Potential Application Of A Real Time Microbial Detector IMD In Aseptic Process

Joe Engle European Business Manager BioVigilant Systems, Inc.

Copyright 2010 BioVigilant Systems, Inc.

Goals of Presentation
Optical instrument (IMD) for instantaneous detection of microbes in the environment Intrinsic fluorescence from certain metabolites differentiates microbes from inert particles Process analytical tool to monitor and control an aseptic environment

Copyright 2010 BioVigilant Systems, Inc.

RMM Defined
Rapid Microbiological Method (RMMs) instrument/procedure designed to obtain test results faster than the traditional culture-based approach
FDA encourages RMMs through the Process Analytic Testing and Quality by Design (QbD) initiatives for continuous, online monitoring RMMs have the potential to enable a paradigm shift from batch product release to parametric release

Copyright 2010 BioVigilant Systems, Inc.

RMM Taxonomy: Detection

Microbial Detection
Growth-dependent Spectroscopy Growth-independent

Spectroscopy

Chemical Byproduct

Intrinsic Fluorescence

Extrinsic Fluorescence

Extrinsic Fluorescence

Intrinsic Fluorescence

Intrinsic Raman

Nucleic Acid (Real-time PCR)

IMD (BioVigilant)

Bactometer
Impedance

Growth Direct (Rapid Micro Biosystems)

(BioMerieux) BacT Alert 3D

CO2

Pallchek (Pall)
Advance (Celsis)

MicroPRO (Advanced Analytical)

MicroSEQ (AB) ScanRDI (Chemunex) REBS (Battelle) MicroCompass (Lonza) Pyrosense

Endotoxin

(BioMerieux)

Milliflex Rapid (Millipore)

(Lonza) Copyright 2010 BioVigilant Systems, Inc.

Pharmaceutical Microbial Testing Parameters

Test Requirements:
Sterility testing Batch release testing

Test Locations:
General Environment
Fill line Sterility isolator Raw materials Bioreactors

Media fills
Environmental Monitoring Investigational testing Process refinement/continuous improvement

Test Substrates:
Test Methods:
At a fixed location Mobile test Air Liquid Surface

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Manual vs. Instantaneous

Colony Counting
Requires incubated sample Examination by eye

IMD-A Technology

Laser shines on microbes Detects fluorescence

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Viable Monitoring
Limitations of conventional air sampling
a periodic test operator can cause biological contamination labor intensive retrospective sample collection efficiency (impaction or impingement), selectivity of media VBNC discrepancies

Benefit: culturing gives positive identification

Copyright 2010 BioVigilant Systems, Inc.

IMD Monitoring
IMD techniques offer incremental benefits, :
Ability to detect VBNC microbes Ability to give immediate results

Ability to synthesize multiple environmental metrics to give clear feedback on an environment/process

but are not a total standalone solution

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Need for IMD Technology

Existence of viable but not culturable (VBNC) bacteria
Over 400 scientific articles covering at least 60 bacterial species have documented this occurrence

Ratio of VBNC:Culturable bacteria can be between 100:1 to 1000:1

See Mann, Nagarkar, Heidelberg, Radosevich

VBNC can be detected employing markers

IMD-A technology chiefly uses 3 biological markers: NADH, Riboflavin, and Dipicolinic Acid (DPA)

Copyright 2010 BioVigilant Systems, Inc.

Optical Method for Particle Sizing

Mie Scattering describes the size-dependent relationship of elastic light scatter from particles whose size wavelength of light (0.405 m)
Intensity of scattered light on detector indicates particle size Well-suited for particles sizes from 0.5 to 50 m Measures the optical diameter seen by the laser

Technique used by most particle counters

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Optical Method for Fluorescence Detection

Intrinsic fluorescence occurs in biological particles
Certain organic compounds absorb light and emit photons at a higher wavelength

Not the same as phosphorescence or chemoluminescence (e.g. ATP Bioluminescence)

Not the same as dye-labeled photofluorescence (e.g. ScanRDI)

NADH, Riboflavin, and DPA primary markers

Bacterial auto-fluorescence has been observed and documented by multiple researchers

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IMD Core Technology

LASER (405 nm) AIR PATH

AIR INLET

FAN ASSEMBLY

INTERROGATION ZONE FLUORESCENCE DETECTOR LASER PATH

PARTICLE SIZE DETECTOR

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BioVigilant IMD-A Products

IMD-A 200-1 IMD-A 220-4

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IMD Validation Approach

BioVigilant
USP <1223> tests (standard for Validation of Alternative Microbiological Methods)

Customers
Follow PAT guidance IQ/OQ/PQ Side by side testing with IMD-A and conventional methods (i.e., currently used air samplers)

Accuracy
Precision Linearity Limit of Detection Limit of Quantification Ruggedness Robustness Range Specificity

DMF submission to FDA

Agreement with FDA

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Aerosol Lab Test Parameters

Comparison data for USP<1223> between IMD instruments and traditional air samplers
5 bacteria x 5 concentrations x 10 replicates = 250 tests

Bacterial titers disseminated in 2700-liter aerosol chamber

IMD 200-1 was compared to the Andersen 6-stage

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Aerosol Lab Test Chamber

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Met/exceeded acceptance criteria

Summary Test Matrix for USP <1223> Test Fuctions Performed at AlburtyLab IMD-A-200-1 vs. Andersen 6-Stage Viable Air Sampler
T1 13% T2 Concentrations T3 20% T4 17% T5 17%

Legend:
Did not meet acceptance criteria Test Fuctions Accuracy Bacterial Species Wet Bacillus atrophaeus Dry Bacillus atrophaeus Escherichia coli Micrococcus lylae Staphylococcus epidermidis Corynebacterium afermentans Wet Bacillus atrophaeus Dry Bacillus atrophaeus Escherichia coli Micrococcus lylae Staphylococcus epidermidis Corynebacterium afermentans Wet Bacillus atrophaeus Dry Bacillus atrophaeus Escherichia coli Micrococcus lylae Staphylococcus epidermidis Corynebacterium afermentans IMD: Wet Bacillus atrophaeus And: Dry Bacillus atrophaeus Escherichia coli Micrococcus lylae Staphylococcus epidermidis Corynebacterium afermentans IMD: Wet Bacillus atrophaeus And: Dry Bacillus atrophaeus Escherichia coli Micrococcus lylae Staphylococcus epidermidis Corynebacterium afermentans Wet Bacillus atrophaeus Dry Bacillus atrophaeus Escherichia coli Micrococcus lylae Staphylococcus epidermidis Corynebacterium afermentans Range IMD: 5.217-11.304 Wet Bacillus atrophaeus And: 20.563-107.730 Dry Bacillus atrophaeus Escherichia coli Micrococcus lylae Staphylococcus epidermidis Corynebacterium afermentans
2

IMD-A-200-1 Passing Rate All # of tests # of passing 138 120 87% Exclude wet Bg 115 114 99%

pass z-test

Precision

RSD 37%

RSD 38%

% passed

Specificity 1

42%

Reduced sampling time (Andersen)

Limit of Detection (Lowest concentration only)

6.140-8.643 39.245-76.010

Lowest concentration only

Justifications: Most failures involve Wet Bacillus atrophaeus

Limit of Quantification (Lowest concentration only)

73.913 576.276

Lowest concentration only

One instance involves <60 sec sampling time for Andersen 6-stage

Linearity

r = 1.00 r2 = 0.86 r2 = 0.87 r2 = 0.94 r = 0.99 r2 = 0.69

IMD: 20.000-73.043 IMD: 73.043-231.304 IMD: 220.870-342.609 And: 198.233-280.989 And: 521.972-892.000 And:1228.410-2345.338
2

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USP <1223> Test Results IMD-A-200-1

Escherichia coli
1.0E+05 1.0E+04

APS

IMD-A-200-1

Andersen 6-Stage

Counts or CFU per Liter

1.0E+03 1.0E+02 1.0E+01 1.0E+00 1.0E-01 0 Titer 1 10 Titer 2 20 Titer 3 30 Titer 4 40 Titer 5 50

Runs

Both IMD-A-200-1 and Andersen trend well with APS particle counter Variability seen in all three instruments within concentration runs In general, 200-1 counts higher than Andersen over all runs

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FDA View on RMM Acceptance Levels

Dr. Brenda Uratani, FDA
2007 PDA Annual Global Conference on Pharmaceutical Microbiology
The FDA expects that higher counts will be recovered when using new RMM technologies, especially if the methods are more sensitive than conventional methods If you use a RMM for air monitoring, the 1 CFU/m3 specification may be changed, because this was based on the less sensitive agar-based method The change must be supported by good science, the data and a GMP evaluation of the new method and the results (i.e., higher counts)

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Risk Analysis Approach to Sampling Location

Risk Priority Number (RPN)
Stopper hopper Vial exit Filling zone
RPN= Severity X Probability X Effect Where: Severity severity of a contamination Probability probability of its occurrence Effectits effect to the product

Example
Description of the location Personnel passing Criteria High Average Low Close Median Far High Average Low Short Median Long Rating point 3 2 1 3 2 1 3 2 1 3 2 1

Proximity to exterior

Frequency of previous excursions Distance to product (drug)

Vial entry port

Examining Risk Priority Numbers in FMEA http://www.reliasoft.com/newsletter/2q2003/rpns.htm

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Environmental Sampling Suggestions

Methods of routine survey of a facility
(1) Single point monitoring: at the HVAC ventilation outlet port (ceiling or floor) (2) Selected crucial points in a room: door, HVAC inlets/outlets, drain, workstations (use risk analysis approach to rate and determine sampling locations)

(3) Grid-pattern survey of a room (see graphs below): divide room into grid, use IMD to test each point; then generate a two-dimensional bio-burden map (lower left)

14 12 10 8 6 4 2 0 1 2 3 4 5 S1 6 7 S3

Door

S4
workstation

S3 S2

1 2 3 4 5 6

S1

Company X Fill Room IMD Biologic Counts 09/09/2008: two workstations at (2, S1) and (6, S1); entrance door at (7, S4)

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Potential IMD Applications

IMD-A 220-4

Applications
Clean Room/Critical Area monitoring RABS monitoring (Restricted Access Barrier System) Aseptic isolator monitoring Bacterial challenge chambers (ingress testing vs. experimental testing) Investigation of excursions

Training for clean room operators

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Summary
Quality by design is a goal for pharmaceutical manufacturing
QbD requires control of the production process

Environmental monitoring is a necessary requirement

IMD is an RMM for real time environmental monitoring Proven theory of operation of the technology IMD requires evaluation and baseline setting in customer application for regulatory approval Adoption of IMD is underway

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Contact Information
Thank you for your attention
BIOVIGILANT : PMT FRANCE :
jengle@biovigilant.com jean-sebastien.bonin@pmtfrance.fr

http://www.biovigilant.com/

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