Recently, many industries from pharmaceutical, biotechnology, medical device, hospital pharmacies and medical disposables have clean room operations of differing sizes and complexities. Clients in these industries have recognized that there is a regulatory compliance to demonstrate clean room performance and controlled product bioburden with monitoring the environmental conditions in their aseptic manufacturing areas. Environmental Monitoring (E/M) is a program designed to demonstrate the control of viable (living microorganisms) and non-viable particles in critical areas. These areas include clean-rooms for drug fill/finish, formulation tank rooms, laminar flow hoods, biological safety hoods, isolators, glove boxes, molding machines, kit assembly lines, Intravenous (IV) compounding areas and sterile packaging. This article will discuss environmental monitoring from a viable particles perspective. Viable monitoring refers to testing for the detection and enumeration of bacteria, yeast and mold. It includes the monitoring of personnel, air and area surfaces for microbial contamination. And differs from non-viable environmental monitoring which is a reference for particle counts measured by a laser counter. Viable counts provide metrics on the potential for contamination of a company’s products as well as demonstrating the veracity that a clean room is functioning as designed and being properly maintained. Surface and air monitoring exhibit the asepsis of the product manufacturing operation,
Companies who have their clean room facilities monitored do so to ensure their desired/required quality standards are met. The areas that are sampled in a manufacturer’s clean room include:
1. Personnel – Personnel are the biggest source of contamination in clean areas. Personnel harbor millions of bacteria, carrying them with them everywhere they go. Gowning is the most effective way to protect the cleanroom environment from ourselves. To asses the effectiveness of the gowning program personnel may be monitored on a regular basis for viable counts. Personnel monitoring employs contact plates to assess microbial contamination of clean room personnel.
2. Air - the air in a clean room is controlled and Monitored on a regular basis (e.g., daily, weekly, quarterly) for particle counts, viable counts, temperature and humidity. HEPA filters are used to control the viable and non-viable particulate counts within the air. HEPA filters have the capability to filter out particulates down to 0.2 ?m in size. These filters usually run continuously at a calibrated flow rate in order to maintain the required air quality within the room. Humidity is usually kept at a low level in order to help prevent the proliferation of microbes within the room such as bacteria and mold, which tend to prefer damp conditions in order to replicate.
3. Surfaces (including floors, walls, equipment, etc.) are cleaned and monitored on a regular basis for viable counts by using specially designed contact plates that contain a growth medium called Trypticase Soy Agar (TSA) and Sabouraud Dextros Agar (SDA). The TSA is a growth medium designed for bacteria and the SDA and a growth medium designed for mold and yeast. TSA and SDA are typically incubated at different temperatures, TSA at 30-35 C which is mainly the optimal growing temperature for most environmental bacteria, and 20-25 C which is the optimal growing temperature for most mold and yeast species.
Two methods of Air sampling in a Clean Room
1. Air Samplers (active air sampling) – Air samplers draw in predetermined volumes of air. The air is drawn over a sterile media plate, which is later incubated to reveal the number of viable organisms per cubic feet or liter. Currently agar impaction is the method of choice throughout the industries. Using a specially designed, and calibrated piece of equipment which holds the media plate under a perforated lid and draws in a known amount of air one can accurately measure the amount of viable bacteria within the air.
2. Settling plates (passive air sampling) - Petri dishes containing sterile growth media are exposed to the environment for a specific period of time, usually between 30-60 minutes but can be exposed up to four hours before compromising the integrity of the media itself. Viable microorganisms which settle onto the media surface will grow after the plates are incubated. However, passive air sampling is tending to be phased out because it does not reflect microbial contamination with an accurately measured volume of air.
Two methods for surface monitoring in a Clean Room
1. Contact Plates – as mentioned above are special Petri dishes which contain sterile growth medium prepared in a manner so the surface of the media protrudes above the rim of the plate. The contact plate is pressed against any flat surface the needs to be sampled. Any viable microorganisms on the surface will stick to the agar surface and will grow upon proper incubation. This technique reveals the number of viable microorganisms on a surface.
2. Swabs - are sterile and stored in a suitable sterile liquid. The swabs are rubbed over the test surface. The microbiologist can determine the type of microorganisms on the swab by subculturing it to media. Swabs are used for surfaces that are not flat, and can be used to sample hard to reach areas of machinery that could not be sampled with a contact plate. Swabbing is more qualitative than quantitative.
How Personnel are monitored in a Clean Room
Contact Plates - Personnel in critical areas may be monitored for microbial contamination utilizing contact plates. The contact plates monitor areas of the body that may interact with the sterile field or product exposure areas. These may include gloved hands, forearms, or other areas. Personnel monitoring is a good indication of how well personnel are gowning when they enter the clean room. Many companies utilize this testing for proficiency based training programs for clean room personnel.
Environmental organisms recovered from manufacturing areas should be identified to show what organisms are present, and what may be contaminating the product. Knowing will provide the company with important information in monitoring and preventing potential future contamination pitfalls. Microbial identification is another service which E/M Associates, Inc offers. It is important that proper disinfectants are used on a routine basis to keep the level of house organisms in check. A disinfectant study should be implemented to demonstrate the effectiveness of the sanitizers used against "house organisms." This effectiveness study exhibits to regulatory agencies that the company is using the correct sanitizer at appropriate dilutions and contact times to combat potential contamination.
There are many factors one should consider when determining what locations in a clean room to sample. These factors include potential product exposure areas, processing parameters, HEPA locations, equipment design and validation criteria. Frequency of monitoring depends on product and quality requirements. Sampling frequency may be subject to change depending on trending analysis and changes in equipment, processing or number of personnel. A sampling plan describing procedures and identifying sample sites, sampling numbers and sample frequency should be developed and properly documented in order to demonstrate that there is a constant level of control over the environment within the clean room facilities.
Alert and Action levels should be implemented based on your products, the intended use of the clean room and the classification of the clean room. There are three classifications for clean room facilities and each has it’s own Alert and action levels. The three classifications are ISO 5 (Grade A), ISO 7 (Grade C) and ISO 8 (Grade D). ISO 5, having the lowest Alert and Action levels of environmental contaminants and ISO 8 having the highest. An Alert is an indication that the level of microbial growth may me reaching an undesirable level. If an Action Level occurs, sanitation of the area should begin immediately as well as an assessment of what caused the Action level to be reached. Monitoring should be increased until microbial counts return to a desired level.
A well developed Environmental Monitoring Program is a proactive way to assure the cleanliness of the manufacturing facility is maintained.
Current Guidelines for Viable and Non-Viable Specifications within Clean Rooms
There are differences across the major guidance documents regarding the limits for both viable and non-viable particulates. The ISO 14644-1 deals entirely with non-viable particulate sizes for various clean-room classifications. The European Commission Annex one and the United States Pharmacopeia <1116> focus on both viable and non-viable particulates within grades A (class 100), B (class 1000), C (class 10,000), and D (class 100,000). The tables below compare the guidance documents and help to display the inconsistencies between all the main clean-room classifications.
1 United States Pharmacopeia 32nd Edition
2 European Commission Annex 1, Volume 4.
3 ISO 14644-1