Introduction
Sterility is a key quality attribute for a class of medicines required to be sterile. The consequences of non-sterility are direct patient harm. The degree of harm is dependent upon the route of administration and the types and numbers of microorganisms, as well as the health and immune state of the patient. The likely outcomes of the administration of a non-sterile product are disability or death. For this reason contamination control is of utmost importance for the manufacture of sterile products and this is especially important for products that are filled aseptically, where terminal sterilization is not possible. Aseptic manufacture involves the production of drug products which are not subject to a sterilizing step; instead, sterility is assured through the prevention of microbial ingress.
To assess the risks of non-sterility each organization should develop a contamination control strategy. This requires an assessment, ac-knowledgement and remediation process for contamination risks. A contamination control strategy will be multifaceted and complex; as a means of addressing some of the basics and in raising some points for consideration, this article discusses the key starting points to be included in contamination control strategy for aseptically produced products.
Sterility
The importance of a sound contamination control strategy for aseptic processing has been exemplified by advancements in microbiology which, in turn, affect our understanding of sterility and sterility assurance. Advances in metagenomics, used for the Human Microbiome Project, have shown great diversity of microorganisms found in association with the human body, residing in distinct ecological niches. Moreover, the role these organisms play with health and disease is highly complex.1 Many of these organisms can only be determined through piecing together genetic material.
This leads to the concern that the assessment of contamination in pharmaceutical facilities remains reliant upon the recovery and enumeration of microorganisms by culturing (onto solid and liquid media). This form of assessment underpins the pharmacopeia me-thods for sterility testing and environmental monitoring. These tests are limited by the fact that many (if not the majority) of the microorganisms within the environment are metabolically active but non-culturable2 (either permanently or they enter this state transitorily, including common human comensurables like Micrococcus luteus). Monitoring limitations are exacerbated through the small samples sizes used with each test;3 plus other factors such as the culture medium selected and the temperature and time selected for incubation, which will affect microbial recovery. Although advances have been made with rapid sterility tests and, for environmental monitoring with spectrophotometric technologies to allow for the differentiation between inert and biologic particles, these technologies remain under the category of ‘emerging’ rather than being fully developed.4
It is for these reasons that a contamination control strategy needs to be constructed by manufacturers of aseptically filled products to put most of their resources into; with a greater focus on control rather than simply monitoring using methods with inherent limitations. Importantly there can be little comfort gained from a series of zero counts recovered from environmental monitoring or sterility test passes if there are inadequacies with contamination control.
Contamination Control Strategy
The objective of a contamination control strategy, for aseptically filled products, is sterility assurance and the production of a sterile product (a product absolutely devoid of viable microorganisms). This outcome is intrinsically dependent upon a process specifically purposed to impart that desired state, consistently.5 Unlike terminal sterilization, where the Sterility Assurance Level can provide a statistical understanding of the probability of non-sterility; with aseptic processing, where the imperative is to prevent microbial ingress, the same statistical assurance cannot be provided. Thus the manufacturer is therefore reliant upon a good contamination control strategy.
Aseptic Processing
The process of producing a sterile product by aseptic processing is either through conventional filling or by blow-fill-seal. With both, a product is sterile filtered into a sterile container (sterile stainless steel vessel or plastic biocontainer bag) and filled into depyrogenated containers (glass vials, syringes, sealed plastic capsules and so on) and then sealed (such as cap and oversealer being placed onto a vial or through molding). The bringing together of the sterile product and the container is performed under ISO 14644:2015 Class 5 conditions. Within this simplified narrative of aseptic process there are several elements that need to be considered when devising a contamination control strategy. These are discussed below.
Good Facility Design
All sterile product manufacturing must, according to regulations, be undertaken within a classified cleanroom environment. This is in order to minimize product contamination (for if the product becomes contaminated, the level of contamination may be to the extent that the contaminating microorganisms are resistant to a sterilization process; cannot be removed by filtration; or, in the case of aseptic processing, contaminate the product during aseptic filling). Spending time on appropriate design is important, including having the shortest product flow paths possible to ensure there are airlocks in place between cleanrooms of different grades and for changing rooms.
Good design relates to both selecting a suitable grade of cleanroom together with a design intended to minimize contamination. This includes the use of appropriate construction materials and spending time on the suitability of the layout, covering elements like process and material flows. With this the layout should promote the orderly handling of materials and equipment, the avoidance of mix-ups, and the prevention of contamination of equipment or product by chemical substances, previously manufactured products, and microorganisms.
Most microorganisms within cleanrooms derive from people (al-though water, as a natural environment and as a vector, together with transfer in via equipment or faulty air handling systems, also present microbial challenges). In minimizing the risks from people it is not simply sufficient to have a cleanroom of an appropriate grade, the cleanroom must have suitable air change rates, air mixing and be able to recovery rapidly after a contamination event. These should be factored in during the design phase.
Weaknesses in the design, or at least areas that are not as robust as they should be, need to be assessed when devising the environmental monitoring program. Risk assessment is useful for determining the critical control points together with the relative risks of air and surface contamination in relation to potential transfer onto critical surfaces or ingress into product.6
Personnel Training and Gowning
With people representing the primary source of contamination effective training of operators is paramount. Operator training should be continuous, covering theoretical, practical and cGMP aspects; with the curriculum including microbiology and hygiene. Training should address everything from standing appropriately when not engaged in activities (with hands raised) to performing interventions in an aseptic manner into the aseptic core (this is something that needs to be practiced and then demonstrated through media simulation trials). While this latter activity requires highly developed aseptic techniques,7 there is no such thing as a truly safe intervention. The appropriate focus of the strategy should with minimizing the need for interventions more so than making interventions easier to perform. This can be addressed through better functioning machinery and with the use of barrier technology, particularly isolators where gloveport manipulations replace open door interventions.
Effective gowning must not be overlooked to avoid contamination being transferred from the operator onto the sterile gown worn in the aseptic area. Weak areas with gowning including the donning of gloves and the placing of goggles for both carry a risk of transfer of skin bacteria onto the gown. The suitability of gowning should be assessed through regular gown qualifications, which need to be assessed both visually and through microbiological sampling.
Cleaning and Disinfection
While cleanrooms can be designed effectively and the people working within them controlled, contamination will still arise not least from the shedding of skin detritus from operators. Regular cleaning (using a detergent to remove soil) and disinfection (to inactivate microorganisms through cellular destruction) is required. It is typical to use two disinfectants on rotation, one of which is often a sporicide (capable of destroying bacterial endospores and fungal spores). For batch filling, cleaning and disinfection of cleanrooms must take place before and after each run (and also of the conventional filling area should a closed RABS or isolator not be used). With other areas, cleaning and disinfection frequencies must be established through the review of empirical data as collected through a field trial.8
Robust Sterilization
The use of sterile items, from vessels to closures, needs to be practiced and controlled. Increasingly such items are of a bespoke design and are introduced as sterile and ready-to-use; however, there remains a strong reliance upon in-house sterilization and this is most commonly undertaken using an autoclave. To assess the effectiveness of different autoclave loads, as used with different cycles, these need to be evaluated thermometrically and using biological indicators prepared from Geobacillus stearothermophilus.
Again design is important. When developing steam sterilization cycles the specification needs to be decided at the outset. The required assurance of sterility is a minimum Sterility Assurance Level (SAL) of 10-6; it is more common for autoclaves which sterilize critical loads to be operated to achieve an SAL of 10-12 (in order to obtain ‘overkill’).9
Use of Single-Use Sterile Disposable Items
The adoption of single-use sterile disposable items has helped to move aseptic processing forwards, through reducing the reliance upon autoclaves and helping to guard against both a failure with a sterilization cycle and a control breakdown when a critical path step is undertaken, such as an aseptic connection. Single-use items are typically sterilized using gamma rays (electromagnetic irradiation). Ethylene oxide gas remains an alternative sterilization process, although this is not, as yet, used to the same extent as irradiation.10
Types of single-use technologies relevant to aseptic processing include tubing, capsule filters, single-use ion exchange membrane chromatography devices, single-use mixers, and bioreactors, product holding sterile bags in place of stainless steel vessels (sterile fluid containment bags), connection devices and sampling receptacles. Perhaps the most useful innovation has been the aseptic connector. These connectors allow for a totally enclosed and automated process, enabling a connection to be performed, by the joining of two components together, in an environment that does not require unidirectional airflow cabinets to be used. This concept allows liquid sterile products to be transferred simply and safely, towards or from contained areas, via a small scale rapid transfer ports. Additionally such devices shorten the time required for the connection.
Depyrogenation
Concern with pyrogens in aseptically prepared products (especially bacterial endotoxin in relation to parenteral products) requires that the material into which the sterile bulk is dispensed is depyrogenated. For glass vials this is via a depyrogenation tunnel (typically dry heat). Endotoxins can cause, to varying degrees depending upon potency and target site, endotoxemia (the presence of bacterial toxins in the blood) and septic shock (the prolonged presence of bacteria and bacterial toxins in the body). Therefore, depyrogenation of glassware is important in the production of parenteral pharmaceuticals as residual pyrogens could ultimately be injected into a patient resulting in an adverse reaction. This is especially important as endotoxins are heat stable, making them resistant to most conventional sterilization processes and thus necessitating separate tests for viable cells and endotoxin.
The assessment of depyrogenation involves a formal study using thermometric measurements and the use of endotoxin indicators. The endotoxin assessment involves the introduction of purified endotoxin, of a high potency, and post-process testing to assess if a minimum of a three-log reduction has been achieved. Critical aspects include using a representative number of challenge vials and positioning vials in representative locations, close to where thermometric measurements have indicated there could be cold spots. The other important aspect is with the tunnel cycle design where the optimal time and temperature combination needs to be selected to inactivate the endotoxin.11
Aseptic Filling and Barrier Technology
The contamination control strategy should focus on protecting the product from a microbial contamination event during the point of greatest risk. With aseptic filling the sterile product is filled into depyrogenated glassware and fitted with a sterile stopper and then oversealed. The most vulnerable step is with the dispensing of the product, via filling needle, into the vial. Regulations require this to be undertaken in ISO 14644-1:2015 Class 5 conditions (with particle and microbial control). In terms of the design space this can be conducted in a UDAF device or Rapid Access Barrier System (RABS) contained within an ISO Class 7 cleanroom, or it may be performed inside a barrier isolator.12 With these design space choices there is a cascade of control in terms of automation and consistency of the decontamination process (with closed RABS and isolators undergoing decontamination with a gaseous agent like hydrogen peroxide); and with the barrier between the critical area and the outside environment (including personnel). Here the isolator provides a complete barrier.
While the contamination strategy would direct the user to select an isolator, and these are conceptually superior to the cleanroom, they nevertheless carry weaknesses that need to be considered. The decontamination cycle must be suitable, in terms of safety and provide demonstrable biological kill (which requires a biological indicator assessment). Although 10-6 log-kill decontamination cycles can be developed some regulators, especially those from Europe, expect product contact parts to be sterile (i.e. single use, or to have undergone a separate sterilization cycle). Additionally leakage needs to be assessed, of both the isolator system and of gloveports. When personnel use gloves, these represent one of the most significant risk areas.13
In addition, the airflow within the isolator (as with any aseptic filling zone) needs to be visually assessed, using smoke or fog, in terms of having a suitable velocity and direction so that any contamination that might gravitate towards a critical area like point of fill is directed away. Additionally, the study of air patterns is particularly useful for the selection of environmental monitoring locations.
Media Simulation Trials
The media simulation trial provides the means to challenge the aseptic processing assurance system. With media simulation trials, a microbiological growth medium is used in place of the product and filled as if it was product under the ordinarily processed conditions. Media fills start with the beginning of filling operations (immediately after the line setup), during and after manipulations and interventions, and until the last vial has been filled.
As part of the contamination control strategy it is important that media fills are representative of conditions during processing and that they reflect the greatest challenges. With aseptic processing the greatest challenge is microbial ingress, either as a result of transfer (such as an operator performing an intervention) or deposition from a microbial carrying particle. This requires an assessment of ‘worst case’ parameters that might lead to a microbial contamination event occurring. Such parameters include vial size, vial neck diameter, line run speed, stoppages, and the number and complexity (including time) of personnel interventions and manipulations.14
Summary
This article has introduced some of the important elements that make up a contamination control strategy for aseptically filled products. There are, of course, other elements such as cleaning validation and the environmental monitoring strategy; the purpose here was to focus on the core parts of the contamination control strategy and to highlight areas that are sometimes overlooked.
Some of the topics selected are being strengthened by advances in technology and it is with further technological advances that the industry must continue to lend its support. Any over-reliance upon microbiological tests, which have not kept pace with the revelations about the diversity of non-culturable but active microorganisms, should be avoided for these do not provide sufficient assurance of product sterility. The key message of this article is to focus on strengthening control.
References
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Author Biography
Dr. Sandle is the Head of Microbiology at Bio Products Laboratory Limited (a pharmaceutical organization). Dr. Sandle is a chartered biologist (Society for Biology) and holds a first class honors degree in Applied Biology; a Masters degree in education; and obtained his doctorate from Keele University.
Dr. Sandle has over twenty-five years experience of designing and operating a range of microbiological tests (including sterility testing, endotoxin LAL methodlogy, microbial enumeration, environmental monitoring, particle counting, bioburden, isolators and water testing). In addition, Dr. Sandle is experienced in microbiological and quality batch review, microbiological investigation and policy development.
Dr. Sandle is an honorary consultant with the School of Pharmacy and Pharmaceutical Sciences, University of Manchester and is a tutor for the university’s pharmaceutical microbiology MSc course. Dr. Sandle serves on several national and international committees relating to pharmaceutical microbiology and cleanroom contamination control (including the ISO cleanroom standards). He is chairman of the Pharmig LAL action group and serves on the Blood Service cleaning and disinfection committee. He has written over two hundred book chapters, peer reviewed papers and technical articles relating to microbiology; and delivered papers to over forty conferences.
Dr. Sandle is the editor of the Pharmaceutical Microbiology Interest Group Journal and runs an on-line microbiology website and forum (http://www.pharmamicroresources.com/). Dr. Sandle is an experienced auditor and frequently acts as a consultant to the pharmaceutical and healthcare sectors.