Rapid Method for Enumeration of Microbial Counts - Real Time Air - - PowerPoint PPT Presentation

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Rapid Method for Enumeration of Microbial Counts - Real Time Air - - PowerPoint PPT Presentation

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Rapid Method for Enumeration of Microbial Counts - Real Time Air Monitoring Roni Cohen, PhD Head of the Microbiology and Chemistry Division Hy Laboratories, Rehovot PDA Seminar, November 28, 2016 1 RMM Rapid Microbiology Methods Rapid

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Rapid Method for Enumeration of Microbial Counts - Real Time Air Monitoring

Roni Cohen, PhD

Head of the Microbiology and Chemistry Division

Hy Laboratories, Rehovot

PDA Seminar, November 28, 2016

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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

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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

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

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3 Parameters: Can discriminate !

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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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  • 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

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Can A Product be released ?? Merck …

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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,
  • smotic, 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

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Oliver, 2010, FEMS Microbiology Reviews

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HyLabs – ISO 8 AirLock Real data

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Exposed for 5 hours

Conventional isolation MALDI-TOF-MS Identification Brevibacterium celere Staphylococcus haemolyticus Pseudomonas oryzihabitans Alternaria alternata

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HyLabs – ISO 8 AirLock Experiment

??

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 RMM - conventional microbiology methods are limited by slow microbial growth  ICH Q8 & Q9 – “Implementation “real time” quality control, leading to a reduction

  • f 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 !!