Online measurements of glucose, lactate, glutamine, and glutamate are possible

through miniaturized versions of the well established electrochemical enzyme-based

sensors [44]. In addition, it is common that stand-alone analysis systems are coupled

to sampling units [45]. The measurements are often based on the hydrogen peroxide

concomitantly produced upon oxidation of the metabolites catalyzed by membrane

immobilized specific oxidases. The oxidation of the hydrogen peroxide on a platinum

anode generates a nanoamperometric signal proportional to the metabolite present in

the sample. Potassium, sodium, and ammonium ions are measured by ion selective

sensors. Examples of current commercially available systems for animal cell culture

are the YSI 2940-2980 Multichannel Online Monitor, the Sartorius BioPAT^® Trace

(currently limited to glucose and lactate) or the Bioprofile Flex2 online autosampler

from Nova Biomedical. These systems are also capable to monitor multiple bior-

eactors. These measurements have enabled process control in high cell density cul-

tures to adjust perfusion rates to maintain constant glucose and lactate concentrations

[46]. In addition, online chromatography systems are available that are mainly used in

research laboratories. For example, using anion exchange chromatography and am-

perometric detection, glucose and 19 amino acids were measured in CHO cell cultures

[47]. In comparison to chromatography methods, enzymatic methods for the relevant

metabolites in cell culture require less time to generate specific results, do not require

exhaustive sample preparation and are less prone to interferences. Clearly, all the

methods addressed above require either manual sampling or automatic sampling

systems. These consist of pumps and a valve system that withdraws a cell-free sterile

sample (through sampling probes suited either with filter or dialysis elements) from

the bioreactor and dilutes it before injection either into a sampling port or onto a

column. In addition, there is a growing number of non-invasive technologies that are

based on spectroscopic methods (near-infrared spectroscopy [NIR], mid-infrared

spectroscopy [MIR], fluorescence), but still need considerable effort to fully exploit

their potential, especially regarding data mining and data analysis [48,49].

Nevertheless, these techniques seem to constitute the future in bioprocess monitoring

gln

Time (h)

lac

amn

glc

I

II

III

(mM/mM)

Metabolite conc. (mM)

FIGURE 5.3 Typical time course for metabolites of animal cells in batch culture (left) and

typical stoichiometric ratios for different metabolic states (right) (adapted from [ 39]). When

the availability of glucose or glutamine is limited, some cell lines shift their metabolism

towards lactate consumption [ 42]. In state I, a high uptake rate of glucose and glutamine is

necessary for rapid cells proliferation. In states II and III, along with lactate, glucose, and

glutamine are consumed, but at lower uptake rates than in state I, mainly for cell maintenance

[ 43]. Glc: glucose; gln: glutamine; amn: ammonia; lac: lactate.

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