PDA Pharmaceutical Microbiology Conference 2024
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PDA Pharmaceutical Microbiology Conference 2024 Posters

Poster Summary/Abstract Information

  • Comparative Evaluation of Microbiologics EZ-Accushot and bioMerieux Bioballs for Media Qualification Testing

    Dustin A. Baird, MS
    Dustin A. Baird, MS
    Clinical Laboratory Scientist
    National Institutes of Health (NIH)
    Abstract
    The NIH Sterility Lab has used the Microbiologics EZ-Accushot organisms for growth promotion testing of solid media and BacT/ALERT bottles since 2018. The product is advertised to provide 10-100 CFU per inoculum; however, low recovery trends (sometimes < 10 CFU) have been observed when growth promoting routine environmental monitoring media following manufacturer’s instructions. Furthermore, Aspergillus brasiliensis has failed detection in BacT/ALERT bottles on several occasions. This study provides a historical review of 6 years’ worth of data spanning numerous organism lot numbers, 6 USP QC organisms, and multiple analysts to compare lot to lot recoveries with manufacturer’s published COA. Additionally, we performed a parallel evaluation of the Microbiologics EZ-Accushot organisms with bioMerieux Bioballs. In summary, no average CFU counts fell within the published COA 95% confidence interval for EZ-Accushot compared with Bioballs (81%). Notably, the published 95% CI range was narrow for EZ-Accushot (up to 4 CFU) compared with Bioballs (up to 27.7 CFU). Faster time to detection was observed for A. brasiliensis Bioballs in BacT/ALERT bottles (average 1.89 days) compared with EZ-Accushot (2.49 days). Importantly, culture conditions (media type, incubation temperature, and duration) listed in both manufacturer’s COAs differed to routine practices defined in USP< 61>.
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  • Moving forward: Rapid Detection of Bacteria and Fungi in Cell Therapy Products

    Darren J. Bauer
    Darren J. Bauer
    Staff Scientist
    Thermo Fisher Scientific
    Abstract
    This abstract presents the findings of a study on the detection of live bacteria and fungi in cell therapy products using the Applied Biosystems SteriSEQ Rapid Sterility Testing Kit. Given the nature of cell therapy production processes and limited shelf life of cell therapy products, it’s imperative to detect and prevent contamination as early as possible in the workflow, such as testing raw materials or in-processing samples. By implementing rapid sterility testing, potential contamination can be revealed earlier and addressed promptly, helping to reduce the risk of product loss and unexpected production delays and outcomes. This not only enhances the overall efficiency of the workflow, but helps to maintain the integrity and efficacy of the cell therapy product. The study involved the use of six species listed in USP chapter < 71>, to determine LOD as well as compatibility with cell culture matrices containing 10^6 mammalian cells. One of the known challenges to sterility testing is confirming the presence of live species contamination. Additionally, we explored a potential method to reduce false positive results. The combined data demonstrates the kit’s swift detection capabilities and offers a promising approach for helping to ensure the safety and quality of cell therapy products.
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  • A NEW Real-Time RT-PCR-Based Mycoplasma Detection Kit to Meet the Current Revision of Ph. Eur. Chapter 2.6.7 and USP <77> Draft

    Thomas W. Bauman, Jr., BS
    Thomas W. Bauman, Jr., BS
    Field Application Specialist - Microbiology
    Sartorius Corporation
    Abstract

    Mycoplasma testing is a critical quality attribute (CQA) of cellular therapeutics. Since microbial contamination of cell therapy products can potentially result in the recipients' deaths, Mycoplasma testing is a critical component of the release testing process for any cell therapy product. The compendial Mycoplasma test takes up to 28 days before a contamination can be ruled out with certainty, which is too long for autologous cell therapies intended to treat terminally ill patients. As a result, there is a high demand for growth-independent rapid assays.

    Currently the regulatory landscape for NAT based Mycoplasma detection is changing, USP  < 77> is drafted and Ph. Eur. chapter 2.6.7 under revision, setting new requirements on the detection capability of a respective assay. Therefore, a detection system with a highly efficient automated or manual DNA extraction protocol followed by a real-time RT-PCR assay has been developed, and a validation study was set up to meet the requirements of the current revision of Ph. Eur. chapter 2.6.7 and USP  < 77> Draft


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  • Alternatives to Sterility Testing of Cell and Gene Therapies

    Zachary T. Beck, BS
    Zachary T. Beck, BS
    Senior Microbiologist, Group Leader III
    Eurofins BPT
    Abstract
    As the pharmaceutical industry continually evolves, so do the products they produce.  Viral Vectors, Cellular Therapies, and Viral Vaccines are becoming increasingly predominant as they are a more naturally sourced treatment of illness and disease.  However, these types of treatments can present challenges to pharmaceutical developers because of their unique make-up, as most of these products are made for specific, individual patients, and sometimes originate from a patient’s own body.  Along with this, most of these products are integral, requiring short testing timelines, as they may be needed as soon as possible for critically ill patients, and most compendial methods aren’t conducive to these tight timelines.  In these instances, alternative methods may be the only option in getting these products to patients in dire need.  A rapid contamination check may be an alternative option to compendial sterility testing to ensure that these products are administered to patients in time.  There are important things to consider when evaluating this alternative testing and the outsourcing of this testing, to ensure your product is going through appropriate GMP and compliant testing.
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  • Assessment of Differential Pressures Across Sterilizing Filter Membranes with Various Test Solutions

    Dr. Candau-Chacon’s presentation from the 2023 PDA Pharmaceutical Microbiology conference “Sterile Filtration of Biological Products” discussed the importance of measuring differential pressures during sterile filtration process validation. I was inspired to develop this innovation to help pharmaceutical manufacturers anticipate upstream and differential pressures of varying process solutions during sterile filtration with different filter membranes.
    Zachary Bendiks, PhD
    Zachary Bendiks, PhD
    Senior Scientist
    Meissner Corporation
    Abstract
    FDA recommends monitoring differential pressure across filter membranes during sterile filtration process validation. However, few resources are available to help pharmaceutical manufacturers anticipate expected differential pressures during sterilizing filtration of different solutions. To address this gap, Meissner evaluated differential pressures across different filtration membranes using various test solutions at increasing pump speeds. Specifically, we investigated differential pressures across sterilizing-grade PVDF, PES, and PTFE membrane discs, either in series or with downstream 0.4 μm PES analysis discs commonly used in bacterial retention testing. The test solutions employed for this study include saline, grapeseed oil, FBS, and DMEM cell culture media with 10% FBS. These solutions were chosen based on their differing physicochemical properties and their relevance to the pharmaceutical industry. This work will serve as a reference for pharmaceutical manufacturers and help them anticipate differential pressures across sterilizing filter membranes at different pump speeds based on the physicochemical properties of their drug products.
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  • The Human Element: Mitigating Human Error with Digital Solutions in Environmental Investigations

    Alexandra Bezilla, PMP
    Alexandra Bezilla, PMP
    Project Manager and Technical Specialist
    SherpaPharma
    Abstract
    This poster presentation explores the intricate realm of human error within environmental monitoring activities, focusing on the pivotal role of digital solutions to successfully mitigate its likelihood of occurrence. Beneath regulatory and industry scrutiny, we examine the multifaceted nature of the human element in environmental deviation management. We highlight the transformative impact of digital solutions in addressing root causal factors associated with human error. By utilizing technological interventions such as digital environmental monitoring systems and software tools, organizations can significantly enhance their ability to prevent, detect and address deviations attributed to human error. Our discussion underscores the significance of leveraging technology to enhance the reliability and effectiveness of environmental monitoring processes.
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  • Training Program Development: Microorganism Recovery in Broad Spectrum Test Samples and in Neutralization Broths with Three Different Microbiologists (Accuracy and Precision Evaluation)

    Richard Boehler Jr, MSc.
    Richard Boehler Jr, MSc.
    Microbiology Manager
    Certified Laboratories
    Abstract
    Accuracy and Precision are two key test parameters. During a training program data was collected.  The microorganisms were utilized to spike three different cosmetic categories (baby sunscreen lotion, biotin collagen shampoo and body wash). Positive control data was also collected and compared. After cosmetic neutralization a small population of microorganisms were used as separate inoculations.  The recovery of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Aspergillus brasiliensis was determined. Tryptic Soy Agar and Potato Dextrose Agar were used for enumeration.  The test plates were incubated at 32.5℃ and 22.5℃, respectively for 3 and 5 days. Comparing each microorganism “positive control” to the average colony forming units recovered in each cosmetic, the plate count test method proved to be very accurate.  The percent recovery range for bacteria in cosmetic, compared to the positive controls was 96.8% to 100.4%.  The yeast and mold recovery range exhibited 88.8% to 95.8%.  The precision was determined by comparing the results between two different microbiologists using five different microorganisms, and fifteen dilutions per test run.  Good precision was observed with little variability.  Both microbiologists recovered mold in cosmetic samples at a 109.8 % rate. The training program emphasized the importance of aseptic technique.
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  • Compatibility of Organic Solvents with the Endosafe Nexgen-PTS for Bacterial Endotoxin Testing of Water-Insoluble Pharmaceutical Products

    Hannah Bolinger, PhD
    Hannah Bolinger, PhD
    Research Scientist
    Gilead Sciences
    Abstract
    Parenteral products must undergo testing for bacterial endotoxins (BET) per USP < 1>. Due to the assay’s reliance on enzymatic activity, aqueous solutions near neutral pH are most suitable. However, some products are not soluble in aqueous and rely on solubilization in organic solvents. This study evaluates organic solvents’ compatibility with the Limulus amoebocyte lysate assay for detecting endotoxin. Six organic solvents (N-methylpyrrolidone, 10:90 methanol:dichloromethane, acetonitrile, dimethyl sulfoxide (DMSO), ethanol, and methanol), and five solubilizers (KLEPTOSE®, mannitol, polyethylene glycol, polysorbate 20, and polysorbate 80) were evaluated for use per USP < 85>. Materials were evaluated at multiple dilutions on a Charles River Endosafe nexgen-PTS. All solvents and solubilizers were compatible with BET at dilutions of 1:25 to 1:200, with the exception of the polysorbates, which were incompatible with BET at dilutions below 1:1000. Additionally, N-methylpyrrolidone produced such a low spike recovery at 1:100 dilution that it was not evaluated further. Published data on the compatibility of organic solvents with the LAL assay for endotoxin testing is lacking. This data supports understanding how to test insoluble compounds for endotoxins and will provide useful insights for developing sensitive methods for materials with low solubility in aqueous system.
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  • Application of Fourier-Transform Infrared Spectroscopy (FT-IR) for Staphylococcus Epidermidis Typing as a Tool for Contamination Control Strategy in a Pharmaceutical Industry Facility

    Marcelo Luiz L. Brandao, PhD
    Marcelo Luiz L. Brandao, PhD
    Public Health Researcher
    Fiocruz
    Abstract
    The typing of micro-organisms in pharmaceutical factories often relies on expensive and time-consuming molecular techniques. So, the implementation of cheap, fast and reliable typing methods in the routine would speed up the investigation procedures improving the contamination control strategy. The Fourier-transform infrared (FT-IR) spectroscopy is a method that generates spectra, that enables to micro-organisms typing within 3 h. This study aimed to evaluate the FT-IR for typing S. epidermidis strains isolated from an immunobiological pharmaceutical industry in Brazil. Fifty strains were evaluated by FT-IR using IR Biotyper®. A dendrogram was created with the raw data to cluster the separation spectrum and the cut-off value was automatically calculated. Forty-four FT-IR profiles were obtained, a ratio of 1.14 strain/profile. From the five clusters formed, Cluster 1, 2 and 3 (6 strains) were isolated from environmental monitoring of air and operators (EMO). Cluster 4 (3 strains) were isolated from EMO and bioburden assays, suggesting that the environment could be the main source of bacterial contamination in the product analyzed in bioburden assay. Cluster 5 (2 strains) were isolated from EMO and a cell culture lineage used in quality control assays, suggesting that the environment could also be the main source of cell contamination.
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  • Environmental Monitoring Review and Trending: Two Case Studies on the Importance of Assessing Sampling and Results

    Mariana Brena, MS
    Mariana Brena, MS
    Microbiology Manager
    Cambrex
    Abstract
    Environmental monitoring is one of the key aspects of ensuring that facilities are operating in a state of control, detecting contamination before there is impact to products. In two case studies, we review situations where existing sampling and trending explained out of specification sample results and one where the existing sampling scheme did not detect potential contamination prior to positive environmental controls. Through a formal risk assessment performed by Cambrex, it was determined that a water system took longer to polish the water and remove impurities post-sanitization, which was resulting in out of specification results for Total Organic Carbon (TOC) testing. Careful review of testing and maintenance events resulted in an update to criteria required to release the system for use post-sanitization. In a second scenario, Cambrex performed an investigation to understand how fingertip samples recovered growth that was not detected during routine environmental monitoring. It was determined that the point of entry of the microorganisms was likely through carts that were not being sampled frequently enough, prompting an updated risk assessment of the routine environmental monitoring sampling scheme. Both cases demonstrate the importance of evaluating individual test results, historical results, and recent events related to the process under consideration.
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  • Contamination Control Strategy (CCS), Check! Now What? Lifecycle Management of CCS

    Hilary A. Chan, MS
    Hilary A. Chan, MS
    Global Sterility Assurance and Microbiology Lead
    Takeda
    Abstract
    Establishing a Contamination Control Strategy (CCS) is a requirement per EU Annex 1 (Aug 2022).  Once a CCS is established, the challenge becomes; “how do I ensure appropriate lifecycle management and effectiveness monitoring for my CCS?”  According to Annex 1, “The CCS should be actively reviewed and, where appropriate, updated and should drive continual improvement of the manufacturing and control methods. Its effectiveness should form part of the periodic management review.”  This presentation will provide examples of how a pharma company is integrating CCS review into existing quality management system processes to ensure CCS remains updated and accurate.  Additionally, the use of tools to automate and digitize monitoring of the effectiveness of CCS will be presented.
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  • Data Analysis / Trending / Pattern recognition for Annex 1 Contamination Control Strategy

    Susan B. Cleary, B.CS, EMBA
    Susan B. Cleary, B.CS, EMBA
    Director Product Development
    Novatek
    Abstract
    Regulations are evolving, for example EMA Annex 1, earlier versions did not mention trends, the current draft version mentions many times.  WHO, and FDA also talk about trending, root cause analysis, investigation, and using the data for these purposes.  PDA technical report 13, the new revision, even states that automation for data management for environmental mentoring is “essential”.  Trending the data is now a regulatory requirement but what trends should we use, and when?  This presentation will include a review of key regulations as they relate to the need to use our data for trending including how often and what events should trigger trending, root cause analysis, and investigations.  Also, which trend tools should we use for the different contamination control processes, cut off method, Control charts (Shewhart, etc..), Quantiles, Percentiles, Weibull Distribution, Scatter plot, Regression analysis for slope (upward/downward trends). 
    The last 20 years we have been collecting data and regulations like Annex 11/21 CFR part 11, and more recently the data integrity guidelines, have facilitated security and confidence the data is accurate and reliable.  Now this regulatory evolution is mandating the use of the data for the betterment of our processes, process control, product quality and patient safety.
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  • MALDI Biotyper® Database Expansion with a Possible New Species of Genus Aliidiomarina Isolated in an Immunobiological Pharmaceutical Industry Facility: New Approaches to Contamination Control Strategy

    Luciana V. Costa, PhD
    Luciana V. Costa, PhD
    Health Public Technologist
    Fiocruz/Bio-Manguinhos
    Abstract
    Microbial identification is extremely relevant for parenteral drug pharmaceutical industry as it is pre-requisite for Contamination Control Strategy (CCS) implementation. However, some microbial identification methodologies are not appropriate to identify environmental microorganisms. On the other hand, pharmaceutical production environments present considerable potential for new species discovery, because their microbiota is little explored and studied. Matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) is a fast and accurate methodology for microbial identification, despite many environmental strains not being covered by its database. Aliidiomarina is a genus composed by Gram-negative heterotrophic, aerobic and motile rods. The aim of this study was to expand the MALDI Biotyper® database for the identification of an Aliidiomarina strain isolated in an immunobiological pharmaceutical facility. B602/23 was isolated in 2023 and was not identified by VITEK®2 (biochemical method) nor MALDI Biotyper®. The strain was submitted to full 16S rRNA gene sequencing, whose result was Aliidiomarina quisquiliarum (97.20%), indicating a possible new species, as the cut off for species identification is 98.70%. B602/23 was added to MALDI Biotyper® database as Aliidiomarina sp., by creating a Main Spectrum and analyzed again, so that it could be identified as Aliidiomarina sp. with a score of 2.35.
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  • Navigating Risk-Based Approaches with Annex 1: Prevalence of Spore-Forming Microorganisms and Molds Recovered During Routine Environmental & Process Monitoring

    Ryan Cox
    Ryan Cox
    Associate Scientist
    Charles River Laboratories - Accugenix
    Abstract
    Environmental monitoring (EM) assesses the level of microbial control within a facility. The relevance of EM is apparent in Annex 1 with the implementation of more stringent manufacturing regulations focused on tracking and trending a facility’s microflora. To evaluate common, often challenging, microflora for contamination control, we analyzed over 500,000 global samples identified by sequencing 16S and ITS2 gene regions and using MALDI-TOF, a proteotypic method. Of the top 50 species, fungi make up 64% - 70% of the organism diversity, followed by spore-forming bacteria (4% - 12%) and gram-negative bacteria (6% - 10%). The study identified four top genera: Aspergillus, Penicillium, Staphylococcus, and Cladosporium, three of which are filamentous fungi. Thirty-three unique genera compose the top 50 frequently occurring species list of all geographical regions. Identification accuracy is influenced by technology, library quality, and data analysis methods. Annex 1 states monitoring systems should include scientifically sound, modern methods that optimize detection of contamination. Using outdated or minimally diverse libraries more often yields no identification or misidentification to species or genus level. Difficult to control organisms are ubiquitous. It is incumbent on all sterile manufacturers to routinely evaluate EM data to improve traceability and maintain confidence in microbial control strategies.
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  • Use of Endotoxin Detection Methods to Demonstrate Equivalency of a New 96-well Microplate Reader

    Melissa Cramer
    Melissa Cramer
    Laboratory Manager
    Charles River Laboratories
    Abstract
    Within pharmaceutical manufacturing, when a new iteration of equipment is on-boarded it is essential to ensure equivalent data is generated by both pieces of equipment (existing and new). The BioTek ELx808IU 96-well microplate reader has been discontinued and the Endosafe™ PRS 3 Microplate Reader was selected as the replacement. Prior to the launch of PRS 3, the microplate reader was evaluated for performance by testing the linearity, optical density accuracy, alignment, kinetic noise, and temperature uniformity. In addition, the equivalency of the two instruments was evaluated using four endotoxin detection methods: endpoint chromogenic assay, kinetic turbidimetric assay (KTA), kinetic chromogenic assay (KCA), and Trillium™ recombinant cascade reagent kinetic assay (rCR). Pre-approved acceptance criteria that included validity under current BET guidelines, 5% variability for temperature range, and 2% variability for accuracy and linearity for universal test plates were met. The results of overall performance and equivalence tests demonstrated the equivalence of the two microplate readers for all endotoxin detection methods.
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  • Early Detection of Microbial Contaminants in Cell-Based Products Using the Milliflex® Rapid System Combined with a Selective Lysis Solution

    Cecile Delbos, M.S.
    Cecile Delbos, M.S.
    Innovation Pharma director
    Merck KGaA
    Abstract
    The Milliflex® Rapid System is a proven automated ATP-based solution used for rapid bioburden and sterility testing. Combining membrane filtration, ATP bioluminescence and imaging, it provides microbial enumeration in significantly less time than traditional methods.  Detecting microbial contamination in cell-based products remains a challenge due to filterability issues and interference from mammalian ATP. To address this, a sample processing method has been developed using a selective cell lysis solution and a free ATP removal enzyme effective at both low and high mammalian cells densities.  The processing of Chinese Hamster Ovary (CHO) cells ranging from 106 to 107 cells/mL with lysis solution eliminates  the mammalian ATP background in 15 minutes. This requires a ratio of 4 mL of lysis solution per mL of sample at 106 cells/mL or 9 mL of lysis solution per mL of sample at 107 cells/mL.  CHO cells were inoculated separately with 6 microorganisms including Cutibacterium acnes ATCC 6919 and then processed with the lysis solution before enumeration with the Milliflex® Rapid System. Results showed a rapid enumeration of the 6 microorganisms with  good recovery according to the control.
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  • A Cleanroom Surface Treatment that Provides on Demand Hydrogen Peroxide at the Nanoscale for Microbe Contamination Control

    Christina H. Drake, PhD
    Christina H. Drake, PhD
    CEO
    Kismet Technologies
    Abstract
    An 8-week study was conducted in the transfer room of an ISO 7 cleanroom facility with a surface treatment that provides on demand hydrogen peroxide at the nanoscale when bacteria, fungus or mold encounter the treatment. A single treatment of various surfaces including floors and tables in the transfer room between cleanroom levels in a pharmaceutical plastics manufacturing environment were evaluated. This facility is cleaned daily with IPA, with a weekly disinfection rotation of Vesphene, LpH III and Spore-klenz. Bacterial loads on the various surfaces were determined by swabbing and plating with MC-Media Pads. The treated surfaces provide reduced bioburden compared to the current cleaning protocol alone in the transfer room. The coating showed efficacy out to 8 weeks post application with 86% microbe reduction on the cleanroom floor and 96% microbe reduction on a transfer room table. This surface treatment will help ensure that a cleanroom maintains its ISO rating and adheres to the strict guidelines that define that rating. In addition, the decreased number of microbes will help decrease the risk of rare but possible contamination of products produced in this facility. The coating shows superior performance in reducing these surface microbes for up to 8 weeks.
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  • Application of a High Throughput Automated Colony Counting System Powered by AI to Environmental Monitoring

    Phil Duncanson, PhD
    Phil Duncanson, PhD
    Chief Microbiologist
    AstraZeneca
    Abstract
    A system applied to clinical microbiology was adapted for the high throughput assessment of environmental monitoring plates collected from a parenteral manufacturing site. Proof of concept and industrialization of the instrument necessary to ensure a robust counting system where false negatives results could not be tolerated. Here we describe the side-by-side comparison of the system compared side by side to qualified microbiologists in routine use over multiple months.
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  • Cloud Based and Hosted Laboratory Information Systems: Strategies to Balance their Advantages and Disadvantages

    This poster explores the benefits and risks of a Cloud-based or Hosted LIMS. Clients are moving in this direction and this innovation helps them make informed decisions.
    Jim Elliott
    Jim Elliott
    Computer Systems Validation Director
    Trinity Consultants Life Sciences
    Abstract
    Cloud-based and hosted Laboratory Information Systems (LIMS) offer a range of benefits including scalability, ease of implementation, and improved collaboration and real-time data access among geographically dispersed operations; however, security, infrastructure outages, and system and data stewardship issues pose challenges. This poster explores the benefits of a Cloud-based or Hosted LIMS as well as options for ensuring system and data security, approaches to mitigate the impact of infrastructure disruptions, and balancing responsibilities between the LIMS Vendor and the End User Company. By carefully considering and implementing the appropriate options, companies can leverage the benefits of cloud-based and hosted LIMS solutions while minimizing associated risks.
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  • World’s Largest Microbial Surface Sampling Dataset – What It Means for Personnel and Contact Plate Qualification

    Joshua D. Erickson, M.S.
    Joshua D. Erickson, M.S.
    President & CTO
    Stratix Labs Corporation
    Abstract
    Monitoring the level of bioburden on surfaces in cleanrooms is a critical component of an effective contamination control strategy. The recent revisions to Annex 1 underscore the importance of understanding recovery efficiency as it relates to your environmental monitoring program. Here we present the results from the world’s largest surface sampling performance dataset to date that includes >1,000 sampling events across more than 300 test sites. This dataset reveals novel insights into the impacts that personnel surface sampling technique and contact plate devices have on environmental monitoring results. There is a clear need for each person collecting surface samples to demonstrate their qualification to recover microbes from a surface as part of their annual environmental monitoring training. In addition, this dataset reveals the impact that contact plates can have on microbial recovery efficiency from a surface and the need to properly qualify new lots of contact plates in the way they are used in the cleanrooms.
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  • Evaluation of ATP-Bioluminescence Detection as a Supportive Technology for Bioburden and Sterility Testing Processes

    Madaisabel O. Fuentes Arias, MS
    Madaisabel O. Fuentes Arias, MS
    Contractor-Scientist I
    U.S. FDA
    Abstract
    A challenge facing the biomanufacturing industry is the lengthy timeline for quality control testing that delays critical go/no-go decision making for the rapid release of drug products. The recommended USP methods for bioburden and sterility testing are surface-spread plating method and direct inoculation method, respectively. These compendial methods are reliable; however, results take approximately 5-7 days for bioburden testing (USP< 61>) and no less than 14 days for sterility testing (USP < 71>). ATP-bioluminescence detection is a rapid microbial method (RMM) that can reduce the time to result for both bioburden and sterility testing, taking 18-24 hours and 6 days, respectively. This study aims to evaluate the performance of ATP-bioluminescence in comparison to compendial methods to understand the implications of using this technology in bioburden and sterility testing processes.  The research conducted consisted of simulating contamination to assess the detection capabilities of the ATP-bioluminescence assay. Results from compendia and ATP-bioluminescence detection were analyzed for comparability. Findings from this study will provide insight on the use of this platform as an alternative tool for in-process testing.
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  • Environmental Monitoring: Developing Tests to Challenge Artificial Intelligence Models for Colony Counting Using the APAS Independence

    Steven Giglio, PhD
    Steven Giglio, PhD
    Scientific Director
    Clever Culture Systems
    Abstract
    Healthy scepticism surrounds the use of automated systems to reliably detect and count colonies on an agar plate. Compendial guidelines covering the assessment of alternative microbiology methods are well established (e.g. USP< 1223>) which provide measurements of method suitability. The appetite for technology interrogation, however, is high and driven by a desire to understand any limitations as part of risk profiling for an application. One of the most common concerns for automated colony counting systems used in Environmental Monitoring is the ability to detect colonies on the perimeter and labels on the plate, for which there are no established methods or acceptance criteria. When performing primary validation for the APAS Independence, this particular challenge test was developed as part of robustness examinations. Bespoke AI application tools were used for microbiologists to annotate colonies on the APAS Independence images which were then objectively and computationally compared to the APAS Independence result, on a colony-by- colony basis and stratified by location (edge, label, non-edge/non-label). This is a unique approach to a unique challenge test developed for this type of colony counting technology. The high level of colony detections by the APAS Independence underpins performance across all validation tests in a statistically defensible manner.
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  • Mitigating Risk for Transfer of Living Materials into Closed Systems

    Cody Glisson
    Cody Glisson
    Sr. Business Development Manager
    Ecolab Life Sciences
    Abstract
    One of the biggest challenges for developers and manufacturers of cell and gene therapies is to design a robust process to transfer living material into closed systems such as isolators, without introducing contamination into the grade A/ISO 5 area or damaging them from the bio-decontamination process. Different processes (automated and manual) and strategies are being taken into consideration by various cell therapy manufacturers, as well as some novel strategies to overcome this challenge. Use of isolators with automated bio-decontamination systems are becoming a first intent consideration for robust process design and should be integrated with procedures that ensure protection of living material viability.
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  • Decentralized Manufacturing: Compliance in the Future of Production

    Mark C. Hallworth
    Mark C. Hallworth
    Senior GMP Scientist
    Particle Measuring Systems
    Abstract

    Decentralized manufacturing has emerged as a transformative model in the production of vaccines, biologics, personalized therapies, and various pharmaceutical and biomedical products. The unique advantages also present distinct challenges in sterility assurance, particularly within the realm of contract manufacturing. This discussion explores the dynamic landscape of decentralized manufacturing within the pharmaceutical industry, focusing on its application in the production of critical medical products.

    Decentralization enables regional responsiveness, reduces transportation costs, and enhances supply chain resilience, fostering agility in addressing global health needs. However, this model introduces complexities in ensuring sterility assurance, given the diverse environments and regulatory frameworks across decentralized facilities. Contract manufacturing specifically emphasizes these challenges, as outsourcing processes can lead to gaps in quality control and oversight.

    The examination of gaps in sterility assurance within decentralized manufacturing, including variations in cleanroom standards, environmental monitoring protocols, and personnel training is needed to maintain compliance. Understanding and addressing the unique challenges is crucial for maintaining product quality, ensuring patient safety, and advancing access to critical therapies.

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  • Quantifying and Assessing Cleaning and Disinfection Residues

    Madison Hoal
    Madison Hoal
    Global Technical Consultant Manager
    Ecolab Life Sciences
    Abstract

    Joint presentation - Ecolab and HiTech Health (CDMO for CGT)

    The purpose of this presentation is to review the impact cleaning and disinfectant residues can present to an ATMP cGMP manufacturing facility and discuss how to assess and manage these residues.

    The term “disinfectant residue” can illicit different responses.  For some, they pose a risk potential product contamination, or can alternatively be seen as evidence that cleaning and disinfection has been performed. For others it could be a sign of lack of control.  With the most recent revision to Annex 1 there has been a renewed focus on control and removal of disinfection residues.  This presentation will discuss how to minimize disinfectant residues by regime design and investigate the methodology associated to quantifying residues beyond subjective visual assessment.

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  • Ready to Use Quantitative Microorganisms for Compendial Quality Control Testing

    Jyoti K. Jha, PhD
    Jyoti K. Jha, PhD
    Senior Scientist
    ATCC
    Abstract
    Just-in-time availability of  quantified microbial standards are of significant value to improve end-user workflow and turn-around times involving compendial release assays in manufacturing of biopharmaceutical products, cosmetics , food safety, and wastewater  testing. Quantitative preparations of microbial controls are traditionally produced from the dilution of saturated growth culture; however, such techniques demand significant efforts to obtain desired concentration. Traditional techniques also require revalidation of the cultures at a defined interval to ensure compliance to prescribed standards. While multiple companies have quantitative microbial products on the market that are based on microbial materials sourced from ATCC, they either require longer and more involved assay preparation time or require lower storage temperature (-10°C to -20°C). In order to satisfy the need for quantitative compendial controls  that deliver a fast turnaround time with minimal handling, we have developed a new portfolio of single-use, ready to use products for quality control strains in a discretely quantitative pellet  . These quantitative controls are directly from a USP-cited strain source-of-origin and demonstrate consistent lot-to-lot quantitation, immediate rehydration at room temperature, and stable storage at refrigeration temperature  (2-8°C).
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  • Improved Time to Detection of Slow-Growers Microorganisms with an Automated Growth-Based Method

    Improved Time to Detection of Slow-Growers Microorganisms with an Automated Growth-Based Method improves sterility testing duration with an automated growth-based method. CGT manufacturers are seeking for faster methods to deliver treatment to patients. Time and performances are key criteria for their sterility testing. In an effort of continuous improvement, we investigated ways faster time impacts our automated growth-based method. A CGT manufacturer will be able to evaluate those and decide on implementation for a faster time for results.
    Caroline Kassim Houssenaly, PhD
    Caroline Kassim Houssenaly, PhD
    R&D Biosciences Manager
    bioMérieux
    Abstract
    Automated Growth-based methods are used for rapid microbial contamination detection in pharmaceutical products. Cell and gene therapy (CAGT) manufacturers are widely adopting such methods as compendial following EP 2.6.27 and soon USP  &#60; 72> or alternative to the compendial USP  &#60; 71> / EP2.6.1. For short shelf life products, every hour gained on release testing is crucial to deliver quickly therapies to patients in need. The BACT/ ALERT® 3D (BTA) Solution provides an automated non-destructive growth-based rapid microbial method. Thanks to a combination of culture bottles and incubation temperature, BTA is capable of detecting a variety of aerobic and anaerobic microorganisms including fungi. Although most relevant microorganisms are detected in less than 72h, the current practice is the release of product after 7 days incubation, mainly due to the risk of contamination with slow grower microorganisms such as Cutibacterium acnes. In order to improve the detection of ‘slow growers’, different culture media additives and incubation conditions were evaluated. Our new data shows that the time to detection can be significantly improved and reduced significantly. Automated growth-based methods remain an important technology for CAGT manufacturers and our new data shows that there are still room for improvements from current practices.
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  • Exploring the Application of ATP Bioluminescence Method (Rapid Microbiological Method) to Microbial Enumeration Testing in Pharmaceutical Water Monitoring

    Marika Katsuda
    Marika Katsuda
    Associate Researcher
    Daiichisankyo Co., Ltd.
    Abstract
    Microbial enumeration tests in water monitoring commonly utilize the culture method, which typically requires up to a week to obtain results. To enable prompt resumption of production in the event of system abnormalities and ensure timely water quality management, we explored the ATP bioluminescence method as an alternative to the culture method, offering results within a few hours.
    We demonstrated analytical validation for the ATP method as an alternative approach and evaluated its non-inferiority compared to the culture method. We also established and assessed the validity of action level and alert level for ATP method in pharmaceutical water. The results suggest that the ATP method is comparable to the conventional method and indicate the potential appropriateness of the established action level and alert level thresholds.
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  • Qualification Versus Claims

    Dan A. Klein, MA
    Dan A. Klein, MA
    Sr. Technical Service Manager
    STERIS Corporation
    Abstract
    Regulations, both within Europe and the United States, require disinfectant manufacturers to test disinfectants prior to sale.  Regulations also require end-users to qualify disinfectants using a sound scientific approach.  As regulations and methods continue to grow and evolve, it can be difficult to understand the requirements for the disinfectant supplier relative to the end user.  This presentation will aid in understanding US, EU and rest of world regulations and testing requirements including why new methods are slow to evolve.  It will also address the testing role, if any, of new and novel microbiological test methods versus older “Pasteur” microbiology tests while discussing what and how to test and other best practices to aid in the successful implementation of a rotational disinfectant program.
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  • Vaporized Hydrogen Peroxide Decontamination – Evaluation of Log Reduction for Biological Indicators and Other Considerations for Cycle Development

    Vaporized Hydrogen Peroxide Decontamination - Evaluation of Log Reduction for Biological Indicators and Other Considerations for Cycle Development is something I spent a lot of time engaging in and learning from with unexpected results along the way. Understanding the complexities of Biological Indicators and their interaction with VHP is very important to understand and plan accordingly when performing cycle development studies. As the industry moves more toward decontamination and sterilization utilizing methods such as VHP, the information presented within my poster can help others understand the key factors to consider during cycle development of isolator systems, RABS, etc. and to achieve an optimized cycle from the start.
    Ashley C. Lang, MS
    Ashley C. Lang, MS
    Principal Consultant
    Lang Quality Consulting Services, LLC.
    Abstract
    Vaporized Hydrogen Peroxide (VHP) decontamination is becoming widely used in the pharmaceutical industry.  As part of the cycle development process, biological and chemical indicators are used as the standard measure to confirm that successful decontamination has been achieved.  Based on the current precedence for the use of biological indicators in processes such as steam sterilization (autoclaves), it is expected that a 6-log reduction is reached in order to demonstrate a sufficiently sterilized environment.  Current guidance documents such as PDA Technical Report 51 mentions that “for surface decontamination with an agent such as VHP, that the cycle may be tailored to achieve a less than 6-log reduction, given a supporting rationale is provided.”  What factors should be considered during VHP cycle development in conjunction with the level of kill desired?  What biological indicator is appropriate to select?
    Another option that may be explored is the use of enzymatic indicators (EI) to demonstrate the success of a decontamination cycle.  Some positive improvements EIs can provide over Bis is rapid results, reduction in overall cycle development process time, and cost effectiveness, all while achieving a successful cycle.
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  • Terminal Pharmaceutical Sterilization Using E-beam.

    We’ve made tremendous progress in terminal sterilization of drug products using some innovative dose delivery techniques. We are excited to offer this option to reduce risk compared to aseptic processing. It will help the industry with risk reduction, contamination control, and overall safety of the drug supply.
    Rose LaRue-Slater
    Rose LaRue-Slater
    Director of Sales
    Steri-Tek
    Abstract
    Case studies on pharma products that have undergone radiation dse tolerance studies in e-beam.
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  • Foster Environmental Monitoring Results with Advanced Automation Systems

    Laurent C. Leblanc
    Laurent C. Leblanc
    Senior R&D MAnager
    bioMérieux
    Abstract

    The Environmental Monitoring program is an important GMP control in the pharmaceutical manufacturing operations. It must rapidly detect deviations from established alert / action limits that can compromise the facility’s state of control. The trend analysis highly depends on the methodology used to recover environmental microorganisms, this includes the type of culture media (Trypticase Soy base or Sabouraud base) but also the incubation time and temperature to grow the stressed clean rooms organisms.

    The current practices are still very manual and the microbial results are often the bottle neck in term of time to result compared to chemicals analysis. The dynamic in the pharmaceutical world around Pharma 4.0 fostered by the new Annex 1 revision, implies the adoption of new advanced and rapid solutions. Digitalization and automation are at the center of these evolutions.

    In this presentation we will see the benefits of EM automation and how this technology improves the level of contamination control by allowing a very rapid response to deviations, but also how this can help to improve result times.

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  • Validation of Rapid Microbiological Testing Methods Using Cryopreserved and Live Cell Therapeutics Mimic Samples

    Da-Hye Lee, PhD
    Da-Hye Lee, PhD
    Senior Research Scientist
    Korea Research Institute of Standards and Science
    Abstract
    Mold contamination in manufacturing facilities can be a serious issue for pharmaceutical and life science companies. Not only does it endanger patient safety, but it can also cause product recalls, financial and reputational harm, and regulatory ramifications. Rapid, accurate, and reliable microbial identification provides understanding of the environmental quality and acting on to avert risk and contamination. In this study, we analyze two fungal genera: Aspergillus and Penicillium that are frequent fungal contaminants, producing mycotoxins and with the ability to cause human infections. Identification of organisms within these genera can be tricky across different platforms due to species diversity. A total of 14,687 Aspergillus and 14,827 Penicillium samples were identified across Accugenix® global labs in 2023, 23.5% of these samples were identified using proteotypic method and the remaining by genotypic. More than 100 unique species groups were identified, for each genus, during the process. We further evaluate the potential of these organisms to be conclusively identified using three different identification platforms and compare species diversity across multiple databases. The distribution of these critical organisms across Accugenix® global labs emphasizes the necessity for robust cross functional approach to identify the organisms confidently to strategically nullify any contamination risks.
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  • Comprehensive Approach for Annex 1 Gap Assessments

    Satya T. Makwana, MS
    Satya T. Makwana, MS
    Quality Assurance
    Resilience
    Abstract
    Since the current version of European Union (EU) GMP Annex 1 took effect on 25 August 2023, sites across the world have begun to assess their adherence to the Annex 1 regulations to meet the European market patient demand. Here at Resilience Research Triangle Park, we have taken a comprehensive approach to assessing three-hundred and sixty (360) Annex 1 requirements against our processes and procedures.  Each gap that was identified through the assessment was categorized by topic area (i.e.-single use systems, equipment, utilities etc.) and ranked as either Major or Minor according to criticality to properly assign resources and timelines for mitigation. Through this assessment we will share our strategy for methodically assessing the alignment between Annex 1 and our current processes and procedures.
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  • A Metagenomic Analysis with Oligotrophic Enrichment Approach for Detecting Specified Microorganisms

    Bernard S. Marasa, PhD
    Bernard S. Marasa, PhD
    Pharmaceutical Scientist
    U.S. FDA
    Abstract
    In pharmaceutical manufacturing, benefit is conferred in detection of specified microorganism (i.e., Burkholderia cepacia complex (BCC), E. coli, Pseudomonas aeruginosa, Salmonella enterica) not readily identified by culture-dependent methods. It's logical to test for the presence of “specified microorganism” using metagenomic analysis before culturing a “specified organism”, especially when the organism isn't easy to culture. We developed a metagenomic analysis during enrichment to identify specified organisms. The enriched bacterial community consisted predominantly of Bacillus spp. and Stenotrophomonas spp., each contributing about 97-99% to total taxon abundance in TSB and 1/10× TSB. The specified microorganisms that were observed were Clostridium spp., Burkholderia spp., and Staphylococcus spp. (0.04 - 0.07%) in TSB, otherwise Burkholderia spp., Pseudomonas spp., Salmonella spp., Staphylococcus spp. and Escherichia spp. (0.01 - 1.73%) in 1/10× TSB. PreQ0 biosynthesis (PWY-6703) and guanosine ribonucleotides de novo biosynthesis (PWY-7221) were the most abundant pathways in 1/10× TSB-24 h. BCC chiefly contributed to the toluene degradation (PWY-5180 and PWY-5182) pathways. Initial results demonstrate the potential of the metagenomic approach during enrichment in water-based environments. These results indicate that a metagenomic enrichment approach to evaluating water samples can be useful to monitor specified organisms over time, including oligotrophs such as BCC in 1/10× TSB.
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  • Rapid Robust Mycoplasma Detection System for Release Testing of Cell And Gene Therapy Products Containing Mammalian Cells in 1-Hour

    Ambili Menon, PhD
    Ambili Menon, PhD
    Sr. Staff Scientist
    bioMérieux
    Abstract
    Sterility testing process of cell & gene therapy (CGT) products is an essential component to ensure patient safety. Compendial and alternative Nucleic acid testing (NAT) methods are being utilized to ensure the absence of Mycoplasma as a release requirement. Compendial methods require 28 days of testing and are not suitable for short shelf-life products.
    The BIOFIRE® Mycoplasma is a closed system sample-to-answer nucleic acid test that reports the presence/absence of over 130 mycoplasma species in less than an hour, the system is well suited as a release test for short shelf-life CGT products.
    USP and the upcoming European pharmacopeia release testing guidelines for CGT product require the presence of cellular matrix in the test article.  To align with these regulatory guideline two new protocols have been developed: 10mL Single centrifugation sample preparation protocol and Low volume (~1.7mL) sample preparation protocol. Both protocols allow inclusion of mammalian cells and achieve appropriate sensitivity of ≤10 CFU/mL needed for release test.
    These protocols were also evaluated on CGT customer samples with multiple Mycoplasma strains. These results thereby demonstrate BIOFIRE® Mycoplasma is compatible as a release test method for CGT products and high sensitivity is achieved providing mycoplasma results in less than one hour.
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  • An Integrated Lifecycle Approach to Contamination Control Strategy (CCS) Using Quality Risk Management (QRM) Principles with a Case Study

    An Integrated Lifecycle Approach to Contamination Control Strategy (CCS) Using Quality Risk Management principles is a case study responding to the increasing emphasis on quality and compliance in pharmaceutical manufacturing from Eudralex Vol 4 Annex 1 on contamination control in the pharmaceutical industry and incorporating Quality Risk Management (QRM) principles to develop a robust Contamination Control Strategy (CCS). It can potentially empower industry professionals with the knowledge, tools, and practical insights needed to strengthen contamination control strategy and how QRM can be used as the foundation to build the CCS, enhance product quality and safety, and drive continuous improvement in pharmaceutical manufacturing. Inclusion of a case study in the presentation offers a practical application of QRM principles and CCS development. Industry professionals can learn from real-world examples, gaining insights into how these strategies are implemented, the challenges encountered, and the outcomes achieved. This practical perspective can help professionals contextualize theoretical concepts and apply them in their own manufacturing areas.
    Darshana Patel
    Darshana Patel
    Associate Director, Micro Quality & Sterility Assurance
    Merck & Co., Inc.
    Abstract

    Contamination control is a critical aspect of ensuring that a pharmaceutical manufacturing process is designed to meet product quality, safety, and efficacy as well as ensuring compliance with regulatory expectations (Annex 1). By adopting a lifecycle approach and incorporating QRM principles, organizations can proactively identify and mitigate contamination risks, ensuring production of safe and high-quality products. Quality Risk Management (QRM) provides a systematic framework to identify, assess, control and communicate risks associated with contamination.  Using a cohesive structure of QRM process and integration of key components supports the development of a comprehensive Contamination Control Strategy (CCS). CCS should consider all key elements of sterile product manufacturing/aseptic preparation, including control measures required to prevent /reduce risk of microbiological, non-viable particulate, extraneous matter, cross contamination, and endotoxin/pyrogenic contamination. A life cycle management strategy should be established for CCS and consideration should be given to design, development, manufacturing process controls, and ongoing continuous improvements of the product.

    This presentation will include a case study that emphasizes understanding of how QRM principles can be applied to develop an effective CCS.

    This presentation will cover the following elements:

    • Regulatory expectations
    • QRM Process
    • Elements of CCS
    • Case Study
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  • Good Practice in LER Hold Time Study: The Choice of the Endotoxin

    Good Practice in LER Hold Time Study: The Choice of the Endotoxin addresses the choice of endotoxin to be used in LER hold time studies based on my experience with the potential impact of endotoxin choice on hold time study results. This can help the industry by providing scientific data to make informed decisions on the use of CSE/RSE endotoxins in LER hold time studies.
    Alessandro Pauletto, MSc
    Alessandro Pauletto, MSc
    Global LER Business Development Manager
    bioMérieux
    Abstract
    Before PDA TR82 defined the use of CSE and RSE in low endotoxin recovery (LER) hold time studies, there had been debate for many years as to which endotoxin was most appropriate to use. Naturally occurring endotoxin (NOE) or standard endotoxin (CSE/RSE)? Little had been discussed about the differences between CSE and RSE. In this study, we investigated the possible impact that CSE and RSE may have on the results of a LER hold time study
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  • Replacing Traditional Aseptic Process Simulations with Qualification of Cell and Gene Therapy (CGT) Supernatant

    The goal of this study is to demonstrate that a cell therapy manufacturing process that is contamination-indicating could negate the need for an APS every six months since the manufacturing process can serve as a re-qualifying APS run each time it is executed. The current guidelines for APS have not been updated to represent the growing field of Cell and Gene Therapy (CGT). We believe this study can begin the discussion of updating these guidelines to better represent CGT and can reduce the need for costly APS runs and allows more patients to receive lifesaving treatments by reducing manufacturing operation delays caused by APS runs.
    Bernardo Perez
    Bernardo Perez
    QC Associate
    Century Therapeutics
    Abstract

    Aseptic process simulations (APS) are traditionally performed using Tryptic Soy Broth (TSB) as a surrogate for finished product to qualify aseptic manufacturing operations. In this study, the supernatant from cell processing media was examined for bacterial and fungal growth viability to determine equivalency with TSB. With the use of cell processing media in Cell and Gene Therapy (CGT) manufacturing, can qualifying the supernatant collected from the process eliminate the need for an APS run?

    Supernatant was collected from cell processing media and incubated at same incubations conditions required for the APS post sterility check (Test A - 7d 20-25°C/7d 30-35°C) and at use conditions (Test B – 14 d at 35°C/5%CO/5%O2). Post incubation, growth promotion testing was performed using ATCC cultures (Ab+, Bs+, Ca+, Ec+, Sa+, Pa+ and Se+), which resulted in growth for Tests A-B and the positive inoculum control. Results concluded that the cell processing supernatant examined was equivalent to TSB, thereby implying that each cell therapy manufacturing run is aseptically self-validating. As more practical approaches emerge for APS qualifications in CGT manufacturing, this data can be used to implement alternate options to qualify a CGT manufacturing process and help establish guidelines for future cell therapy APS qualifications.

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  • Process Improvement in Microbial Identification Through Use of MALDI-TOF and Axcess: Case studies in Microbial Contamination Response

    Amy E. Perkins-McKenna, MS
    Amy E. Perkins-McKenna, MS
    QC Manager, Microbiology, Contamination Control
    AGC Biologics
    Abstract
    Microbial identification is a critical element within a contamination control strategy. In-house identification can improve lot release process flow, investigation root cause analysis and contamination response. Through use of a Bruker MALDI-TOF paired with Charles River’s Axcess system, these case studies demonstrate improved response times and reduction of turn around to support lot release in a multi-product manufacturing facility. Case studies and data analysis pre- and post-implementation of in-house identification highlight the benefit of implementing such a system.
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  • The Challenges of Floor Cleaning and Disinfection

    James Polarine Jr. MA
    James Polarine Jr. MA
    Senior Technical Service Manager
    STERIS Corporation
    Abstract

    Maintaining and improving cleanroom manufacturing operations is required to assure quality production of products. In today’s world, there are many challenges facing Cleanrooms and Controlled Environments and their support areas. Proper cleaning and disinfection of cleanrooms and controlled environments is key to maintaining these facilities at the level they were designed. As an industry, we face challenges to surface cleaning and sanitization. These include:

    1. Manpower shortages to maintain these facilities
    2. Need for consistency of cleaning to maintain the contamination levels
    3. Management of cleaning and operations requiring strict attention to details and techniques
    4. Adequate time for cleaning and other ancillary operations
    5. Rising cost of operations
    6. Safety
    7. Changes in regulations
    8. Proper use of disinfectants and rinsing

    Each of these challenges can impact the ability to maintain control of a cleanroom. The focus of this presentation is on proper disinfectant use including understanding of the selection, contact time, rotation and residue. The presentation includes multiple site data from recent case studies focused on the impact of rinsing on environmental monitoring results.

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  • Positive Control Verification of an Alternative Microbial Method Using the Sievers Soleil Rapid Bioburden Analyzer

    Meg Provenzano, BS
    Meg Provenzano, BS
    Product Manager Biodetection
    Veolia
    Abstract
    What if your rapid bioburden results could correlate to traditional plate counts?
    The Sievers Soleil Rapid Bioburden Analyzer is an innovative, at-line rapid microbial method (RMM) instrument designed to monitor bioburden at ultrapure levels utilizing fluorescence based stains with results in less than 45 minutes. The Soleil is easy to use, portable, flexible, rapid, and equivalent to compendial plate count methods as demonstrated by data presented here. Soleil represents a significant improvement over existing agar plate methods by enhancing user capabilities and ease of use without  sacrificing essential characteristics of that technology. With the Sievers Soleil Rapid Bioburden Analyzer, firms can make actionable decisions quickly to mitigate risks and improve manufacturing agility. The Sievers Soleil allows users to monitor bioburden levels throughout the manufacturing process from raw materials to in-process samples to final product. With the 2023 publication of Annex 1, more manufacturers are adopting Rapid Alternative Methods as a means of process monitoring.
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  • A Demonstration of Patient Safety and the Importance of the Detection of Natural Environmental Endotoxins for Recombinant Cascade Reagents

    Allan J. Romberg
    Allan J. Romberg
    Research Scientist I
    Charles River Laboratories
    Abstract
    With advancements in science and sustainability initiatives, new animal-free technologies have emerged as a replacement for natural Limulus Amoebocyte Lysate (LAL). Recently, Charles River Laboratories (CRL) launched Trillium™, a Recombinant Cascade Reagent (rCR) that utilizes three recombinant proteins involved in the detection of bacterial endotoxins that are naturally found in Limulus polyphemus. For optimal development of Trillium™, CRL performed a direct comparison of the eight-rabbit pyrogen test (RPT) per USP  &#60; 151> and the Bacterial Endotoxin Test (BET) per USP  &#60; 85> of naturally contaminated pharmaceutical water samples and carbohydrates using multiple rCR formulations. Patient safety factor was calculated through the ratio of endotoxin concentration in EU/mL from RPT positive samples to the established pyrogenic threshold of 5.0 EU/Kg. Three (3) rCR formulations were included, with varying amounts of recombinant factors and detergents. Results show that Trillium™ has a patient safety factor that is equal to or greater than natural LAL, and that slight changes in formulation can negatively affect the detection of Natural Environmental Endotoxins (NEE) while still recovering RSE within BET acceptance criteria.
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  • Application of Targeted Amplicon Next Generation Sequencing Using Curated Ribosomal Sequence Databases for Identification of Complex Microbial Samples.

    Lauren R. Salvitti, PhD
    Lauren R. Salvitti, PhD
    R&D Manager
    Charles River Laboratories, Microbial Solutions
    Abstract
    Growth based methods for isolating microbes as pure subcultures, followed by characterization and/or identification (ID), is the standard practice in the industry for microbiome profiling applications, such as environmental monitoring and in-process testing. However, these methods can be time consuming if the subcultures contain mixed species, or are not compatible with current phenotypic, proteotypic and genotypic ID methods resulting in a failure to generate actionable information. Targeted Amplicon Next Generation Sequencing (TA-NGS) provides an effective solution to identify microbes when current methods fail or are inadequate. TA-NGS amplifies either the bacterial 16S ribosomal gene or the fungal internal transcribed spacer gene – both well-established targets for microbial ID. By leveraging the high throughput nature of TA-NGS, thousands of sequence reads are produced per sample, allowing for resolution of mixed species samples when matched against a curated database for identification. We will present an end-to-end framework of the wet lab and bioinformatics and discuss best practices. Successful identification results were generated for a variety of bacterial and fungal samples (~150) including pure isolates, mixed species, and isolates with sequence variants.
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  • Real-Time Response from an Online Microbial Analyzer to Monitor the Health of the Water System

    Good Manufacturing Practice (GMP) guidelines are essential in the pharmaceutical industry to maintain product quality and safety. To help ensure compliance with GMP principles, companies seek new Process Analytical Technology (PAT) instruments to improve safety and quality controls and identify compliance challenges before Out-Of-Specification (OOS) conditions arise. In the specific case of microbial enumeration in compendial water systems, the analytical response time of the traditional plate count method is measured in days. A more rapid response in identifying that compliance is being met is an opportunity to complement plate counts for bioburden monitoring. In this poster, we will talk about how the 7000RMS analyzer, when used in parallel with plate-count data, allows users to make informed decisions regarding the operations of their water system. I was inspired to present at the PDA Microbiology conference, people come from all aspects of the pharmaceutical companies to learn how they can improve their manufacturing conditions and monitor their systems so they can ultimately produce life-saving drugs with better confidence and less cost. Risk management is a top priority for the audience in this conference, and hence, we wanted to present to them how they can manage their risk with bioburden analysis of their source water by using the cutting-edge technology of real-time microbial detection analyzer from Mettler Toledo. This work will help users of this device to better manage their overall water system by giving them continuous real-time data, which will help them to assess the continuous bioburden measurements as a response to the operation of their water system, process and contamination control strategies of the water system, real-time response to adverse events in the water system, reduce time and cost associated with root cause analysis with the Rapid Microbiological Method, and continuous monitoring to provide process understanding and improvements of water system operation.
    Arundhati Samanta, MA, MBA
    Arundhati Samanta, MA, MBA
    Senior Global Product Manager
    Mettler Toledo
    Abstract
    The poster explores the benefits of using an online microbial detection analyzer for water systems. By providing real-time measurements without the need for sample collection and preparation, the analyzer offers increased transparency and better process control while reducing operation costs associated with water monitoring. The online microbial detection analyzer provides continuous measurements responding to water consumption and relevant activities in the water system. The measurements serve as a microbial profile of the water system, enabling efficient identification and resolution of adverse events. The online microbial detection analyzer also enables early detection of microbial contamination, reducing the risk of production loss. The analyzer is highly sensitive and can detect small changes in microbial populations that traditional plate counting may miss. The real-time monitoring also allows for timely corrective actions, ensuring the water system remains in optimal condition. Traditional plate counting requires 5-7 days for results and does not provide a true understanding of the water loop profile. Using online analyzers in conjunction with plate counting can lead to better overall contamination control strategies. Overall, the use of an online microbial detection analyzer offers a more comprehensive and efficient approach to water monitoring and management.
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  • Use of Naturally Occurring Endotoxins (NOE) for the Release of No-Biological Drug Product Affected by Low Endotoxin Recovery (LER) in LAL Kinetic Assay

    Roberto Santucci
    Roberto Santucci
    Microbiology Quality Control Validation Specialist
    Thermo Fisher Scientific
    Abstract
    Low Endotoxin Recovery (LER) studies are intended to determine any potential endotoxin masking effect of sample matrices tested using the Limulus Amebocyte Lysate. The failure in endotoxin recovery from drug products (DP) may lead to endotoxin contamination not detected at release and pyrogenic products distributed for commercial uses. According to PDA Technical Report 82, LER study is performed spiking DP with Reference Standard Endotoxin (RSE) as preferred standards. Naturally Occurring Endotoxins (NOE) may be used as further assessment. According to the regulation LER study is mandatory only for biological DP. In the present study a no-Biological DP showed LER phenomenon. The product spiked with RSE showed a spike recovery below 50% at 24 hours. The most common mitigation strategies (i.e. use of MgCl2, MgSO4 and dispersing agent) were found to be not suitable for LER troubleshooting. Further investigations on this LER effect have highlighted that the masking activity was related exclusively to the Active Pharmaceutical Ingredient. No LER effect has been observed spiking the DP with NOE at time zero, 24 and 48 hours. LER effect observed in no-Biological DP and the use of NOE as concrete investigation option might lead to a batch release in a non-regulated scenario.
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  • Accurate Identification of Microbial Contaminants: Case Study of Aspergillus and Penicillium

    Cindy G. Serrato Zavala
    Cindy G. Serrato Zavala
    Research Associate I
    Charles River Laboratories
    Abstract
    Mold contamination in manufacturing facilities can be a serious issue for pharmaceutical and life science companies. Not only does it endanger patient safety, but it can also cause product recalls, financial and reputational harm, and regulatory ramifications. Rapid, accurate, and reliable microbial identification provides understanding of the environmental quality and acting on to avert risk and contamination. In this study, we analyze two fungal genera: Aspergillus and Penicillium that are frequent fungal contaminants, producing mycotoxins and with the ability to cause human infections. Identification of organisms within these genera can be tricky across different platforms due to species diversity. A total of 14,687 Aspergillus and 14,827 Penicillium samples were identified across Accugenix® global labs in 2023, 23.5% of these samples were identified using proteotypic method and the remaining by genotypic. More than 100 unique species groups were identified, for each genus, during the process. We further evaluate the potential of these organisms to be conclusively identified using three different identification platforms and compare species diversity across multiple databases. The distribution of these critical organisms across Accugenix® global labs emphasizes the necessity for robust cross functional approach to identify the organisms confidently to strategically nullify any contamination risks.
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  • MylcMAT™: Pyrogen-Detection System Using Immortalized Human Monocyte Cell Line

    We developed a new monocyte-activation test (MAT) system, MylcMATTM ver.2, that is useful in evaluating pyrogen-contamination in the sample of interest, in less than five hours. Generally, the current MAT systems measuring inflammatory cytokines as indicators requires two days to obtain the results. In this study, we tried to develop a new MAT system that complete the evaluation within a short period. Also, using MylcMATTM ver.2, you will know immediately if your samples contain pyrogen. A sequential measurement of double parameters (two inflammatory cytokines) for pyrogen detection in the sample of interest is also possible as an option, leading to more reliable results.
    Jun Shimizu, PhD
    Jun Shimizu, PhD
    Director/Executive Officer
    MiCAN Technologies Inc.
    Abstract
    Monocyte-activation test (MAT) utilizes human peripheral blood mononuclear cells (PBMCs) and/or monocyte cell lines for the detection of pyrogens. We have the platform to establish immortalized human monocyte cell lines, Mylc, from PBMCs or iPS cells. We have previously reported a novel MAT using Mylc cell line, MylcMAT™. Mylc cells were cultured with LPS or several non-endotoxin pyrogens (NEPs: Pam3CSK4, Poly(I:C), Flagellin, HKSA). The amount of IL-6 in 20-hr culture supernatants (SNs) was measured by ELISA, and the reactivity against LPS (LOD = less than 0.025 EU/mL final) and NEPs examined was confirmed. Here we report the improved MylcMAT™ ver.2 using 3-hr culture SNs instead of 20-hr SNs, with 1.5-hr cytokine (TNFα, another inflammatory cytokine)-detection system, instead of ELISA, namely less than 1-day assay. MylcMAT™ ver.2 maintained low LOD (less than 0.025 EU/mL final) and reactivity against several NEPs examined. Both MylcMAT™ original and ver.2 are utilizing freshly thawed Mylc cells, therefore, are less laborious and without the need to maintain cells. This MylcMAT™ will be useful in evaluating pyrogen-contamination in the sample of interest within a short-time.
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  • Gaining Efficiencies in Bacterial Endotoxin Testing with the Adoption of Annex 1

    Hayden Skalski
    Hayden Skalski
    Global Product Specialist
    Veolia Water Technologies and Solutions
    Abstract
    Annex 1 encourages pharmaceutical companies to adopt new and innovative technologies in order to streamline their manufacturing processes. As well, companies are continually looking to create more sustainable laboratories. Using microfluidics and centripetal force, a new BET platform allows for assay set up in 85% of the time it takes to set up a traditional 96-well microplate; uses up to 90% less LAL; and is fully automated. It increases efficiency and assures precise and accurate results, allowing manufacturers to meet Annex 1 and sustainability goals while remaining in full compliance with regulations to assure patient safety. In this session, we’ll also review a case study illustrating how a manufacturer can successfully transition to the new BET platform.
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  • Comparability Study between the MycoSEQ™ Plus Mycoplasma Detection Kit and Compendial USP<63> Test

    I was inspired to do this because the compendial mycoplasma test requires 28 days to complete. There are significant advantages of validating alternative, rapid methods, including improved efficiency, the ability to respond to contaminations faster, and shorter lot release times. The tMycoSEQ Plus Mycoplasma Detection Kit could be a suitable alternative to the compendial mycoplasma test. Once validated for a product, this assay would provide greater efficiencies in mycoplasma testing, particularly in the time to results and subsequent lot release. For short shelf-life products like cell and gene therapies, this enables patients to be treated sooner.
    Kevin Susice, MS
    Kevin Susice, MS
    Project-Based Scientist
    Bionique Testing Laboratories
    Abstract

    There is high demand for rapid, automated alternative microbial detection methods to streamline workflows and reduce turnaround times. The MycoSEQ™ Plus Mycoplasma Detection Kit is a probe-based qPCR assay that can improve workflow efficiency and shorten turnaround times compared to compendial culture-based methods. According to the manufacturer, “[this], streamlined workflow typically delivers actionable results in less than 5 hours, enabling early detection of mycoplasma contamination.”

    We performed studies to assess comparability between the MycoSEQ Plus lot-release method, for supernatant or spent media production harvests, and the compendial USP &#60; 63> method.  The detection of seven species of viable mycoplasma from CHO bioreactor bulk clarified harvests (removal of cells by centrifugation and filtration) was evaluated at ≤ 10 CFU/mL using both testing approaches. Due to volume constraints, the methods tested different preparations of the test article.

    The results demonstrated that the non-spiked sample was negative for mycoplasma and all seven species were detected in the test article by both methods, thereby establishing comparability.

    MycoSEQ Plus delivers results in hours versus the 28 days required for the compendial method, offering an efficient workflow with simplified sample prep and built-in assay controls.

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  • An Annex 1 Compliance Approach Using Site Internal Audits

    Margaret A. Targove, PhD
    Margaret A. Targove, PhD
    Director, Quality Assurance
    Merck & Co., Inc.
    Abstract
    Execution of Annex 1 focused site audits, using a comprehensive protocol that mimics inspectors' expected challenges, offers the following benefits: A) Deep-dive auditor/site personnel upskilling in Annex 1 requirements; B) Applying effective audit techniques to detect Annex 1 compliance concerns; C) Strengthening of partnerships between Quality and Operations (microbial/sterile SMEs); D) Management evaluation of compliance status across sites; E) Trend analysis of failure modes across sites for holistic CAPA implementation.
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  • Factors to Consider in a Risk-Based Approach to Dispositioning Underdosed Radiation-Sterilized Product

    Zabrina Tumaitis-Namba
    Zabrina Tumaitis-Namba
    Technical Director
    Sterigenics
    Abstract
    For radiation sterilization, what is considered conforming product with respect to delivery of the minimum sterilization dose? For example, is 24.9 kGy conforming to a 25 kGy minimum sterilization dose? Logically, it seems that a 24.9 kGy dose does not incorporate any potential negative impact to patient safety. However, where is the cut-off point?

    Answers to these questions will be provided through a mathematical assessment including reverse calculating the corresponding sterilization dose based on the verification dose applied. There will be a discussion on rounding SAL values regarding whether the SAL must be rounded to one tenth of the exponent, or to a whole number exponent. Additionally, other factors will be discussed that could be addressed in a risk assessment such as how the dose substantiation data and PQ dosimetry can be leveraged to better understand the potential risk to the patient.

    If an industry-wide rationale can be established to easily assess product that is slightly underdosed, it could result in significant cost and time savings when these events occur as well as being able to provide these health care products to patients.
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  • Meaningful & Measurable Risk Assessment Tools for Environmental Monitoring Site Selection Program

    Meaningful & Measurable Risk Assessment Tools for Environmental Monitoring Site Selection Program focuses on risk assessment methodologies for environmental monitoring, specifically aimed at determining appropriate sampling sites in classified areas such as an aseptic processing facility. Environmental monitoring remains a critical focus in our industry, with many companies struggling to implement risk assessment methodologies and tools that are straightforward, manageable, and reproducible year over year. This poster provides practical tools and insights that can be immediately applied to EM programs throughout the industry, while also explaining the importance of the sequence of risk assessment activities in achieving successful outcomes. Sharing information is the best way to ensure industry alignment and allows everyone to achieve the level of quality and compliance needed for product safety. This poster represents a refined approach for EM risk assessment methodologies that has been proven successful over several projects in different markets in the past three years. Sharing detailed process steps, scoring tools and visuals can encourage other microbiologists to take on this method, update their procedures and ultimately, ensure that a robust EM program is in place at their facility.
    Vanessa Vasadi Figueroa, MA
    Vanessa Vasadi Figueroa, MA
    Chief Microbiologist
    VVF Science
    Abstract
    The role of Environmental Monitoring has evolved alongside the manufacturing processes and filling technologies its aims to monitor, and so should the risk assessment tools we implement for establishing this important program. Sample site selection, appropriateness of sampling methods, sampling volumes and sampling frequencies are all important components of contamination control for a facility and must be evaluated as appropriate using a robust risk assessment. The types of environmental monitoring required for a robust program will vary based on the type of operation, frequency in which that operation is performed, and the level of risk associated to the process. Learn how to develop a meaningful risk assessment and include measurable risk rankings for 6 applicable categories in biopharmaceutical manufacturing. The process for scoring each of the 6 categories, systematic evaluation of contamination probability and example outcomes will be shared for theoretical EM sites mapped throughout an ISO 5 and 7 cleanroom area, thus ensuring adequate criteria and fair assessment are applied in each case. The methodology for this risk assessment tool will be demonstrated as suitable for environmental monitoring programs during initial site qualification, when evaluating EMPQ results, or when periodically updating the requirements for monitoring during routine operations.
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  • Validation of the Mango System vs Compendial USP <61> Bioburden Testing

    Sophia Wei
    Sophia Wei
    Scientist I
    Mango
    Abstract
    In April 2024, a top-3 pharmaceutical company conducted a study to compare the Mango system with the compendial USP  < 61> bioburden test. 5 microorganisms were tested at concentrations of 10 and 100 colony-forming units (CFUs). Results suggested the Mango system’s non-inferiority to the compendial method. Unstressed yeasts and bacteria were detected within 5 hours, and Aspergillus mold within 10 hours.
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  • Recombinant Factor C Characterization Studies Confirm the Published Structure and Function of Manufactured rFC

    I wanted to tell the story of the work we have done that gives confidence in the reagents we produce for QC customers.
    Kevin I. Williams, BS
    Kevin I. Williams, BS
    Senior Scientist
    bioMérieux
    Abstract
    With the recent increase in interest and use of rFC, users can gain confidence from understanding the characterization studies that have gone into assuring the zymogen's correct structure and function.  Extensive studies have been performed to confirm the structure and utility of manufactured recombinant factor C zymogen.  These complex studies go well beyond the routine release of each protein's historical batch record.  Historical batch Western blots are shown side by side as well as verification of the amino acid structure that was found to agree with that given by Muta et al. in1991.  The glycosylation pattern has also been studied and the results are shown.  Whereas LAL has been heavily regulated since its adoption because it is a blood product, rFC relies on manufacture and adopting user dedication to assure consistent quality.
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