When we are confronted with a contamination problem, as in a unsuitable case of a MFT, it is valuable to make speak the microbial agent which is involved. This one is indeed able to reveal us the moment when it appeared in the system, thanks to the bacterial kinetics rules. This information will make it possible to determine, even to confirm the event which allowed its introduction into the system. The example which follows in is a realistic illustration.
MFT protocol (summarized): use of a storage tank containing 25 L of trypcase soya broth, equipped with a recirculation system (the simulated product is a suspension) and with a filling machine on line (filled volume : 1 ml per unit). Medium filling over 3 days taking into account the stopping. Regrouping of units during the same sequence of filling. Incubation by sequence of filling.
MFT situation: After incubation, a huge number of contaminated units is noted (see table hereafter)
|
Filling Day
|
Filled units showing microbial growth
|
|
1
|
0 unit
|
|
2
|
337 units
|
|
3
|
100 % of filled units
|
Data analysis without microbial kinetic data:
At this investigation stage, the best assumption is an contaminating episode occurred on the storage tank,before restarting operations of day 2.
The recirculation product system could be one of the potential tracks because a leakage was observed.
The germ found inside this device is similar to the germ isolated in the filled units.
Data analysis with microbial kinetic data:
Thanks to the filling units traceability as required by the MFT protocol, it was possible to re-examine the data of D2 on a kinetic level. The appearance and propagation mode of the contaminating agent are then studied.
|
Time spent
after D2 (min)
|
Contaminated
units
during this period
|
Units filled
during
this period
|
% of
contaminated units
|
|
0
|
4
|
1226
|
0,33
|
|
180
|
14
|
589
|
2,38
|
|
240
|
20
|
611
|
3,27
|
|
280
|
39
|
611
|
6,38
|
|
340
|
114
|
1231
|
9,26
|
|
420
|
146
|
902
|
16,19
|
|
|
Total : 338 u
|
Total : 5170 u
|
|
With these data it is possible to plot a curve (hereafter) and to calculate the germ generation time (around 90 min).
At the D2 early beginning, only 4 units are contaminated out of approximately 1200 ml of filled medium; The bulk contamination not filled at the same time is consequently estimated at 50 CFU.
The stoppage between end of D1 and D2 is 16 hours long. No operation is carried out during this phase (the recirculation system is stopped). The first operation of D2 is to start up of the pump.
These 3 data (duration of the stoppage duration, generation time of the contaminating agent, number of bacterial cells present in the tank) confirm the assumption of a “massive” contamination into the tank at the time when the operator restart the recirculation system ; The investigation must focus effort on this equipment.
The operating process analysis, especially of the recirculation system, allows to split out the contaminating episode in several steps:
- 1st step (D1): storage tank is in overpressure, so a passage of a very low volume of media towards the interior of the recirculation system is possible; setting in contact media with the spores present inside the device.
- 2nd step (between D1 and D2, during 16hrs): Filling break and recirculation system stopped. Tank is still in overpressure; beginning of the spore germination and proliferation into the media contained in the recirculation system.
- 3rd step (beginning of D2): Restarting of the equipment involves a depression, generating a tank content inoculation by return of media involving approx. 50 CFU; dispersion of the germs is ensured by media recirculation.
- 4th step (throughout D2): microbial proliferation in the tank, number of contaminated units is proportional to the micro organism multiplication speed
- 5th step (break between D2 and D3): Tank contamination is going on
- 6th step (D3): the contamination in the tank is such as all the units filled will be contaminated
This study case shows that it is any beneficial to foresee prior to start a MFT, a precise traceability of the filled units according to time.
In the case of failed MFT , the investigation group will then have the opportunity of using bacterial kinetic data.
The team will may find a great support to include a microbiologist who will able to entrusting this data decoding. This time spent to give-extra paper , at the time of MFT performance, will offer quantitative information which will strongly consolidate assumptions developed during the investigation. In some situations, this kind of data will make it possible to exclude without ambiguity a process problem (e.g.: batch of non sterile tryptase soya powder used for MFT).
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