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Rapid Method for Enumeration of

Microbial Counts - Real Time Air Monitoring

Roni Cohen, PhDHead of the Microbiology and Chemistry Division

Hy Laboratories, Rehovot

PDA Seminar, November 28, 2016

1

RMM – Rapid Microbiology Methods

Rapid Sterility testing (BacT/ALERT)

MALDI-TOF MS

Real time viable air sampling

Real time viable water sampling

HB & L – laser detection (TPN, cell therapy)

Also – Flow cytometry, ATP, Molecular biology methods

2

Environmental Monitoring Techniques

Settling Plates

Contact Plates

Active Air Sampler

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Not a REAL TIME sampling

5 Days Incubation !!

Under estimation (VBNC, injured)

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The Need: Continuous microbiological control

during pharmaceutical manufacturing

BioVigilant IMD-A

BioTrak, TSI

BioLaz, PMS

Real Time Air Sampling Instruments

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Rapid Methods for Enumeration of

Microbial Counts - Real Time Air Monitoring

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ISO 21501-4 compliant particle counter 28.3 lpm

Three Portable Instruments in One

Particle Counter

Viability Detector

Particle Concen-

trator

Collection filter

HEPA filter

37mm Collection filter for speciation of analyzed particles

Viability detector- Laser Induced Fluorescence

7

ISO 21501-4 compliant particle counter 28.3 lpm

Same Functionality as an Airborne Particle Counter

Particle Counter

Viability Detector

Particle Concen-

trator

Collection filter

HEPA filter

8

Real-Time Aerosol Optical Spectroscopy

Scattered Light

Fluorescence A

Fluorescence B

9 e.g. NADH – Excitation 340 nm, Emission 450 nm)

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3 Parameters: Can discriminate !

Real-Time Aerosol Optical Spectroscopic

Viable Cell Metabolites

Microorganisms and simulants

Nonviable particles with fluorescent properties

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Particle Analysis and Speciation

Load Collection Filter

Load Agar Plate

Insert Filter Holder Lock Filter Holder

Incubate and identify

Integrated Collection Filter

Up to 9 Hrs continuous air flow

12

Application use of

Real Time Air Sampling

Why do we need it ??

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Human are the biggest source of contamination in controlled areas

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Particle Counter

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Root Cause Investigation

In Real Time

ISO-7 Room

Total particle peak @~11:00

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TCNT – Total Particle Count

VCNT – Total Viable Count

Viable peak @ ~12:30

There is a Viable & Non -viable – but

not aligned

The Settle Plate were POSITIVE

A correlation with the operator’s

activities log is needed

23Can A Product be released ?? Merck …

Why NO for Real Time Air Sampling?

Companies generally are not committed to technical changes unless forced to do so

We will see things we are never seen before and be forced to reject product

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We observe zero CFU on agar plates when in fact, viable organisms are present

Bacteria that are in a state of very low metabolic activity and do not divide, but are alive and have the

ability to become culturable once resuscitated

Most organisms found in manufacturing, in-process samples and raw materials are stressed or

injured and current media and incubation conditions are not optimal for their growth

Viable but non-culturable (VBNC)

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Bacteria can enter the VBNC state as a response to stress, due to adverse nutrient, temperature,

osmotic, oxygen and light conditions, Air Samplers

Sometimes, VBNC bacteria can remain in that state for over a year

It has been shown that numerous pathogens and non-pathogens can enter the VBNC state, and

therefore it has significant implications in pathogenesis

Oliver, 2010, FEMS Microbiology Reviews26

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HyLabs – ISO 8 AirLock Real data

Exposed for 5 hours

Conventional isolation

MALDI-TOF-MS Identification

Brevibacterium celereStaphylococcus haemolyticusPseudomonas oryzihabitansAlternaria alternata

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HyLabs – ISO 8 AirLock Experiment

??

RMM - conventional microbiology methods are limited by slow microbial growth

ICH Q8 & Q9 – “Implementation “real time” quality control, leading to a reduction

of end-product release testing

FDA Pharmaceutical cGMPs for the 21st Century – “Manufacturers are

encouraged to use the latest scientific advanced in manufacturing and

technology. Move towards a model of continuous improvement and real time

quality assurance

Why YES for Real Time Environmental Monitoring Air Sampling ?

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Trending of microbial content in the manufacturing environment

Instantaneous notification of microbial contamination events

Enabling segregation of potentially exposed product

Rapid initiation of root cause investigations

Operator training & certification, air exchange optimization, risk analysis

Potential to identify VBNC

Potential for real-time room or product release

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Why YES for Real Time Environmental Monitoring Air Sampling ?

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Say YES to RMM

Microbiology testing is no longer the rate

limiting step for product release

Thank you !!