Claus Tollnick 1, Matthias Beuse 2
Glucose is by far the most important nutrient source for microorganisms in biotechnological processes. This carbon source is used in almost 95% of all cultures. The preparation of increasingly more complex products by in some cases genetically engineered organisms in highly sophisticated plants requires comprehensive monitoring of the experimental parameters. Both quality assurance and systematic cost accounting demand not only the analysis of physical parameters such as temperature, pH and pO2, but also the control and monitoring of chemical variables of the production process, for example glucose.
The following article demonstrates an industrial application of a glucose on-line system in industrial process development.
Problem definition
Instrumental glucose determination nowadays plays an important part in routine analysis. Many laboratory instruments, however, at present are suitable only for off-line use, i.e. the sample must be provided manually and the measurement be started manually. On the other hand, aspects of major importance in bioprocess engineering include not only rapid glucose detection but also sterile sampling. These problems in the form of "liquid handling" militate against the wider use of glucose measuring systems in biotechnology. The on-line ProcessTRACE system from Trace Biotech AG now allows reliable determination of glucose in bioreactors to be performed. It is employed industrially for on-line measurements of glucose at Hoechst Marion Roussel Deutschland GmbH.
Flexible glucose measurement
ProcessTRACE is primarily used for on-line monitoring of industrial or laboratory cultures which employ glucose as a carbon source. Irrespective of the type of culture (batch, fed-batch, continuous culture), continuous glucose analysis using ProcessTRACE can be carried out in virtually all cell and microorganism cultures.
The individual analysis is based on the flow diffusion analysis method which employs time-dependent sample enrichment (Figure 1). In this arrangement, the measuring section and the sample or sample inlet are separated from one another by a membrane. The measuring section is referred to as the acceptor and the sample side as the donor.
Figure 1: Principle of the flow diffusion analysis method
In an analysis, the acceptor stream is stopped for a defined time behind the membrane, this being effected by valves being switched over to open up a bypass section. The analytes in the sample (in this case glucose) meanwhile diffuse through the membrane and accumulate in the acceptor chamber. Thus, with time, the concentration of the dialysable sample constituents in the liquid segment of the acceptor will rise.
When this segment is released by the valves switching back, it flows to the detector and there, after enzymatic reaction of the glucose, generates a signal (amperometric measurement). The signal intensity is a yardstick for the concentration of the sample. The major advantages of this measuring method, compared with flow injection, are firstly the very wide measuring range, achieved simply by varying the enrichment time, and secondly the long service life of the sensor owing to the separation of sample and buffer.
Reliably sterile sampling
The analysis of glucose in culture media requires a sample to be drawn under sterile conditions from the bioreactor (fermenter), while ensuring reproduceability and relevance of the sample taken. Three sampling options are available in conjunction with ProcessTRACE: filtration probe, dialysis probe and bypass module. All the systems enable reliably sterile sampling directly from the bioreactor.
The filtration probe is ESIP probe 5441 from Eppendorf-Netheler-Hinz GmbH, distributed by Trace Biotech AG. The probe allows cell-free substrate samples to be drawn from bioreactors. It is secured in the interior of the bioreactor by means of a socket and is sterilized in situ together with the medium.
Alternatively, the sample can be delivered from the bioreactor to the analytical instrument via a dialysis probe, the glucose being transferred via a diffusion membrane into a buffer stream and being transported to the measuring cell, with zero-volume sampling. The probe is therefore particularly suitable for minifermenters in the field of research and process development. The functional principle of the probe is shown by Figure 2.
Figure 2: Functional principle of the dialysis probe, a volume free sampling method
The dialysis probe in its entirety is made of alloy steel V4A (DIN 4571) in accordance with DIN EN 1672-2 (hygienic requirements). It is thus suitable for any type of culture. Operation under sterile conditions is greatly promoted by the use of an extremely robust diffusion membrane and thermally stable sealing rings. The bioreactor can be fitted with the probe either via the reactor cover or via a socket at its side.
Field of application illustrated by an industrial process development example
A typical example of how ProcessTRACE can be used in practice can currently be found at Hoechst Marion Roussel Deutschland GmbH. There the glucose on-line system is employed in process development, with the aim of virtually comprehensive monitoring of the glucose concentration in a fed-batch reactor. The microbial break-down of the glucose is tracked over a range from 37 to 0 g/l. A feeding strategy is designed to control the concentration to a constant value. Delivery of the sample via the filtration probe ESIP allows ProcessTRACE to determine the glucose concentration at three minute intervals. Figure 3 shows how the glucose concentration of a fed-batch culture changes over time.
Figure 3: Control of the glucose concentration during a fed-batch cultivation: Comparison of the on-line ProcessTRACE data versus the off-line data (original data of HMR Deutschland GmbH)
Clearly visible is the batch phase from the initial concentration of 37 g/l down to 0 g/l. The refeed initiated before all of the glucose has been consumed causes the concentration to rise back slowly to 5 g/l. Manual correction of the feed gives rise to fluctuating concentrations in the reactor.
The advantage of an on-line glucose system becomes particularly clear if we look at the off-line data (red). The higher data density due to the on-line measurement precludes any misinterpretation of the progress of the culture. The off-line data on their own do not suffice for monitoring or controlling the process in dynamic domains.
Figure 4 shows a glucose feed with precise control of concentration between 0 and 0.5 g/l. After a brief transient phase, a relatively constant glucose concentration is observed over a period of several hours, while the feed rate increases slightly and
Figure 4: Glucose feeding during a fed-batch cultivation between 0 and 0.5 g/l using ProcessTRACE in combination with a process control system (original data of HMR Deutschland GmbH)
the dissolved-oxygen level shows a distinct drop from 42 to 6.6%. A sampling interval of three minutes was sufficient to ensure a constant glucose level for the microorganisms in the bioreactor.
1 Hoechst Marion Roussel Deutschland GmbH,
Industriepark Hoechst, D-65926 Frankfurt
2 TRACE Biotech AG,
Mittelweg 7, D-38106 Braunschweig
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