The Latest RBD 3000 Applications The Latest RBD 3000 Applications Advanced Analytical User’s Meeting November 30, 2006 Chicago, IL

Overview Contamination Level Test –For materials that contain low-level bio-burden –Determines that material meets microbial specification –Utilizes the Most Probable Number (MPN) protocol –Useful for complex matrices Antimicrobial Effectiveness Test –Rapidly screens candidate preservative systems –Tracks an increase in dead cell count

Contamination Level Test – Feasible in a Variety of Complex Matrices Company A – In-process pharmaceutical product Company B – Various nutraceutical finished products –Juice, Body Butter, Fiber Company C – Liquid nutraceutical finished product Company D – Various alcoholic beverages

CLT – Protocol for <100cfu/g specification

CLT – Results Company A In-process Product: Specification <1000cfu/g Sample DilutionTheoretical ResultDetection by RBD 3000Growth on YM Petrifilm TM 1: :1, :10, Sample Dilution RBD 3000 Result (# pos./# neg.) Plate Result (# pos./# neg.) Microbial level determined by RBD 3000 (cfu/g) 1:103/0 >1 1:1003/0 >10 1:1,0003/0 >100 1:10,0001/2 <1,000 Sample Dilution RBD 3000 Result (# pos./# neg.) Plate Result (# pos./# neg.) Microbial level determined by RBD 3000 (cfu/g) 1:103/0 >1 1:1003/0 >10 1:1,0003/0 >100 1:10,0003/0 >1,000 Spiked Below Specification Spiked Above Specification Product contains >100cfu/g and <1000cfu/g; product is within specification Actual spike was 559 cfu Ps. aeruginosa/g product Product contains >1000cfu/g; product fails Actual spike was 3920 cfu Ps. aeruginosa/g product

CLT – Results Company B Product contains >100cfu/g; product fails Actual spike was 110cfu C. albicans/mL juice Sample DilutionTheoretical ResultDetection by RBD 3000Growth on YM Petrifilm TM 1: :1, :10, Taken from poster presentation at ASM 2006 A Rapid Most Probable Number Test for Yeast in Nutraceutical Products, K.A. Molitor and A.M. Steger, Advanced Analytical Technologies, Inc., Ames, IA and D. Wright and M. Roblin, Morinda, Inc., Orem, UT Sample Dilution RBD 3000 Result (# pos./# neg.) Growth on YM Petrifilm TM (# pos./# neg.) Microbial level determined by RBD 3000 (cfu/g) 1:1002/1 >10 1:1,0000/3 <100 1:10,0000/3 <1000 Sample Dilution RBD 3000 Result (# pos./# neg.) Growth on YM Petrifilm TM (# pos./# neg.) Microbial level determined by RBD 3000 (cfu/g) 1:1003/0 >10 1:1,0002/1 >100 1:10,0001/2 <1000 Body Butter & Juice Products: Specification <100cfu/g or mL Juice: Spiked Above Specification Body Butter: Spiked Below Specification Product contains >10cfu/g and <100cfu/g; product is within specification Actual spike was 35 cfu C. albicans/g body butter

Contamination Level Test – Benefits 18-24hr time-to-result Detects lower levels of potential microbial contamination than the traditional plate count method in complex/opaque matrices Results are confirmed by the “Most Probable Total Count by Multiple-Tube Method” in USP Chapter, Microbial Limits Test

Antimicrobial Effectiveness Test

Used to screen candidate preservatives for liquid products Pharmaceutical and Cosmetic Industries routinely perform –New & reformulated products Problems with the current method: –Compendial method takes 35 days –Requires significant labor & materials –Not all types &/or formulations of antimicrobials are screened due to time & labor constraints –Delays development of suitable preservative systems –Delays product time to market

Antimicrobial Effectiveness Test – Solution Use the RBD 3000 to rapidly screen candidate preservative systems Benefits: –Shorter time to result –Significant labor and materials savings –Evaluate more types/formulations of antimicrobials –Develop better preservative systems –Speed product time to market

Antimicrobial Effectiveness Test – Protocol Challenge 0.01% (w/v) Benzoic Acid with 10 5 – 10 6 cfu/mL Perform challenge studies with Ps. aeruginosa, S. aureus, and C. albicans Use RBD 3000 to determine titer of inoculum Sample at 0, 2hr, 4hr, 6hr, 8hr & 24hr post-inoculation –Dilute samples 1:10 in phosphate buffer for RBD 3000 Biomass & Dead Cell counts using the Biomass and Dead Cell Test Kits –Dilute samples in phosphate buffer + 4% Tween 20 for TSA plate count comparison

0.01% Benzoic Acid Challenged with Ps. aeruginosa – Test 1

0.01% Benzoic Acid Challenged with Ps. aeruginosa – Test 2

0.01% Benzoic Acid Challenged with S. aureus

0.01% Benzoic Acid Challenged with C. albicans

Antimicrobial Effectiveness Test – Action Items Test other preservative systems Determine utility in various liquid product formulations Demonstrate feasibility with A. niger spores

Summary Contamination Level Test –Provides a rapid & sensitive detection method for determining if materials meet particular microbial specifications Antimicrobial Effectiveness Test –Provides a rapid method for screening candidate preservative systems