BioLaz Real Time Microbial Monitoring in Aseptic Pharmaceutical Manufacturing. March 2013

Transcription

1 BioLaz Real Time Microbial Monitoring in Aseptic Pharmaceutical Manufacturing March 2013

2 Introductions Gilberto Dalmaso, PMS Global Aseptic Processes Development Manager

3 Who We Are: Particle Measuring Systems Inc. HQ in Boulder, Colorado, USA Founded in 1972 Acquired by Spectris plc in 1996 Global leader in contamination control instrumentation Inventors of laser-based particle counting Over 40 patents

4 Our Profile: Cross- industry specialists for those cleaner-than-nature environments and materials Product lines Particle counters Microbial air samplers Molecular gas analyzers Software and systems Services What we do Design Manufacture Service Educate

5 Market Focused Life Sciences Division Pharmaceutical Biotechnology Medical devices Tissue banks, hospitals Food & beverage Electronics Division Semiconductor Flat panel display Hard disk drive Aerospace Industrial

6 Microbiological Monitoring Today.

7 EU GMP Limits AT REST IN OPERATION GRADE Max Particle concentration > size Microbiological 0.5um 5.0um 0.5um 5.0um Air Sample cfu/m^3 90mm settle plate cfu/4hr A <1 <1 B C D Not defined Not defined

8 Typical active air sampling portable samplers: Samples taken at beginning or end of batch manufacturing Risk of contamination from personnel if sampling during filling Typically 1 m 3 sample in 10 minutes

9 Limitations of portable viable samplers: At best do not monitor, but like portable particle counters, take intermittent samples Media desiccation limits sample duration to 1-2 m 3 (10-20mins sample time) Introduce people into sterile areas (risk) Great risk the introduction in grade A for exhaust air No real-time indication of bacteria

10 Real Time Microbial Monitoring

11 Fixed point microbiological monitoring Located at many of the same points identified by risk analysis for the non-viable (particle) monitors Sampling ideally during every batch Slower sampling an advantage - to represent conditions during a greater proportion of each batch, before media becomes desiccated

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13 Limitations of Impaction technologies: Still an impaction based technology using agar medium, with its desiccation / biological recovery limitations No real time indication of bacteria, therefore no real-time process control

14 FDA PAT initiative Industry is pushing for continuous methods to control the aseptic process to ensure quality by design (QBD) Ideally this requires a means of detecting viable organisms real-time Non-viable particle counting fits with PAT, but cannot tell the difference between live particles and inert ones

15 Rapid Microbial Methods

16 BioLaz Base Technology

17 Cell Structure All living cells comprise of specific chemical compounds that allow for the metabolism of energy. A catalyst of those reactions is the co-enzyme NADH.

18 Cell Fluorescence NADH has a fluorescence of 460nm when excited at 405nm This fluorescence is a clear indicator of cell activity and is directly associated with living cells, i.e. those metabolizing. Other cell components also exhibit a fluorescent emission including NADH, Riboflavin and Dipicholinic acid in spores

19 Optical Fundamentals The sample is pulled through the optical chamber and illuminated by the UV laser source. Elastic light scatter occurs when a particle enters a laser beam. The trigger from the elastic light scatter activates the fluorescence circuit to detect any fluorescence from that particle at a wavelength shift. Filters ensure only the active component of fluorescence is detected and the scattered light does not interfere with the signal

20 Bio-fluoresence Technology 405nm laser light illuminates sample air flow through the sensor ALL particles, viable and nonviable, scatter light Living organisms fluoresce (NADH (nicotinamide adenine dinucleotide), Riboflavin and Dipicholinic Acid in viable organisms will fluoresce at ~ 460nm) BioLaz simultaneously detects both scattered light and fluorescence Nonviable particles only scatter light Viable particles scatter light and fluoresce 1 fluorescent particle = 1 Bio-Count

21 Data Output 4 channels Small Particles (c.f.>0.5um) Large Particles (c.f.>5um) Small Biological Particles (c.f.>0.5um) Large Biological Particles (c.f.>5um)

22 Our Approach Step 1: Can we validate instrument performance to USP and EP? Step 2: Validate that common chemicals and materials in the aseptic environment do not interfere Step 3: Integrate BioLaz into our current environmental monitoring system Step 4: Establish operational levels in aseptic environments Step 5: Dialogue with regulatory bodies to establish criteria for operational use

23 USP / EP Validation

24 Validation Test Overview Objective To validate the BioLaz according to USP <1223> Validation of Alternative Microbiological Methods and EP Alternative Methods for Control of Microbiological Quality Use of an Independent Test Facility Edgewood Chemical and Biological Center (ECBC) Facility has expertise in single biological particle generation and recovery Known microorganism Known particle size Known concentration of challenge

25 USP <1223> and EP Not required Required

26 Validation Protocol

27 Validation Test Setup

28 Biological Particle Generator Ink Jet Aerosol Generator (IJAG) Adapts ink jet printer technology for the production of well controlled and well characterized aerosols Three components: Dispenser Controller Computer High-speed images of particle ejection (in air) by IJAG Left to right = time (frames) Top to bottom = distance

29 Initial Microorganisms Tested: 4 organisms tested using a single particle test method Bacillus Subtilis (Bs), vegetative form Bacillus Globigii (Bg), spore form, washed Bacillus Globigii (Bg), spore form, unwashed Bacillus Thuringiensis (Bt), spore form, washed Supplemental testing of 4 organisms using the traditional bio-aerosol test method (high concentration aerosol challenge) Staphylococcus epidermidis Bacillus Thuringiensis Aspergillus Niger Yeast

30 Test Methodology 5-min Blank t=0 5-min Blank One complete test cycle was: 1) 5 minutes blank at Zero Concentration 2) 5 minutes at Concentration 1 (One particle every 12 seconds: 25 total) 3) 2 minutes blank 4) 5 minutes at Concentration 2 (One particle every 9 seconds: 33 total) 5) 5 minutes blank Blank periods provide clean separation and verify zero detection capability Test cycle was completed separately for BioLaz then slit-to-agar sampler Cycle was repeated five times for each organism at 5 µm particle size Sixth test was then run at 3 µm size 5-min Conc. 1 2-min Blank Conc 1 Blank Conc 2 5-min Conc. 2

31 Test Results Summary (concentration 1) BioLaz passed all USP<1223> and EP requirements Above summary is for parameters deemed most critical to success

32 Test Results Summary (concentration 2) BioLaz passed all USP<1223> and EP requirements Above summary is for parameters deemed most critical to success

33 Example of Individual Bs Test Cultured Plate CFU Results BioLaz Bio-Count Results

34 Bs Replicate Data Summary

35 Bs Linearity

36 Bs Validation Table

37 Initial EP /USP Validation Conclusions: Meets or exceeds the guidelines established in USP <1223> and EP for Validation of Alternative Microbiological Methods Capable of single Bio-Count detection and quantification One Bio-Count is representative of CFU The accuracy at multiple concentrations with different species of microorganisms passes the USP and EP requirements The instrument does not require special handling, training or logistics to operate

38 Our Approach Step 1: Can we validate instrument performance to USP and EP? Step 2: Validate that common chemicals and materials in the aseptic environment do not interfere Step 3: Integrate BioLaz into our current environmental monitoring system Step 4: Establish operational levels in aseptic environments Step 5: Dialogue with regulatory bodies to establish criteria for operational use

39 Can common disinfectants / cleaning materials give false positive results?

40 Additional Testing False positive testing: Conducted additional testing with formalized method using chemicals found in clean rooms: Isopropyl alcohol 70% Hydrogen peroxide 6% Sodium Hypochlorite 6% Quaternary Ammonium Phenolics Peracetic acid/ hydrogen peroxide Decon-Clean Conduct additional testing with formalized method using materials found in clean rooms: Latex/Nitrile gloves Cleanrooms garments Cleanroom ISO 5 wipes

41 Shield Medicare Biocides Date Description Date Time Large Bio Small Bio Large Particle Small Particle Biocide A 1 spray 07/11/20129:12:56 AM 9:13:26 AM Biocide A 1 spray x2 9:13:36 AM 9:14:36 AM Biocide A 2 sprays 9:14:46 AM 9:15:46 AM Biocide A 2 sprays x2 9:15:56 AM 9:17:36 AM Biocide A Nebulized 2:00:56 PM 2:12:06 PM Biocide B 1 spray 9:18:56 AM 9:19:36 AM Biocide B 1 spray x2 9:19:46 AM 9:20:16 AM Biocide B 2 sprays 9:20:26 AM 9:21:06 AM Biocide B 2 sprays x2 9:21:16 AM 9:21:46 AM Biocide B Nebulized 2:16:16 PM 2:27:37 PM Biocide C 1 spray 9:24:26 AM 9:24:56 AM Biocide C 1 spray x2 9:25:06 AM 9:25:46 AM Biocide C 2 sprays 9:25:56 AM 9:26:36 AM Biocide C 2 sprays x2 9:26:46 AM 9:28:36 AM Biocide C Nebulized 2:31:27 PM 2:42:17 PM Biocide D Nebulized (Not available in trigged spray bottle) 2:46:07 PM 2:57:17 PM Biocide E 1 spray 9:30:56 AM 9:31:36 AM /11/2012 Biocide E 1 spray x2 9:31:46 AM 9:32:16 AM Biocide E 2 sprays 9:32:26 AM 9:33:07 AM Biocide E 2 sprays x2 9:33:16 AM 9:34:36 AM Biocide E Nebulized 3:00:37 PM 3:11:08 PM Biocide F 1 spray 9:35:37 AM 9:36:17 AM Biocide F 1 spray x2 9:36:27 AM 9:36:47 AM Biocide F 2 sprays 9:36:57 AM 9:37:47 AM Biocide F 2 sprays x2 9:37:57 AM 9:39:17 AM Biocide F Nebulized 3:14:58 PM 3:26:28 PM /30 sterile IPA 1 spray 9:40:07 AM 9:40:47 AM /30 sterile IPA 1 spray x2 9:40:57 AM 9:41:27 AM /30 sterile IPA 2 sprays 9:41:37 AM 9:42:17 AM /30 sterile IPA 2 sprays x2 9:42:27 AM 9:42:57 AM Sterile IPA Nebulized 3:29:58 PM 3:40:58 PM denatured ethanol 1 spray 9:46:47 AM 9:47:17 AM denatured ethanol 1 spray x2 9:47:27 AM 9:47:57 AM denatured ethanol 2 sprays 9:48:07 AM 9:48:47 AM denatured ethanol 2 sprays x2 9:48:57 AM 9:50:07 AM Denatured Ethanol Nebulized 3:44:48 PM 3:54:38 PM DI water sterile 1:55:56 PM 3:57:08 PM

42 Veltek Biocides Description Date Time Large Small Bio Bio Large Particle Small Particle Decon Spore Nebulized 08/11/2012 8:08:01 AM 8:19:01 AM Undiluted: Not available in trigger spray Decon Phene 1 spray 08/11/2012 9:34:05 AM 9:34:55 AM Decon Phene 1 spray x2 08/11/2012 9:35:05 AM 9:36:05 AM Decon Phene 2 sprays 08/11/2012 9:36:15 AM 9:37:25 AM Decon Phene 2 sprays x2 08/11/2012 9:37:35 AM 9:41:35 AM Decon Phene Nebulized 08/11/2012 8:23:21 AM 8:33:54 AM Steri Perox 1 spray 08/11/2012 9:28:45 AM 9:29:35 AM Steri Perox 1 spray x2 08/11/2012 9:29:45 AM 9:30:35 AM Steri Perox 2 sprays 08/11/2012 9:30:45 AM 9:31:55 AM Steri perox 2 sprays x2 08/11/2012 9:32:05 AM 9:32:55 AM Steri perox Nebulized 08/11/2012 8:38:04 AM 8:48:54 AM Decon clean Nebulized 08/11/2012 8:53:14 AM 9:03:54 AM Undiluted: Not available in trigger spray Decon quat Nebulized 08/11/2012 9:08:04 AM 9:18:54 AM Undiluted: Not available in trigger spray DI Water Nebulized 08/11/2012 8:04:01 AM 9:26:45 AM

43 Biocide testing: Nebulized Biocides cause negligible background interference Direct spray interference levels unlikely to be problematic in operational conditions in Class A & B

44 Materials tests Nitrile Gloves Cleanroom Gowns Wipes, cloths The presence of whiteners or cellulose can cause some level of fluorescence breakthrough, but these were only seen whilst directly injecting material into the Biolaz inlet and tests in normal cleanroom usage suggest this is not a significant source of counts.

45 Our Approach Step 1: Can we validate instrument performance to USP and EP? Step 2: Validate that common chemicals and materials in the aseptic environment do not interfere Step 3: Integrate BioLaz into our current environmental monitoring system Step 4: Establish operational levels in aseptic environments Step 5: Dialogue with regulatory bodies to establish criteria for operational use

46 Integration with a Monitoring System Technical Area FacilityPro Ethernet LAN IT Vacuum Pumps (hot swap control) SCADA server SCADA clients Vacuum Ethernet Redundant server External DB (Oracle, SQL,...) Analog & Digital I/O BioCapt microbial samplers BioLaz real-time microbial monitor IsoAir particle sensors Env. sensors Alarms Critical Cleanrooms (A/B) HMI Clients data download ports Lasair III particle counters MiniCapt microbial samplers Background Cleanrooms (C/D)

47 System Integration Biolaz is not seen as a substitute for conventional microbiological methods, but a process control device for real-time microbial indication By incorporating Biolaz data along with particle counts and other environmental parameters, it becomes a tool to assist in determining interventions or process checks, based on gathered data

48 5 Steps of Biolaz Integration 1) Qualification of instrument for alternative microbial methods. Completed, with full supporting validation for USP/EP qualification from PMS 2) Installation Qualification of instrument. 3) Operational Qualification of instrument. 4) Performance Qualification of instrument. 5) Integration into Environmental Monitoring program for release of product.

49 Installation Qualification Probe & Tubing Computer Data Cable Instrument mounting Orientation External obstructions to cleanroom Location relative to sample point Sample Probe & Tubing Orientation of sample probe isoaxial +/- 15% Tubing length not to exceed 1.0m (ISO ) Tubing materials, connectors Power Power Supply 110 or 240V fed from where? Circuit number, conduit requirements, isolation of power Communications RS232 or Ethernet Cable type, low smoke, CAT5, shielding Convertor devices for long distances Hubs for multiple instruments within a facility

50 Operational Qualification Hardware Errors Power loss Sample probe blocked Data cable broken Laser / Flow failure Alarm Response System colour changes on screen External alarm devices / SMS text paging responses and configuration Data formats available Historical data Time Plots Data Reports and data export Security of SCADA package 21CFR 11 E records & E Sigs Archived data retrieval

51 Performance Qualification Verify against current practices and set statistically appropriate alarm and alert limits EU GMP Annex 1 PIC/S interpretation of Annex 1 This is a sensor like particle counting so Appropriate alarms should be applied = alarm..? = alarm Cubic Meter Monitoring flow is 4 LPM, to take a 1m 3 sample it would take apx 4 hours, therefore 2m 3 per shift, this is in line with current practices

52 BioLaz Benefits Real Time Alarm on viable organisms When used in conjunction with a non-viable particle counting system (FMS), Biolaz can indicate whether a contamination event contains live organisms allowing rapid response When results from both non-viable monitoring and BioLaz indicate all is in limits, greater certainty of batch release is assured. Consistent with the PAT approach

53 Incorporation into Environmental Monitoring Program Once statistically valid, action and alert limits can be set and these correlated against: Traditional particle counting Microbial samples active air + settle plates Media fill finished product quality data Reviewed on a routine basis ensuring creditable alarms: Not so many alarms as to cause a nuisance Not so few as to not be able to demonstrate control Review of EM program to monitor number of traditional samples required to demonstrate control 99.9% of plates read zero A reduction of half would significantly alter the workload on microbiology groups NOT proposing to not do plates speciation, fail safe, maintenance, etc. BioLaz Real-time Microbial data is included into environmental monitoring records for product release

54 Applications

55 Monitoring Applications Filling lines Classic hard and soft wall filling lines Isolator and RABS technologies Grade B background zones Sterility test isolators Critical to ensuring product environment was suitable prior to test false fail. Biosafety cabinets Critical activities of manual formulations lasting a short period, unsuitable for traditional techniques Aseptic transfers Short duration activities lasting a few minutes, connection of aseptic piping to fill line from vessel.

56 Monitoring Applications Biotech & Radioactive products Where the product is formulated for specific patients requiring a short time from product to patient. Typical cycles are <72 hrs, insufficient for results from traditional methods. Portable monitoring Routine monitoring of Grade B and little used Grade A environments, used in conjunction with portable particle counters. Regular use in Grade C and D This is where the frequency of monitoring can be increased as enumeration becomes more important than speciation. Allows for identification of seasonal challenges.

57 BioLaz Benefits Faster batch release Partitioning of finished product (based on timing of alarms) Immediate notification for alarm response Verify biological levels are acceptable prior to filling or sterility testing Reduced operator error Paperless data management + + = Compliance and Cost Reduction Traditional microbial Real-time microbial Real-time particle

58 Conventional Microbiology + RMM You still need to perform conventional viable sampling as organism identification is required by GMP. This requires agar cultivation and speciation. Current Annex 1 limits are set for traditional air sampling methods. No RMM levels are yet in place.

59 Our Approach Step 1: Can we validate instrument performance to USP and EP? Step 2: Validate that common chemicals and materials in the aseptic environment do not interfere Step 3: Integrate BioLaz into our current environmental monitoring system Step 4: Establish operational levels in aseptic environments Step 5: Dialogue with regulatory bodies to establish criteria for operational use

60 Biolaz in a Working Aseptic Manufacturing Facility

61 0:00:07 0:34:27 1:08:48 1:43:11 2:17:31 2:51:52 3:26:12 4:00:33 4:34:54 5:09:14 5:43:35 6:17:55 6:52:16 7:26:36 8:00:59 8:35:23 9:09:56 9:44:19 10:18:40 10:53:00 11:27:21 12:01:41 12:36:02 13:10:23 13:44:43 14:19:04 14:53:24 15:27:45 16:02:05 16:36:26 17:10:46 17:45:07 18:19:39 18:54:00 19:28:24 20:02:44 20:37:24 21:11:45 21:46:06 22:20:26 22:54:47 23:29:07 BIOLAZ GRADE A - AT REST Small Bio Large Bio

62 0:00:07 0:35:17 1:10:28 1:45:41 2:20:51 2:56:02 3:31:13 4:06:23 4:41:34 5:16:44 5:51:55 6:27:05 7:02:16 7:37:26 8:12:39 8:48:05 9:23:19 9:58:29 10:33:40 11:08:51 11:44:01 12:19:12 12:54:22 13:29:33 14:04:43 14:39:54 15:15:05 15:50:15 16:25:26 17:00:36 17:35:47 18:11:09 18:46:20 19:21:34 19:56:44 20:32:02 21:07:25 21:42:36 22:17:46 22:52:57 23:28:07 BIOLAZ GRADE A - AT REST Large Particle Small Particle 4 2 0

63 :00:02 0:32:56 1:05:46 1:38:40 2:11:30 2:44:21 3:17:11 3:50:02 4:22:53 4:55:43 5:28:36 6:01:26 6:34:17 7:07:07 7:40:00 8:12:50 8:45:41 9:18:35 9:51:25 10:24:16 10:57:06 11:29:57 12:02:47 12:35:38 13:08:28 13:41:24 14:14:14 14:47:05 15:19:56 15:52:49 16:25:45 16:58:36 17:31:26 18:04:17 18:37:07 19:09:58 19:42:48 20:15:39 20:48:29 21:21:20 21:54:24 22:27:15 23:00:05 23:32:56 Large Bio Small Bio BIOLAZ GRADE A IN OPERATION

64 BIOLAZ GRADE A IN OPERATION :00:02 0:33:36 1:07:06 1:40:40 2:14:10 2:47:41 3:21:11 3:54:42 4:28:13 5:01:43 5:35:16 6:08:47 6:42:17 7:15:48 7:49:20 8:22:51 8:56:24 9:29:55 10:03:25 10:36:56 11:10:26 11:43:57 12:17:27 12:50:58 13:24:34 13:58:04 14:31:35 15:05:05 15:38:36 16:12:15 16:45:45 17:19:16 17:52:46 18:26:17 18:59:47 19:33:18 20:06:49 20:40:19 21:13:50 21:47:34 22:21:05 22:54:35 23:28:06 Large Particle Small Particle

65 0:00:01 0:31:21 1:02:42 1:34:02 2:05:23 2:36:43 3:08:04 3:39:24 4:10:45 4:42:05 5:13:26 5:44:46 6:16:07 6:47:27 7:18:48 7:50:08 8:21:29 8:52:49 9:24:10 9:55:30 10:26:53 10:58:13 11:29:34 12:00:54 12:32:15 13:03:35 13:34:56 14:06:16 14:37:37 15:08:57 15:40:18 16:11:38 16:42:59 17:14:19 17:45:40 18:17:00 18:48:21 19:19:41 19:51:02 20:22:22 20:53:43 21:25:03 21:56:24 22:27:44 22:59:05 23:30:25 BIOLAZ GRADE B - AT REST 2 1 Large Bio Small Bio 0

66 0:00:01 0:31:21 1:02:42 1:34:02 2:05:23 2:36:43 3:08:04 3:39:24 4:10:45 4:42:05 5:13:26 5:44:46 6:16:07 6:47:27 7:18:48 7:50:08 8:21:29 8:52:49 9:24:10 9:55:30 10:26:53 10:58:13 11:29:34 12:00:54 12:32:15 13:03:35 13:34:56 14:06:16 14:37:37 15:08:57 15:40:18 16:11:38 16:42:59 17:14:19 17:45:40 18:17:00 18:48:21 19:19:41 19:51:02 20:22:22 20:53:43 21:25:03 21:56:24 22:27:44 22:59:05 23:30:25 BIOLAZ GRADE B - AT REST Large Particle Small Particle 0

67 BIOLAZ GRADE B IN OPERATION :00:09 0:31:03 1:01:53 1:32:44 2:03:34 2:34:25 3:05:15 3:36:05 4:06:59 4:37:49 5:08:40 5:39:30 6:10:21 6:41:11 7:12:02 7:42:52 8:13:43 8:44:39 9:39:47 10:10:38 10:41:28 11:12:19 11:43:09 12:14:00 12:44:50 13:15:41 13:46:31 14:17:22 14:48:14 15:19:05 15:49:58 16:20:48 16:51:39 17:22:29 17:53:19 18:24:10 18:55:00 19:25:51 19:56:41 20:27:32 20:58:22 21:29:13 22:00:06 22:30:56 23:01:47 23:32:40 Large Bio Small Bio

68 BIOLAZ GRADE B IN OPERATION :00:09 0:31:03 1:01:53 1:32:44 2:03:34 2:34:25 3:05:15 3:36:05 4:06:59 4:37:49 5:08:40 5:39:30 6:10:21 6:41:11 7:12:02 7:42:52 8:13:43 8:44:39 9:39:47 10:10:38 10:41:28 11:12:19 11:43:09 12:14:00 12:44:50 13:15:41 13:46:31 14:17:22 14:48:14 15:19:05 15:49:58 16:20:48 16:51:39 17:22:29 17:53:19 18:24:10 18:55:00 19:25:51 19:56:41 20:27:32 20:58:22 21:29:13 22:00:06 22:30:56 23:01:47 23:32:40 Large Particle Small Particle

69 BIOLAZ evaluation Summary data 09 dec. 10 dec. 11 dec. 12 dec. 13 dec. 14 dec. 15 dec. 16 dec. 17 dec. 18 dec. volume large bio large particle small bio small particle Grade total (5,76 m 3 ) total m 3 0 0,52 0,86 92 grade A at rest total (5,76 m 3 ) grade A in total m 3 0,86 1,74 1, operation total (5,76 m 3 ) grade A in total m 3 0,86 1,91 1,21 105,73 operation total (5,76 m 3 ) grade A in total m 3 1,04 2,08 1,74 155,38 operation total (5,76 m 3 ) total m 3 0,34 0,34 2, grade A cleaning total (5,76 m 3 ) grade B in total m 3 0,86 3,12 5,9 155,9 operation total (5,76 m 3 ) total m 3 0 0,52 0,35 153,6 grade B at rest total (5,76 m 3 ) total m 3 0,52 0,52 0,69 96,87 grade B at rest total (5,76 m 3 ) grade B in total m 3 2,25 16,49 13,02 771,52 operation total (2,4 m 3 ) grade B in total m 3 0, , operation

70 Summary Grade A at rest 0.5 bio-counts / m3 Grade A operational - 2 bio-counts / m3 Grade B at rest 0.5 bio-counts / m3 Grade B operational 10 bio-counts / m3

71 Calibration

72 Calibration Nebulizer with PSL s and fluorescing particles Reference Particle Counter Reference Unit Unit Under Test (UUT) Ethernet cables Calibration based on comparison to reference unit. Nebulized 0.7 & 1.5 m PSL s for particle channels Nebulized BHA Green Visolite calibration spheres for fluorescent channels. Specific number of counts in the large channel (>2 m) and specific number in small channel (0.5-2 m) essentially splitting the sizing threshold. System verified as operational using a mixture of Visolite, Riboflavin and 0.7 m particles Calibration performed at customer site or back at laboratory.

73 Sensor Calibration The software automatically starts generating calibration particles into both sensors and adjusts settings on the sensor to match the particle counts, size ratio, and fluorescence signals to the referee sensor. Each adjustment is represented as a vertical black line on the graph showing where the change was made. Once that data point is properly calibrated, the vertical line turns green. Threshold boxes turn green when calibration is successful These thin grey lines show the upper and lower tolerance levels for a successful calibration. The Calibration Actions box shows a text display of the calibration process as it s occurring page 73

74 Calibration Certificate Instrument details Serial number Model number Calibration Details Sensor thresholds Response to calibration media Comparison against reference Details of media used Expiration date 12 month expiration

75 BioLaz Summary Real-time, continuous microbial monitoring is now possible System integration with traditional monitoring systems provides no-risk qualification path Small 316L stainless steel enclosure No maintenance or consumables Annual calibration as with existing environmental sensors Validated to USP and EP requirements

76 Our Approach Step 1: Can we validate instrument performance to USP and EP? Step 2: Validate that common chemicals and materials in the aseptic environment do not interfere Step 3: Integrate BioLaz into our current environmental monitoring system Step 4: Establish operational levels in aseptic environments Step 5: Dialogue with regulatory bodies to establish criteria for operational use

77 Questions?