When it comes to bioreactor contamination, there are exactly two failure modes:
For validated GMP systems, the sterilization procedure is usually automated with several critical manual steps. The faithful execution of the sterilization procedure will rarely result in the "Failure To Kill."
Some microbial contaminants are harder to kill than others. Gram-positive bacteria, which contains a hardy outer shell - "peptidoglycan" - can withstand higher temperatures for longer than gram-negative bacterial.
Also, gram-positive bacteria have the ability to form-spores where they reduce themselves into viable, but dormant form. The spores lie dormant until more favorable environmental conditions emerge.
Most sterilizing procedures are validated to inactivate spores, but if your bioreactor contaminations are predominantly gram-positive, hard-to-kill, spore-formers only, then look for "Failure To Kill" as one of the components of the bioreactor contamination.
During contamination responses, people often visually inspect elastomers and valve diaphragms for nicks and cuts as there is wear and tear on this material over the course of use.
Filters are often suspected and post-use integrity tests are done to ensure that the filter integrity was not breached.
When more media or feed is needed, the sterile boundary may be extended. This is when a second envelope is created near the bioreactor, sterilized and then opened next to the bioreactor.
Maintaining the integrity of the sterile boundary often requires positive pressure. The failure to maintain an outwardly flow can often result in the deficiency of maintaining integrity.
In fact, when steam is rapidly cooled, the pressure will drop suddenly often creating a vacuum that can suck "bugs" into the envelope.
Consult A Bioreactor Sterility Expert
- Failure to Kill (the bugs)
- Failure to Keep (the bugs) Out
The way it works is this:
For the volume of space (say a bioreactor) you want sterile, you start with a clean tank free of residue. You do this by executing a Clean-In-Place (CIP).
Once your reactor is clean, you seal off orifices of your envelope. Once this envelope is sealed, you sterilize it with steam (SIP), creating a sterile envelope. Once the interior of this envelope bug-free, you try to keep it that way.
For the volume of space (say a bioreactor) you want sterile, you start with a clean tank free of residue. You do this by executing a Clean-In-Place (CIP).
Once your reactor is clean, you seal off orifices of your envelope. Once this envelope is sealed, you sterilize it with steam (SIP), creating a sterile envelope. Once the interior of this envelope bug-free, you try to keep it that way.
Failure To Kill
"Failure to Kill" is a deficiency in creating a sterile boundary.... the failure to reach sterilizing temperatures, thereby leaving viable microbes inside of the envelope. Steam-In-Place procedures can fail for a variety of reasons, including:- Mis-calibrated temperature probes
- Failure to maintain sterilizing temperature
- Failure to reach sterilizing temperature
- Dirty Surface
- Inaccessible Process Surface
For validated GMP systems, the sterilization procedure is usually automated with several critical manual steps. The faithful execution of the sterilization procedure will rarely result in the "Failure To Kill."
Some microbial contaminants are harder to kill than others. Gram-positive bacteria, which contains a hardy outer shell - "peptidoglycan" - can withstand higher temperatures for longer than gram-negative bacterial.
Also, gram-positive bacteria have the ability to form-spores where they reduce themselves into viable, but dormant form. The spores lie dormant until more favorable environmental conditions emerge.
Most sterilizing procedures are validated to inactivate spores, but if your bioreactor contaminations are predominantly gram-positive, hard-to-kill, spore-formers only, then look for "Failure To Kill" as one of the components of the bioreactor contamination.
Failure To Keep Out
"Failure to Keep Out" is a deficiency in maintaining the integrity of the sterile boundary. The non-moving-parts of a sterile boundary includes the physical vessel, elastomers, valve diaphragms, and sterile filters.During contamination responses, people often visually inspect elastomers and valve diaphragms for nicks and cuts as there is wear and tear on this material over the course of use.
Filters are often suspected and post-use integrity tests are done to ensure that the filter integrity was not breached.
When more media or feed is needed, the sterile boundary may be extended. This is when a second envelope is created near the bioreactor, sterilized and then opened next to the bioreactor.
Maintaining the integrity of the sterile boundary often requires positive pressure. The failure to maintain an outwardly flow can often result in the deficiency of maintaining integrity.
In fact, when steam is rapidly cooled, the pressure will drop suddenly often creating a vacuum that can suck "bugs" into the envelope.
Summary
There are exactly two failure modes that cause bioreactor contamination. While failures can be a combination of both, it is important for the bioreactor contamination response to simplify and clarify these two modes so that the true root cause is easily enunciated and therefore found.Consult A Bioreactor Sterility Expert
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what are the signs of contamination of bioreactor
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