International PBI S.p.A.

Question:
How can I simplify the work, reduce sampling errors of operator, and apply the Good Sampling Practice while performing microbiological air monitoring in different environments, using an air sampler?

Answer:
Zenone DallaValle, International Pbi
-Introduction
Microbiological contamination levels may be extremely different from one site to another.
It is necessary to collect the appropriate volume of air in each environment avoiding operator’s mistakes.
-The “Configuration” function
The “CONFIGURATION” function of “SAS Super ISO” air sampler has been added with the purpose to give to the operator that works in different environments the chance to easily set the sampling parameters (sampling volume, type of sampling, operator name, sampling site). with The aim is:
(a) to reduce the possible errors;
(b) to apply the Good Sampling Practice.
This function allows to preset 8 different instrument configurations that the operator can easily recall from the MENU using the option “CONFIGURATION”.
The operator moving from one site to another doesn’t need, in this way, to re-set the instrument.
Using the “SAS Super ISO”, he just needs to recall the programmed configuration saving time and avoiding possible confusion and / or mistakes.

Question:
How the ISO, EC GMP, USP environmental classifications match?

Answer:
The classification is reported in the following table:

ISO	EC GOOD MANUFACTURING PRACTICE	USP	PURPOSE AND ENVIRONMENTAL AIR MONITORING FREQUENCY
5	A	100	Absence of micro-organisms during each shift / lot
6	B	1,000	Micro-organisms identification to built a database trend of the microbial population (during each shift / lot in the filling area)
7	C	10,000	Be-weekly monitoring
8	D	100,000	Monthly monitoring

Question:
What is the difference between the “Pyramid” and the “Politecnic” for the control of the “SAS” microbiological air samplers?

Answer:
The “Pyramid” and the “Politecnic” are the user-friendly systems to monitor the performances of the “SAS” microbiological air samplers.
The “Pyramid” is used by the operator to evaluate weekly, monthly or quarterly if the air sampler works regularly and it is not damaged by incorrect use or falling down.
The “Politecnic” is an official calibrated system and it s used by the distributor or official Agencies to calibrate and validate the volume of aspirated air of the sampler.

Question:
What is the purpose of the ISO/CD 14644-9: Cleanrooms and associated controlled environments - Part 9: Classification of surface particle cleanliness?

Answer:
This ISO standard describes the classification of the particle contamination levels on solid surfaces in Cleanroom applications.
Recommendations on testing and measuring methods as well as information about surface characteristics are given in informative annexes.
The standard applies to all surfaces in Cleanroom such as walls, ceilings, floors, working environments, tools, equipments and devices.
The surface particle cleanliness classification is limited to particles between 0.05 um e 500 um.
The aspects of chemical and biological surface contamination will be specified in further standards of this series.

Question:
Which stand/tripod for air samplers should be used in Clean Room?

Answer:
-Introduction
The stand or tripod used in Clean Room for the microbiological air samplers like “SAS” should be in stainless steel to avoid the particles emissions and to facilitate the cleaning and disinfection operations.
-The available stand / tripod
(a) The stainless steel Clean Room floor tripod has a fixed high of 168 cm.
(b) The stainless steel Clean Room wall / surface / cart stand is specific to fix the “SAS” air samplers to a wall or a wheel chart.

Question:
How to test the sterility of compressed and air gases used in Clean Room?

Answer:
-Introduction
The compressed gas and air used in Clean Room should be sterile. The microbiological sampling is made using the “Pinocchio Super II”.
-The working principle of “Pinocchio Super II”
A compressed air source is connected to the flow meter regulated to achieve the required flow rate (e.g.:100 litres of air / minute). The sampling period is timed to obtain the required total sample volume (e.g.:1000 litres of air). The impact on agar is the method of sampling. At the end of sampling time the plate with the media is incubated and then the CFU counted on the surface of the agar.
-The advantages of “Pinocchio Super II”
The advantages of “Pinocchio Super II” are:
• It follows the official impaction method recommended in USP.
• Simplicity.
• No electrical power (it is therefore “intrinsically safe”).

• No electronic parts (it is therefore “intrinsically safe”).
• No specialised servicing needed.
• Easy to use in the hand of operator thank to the complete documentation with pictures, clear installation diagrams and detailed User Guide.
• Easy to clean and sterilize.
• All parts coming into contact with gas sample may be steam sterilized.
• Convenient light weight carrying case for easy storage and transport to one sampling point to another.
• The validation is performer just on the air flow metre, avoiding the delivery of all the instrument.
• Application Notes, Standard Operating Procedures (SOP), and the I.Q., O.Q., P.Q. are available.

Question:
Where to find useful “Clean Room Recovery Times” information?

Answer:
Several useful suggestions are reported In “Recent Sci-Tech Discussion Group” on pages 14-16 of the document “PDA Letters” June 2008.
The question was: “Are there any regulation or reference that defines the performance of recovery time in Cleanrooms? So far I have checked EU Annex 1, but nothing is mentioned about the specific test. Any hint or comment will be welcomed.”
Several “respondents” give their point of view.
- www.pharmweb.net/pwmirror/pwq/pharmwebq2.html
- www.pda.org/pdaletter

Question:
How to test the sterility of compressed gas and air in Clean Room by a simple system?

How to test the sterility of compressed gas and air in Clean Room by a simple system?

Answer:
The “Pinocchio Super II” system has the advantage of robustness, simplicity and portability.
Robustness because it is totally made in stainless steel; simplicity because the system is ready to use in a few seconds and its components are elementary; portability because it is transferred from a place to another place using just a single handle. Furthermore the air flow meter of “Pinocchio Super II” can be officially certified, according to the inspection authorities, to guarantee the volume of the aspirated air.

Question:
Are the “SAS Super” microbiological air samplers conform to the ISO 14698?

Answer:
The best answer is to consider each single request of the ISO 14698.

-Summary
Glossary
Introduction
Active air sampling systems
The sampler characteristics in function of the area to be monitored
Impact agar system characteristics
Air samplers efficiency evaluation
Air sampler selection for special applications.
Conclusions

-Glossary
Agar, Bacteria, Bio-contamination, Active air sampler, CleanRoom, Disinfections, Efficiency , Microbiological indigenous population, Unidirectional air flow, Impact, Microbiological monitoring, Microbiological population , Standard ISO, Sterilization, CFU, Risk zone

-Introduction
The Standard ISO that involves the Microbiological Environmental Monitoring in CleanRoom are ISO-14644/1-2-3 for Clean Room classification and ISO 14698/1-2 for microbiological monitoring of contamination in Clean Room.

-Active microbial sampling devices
ISO 14698/1 - A.3.4.1. General
“The use of active air sampling devices in risk zones is essential to the assessment of the microbial quality of air. There are several types of active devices commercially available, each having its own limitations.”
It is therefore the microbiologist that decides the most suitable air sampler according to his needs, environment, etc.

- The sampler characteristics in function of the area to be monitored
ISO 14698/1 - 5.3.2. Sampling device
“A sampling devices shall be selected according to the area being monitored. The selection for a particular application shall take into consideration the following factors”:
The sampler should be selected in function of the area to be monitored.
“a) type of viable particles for which to sample”;
It is the microbiologist that decides which are the micro-organisms to be monitored: total microbial population or specific microbial population, according to the updated norms and his local experience
“b) sensitivity of the viable particles to the sampling procedure”;
Using the impact agar samplers, it is important to use fresh prepared plates and with the correct amount of media per plate
“c) expected concentration of the viable particles”;
It is the microbiologist that, depending from the type of environment, decides the volume of air to aspirate to avoid the dryness of the agar medium and to obtain a correct number of CFU to be counted
« d) indigenous microbial flora »;
The microbiologist should perform a preliminary air monitoring to know the indigenous microbial population of his environment. This results will be the base of his subsequent routine activity.
“e) accessibility of the risk zones” ;
The air sampler should be really portable
“f) ability to detect low levels of contamination”;
The low level of contamination can be detected with prolonged and interval sampling cycles
“g) ambient conditions in the risk zone being sampled”;
A suitable air sampler should be used in explosion risk area
“h) time and duration of sampling”;
The microbiologist should decide time of sampling in function of the risk experience, trends, etc.
“i) sampling method, material and properties of the sampling medium”;
The microbiologist should prepare a detailed protocol specifying the type of medium, etc. considering the standards involved, guidelines, expected risks and future possible problems
“j) effect of the sampling device on the process or environment to be monitored”;
The sampler should not interfere with the environmental conditions (e.g.: unidirectional air flow, temperature, working activity, etc.)
“k) collection accuracy and efficiency”
A comparative documentation should indicate accuracy and efficiency for micro-organisms collection
“l) incubation and viable particle detection and evaluation method”
The microbiologist should follow the protocol reported in norms, guidelines, and his experience
“m) type of information to be obtained (e.g.: qualitative or quantitative aspects)”
The microbiologist should select the information useful for his activity

-Guidance on measuring airborne biocontamination (Annex A)
ISO 14698 - A.3.2. Impact and impingement samplers
“Because of the number and variety of impact and impingement samplers available for the detection of viable particles, the device selected for use should have the following characteristics:
c) Impact velocity of the air hitting the culture medium that is a compromise between.
1) being high enough to allow the entrapment of viable particles down to approximately 1 um, and
2) being low enough to allow the entrapment of viable particles by avoiding mechanical damage of the break-up of clumps of bacteria or micro-mycetes”
The speed of the air on the agar surface produces by impact small cavity in correspondence of each hole of the aspiration head of the sampler. These cavity are the confirm of the correct conditions of impact.
d) “sampling volume that is a compromise between being large enough to detect very low levels of bio-contamination and being small enough to avoid physical or chemical degradation of the collection medium”
The possibility to sample from 10 to 1999 litres of air offers to the microbiologist a complete range to be chosen
“In areas of high bio-contamination, the impaction method and sample volume should be selected in way appropriate to achieving separate colonies, to allow the results to be interpreted”
The microbiologist, in case of expected high contamination, should program short aspiration times (low volumes of air) to obtain a countable CFU onto the agar plate
“The device should meet the following minimum requirements”_
-sufficient flow rate to collect 1 cubic meter of air in a reasonable time, without significant drying of the sampling medium;
The volume of aspirated air could be 100 litres (e.g.:10 minutes for 1000 litres of air) or 180 litres ( 6 minutes for 1000 litres of air)
-appropriate air impact speed to the culture medium”
The correct impact speed is confirmed by the presence of small cavities on the agar surface under each hole of the aspirating head

- Air samplers efficiency evaluation
The Annex “Guidance for the evaluation and qualification of the efficiency of air sampler in bio-contamination control in Clean Room” ISO DIS 14698 presents the comparison method using a membrane system sampler
The “SAS” comparative efficiency test were performed using a membrane system sampler

- Air sampler selection for special applications
“Because of the number and variety of microbial air sampling systems commercially available, the selection for a particular application should consider, as a minimum, the following factors:
j) “type and size of viable particles to be sampled”;
It is the microbiologist that decides which are the micro-organisms to be monitored: total microbial population or specific microbial population, according to the updated norms and his local experience
k) “sensitivity of the viable particles to the sampling procedure”;
Using the impact agar samplers, it is important to use fresh prepared plates and with the correct amount of media per plate
l) “expected concentration of the viable particles”;
It is the microbiologist that, depending from the type of environment, decides the volume of air to aspirate to avoid the dryness of the agar medium and to obtain a correct number of CFU to be counted
m) “ability to detect high or low levels of bio-contamination”;
The low levels of contamination can be detected with prolonged and interval sampling cycles. The high levels of contamination can be detected with short sampling cycles
n) “appropriate culture medium”;
Is the microbiologist that, depending from his needs connected to the local environment and / or official norms, decides the correct type of medium to be used
o) “time and duration of sampling”;
The microbiologist should decide time and volume depending from his experience, trend and local situation (microbial population, continuous or interval sampling)
p) “ambient conditions in the environment being sampled”;
If the sampling is requested in a risk area, it is necessary to use a suitable sampler for the specific area (e.g.: aspiration chamber separated from the command unit or explosion proof sampler)
q) “disturbance of unidirectional airflow by the sampling apparatus”;
The aspirated air inside the air sampler and then expulsed should not alter the air unidirectional flow of the environment. The picture reported in the Application Note N.99/46 shows how the “SAS” air samplers do not interfere with the unidirectional air flow
r) sampler properties such as:
1) “appropriate suction flow rate for low levels of viable airborne particles”:
The air flow aspirated by SAS is suitable for low level of contamination monitoring
2) “appropriate impact / airflow velocity”
The correct air flow velocity of SAS has been calculated and experimented to reach the most suitable impact on agar surface during the last 20 years
3) “collection accuracy and efficacy”;
The text of the Application Note N.99/70 shows the correct “SAS” performances
4) “easy of handling (weight, size) and operation (easy of use, auxiliary equipment, dependance on vacuum pump, water, electricity, etc.)”
The “SAS” air sampler is handy, compact, and complete of accessories. It is autonomous, without vacuum external pump, water and main electricity (the battery has an autonomy of about 7 hours)
5) “easy of cleaning and disinfections or sterilization”;
The cleaning and disinfections are easy because the body is a single piece without fissures

“The exhaust air from the sampling apparatus should not contaminate the environment being sampled or be re-aspirated by the sampling device”
The pictures reported in the Application Note N.99/46 clearly show how the “SAS” air sampler do not modify the unidirectional air flow , do not contaminate the environment and the air is not re-aspirated.
-Conclusions
The reported information confirm the “SAS” microbiological air samplers conform to the ISO 14698.

Question:
How to wipe a surface in Clean Room using the wiper?

Answer:
A specific technique should be applied for cleaning, disinfection, residue removal, and drying of surfaces using wipers. Always begin cleaning from the area of the surface with the expected lowest contamination to the area of highest contamination.
The wiper should be fold into four distinct quadrants.
Always present a clean wiper surface to remove the contamination with each unidirectional stroke. Each pass of the wiper should overlap the previous by about 10/20%. The wiper should be refolded after each pass to present a clean surface.
Do not wipe in a circular pattern, as this will lead to the re-depositing of contaminants onto freshly cleaned surfaces.

Question:
How the maintenance and calibration of the air sampling devices should be set up to apply the Good Laboratory Practice (GLP) and Good Manufacturing Practice (GMP)?

Answer:
We report the text of the Chapter 12 “Quality Assurance” of the European Standard EN 14042 – “Workplace atmosphere – Guide for the application and use of procedures for the assessment of exposure to chemical and biological agents”.
“It is good practice to set up a Quality Assurance scheme for the maintenance and calibration of the samplers. This includes:
a) the establishment of a Standard Operating Procedure (SOP);
b) for re-usable device, a log of usage;
c) keeping a record of the traceability of the calibration;
d) retaining the raw data as required by the quality or other system;
e) using a unique and durable sampler numbering system for the re-usable devices;
f) depending on the measurement task, taking an appropriate number of field blank and replicate samples (e.g.: 10%);
g) an appropriate level of internal and external Quality Assurance.”

Question:
What is the difference in Clean Room classification between Annex 1 Eudralex and ISO 14644-1?

Answer:

	At Rest	At rest
Grade A	ISO 4.8	ISO 4.8
Grade B	ISO 5	ISO 7
Grade C	ISO 7	ISO 8
Grade D	ISO 8	not defined

Question:
Which are the news in the CE GMP Revision Annex 1 (14 February 2008) about “Media Simulation”?

Answer:

When filling fewer than 5.000 units	No contaminated units should be detected
When filling from 5000 to 10000 units	c) one contaminated unit should result in an investigation, including consideration of a repeat media fill d) two contaminated units are considered cause for revalidation, following investigation
When filling more than 10000 units	c) One contaminated unit should results in an investigation d) Two contaminated units are considered cause for revalidation, following investigation

Question:
According to the CE GMP Annex 1 Revision (14 February 2008), which are the maximum permitted number of particles per cubic metre equal or greater than 0,5 um e 5,0 um. in Grade A, B, C, D.?

Answer:

According to the CE GMP Annex 1 Revision (14 February 2008), which are the maximum permitted number of particles per cubic metre equal or greater than 0,5 um e 5,0 um. in Grade A, B, C, D.?

	At rest	At rest	In Operation	In Operation
Grade	0.5 um	5.0 um	0.5 um	5.0 um
A	3520	20	3520	20
B	3520	29	352000	2900
C	352000	2900	3520000	29000
D	3520000	29000	not defined	not defined

Question:
How to position the microbiological air sampler inside a Clean Room?

Answer:
The microbiological monitoring of the air inside the Clean Room by an air sampler needs different solutions depending from the type of production, available space, inside environment, sampling frequency, personnel, etc. It is important to adopt the correct tripod or holder.
Different solutions are adopted:
(a) Tripod >>>
Four different models are available:
a1. Tripod “Stainless Steel Clean Room Floor Tripod“ for floor, totally made in stainless / steel to avoid particulate emission;
a2. Tripod “Compact Floor Tripod“ for table, extensible;
a3. Tripod “Normal Floor Tripod” for floor, extensible from 100 cm up to 280 cm.
a4. Tripod “Table tripod“ for table, not extensible.
(b) Support
b1. Support “Stainless Steel Wall / Surface / Cart Tripod“ for wall, table o mobile cart, totally made in stainless / steel.

Question:
How to convert in Clean Room the alert and action microbiological limits from "Passive Air Monitoring" to "Active Air Monitopring"?

Answer:
Zenone Dall Valle, International Pbi
-Introduction
The answer is not simple and it requires some explanations.

Passive air monitoring (Petri plates) and active air monitoring (Air Sampler) provide DIFFERENT information about the microbiological quality of the environment:

Following USP indication, Petri dishes provides only QUALITATIVE information (presence or absence) of microorganism sedimentation in a specific place. Note also, that many air borne micro-organisms do not sediment since they are so small that they are to subjected to the gravity force.

Beside, active air monitoring provide both QUALITATIVE and QUANTITATIVE microbiological information since it is possible to correlate the CFU to the volume of air ( results are shown as CFU/m³ of aspirated air).

The two monitoring systems are therefore different and they can not be correlated each other.

- Alert and Active level

To asses alert and active level using an Air Sampler, you should consider the classification of the specific environment rather than to extrapolate the indication by a comparison to the passive air monitoring.

The below reported tables may be useful for this purpose.

Particles Air Classification

Clean Area Classification (0.5 um particles/ft³)	ISO Designation	≥ 0.5 mm particles/m³	Microbiological Active Air Action Levels (cfu/m³)	Microbiological Settling Plates Action Levels (diam. 90mm; cfu/4 hours)
100	5	3,520	1	1
1000	6	35,200	7	3
10000	7	352,000	10	5
100000	8	3,250,000	100	50

Please note that the passive air monitoring is complementary to the active air monitoring, and by itself , it is not adequate to asses the microbiological air quality.

Reference: http://www.fda.gov/cder/guidance/5882fnl.htm

Recommended microbiological limits for “Clean Areas”

Grade	Air Sample (CFU m³)	Contact Plates (CFU Plate)	5 Gloves Finger Print (CFU Plate)
A	< 1	<1	<1
B	10	5	5
C	100	25	-
D	200	50	-

Reference: European GMP guidelines. January 1998.

Question:
How to decontaminate and control the result of decontamination in isolator?

Answer:
-Glossary
Biodecontaminant, biodecontamination cycle, cleanroom, D-value, biological indicator, chemical indicator, isolator, hydrogen peroxide, peracetic acid, spore, sporicide, sterilisation, total kill, validation, VHP.
-Introduction
The use of isolators is day by day more frequent in the pharmaceutical and food fields, in total or partial replacement of Clean Room.
The official definitions of isolator for pharmaceutical use are reported in the following documents:
-PICS 014-1 “Isolators used for aseptic processing and sterility testing” (5.1 e 5.2.)
-PDA Technical Report 34 “Design and validation of Isolator Systems for the manufacturing and testing of health care products”.
-USP <1208> “Sterility testing validation of isolators systems”.
The biodecontamination cycle of the isolator and the related control are fundamental for its correct use.
-Isolator biodecontamination
The used and more frequent typical germicidal agents are peracetic acid and hydrogen peroxide (VHP). The biodecontaminants are toxic and therefore should be use with precaution.
The biodecontamination parameters of the gaseous cycle are its concentration, permanence time, final aeration time. It is also very important to consider the amount of items inside the isolator to avoid non homogeneous concentration of the sporicide gas and its presence at the end of the decontamination period.
-The use of chemical indicators
The chemical indicators should be inserted in different positions inside the isolator to find areas that are not adequately treated by the sporigen gas.
-The use of biological indicators
The sterilization should reach the reduction of the microbial population of at least 6-log.
The biological indicator to be used is Geobacillus stearothermophilus ATCC12980
with a starting population of 106 spores and a D-value between 1 and 2 minutes.
The used method is the “total kill”. The ideal “carrier” for the microbial population of the biological indicator is the stainless steel.
-The periodic microbiological monitoring of the isolator
The isolator should be regularly monitored using an active microbiological air sampler like “SAS-ISO” using contact plate (RODAC) o Petri dish.
The advantages of the use of “SAS-ISO” in isolator are: very compact, easy to use, stainless steel construction, robustness, sporicide resistance.

Question:
Which are the physical and microbiological specifications for particulate and microbial counts (CFU) in Class 100 Clean Room according to US FS209E, USP <1116>, EU e ISO 14644-1?

Answer:
Maximum values are given

Document Documento	US FS209E	USP<1116>	EU (at rest,static)	EU (operational, dynamic)	EU (operational, dynamic)	ISO 14644-1
Clasification Classificazione	M 3.5 (100)	M 3.5	A and B	A	B	5
Frequence Frequenza	Not stated	Each operational shift	Not stated	Frequent, using a variety of methods	Frequent, using a variety of methods	Not stated
Total Particulate Count Conta particellare	3.500/m3 (>0.5 um) 100/cu.ft	100/cu.ft (>0.5 um)	3.500/m3 (equal to or above 0.5 um) 0/m3 (>5 um)	3.500/m3 (equal to or above 0.5 um) 0/m3 (>5 um)	350.000/m3 (equal to or above 0.5 um) 2.000/m3 (>5 um)	3.520/m3 (equal to or above 0.5 um) 29/m3 (5.0 um)
Airborne viable Conta microbica aria	Not stated	0.1 CFU per cu.ft	Not stated	<1 CFU/m3 Settle plate 90 mm <1 CFU/4 hr	<10 CFU/m3 Settle plate 90 mm 5 CFU/4 hr	Not stated
Surface viable Conta microbica superfici (escluso pavimenti)	Not stated	3 CFU per Contact Plate (20-24 cm2)	Not stated	<1 CFU per Contact Plate (no distinction for floors and walls)	5 CFU per Contact Plate (no distinction for floors and walls)	Not stated
Surface viable floors Conta microbica pavimenti	Not stated	3 CFU per Contact Plate	Not stated	<1 CFU per Contact Plate	5 CFU per Contact Plate	Not stated
Personnel gown Indumenti del personale	Not stated	5 CFU per Contact Plate	Not stated	Not stated	Not stated	Not stated
Personnel gloves Guanti del personale	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 unidirectional speed Velocità unidirezionale dell’aria	Not stated	Not stated	0,45 m/s + 20%	0,45 m/s + 20%	Not appropriate	Not stated
Differential Pressure Frequency Frequenza pressione differenziale	Not stated	Each shift	Not stated	Continuous	Continuous	Not stated

Question:
How to investigate for “OOS” results in environmental monitoring?

Answer:
The suggestions were given by Dr. Francesco Boschi at “pbi” Seminar about the environmental monitoring in Clean Room.
“Investigation for “OOS” results
If there are significative variation in the trend, the following investigations are necessary:
(a) Identification of isolated environmental micro-organisms
(b) Revision of the trend during the last 6-12 months in the specific area
(c) Particle monitoring
(d) Monitoring and Servicing of the air conditioning or HVAC System
(e) Sampling improvement
(f) Revision of the sterilization processes
(g) Revision of the cleaning and disinfection processes
(h) Revision of staff training about cleaning, disinfection, sterilisation
Corrective actions in case of “OOS”
(i) Environmental Sampling procedure revision
(l) Re-environmental monitoring of the considered area
(m) Use of other specific microbiological media
(n) Revision of production procedures
(o) Revision of cleaning and disinfection procedures
(p) Challenge of isolates vs products
(q) Challenge of isolates vs disinfectants
(r) More appropriate staff training
(s) Further controls on products.

Question:
Which are the physical and microbiological specifications of USF 209E, USP<1116>, EU, ISO 14644-1 for Class 10.000 environments?

Answer:

Document	US FS 209E	USP <1116>	EU (at rest, static)	EU (operational, dynamic)	ISO 14644-1
Classification	M5.5 (10.000)	M5.5	C	C	7
Frequency	Not stated	Each operating shift	Not stated	Not stated	Not stated
Total Particle Count	353.000/m3 (>0.5 um) 10.000/cu.ft	10.000/cu.ft. (>0.5 um)	350.000/m3 (equal to or above 0.5 um) 2.000/m3 (>5 um)	3.500.000/m3 (equal to or above 0.5 um) 20.000/m3 (>5 um)	352.000/m3 (equal to or above 0.5 um) 930/m3 (5.0 um)
Airborne viables	Not stated	0.5 CFU per cu.ft.	Not stated	100 CU/m3 Settle plate 90 mm 50 CFU/4 hr	Not stated
Surface viable (except flor)	Not stated	5 CFU per contact plate	Not stated	25 CFU per contact plate	Not stated
Surface viable (floors)	Not stated	10 CFU per contact plate	Not stated	Not stated	Not stated
Personal gown	Not stated	20 CFU per contact plate	Not stated	Not stated
Personal gloves	Not stated	10 CFU per contact plate	Not stated	Not stated
Frequency of ∆P monitorig	Not stated	Each shift (1); 2x/week (2)	Not stated	Not stated

Note:
∆P = Differential Pressure
(1) = Adjacent to Class 100
(2) = Support areas - Product

Question:
Is the “SAS” microbiological air samplers family conforming to the ISO 14698-1 Standard request concerning the collection of micro-organisms?

Answer:
According to ISO 14698-1, the microbiological air samplers should be able to collect micro-organisms present in the air of Clean Room up to 1 micron in size.
Considering that:
(a) the SAS air samplers are able to collect 100% particles over 4 microns and about 60% below 4 microns;
(b) the concentration of microbiological particles of 1 microns in indoor environments is extremely low (the d50 – the particle diameter at which the sampler efficiency drops below 50% - is approx. 0.5 microns) the collection efficiency of SAS in Clean Room could be considered close to 98/99%.

Question:
Why to use the “DispoHead” certified sterile, disposable, aspirating head with “SAS” microbiological air samplers in Clean Room air monitoring or Indoor Air Quality?

Answer:
Clean Room
The air monitoring in Clean Room using an “active” sampler should be performed with sterile aspirating head and plates (RODAC or Petri dish). The aspirating head should ideally be sterilized by autoclaving and the sterilization should be “certified”. Other methods (e.g.: chemical sterilization) doesn’t guarantee (and cannot be certified) that the goal has been reached.
According to the requests of the official inspectors (e.g.: FDA), the sterilization document should be available.
It is for these reason that international pbi developed the “DispoHead” disposable aspirating head. Each aspirating head in identified by lot number and sterilization certificate and has the same physical performances of the aluminium or stainless steel heads.
The “DispoHead” is disposable but can be used several times in Clean Room in the same day or for the same group of sampling daily activity (or shift) in the same closed environment.
Indoor Air Quality
The “Dispohead” is “imperative” in the Indoor Air Quality activity when legal disputes are on.

Question:
According to the FDA “Guidance for Industry Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice” dated September 2004, which characteristics an air sampler for the active air monitoring should have?

Answer:
We report the text of chapter “A. Environmental Monitoring – Monitoring Methods. b. Active Air Monitoring”:
“Assessing microbial quality of air should involve the use of active device. We recommend that such devices be used during each production shift to evaluate aseptic processing areas at carefully chosen locations. Manufacturers should be aware of a device’s air monitoring capabilities, and the air sampler should be evaluated for its suitability for use in an aseptic environment based on collection efficiency, cleanability, ability to be sterilized, and disruption of unidirectional airflow.
The user should assess the overall suitability of a monitoring device before it is placed into service. Manufacturers should ensure that such devices are calibrated and used according to appropriate procedures.”
All “SAS Surface Air System” air sampler produced by International pbi have the requested specifications.
The “Certificate of Conformity” document confirms that the “SAS” air samplers fulfil the FDA requirements.

Question:
Which are the microbiological specifications for CFU counts in Clean Room according to EU and USP?

Answer:

Sample	Class 100 USP <1116>	EU Grade A	Class 1000 USP <1116>	EU Grade B	Class 10.000 USP <1116>	EU Grade C	Class 100.000 USP <1116>	EU Grade D
Air Sample/cubic meter 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 visibles/plates (except floors)	< 3 CFU	NS* <1 CFU**	NS	NS* <5 CFU**	< 5 CFU	<25 CFU	NS	NS* < 50 CFU**
Surface viables/plate (floor)	< 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	< 3 CFU	NS	NS	NS	< 20 CFU	NS	NS	NS

Note:
* = At Rest
** = Dynamic
NS = Not Stated
CFU = Colony Forming Unit

Question:
Which are the applications of all “SAS” instruments and accessories for the microbiological monitoring of the air?

Answer:

Environment	Application	Instrumentation
Clean Room	Portable sampling in Clean Room Grade A and B	SAS Super ISO 100 or SAS Super ISO 180
Clean Room	Tripod without particle emission	Stainless Steel Tripod or Wall Stand
Clean Room	Wall fixed sampling in Clean Room Grade A and B	SAS ISO Network
Clean Room	Bar-code Identification	SAS ISO Barcode
Clean Room	"Contemporaneus Double" sampling in Clean Room	Duo SAS Super 360
Isolator	Sampling with outside command	SAS Isolator
Explosion Proof Environment	Sampling in Hazard Risk Areas	SAS Super ISO EXPR
Bioterrorism	Sampling of pathogens for PCR Tests	SAS PCR
Compressed air and gas	Sterility evaluation of gas used in Clean Room	Pinocchio Super II
Biological Safety Cabinet	Sterility Cabinet Evaluation	SAS Super ISO
Laboratory	In House Physical Control of air sampler	Pyramid
Laboratory	Official Physical Control of air sampler	Politecnic
Food, Dairy, Beverage Industry	Hygienic evaluation of production premises	SAS IAQ or SAS ECO
Hospital	Operating theatre and high risk patient area monitoring	SASuper ISO 100
Indoor Air Quality	Public areas and building monitoring	Duo SAS Super 360 or SAS IAQ
Waste Water Treatment Plant	Surrounding area monitoring for staff and inhabitant safety	SAS ECO
HVAC	Heating Ventilation Air Conditioning	SAS Super ISO + Tripod
Clean Room and IAQ	Certified sterile, semi-disposable aspeirating head	Dispo-Head
School and Univesity	Hygiene training and education	SAS ECO
Biological Research	Microbiological Tests	SAS Super ISO
All environments	Certified sterile, empty Contact Plate (RODAC)	Surfair Plate
All environments	Certified sterile, medium filled Contact Plate (RODAC)	Agar Contact
All environments	Sterile disinfectant for aspirating head	Bacti Spray
All environments	Carrying case for Clean Room	Bio Safe Carrier
All environments	Soft Carrying case for local sampler transfer	Pratica Soft
All Environments	Impact Proof Carrying case for sampler shipping	Light Case
All instruments	Physical Calibration	Official Calibration according to NIST
All instruments	Microbiological calibration	Official Calibration according to ISO

Question:
Which is the terminology that the operator of clean room should know?

Answer:
Contamination Control Monitoring – IEST RD CCO11.2

Glossary of terms

A
Adhesion - The force exerted across a surface of contact between liquids and solids, or solids and solids, that resists their separation
Aerosol - A gaseous suspension of fine solids or liquid particles
Aerosol air generated - The aerosol that results when the pneumatic force of a high-velocity stream of air breaks up a specific room-temperature liquid into droplets
Aerosol photometer - An instrument that measures mass concentrations of aerosol by using the forward light-scattering principle.
Airborne particle - Particles suspended in moving or stationary air
Airborne particulate cleanliness class - A numerical designation that signifies a maximum allowable concentration of airborne particles of a specified particle size. As established by FED-STD-209, the numerical value of a class is related to the concentration of particles in the size range of 0,5 um and larger
Airflow - Movement of air in a given direction with respect to a specified reference plane or other designated physical reference. The rate of airflow is expressed in terms of volume per unit time
Airflow, parallel - Unidirectional airflow, as demonstrated by a measured deviation of not more than 14 degrees from straight line flow.
Airflow, uniform - A unidirectional airflow pattern in which the point-to-point readings are within +/- 20% of the average air flow velocity for the total area of the unidirectional flow work zone
Anemometer - An instrument for measuring airflow velocity
Anisokinetic Sampling - The condition of sampling in which the mean velocity of the air entering the inlet of the sampling probe. Because of particle inertia, anisokinetic sampling can cause the concentration of particles in the sample to differ from the concentration of particles in the being sampled
Aseptic processing - Production activities that exclude all extraneous living matter, especially microscopic forms such as bacteria and fungi, from the production units being assembled, filled, packaged, or built

B
Bacterial spore - A resistant body produced by a vegetative bacterial cell. These spores may be resistant to heat, ultraviolet light, and may remain dormant for extended periods of time
Bactericide - A substance or formulation capable of destroying bacteria
Bacteriostat - A compound or formulation that retards or prevent the growth of bacteria. Bacteriostats act reversibly; when they are removed, cells resume their normal growth patterns
Bioburden - A measure of the number of micro-organisms resident or within products and inanimate objects. A common synonyms is micro-count

C
Certificate of Compliance - A written statement signed by a qualified person and accompanied by substantiating information, attesting that the items or services are in accordance with specified requirements
Certification - The culmination of procedures leading for validation, expressed in the form of written evidence (certificate of compliance) that verification has been accomplished in accordance with specified testing procedures such as FED-STD-209
CFU - Colony Forming Unit - An individual or an aggregate of many microbial cells which, when cultivated on solid media, will develop into a single visual colony
Challenge Aerosol - An aerosol derived from the selected aerosol source material and used as the test challenge for filter medium testing
Chemical compatibility - The ability of a material to withstand chemical contact with minimal interaction
Chemical degradation - Changes in materials that occur when they come into contact with chemicals. Degradation may take such forms as swelling, loss of tensile strength, or loss of abrasion resistance
Class (airborne particulate) - The cleanliness class as defined by FED-STD-209
Clean Air Device - A clean bench, clean work station, download module, or other equipment designed to control particulate air cleanliness in a localised working area and incorporating, as a minimum, a HEPA filter and a blower
Clean Air Device (laminar flow) - This term is no longer in use. See clean-air device, unidirectional flow
Clean Air Device (unidirectional flow) - A clean work station or other device that incorporates one or more HEPA or ULPA filters and one or more motor-blowers for the purpose of supplying unidirectional-flow clean air to a controlled work space
Clean Air System - A system designed to maintain the cleanliness of air in a clean room or clean zone at or below a specified airborne particulate cleanliness class
Cleaning agent - A liquid that enhance the removal of contaminants from surfaces, by such mechanism as dissolution, reducing surface tension, and neutralising electrostatic charge. Cleaning agents can also be disinfectants.
Cleanroom - A room in which the concentration of airborne particles is controlled and which contains one or more clean zones
Cleanroom facility as-built - A cleanroom that is complete and ready for operation, with all services connected and functional , but without production equipments or personnel within the facility
Cleanroom facility at-rest - A cleanroom facility that is complete and has the production equipments installed and operating, but is without personnel within the facility
Cleanroom facility operational - A cleanroom facility in normal operation with all services functioning and with production equipments and personnel, if applicable, present and performing their normal work function in the facility
Cleanroom, laminar air flow - This term is no longer in use. See cleanroom, unidirectional
Cleanroom mixed airflow - A hybrid cleanroom consisting of a combination of an unidirectional airflow cleanroom and a nonunidirectional-flow cleanroom
Cleanroom non unidirectional airflow - A cleanroom in which airflow exist with a multiplicity of flow directions or multiple-pass circulations or both
Cleanroom turbulent airflow - This term is no longer in use. See cleanroom, nonunidirectional airflow
Cleanroom unidirectional airflow - A cleanroom in which airflows have generally parallel streamlines operating in a single direction and with uniform velocity over the cross section
Contaminant - Any unwanted substance present in or a material or an surface within a clean zone
Contamination - The result of the addition of contaminants to a material or to any surface within a clean zone.
Contamination control - Any organized effort taken to reduce the level of contamination
Controlled environment - An environment in which parameters such as temperature, pressure, humidity, contaminant level are controlled within specified limits

D
Decontamination - The removal of contaminants present in or on a material
Decontamination factor - The ratio of the concentration of contaminant before and after decontamination, cleaning or treating
Disinfectant - An agent, chemical or physical, designed to reduce the population of micro-organisms

F
Filter - A device used to remove particulate matter or impurities from a fluid (liquid or gas) flowing through it
Filter, mechanical - Any device that remove particles from a fluid by mechanism of impaction, settling, screening, inertia, diffusion, or any combination thereof
Filter, membrane - A filter, generally thin and manufactured from polymeric plastics or from metal, the open structure of which is comprised of pores or capillaries in a controlled range of sizes
Flora - The type of microbiological organisms found in an environment
Fumigation - A process of decontamination in which the decontaminant is administered in gaseous, smoke or aerosol form at ambient pressure
Fungi - Non photosynthetic micro-organisms possessing cell wall and usually growing as amass of branching interlacing filaments known as mycelia. The mycelial forms are called moulds, and the nonmycelial forms are called yeast. Fungi are usually larger than 0,4 um in size
Fungicide - A substance that is capable of destroying fungi
Fungistat - A substance that retards or prevents the growth of fungi. The effects of fungistats are reversible; when the fungistats are removed, the fungi resume their normal growth patterns

G
Garments for cleanroopm - Specially designed items of clothing that are worn to prevent or reduce the dispersion of contaminants that may be shed or released by personnel (footwear, shoe cover, gloves, head covers)
Gram-negative bacteria - Bacteria that do not retain the purple colour of the crystal violet dye when treated with Gram’s stain microscopic technique
Gram-positive bacteria - Bacteria that retain the purple colour of the crystal violet dye when treated with Gram’s stain microscopic technique
Gravimetric analysis - A standard analytical technique used to determine the mass of a specified material and commonly used to quantify the amount of non volatile residue in a solvent

H
HEPA - High Efficiency Particulate Air filter

I
Impaction - Physical contact of particles with other surfaces in response to the application of an external force. The term is occasionally used synonymously with impingement
Impactor - A sampling device capable of removing particles from a suspension using he principle of impaction (impingement)
Impactor, cascade - A “cascade impactor” refers to a specific instrument that employs several impactions in series to collect particles of successively smaller sizes
Impingement - A sampling procedures whereby particles are removed from a gaseous suspension by forcibly directing the flowing gas against a collection surface
Impingement dry - Impingement in which the particle-colleting surface is a solid or a solid coated with an adhesive
Impingement wet - Impingement in which the particle-collecting surface is a liquid or is located beneath the surface of a liquid bath
Isoaxial - A condition of sampling in which the direction of the air flow into the sampling probe inlet is the same as that of the unidirectional airflow being sampled
Isokinetic sampling - The condition of isoaxial sampling in which the mean velocity of the air entering the sampling probe inlet is the same as the mean velocity of the unidirectional airflow at that location

L
Laminar air flow - The term is no longer in use. See unidirectional airflow
Launderability - The ability of an article such as glove or garment to be laundered to a specific level of cleanliness
Leak Test - A procedure for evaluating the potential for particle intrusion into a clean zone

M
Microbiological Clean Area - A defined space with a filtered air supply that is designed to accommodate the performance of activities for which the control of viable particles is essential
Monitoring - The routine determination of airborne particle concentrations, as well as other relevant parameters, in Clean Rooms and clean zones

N
Non corrosive - Term applied to substances that have a minimal effect on the integrity or appearance of surfaces with which they come in contact
Non ionic - A term used to describe materials that do not posses a net electrical charge
Nonunidirectional airflow - Airflow that does not qualify as unidirectional because it has a multiplicity of flow directions or multiple pass circulation, or both

P
Particle - An object that is solid, liquid or both, usually between 1 nanometer and 1 millimetre in size
Particle burden - The number of particles in the relevant size range per unit area of surface
Particle concentration - The number of particles per unit volume of fluid
Particle count - The number of particles detected (or reported) in a given volume of fluid
Particle counter, airborne - An instrument for continuous counting of airborne particles larger than a given threshold size. The sensing means may be optical, electrical, aerodynamic, etc.
Particle deposition - The portion of the transient particle population that does not remain airborne, typically with respect to a specified period of time, under the influence of ambient airflow conditions, air and surface temperature, and localised electrostatic charges
Particle diameter, mass median - A measure of median particle diameter based on the particle mass
Particle size - The apparent maximum linear dimension of a particle in the plane of observation, observed with an optical microscope or the equivalent diameter of a particle detected by automatic instrumentation
Particle size distribution - The relative mass or number of particles present in a series of discrete subdivision of the overall range of designed particle sizes
Particulate - An adjective referring to particles (e.g.: particulate matter)
Pathogen - A micro-organism capable of causing disease
Plenum chamber - An enclosed, positive pressure section of a gas supply system, designed to ensure even distribution of flow. Examples include: (a) the point of origin for air distribution in air conditioning and ventilation; (b) the supply source for a filtration system to create even distribution of the gas to each filter unit
Precision - The closeness of agreement between values obtained by repeated measurements of the same quantity under the identical conditions; also known as repeatability or reproducibility
Pre-filter - A cleaning unit installed upstream of another unit to protect the latter from high concentration of contaminants. Pre-filters are usually less efficient and less expensive that the units they protect
Prep-Area (Decontamination area) - A cleanroom or clean zone in which preliminary cleaning or other preparatory procedures are performed before entry into the operational cleanroom. The prep area typically is less clean than the operational cleanroom
Pressure differential - The difference in pressure between two points of a system, which may be separated by a solid or fluid barrier
Pressure gauge - The amount by which the total absolute pressure exceeds the ambient atmospheric pressure
Pressure, total - The sum of static pressure and velocity pressure at the point of measurement
Pressure, velocity - The impact pressure associated with the velocity of a flowing fluid. It is related to the kinetic energy per unit volume of the flow
Primary surface - A surface that is in direct contact with the product
Pyrogen - A substance capable of producing a fever. Common pyrogens are endotoxins, a class of lipopolysaccharides that comprise the outer wall of Gram-negative bacteria and are themselves by-products of cell metabolism

R
Re-circulated air - The portion of the clean room air supply that has been re-circulated by the air-handling equipment
Recommended Practice - A published document that provides technical guidance, philosophy, or preferred procedures regarding a given topic
Residence time - The finite period of time during which a material is contained within a handling or processing system, wherein component of the material may be subjected to change.

S
Sampling - A process consisting of the withdrawal or isolation of a fractional part of a whole. In air or gas analysis, the separation of a portion of an ambient atmosphere with or without the simultaneous isolation of selected components
Sampling, continuous - Sampling without interruption throughout an operation or for a predetermined time
Sampling, instantaneous - Sampling of a period of time that is negligible in comparison to the duration of the operation or to the total period of time being studied
Sampling, intermittent - Sampling successively for a limited period of time throughout an operation or for a pre-determined period of time. The duration of sampling periods and of the intervals between are not necessarily regular and are not specified
SAS Surface Air System - Microbiological air sampler
Secondary surface - A surface that is not in contact with the product, but which indirectly affects the product
Simultaneous counting system - A system that uses a pair of detectors to simultaneously determine the concentrations of the test aerosol both upstream and downstream of the filter medium
Sporicide - A substance or formulation capable of destroying bacterial spores
Standard air - Air at 50% relative humidity, with a temperature of 21°C and pressure of 760 mm Hg
Sterilisation - A physical or chemical process that completely destroys or eliminates all forms of microbial life

T
Tunnel - A mini-environment in which an enclosure separates the tools and wafer-handling robot aisle from the general clean room; or an enclosed single-wafer track for transport into and between tools

U
Unidirectional air flow - Air that flows in a single pass in a single direction through a clean room or clean zone with generally parallel streamlines. Formerly referred to as laminar airflow

V
Validation - Establishment, by means of a specified testing program, that a system or facility under test is capable of performing as intended
Verification - The procedure for determining compliance of air in a clean room or clean zone to a specified airborne particulate cleanliness class limit or U descriptor; also the result of determining such compliance
Viable particle - A particle capable of reproduction ; a living organism
Virus - An obligate intracellular parasite, typically in the size range of 0.01 to 0.1 um, that can infect and cause disease in man, animals, plants, and insects

W
Wiper - A piece of fabric or cloth designed for cleaning and removing liquids from critical surfaces

Question:
Which are the most important references to consider for the environmental monitoring in Clean Room and Controlled Environments?

Answer:
(A) Parenteral Drug Association (PDA), Technical Report No.13. “Fundamentals of an Environmental Monitoring Program”.
(B) United States Pharmacopeia and National Formulary, USP <1116> -“Microbiological Evaluation of CleanRooms and Controlled Environments”.
(C) International Standard Organization ISO 14644-1 – “Classification of Air Cleanliness”
(D) European Union (EU) “Annex on the Manufacture of Sterile Medicinal Products”
(E) U.S. Food and Drug Administration (FDA) “Guideline on Sterile Products Produced by Aseptic Processing”.

Question:
Which documents should be prepared to demonstrate the correct application of the environmental monitoring in a Clean Room?

Answer:
Documentation for a robust Environmental Monitoring System should include the following:
▪ Standard Operatine Procedures (SOP)
▪ Environmental Monitoring test procedures
▪ Instructions and service manuals, , calibration procedures and logs
▪ Cleaning and sanitization data sheets
▪ Sample site maps
▪ Alert and Action Levels
▪ Test results, date of results, incubation temperature deviation, and quality review
▪ Microbial Identification
▪ Environmental trending spreadsheets, histograms, etc.
▪ Deviation reporting and / or Company “Corrective and Preventive Action” (CAPA) procedures.
All the documentation should be completed according to Good Documentation Practices (GDP).

Question:
Which news are present in the Annex 1 of the EU “Manufacture of Sterile Medicinal Products”?

Answer:
The revised Annex 1 provides supplementary guidance on the application of the principals and guidelines of GMP to sterile medicinal products.
The guidance has been updated in four main areas:
-classification table for environmental cleanliness of clean rooms and associated text
-media simulation
-bioburden monitoring
-capping of freeze-dried vials