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Adeline Siew is editor for Pharmaceutical Technology Europe. She is also science editor for Pharmaceutical Technology.
Experts describe best practices for sterility assurance in parenteral drug manufacturing. This article contains bonus online-exclusive material.
Maria Toutoudak/Getty ImagesSterility assurance is of paramount importance in parenteral drug manufacturing. These products are administered directly into the bloodstream, bypassing the body’s natural defenses. Contamination of parenteral drug products, therefore, can have serious consequences on the patient. To gain further insight into best practices for sterility assurance, Pharmaceutical Technology spoke to James Agalloco, president of Agalloco & Associates and member of Pharmaceutical Technology’s editorial advisory board; Tim Sandle, head of microbiology at Bio Products Laboratory; and Benoît Verjans, scientific advisor at Aseptic Technologies and member of Pharmaceutical Technology’s editorial advisory board.
PharmTech:What are the most common sources of contamination that can arise in sterile manufacturing or aseptic processing?
Agalloco (Agalloco & Associates): The most common source of contamination has been the operator. We’ve all known this for years and there’s been a steady progression of technology advances to reduce the problem that the presence of the worker presents. It started with curtains, and has progressed to closed restricted access barrier systems (RABS) and isolators. The next advancement in this area will be the use of robotics and automation to further reduce the impact of personnel.
Sandle (Bio Products Laboratory): Parenteral drug products are required to be free from three things—viable microorganisms, pyrogenic substances (which essentially means a low-level of bacterial endotoxin), and visible particulates.
There are different sources of microbiological contamination within clean environments. They can be divided into water, air, surfaces (both within the room and from equipment), and personnel. The main risk from water sources is to product formulation and the activities up to and including final sterilization. In my experience, the greatest concern comes from wet equipment, allowing water-borne bacteria such as Pseudomonads to grow.
Water is a double concern because it is a vector for contamination and a growth source for microorganisms. We cannot avoid water in cleanrooms. Water is a common feature in pharmaceutical processing (e.g., as an ingredient, a cleaning agent, a diluent for disinfectants, and steam supply).
Other sources that affect aseptic processing include improperly designed clean air devices and air-flows that can direct microbial-carrying particulate contamination towards the exposed product. Importantly, these microorganisms will come from people, who are the biggest contamination risk within cleanrooms. As with water, we cannot wholly exclude people from cleanrooms, although we can reduce their contact with critical zones through the use of barriers such as isolators.
People can also pose a risk through touching, such as moving a contaminated object from a less clean area to a cleaner one, or more directly via their gloved hands. This risk often arises through improperly sanitized hands, where hands are either not sprayed frequently enough or the hand-rubbing technique, once the glove alcohol has been sprayed on, is not carried out effectively. Another important area is training; especially on aseptic techniques, gowning, and cleanroom behaviors.
Verjans (Aseptic Technologies): There are two distinct categories of contamination of injectable drugs. Some contaminations are a result of bad practices and in this case, multiple containers are usually affected. These contaminations are identified through outbreak episodes that affect several patients. Recent examples include the contamination from a cracked glass container that affected eight newborn babies in the Mainz hospital in Germany, of which three died; and the contamination case involving approximately 700 people in the US due to corticosteroids infected with a fungus triggering meningitis (1, 2). For the second case, it is obvious that bad practices led to the production of heavily contaminated batches by the compounding pharmacy.The other group of contamination is more insidious because it affects one vial from time to time. The source of contamination is a living organism that managed to penetrate the container at a certain moment and, if not detected, may trigger disease episodes such as septic shock. Because it is a single event, this type of contamination is often classified in the group of nosocomial diseases. A rough estimate of outbreak analyses and nosocomial disease epidemiology suggests that one out of 100,000 containers has a contaminated drug, resulting in approximately 30,000 contamination accidents in the US due to contaminated drugs (3).
Contamination of drugs may come from various sources but in most cases, we can identify a human being behind the contamination. It can be due to multiple sources of mistakes such as gowning mistake, whereby living organisms are carried on the gowning; lack of maintenance of a protected environment; contamination of product contact parts during manufacturing; or disrespect of procedures during sanitization.
PharmTech:What are the limitations or challenges to current sterilization methods?
Agalloco (Agalloco & Associates): The obstacle we face is the expectation for higher F0 values, increased doses, and tighter filters. There a belief that if we just make the process a little more lethal or more robust, it will be better. That ignores the whole other side of the process—what it does to the materials we are processing. There is degradation, increased particles, extractables, less mechanical strength and other impacts that oversterilization can cause. There needs to be more consideration of the negative consequences of what sterilization does. We only need kill or remove the bioburden once. Overprocessing is rampant and aside from making things look better on the surface, it’s actually not something we should be doing. The half-cycle approach to sterilization should be used rarely and unfortunately its use is becoming more prevalent rather than less.
Sandle (Bio Products Laboratory): The main limitation with any sterilization method relates to the validation, the way it has been executed, and the way the validated sterilization technology is used in practice. One only has to look at the major pharmaceutical contamination scandals of the past 40 years to see this limitation, from the Devonport incident in the early 1970s (which was partly the basis of modern GMPs) (4) to the issues surrounding the New England Compounding Center last year, where three lots of methylprednisolone acetate, intended to be injected into the spinal cord as a treatment for arthritis, were contaminated with Exserohilum rostratum (5). This incident led to more than 700 reported infections and some 48 deaths, based on figures from the US Centers for Disease Control and Prevention. In both these cases, the sterilization equipment was involved and it was not operated correctly, which is largely a practical matter of engineering and systematic checks (6, 7).
Besides validation issues, various factors (e.g., economic, space, time-to-release) drive the use of different sterilization technologies. The factor, however, is the product and whether it is compatible with the technology. The lower-risk technologies are terminal sterilization methods, especially for medical devices that can be gamma irradiated or treated with ethylene oxide.
Terminal sterilization is most commonly carried out using steam (moist heat). Risks are often low provided the cycles have been validated thermometrically and biological indicators have been used to show that sterility-assurance levels are at 10-6 as a minimum. A number of quality attributes must, however, be carefully checked for each run. Most important is air removal. It is crucial to ensure that all of the trapped air is removed from the autoclave before activation as hot air is a very poor medium for achieving sterility.
The biggest challenge is aseptic filling. There are complications around product filtration relating to the validation of the product through the filter (where the filter needs to be challenged with 10,000,000 cells of a diminutive bacterium); product bioburden; and issues relating to filter failure (for which post-use integrity checks are crucial). There are also the complications of bringing together a sterile product and sterile components (vials, stopper, crimpers) and attempting to fill thousands of vials under a clean air zone.
Verjans (Aseptic Technologies): We need to distinguish between terminally sterilized and aseptically filled products. For the first category, products are sterilized shortly after fill–finish, therefore eliminating contamination that could potentially put patients at risk. On the contrary, for aseptically filled products, there is a real concern of contamination because the last safety barrier provided by terminal sterilization is not there. From now, I will exclusively talk about aseptic processing, in particular fill–finish.
The following is a list of contamination sources, among others:
To have safe aseptic processing, it is mandatory to address all these aspects carefully, which therefore makes aseptic processing perhaps the most complex pharmaceutical manufacturing process. The challenges are to:
PharmTech :Can you identify recent advances in equipment design, operation, filtration, or processes that are addressing some of these problems?
Agalloco (Agalloco & Associates): The operational improvements made by increased use of closed RABS and isolators are well known. Increased use of robotics and automation are making aseptic processing safer. Other technologies such as closed-vial filling, gloveless isolators, and single-use systems will further enhance performance of aseptic manufacturing. Understanding the importance of bioburden destruction as opposed to biological indicator destruction would help as well.
Sandle (Bio Products Laboratory): Most of the technologies that we are using have been around for quite a long time. Scientists experimented with heat under pressure to improve food preservation in the mid-19th century, for example. Many other sterilization processes began to be more widely used post-World War II, such as irradiation.
Cleanroom technology did not advance greatly until the late 1990s. This pace of transformation has accelerated more quickly in recent years, notably with barrier technology. Aseptic filling risks have been lowered through the use of isolators and RABS. RABS create a physical and aerodynamic barrier to protect the product, but they are not all enclosing. Isolators are the most effective as they create a complete barrier (isolation) between the products and people. Where isolators can be placed around filling machines, it allows for the entire space to be decontaminated using hydrogen peroxide (either as a vapor or in the ionized state). This approach allows for the sporicidal sanitization of all exposed surfaces. Isolators are not risk free, however, due to issues such as air leakage.
There has been some conceptual changes with cleanroom design, using computer-aided engineering software that can help pinpoint contamination risks. There are also big advancements in the use of risk management, supported by initiatives from regulators such as FDA. Risk assessment tools such as HACCP (hazard analysis and critical control points) have become more common.
Also with cleanrooms, various items of equipment and surfaces are now manufactured with antimicrobial coatings. One example is the incorporation of silver, which is effective against a range of microorganisms, into implements such as forceps. With processing, the most important recent advances have come from single-use disposable technologies. Such technologies have reduced risks by allowing pharma organizations to move away from equipment that need to be sterilized or consumables that are recycled or pose a risk with their transfer into cleanrooms. Single-use items are typically sterilized using gamma rays, which kill microorganisms by destroying cellular nucleic acid.
Examples of single-use systems include aseptic connections for the connection of a vessel or filter to another item of equipment for the transfer of fluid. Here, a big contamination risk is from the hand of the operator; the connector effectively eliminates this risk. Another example relates to disposable bag technologies for holding the product. These technologies have huge potential economic benefits since plastic technology can reduce validation and clean-in-place requirements, lower the requirements for pure water, clean steam, and water for injection (WFI), as well as cut costs (e.g., from reduced set-up times). Other examples include consumables and disposable filling manifolds, each of which reduces the need for operator involvement.
Verjans (Aseptic Technologies): In the last decade, multiple improvements have been introduced to mitigate the risk of contamination. The first one is to improve gowning of operators moving from classical laboratory coats in the fifties to fully gowned operators. The second one is to use filters with extremely good efficacy in retaining living organisms, even the smallest ones and the mobile ones. The quality of these filters is improving regularly. The third one is to separate the operators from the processing area. Processing equipment can now be protected by advanced barriers such as the RABS and the most advanced ones such as the closed RABS and the isolators. The isolators offer complete separation of the processing area from the environment, combined with an automated sanitization system using sporicidal agents, which are usually hydrogen peroxide.
Beside these improvements, there is a new category of improvements that consists of the reduction of exposure to the environment. Reducing the time when a container is open reduces the probability of having a living organism penetrating into the container. The same concept applies to contact of the inside part of the container. For example, a stopper or a plunger being in contact during a significant period of time with a stopper bowl and various ramps may accidentally capture a living organism and bring it inside the container when closing it. Two new technologies that aim to reduce this exposure include:
These technologies have demonstrated reduction in contamination risks by at least 2 log (100 times) compared to the classical glass vial filling process (8).
PharmTech :What are the limitations/challenges to current testing methods for microbial control?
Agalloco (Agalloco & Associates): We’ve exhausted the ability of microbial sampling and test methods to help us. The expected quantities of microorganisms are at or below the threshold of detection for most sampling methods. The only acceptable result in Class 100 (Grade A) is less than 1 colony-forming unit (CFU). There are problems with this because it suggests that aseptic processing has to be conducted under essentially sterile conditions, which is not possible, especially with the manned filling technologies in use. Aseptic processing can be successfully performed in less than sterile condition, and that creates severe tensions between what we can provide in the way of environmental and process control, and the extreme regulatory expectation of those same controls. Rapid microbial methods aren’t the answer, because they only provide the results somewhat sooner.
Sandle (Bio Products Laboratory): Monitoring methods are divided into viable monitoring and nonviable particle monitoring. The objective of viable environmental monitoring is to enumerate the numbers of microorganisms present at a location within a cleanroom, to allow incidents to be recorded and, ideally, to permit species level identification. This type of monitoring is undertaken using a range of different air and surface counting methods, namely active air-sampling using volumetric air-samplers; so-called passive air monitoring using settle plates; the surface methods—contact plates and swabs; and the monitoring of personnel in terms of gloved hand prints and suit gown plates, taken on exit from the cleanroom suite.
Concerns with these classic methods was highlighted in the recent update to the USP chapter <1116>, which argued that we need to get away from seeing these methods as somehow “super accurate” such as an analytical instrument in a chemistry laboratory (9). The methods are limited because they can only be used periodically and thus serve as “spot checks” only. They cannot pick up all the culturable microorganisms present for example, due to weaknesses in collecting all the microorganisms that adhered to surface when using a contact plate. Recovery is also affected by temperature and agar variations.
As another example, with active air-samplers, these devices are only designed to pick up 50% of the viable particles that are drawn in. There are risks with the method of drawing the air in, such as by impaction or through centrifugal forces, damaging or stressing the microorganism to the extent that it will not grow. It has been estimated that many of the micororganisms present in cleanrooms will not grow using the conventional methods. These are termed the viable but nonculturable (VBNCs) organisms.
These same issues also affect in-process bioburden monitoring, used to measure contamination build-up in process areas, even with end-product sterility tests. There are, however, things that can be done to improve detection. With settle plates, it is important that the plates are tested to show that after exposure, due to the inevitable weight loss from drying out, they can still grow microorganisms. With contact plates used on surfaces, these plates should contain neutralizers to ensure that any residues from cleaning agents do not mask any microorganisms present. With swabs, the method will always be limited. However, there are new types on the marketplace that give better recoveries. Finally, with active air-samplers, tests should be conducted to show that the sampler does not disrupt the air-flow, especially at ISO Class 5.
The use of risk assessment and putting together a well-thought of environmental monitoring plan can also help. Monitoring should be orientated towards the main activities within an area and directed to where product is exposed. Historical data can help to set appropriate monitoring frequencies.
Verjans (Aseptic Technologies): Let’s compare between large particle detection in containers and environmental monitoring. Particle detection is a systematic monitoring that screens all containers. The efficacy of the particle-monitoring process, even if not 100% perfect, is good enough to eliminate all or almost all containers containing a large particle, which is a potential source of embolism for the patient. This approach is not yet feasible with small living organisms and one way to address the contamination risk issue is to have environmental monitoring. This control is essential but presents the disadvantage of being based on samples. For example, contact plates and active air sampling are only targeting one sample of air. Therefore, the probability of detecting bacteria in the processing environment remains low.
It has been estimated that approximately 28 dm³ of air are in contact with each 2R glass vial (8). Therefore, classical microbial air monitoring systems collecting 1 m³ of air are only representative of 35 vials. Knowing that a batch may represent few hundreds of thousands of vials, statistical calculation demonstrates that the probability of detecting a CFU during microbial environmental monitoring is much lower than having one or few contaminated vials.
Another aspect is that monitoring is limited in terms of location. Monitoring is usually done at positions where the impact of a contamination may be serious such close to the filling needle, close to the stopper or plunger bowl, or close to the stoppering area. Nevertheless, it is difficult to monitor everything, which hence, leaves room for contamination in an area other than the scrutinized ones.
PharmTech :Can you identify recent advances in process analytical technology (PAT) or other analytical testing methods (such as rapid microbial testing) that can help improve testing for microbial control.
Agalloco (Agalloco & Associates): We have exhausted the current technologies for confirmation of classical environmental controls for aseptic processing. New technologies with the ability to detect in near real-time the presence of viable microorganism’s are becoming available. These technologies have promise, but the concern I have with respect to their use is that while they will detect what cannot be detected using the classical practices, they will confirm the presence of viable microorganisms at a lower level of sensitivity. That is not necessarily good, in that it may result in higher expectations for even better control of environmental controls. The sterile products we manufacture are certainly safe at the microbial levels that we have already established. We do not need ‘sterile’ environments for the manufacture of aseptic products, and striving to do so will result in substantially increased cost without any meaningful improvement in patient safety.
Sandle (Bio Products Laboratory): Rapid microbiological methods are being developed in earnest by most of the major vendors, although adoption by the pharma industry remains slow, but I suspect that will change over the next five years. In relation to PAT, measuring critical process parameters during manufacturing, as well as current quality risk management and spectrophotometric methods look very promising. There is essentially a development of the particle counter, where air is drawn in through an instrument and passed through a laser. The instrument software is able to differentiate the particle size and to count the number of particles.
The newly developed biophysical counters record biological events. How exactly these ‘events’ equate to microorganisms is unclear, although several industrial research projects are ongoing. The advantage with these real-time viable counters is that they are fully compatible with the incident rate approach outlined in USP <1116> in 2012, which is attempting to direct regulatory thinking to being more concerned with how often a microorganism is seen rather than the magnitude of the count. They are also very fast.The downside is that not all of the instruments allow for microbial identification, and those that do cannot recover everything. There is also a danger that they react to nonmicrobial events and autofluoresce due to clothing fibers or disinfectant sprays.
Other advances with rapid methods are focused on the time-to-result. Most cultural based methods require incubation times of five-to-seven days. Technology has been developed for testing process water. One system uses an ATP-releasing reagent, added to microbial cells, followed by the addition of luciferin and luciferase. The system then scans for fluorescence.
With in-process bioburden, there is a method which uses digital imaging technology to automatically enumerate micro-colonies. This method has cut the time-to-result down by half. An alternative is quantitative real time polymerase chain reaction (qPCR), which allows amplified microbial DNA to be detected as the reaction progresses in real time. However, there is concern whether the methods can detect a wide range of microorganisms, especially when they are bound to the product.
Other areas have centered on accuracy, most notably with microbial identification methods. Knowing the correct ID is important for corrective and preventive action (CAPA) investigations. For me, the most exciting developments here are with MALDI-TOF, which is almost instantaneous, and the genotypic methods that can pin-point different strains. With each method, there are difficulties with validation, cost, and in gaining regulatory acceptance. Nevertheless, the case for implementation will soon be sufficiently strong and I'm sure we will see many of these methods in place five to ten years from now.
Verjans (Aseptic Technologies): PAT is defined as mechanisms to design, analyze, and control manufacturing process by measuring critical process parameters. It can be separated into three categories: PAT on the environment, PAT on the quality, and PAT on the equipment operation. All three have their own importance and may contribute to the quality of the product.
PAT on the environment allows release of the production in a timely manner and minimize risk of missing a deviation. For example, a particle counter may be connected to the filling equipment and may dispense a warning signal when exceeding an alert level or an action level. With appropriate procedure, the operator may act to correct the issue if possible, or he may interrupt the production of the batch if the problem cannot be solved without being disruptive. The value of such rapid information is increasing with the price of the product because it reduces the risk of batch rejection thanks to correction of a deviation.
PAT on particle monitoring can be easily standardized. PAT on viable monitoring is more complex as most of approved methodologies are based on bacterial growth. Nevertheless, a new technology emerged recently, combining viable monitoring and total particle monitoring. This technology combines classical particle count with laser-induced fluorescence and therefore detects all particles that contains excitable molecules such as nicotinamide adenine dinucleotide (NADH) and riboflavines. These molecules are characteristic of living organisms so this technology is able to detect in real time the presence of a living particle (10).
PAT on product quality relies on container inspection. Besides the classical particle count, new technologies include vacuum testing, high-voltage testing, and headspace analysis (to detect e.g., lack of closure integrity, poor nitrogen flush, or inappropriate lyophilization).
PAT on equipment operation allows the elimination of badly/poorly processed containers. One of the PAT processes that has been used for long is weight measurement. What is changing in recent years for weight measurement is the move from sample measurement to 100% measurement, even on machines with high-speed production.
Other more sophisticated PAT on equipment operation are emerging in parallel with new processing technologies. For example, in the case of the closed-vial technology, the container-closure integrity is ensured by laser re-sealing of the piercing trace left by the needle in the stopper. To ensure that the laser is effective, all the vials are checked for laser shot, both in terms of shot time and shot intensity. This measurement process is directly coupled with the automation system, leading to automatic reject of containers with laser beam out of specification. By optimizing PAT, the pharmaceutical industry improves its capability to ensure that only well-processed containers are released for use to the patient.
1. The Local, “Dirty bottle likely source of bacteria in Mainz infant deaths,” Press Release, Aug. 27, 2010.
2. CBS News, “Lethal medicine linked to meningitis outbreak,” Press Release, Mar. 10, 2013.
3. R.P. Vonberg and P. Gastmeier: J. Hosp. Infect., 65 (1) 15-23 (2007).
4. C.M. Clothier, Clothier Report, Report of the Committee appointed to inquire into the circumstances, including the production, which led to the use of contaminated fluids in the Devonport section of Plymouth General Hospital, London: Her Majesty’s Stationery Office, 1972.
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6. B. Cox, A Lesson on Outsourcing: The NECC Fungal Meningitis Outbreak, The Gold Sheet, 46 (11) 1-6 (2012).
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8. B. Verjans and C. Reed, Biopharm. Intl., 25 (3) 46-58 (2012).
9. S.V.W. Sutton, J of GXP Compliance, 16 (4) 59-63 (2012).
10. D. Niccum and P. Hairston in Environmental Monitoring: A Comprehensive Handbook, Volume 6, Jeanne Moldenhauer, Ed. (PDA, Bethesda, MD 20814, 2012), pp. 219-240.