Key Metabolites 13241 Introduction

D-Glucose, L-lactate, ammonia and L-glutamine, as well as several other L-amino acids, have long been recognized as being key metabolites in animal cell cultivation and have thus been routinely monitored, first off line, using enzymatic and HPLC methods, and now increasingly using on-line techniques. There are of course numerous other chemical species that play a role in the metabolism of animal cells, such as vitamins, lipids, peptides, various growth factors, etc. These species are rarely, if ever, monitored in large-scale processes, due to technical difficulties and a poor understanding of their actual role. Similarly, on-line analysis of off-gases (O2, CO2) is rarely performed, unlike in microbial fermentation (see however some examples in Behrendt et al. 1994 and Eyer et al. 1995). Therefore the discussion below focuses on the on-line monitoring of the aforementioned key metabolites; the main techniques are reviewed below and summarized in Table 13.2 with some of the key references from literature.

13.2.4.2 Biosensors and flow-injection analysis

The requirement for on-line measurement of metabolites and complex biological species has driven the rapid development, over the past decades, of biosensors. A biosensor is a special class of sensor (see Section 13.2.1.1), which combines a biological sensing element for the analyte of interest with a transducer, to produce a correlated and measurable signal (electrical, optical, etc.). Biosensors are often miniaturized and are typically characterized by a high selectivity and a fast, reversible response, which makes them particularly suitable for high-throughput and automated measurements. A detailed review of the different types and principles can be found elsewhere (Glazier & McCurley 1995; Mulchandani & Bassi 1995; Schugerl 2001).

For the measurement of cell culture metabolites, the most widely used class of biological sensing elements are enzymes, such as oxidoreductases, transferases, hydrolases and lyases, because of their high specificity and their catalytic (amplification) properties (with the exception of ammonia, which is more often detected with non-biological sensors; see Table 13.2). To ensure operational stability and a useful lifespan whilst in use, enzymes are generally immobilized, either by physical entrapment in beads or polymeric films, or by chemical binding to membranes, beads or an electrode. The most common transducers of enzyme-based biosensors are either electrodes (poten-tiometric and amperometric) or optical probes (often called optrodes). Numerous combinations of enzymes and transducer are possible, the choice depending, among other things, on the composition (interfering factors) of the sample to be analysed. This is illustrated below with immobilized oxidases, which are the most widely used enzymes in biosensors, thanks to their availability. For the detection of an analyte, the general reaction, when oxygen is the acceptor, is as follows:

The concentration of analyte (XH2) can then be determined in different ways:

(i) Measurement of the hydrogen peroxide produced, by:

• amperometric method, e.g. Pt anode at 0.5 - 0.8 V;

• potentiometric method, e.g. via formation of fluoride from 4-fluoroaniline and detection by a field-effect transistor;

• optical probe, via the chemiluminescent reaction of H2O2 with luminol, catalysed by per-oxidase.

(ii) Measurement of the oxygen consumed, by:

• amperometric method;

• potentiometric method;

The advantages and disadvantages of the various determination methods are discussed elsewhere (Luong et al. 1993; Mulchandani & Bassi 1995).

Although it is desirable, for on-line monitoring, to use biosensors as in situ probes, applications are very limited due to several aforementioned technical difficulties (see Section 13.2.1.1). These difficulties are particularly severe in the case of biosensors, since the biological element is not very

Table 13.2 List of the main techniques for on-line monitoring of key metabolites.

Detection

Assay cycle

References

Metabolite Main instrument

Measurement principle

limitJ 1

timeJI

Technique/Main Device

Detection principle

(mM)

(min)

Glucose FIA

Immobilized glucose oxidase11

Amperometric detection of 11 O or of a mediator

0.001-0.1

2-20

Male et al. 1997; Meyerhoff et al. 1993; Ozturk

et al. 1997b; Renneberg er al. 1991; White et

al. 1995

Chemiluminescence detection of 11 O

0.001

2-10

Blankenstein et al. 1994

Fluorescence detection of O ,

0.05

2-10

Dremel et al. 1992

Immobilized glucose dehydrogenase

Fluorescence detection of NADH

0.1

1

Spohn et al. 1994

HPLC

Anion-exchange column

Integrated pulsed amperometric (IPAD) or

1

30-60

Favre et al. 1990; Kurokawa et al. 1994; Larson

refractive index detection

era/. 200241

In-situ probe

Immobilized glucose oxidase11

Amperometric measurement via mediators

3

1-3

Bilitewski et al. 1993

Near-infrared spectroscopy

0.1-1

1-3

Arnold et al. 2003; Chung et al. 1996; Rhiel et al.

2004; Riley et al. 2001; Yano and Harata 1994

Mid-infrared spectroscopy

0.5

1

Rhiel era/. 2002b

Lactate FIA

Immobilized lactate oxidase11

Amperometric detection of H^O^ or of a mediator

0.025

2-10

Ozturk et al. 1997b; Renneberg et al. 1991

Chemiluminescence detection of 11 O

0.001

2-10

Blankenstein et al. 1994

Fluorescence detection of O ,

0.1

2-10

Dremel et al. 1992

Immobilized lactate dehydrogenase

Fluorescence detection of NADH

0.1

3

Spohn et al. 1994

HPLC

Anion-exchange column

Refractive index detection

1

30

Favre et al. 1990

In-situ probe

Near-infrared spectroscopy

0.2-2

3

Arnold et al. 2003; Chung et al. 1996; Rhiel et al.

2004; Riley et al. 2001; Yano and Harata 1994

Mid-infrared spectroscopy

0.5

1-3

Rhiel era/. 2002b

Table 13.2 Continued

Glutamine

FIA

Immobilized glutaminase + glutamate

Amperometric detection of H ,0, or of a mediator

0.03-0.1

2-10

Renneberg et al. 1991; White et al. 1995

oxidase51

Chemiluminescence detection of ELO^

0.001-0.01

2-10

Blankenstein et al. 1994; Cattaneo and Luong

1993

Immobilized glutaminase + glutamate

Fluorescence detection of NADH

0.1

1

Spohn et al. 1994

dehydrogenase

Immobilized glutaminase + gas

Spectrophotometric detection of the pH change of

0.05

2-10

Stoll et al. 1996a

permeable membrane

an indicator

Potentiometrie detection of NH4 (ion-selective

0.1

2-10

Campmajo et al. 1994; Meyerhoff et al. 1993

electrode)

HPLC

Anion-exchange column

Integrated pulsed amperometric (IPAD) or

0.01-1

30-60

Favre et al. 1990; Kurokawa et al. 1994; Larson

refractive index detection

et al. 200241

In-situ probe

Near-infrared spectroscopy

0.05-0.5

1-3

Arnold et al. 2003; Chung et al. 1996; Rhiel et al.

2004; Riley et al. 2001; Yano and Harata 1994

Glutamate

FIA5'

HPLC

Anion-exchange column; integrated

0.01

30-60

Larson era/. 200241

pulsed amperometric detection

(IPAD)

In-situ probe

Near-infrared spectroscopy

0.6

1-3

Chung et al. 1996

Ammonia

FIA">

Gas-permeable membrane

Chemical reaction with ammonia + fluorescence

2-10

Spohn et al. 1994

detection of resulting product

Immobilized glutamate dehydrogenase

Chemiluminescence detection of ELO^

0.001

5-10

Blankenstein et al. 1994

+ glutamate oxidase

Potentiometrie detection of NH4 with

0.01

na

Saez de Viteri and Diamond 1994

ion-selective electrode array

In-situ probe

Near-infrared spectroscopy

0.04-1

1-3

Arnold et al. 2003; Chung et al. 1996; Riley et al.

2001; Yano and Harata 1994

2001; Yano and Harata 1994

1) The values represent only an order of magnitude, since different methods have been used to determine the detection limit in the cited references. The maximum measurable concentration is not given since it is not considered a critical parameter for these techniques.

2) Cycle time for the injection and analysis of one sample or standard (FIA, HPLC); the frequency during on-line monitoring of a culture was often lower, due to the injection of replicates and of standards for recalib ration. For an in-situ probe, the cycle time corresponds to the time for the collection of spectra.

3) See text for additional references on all the detection methods with oxidase-based sensors.

4) Simultaneous detection of glucose and 17 amino acids.

5) See the FIA methods for glut amine which are based on glutamate-oxidase or dehydrogenase.

6) See also the FIA methods for glut amine which are based on glutaminase and detection of the ammonia produced.

stable, and thus is difficult to sterilize and to use without frequent recalibration. An additional barrier is that the optimal physico-chemical environment for biosensors (pH, temperature, ionic strength, etc.) is often different from that of the culture medium, which, additionally, may contain interfering species. In the case of multistep enzymatic reactions, these conditions must actually be modified at each step. Even a relatively simple sensor such as the ammonia-selective electrode (Table 13.2) cannot be used accurately in situ, due to strong interference from other ions. As a consequence, in situ applications of biosensors are very limited. One successful example with glucose has been reported for bioprocess monitoring (Bilitewski et al. 1993).

The second, more practical use of biosensors for on-line monitoring is ex situ, in a flow-injection analysis (FIA) system. The FIA technique was first described by Ruzicka and Hansen (1975); principles and applications to bioprocess monitoring are summarized below and reviewed in detail elsewhere (Schugerl 2001; Schugerl et al. 1996; van der Pol et al. 1996). In brief, a known volume of sample (or standard) is injected into a constant flow of a buffer, then mixed with streams of different reagents or subjected to various physico-chemical treatments, and finally passed through a (bio)sensor. The sensor continuously records the change in absorbance, electrical current, potential or any other physical characteristics of the liquid stream caused by the injection of the sample. This measurement can be correlated with the amount of the species to be analysed in the sample. An essential component of FIA is a sterilizable sampling device that allows the injection of a cellfree sample from the bioreactor (see Section 13.2.1.1). One of the main characteristics of FIA - at least in its original form - is that no complete reaction, equilibrium or even homogeneous mixing is required in the system; this is acceptable provided that the process is well automated and that all operating parameters such as volume, flowrate and mixing time are highly reproducible. Thus, the main advantage of FIA is that relatively fast measurements (typically within a couple of minutes) can be performed despite the complexity of the detection process. Reactions that would not go to completion or not rapidly reach an equilibrium can be employed. Therefore FIA measurements, although not continuous, can be generated at a high frequency and are suitable for on-line monitoring of animal cell cultures. Other advantages offered by FIA for bioprocess monitoring are:

• FIA can incorporate an extremely large variety of chemical and biological reactions, together with physical methods, for which many different types of detection system can be used; thus, a very broad range of chemical species can potentially be measured.

• Small sample volumes are required.

• Dilution by a large factor (e.g. 1000) can be achieved very rapidly (e.g. within 1 min) with high precision (<1 %) (Sonnleitner 1997; van der Pol et al. 1996).

• One instrument can contain an array of biosensors and can thus be used for the 'simultaneous' (actually often sequential) determination of several species with one single injection.

• Frequent automatic recalibrations can be performed easily.

Despite the separation of the sensing and detection elements from the bioreactor content, FIA-based monitoring is often affected by problems of interfering species. Various strategies have been developed to circumvent these effects (Luong et al. 1993; Mulchandani & Bassi 1995). In brief, these strategies include:

• The use of an electron acceptor other than oxygen in oxidase-based sensors.

• The removal or degradation of interfering species by use of an ion-exchange column or enzymatic microreactor upstream of the sensor.

• A differential measurement, with two detectors, and a subtraction of the signal due to the interfering species only.

• The protection of the sensor with a selective membrane.

• The careful control of the physico-chemical environment (e.g. pH, pO2).

D-Glucose, L-lactate, L-glutamine, L-glutamate and ammonia have been measured successfully in different FIA systems over complete culture runs (Table 13.2). Reported detection levels for all these metabolites were in the range 0.001-0.1 mM. This broad range reflects the diversity of techniques and instruments, the different methods applied for determining the detection level, and the different degrees of assay optimization. The maximum measurable concentration is usually not critical, since the injection volume can be easily adapted or a highly reproducible dilution of the sample can be performed prior to detection. Due to the interdependence of glutamine, glutamate and ammonia via the glutaminase- and glutamate oxidase-catalysed reactions, detection methods for glutamine require the elimination - physically, chemically or by calculation - of the contribution of glutamate and ammonia; when properly performed, this operation actually allows the simultaneous quantification of all three species. Blankenstein et al. (1994) for instance, reported the simultaneous monitoring of all five aforementioned metabolites in a fluidized-bed reactor over 15 days using a multichannel FIA instrument (Figure 13.4). Although the time for injection and analysis of one species was about 2 minutes, the complete analysis time for all species was 42 minutes, due to injections in triplicate and washing steps before and after each species. Other amino acids can actually be detected using the same principle, for instance with immobilized oxidases. However, as each species requires its own biosensor, the FIA technique tends to becomes very slow and complex for the determination of a large number of species. Commercial instruments are now available with sensors based on immobilized enzymes, to detect the main metabolites in cell cultures (for instance the YSI analyser, YSI, Inc.). These instruments are actually a variant of FIA systems, since measurement is typically performed under equilibrium instead of dynamic conditions. Recently, genetically engineered binding proteins containing a fluorophore have been successfully tested in biosensors to measure D-glucose and L-glutamine off line (Ge et al. 2003). The high sensitivity (|iM range) and the very small sample volume requirement (<1 |il) make these biosensors attractive alternatives to enzyme-based ones for bioprocess monitoring. The use of biosensors with FIA at pilot- and large-scale is, however, still limited; the modest benefits of on-line monitoring over well-established off-line techniques do not compensate for the additional complexity of the fluid handling system and the lack of robustness of many biosensors.

13.2.4.3 Infrared spectroscopy

Near-infrared (NIR) spectroscopy is another promising technique for detecting metabolites. A sample is exposed to a beam of NIR light through a quartz window and the amount of light absorbed at various frequencies can be correlated to the concentration of some species in the sample. The method offers several advantages. It is non-invasive and can be performed with an in situ fibre-optic probe (with all the advantages mentioned in Section 13.2.1.1, particularly the fast response time). The signal is also very stable, and little maintenance is required during operation. Finally, several species can be determined simultaneously with only one sensor (as opposed to one for each species in FIA techniques). Riley et al. (2001), for instance, were able to monitor 19 components in serum-containing cell culture media, among which 15 were amino acids, including glutamine. The main difficulty of this technique is that the NIR signal may be affected by several additional (unknown) species in the media; extensive chemometric algorithms, such as partial-least squares regressions, must therefore be applied first, with a set of off-line samples from several previous cultures, to build calibration models (Riley et al. 1998; Rhiel et al. 2004). Consequently, this technique is particularly suitable for the routine monitoring of cultures that are always operated under similar conditions. However, when new conditions are tested, changes in the cell metabolism may not be detected properly.

Substrate glucose/glutamine

Product

Substrate glucose/glutamine

Product

Figure 13.4 Example of a multichannel FIA instrument for on-line monitoring of glucose, lactate, glutamine, glutamate and ammonia in a fluidized-bed bioreactor. (a) Schematic diagram of the FIA instrument. A microfiltered sample from the bioreactor or a standard (selection via a six-way valve (6WV-1)) is transported to a dialysis unit, which is used to remove high molecular weight compounds (proteins) and to dilute the sample further in the stream of an 'acceptor' (buffer solution). A second six-way valve (6WV-2) directs the sample to one of five microreactors with immobilized enzymes. All of them lead to the formation of H2O2, which is measured downstream via a chemiluminescent reaction, after addition of a luminol solution and passage through a sensor with immobilized peroxidase (POD). All operations of the FIA are performed automatically. A complete analytical run, with injections in triplicate and washing steps, takes 42 min. IV = injection valve, CL = chemiluminescence (b) Comparison of glucose measurement on line, with the FIA instrument (—), and off-line, with a commercial glucose analyzer (■), during a continuous culture of immobilized hybridoma cells. X-axis: process time (h); Y-axis: glucose concentration (g/l) (Reproduced with permission from Blankenstein et al. (1994).)

Figure 13.4 Example of a multichannel FIA instrument for on-line monitoring of glucose, lactate, glutamine, glutamate and ammonia in a fluidized-bed bioreactor. (a) Schematic diagram of the FIA instrument. A microfiltered sample from the bioreactor or a standard (selection via a six-way valve (6WV-1)) is transported to a dialysis unit, which is used to remove high molecular weight compounds (proteins) and to dilute the sample further in the stream of an 'acceptor' (buffer solution). A second six-way valve (6WV-2) directs the sample to one of five microreactors with immobilized enzymes. All of them lead to the formation of H2O2, which is measured downstream via a chemiluminescent reaction, after addition of a luminol solution and passage through a sensor with immobilized peroxidase (POD). All operations of the FIA are performed automatically. A complete analytical run, with injections in triplicate and washing steps, takes 42 min. IV = injection valve, CL = chemiluminescence (b) Comparison of glucose measurement on line, with the FIA instrument (—), and off-line, with a commercial glucose analyzer (■), during a continuous culture of immobilized hybridoma cells. X-axis: process time (h); Y-axis: glucose concentration (g/l) (Reproduced with permission from Blankenstein et al. (1994).)

Mid-infrared spectroscopy (MIR) has recently been developed as a promising variant to NIR for animal cell cultures, since it has a higher sensitivity and selectivity for the metabolites of interest in the so-called 'fingerprint' spectral region (1800 - 800 cm-1). A difficulty, however, is the strong water absorbance in this region. So far, only a few applications for the monitoring of animal cell cultures have been reported (for instance, Rhiel et al. 2002b). However, the development of robust calibration models (Rhiel et al. 2002a), combined with long-term stability (Rhiel et al. 2002b) reported 2.3 years of operation without maintenance, except for daily replenishment of liquid nitrogen for detector cooling) make this technique an attractive basis for future applications.

Compared with FIA, NIR and MIR measurements are less accurate, with a typical detection level of 0.1 - 1 mM for the cell culture key metabolites. However, these techniques are currently undergoing very rapid development such that significant improvements can be expected in the near future. The maximum measurable concentrations are not critical parameters since they are significantly higher than the highest levels found in cell cultures.

13.2.4.4 HPLC and miscellaneous techniques

HPLC, although relatively expensive, is an attractive alternative to biosensor-based techniques, in particular when several species have to be analysed simultaneously. With the proper sampling device, an HPLC instrument can be readily used for on-line monitoring of a bioreactor. Several applications have been reported for glucose, lactate and most of the amino acids present in animal cell cultures, with a complete analysis time in the range of 30-60 min (for instance Larson et al. 2002).

Several other (complex) instruments or techniques, typically developed for off-line analysis, can in principle be used to monitor a culture on line or in real time. NMR, for instance can detect a broad range of metabolites in cell cultures including intracellular species, and can be used for metabolic flux analyses (Forbes et al. 2000). Very sensitive calorimeters have also been developed, to monitor continuously the heat signal generated by cultivated cells, which is a measurement of their catabolic activity (Kemp & Guan 1997). The complexity and cost of these tools, however, have limited their use to research and development applications.

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