4. Create as leak-free a system as possible. All systems should undergo a leak test prior to and after sterilization. Leaks are bad for several reasons:
• Pressurized air can leak into the system and void the saturated steam conditions, or formation of a slight vacuum during cool down can bring in organisms after sterilization.
• Also, during the fermentation, the leak may fill with media, thus providing a stagnant “fuse” through which foreign organisms can grow into the fermenter.
• Steam and condensate can leak out of the system and cause safety hazards.
The last system I designed had a computer-aided three-stage leak test performed after the steam sterilization phase. The test ensured leaks into and out of the system were minimized and also that key valves would not leak through when closed, to eliminate risk to the product that would be contained in the system.
Leaks occur. No leak is good, but not all leaks cause contamination. Leaks create a higher chance of contamination or decrease sterility assurance. My experience in the fermentation pilot plant showed that all leaks are not created equal when it comes to contamination. When sterile air was carried into a fermenter from an outside source and a leak was detected in the air line, a high rate of contamination occurred. I suspect a venturi effect caused non-sterile air from the outside to be pulled into the fermenter, contaminating it. However, an outward leak from a nozzle at the top of the tank caused a low rate of contamination.
5. Post-sterilization, follow steam with sterile air and maintain a positive pressure. After the steam sterilization cycle is complete, introduce sterile air into the system. If this is not done, when the steam flow is stopped, the steam will condense and begin to create a vacuum in the system. This vacuum may pull in non-sterile air from outside via a leak, resulting in a potentially high occurrence of contamination. Once air pressure is present in the system (I recommend 3 to 5 psig), maintain that pressure. It serves as a sterility assurance measure, increasing the likelihood that, if a leak occurs, the flow will be outward, not inward.
6. Design the system to be easily cleaned. In pharmaceutical applications, this is a pretty obvious statement, because the product needs to be kept clean. When the work is done manually, which often is the case for fermenters, improper cleaning can create steam sterilization problems:
• Layers of debris, media or product can make heating slower than expected or keep steam from penetrating to sterilize the material or system.
• Debris can cause steam traps or orifices used for sterilization to not work properly.
7. Look for unexpected temperature variations. A large article could be written on just this subject; here we can only describe the basics.
When sterilization of the system is to be validated, myriad thermocouples are placed in the system in conjunction with the permanent temperature elements to study the sterilization process. If two properly calibrated temperature elements in the same part of the system vary from one another significantly, then air, superheated steam or excess condensate probably is present.
Always suspect and verify temperature element calibration first. Once temperature elements are confirmed to be accurately calibrated if:
• Lower than sterilization temperatures are seen in low spots, suspect condensate problems.
• Lower than sterilization temperatures are seen in high spots, suspect air problems.
• Temperatures are too high for the pressure present, then suspect superheated dry-steam problems.
Unfortunately, when too-low temperatures are observed, it is not always possible to tell the difference between trapped air and excess condensate. In the case of condensate, as long as it meets temperature and time requirements, sterilization will occur. However, if air causes the temperature variation, then assume sterilization will not occur. Developing the ability to detect temperature variations is important and takes experience. Other devices can detect that air is present in a system, but temperature variation with thermocouples can actually help locate the source of the problem.
Hard-and-fast rules on how much temperature variation indicates a sterilization problem have not been provided, but the concepts presented apply. Consider the actual temperature elements used, with their given accuracies, when creating a standard for determining when temperature variation indicates a sterilization problem.
Let’s now look at five common mistakes.
1. Don’t create deadlegs. When designing piping for steam sterilization, it is all too easy to create areas where air pockets form and, thus, steam does not fully penetrate and the proper temperature is not achieved. These so-called deadlegs are a concern because, if air is present, sterilization probably is not achieved. The only ways to truly detect a deadleg is with temperature measurements or indicator spore strips failing to be killed.
During validation, always place a thermocouple in all suspected deadleg locations to ensure the piping is not a deadleg. My rule of thumb for a suspected free-draining deadleg in a non-vacuum pump system is a length-over-diameter (L/d) ratio greater than 2.5. This ratio is the length of pipe with no steam flow, from the edge of the pipe with steam flow to the end of the pipe that has no flow, divided by the inner diameter of the pipe without steam flow (Figure 1).
|The length-over-diameter ratio provides a useful tool for spotting potential deadlegs.|