available as a rate, a solution is to determine it
according to Equation (3). The species concentration, S, is
measured off-line and a curve-fitting function is applied to
the data as a function of time. The metabolic rate is then
calculated from the analytical derivative of this function.
Among several such methods, two asymmetrical sigmoidal
curve-plotting functions were reported for batch cultures:
the generalized logistic curve and the Richards curve (Seber
and Wild, 1989; Stoll, 1995). In our case the Richards curve
[Eq. (4)] gave the best results (correlation coefficients
greater than 0.998) and was thus used. It contains four fit-
Figure 2. Variation of the total cell concentration determined by crystal ting parameters: a, b, c, and d.
violet nuclei-counting (䊊) and from predictions based on the yield coef-
ficient method through measurement of glucose (dots), oxygen (line) and f(t) ⳱ a(1 + (b − 1)exp[−c(t − d)])1/(1−b) (4)
secretory component (䊉) during a batch culture of free-suspended CHO
The metabolic rate, which is measured directly or obtained
SSF3 cells.
by curve fitting, has then to be divided by the corresponding
specific rate to calculate the cell concentration, according to
on-line measurement of the metabolite concentration to pre- Equation (2). Two approaches were used to calculate the
dict on-line cell concentration. Thus, because the secretory specific rates: a calibration by an integral method and a
component could only be analyzed off-line by an ELISA calibration by a differential method (Stoll, 1995).
technique, the resulting cell concentration prediction (Fig.
2) is then also off-line. On-line immunoassays has been Calibration by an Integral Method
established (Hitzmann et al., 1995; Middendorf et al., 1993)
and many techniques have been developed to measure on- The calibration by an integral method assumes that the spe-
line most of the metabolites (Bradley et al., 1991; Locher et cific rate is constant during part of, or throughout, the cul-
al., 1992; Olsson and Nielsen, 1997), however, they cannot ture. This hypothesis allows the integration of Equations (2)
be adapted to all culture systems. and (3) and their reformulation as follows:

兰 Xdt
t
St − S0 = ⳲqS (5)
0
Metabolic Rate Method
The species concentration is measured either directly or
A kinetic approach can be used to build a model that is able obtained by integration of the corresponding metabolic rate.
to follow any phase of the culture—not only the growth The integral term can be calculated numerically, for ex-
phase as with the yield coefficient model. In batch culture ample, using the trapezoidal method. qS is then determined
the cell concentration (X) is determined by the metabolic from the slope of a plot of the species concentration against
rate of a species (RS) divided by the specific metabolic rate this integral term as a function of time.
(qS): A calibration of the specific oxygen-uptake rate, qO2, by
RS integral method for a batch culture of free-suspended CHO
X=Ⳳ (2) SSF3 cells is presented in Figure 3. The concentration of
qS
consumed oxygen was obtained by integration of the oxy-
dS gen-uptake rate (∫RO2dt). A linear correlation established
RS = (3) during the exponential-growth phase of the culture allowed
dt
the determination of a value for qO2 : 3.24 10−13 mol cell −1
The species S stands either for a product or a substrate. h−1. The corresponding correlation coefficient is high:
Thus, once qS has been calibrated, cell concentration can be 0.997. Thus, qO2 can be considered as constant during the
determined on-line if a technique enabling on-line monitor- exponential-growth phase. However, the slope of the curve
ing of RS is available. For example, a high-sensitivity calo- in Figure 3 decreases to a lower value after the exponential-
rimeter (Bio-RC1, Mettler-Toledo AG, Greifensee, Switzer- growth phase, as cell growth ceases. The previously deter-
land) has been recently developed within our institute mined qO2 is then no more valid for this part of the culture.
(Marison et al., 1998) and allows monitoring on-line the A batch culture of free-suspended CHO SSF3 cells was
heat-production rate of the cells. Another potential tech- performed under the same conditions as the culture used
nique is the on-line measurement of the volumetric oxygen- above for the calibration of qO2. The cell concentration was
uptake rate in cell cultures (Ducommun et al., 2000). determined on-line according to Equation (2) by dividing
However, techniques enabling on-line monitoring of the on-line monitored oxygen-uptake rate, RO2, by the value
metabolic rates are generally more difficult to perform than of qO2 which had been established during the calibration

518 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 72, NO. 5, MARCH 5, 2001


determination of qO2 is based on the exponential-growth
phase only, thus the death phase cannot be quantitatively
predicted.
Cell concentration could be predicted very precisely by
the integral method during the whole exponential-growth
phase of the culture, as shown in Figure 4, because qO2 was
constant during this period. However, specific rates may
vary continuously throughout the culture process. For CHO
SSF3 no satisfying linear correlation was found during the
calibration culture between the measurement of glucose
concentration and the integral term of Equation (5). Thus,
the specific glucose-consumption rate, qGlucose, is not con-
stant and cannot be calibrated using the integral method. An
approximate value of qGlucose was, however, tested (qGlucose:
Figure 3. Calibration of the specific oxygen uptake rate, qO2, by the
4.74 10−13 mol cell−1 h−1, correlation coefficient: 0.958) but
integral method for a batch culture of free-suspended CHO SSF3 cells. qO2
is determined from the slope of the linear correlation applied during the results led to incorrect biomass predictions, as shown in
exponential-growth phase of the culture. Figure 4. Hence, if glucose concentration has to be consid-
ered as a basis for the determination of cell concentration—
in many cases on-line measurements of RO2 may not be
culture (Fig. 3). The predicted cell concentration resulting available—a new method enabling the calibration of non-
from RO2 measurement is shown in Figure 4. Results are in constant specific rates has to be developed.
very good agreement with the direct off-line data measured
by crystal violet nuclei-counting during the whole growth
phase of the culture. This model also allows prediction of Calibration by a Differential Method
the onset of the death phase at the end of the culture, which
was not possible with the yield-coefficient method. How- Calibration by a differential method enables the determina-
ever, this death phase is predicted some 20 hours earlier tion of nonconstant specific rates. The metabolic rate is
than it is measured with the direct technique. This difference determined by on-line measurement, or by curve-fitting, and
can be due to the fact that indirect techniques measure cell- is then divided by the cell concentration, according to Equa-
metabolic activity rather than cell concentration. They are, tion (2). The nonconstant, specific-glucose-uptake rates de-
as discussed above, purely descriptive and changes in the termined using the differential method in free-suspended
specific metabolic rates—compared to those determined and immobilized CHO SSF3 cell cultures are shown as a
during the calibration culture—may affect directly the de- function of time in Figure 5. The glucose-uptake rate was
termination of the cell concentration and lead to incorrect calculated from the analytical derivative of a Richards curve
predictions. Moreover, the calibration of qS by the integral [Eq. (4)], and cell concentration was determined by crystal
method can only be used in phases of the culture where the violet nuclei-counting. The resulting specific uptake rates
specific rate can be considered as constant. In Figure 3 the are shown to vary in both cases during the whole culture
process, as suggested previously. Thus, accurate cell-
concentration determination, based on indirect glucose mea-

Figure 4. Variation of the total cell concentration determined by crystal


violet nuclei-counting (䊊) and from predictions based on the metabolic
rate method through measurement of oxygen-uptake rate (dots) and glu- Figure 5. Variation of the specific glucose-uptake rate determined by the
cose concentration (line) during a batch culture of free-suspended CHO differential method during batch cultures of free-suspended (䊊) and im-
SSF3 cells. mobilized (䊉) CHO SSF3 cells.

DUCOMMUN ET AL.: ON-LINE DETERMINATION OF ANIMAL CELL CONCENTRATION 519


surement, requires the knowledge of the specific consump- which glucose concentration is also used as one of the pa-
tion rate at any time during the culture. rameters (Zeng and Deckwer, 1995). In this case, the spe-
However, a knowledge of the specific rate as a function cific glucose-uptake rate was modeled with three parts: a
of time during the calibration culture may not be enough to growth dependent part, a part due to glucose excess, and
predict cell concentration during other cultures, because another to glutamine excess.
changes in inoculum density or in the duration of the lag Similar results were not found for immobilized cell cul-
phase would lead to incorrect specific rate predictions. tures. In this case, no correlation was observed with glucose
Thus, a reference other than time would be more appropriate concentration. However, a linear correlation was found
for specific rate determinations. A relationship between spe- (Fig. 7) between the specific glucose-uptake rate and the
cific glucose-uptake rate and cell concentration has been concentration of consumed oxygen, cConsumed oxygen (corre-
proposed in the literature (Pörtner et al., 1994). In this study, lation coefficient: 0.994):

冋 册
no correlation between specific glucose-uptake rate and cell
mol
concentration was observed. However, another approach is qGlucose = 9.26 ⭈ 10−13
cell ⭈ h

冋 册
required because cell concentration is indeed the unknown
parameter. Other relationships were investigated and a lin- mol
− 3.71 ⭈ 10−7 ⭈ cConsumed⭈oxygen
ear correlation was found (Fig. 6) between the specific glu- mL
cose-uptake rate and glucose concentration, cGlucose (corre- (7)
lation coefficient: 0.999):
Mass-transfer limitations might be expected in immobilized

qGlucose冋 mol

cell ⭈ h
= 1.78 ⭈ 10−14
cell cultures, as the cells tend to accumulate at the surface of
the microcarriers (Cytopore 2) to form multicell layers.
Oxygen might then also become a limiting substrate and
+ 8.11 ⭈ 10−14 ⭈ cGlucose关mM兴 (6) regulate the specific glucose-uptake rate. These time-
Glucose is the limiting substrate in batch culture of free- independent models, correlating specific glucose-uptake
suspended CHO SSF3 and it was totally exhausted by the rate as a linear function of glucose [Eq. (6)] and oxygen [Eq.
end of the exponential-growth phase. A regulation of the (7)] concentration, offer the advantage of predicting cell
specific rates by the limiting-substrate concentration in the concentration independently of the inoculum density. More-
culture medium could explain the decrease of the former. over, they allow determination of the specific glucose-
Besides, glucose concentration has been stated in the litera- uptake rate at any time during the culture by measuring the
ture as the key parameter affecting glucose metabolism of limiting substrate concentration using the on-line methods
animal cells (Zeng et al., 1998), and specific glucose-uptake commonly available.
rates have been reported to be a function of the residual The time-independent model [Eq. (6)] was applied, for
glucose concentration in the culture (Frame and Hu, 1991; validation purposes, to a second batch culture of free-
Ljunggren and Häggström, 1994). However, as discussed suspended CHO SSF3 cells initiated with a different inocu-
previously, indirect techniques are reduced to a purely de- lum cell density. On-line monitoring of the limiting-
scriptive role and the linear model [Eq. (6)] is only valid for substrate concentration enabled continuous determination of
the biological and bioreactor systems that were used during qGlucose. A Richards curve [Eq. (4)] was applied to differ-
its establishment. A more general model has been proposed entiate cGlucose with respect to time, and thus cell concen-
for the determination of the specific glucose-uptake rate in tration could be determined using Equation (2). The pre-
dicted cell concentration was then compared to off-line

Figure 6. Time-independent model correlating the specific glucose- Figure 7. Time-independent model correlating the specific glucose-
uptake rate to the glucose concentration for a batch culture of free- uptake rate to the concentration of consumed oxygen for a batch culture of
suspended CHO SSF3 cells. immobilized CHO SSF3 cells.

520 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 72, NO. 5, MARCH 5, 2001


measurements made using the reference crystal violet nu-
clei-counting method (Fig. 8). The results obtained in this
way are in very good agreement with the off-line reference
method during the whole of the exponential-growth phase.
At the end of this phase the specific glucose-uptake rate
tends to very low values, however, the model is still able to
qualitatively illustrate the decline phase. Prediction of cell
concentration by the integral method using an average value
of qGlucose (4.74 10−13 mol cell−1 h−1, correlation coeffi-
cient: 0.958), as already shown in Figure 4, is also included
in Figure 8 for comparison and illustrates the necessity to
apply a correlation using nonconstant specific glucose-
uptake rates (differential method) instead of constant ones
(integral method).
Another validation culture was performed with CHO Figure 9. Variation of the total cell concentration determined by crystal
violet nuclei-counting (䊊) and from predictions based on metabolic rate by
SSF3 cells immobilized on Cytopore 2 (Fig. 9) and, despite integral (plain line) and differential (bold line) method through on-line
an important lag phase, the time-independent model [Eq. measurement of glucose and oxygen during a batch culture of immobilized
(7)] is able to determine accurately qGlucose and thus, the CHO SSF3 cells.
immobilized cell concentration during the exponential-
growth phase. At the end of this phase the specific glucose-
uptake rate tends to zero, which induces an infinite value for and were able to predict cell concentration despite noncon-
the cell concentration, according to Equation (2). Prediction stant, specific glucose-uptake rates. The results were not
of cell concentration by the integral method using an aver- affected by changes in the lag-phase duration nor in the
age value of qGlucose for immobilized cell culture (7.81 inoculum concentration, compared to the calibration cul-
10−13 mol cell−1 h−1, correlation coefficient: 0.998) is also tures. On-line monitoring of the substrate was therefore suf-
included in Figure 9 as a comparison. As for free- ficient to enable on-line prediction of the cell concentration.
suspended-cell culture, the data that are predicted by the These results were confirmed off-line by crystal violet nu-
integral method are inaccurate and a calibration of Equation clei-counting and correlated well during the whole expo-
(2) with nonconstant specific glucose uptake rates has to be nential-growth phase, until glucose was depleted, despite
considered. the fact that specific rates varied with time. This technique
offers the potential to apply such an indirect approach to a
wide range of processes with either free-suspended or im-
CONCLUSIONS mobilized cells for which other on-line direct methods are
not available, and could be used to form the basis of on-line
An indirect approach, based on time-independent models control systems to maintain desired cell concentration or
relating the specific rates to a substrate concentration, has productivity by regulating the nutrient feed or perfusion
been developed. The models could be used in batch cultures rates.
of both free-suspended and immobilized CHO SSF3 cells,

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