Disruptive Technologies Product Range and Manufacturers Represented

2. Disruptive Technologies Product Range and Manufacturers Represented • Biolgical Sample Preparation • Nucleic acides, protein and small molecules extraction from hard-to-lyse tissue samples (Pressure Biosciences) • Dissolution/Formulation • Dissolution baths, friability and disintegration instruments (Distek) • Physico Chemistry • HT Log P and pKa analyzer (AATI) • Kinetic solubility instruments (Analiza) • Rapid microbiology with MicroPRO (AATI) • Service • Agilent Channel Partner to service their range of HPLC, UV spectrophotometers and CE systems • Preparative • OPLC chromatography solutions for semi preparative applications (OPLC systems, pumps, sample applicator, video imaging and densitometry instruments, reagent sprayer (OPLC-NIT) • Flash chromatography (Gyan) • Automated SPE system (HTA) • Analytical • HT oligonucleotides purity analyzer (AATI) • HT proteins analyzer (AATI) • HT DNA analyzer (AATI) • HT Chiral analyzer (AATI) • Spotter for MALDI and tissue MALDI imaging (SunChrom) • Type-C silica hydride HPLC columns Flat sorbent beds for OPLC (MicroSolv) • Accessories and consumables for CE and HPLC (MicroSolv) • Validation kits for HPLC systems (MicroSolv)

3. Who are we? Our History: Founded 1997, 4 Scientific Co-founders, Privately Funded Our Business: Rapid Microbial Detection Technology Capillary Electrophoresis (January 2007) Our Markets: Pharmaceutical, Personal Care Products, Fermentation, Environmental, Drug Discovery, Oligonucleotides Production. Our Solution: Replace current microbial detection methods (requiring 24 – 72 hours). High throughput quality analysis of oligonucleotides and fast pKa and Log P determinations of chemical compounds. Our Products: MicroPRO Instrument, Custom Kits. OligoPRO and pKa PRO analyzers.

4. Ames, Iowa

5. Overview of Microbial Detection Presentation • How the Technology Works • Standard Labeling Protocol • The MicroPRO Instrument • Water Monitoring • Bacteria, Yeast and Mold in 24 hours in Products • Surface/Environment Monitoring Using Swabs • Fermentation Products • Challenge Tests in Products

6. Basics of Flow Cytometry

7. Basics of Flow Cytometry • Laser-based irradiation of cells • Fluorochromes bound to cells provide information on cell state (e.g., live, dead, spores, vegetative) • Light scattering provides relative size information • System composed of fluidic, optic and electronic components • Advantages: Rapid and quantitative analysis of individual cells

8. Fluidic System Sample delivery (0.05mL/min) Sheath delivery (16-18mL/min) • Quantitative cell delivery • Hydrodynamic Focusing • Single File Passage through detection region Sheath flow Labeled bacteria Core flow

9. Optic System Labeled microorganism Scatter Detector Scatter signal Laser Beam – shaped and focused; 635 nm laser excitation Fluorescence Detector Fluorescence signal High performance optical filters Fluorescence plus Scatter = One Count

10. Electronic System • Signal processing component • The Micro PRO™ triggers on fluorescence • Fluorescent event above the threshold is processed, along with the corresponding scatter event, and is plotted and recorded as a count = 1 count Detector output = 0 count Fluorescence Threshold Level Time

11. Signal Processing Microbe A cumulative Microbe A + B Microbe A + B +C

12. Example of Micro PRO Intensity Plot Cell size Amount of label

13. Results Fluorescence Intensity vs. Counts Intensity Plot Scatter Intensity vs. Counts Box = 9907 counts/0.25mL 97.4% of the counts are in the box

14. MicroPRO Standard Labeling Protocol

15. RBD Total Cell Count / mL Biomass Labeling Protocol (Membrane Permeable – all Cells are Stained) • Ideal for enumerating Live & Dead microbes in: • Fermentation cultures • Spore preparations

16. Ideal for enumerating Viable microbes in: • Process water monitoring • Presence/Absence testing • Fermentation cultures • Final product testing • Pure cultures TVO Labeling Protocol (Only Viable Cells are Stained)

17. Antibody-Specific Detection Labeling Protocol Mixed Sample • Ideal for enumerating microbes for/in: • Specific pathogen detection • Mixed cultures • High background matrices

18. MicroPRO System

20. Auto-Sampler Auto-Prep • Holds up to 42 samples • Adds reagents • Mixes sample • On-board automated cleaning and bubble removal

21. Analysis on the Micro PRO™ 2 1 Load sample vials and syringes Select Tray SetUp Reagent additions and sample injection performed automatically as defined in the Method Micro PRO™ Output: Pass/Fail & Counts/mL 3 4

22. MicroPROVarious Results/ Applications

23. Purified Water Monitoring

24. Sample Preparation • Collect sample; • Dispense 3mL sample into 5mL snap-cap tube • Load sample into MicroPRO Sample Tray with capped syringe • Select pre-defined (or create new) Analysis/Tray Sequence • MicroPRO count result in 5 minutes

25. 1 2 3 Background / Neg.Ctrl Sample 3 Sample 7 Box = 0 counts/0.25mL Box = 2 counts/0.25mL Box = 22 counts/0.25mL

26. Purified Water Monitoring: MicroPRO TVO & R2A Plate Counts action warning

27. Purified Water System From: Hasher-Homesley, P.1, 2006. R&D Applications for the RBD3000. 1Johnson & Johnson Vision Care. Rapid Microbial Methods User’s Meeting, Chicago, IL

28. Product Research Lab Water Testing Kozak, K.1, 2006. Rapid Microbiological Testing in Support of Product Development. 1Procter and Gamble. Rapid Microbial Methods User’s Meeting. Chigaco, IL

31. Product Testing Presence/Absence

32. Traditional methods USP <61> Bacteria 1:10 dilution of product 1 ml in each of two Petri dishes with Soybean Casein Digest medium melted <45 C Incubate 48 to 72 hours at 30 C Count If zero counts, results are expressed as less than 10 cfu/ml Yeast and Mold 1:10 dilution of product 1 ml in each of two petri dishes Saboraud Dextrose Agar Incubate 5 to 7 days at 20-25 C Count If zero counts, results are expressed as less than 10 cfu/ml

33. Advanced Analytical Method Bacteria, Yeast & Mold Incubate 24 to 48hours at 30°C Transfer substrate tube A to Tube B; vortex; 0.1mL to Tube C 1:10 dilution of product 1ml in Tube A Micro PRO™

34. Product Test Kit 35mm filter mold Micro PRO™ Transfer swab from Tube A to B Tube A - GEM Add product, enrich Tube B - PB Add swab, mix Tube C - PB Add 0.1mL from Tube B

35. Experimental Procedure • Prepare 1:10 product suspension in buffer or neutralizing growth enhancement media • Add 1mL 1:10 product suspension to Tube A & neutralize 30 min • Spike with <100 cfu Escherichia coli ATCC 8739 or 25922, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, Candida albicans ATCC 10231, Aspergillus niger ATCC 16404 • Prepare non-spiked product controls • Enrich at 30oC for 24-48 hours • Transfer substrate from Tube A to Tube B and vortex • Transfer 0.1mL from Tube B to Tube C and load in the Micro PRO™ • Plate samples post-enrichment

36. Analysis on MicroPRO 2 Select Method Load sample vials and syringes 1 Reagent additions and sample injection performed automatically as defined in the Method Results 3 4

37. Micro PRO™ Output

38. Results Micro PRO™ output shows few to no counts within the area definitions (product baseline) Pass result indicates that the sample does not contain microbial contamination

39. Results Micro PRO™ output shows many counts within the area definition (>3X product baseline) Fail result indicates that the sample contains microbial contamination Bacteria

40. Yeast Mold

41. Results Summary

42. Results Summary

43. Results Summary

44. Negative Control Positive Ps. aeruginosa

45. Negative Control Positive C. albicans

46. Negative Control Positive A. niger

47. Results • Approximately 300 samples were run parallel to standard cultural methods. Results from RBD are equivalent to the plate method.

48. The Advanced Analytical Solution Provides • One test for the detection of bacteria, yeast & mold in a variety of matrices • Rapid screening for microbial contamination with 24 hour results for a majority of the products tested • Objective Pass/Fail results requiring no additional interpretation • Pass/Fail criteria that generate no false positives or false negatives • Versatile system for quantification of microorganisms in purified water, surface swabs, and pure cultures

49. Environmental Monitoring (swabs)

50. Environmental Swab Protocol • Place a swab sample in a 5mL snap-cap tube containing 900µL filtered, sterile PB • Break the swab handle over the rim of the tube • Replace snap-cap and vortex swab and buffer for 30 seconds • Press the swab against the side of the tube to express extra liquid • Bring the volume to 3mL with filtered, sterile PB • Analyze samples and controls on the MicroPRO