Environmental Monitoring Overview GOOD TABLES
Transcript of Environmental Monitoring Overview GOOD TABLES
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”