Heinrich C, Beckmann T, Büntemeyer H, Noll T. Utilization of multifrequency permittivity measurements in addition to biomass monitoring. BMC Proceedings. 2011;5(Suppl 8)

CA Boulton

Christoph Heinrich

Heino Büntemeyer

S Ansorge

T Noll

Thomas Noll

Tim Beckmann

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Utilization of multifrequency permittivity measurements in addition to biomass monitoring

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Beckmann Tim

Büntemeyer Heino

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MEETING ABSTRACT Open Access
Utilization of multifrequency permittivity
measurements in addition to biomass monitoring
Christoph Heinrich*, Tim Beckmann*, Heino Büntemeyer, Thomas Noll
From 22nd European Society for Animal Cell Technology (ESACT) Meeting on Cell Based Technologies
Vienna, Austria. 15-18 May 2011
Introduction
In recent years measurement of permittivity signal has
been increasingly used for online biomass monitoring of
cell cultures. Breweries use it as an established method
for fermenter inoculation and bioprocess control for
instance [1]. In the case of animal cell cultures the cor-
relation between permittivity and viable cell densities
determined offline varies along cultivation time. Hence,
several authors have used the permittivity signal as an
indirect method for measuring oxygen and glutamine
consumption as well as intracellular nucleotide phos-
phate concentrations [2,3]. The latest generation of bio-
mass monitoring devices allows parallel measurement of
permittivity at a range of frequencies leading to an
improvement in the correlation between biomass and
permittivity and providing a tool to further explore
other aspects related to the physiological state of the
cells.
Material and methods
In this study 10 different cell lines, among them indus-
trial cell lines as well as cell lines distributed by ATCC,
were cultivated. Cell type specific chemically defined
and animal component-free media (TeutoCell AG) were
used. Batch, fed-batch and perfusion bioreactor cultiva-
tions were carried out in controlled benchtop vessels
(Sartorius AG or Applikon Biotechnology). The i-Bio-
mass 465 sensor (FOGALE nanotech) was used for
online multifrequency permittivity monitoring. In addi-
tion the permittivity signal was used to implement a
fully automated cell bleed to maintain a constant viable
cell density in a perfusion process. Furthermore, a fed-
batch feed strategy was introduced to keep the substrate
concentration at a certain level. Cell density and viability
were determined using a CEDEX system (Innovatis-
Roche AG). Glucose and lactate were measured with an
YSI 2700 Biochemistry Analyzer (YSI Life Sciences).
Amino acids were quantified using an in-house devel-
oped HPLC method.
Results
The FOGALE i-Biomass 465 sensor was used to moni-
tor the viable cell density of different human, CHO and
hybridoma cell cultures online. A good correlation of
the permittivity signal and the offline measured viable
cell density for the growth phase was verified (R > 0.99),
but pH-shifts and increased cell aggregation had a nega-
tive impact on the correlation. The linear factor to cal-
culate the viable cell density from the online
permittivity signal varied between 4.5·105 cells/(pF/cm)
and 12.0 cells/(pF/cm). A clear relation between cell
type (CHO, human or hybridoma) and the linear factor
could not be established from the available data.
Subsequently, the online biomass monitoring system
was used to carry out a 1 L spin-filter perfusion process
with constant viable cell density at a predefined setpoint.
The application of a permittivity closed-loop controlled
cell bleed resulted in a steady concentration of 107
viable cells/mL during perfusion, at a dilution rate of 1.0
d-1. As soon as this threshold was reached, the cell
bleed was automatically started and controlled based on
the online signal of the i-Biomass 465 sensor.
In addition to the correlation with viable cell density,
a linear relationship (R2 > 0.96) between the online i-
Biomass 465 signal and the concentrations of numerous
components, e.g. glucose, lactate, asparagine, glutamine,
tyrosine, threonine, methionine, lysine, phenylalanine,
serine, leucine and isoleucine, was found during the
exponential growth phase of CHO-K1 and CHO DP-12
cultivations. The results indicated that the number of
* Correspondence: che@zellkult.techfak.uni-bielefeld.de
Institute of Cell Culture Technology, Bielefeld University, 33615 Bielefeld,
Germany
Full list of author information is available at the end of the article
Heinrich et al. BMC Proceedings 2011, 5(Suppl 8):O10
http://www.biomedcentral.com/1753-6561/5/S8/O10
© 2011 Heinrich et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
correlating substrates depended on the used cell line
(CHO, human or hybridoma) and the process strategy
(constant pH or pH-shift). Since, the established sub-
strate correlations were more robust against process var-
iations, they were investigated as a basis for a closed-
loop feeding strategy in fed-batch cultivations. Com-
pared to a pre-defined feeding schedule or to inter-
mitted feeding this would have the advantage of
avoiding nutrient limitations and substrate accumulation
that might occur due to unexpected high or low cell
growth. Also, feeding would be independent of human
surveillance. The successful application of a completely
automated permittivity-controlled feeding strategy was
proved in two fed-batch runs with CHO DP-12 (ATCC
CRL-12445) cells, as shown in Figure 1.
The feeding of both runs was controlled only through
the permittivity signal in order to maintain the aspara-
gine concentration at a certain level. Asparagine was
chosen due to its central role in cell metabolism and to
the fact that it is usually a limiting substrate in CHO
DP-12 cultures. These proof-of-concept runs demon-
strated that permittivity-based automated feeding can be
a valuable tool for the optimization of fed-batch process
parameters, such as feeding start, flow rate and
composition.
Conclusions
For suspension cultures with single cells and high viabi-
lity a linear correlation (R2 ≥ 0.98) of the permittivity
signal with the viable cell density measured offline was
obtained, at least for the exponential growth phase.
Based on this correlation, a closed-loop controlled cell
bleed was implemented in a perfusion process in which
the permittivity signal was used to keep the viable cell
density at a constant level of 107 viable cells/mL.
Furthermore, a linear correlation with the i-Biomass 465
signal was observed for several substrates independent
of the correlation between viable cell density and per-
mittivity. Based on these results, a closed-loop con-
trolled feeding was successfully established resulting in a
fully automated fed-batch process.
Acknowledgements
We would like to thank FOGALE nanotech for providing the i-Biomass 465
system and TeutoCell AG for supplying the cell culture media and feed
solutions.
Published: 22 November 2011
References
1. Boulton CA, Maryan PS, Loveridge D: The application of a novel biomass
sensor to the control of yeast pitching rate. Proc 22nd Eur Brew Conv
Zurich European Brewing Convention Oxford: Oxford University Press; 1989,
653-661.
2. Noll T, Biselli M: Dielectric spectroscopy in the cultivation of suspended
and immobilized hybridoma cells. J Biotechnol 1998, 63:187-198.
3. Ansorge S, Esteban G, Schmid G: Multifrequency permittivity
measurements enable on-line monitoring of changes in intracellular
conductivity due to nutrient limitations during batch cultivations of
CHO cells. Biotechnol Prog 2010, 26:272-283.
doi:10.1186/1753-6561-5-S8-O10
Cite this article as: Heinrich et al.: Utilization of multifrequency
permittivity measurements in addition to biomass monitoring. BMC
Proceedings 2011 5(Suppl 8):O10.
Figure 1 Total viable cell count, viable cell density and asparagine concentration of the two proof-of-concept CHO DP-12 fed-batch processes
(initial working volume: 1 L; 37°C; 40% DO; pH 7.1).
Heinrich et al. BMC Proceedings 2011, 5(Suppl 8):O10
http://www.biomedcentral.com/1753-6561/5/S8/O10
Page 2 of 2


Beckmann, Tim

Büntemeyer, Heino

Noll, Thomas 

Hauser, Hansjörg

Publications at Bielefeld University

Dielectric spectroscopy in the cultivation of suspended and immobilized hybridoma cells.

Multifrequency permittivity measurements enable on-line monitoring of changes in intracellular conductivity due to nutrient limitations during batch cultivations of CHO cells. Biotechnol Prog

The application of a novel biomass sensor to the control of yeast pitching rate. Proc 22nd Eur Brew Conv Zurich European Brewing Convention Oxford:

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Utilization of multifrequency permittivity measurements in addition to biomass monitoring

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