the validation of rapid microbiological methods: a case study...

10/31/2014

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Michael J. Miller, Ph.D. President

Automated and Rapid Microbiological

Methods: Selection and Validation

Rapid Microbiological Methods. Copyright © Michael J. Miller, Ph.D. 2014.

Microbiology: Past

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• 1683. Anton van Leeuwenhoek observes bacteria

• 1861. Pasteur disproves spontaneous generation

• 1876. Koch defines pure culture and colony

• 1881. Fanny Angelina Hesse introduces agar-agar

• 1884. Hans Christian Joachim Gram develops the Gram stain

• 1887. Julius Petri invents glass plates for bacterial growth


• Still using 19th-Century methods…counting colonies

on agar plates and Gram staining

Microbiology: Today

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• Classical methods are limited by slow growth rates

• Variability of microbes in their response to culturing

• Most microorganisms in the manufacturing environment,

in-process samples and raw materials are starved,

stressed or injured, and current media and incubation

conditions are not optimal for the resuscitation and

growth of these microorganisms

• Many times we will observe zero colony forming units

(CFU) on agar plates when in fact, viable

microorganisms are present

Microbiology: Today

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• Significant opportunities exist for improving the

efficiency of manufacturing and quality

assurance through the application of modern

process analytical tools

• There are regulatory initiatives that are

recommending changes in the way we approach

microbiology testing

• These include Rapid Microbiological Methods

(RMMs) and automated technologies

Opportunities

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• Novel technologies that provide microbial detection,

quantification and identification results much faster

than conventional methods

• Increased accuracy, reproducibility and sensitivity

• Automated, miniaturized and high-throughput

processing

• Improved sampling, data handling and trend

analysis

• For some technologies, results in real-time

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Rapid Microbiological Methods

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• Many RMMs do not require microbial growth

• Enhanced detection of single cells, stressed or

injured microorganisms

Healthy or stressed viable but non-culturable (VBNC) cells

Cells induced into dormancy at the beginning of the

stationary phase following environmental stress

• Improved microbial identification and strain

differentiation

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• Applications

Bioburden (raw materials, in-process, finished

product)

Sterility testing

Environmental monitoring

Process water testing

Microbial identification

Mycoplasma

Microbial Limits Testing

• Enumeration and presence/absence


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• Overview of currently available RMMs that can be

used for Microbial Limits Testing

Enumeration

Presence/absence testing

Technology

Workflow

Case studies

• Validation strategies

• PDA Technical Report #33

Agenda

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• It is important to understand what technology platforms

are available, in order to appropriately match the RMM

with its intended application

• Consider the technical or method requirements

Do you need to detect, enumerate and/or identify microorganisms?

Is the RMM compatible with your samples or product?

Do you need to detect different type of microorganisms?

What is the required level of sensitivity or limit of

detection/quantification?

What sample sizes are required?

Data management requirements?

Operator qualification requirements?

RMM Technologies

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• RMMs can provide qualitative, quantitative and/or

microbial identification data

• Qualitative Information on the presence or absence of all microorganisms or

the presence of specific microbial species

• Quantification The number of microorganisms present in a sample

• Microbial identification The identity of at the Genus, species and/or strain level

We will not discuss ID systems today

RMM Technologies

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• Based on a wide variety of detection principles

The use of viability stains and laser excitation for the

detection and enumeration of microorganisms without

requiring cell growth

The detection of cellular components or markers (e.g., ATP

and endotoxin)

Optical spectroscopy, such as light scattering

The amplification of nucleic acids and detection of specific

genetic sequences (e.g., PCR)

The use of fluorescence techniques to rapidly detect the

growth of microorganisms on conventional media

Micro-Electro-Mechanical Systems (MEMS), such as

microarrays, biosensors, Lab-On-A-Chip and nanotechnology

RMM Technologies

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• No endorsements during this presentation

• Order of RMMs discussed is random

• More than 60 different RMMs have been

implemented or reviewed by various industries; we

will review some of them

• For an in-depth review of RMM technologies,

workflow, and other relevant information, see the

RMM Product Matrix at rapidmicromethods.com

Disclaimer

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Enumeration

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• Digital imaging technology that

enumerates micro-colonies in one-

half the time to visualize colonies

• The sample is filtered and the filter

is placed onto a flat agar medium

cassette with an optically clear lid

• A light emitting diode (LED)

excites micro-colonies to

autofluoresce, which are

enumerated by a CCD imaging

system

Rapid Micro Biosystems

Growth Direct

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• Cells fluoresce in the yellow-green spectral region when

illuminated with blue light due to oxidized flavins

Photosensitive pixels in the CCD camera chip detect auto-

fluorescing micro-colonies


Growth Direct

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• The system automatically incubates and analyzes

each cassette over time

Particles that do not grow in size over time are ignored

• Non-destructive – can continue to incubate media to

obtain colonies for microbial identification

• Considered an automated version of the existing

compendial method

• Bioburden and environmental monitoring

• One or two temperatures

• Capacity: up to 350 plates


Growth Direct

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Growth Direct

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0 hr 6 hr 7 hr

8 hr 9 hr 10 hr

11 hr 12 hr 13 hr

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• Fluorescent staining and laser excitation of micro-

colonies on a membrane

• Applicable for all filterable samples, including water, in-

process and finished product

• Non-destructive – can continue to incubate media to

obtain colonies for microbial identification

EMD Millipore Milliflex Quantum

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• Filter the sample, place the membrane onto an agar

cassette and remove the funnel

• Incubate for an appropriate time period


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• Saturate the staining cassette with a non-fluorescent

substrate, remove the agar cassette from the incubator,

place the membrane onto the staining cassette and

incubate for 30 minutes at 32.5°C


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• Microorganisms retained on the membrane will take

up the non-fluorescent substrate

• Within viable and culturable cells, the non-

fluorescent substrate is enzymatically cleaved

• The cleaved substrate liberates free fluorochrome

into the microorganism cytoplasm

• As fluorochrome accumulates inside the cells, the

signal is naturally amplified


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• Following incubation, the membrane is placed into the reader

and exposed to the excitation wavelength of the dye

• Fluorescent micro-colonies can then be counted in the

instrument window or on a computer via a camera


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• Following staining and counting of micro-colonies,

the membrane can be placed onto the agar cassette

and re-incubated to allow larger colonies to form

which can then be used for microbial identification

(non-destructive method)


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• Bioluminescence is the generation of light

by a biological process

• 1947: William McElroy discovered the

mechanism by which bioluminescence

occurs

• Observed in the tails of the American

firefly Photinus pyralis

• Specific enzyme reaction catalyzing the

consumption of ATP (Adenosine

Triphosphate)

ATP Bioluminescence

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• In the presence of the substrate luciferin, the

enzyme luciferase will use the energy from ATP to

oxidize luciferin and produce photons (hv; light at

a wavelength of 562nm)

ATP Bioluminescence

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Luciferin + ATP + O2 AMP + PPi + CO2 + Oxyluciferin + Luciferase

Mg++


• Because all living cells store energy in the form of

ATP, it can be used as a measure of organism

viability

• Capture microorganisms, release ATP from within

the cells, and measure the amount of

bioluminescence generated

• Instruments utilize a luminometer equipped with a

photomultiplier tube to detect the photons

ATP Bioluminescence

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• The concentration of ATP required for measurement

is about 200 attomoles, which is equivalent to one

yeast or mold cell or approximately 100 bacterial

cells, depending on their metabolic state.

May require up to 1000 bacterial cells

• When low numbers of cells are expected, an

enrichment step in media is required to allow the

cells to multiply and produce a sufficient level ATP

for detection

ATP Bioluminescence

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• Handheld system that

measures ATP on directly on

surfaces, on a membrane filter

or liquid samples

• If sufficient cells (and ATP) are

present, ATP measurements

are obtained within minutes

• When low counts are expected,

incubate sample/membrane in

liquid media (18-24 hrs). The

media is then filtered

Pall Pallchek

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• Add luciferin and luciferase

reagents to the membrane or

surface

• Place instrument over sample

• Results are provided as relative

light units (RLU), which can be

correlated with an estimation of

cell count

• Appropriate for

presence/absence testing and an

estimation of cell counts

Pall Pallchek

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• GSK received FDA approval to use the Pallchek system

for the early release of a non-sterile prescription nasal

spray product (up to four days earlier than conventional

methods)

• They were the first pharmaceutical company to obtain an

approval under the FDA PAT initiative

• The firm used a comparability protocol and implemented

the technology under a CBE-0

• Filtered the product, enriched overnight and tested the

filter

• They used a 2-tiered approach for product release

Case Study for Microbial Limits

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Case Study

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• Utilizes a filter membrane to capture individual cells,

allow them to grow into micro-colonies and provide an

actual cell count

• Pass sample through 0.45 micron PVDF membrane

• Can rinse filter to reduce bioluminescence inhibition or

interference

Millipore Milliflex Rapid

Microbiology Detection System

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• For bacterial detection, incubate on appropriate

medium to form micro-colonies (e.g., 18 hrs)

Growth is not required for yeast or vegetative mold

• The filter is then placed into the AutoSpray station,

where ATP releasing agent and bioluminescence

reagents are applied



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• The filter is then transferred to the Detection Tower

• The detection tower intensifies bioluminescence from

each cell (or micro-colony) thousands of times



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• Light signals are

captured with a CCD

camera; an image

processor analyzes the

signal and provides a

cell count

• Each image

theoretically arises from

a single cell

• May be non-destructive

(continue to grow into a

CFU)



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• Because viability-based RMMs do not rely on microbial

growth, microorganisms that are stressed, starved,

difficult to culture, or viable but non-culturable (VBNC)

may be detected and enumerated

• Could result in a higher count compared with

conventional methods

In these cases, a correlation between the RMM counts

and the conventional counts can be developed

The RMM counts can then be used to set new

acceptance or specification levels

Viability-based Technologies

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• Counting individual cells as they

pass through a laser beam in a very

narrow flow cell

• Microorganisms are labeled with a

viability stain and then passed

through a laser

• The laser causes the stain to

fluoresce

• Low sample volumes (1 mL or less)

• Sensitivity is 10-50 cells

• Bioburden testing of liquids and non-

filterable material

Flow Cytometry

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• Automated enumeration of bacteria, yeast, mycoplasma

and spores (bacterial and mold) as early as 4 minutes

• Accurate detection between 10 – 106 organisms per mL

• Fully automated, robotic arm processes samples

• Up to 42 samples can be analyzed automatically

BD Biosciences FACSMicroCount

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• Nucleic acid dye labels live & dead cells; BRAG3

labels live cells

• The labeled organisms pass through the flow cell

and a 635 nm red diode laser


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• The scatter and fluorescence intensity for each individual

microorganism are displayed, as well as counts per mL


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• Counting individual microorganisms that have been

captured onto a filter membrane

• Microorganisms are stained and exposed to a laser

• The laser will cause the viability stain to fluoresce

• Sample volumes are higher than those used in flow

cytometry (e.g., > 100 mL), but sample must be

filterable

• Sensitivity down to a single cell

• Appropriate for bioburden testing, environmental

monitoring and sterility testing

Solid Phase Cytometry

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• Organisms are stained with a non-fluorescent substrate

• Within the cytoplasm of metabolically active cells, the

substrate is enzymatically cleaved (by esterase) to release

a fluorochrome

• The fluorochrome will fluoresce when excited by a laser

• Cells with intact membranes will retain the fluorescent label

AES Chemunex ScanRDI

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Enzyme

Viability substrate

Free fluorochrome


• All viable bacteria, yeast and spores (bacterial and

mold) are detected within 2 hours, with single cell

sensitivity

• Accurate detection between 1-105 bacteria and 1-

104 for yeast and spores


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• Filter the sample through a 0.4 µm polyester

membrane

• Label with viability substrate, incubate

• Place membrane into laser scanning chamber


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• The membrane is scanned by an

argon laser at 488 nm

Scan lines are 2.2µm apart to ensure

overlap from previous scan

• Photo-multiplier tubes detect

emitted fluorescent light in 3 min

• Algorithms and discrimination

processes determine if the

fluorescent signals originate from

labeled viable microorganisms or

from an auto-fluorescent particle


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• Auto-fluorescent particles, membrane fluorescence

and background noise are rejected and a total viable

count is displayed


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Case Study

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Bausch & Lomb Purified Water Testing

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Presence/Absence

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• Detects target microorganisms by monitoring

changes in color or fluorescence in selective media,

and/or by monitoring the generation of CO2

BioLumix

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• Each vial contains a broth

medium and/or other reagents

specific for the target organism

with unique dyes in which target

microorganisms grow and are

detected by changes in color or

fluorescence

• These changes, expressed as

light intensity units, are detected

by an optical sensor

BioLumix

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• Disposable two-zone vials

contain an incubation zone

(top of vial) for the sample

and microorganism, and a

reading zone (bottom of

vial)

• The two-zones eliminates

masking of the optical

pathway by the product and

by microbial turbidity

BioLumix

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• One bacterial cell is usually

detected within 8-18 hours, a

single yeast cell is detected in

20-30 hours, and mold requires

35-48 hours

• The threshold for bacteria is

100,000 cells/ml and the

threshold for yeast/mold is

10,000 cells/ml

• The time to detection depends

on the initial concentration of

organisms in the product sample

BioLumix

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• Detection of specified microorganisms

• Tests include total aerobic count, yeast & mold,

coliforms, E. coli, lactic acid bacteria,

Enterobacteriaceae, Salmonella, Pseudomonas,

and Staphylococcus

BioLumix

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• The system can be used to screen for an

estimation of organisms in a test sample that are

above or below a certain quantitative specification

• Dilute the test sample to a level that represents the

specification level (e.g., 1:100 dilution for a spec of

not more than 100 cfu)

• No response means that there were less than 100

cfu in the sample

• A positive response means that there were greater

than 100 cfu in the sample

BioLumix

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PCR for Presence/Absence

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DNA is extracted and heated to separate the double strands

DNA primers (short, synthetic sequences) are added, which bind to unique target

sequences on the template DNA, if they are present

Heat-stable DNA polymerase and nucleotide bases (A,T,G,C) are added. The primer is

elongated, producing two new complete copies of the template DNA strands

Repeating the process results in millions of copies of target DNA; a probe is used to

detect the DNA sequence

Heat to

90°C

Lower

heat to

55°C

Raise

heat to

70°C


• Heat to denature DNA; anneal primer

• At the same time, a probe anneals to

another region

• The probe contains a fluorescent

reporter dye at one end and a quencher

dye at the other end. There is little

fluorescence when the probe is intact.

• As the primer extends, the probe is

cleaved, the two dyes separate and the

fluorescent signal increases

• Fluorescent signal increases with each

PCR cycle

Taqman Probe to Detect Sequence

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Taqman Probe-based Assay

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• qPCR to simultaneously detect multiple organisms in

the same sample

• Different primers and probes/dyes for each

organism/DNA sequence

• Taqman probe technology

Pall GeneDisc

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• Samples are filtered, media is added to the filter

cartridge, and the membrane is incubated for 6-16 hours

Required to eliminate false positives from residual DNA

• Organisms on the membrane are lysed (sonicated for 8

min) and heated (100° C for 19 min) to release the

DNA

Pall GeneDisc

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• The purified DNA and a Master Mix

(polymerase and deoxynucleotides)

are added to the upper hub of a

GeneDisc plate

• The plate is inserted into the

instrument, and the disk rotates

through 4 different heating and

cooling sections during the PCR

amplification and detection process

Pall GeneDisc

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• Primers will amplify specific DNA sequences, if

present

• Fluorescent signals increase during DNA

amplification

Pall GeneDisc

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• Up to 6, 9 or 12 test samples may be assayed per disc

Depends on disc; includes positive and negative controls

8 modular system units have the capability of testing up to 96

samples per run (~1 hour)

• Plate for Compendial Specified Microorganisms

Escherichia coli, Salmonella spp., Pseudomonas aeruginosa,

Staphylococcus aureus, Candida albicans, Aspergillus

brasiliensis (A. niger)

• Food Testing and Environmental Testing

STEC, non-STEC and E. coli O157, Salmonella, Listeria

Legionella, Pseudomonas, Enteroccocus, Cyanobacteria, E. coli

Pall GeneDisc

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• New technologies are being developed that

combine both enumeration and

presence/absence testing

Combination Technologies

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• Viability staining and automated image analysis using

dark field illumination

• Confocal Raman laser beam (532 nm) is then

automatically aligned with the viable particle locations

Rap.ID Bio Particle Explorer

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• Particles ≥ 500nm are examined for shape and size

• Spectral signatures from viable particles are generated

and compared with a library of known microorganisms

• Rapid enumeration and ID with single cell sensitivity


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• Particles are collected on metal foil using impaction or

filtration methods for airborne or liquid samples

• Viability staining and particle enumeration in 4 minutes

• Identification of a single viable cell is 1-5 seconds

• 150 bacteria and spore entries in database,

customizable

• 300-600 individual ID’s per hour

• >150 samples per 8 hours

• Non-destructive for further analysis


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Q&A

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Validation

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• USP <1223>, Validation of alternative microbiological

methods (UNDER REVISION)

• Ph. Eur. 5.1.6, Alternative methods for control of

microbiological quality (UNDER REVISION)

• PDA Technical Report #33, Evaluation, Validation

and Implementation of Alternative and Rapid

Microbiological Methods (REVISED 2013)

Validation Guidance

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• Proof of Concept (POC) or feasibility testing

• Assessment of supplier capabilities / supplier audit

• Review business benefits; conduct Return on

Investment analysis

Pre-Validation Activities

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• Validation of the Equipment, Software and Method Responsibilities

Risk Assessment

Validation Planning

User Requirements Specification (URS)

Design Qualification (DQ)

Functional Design Specification (FDS)

Requirements Traceability Matrix (RTM)

SOPs and Technology Training

System Integration, IT, LIMS

Installation Qualification (IQ)

Operational Qualification (OQ), computer system validation

TR33 - Validation

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• Validation of the Equipment, Software and Method Performance Qualification (PQ)

• Method validation and suitability testing

Ongoing Maintenance and Periodic Reviews

• Preventive maintenance, calibration, software updates

• Method Validation Quantitative and qualitative methods

Standardized cultures

Actual product or samples (equivalence/comparative testing)

Testing procedures, acceptance criteria and recommended

statistical analyses

TR33 - Validation

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• Method Validation Criteria

Accuracy

Precision; repeatability

Specificity, stressed organisms and mixed cultures

Limit of Detection

Limit of Quantification

Linearity

Range

Ruggedness; intermediate precision, reproducibility

Robustness

Equivalence/comparative testing

TR33 - Validation

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• Suitability Testing

False positive and false negative testing

Recommended procedures, acceptance criteria and

statistical analyses

• Validation – Additional Considerations

Automated methods; extensions of compendial tests

Unique Methods; Additional or Modified Validation

Strategies

Guidance on changing existing acceptance criteria

Technology transfer

TR33 - Validation

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• FDA, EMA, Australian TGA, Japanese PMDA, WHO

all accept RMMs and encourage their use

• Policies have been implemented that provide a

framework for validating and implementing RMMs

• RMMs have been approved for use, even for sterility

testing of finished pharmaceuticals

A Note About the Regulators

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• Rapid methods continue to gain momentum

• Regulators encourage their use

• Companies have validated and implemented RMMs

• RMMs have provided quality and efficiency benefits

• New technologies continue to be introduced and

they are getting better

• Resources are at your fingertips

The Path Forward

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• Many papers and publications

PDA Encyclopedia of Rapid Microbiological Methods

• Dedicated RMM seminars, training and conferences

• Discussion forums, e.g. “Rapid Micro Methods”

LinkedIn Group

• Websites, e.g., rapidmicromethods.com

Lists of RMM references

Technology and application matrix

RMM news blogs

Guidance on regulatory acceptance, validation and ROI

Additional Resources

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http://rapidmicromethods.com

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Michael J. Miller, Ph.D.

Web: microbiologyconsultants.com

email: [email protected]

LinkedIn:

http://www.linkedin.com/in/drmichaelmiller

phone: 72743 72743 (RAPID-RAPID)

Thank You!

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Q&A

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the validation of rapid microbiological methods: a case study...

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