What is the Future of Pharmaceutical Microbiology?

Tony Cundell, Ph. D.

Consulting Microbiologist,

Scarsdale, New York

September 2015 bioMerieux Meeting 1

Future Trends

September 2015 bioMerieux Meeting 2

• In my opinion, what are the future trends in pharmaceutical microbiology?

• Knowing these trends, how can we response to them?

Trends in Pharmaceutical Microbiology Some recent industry trends that I have noticed include:

• Replacement of Growth-based Microbiology Testing Methods with Non-growth Methods.

• More Emphasis of Quality Risk Management and less on Microbiological Testing.

• Integration of the Human Microbiome Findings into Clinical and Pharmaceutical Microbiology.

• Movement of Microbial Testing from the Laboratory to the Factory Floor or the Patient Bedside.

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Trends in Pharmaceutical Microbiology

Industry Trends (Continued):

• Further Implementation of Advanced Aseptic Processing and Robotics

• Replacement of Overkill Sterilization Cycles with Bioburden-based Cycles

• Outsourcing of Routine Microbiological Testing to Contract Testing Laboratories

• Re-evaluation of Pyrogenicity and the role of Bacterial Endotoxins Testing

• Digital Disease Detection

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Emerging Microbiological Testing Technologies

• Given the time limitations, this presentation will survey the emerging microbiological testing technologies suitable for the pharmaceutical industry.

• In terms of my background I am an internationally-recognized expert in rapid microbial methods who chaired the PDA task force responsible for the 2000 Technical Report Number 33 on the evaluation, validation and implementation of advanced microbiological methods.

• Also I am member of the 20015-2020 USP Microbiology Expert Committee.

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Areas of Discussion

• Rapid microbial methods suitable for air, surface and water monitoring, pharmaceutical ingredient, intermediate and final product testing, and microbial identification will be highlighted and discussed.

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Compendial Guidance

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• USP General Notices

• USP <1223> Validation of Alternative Microbiological Test Methods

• Ph. Eur. 5.1.6 Alternative Methods for the Control of Quality.

Other Guidance

• ICH Guidance Q2 (R1) Validation of Analytical Procedures: Text and Methodology, May 1997

• ASTM Standard E2935 Standard Practice for Conducting Equivalence Testing in Laboratory Application, 2013e

• PDA TR#33 (Revised) The Evaluation, Validation and

Implementation of New Microbiological Methods 2014

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Relocation of Microbial Testing

• The movement of microbial testing from the laboratory to the factory floor is under way.

• This trend was influenced by the adoption of online water monitoring for conductivity and Total Organic Carbon.

September 2015 bioMerieux Meeting 9

Relocation of Microbial Testing

• The advantages of factory-floor microbial monitoring of utilities, clean rooms and intermediates are that it promotes better in-process control, eliminates taking samples and conveying them to the lab for testing, employs rapid microbial methods, and reduces product failure.

September 2015 bioMerieux Meeting 10

Relocation of Microbial Testing

• Examples of factory-floor testing include LASER-induced fluorescence bioburden monitoring of air, water and in-process intermediates, hand-held cartridge-based endotoxin testing equipment, and in-process sterility testing.

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Origins of Traditional Methods

• C’est un grand progrês, Monsieur” Louis Pasteur to Robert Koch (Pictured below), on observing his demonstration of the plate technique, at the Seventh International Medical Congress, London, August 1881.

September 2015 bioMerieux Meeting 12

Replacement of Traditional Microbial Tests

• Growth-based microbiology methods, i.e., traditional plate counts, sterility testing and microbial identification methods are being replacement with non-growth methods, i.e., Scan RDI (solid phase cytometry), D-count (flow cytometry) and Vitek MS (MALDI TOF mass spectrometry) marketed by bioMerieux.

September 2015 bioMerieux Meeting 13

Water Monitoring • Pharmaceutical companies routinely monitor

incoming potable water, and their systems producing and distributing Purified Water, USP and Water For Injection, USP .

• Typically, product is not released to inventory until the water monitoring results are reviewed and found to be satisfactory.

• Although the standard plate count and membrane filtration methods are satisfactory, incubation times of up to 5 days delays the detection of adverse trends and product release.

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Candidate Methodologies

Candidate rapid water monitoring systems and their technology include:

• PallChek System (ATP bioluminescence) • Milliflex Rapid Detection System (ATP

bioluminescence) • Scan RDI Microbial Detection System (Solid Phase

LASER Scanning Cytometry)* • RBD 3000 System (Vital Staining Flow Cytometry) • Growth Direct System (Advanced Imaging of

Microcolonies) • IMD-W (LASER-induced fluorescence)

* Marketed by bioMerieux

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Microbial Limit Testing

• Many non-sterile pharmaceutical and over-the-counter drug products are subjected to Microbial Limits Testing prior to release

• The USP microbial enumeration and absence of specified micro-organism tests are protracted and delay product release.

• Since many products have little or no bioburden is it possible to screen products for release?

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Candidate Methodologies Candidate rapid microbial enumeration tests and their

technology include:

• Celsis Rapiscreen (ATP bioluminescence)

• PallChek System (ATP bioluminescence)

• Milliflex Rapid Detection System (ATP bioluminescence)

• D-Count System (Flow Cytometry)*

• Growth Direct System (Advanced Imaging)

• Scan RDI (Solid Phase Cytometry)*

• PTS-Micro (Solid Phase Cytometry)

• Sieve-ID MuScan (Solid Phase Cytometry)

* Marketed by bioMerieux

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Microbial Limit Testing

Candidate rapid absence of specified microorganism tests and their technology include:

• Vitek MS (MALDI – TOF Mass Spectrometry)*

• BAX Microbial Detection System (PCR DNA amplification based on primers and probes for each specified microorganism)

• Roche LightCycler PCR Detection System (PCR amplification based on primers and fluorescence hybridization probes)

* Marketed by bioMerieux

September 2015 bioMerieux Meeting 18

Microbial Identification

• With the introduction of MALDI TOF mass spectrometric microbial identification there is an opportunity to establish a new paradigm for routine microbial identification.

• With the materials and labor cost for identification being less than U.S $1.00, all bacterial colonies can be identified directly from the primary isolation plates used for facility, water and product testing to continuously evaluate the microbiota associated with your manufacturing plant.

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Cost Comparison of Different Bacterial Identification Systems

Identification

Instrumentation

Capital Cost Estimated Cost per

Test

Turnaround Time

Gram Stain Microscope $0.8 10 minutes

API Strips Incubator $5 Overnight

Omnilog $80-100K $10-20 4-6 hours

VITEK Compact 2 $80-100K $8-12 5-6 hours

Vitek MS (MALDI

TOF Mass

Spectrometer)

$180K $1-2 10-20 minutes

ABI MicroSeq

(rRNA base

sequencing)

$150K $80-120 (U.S.

contract lab)

3-6 hours

MALDI TOF Mass

Spectrometer plus

ABI MicroSeq

$330K $35 (U.S. contract

lab)

5 days

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Vitek MS Work Flow

Profiling results from different Bacillus strains

-6000

-4000

-2000

0

2000

4000

6000

a.i.

B. globigii

B. licheniformis

B. subtilis

B. thuringiensis

?

Unknown microrganism

Select a colony

Transfer a thin-layer onto a MALDI target plate

Generate MALDI-TOF profile spectrum

MALDI-TOF Pattern Matching

Identified species Bacillus subtilis

Microbial Limit Testing

Candidate rapid absence of specified microorganism tests (Continued):

• DiversiLab (DNA barcoding and analysis for strain typing)*

• RiboPrinter Microbial Characterization System (Based on rRNA gene restriction pattern determinations for strain typing)

• Gen-Trak Bacterial Detection System (DNA Hybridization).

• Bactometer (Impedence to detect growth in selective media)

* Marketed by bioMerieux

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Sterility Testing

Candidate rapid sterility tests and their technology include:

• Scan RDI Microbial Detection System (Solid Phase LASER Scanning Cytometry)*

• BacT/ALERT Microbial Detection System (CO2 sensor)*

• BACTEC System (pH sensor)

• Milliflex Rapid (ATP Bioluminescence)

• easyMAG* & ABI 7500 System (RT-PCR) * Marketed by bioMerieux

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Other bioMerieux Innovations

• Previ (Automated Gram staining system)

• AirIdeal 3P Traceability (Active air samplers, with an integrated network-based system including 2P bar-code scanners and remote embedded software)

• BioBall (High-precision QC microorganism inocula)

• LabGuard 3D (24/7 environmental monitoring system recording temperature, static pressure, humidity and CO2 concentration)

• BacT/ALERT Virtuo (automated blood culturing)

• Advencis (Advanced image processing to read agar plates)

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RMM Advantages

• In a recent Wall Street analyst briefing the Charles River Laboratories Executive Jim Jordan emphasized Risk Reduction, Time to Result and Cost Saving when discussing their Celsis acquisition.

• These concepts are applicable to bioMerieux’s microbiology product line.

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Risk, Time and Cost Continuums

Parameter RMMAdvantagesRiskMitigation ImprovedDetection

OnlineversusSample-basedTestingContinuousversusPeriodicTestingIn-processversusLaboratory-basedTestingReal-timeDecisionMaking

TimetoResult RealTime(Rapid)1-3HoursTurnaroundOvernightTurnaround24to48Hours5to7DaysTurnaroundAtleast14DaysTurnaround(Slow)

Cost LowversusHighCapitalCostNoReagentsversusReagentsSamplingversusOnlineTestingLowversusHighCalibrationCostsLowversusHighMaintenanceCostsLowversusHighAnalystSkillsandTraining

Technology GrowthDependentversusGrowthIndependent

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Unmet Stakeholders Needs

• The sterility testing needs for short-lived products such as compounded sterile preparations, cell based therapies and radiopharmaceuticals are not being meet by the current USP/Ph.Eur./JP sterility test what has at least a 14-day incubation period.

• These products are manufactured and infused into patients before the completion of the sterility test.

• Technologies currently marketed by bioMerieux, i.e., ScanRDI and BacT/ALERT System, are available to meet these needs.

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BioMerieux Sterility Testing Options

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TestAttribute USP<71>Sterility

TestScanRDISystem BacT/ALERTDual

TemperatureSystem

Growth-basedmethod

Yes No Yes

Technology Classical SolidPhaseCytometry

CO2sensor

Media Soybean-caseindigestandthioglycollatebroth

Resuscitationstep

Proprietaryaerobicandanaerobicbroth

Samplevolume AsspecifiedinUSP<71>Table2

Variable 10mL

Timetoresults

Atleast14days 1-2hours 5-7days

Verification Subculture Fluorescencemicroscopy

Subculture

ValidationRequirement

Suitabilitytestingonly

EquivalencytoUSP<71>

EquivalencytoUSP<71>

September 2015 bioMerieux Meeting 29 November 14, 2011 Eastern Analytical Symposium,

Somerset, NJ 29

BacT/Alert Microbial Detection

System

September 2015 bioMerieux Meeting 30 November 14, 2011 Eastern Analytical Symposium,

Somerset, NJ 30

BacT/Alert Microbial Detection

System •LED illuminates sensor

• Continuous monitoring: bottles are

read every 10 min.(144 times/day)

• Photodiode collects reflected light

• Signal transmitted to computer

• “Reflectivity Units” plotted over time

Scan RDI System

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ScanRDI System

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Conclusions

• Although pharmaceutical microbiology is a relatively conservative scientific discipline, due recent advances it is a most exciting time to be a microbiologist.

• Lets embrace these advances for the benefit of our companies and users of our pharmaceutical products.

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