Biocontamination control in pharmaceutical production

Transcription

1 White Paper Data Sheet Biocontamination control in pharmaceutical production and compliance to ISO with validated active microbial air samplers Tim Sandle, Ph.D., Head of Microbiology at Bio Products Laboratory (UK), Tony Ancrum, Global Product Manager for Viable Air Monitoring, Merck Millipore, Anne Connors, Field Marketing Manager, Merck Millipore, Introduction Biocontamination refers to biological contamination of products by bacteria and/or fungi, as well as the toxic by-products of these microorganisms, such as endotoxin and mycotoxins from Gram-negative bacteria and fungi, respectively. When designing a biocontamination control strategy, which is based on the manufacturing process, there are three components to take into consideration, each of which requires risk assessment: 1) designing process systems to avoid contamination; 2) monitoring process systems to detect contamination; and 3) reacting to contamination events and putting proactive measures in place. The design of process systems is where maximal effort should be placed. Although often overlooked by some laboratories, the international standard on biocontamination control, ISO 14698, is an important resource for the development of a biocontamination strategy. There are two parts to this standard: Part 1 covers general principles and methods of biocontamination control; Part 2 covers the evaluation and interpretation of biocontamination data. Both parts of ISO are currently undergoing revision. It is important to clarify the distinction between the cleanroom standard ISO from ISO ISO is a 12-part cleanroom certificate standard that is focused on airborne particulates. This standard covers cleanroom design, HEPA filter specification, pressures, and how to monitor a cleanroom in order to assess the cleanroom class. ISO focuses on the ongoing assessment of cleanrooms for viable contamination. EMD Millipore Corp. is a subsidiary of Merck KGaA, Darmstadt, Germany

2 Despite good design and following available guidances, cleanrooms are at risk for several sources of contamination, of which people are the greatest source. Some studies estimate that people can contribute up to 70% of microorganisms found within a standard cleanroom. Second to people, water is a key source of contamination. The challenge with water is that it not only allows contamination to spread, but it also helps microorganisms to grow. Microorganisms are carried in air streams until they are deposited on a surface. Unless they have recently been disinfected, most surfaces will have contamination on them. The risk arises when the contamination moves from a less critical to a critical location, so it follows that using clean utensils and having clean gloves is very important to minimize contamination transfer. Contamination Control To minimize contamination from people, proper gowning is essential to curtail the amount of shedding of skin matter and microorganisms that a person can deposit within a cleanroom. Localized protection, such as isolators and unidirectional airflow cabinets, should also be established around the product to minimize contact with people. Good cleanroom design includes high-efficiency particulate air filters (HEPA), pressure cascade, and air distribution. Cleanrooms must also be cleaned and disinfected regularly, and transfer of items in and out of the cleanroom must be controlled. Once good design principles are in place, an environmental monitoring program should be designed in order to provide information about the state of control of the facility. It is important to note that environmental monitoring does not replace good environmental control (the design of cleanrooms and operational practices); environmental monitoring only provides a snapshot of time. Individually counts are rarely significant, but it is the trends over time that are important: as counts, as frequency of incidents, and as microflora. The presence of microflora, such as waterborne bacteria or organisms that are hard to kill with disinfectants, may indicate the breakdown of control. ISO Part 1 Monitoring Program The international standard ISO can assist in the development of an environmental monitoring program. Program development involves asking a series of questions each of which requires a risk-based response. Not everything can be monitored and therefore consideration must be given to when and why monitoring should occur. When developing a program, the sampling plan should take into account the cleanliness level required at each site to be sampled as well as which types of samples are appropriate (air and/or surface samples). The plan should also define whether this is a quality control activity or an activity delegated to production. Additionally, it must be determined how to trend the data and how to set limits. Sampling Types The measurement of airborne particle counts is a key part of environmental control. Airborne particles are measured using optical particle counters, whereby air is drawn through the instrument and moves through a laser. The laser calculates the number and size of airborne particles present. In pharmaceuticals, there is either one or two particle sizes to look at, depending upon the region of the world. The US Food and Drug Administration requires particles that are of equal or greater size than 0.5 micron from one cubic meter of air to be counted. In addition to this requirement, EU GMP also requires the monitoring of particles equal and greater to 5 micron. The reason for the 5 micron size requirement is that this is closer to the size of skin cells, which is the most common type of contamination floating within a cleanroom. The limits will be according to the class of cleanroom, and out-of-limits particles may indicate a problem, for example, with an air handling system.

3 Viable monitoring methods all use either a general purpose culture medium like trypotone soya agar (TSA), at a dual incubation regimen of C followed by C, or two different culture media that are used at two different temperatures, of which one of the media is selective for fungi (e.g. Sabouraud Dextrose Agar, SDA). Importantly, the choice of culture media, incubation times, and temperatures all require assessment and validation. There is no clear regulatory guidance on which agar to use, and for how long to incubate. It is also important that before use, each lot of culture media must be verified and compared to the manufacturer s certificate of analysis. There is a requirement to monitor different surfaces, either those close to the product, which may indicate contamination, or other samples to indicate cleaning and disinfection efficacy. Surface monitoring includes testing these various surfaces, including product contact surfaces, floors, walls and ceilings, for microorganisms. The two common methods used are contact plates and swabs. In general, contact plates get better recovery; however, swabs are frequently used for curved and thin surfaces, such as a window frame. The culture media should ideally contain a suitable disinfectant neutralizer to reduce the risk of disinfectant residues that remain on surfaces. Air monitoring methods include active air samplers and settle plates. Active air samplers generally fall into three designs, which affects how they collect airborne particles: still to agar, membrane filtration, and Anderson impaction. The standard requirement is to sample one cubic meter of air and capture the microorganisms onto an agar surface. It is important to know the particle collection size efficiency of an air sampler, known as the D50 value, as well as the biological collection efficiency. Settle plates, in contrast, detect microorganisms that fall out of the air due to gravity. They are useful when placed in the right location, especially within the unidirectional airflow cabinet, and smoke studies are useful for helping to select the best locations. Settle plate results are typically expressed as the number of microorganisms collected per time of exposure. Care must be taken when using settle plates because they are quite easy to cross-contaminate, especially in grade A or ISO class V conditions. Sampling Locations The monitoring plan should also note the choice of sample locations based on the nature of the work to be carried out and the impact that cleanroom operators and equipment (both fixed and portable) will have. Using a risk assessment tool such as Hazard Analysis Critical Control Points (HACCP) helps to construct workflows. Through these workflows, the areas of greatest risk can be pinpointed, the appropriate sample types and locations can be selected, and the basis of a sample map can be formed. Sampling Number, Time and Frequency Next, the number of samples to be taken during a production run can be determined. The time of sampling should also take account of testing after a cleandown testing at the end of a shift, testing at times of highest operator activity or high levels of materials in the area. The frequency of sampling should be assessed based on a risk management approach. Factors to consider may include room activities, product exposure risk, room temperature, process stage, duration of process activities, and water exposure. ISO Part 2 Trending Data In order to identify patterns and possible reasons for a given trend, it is useful to include appropriate information with tables and graphs. Such information includes locations, dates, times, identification results, changes to room design, operation of new equipment, shift or personnel changes, seasons and HVAC problems (e.g., an increase in temperature). It is also important to regularly profile the microorganisms found during environmental monitoring, some of which must be speciated using identification techniques. The species should be profiled regularly for changes in profile, the presence of unexpected or objectionable microorganisms, and cross-comparison (e.g. comparing surfaces to people or differently graded cleanrooms).

4 Planned Revision to ISO There are some issues with the existing standard, particularly how it fits in with EU GMP and FDA guidance documents (e.g. guidance on aseptic filling). There is also a lack of working examples for constructing an environmental monitoring program. Additionally, rapid microbiological methods, such as spectrophotometric counters, are not included. Therefore, there is clearly a need for a revised standard, and this process has been ongoing since January It is important for industry to contribute towards this process. A revised standard is expected to provide the following: Detail on how to classify airborne biocontamination in cleanrooms, including methods of measurement and the validation of sampling methods Detail concerning approaches to classify and monitor cleanroom surfaces Setting of microbial monitoring limits Risk management and assessment techniques Consideration of the relationship between enumeration and the types of isolates detected (objectionable microorganism) Consideration of some of the weaknesses of environmental monitoring sampling methods, such as active (volumetric) air-samplers, which could be outlined Introduce a possible classification scheme for maximum permitted viable counts in a similar way to ISO s tables for particle counts Air Sampler Validation The MAS-100 family of air samplers (Merck KGaA Darmstadt, Germany) is specially designed for monitoring microbiological contamination in aseptic production areas and isolators with the rigorous requirements of the pharmaceutical industry. All MAS-100 instruments are tested and designed to ISO annex B (guidance in validating air samplers). The MAS-100 instrument family was recently independently validated according to this international standard. The test facility incorporated a cleanroom with a volume of 20 cubic meters, and a 10-minute flush of clean, horizontal flow of clean air after sampling. The setup allows for quick turnover of the room between bacterial aerosol challenges. There are two critical factors in air sample validation: physical efficiency and biological efficiency. Suspensions of washed Bacillus atrophaeus spores (NCTC 10073) in distilled water were prepared and used as the source in physical and biological efficiency testing. Suspensions of spores (1 x 10 5 colony forming units (cfu) per ml) in 0%, 0.007%, 0.07%, 0.7% and 7% of potassium iodide (KI) in 80% aqueous ethanol were prepared for use in the physical efficiency testing. The B. atrophaeus solution was then dispensed using a spinning top aerosol generator to produce an aerosol of controlled particle size. This calibrated instrument can determine the actual amount of particles captured by the instrument with high accuracy. Units are placed in semi-circle at the same distance from STAG Units are placed at the same height as each other, a small fan is placed below the STAG and a larger fan above to ensure even distribution Figure 1. Test Installation According to ISO 14698

5 Physical Efficiency For the physical efficiency testing procedure, membrane filtration (diameter of 80 mm, pores of 0.8 microns) was used as a reference method. The filtration system was connected to a vacuum pump and flow was regulated by a mass flow meter. The reference method membranes were then transferred to gross medium and incubated for at least 18 hours at 37 C before examination. The MAS-100 air samplers and the reference method equipment were equidistant from the aerosol generator to ensure consistency in sampling, and appropriate distribution of the bioaerosol within the environment chamber (Figure 1). When evaluating physical efficiency, the impaction speed of the air sampler is an important parameter. The speed should be high enough to aspirate particles down to one micron, and should be adapted in a way that viable particles are not damaged when they are impacted on the agar plate. Also, physical efficiency is the same whether the particle is a microorganism, carries a microorganism, or is an inanimate particle. Physical viable particles aspirated by air sampler = Efficiency (%) viable particles aspirated by reference method All validated MAS-100 instruments perform similarly across the range of the portfolio (Figure 2). The MAS-100 family performs well above the d50 cutoff value according to ISO, and becomes even more accurate as the particle sizes increase. Since all the air samplers have a standard flow of 100L per minute, these data support using a variety of MAS-100 systems in various applications while maintaining consistency in results. Average % Physical Efficiency Particle Size (Mass mean diameter, Microns) Biological Efficiency For biological efficiency testing, a three-jet Collison nebulizer (manufactured by Mesa Labs) operating at a pressure of 26 pounds per square inch (psi) was used to generate the mixed microbial aerosol. The spray suspensions were made up using a stock 10 5 sterile aqueous dilution of the B. atrophaeus spore suspension and suitably diluted recent liquid culture of Staph epidermidis. After the bacterial solution was prepared, the nebuliser was placed in the controlled test chamber. The biological efficiency testing was carried out in a Class III microbiological safety cabinet that allows for greater aerosol control during testing. Because of the nature of cultured microorganisms, biological efficiency results are typically lower than physical efficiency results, leading to extra control measures being put in place. Biological = ratio Se/Ba sampled by the air sampler x 100 Efficiency (%) ratio Se/Ba sampled by reference method The biological efficiency of the MAS-100 range was compared to a commonly used reference Instrument method. Figure 3 contains the results of each type of MAS-100 instrument that was tested, and their corresponding biological efficiency results. These results support the ISO statement that there should be a compromise between the ability to collect particles (physical efficiency) and recover viable microorganisms (biological efficiency). These results indicate great recovery, even at an accelerated impaction rate over the reference method. Instrument % Efficient MAS 100 ISO MH 1 meter MAS 100 ISO MH 9 meter MAS 100 ISO NT MAS 100 NT MAS 100 VF Figure 3. MAS-100 Family ISO Microbiological Efficiency results MAS-100 ISO MAS-100 NT MAS-100 VF MAS-100 ISO MH 1 m MAS-100 ISO MH 9 m Figure 2. MAS-100 Family ISO Physical Efficiency results

6 Selecting an Agar Plate According to ISO 14698, certain criteria should be considered when selecting an appropriate growth medium. It should be a broad, non-selective medium, with appropriate additives or neutralizers to minimize antimicrobial activity of disinfectants or antibiotics. It is also important to evaluate the packaging of the plates used, depending on the level of cleanliness expected in the sample area. Using double- or triple-wrapped plates can help. Additionally, it is recommended to use internally sterilized media, such as gamma-irradiated media, in aseptic areas. The agar should contain the right neutralizers for their intended application, whether the objective is to neutralize VHP, disinfectants, or overcome antibiotic activity from residual products. Following incubation of the sample plates, the colonies of the reference media and ICR media (Merck Millipore), in combination with the MAS-100 air samplers, were evaluated. A third-party laboratory reported both larger colonies and good morphology, and also noted that the strains grew slightly faster using ICR media. Conclusion The international standard ISO can serve as a resource for laboratories when developing a biocontamination control strategy, which should focus on product quality, efficacy and patient safety. Environmental control, or the design of process systems to avoid contamination, is the most important component of any biocontamination strategy. Environmental monitoring is a useful program to verify environmental control. A successful environmental monitoring program must identify and encompass all the possible sources of contamination including people, air, surfaces and water spillages. Environmental monitoring data should be reproducible, taking both physical and biological efficiency into account. The selection of high quality media also has an impact on the quality of environmental monitoring results. The combination of instruments and media chosen must be designed for different clean environments, and the instruments selected for each area should be matched to give the same result using the same media so easy baseline measurements can be achieved. To place an order or receive technical assistance In the U.S. and Canada, call toll-free 1(800) For other countries across Europe, call +44 (0) For other countries across Europe and the world, please visit For Technical Service, please visit EMD Millipore and the M logo are registered trademarks of Merck KGaA, Darmstadt, Germany. BM / EMD Millipore Corporation, Billerica, MA USA. All rights reserved.