For a better overview, we have divided these into 5 groups:
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.
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.
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.
Dialysis mode | |||||
Interval | Buffer Consumption | 10 L for: | |||
min | µl/min | ml/h | ml/d | h | d |
1 | 835,6 | 50,1 | 1203,2 | 199 | 8,3 |
5 | 835,6 | 50,1 | 1203,2 | 199 | 8,3 |
10 | 614,4 | 36,9 | 884,7 | 271 | 11,3 |
20 | 356,3 | 21,4 | 513,1 | 468 | 19,5 |
30 | 270,3 | 16,2 | 389,3 | 617 | 25,7 |
60 | 184,3 | 11,1 | 265,4 | 904 | 37,7 |
120 | 141,3 | 8,5 | 203,5 | 1179 | 49,1 |
360 | 112,6 | 6,8 | 162,2 | 1480 | 61,7 |
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 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 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.
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.
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.
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.
The table shows the buffer consumption depending on the interval:
Dialysis mode | |||||
Intervall | Buffer Consumption | 10 L for: | |||
min | µl/min | ml/h | ml/d | h | d |
1 | 835,6 | 50,1 | 1203,2 | 199 | 8,3 |
5 | 835,6 | 50,1 | 1203,2 | 199 | 8,3 |
10 | 614,4 | 36,9 | 884,7 | 271 | 11,3 |
20 | 356,3 | 21,4 | 513,1 | 468 | 19,5 |
30 | 270,3 | 16,2 | 389,3 | 617 | 25,7 |
60 | 184,3 | 11,1 | 265,4 | 904 | 37,7 |
120 | 141,3 | 8,5 | 203,5 | 1179 | 49,1 |
360 | 112,6 | 6,8 | 162,2 | 1480 | 61,7 |
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