pet: purpose, limitations, and next generation...
TRANSCRIPT
PET: purpose, limitations, and next generation testing
Lori Daane, Ph.D.
October 23, 2017
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Why talk about PET? Challenges of Cosmetic Microbiology Today
Constant pressure on conventional preservative systems
Consumer & marketing pressure to move toward natural preservatives or preservative-free
Increased pressure to get products to market faster
More reliance on third party manufacturers for development & manufacturing
Fewer experienced microbiologists
Pressure to do more with less: need for improved laboratory efficiency
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Agenda
Purpose of Preservation
Methods of Preservation
Current Guidelines
Limitations of PET
Next Generation PET
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Manufacturers Responsibility
Ensure that the product, as purchased, is free from the number and types of microorganisms that could affect product quality and consumer health
Ensure that microorganisms introduced during normal product use will not adversely affect the quality and safety of the product
Source: Steve Schnittger
Estee Lauder Company
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R&D
Adherence to Two Programs
Proper Preservation and Protection of the Marketed Product
Manufacturing Adherence to GMP’s and Quality – including an effective “Environmental Monitoring Program”
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Preservation and Product Protection
This is what it would look like without proper preservation!
Source: Steve Schnittger
Estee Lauder Company
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Methods of Preservation
Solely Chemical
Multifunctional ingredients
Physiochemical (ISO 29621)
Packaging
Over-preserve
Under-preserve
Fail Safety test
Fail Challenge test
Must find balance of the minimum effective
preservative system concentration
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What is Preservative Efficacy Testing?
An in-vitro challenge test designed to determine the ability of the preservative system to prevent microbial growth through the shelf-life of the product
It is not meant to be a simulation of a real world situation nor is it meant as a guarantor that a preservative system will never allow contamination to grow in a product
It is not predictive of consumer contamination potential
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ISO 11930
USP <51>
EP 5.1.3
PCPC (formerly CTFA)
ACI (formerly SDA)
Current PET Guidelines
Most companies follow a
hybrid approach with
modifications and use
« worst case scenario » for
global harmonization.
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Neutralization Efficacy
Ability of the neutralizing medium to neutralize antimicrobial activity of the tested formulation without inhibiting the test microorganisms
Preservation Efficacy
The evaluation of the preservation of a cosmetic formulation
Inoculation with calibrated inocula of relevant strains
Measuring the number of surviving microorganisms at defined intervals during a period of 28 days
Components
Media prep
Culture prep
Dilution
Plating
Incubation
Counting
Calculating/Data recording
Arduous planning!
Components of PET
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SCREENING
~70% PET
Limited time points
Less standardized
Performed on:
Experimental Formulas
Not intended to give definitive information
STANDARD
~30% PET
Full 28 days
More standardized
Performed on:
Experimental Formulas
Final Formulas
Scale-Up Batches
Production Batches
Production Filled Pieces
Two Main Types of PET
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Preservation Efficacy
Dispense 20 g or 20 mL test formulation into sterile container
Add 0.2 mL of each organism for final [C] of 105 - 106
Mix thoroughly and incubate @ 22.5 +/- 2.5oC
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Preservation Efficacy
Add 1 mL or g of sample to 9 mL of neutralizing medium
Incubate @ RT 14-45 mins
9mL
Neutralizing
Medium
T = X
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Preservation Efficacy
9 mL Diluent
(Tryptone Salt)
Perform ten-fold serial dilutions beginning with 1 mL sample from neutralizing medium into 9 mL diluent
10-1
10-2
10-3
10-5
10-4
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Preservation Efficacy
Plate 1 mL each dilution in duplicate using appropriate medium and incubate
48-72 h B&Y
3-5 days Mold
Pour plate preferred
Spread and membrane filtration also acceptable
SDA PDA TSA TSA TSA
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Preservation Efficacy
Repeat at each timepoint (Days 0, 7, 14, 28)
Many companies also perform at Day 2
50 plates, 100 tubes per standard PET
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Count colonies and calculate log reduction
Preservation Efficacy Results (Bacteria)
USP
PCPC
In-house -1.0
-3.0
-4.0
-2.0
0
Log reduction
Source: Steven Schnittger,
Estee Lauder Companies
0 24h 7d 14d 21d 28d
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Preservation Efficacy Results (Bacteria)
USP
PCPC
In-house -1.0
-3.0
-4.0
-2.0
0
Log reduction
0 24h 7d 14d 21d 28d
USP (category 2): > 2 log reduction from initial count at 14 days with no increase from 14 day count at 28 days
PCPC > 3 log (99.9%) reduction within 7 days following each challenge and no increase for duration of test
In-house > 3 log reduction within 2 days with no increase for duration of test
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2 pools, 8 weeks, neat & 5-fold dilutions, <20 CFU/g at day 1 and 7
3 pools, 2 weeks, 100% kill by day 7
4 pools, 13 weeks, rechallenge at week 6, <0.2% survival
Single inoculations, 12 weeks, rechallenge at week 3, <10 CFU/g at weeks 3 and 12
4 pools, 4 challenges over 4 weeks, 99.99% reduction after each challenge
PET Survey of In-House Methods
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Type of product
Area where product is to be applied
Type of component
Rinse off vs. Leave on
History of Product
Contains Micro Susceptible Raw Materials
Acceptance Criteria (Risk Assessment)
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Microorganisms
ATTC cultures >70 years from original isolation
Pure cultures
Wimpy & not relevant to product & facility
“Fighting the last war”
Changing products, packaging, raw materials, geography, usage
Emerging pathogens (microbial recovery/discovery)
Preservation of products to meet the criteria you are testing against
PET Limitations
Sources: Phil Geis, Geis Microbiological Quality
Steve Schnittger, Estee Lauder Companies
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PET Limitations: Rebound Effect
USP
PCPC
In-house -1.0
-3.0
-4.0
-2.0
0
Log reduction
0 24h 7d 14d 21d 28d
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Traditional PET Summary
Tedious work with lots of steps
Arduous planning
Performed by trained microbiologists
A LOT of plates, tubes, counting
Often a bottleneck
Marketing wants results yesterday
Must often outsource to make deadlines
Necessary for innovation
Critical for bringing new products to market
Driven by customer demand
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How to Simplify PET?
Enumerations
Colony counters
Pour plating
Spiral plating
Homo- genizers
Gravimetric Dilutors
Ready-to-use strains
Direct enumeration Automation TEMPO® CT
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TEMPO® CT Workload Reduction
Media Preparation
Microbial Preparation
Serial Dilutions
Plate Inoculations
Pour Plating
Incubation
Plate Counting
Analysis
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&&
Two Step Automated Process
Filling and sealing enumeration cards
1
Automatic reading
2
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Safe Data Management
Automated traceability (Bar code for reagents) Software 21 CFR PART 11 LIMS connectivity
DATA INTEGRITY
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Standardized Method
Optimized and Miniaturized MPN Method
2.25 µl
22.5 µl
225 µl
Direct enumeration
dilution 1
1/10 dilution 1/100 dilution
1 CARD = 6 Tubes + 6 Plates
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Reduces Waste and Improves Productivity
CT analysis capacity from 1 to >500 enumerations / day 1 card = 1 enumeration
20 enumerations in less than 5 minutes
Automated results in CFU/g or ml of product
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Quicker decision on a formulation Test more formulations at the same time Incubation Time reduction (gain of 1 to 3 days)
D+1 for Bacteria vs 2-3 days Trad. method D+2 results for YM vs 3-5 days Trad.methods
Development Cycle Time Reduction
Trad Method 3-5 days
Trad Method 2-3 days Bacteria 1 day
Yeasts/Molds 2 days
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Tempo® CT Implementation Process
IQ
• Installation Qualification • Validation of the installation of the system (systems components,
electrical requirements, computer qualification, environmental conditions)
OQ
• Operational Qualification • Verification that all the instrument functions operate as expected
(calibration) and verification of training records
PQ
• Performance Qualification • Verification of the equipment performances for the intended use
• PQ1 : quantification performances of the method on strains
• PQ2 : equivalency with the reference method on representative products
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Validation Strategy
USP <1223> VALIDATION OF ALTERNATIVE MICROBIOLOGICAL METHODS
Quantitative method performances (PQ1)
An in-depth validation study providing performances
of the alternative method to the users
On pure strains
ISO 11930 EVALUATION OF THE ANTIMICROBIAL
PROTECTION OF A COSMETIC PRODUCT
Equivalency of challenge tests results towards the reference
method (PQ2)
On cosmetic products
Accuracy Linearity
Precision Operational range
Specificity Repeatability
Detection limit Robustness
Quantification limit Ruggedness
Study of results equivalency between traditional and Tempo method on over 125 cosmetic product formulations.
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Tested on Wide-Range Of Products
Creams
Lotions
Serums
Masks
Sunscreen Lotions/Sprays
Whitening Lotions w/SPF
Facial Scrubs
Cleansing Foam
Liquid Soaps
Mascara
Eyeliner
Liquid & Pressed Powder Foundations
Make-up Remover/Cleanser
Toothpaste
Shower Gels
Shampoo
Conditioner
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Validated on Wide-Range of Microorganisms
Microorganisms Reference
Staphylococcus aureus ATCC 6538 / NCTC 10788
Pseudomonas aeruginosa ATCC 9027 / ATCC 27853 / NCTC 12924
Escherichia coli ATCC 8739 / ATCC 25922 / NCTC 12923
Candida albicans ATCC 10231 et NCPF 3179
Aspergillus brasiliensis NCPF 2275
Other tested Microorganisms Reference
Burkholderia cepacia (plant isolate)
Bacillus subtilis ATCC 6633
Candida parapsilosis (plant isolate)
Enterococcus faecalis ATCC 33186 / ATCC 29212 / CIP 103214
Enterobacter gergoviae CIP 761T
Klebsiella pneumoniae ATCC 13883
Lactobacillus lactis ATCC 19435
Pseudomonas putida (plant isolate)
Serratia marcescens (plant isolate)
Staphylococcus epidermidis ATCC12228 / CIP6821
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Traceability/Standardization
TEMPO CT Improves Efficiency Next Generation PET
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More Tests; Less Waste 1
Equivalent to Traditional Method
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