Environmental Monitoring

FDA 2004 Guidance for Industry
EU Annex 1
USP <1072>
ISO 13408-1 and 14698
USP general information chapter <1116> “Microbial Evaluation and Classification of Clean
Rooms and Other Controlled Environments”

PDA- 13 (Fundamentals of an Environmental Monitoring Program)

Colony Forming Unit (CFU):
A single macroscopic colony formed after the introduction of one (or more) microorganism(s) to
a microbiological growth medium.

Continuous Monitoring:
A process of data collection where conditions are monitored continuously throughout the
operation. In most U.S. applications, this definition implies “during production.” For ISO
applications, this means twenty-four hours per day, seven days a week.

Frequent Monitoring:
A process of collecting data where conditions are monitored at least once per hour. In most U.S.
applications, this means during production. In most ISO applications this means twenty-four
hours per day, seven days per week.

Disinfection:
The chemical or physical inactivation of pathogenic microorganisms on inanimate surfaces.

D-value:
The time in minutes at a specific temperature required to reduce the microbial population by 90%
(or one [1] log) in defined conditions, e.g., method of sterilization (dry heat versus steam), solute,
carrier, etc. It may also be referred to as thermaldeathtime or lethality.

Heat Shock:
A process where bacterial cultures are subjected to a heat stress (usually 80°C-100°C) which
causes heat-resistant spores to form if the culture can produce spores.

Parametric Release:
A sterility release procedure based upon effective control, monitoring and documentation of a
validated sterilization process cycle in lieu of release based upon end-product sterility testing.

Static Monitoring:
Monitoring of the environment in the absence of normal operations, i.e., no equipment operating
and no personnel are present - at rest. For Medicines Control Agency (MCA) regulated
companies, this includes the equipment operating when no personnel are present. Per the EU and
ISO standards this is synonymous with at rest.

Terminal Sterilization:
A process where the drug product is sterilized in its final container.

Trend Analysis:
A review performed either routinely or in response to an alert or action condition. This review
provides an analysis of specific environmental monitoring data.

ISO/TC 198 provides Guidance for Sterilization of Health Care Products and ISO/TC 209 provides Guidance for the Classification of Airborne Particulate for Clean Rooms and Associated Controlled Environments. Copies of these documents can be obtained from American National Standards Institute (ANSI).


Chemical agents that are capable of destroying microbial contamination are normally categorized into three classifications that are based on the agents’ ability to destroy microorganisms. The three categories are sanitizers, disinfectants and sporicides, based on the strength of the agent and the
time period that the surfaces remains wetted (dry time).

Normal wetted times on hard surfaces in clean room operations normally range from 2-10 minutes.

Sanitizers reduce some level of bacterial contamination and are the least effective agents. Common sanitizers include isopropyl alcohol (IPA), ethyl alcohol (or ethanol) (EtOH), and low active levels of hydrogen peroxide (below 3%) (H2O2). Sanitizers are effective against some level of vegetative cells but are ineffective against more resistant vegetative bacteria, mold and spores.

Disinfectants reduce higher levels of vegetative bacteria and molds than do sanitizers. Common disinfectants include phenols, quaternary ammoniums, and hydrogen peroxide (above 3%). Disinfectants have a very limited ability, if any, to destroy bacterial and mold spores.

Sporicides are effective against all microorganisms provided the wetted time is achieved. This includes vegetative organisms and spores. Common sporicides include sodium hypochlorite, peracetic acid and hydrogen peroxide. Sporicides are usually corrosive to equipment (e.g.,
acidified bleach or peracetic acid and hydrogen peroxide on stainless steel) and should be used
sparingly at a reduced frequency than sanitizers and/or disnfectants. Selection of agents should
incorporate and evaluation process that validates the required contact time, type of microorganisms
that are to be eliminated (if possible and environmental isolate), confirmation of efficacy, type of
surface to be treated, toxicity, residue, and means of application.

For aseptic processes, the Quality unit should review the trends at a frequency of daily, weekly
monthly quarterly, as appropriate.

Staphylococcus species are commonly found on skin and
Pseudomonas species are usually associated with water.

Water Monitoring-
Since some organisms can survive and proliferate in ambient water, the microbiological attributes are critical. For clean steam monitoring, the BET and chemical purity as measured by TOC and Conductivity are critical.

Microbiological examination of water should be initiated as soon as possible after collection of the sample; in accordance to validated procedures. If immediate processing is not possible, refrigerate samples at 2°-8°C upon receipt in the laboratory. Time elapsing between collection and examination should not exceed 24 hours.

Culture medium used for microbiological testing of purified water and WFI, should be capable of detecting and enumerating the aerobic, mesophilic microbial flora including those that require low nutrient media such as R2A regardless of the type of product manufactured.

Acceptance Levels For:
1. Source (Potable) Water= Less than or equal to 500 CFU/ mL
2. Purified Water= Less than or equal to 100 cfu / mL
3. Feed water to WFI and Clean Steam Systems= Less than or equal to 100 cfu / mL
4. Water for Injection= Less than or equal to 10 cfu / 100 mL

NVPC Monitoring-

In the United States, the FDA requires that non-viable particles in the range of 0.5 microns or larger be monitored during routine manufacturing operations in a sample volume of one cubic foot. Requirements outside of the United States (e.g. EU Annex 1) includes monitoring greater than or equal to 5.0 micron particles, in addition to 0.5 microns in a sample volume of 1 cubic meter in certain classified areas.

Non-viable routine monitoring should be performed approximately one foot away from the work site when possible. However, sampling should not be intrusive to the process.

Viable Monitoring-

The Inertial Impaction Theory is based on the principle that air located above the sampling device is pulled into the sampler by a vacuum or fan source at a dramatically increased air speed (from that of ambient air) through varying orifice sizes and deposited to the nutrient media, gelatin or filter surface, if media is used. The inertial impaction principle assumes that the increase in speed of the microorganism through the orifice will deposit the cell to a deposition surface located a very short distance from the ending of the orifice canal. This principle is very sound and has been proven for years in the capture of microorganisms and particulates in a multitude of industries. Caution exists in the inertial impaction samplers when airflow speed reach a level whereby the airflow can damage the nutrient media deposition surface either from speed of air or from prolonged exposure time periods. While most inertial impaction samplers relieve exhaust air away from the process, additional caution should be considered when discerning the route of the exhaust air so as not to compromise laminar flow or adversely affect product or components above the product fill site or in what can be called the critical zone.

The Centrifugal Theory operates on the principle that air is drawn into the unit by means of an impeller and the particles are deposited on the surface of a solid nutrient collection medium (strip) by centrifugal force. The theory provides that air located above or beside the sampling device is pulled into the sampler by a fan source that spins creating a centrifugal effect to the incoming air. In this scenario, microorganisms enter a cone whereby airflow propels air (with particulates and microbes) to the exterior wall of the device. The spinning effect occurs at the initial intake and microorganisms are in effect displaced from the center cone of air to the nutrient media located in the outer perimeter of the cone. Caution should exist for the disturbance of laminar flow air near the air intake so as not to compromise laminar flow or adversely affect product or components above the product fill site. Additional caution should also be taken to assure that exhaust air from the sampled air is not emitted toward product or components above the product fill site or in what can be called the critical zone.

Basic concepts for the implementation of any microbial air sampling device include:
• The sampling process must never compromise the microbial attributes of the product
• For Class 100 (Grade A, ISO 5) areas, the sample should be taken within a 12” x 12” area near the fill zone. A minimum of one sample site is required and additional sites may be required depending on the criticality of the process.
• For Class 10,000 (ISO 7) and Class 100,000 (ISO 8) areas, a minimum of one sample site is required and additional sites may be required depending on the complexity of the zones adversely affecting the Class 100 (Grade A, ISO 5) zone.

• Required total volumes for sampling of the clean room conditions include:
o For Class 100 (Grade A, ISO 5) areas: minimally 1 cubic meter (36 cubic feet, 1000 liters)
o For Class 10,000 (Grade B, ISO 7) and for Class 100,000 (Grade C, ISO 8) areas: minimally 10 cubic feet (281 liters)

• Required total volumes for sampling of compressed air and gasses include:
o For Class 100 (Grade A, ISO 5) point of use samples (that may affect the clean room conditions): minimally 1 cubic meter (36 cubic feet, 1000 liters)
o For Class 10,000 (Grade B, ISO 7) and for Class 100,000 (Grade C, ISO 8) areas: minimally 10 cubic feet (281 liters)

Compressed Gas Monitoring-
Nitrogen gas (heavier than air) is frequently used as a blanket usually in tunnels. Argon may also be used and it is lighter than air.

It is common practice to use a 0.2 micron filter on the compressed gas line prior to entry into the room. Sampling is normally conducted after the 0.2 micron filter.

Any gas or air expelled into the environment should be at a level similar to that of the room.

Compressed Air/Gas that is dispelled into the ISO 5/Grade A/Class 100 aseptic environment and
must be tested for non-viable and viable particulates, including anerobes (if appropriate), as part
of the normal room air on a frequency that would assure that the air/gas does not adversely effect
the environment.

Surface Monitoring-

Contact Plates- This is the most commonly used sampling method, due to the ease of use. It provides for quantitative results. The plates are usually filled with media that contains a neutralizer. The plates are filled so that the media forms a dome to ensure a convex surface to recover the microorganisms. The type of media containing disinfectant neutralizers and incubation conditions must be qualified.

Swabs-
This method is employed for equipment and irregular surfaces that are not suitable for contact plates. This method can be used on flat surfaces, provided a template is used to define the sample size – usually approximately 2 inches x 2 inches (approximately 25 cm²).

Types of swabs that can be used for this method include Non-absorbent cotton, dacron and calcium alginate materials with the appropriate diluent. The cotton and dacron swab can be used to provide qualitative results by placing the used swab into broth media.

Surface Rinse Method-
This method is best used for a large surface area where the interior surface bioburden determination is needed. This includes kettles, equipment trains, and tanks.

MICROBIOLOGICAL EVALUATION OF CLEAN ROOMS AND OTHER CONTROLLED ENVIRONMENTS

Media & Incubation conditions: Use Triple wrapped Gamma irradiated Soyabean casein digest agar media plates or prepared and pre-incubated inhouse Soyabean casein digest agar plates supplemented with neutralizers. Incubate the media plates in inverted position at 20 º C to 25 º C for NLT 72 hours and further at 30 º to 35 º C for NLT 48 hours (Maximum allowable incubation period is 72 hrs. + 2days at 20°-25°C & 48 hrs. + 2days at 30 º to 35 º C).

Microbiological monitoring shall be performed post cleaning & sanitization activities during Qualification/ requalification/ installation/ renovation/ smoke study activities.

Perform negative control by carrying one plate from each type (90mm & 55mm) from the monitoring area along with monitoring plates. Negative control plates shall be held in the petri can /SS bin in the aseptic area. End of the activity, transfer the plates and incubated along with sampled plates.

Soyabean casein digest agar media plates supplemented with neutralizers shall be used for environmental monitoring.

Pre-incubation is not required for gamma ir-radiated agar plates.

Open the final wrap of ready to use media plates in the same grade it is intended to be monitored or sanitize the plates before transfer from lower grade to higher grade.

In case any accessory falls on exposed media plate on media surface, collect the media plate and proceed for incubation and expose new plate at that location. Discontinue the usage of fallen accessory.

PASSIVE AIR SAMPLING (SETTLE PLATE):

Transfer the pre-incubated Soya bean casein digest agar media plates in petri plate carriers to the location of exposure.

Use slanted SS-stands approximately 45º angle to expose media plates for return grills and use flat SS-Stand for remaining areas as applicable.

Settle plates shall be exposed for maximum period of 4 hrs. 30 minutes.

ACTIVE AIR SAMPLING:

Grade A: Filling station & IOS locations are monitored once in a shift during operation. Operations include machine assembly, product filling, loading to Lyophilizer and unloading from the Lyophilizer.

Active air sampling shall be performed only during batch operation shift in critical zones like filling and sealing activity. Do not perform sampling in static conditions such as breaks for tea, Lunch, shift change and during break downs where the filling activity found suspended. Critical zone includes filling & bunging cabinets and Lyo loading and unloading pathways.

Perform active air sampling in product pathway (filling, bunging, lyo loading) on priority and perform other active air sampling concurrent to batch activity.

SWAB SAMPLING:

Swab the surface approximately 25 cm2 to 30 cm2. Swab unidirectional to cover the entire surface. If area of sampling accessories is less than 25 cm2 to 30 cm2 swab entire surface.

On receipt of the swab samples microbiological analysis of swab samples shall be initiated as soon as possible, if for any reason microbiological analysis of swab samples is not initiated immediately the
swab samples should be stored in refrigerated condition at 2°C to 8°C for a maximum period of 12 hours before initiating the analysis after receipt of the samples.




Yeast and molds should be absent in Grade A & Grade B areas.

After completion of assembly & any open door interventions during operations, monitor personnel for finger dabs (left & right) & forearms (left & right).

Monitor personnel for finger dabs (left & right), forehead & chest during each exit from filling and sealing activity days.

Settle plate shall be exposed 30 minutes prior to initiation of assembly (pre-start up activity) and it will be collected at the end of assembly in filling and sealing stations (If assembly activity is not initiated or completed within 4hrs of settle plate exposure, the exposed plates shall be replaced with another set of plates for assembly activity). New set of plates shall be exposed after assembly activity
and it shall be replaced after every 4 hrs. till completion of batch activity.

Once in a month perform microbiological monitoring by active air sampling in grade A filling & sealing zone and filling room grade A during batch filling activity. Incubate the monitored media plates anaerobically at 30-35°C for 72 hours (maximum allowable incubation period is 72 hrs +2 days).

Perform the monitoring of filling area Return air grills (RAG’s) and filling room wall surface as per schedule/plan.

SETTLE PLATE MONITORING (CFU/90 mm Plate/ 4 Hours)
Media Used: Soyabean Casein Digest Agar with Polysorbate 80 and Glycerol