124
Bioprocess monitoring
Peter Harms, Yordan Kostov and Govind Rao*
Electrochemical sensors for pH and dissolved oxygen remain the
most commonly used in bioprocess monitoring, but continued
research has resulted in improved optical sensors. Optical
sensors for dissolved oxygen and dissolved carbon dioxide are
now commercially available. Advances in optics and electronics
are further driving down the costs of these sensors. In the near
future, bioprocess optimization will change paradigms as
massively parallel, fully instrumented bioreactors become
available and high-throughput bioprocessing becomes a reality.
Addresses
Department of Chemical and Biochemical Engineering, UMBC,
1000 Hilltop Circle, Baltimore, MD 21250, USA
*e-mail: grao@umbc.edu
Current Opinion in Biotechnology 2002, 13:124–127
0958-1669/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
Abbreviations
FIA flow injection analysis
GFP green fluorescent protein
Introduction
Process development accounts for a significant fraction of
the cost of bringing a drug to market and there is a strong
economic incentive for improved monitoring tools. In theory,
early process development stages have a goal of understanding
and optimizing the process by measuring as many parameters
as possible. Later on in production, only critical parameters
may be measured to enable proper control of the process and
ensure high quality and yield; however, in practice, the paucity
of suitable sensors and tools for online monitoring has not
allowed this ideal to be widely implemented.
Although there is vigorous research being done on sensor
development, only a small fraction of new sensors are suitable
for use in fermentations. Bioprocesses are remarkably harsh
environments for sensors, as the growing culture can infiltrate
sensors and thus invalidate their results. Further, culture media
is often poorly defined with many substances that interfere
with sensor readings. Sensors used in situ should not contami-
nate the bioprocess, so they need to be sterilizable and their
components must not leach out into the culture. Another
challenge is that bioprocesses may run for weeks, during which
time the potential sensor must be able to perform without
recalibration. Sensors that only marginally meet these require-
ments are often disqualified for not being valid. Because of
these hurdles, sensors need a certain level of maturity before
they can be easily used to monitor bioprocesses and some of
these sensors are described herein.
Online in situ measurements
It is important to measure critical process variables online,
especially parameters that are used in closed control loops.
There are often several means to measure these common
parameters, with advantages of each method. In practice,
temperature, pH and dissolved oxygen are the most widely
measured and controlled in situ parameters. Sensors for these are
typically inserted into specially designed ports on the vessel.
As fermenters increase in size, mixing problems become
commonplace [1••] and probe location becomes a problem.
To accurately profile large fermenters, probes may be needed
in several locations as the environmental heterogeneity can
adversely affect productivity [2]. This effect can be approxi-
mated in the laboratory scale [3] by combining two
bioreactors and can be reduced in the large scale by using
multiple feed zones [4••].
pH
The most commonly measured parameter is probably pH.
Optimal cell growth depends heavily on tight pH control and
many cells produce acids as a metabolic by-product. Steam-
sterilizable glass electrodes remain the state-of-the-art for use
in bioreactors. Their low mechanical stability has spurred
research in optical sensors based on absorbance or fluores-
cence from pH-sensitive dyes. Optical sensors typically suffer
from a narrow operating range, but this limitation is balanced
by increased sensitivity near the pKa of the dye. Although a
single optical sensor generally cannot be used to monitor all
bioprocesses, a fermentation can successfully be monitored by
a sensor matched to the pH range of the process (HR Kermis,
Y Kostov, P Harms, G Rao, unpublished data). Optical sensors
with increased ranges, based on novel polymers, have recently
been developed but have yet to be tested in fermentations [5,6].
Dissolved oxygen
Oxygen is also very commonly measured, because it is
often a limiting substrate for cell growth owing to its low
solubility in water. Oxygen can be measured in the exhaust
gas to monitor the oxygen uptake rate of the culture, and
dissolved oxygen in the liquid phase is of great impor-
tance. The steam sterilizable Clark-type electrode remains
one of the most important sensors for bioprocess monitor-
ing [7•]. A second means of measuring dissolved oxygen is
based on quenching of fluorescence [8], and these sensors
have recently been commercialized [9,10]. These two
methods of oxygen measurement may be more comple-
mentary than competitive, as electrochemical sensors
perform best at high oxygen concentrations, whereas optical
sensors are optimally sensitive at lower concentrations
(generally <50% of air saturation). A further distinction is
that, unlike optical sensors, electrochemical sensors actively
consume oxygen, which may influence some measurements.
Cell mass
Cell mass is another crucial process indicator. Often the
cell mass will be brought up to a certain level before

Figure 1
Concept for high-throughput bioprocessing.
The system would consist of a modified well-
plate with sensor patches, as shown, and
Acid
would be designed to operate in a laminar flow
hood. Alternatively, individual microbioreactors
designed as miniature bioreactors could be
employed. The key enabling technology is the
use of low-cost light-emitting diode (LED) light LEDs
sources and compact photodetectors. The
sensor patches have the appropriate analyte-
sensitive fluorophores immobilized in them and
are disposable.
O2 sensor
Cells growing in
nutrient medium
inducing product formation or harvesting the cells. There
are many methods to measure cell mass, each with its own
set of limitations. The reference for cell-mass sensors is
generally dry cell weight. A sample is taken, washed and
dried for several hours at around 105°C before being
weighed. This method obviously cannot be used for online
measurements. Instead, cell mass can be measured indi-
rectly by electrical or optical means. Electrically, cell mass
is often related to capacitance or permitivity [11], which
measures the fraction of fluid enclosed by polarizable
membranes [12]. Optically, cell mass is often measured by
light absorbance, scatter or some combination of the two.
Scatter alone can measure cell mass, but the function is not
monotonic so care must be taken at high masses [13].
Absorbance or optical density is a better measure than
scatter, but simple sensor designs choose between range
and precision. These sensors are also vulnerable to inter-
ference from particulates and gas bubbles. Cell mass is
also often predicted by software sensors [14], which are
covered later in this paper.
Dissolved carbon dioxide
Carbon dioxide is of importance because it is a product of
cell respiration and can pass through cell membranes to
influence the pH inside cells. It is of particular importance
to animal cell cultures, which generally require carbon
dioxide supplementation for part or all of the bioprocess.
Unfortunately, sterilizable sensors for dissolved carbon
dioxide have a history of problems. Severinghaus-type
electrodes consist of a bicarbonate buffer separated from
the fermentation media by a carbon dioxide permeable
membrane. Carbon dioxide causes a pH change in the
buffer that is monitored by an electrode. The membrane
can be sterilized in place, but not the buffer or electrode.
A membrane failure would contaminate the fermentation.
These sensors commonly have problems with drift and
interference from changes in culture media composition. A
new sensor is available that can be cleaned and sterilized
Bioprocess monitoring Harms, Kostov and Rao 125
Base Nutrient
Robot
96-well plate
Approximately 250 µl volume
pH sensor
Optical density
Current Opinion in Biotechnology
in place, which uses the ratiometric fluorescent dye HPTS
(8-hydroxypyrene-1,3,6-trisulfonic acid) [15•].
One step further is the use of a fluorescence-based pH
sensor immobilized in a gas-permeable polymer. This
sensor is solid state, removing the bicarbonate buffer [16];
however, the instrumentation required to measure this
sensor remains expensive.
Spectroscopy
Many biological species are optically active and can be
quantified spectroscopically. For example, NAD(P)H
sensors are well known. With advances in electro-optics
and computing power, two-dimensional fluorescence
spectra are possible that provide information about many
fluorescent species simultaneously [17]; however, the data
are difficult to interpret because of spectral overlap and
complex interferences. Neural networks can be used to
reduce the data into meaningful information [18].
Near spectra [19] and mid-infrared spectra [20,21] can also
provide a wealth of information. Glucose, fructose, glutamine,
glutamate, proline, ammonia, carbon dioxide and phos-
phate are among the components that can be measured;
however, the high cost and difficulty of calibration prevent
widespread use of this method.
One approach to measure recombinant protein products
spectroscopically is to attach the gene for green fluorescent
protein (GFP) to the gene of the protein of interest. This
allows the product concentration to be determined by the
fluorescence intensity of the GFP in the culture; however,
GFP fluorescence lags 95 min behind product formation
and producing GFP can be a metabolic burden, reducing
product formation [22•]. Newer variants of GFP are
available with shorter maturation times. These and the
availability of variants of other colors open up more options
for real-time monitoring of gene expression [23]. Yet
126 Biochemical engineering
another application is the cloning of GFP downstream of
stress promoter probes, which could serve as diagnostic
indicators of fermentation process conditions to minimize
the triggering of stress responses [24].
Alternative measurements
Flow injection analysis
With flow injection analysis (FIA), almost any other concen-
tration can be measured [25•]. Systems are commercially
available that can measure pH, partial pressures of oxygen
and carbon dioxide, sodium, potassium, calcium, glucose,
lactose, ammonium, lactate, galactose, glutamate, glutamine,
choline, ethanol, hydrogen peroxide, starch and sucrose [26].
FIA is sampled rather than an in situ method, but analysis
times are commonly only a few minutes. As a sampled
system, sensors for FIA do not need to be sterile, so biosensors
and similar ‘fragile’ sensors can be used, as well as more
conventional sensors. Although biosensors can have attractive
selectivities for almost any conceivable substance [27], they
often have problems of drift during long bioprocesses as their
biological components degrade [28]. Recent developments
that utilize fluorescent labels on analyte-sensitive proteins
open up exciting avenues for online monitoring [29–31], but
are far from being commercially available.
Virtual measurements
When sensors do not exist or do not perform satisfactorily,
the analyte of interest can sometimes be estimated by
computation [1••]. This approach is often taken for
measuring cell mass [14] and final product. In many cases,
simple mathematical models can give accurate enough
information about these species based on several simple
measurables [32,33]. These ‘software sensors’ can also be
built to identify possible sensor faults [34]. Biological
systems are often too complex to represent with simple
models, making neural networks, expert systems and
other artificial intelligence approaches useful; however, an
artificial intelligence is only as good as its training set and
the limits of any model need to be recognized [35].
High-throughput bioprocessing
A new field of special interest is highly parallel bioprocessing
for early process development studies. This could do to
process optimization what high-throughput screening is
doing to drug discovery efforts. One approach is to add
instrumentation to shake flask studies, building on an
existing massively parallel technology base [10,36,37]. A
second approach is to scale-down and simplify bioreactors
into a new format that can be made highly parallel [38,39•].
Scale-down is helpful here to reduce raw material costs and
simplification is necessary to reduce set-up and breakdown
times. Some existing products for high-throughput screening
could also be easily adapted for parallel bioprocessing, such
as 96-well plates with fluorescence-based oxygen sensors in
each well [40] or a system that can measure oxygen, pH, carbon
dioxide or temperature in 96-well plates [41]. Optical sensors
have a distinct advantage over electrochemical ones in this
case, as they are more readily miniaturized.
One can envision the next generation of this technology as
not just monitoring, but also controlling critical parameters,
such as dissolved oxygen, pH and feed-rate, by robotic or
microfluidic dispensers. Figure 1 is a conceptual sketch of
one such microbioprocessor that would meet this goal.
This would allow rapid optimization of process parameters
as well as control strategies and would simultaneously
provide information that would simplify process validation.
Conclusions
Because of the stringent requirements of sensors used to
monitor bioprocesses, the sensors frequently used are time-
tested and new sensors are incorporated slowly. In practice,
this has resulted in decades of old sensor technology (e.g. to
monitor pH and dissolved oxygen) remaining the mainstay
in process monitoring. Furthermore, most fermentations in
process development are performed in shake or spinner
flasks with no online monitoring. There are promising devel-
opments in optical sensors and low-cost instrumentation.
Hopefully, the high-throughput screening paradigm will
extend to process optimization in the next few years. This
should significantly increase process productivity while
decreasing time-to-market. After that, microfluidic technolo-
gies are likely to provide low-cost sensing [42•] and may
enable the full use of metabolic engineering. Sometime in the
future, advanced separation techniques coupled with mass
spectrometry will provide real-time proteome analysis.
These advances will greatly expand the understanding of
bioprocesses and potentially lead to customization. An excit-
ing future post-genomic and post-proteomic possibility is
one of individualized patient therapy. Here the patients’ own
cells may serve as the source of therapeutic antibody or
cytokine. This would require the use of a customized
microscale bioreactor to produce just enough drug for the
patients immediate use.
Acknowledgements
Funding from National Science Foundation, National Institutes of Health,
Fluorometrix, Genentech, WR Grace, Merck and Pfizer over the years have
contributed to our understanding of bioprocess monitoring.
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