pet: purpose, limitations, and next generation...

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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