Environmental Monitoring (EM) – New Approach Guide

This Guideline applies to routine Environmental Monitoring (EM) activities in classified manufacturing areas for viable and non-viable particulates, aseptic area personnel gown and glove samples, and for utility systems supplying those areas, such as compressed air, process gases, clean steam and water systems



    • This Global Quality Standard (GQS) provides the requirements for development and management of Environmental Monitoring (EM) Programs for sterile and non-sterile production.

2.0   SCOPE :

    • Guidance is provided for set-up of the program, criteria for selection of monitoring sites based on preceding qualification studies, Environmental Monitoring (EM) personnel training, frequency of monitoring, acceptance limits, methods for tracking, trending and reporting data, and investigation of exceeded limits and adverse trends.
    • This Guidance applies to routine Environmental Monitoring (EM) activities in classified manufacturing areas for
      • Viable and non-viable particulates,
      • Aseptic area personnel gown and glove samples, and for
      • Utility systems supplying those areas, such as compressed air, process gases, clean steam and water systems.
    • This Guidance applies to routine monitoring of environmental conditions in production areas, including, as applicable,
      • Temperature,
      • Differential pressure,
      • Relative humidity, and
      • Non-viable particulate levels,
      • To verify acceptable conditions before beginning daily operations.
    • This Guideline for Environmental Monitoring (EM) provides guidance for response to environmental alarms following predefined alert/action limits and excursions during operations within classified manufacturing areas, utility systems supplying those areas, such as compressed air, process gases and water systems.
    • This Guideline provides guidance for monitoring and responding to alarms following predefined action/alert limits in refrigerators, incubators, stability chambers and other environmental chambers.

    • This standard does not apply to-
      • Establishment of alarm (alert/action) limits for temperature, humidity or differential pressure,
      • Operation of environmental system controls, utility system operation or to setting up and operating Environmental Monitoring (EM) systems, Equipment, Facilities and Utilities Validation.
      • Requirements for routine operation of Environmental Monitoring (EM) systems are included in the SOP for Manufacturing Controls.
    • Research & Development (R&D) functions are excluded from the scope of this GQS.


    • Critical utility systems shall be qualified, including, but not limited to:
      • Water,
      • Clean steam,
      • Compressed air and gas systems, and
      • HVAC systems, as applicable to the specific facility.
    • Integrity of HEPA filters shall be tested and certified in classified areas of sterile and non-sterile facilities.
    • Sterile and non-sterile product manufacturing facilities shall implement procedures and training for routine and non-routine cleaning and sanitization of processing areas, including classified areas.
    • Sterile product manufacturing facilities shall also implement a program validation of sanitizer effectiveness, preferably with the inclusion of plant isolates as challenge organisms along with standard ATCC organisms.
    • Procedures shall be in place and effective for routine operation, maintenance, and calibration of equipment used to perform environmental sampling and testing at all facilities.
    • Equipment used for Environmental Monitoring (EM) must be calibrated and validated before use.
    • Refer to Attachment I for gowning qualification and the training and certification process requirements for personnel working in aseptic manufacturing areas.
    • Media for Environmental Monitoring (EM) shall be qualified and procedures in place for media preparation, sterilization, growth promotion, addition of antibiotic neutralizing agents (where applicable), sterility checks (pre-incubation), release, storage and disposal at all facilities that prepare or purchase prepared media for Environmental Monitoring (EM).
    • If the routine environmental monitoring sampling is performed by non- microbiologist personnel (e.g. operators from production) .
    • The following system shall be in place and documented:

      • Formal delegation program & documentation
      • Training program for environmental monitoring
      • Quality oversight evaluation
    • Environmental Monitoring (EM) procedures shall be current and effective, for sterile and non-sterile product facilities, and include:
      • Sampling plans, including frequency, location and methodology, graphic maps of sample locations, and sample frequency at each location.
      • Methods for handling and sanitizing documentation within aseptic areas, including sampling maps, forms and procedures.
      • Operation, calibration, and maintenance of Environmental Monitoring (EM) sample and test equipment.
      • Sanitization and transfer of Environmental Monitoring (EM) sampling equipment and utensils into and within aseptic processing areas.
      • Sampling procedures and methods for viable and non-viable particulates, as appropriate.
      • Laboratory procedures for sample handling, testing, data interpretation, storage, and recordkeeping.
      • Procedures for interpreting Environmental Monitoring (EM) media plates from powder filling operations shall include criteria for discrimination of microbial colonies versus powder on the media surface.
      • Setting alert and action levels and periodic reevaluation of the same based on trends.
      • Reporting and investigating out-of-specification Environmental Monitoring (EM) results and adverse trends and effectiveness verification of corrective action. (Refer to Guideline for Handling of Out-of-Specification (OOS) Results).
      • Verification of environmental conditions before beginning daily operations.
      • Environmental Monitoring (EM) Data trending and reporting.
    • Training Personnel:

    • Personnel that perform Environmental Monitoring (EM) sampling and testing shall be trained to current procedures for Training Management.
    • Environmental Monitoring (EM) sampling personnel entering classified areas shall be trained to procedures that govern required hygiene, gowning, and access requirements.
  • Environmental Monitoring (EM) Requirement in Sterile Manufacturing Facility :

    • Personnel performing Environmental Monitoring (EM) in sterile product facilities shall be trained and qualified to work in classified areas, including aseptic processing areas, and have been qualified for aseptic gowning.
    • Personnel performing Environmental Monitoring (EM) shall be trained and qualified to perform the following sampling processes for viable and non-viable particulates:
    • Viable particulate monitoring:

    • Active air samples
    • Passive air samples – Settle plates
    • Surface contact plates
    • Surface swabs
    • Personnel gown and glove contact plates
    • Non-viable particulate monitoring:

    • Continuous monitoring with a computerized data collection system
    • Discrete samples using portable particle counters with local printouts or data storage.
    • Aseptic processing personnel qualification requirements:

    • All personnel that enter the aseptic processing area, including those that only enter periodically and outside personnel approved by Quality, shall be qualified through a formal training program.
    • Assessment of their ability to consistently perform aseptic gowning shall be qualified initially and at least semi-annually (each 6-months) using contact-plate samples from gowns and gloves.
    • Attachment I contains training and certification requirements and Attachment II provides aseptic technique training guidelines. Site training programs shall include requirements from both attachments.
    • Personnel access o aseptic processing areas shall only be granted after successful completion of aseptic processing training and certification.

    • Sample site locations shall be determined during initial startup and commissioning of classified areas using risk analysis.
    • Sample sites shall be based on the risk for product contamination:

      • Perform risk analysis during initial establishment of the Environmental Monitoring (EM) Program, when changes to the process, practices, or environment occur, and during investigation into excursions and adverse trends that may impact product quality. (Refer to Attachment III for Example of Risk Assessment
        Template for Sterile Products).
      • Determine locations for air and surface sampling based on the risk for product contamination.
      • High risk areas may include locations in proximity of the exposed product, containers, closures, and product-contact surfaces.
      • Evaluate activities, practices, and materials that present a potential risk for contamination of the environment where product and primary packaging components are exposed.
      • Product-contact surface contamination shall be evaluated to assess potential risk factors that may include, but are not limited to:

      • Frequency and duration of human activities and interventions.
      • Sterility of components and product-contact surfaces.
      • Air flow characteristics and differential pressures.
      • Impact of process equipment and operator movement / activity on unidirectional air flow.
      • Efficiency of the barrier and other control systems designed to protect product
      • Sites or processes in which microbial contamination would most likely have an adverse effect on product quality.
      • Sites that represent the most inaccessible or difficult areas to clean and disinfect.
      • Understanding of modes of microbial dispersal in the environment and related causes and sources of microbial load.
      • Risk of monitoring activity for contamination of product.
      • Review of historical data for high risk area.
      • Personnel, materials, and process flow.

      • Proximity to exposed components, product, or product-contact surface.
      • Potential of critical zone contamination from adjacent lower-grade areas.
      • Potential for interventions, including the manipulations that may be associated with Environmental Monitoring (EM), and which may adversely affect the critical zone environment.
    • Evaluate airflow visualization (“smoke”) studies under dynamic conditions to assess risk of airflow disruption within critical zones and to assist in determining potential sites for viable and non-viable particulate monitoring.
    • Rationale for selection of all sample sites shall be prepared and included in the area qualification report. Risk assessments shall be included in the See Attachments III and IV for risk assessment templates.
    • The minimum number of sample sites in a classified area shall be determined from Table 1, as published in ISO 14644-1:2015(E),
    • Table A.1. The number of sampling sites recommended represents the minimum. Based on risk assessment, additional sites may be needed.
    • Select sample sites, so that they evaluate the impact of personnel movement and work within the area, particularly during interventions and manipulations within critical zones where sterile product, containers, closures, and product-contact surfaces are exposed to personnel.
    • Active air samples collect a large volume of air in a short period of time, and could disrupt air flow,
    • Therefore the location and timing of sampling shall be considered to avoid risk of this disruption.
    • If the risk of airflow disruption from active air samplers is determined to be unacceptable in a critical location, settling plates shall be considered as an alternate monitoring method at that location.

Table 1: Sample Locations Related to Clean Rooms: Total Particulate Count

Area of Clean Room (m2) less than or equal to

Minimum number of sample locations to be tested (NL)


























































Use Formula A.1

Formula A.1:


Where NL is the minimum number of sampling locations to be evaluated, rounded up to the next whole number.

A is the area of the cleanroom in m2.

  • Surface monitoring locations shall be selected to:

    • Assess microbial load on product-contact (equipment) and non-contact surfaces (floors, walls, benches, and non- product contact equipment).
    • Product-contact surface monitoring shall only be performed at the conclusion of critical operations in aseptic manufacturing environments to avoid contamination of the surface during monitoring.
    • Select non-product contact surface monitoring locations to assess the efficacy of cleaning/sanitizing/disinfecting practices within the aseptic processing area, see Table 2.
    • Table 2 lists example sample sites.
    • The number of sample sites and locations shall be based on documented risk assessments.

Table 2: Example Viable Monitoring Sites (for reference only)

Environmental air (filling line) Near open or filled containers
Room air Proximal to work area
Surface (facility) Door     handles,     walls,     curtains, floors, ceilings, intercoms
Surface (equipment) Filling line, control panels, stopper bowl, filling needles (post fill)
Operator     on     filling     line     or operator glove in an isolator Finger (glove) impressions, at a minimum of five fingers of both hands
Laminar airflow (e.g., hood) Near    high-activity    areas,    finger (glove) impressions

    • Sample frequency for aseptic processing environments shall be defined in written procedures.
    • Table 3 lists specific functions typical in each Graded area for sterile product manufacturing.
    • Table 4 lists minimum frequencies to be implemented at each site based on the most frequent sampling recommended in the referenced guidance document.
    • Environmental Monitoring (EM) sampling in aseptic processing isolators, including sterility test isolators, is listed in Table 5
    • The only guidance for sample frequency in grade D environments is from EU Annex 1, PIC/S and WHO Annex 4 where frequency should be risk based, see Table 3.
    • Although sampling frequency is not specified, risk of contamination carry-over to cleaner areas from grade D, ISO 5 areas is greater in aseptic manufacturing facilities than in non-aseptic facilities.
    • Based on the higher risk, airborne viable and non-viable particulates should be sampled regularly to provide assurance that contamination remains Sampling on at least a monthly basis is recommended when the area is in use.

Table 3: Classified Areas for Specific Functions in sterile product facilities

EU Grade

Typical Application

    • Critical areas where sterile drug substances (API), drug products, and sterile filling components may be exposed.
    • For example: aseptic preparation of sterile solutions and suspensions without subsequent sterile filtration or terminal sterilization, Aseptic filling and stoppering, and stoppered vials, transfer of partially closed aseptically-filled containers to a lyophilizer.
    • Crimping/capping may be conducted outside the aseptic filling area,
    • In which case vials must remain under Grade A microbial conditions until leaving the filling area and then be protected by Grade A air supply until the cap has been crimped.
    • Particulate monitoring is not required in crimping/capping areas due to high particulate levels generated by the operation.
    • Background environment for aseptic filling of sterile drug substances and products and for sterility testing performed in a Grade A cabinet.
    • Preparation of aseptically-filled solutions to be filtered. Filling and stoppering of products to be terminally sterilized.
    • Note that terminally- sterilized drug product that is sterilized using a bio burden dependent non-overkill cycle or that is filled on a line that is common to aseptically-filled products must be treated in a manner similar to aseptically-filled products.
    • Background environment for aseptic filling isolators and sterility test isolators.
    • Preparation of terminally sterilized solutions for subsequent filling.
    • Wash rooms for equipment, and utensils used for manufacturing of sterile drug substances (API) and products.
    • Access corridors, office areas and material holding areas adjacent to manufacturing areas where materials, components, and product are not exposed.

Table 4: Minimum Environmental Monitoring (EM) Sample Frequency Requirements/ Guidance

Monitoring Guidance Frequency Airborne Total & Viable Particles Surface Viable & Personnel – Clean Room and RABS
US    FDA    Aseptic    Processing Guidance


Class 100 (Grade A) continuous each production shift. Gloves daily or each lot and after aseptic interventions.
USP <1116>


ISO 5 or better (Grade A): Surface monitoring at the end of the operation.

Aseptic area adjacent critical zone: All sampling each operating shift.

EU Annex 1, PIC/S and WHO Annex 4 A.: In Operation, continuous total particulate monitoring required for critical operations. Frequent viable sampling.

B: In operation, frequent particle monitoring is required.

C, D: Monitoring on risk basis. (WHO states weekly grade C and Monthly Grade D in- operation monitoring for Vaccine manufacturing facilities).

Surfaces and personnel should be monitored after critical operations.

Table 5: USP <1116> Sample Frequency Recommendations for Isolators

Sampling Area/Location Frequency of Sampling
Critical zone (ISO 5 or better)
Active air sampling Once per day in operation
Surface monitoring At the end of the campaign
Non-aseptic areas surrounding the isolator
All sampling Once per month

    • Appropriate alert and action limits shall be set for total particulate and microbiological monitoring.
    • Site procedures shall be in place for investigation and corrective actions when limits are exceeded, or where there are indications of an adverse trend.
    • For Grade A environments, where viable counts are expected to approach 0 CFU, and only action level is required because there is no meaningful difference between alert and action levels.
    • Refer to Tables 6 and 7 for total particulate and viable particulate requirements stated in the EU Annex
    • “The site’s alert and action levels may be tighter than those recommended in Annex 1 based on historical data, and should be the result of reasonable performance assessment after periodic and regular review of the data”.
    • PDA TR13 provides several approaches to setting limits depending on the distribution of viable particulates.
    • Cut-off Value Approach:

      • All the test data for a particular site, or group of similar sites, are arranged in a histogram and the alert and action levels are set at values whose monitoring results are, respectively, 1% and 5% higher than the level selected.
      • Other percentiles may be used in establishing levels.
      • A variation is to take the last 100 monitoring results and use the 95th and 99th percentile values as the alert and action levels.
    • Normal Distribution Approach:

      • The mean and standard deviation of the data are calculated and the alert and action levels are set at the mean plus two (2) and three (3) times the standard deviation, respectively.
      • This approach is only used for high counts and when the data is normally distributed.
      • A Poisson distribution is used for low counts.
    • Non-Parametric Tolerance Limit Approach:

      • As EM data, especially in clean rooms, is typically not normally distributed (i.e., favors lower counts and/or zero counts),
      • A non-parametric Tolerance Limits approach to setting alert and action levels are recommended.
      • These limits allow assertion with confidence at least 95% (K=0.95) that 100(P) or 99% of a population lies below the value, depicted by the stated action limits, for the respective For Distribution- Free Tolerance Limits, Minimum Sample Size are N=60 for 95/95 (Alert Limit) and N=300 for 95/99 (Action Limits).

Table 6: Maximum Total Particulates from EU Annex 1.

Grade Maximum permitted number of particles per m3 equal to or greater than the tabulated size

At Rest

In Operation

0.5 µm

5.0 µm 0.5 µm

5.0 µm



20 3520 20



29 352000 2900
C 352000 2900 3520000


D 3520000 29000 Not defined

Not defined

Table 7: Maximum Viable Particulates from EU Annex 1.

Recommended limits for microbial contamination (a) during operation
Grade Air sample cfu/ m3 Settle plates (diameter 90 mm) cfu/4 hours (b) Contact plates (diameter 55 mm) cfu/plate Glove print 5 figures cfu/glove
A <1 <1 <1 <1
B 10 5 5 5
C 100 50 25
D 200 100 50
  • Excursion and Recovery Rates of Environmental Monitoring (EM) Results:

    • Excursion and recovery rates are related, but different measures of the microbial quality of the environment.
    • Once alert and action levels are derived, the excursion rate measures the number of samples exceeding the defined alert and action levels.
    • Recovery rates measure the overall microbial recovery (percent of samples with growth) in a given classified area, regardless of actual colony count.
    • Though alert and action levels have been eliminated from USP <1116> in favor of recovery rates, each site shall monitor both excursion rate and recovery rate because official regulatory guidance from the EU and US still retain GMP requirements for alert and action levels, see Tables 7 and 8.
    • Predefined excursion rates shall be developed from trend analysis and each site shall develop recovery rates for each classified area.
    • Table 8 lists USP <1116> recommended initial recovery rates that may be assigned for new aseptic processing areas.
    • If site recovery rates have already been developed for similar areas, then it is acceptable to continue using those rates.

Table 8: Suggested Initial Contamination Recovery Rates in Aseptic Environments.

Environmental Monitoring (EM) - Chart


    • EM shall be performed during all aseptic manufacturing operations, including aseptic process simulations (media fills).
    • In addition, during media fills, duplicate plates shall be incubated
    • Active Air Sampling:
    • Active air samples are collected via suitable air sampling The device shall be qualified, calibrated, and properly sanitized/sterilized for use in classified areas.
    • Operation, calibration, and maintenance of the device shall follow manufacturer’s recommendations.
    • Devices may be located directly in the clean zone or remotely with a suitable sampling nozzle and tubing.
    • When remote sampling is performed, minimize the length of tubing and the number and angle of bends to avoid under recording of viable particulates.
    • Internal diameter of tubing must be such to provide air flow within the vendor’s specified range.
    • A single medium type or separate media for bacteria and fungi may be used.
    • Care must be exercised when placing  the agar medium on the sampling device to avoid contamination.
    • During room classification, sample volumes not less than 1 m3 are required.
    • The volume of air sampled  for routine monitoring shall be qualified.
    • Passive Air Sampling – Settle Plates:

    • Non-selective agar, such as Soybean Casein Digest Agar, in 90 mm plates shall be standard for settle plates.
    • Other media may be used with justification.
    • Settle plates shall not exceed an exposure time of four hours.
    • Validation that four hours is appropriate shall be performed by challenging exposed plates with low levels of challenge organisms that are appropriate for the media.
    • For processes that extend beyond the validated exposure time, additional (new) plates shall be used.
  • Surface Sampling:

    • Each surface sample location shall be evaluated for geometry, e.g. curved, flat, textured, etc., and presence of sanitizing agents or other potentially inhibitory substances.
    • Agar media used for surface sampling shall be qualified for recovery of low levels of standard challenge organisms and expected environmental organisms
    • Appropriate neutralizing agents may be added to the agar or rinse or diluent when sampling surfaces that have been chemically
    • For examples of neutralizing agents for various classes of disinfecting agents see Table 9.
    • The efficacy and toxicity of neutralizing agents must be evaluated against the disinfectants used in the environment and the classes of organisms that may be present.
    • Methods for sampling surfaces for microbial contamination:

    • Flat surfaces and large curved surfaces that allow for good contact of the agar surface should be sampled using contact plates (RODACS),
    • But may be sampled using For contact plate sampling, remove the lid and press the agar surface against the surface with a gentle rocking motion to ensure contact of the surface with maximum agar area.
    • Irregular surfaces require a swab method or flexible agar film device.
    • Swabs are typically moistened with sterile water, peptone, buffer or transport media and a defined surface area, typically 25 cm2 (ca. 2 in X 2 in) is swabbed.
    • The sample may be directly plated or placed in a transport medium for later testing in the Microbiology Lab.
    • Transport media contains constituents that permit the organism to retain its viability without allowing proliferation.
    • Use of transport medium is recommended as it allows for a more accurate quantitative result.
    • Sanitization Procedure after surface monitoring:

    • Media residue should be removed from the sample site immediately after taking the imprint using an appropriate disposable wipe or swab impregnated with a suitable sterile disinfectant (e.g. sterile non-shedding disinfectant swab or wipe with sterile 70% Isopropyl Alcohol).
    • Otherwise the nutrient could create favorable conditions for microbial growth.
    • Rinse methods shall be used for evaluating areas that are difficult to sample by contact plate or swabs,
    • e.g., tubing or other hard-to- reach surfaces or for large surface areas, such as kettles and tanks, where the bio burden of the vessel is evaluated.
    • A sterile rinse diluent is added to, or flowed through the area to be sampled and collected for analysis.
    • For large volumes of rinse, a membrane filtration method shall be used to process the sample prior to plating the filter.

Table 9: Neutralizing agents for different classes of sanitizing agents/disinfectants (for reference only)

Interfering Substance Potential Neutralizing Agents/Method
Glutaraldehyde, mercurials Sodium hydrogen sulfite (Sodium bisulfite)
Phenolics, alcohol, aldehydes, sorbate Dilution
Aldehydes Glycine
Quaternary ammonium com- pounds (QACs), parahydroxy- benzoates (parabens), bis-biguanides Lecithin
QACs, iodine, parabens Polysorbate
Mercurials Thioglycollate
Mercurials, halogens, aldehydes Thiosulfate
EDTA (edetate) Mg+2 or Ca+2 ions
    • Non-Viable Particulates:

    • Portable particle counters with a short length of sample tubing shall be used for classification purposes because of the relatively higher rate of precipitation of particles ≥5.0 µm in remote sampling systems with long lengths of
    • Isokinetic sample heads shall be used in unidirectional airflow systems.
    • Computerized data collection systems shall be validated per written computerized system procedure.
    • Length of tubing and the number and angle of bends shall be minimized to avoid under recording of particulates.
    • Tubing type and length shall meet vendor specifications to assure maximum recovery of particulates in accordance with ISO 14644-1:2015(E).
    • Personnel gown and glove monitoring:

    • Sample locations shall at a minimum, include each forearm, chest, forehead, and glove fingertips.
    • Glove monitoring shall be performed for every person at least daily, or in association with each lot. Sampling upon each exit from the area should be considered.
    • Personnel monitoring shall be performed at the conclusion of critical operations and before sanitizing gloves.
    • Retraining shall be performed for operators having OOS monitoring results that exceed a pre-defined frequency.
    • Annual personnel requalification is sufficient for those automated operations where personnel involvement is minimized and monitoring data indicate environmental control.
    • Other gowning sites may be sampled according to an appropriate sampling frequency based on risk to operations, such as for operators involved in repeated or more complex aseptic manipulations; sampling sites shall be justified.

    •  Environmental sampling for yeasts and molds shall be performed in all classified areas at all routine sample sites at least semi-annually.
    • These surveys may be performed using Sabouraud’s Dextrose, or equivalent, agar or Soybean Casein Digest Agar with incubation at 20°C-25°C.
    • Yeast and mold data shall be trended to assess effectiveness of the Cleaning and Sanitization Program for controlling yeast and mold populations.
    • Adverse trends may require additional action, such as investigation to identify sources, additional cleaning / sanitization activities and possible modification of the Sanitization.
  • EM Media Preparation for Sterile product facilities only:

    • Isolates from classified areas, particularly ISO-5/Grade A areas, shall be used in growth promotion and suitability studies, as well as standard ATCC organisms specified in Pharmacopeia for the specific media being challenged.
    • Obligatory microorganisms isolated from the local flora should be used and should be representative of the qualitative trends evaluation made from the identified microorganisms originated from the environment.
    • Storage of environmental isolates should be accomplished through cryopreservation, since there exists no standardized reservoir, e.g. ATCC for these entities.
    • Each identified microbial strain should have unique isolate number that should be allotted as per site procedure after identification of microbial strain.
    • Microbial isolates should be preserved in cryo vials (for bacteria use soybean casein digest medium + 15-20% glycerol and for fungi use sabouraud dextrose broth + 15-20% glycerol) at low temperature (-15 to -70°C) for extended time.
    • The microbial isolate observations  of  colony morphology,  cell characteristics and photograph can be preserved as data base that should be served as a reference for the easy identification of the microbial cell whenever they recur.



Janki Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an ultimate pharmaceutical blogging platform. Email: [email protected]

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