POLL QUESTION #1 · 7/28/2020 · 13 EVERY STEP OF THE WAY Charles River Biologics – Ballina, Ireland GMP Laboratory for Drug Product Release is established in Ireland for 15 years

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


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


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


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


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


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.


POLL QUESTION #2

Contract Testing, Laboratory Details

Charles River Biologics, Ballina, Ireland

CELSIS STERILITY EQUIVALENCYContract Lab Facility & Details


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


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


POLL QUESTION #3


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


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

preh

ensi

ve G

MP

Serv

ice

for v

alid

atio

n, in

clud

ing

prot

ocol

sup

port

for

met

hod

trans

fer.

Cel

sis

Adva

ntag

e fo

r Ste

rility

Mem

bran

e Fi

ltrat

ion

Full

do

cum

enta

tion

pack

age

to in

clud

e pr

imar

y va

lidat

ion

repo

rts a

nd p

roto

cols

for

dem

onst

ratin

g m

etho

d su

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


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


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


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


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


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


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.


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


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


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]

[email protected]

Address:251 Ballardvale StreetWilmington, MA01887

Website: www.criver.com

Email:[email protected]

Phone:877.CRIVER.1

mailto:[email protected]

Documents

POLL QUESTION #1 · 7/28/2020 · 13 EVERY STEP OF THE WAY Charles River Biologics – Ballina, Ireland GMP Laboratory for Drug Product Release is established in Ireland for 15 years