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Eng. Life Sci. 2015, 15, 469–488 www.els-journal.

com

Philipp Biechele1 Review


Christoph Busse1
Dörte Solle1 Sensor systems for bioprocess monitoring
Thomas Scheper1
Kenneth Reardon2 The ability to measure all process variables is of great importance in the field of bio-
process monitoring and control, and continuous, real-time measurements are highly
1
Institute of Technical Chemistry, desired. The more complete and real-time the measurements are, the more stable,
Leibniz University, Hannover, reproducible, and efficient the process can be, leading to reproducibly high-quality
Germany products. This additional information allows the operator to better document the
2
Department of Chemical and entire process. The process analytical technologies initiative of the US Food and Drug
Biological Engineering, Colorado Administration is strongly related to the analysis and control of biopharmaceutical
State University, Fort Collins, processes. The aim of the initiative is to create processes, generating products of
CO, USA ensured quality by measuring quality-related process variables. The quality of the
product is enhanced by a deep understanding of the process, which is enabled by an
effective and suitable sensor system. The aim of this review is to provide an overview
of current and emerging sensors for bioprocess monitoring. Sensors directly inter-
faced to bioreactors for measuring important variables from the gas phase, such
as oxygen and carbon dioxide concentration, are discussed, as well as sensors for
the monitoring of the biomass concentration and morphology and of the changing
medium composition. Furthermore, sensor systems are discussed. These involve sen-
sors (especially biosensors) that are not implemented directly inside the bioreactor
but rather are used in conjunction with sample-taking systems such as flow injection
analysis. A major focus is given to spectroscopic sensors, which are noninvasive and
offer interesting options for a simultaneous analysis of various compounds. Since
data handling is extremely important for this kind of sensor, chemometrics are also
included. Soft sensors are discussed as technology that allows a user to incorporate
more process data as it become available. Finally, the current state of disposable
sensor technology is presented. These sensors are needed for the growing area of
disposable and continuous biomanufacturing.

Keywords: Bioprocess monitoring / Cell culture / Process analytical technology / Process


control / Sensor systems

 Additional supporting information may be found in the online version of this article at
the publisher’s web-site

Received: February 12, 2015; revised: March 19, 2015; accepted: March 23, 2015
DOI: 10.1002/elsc.201500014

1 Introduction
Most bioprocesses are three-phase systems. The cells are dis-
Correspondence: Dr. Dörte Solle (Solle@iftc.uni-hannover.de), In-
persed as a solid phase in a liquid medium phase, which is aerated
stitute of Technical Chemistry, Gottfried Wilhelm Leibniz University
by a gas phase [1]. The interactions among these three phases
Hannover, Callinstraße 5, D-30167 Hannover, Germany
are complex. Biological components often react very sensitively
Abbreviations: ATP, adenosine-triphosphate; ATR, attenuated total re-
to environmental changes (e.g., pH, temperature, pO2, nutri-
flection; FDA, Food and Drug Administration; FIA, flow injection anal-
ysis; IR, infra-red; ISFET, ion-sensitive field-effect transistor; ISM, in ents), which may result in adverse effects on the activity of the
situ microscope; MIR, mid infra-red; NAD(P)H, nicotinamide-adenine- cells or the reproducibility of the process. Detailed analysis and
dinucleotide-phosphate; NIR, near infra-red; pO2 , partial pressure of
oxygen; pCO2 , partial pressure of carbon dioxide; OUR, oxygen up-
take rate; PAT, process analytical technology; PCA, principal component PTR, proton transfer reaction; PTFE, polytetrafluorethylene; RQ, respi-
analysis; PCR, principal component regression; PLS, partial least square; ratory quotient


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monitoring of these three phases, combined with deep process ture, viscosity, stirrer speed), chemical variables (e.g., pH, pO2 ,
knowledge, is necessary to control and optimize cultivation pro- nutrients, metabolites), and biological variables (e.g., biomass
cesses for high product concentration and quality as well as for concentration, cell metabolism). For each type of variable, sev-
documentation purposes. eral sensing methods are available [2–5] (Fig. 1).
Over the last few years, the process analytical technology Sensors interfaced directly to a bioreactor must be sterilizable
(PAT) initiative of the Food and Drug Administration (FDA) (typically this means resistance to steam or γ-radiation), as well
has been introduced in biotechnology, biopharma production, as temperature and pressure stable. The sensor should not be
and the food industry. By combining process analysis, process affected by fouling or interfere with the medium. Therefore, not
knowledge, and process modeling, a “built-in” quality in bio- every analysis method from the laboratory can be used for in-line
processes is enabled. monitoring of bioprocesses in an industrial setting.
Sensors provide important information for all unit operations For each bioprocess, the appropriate analysis quality at-
in a biological production facility. However, the primary target of tributes such as accuracy, selectivity, and sensitivity must be
sensor development and application has been for bioreactors in fulfilled. Accuracy refers to the true value of the analyte [6]. It
which cells convert various substrates to additional biomass and involves the degree of accordance between the measured and
desired products. In most bioprocesses, the bioreactor is the most true values. If the accuracy of the analysis is high, the difference
complex unit operation and is the one that governs the success between true and measured value is very small.
of the overall process. Thus, while the sensors and concepts Selectivity describes the ability to measure a certain variable
described here have application for bioprocesses broadly, this in an environment of other components or background noise.
review focuses on their application to bioreactors. Sensors with high selectivity are limited to a special group of
There are many types of sensors, but only a few are com- analytes or even to one specific analyte of interest.
monly used in the biotechnology industry. Others are complex, Sensitivity refers to the magnitude of the change of the mea-
expensive, labor intensive, and not compatible with standard sured signal per unit change of analyte concentration. It is de-
sterilization procedures. Thus, there is a need for noninvasive scribed by terms like the level of detection or level of quantifi-
and versatile sensors. Optical and spectroscopic sensors meet cation. An analysis with a sensor that has high selectivity and
these requirements. They offer the possibility to monitor vari- sensitivity is able to determine and quantify small amounts of
ous compounds simultaneously without direct contact with the the desired analyte despite the presence of other substances.
bioprocess fluids. Multiplexing and interfacing to any type of Alongside the analysis quality attributes, sensor quality at-
bioreactor and biological system are possible. However, these tributes, such as repeatability, robustness, stability, and linearity
sensors require complex data handling via chemometric models are important. Repeatability describes the agreement among dif-
to derive valid process information. The variety of such sen- ferent measurements of the same variable by the same operator
sors described in research is huge and they will be transferred under same conditions at different times, and includes the mea-
to broader application in industrial biotechnology in the near surement noise. Robustness refers to the ability of a sensor to de-
future. liver a consistent signal, independent of process conditions (e.g.,
temperature, background), and the length of operation. Stability
indicates the ability of a sensor to provide reproducible results for
2 Principles of bioprocess monitoring a certain period of time, retaining sensitivity and selectivity [7].
Finally, linearity is the relative deviation of an experimentally
2.1 Process analytical technology initiative determined sensor response from a straight line [7].
Another important sensor characteristic is the response time.
PAT is a regulatory initiative introduced by the FDA to improve It is based on a defined system and monitoring time delay and is
the understanding of production processes and to increase the specific to the sensor. The response time should be small relative
quality of the final product by building this quality into the entire to the important process dynamics; otherwise, efficient control
production process. Tools, like multivariate data analysis, bio- of the process is not possible [8].
process modeling, design of experiments approaches, and new In summary, the ideal sensor system for a certain process
sensor technologies, are incorporated by PAT to reach defined variable must provide high selectivity, sensitivity, robustness,
quality goals and to ensure adequate documentation of the entire repeatability, and stability, and have a low detection limit and
process. An always-growing pool of process information results linearity with short response times and a long life.
in the generation of deeper process knowledge, which helps to
safely handle all quality-related variables. The ability to monitor
and control these critical process parameters leads to a holistic 2.3 Sensor application types
process control. This ensures that quality inside defined ranges
can be ensured during all manufacturing steps. This allows the There are different ways to connect sensors to a bioprocess
real-time release of products via process validation. (Fig. 2). A sensor that is directly interfaced and in contact with
the process fluids is called an in line or in situ sensor. (The termi-
nology in-line will be used in this article.) The place at which an
2.2 Sensors in-line sensor is located is important. If the culture broth in the
process vessel is homogeneous, the sensor can be located any-
In general, there are three types of variables to measure for bio- where in the bioreactor and the sensor signal will represent the
process monitoring: physical variables (e.g., pressure, tempera- actual state of the bioreactor. However, the system might not be

470 
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temperature thermostat, thermistor, thermal element

foam resistance
physical variables viscosity viscosimetry

pressure pressure gauge

oxygen electrochemical, semicunducting, optical


carbon dioxide electrochemical, semicunducting, optical
chemical variables substrates and biosensors, electrochemical, spectroscopic
metabolites
pH-value
electrochemical, semicunducting, optical
volatile gases
biosensors, HPLC, GC/MS

biomass spectroscopic, impedance based, in-situ-


microscopy
biological variables cell morphology in-situ- microscopy, flow cytometry

cell metabolism
Figure 1. Bioprocess variables, exam-
fluorescence measurement
ples of sensing techniques.

A not represent the status of the medium composition inside the


in-line at-line
bioprocess.
If in-line and at-line sensors measure a quantity (e.g., pH
filtration value) continuously, the analysis can be regarded on-line. In this
probe /
case, the response time of the sensor signal, including the flow
sampling unit
rate of the sampling system or repeated measurements to increase
in-line probe the S/N, must be small in comparison to the process dynamics.
If the response time is in the range of minutes, this could be ac-
ceptable for mammalian cell cultivations but not for Escherichia
coli cultivations. If sensor data are generated at different time
B intervals, the definition of on-line analysis is problematic. In this
in-line
quasi on-line case, the term “quasi on-line analysis” could be used. If sensor
data are available without any time delay, real-time monitoring
signal

signal

is possible. All other measurements are considered to be off-line.


Invasive and noninvasive sensors are classified by the interac-
time time tion of the sensor and the bioprocess. If the bioprocess medium
interacts with the sensor as it is done for biosensors, the sensor
Figure 2. Conceptual definitions of types of sensor systems and is called invasive. If no interaction between sensor and medium
measurements. (A) In-line and at-line sensor systems. In-line occurs, the sensor is noninvasive (optical sensors).
means that the sensor measures inside the bioreactor; at-line
means that the sensor operates outside the reactor and that sam-
pling is necessary. (B) On-line versus quasi on-line measurement.
While on-line signals are available continuously (ideally without
time delay), sequential data from sensor devices are regarded as 3 Bioprocess monitoring systems
quasi on-line.
Bioprocess variables are of a chemical, physical, or biological
nature and can be measured in the gas, liquid, and solid phases
of a bioprocess. Because the characteristics required of sensors to
homogeneous, and for these situations, more sensors or sensor measure these variables depend upon the phase in which they are
arrays are necessary to fully describe the process state. used, information is organized here according to the bioprocess
If the sample is withdrawn via a filtration module and an- phase.
alyzed outside the bioreactor, the analysis is termed at-line. In
principle, any analytical system can be used in an at-line for-
mat to analyze a sample stream from a bioprocess. The response
time depends on the flow rate of the sample from the bioreactor, 3.1 Gas-phase monitoring
the dead volume of the filtration probe, and the duration of the
analysis in the instrument. At-line systems can have problems In bioprocesses, the monitoring of several gases, especially oxy-
from filtration effects such that the sample that is analyzed might gen and carbon dioxide, is important. The changes in the


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concentrations of these gases provide information about cell the proton-transfer-reaction-MS (PTR-MS), which is based on
growth, metabolism, and productivity. chemical ionization, is a good alternative [14]. MS produces
For oxygen analysis in bioprocesses, semiconducting, electro- a fingerprint of the whole gas phase, including the analysis of
chemical, and paramagnetic sensors are commonly used. Semi- volatile components like methanol or ethanol. However, for spe-
conducting devices (i.e. λ-probes) are based on the ionic con- cific process gases and volatile components, dedicated sensor
ductivity of a semiconducting oxide such as zirconia, which is systems are available. A common example for the analysis of
heated to temperatures of about 700–800°C [9]. The difference ethanol or methanol is the Figaro sensor, based on tin dioxide
between the oxygen concentration in the off gas and an external semiconducting material [17, 18].
air reference results in an electrical resistance change correlating
to the oxygen concentration [10]. Due to the strong heating of
the semiconducting component, the danger of explosion has to 3.2 Liquid-phase monitoring
be taken into account if explosive gases are present.
Electrochemical galvanic cells comprise an anode and a cath- The concentrations of various nutrients, metabolites and prod-
ode, typically made of silver and lead. The electrode space is filled ucts, as well as dissolved gases like oxygen and carbon dioxide,
with an electrolyte and is separated from the gas space via a gas must be monitored in the liquid phase. This monitoring can be
permeable membrane (e.g. PTFE). Oxygen passes through the performed with in-line sensors or via the large variety of analyt-
membrane into the cell and diffuses through the electrolyte to the ical systems available in chemical and biochemical laboratories
cathode where it gets reduced. Electrochemical oxygen sensors that can be interfaced via appropriate sampling devices as at-line
operate at ambient temperature, so the presence of explosive sensor systems.
gases does not pose a problem. During measurements, partial
pressure and temperature changes of the environment must be
taken into account. Hence, most commercially available exhaust 3.2.1 Sampling modules
gas analyzers incorporate automatic pressure and temperature At-line analysis requires frequent and aseptic sampling. Probes
adjustment. are available for cell-free and cell-containing sampling. Both
Another well-known approach to measure gas-phase oxygen probe types must provide a representative, stable sample stream
is the use of paramagnetic sensors [11, 12]. These sensors are with minimal time delay [2, 19, 20].
based on exploiting the strong affinity of paramagnetic gases to Cell-containing sampling can be performed manually, using
a magnetic field resulting in a positive magnetic susceptibility. In either repetitive sterilizable check valves and exit lines or dip-
most bioprocesses, oxygen is the only paramagnetic gas, so these tube systems with pumps or overpressure of the reactor. There
sensors are highly selective [10]. are also automated systems [21, 22] that use multivalve systems
While oxygen is consumed during the process, cells produce to flush tubing with cleaning solution and hot steam for steriliza-
carbon dioxide due to metabolism. To determine the carbon tion after each sample-removal step [23]. Cell metabolism con-
dioxide concentration in the gas phase, dual-beam infrared mea- tinues after sampling, which leads to changes in cell-containing
surement is conventionally used due to the strong adsorption of aliquots prior to analysis. So, sampling procedures must include
carbon dioxide in the infrared region. A typical, nondispersive defined quenching steps (e.g. cooling) that stop these reactions
infrared detector (NDIR) comprises two identical reference and rapidly [24–29]. This is particularly important if metabolic stud-
sample tubes, through which monochromatic infrared light is ies are being conducted [20, 30–32]. Håkanson et al. present a
passed. The difference in absorption of infra-red (IR) light be- general sampling system based on the use of a coaxial catheter,
tween the sample and reference (usually nitrogen) is measured by enabling the direct addition of metabolism inhibitor solutions
the detector and can be correlated to the gas concentration [13]. to fermentation samples [33].
Because water also adsorbs IR light, it must be removed via a In-line filtration probes provide cell-free samples. They form
gas cooler or dryer prior to measurement [14]. Furthermore, the sterile barrier between the bioprocess and the analysis system.
the analysis of carbon dioxide via this approach is affected by There are different in-line sterilizable systems that use ceramic,
other IR-active gases such as methane [15]. For cultivations in micro-, or ultrafiltration (e.g. polypropylene) membranes [2,
carbonate-buffered media, the carbon dioxide measurement in 19, 34, 35]. Another possibility for cell-free in-line sampling is
the off gas cannot be correlated to the cell respiration directly. provided by dialysis filtration probes. These probes contain a
More information on interpreting the carbon dioxide data is dialysis membrane inside the stainless steel tube, which forms
given by Frahm et al. [16]. the sterile barrier. A carrier buffer stream circulates at the inner
Optodes have also been described in the literature for exhaust permeate site of this membrane [20, 36, 37].
gas analyses. Since these sensors are more often used to measure To prevent fouling, these probes can be installed inside the
oxygen and carbon dioxide partial pressures in the liquid phase, turbulent flow region of the bioreactor. If this is not sufficient,
the operating principle of these sensors will be discussed later. cross-flow ultrafiltration modules in bypass mode can be used.
In anaerobic processes (e.g., biogas production or yeast
ethanol cultivations), monitoring the levels of gases such as
methane and hydrogen is important. A general approach to an- 3.2.2 Biosensors
alyze the composition of the gas phase in bioprocesses is to use Biosensors for many different analytes have been developed
MS, because of its great versatility. Instruments with electron [38–41]. Some biosensors can be sterilized by γ-irradiation.
impact ionization technology and a quadrupole mass filter can A typical biosensor comprises three parts, a biological
cover all relevant gases. If particularly high sensitivity is needed, detection component that is immobilized on a signal transducer

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unit, followed by an amplification and signal conversion unit. 3.2.3 Classical electrochemical sensors
The biocomponent recognizes the analyte either through a cat- Electrochemical sensors detect changes of electrical potentials
alytic mechanism (e.g. cells, organelles, enzymes, enzyme com- or defined charge changes, due to chemical reactions. The most
ponents, whole organisms, or tissues) or via binding (e.g. anti- important chemical variables monitored in-line with electro-
bodies, membrane receptors, protein channels, or nucleic acids) chemical sensors are the partial pressure of dissolved oxygen
[38, 42–44]. Major future trends for biosensor technology com- (pO2 ), the partial pressure of dissolved carbon dioxide (pCO2 ),
prise the development and use of new biocomponents, like ther- and the pH value [2, 60, 82].
mostable enzymes or aptamer structures (as thermostable alter- Furthermore, these sensors can be divided into three classes:
natives to antibodies). Aptamers are short, single-stranded DNA conductometric, potentiometric, and voltammetric.
or RNA oligonucleotides forming a unique three-dimensional
structure that is highly specific to the ligand [45–49].
The transducer unit determines the changes caused by the 3.2.3.1 pO2
selective interaction of the biological detection component and The dissolved oxygen concentration in culture media depends
the analyte. This unit can use electrochemical, optical, calori- on several variables, including agitation, aeration rate, and com-
metrical, or piezoelectrical principles. Often, classical oxygen position of the gas phase, pressure, temperature, and medium
electrodes [50], or pH electrodes [51] are used. composition. Considering these variables, it is desirable to con-
General reviews of biosensors and their application for bio- trol the oxygen supply to a bioprocess based on the data of the
process monitoring are available in the literature [38, 52–54]. oxygen sensor.
Other reviews focus on specific biosensor classes, including Membrane-covered amperometric electrodes, known as
calorimetric [52, 55–57], optical [39, 41], and electrochemical Clark-type electrodes, are the most common type used [60, 83,
biosensors [38, 43, 44]. 84].
The most common biosensor application is the determination
of glucose concentration. pH or pO2 sensors can be used as trans-
ducing units for glucose biosensors. For example, the enzymes 3.2.3.2 pH
glucose oxidase and catalase, immobilized on a classical oxy- The pH must be controlled in nearly all bioprocesses to keep
gen electrode or optical oxygen sensor, can be used to measure the cells at optimal cultivation conditions. Electrochemical tech-
glucose concentration by oxygen consumption [50]. Another niques for pH monitoring are based on potentiometric and am-
approach uses the enzymes glucose/fructose oxidoreductase and perometric measurements, with potentiometric sensors more
gluconolactonase, immobilized upon a pH electrode, which de- commonly used. A working (measurement) electrode (working
termines the production of hydrogen ions resulting from the potential) is correlated to a reference electrode (reference poten-
enzymatic turnover in presence of glucose [51, 58, 59]. tial). Several different designs exist for both, Henkel et al. review
While biosensors provide the opportunity to monitor glucose possible electrodes, materials, and combinations [85].
and other important analytes with specificity, biosensor systems Other systems for pH measurement exist and have been also
have some challenges. In case of biosensors based on pH changes reviewed by Henkel et al. [85]. Ion-sensitive field effect transis-
from the detection reaction, the sensor signal depends on the tors (ISFETs) are promising alternatives to common glass-type
buffer capacity and pH value of the reaction solution [51]. In sensors. Current changes are measured, caused by changes of
case of biosensors based on oxygen-consuming reactions, oxy- the ion concentration in the solution. For measurement of the
gen must be available in sufficient concentration, otherwise it pH value, proton-sensitive ISFETs are used, which combine a
would be limiting for the enzymatic turnover [59]. In both cases, proton selective membrane and a measurement amplifier. Two
the use of an additional reference sensor (for pH or oxygen) is electrodes are grounded on a substrate. An electrical current
often required. The activity of enzymes used as biological com- flows between these electrodes. This current is influenced by a
ponents also depends on the surrounding pH value. Drifting third electrode (gate electrode) comprising proton-sensitive ma-
effects in long-term use of biosensors may also be problem- terial (e.g. SiO2 , Si3 N4 , or Ta2 O5 ), which is electrically isolated
atic. So, measurements are only accurate if the reaction environ- from the drain and source electrodes. The gate electrode is con-
ment is controlled within a defined range or if the influencing nected to the culture broth and changes its electrical potential
factors can be accounted for in the calculation of the analyte depending on the pH value. This potential influences the current
concentration. between source and drain. [4,85–88]. A conceptual schematic of
Flow injection analysis (FIA) provides a possibility to create an ISFET is provided in the Supporting Information, Fig. S1.
such a defined reaction environment (e.g. optimal pH and/or These systems are easily miniaturized and many sensors can
dissolved oxygen level). Samples can be diluted and precondi- be placed within a small space. The drawbacks of this system are
tioned by the addition of reagents to the FIA carrier stream before that they are light sensitive, they cannot be used at high pH, and
analysis [60–62]. Drifting effects can also be countered by the they require a reference electrode [85].
use of FIA by frequent recalibration [2].
In general, FIA is a fast, accurate, and versatile tool to measure
specific analytes and to automate (bio-)chemical analyses [39]. 3.2.3.3 pCO2
Many examples have been described in the research literature for Carbon dioxide is an indicator for the respiratory activity of cells
FIA-based biosensor systems [63–66]. in bioprocesses and is used to control the pH in mammalian cell
Literature, relating to different applications of biosensors in cultivations. Dissolved carbon dioxide concentrations can be
process monitoring is listed in Table 1 [65, 67–81]. measured by the Severinghaus principle, in which pH electrodes


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Table 1. Literature covering applications of at-line biosensors

Sensor principle Sensor type Measured variable Author

Electrochemical Amperometric, platinum electrode Glucose, glutamine, glutamate [67]


Amperometric, ferrocene electrode Glucose [68]
Amperometric, oxygen electrode Glucose [69, 70]
Ethanol [70, 71]
Glutamine [72]
Mono-, disaccharides, lactate, amino acids [73]
Total sugars [74]
Optical Fiber-optic-based oxygen sensor Glucose, lactate [75]
NAD(P)H-fluorosensor Glucose, fructose [76]
Glucose, ethanol [77]
pH optode pH, urea, penicillin concentration [65]
Chemiluminescence fiber optode Glutamine [78]
Calorimetric Enzyme thermistor Glucose [79]
Enzyme thermistor Penicillin, glucose, ethanol [80]
Enzyme thermistor Different IgGs [65]
Piezoelectric AT-cut piezoelectric crystal sensor IgGs [81]

are modified with a carbon dioxide permeable membrane such


as polytetrafluoroethylene or silicon [14, 89].

3.2.4 Optical sensor systems: Chemosensors


Optical chemosensor systems, also called optodes, are based on
the interaction of a matrix-embedded indicator and the ana-
lyte [90]. The indicator is immobilized at the sensor tip and is
illuminated by a light source (e.g. light emitting diode) through
a fiber optical cable. A change in the optical properties of the
indicator, such as photoluminescence intensity, absorption, or
reflection, is correlated with the analyte concentration. A light
detector (e.g. photodiode) is connected to the other end of the
fiber [3,91]. The light excitation and emission are separated by a
dichroic mirror or optical filter so that both wavelengths can be
measured using only one fiber. The general setup is illustrated in
Fig. 3.
In addition to in situ probes for bioreactors using standard
ports, disposable sensor patches are available (Fig. 3B). Sen-
sor patches allow a modular setup and are very well suited for Figure 3. (A) General setup of a fiberoptic chemosensor. (B) Mod-
modern single-use bioreactor systems. They can be presterilized ular setup using sensor patches.
within the disposable containers by γ-radiation, and the place-
ment inside the reactor is flexible. These patches can be con-
nected to optical fibers and external equipment via transparent
material such as glass windows. The most common chemosen- matrices are used for immobilization of the dye, to avoid cross-
sors in bioprocess monitoring are those for monitoring the par- sensitivity to ions that can also quench fluorescence. Amao re-
tial pressure of gaseous or dissolved oxygen, carbon dioxide, and viewed several polymers as a possible matrix of optical oxygen
pH. sensors and various dye probes for oxygen sensing [93].
In contrast to conventional amperometric sensors, optodes
do not consume oxygen during measurement, which allows their
3.2.4.1 pO2 use in small volumes and in diffusion-limited cases at low oxygen
Optical measurement of the partial pressure of oxygen is based concentrations below 5% [94]. In addition, optical sensors can
on the fluorescence quenching of an indicator by molecular oxy- easily be miniaturized [89]. Commercially available probes and
gen [83, 90, 92]. Oxygen-sensitive indicators are complexes of sensor patches can be autoclaved without any loss of sensitivity
ruthenium, palladium, or platinum such as Tris-4,7-diphenyl- but their long-term stability is limited due to photobleaching
1,10-phenanthrolin-ruthenium(II) [14, 91]. The fluorescent in- during use. The impact of photobleaching can be reduced by
dicator is immobilized in an oxygen-permeable polymer matrix using lifetime instead of intensity measurements and by chem-
such as silicone and attached to the tip of an optical fiber or the ical modifications such as the use of multiple fluorinated plat-
transparent window of the probe [90, 93]. Usually, hydrophobic inum porphyrin to reduce the drift of the signal. Hanson et al.

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published a comparison of optical dissolved oxygen sensors with methods for bioprocess monitoring are focused on the spectral
traditional electrochemical probes in mammalian cell cultiva- range from UV to mid infra-red (MIR), including fluorescence
tions. They showed that optical sensors enabled small-scale and Raman spectroscopy. Various bioprocess variables can be
monitoring and that the measurement results are comparable measured in different spectral ranges as reported in the lit-
to standard electrochemical probes [95]. erature [103–120] (Fig. 4 and Supporting Information, Table
S1). Several review articles on infrared and fluorescence spec-
troscopy [89, 94, 110, 121–124] are summarized in Supporting
3.2.4.2 pH Information, Table S2, sorted by the type of spectroscopy with a
Fiber optic pH sensors are based on absorbing or fluorescing short description of the topics covered in each.
pH indicators. Frequently used pH-sensitive indicators for ab- The advantages of spectroscopic sensors are that no sam-
sorbance are cresol red [96], phenol red, bromophenol blue, pling is needed (except for calibration), there is no interaction
and chlorophenol red [97]. These indicators are immobilized on between the sensor and analytes, and several different process
solid substrates or in polymers that are mounted directly on a variables can be determined simultaneously. They can easily be
transparent carrier (e.g. glass or optical fibers) [89,91,98]. Thus, multiplexed and instantaneously deliver signals [122].
the instrument setup is similar to oxygen optodes. Chemometric data analysis is required for spectroscopic bio-
pH optodes can easily be miniaturized and have response process monitoring to extract relevant process information from
times in the range of milliseconds. However, they suffer from the generated data. Some spectroscopic measurements are sus-
cross sensitivity to ionic strength, a limited dynamic range, ceptible to scattered light, so their use in glass reactors or mi-
and the loss of sensitivity during sterilization or cleaning pro- crowell plates is limited.
cedures [89, 90]. To increase the dynamic range, Gupta and
Sharma [97] presented a long-range fiber optic pH sensor based
on evanescent wave absorption. It was equipped with three im- 3.2.5.1 IR spectroscopy
mobilized pH-sensitive dyes on the surface of an optical fiber Infrared spectroscopy includes spectral areas of near infrared
core by sol-gel technology. Covalently bound dyes have been (NIR, 740–1300 nm) and mid-infrared (MIR, up to 15 000 nm).
proposed for better temperature resistance, so that in situ steril- Everything with a wavelength above 15 μm belongs to the far
ization might become possible in future [99]. Nowadays only γ- infrared, but this range is rarely used for bioprocess monitoring.
sterilized optical pH sensors are commercially available and are IR light excites different vibrational modes of molecules. Each
implemented in presterilized disposable reactor systems. Lam organic and inorganic compound has a special spectral IR signa-
and Rao [91] review several possibilities for optical sensing of ture from these vibrations. The more different vibrational modes
pH in bioprocesses. that can be excited, the more information can be gained and the
more specific the determination of analytes can be [6, 122]. IR
spectroscopy offers very fast, robust, and sensitive multi-analyte
3.2.4.3 pCO2 information out of the culture broth of bioprocesses. It is an
Most optical CO2 sensors operate in the same way as the classical on-line and noninvasive process analytical technology, applied
Severinghaus electrodes, which measure the change of pH of in-line by direct beam or optical fiber.
an internal carbonate buffer system due to CO2 presence. They
are typically based on the fluorometric or colorimetric changes
of pH-sensitive indicators that are added to the buffer system MIR spectroscopy MIR radiation excites fundamental rota-
and separated from the analyte solution by a CO2 -permeable tional vibrations of functional groups from organic compounds.
membrane [100]. Molecules such as glucose, lactate, fructose, acetic acid, ammo-
The response time of the sensor is in the range of minutes be- nia, and even antibodies [122] have a characteristic absorption
cause the CO2 diffusion through the membrane is slow. Optical spectrum, known as the molecular fingerprint. These finger-
pCO2 sensors suffer from the same drawbacks as pH optodes, prints are used to identify single components in bioprocesses in
including low stability at high temperatures and dependency a manner that is quantitative, sensitive, and specific.
on the ionic strength of the buffer, which must be exchanged A high degree of water absorption appears in MIR spectra.
frequently [4]. However, in-line measurement in aqueous solutions is possible
To decrease the response time and ionic strength dependence, using appropriate fiber optic probes that incorporate attenu-
Mills et al. [96] established a new scheme for optical pCO2 ated total reflection (ATR) technology and Fourier transforma-
sensors. The pH-sensitive indicator is incorporated into a hy- tion [14, 121, 122]. The measurement principle of ATR probes
drophobic membrane and a lipophilic quaternary ammonium is based on reflection of light at phase interface of two media
hydroxide is used instead of the bicarbonate buffer. The pro- with different refractive indices, the ATR crystal and bioprocess
tonation of the pH-sensitive fluorophore initiates a reversible medium. This results in a very short (only few micrometers) path
reaction, which results in a change of the fluorescence, which length. Therefore, information about cells cannot be detected by
correlates to the CO2 concentration [101, 102]. this technique because cells are too large to enter the measuring
zone.

3.2.5 Optical sensor systems: Spectroscopic sensors


Spectroscopic analyses are based on the interaction of electro- NIR spectroscopy NIR spectroscopy is also based on dif-
magnetic waves and molecule bonds. Common spectroscopic ferent vibrational modes, overtone and combination vibrations


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• glucose; lactate [110]


• glutamine [109]
• glutamine; glutamat [136] • ammonium [112]
• BSA, celldebries [114] • acetate; phenylalanine [119] • pH [112]
• toxic substances [115] • TCD /VCD [138] • biomass [113]

Raman

UV VIS NIR MIR


10nm-400 nm 400 nm-740 nm 740 nm - 1,3 µm 1,3 µm - 15 µm

Fluorescence

• proteins [134] • glucose [103]


• vitamins [132] • lactate [104]
• pyruvate [131] • antibodies [105]
• ATP [131] • pH [106]
• NAD(P)H [129] • fructose [107]
• cell mass [118] • acetic acid [107] Figure 4. Spectral range for bio-
• ethanol / metabolic changes [117] • ethanol [108] process monitoring with acces-
sible variables.

after excitation. Important targets are the O-H bonds of alco- spectroscopy does not currently play a major role for biopro-
hols, C-H bonds of aliphatic, and aromatic carbon compounds, cess monitoring [126]. Direct readout high resolution at-line
and N-H bonds of proteins. The NIR range is thus suitable for UV spectrophotometer systems have been reported [114]. As
monitoring of substrates such as glucose and lactate, biomass, with all spectroscopic methods, UV/VIS spectroscopy requires
and the products of a bioprocess [89, 122]. chemometric-based calibration and data treatment.
As a result of the lower energy of the NIR and the resulting
overtone vibrations, the bands are much broader, often overlap-
ping, and not as specific as in MIR spectroscopy [121]. Thus, to 3.2.5.3 Fluorescence spectroscopy
obtain a higher S/N, a longer path length is used [121–123]. NIR Many important molecules for bioprocesses monitoring and
spectroscopy has a more qualitative character, compared to the control fluorescence. This includes proteins with aromatic
more precise and quantitative MIR spectroscopy. Glucose levels amino acids (tryptophan), NAD(P)H (biomass), adenosine-
can be monitored by MIR spectroscopy more sensitively than via triphosphate, pyruvate, vitamins, pyridoxines, coenzymes, and
NIR spectroscopy [104]. NIR spectroscopy offer a more global flavins [89, 116, 127–131]. After excitation of these fluorophores
view to a bioprocess, for example, by batch trajectory [125]. Due with light of a specific frequency, molecules are elevated to
to its higher robustness, NIR spectroscopy is better applied for a higher energy level. As these molecules fall back to their
monitoring industrial production processes. MIR spectroscopy base energy level, photons are emitted at other frequencies.
is well suited for process development and optimization due to So, each fluorescence-active compound has a specific pair of
its multiplexing technology and the fact that fragile ATR fibers excitation and emission wavelengths. Simultaneous measure-
are used. ment of several different fluorophores in the culture broth is
possible using modern approaches like 2D fluorescence spec-
troscopy [94, 117, 127, 132–134]. Fluorescence probes can either
3.2.5.2 UV/Vis spectroscopy be implemented into a bioreactor directly as fully sterilizable in-
UV/Vis spectroscopy uses ultraviolet and visible light (10– line devices, or via optical windows. Chemometric approaches
740 nm) to excite electrons of molecules. The observable transi- are indispensable to obtain valid process information.
tions take place at unsaturated bonds, such as in aromatics [115].
A variety of analytes, substrates, metabolites, and products can
be determined with UV/Vis spectroscopy. 3.2.5.4 Raman spectroscopy
UV/Vis spectroscopy has a high sensitivity, and high resolu- Raman spectroscopy is another form of vibrational spectroscopy.
tion spectrophotometers can be compact, inexpensive, and ro- It is based on shifted wavelength scattering of molecules,
bust, making these instruments interesting for industrial process after excitation by monochromatic light, usually produced
applications [89]. The use of CCDs or photodiode arrays makes by adjustable lasers [42]. Several analytes, including glucose,
UV/Vis spectroscopy even more attractive. However, UV/Vis lactate, acetate, formate, glutamine, and glutamate, can be

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measured [89,119,135–139]. In general, each interaction of pho- A


ton and molecule leads to the absorption and then emission 90° scattering
of light energy. In some cases, in addition to normal absorp- 180° reflection
tion and emission effects, in-elastic scattering effects can oc-
cur [89, 127, 140], resulting in Stokes and anti-Stokes scattering.
For Stokes scattering, the emitted photons have lower frequen-
cies. In anti-Stokes scattering, molecules are already excited and
the emitted photons have higher frequencies [141]. This inelas- incident
light transmission
tic scattering occurs only a few times, and then the re-emitted
frequencies are measured by a detector and a monochromator.
The use of Raman spectroscopy is limited by the strong flu-
turbid sample
orescence activity of several biological molecules in the culture
broth [142]. This fluorescence signals overlay the Raman bands.
To avoid fluorescence, low energy lasers can be used, but then
B
heating effects can occur [89,127,140]. Also, interferometric de-

sensor signal
linear response
tection [94] or shifted subtracted Raman spectroscopy [12] helps
to reduce the noise and fluorescence interferences.

180° reflection
3.3 Biomass monitoring

The most important variable in bioprocesses is the biomass, 90° scattering


representing the solid phase in the complex, three-phase system.
The biomass can be characterized by its concentration (mass transmission
per volume or cells per volume) or, more importantly, by its
biomass concentration
metabolic activity. Different analytic methods exist to determine
the biomass concentration and the biomass activity. Figure 5. (A) Different methods for measuring turbidity. (B) Sen-
sor signal as function of biomass concentration for turbidity mea-
surement methods.
3.3.1 Biomass concentration analysis
Well-established off-line methods for biomass concentration de-
termination are based on gravimetric, microscopic, or optical OD sensors must be calibrated for each biological system and
density measurements. These are the typical benchmark meth- do not allow differentiation between viable, dead, or inactive
ods in bioprocess engineering. However, in-line techniques also cells. Moreover, the measurements are affected by air bubbles
exist. The common ones are turbidity sensors, impedance sen- and noncellular particles, which may have an influence on the
sors, and in situ microscope systems. biomass concentration analysis [127, 144]. Inclusion of a de-
gassing chamber or the application of a very fine mesh on the
sensor tip that is permeable for cells but not for bubbles can
3.3.1.1 Turbidity sensors minimize this negative influence.
The measurement of the optical density (OD), also known as OD sensors have been used in many microbial, fungi, and
turbidity, is one of the easiest, most frequently applied technolo- mammalian bioprocesses [144–147]. Six different commercially
gies for biomass monitoring. Various in-line optical fiber probes available OD probes were tested by Wu for monitoring of mam-
are available based on transmission, absorption, reflection, or malian cell cultures. Backscattering probes show better results
scattering of light [14, 127]. For low-to-moderate turbidity cul- at cell concentrations up to 20 × 106 cells/mL in comparison to
tivation broths, probes based on transmission or 90° scattering transmission probes [148].
are best (Fig. 5 [143]). Reflection measurement is primarily used
for high cell densities. It allows construction of a gap-less probe,
in which emission and detection are combined in a single optical 3.3.1.2 Impedance sensors
fiber. The impedance of a cell suspension can be measured via a si-
The selection of an appropriate light source and wavelength nusoidal electrical field. This field is applied to two electrodes
depends on the size of the organisms observed. For objects between which the sample is placed. The conductivity and the
smaller than 3 μm, visible light should be used, while near in- electrical permittivity describe the electrical properties of the
frared light is better for larger objects such as mammalian cells. sample. Biomass concentration affects both parameters, and the
Most sensors use wavelengths between 840 and 910 nm, because frequency-dependent permittivity is the more sensitive. Air bub-
most culture media absorb very little light in this region [144]. bles and other nonbiological particles have no plasma membrane
The light source of the sensor can be a light emitting diode, a and the membrane of dead cells is ruptured, hence they do not
laser (diode), or a filtered halogen light. The attenuated light have a significant influence on the electrical properties. This is
intensity is typically measured by a photodiode. a large advantage over other methods, such as optical density


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measurement [14, 149–151]. However, bubbles dilute the sam- 3.3.2.1 Calorimetry
ple and changes in the aeration would affect the measurement. Calorimetry refers to the measurement of heat that is released
Also, the temperature and ionic strength of the culture medium by the biological activity of the organisms. The challenge of
might have additional effects. this method is that the overall heat production of the process is
Modern impedance sensors use frequency scanning tech- measured, so heat originating from the cellular metabolism and
niques in a range of 100 KHz to 20 MHz (dielectric spectroscopy) growth must be calculated separately. Thus, simple adoption
to estimate cell size and volume in addition to biomass concen- to different process types and procedures is limited [165]. Von
tration [149]. Kaiser et al. used a radiofrequency impedance Stockar and Marison have been pioneers in this field [166–170].
based biomass monitoring system to determine the entire viable
cell volume [152]. Moreover, metabolic information about cul-
ture condition can be assessed via impedance measurements. For 3.3.2.2 NMR spectroscopy
example, the differentiation between growing, sporulating, and Alternatively, NMR spectroscopy allows the direct measurement
mature spores of Bacillus thuringiensis during endotoxin pro- of different intracellular components such as metabolic interme-
duction was tracked using impendence data [153]. Impedance diates, amino acids, ammonia, intracellular pH, Ca+ and Na+ ,
measurement works best with large cells like CHO cells but is adenosine-triphosphate, and NAD(P)H [2, 171, 172]. Brecker
limited by the conductivity of the culture medium and must et al. used H-NMR to observe the metabolic shift from citrate to
be calibrated for each biological system [154]. Currently, several glucose metabolism of E. coli [173]. In addition, the monitoring
sensors designed for standard bioreactors ports are commercially of mammalian cell cultures by NMR was presented by Bradley
available, and new approaches for direct application in modern et al. [174]. Important drawbacks of this technology are the high
disposable reactor systems have been developed [155]. prices for the hardware, the general technical setup, the need for
high biomass concentrations, small measurement volumes, and
long measurement periods [175].

3.3.1.3 In situ microscopy


The application of microscopy systems directly in a bioreactor is 3.3.2.3 Oxygen uptake rate and respiratory coefficient
called in situ microscopy (ISM). It is possible to acquire pictures The oxygen uptake rate (OUR) is a robust indicator for the
of the suspended organisms and to analyze the cell concentra- determination of cellular activity. As one of the fundamental
tion, cell size, cell distribution, and morphology automatically physiological characteristics of aerobic culture growth, it has
by image-processing algorithms [156]. been used frequently for optimization of bioprocesses [176–
A typical ISM device comprises a CCD camera, an objective, 178]. Various methods for the determination of the OUR based
and a light source, all of which are separated from the culture on oxygen balance in the liquid phase or the whole reactor,
broth by two sapphire windows. The gap between these win- as well as with variable (dynamic) or stable dissolved oxygen
dows constitutes the sampling zone, and is variable in height partial pressure, are known [179, 180]. When carbon dioxide
and thickness. Two stepper motors control image focusing and sensors are available in the gas or liquid phase of the process, the
adjustment of the sampling zone height, depending on the or- carbon dioxide production rate can be calculated accordingly.
ganisms observed and their concentration [157]. The complete The respiratory coefficient (RQ), defined as the carbon dioxide
setup is shown in Supporting information, Fig. S2. production rate divided by the oxygen consumption rate in units
Several bioprocesses have been monitored using ISM sys- of mol mol−1 , can be calculated from the OUR and carbon
tems [1,157–162]. A recent paper reports on the combination of dioxide production rate data. RQ allows one to make inferences
an ISM with MIR measurement to obtain reliable estimates of about substrate utilization rates of the culture and has been
the process state of a CHO cell cultivation, as well as the estab- proposed as a valuable parameter for monitoring and controlling
lishment of a process control loop for substrate feed and optimal cell cultures [14, 181, 182].
harvest [163]. Table 2 presents a summary of in situ measurement tech-
niques for biomass monitoring and relevant literature [145,
149, 152, 157, 158, 160–162, [164, 165, 167, 168, 174, 176–179],
[181–187]].
3.3.2 Biomass activity analysis
In addition to the biomass concentration, the concentration of
viable, metabolically active cells is of special interest in bio- 3.4 Chemometrics
processes because only those are able to grow and produce the
desired product. Some sensors for biomass concentration also Several sensor technologies, and spectroscopic measurements in
allow the determination of bioactivity. For example, turbidity particular, provide an enormous amount of data, especially when
probes allow inferences about cell size and morphology if the spectra are generated via in-line sensors at high frequency. The
changes in sensor signal occur much faster than cell growth or spectral data must be correlated to important process variables,
death. Also, impedance sensors can be used for the observation like substrate concentration or to the actual process status in a
of lipid storage in yeast [164], and image analysis by ISM sys- mathematical model. Afterward, the model can be used to pre-
tems makes information on cell size and morphology assessable. dict the variables or the process status from on-line spectroscopic
However, special systems and techniques have been developed data. For these correlations, multivariate data analysis must be
to analyze specifically the metabolic activity of cells. applied because the relevant information is distributed over the

478 
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Table 2. In situ measurement techniques and target values for biomass monitoring

In situ method Target value Reference

Concentration Morphology Activity

ISM X X X [157, 158, 160–162, 183, 184]


Optical density X X [145]
Impedance X [149, 152, 164, 185]
Calorimetry X X [165, 167, 168]
NMR spectroscopy X [173, 174]
OUR / RQ X X [176–179, 181, 182, 186, 187]

entire dataset. It cannot be found in a subset of a spectrum nor variables, represented by the most relevant changes in the spectra
in only one spectrum [188, 189]. Multivariate data analysis is [200].
one part of chemometrics, the science of extracting information These qualitative methods can be used to monitor a bio-
from chemical systems by data-driven methods. It uses many process as defined by PAT [201, 202]. High process repro-
methods from multivariate statistics, applied mathematics, and ducibility and product safety can be provided by this type
computer science. These chemometric methods are used to pro- of process supervision [203]. A process target line, trajec-
vide interpretable information from the enormous amount of tory, or so-called “Golden Batch” can be identified out of
spectroscopic data of a bioprocess. similar ideal process runs [204–207]. Different measurements,
Before spectral data can be analyzed or correlated, the ho- multivariate spectroscopic data, or univariate classical process
mogeneity of the dataset must be confirmed. For example, data can be pooled [208] to generate a holistic view of the
the comparability of different cultivation datasets should be bioprocess.
established for successful chemometric model transfer. Fil- In contrast to those qualitative methods, quantitative models
tering [190], centering, normalization, standardization, devi- are needed to describe correlations between single analytes and
ation [191], wavelength selection [192, 193], weighing, and spectral data. Multilinear regression (PCR) on principal com-
scaling are common preprocessing methods for multivariate ponents is used to predict a process variable from the spectra.
data [6, 194]. Data preprocessing is a sensitive and powerful However, partial least square regression (PLS) provides better
tool for spectroscopic data [195]. The method of choice de- results in most cases. PLS considers the covariance of spectral
pends upon the spectral data. For example, deviations with re- changes to the variable concentration, instead of process vari-
spect to wavelength are common for IR spectroscopy but un- ance [209]. Herewith, the values of different variables can be
usual for 2D fluorescence. Further, data preprocessing can fo- predicted from a spectroscopic measurement by adapted chemo-
cus relevant information in the process data [196]. For exam- metric models for each variable [111,210]. For a PLS calibration,
ple, scattered light containing information about biomass or representative process data, including both spectral data and the
turbidity, can disturb quantitative data analysis. This informa- corresponding reference values, are needed. The data should be
tion can be excluded by normalization or baseline correction, distributed over the entire process, describing the variance in-
while information about biomass is eventually eliminated by that side a single process run as well as different process runs. Both
approach. variabilities should be considered for calibration, in order to cal-
After data preprocessing, multivariate data analyses are per- culate a reliable PLS model with satisfying prediction quality to
formed to extract qualitative or quantitative process informa- unknown process data. There has been a considerable effort to
tion from the spectral data. Many established applications are produce such a representative data basis for process variables and
based on principal component analysis (PCA), an approach that corresponding spectroscopic data [211, 212]. Pilot tests, spiking
reduces high-dimensional data to fewer, linearly independent experiments, and data from experimental design allow one to
components. This method assesses the principal components by construct premodels, which can be adjusted and validated by
spectral variance, which is induced by changes during the pro- process data [213, 214].
cess. PCA is commonly used to study the structure, variance, or Neural networks are a completely different concept to pro-
distribution of data sets and to identify outliers. With PCA, a vide quantitative on-line prediction of process variables based
classification of raw materials, batches, or the process status is on spectroscopic methods [215,216]. For highly complex data or
also possible [197]. relationships, NNs provide good results, but no causal conclu-
Supported vector machines are also used for classification of sion to the measurement data is possible.
spectral data. Supported vector machines separate the data by Clearly, spectroscopic measurements require chemometric
as much as possible [198, 199]. These qualitative methods use data analysis to offer continuous, on-line prediction of biopro-
only spectral or on-line data to find similarities or differences. cess variables or an estimation of the actual process status. In
They are able to describe the main process changes that can be particular, the process of model construction is sensitive and
seen in spectral data. Insertion of constraints to a PCA offers the extensive. However, based on this, broad on-line monitoring in
possibility to monitor the course of different unspecific process terms of the PAT is feasible [217–221].


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3.5 Soft sensors to the special requirements of different processes, and improved
process repeatability is often reported, making it valuable for
A soft sensor combines a measuring part (sensor) and a software- satisfying the goals of PAT [4, 235].
based estimation algorithm for on-line monitoring [222]. The Modern single-use reactor systems necessitate a new sensor
sensor delivers data from directly measureable variables (e.g. philosophy to meet the special requirements of these systems.
pressure, pO2 ) or complex multivariable measurements (e.g. Conventional sensors were mostly built as reusable devices for
NIR spectroscopy, in situ microscopy). Based on the sensor long-term operation. Such sensors cannot be inserted directly
data, the software part predicts process variables that cannot be into single-use bioreactors, because these reactors form a closed,
measured directly, such as substrate concentration and specific sterile system.
growth rates [223–225]. Soft sensors are able to pool, condense, New sensors for disposable reactor systems must demonstrate
and process all data from several probes, measuring different the same characteristics as well-established classical sensors. But
process variables [8]. single-use sensors must be γ-irradiation sterilizable instead of
In general, there are two types of soft sensors described in the being steam sterilizable. In comparison to reusable devices, the
literature: model-driven and data-driven. Data-driven soft sen- lifetimes of such single-use sensors can be shorter, but still must
sors are based on chemometric models, informed by historical be long enough for long-term, continuous production processes
process data to predict other process variables on-line. This type (up to 2 months). They must be precalibrated and should be
of soft sensor uses chemometric techniques such as PLS, PCA, installed within the single-use reactor by the manufacturer. Fur-
and NNs to describe the internal relationships between measured thermore, these sensors must be cheap (owing to their single-use
process data and various process variables [224,226,227]. For ex- nature), small, and modular [89,94]. The development of a stan-
ample, multi-wavelength fluorescence sensors can be used to de- dardized sensor port in the single-use bag would facilitate the
termine biomass, glucose, and ethanol [209]. NIR spectroscopy design, fabrication, and application of disposable sensors for a
can be used to determine biomass and the specific growth rate mechanically stable integration.
[224]. Another approach, using 2D fluorescence spectroscopy, Optical sensors (primarily spectroscopic and chemo-optical
enables the detection of ethanol production, hence predicting sensors) and semiconductor devices (e.g. ISFETs) are well suited
the metabolic state of Saccharomyces cerevisiae cells [117]. for such purposes [4]. Sensor patches or other measurement
The model-driven soft sensors are mainly based on mass and systems can be connected with reusable external equipment.
energy balances, known as the “First Principles” of processes. Therefore, the material of disposable reactor systems must be
First Principle Models describe the physical and chemical pro- permeable to the sensor signal. Glass windows are a common way
cess principles and backgrounds (e.g. using mass preservation of transmitting optical signals from the inner space of the reactor
principles or energy balances) [223, 228]. Sensors based on the (where a patch detects the analyte) to connected external devices,
use of Kalman filters or extended Kalman filters are commonly such as optical fibers and detectors. An alternative concept is to
applied in this field of soft sensing [229, 230]. As an example, design a novel sensor port or adapter that allows the connection
Sundström et al. used four software sensors based on standard of classical sensors to single-use systems.
on-line data from fermentation processes and simple mathemat-
ical models to monitor different state variables (biomass concen-
tration, specific growth rate, the oxygen transfer capacity of the 4 Concluding remarks
bioreactor, and the ratio between oxygen and energy substrate
consumption) in E. coli fed batch fermentations [225]. Some sensor technologies used for bioprocess monitoring are
Unforeseen process changes that result in high process vari- relatively old and well established (e.g. Clark electrode). These
ances cannot be balanced by multivariate data analysis in every systems have good, reliable performances. However, to meet the
case [210, 231, 232], but some multivariable applications can be goals of the PAT initiative that was introduced by the FDA more
stabilized by soft sensors [228, 233, 234], adjusting the on-line than 10 years ago, modern sensors are needed for a total process
prediction by a mathematical process model. Data-driven and overview, aiming a steadily ongoing improvement of processes
knowledge-based soft sensors can be combined with the Kalman by on-line monitoring. Newer systems, including optical sensors
filter approaches for this purpose. and FETs, are available but still not commonly used for biopro-
In summary, soft sensors can be used as appropriate alterna- cess monitoring. One reason for this is that the implementa-
tives to standard hardware sensors to meet PAT and quality by tion of new sensor systems to already approved processes, which
design requirements and to find new ways to detect and measure use well-established sensor technology, would lead to a time-
more variables. Thus, soft sensors facilitate the understanding intensive and expensive re-approval of the process. In the future,
of complex dynamics within bioprocesses and the variability new bio-production processes will allow the use of modern sen-
between single batch processes. sor systems, which provide more relevant process information
about the three phases of a bioprocess.
Off-gas analytics is very common in bacterial and yeast culti-
3.6 Future aspects of bioprocess monitoring vations. The volume fraction of oxygen and carbon dioxide are
determined, and from those the OUR, CER, and RQ values, but
Disposable reactor systems are more and more important in other gases such as methane and hydrogen can also be mon-
biotechnological applications. These systems are presterilized, so itored. While OUR measurement is also performed in animal
no laborious and time-intensive sterilization and cleaning pro- cell cultivations, the CER value cannot be easily determined in
cedures are needed. Production capacities can easily be adapted carbonate-buffered systems.

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