Industry Case Study: A Microbial Investigation of Contamination by Burkholderia multivorans Jim Klein and Geert Verdonk Merck & Co., Inc.
Messages The role of microbiological analysis The role of subject matter experts Coordination with FDA
Product & Process Overview Product for intranasal administration non sterile contains an antimicrobial preservative Purified Water Filling 0-3 days 0-3 days 3 days 3
Prior to the Contamination Event... Method Qualification confirmed suitability of the micro test methods. USP <61>: Microbial Enumeration Tests: Results of Spike-Recovery Testing Test Total Aerobic Microbial Counts (TAMC) Total Yeast Molds Count (TYMC) Pseudomonas aeruginosa spike: 33 recovery: 27, 31, 31 Spike Recovery Results, CFU Bacillus subtilis spike: 36 recovery: 30, 35, 31 Staphylococcus aureus spike: 68 recovery: 65, 59, 69 n.a. n.a. n.a. Aspergillus niger spike: 44 recovery: 24, 28, 28 spike: 27 recovery: 28, 29, 25 Candida albicans spike 27 recovery: 38, 37, 42 spike: 43 recovery: 41, 38, 38
Prior to the Contamination Event... Method Qualification confirmed suitability of the micro test methods. USP <62>: Tests for Specified Microorganisms: Results of Spike Recovery Testing Test Challenge Organism Detection Medium Absence of Pseudomonas Pseudomonas aeruginosa Absence of bile tolerant gram - microorganisms Pseudomonas aeruginosa Absence of Salmonella Salmonella enterica Absence of S aureus Staphylococcus aureus Absence of E coli Escheria coli CMA VRBGA XLDA MSA MacConkey Result pass pass pass pass pass
And So It Begins... Batch 0 fails micro - Batch +1 already in production Batch +1 passes Batch +2 fails micro Investigation Initiated Production Halted Pertinent Batch Release Tests: Total Aerobic Microbial Count (TAMC) and Total Yeast Molds count (TYMC) USP <61> Specified Microorganisms USP <62> absence of Pseudomonas aeruginosa absence of bile tolerant gram negative absence of E. coli absence of Salmonella absence of S. aureus For each test, a single composite of samples from across the batch was tested.
Release Test Results TEST RESULTS Total Aerobic Microbial Count (TAMC) Total Yeast and Mold Count (TYMC) Absence of Staphylococcus areus Absence of Pseudomonas aeruginosa Absence of Escherichia coli Absence of Salmonella Species Absence of Bile-tolerant gram negative bacteria Batch 0 Batch +1 Batch +2 20 CFU/ml (Burkholderia multivorans) 20 CFU/ml (Burkholderia multivorans) <10 CFU/ml <10 CFU/ml <10 CFU/ml <10 CFU/ml Absent Absent Absent Present (Burkholderia multivorans) Absent Present (Burkholderia multivorans) Absent NA NA Absent NA NA Present (Burkholderia multivorans) Absent The contamination was detected by several different test methods. Present (Burkholderia multivorans)
Response: Micro Elements Investigation Initiated Production Halted Subject Matter Experts Engaged Expanded Micro Test Plan Investigational testing initiated on batches preceding Batch 0 Investigational Testing using : Total Aerobic Microbial Count (TAMC) and Total Yeast Moulds count (TYMC) USP <61> Specified Microorganisms USP <62> absence of Pseudomonas aeruginosa absence of bile tolerant gram negative For each test, five separate results from sequential intervals across the filled batch.
Subject Matter Experts Engaged Site: Strong experience with critical investigations Extensive process understanding Company-Level Subject Matter Experts: Expertise in industrial microbiology and related subjects Primary responsibility is Sterile Product Manufacturing Process Engineering Lab Operations Equipment Engineering Quality Operations Microbiology Water Systems Sterile Manufacturing Technical Operations Together: An Exceptional Team!!!!
Subject Matter Experts Key Contributions: assessment of investigation hypotheses review of the purified water system characterization of the identified organism (detail follow) expanded analytical test plan (details follow)
Microbial Characterization Burkholderia multivorans: member of the Burkholderia cepacia complex classified as an objectionable organism common in potable water biofilm former grows on very low nutrient, e.g. distilled water resistant to multiple disinfectants and antibiotics Burkholderia cepacia poses a special risk for pharmaceutical manufacturing: Burkholderia cepacia: This Decision is Overdue; PDA Journal of Pharmaceutical Science and Technology, 2011, 65 535-543.
Supplemental to Method Qualification... Supplemental testing using Burkholderia multivorans confirmed suitability of the methods with the specific organism of concern. USP <61>: Microbial Enumeration Tests: Results of Spike Recovery Testing Test Total Aerobic Microbial Counts (TAMC) Spike Recovery Results, CFU Bukholderia multivorans spike: 81 recovery: 84, 91, 91 USP <62>: Tests for Specified Microorganisms: Results of Spike Recovery Test Absence of pseudomonas Absence of biletolerant gram - microorganisms Challenge Organism Burkholderia multivorans Detection Medium CMA VRBGA Result pass pass Burkholderia multivorans
Investigational Testing...Batch 0 Batch 0 : Investigational Testing (5 Separate Intervals) TEST RESULTS Sequence of Filling Total Aerobic Microbial Count (TAMC) Total Yeast and Mold Count (TYMC) Absence of Pseudomonas aeruginosa Absence of bile-tolerant g- bacteria Interval 1 Interval 2 Interval 3 Interval 4 Interval 5 <10CFU/ml <10CFU/ml 10 CFU/ml 70 CFU/ml TNTC <10CFU/ml <10CFU/ml 40 CFU/ml 20 CFU/ml TNTC <10CFU/ml <10CFU/ml Present Present Present <10CFU/ml <10CFU/ml Present Present Present Initial lead hypothesis: contamination was being introduced midway through the filling operation.
Investigational Testing...Batch +2 Batch +2 : Investigational Testing (5 Separate Intervals) TEST RESULTS Sequence of Filling Total Aerobic Microbial Count (TAMC) Total Yeast and Mold Count (TYMC) Absence of Pseudomonas aeruginosa Absence of biletolerant g- bacteria Interval 1 Interval 2 Interval 3 Interval 4 Interval 5 <10CFU/ml <10CFU/ml <10CFU/ml <10CFU/ml <10CFU/ml <10CFU/ml <10CFU/ml <10CFU/ml <10CFU/ml <10CFU/ml Absent Absent Absent Present Present Absent Absent Absent Present Present
Investigational Blank Run An investigational batch was manufactured with: No active ingredient Extra sampling throughout for microbial testing The results (next slide) were informative: contamination detected before filling: after tanks were rinsed with Purified Water, prior to charging of materials contamination did not increase as the batch progressed, but dropped to undetectable levels before increasing again growth was higher in samples without preservative
Investigative Blank Run Post Rinse TNTC Purified Water 0 Post Rinse End of Processing TNTC TNTC Mid-processing 0 Mid-processing 3520 cfu/ml PRESERVATIVE ADDITION End Processing 0 End Processing 0 Start 190 cfu/ml Filling Interval 1 560 cfu/ml Interval 2 390 cfu/ml Interval 3 460 cfu/ml
TAMC (CFU/mL) 1,000,000 Laboratory Growth Studies Growth studies in final product matrix reconciled all observations: Contamination was being introduced early......then reduced when contacted with preservative...then recovered over a time-scale of several days 100,000 10,000 Preservative Effect 1,000 100 10 1 Acclimation and Exponential Growth Low Inoculum High Inoculum TNTC 28 days 21 days 14 days 11 days 7 days 6 days 5 days 4 days 3 days 48 hours 24 hours 0 hours Spike
Micro Test Plan: Genotyping Genotyping results further supported the hypothesis of a biofilm vs in-line contamination. Isolates from each contaminated batch AND the blank production run were analyzed by Riboprinting ( RiboPrinter ) The results established that all isolates originated from the same source RiboPrinter pattern results using EcoRI marker Batch 0 Batch +2 Blank- Run
Coordination with FDA Field Alert issued to FDA Investigational testing detected contamination in 5 additional batches beyond the initial two All seven contaminated batches remained within company control none had been released to the market Investigation was at an early stage Company then initiated a call to FDA to offer additional information and consultation Teleconference occurred shortly after FAR submission
Highlights: Teleconference with FDA FDA Areas of Inquiry: microbiological test methods and results assessment of root cause disposition of batches assurance that marketed batches were free of contamination Company Input: addressed the FDA topics of inquiry listed above provided an overview of the investigation methodology, status and preliminary findings committed to provide an interim investigation report submitted approximately 5 days later
Final Findings & Outcome Root cause: Burkholderia multivorans had formed a biofilm in a section of the purified water system deficiencies in the design, operation and monitoring of the water system deficiencies were present at only one specific Point of Use location rest of the system found to be sound
Batch Final Investigation Results for All Release Testing Investigational Testing +2 Fail Fail +1 Pass Pass 0 Fail Fail -1 Pass Fail -2 Pass Fail -3 Pass Fail -4 Pass Fail -5 Pass Fail -6 Pass Pass -7 Pass Pass -8 Pass Pass -9 Pass Pass -10 Pass Pass -11 Pass Pass -12 Pass Pass -13 Pass Pass -14 Pass Pass -15 Pass Pass -16 Pass Pass Tested Batches Batch Release Testing 37 total batches tested represents 8 months of manufacturing 7 found contaminated Investigational Testing -17 Pass Pass -18 Pass Pass -19 Pass Pass -20 Pass Pass -21 Pass Pass -22 Pass Pass -23 Pass Pass -24 Pass Pass -25 Pass Pass -26 Pass Pass -27 Pass Pass -28 Pass Pass -29 Pass Pass -30 Pass Pass -31 Pass Pass -32 Pass Pass -33 Pass Pass -34 Pass Pass
Lessons Learned Successes: Utilization of Subject Matter Experts Use of Micro Analytics as an Investigative Tool Engagement with FDA Learnings: Speed requires: 1. Rapid decision to deploy expanded micro analytics and SMEs 2. Ready availability of micro analytical capabilities and SMEs
Acknowledgements The following individuals were key contributors to this work: Daniel Knapp Pericles Lagonikos Len van Lin Lim Meng Lip Anthony Mui Dan O Mahony Fergal Quinn David Read Carlo Shiu Jan Jaap Schot Lydia Troutman