Validating the Micro PRO™ Technology. Overview of Today’s Presentation Validation Resources Micro PRO™ Applications and Corresponding Validation Parameters.

Validating the Validating the Micro PROMicro PRO™™ Technology Technology

Overview of Today’s PresentationOverview of Today’s Presentation

• Validation Resources• Micro PRO™ Applications and Corresponding

Validation Parameters• Qualitative Validation of Personal Care

Products• Water Validation• Ruggedness• Robustness

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

Rapid Method Validation ResourcesRapid 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, <1223> Validation of Alternative Microbiological Methods (Aug. 1, 2006), p. 3807.

Validating the Validating the Micro PROMicro 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

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

Validation Parameters by Type of TestValidation Parameters by Type of Test

Parameter Qualitative Tests Quantitative Tests

Accuracy

Precision

Specificity

Detection Limit

Quantification Limit

Linearity

Operational Range

Robustness

Repeatability

Ruggedness

Micro PRO™ RuggednessRuggedness

• 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)

Micro PRO™ RobustnessRobustness

• 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

Qualitative Validation for Personal Qualitative Validation for Personal Care ProductsCare 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

Qualitative Validation for Personal Qualitative Validation for Personal Care ProductsCare 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

Qualitative Validation for Personal Qualitative Validation for Personal Care ProductsCare 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

Personal Care Product Spiked ResultsPersonal 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

Quantitative Validation for Purified WaterQuantitative 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

Results – Defining the Analysis BoxResults – Defining the Analysis BoxMicro PRO Intensity PlotsMicro 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. aeruginosa Serratia marcescensE. coli

Clean Water TVO:Clean Water TVO: Ps. aeruginosa Ps. aeruginosa SpikeSpike

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) ~101 cfu/mL ~102 cfu/mL ~103 cfu/mL ~104 cfu/mL

SampleRBD 3000

Counts/0.25mLRBD 3000

Counts/mL*Plate

Counts/mL

RBD 3000

log10 Counts/mL

Plate Counts

log10 cfu/mL

Filtered DI H2O (Bkgd) 4 -- 0 -- --

~101 Ps. aeruginosa 15 48 34 1.68 1.53

~102 Ps. aeruginosa 80 334 340 2.52 2.53

~103 Ps. aeruginosa 921 4,035 3,800 3.61 3.58

~104 Ps. aeruginosa 8,616 38,157 35,000 4.58 4.54*RBD 3000 counts/mL are background corrected and have been adjusted for reagent additions.

Questions?Questions?

Megan Pimsner

[email protected]

(312) 440-3390