1. Validating the Micro PRO™ Technology

2. Overview of Today’s Presentation Validation Resources Micro PRO™ Applications and Corresponding Validation Parameters Qualitative Validation of Personal Care Products Water Validation Ruggedness Robustness

3. Advanced Analytical Validation Resources Micro PRO™ Installation and Operation Qualification Micro PRO™ Software Test Procedures Micro PRO™ validation protocols are available for: –Personal care product testing –Purified water monitoring –Swab testing

4. Rapid Method Validation Resources Parenteral Drug Association, Technical Report #33: Evaluation, Validation, and Implementation of New Microbiological Testing Methods (PDA, Bethesda, MD, 2000). European Pharmacopoeia, “5.1.6 Alternative Methods for Control of Microbiological Quality,” PharmEuropa, Suppl. 5.5, pp 4131- 4142. United States Pharmacopoeia, Validation of Alternative Microbiological Methods (Aug. 1, 2006), p. 3807.

5. Validating the Micro PRO™ Quantitative Validation: –Estimation of the number of viable microorganisms in a sample Quantitative Applications on the Micro PRO™: –Purified Water Monitoring –Swab Testing –Pure Culture Enumeration –Challenge Tests

6. Validating the Validating the Micro PRO™ Qualitative Validation: –Presence or absence of viable microorganisms in a sample –Micro PRO™ can be used to screen samples for contamination Qualitative Applications on the Micro PRO™: –Raw Material Testing –In-Process Testing –Finished Product Testing

7. Validation Parameters by Type of Test ParameterQualitative TestsQuantitative Tests Accuracy  Precision  Specificity Detection Limit Quantification Limit  Linearity  Operational Range  Robustness Repeatability Ruggedness

8. Ruggedness Micro PRO™ Ruggedness Described as the “degree of precision of test results” from different operators, instruments and reagent lots We tested several pure cultures and performed an ANOVA analysis of the results The differences between operators, instruments and reagent lots had no significant effects on the results (counts/mL)

9. Robustness Micro PRO™ Robustness Described as the ability of the rapid method technology to remain unaffected by small but deliberate changes to the method parameters We tested several robustness parameters including: –Nucleic Acid Dye incubation times –BRAG3 incubation times –Altering the concentration of Nucleic Acid Dye –Vortex step vs. no vortex –Filtration of the reagents vs. no filtration –High and low reagent volumes –High and low samples volumes

10. Qualitative Validation for Personal Care Products We have completed several studies that: –Demonstrate performance characteristics of the Micro PRO™ –Help us advise our clients on how to validate the Micro PRO™ Based on our internal studies, personal care product validation has been divided into two phases

11. Qualitative Validation for Personal Care Products Phase 1: Determination of Pass/Fail Criteria –Data collected on 3 lots of product –Group similar baselines Advanced Analytical tested 11 products –8 Products had baselines < 200 counts/mL with an average baseline of 130 counts/mL –Toothpaste and stomach relief products required their own baselines because they are high background matrices –Detection of microorganisms in high background matrices has been proven in Phase 2

12. Qualitative Validation for Personal Care Products Phase 2: Microbial Recovery from Products –Neutralization of product –Growth of microorganisms in incubation conditions –Use of ATCC microorganisms and environmental isolates We spiked the 11 product types –Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus –Incubated the controls and spiked samples for 24 hours at 30°C

13. Personal Care Product Spiked Results All spiked sample results were at least 4x the grouped baseline counts/mL Product results with high background matrices were also at least 4x the baseline counts/mL

14. Quantitative Validation for Purified Water Establish Box Parameters Using Pure Cultures –P.aeruginosa, Serratia marcescens, S.aureus, isolates from in-house water system Establish Background Counts Using Sterile Water Determine Sampling Points Baselines Paralell Testing; Traditional Method X Micro PRO

15. Results – Defining the Analysis Box Micro PRO Intensity Plots Faucet Isolate MixR. pickettii After the box parameters were set using ATCC strains, a mix of isolates from Faucet A and Faucet B were analyzed on the MicroPRO to verify the placement of the analysis box Staph. aureusPs. aeruginosaSerratia marcescens E. coli

16. Clean Water TVO: Ps. aeruginosa Spike Box = 4 counts/0.25mL Box = 15 counts/0.25mL Box = 80 counts/0.25mL Box = 921 counts/0.25mL Box = 8,616 counts/0.25mL Filtered DI H2O (Bkgd) ~10 1 cfu/mL ~10 2 cfu/mL ~10 3 cfu/mL ~10 4 cfu/mL Sample RBD 3000 Counts/0.25mL RBD 3000 Counts/mL* Plate Counts/mL RBD 3000 log 10 Counts/mL Plate Counts log 10 cfu/mL Filtered DI H2O (Bkgd)4--0 ~10 1 Ps. aeruginosa154834 1.681.53 ~10 2 Ps. aeruginosa80334340 2.522.53 ~10 3 Ps. aeruginosa9214,0353,800 3.613.58 ~10 4 Ps. aeruginosa8,61638,15735,000 4.584.54 *RBD 3000 counts/mL are background corrected and have been adjusted for reagent additions.

17. Questions? Megan Pimsner mpimsner@aati-us.com (312) 440-3390