Contamination - focus on the risks

Sterile is a powerful word, with harsh legal implications surrounding non-compliance. Global regulatory authorities would define sterile as "free of viable organisms", and sterility assurance has become one of the most scrutinised areas of pharmaceutical and medical device manufacture.

The favoured method of manufacture of sterile pharmaceutical products includes a terminal sterilisation process, such as steam sterilisation or irradiation. However, many therapeutic agents would not withstand terminal sterilisation, and so aseptic manufacture and aseptic filling processes are required. Without a terminal sterilisation step the risk of contamination, especially from bacteria, bacterial spores and mycoplasma, is increased. Aseptic processing used to produce sterile parenteral drug products and active pharmaceutical ingredients (APIs) involves the handling of pre-sterilised products in a highly controlled environment. Unlike terminal sterilisation, which uses biological indicators (BIs) to provide assurance of sterility, aseptic processing involves maintaining a high level of process control, with careful handling of products until they are sealed within their final containers. Many efforts are made to minimise the risk of contamination, for example: • filling and support areas are engineered to minimise contamination; • air in critical areas is supplied at point-of-use as high-efficiency particulate air (HEPA)-filtered, with laminar flow at a velocity sufficient to sweep particles away from the filling and closing areas; • positive air pressure is used to prevent ingress of airborne contamination, and anything that can be sterilised must be rendered sterile before being taken into the clean area where the process is performed; • cleaning is thorough and validated; • human interventions are minimised; • sanitisation and disinfection practices are fixed and validated; • monitoring is carried out to ensure the process and environment are under control. Despite such measures, contamination is an ever-present threat since there will always be a risk that organisms may be introduced into the process from inefficiencies in air filtration systems, from surfaces and from equipment used in the process. The largest source of potential viable contamination comes from people – the operators running the filling process. Aseptic processing is a process being operated in a controlled, but not sterile environment. The probability of non-sterility cannot be calculated. Instead, the industry works to recognised, accepted contamination levels, and the probability of viable contamination is recognised and calculated. Routine sampling for sterility testing is not sensitive enough to detect such low-level contamination: sample numbers are too small and only gross contamination is likely to be detected. Evaluating contamination Pharmaceutical manufacturers therefore need other means of guaranteeing the quality of their product. This is why process simulations (media fills), supported with environmental monitoring and other related processes, are required. These are used to demonstrate control of the process to the industry standard for allowable contamination levels. Media fills utilise culture media in place of drug product to evaluate the risk of contamination levels. However, such media fills are a snapshot in time, and subtle changes can bring about changes in contamination levels. It is therefore of paramount importance that process simulations are designed to give an accurate representation of the aseptic process. In September 2004, the US Food and Drugs Administration (FDA) replaced its 1987 guidelines with an updated document: "Guidance for Industry – Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practices". Europe quickly followed suit, updating the "EC Guide to Good Manufacturing Practice, Annex I, Manufacture of Sterile Medicinal Products". Within these documents the guidance on aseptic process simulations has undergone significant change. Process simulation The new guidelines pay particular attention to process simulations for the validation of aseptic processing. The media fill should be designed to accurately mimic, as closely as possible, the aseptic processes used in practice. Media fill design is one element within the overall considerations to be made in the validation of an aseptic process. It is rapidly becoming an area requiring more work and focus to satisfy the regulators. A broad understanding of the aseptic process is required. Areas of focus include: • facility and room design; • design of the filling machine; • process flow; • heating, ventilation, and air conditioning design and validation; • utility design and validation; • response to deviations; • trends in environmental monitoring data; • contamination control programme; • QA and QC systems; • process simulations; • personnel training and qualification. An appreciation of the many factors influencing the validation programme allows a process simulation to be designed effectively. Key elements in the simulation to be taken into account include: • type of drug products being filled; • lot and batch sizes; • container and closure configuration; • fill volume; • line speed; • operator shifts and fatigue; • filling line configuration; • sterile hold times; • number of units filled (production versus simulation); • number and frequency of runs; • acceptance criteria; • run duration; • interventions – atypical and typical; • other elements that could impact upon sterility assurance. Worst case conditions are used in many forms of validation, including process simulations. This does not mean deliberately contaminating controlled areas before the media fill, but undertaking the simulation at the limits of a normal process. Media selection The selection of the correct growth medium to be used in the process simulation is a very important step. The medium needs to support the growth of a wide variety of micro-organisms, including aerobic bacteria, yeasts and moulds. The broad range of organisms being looked for is consistent with organisms tracked through the plant environmental monitoring programme. The FDA guidance notes the use of soybean casein digest medium, also known as Tryptone Soya Broth. With concerns about prion contamination from components of animal origin found within such media, it is vital that the medium supplier can provide the necessary certifications and documentation for confirming materials are sourced from "BSE-free" countries. For example, Oxoid Cold Filterable Tryptone Soya Broth, product code CM1065, is a highly nutritious general purpose medium ideal for microbiological media fills. An alternative approach would be to use a medium derived from vegetable materials, such as Oxoid's Cold Filterable Vegetable Peptone Broth (VG0104), which is suitable for use in place of Tryptone Soya Broth. The guidance also indicates that if the product is being filled in anaerobic conditions, usually in a nitrogen environment, an anaerobic medium (such as fluid thioglycollate) is used. As already noted, the new guidelines recommend that media fills mimic the actual aseptic process as closely as possible. One of the main implications of this is where the culture medium is introduced into the process. In the past, manufacturers have made up and sterilised the medium outside of the controlled area and introduced it directly into the filling line, but in order to mimic the process more closely the culture medium should be filtered into the process, just as would occur to a liquid pharmaceutical product. However, this creates several concerns, such as: • dried culture media is usually supplied in a non-sterile form and carries a high bioburden, preventing it from being taken directly into a controlled area. It would be preferential to source media that has been irradiated; • for liquid fills, many holding vessels upstream of filtration do not have the capability to heat culture media to a temperature to adequately dissolve the powder into a solution. Even those that have this ability take up time and energy in heating and cooling. Sourcing a medium that dissolves at ambient temperature would remove such obstacles; • mycoplasma can also be a concern with culture media, so assurance of eradication of mycoplasma would be favoured; • broths traditionally used for media fills do not have good filterability characteristics, and could "blind" the sterilising filters. This would invalidate the process simulation, but would be advantageous to understand the filterability profile (such as Vmax or Vcap) of the microbial growth medium, to ensure filter sizing can tolerate it. Growth promotion testing must be undertaken on the growth medium used for process simulations. There is some confusing guidance as to when to perform this: the FDA guidance document does not specify the timing of the test and the EU Annex 1 document does not even ask for a growth promotion test. However, both the PICS (Pharmaceutical Inspectorate Cooperation) and ISO documents ask that growth promotion testing is performed upon conclusion of the incubation period (usually 14 days). While the latter initially seems to be the more sensible option, it also increases the holding time prior to release, as product is waiting for the growth promotion tests to be incubated and analysed. Many pharmaceutical manufacturers prefer to run growth promotion testing in parallel with the media fill samples. Randomly removing samples from the process simulation run has little basis for detecting contamination. Contamination of the filling line being challenged is a random event, and such samples are unlikely to show all of the contamination present. Growth promotion tests In order to meet the various regulatory guidance "half-way", a compromise would be to fill additional units at the end of the process simulation and use these for the growth promotion test. These can then be incubated under identical conditions as the process simulation samples. This approach ensures that both the process simulation units and growth promotion test units are separated, and that the overall time of the process simulation project is reduced. Any units that are incubated should be inspected prior to incubation. Any defects that compromise the container closure, or non-integral units are rejected. All rejections should be documented, with reasons for rejection and the number of units rejected noted. Incubation is then performed within parameters that have been accepted by the global regulatory authorities to allow the growth of bacteria, yeast and moulds – 14 days at 20°C–35°C (+/- 2.5°C). Units are inverted and incubated for the first half of the incubation period, and then returned to an upright position for the remainder. Also, isolates that are seen in the plant environmental monitoring pro-gramme need to be picked up by a media fill run, and data confirming this should be made available. Through the thorough design and detailed execution of a process simulation, and the use of a high quality growth promotion medium, the meticulous challenge of the aseptic process is tackled. The risk of contamination is established and corrective measures can be drawn up. With worries about mycoplasma, dissolution characteristics, filterability and bioburden, the selection of the correct growth medium is a vital part of this process.