Abstract
Terminal sterilization is considered the preferred means for the production of sterile drug products, as it affords enhanced safety for the patient because the formulation is sterilized in its sealed, final container. Despite the obvious patient benefits, the use of terminal sterilization is artificially constrained by unreasonable expectations for the minimum time-temperature process to be used. The core misunderstanding with terminal sterilization is the notion that destruction of a high population of a resistant biological indicator microorganism is required. More contemporary thinking on sterilization acknowledges that the bioburden is the actual target in sterilization and its destruction must be assured. In the application of low-temperature moist heat for terminal sterilization, especially subsequent to aseptic processing, establishing the pre-sterilization bioburden to consider has proven challenging. Environmental monitoring survey data has determined the identity of potential microorganisms but not their resistance to sterilization. This review article provides information on the moist heat resistance of vegetative and sporeforming microorganisms that might be present. The first paper in this series provided the overall background and described the benefits to patient, producer, and regulator of low-temperature moist heat for terminal sterilization. The second paper outlined validation and operational advice that can be used in the implementation. This final effort concludes the series and provides insight into potential bioburden and its resistance.
LAY ABSTRACT: Terminal sterilization is considered the preferred means for the production of sterile drug products as it affords enhanced safety for the patient as the formulation is filled into its final container, sealed and sterilized. Despite the obvious patient benefits, the use of terminal sterilization is artificially constrained by unreasonable expectations for the minimum time-temperature process to be used. The primary consideration in terminal sterilization is the reliable destruction of the bioburden. The earlier manuscripts in this series described the principles and implementation of low temperature terminal sterilization processes where the sterilization conditions would destroy the expected bioburden present. To accomplish that reliably knowledge of the bioburden expected resistance to moist heat is necessary. This review article will identify publications where that data can be found.
- Terminal sterilization
- Aseptic processing
- Sterilization
- Biological indicator
- Bioburden
- Probability of a non-sterile unit (PNSU)
- Regulation
- Sterility assurance
Introduction
The earlier papers in this series described the core principles and implementation steps needed for the use of low-temperature moist heat sterilization processes subsequent to aseptic processing (1, 2). As those articles were being prepared for publication, comments were received that raised concerns regarding the bioburden potentially present in these projected low-temperature moist heat processes. Environmental results from conventional cleanrooms have been discussed by many different authors (3⇓–5). This article addresses cleanroom bioburden, focusing on the moist heat resistance of microorganisms rather than their identification and origins.
Environmental monitoring of processing environments used for aseptic processing has been a focus of attention since the late 1970s, when validation of processes became a requirement. The vectors for their introduction, the ability to quantify populations, their identification and their rapid detection have received considerable attention with regard to aseptic processing. Maintaining the aseptic environment is understood to be a holistic activity, with the majority of attention directed at personnel because they are understood to be the primary source of microorganisms in the cleanroom. Sterilization processes for the product, equipment, and components introduced in the aseptic environment are acknowledged as perhaps the least likely source of microbial ingress. This is true because the validated sterilization processes have demonstrated lethality against resistant biological indicators and are understood to be even more effective against the native bioburden. This is so widely acknowledged that bioburden monitoring is considered by many to be irrelevant with overkill sterilization processes.
For the successful application of low-temperature moist heat sterilization processes, knowledge of the moist heat resistance of the bioburden is essential. Unfortunately, there is a paucity of moist heat resistance data available for microorganisms other than spores. (There is extremely limited resistance data for non-spore formers to other sterilization process.) The healthcare industry knows a great deal about non-sporeformers, apparently except how difficult (or easy) it is to kill them.
Bioburden Expectations
Low-temperature moist heat sterilization is best applied where there are robust controls over the bioburden potentially present in the containers. This is best accomplished when the sterilization process is performed subsequent to aseptic filling. A non-aseptic filling process could be utilized, but this would require rigorous control over microorganisms in the product, the container, and the environment. These include a variety of design and operational practices that collectively mitigate the microbial contamination risk (4). The measures taken to control potential bioburden in aseptic (and near aseptic) processing environments and materials are well established and rigorous (5, 6). The USP chapter on environmental monitoring expectation suggests a maximum non-zero result in not more than 1 sample out of every 100 in ISO 5 environments (7). Given the many constraints and performance expectations, it should be apparent that the vast majority environmental samples taken in conjunction with aseptic processing will have no detectable microbial population. While this is certainly the case in compliant aseptic operations, if enough samples are taken, some contamination will inevitably be detected. The typical microflora detected in pharmaceutical cleanrooms are Gram-positive cocci; Gram-positive rods (non-sporeforming); sporeforming bacilli; Gram-negative rods, yeasts, and molds. Sandle summarized the recovered microflora from ISO 5 (Grade A and B) manned environments in the United Kingdom over an 8 year period (see Table I).
Microbial Recovery in ISO 5 (11)
From a sterilization perspective, these results can be simplified to 87% vegetative cells and 13% sporeformers, which may or may not be present as actual spores. The potential bioburden for a terminally sterilized product manufactured in manned aseptic facilities would be expected to have a similar distribution of microflora. (The actual bioburden transferred to the containers may be nil, because the environmental sampling results summarized included many locations remote from the exposed materials that become the filled product container.) The distribution of microorganisms from isolator-based aseptic filling has not been reported because the contamination rate in these systems is too low to accurately report. Thus, it should be acknowledged that the expected number of microorganisms present in aseptically filled containers prior to sterilization will be nil, or in other words, aseptically filled product containers are already “sterile” before the low-temperature sterilization process starts. (This seems to eliminate the need for a subsequent low-temperature sterilization process; however, as outlined in Part 1 of this series, there are substantive benefits to the use of this process subsequent to an aseptic fill.) The typical controls associated with aseptic processing are such that contamination levels are believed to be quite low, estimated at better than 1 in 10,000 based upon media fill results. Nevertheless, it is useful to consider what microbial resistance any potential bioburden might possess.
Moist Heat Resistance of Non-Sporeforming Bioburden
Moist-heat resistance of non-sporeformers is widely acknowledged to be minimal, and much of the reported data is derived from the food industry, which has extensive experience with low-temperature processing. Tables II⇓⇓–V provide moist heat resistance data on various pathogenic microorganisms.
Moist Heat Resistance of Selected Pathogens (Pflug) (8)
Moist Heat Resistance of Pathogens (webRFA) (9)
Moist Heat Resistance of Various Microorganisms (Sorqvist) (10)
Moist Heat Resistance of Various Microorganisms (Stumbo) (11)
The data presented above is only a small example of the information available within the food industry on moist-heat resistance of non-sporeformers. Processes operating at 80 °C and higher temperature as described in the first parts of this series are more than adequate to destroy the microorganisms listed in Tables II⇑⇑–V and other non-sporeforming bacteria, all of which possess minimal moist heat resistance.
Moist-Heat Resistance of Spore Bioburden
There are elements of potential spore bioburden in filled containers that a require examination.
The sporeforming bacterial strain that comes to mind first with respect to steam sterilization, Geobacillus stearothermophilus, is an unlikely contaminant in any pharmaceutical facility. G. stearothermophilus, the predominant biological indicator for steam sterilization, is thermophilic and grows at temperatures in excess of 55 °C. It and the other Geobacilli extremophiles have not been recovered in pharmaceutical environments (3).
While Clostridia are acknowledged concerns with respect to patient safety, the origins of that concern lie with the food industry. It was the deadly toxin derived from Clostridium botulinum that shaped much of the initial sterilization work in the food industry. Clostridia are able to survive and propagate in environments with less than 0.2% oxygen, and thus their detection in a conventional cleanroom, restricted access barrier system, or isolator is improbable (12).
The growth-based methods used for environmental and product microbial monitoring cannot establish that sporeforming bacteria are actually spores at the time of their collection. This is significant because while strains Geobacilli, Bacilli, and Clostridia are moist heat–resistant when present as spores, in their vegetative state their moist heat resistance is unremarkable (13). A worst-case assumption that they are in the spore state is commonly made when estimating their resistance.
The Bacillus species is the primary spore-forming isolate in cleanroom environments (3). The resistance of many of the different strains of Bacilli have been studied and extensively identified (14). The referenced data indicates that the vast majority of Bacilli have D110 values of less than 1 min. (The referenced tables include data for several Clostridia strains as well.) Mold and fungal spores are known to be substantially less resistant to moist heat than bacterial spores, and their survival is almost as unlikely as the survival of a vegetative cell (15, 16).
Most importantly, these processes are most useful where they are performed subsequent to aseptic processing. (When used subsequent to a non-aseptic fill, monitoring and control over the of pre-sterilization bioburden is essential.) The aseptic fill process would be executed with the standard controls. When satisfied these controls demonstrate a “sterile” product is manufactured without the addition of a following terminal process, so the pre-sterilization bioburden in these applications should be understood as “nil”, eliminating risk of survival entirely.
Conclusion
The use of low-temperature moist heat sterilization subsequent to aseptic processing provides an added margin of safety compared to the aseptic process alone. When understood as a viable alternative to artificially high F0 processes associated with the destruction of high populations of G. stearothermophilus, it is also appropriate for terminal sterilization processes where aseptic filling is not performed.
The multiple benefits to patient, producer, and regulator can be realized by consideration of this novel approach to establishing sterility. Closing the process gap by using a combination of aseptic processing (or well controlled non-aseptic filling) and subsequent terminal sterilization is both achievable and safe. The key to its adoption lies in acknowledgement of a few fundamental concepts captured in USP's recently revised sterilization content (17).
Knowledge and control of the pre-sterilization bioburden is a necessary component of sterilization processes.
Sterilization processes must reliably kill the bioburden present, nothing more.
Overkill is a term intended to apply to the bioburden and not the biological indicator.
The purpose of the biological indicator is to measure the process, not to define it.
Conflict of Interest Declaration
The author declares that he has no competing interests.
Acknowledgements
The assistance of Dr. James Akers and Mr. Russell Madsen in the preparation of this article is greatly appreciated.
- © PDA, Inc. 2017
