Advances in cleanroom technologies

Originally published by Express Pharma in 2010: http://www.expresspharmaonline.com/20100930/expressbiotech13.shtml There have been a number of advances in new cleanroom technologies, which have helped to both reduce the risk of contamination and to streamline process operations. The majority of these technological developments have been orientated towards the manufacturing of sterile products, particularly aseptically filled products. Tim Sandle, Head of Microbiology, Bio Products Laboratory, UK and Madhu Raju Saghee, Corporate Quality Assurance, Gland Pharma review some of the more significant advances in cleanroom technology The pharma manufacturing environment is based around a series of rooms with specially controlled environments. These are termed cleanrooms. A cleanroom or zone, on one level, is simply is a room that is clean. A more specialised meaning is defined by the International Organization for Standardization (ISO) in the international cleanroom standard ISO14644 where clean is defined by a maximum permitted concentration of airborne particles according to an assigned class. The key aspect is that the level of cleanliness is controlled. The regulatory requirements for cleanrooms are detailed by EU GMP or the FDA guidelines.

Tim Sandle, Head of Microbiology, Bio Products Laboratory, UK

Cleanrooms have been used for the preparation of pharma preparations since the 1960s. The genesis of cleanrooms was after World War II when advances in HEPA (high efficiency particle air) filters were applied by NASA to further the space programme. The technology was later adopted by the electronics industry and then by healthcare. Therefore the basic cleanroom design, with control of airflow, air filtration, air velocity, air change rates and so on, has been a stable part of the preparation of pharmaceuticals for the past fifty years. Cleanroom technology did not advance greatly until the late 1990s. This pace of transformation has accelerated more quickly in recent years. In the 21st century, there have been a number of advances in new cleanroom technologies, which have helped to both reduce the risk of contamination and to streamline process operations. The majority of these technological developments have been orientated towards the manufacturing of sterile products, particularly aseptically filled products. Furthermore, given that most new drug development is centered on biopharmaceutical rather than chemicals, the technological innovations have been primarily for drugs produced as injectable liquids rather than solid compounds. The technological drive has arisen through pharma organisations facing incessant pressures to enhance efficiency, reduce costs and minimise risks, including cross-contamination between batches. In light of these different influencing factors this article reviews some of the more significant advances in cleanroom technology being embraced by pharma organisations. Clean air devices
Madhu Raju Saghee, Corporate Quality Assurance, Gland Pharma. He can be reached at madhu@ glandpharma.com.

There have been several innovations within clean air devices situated inside cleanrooms. The most technologically advanced of the clean air devices are isolators. Other applications include restricted access barrier systems (RABS) and weighing stations. Each of these clean air devices are grouped under the category of barrier technology. Barriers range from simple screens or

demarcated zones, through RABS to full physical barriers in the form of isolators. We all in the industry acknowledge that the people are the primary sources of contamination, most of the contemporary cleanroom technologies are centered in separating the operator from the process as shown in figure: 1 Isolation technology Isolators, or microenvironments, are increasingly being used in place of conventional cleanrooms for aseptically filled products. Isolators are used within a cleanroom to provide the highest level of protection against contamination. Isolators in 2010 are really a rather mature technology and choosing to use them is not a brave step into the future. In fact, by most any reasonable technical measure, isolators at this stage of development can, believe it or not, be considered a mature technology. An isolator is: a containment device which utilises barrier technology for the enclosure of a controlled work space. Thus isolators provide a separated environment in order to avoid contamination during processing. Isolators, by their design, are less prone to contamination than conventional cleanrooms. However, although isolators reduce the chance of contamination they are still at risk from contamination, as noted by the FDA: barrier isolators cannot prevent contamination caused by GMP deficiencies such as poor aseptic procedures and inadequate training ofoperators Most isolators consist of a either a solid wall envelope or a flexible wall which serve to separate the inside of the isolator from the surrounding environment as shown in figure: 2. Most pharma isolators, when operative, are at positive pressure relative to the room and have a HEPA filtered air-flow (there are some examples of hermectically sealed negative pressure isolators for applications where the drug poses a risk to the operatives, an example would be a cytotoxic drug manufactured in a hospital pharmacist unit). The pressure level of the isolators is demonstrated by pressure alarms and readings. The internal environment inside most isolators is sanitised using hydrogen peroxide vapour (a surface disinfectant), although some isolators are sanitised using other chemical like peracetic acid. Isolators are said to disinfect or sanitise because absolute sterility cannot be demonstrated (sanitisation, in this context, is the reduction of a number of micro-organisms within the clean environment as demonstrated through the use of biological indicators in validation studies. The biological indicators are a preparation of microorganisms in the endospores state).

Figure 1: Picture showing on how process separation and product protection interact (adopted from Bioquell U.K Ltd).

Isolator systems function to achieve: Isolation between the operator and the process;

A non-corrosive, drainable, and easily cleaned enclosure; Doors to provide access to the equipment inside the Isolator; Glove ports to allow access to machinery and product during testing; Rapid gassing port to introduce materials, product and equipment to the environment; Infeed opening to introduce testing materials and product to the system; Exit opening to allow finished product and test materials to exit the system.

In achieving these functions a greater level of contamination control is achieved than would be possible in a conventional cleanroom. Restricted access barrier systems (RABS) RABS systems are similar in basic design to isolators. Nevertheless there is a fundamental issue that separates RABS from isolators. RABS provide product protection and contamination control by a combination of a physical and aerodynamic barrier over the critical process zone and operate in cleanroom environments of EU Grade B (ISO Class 7). This is the same grade of cleanroom as used to house the older style uni-directional airlow (UDAF) cabinets common in conventional cleanrooms. In contrast, due to the complete separation of the Figure 2: Three Gloved Isolator (Photo Courtesy: La Calhne) environmental to the room isolators can be housed in lower trade cleanrooms. Other important differences between a RABS system and an isolator relate to the degree of closure (RABS are not necessarily full separative devices) and with disinfection (with RABS this is typically manual and with an Isolator this is automated and can be rated using biological indicators of a known population and D-value). These differences aside, the restricted access barriers provide further zoning, by screened barriers and HEPA clean-air filtration above, such that an EU GMP Grade A (ISO Class 5) critical zone can be established. The RABS is superior to a conventional UDAF because the RABS system provides a high level of protection against contamination from operator intervention, under validated conditions, due to the operator accessing the internal environment through gloveports. The construction of an efficient RABS minimises the number of process operations including open door access and manual intervention, which would occur, for instance, with a UDAF covering a filling line. Cleanroom weighing stations Another type of separative device is the cleanroom weighing station. Cleanroom weighing stations are portable devices with controlled environments which prevent contamination of the sample or product but are also sufficiently stable to prevent the air from disrupting the calibrated balance. The construction of such devices, in stainless steel, makes them easy to clean. Clothing

Personnel pose the biggest contamination risk to cleanrooms. However, with most cleanrooms the presence of personnel cannot be avoided other than through the use of restrictive barriers (as with the clean air devices discussed above). With most cleanroom operations the control of personnel is achieved through restrictions on numbers, good quality training aimed at imparting appropriate training and through the use of suitable cleanroom clothing. With cleanroom clothing the primary objective of cleanroom garments is to form a particulate barrier to prevent contaminants carried by personnel from entering the controlled environment and making contact with equipment or product. In terms of general design the full-piece cleanroom suit, mask and gloves has not altered significantly since the 1960s. However, there have been recent advances with laundry technologies and the manufacture of state-of-the-art fabrics applicable for cleanroom wear. Polyester traditionally has been considered the most practical fabric for reusable cleanroom wear because of its non-lint generating filaments. However, the fabric fibers which constitute polyester can break down through gamma irradiation, especially after repeat applications. This has led to advances with new synthetic blends, some which have anti-microbial finishes and others are electrostatic which helps to prevent some particles from being attracted towards the person wearing the gown. Not only the actual material and its properties are important. The filtration efficiency of the fabric together with the experienced comfort must be considered very thoroughly. Filtration and comfort goes hand in hand, but in a contradictory way, i.e. the better the filtration efficiency the poorer the comfort experienced by the user, especially from a thermal point of view. This antagonistic situation is dependent on the way by which the textile fabric is woven: The tighter the weave, the poorer the breathing capacity of the material, the less comfortable to use, and vice versa. In relation to gowning, some changing areas which lead into cleanrooms have utilised air-shower technology as a means to reduce the particulate contamination on operators generated from the lower cleanliness levels common to cleanrooms. Air-showers are specialised antechambers which personnel pass through before entering a cleanroom in order to blow off excess dust particles. Disposable technology and single-use systems Arguably the most significant advances with cleanroom technology in the past two to three years have been with single-use disposable technologies. Such technologies have reduced risks by allowing pharma organisations to move away from equipment which needs to be sterilised or consumables which are recycled or pose 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 sterilization, reduces the use of chemicals, reduces storage requirements, reduces process downtime and increases process flexibility, and avoids cross contamination. Single-use items are typically sterilized using gamma rays (electromagnetic irradiation), which kill microorganisms by destroying cellular nucleic acid. Aseptic connections A critical cleanroom step is the aseptic connection, especially for aseptically filled products. Types of aseptic connections include the connection of a vessel or filter to
Figure 3: BioVigilant system for real time microbial detection (courtesy of BioVigilant Systems, Inc.)

another item of equipment for the transfer of fluids. The major risks arising from this stem from the external environment and from any microbial contamination which could be transferred from the operators hand. 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 laminar flow hoods or other capital equipment to maintain sterility. This principle allows liquid sterile products to be transferred simply and safely, towards or from contained areas, via a small scale rapid transfer ports. These devices shorten the time required for the connection. Companies who manufacture such devices include Pall (Kleenpack), Millipore (Lynx) and Bioquate (DAC). Disposable product holding systems In line with advances with aseptic connections there is a drive towards the adoption of disposable bag technologies in biopharma production and away from fixed, stainlesssteel equipment. This change has arisen because such technologies can reduce validation and clean-in-place requirements, lower the requirements for pure water, clean steam and water for injection (WFI), and cut costs (such as from reduced set-up times). For example, the cleaning and sterilisation down-times for stainless steel vessels, transfer lines, or filter housings might require 810 hours and copious amounts of cleaning solutions and water-for-injection (WFI), none of which is required with single-use approaches. The single-use technology takes the form of plastic bags or packs used to process or store product. The first disposable units were probably filter capsule devices, which could filter small volumes without the need of filter housing and associated cleaning. The next development was single-use sterile bags to replace glass bottles, plastic carboys, or stainless-steel containers for small-volume storage, transport of biological solutions, and growth media. A later development was with disposable mixing systems which 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. For the future, rapidly developing connectivity will enhance the development of connected, integral systems and potentially total disposable processes. Consumables The transfer of materials into and out of a cleanroom or clean air device is, as noted in EU GMP, one of the greatest contamination risks. Most items which are untreated by sterilisation or disinfection are contaminated with microorganisms. This phenomenon creates a problem as many items, particularly plastics cannot be sterilised. Although such items can be disinfected, where contamination originates from spore bearing microorganisms the contamination cannot be removed by many of the standard cleanroom disinfectants. This risk has helped to trigger the recent advance in disposable, plastic irradiated consumables for cleanrooms. These are typically presented in sterile multi-packs. These are bags, often constructed from polythene or other low particulate material, which contain consumables like connectors and syringes. The packs are normally double or triple bagged so that any contamination on the outside is removed prior to transfer into a critical area. Filling machines The technology related to product filling machines in cleanrooms continues to advance with systems designed to increase throughput and to decrease the big risk of operator intervention. The most robust machines combine in-line filling and stoppering with

continuous motion positive in-line transport systems, which are particularly suitable for filling liquid solutions into cylindrical vials and for rubber stoppers insertion. The construction of modern filling machines means that they can be exposed to the VHP sterilisation gas agent required for isolators, whilst the flexible nature of contemporary design allows machines to be positioned under RABS or isolators. Many of the current technologies utilise lasers to measure fill sizes, headspace gaps and closure seals. Furthermore, in keeping with the earlier discussion on single-use disposable technologies many filling machines use peristaltic pumps with single-use tubing or tubing sets and disposable filling needles and manifolds. The advantage of peristaltic pumps is that the piston pumps associated with older models of filling machines have many moving parts which can not only break down but are also a common source of airborne particle counts. With these technologies careful calibration is required to ensure accurate and consistent fill volumes. Nevertheless providing that these difficulties of measurement are overcome the many advantages of sterile disposable design can be successfully applied to filling lines. Cleanroom decontamination There are different techniques used to decontaminate cleanrooms. Traditionally this has involved either manual cleaning or disinfection by operators using mops and buckets, or decontamination with fumigation units and chemicals like formaldehyde (which, in the gaseous form, creates significant health and safety concerns). More advanced means for cleanroom decontamination involve the use of vapourised hydrogen peroxide (VHP). VHP is created by liquid hydrogen peroxide being placed into special generators which, when operated, create a gas. VHP is an oxidising agent and readily kills most microorganisms including spore forming bacteria like Bacillus. Gas generators can be placed into cleanrooms at known locations. This takes some time and effort in mapping the cleanroom (which additionally requires some form of validation using chemical and biological indicators). More advanced applications of VHP generators site the generator in the HVAC system so that the gas vapour can be evenly distributed into all areas of the cleanroom. Comprehensive validation studies are required to develop the appropriate cycles (which move from dehumidification, through conditioning, to decontamination and finally aeration), however once the validation studies have been completed the time savings are considerable and the effectiveness of decontamination is greater than comparable manual cleaning methods. Cleanroom surfaces and flooring Surfaces and equipment Various items of equipment and surfaces are now manufactured with antimicrobial coatings. This is more commonplace in the US than in Europe, although the number of items available is increasing. One example is the incorporation of silver which is effective against a range of micro-organisms. An advantage of silver ions 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. One of the risks 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. In an article by Sandle, the author provides empirical evidence that microbial contamination is significantly reduced by using polymeric flooring to reduce contamination. Energy efficiency 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 BS EN 16001, a new European energy standard are increasingly used (both standards are likely to be amalgamated into international energy standard ISO 5001. Care must be taken when adopting such standards in relation to contamination control for the enthusiasm of 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. Environmental control 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). The FMS technology in the 1980s and 1990s connected particle counters to a manifold system. Such a system meant that particle counters were only operated intermittently and that there was a risk, given the need to locate vacuum pumps centrally, that particles could be lost as they were drawn from the counter down lengths of tubing. The FMS systems of the Twenty-First Century 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. Other recent developments in environmental control are real-time microbiological airsamplers (so-called instantaneous microbial detection counters) as shown in figure-3. These systems are capable of instantaneously detecting the presence of extremely small particles using optical technology and determine, on a particle-by-particle basis, the size of each particle, the total quantity of each size of particle, and if each particle is biological or inert. The advantage of such systems is that they can alert cleanroom operators to the start of a microbiological contamination problem so that action can be taken and the potential loss of a product batch avoided. Conclusion Cleanroom technologies have evolved impressively over the past twenty years to offer a greater range of options and easier means to achieve those options, and better productivity and cost-effectiveness. The many benefits of advanced technology, however, require careful planning, validation and investment so that they can satisfy external regulators and senior management.