E/M Associates, Inc.

P.O. Box 2077

Salem, NH 03079

978.686.0215

ENVIRONMENTAL MONITORING OVERVIEW

Environmental Monitoring (E/M) is a surveillance system for microbiological control of cleanrooms and

other controlled environments. It is a process which provides monitoring, testing and feedback to the

microbiological quality levels in aseptic environments. Environmental surveillance monitors the

effectiveness of various controls for microbiological contamination of the environments. Sources of

contamination in the cleanroom can come from air, personnel, equipment, cleaning agents, containers,

water and compressed gases amongst other things. Sound monitoring required understanding the

various stringent regulatory specifications by various organizations such as the Food and Drug

Administration (FDA), International Standards Organization (ISO), Parenteral Drug Associates (PDA),

European Union (EU) and United States Pharmacopeia (USP). The following table is a synopsis of the

regulatory publications. It provides a summary and comparison of various parameters. The tables are

separated by cleanroom classification and are found in Parenteral Drug Associates (PDA) Technical

Report No. 131.

Table 1. Class 100 Monitoring Table (max. values are given). COUNTY

DOCUMENT

U.S.

209E

U.S.

USP <1116>

EU

(at rest, static)

EU

(operational,

dynamic)

EU

(operational,

dynamic)

ISO

14644-1

CLASSIFICATION M 3.5 (100) M 3.5 A and B A B 5

FREQUENCY Not stated Each

operational

shift

Not stated Frequent, using a

variety of

methods

Frequent, using a

variety of methods

Not stated

TOTAL

PARTICULATE

COUNT

3.500/m3

(>0.5μm)

100/cu. ft.

100/cu. ft.

(>0.5μm)

3.500/m3

(equal to or

above 0.5μm)

0/m3 (>5μm)

3.500/m3 (equal to

or above 0.5μm)

0/m3 (>5μm)

350,000/m3 (equal

to or above 0.5μm)

2,0000/m3 (>5μm)

3,520/m3

(equal to or

above 0.5μm)

29/m3 (5.0

μm)

AIRBORNE

VIABLES

Not stated 0.1 CFU per

cu. ft.

Not stated <1 CFU/m3

Settle plate 90

mm

<1 CFU/4 hours

<10 CFU/m3

Settle plate 90 mm

5 CFU/4 hours

Not stated

SURFACE VIABLES

(except floors)

Not stated 3 CFU per

contact plate*

Not stated <1 CFU per

contact plate (not

distinction for

floors and walls)

5 CFU per contact

plate (not

distinction for

floors and walls)

Not stated

SURFACE VIABLES

(floors)

Not stated 3 CFU per

contact plate

Not stated <1 CFU per

contact plate (not

distinction for

floors and walls)

5 CFU per contact

plate (not

distinction for

floors and walls)

Not stated

PERSONNEL GOWN Not stated 5 CFU per

contact plate

Not stated Not stated Not stated Not stated

PERSONNEL

GLOVES

Not stated 3 CFU per

contact plate

Not stated Glove print

5 fingers

<1 CFU per glove

Glove print

5 fingers

5 CFU per glove

Not stated

AIR VELOCITY

UNIDIRECTIONAL

Not stated Not stated 0.45 m/s

+ 20%

0.45 m/s

+ 20%

Not appropriate Not stated

FREQUENCY OF ΔP

MONITORING

Not stated Each shift Not stated Continuous Continuous Not stated

ΔP = Differential pressure *Contact plate areas vary from 24-20 cm2

Comment: Fed-Std-209E indicates that SI names and units are preferred for naming and describing the classes, but the English (U.S. customary) units may

be used. With the publication of ISO 14644-1 and 14644-2, it is expected that Fed-Std-209E will be retired by the end of 2001.

Grade A Terminally sterilized: Filling of terminally sterilized products, when usually at risk.

Aseptically prepared: Aseptic preparation and filling. Handling of sterile starting material and components. Transfer of partially

closed containers and open trays.

Grade B Background for grade A. Transfer of partially closed containers in sealed trays.

Table 2. Class 10,000 Monitoring Table (max. values are given). COUNTY DOCUMENT U.S.

FS 209E

U.S.

USP <1116>

EU

(at rest, static)

EU

(operational, dynamic)

ISO

14644-1

CLASSIFICATION M 5.5 (10,000) M 5.5 C C 7

FREQUENCY Not stated Each Operating

Shift

Not stated Not stated Not stated

TOTAL PARTICULATE

COUNT

353,000/m3

(>0.5μm)

10,000/cu. ft.

10,000/cu. ft.

(>0.5μm)

350,000/m3 (equal

to or above 0.5μm)

2,0000/m3 (>5μm)

3,500,000/m3 (equal to

or above 0.5μm)

20,0000/m3 (>5μm)

352,000/m3

(equal to or

above 0.5μm)

930/m3 (5.0

μm)

AIRBORNE VIABLES Not stated 0.5 CFU per cu. ft. Not stated 100 CFU/m3

Settle plate 90 mm

50 CFU/4 hours

Not stated

SURFACE VIABLES

(except floors)

Not stated 5 CFU per contact

plate*

Not stated 25 CFU per contact

plate

Not stated

SURFACE VIABLES

(floors)

Not stated 10 CFU per

contact plate

Not stated Not stated Not stated

PERSONNEL GOWN Not stated 20 CFU per

contact plate

Not stated Not stated Not stated

PERSONNEL GLOVES Not stated 10 CFU per

contact plate

Not stated Not stated Not stated

FREQUENCY OF ΔP

MONITORING

Not stated Each shift1

2x/week2 Not stated Not stated Not stated

ΔP = Differential pressure *Contact plate areas vary from 24-20 cm2

Comment: Fed-Std-209E indicates that SI names and units are preferred for naming and describing the classes, but the English (U.S. customary) units may

be used. With the publication of ISO 14644-1 and 14644-2, it is expected that Fed-Std-209E will be retired by the end of 2001.

Grade C Terminally sterilized: Preparation of solutions, when usually at risk. Filling of products

Aseptically prepared: Preparation of solutions to be sterile filtered.

1Adjacent to Class 100 2Support Areas – Product

Table 3. Class 100,000 Monitoring Table (max. values are given). COUNTY DOCUMENT U.S.

FS 209E

U.S.

USP <1116>

EU

(at rest, static)

EU

(operational, dynamic)

ISO

14644-1

CLASSIFICATION M 6.5 (100,000) M 6.5 D D 8

FREQUENCY Not stated Twice Weekly Not stated Not stated Not stated

TOTAL PARTICULATE

COUNT

3,530,000/m3

(>0.5μm)

100,000/cu. ft.

10,000/cu. ft.

(>0.5μm)

3,500,000/m3

(equal to or above

5μm)

20,0000/m3

(>5μm)

Not defined 3,520,000/m3

(equal to or

above 0.5μm)

29,300/m3 (>5

μm)

AIRBORNE VIABLES Not stated 2.5 CFU per cu. ft. Not stated 200 CFU/m3

Settle plate 90 mm

100 CFU/4 hours

Not stated

SURFACE VIABLES

(except floors)

Not stated Not stated Not stated 50 CFU per contact

plate

Not stated

SURFACE VIABLES

(floors)

Not stated Not stated Not stated Not stated Not stated

FREQUENCY OF ΔP

MONITORING

Not stated Weekly Not stated Not stated Not stated

ΔP = Differential pressure *Contact plate areas vary from 24-20 cm2

Comment: Fed-Std-209E indicates that SI names and units are preferred for naming and describing the classes, but the English (U.S. customary) units may be used. With the publication of ISO 14644-1 and 14644-2, it is expected that Fed-Std-209E will be retired by the end of 2001.

Grade D Terminally sterilized: Preparation of solutions and components for subsequent filling. Aseptically prepared: Handling of components after washing.

1Adjacent to Class 100 2Support Areas – Product

Please note Federal Standard 209E has been retired and superseded by ISO 14644-1.

As one can plainly view, there are many differences in both terminology and measurement parameters.

This can cause confusion by personnel developing environmental monitoring progress. Acceptance

criteria for microbiological contamination are different for air surfaces, and personnel. This difference

forces companies to look at setting appropriate surveillance based on where they may be selling their

products. Cleanroom Classifications differ between the United States and Europe ISO classifications.

Rank cleanliness of cleanrooms from Class 1 thru 9 based on particle size of interest. The table below

illustrates ISO Standard in comparison to the cancelled Federal Standard 209E.

From USP30 <797> International Organization of Standardization (SIO) Classification of Particulate

Matter in Room Air [Limits are in Particles 0.5μm and Larger per cubic meter (Current ISO) and cubic

feet (former Federal Standard No. 209E, FS209E.]

Class Name Particle Size

ISO Class U.S. FS209E ISO, m3

FS 209E, ft3

3 Class 1 35.2 1

4 Class 10 352 10

5 Class 100 3520 100

6 Class 1000 35,200 1000

7 Class 10,000 352,000 10,000

8 Class 100,000 3,520,000 100,000

Adapted from the Federal Standard No. 209E General Services Administration, Washington, DC 20407

(September 11, 1992) and ISO [4644-1: 1999 Cleanrooms and Associated Controlled Environments –

Part 1: Classification of Air Cleanliness. For example, 3520 particles of 0.5μm per m3 or larger (ISO

Classes) is equivalent to 100 particles per ft3 (Class 100) (1m

3 = 34.314ft

3).

The European Union Guidance documentation, “The Rules Governing Medicinal Products in the

European Union,” takes cleanrooms and further breaks them down to their conditional state specifying

either (1) at rest, static, or (2) operational, dynamic. Each state is further segregated based on air quality

from Grade A thru Grade D, with Grade A being the cleanest. Associated with each grade are the

maximum allowable viable and non-viable particulates. Grade A Areas are associated with high risk

applications such as fill/finish operations. Grade B is usually preparation areas for fill/finish operations

where open containers may be present. Grade C are clean zones for less critical applications such as

media and buffer preparations. Grade D areas are the least clean and involve cleaning, wash or other

preparatory requirements.

In the United States, the classification of aseptic manufacturing is classified as either Level I, Level II or

Level III and the classification refers to the microbiological risk of contamination; with Level I being

associated with areas that have open product exposure and Level III associated with ancillary functions

such as cleaning or other preparation activities.

Most major biotechnology, pharmaceutical, and medical device companies today market their products

in both the United State and in Europe. In order to compete in the appropriate country, they must ensure

that the manufacturing environment can meet all of the regulations set by the USP, ISO 14644-1 and

European Commission annex one. Current efforts to harmonize these specifications have not been

finalized. However, there are slight inconsistencies among these different documents which may add

unnecessary confusion and difficulty to the already extensive process of validating a pharmaceutical,

biotechnological, medical device manufacturing facility.

One of the most critical aspects within an aseptic manufacturing facility is the amount of viable

microorganisms and non-viable particulates within a controlled area. However, this is where the

different guidance documents fail harmonize and integrate into a unified environmental monitoring

specifications.

In order to control the levels or amount of microorganisms and non-viable particulates within the

different areas used for manufacturing purposes most aseptic processing identify the zones within the

facility into different grades based on the levels of cleanliness. The different grades of the rooms are

specifically designed and set up in an order to keep microorganisms and particulates away from any

sensitive, exposed parts of the manufacturing process where the product may be susceptible to

contamination. For newer companies and possibly even for some established ones, trying to decipher

the different terminology of the various grades described in the guidance documents can be one of the

first hurdles in setting appropriate specifications.

Whether manufacturing for the United States and/or European drug market, the specifications for non-

viable particulates are mainly located within three separate guidance documents; USP <1116>, ISO

14644-1 and the European Commission annex one. However, the three sets of guidance are not

completely harmonized.

Specifications: Limits for Particulates per cubic meter of air

Particle Size

ISO

14644-1

Class 5

USP

<1116> Class

100

EU

Grade

A

ISO

14644-1

Class 6

USP

<1116> Class

1,000

EU

Grade

B

ISO

14644-1

Class 7

USP

<1116> Class

10,000

EU

Grade

C

ISO

14644-1

Class 8

USP

<1116> Class

100,000

EU

Grade

D

.1μm 100,000 NS NS

1,000,000 NS NS

NS NS NS

NS NS NS

.2 μm 237,00 NS NS

237,000 NS NS

NS NS NS

NS NS NS

.3 μm 10,200 NS NS

102,000 NS NS

NS NS NS

NS NS NS

.5 μm 3,520 3,530 3,500* 3,500**

35,200 35,300

3,500* 350,000**

352,000 353,000

350,000* 3,500,000**

3,520,000 3,530,000

3,500,000* NS**

1 μm 832 NS NS

8,320 NS NS

83,200 NS NS

832,000 NS NS

5 μm 29 NS 0*

0**

293 NS

0*

2,000**

2,930 NS

2,000*

20,000**

29,300 NS

20,000*

NS**

* At rest

** Dynamic

NS – Not stated

As one can see there are many discrepancies about the particulate limits for different classifications

among the different guidance documents. The ISO 14644-1 covers the most ground in all the different

classifications; however, the majority of the aseptic manufacturing companies within the U.S. and

European market are mainly concerned with the 0.5 micron and 5.0 micron size particulates. The ISO

14644-1 and the European Commission both list specifications for the 0.5 micron and 5.0 micron size

particulates but the USP <1116> does not specify any limits for the larger 5.0 micron size particulate. It

only deals with the 0.5 micron particulate size. Another inconsistency is that the European Commission

has specifications for both dynamic and at rest (static) conditions where the USP <1116> and the ISO

14644-1 have just one general specification for each limit. Having separate specifications for static and

dynamic conditions does make sense due to the fact that adding people and activity to a room will

normally result in adding more particulates to the environment as well. Another discrepancy involves

the particulate limits within each classification. The European Commission (EC) is generally the most

conservative. Lastly, and arguably the largest issue, concerning the particulate limits for these classified

areas is the zero specification for the 5.0 micron size particulate per cubic meter of air in the EU Grade

A and Grade B at rest. The zero specification is not practical because this number is too small to be

statistically significant and is virtually impossible to obtain due to the fact that the distribution of

particulate matter within a classified area is not homogeneous. Furthermore, false counts, associated

with electronic and other background noise within the room can cause the particle counter device to

measure trace amounts of larger particulates. Due to this issue there have been discussions amongst the

European Commission to consider a limit of 20 5.0 micron size particulates per cubic meter of air within

these classifications.

Viable counts, colony forming units (CFU) specifications, concerning aseptic manufacturing companies

within the U.S. and European market are listed in the USP <1116> and the European Commission

Annex One. The ISO 14644-1 is for non-viable particulates only. Again, there are discrepancies and

inconsistencies regarding the acceptable limits with the different classifications of rooms.

Specifications: For Viable Counts - Colony Forming Units (CFU)

Sample Type

USP

<1116> Class

100

EU

Grade

A

USP

<1116> Class

1,000

EU

Grade

B

USP

<1116> Class

10,000

EU

Grade

C

USP

<1116> Class

100,000

EU

Grade

D

Air sample / M3 Air <3 CFU NS*

<1 CFU**

NS

NS*

≤10 CFU**

<20 CFU

NS*

≤100 CFU**

<100

NS*

≤200 CFU**

Settling Plates / 4 hr.

exposure NS

NS*

<1 CFU**

NS

NS*

≤5 CFU**

NS

NS*

≤50 CFU**

NS

NS*

≤100 CFU**

Surface Viables (except floors) / plate

≤3 CFU NS* <1 CFU**

NS

NS* ≤5 CFU**

≤5 CFU ≤25 CFU

NS

NS* 50 CFU**

Surface Viables (floors) / plate

≤3 CFU NS* <1 CFU**

NS

NS* ≤5 CFU**

≤10 CFU NS

NS NS

Gloves / plate ≤3 CFU NS*

<1 CFU**

NS

NS*

≤5 CFU**

≤10 CFU NS

NS NS

Personal Gown

/ plate ≤5 CFU NS

NS NS

≤20 CFU NS

NS NS

* At rest

** Dynamic

NS – Not stated

The table above displays the inconsistencies between the two guidance documents throughout all the

different classifications. First off, one can see that some items are specified in one guidance and not in

the other. Also, the table exhibits the different acceptable limits for each classification specified by each

of the guidance documents. In the Grade A (USP Class 100) area the European Commission is more

conservative than the USP <1116>; however, this is not the case in Grades C (USP Class 10,000) and

Grade D (USP Class 100,000) where USP <1116> has the more conservative limits. Either way the

discrepancies displayed in the above table are very significant in each of the classified areas.

One can see how this guidance can be very confusing and difficult to interpret, therefore the pressing

need for these guidance documents to be harmonized as soon as possible. Currently, our clients are

advised to go with the most conservative specification which will ensure that all acceptance criteria are

met. However, this certainly increases overall quality and facility costs.

Environmental monitoring evaluates existing cleanrooms, HVAC systems, personnel, cleaning and

sanitization activities. It monitors both viable and non-viable particles. Viables would include

microorganisms such as bacteria, yeast and molds. Non-viables would be air particulates of various

sizes. For viable counts, environmental monitoring programs test air, surfaces and personnel. Viable

particles are living microscopic organisms present in the aseptic environment. Is especially quantifies

bacteria, yeast, and mold in the air and on surfaces. Testing usually includes surface monitoring,

personnel monitoring and air monitoring. Companies check their clean room environments are testing

routinely to insure they meet required standards.

Monitoring of air can be by rotary centrifuged sampling on settling plates. Rotary Centrifugal Air

Samplers (RCS) are also used. These instruments measure an exact amount of air with a quantifiable

number of viable microorganisms. The most widely used organisms are media impaction instruments.

Contamination can be measured per cubic foot or cubic meter of air. Rotary Centrifugal Air samplers

are active monitoring devices that do correlate microbial contamination with measured air flow. They

include:

Slit to Agar (STA)

Seive Impactors

Cetrifugal Impactors

Liquid Impingement

Filtration

Gravitational or settling plates are Petri dishes that contain sterile growth media. They are passively

exposed to the environment, usually for 30-60 minutes. Viable microorganisms that settle onto the

media surface will grow when the plates are incubated. Settling plates offer ease of use and remain cost

effective. However, they do not directly correlate microbial contamination with measured air volume

and do not provide a quantitative measurement of air contamination.

Surface monitoring involves contact or rodac plates which contain sterile growth medium in 50mm

plates or a sample area of 25cm2. The agar protrudes above the sides of the plate. This convex contact

plate is pressed against any flat regular surface that needs to be sampled. Any viable microorganisms on

that surface will adhere to the agar and grow upon proper incubation. This technique replicates the

number of viable microorganisms on a surface. Contact plates are easy to use and widely available, but

they may not be appropriate for irregular surfaces. Neutralizers are usually in the media to minimize

inhibitory effects of disinfectants used in the cleaning process. Contact plates residue must be removed

immediately after testing the sample site.

Swabs are used for sampling surfaces that are not flat, such as tubing or equipment. Swabbing is

qualitative and is technique dependent. A 2x2 sq. in. sample site approximately 25cm2 surface area is

swabbed by using a back and forth technique and rotating 90° and repeating. The swabs are then

streaked onto microbiological agar plates for identification. Swabs can also be quantified by using

transport media in pour plates or membrane filtration techniques. Swab technique can vary greatly from

technician and training should be enforced.

Personnel working in a clean room need to be monitored for microbial contamination. Personnel

monitoring is an indication of gowning proficiency. Sampling sites are located on both gloves and

gowns. Touch plates (contact plates) can be used to dynamically monitor technician’s hands

immediately after a critical process, such as sterility. Personnel hygiene training should be conducted

for all aseptic processing personnel. Personnel monitoring is a good tool to assess cleanroom gowning

techniques.

Surface rinse methods are another monitoring technique for fermentation vessels, hold tanks and kettles.

It is used for large surface areas and tested by membrane filtration.

For non-viable measurements, particle counts measure airborne particulates. These counts help qualify

the cleanroom or controlled environments by demonstrating control of potential contamination. Some

particle count systems offer continuous monitoring (24/7). The equipment uses light scattering

technology based on the principle of passing anaerobia through a light source. Selection of the

appropriate particle counter is based on size range, sensitivity and flow rate. Most specifications for

particle count are based on 0.5 micron size associated with the overall size of bacteria. As previously

discussed, the European Union (EU) is also concerned about larger particles which are felt to possibly

carry multiple microorganisms.

Samples sites for environmental monitoring are dependent on the manufacturing process. Evaluation of

sample sites is based on the risk of microbiological contamination. Sites should be clustered at areas

where the product is exposed to the environment such as (a) filling lines for parenterals especially at fill

heads; (b) inoculation vessels for fermentation activities or (c) loading of the freeze dried for lyophilized

product. Sample sites should include areas that may be inaccessible or difficult to clean. This may

include stopper bowls, chromatography columns, transfer lines or fill nozzles. Other site selection

factors may include areas that have a greater impact to add to bioburden levels. This may involve water

point of use valves, compressed air lines or simply sites such as door handles. Sample site selection is

customized to the product produced by the company and unique to the individual manufacturing facility.

Sampling sites selection can also be done uniformly by using grid profiling usually associated with

particle counts.

Sample frequency is also dependent on multiple factors such as product type, equipment used, facility

layout, type of processing, etc. The high quantity of hands-on processing by personnel will necessitate

increased frequency of monitoring activities. Changes in sample frequency are dictated by changes in

facility design, construction activities, processing changes, microbiological trends and new equipment

acquisition. Frequency changes should be accompanied by documentation summarizing potential

upcoming activities, a summary of historical monitoring data and be reviewed by a qualified

microbiologist.

Action and alert levels are parameters designed to signal drifts in data from historical performance

measurements. They are meant to point out changes in data before the quality of the product is affected.

Alert levels are set below action levels and based on quality levels may signify a potential deviation

from normal case scenarios. Alert levels are usually associated with increased formal communication to

quality and manufacturing management. Action levels are based on accepted regulatory specifications

such as FDA, EU, ISO, PDA or USP as previously discussed. Action level deviations require a

corrective action and root cause analysis as detailed in a companies Corrective Action and Preventative

Action (CAPA) Standard Operating Procedures (SOP).

A hot topic in today’s environmental monitoring is E/M Data Management. This includes data

collection, analysis and interpretation. Based on the larger number of E/M sites, sampling frequency

and conditional states, many companies are utilizing computer data informational programs specifically

for E/M. Image scanner and label systems help improve data collection. Trending analysis can also be

obtained from both manual and computer based programs. Trends can be depicted by histograms

showing action levels. Data distribution will be different due to cleanroom classification, processing

activities, the amount of human intervention, seasonal effects and cleanroom conditional states (i.e.

dynamic vs. static). Interpretation of trend analysis is based on statistical process control (SPC). SPC

may show increases or decreases over time (i.e. seasonal effect), change in floras, possible patterns or

clusters and process outlined. Risk analysis techniques can make determinations to assess potential

contamination or other consequences with a process outline.

Characterizing E/M isolates to species levels helps categorize “House Organisms”. The “House

Organisms” profile will be useful in future investigational analysis involving sterility test failures,

disinfectant efficacy challenges and positive media tables. Vegetative cells can be characterized by

gram stain, colony morphology, sporulation, selective medias or automated identification systems.

Sporulating organisms such as Bacillus species and/or molds are of particular concern due to their high

resistance. Characterizations of isolates can be helpful in determining possible source of contamination.

For example, a gram positive coccus may indicate human intervention (skin bacteria).

Environmental monitoring methods need validation to demonstrate accuracy and robustness. Equipment

such as air samples and particle counters should have installation, operation and performance

qualifications (i.e. IQ, OQ, PQ). These validation tests should verify and document that the equipment

performs consistently over time. For microbiological medias utilized in air and surface monitoring,

growth promotion should exhibit acceptable recovery of challenged microorganisms including bacteria,

yeast and molds. If medias are being used with neutralizers such as polysorbate 80 or lecithin, then

neutralizer efficacy testing should be done. Neutralizers are put into media to cancel out the effects of

sanitizers or disinfectants that are present from tough cleanroom cleaning programs. A neutralizer

efficacy test tests the effectiveness of the neutralizer in counteracting the active microbial inhibitory

effects of the sanitizer. Even swab methodology can be validated for consistency and recovery.

Other aspects of a strong surveillance program include:

1. Water Testing

2. Compressed Air Testing

3. Media Fill

4. Terminal Sterilization

5. Utilities

Water is ubiquitous in an aseptic processing facility. It includes uses as a raw material, buffer, rinse

agent, etc. Microbiological quality of water is of particular importance. Feed water, pre-treatment,

reverse osmosis (RO), deionization (DI) and water for injection (WFI) need to be tested and assessed for

microbial counts. United States Regulatory Guidance is provided by USP <1231> “Water for

Pharmaceutical Purposes”, American Public Health Association (APHA), “Standard Methods for

Examination of Water and Wastewater” and Environmental Protection Agency (EPA), “National

Primary Drinking Water Regulations.” Water systems should be monitored at points of use, storage

tanks and loop circulation. Water sampling is critical and sampling personnel should be trained in

aseptic technique. Microbiological medias, such as R2A, should be selective for slow growing, injured

bacteria which may be present in the very harsh environment of a pharmaceutical water system. pH,

conductivity, total oxidizable carbon (TOC) and water chemistry are routinely tested. Water results

should be trended to look for seasonal effects especially in the feed water source. These seasonal effects

may change salt contamination, pH and in microbiological flora which can have an effect on product

quality.

Compressed air systems are used in cleanroom operations to overlay products, pressurize tanks, dry

equipment and energize equipment. Compressed air including air nitrogen, CO2, etc. should be filtered

with hydrophobic vent filters. Testing for both viables and non-viables should take place. Use pressure

reduction orifices to provide a steady stream of air. Insure validation of media especially paying

attention to desiccation.

Media fills are product simulation studies using general bacterial growth media like Trypticase Soy

Broth (TSB). This broth is usually inserted into a production fill finish process at the end of a normal

manufacturing campaign. This end-run demonstrates “worst case scenario” testing. The TSB is filled

into the same product container/closure system in statistically significant quantities (i.e. >1000 units)

and incubated for 14 days. The units are then inspected for turbidity or microbial growth. Sterility of all

units validates the entire fill/finish system including tanks, lines, conveyors, containers, fill lines,

closures, personnel and cleanroom facility. Media fills should be done frequently as specified by quality

systems specifications and be done to validate any changes to the fill/finish operation.

Terminal sterilization may be used in aseptic processing industries to sterilize raw materials, equipment,

buffers and containers. Autoclaves or steam in place (SIP) systems should be monitored on a routine

basis for temperature time and pressure. The used of biological indicators (BIs) will demonstrate

appropriate lethality for sterility assurance level (SAL). Terminal sterilization system assists in

controlling overall bioburden and endotoxin levels in final product quality and should be monitored for

consistency of operation.

Product bioburden is another aspect in some environmental monitoring programs. Bioburden testing

provides microbial surveillance of various components in the manufacturing process including raw

material, water, components, buffer prep, equipment and upstream processing. The bioburden levels can

be cumulative and can overwhelm filtration in aseptic processing. Knowing these microbial loads can

build appropriate process controls to improve final product quality. Bioburden methodology includes

pour plates, spread plates, membrane filtration, most probable number (MPN), water activity (Aw) and

some automated rapid microbiology systems. All bioburden methods should be validated for plate count

recovery.

HVAC systems in controlled environments should also undergo validation under both static, at rest,

testing and dynamic, at operation, testing. These two conditional states will demonstrate the consistency

of the HEPA filtration over time at both baseline and stressed conditions. Dynamic operational or

stressed conditions should include the personnel typically working in the cleanroom and the equipment

in operation. Typical tests include air and surface monitoring for viable counts, particle counts,

temperature humidity control, HEPA leak tests, velocity and smoke tests for laminar flow. Utilities such

as electrical, gas and steam should be validated upon initial start-up and consistently monitored.

Documentation for a strong environmental monitoring system should include the following:

A. Standard Operating Procedures (SOPs)

B. E/M Test Procedures

C. Equipment Use, Maintenance and Calibration Procedures and Logs

D. Cleaning and Sanitization Data Sheets

E. Sample Site Maps

F. Alert and Action Levels

G. Test Results, Date of Results, Incubation Temperature Deviation and Quality Review

H. Microbial Identifications

I. Environmental Trending Spreadsheets, Histograms, etc.

J. Deviation Reporting and/or CAPA Procedures

Documentation should be completed using Good Documentation Practices (GDP).

Overall, a good environmental monitoring system will provide management with process feedback to

compliance activities. Environmental monitoring primarily focuses on microbiological control.

Surveillance of the aseptic processing environment confirms overall production quality and provides

significant insight into potential contamination risk. Environmental specifications can be dictated by

where aseptic products are being marketed. Regulatory guidelines offer various measurement

parameters but are inconsistent with acceptable limits. This can cause confusion in the development and

data interpretation of environmental monitoring programs. In some cases limits are not specified or are

totally different. It is important for quality management to be aware of these discrepancies in meeting or

attempting to meet compliance standards. US and EU harmonization of both viable and non-viable

counts in various cleanroom conditional states is needed to provide better consistency and quality levels.

References Parenteral Drug Association PDA Technical Report No. 13 “Fundamentals of an Environmental

Monitoring Program”

United States Pharmacopeia and National Formulary USP30

<1116> “Microbiological Evaluation of

Cleanrooms and Controlled Environments”

International Standards Organization (ISO) 14644-1 “Classification of Air Cleanliness”

European Union (EU) Annex on the Manufacture of Sterile Medicinal Products

Food and Drug Administration (FDA) “Guideline on Sterile Products Produced by Aseptic Processing”