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Application Notes and White Papers - Particle Contamination

Particle Counting and the FDA

By Joe Gecsey, Product Manager, Pacific Scientific

This article explores the FDA's approach to monitoring particle contamination and the need to protect particle counters in humid or washdown environments. The article was compiled frorn conversations with the FDA and operators of sterile fill lines.
THE IMPACT OF VARIOUS government regulations on actual manufacturing processes are not always obvious. The current government requirements for monitoring sterile fin lines in cleanrooms requires a specific method of particle counting that is somewhat different than Federal Standard 209D. 'Me particle counting system will have to be compatible with the manufacturing environment, sample the correct locations at the right size. and fulfill the FDA's requirement for batch information.
The FDA currently requires the manufacturer to show that the process area was in control only during the actual filling operation. Continuous monitoring of the whole filling process is preferred, but not required. Monitoring Is not required when the room is not in use. It is good practice to monitor during set-up to be assured of the quality of the air, but it is not required.
If the product or personnel are changed during a shift, a new monitoring program must be started. The particle counts taken during the morning do not reflect the operating conditions later in the day if a new product or new personnel have been introduced. Changing shifts would also require a new verification of the mom, even if the same product was being made. The monitoring procedure must occur during the actual filling operation, with the equipment and personnel operating in their typical modes. This approach makes good sense in a majority of cleanroom operations.
The monitoring of microorganisms in the product and in the environment is still the main focus for assuring product sterility: monitoring of particles - living or not-is useful as an additional indicator of control and as a "real-time" indicator of potential contamination.

Federal Standard 209D
Although the FDA uses Federal Standard 209D as a general basis for their guidelines. the FS-209D Is not an absolute set of rules for the pharmaceutical industry. One of the essential differences between the FDA's philosophy and FS-209D is that FS-209D allows averaging of counts across sample points. During batch filling, averaging data across multiple sampling points is not acceptable to the FDA. To keep proper records for a sterile fill area. the particle counts obtained at each critical point must be maintained as a separate record. The intent here is to monitor the air approaching the filling line, not to characterize the general particle level of the room.
The "Guideline on Sterile Drug Products produced by Aseptic Processing" states that the sample points should be within one foot of the fill line. The FDA will accept a greater distance if It can be shown that sampling within one foot will produce erroneous counts due to some processing condition. such as over spray from a highspeed filling needle or a powder filling operation.
Also In contrast to FS-209D. the "Guideline ... for Aseptic Monitoring" requires monitoring at 0. 5 microns: monitoring at other size ranges, for example 1.0 or 0.3 microns, must be well justified for the particular filling process. For aseptic areas. the counts at each sample point must be below 100 counts per cubic foot for all particles 0.5 microns and larger. Ibis generally equates to an FS 209D "Class 100." but the FDA specifically requires monitoring at "0.5 micron and larger" sizing. Using alternate size equivalents is not acceptable unless the manufacturer can justify an alternate size based on unique processing requirements.

Establishing Quality and Control of the Processing Area Using Trends
At a given sample point, repeated monitoring will provide a typical "baseline" of values during the manufacturing operation. "Alert" and "Action" levels can be set relative to the empirically determined baseline count. Using 1 -sigma and 3-sigma points derived from the baseline count might be appropriate. The FDA is comfortable with averaging data at a given sample site: that is. each and every sample does not have to be below the "Alert" level to continue the processing the batch. However, a consistent trend away from the baseline should be a signal that the area is out of control.

Selection of Sample Points
Within the sterile fill area. samples must be taken in the immediate vicinity of the fill heads, and preferably near where the containers enter the sterile area. In general. you should monitor at points where the product Is exposed to potential contamination and where an operator might frequently be present.
Outside of the sterile fill area or other "critical" areas. the FDA recommends frequent monitoring to ensure that the HVAC filtration system is in order, but the frequency of monitoring can be relaxed to between daily and once-per-week. Again. the intent of gathering this data is to show that the area is generally in control.

Tubing Extensions
Short extensions - up to a maximum of roughly 10 feet - from the sample point to the sensor are generally acceptable, assuming that the tubing has a minimum of turns or curves and that the curves have a generous radius. Due to the statistically low number of particles within a sample under "Class 100" conditions, it is best to limit the use of tubing, which causes some entrapment or fragmentation of particles. If the tubing must be longer than 10 feet. then the loss factor for that given tubing must be determined and a correction factor must be used to adjust the counts obtained during filling procedures. In general, the use of manifolds for sampling in extremely clean areas (e.g. Class 10) Is strongly discouraged by the FDA.

Saving Data
In general. for most sterile products. the particle count data should be saved for a period extending one year past the expiration date of the product. There is no restriction on the form of the data storage: it must simply be retrievable for inspection within a reasonable time.

Equipment Considerations
Sterile fill areas are often aseptically washed. However, particle counters must be protected from moisture. Since tubing length restrictions require the particle counter/sensor to be in the washed area, the equipment must be housed in a moisture and splashproof container. such as a NEMA 4X rated enclosure.

Summary of ASHRAE Standard 52.2P

By Paul F. Johnstone

Subject- A.S.H.R.A.E. Standard 52.2P Purpose: To summarize the standard where it applies to particle counting and define the role of Particle Counters in 'this application. General: Prior to 1996 the ASHRAE standard (52.1 -1992) utilized a gravimetric method of measuring airborne particulate which weighed accumulated particles, expressed in micrograms per cubic meter. In 1996 'the standard was amended to include 'the use of Particle Counters as a method of particle count and size measurement.

Section 1) Description and Summary of the Standard
This standard addresses air cleaner performance characteristics of importance to the users; the ability of a device to remove particles -from the airstream and it's resistance to airflow. Air cleaner testing is conducted at airflow rates of not less than 510 cfm nor greater than 3180 cfm. A sample of air from a general ventilation system contains particles with a broad range of sizes having varied effects, sometimes dependent on particle size. Coarse particles for example, cause energy waste when they cover heat transfer surfaces. Fine particles cause soiling and discoloration of interior surfaces and furnishings as well as possible health effects when inhaled by occupants of the space. When air cleaners are tested and rated for efficiency in accordance with this standard, there is a basis for comparison and selection for specific tasks. The test procedure uses laboratory generated potassium chloride particles dispersed into the airstream as the test aerosol. "A particle counter measures and counts the particles in 12 size ranges both upstream and downstream for the efficiency determinations.

I. Purpose
This standard establishes a test procedure for evaluating the performance of air cleaning devices as a function of particle size.

2. Scope

2.2 The standard defines for generating the aerosols required for conducting the test. "The standard also provides a method for counting airborne particles of 0.3µ to 10.0µ in diameter upstream and downstream of the air cleaning device in order to calculate removal, efficiency by particle size."

2.3 This standard also establishes performance specifications for the equipment required to conduct the tests, defines the methods of calculating and reporting the results obtained from the test data and establishes a performance classification system which can be applied to air cleaning devices covered by this standard.

3. Definitions and Acronyms
3.1 Definitions Correlation Ratio, R: The ratio downstream to upstream particle counts determined from "the average of at least three samples taken during a "no-filter" or "0% efficiency" test". This ratio is used to correct for any bias between upstream and downstream sampling systems and it's derivation is described in 10.3.

Isokinetic Sampling Is that in which in the sampler inlet is moving at the same velocity and "direction" as the flow being sampled.

Test Aerosol: Poly disperse solid-phase (i.e., dry) Potassium Chloride (KCL) particles generated from an aqueous solution, used- in this standard to determine the particle size efficiency of the device under test. Generation of the aerosol is described in 5.3.

Device: Throughout 'this standard the word "device" means air cleaning equipment such as a filter.

3.2 Acronyms ASHRAE: American Society of Heating, Refrigerating, and Air Conditioning Engineers, Inc. CV: Coefficient of Variation HEPA: High efficiency particulate air PSE: Particle size removal efficiency ULPA: Ultra-low penetration air 4 Test Apparatus

4.2.2 Room air shall be used as the test air source. The temperature of the air at the test device shall I be between 50 and 100°F with a relative humidity of between 20% and 65%.

4.3.1 The test aerosol shall be poly disperse solid-phase KCL particle generated from an aqueous solution. The aerosol generator shall provide a stable challenge aerosol of sufficient concentration over the 0.3 to 10µ diameter size range to meet the requirements of section 10 without overloading the aerosol particle counter.

4.3.2 The aerosol generation system shall be designed to ensure that the KCL particles are dry prior to being introduced into the test duct. After drying, the aerosol must be electrostatic charge-neutralized by passing it through an aerosol neutralizer with a Kr85 radioactive source.

4.4.1 The upstream and downstream sample lines must be made of rigid electrical ly-grounded metallic tubing. The use a short length (2" max.) of straight, flexible, "electrical ly-dissapative" tubing to make the final connection to the particle counter is acceptable.

4.6 Particle Counter

4.6.1 The particle counter shall be capable of counting and sizing individual KCL particles in the 0.3 to 10µ diameter size range "The counting efficiency shall be 50% min. for 0.3µ KCL particles".

4.6.2 The particle counter shall measure and then group the test aerosol particles in 12 size ranges. The range boundaries, based on the physical size of the KCL aerosol, shall conform to the table 4-1. The particle counter's correlation of measured response voltage to physical size shall be monotonic for KCL particles from 0.3 to 10µ such that only one size range shall be indicated for any measured response.

4.6.3 The primary size calibration of the particle counter shall be to report the actual physical size of the KCL particle. A secondary calibration shall be performed on the particle counter using PSL particles. The secondary calibration data shall be converted to primary calibration data. "The particle counter manufacturer shall determine the secondary to primary conversion for the instrument type by calibrating an instrument of that type with both mono disperse KCL and PSL. The change in indicated particle size when making the secondary to primary conversion shall not exceed 20%." Exception: "An exception to 4.6.3 is permitted for light-scattering particle counters. If this exception is taken, the manufacturer shall determine the secondary to primary conversion by calculating the MIE scattering response for KCL and PSL, assuming spherical particles and considering the light source and collection angles of the instrument. The change in indicated particle size when making the secondary to primary conversion shall not exceed 20%".

4.6.4 The secondary calibration shall be performed at least once a year. The primary calibration shall then be updated using the conversion calculation and the secondary calibration.

4.6.5 A minimum of 90% of all observed counts shall register in size ranges 1 and 2 of table 4-1 when the particle counter is challenged with mono dispersed 0.40µ diameter particles. Secondary calibration data shall be used to demonstrate compliance with this requirement. Refer to 4.6.4. 

Table 4-1 Particle Counter Size Range Boundaries


 Size Range Lower limit (microns)   

 Size Range Upper limit (microns)   

 Geometric Mean Particle Size (microns)

















































4.6.6 The particle counter shall have less than 10% coincidence loss at a particle counting rate of 300,000 particles per minute and shall have a minimum viewed volume flow rate of 0.10 cfm. This flow rate shall not change with a 4.0" of water in the pressure of sampled air.

4.6.7 The measured particle concentration shall be a maximum 5.66 particles/ft3 when the particle counter is sampling air with a HEPA or ULPA filter on it's intake.

5 Apparatus Qualification Testing

5.1 Apparatus qualification tests shall verify quantitatively that the test rig and sampling procedures are capable of providing reliable particle size efficiency measurements. The tests shall be performed in accordance with table 5-1.

5.3 Aerosol concentration uniformity in the test duct

5.3.1 The uniformity of the challenge aerosol concentration across the duct crosssection shall be determined by a nine point traverse by using the grid points as shown in figure 5-1. The traverse shall be made by either (A) installing nine sample probes of identical curvature, diameter, and inlet nozzle diameter but of variable lengths or (B) repositioning a single probe. The inlet nozzle of the sample probe(s) shall be sharp edged and shall maintain isokinetic sampling within 10 % at 1990 cfm. The same nozzle diameter shall be used at all flow rates.

5.3.2 The aerosol concentration measurements shall be made with a particle counter meeting the specifications of 4.6. A one minute sample shall be taken at each grid point with the aerosol generator operating. After sampling all nine points the traverse shall be repeated four more times to provide a total of five samples from each point. The five samples shall then be averaged for each of the 12 particle size ranges. The traverse measurements shall be performed at air flows of 510, 1990, and 3180 cfm.

5.3.3 The CV of the corresponding nine grid point particle concentrations shall be less than 10% for each air flow in each of the 12 particle counter size ranges.

5.4 Downstream Mixing of Aerosol

5.4.1 A mixing test shall be performed to ensure that all aerosol that penetrates the air cleaner is detectable by the downstream sampler. The mixing test shall be performed at air flows of 510, 1990, and 3180 cfm. The point of aerosol injection immediately downstream of the device section shall be traversed and the downstream sampling probe shall remain stationary in it's normal center-of -duct sampling location.

5.4.2 A HEPA filter shall be installed to obtain a smooth airflow at the output of the device section. An aerosol nebulizer shall nebulize a KCI/water solution ( prepared using a ratio of 300g of KCL to 1 1000ml water ) into an aerosol of primarily submicron sizes. A rigid extension tube with a length sufficient to reach each of the injection points shall be affixed to the nebulizer outlet. A 900 bend shall be placed at the outlet ofthe tube to allow injection of the aerosol in the direction of the airflow. The injection probe shall point downstream. The aerosol shall be injected immediately downstream (within 10" ) of the HEPA filter at pre selected points located around the perimeter of the test duct and at the center of the duct as indicated in figure 5-2. The flow rate through the nebulizer and the diameter of the injection tube outlet shall be adjusted to provide an injection air velocity within + 50% of the mean duct velocity.

5.43 Sampling sequence: A one minute sample from the downstream probe shall be acquired with the nebulizer operating and the injection tube positioned at the 'first injection grid point. The injection point shall then be moved 'to the next grid point location. A new one minute sample shall be obtained after waiting a minimum of 30 seconds. The procedure shall be repeated until all nine grid points have been sampled.

5.4.4 The aerosol injection traverse shall be repeated two more times to provide triplicate measurements at each grid point.

5.4.5 The downstream aerosol concentration shall be measured as total aerosol concentration > 0.3um. The CV of the corresponding nine downstream grid point particle concentrations must be less than 5% for each airflow.

5.5 Aerosol Generator Response Time

5.5.1 Measure 'the time interval for 'the aerosol concentration to go from background level to steady test level. The test shall be performed at an airflow rate of 1990 cfm with 'the particle counter sampling from the upstream probe. Similarly, measure the time interval for the aerosol to return to background level after turning of the generator.

5.5.2 Record 'the time interval needed for the aerosol concentration to reach it's steady test value after being activated, and also record the time interval for the
concentration to return to background level after deactivating the aerosol generating system. These time intervals shall be used as the minimum waiting time between (a) activating the aerosol generator and beginning the particle counter sampling sequence and (b) deactivating the aerosol generator and beginning the particle counter sampling sequence for determination of background aerosol concentrations, respectively.

5.6 Concentration Limit of the Particle Counter

5.6.1 A series of controlled 50% dilution tests shall be performed over a range of challenge aerosol concentrations to ensure that the concentration used in the tests does not overload the particle counter. The lowest concentration level shall be less than 1% of the instrument's stated concentration limit. The tests shall be performed by one of the following methods: (a) Achieve the 50% dilution by alternating the test duct airflow from 996 to 1990 CFM while operating the aerosol generator at a constant output. The aerosol samples shall be obtained from the upstream sampling location only. (b) A controlled 50% dilution of the sample pie stream to the particle counter.

5.6.3 The tests shall be performed over a sufficient range of challenge concentration levels to demonstrate 'that the particle counter is not overloaded at the intended test concentration.

5.6.4 A minimum of three samples shall be obtained for the undiluted and 50% dilution levels. These samples shall be obtained by alternately sampling the undiluted and 50% diluted level. The dilution ratio shall be computed as the ratio of the average diluted concentration to the average undiluted concentration. The dilution ratio calculation shall be based only on the 0.30 to 0.40um channel of the particle counter. The measured dilution ratio in the absence of coincidence error shall be within 10 percentage points of the expected 50% dilution.

5.7 100% Efficiency Test

5.7.1 An efficiency test shall be performed using a HEPA or ULPA filter as a test device to ensure that the test duct and sampling system are capable of providing a 100% efficiency measurement. The test procedures for determination of PSE given in Section 10 shall be followed, and the "test shall be performed at an airflow of 1990 cfm .

5.7.2 The computed PSE values shall be greater than 99% for all particle sizes.

5.8 0% Efficiency Test

5.8.1 An efficiency test shall be performed without a test device in place to check the adequacy of the duct, sampling, measurement and aerosol generation systems.

5.8.2 The test procedures for determination of PSE given in Section 10 shall be followed.

5.8.3 A total of fifteen 0% efficiency tests shall be performed; five at each of three flow rates: 510, 1990, and 3180 cfm. The average of the five efficiency values at each particle size shall be computed for each flow rate. The average efficiency for each particle size shall be between -10% and 20% for all particle sizes.

5.10 Particle Counter Zero

5. 10. 1 The zero count of the particle counter shall be verified to be < 10 total counts per minute in the 0.30 to 10um size range when operating with a high efficiency filter attached directly to the instrument's inlet.

5.11 Particle Counter Sizing Accuracy

5.11.1 The sizing accuracy of the particle counter shall be checked by sampling an aerosol containing monodisperse polystyrene spheres of a known size. A relative maximum particle count shall appear in the particle counter sizing channel which encompasses the PSL diameter.

The remainder of the standard consists of tests for airflow, filter efficiency, dust loading, etc.

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