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Published on 1 March 2004

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Isolators: maintaining sterility standards


Brian Midcalf
BPharm FRPharmS
Pharmaceutical Isolator Working Party Chairman

In the last issue, we gave an overview of isolator use from an American perspective, and then looked at the different materials used in isolator construction and why they might be chosen. We now look at the standards controlling isolator manufacture and maintenance, and at decontamination processes used to maintain sterility.

Separative device standards
Mike Foster of Bassaire Ltd brought delegates up to date with the latest information appertaining to ISO 14644-7 Cleanrooms and Associated Controlled Environments – Part 7: Separative Devices, which focuses on clean air hoods, glove boxes, isolators and mini-environments. The standard is supported by the other sections in the 14644 family of documents. A “continuum of devices” shows a progressive list by increasing integrity.

ISO 14644-7 is separate from BS EN 12469:2000, the latest standards for microbiological safety cabinets, and it is not application-specific. Mike defined a “separative device” as a device that separates people from processes using elements of quantifiable performance, such as contained enclosures, transfer devices or systems and access devices, and uses HEPA (high-efficiency particulate air filtration) and ULPA (ultralow particulate air) filters.

The standard was developed under the umbrella of the ISO TC209 14644 series of clean air standards, and the document received peer review at draft international standard (DIS) stage. Comments were incorporated on 4/5 October 2001, and the standard has advanced to final draft international standard (FDIS) stage, with publication expected in the near future.

The standard covers the requirements of pharmaceutical isolators, semiconductor mini-environments, clean air cabinets, glove boxes and gas environments and restrictive access barrier systems. Pharmaceutical isolators are used for sterility testing, semiconductor mini-environments and pharmaceutical filling lines, and may be a clean air cabinet, vertical flow clean air enclosure, high-integrity gas enclosure or high-integrity gas glove box.

Transfer devices are covered in a separate section of the standard (Annex D). Consideration needs to be given to interlocks, siting and installation, air classification of the room and operational ergonomics.

The standard is divided into four sections:

  • Annex B deals with air-handling systems, gas systems and gas recirculation systems.
  • Annex CAccess devices relates to access devices, gloves, glove systems, glove ports and glove port bungs, glove changing procedures and half suits.
  • Annex D – Transfer devices addresses transfer devices listed from low- to high-integrity, which are similar to the UK PIWP (Yellow guide) with additions.
  • Annex E – Leak testing (informative) contains procedures and considerations, test equipment used, quantitative leak rates and equation developments.

Test methods are as described in ISO10648-2 (oxygen, pressure decay and pressure maintenance) and ISO14644-7 (Parjo comparator test and glove testing). Mike concluded with a warning that things often don’t go wrong gradually; they go wrong all at once.

Decontamination and contamination control
Gerald McDonnell of Steris delivered a paper on decontamination and contamination control using sporicidal gassing. He noted that a variety of methods can be used for decontamination and contamination control. Liquid-based formulations are applied manually or semiautomatically (by fogging). These formulations will vary in their antimicrobial efficacy, material compatibility and safety. It can be difficult to ensure reproducible coverage with liquid-based methods, and application can be time-consuming and labour-intensive. Fumigation processes are more widely used for isolator decontamination, and primarily use formaldehyde and hydrogen peroxide vapour. Other methods include condensed hydrogen peroxide, peracetic acid, chlorine dioxide and ozone. The hydrogen peroxide system is based on condensation-free conditions and is very effective in practice. Relative humidity in the chamber is controlled so that condensation is prevented.

Gerry also delivered a paper later in the conference discussing microbiological kill mechanisms and the chemical inactivation of potentially hazardous agents by strongly oxidising decontamination agents.

David Watling of Bioquell Pharma presented a talk on the role of hydrogen peroxide as a gaseous surface biodecontaminant. He provided delegates with a theory on gassing cycles and parameters, and how biodecontamination in microbiological safety cabinets, isolators, restricted access barriers (RABs), rooms or full zones can occur.

The process is essentially a surface activity, and consideration should be given as to how the hydrogen peroxide arrives at the surface of the micro-organism. Has water a role to play, can dry gaseous hydrogen peroxide kill microorganisms and will liquid hydrogen peroxide kill microorganisms?

Factors relating to gaseous and liquid concentrations and solution vapour pressures were described. The calculations used to interpret the conditions were shown and the theory of microcondensation was explained. This research formed the basis of the current hydrogen peroxide vapour sanitising or sterilising system, which can be used for direct injection into single or multiple isolators, or RABs. The system can be a mobile system with heating, ventilating and air conditioning (HVAC) aeration, a fixed installation with a gas generator in a technical area or a room biodecontamination service (RBDS), which is “infinitely” scalable. It takes into account dew point variables (eg, temperature, relative humidity, gas distribution and gas concentration), and dew point is often reached without visible condensation.

The technical nature of this paper stimulated much discussion during the conference and, although not everyone was in agreement with the theory, it seemed to provide a good, logical explanation for the way in which gaseous hydrogen peroxide works, with this technique becoming more frequently used in the industry and smaller-scale establishments.

Biological indicators
Graham Steele, consultant microbiologist, discussed biological indicators and their use in the sporicidal gassing of surfaces within separative enclosures. His paper reflected the work of the Parenteral Drug Association (PDA) biological indicator working group.

Graham defined a biological indicator as an inoculated carrier contained within its primary pack, ready for use and providing a defined resistance to the specified sterilisation process. The organisms used in biological indicators should have a higher resistance to the sterilising agent than the bioburden (the microbiological contamination level on the material to be decontaminated), sporulation should occur efficiently on readily prepared and defined media, and they should retain their resistance characteristics over long periods during storage. Desirable attributes of organisms for use in biological indicators include homogeneous dispersal with little tendency to form aggregates or clumps; in addition, survivors should germinate readily without the need for germination inducers, and should give easily counted colonies on the recovery medium.

There are a number of “levels of standardisation”, which include intra-assay, interassay, interlaboratory and intermethod assessments. Biological indicators provide a means to assess directly the microbial lethality of a sterilisation process and consist of a defined population of test organisms presented so as to allow their recovery following the sterilisation procedure. Variability in biological indicator performance arises during manufacturing, handling and storage, as well as through differences in presentation during the gassing cycle and from the recovery environment.

Graham went on to identify anomalous residual viability of biological indicators exposed to sporicidal gassing cycles. Some deviations from ideal death kinetics and tailing effects can occur as a result of faulty experimental equipment or procedures. This may be due to the presence of large clumps of cells in the population; it may also reflect cell-to-cell variations in resistance to the stress, or may be due to heterogeneity in the population. A number of factors affect the resistance characteristics of biological indicators. These are either genotypic or phenotypic variations. The origins of genotypic variations in spore resistance include mutations to the genomes of cells within a pure population, population heterogeneity due to strain impurity and gross contamination leading to more than one species being present. Phenotypic variations relate to variations in the growth and temperature of sporulation media used to prepare the spores, with respect to limiting carbon and nitrogen or the presence of divalent cations, such as Mn2(+) and Ca2(+).

Investigations on the carriers used for the spores as biological indicators showed remarkable variations, and some producers were more consistent than others in this preparation. The different carriers investigated were steel, polymer and paper.

Faults found included spores deposited at the edge of the carrier, an encrusting spore layer, occlusion of spores, and a high proportion of vegetative cells. The chosen carrier material should reflect the surface characteristics of the principal exposed surfaces of the isolator, must not affect the viability of the indicator organism, must be unaffected by the sporicidal gassing agent, should not retain the sporicidal agent and must not interfere with the chosen recovery method for enumeration of the spores loaded on to it.

Reporting on the biological indicator PDA taskforce, Graham indicated that the remit was to investigate the extent of the problems associated with biological indicator use in isolators, to collate evidence from interested parties and draw up recommendations for the production, control and use of biological indicators for sporicidal gassing cycles.

The draft PDA monograph entitled Biological Indicators for Sporicidal Gassing of Separative Enclosures includes: a user requirement specification for a biological indicator; guidelines for the preparation of biological indicators; the use of biological indicators in monitoring gassing cycles (including the setup, collection and recovery of exposed biological indicators); and documentation, audit and standard operating procedures and comments on variables in the use of biological indicators. Some feedback from the PDA has been received, and a number of action points have been identified.

Wander ter Kuile of Vokes Ltd described the filtration process and illustrated his talk with examples of the importance of filtration. He described a filter as a device that removes particulate from an airstream as the particulate- laden air passes through it. He then described the six separate mechanisms that act in combination or predominate depending on the filter type and the form of contaminant. These filtration methods are gravitational , straining, interception, inertia, diffusion and electrostatic effects.

Filter materials include coarse glassfibre, nonwoven polyesters, fine glassfibre, spun-bonded or meltblown synthetics and microfine glassfibre paper. Classical HEPA filters can be cartridge, deep-pleated or multiwedge construction and are mostly used in ducts or terminal devices.

The different methods for the testing of HEPA filters were discussed. Wander noted that filters are a specialist product used in air handling systems to protect sensitive processes, delicate equipment, personnel or the environment and, as such, they provide the means of controlling air quality to exacting standards. Test methods are part of the production process, and each filter is individually certified to enable traceability.

The commonly used standards are BS 3928:1969 and BS 5295:1989. BS 3928 is a volumetric method using a relatively large particle size that measures only  overall efficiency. It uses the sodium flame test with an aerosol of sodium chloride 2% solution, and efficiencies of up to 99.999% are detected using photometry.

BS 5295 is used as a leak test but is not sufficiently accurate for the performance range of filters needed. It uses dispersed oil particulate (DOP) with a challenge of mineral oil aerosol of 0.3mm particles. Loading of the filter can be higher than BS 3928, and the efficiency is also to 99.999%. The standard is being superseded.

A further standard, BS EN 1822:1998, tests and classifies HEPA and ULPA filters using a synthetic oil aerosol of 0.12–0.3mm size range. Efficiency is measured at the maximum penetrating particle size (MPPS). The most important criteria within an air filter system is the installation of the correct grade and the quality of filters. These filters will require scheduled maintenance and replacement. A poorly fitted, damaged or missing filter will allow the passing of unfiltered air, which will defeat the whole object of their installation.

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