This month’s Cleaning Memo covers bioburden limits (microbial) for clean hold time (CHT) protocols. The CHT is the time from the completion of the cleaning process (which may include the drying step) until the equipment is to be used again for subsequent product manufacture. We will first cover the possibility or probability of microbial proliferation during the CHT, then move to how to set limits, and then discuss options to avoid a formal CHT protocol entirely. Finally we will cover other related issues for the CHT.
The most significant regulatory concern with microbial proliferation is the likelihood of microbes reproducing if the equipment is stored wet. The general conditions for microbial growth are water, oxygen, and a nutrient source (assuming that there are viable microorganisms that might proliferate). We should avoid the common assumption that microbes can double in a very short time (like a few hours). The conditions that are typically present in cleaned pharmaceutical equipment are not conducive to rapid growth. While the oxygen content may be high due to exposure to air, the nutrient levels present (from drug product or cleaning agent components) should be relatively low such that rates of proliferation are much lower (as compared to proliferation rates in the presence of nutrient-rich media). Furthermore, if the equipment is dry (very low water levels), proliferation is not likely to occur. The opposite may be true – under dry conditions viable microorganisms may either die or become non-viable. Understanding the possibilities of the rates of proliferation in the cleaned equipment is critical to appropriately addressing bioburden limits for the CHT.
We will start this section with a repeat of what we covered last month dealing with the accuracy and precision of microbial enumeration methods. Analytical methods for chemicals residues are generally accurate and precise (such as “plus or minus 5%”). Furthermore, recovery of chemical residues from equipment surfaces is generally high (such as 70% or more). For typical bioburden methods involving growth on suitable media, this is not the case. The reason for this is that these growth/enumeration techniques measure CFUs (colony forming units). During the sampling and application to the growth medium, one “colony forming unit” taken from a surface may be “disrupted” to form two or more CFUs. As a general rule, a difference of one log (a factor of 10 times) is required to see a significant difference in bioburden. Furthermore, most microbiologists will state that one cannot specify with any confidence the accuracy and precision of any result below 20 CFU per 25 square centimeters in a traditional growth method. The situation is more complex if the evaluation involves either swab sampling or rinse sampling. In both cases, the action involved in sampling can disrupt the colonies on the surface resulting in higher CFU values in the measured test results.
So you might now ask “Okay, how are limits set for the CHT, which is the topic of this Cleaning Memo?” Well, if the equipment is relatively dry, and if the microbial levels measured at the end of the CHT also meet the bioburden limit used at the end of the cleaning procedure, then there is no significant proliferation during the CHT. I am not saying the measured microbial level found at the end of the cleaning procedure is not exceeded at the end of the CHT measurement. Here is an illustration. Let’s suppose the “end of cleaning procedure” limit is 10 CFU per 25 square centimeters, but the measured result at the end of the cleaning procedure is only 2 CFU per 25 square centimeters. As long as the measured result at the end of the CHT is 10 CFU or less per 25 square centimeters, then I should be happy with that measured result. After all, if 10 CFU were acceptable at the end of the cleaning procedure, that same limit should also be acceptable at the end of the CHT.
Note for this example just given that it was stated “if the equipment is relatively dry”. What if the equipment is not relatively dry? An option in that case is to include a criterion that a change from the measured bioburden at the end of the cleaning process to a measured bioburden of no more than a one-log change at the end of the CHT would be considered lack of significant proliferation (utilizing there general principle mentioned earlier that a one-log difference would be a significant change). Some might not want to allow this, and just revert to using the same limit at the end of the cleaning procedure. Part of the reason is that if the measured bioburden increases by exactly one log at the end of the CHT, what is the bioburden at the beginning of the start of manufacture of the next product? Assuming that there will be an elapsed time of several days between sampling the equipment and actually counting the CFU, then it is possible that the bioburden at the end of “CHT plus several days” will be more than a one log change. This results in what is called a “Catch 22” (Google the term if it is not familiar). So, the safest approach might just be to apply the bioburden limit at the end of the cleaning procedure and use it also for the bioburden limit at the end of the CHT.
After all that discussion in the prior section, how is it possible to avoid doing a formal protocol? The answer goes back to what we said was the primary objective of a CHT protocol. That objective is to demonstrate that there is no significant proliferation of microorganisms during the CHT. Well, if the measured bioburden at the end of the cleaning procedure is no more than the specified limit and if the equipment is dry at the end of the cleaning procedures, then we could apply the scientific principles (discussed earlier) of what is needed for microorganisms to grow. Absent any introduction of microorganisms into the cleaned equipment during the CHT, any measured bioburden at the end of the CHT should also be less than the bioburden limit at the end of the cleaning procedure.
So the keys to successfully applying this scientific rationale are to establish (1) that the equipment is dry at the end of the cleaning procedure and (2) that the system is closed such that microorganisms and/or water are not introduced in to the equipment during the CHT. This rationale should be captured in a report approved by QA, Validation, and Production. However, there should be a specified maximum CHT that is reasonable and believable. This does not mean that the CHT can be an extremely long time, such as several months. The selected maximum time should be a practical time as to when the equipment would be unused for manufacturing another product. Note that is you expect the equipment will not be used for a period of several months, it might be better to reclean it again with the previously validated cleaning procedure and then restart the CHT expiry period. Remember that in this situation, you should carefully explain your selected approach and then be prepared to defend it.
Some may ask how to establish that the equipment is in fact dry. One way might be to do analysis for trace amount of water by swab sampling (using a dry swab) for key areas in the equipment where water might accumulate if drying were inadequate. Another appropriate approach might be measuring water in the flowing air (flowing dry air) during the drying process. There may be other feasible options.
One issue is how bioburden is sampled both at the end of the cleaning process and the end of the CHT. If swab sampling is done, it is not acceptable to sample the identical surface area both before and after the CHT. It may be possible to sample adjacent equivalent locations. It should also be realized that swab sampling involves some operator interventions, which may compromise the microbial integrity of the equipment during the CHT. If rinse sampling for bioburden is done, the rinse is more typically a sample of the final portion of the process rinse. For rinse sampling done at the end of the CHT, the question arises as to whether the two sampling times (at the end of the cleaning process and at the end of the CHT) are truly comparable in terms of volume used and temperature. If the final process rinse were at an elevated temperature (done to help with proper drying of the equipment), then it might not be acceptable to also perform the sampling rinse at the end of the CHT at that same elevated temperature. The complicating factor with this is that any microorganisms which could have significantly increased during the CHT would be killed by the elevated temperature of a sampling rinse at the end of the CHT. These issues complicate formal CHT protocols, which is why I like the option of providing a scientific rationale why a formal protocol is not required.
A second issue is for situations where the equipment is steam sterilized. The more typical situation here is to clean the equipment using the cleaning procedure followed by a SIP process. The time between the end of the cleaning procedure and the beginning of the SIP process is the CHT. The time after completion of the SIP process is critical, but is not strictly a CHT. If the equipment is sterilized by the SIP process, then it is not likely there would be microbial proliferation after the SIP unless there was a breach such as to allow entrance of external microorganism=s into the equipment. This maintenance of sterility integrity can best be done ay monitoring continuous positive pressure in the equipment which is continuously monitored. The point is that the issue for CHT in this situation is to focus on the time between the end of the cleaning process and the beginning of the SIP process.
A third issue involves storage of smaller items that are placed in bags or trays for the CHT period. The first concern here is that these smaller parts must be dry before being transferred into a bag or tray. A second concern is the possibility of external contamination of the small part during the transfer into the bag/tray. Sterile gloves and aseptic handling should be considered here. A third concern is that the bag or tray should be closed to prevent external ingress of microorganisms and/or water into bag/tray. A fourth concern is that the bag/tray should be stored in a controlled area at ambient temperature.
A fourth issue is the possibility of grouping equipment for a CHT protocol, and only performing formal protocols on the “worst case” equipment. Not that the use of the term “worst case” here does not necessarily mean the equipment that is most difficult to clean for the cleaning procedure. An additional consideration is which equipment is more likely to retain pooled water and/or moisture following completion of the cleaning procedure (which would include the drying step). This concern may be related to the complexity of different pieces of equipment. For example, if I wanted to group together a wet granulator and a dry blender, I might choose the wet granulator both because of the difficulty of cleaning (more likely to leave residue behind which could serve as a nutrient source) as well as equipment complexity/geometry (more likely to leave moisture behind if drying were inadequate).
A fifth issue is whether three protocol runs should be made on each equipment item in an equipment train. This gets us back to the issue of grouping for CHT protocols. There are at least two possibilities here. One is to perform three runs of a given protocol on the worst case item in the group (if a worst case can be clearly identified). A second to perform a total of three runs, with each run done on a different item. Either, of course, involves a suitable risk assessment and rationale.
Next month we’ll cover routine bioburden monitoring (apart from validation protocols).
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