FAQ

Online measurements provide readings in a short time and allow for more accurate monitoring and, if necessary, control of the process. Frequent manual sampling can be omitted. This reduces the risk of contamination and minimizes the time and effort involved. The resulting concentration / time profile of the cultivation additionally represents a good documentation of the overall process.

TRACE C2 allows the simultaneous determination of glucose and lactate with the aid of a selective biosensor.

The measuring range for glucose is 0.1 - 40 g/L. Lactate can be measured between 0.05 and 5 g/L.

Yes, Ethanol and Methanol. In the future, however, more applications will be available.

The TRACE C2 is connected to the reactor via sampling probes. These are available in various diameters and lengths and can be connected to a 25 mm side port or 12/19 mm top plate port as needed. For disposable bioreactors, sampling via probe port is available.

You can basically choose between two types of sampling: filtration and dialysis. In  filtration mode cell-free medium is removed from the reactor via a membrane probe and measured using a biosensor. Alternatively, a dialysate can be removed from the bioreactor.

Using the dialysis probe, representative samples can be obtained from a bioreactor without having to remove any liquid. This method is therefore very well suited for small volume reactors. With the help of a semipermeable membrane only the low molecular weight substances are separated by diffusion from the medium.

Filtration allows continuous recovery of a cell-free sample from the bioreactor. The microporous filtration membrane consists of a robust and pressure-resistant plastic body on the filtration probe. This also makes possible the use in very large reactors of several m³ content.

With the single use probe, dialysis sampling is available, which is integrated into a reactor port. This disposable sampling is supplied as a sterile kit or can be delivered as part of custom disposable reactors.

• No, not in dialysis sampling.
• Only when using the sampling method filtration medium is consumed. With continuous measurement, approx. 1 mL of medium per minute or 1.5 L per day is required.

A tubing set containig the whole liquid path, all pumps and valves as well as the sensor. This is combined with calibration solutions and transport buffer solution.

The PID is programmed for only one leading parameter. This can be glucose or lactate or ethanol or methanol, depending on the analytical method choosen.

One device at the same time. We have an internal built-in pump for feeding or alternatively external pumps (like Watson Marlow) can be connected via analog or digital connection.

No.

Basically all microorganisms and cell lines can be measured with both types of probes. But especially filamentous fungi often form a highly viscous mass during fermentation. In these cases, the dialysis probe is to be preferred, as it is better suited for highly viscous media.

The tube set is a disposable item. The tube set should therefore be replaced after each cultivation or fermentation.
If the operator decides to run the tube set several times, we recommend to use the cleaning solution.

• The supplied transport buffer concentrate (0.5L) gives 10 L transport buffer. It is sufficient for continuous measuring operation for approx. 8 days.
• The table shows the buffer consumption depending on the interval:

All solutions are stable for 15 months. Diluted solutions are stable for a maximum of 4 weeks. If necessary, sterile filtered and well sealed, the solutions can be used longer.

The spread of the glucose reading around the mean is less than 1,5% at 4 g/L.
The spread of the lactate reading around the mean is less than 1,5% at a value of 2g/L.

The TRACE C2 is primarily an online meter for continuous measurements. However, individual samples (for example for reference measurements) can also be measured offline.

Measurements of TRACE C2 and MultiTRACE are based on a biosensor, which is integrated in a preconfigured tubing set. There are no reagents needed, except a transport buffer solution for the transport of the analyte to the biosensor. The buffer is supplied as a concentrate (20x) which gives 10L transport solution. This lasts in continuous operation for 10 days (dialysis mode) or 11 days (filtration mode). The system automatically calibrates itself in customer defined intervalls. We use two calibration standards. Each bottle (500mL) lasts for approx. 120 calibrations. 

The temporal resolution of the readout is depending on the sampling method. If you use filtration sampling the resolution is one per minute. With dialysis as sampling method the resolution is two minutes. Typically, customers set the system to one calibration per day which is enough to compensate for drifting effects or sensor deterioration. However, the calibration frequency can be increased if required. There is no extra workload since the calibration is fully automated.

You are right, there is a time lag between sampling and display of result, which is the sum of the following processes:

a) sample transport (only in filtration method): time during which the medium travels from the reactor to the diffusion module in the tubing set;
b) accumulation time: time during which the analyte passes the diffusion membrane and accumulates in the holding chamber;
c) transport of the accumulate to the measurement cell (biosensor) and
d) passage of the measurement cell and peak detection.

For the filtration method we have a) = 60s; b) = 30s; c) & d) = 30s adds up to 120s and for dialysis method we have b)=45s and c)&d)=75s adds up to 120 s. As a consequence in filtration mode the signal represents the situation in the reactor 2 minutes ago with an integral of 30 s and  the dialysis method represents the situation in the reactor 2 minutes ago with an integral of 45 s.

• TRACE C2 offers automated simultaneous glucose/lactate monitoring at frequent intervals (every 1 or 2 minutes). Other analytes available are ethanol or methanol.
• The measurement principle is based on enzymatic biosensors, thus a true and selective molecular measurement is the basis for every analysis.
• The analyte is sampled by a probe within the bioreactor and automatically transported to the biosensor. The sensor is located outside the reactor integrated in a tubeset.
• The sampling technologies that TRACE offers are filtration and dialysis. The respective stainless steel probes are similar to conventional probes for pH or pO₂ measurement. Additionally, a dialysis probe for single-use reactors is available.
• The device is also available as MultiTRACE or C2/M4 for parallel monitoring of up to four bioreactors.

• Manual sampling and sample drawing always pose greater risk than sampling via sterile membrane probes.
• TRACE sampling probes for filtration and dialysis have been proven as stable and reliable sampling devices in GMP as well as large scale processes. Due to their aseptic stainless steel design, the probes can be sterilized like conventional probes. Both probes are pressure resistant up to 6 bar (87 psi).
• A great advantage of the TRACE dialysis sampling technology is that it does not consume any media of the process, making closed loop control available for very small bioreactors.

TRACE C2 is a small-footprint device that uses preconfigured single-use tubing sets. This renders cleaning unnecessary. The sets come with respective calibration and flushing media, which, when connected to the device, allow for automated calibration and recalibration at customer specified times or intervals.

• TRACE C2 allows for direct feedback control due to an integrated PID controller. With an integrated pump the instrument can directly feed small and medium sized reactors. For larger reactors or high glucose consumption rates larger external pumps (e.g. Watson Marlow 5xx series) can be directly connected via the digital or analog port.
• Alternatively, if a SCADA system is available for bioreactor control, the measurement results can be instantly transferred via analog (0-10V / 4-20 mA) or via digital (Modbus, OPC) signals.

TRACE technology and support is readily available worldwide through a network of established partners like Sartorius, Eppendorf/Dasgip, BlueSens and PROAnalytics.

TRACE technology is in continuous use since 1997 in GMP production of large scale processes at insulin production at Sanofi (former Aventis/Hoechst). Several other customers use the technology in their GMP facilities, however, since this is mostly confidential, there are only few publications available. Contact to reference customers will be given by TRACE on demand.

Yes, you can use the MultiTRACE or TRACE M4 as a peripheral to the C2.

When using the dialysis probe, the transport solution as such cannot penetrate into the fermentation medium. Since the dialysis membrane is permeable to very small molecules, individual ions from the solution can penetrate into the fermentation medium. The transport solution contains only physiological salts. On request, we will provide our customers with a material certificate of the solution.

If you want to perform a control based on the measured values, you should adapt the measuring frequency to the dynamics of your process. With the filtration, 60 measurements per hour and dialysis 30 measurements per hour are possible, which can also track fast microbial processes very well. For slow growth processes (e.g., cell cultures), perhaps 2 measurements per hour may be sufficient for control.

TRACE C2 can be set to any time interval so that even large measurement intervals are possible. Here, too, the automatic measurement is worthwhile, especially at night and at weekends, when there is little personnel available for manual sampling.

The measured values are displayed graphically using the supplied PC software and saved in text form. Parallel to this, the current measured values are available as analog signals (0..10 V, 0..20 mA and 4..20 mA).

The sensor is integrated into the disposable tubing set and will be replaced with it. The lifetime of the sensor is at least 5000 measurements. In continuous operation, at least 14 days can be covered.

The device calibrates itself at freely selectable intervals. The calibration standards provided are automatically measured. It is also possible to trigger the calibration manually if necessary, or to do without it altogether.

As a rule, one calibration per day is sufficient. We recommend a more frequent calibration in strongly changing environmental conditions (for example, temperature differences day / night). At large time intervals (e.g., measurement every 6 hours), calibration may also be performed prior to each measurement.

Select the concentrations of the two calibration standards so that the concentration range that is important for you (for example for controls) is between these concentrations. Higher concentrations are usually still determined sufficiently good, but with less accuracy.

It is possible in principle to start with high glucose concentrations and control them in the lower concentration range. In this case, it is recommended to calibrate with two low standards (e.g., 4 g / L and 0.5 g / L glucose). The course of the high glucose concentrations will then be recorded, but the measured values in this area will deviate from the actual values. In the control area, however, the measured values will then be recorded exactly.

The device can be connected to a process control system via the analogue outputs on the rear panel. The following outputs are available: 0..10V, 0..20mA and 4..20mA.

Basically, the TRACE C2 is also suitable for industrial scale. It will, however, be necessary to adapt the device to the particular environmental conditions (for example put it in a splash-proof cabinet).

• Steam sterilization is possible with both probe types (dialysis and filtration). However, it must be ensured that the respective membrane is wetted with liquid during sterilization.
• Dry sterilization is not possible due to the high temperature load with either of the two types of probes.

It is recommended to carry out steam sterilization at 121 ° C and 1 bar overpressure. If higher temperatures are reached by up to 10 ° C in the short term, this is no problem for the membranes.

For highly viscous media, the dialysis probe is better suited than the filtration probe. However, it must always be tested whether the dialysis probe is suitable for the viscous medium.

When storing after use, 3 factors are crucial:

1) Disinfection - this is easily done by the cleaning solution. But it is also important that the adapter lines (UNF LUER) are flushed! They will be connected to the tubeset again later and should not be contaminated or blocked. We use the syringe, first with cleaning solution, then water, then air.

2) Blockage due to crystal formation of buffer residues - ie: wash well and with plenty of dest / deion water (also runs automatically in cleaning mode)

3) Gluing the silicone or pump hoses - we recommend taking the hoses out of the valve blocks for longer periods of storage. A channel is always closed and the corresponding silicone tubes are glued dry in some cases and have not opened again by itself when re-priming. We did not make any such observations with the pumps, but for safety's sake we could take the cassettes off the axles.

If the set is so disinfected and rinsed it can simply be left standing. It then dries up slowly and must of course be rinsed again during startup. Maybe after a long break it will take longer to get fit. For a short storage (1 week) that works well several times.

We would advise against cooling the set. This could rather lead to problems with e.g. condensation and possibly then growth. Dry and room temperature is better.

In dialysis mode, probe tubes with lengths of up to 8 m are possible. The measuring time is extended from 2 to 4 minutes. For conversion, however, the device must be sent.

The higher temperature in the medium is no problem. The signals will be higher and thus the linear range might be lower.

• Yes, there are other success criteria. If the deviation of three calibration measurements is too high, the system will show you “calibration not plausible”.
• This deviation can be caused by an old tubing set or if you have a leakage in the tubing set. You have to check your tubing set and the placement of the silicon tubes inside the valves.

The higher the lactate concentration is, the higher the signals of the sensor are and the better the referencing will be. For low concentrations I would advise to start referencing only above conc. of 0.2 g/L lactate.

• We tested the dialysis mode at 3.5 bar (50.76 psi) pressure in the reactor for test duration of 6 days. The membrane showed no rupture or leakage during that time period (not tested longer).
• As expected, there is an increase in signal with increase in pressure (approx. 10% per bar). This would be compensated by the reference factor.
• We tried to perform a burst test, but the membrane did not burst even up to 6 bar (87psi), no matter whether a tubing set is connected to the probe or the probe is left open on the connectors.

1 equates to 100%

The table shows the buffer consumption depending on the interval: