A Brief Overview on Active Air Sampling Procedure for Environment Monitoring

International Journal of Trend in Scientific Research and Development (IJTSRD)
Volume: 3 | Issue: 3 | Mar-Apr 2019 Available Online: www.ijtsrd.com e-ISSN: 2456 - 6470
@ IJTSRD | Unique Paper ID – IJTSRD22987 | Volume – 3 | Issue – 3 | Mar-Apr 2019 Page: 732
A Brief Overview on Active Air Sampling
Procedure for Environment Monitoring
Juhi Rastogi1, Faiz Hashmi2
1QC Microbiologist, Nexgen Scientific, Baddi, Himachal Pradesh, India
2QC Microbiologist, Scott-Edil Pharmacia, Baddi, Himachal Pradesh, India
How to cite this paper: Juhi Rastogi |
Faiz Hashmi "A Brief Overview on
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Environment Monitoring" Published in
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ABSTRACT
In this paper, we are going to discuss the ‘Active Air Sampling procedureforEM’.
EM stands for Environment Monitoring. Environment monitoring is performed
in the pharmaceutical manufacturing plants to monitor the contamination of
viable and non-viable particle count. Viable particle count can be observed
through the ‘Settle Plate method, ActiveAir Sampling, SurfaceMonitoring (contact
plate & swab test), and Personnel Monitoring method’. Non-viable particles are
dust particles and other non-living particles.
Active Air sampling is performed by microbiologist in the production &
manufacturing area using the equipment known as ‘Air Sampler’. A media plate
of SCDA (Soybean-Casein Digest Agar) prepared under sterile condition by the
microbiologist. The media plate is then allowed to adjust under the ‘AirSampler
hood’ and then it is used for sampling purpose. Air sampler captures 1000L air
as per validated time in a 1cubic meter of volume and therefore air sampling is
thus performed in the middle of the surrounding area. The sampled plateisthen
incubated, and after then the required incubation is provided, and the plate is
thus analyzed to determine whether our manufacturing area meets the level of
expected counts or it crosses the required limit; and, on this basis, the reporting
is thus generated on regular basis.
KEYWORDS: Environment Monitoring, Clean Room, Grades and their respective
limits, Checkpoints of Media, Air Sampler and its operation, Incubation of the
sampled plates, Trend chart analysis
INTRODUCTION
Active Air Sampling for Environment Monitoring is
performed in the pharmaceutical manufacturing plants.The
purpose here is to monitor the contamination present in the
environment which ultimately causes contaminate in the
medicines i.e. being manufactured in specified areas. The
contaminants can benon-viableparticlesand viableparticles
as well.
Active Air sampling is done by microbiologist in the
production & manufacturing area using the equipment
known as ‘Air Sampler’. A media plate of SCDA (Soybean-
Casein Digest Agar) which is prepared under the sterile
condition by the microbiologist. The media plate is then
allowed to adjust under the ‘Air Sampler hood’ and then it is
used for sampling purpose. Air sampler captures 1000L air
per 10 minutes in a 1cubic meter of volumeandthereforeair
sampling is thus performed in the middle of thesurrounding
area. The sampled plate is then incubated and after the
required incubation, the plate is thus analyzed to determine
whether our manufacturing area meets the levelofexpected
counts or is it crosses the required limit; and, on this basis
the reporting is thus generated on a regular basis, weekly
basis, monthly basis or twice a month.
ENVIRONMENT MONITORING
Environment Monitoring is the monitoring of surroundings
in the production and manufacturingareas. EMis performed
by various procedures, those are:
 Settle Plate Method:
The 90mm Petri plates are being prepared with 20ml-25ml
of SCDA (Soybean casein digest agar), after the pre-
incubation of 24 to 48 hours the Petri plates are allowed to
be exposed for the 4 hours in the manufacturing area and
then it is incubated at 20°C-25°C for 72 hours and
observation is being recorded. Later the same plate is then
incubated at 30°C-35°C for 48 hours and later the
observation is being recorded.
 Active Air Sampling Method:
of SCDA (Soybean casein digest agar), after the pre-
incubation of 24 hours the Petri plates are allowed for
sampling and the Active Air Sampling procedure is to be
started and to be continued up to 10 minutes or as per the
time validated by each industry and then it is incubated at
20°C-25°C for 72 hours and observation is being recorded.
Later the same plate is then incubated at 30°C-35°C for 48
hours and later the observation is being recorded.
IJTSRD22987

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 Surface Monitoring methods:
The surface monitoring procedure is performed to monitor
the contaminants which stick to the surface of the working
machine, laminar air flows, conveyor belts, door surface,
floor surface, window glass wall surface. Then itis incubated
at 20°C-25°C for 72 hours and observation is beingrecorded.
Later the same plate is then incubated at 30°C-35°C for 48
hours and later the observation is being recorded.
Surface Monitoring is performed by the following two ways:
 Contact Plate Method:
The 55mm Petri plates arebeing preparedwith 10ml-
12ml of DNA (De-Engley Neutralizing Agar), after the
pre-incubation of 24 – 48 hours, the Petri plates are
allowed to get in contact with the working machine,
laminar air flows, conveyor belts, door surface, floor
surface, window glass wall surface, etc. Then it is
incubated at 20°C-25°C for 72 hours and observation
is being recorded. Later the same plate is then
incubated at 30°C-35°C for 48 hours and later the
observation is being recorded.
 Swab Test Method:
Normal saline is used for this purpose.Swab sticks are
dipped in normal saline in the swab collection tubes
and this configuration is used for surface monitoring
by swab test. All those locations where sample
collection by the contact plate is not possible, swab
test are then performed for all those locations, like a
hopper, LAF corners, door handle wall corners, etc.
The collected samples are then is being solidified by
SCDA (Soybean casein digest agar) via pour plate
techniques. Then it is incubated at 20°C-25°C for 72
hours and observation is being recorded. Later the
same plate is then incubated at 30°C-35°Cfor48-hour
sand later the observation is being recorded.
 Personnel Monitoring Method:
of DNA (De-Engley Neutralizing Agar), after the pre-
incubation of 24 – 48 hours the Petri plates are allowed to
get in contact with the personnel working in the
manufacturing area. The contact plate is being taken from
various locations like forehead, chest, armpits, elbows,
booties, and fingers. Then it is incubated at 20°C-25°C for72
hours and observation is being recorded. Later the same
plate is then incubated at 30°C-35°C for 48 hours and later
the observation is being recorded.
CLEAN ROOM AND ITS CLASSIFICATION
A cleanroom is a facility ordinarily utilized as a part of
specialized industrial production or scientific research,
including the manufacture of pharmaceutical items and
microprocessors. Cleanrooms are designed to maintain
extremely low levels of particulates, such as dust, airborne
organisms, or vaporizedparticles. Cleanrooms typicallyhave
a cleanliness level quantified by the number of particles per
cubic meter at a predetermined molecule measure. The
atmosphere outdoor air in a typical urban area contains
35,000,000 particles for each cubic meter in the size range
0.5 μm and bigger in measurement, equivalent to an ISO 9
cleanroom, while by comparison an ISO1cleanroom permits
no particles in that size range and just 12 particles for each
cubic meter of 0.3 μm and smaller.
Table 01: The Area Classification of Inject-able Plants in general
Room name Grade
Change Room-I of filling area Grade D ( ISO -8)
Change Room-I of Manufacturing area Grade D ( ISO -8)
Change Room-I of Sampling area Grade D ( ISO -8)
Change Room-II of sampling area Grade C ( ISO -7)
Change Room-II of dispensing area Grade C ( ISO -7)
Change Room-II of Manufacturing room Grade C ( ISO -7)
Change Room-II of washing room Grade C ( ISO -7)
Change Room-II of preparation room Grade C ( ISO -7)
Staging room Grade B ( ISO -6)
Cooling Zone Grade B ( ISO -6)
Filtration Room Grade B ( ISO -6)
Corridor area of filling Grade B ( ISO -6)
Blending room Grade B ( ISO -6)
Mobile LAF Grade A ( ISO -5)
Filtration LAF Grade A ( ISO -5)
Garment Cabinets Grade A ( ISO -5)
Passbox Grade A ( ISO -5)
RLAF Grade A ( ISO -5)
GRADES AND THEIR RESPECTIVE LIMITS
Table 02: Limits According to EU guidelines
Grade
Active Air
Sampling
Settle Plate (90mm),
cfu/4hrs
Contact Plate (55mm),
cfu/plate
Glove print 5 fingers,
cfu/glove
A <1 <1 <1 <1
B 10 5 5 5
C 100 50 25 n.a.
D 200 100 50 n.a.
According to EU guidelines

Table 03: Limits According to FDA guidance (2004)
Clean area classification
(0.5µm particles/ft3)
Active Air
Sampling, cfu/m3
Settle Plate
(90mm), cfu/4hrs
Glove Print 5
fingers, cfu/glove
100 ISO 5 1 1
1000 ISO 6 7 3
10000 ISO 7 10 5
100000 ISO 8 100 50
According to FDA guidance (2004)
Table 04: ISO 14644-1 Cleanroom Standards
Class
maximum particles/m3 FED STD 209E
equivalent>=0.1 µm >=0.2 µm >=0.3 µm >=0.5 µm >=1 µm >=5 µm
ISO 1 10 2
ISO 2 100 24 10 4
ISO 3 1,000 237 102 35 8 Class 1
ISO 4 10,000 2,370 1,020 352 83 Class 10
ISO 5 100,000 23,700 10,200 3,520 832 29 Class 100
ISO 6 1,000,000 237,000 102,000 35,200 8,320 293 Class 1,000
ISO 7 352,000 83,200 2,930 Class 10,000
ISO 8 3,520,000 832,000 29,300 Class 100,000
ISO 9 35,200,000 8,320,000 293,000 Room Air
Table 05: BS 5295 Cleanroom Standards
maximum particles/m3
Class >=0.5 µm >=1 µm >=5 µm >=10 µm >=25 µm
Class 1 3,000 0 0 0
Class 2 300,000 2,000 30
Class 3 1,000,000 20,000 4,000 300
Class 4 20,000 40,000 4,000
MEDIA AND ITS PREPARATION
Checkpoints of the media:
 Name of Media
 Media Lot No/Batch No.
 Media Mfg. Date and Expiry Date
 Receipt of Certificate of Analysis
 Is Physical condition OK
 Is Label claim matched with INDENT and CoA
 Color
 Lumps
 pH (before and after sterilization)
 solubility
 GPT/GIT
Preparation of media
The purified water are used for the media preparation. The quantity of dehydrated media required for the volume of mediato
be prepared is to be calculated. The calculated quantity of media on a calibrated weighing balance is to be weighed and then is
transferred into the conical flask/ Bottle/ Tubes containing a small part of required water quantity. The remainingquantityof
water is added into the conical flask/ Bottle/ Tubes. The media is then dissolvedbyswirlingtheflaskorboil/heat themedia(as
per manufacturer recommendation if required) on a water bath or hot plate to dissolve completely. The pH of the unsterilized
media is then checked and recorded the verified pH of the media. If the pH varies, it is then adjusted by the addition of 0.1N
Hydrochloric acid or 0.1N Sodium hydroxide before sterilization so as to pH remains under limit after sterilization. If thepHof
media is satisfactory then set the cotton plug in the flask / bottle / tube mouth. Before sterilization, chemical indicatorshallbe
placed in the autoclave. The test tubes/Flask /Bottles shall be labeled with 3M tape with the name of the media lot No. The
media is then sterilized in steam sterilizer by operating the autoclavestandard process 1(121oC)&standardprocess 2(115oC)
for 20 minutes. For Heat Labile Media boil the media up to 80ºC up to dissolved. The sterilized media is then unloaded from
autoclave in the Cool Zone area. Allow the media to cool according the room temperature. Check the pH of sterilized media by
taking a broth media tube.
Preparation of Plates
SCDA (1% glycerol added) media is used for active air sampling. The media shall be prepared and autoclaved respectively.
Approximately15-20 ml of liquefied SCDA (1% glycerol added) media is being under LAF and cooledupto40⁰C-450C in allthe
Petri plates required as per the exposure location. The Petri plates is then covered and the poured media is allowed tosolidify.
After the solidification, all media containing Petri plates are incubated for the purpose of pre-incubation in the incubator at
32.50C± 2.5 0C for 24 - 48 hours in an inverted position for detecting any contamination during plate pouringoperations.After

the pre-incubation, examine all Petri plates for microbial growth. Petriplates without contamination andhavingnoairbubbles
shall be selected for active air sampling.
Result Calculation
Calculate the result as per the formula is given here.
Where;
X = CFU/m³
V = Volume of air sampler
r = CFU counted on 90 mm plate
Pr = Probable count obtained by positive hole correction against r value.
AIR SAMPLER AND ITS OPERATION
Preparation of Air Sampler and its Accessories
The Sterilized/ Depyrogenate hood and labeled Petri plates are to be kept in thehatchboxas perrequirement. The Air Sampler
and hatch box are being carried into the aseptic area by mopping with 70% IPA.
Working of Air Sampler:
The pre-incubated sanitized labeled SCDA (1% glycerol added) plateis beingplacedin thefeeder cone circularclamp assembly
in position. The air sampler is then placed in the area where air sampling is being done. The “ON” switch is then turned on
which is provided on the back side of the air sampler which shows power “ON” by glowing lamp. The following procedure is
then followed for the sampling purpose:
 Initial display shall be observed.
 Press “START ON” the control panel to obtain 1000 Ltr air
 Instrument shall be automatically OFF after sucking 1000 Ltr of air.
 Then open the SS hood and remove the plate aseptically.
 For next location, sanitize the stainless steel Hood and carry within air Sampler trolley.
Cover the plate with their upper lid then incubate for first 72 hours at 22.5 0C ± 2.5 °C and further 48 hours at 32.50C ± 2.5 °C
for fungal and bacterial growth respectively.
OBSERVATIONS
Table 06: TAMC < Action limit or Alert limit signifies that the area is pass.
Grade-B | Method: Active Air Sampling
Date Observation after 72hrs. Observation after 48hrs.
1/3/2019 <1 1
2/3/2019 <1 2
3/3/2019 <1 1
4/3/2019 <1 2
5/3/2019 <1 2
6/3/2019 <1 1
7/3/2019 <1 2
Table 07: TAMC > Action limit or Alert limit signifies that the area is fail.
Grade-B | Method: Active Air Sampling
Date Observation after 72hrs. Observation after 48hrs.
1/3/2019 <1 2
2/3/2019 <1 3
3/3/2019 <1 4
4/3/2019 <1 3
5/3/2019 <1 2
6/3/2019 <1 3
7/3/2019 <1 3
TREND CHART ANALYTICAL DATA
Table 08: TAMC < Action limit or Alert limit signifies that the area is pass.
Demonstration of One week Trend Chart for Active Air Sampling in Grade-B Area
Date 1/3/2019 2/3/2019 3/3/2019 4/3/2019 5/3/2019 6/3/2019 7/3/2019
TAMC 1 2 1 2 2 1 2
Limits 10 10 10 10 10 10 10
Action Limits 8 8 8 8 8 8 8
Alert Limits 6 6 6 6 6 6 6
Minimum 1
Maximum 2
Average 1.571428571

Chart 01: TAMC < Action limit or Alert limit signifies that the area is pass.
Table 09: TAMC > Action limit or Alert limit signifies that the area is fail.
Demonstration of One week Trend Chart for Active Air Sampling in Grade-B Area
Date 1/3/2019 2/3/2019 3/3/2019 4/3/2019 5/3/2019 6/3/2019 7/3/2019
TAMC 9 8 10 11 12 11 9
Limits 10 10 10 10 10 10 10
Action Limits 8 8 8 8 8 8 8
Alert Limits 6 6 6 6 6 6 6
Minimum 2
Maximum 12
Average 10
Chart 02: TAMC > Action limit or Alert limit signifies that the area is fail.
REFERENCES
[1] USP, “USP Microbiological Control and Monitoring of
Aseptic Processing Environments,” USP 35 vol. 1
2012a, 2012: pp. 697-707.
[2] Guidance for Industry – Sterile Drug Products
Produced by Aseptic Processing - Current Good
Manufacturing Practice, U.S. Food and Drug
Administration, 2004
[3] “Manufacture of Sterile Medicinal Products” In
EudraLex – The Rules Governing MedicinalProducts in
the European Union, Volume 4 EU Guidelines to Good
Manufacturing Practice – Medicinal Products for
Human and Veterinary Use – Annex 1: Manufacture of
Sterile Medicinal Products, European Commission,
2008
[4] Dalmaso, G., and Denoya, C. “Microbial Control and
Monitoring in Aseptic Processing Cleanrooms”
Controlled Environments (2015)
http://www.cemag.us/articles/2015/01/microbial-
control-and-monitoring-asepticprocessing-cleanrooms
[5] ISO International Standard 14644 Part 1,International
Organization for Standardization, May 1999
[6] https://en.wikipedia.org/wiki/Cleanroom

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