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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
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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
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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
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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
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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 ?
31
Say YES to RMM
Microbiology testing is no longer the rate
limiting step for product release
Thank you !!