Microbial Identification for the Environmental Monitoring Arena Using MALDI-TOF

Microbial Identification for the Environmental Monitoring Arena Using MALDI-TOF Anne M. Russell, Ph.D. Senior Staff Scientist EMD anne.russell@crl.com

Presentation Overview 1. Accugenix - History and Market 2. MALDI-TOF Theory 3. MALDI-TOF and Microbial IDs 5. Validation Due Diligence Fitness for Use Qualification 6. Library Development Process Validation Investment Operational Impact 7. Questions 2

Accugenix - History and Market Premier, cgmp compliant, contract laboratory for microbial ID serving the pharmaceutical, medical device, nutraceutical, personal care and cosmetic industries for more than 20 years Global leader with expertise in sequence-based identification and strain typing and mass spectrometry-based identification Build own validated, proprietary identification libraries with real time updates reflecting taxonomic changes and novel organisms of significance to our customers Continually develop and apply innovative technologies Provide the most accurate and reliable identification and characterization of microorganisms found in manufacturing environments 3

Accugenix - History and Market Introduced the Axcess System and Tracking &Trending features in the Webportal - 2014 Charles River France, South Korea and India offering Accugenix Services - 2013 Introduced the Customer Webportal myaccount.accugenix.com Genotypic strain typing methods of MLST/SLST Acquired by Charles River - 2012 Proteotypic ID and real time library updates introduced - 2010 Accugenix, GmbH was established - 2008 Officially changed name to Accugenix & discontinued FA ID service - 2004 2009 - Fungal ID using ITS2 region introduced 2005 - Introduced validated, proprietary identification libraries 2002 - Strain typing using the RiboPrinter introduced 1999 - Accugenix created with introduction of sequence-based ID 1990 - Acculab, Inc. established, specializing in fatty acid-based microbial ID 4

Accugenix - History and Market Delaware, USA (Accugenix ) S. Carolina, USA (Endosafe ) Lyon, France Ahmedabad, Bangalore, Hyderabad, Mumbai Incheon, S. Korea Zhanjiang, China Singapore New South Wales, Australia 5

Accugenix - History and Market Environmental Monitoring (EM) A surveillance system that: Provides a baseline profile of a manufacturing environment Promptly identifies the sites at risk of contaminating the product Acts as an early warning system to detect possible environmental contaminants that that may impact the product Statistical data analysis over time demonstrates deviations and conformance to established limits document control Trending is performed by comparing the number of isolated colonies as well as evaluating recurrences of the same species Essential to use accurate & reproducible methods for ID 6

Accugenix - History and Market Federally Mandated Regulations: Product & Process Control 21 CFR 211.165(b) There shall be appropriate laboratory testing, of each batch of drug product required to be free of objectionable organisms 21 CFR 211.84(d)(6) Each lot of a component, drug product container, or closure with potential for microbiological contamination that is objectionable in view of its intended use shall be subjected to microbiological tests before use. Required to set (21 CFR 111.70) and monitor ( 111.75) in-process specifications for any point in the manufacturing process where control is necessary and set limits on those types of contamination that may adulterate your finished dietary supplement products 111.75 For each batch, verify that the product or incoming components/ ingredients meet the specifications for identity, purity, strength and composition 111.320 You must identify, verify and use an appropriately scientifically valid testing method for each established specification to determine whether the specification is met 7

Accugenix - History and Market Importance of Identifications It is extremely important to be able to accurately identify the organism to the species, and many times, to the strain level Permits tracking of the potential origin of the contamination Avoids delays in product release and completion of investigations The ID of microorganisms can be done through different processes, each with its own level of accuracy and reproducibility 8

MALDI-TOF Theory Theory and Instrument MALDI-TOF MS Matrix-assisted laser desorption/ionization Time-of-Flight Mass Spectrometry Ion Source (MALDI) Mass Analyzer (TOF) Detector (SEM) Secondary Electron Multiplier Analysis (Mass Spectra) Process Sample preparation co-crystallization Ionization Electrode Acceleration Ionic Separation Detection Analysis laser Matrix molecule Analyte molecules 9

MALDI-TOF and Microbial IDs Library Comparison Sample Preparation Spectra Unknown (Ribosomal proteins) Escherichia coli Enterobacter aerogenes Sample Spotting Identification Spotted Target Plate MALDI-TOF analysis Bacillus niacini 10

MALDI-TOF and Microbial IDs Library Comparison Peak Lists and Match Factors Unknown Sample Library Reference Entry: Enterobacter aerogenes Match Factor = 2.84 Library Reference Entry: Brevundimonas diminuta Match Factor = 1.56 Normalized logarithmic value of: The number of peaks in the known library entry that match to peaks detected in the unknown sample. The number of peaks thin the unknown sample that match to peaks in the known library entry. The symmetry of the intensity of peaks that match between the known library entry and unknown library entry. 11

MALDI-TOF and Microbial IDs Requires a live, healthy organism Time and temperature constraints Sample and reagent preparation Limited entries in the reference libraries May require additional tests for an identification 12

Validation Due Diligence Fitness for Use Study Installation Qualification / Operational Qualification Instrument Software Performance Qualification Software Process Validation Instrument and Methods 13

Validation Due Diligence Comparison between MALDI-TOF and existing platform(s) Labor: Method: Hands-on processing time, operator training Supports ID of a broad-range of microbes Reporting: Sample throughput, Automated, Management Regulatory: Auditable, 21 CFR Part 11 Capital: Financial: Equipment (MALDI and ancillary) ROI, Upfront investment (labor from various departments to develop new service), Long-term investment (operations labor, equipment maintenance) 14

Validation Due Diligence Vendor Comparison Vendor: Library: Reporting: Workflow: Quality System, Training, Technical Service Size, Breadth and Development (incremental and entry independence) LIMS Integration, Development (taxonomic updates) Hands-on processing time, batch size (fixed or flexible), Throughput (see scenarios) Scenario 1: 1 plate of 96 spots per plate = 1 Run 10 min Scenario 2: 4 plates of 48 spots per plate = 1 Run 15 min 96 spots 60 min 10 min 96 spots 60 min 192 spots 180 min (3hrs) 15

Validation Fitness for Use Study Initiated Fitness for Use Studies Baseline for Accuracy, Precision, Robustness, Ruggedness, Equivalence: Approximately 1,900 sample spectra were analyzed. Study included 146 strains comprising knowns and unknowns representing organisms frequently encountered and taxonomically diverse species Examine sample storage requirements, age, sample preparation methods, target cleaning All organisms were processed on the MALDI-TOF and identified with our AccuGENX-ID 16S rrna sequencing service Identification of organisms were also generated on a VITEK 2 Compact Evaluation of the data set to ascertain the appropriate interpretation criteria to maximize accurate species-level identification 17

Validation Fitness for Use Study OEM s Highly Probable versus Probable Species? User Determined Interpretation Required 18

Validation Fitness for Use Study Accuracy: Sequencing ID compared to MALDI-TOF ID (n = 1,204) ID Agrees No ID ID Disagrees No Spectra The species-level cutoff supporting the most species-level IDs with the fewest report disagreements with sequencing IDs and no identifications = 1.75. 19

Validation Fitness for Use Study Identifications with OEM software recommended match factor Identifications with Accugenix optimized match factor set to 1.75 for species-level identifications Both results were generated against the original OEM library release 20

Validation Fitness for Use Study These samples would be sequenced under the AccuPRO-ID service 21

Validation Fitness for Use Study As a result of the Fitness for Use Studies Established novel interpretation guidelines System Integration: Customized the software for reporting and interfacing with LIMS Training, SOP Development and Update Mitigated risk of unidentified samples by incorporating sequencebased identifications into the AccuPRO-ID service offer 22

Validation Instrument IQ Instrument Installation Qualification Vendor Supplied Site Preparation Design Shipment Verification System Installation Computer System setup and MALDI-TOF communication Instrument Method Availability Instrument Setup Verification System backup Customer Familiarization with Instrument Operation System Maintenance 24

Validation Instrument OQ/PV Instrument Operational Qualification / Performance Verification Vendor Supplied Instrument Preparation Data Acquisition and Analysis Data Analysis and Acceptance Instrument Acceptance Report Vendor Supplied Site Preparation Supplies Instrument Method Functionality (Performance Verification) Setup Finalization Customer Familiarization Software, Sample Preparation, Safety 25

Validation Software IOQ/PQ Software Installation and Operational Qualification CRL Executed Remote Client Installation Remote Client Operation Software Performance Qualification CRL Executed Software Application and Subsystem interoperability 26

Validation System PQ System Performance Qualification CRL Executed Method Validation Test Set: 31 known organisms 2 sample processing methods 1 target cleaning method Same organisms processed over 3 days Same organisms processed by 3 operators Acceptance Criteria: Accuracy, precision, robustness & equivalency based on results from the Fitness for Use study Software / Reporting Test Set: 5 samples that represent 5 different report categories Acceptance Criteria: Results represented each of the 5 categories 27

Validation Results Successfully validated MALDI-TOF identifications for use in a cgmp environment Launched the AccuPRO-ID service offer AccuPRO-ID is a polyphasic approach to microbial identifications with MALDI-TOF and 16S sequencing, which increases quality by decreasing variability AccuPRO-ID provides a lower cost identification and a lower risk of sample mis-identifications than phenotypic methods AccuPRO-ID s is an economical, accurate and reproducible way to identify samples for non-critical EM samples Initiated MALDI-TOF library updates 28

Library Development Process Fitness for Use Results: Development of library entries could help ID these samples at MALDI, rather than be sequenced under the AccuPRO-ID service 29

Log (Frequency of Occurrence) Library Development Process Library Comparison 16S FOO 10,000 90% 95% 99% 100% 1,000 45 100 55 10 1 1 501 1001 1501 2001 2501 Species Count n = 85,557 Represented in the OEM Library Not Represented in the OEM Library n = 987 55% of most frequently identified bacteria are contained in the OEM Library 30

Log (Frequency of Occurrence) Library Development Process Library Comparison ITS FOO 10,000 90% 95% 99% 100% 1,000 16 100 84 10 1 1 251 501 751 1001 Species Count n = 25,930 Represented in the OEM Library Not Represented in the OEM Library n = 446 16% of the most frequently identified fungi are contained in the OEM Library 31

Log (Frequency of Occurrence) Percent Library Development Process Library Comparison ITS FOO 10,000 90% 95% 99% 100% 100 ITS 1,000 80 OEM Library 60 100 40 10 20 1 0 1 251 501 751 1001 Species Count n = 25,930 n = 446 Species and type of fungi contained in the OEM library are not representative of the most frequently encountered fungi observed. 32

Library Development Process 33

Log (Frequency of Occurrence) Library Development Process Prioritize development of library entries by identifying the most frequent Fall Through species first 1,000 90% 95% 99% 100% 100 10 1 1 201 401 601 801 1001 1201 Species Count n = 25,930 34

Library Development Process General Background Library entries (Main Spectrum, MSPs) consist of type strains AND non-type strains to cover the interspecies variability in mass peak patterns, ~mega spectrum Library Maintenance a. Identify Fall Through Organisms b. Verify Identity via 16S sequencing using Accugenix methods and validated bacterial ID database c. Collect MALDI spectra (20-48) Build MSP: An average of all spectra to create mega spectra; a list of mass peaks and intensities m/z S/N Res. Intens. Rel. Inten Area 2066.97 6.2 543 1213.67 29 6228 3261.15 4.7 833 883.67 21 4507 3416.68 6.1 858 1144.19 27 7444 3681.37 3.4 817 615.27 15 3235 3754.64 11.6 793 2068.41 50 12372 3762.31 4.6 1340 819.26 20 4106 4145.95 7.1 863 1224.47 29 7067 4836.29 8.9 1002 1453.08 35 12129 4905.42 4 955 657.8 16 5162 5107.38 5.3 1103 847.67 20 5663 5126.64 25.9 1013 4163.63 100 33200 5133.95 6.1 751 974.31 23 9421 5163.33 5.7 1133 920.3 22 7178 5488.37 4.5 1297 694.95 17 4817 6523.66 9.2 983 1213.67 29 9805 7510.51 31.4 1070 3456.94 83 30673 7526.67 8.2 1053 906.51 22 9398 7539.42 6.6 952 725.59 17 12926 8291.84 9 1054 875.75 21 9124 10251.02 26.4 1115 1704.18 41 29594 35

Library Development Process Library Maintenance (continued) d. Perform MSP challenge Robustness and Accuracy Test new MSP against the existing library New MSP should not match to other MSPs already in the library no duplicates Test one spectra acquired from the candidate that was not used to create the entry against the library containing the new entry(ies) Spectra should match the new MSP Test original unknown s spectra against the library containing the new entry(ies) Spectra should match the new MSP e. Create Library Entry Form to document identity, validity and testing of the new MSP f. Update and Release the Accugenix MALDI Library 36

Total Number of Library Entries Library Development Process 8000 7000 6000 5000 4000 MALDI Library Update History As of July 2, 2014 (v.14.08) Library (Entries) Total Entries = 7,169 OEM = 5,627 Accugenix = 1,542 OEM = 78.5% Accugenix = 21.5% 3000 2000 1000 0 Total Entries BioTyper OEM Entries Entries Accugenix Entries Accugenix entries currently makeup 21.5% of all entries in the library 37

Library Development Process As of July 2, 2014 (v.14.08) Entries Accugenix OEM Library (Entries) Total Entries = 7,169 OEM = 5,627 Accugenix = 1,542 OEM = 78.5% Accugenix = 21.5% Species Accugenix OEM Library (Species) Total Species = 3,088 OEM = 1,728 Accugenix = 1,089 Shared = 217 Shared OEM = 56% Accugenix = 35% Shared = 9% While Accugenix entries currently only makeup 21.5% of all entries, they also represent 35% unique species. 38

Log (Frequency of Occurrence) Library Development Process Library Comparison 16S FOO 10,000 90% 95% 99% 100% 100 14% 1,000 100 80 60 40 45% 10 20 1 1 501 1001 1501 2001 2501 Species Count n = 85,557 Accugenix entries target the most frequently encountered species, effectively tailoring the library for environmental monitoring. 0 OEM Accugenix 14.08 Present Missing n = 987 39

Library Development Investment Time Years Labor 1.5 full time employees dedicated to MALDI library development Assistance from operations (data generation and sequencing analysis) Supply Costs (reagents and consumables) Sequencing MALDI Quality System cgmp compliance: SOP development and execution, library maintenance, auditable 40

Percent Library Development Performance 20% of identifications are only generated because of an Accugenix entry Do Accugenix entries improve reporting worldwide? Accugenix entries are the top match on more than 40% of reports (increase in accuracy) 45% 40% 35% Accugenix (Top) Accugenix (Only) 30% 25% 20% 15% 10% 5% 0% Accugenix entries have more impact on reporting than recent OEM library releases (Accugenix library tailored for EM) 41

Percent Library Development Performance Do Accugenix entries improve reporting worldwide? 45% 100% 40% 35% 80% 39% 41% 61% 30% 25% 20% 15% 10% 5% 0% 60% 40% 20% 0% US EU S. Korea Accugenix entries improve reporting performance locally and internationally. 42

Library Development Performance Do OEM Library Updates improve operational performance? Do Accugenix entries improve operational performance? From Fitness for Use Study 100% 80% 60% 40% 28 27 15 No ID Reportable ID 20% 0% OEM originial OEM latest Accugenix Accugenix library entries have a greater impact on improving operational performance than OEM library releases From operations data (n = 72,115) 43

Library Development Performance Do Accugenix entries improve operational performance worldwide? 100% 80% 15 15 23 38 60% 40% No ID Reportable ID 20% 0% All Sites US EU S. Korea Accugenix entries improve operational performance locally and internationally. 44

Library Development Performance How does library development impact tracking and trending? Total Samples = 61,223 Total Species = 1,303 Accugenix entries increasing the operational performance of the MALDI platform by ~15% translates into 15% more sample results being available for tracking and trending without requiring a secondary identification technology. 45

Conclusion Take Home Messages Reviewed the impact of the library on Accugenix MALDI-TOF performance and our own tracking and trending. Why should you care? Accugenix services assist with customer Tracking and Trending, which is an integral part of a well-designed and executed EM program as it provides critical information about your manufacturing environment on a routine basis By increasing reporting through library development, significant changes in microbial flora can be identified sooner thereby enabling a faster review of the ongoing monitoring data and used in investigations of the excursion, affect the mitigation process and aid in root cause determination Detect the emergence of indicator organisms and Allow excursions from normal operating conditions to be identified quickly and reliably Validation of a clear species-level cutoff when analyzing MALDI-TOF IDs provides accurate and consistent species-level identifications that will result in more confidence in the T&T information 46

Conclusion Take Home Messages Tracking a genus-level (e.g. OEM reporting) provides good information but not detailed information Accugenix MALDI-TOF reporting provides species-level identifications, enabling the capture of bad bugs and detailed level analysis of your environment 47

Thank you and Questions Anne M. Russell, Ph.D. Senior Staff Scientist EMD anne.russell@crl.com 48