Dr Tim Sandle, Head, Microbiology at Bio Products Laboratory and visiting tutor at the Department of Microbiology, University of Manchester, UK discusses some innovations relating to cleanroom and clean device operations, together with personnel control and environmental monitoring
Pharmaceutical manufacturers of both sterile and non-sterile products, and medical devices, are required to demonstrate that manufacturing processes and procedures minimise any potential contamination to the product from the manufacturing environment. Contamination can arise from a number of sources: water, air, surfaces and personnel, each of which poses a potential risk to product.
These risks of contamination are avoided by putting environmental controls in place (through correct grade of air-supply, satisfactory cleaning and disinfection practices and so on). Where controls cannot off-set every contamination risk, and also as a means to demonstrate the level of control, environmental monitoring programmes are devised and put into action(1).
The primary protection from contamination is through well-constructed and maintained cleanrooms. This is supported by trained personnel, following strict gowning protocols, and cleaning and disinfection(2). Once environmental control has been accomplished, verification is undertaken through environmental monitoring (for both particulates and viable microorganisms).
Cleanrooms and clean air devices are typically classified according to their use (the main activity within each room or zone) and confirmed by the cleanliness of the air by the measurement of particles. The primary objective of cleanrooms in pharmaceutical processing is to minimise and control microbial and particulate contamination. There are many sources of contamination. There are four principles applying to control of airborne microorganisms in cleanrooms. These are(3):
- Filtration (through the use of HEPA filters). The air entering a cleanroom from outside is filtered to exclude dust, and the air inside is constantly re-circulated through HEPA (High Efficiency Particulate Air) filters (alternative filter are ultra-low penetration air (ULPA) filters). This is controlled through a HVAC (Heating, Ventilation and Air Conditioning) system.
- Dilution (to ensure that particles generated in cleanrooms, in addition to those which pass the filters, are carried away by diluting the area with new “clean” air).
- Directional Air Flow (to ensure that air blows away from critical zones, as particles and microorganisms cannot “swim upstream” against a directional air flow). This is achieved through pressure differentials.
- Air Movement (rapid air movement is important for as long as particles and microorganisms stay suspended in the air they are not really a problem, for it is only when they settle out that they become an actual cause of contamination).
Innovations with cleanrooms
There have been several advancements or changes in approach relating to cleanrooms. These include the use of modular cleanrooms and studies in energy efficiency, designed to make cleanrooms cost effective whilst still maintaining contamination control principles.
Modern approaches to cleanroom design is aimed at ensuring that the cleanroom is designed at optimising contamination control. It is important to dedicate time in designing cleanrooms and the equipment located in cleanrooms for, if there is a design fault in one part, this will affect the items of equipment and if there is a fault in conception stage this will be expensive and time consuming to rectify.
For cleanroom design, modern approaches utilise Computer Aided Engineering software for the design process, such as Building Information Modelling (BIM) software. Such software covers geometry, spatial relationships, light analysis, geographic information, quantities and properties of building components (for example manufacturers’ details)(4). Systems, assemblies and sequences can be shown in a relative scale with the entire facility or group of facilities. When designing modern cleanrooms, the following approach should be adopted:
- The type and function of the cleanroom should be established. This should include the required cleanroom grades or classes and how cleanrooms of different grades will interact (including requirements for air-locks and pressure cascades).
- The most important aspect is drawing up the process flow. Here the cleanroom management, together with engineers and quality assurance personnel, should map the path that equipment, product and operators will take in the cleanroom.
- Established quality risk management tools like HACCP (hazard analysis and critical control points) or FMEA (failure modes and effects analysis) can be used for this purpose. Areas which pose a contamination control risk should be noted and attempts should be made to design these risk areas out (the principles of quality by design). Other considerations can also be included at this stage, including whether there is adequate clearance under door frames for equipment to pass through.
- In the design, there should be sufficient space for equipment and connections.
- The cleanroom should be constructed from a material which is compatible with different cleaning and disinfection solutions.
- Ideally, a mock-up of the cleanroom should be constructed. This is particularly important for testing the process, product and personnel workflow. In terms of understanding contamination control it is essential to understand what objects are passed from one class of cleanroom to another.
Modular cleanrooms and bespoke design
Modular cleanroom are cleanrooms that are assembled from prefabricated modules. This process of cleanroom construction differs from standard (or ‘common’) cleanrooms in that:
- Common cleanrooms are assembled at the construction site from many elements
- For modular cleanrooms a significant part of assembling works is done at the factory that produces modules. Only assembling of complete modules remains for the customer’s site.
Common cleanrooms are tailor made cleanrooms. Their design follows specific layouts that are drawn by the technologist from understanding specific processes. Whereas modular cleanrooms are often designed to fit into existing spaces. With modular cleanrooms other restrictions can appear. This is because the cleanroom construction process is separated into two parts, which are executed in two different places: the modules manufacturer and the customer’s site. This can present certain difficulties in terms of transport and later assembly (5).
Some types of equipment and surfaces can be manufactured with antimicrobial coatings. One example is the incorporation of silver or copper which are effective against a range of micro-organisms. An advantage of silver ions, for example, is that although they have antimicrobial properties, silver is rarely toxic against human cells. Examples of the application of silver include implements like forceps. Also in relation to surfaces, the incorporation of wipeable surfaces onto equipment allows for the easier cleaning and disinfection. Some of these innovations include polythene covered computer keyboards.
Cleanroom technologies are not only directed towards contamination control. The energy efficiency of cleanrooms is currently of great importance for companies who wish to save costs and to reduce the amount of carbon generated. To address this International Standard ISO 14001, which describes environmental management and practices and EN 16001, a European energy standard, are becoming increasingly used (both standards are likely to be amalgamated into international energy standard ISO 5001). Despite the appeal of controlling energy consumption, care must be taken when adopting such standards in relation to contamination control for actions to alter the operation of HVAC (heating ventilation and air conditioning) parameters can have an impact upon the level of non-viable particles and viable counts. Therefore microbiologists should always be involved in any energy saving projects.
The use of barrier technology protects critical cleanroom operations. Within many cleanrooms unidirectional airflow (UDAF) units are found. A UDAF is classified as a minienvironment; an alternative term is ‘separative devices’ (separative devices range from open to closed systems and include isolators and Rapid Access Barrier Systems (RABS)). These are localised environments created by an enclosure to isolate a product or process from the surrounding environment. The advantages in using a minienvironment include the following:
- Minienvironments may create better contamination control and process integration.
- Minienvironments may maintain better contamination control by better control of pressure difference or through the use of unidirectional airflows.
- Minienvironments may potentially reduce energy costs.
Of these types of micro-environments, the most widely used for contamination control in relation to aseptically filled products are isolators.
Isolators intended for aseptic processing are required to be operated under positive pressure and are subjected to decontamination process before start of the batch processing. Modern isolators more often use vapourised hydrogen peroxide, although alternatives are available including peracetic acid or chlorine dioxide(6). These methods can also be deployed for the decontamination of cleanrooms.
The key principles for isolator use are (7):
- The air exchange between the isolator and with the surrounding environment must occur only through a microbial retentive filter such as HEPA or /Ultra Low Penetration Air (ULPA).
- The positive pressure aseptic processing isolator must be decontaminated in a reproducible and quantifiable manner to ensure the sterility assurance level of 10-6. This is assessed through the use of biological indicators of a suitable population, species and resistance. For vapour phase hydrogen peroxide systems, geobacillus stearothermophilus is normally used.
- Entire activity / handling of materials inside an isolator shall be achieved remotely; any part of human body cannot enter the isolator.
- Asepsis shall be maintained for each unit operation and for material transfers. Any material entering the isolator must either be decontaminated inside the isolator or shall be sterilised and taken inside via a rapid transfer port.
Disposable sterile plastic technology
A major advance with cleanroom technology is with single-use sterile disposable technologies. Such technologies have reduced risks by allowing organisations to move away from equipment which needs to be sterilised (such as stainless steel vessels). It also negates the need to use consumables that are recycled or which present a risk with their transfer into cleanrooms, to disposable and single-use sterile items. The advantages of this technology is that it eliminates the need for cleaning, eliminates the need for the pharma company to perform in-house sterilisation, reduces the use of chemicals, reduces storage requirements, reduces process downtime and increases process flexibility, and avoids cross contamination. Single-use items are typically sterilised using gamma rays (electromagnetic irradiation), which kill microorganisms by destroying cellular nucleic acid(8).
A variant on single use technology is the aseptic connector. Innovations in aseptic connection technology have led to the development of single use connector systems. These are based on the so-termed alpha-beta principle which allows the connection to be performed in an environment this does not require unidirectional airflow or other capital equipment to maintain sterility.
Disposable holding devices
Plastic technology has also led to an array of sterile plastic holding devices. An example is with disposable mixing systems. These can be connected to capsule membrane filters and a hold bag. These interconnected disposable systems have a considerable advantage in that they are gamma sterilised and ready to use.
Given that people are one of the primary sources of microbial contamination in cleanrooms (through shedding of skin flakes, many of which contain microorganisms), attention has been paid to gowning. While behaviours and techniques for gowning can be addressed through training and procedures, aspects of the gown design require attention.
Manufacturing gowns using continuous strong fibres of pure high-density materials, like polyethylene, fabrics can be constructed that are low linting and free of inherent contaminants that could represent a risk in critical environments. Cleanroom managers are more often requesting certification for gowns to show that they are low in particulates. For gowns that are re-laundered, stipulating the maximum number of times that a gown can be washed and irradiated is important.
A risk to cleanrooms arises from personnel transferring contamination into the area via footwear or through equipment transfer (such as trolley wheels). One way to minimise contamination is to use special mats which are designed to remove dirt, particles and micro-organisms. Where such mats are used they have traditionally been sticky-mats. Although these are fairly effective, more efficient contamination control can be achieved from polymeric flooring.
Polymeric flooring is an especially designed ‘plastic’ which works through electromagnetic forces causing particles to be attracted from surfaces like footwear, and retained on the surface of the mat. This mechanism ensures that any contamination residing on the mat is not passed back onto the personnel who walk across it.
The most significant advancement with microbiological monitoring methods has been the advent of Real Time Laser-Induced Fluorescence Systems. These instruments continuously monitor both inert particulates and viable microorganisms in real time. They are very sensitive, where the limit of detection can be down to 1 microbial cell. It provides both total particulate and viable counts.
The instruments are based on optical spectroscopy. This is an analytical tool that measures the interactions between light and the material being studied. These instruments work by elastic light scattering. This measures two things (9):
Particle counts: where the size of a scattering particle, as it passes through a light beam, is comparable to a certain wavelength of light. The intensity of the scattering is dependent upon the size of the particle. Such systems will detect and quantify particles within a 0.5 to 20 um range.
Microbial counts: a 405 nm laser that intersects the particle beam, so that as a particle passes through the inelastic scattering measures the intrinsic fluorescence of the particle, from the metabolites (such as NADH and riboflavin) inside microorganisms.
There is a growing trend within the pharma industry towards the use of Process Analytical Technology (PAT). The goal of process analytical technologies (PAT) are to improve consistency of product quality, provide “right first time” manufacturing (to reduce costs and reduce cycle time), reduce the regulatory delays associated with changes in manufacturing, and improve the safety of chemical processes. PAT could also be used to reduce the end-of-line laboratory testing. The ‘real time’ counters fit well with this paradigm.
With more classical environmental monitoring methods (the use of agar plates), it is now commonplace to be able to track the use of the media through barcoding. Scanning bar codes allows the information about the plate, such as the media batch number and expiry time, to be transferred to a Laboratory Information Management System (LIMS). To ensure that the sample is not at risk to adventitious contamination, several types of Petri dishes now come equipped with lockable lids.
Arguably the most important aspect of environmental control in a cleanroom is the control of airborne particulates as this is a direct indicator of cleanroom contamination. Particles in the air are measured through particle counters. The most efficient means of monitoring particles is by linking particle counters to a facility monitoring system (FMS). It is consist of discrete particle counters, each with individual pumps, and the data is sent using wireless ethernet to a central data capture system. Modern particle counters have the advantage that they meet the more rigorous demands of the new international standard for particle counter calibration (ISO 21501). In the event of a counter breakdown a spare counter can quickly replace the malfunctioning counter due to ‘plug-and-play’ features and the particle counting software will record the serial number for audit purposes. This feature is important for aseptic filling where continuous particle counting is a GMP requirement.
1. Sandle, T. and Saghee, M.R. (2013). ‘Cleanroom certification and ongoing compliance’. In: Sandle, T. and Saghee, M.R. Cleanroom Management in Pharmaceuticals and Healthcare, Euromed Communications: Passfield, UK, pp169-184
2. Sandle, T. (2012a). ‘Application of Disinfectants and Detergents in the Pharmaceutical Sector’. In Sandle, T. (2012). The CDC Handbook: A Guide to Cleaning and Disinfecting Cleanrooms, Grosvenor House Publishing: Surrey, UK, pp168-197
3. Halls, N. (2004): ‘Effects and causes of contamination in sterile manufacturing’ in Halls, N. (ed.): Microbiological Contamination Control in Pharmaceutical Cleanrooms, CRC Press, Boca Raton, pp1-22
4. Sandle, T. (2013). Application of Quality Risk Management to cleanroom design, Clean Air and Containment Review, 13, pp24-25
5. Sandle, T. (2014) Modern Approaches to Pharma Cleanroom Design, Controlled Environments, 17 (1): 8-10
6. Mau, T., Hartmann, V., Burmeister, J., Langguth, P. and Häusler, H. (2004) Development of a sterilizing in-place application for a production machine using Vaporized Hydrogen Peroxide, PDA J Pharm Sci Technol. 58(3):130-46
7. Midcalf, B, Neiger, J. and Sandle, T. (2013). ‘Fundamentals of pharmaceutical isolators’. In: Sandle, T. and Saghee, M.R. (Eds.) Cleanroom Management in Pharmaceuticals and Healthcare, Euromed Communications: Passfield, UK, pp185-226
8. Sandle, T. and Saghee, M.R. (2012). “Application of Sterilization by Gamma Radiation for Single-Use Disposable Technologies in the Biopharmaceutical Sector”, Pharmaceutical Technology, Supplement: Bioprocessing and Pharmaceutical Manufacturing, May 2012, S20-S27
9. Sandle, T. (2012b). Real-time counting of airborne particles and microorganisms: a new technological wave?, Clean Air and Containment Review, Issue 9, pp4-6