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

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 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 3

 Not a REAL TIME sampling  5 Days Incubation !!  Under estimation (VBNC, injured) The Need: Continuous microbiological control during pharmaceutical manufacturing 4

Real Time Air Sampling Instruments BioLaz, PMS BioTrak, TSI BioVigilant IMD-A 5

Rapid Methods for Enumeration of Microbial Counts - Real Time Air Monitoring 6

Three Portable Instruments in One Particle ISO 21501-4 compliant particle counter 28.3 lpm Counter Particle Concen- trator Viability detector- Laser Induced Fluorescence HEPA filter Viability Detector Collection 37mm Collection filter for speciation of analyzed filter particles 7

Same Functionality as an Airborne Particle Counter Particle Counter ISO 21501-4 compliant particle counter 28.3 lpm Particle Concen- trator HEPA filter Viability Detector Collection filter 8

Real-Time Aerosol Optical Spectroscopy Scattered Light Fluorescence B Fluorescence A 9 e.g. NADH – Excitation 340 nm, Emission 450 nm)

3 Parameters: Can discriminate ! 10

Real-Time Aerosol Optical Spectroscopic Viable Cell Metabolites Microorganisms and simulants Nonviable particles with fluorescent properties 11

Particle Analysis and Speciation Integrated Collection Filter Lock Filter Holder Load Collection Filter Insert Filter Holder Up to 9 Hrs continuous air flow Load Agar Plate Incubate and identify 12

Application use of Real Time Air Sampling Why do we need it ?? 13

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Human are the biggest source of contamination in controlled areas 16

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Particle Counter 19

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Root Cause Investigation In Real Time  ISO-7 Room  Total particle peak @~11:00  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 TCNT – Total Particle Count VCNT – Total Viable Count 22

Can A Product be released ?? Merck … 23

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 24

Viable but non-culturable (VBNC)  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  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 25

26 Oliver, 2010, FEMS Microbiology Reviews

HyLabs – ISO 8 AirLock Real data 27

HyLabs – ISO 8 AirLock Experiment Exposed for 5 hours Brevibacterium celere Conventional isolation Staphylococcus haemolyticus MALDI-TOF-MS Identification Pseudomonas oryzihabitans Alternaria alternata ?? 28

Why YES for Real Time Environmental Monitoring Air Sampling ?  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 29

Why YES for Real Time Environmental Monitoring Air Sampling ?  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 30

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