In Situ Monitoring of Cell Behavior Using Advanced
Dielectric Spectroscopy (DS)
An Integrative Approach To Improve Product Quality and Quantity
Overview
The bioprocessing industry demands an online monitoring
technique that accurately provides real-time cell-level
information and guidance in situ, in the bioreactor. Of
particular importance is the ability to perform online
monitoring of viable cell volume (VCV), which is essential to
the development and control of bioprocesses. The availability
of dielectric spectroscopy (DS) probes has enabled successful
adoption of this technology as a key noninvasive method to
measure VCV for cell-culture processes. Bend Research has
developed methods incorporating DS measurements and the
reduction of large DS data sets to obtain measurements of
both VCV, as well as cell state throughout the bioreactor
growth curve.
Background
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Other measurements (e.g., metabolite
concentrations) must be normalized to the amount
of cells present to yield useful information on cell
state
Consumption rates of feed nutrients are determined
by both the amount of cells and the state of those
cells
Productivity and product quality are typically related
to the growth profile of cells (e.g., the cell growth
profile is indicative of culture performance)
DS, or frequency-scanning capacitance, is a method for
measuring cell mass and cell state in situ and in real-time. DS
probes measure the passive electrical properties of cells.
These electrical properties are determined by biological and
physiochemical properties of the cells such as size, shape,
intracellular conductivity (related to osmolality, pH, redox
state); membrane charge (related to glycocalyx,
www.BendResearch.com
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Bioreactor
On-line knowledge of both the amount of cells, as well as the
state of those cells, is critical for developing, monitoring, and
controlling bioprocesses for the production of therapeutic
proteins. Real-time DS data are important for the following
reasons:
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transmembrane proteins, membrane permeability); and
morphology (clumping, shape). In the context of a bioreactor,
DS measurements are intimately linked with the changes in
cell biology associated with changing process conditions.
Figure 1. Schematic Showing Dielectric Probes in Bioreactor
Novel DS based applications enable noninvasive real-time
“observability” of cell health inside a bioreactor, as well as
samples from shake flasks. These technologies provide
valuable data not accessible by other technologies and report
true “cell-level” properties within a population of cells. The
data can be obtained for a range of mammalian cells such as
CHO and NS0 cells, as well as microorganisms, such as yeast,
and bacteria. These DS applications, coupled with models and
data analysis techniques can be used to:
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aid in development-scale bioreactor experiments for
design-space generation that incorporate “cell-level”
observations
increase process understanding at the cell level,
guide media development
monitor cell state in larger scale bioreactors, and
implement process-control strategies based on cell
behavior
Advanced Dielectric Spectroscopy
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Advanced Dielectric Spectroscopy for In Situ Cell
Monitoring
ratio correction results in accurate predictions of VCV during
the final stages of cell culture (Figure 3).
DS utilizes the fundamental biophysics of cells to directly
measure cell state. It is well established that cell biology
changes dynamically in response to changes in process
parameters (e.g., feed components, shear, temperature).
These changes in cell character and phenotype are difficult to
detect and quantify in a noninvasive, real-time manner and
current analytical techniques that interrogate cell state rely
on cell/media sampling. Cellular analysis utilizing DS: is
noninvasive; is frequent (120 samples/hour); poses minimal
risk of media contamination; and does not rely on human
involvement during sampling.
The frequent, noninvasive, cell-level nature of DS
measurements enables real-time feedback of changes in cell
behavior. This real-time feedback, or observability results in
“actionable guidance” for use in closed-loop process-control
strategies based on cell behavior, rather than open-loop
strategies based on recipes (Figure 2). The data can also allow
real-time feedback regarding cell-specific responses to
process and media conditions. At Bend Research, we are
refining existing DS detection capabilities and collaborating
with leaders in the field to refine the hardware and software
associated with these instruments.
Figure 2. Use of Dielectric Spectroscopy with Other Process
Measurements Enables Enhanced Cell-Specific Process
Control
Case Study 1:
Normalization of Measurements To Cell Volume
Bend Research has demonstrated the feasibility of using
full- spectrum DS measurements to predict VCV in
bioreactors more accurately than with single-frequency
permittivity methods. We do this by accounting for changes
in cell physiological state that occur in the death phase. As
cell viability drops, transformations in the cell lead to
variations in capacitance. This often causes cell viability
measurement by DS to deviate from actual. With frequency
scanning, it was discovered that distinct cell populations
could be identified and quantified. Applying a unique area
www.BendResearch.com
Figure 3. Cell Volume Measurements Using Uncorrected
and Corrected Dielectric Spectroscopy Biovolume Results.
A novel method has been presented in which changes in the
state of the cell population are quantified using frequencyscanning data and used to predict VCV more accurately. The
ability to accurately measure VCV online in the bioreactor,
without sampling, makes possible the implementation of
process-control schemes (e.g., cell-rate defined feeding) that
would be more difficult or impossible using other
technologies. This initial work demonstrates the possible
utility of using commercial frequency-scanning probes for a
use beyond measuring VCV: the online monitoring of cell
state in the bioreactor.
Additional work remains to investigate the specific
physiological changes that are detected by DS in the death
phase. Such fundamental understanding will likely promote
understanding of other important cellular processes that are
of interest to the biopharmaceutical industry. The robustness
of this area ratio algorithm method also remains to be
determined. Changes in media composition, mode of cell
death, and cell type remain important variables to consider.
Case Study 2:
Apoptosis Detection using Dielectric Spectroscopy
Complex cellular processes, such as apoptosis, cause specific
changes in cellular biology, which can be detected by DS.
Bend Research has investigated the use of DS to detect the
cellular changes associated with the activation of Caspase 3, a
key marker of apoptosis.
To determine the applicability of DS for observing cellular
processes such as apoptosis in situ, two fed-batch bioreactors
were run in parallel, each an identical fed-batch process
growing CHO cells. The cultures were monitored using
frequency-scanning dielectric probes. The experimental
Advanced Dielectric Spectroscopy
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Control (no Staurosporine)
S
Timeseries
of Normalized
NormalizedCapacitance
Capacitancevs.
vs.
Frequency
Timeseries of
Frequency
Capacitance
Normalized
Capacitance
Normalized Capacitance
As the resulting dielectric spectra in Figure 4 show, a
pronounced shift in the shape of the dielectric spectrum is
observed progressively in the experimental culture as
apoptosis occurs. The shape change was quantified over time
using physically relevant models (i.e., a single term Cole-Cole
model) to fit the dielectric data. The model outputs were
related quantitatively to activation of Caspase 3.
Click here to view the webinar
titled “Fundamentals and Application
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of Dielectric Spectroscopy”:
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http://www.youtube.com/watch?v=qkpA0FwfBeI&feature=youtube
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About Bend Research
For more than 35 years,0.2Bend Research has worked with
clients to solve their most difficult scientific and technical
0
problems, advancing new medicines that improve human
health. This success is based
on a solid understanding of
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10
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scientific and engineering10 fundamentals,
enabling
Bend 10
Frequency(kHz)
(kHz)
Frequency
Research to develop, progress, and commercialize
pharmaceutical technologies. The firm’s innovative drugdelivery solutions grow from a solid base of scientific and
engineering fundamental understanding.
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Bend Research provides formulation and dosage-form
support, assists in process development and optimization,
manufactures clinical-trial quantities of drug candidates in its
cGMP facilities, advancing promising drug candidates from
conception through commercialization. It is a leader in novel
formulations, including solubilization technologies such as
spray-dried dispersions and hot-melt extrusion formulations,
as well as controlled-release, inhalation, and biotherapeutics.
Contact
Bend Research is looking for partners to collaborate in the
development of process analytical tools. To discuss potential
application in your laboratory, please contact:
Lisa Graham
Bend Research Inc.
Toll Free Phone: 1-800-706-8655
E-mail: lisa.graham@bendresearch.com
www.BendResearch.com
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(a)
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Frequency(kHz)
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Timeseries of Normalized Capacitance vs. Frequency
Capacitance
Normalized
Capacitance
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Frequency (kHz)
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(kHz)
Capacitance (pF/cm)
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(no
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This case study shows theControl
utility of DS
to quantify
cell
behavior in situ, in theTimeseries
bioreactor,
and
also
shows
that
of Normalized
NormalizedCapacitance
Capacitancevs.
vs.
Frequency
Timeseries of
Frequency
dielectric spectra can be quantified using physically relevant
models and related to fundamental biological processes.
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Normalized Capacitance
culture was treated with 1-µM staurosporine, a known
apoptosis initiator in mammalian cells.
Timeseries
(c)
Frequency (Hz)
Figure 4. Raw Dielectric Spectra Showing Capacitance as a
Function of Frequency for Experimental Cultures Before (a)
and After (b) Staurosporine was Added to the Culture: Blue
(Time=0 Hr) To Red (Time=60 Hr). The Culture Exhibits a
Pronounced Shape Change over the Course of the
Experiment, Quantified By Fitting a Two-Term Cole-Cole
Model to the Data (c)
Advanced Dielectric Spectroscopy
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