New Regulations Concerning Airborne Particle Counters (ISO 21501 and ISO 14644) Bob Latimer, Tony Harrison 2008 Cleanrooms Worldwide eVent My name is Bob Latimer. I have been working with Particle counting technologies and applications for almost 20 years and I currently the market manger for Hach Ultra’s Electronics business. I should add that I produced this presentation jointly with a colleague, Tony Harrison. Tony is an active participant within the Pharmaceutical Industries various standards committees and working groups. Let me begin by thanking everyone for attending our presentation today. I see that we have audience from many parts of the world and I sincerely appreciate all who made the effort to join us outside of their normal working day. I have with me today a panel of experts from within Hach Ultra. Ken Girvin, Tang PingShen, Ernie Sanford and Gene Zeppetello and Jarret Young. These folks represent a number of dept. here at Hach Ultra including engineering, metrology and technical support. The guys will be available throught my presentation to answer posted questions. So please feel free to use this feature throught the presentation NEXT SLIDE

Agenda cGMP and EU GMP Guidelines ISO 21501 Key Elements What is ISO 21501 Replacing Air Particle Sensors and Calibration – The Basics Counting Efficiency Resolution Signal to Noise Ratio and False Count Rate Summary Today we will be talking talking primarily about ISO 21501, what it is and what is means. However prior to that we will touch on how this new ISO standard connects to cGMP and EU GMP and ISO14644. We will also take a brief overview of particle counting basics with regards how they work, how they are calibrated. NEXT SLIDE

Aseptic Pharmaceutical Manufacturing The cGMP and EU GMP guidelines specify particle count limits based upon cleanroom classifications such as Grade A, B, etc. Both EU GMP and cGMP reference ISO 14644-1 for determining cleanroom classification. The next revision of ISO 14644-1 will refer to ISO21501-4, a calibration standard for air particle counters, ratified February 2007. ISO 21501 already cross references back to ISO 14644-1 cGMP EU GMP The aseptic manufacturing of pharmaceutical products in the US and Europe are supported by Good Manufacturing Practice guidelines. In the US we have the cGMP and in Europe the EU GMP. Both guidelines specify particle count classification limits for cleanrooms, for example Grade A, B etc. The EU GMP specifically states that cleanrooms should be classified in accordance with ISO 14644-1. The American cGMP refers to ISO 14644- 1 The next revision of ISO 14644-1 will refer to the new particle counter calibration standard ISO 21501 which we will be covering in detail today. It’s worth noting that ISO 21501 already cross references ISO 14644 stating that “Instruments that conform to this part of ISO 21501 are used for the classification of air cleanliness in cleanrooms and associated controlled environments in accordance with ISO 14644- 1” So in summary, their now exists a very clear link between the standards and guidelines governing Cleanroom Classification and the standards governing the calibration of the equipment used to classify cleanrooms. ISO 21501-4 states – “Instruments that conform to this part of ISO 21501 are used for the classification of air cleanliness in cleanrooms and associated controlled environments in accordance with ISO 14644-1”

Calibration to ISO 21501 Key Elements Particle size calibration using PSL particles traceable to an international standard and within a standard uncertainty < =2.5% The use of a Pulse Height Analyzer (PHA) to determine sensor response for each of the particle counters size channels The intent is to improve the accuracy of particle count data and improve instrument to instrument correlation Most Particle counting manufacturers employ size calibration to a traceable standard although this is now a requirement of ISO 21501. The use of Pulse Height Analysers is also mandatory for determining the voltage response of the particle sensor to different particle sizes and therefore the particle counters size channels. The intention of the new standard is to improve the accuracy of particle counters as well as their correlation. For example when using different units of the same model or different models, or even from different manufacturers. This is achieved within the new ISO calibration standard in a number of ways which we will explore in this presentation.

ISO 21501 – Sub Documents ISO 21501 describes the instruments and calibration requirements. Part 2: Light scattering liquid-borne particle counter Part 3: Light extinction liquid-borne particle counter Part 4: Light scattering airborne particle counter for clean spaces Before we dive in to the material it should be pointed out that the scope of ISO 2150 goes beyond the calibration of Air Particle Counters and includes the two different liquid particle counting technologies of Light Extinction and Light Scattering. This presentation deals only with ISO21501 dash 4 covering Air particle counters for monitoring clean spaces however the scope of Part 2 and 3 is similar.

What Standards/Guidelines Existed Prior to ISO 21501? 1997 1998 2006 2007 JIS B 9921:1997 A Japanese standard which comprehensively deals with OPC design performance, most notably in the area of counting efficiency. ASTM F 328-98 (updated 2003) “Standard Practice for Calibration of an Airborne Particle Counter Using Mono-disperse Spherical Particles” (Officially withdrawn 2007). IEST-RP-CC014.1 “Calibration and Characterization of Optical Airborne Particle Counters” (Provides actual methods to perform the calibration). ISO 21501 It’s perhaps worthwhile having a quick glance at a little of the history to put ISO21501 in context. Much of the content of ISO 21501 is based upon the Japanese standard JIS B 9921 which was released in 1997, the ASTM F 328-98 and recommended practice IEST-RP-CC014.1 There is significant overlap across these documents with varying degrees of emphasis on procedure versus actual limits. Also, parts of the documents allowed some discretion or latitude in the actual procedures. For example, ASTM F 328-98 allowed the operator to interpret the average voltage response to specific particle sizes as either the mode or the median of the particle distribution. ISO 21501, a true standard, clearly defines both procedure and limits.

ISO 21501 Compared to Existing Re-calibration This table highlights the various sections of ISO21501 standard and the guidelines & procedures that came before it. There are two critical aspects to ISO 21501. Firstly, ISO 21501 defines the minimum elements that must appear (and therefore be tested) on an air particle counter calibration certificate - (these are in red). This therefore includes periodic instrument recalibration. Prior to ISO 21501, the critical calibration parameter of “Counting Efficiency” would not normally be conducted on an instrument recalibration, particularly in the field. Minimum content on ISO 21501 calibration certificate. A key difference with ISO 21501 is that key calibration attributes are incorporated within the scope of instrument re-calibration.

MET ONE Air Particle Counters and ISO 21501 Calibration In the next few slides, prior to discussing some of the details of ISO21501 we will take a brief look at how particle sensors work and how they are calibrated. MET ONE Air Particle Counter 0.5µm and 5.0µm

Optical Particle Sensor Configuration Collection Optics Detection Zone At the heart of the Air Particle Counter is the sensor. This is a precision opto- mechanical assembly. Designs vary considerably although the general principle of all light scattering optical particle counters is the same. Basically, air is drawn through the sensor where it intercepts a laser beam. Any particles in the beam will scatter light energy. This scattered energy is collected and focussed onto a detector. Reflector Light Trap

Detecting Particles v t Detector Collection Optics Light Trap In this simplified sketch, we can see that when light scattered by a particle is collected by the photodetector a corresponding electrical pulse is produced. Although the relationship between the magnitude of the pulse and the size of the particle is a somewhat complex relationship, in general, the larger the particle the larger the voltage signal. If we look at the sensor output on an Oscilloscope we would see any particles present in the air stream as a train of pulses of varying sizes. If we were to introduce a sample containing particles of all the same size then we would see a series of voltage pulses of equal magnitude. The PHA or pulse height analyzer is an instrument or device that allows us to build up a graphical image of the particles passing through the sensor. In the case of mono sized particles, such as used for calibration, we would see the actual distribution profile of the particles. In the next slide we will have a closer look at such distributions. Laser Diode Particle Oscilloscope Pulse Height Analyser

The Pulse Height Analyzer (PHA) When a particle sensors output is connected to an oscilloscopes input the particles appear as a series of voltage pulses in time. The amplitudes of the pulses relate to the amount of light energy scattered by the particle and are therefore related to the sizes of the particles. When the sensors signal output is connected to Pulse Height Analyzer (PHA) the particles appear as a distribution with the x-axis being pulse height and the y axis being number of particles. The above picture depicts a typical room air sample. The above picture depicts a PHA response to a mono-dispersed challenge such as PSL calibration spheres.

Calibrating Air Particle Counters: NIST-traceable Particles Mean size 498 nm +/- 5 nm Std Deviation 7.9 nm Std Dev x 100 C.V. (RSD) = Calibrating air particle counters is most commonly achieved by introducing a number of particle standards (known as PSL or Polystyrene Spheres) one by one. ISO 21501 requires that these particles be traceable, for example to NIST and to exhibit an uncertainty level below 2.5% Particles are normally supplied in highly concentrated aqueous suspensions. Specialized aerosol generators are required to deliver dried, relatively low concentrations of these particles to the the sensor being calibrated. The mean diameter and standard deviation are certified. Mean size 490.1 498 Size (nm) NIST-traceable standard particles have normal size distribution. Nominal size and CV are certified.

Selecting the Correct Calibration Voltage ISO 21501 requires that the PHA response to the PSL spheres is interpreted as the median. This is the point where an equal number of particles fall on each side of the bisected distribution. For normal distributions the mode and the median fall at the same point. This is not always the case in particle counting where skewed or irregular distributions are not uncommon. median mode Particle sensors do not always produce perfectly uniform or normal distributions. In addition to the normal distribution of the particles themselves (as quoted on the standards certificate) there is also the impact on the optics of the sensor. Due to this, there has always been a little subjectivity in picking the peak of the distribution, which is the voltage attributable to that specific particle size. ISO 21501 specifically identifies the median voltage as being the calibration point. The calibration voltage for a given PSL introduction

Calibrating Air Particle Counters: Constructing the Calibration Calibration Example The calibration for the sensor is established by finding the peaks of a set of standard particles. Generally, particle standards are chosen that are very close to the actual size channels that the particle counter is set to count and sufficient particle sizes are run to characterizes the full size range of the instrument. Again, ISO 21501 requires that the medians of the distributions be taken as the voltage point attributable to each PSL size. 0.3 0.5 1.0 3.5 5.0 Voltage Calibration requires that a variety of different PSL sizes are run and the median voltages are recorded.

Calibration of Air Particle Counters This is a typical calibration produced for the Met One Model 3400.

ISO 21501: Counting Efficiency The particle concentration (C) displayed by particle counter Concentration (Co) of particles drawn into the inlet of an OPC C.E. is the ratio:  = x 100 (%) C Co Counting efficiency has arguably been the biggest variable impacting the accuracy of airborne counters. Counting efficiency refers to the ability of the instrument to count particles at a specified size. ISO 21501 makes use of the specification for counting efficiency that was defined in the JIS B 9921 standard. Besides the actual traceable size calibration itself, Counting Efficiency is arguably the most important attribute contributing directly to the accuracy of the actual particle counts reported by the particle counter. One significant difference that ISO 21501 brings is that Counting efficiency is required to be verified at both original calibration as well as periodic recalibration in the feild.

Counting Efficiency – ISO 21501 Comparison to 100% Count Standard UUT ISO 21501 Limits 50% +/- 20% (30% to 70%) Unit Under test NIST Traceable PSL Challenge Noise Channel 1 size (um) N Standard Measuring the Counting efficiency accurately requires a 100% counting efficiency standard. In the case of the Met One model 3413 for example – a 0.3um particle counter, Hach Ultra employ 100% Counting efficiency standards to measure counting efficiency of 3413 units. These 100% CE standards are particle counters that have their first channel thresholds set significantly lower than 0.3um such that when challenged with 0.3um PSL, 100% of the distribution is above the standard counters threshold. After the UUT has been size calibrated it can then be checked for 50% CE against 100% CE standard. No Adjustment of thresholds are allowed. The CE test is a check – not an adjustment. If the UUT fails the CE test it fails ISO 21501. NIST-traceable ‘100% Count standard’ Example: Channel 1. Set to recover 100% of the challenge PLS Noise Channel 1 set to 100% efficiency for UUT channel 1 size

ISO 21501 and Counting Efficiency: A Two Part Test At 1.5 x to 2 x the particle counters minimum specified size all particles should be counted in the first channel. ISO 21501 allowed limits are 90% to 110% ISO 21501 Limits 50% +/- 20% (30% to 70%) OPC smallest specified size. Noise The ISO 21501 counting efficiency test is a two part test. The first part involves challenging the sensor with PSL at the same size at the Particle Counters first channel. If the Particle Counter has been calibrated correctly, the median of the particle distribution will be bisected by the Particle Counters channel 1 threshold. As a result, 50% of the challenge particles will be counted. ISO 21501 sets a limit of 50% +/- 20%. The second part of the ISO 21501 Counting Efficiency test requires that particles of size 1.5 to 2 time greater than channel 1 are run. 100% of these particles should be counted in channel 1 with an allowable limit of +/- 10% Channel 1 size (um) Noise Channel 1 size (um) AND for PLS particles of 1.5 times or over up to and including two times the minimum measurable particle size value, shall be 90% to 110% The counting efficiency for PSL particles of the UUT minimum measurable particle size shall be within the range 30% to 70%

ISO 21501: Resolution N Particle size Resolution refers to the ability of a particle sensor to differentiate between similarly sized particles. 0.3µm 0.5µm Resolution – The ability to differentiate between similarly sized particles Affected by instrument quality and alignment of the sensor optics

Resolution – Why It Is Important Particle size 0.3µm 0.5µm A particle sensor with poor resolution, such as the one above, cannot reliably distinguish between different sized particles.

Resolution R% = CV(%) = RSD(%) ISO 21501 requires that resolution be calculated from +/- 1 standard deviation from the median. The higher of the two values for  (SD) is selected. 1 D1 D2 D Peak (median) 61% of peak 2 - P 2 XP √ R% = x 100 (%) where R Resolution  Observed SD in µm P PSL SD in mm XP PSL Size in µm Keeping in mind that the particle distribution obtained on a PHA is attributed to both the actual distribution of the standard particles as well as the influence of the optics. Checking that the resolution is within ISO 21501 specifications requires that the standard deviation of the PSL is subtracted from the total observed standard deviation on the PHA. Resolution % is essentially the residual standard deviation expressed as a percentage of the mean. As an example, a resolution quoted as being 2% at 0.5um implies that a perfect 0.5um particle would produce a voltage response that was equivalent to +/- 0.01um at 1 standard deviation. Or looking at +/- 3 SD's which is 95% of the distribution the 0.5um particle would produce a response somewhere between 0.47um to 0.53um

ISO 21501: False Count Rate – Affected by Signal to Noise Ratio Peak to Valley Ratio The false count rate of the particle counter is impacted by a number of factors. Critically, separation between the channel 1 threshold and the noise level must be sufficiently large to prevent random noise signals crossing the first channel threshold. ISO 21501 does not actually specify limits for signal to noise but it does specify limits for false count rate which we will look at on the next slide. In addition to the ISO 21501 false count rate Hach Ultra measure both peak to valley and signal to noise measurements to internal specifications. Channel 1 Channel 1 Note: ISO 21501 does not specify a minimum S:N ratio although False Count Rate is (next slide). Peak to Valley is described although not specified. Hach Ultra maintains an internal Peak to Valley specification of 2:1

ISO 21501 False Count Rate The ISO 21501 false count spec is less than 1 count in 5 minutes. Hach Ultra test particles counters using zero count filters to ensure that the actual count falls below the 95% upper confidence limit associated with this false count rate.

ISO 21501-4 Summary ISO 21501-4 Parameters Limit Sampling flow rate (volumetric) ± 5% Counting efficiency at channel 1 50% ± 20% Counting efficiency at particle size 1.5 to 2 times channel 1 100% ± 10% Particle size setting error ≤ 10% Instrument resolution (at manufacturers specified size) ≤ 15% Zero count test ≤ 1 count / 5 minutes Maximum particle number concentration (manufacturer specified) Sampling time ± 1% Response rate ≤ 0.5% Calibration interval ≤ 1 year Test Report Must Include as a Minimum: Date of calibration Particle sizes used for calibration Flow rate Size resolution Counting efficiency False count rate

Hach Ultra and ISO 21501 Hach Ultra has completed a full migration to ISO 21501 calibrations. Hach Ultra offers a complete line of ISO 21501 compliant air particle counters. Hach Ultra offers full ISO 21501 field and factory calibration offerings designed to ensure accurate and efficient service that goes beyond the instrument Certified Calibration Service Agreements with your choice of either In-House and\or Service-at-your-Site options. Prescheduled calibration and preventative maintenance services

Hach Ultra and ISO 21501 Calibrations performed by Hach Ultra ISO 21501 certified technicians 60 Global Direct Certified field technicians guarantees prompt qualified support Hach Ultra technicians average over 8 years of Hach Ultra calibration experience per associate 10 ISO 21501 capable factory service centers 1 US based ISO 9001:2000 certified factory service center 3 Strategically located US remote depot service centers to support walk-in and close to customer depot support 5 European remote factory service centers 1 Asian remote factory service center

Hach Ultra and ISO 21501 Customer support available at iso21501@hachultra.com

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