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1 EVERY STEP OF THE WAY
POLL QUESTION #1
Presented By:
Demonstrating Equivalency and Limit of Detection for a Rapid
Sterility Method via ATP-Bioluminescence
PDA Midwest Webinar 28 July 2020Brice Chasey, Sr. Product Manager, Charles River
TOPICS
3 EVERY STEP OF THE WAY
28 July 2020
1 CELSIS® INSTRUMENT & SOFTWARE OVERVIEW
2 REAGENTS, METHOD OVERVIEW & APPLICATION
3 CONTRACT TESTING, LABORATORY DETAILS
4 STUDY DESIGN AND SUMMARY
celsis®Instruments and Software
Overview and Use in Equivalency Project
EVERY STEP OF THE WAY5
BUILT FOR SIMPLICITYCelsis® Rapid Microbial Detection System, based on ATP Bioluminescence
Easy integration
into current test protocols.Use your validated method.Eliminate days of incubation.
Objective resultsreplace manual eye countsor visual turbidity checks with automated, instrument basedanalysis.
Secure data integrityResults controlled with software.Automated reporting, multiusermanagement.
Sterility resultsin 6 days, using compendial consumables
Eliminates errors and ensures locked, traceable results
Automated result interpretation, replace subjectivity of compendial assay
Celsis Advance II™Up to 120 Assays per Hour
Celsis Accel®Up to 30 Assays per Hour
DATA INTEGRITY
6 EVERY STEP OF THE WAY
Objective Evaluation in Celsis Rapid Detection Based on instrument analysis Automation allows walk-away
results Automatic results reporting
and export removes possibility of interpretation or transcription errors
Duplicate cuvettes prepared for each sample compared against duplicate control cuvettes
Celsis.im Software v4
Reagents, Method Overview, & Application
Overview and Use in Equivalency Project
EVERY STEP OF THE WAY8
Celsis® ATP-Bioluminescence Reagents
Two-phase, proprietary enzyme reaction All living organisms also contain the
enzyme adenylate kinase (AK) as part of their biochemical processes
Microbial enzymes convert ADP into ATP Amplification of ATP levels beyond
naturally occurring level
Celsis AMPiScreen®
AMPLIFIED ATP Bioluminescence
IntracellularATP and
components extracted
Contaminated sample
Celsis LuminAMP™ & Celsis LuminEX™
added
Celsis LuminATE®
added50 µL sample
AMPLIFIED light signal generated,
measured by Celsis Advance II™ or
Celsis Accel®
CELSIS APPLICATIONS
9 EVERY STEP OF THE WAY
Celsis® Method Overview
BioburdenSTERILITY
flexibility
Products testing negative: pass Products testing positive: SOP for
enumeration/identification
USP <61>, <62>; EP 2.6.12
Celsis® enrichment reserved for equivalency and identification via Accugenix®
sequencing
Non-destructive Test
Products testing negative: pass Products testing positive: fail
USP <71>, EP 2.6.1
EVERY STEP OF THE WAY10
PROTOCOL – STERILITY TESTINGCelsis AMPiScreen® Method Overview
Direct Inoculation• Protocol can be applied to
different methodology -measure and prepare sample in broth media (FTM and TSB).
Analyze• Pipette 50μL of incubated
sample into duplicate cuvettes and load into instrument.
• After ~1 hr automated analysis, collect results.
Membrane Filtration• Filter sample according to
preparation method and spike organisms per procedure (as applicable).
Incubate 6 days • TSB, FTM at designated
temperatures • Take small aliquot using septum
sampling port on Sterisart canisters.
11 EVERY STEP OF THE WAY
POLL QUESTION #2
Contract Testing, Laboratory Details
Charles River Biologics, Ballina, Ireland
CELSIS STERILITY EQUIVALENCYContract Lab Facility & Details
13 EVERY STEP OF THE WAY
Charles River Biologics – Ballina, Ireland GMP Laboratory for Drug Product Release is established in Ireland for 15 years Sterility
In accordance with USP <71>, ph. Eur 2.6.1, and JP 4.06. Genotypic Identification: AccuGENX-ID®
16S rRNA (Bacteria - BacSeq) ITS2 (Fungi - FunITS)
Goal to select independent lab perform study without bias
Protocol signed by Charles River Microbial – no input afterwards regarding data generation or interpretation
RESULTS INTEGRITY
14 EVERY STEP OF THE WAY
ASSAY AUTOMATION Precisely controls reagent volumes and
reaction timing
OBJECTIVE RESULTS: Interprets results against qualified
parameters & protocols Provides multiple reporting options and
proprietary data files (secure data)
Compliant with 21 CFR Part 11
Validated on Windows system in Charles River, Ireland
Administrator right and users roles/permission structure in place for technicians
Subjectivity vs Objectivity: Celsis.im software
Study Design and Summary
Method Equivalency, Limit of Detection, Specificity
16 EVERY STEP OF THE WAY
POLL QUESTION #3
17 EVERY STEP OF THE WAY
Charles River can now provide customers a pathway to faster implementation with a complete reports & services for Celsis® rapid microbial detection method.
Most labs are challenged with the planning, scheduling, and execution of validation activities.
Even an experienced validation team can spend 6-12 months proving the equivalence of the alternative method
In-house expertise forms the foundation of a strong RMM validation strategy
CELSIS® STERILITY EQUIVALENCY AND VALIDATION Challenges & Needs
EVERY STEP OF THE WAY18
Guidance for Validating an Alternative Microbial Method
PDA Technical Report 33 – Evaluation, Validation, and Implementation of Alternative and Rapid Microbial Methods
US Pharmacopeia <1223> - Validation of Alternative Microbiological Methods
European Pharmacopeia 5.1.6 – Alternative Methods for Control of Microbiological Quality
Generally harmonized
Three guidance documents give direction on how to validate an RMM
ALTERNATIVE METHOD VALIDATION REQUIREMENTSRequirements Required By Definition Charles River Support
POCProof of Concept
Feasibility or principle (i.e., assessing whether the method and accompanying system is suitable for its intended purposes and that it is compatible with the intended product or sample matrix.)
Celsis® Sample Effects and Spiking Studies performed in Applications Lab
Instrument Qualification
Installation Qualification Analytical equipment was installed correctly. Celsis Advance IITM Installation Qualification
Operational Qualification
Analytical equipment meets the manufacturer’s specification for correct operation. Celsis Advance IITM Operational Qualification
Performance Qualification Instrument meets the URS (User Requirements) for performance.
Celsis Advance IITM System VerificationAdditionally, user must complete internal performance qualification requirements as deemed appropriate by the user
Validation of Alternate
Technologies
SpecificityMethod’s ability to detect a range of challenge microorganisms, which demonstrate that the method is fit for its intended use.
Celsis® Sterility Equivalency Project(Membrane Filtration)
Cel
sis
Com
plet
e fo
r Ste
rility
Mem
bran
e Fi
ltrat
ion
Com
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ve G
MP
Serv
ice
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alid
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clud
ing
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ocol
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port
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Cel
sis
Adva
ntag
e fo
r Ste
rility
Mem
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ion
Full
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pack
age
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clud
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repo
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etho
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itabi
lity
Limit of Detection
The lowest number of microorganisms in a defined volume of sample that can be detected, but not necessarily quantified, under the stated experimental conditions.
Equivalence/ Comparative
Testing/ Accuracy
When the test results from two procedures are sufficiently close for the intendeduse of the procedures. Demonstration of equivalence requires a prespecifiedmeasure of how similar the test results need to be. (e.g. statistical test, e.g. non-inferiority).
RobustnessA capacity of the method to remain unaffected by small but deliberate variations in method parameters, e.g., reagent volume, incubation time, or ambient temperature, providing an indication of its reliability during normal usage.
The Celsis AMPiScreen® Rapid Detection Assay Verification of Robustness
RuggednessThe degree of precision of test results obtained by the analysis of the same samples under a variety of typical test conditions such as different analysts (for example, three), instruments, and reagent lots.
The Celsis AMPiScreen® Rapid Detection Assay Verification of Ruggedness
RepeatabilityThe degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of the same suspension of microorganisms and uses different suspensions across the range of the test.
Dependent upon application; support provided as needed.
Method Suitability
Suitability
Demonstrates that the new method is compatible with specific product or sample matrices that will be routinely assayed, each material should be evaluated for the potential to product interfering or abnormal results, such as false positives or false negatives.
Method Suitability Test
Equivalence/ Comparative
Testing
When comparing two test procedures to show equivalent or better performance, statistical evidence is assembled to show equivalence or, in statistical terms, non-inferiority in presence of product.
Equivalence demonstration in presence of product to include relevant environmental isolates
Each customer is responsible for consulting internal quality requirements and Regulatory authority, as appropriate, for validation requirements.
VALIDATION APPROACH & NEED/SOLUTION
20 EVERY STEP OF THE WAY
Non-inferiority to the compendial methodEquivalencyLimit of detection (LOD) the lowest number of microorganisms that can be detected
Specificity Ability to detect a wide range of challenge microorganisms
Repeatability Protocol, test performed repeatedly on multiple samplings of suspension
Ruggedness Resistance to influences from operational or environmental variables
Capacity to remain unaffected by small but deliberate variationsRobustness
Method Suitability Ability to detect challenge microorganisms and remain unaffectedin the presence of product
Celsis® Sterility Equivalency
Study
Parameters selected for Celsis® Sterility Equivalency Study
GOALS FOR CELSIS® EQUIVALENCY STUDYStudy Overview
21 EVERY STEP OF THE WAY
The objective of this study is to demonstrate the equivalence of two microbiological test methods i.e. the Celsis AMPiScreen after 6 days incubation and the Compendial Sterility test method after 14 days incubation.
As part of the Equivalency study the Limit of Detection (LOD) and Specificity of selected organisms were assessed. The limit of detection of the rapid sterility test is the lowest concentration of microorganisms in a test sample that can be detected, but not necessarily quantified, under the stated experimental conditions.
The evaluation of the Equivalency study will be carried out in two phases – 12 total organisms The First Phase used the 6 ATCC organisms
Referenced from USP/EP/JP Pharmacopoeias
The Second Phase of used an additional 6 organisms:
Environmental, typical sterility isolates
3 of these organisms were stressed using validated protocol, per SOP
22 EVERY STEP OF THE WAY
12Aspergillus brasiliensis Mold
Clostridium sporogenesGram Positive spore-forming rods
Gram positive endospore-forming rodsBacillus subtilis
Candida albicans Yeast
Pseudomonas aeruginosaGram negative rods
Staphylococcus aureusGram Positive Cocci
StandardATCCStrains
6StressedOrganisms3
EM Isolates3
organisms
Micrococcus luteusStressed Gram Positive Cocci
Staphylococcus epidermidisStressed Gram Positive Cocci
EM Gram Negative RodBurkholderia cepacia
Penicillium citrinumEM Mold
Methylobacterium extorquensEM Gram Negative Rod
Propionibacterium acnesStressed Gram Positive Rod
CHALLENGING THE SPECIFICITYMicroorganism Panel Used in Celsis® Equivalency Study
Minimize variability.Maximize confidencePre-Qualified TSB and FTM Media from Hardy Diagnostics
Media selection is an important consideration for Celsis® applications
System is compatible with various media types (including compendial TSB, FTM), but Charles River recommends media with consistent lot to lot background ATP content
Each lot of Hardy media is tested on Celsis®
instruments to ensure a consistent ATP-background from lot to lot
Consistent background valuesfor Celsis® testing
EVERY STEP OF THE WAY24
Sartorius Sterisart® NF sterility canistersPRE-QUALIFIED STERILITY CONSUMABLE
An entirely pre-qualified closed-membrane filtration consumable ready for use with Celsis ® instruments.
Use a proprietary septum sample port to extract incubated sterility samples for analysis, preserving the closed-system for paired sample testing.
Reproducible results and confirmed positive results via identification
PROTOCOL PARAMETERS – EQUIVALENCY
25 EVERY STEP OF THE WAY
Study Overview
Hardy Diagnostics TSB & FTM Media used in compendial sterility testing
Culture Medium
Incubation Time
Using Fluid A as the “sample”; organisms spiked into “sample”
Sample
6 days for Celsis AMPiScreen® 14 days for compendial sterility
Test MethodClosed filtration system used - compendial Sterisart NF canisters + septum
APPROACH TO EQUIVALENCE VERIFICATIONStudy Overview
26 EVERY STEP OF THE WAY
Demonstrate the comparability of the two methods in a series of paired samples, with different inoculum levels.
Product (in this case sterile Fluid A) will be inoculated at three different lnocula levels (10 to 100 cfu, 1 to 10 cfu and 0.1 to 1 cfu) and assayed for the presence of microorganisms using both methods:
High inoculum level of 10 cfu
Medium inoculum level of 1 cfu
Low inoculum level of 0.1 cfu Samples are evaluated using Celsis AMPiScreen® and 14 day Compendial Sterility Method
Day 6 - canisters removed from incubation and a portion of the inoculated sample will be removed from the canister and added to a sterile tube. Two 50 µL aliquots of the sample were transferred into duplicate cuvettes and placed into the Celsis Advance II™
Bioluminescence measured and results were recorded as relative light units (RLUs) Samples with mean RLU values 3X the baseline media (TSB, FTM) control were interpreted as positive
The same canisters were re-incubated for the reminder of the 14 day period for the Compendial Sterility Method.
EVERY STEP OF THE WAY27
Positive Signal, Celsis AMPiScreen® - Relative Light Units (RLU) for Different Inoculum LevelsREPLICATE TESTING, MULTIPLE ORGANISMS
1
10
100
1,000
10,000
100,000
1,000,000
10,000,000
100,000,000
1,000,000,000
7155
8
9999
9999
3164
2631
5013
56
9999
9999
9999
9999
2710
20
9999
9999
9999
9999
2109
1094
9999
9999
9999
9999
1 CFU 10 CFU
10replicates
0.1CFU
28replicates
1CFU
10replicates
10CFU
Different inoculum levels weretested in side-by-side assays
EVERY STEP OF THE WAY28
CELSIS STERILITY EQUIVALENCYSummary and Conclusion
Results from all 12 organisms were pooled to give statistical significance to the results obtained.
Compendial testing in accordance with USP <71>, Ph. Eur 2.6.1, and JP 4.06.
Limit of detection was determined to be not significantly different between the two methods. The data used for the equivalence determination were taken for the estimation of detection limits of the two sterility test methods.
Equivalency (Non-inferiority) was demonstrated by measuring the relative rates of agreement and disagreement between the two test methods.
Represents 6-8 months of in-house testing work and >$80,000 in estimated costs of labor and materials
CELSIS® STERILITY EQUIVALENCYCompleted by Charles River Biologics, and offered by Microbial Solutions as Celsis® Complete or Celsis® Advantage
Report (188 pages) Includes: Provided in a regulatory-ready format Test facility details – CRL Biologics Ballina, Ireland Introduction Objective Experimental Design Definitions Standards Test Organism/Incubation Conditions Preparation of lnocula Test Procedure Results Assessment of Results (Statistical Analysis) Acceptance Criteria Any Deviations or unexpected events if applicable Conclusion
EVERY STEP OF THE WAY30
Celsis® Sterility Validation Support Service & Documentation Solutions & Benefits
Decreases implementation time by eliminating the need to demonstrate equivalency to the compendial method
Reduces the need for dedicate personnel, resources, and time to validation
Submission - ready dataset for faster implementation of Celsis® rapid microbial detection for sterility
Enables personnel to focus on other aspects of the validation process
CONTACT USBrice Chasey, Sr. Product [email protected]
Address:251 Ballardvale StreetWilmington, MA01887
Website: www.criver.com
Email:[email protected]
Phone:877.CRIVER.1