Continuous Microbiological Environmental Monitoring For Process Understanding and Reduced Interventions in Aseptic Manufacturing

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Continuous Microbiological Environmental Monitoring for

Process Understanding and Reduced Interventions in Aseptic
Manufacturing
Jeffrey Weber, James Hauschild, Pieta IJzerman-Boon, et al.
PDA Journal of Pharmaceutical Science and Technology 2018,

Access the most recent version at doi:10.5731/pdajpst.2018.008722
Downloaded from journal.pda.org on February 28, 2019
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Title: Continuous Microbiological Environmental Monitoring for Process Understanding and Reduced Interventions in Aseptic Manufacturing.
1 Title: Continuous Microbiological Environmental Monitoring for Process Understanding and
2 Reduced Interventions in Aseptic Manufacturing.
3 Authors
4 1. Jeffrey Weber, Pfizer, 7000 Portage Rd, Kalamazoo, MI 49001 USA

5 2. James Hauschild, Johnson & Johnson, 1000 Route 202 South, Building 930, East Raritan, NJ
6 08869 USA
7 3. Pieta IJzerman-Boon, Merck (Merck, Sharp & Dohme outside the US and Canada), P.O. Box
8 20, 5340 BH Oss, The Netherlands
9 4. Ren-Yo Forng, Amgen, One Amgen Center Drive, Thousand Oaks, CA 91320-1799 USA
10 5. Jeff Horsch, Bristol-Myers Squibb, 1 Squibb Dr, New Brunswick, NJ 08901, USA
11 6. Lisa Yan, Shire, 4501 Colorado Blvd. Los Angeles, CA 90039
12 7. Aditya Prasad, AstraZeneca, 3001 Red Lion Rd, Philadelphia, PA 19114 USA
13 8. Robert ‘Bo’ Henry, Catalent Biologics, 1300 S Patterson Dr, Bloomington, IN 47403 USA
14 9. Marja Claassen, Merck (Merck, Sharp & Dohme outside the US and Canada), P.O. Box 20,
15 5340 BH Oss, The Netherlands
16 10. Philip Villari, Merck (Merck, Sharp & Dohme outside the US and Canada), 770 Sumneytown
17 Pike, Mailstop WP78-210, West Point, PA 19486 USA
18 11. Shebeer Shereefa, Abbvie, 400 Sheridan Rd, North Chicago, IL 60064 USA
19 12. Jane Wyatt, Alexion, Blanchardstown, Dublin 15, Co. Dublin, Ireland
20 13. Jay S Bolden, Eli Lilly, Indianapolis, IN 46285 USA
21 14. Jean-Thierry Pycke, GSK Vaccines, Av. Fleming 20, 1300 Wavre, Belgium
22 15. Dawood Dassu (corresponding author), Biophorum Operations Group, 5 Westbrook Court,
23 Sharrow Vale Road, Sheffield, S11 8YZ, UK. dawood@biophorum.com +447787527067
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24 Abstract
25 This white paper provides recommendations for quality oversight, manufacturing operations, and
26 industry perspective of regulatory expectations to enable aseptic facilities to move toward real-
27 time and continuous microbiological environmental monitoring and thereby reducing
28 interventions and the future replacement of Grade A settle plates and non-remote active air
29 sampling. The replacement of traditional monitoring with bio-fluorescent particle counting
30 systems provides an improvement in process understanding, product safety, and reduces operator
31 manipulations assuring product quality and real-time process verification. The future state
32 pharmaceutical technology roadmaps include gloveless isolators with real-time and continuous
33 monitoring for aseptic manufacturing.
34
35 Keywords
36 Rapid Microbiology, Environmental Monitoring, Bio florescent Particle Counters

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37 Lay Abstract
38 This white paper advocates the use of an alternative and relatively new method of monitoring the
39 air for contamination in biopharmaceutical manufacturing facilities. The alternative method is
40 based on a type of instrument the authors refer to as bio-fluorescent particle counters (BFPC).
41 The BFPC method has the advantage of being able to detect airborne microorganisms
42 continuously and with the actual time of detection recorded. The replacement of traditional
43 monitoring with BFPC systems can provide better data which can be used to improve the
44 understanding of contamination risks in complex manufacturing processes, ultimately providing
45 more confidence in product safety. The authors present data showing the suitability of BFPCs.
46 This immediate result is very useful for picking up early any possible contamination and should,
47 therefore, provide a better way to monitor and control the risk of contamination. As traditional
48 monitoring methods require manual manipulation, an additional advantage of BFPC systems is
49 that they can reduce manual manipulations. Elimination of all interventions is a goal in the
50 industry, because, although they are tightly controlled, interventions are an unwanted potential
51 source of contamination.
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52 Introduction to Bio Fluorescent Particle Counting
53 Pharmaceutical manufacturing requires multiple layers of engineering and process control and
54 monitoring of microorganisms to ensure product quality and patient safety. Aseptic
55 pharmaceutical manufacturing relies on understanding of microbial risks and periodic monitoring
56 to ensure low level bioburden or sterility required in commercial products. Most microbiological
57 tests are indirect measures of product quality, designed to measure process materials and
58 monitoring the surfaces, air and personnel for contamination; rather than direct product testing
59 which consumes the product. Traditional microbial testing has a limited sensitivity due to the
60 sample size (e.g. 20 vials of drug product or 1 cubic meter of air for microbiological testing) and
61 growth-based assays have long incubation periods (3-5 days) and rely on the same techniques
62 developed by Pasteur and Koch more than 100 years ago.
63 This paper provides an overview of the technology, a pathway for implementation, and
64 appropriate responses for positive signals. We will explain the use of a new unit of measure,
65 sample capture, and large data sets to demonstrate monitoring and control of aseptic
66 manufacturing areas. We outline a proposed response for potential false positives (e.g.
67 polymers, electrical noise, etc.) and false negatives (sample capture desiccation), to satisfy
68 quality standards. The goal of the system is to provide improved process knowledge, decreased
69 interventions, and ultimately real-time information for manufacturing areas. Contact plates and
70 surface monitoring are not discussed in this application.
71 There is a new class of alternative and rapid microbiological methods for monitoring airborne
72 microbes to provide rapid alerts to manufacturing risk. Bio-fluorescence particle counters
73 (BFPC) provide continuous size and quantification of internal biomarkers, allowing for the
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74 immediate detection and enumeration of airborne microbes in manufacturing areas. Bio-
75 fluorescence particle counting is a novel method that does not have a compendial analog. Auto-
76 fluorescence (also referred to as biological auto fluorescence) is based on laser excitation of the
77 particle and detecting the fluorescence of biological compounds such as nicotinamide adenine
78 dinucleotide (NADH), riboflavin, and dipicolinic acid, these compounds are universal and found
79 in all microorganisms. Current systems sometimes cannot distinguish between viability or
80 similar compounds (e.g. polymers or solvents) that may fluoresce; to distinguish false positive
81 signals from viable cells the use of sample capture onto media or filters can be used (1). The
82 instruments are validated using the same principles as traditional particle counters.
83 The BFPC system does not measure colony forming units (CFU), rather the systems require a
84 new unit of measure that reflects the universal detection method, the auto-fluorescence unit
85 (AFU). The BFPC systems provide instant and continuous AFU monitoring without the need for
86 reagents or incubation. The challenge of correlating the CFU to AFU is daunting as the majority
87 of microorganisms in our environment are not culturable and therefore, undetectable with
88 traditional plate-count methods; however, such comparisons provide understanding of the
89 technology and aid in the definition of control levels for the measured AFU (2,3,4,5). It is
90 unreasonable to expect an exact agreement of the AFU and CFU given that the detection
91 methods are very different. The AFU, based on molecular detection, may result in a higher
92 numerical value (e.g. more sensitive) when directly compared to the CFU, which is dependent on
93 observation of microorganism’s growth. Therefore, AFU counts greater than CFU counts do not
94 mean that the environment is out of control nor does it imply that there is more risk for
95 contamination.
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96 Opportunities and Challenges of Settle Plates and Active Air Sampling
97 Continuous BFPC monitoring provides the opportunity to reduce operator manipulations of
98 media-based settle plates in critical zones; and thereby reduce the risk of contamination. The
99 pharmaceutical industry has historically relied on settle plates and periodic active air sampling to
100 monitor manufacturing areas; the European Medicinal Agency inspectorate expect the use of
101 settle plates (6). Settle plates and active air sampling have provided acceptable detection
102 controls of microbiological contamination during manufacturing to date; however newer
103 technologies provide the opportunity for real-time and continuous environmental monitoring.
104 Settle plates have fixed exposure times and require operator manipulations in critical zones,
105 when remote sampling is not possible; thereby increasing risk of contamination without
106 improved data quality and aseptic process control.
107 Traditional passive monitoring is performed using Petri dishes containing culture media, which
108 exposed for a given time to collect biological particles which “settle” or deposit on the media and
109 subsequent incubation for colony detection. Settle plate results are expressed in CFU/plate/time.
110 Until now, the strongest support for settle plates is the continuous coverage during
111 manufacturing operations. Typical settle plates are exposed for 4 hours, although it is not
112 unusual to see validations extended to 6 hours to ensure plate viability (e.g. due to desiccation) if
113 used for more than 4 hours (7).
114 Settle plates rely on biological particles “falling” onto the plate; and are positioned according to
115 risk-assessments. Settle plates preferentially select the larger particles due to settling rates
116 compared to smaller particles that may never settle (8). The plates are incubated and the results
117 are retrospectively applied to operations in an all-or-nothing mode that impacts the entire lot with
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118 very limited temporal information. Settle plates are a potential source of contamination, both
119 through the introduction of growth media in an aseptic area and the operator manipulations in
120 high-risk critical zones. In addition, traditional growth media supports microbial growth less
121 than 0.1% species of bacteria from the of bacteria in the world (9). Several guidelines provide
122 guidance for microbial monitoring and particle counting in isolators, cleanrooms and graded
123 manufacturing areas, including: ISO 14644 standards for cleanroom classification and testing;
124 FDA Guidance for Industry PAT: A Framework for Innovative Pharmaceutical Development;
125 USP <1116> Microbiological Evaluation of Clean Rooms and Other Controlled Environments;
126 and EU Annex I – Manufacture of Sterile Medicinal Products. The expectation of Annex I for
127 microbial monitoring specifies using 90 mm settle plates and setting the action limit at the
128 detection limit; with limits for Grade A areas is zero (less than 1 CFU) and the Grade B at 5 CFU
129 per four hours settle plate exposure. There is no risk assessment for the establishment of the
130 current environmental limits and manufacturers have developed capabilities to operate within the
131 requirements. Annex I does not mandate the use of settle plates, using the phrase “such as settle
132 plates”, but the use has become a de facto mandatory requirement. Expectations of Agencies and
133 boards of health differ based on current EM practices; there is the potential to harmonize
134 expectations based on BFPC technologies.
135 The microbial active air sampler addresses the deficiencies of settling plates requiring airborne
136 microorganisms to “settle” on the plates by drawing an air volume across an agar plate and
137 impacting or impinging the particles on growth media. Most active air samplers provide periodic
138 coverage as they require process interventions. The design attempts to correct the passive nature
139 of settling plates, unfortunately the technique still relies on microbial growth, limited time-based
140 resolution and operator interventions.

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141 The manipulation of settle plates and active air sampler plates in critical zones create risk of
142 contamination. The equipment may also inhibit personnel movement, and is contrary to Annex I
143 which indicates “sampling methods used in operation should not interfere with zone protection.”
144 USP <1116> states, “environmental monitoring can both increase the risk of contamination and
145 also give false-positive results” and describes the limited value of having operators place settle
146 plates into critical zones. The replacement of settle plates with an isokinetic probe adjacent to
147 critical operations eliminates the need for operators to be able to place and recover settling plates
148 (Figure 1). Bio fluorescent particle counters reduce manipulations in critical zones; simplify the
149 set up for manufacturing and provide continuous monitoring during manufacturing. Any
150 changes to classified manufacturing areas need to be evaluated with smoke studies and
151 appropriate change control.
152 Role of Bio Fluorescence Particle Counter in Manufacturing
153 These recommendations come from the members of the BioPhorum Alternative and Rapid Micro
154 Methods working group. The goal of the working group is to pave the way for best practice
155 implementation of BFPC in routine aseptic manufacturing. Naturally, as each manufacturer is
156 unique, alternative plans may be more appropriate for some.
157 Currently, the BFPC has been used as an investigational tool which enables manufacturers to
158 rapidly locate sources of microbial risk. BFPCs instantly detect cellular components (e.g.
159 NADH) and do not require incubation and growth for detection, thereby providing a unique
160 capability for environmental monitoring and control. The high sensitivity and universal
161 detection may be considered a business burden as signals and new information may require a
162 response; but the benefits for continuous improvement, process knowledge and patient safety
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163 outweigh this challenge. BFPC systems provide an early warning if there are changes in
164 manufacturing environment as it provides real time data output, enabling faster quality decisions
165 which may reduce a cascade of increasingly serious problems.
166 To mitigate the perceived business risk of higher sensitivity to non-viable or false positive
167 signals; sample capture for identification or discrimination is required for initial equipment
168 validation and recovery studies. A false positive or "false alarm", is a result that indicates a
169 given condition has been fulfilled, when in fact it has not. In the case of BFPC, it has been
170 documented (2, 10) that some polymers and non-viable cells may result in false positive signals.
171 Currently, one vendor offers an integral sample capture based on gelatin filters placed in the air
172 path after the fluorescence detector. There is potential to use commercial off-the-shelf inline
173 liquid impingers or agar plates to capture samples after detection. Due to the discrete nature of
174 microorganisms, it is imperative that the sample capture device filters are placed in series and
175 monitor the same body of air. Aseptic sample capture enables an appropriate response to any
176 AFU signal; to discriminate false positives, viable but non-culturable organisms and culturable
177 microorganisms posing an environmental risk (11). Sample capture may be used for process
178 decisions; therefore, any potential risk for post-sampling contamination should be excluded
179 (Figure 2). Validation studies of microbiological samples at low levels have demonstrated the
180 challenge of performing parallel studies because the microorganism cannot be in two places
181 simultaneously; either within the BFPC or the parallel system. The discrete particle nature of
182 low level contamination and concepts from USP <1116> provide the framework for establishing
183 a frequency and volumetric response during manufacturing. Typically, zero CFUs are observed
184 during manufacturing operations, parallel recoveries in both a BFPC and settle plates are
185 unlikely.
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186
187 Potential Implementation Strategy
188 A stepwise approach for the implementation of BFPC should include evaluation, parallel testing,
189 and full implementation. This approach limits perceived business risk of “unknown” events and
190 demonstrates the current manufacturing areas are in a state-of-control to provide confidence for
191 internal stakeholders. The evaluation period is often in response to an investigation; with the
192 opportunity to perform BFPC testing during non-product manufacturing periods. The data sets
193 should be shared with both engineering and quality control functions to understand the full
194 benefits of the information. Evaluation during non-manufacturing time such as training, water
195 fills and engineering studies provides greater process understanding without product risks.
196 Parallel testing format should have a set timeline or number of samples that will be compared to
197 demonstrate process understanding. The period of parallel testing based on evaluation testing
198 and process complexity can be assessed both within companies and with Agency representatives;
199 this limit speculative discussions (e.g. “what if” questions). Finally, full implementation of
200 BFPC as a method to provide process monitoring and feedback is the goal of the technology.
201 We encourage manufactures to consider the 2004 FDA PAT Initiative that supports the concept
202 of collecting data sets during manufacturing provided the data is NOT used for manufacturing
203 decisions, regardless if the data set is either helpful or detrimental (12). The 2004 FDA PAT
204 guidance states, “…when evaluating experimental online or inline process analyzers during
205 production, it is recommended that risk analysis on the product quality be conducted before
206 installation. This can be accomplished within the facility’s quality system without prior
207 notification to the Agency. Data collected using an experimental tool should be considered
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208 research data, providing manufacturers the ability to collect data during production with limited
209 risk during evaluation of the new method or parallel testing.”
210 Continuous monitoring provided by BFPC provides assurance against microbial risks of isolator
211 integrity changes and environmental controls during manufacturing. This provides rapid
212 feedback of control of the manufacturing isolator during operations. Greater understanding and
213 practical risk assessments for the contamination of the product can be developed by BFPC.
214
215 Data Collection and Presentation
216 Data integrity has become a focus area of regulatory Agencies; BFPC systems provide time-
217 stamped and continuous data enabling quality assurance professionals with the ability to
218 determine the source and impact of any process changes. This capability enables segregation of
219 impacted product for evaluation or investigations. This temporal ability enables operators to
220 understand intervention impact and recovery times within manufacturing areas. The balance of
221 too much data versus instrument response needs to be considered; too frequent data can lead to
222 unnecessary reactions to small perturbations, whereas infrequent sampling does not provide
223 process understanding.
224 Most commercial systems can provide 10 second increments; but this quickly overwhelms the
225 user with almost 3000 data points per 8-hour shift. Many active air particle counters are capable
226 of flow rates of 28.3 L/min, capturing a cubic meter every 35 minutes. Data points every cubic
227 meter do not provide sufficient resolution to understand intervention impacts in the event of a
228 positive signal. Based on current system evaluations, one-minute samples provide sufficient data
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229 granularity to maintain process control and meeting the challenges of business risk. A practical
230 approach is one-minute measurements within a rolling cubic meter block, in other words 35
231 consecutive samples lasting 1-minute each, adding up to a sample of 1m3 of air, with AFU data
232 available for each minute. Sufficient resolution is therefore available for process understanding
233 during operator interventions while aligning with the compendial requirement of cubic meters.
234
235 Grade A Manufacturing Areas, RABS, and Isolators
236 Annex I indicates the purpose of the isolator technology is to minimize the human interventions
237 in process areas, whereas the use of settle plates and non-remote active air sampling plates are
238 direct conflict with this goal. The opportunity to use BFPC in manufacturing isolators and Grade
239 A areas during filling operations when open containers are present reduces high risk activities in
240 critical zones (13). The rapid detection of potential microbial contamination enables the
241 segregation of impacted product as a sub-lot for quarantine without jeopardizing the entire
242 manufacturing lot. Annex I guidance is aligned with the capability of BFPC stating the
243 frequency of monitoring with a suitable sample size that all interventions, transient events and
244 any breach in system integrity would be captured and alarms triggered if the alert limits are
245 exceeded. Agency feedback proposes the same limit of less than one CFU requires that any AFU
246 detected includes sample capture and incubation. We have deliberately taken a conservative
247 approach for the setting of limits; as industry experience with BFPC increases, AFU alert and
248 action limits may be set that are higher than the current action limits at detection limits for
249 traditional methods. Individual company risk assessments, regulatory agreements and predefined
250 quality oversight protocols can enable different risk tolerances based on process capabilities.
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251
252 Data Sets
253 Several pharmaceutical companies provided data sets of which the main objective was to show
254 that the new instrument generates no or few AFU, thereby confirming that the instrument does
255 not introduce counts that affect the state-of-control in classified manufacturing areas, and that the
256 false positive rate is low.
257 Data Set 1 is 10 days of continuous monitoring of Grade A manufacturing area at rest. The goal
258 was evaluation of classified areas without impacting manufacturing capability, no manufacturing
259 activities were conducted during this period. The BFPC system was placed in the manufacturing
260 area and run continuously collecting 402 distinct cubic meters of data, approximately 1 sample
261 every 35 minutes. No inline viable sample collection was performed during this period, due to
262 staffing limitations. Traditional environmental monitoring was used to maintain room
263 classification requirements.
264 Two events with auto fluorescent units (AFU) were observed during the study; more than 99.5%
265 (n=402) of the monitoring had no signal. The first event, with 3 AFU ≥0.5 µm, of which 1 AFU
266 was ≥5.0 µm, was recorded at 21:44 hours. The second event, with 2 AFU ≥5.0 µm, occurred
267 more than two days later at 01:35 (Figure 3a). No readily apparent source of particles was found
268 for the area, as no operators were present. The time-based resolution provided 35 minute
269 continuous AFU updates that is not possible with traditional methods. The need for shorter time
270 resolution data to understand whether an event consisted of a single spike or of multiple smaller
271 signals was identified. The Annex I requirement for viable counts is <1 CFU per cubic meter,
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272 although it is not known how a partial CFU can be measured other than through estimation of
273 large sample volumes.
274 The active air particle counts, measured by BFPC, showed a maximum count of 5 particles ≥0.5
275 µm per cubic meter, of which 4 were ≥5.0 µm (Figure 3b). This spike coincided with the second
276 AFU event with 2 AFUs ≥5.0 µm, but no assignable cause was identified. The particle numbers
277 were low compared to the Annex I limits of 3250 and 20 particles per cubic meter of ≥0.5 and
278 ≥5.0 µm, respectively.
279 Data Set 2 is the evaluation of BFPC monitoring in a manufacturing restricted access barrier
280 system (RABS) suite during a water fills. The RABS suite was operated during water run for
281 training of personnel and to ensure proper set up and function of equipment.
282 The system was operating at 28.3 litres/min for 8095 minutes or 231 cubic meters. The unit was
283 placed within the RABS below a set of operator gloves, Figure 4. The unit was operated
284 continuously and was used to evaluate one-minute counts. The data collection scheme was to
285 provide sufficient resolution to identify sources of variation and also to provide data sets that are
286 comparable to historical values. Sample capture capability was not used due to staffing
287 limitations; additional concerns about aseptic methods for the handling the filters was not
288 evaluated.
289 The viable count measured by BFPC for the suite measured 5 AFU during operations in the area
290 (Figure 5a). The data set provided an opportunity to understand the impact of operator activities
291 and to use the process flow chart described earlier (Figure 2) to provide quality oversight. Using
292 information from log books and card access information; all the AFUs and high particle counts
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293 (Figures 5a & 5b) had readily assignable causes (mopping operations or cleaning/set-up). The
294 activities of mopping and set-up were not performed during filling or when product was exposed.
295 Continuous monitoring during mopping operations demonstrated the activity was not a large
296 source of AFU and non-viable particles. Continuous monitoring enables more accurate risk
297 assessments by understanding the particle loads created by staff operations (e.g. mopping and
298 set-up).
299 Data Set 3: The evaluation looked at the environment inside a sterility test isolator held under
300 positive pressure using room air intake with no activity (at rest). This study was performed to
301 understand the data sets generated and whether one minute increments (28.3 l/min flow rate)
302 provided sufficient process knowledge. Three different sets of data were collected over 24, 48,
303 and 96 hours, respectively. Figure 6 shows the placement of the isokinetic sampling probe in the
304 floor of the isolator. The tubing from the bottom of the probe is connected to a ball valve to
305 separate the BFPC from the isolator during the VHP decontamination process. The BFPC is
306 mounted below the isolator and the location of the instrument below the deck limits the
307 opportunity for the use of the gelatin sample capture filter. The close proximity to the floor
308 presents a high risk for sample cross-contamination during the manipulation of the sample
309 capture device.
310 There was a single AFU observed during the 96-hour run, for which no assignable cause was
311 found. The single AFU was first detected as a particle (≥ 0.5 µ) by the BFPC certified particle
312 counter upstream of the laser induced fluorescence AFU detection step (Figure 7). The isolator
313 was not in use (at rest) and there were no operations in the area. One would expect no sources of
314 AFU while monitoring a VHP decontaminated isolator at rest.

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315
316 Data Set 4: The BFPC evaluation in a Grade A biosafety cabinet was used to perform simulated
317 sterility testing activities to understand real-time AFUs detection and to compare colony forming
318 units (CFU) on the gelatin filter to traditional viable active air sampling methods (Figure 8). A
319 commercial viable active air sampler was configured to collect one 40-minute sample.
320 Additionally, the study compared the BFPC detection of particles to a commercial off-the-shelf
321 non-viable particle counter. The BFPC and traditional non-viable active air particle counters
322 were configured for continuous, one minute, samples. All equipment had the same flow rate of
323 28.3 l/min (1 ft3/min).
324
325 Sampling was performed inside the biosafety cabinet with the BFPC and active particle counter
326 isokinetic probes, and the sampling head of the viable active air sampler, placed within close
327 proximity of each other. The gelatin filter was installed in the BFPC and parallel sampling was
328 initiated on all instruments.
329
330 Simulated sterility testing activities were performed during the sampling to create dynamic
331 conditions. Parallel testing of BFPC, viable active air sampling, and particle counter sampling
332 were performed for 40 minutes to provide simultaneous monitoring. The gelatin filter was
333 aseptically transferred to a tryptic soy agar plate and the viable active air samples were incubated
334 as per standard procedure for environmental monitoring samples collected during routine sterility
335 testing.
336
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337 Sampling was conducted from 15:03 to 15:43. Simulated sterility test preparation activities were
338 performed from 15:20 to 15:30. In addition, a simulated Sterility Test execution was performed
339 from 15:31 to 15:35. The spraying of 70% IPA occurred at the beginning of the simulated test
340 execution to attempt particle generation during the study.
341 Comparison to Traditional Viable Active Air Sampler: Neither the BFPC nor the viable active air
342 sampler detected any airborne viable contamination. Therefore, no comparison could be made
343 between the colonies recovered on the gelatin filter to those on the active air plate since no
344 colonies were present on either plate.
345
346 Comparison to Active Air Particle Counter: The BFPC detected one particle ≥ 0.5 µm in size at
347 15:31, at the time of the spraying of 70% IPA which occurred during the simulation; no other
348 particles were detected by either system.
349 The results of this study demonstrated the BFPC is not prone to false positive viable counts; and
350 the BFPC appeared to be able to distinguish between viable and non-viable particles during the
351 alcohol spraying.
352 Summary of Data Sets: The four sets of data representing more than twenty days of continuous
353 monitoring in Grade A areas provide confidence that the BFPC systems have low particle and
354 AFU counts in these areas, which are in line with the current state-of-control for pharmaceutical
355 manufacturing. The data sets demonstrated commercial systems are robust and can operate in
356 current manufacturing environments. The data demonstrates the value knowing the precise time
357 of events and enables understanding of the impact of activities in manufacturing areas; one
358 minute data points provide resolution to identify potential sources of particles. Finally, the
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359 BFPC based systems may provide active feedback for process understanding – potentially a
360 major step forward in process control.
361 Grade B/C/D areas
362 Grade B/C/D areas have less stringent controls and limits because the risk to the product and
363 patient is determined to be lower. The settle plate microbiological limits during operation for
364 Grade B/C/D are 5, 50, and 100 CFU per 4 hours respectively. The active air sample limits per
365 cubic meter are twice the settle plate limits. The BFPC systems provide both viable and non-
366 viable data simultaneously while enabling the rapid response to shifts in the environment. The
367 current limits for viable counts suffer from averaging during manufacturing, it has been
368 demonstrated that transient events (e.g. door openings, personnel entering) may be an order of
369 magnitude higher than the average during operation, but the length of sampling time prevents
370 accurate time resolved understanding and interpretation. Lower grade areas may provide
371 opportunities for parallel testing that is non-zero compared to Grade A manufacturing and
372 thereby demonstrating statistical comparison to traditional methods. This paper will not discuss
373 the use and impact of BFPC in Grade B/C/D areas, this will be the topic of a future paper.
374 Regulatory Perception
375 Bio Fluorescent Particle Counting is a novel technology less than a decade old; there is little
376 regulatory guidance on the application of the system. Both the Agency inspectorate and
377 pharmaceutical manufacturers are conservative based on the role of pharmaceutical products and
378 patient safety. The hesitation to fully implement the systems is based on industry opinion that
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379 the settle plates and active air samplers provide continuous coverage and large historical data sets
380 require extensive parallel studies to eliminate these process interventions.
381 There is no conventional analog for BFPC and the group has developed a framework of potential
382 questions and responses about the system.
383 • What about suitability (verification) testing of BFPC systems? The BFPC systems are
384 compliant with International Organization for Standardization (ISO) standards for
385 particle counters with an additional fluorescence detector to measure auto fluorescence
386 (14, 15). The BFPC systems are ISO compliant particle counter with fluorescence
387 detectors and appropriate validation methods. Periodic system checks are performed with
388 the BFPC the same as current active air particle counters. The use of actual bacteria to
389 verify the AFU detector response in aseptic manufacturing areas; creates an unacceptable
390 risk. Commercial fluorescent beads have been developed and the National Institute of
391 Standards and Technology (NIST) has evaluated a standard reference material that can be
392 used to demonstrate the system performance. NIST supports the development of a stable,
393 consistent and well-characterized standard reference material (SRM) that can be used as a
394 surrogate for microbial samples.
395 • How will you respond to AFU counts? The conservative approach is to react AFU the
396 same as CFU in aseptic manufacturing. There is an expectation for sample capture to
397 provide incubation and potential identification of any AFU counts. The challenge of
398 stressed or viable but not culturable (VBNC) microorganisms is a valid concern; we
399 would suggest that manufacturers perform reasonable attempts to culture and identify
400 sources of AFU, at least during initial system set-up. Additionally, it would be prudent
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401 that open product that may have been exposed during the AFU event would be segregated
402 for evaluation rather than discarding the product†.
403 • Can we separate the viable and non-viable AFU counts? No, the technology uses
404 biological auto fluorescence to identify NADH and other biological indicators. These
405 indicators are also present in stressed and intact dead cells. Due to the risk, sample
406 capture and incubation of AFU counts is required for potential confirmation.
407 • What are the action and alert levels suggested for Grade A and Isolators? The BFPC
408 system will maintain the same non-viable particle counts and use a limit of no more than
409 1 AFU per cubic meter. Any AFU counts require sample capture and incubation of
410 gelatin filter for potential identification of AFU counts. The initial approach will be that
411 one AFU is equivalent to one CFU, unless a readily assigned cause can be made (e.g.
412 cleaning operations, validated interventions). This conservative approach provides
413 alignment to Annex I limits and ensures that appropriate manufacturing controls are
414 established.
415
416 • What is the regulatory requirement for implementing BFPC? Environmental monitoring
417 is an inspectional rather than registration change for manufacturing. The challenge for
418 companies implementing BFPC will be demonstrating process control. Additionally,
419 education of the inspectorate and collaboration across the industry provides the best
420 approach to improved EM systems.
421
†
As per conversations between Jeffery Webber and a FDA scientist.
MS ID#: PDA/2018/008722
Resubmission
422 • Does the guidance in Annex I section 18: “…such as settle plates, volumetric air and
423 surface sampling”, allow for BFPC to be implemented in manufacturing? There is no
424 official response from the agencies as to the role of this technology; settle plates are
425 historically accepted and have a comfort level for inspectors. The improved process
426 knowledge for the continuous monitoring with no required interventions should satisfy
427 the intent of aseptic EM; ensuring a state-of-control during manufacturing.
428
429 • How does improved process control aid the inspectorate during audits? The systems
430 provide improved data integrity with time-based information and allow the inspectorate
431 to accurately evaluate the impact of operator manipulations in critical zones. With rapid
432 detection, the operator training evaluations may be enhanced. Ultimately, reduced
433 interactions with fewer personnel in the classified areas enables improved controls over
434 the aseptic areas and provides the inspectorate an overview of the activities, impacts, and
435 state of control during manufacturing.
436
437 Business Benefits
438 Many companies purchase a BFPC system during the course of an investigation to rapidly
439 identify the source of contamination. This is a non-compendial role of the system to provide a
440 “microbial sniffer” role that can be used the locate point sources of contamination. The system
441 can also be used to evaluate room recovery after shutdown or maintenance operations. The
442 financial benefits of being able to ensure microbial corrective and preventative actions (CAPA)
MS ID#: PDA/2018/008722
Resubmission
443 are clear; enabling companies to return to manufacturing rapidly and confidently. One company
444 estimates the BFPC systems enable 6 additional manufacturing days capacity annually due to
445 closing investigation quicker and being able to release manufacturing areas faster to production.
446 The cost of the instruments ($50K to 85K USD) may seem high for traditional microbial testing;
447 the insight and process knowledge is transformational for investigation understanding.
448 Previously, investigations would rely on operator memory and training logs to try and identify
449 sources of contamination. The automation and continuous nature of BFPC testing has
450 demonstrated that data integrity and reduced operator interactions support human error reduction
451 initiatives; based on the data in this paper we are able to show the time and activities occurring
452 during operations provide clear benefits over traditional methods.
453 The primary goal for continuous microbiological monitoring is improved process understanding
454 and reduction of operator interventions for the use of settle and active air sampling. The BFPC
455 systems provide improved sensitivity and rate of response to changes in the manufacturing
456 environment. The cost of settle plates is around $1 each; the complete cost of labor, testing,
457 reading, and quality oversight increases the costs to $6 to $10 per plate; several sites reported
458 more than 500,000 EM plates annually. Settle plates and environmental monitoring are highly
459 manual and subjective testing relying on training and documentation to provide meaningful
460 information. Current settle and active air sampling plates are replaced on average every 4 hours;
461 the benefit is reduction of half the EM plates. Additional opportunities for the reduction through
462 reduced settle and active air sampling frequencies using gelatin agar plates are validated for 9-
463 hour continuous sampling; this would cut the number of plates by at least half in the
464 microbiological laboratory.

MS ID#: PDA/2018/008722
Resubmission
465
466 Conclusions
467 Bio fluorescent particle counting provides continuous, sensitive, real-time incisive environmental
468 monitoring enabling reduced process interventions, reduced personnel in critical zones, improved
469 process knowledge and assurance of product quality. The systems have demonstrated the ability
470 to detect low level changes in aseptic manufacturing areas and are ideal for classified
471 manufacturing areas. The systems provide insights into operator interventions and provide rapid
472 information of manufacturing areas supporting product quality and patient safety. The transition
473 from settle plates to BFPC will require characterization of current environmental state-of-control
474 during operations and alignment of regulatory guidance for the classification of manufacturing
475 areas when using new technologies.
476 The data sets and review of BFPC technology provide an introduction for the implementation
477 and use of the technology in aseptic manufacturing. We have evaluated a new unit of measure,
478 sample capture, and large data sets to demonstrate monitoring and control of aseptic
479 manufacturing areas. The goal is to provide improved process knowledge, decreased operator
480 interventions and ultimately real-time information for aseptic manufacturing areas. Finally, the
481 technology is required to support the future manufacturing technology roadmap that may include
482 gloveless isolators and smaller scale manufacturing whilst providing sufficient data to assure
483 patient safety.
484
485
MS ID#: PDA/2018/008722
Resubmission
486 Acknowledgements
487 We thank Constanze Reinhard of Lonza, a valued team member, who sadly passed away in 2017,
488 for her contribution to the development of the ideas in the paper. Many improvements were
489 suggested by many subject matter experts who reviewed the manuscript. Any remaining errors
490 are our own and should not tarnish the reputations of these reviewers.
491 The work was facilitated BioPhorum which has become an open and trusted environment where
492 senior leaders of the biopharma industry come together to openly share and discuss the emerging
493 trends and challenges facing their industry. BioPhorum currently comprises more than 50
494 member companies and 2000 subject matter experts in six forums: Drug Substance, The
495 Development Group, Fill Finish, The Technology Roadmap, BioPhorum IT Group, and
496 BioPhorum Supply Partners. More information can be found at www.biophorum.com
497 Conflicts of Interest Declaration
498 The authors declare that they have no competing interests.
499
MS ID#: PDA/2018/008722
Resubmission
500 References
501 1. Gaigalas AK, Choquette S, Zhang Y-Z. Measurement of Scattering and Absorption
502 Cross Section of Dyed Microspheres, Journal of Research of the National Institute of
503 Standards and Technology, Vol 118 (2013)
504 2. Hill et al., “Real-time Measurement of Fluorescence Spectra from Single Airborne
505 Biological Particles”, Field Analytical Chemistry and Technology, 3, 221–239 (1999)
506 3. Staley JT, Konopka A, “Measurement of in situ activities of nonphotosynthetic
507 microorganisms in aquatic and terrestrial habitats.” Annu Rev Microbiol.;39:321-46
508 (1985).
509 4. Muller, M., and Davey, H. Recent Advances in the Analysis of Individual Microbial
510 Cells. Cytometry, A, 83-85 (2009)
511 5. Oliver, J.D. Recent findings on the viable but nonculturable state in pathogenic bacteria.
512 FEMS Microbiol. Rev., 34:415-425 (2010)
513 6. EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and
514 Veterinary Use, Annex I Manufacture of Sterile Medicinal Products, 25 Nov (2008).
515 7. Andon, BM, “Active air vs. passive air (settle plate) monitoring in routine environmental
516 monitoring programs.” PDA J Pharm Sci Technol. 60(6):350-5 Nov-Dec (2006).
517 8. Buddemeyer, Julie, “Selecting an Active Air Sampling Methodology”. Controlled
518 Environments Magazine (July 1, 2005), retrieved from
519 http://www.cemag.us/article/2005/07/selecting-active-air-sampling-methodology
520 9. Cundell, Tony, “The limitation of the Colony Forming Unit in Microbiology”. Eu Pharm
521 Rev. Vol 20, Issue 8, 11-13 (2015).
522 10. Heinz Langhals et al, “High performance recycling of polymers by means of their
523 fluorescence liftetimes”. Dept of Chemistry, LUM University of Munich, Munich,
524 Germany. August 2014 (365 nm excitation)
525 11. Eaton, T, Davenport, C, Whyte, W. Airborne microbial monitoring in an operational
526 cleanroom using an instantaneous detection system and high efficiency microbiological
527 samplers. Eur. J. Parenter. Pharm. Sci., 17(2),61-69 (2012).
MS ID#: PDA/2018/008722
Resubmission
528 12. Guidance for Industry PAT – A Framework for Innovative Pharmaceutical Development,
529 Manufacturing and Quality Assurance. US Dept. of Health and Human Services, FDA,
530 Sept 2004.
531 13. Akers, J & Agalloco, J. “Clean Rooms, RABS and Isolators: Validation and Monitoring
532 in the Diverse World of Aseptic Processing”. American Pharmaceutical Review. May 11
533 (2011).
534 14. International Organization for Standardization [ISO 21501-4] Determination of Particle
535 Size Distribution – Single Particle Light Interaction Methods, Part 4: Light Scattering
536 Airborne Particle Counter for Clean Spaces (2007).
537 15. International Organization for Standardization [ISO 14644-1] Cleanrooms and
538 Associated Controlled Environments, Part 1: Classification of Air Cleanliness by Particle
539 Concentration (2015).
540
MS ID#: PDA/2018/008722
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541 List of tables and Figures
542
543 Figure 1. Replacement of standard settle plates with active bio fluorescent particle counting
544
545 Figure 2. Flowchart for the response to AFU signals captured during manufacturing. The
546 establishment of quality oversight response needs be developed prior to implementation.
547
548 Figure 3a. 10 days (402 cubic meters) of bio-fluorescent particle counting in an aseptic
549 manufacturing facility. The Annex I limit is <1 CFU per cubic meter. The graph illustrates a
550 total of 5 AFU over the course of 402 measurements. Each tick mark on the horizontal axis
551 represents one cubic meter.
552
553 Figure 3b. 10 days (402 cubic meters) of BFPC continuous active air particle counting in an
554 aseptic manufacturing facility. The Annex I limits are 3250 particles per cubic meter of ≥0.5 µm
555 and 20 particles per cubic meter of ≥5.0 µm. Each tick mark on the horizontal axis represents
556 one cubic meter.
557
558 Figure 4. Placement of the instrument within the RABS.
559
560 Figure 5a. 5.6 days (231 cubic meters) of bio-fluorescent particle counting in an aseptic
561 manufacturing facility. The Annex I limit is <1 CFU per cubic meter. The graph illustrates a
562 total of 5 AFU over the course of 8095 minutes, 28.3 liters per minute. The following assignable
563 actions were performed during events: A – mopping in the manufacturing suite, B,C,D –
564 cleaning operations and set-up, E – unknown. Each tick mark on the horizontal axis represents
566
567 Figure 5b. 5.6 days (231 cubic meters) of BFPC continuous active air particle counting in an
568 aseptic manufacturing facility. The Annex I limits are 3250 particles per cubic meter of ≥0.5 µm
569 and 20 particles per cubic meter of ≥5.0 µm. Each tick mark on the horizontal axis represents
MS ID#: PDA/2018/008722
Resubmission
571
572 Figure 6. Location of the isokinetic sampling probe in the sterility test isolator floor. The ball
573 valve is to separate the isolator from the BFPC system for VHP decontamination. The BFPC is
574 placed below deck and therefore sampling capture was not used.
575
576 Figure 7. BFPC continuous active air particle counting for 24, 48, and 96 hours (41, 82, 164
577 cubic meters) in a sterility testing isolator at rest. Each tick mark on the horizontal axis
578 represents one cubic meter.
579
580 Figure 8. Data in the biosafety cabinet with bio-fluorescent particle counter and traditional
581 active air particle counter.
582
583 Table 1. Summary of the 4 different data sets.
584
1
BFPC system Traditional Assignable Summary

Data Location Data set
Monitoring Environmental cause? comments
set period
Monitoring

Particles AFU Particles CFU
1 Grade A - 10 days ≥0.5 µm: 5 AFU No data 0 CFU No assignable Data resolution of 1
At Rest continuous max 5/m3 collected. (Settle cause was found: m3 insufficient to
of which
monitoring. plates) evaluate source of
≥5.0 µm: 3 ≥5.0 No personnel in
AFUs, 1 minute
402 m3 max 4/m3 µm the
resolution would be
(sequential) manufacturing
No required.
area
sample
capture 0 CFU measured
on with traditional
gelatin methods
1 (4)
filter.
2 Grade A - 5.6 days ≥0.5 µm: 5 AFU No data 0 CFU - 1 AFU were Continuous
Restricted continuous max collected (Settle mopping monitoring gives

of which
Access monitoring. 1890/m3 plates) activities more insight in
0 ≥5.0
Barrier - 3 AFU and particle loads created
≥5.0 µm:

231 m3 µm
Systems max particle by staff operations.
max 159/m3
(RABS) No counts were
(only once
sample cleaning/set-up
Water Run above the
capture - 1 unassigned:
Annex I
on no operations,
limit of
gelatin no product
20/m3)
filter. exposed
3 Sterility 24, 48 and 96 Total 24, 48, No data were collected No assignable System is reliable
Testing hours (max/m3): 96 hr: with traditional cause was found. and provides
Isolator - At continuous 0, 0, 1 methods. continuous coverage.

≥0.5 µm:
2 (4)
Rest monitoring. 24 hr: 333 AFU The low counts
(33) provide confidence

41, 82, 164 No
48 hr: 1591 that the sterility
m3 sample
(98) isolator is properly
capture
96 hr: 439 assembled.
on

(27)
gelatin
≥5.0 µm: filter.
24 hr: 11 (3)
48 hr: 2 (1)
96 hr: 3 (1)
4 Sterility 40 minute ≥0.5 µm: 0 AFU 0 0 CFU 1 Particle was Low AFU and
Testing continuous 1 (Particle (Viable measured at the particle counts in line

Gelatin
monitoring. Counter) Active Air time of alcohol with the high level of
Grade A ≥5.0 µm: filter:
Sampler) spraying. control in a biosafety
Biosafety 1.13 m3 0 0 CFU
cabinet.
Cabinet
3 (4)
BFPC system not
prone to false
positives.
BFPC system

seemed to correctly
identify 1 particle as
non-viable during
alcohol spray.
2
3
4 (4)
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Continuous Microbiological Environmental Monitoring For Process Understanding and Reduced Interventions in Aseptic Manufacturing

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Continuous Microbiological Environmental Monitoring for

PDA Journal of Pharmaceutical Science and Technology 2018,

1 Title: Continuous Microbiological Environmental Monitoring for Process Understanding and

2 Reduced Interventions in Aseptic Manufacturing.

4 1. Jeffrey Weber, Pfizer, 7000 Portage Rd, Kalamazoo, MI 49001 USA

27 time and continuous microbiological environmental monitoring and thereby reducing

29 sampling. The replacement of traditional monitoring with bio-fluorescent particle counting

33 monitoring for aseptic manufacturing.

36 Rapid Microbiology, Environmental Monitoring, Bio florescent Particle Counters

39 air for contamination in biopharmaceutical manufacturing facilities. The alternative method is

44 understanding of contamination risks in complex manufacturing processes, ultimately providing

48 monitoring methods require manual manipulation, an additional advantage of BFPC systems is

52 Introduction to Bio Fluorescent Particle Counting

54 monitoring of microorganisms to ensure product quality and patient safety. Aseptic

55 pharmaceutical manufacturing relies on understanding of microbial risks and periodic monitoring

62 developed by Pasteur and Koch more than 100 years ago.

70 surface monitoring are not discussed in this application.

72 microbes to provide rapid alerts to manufacturing risk. Bio-fluorescence particle counters

74 immediate detection and enumeration of airborne microbes in manufacturing areas. Bio-

79 in all microorganisms. Current systems sometimes cannot distinguish between viability or

88 traditional plate-count methods; however, such comparisons provide understanding of the

96 Opportunities and Challenges of Settle Plates and Active Air Sampling

97 Continuous BFPC monitoring provides the opportunity to reduce operator manipulations of

102 controls of microbiological contamination during manufacturing to date; however newer

106 improved data quality and aseptic process control.

113 used for more than 4 hours (7).

134 expectations based on BFPC technologies.

140 resolution and operator interventions.

151 appropriate change control.

152 Role of Bio Fluorescence Particle Counter in Manufacturing

155 implementation of BFPC in routine aseptic manufacturing. Naturally, as each manufacturer is

156 unique, alternative plans may be more appropriate for some.

165 which may reduce a cascade of increasingly serious problems.

187 Potential Implementation Strategy

209 risk during evaluation of the new method or parallel testing.”

215 Data Collection and Presentation

223 process understanding.

235 Grade A Manufacturing Areas, RABS, and Isolators

252 Data Sets

256 false positive rate is low.

263 classification requirements.

273 large sample volumes.

278 ≥5.0 µm, respectively.

309 capture device.

314 AFU while monitoring a VHP decontaminated isolator at rest.

323 28.3 l/min (1 ft3/min).

328 initiated on all instruments.

340 execution to attempt particle generation during the study.

344 colonies were present on either plate.

348 particles were detected by either system.

351 alcohol spraying.

360 major step forward in process control.

361 Grade B/C/D areas

374 Regulatory Perception

380 require extensive parallel studies to eliminate these process interventions.

382 questions and responses about the system.

394 surrogate for microbial samples.

402 for evaluation rather than discarding the product†.

412 cleaning operations, validated interventions). This conservative approach provides

418 companies implementing BFPC will be demonstrating process control. Additionally,

420 approach to improved EM systems.