PDA Pharmaceutical Microbiology Conference 2025 Posters

There is ongoing debate regarding the most appropriate type of endotoxin to use in Low Endotoxin Recovery (LER) Hold Time Studies. PDA Technical Report 82 (TR82) recommends Reference Standard Endotoxin (RSE) and Control Standard Endotoxin (CSE) as the primary choices. Natural Occurring Endotoxins (NOEs) may be included as supportive data. However, their relevance remains controversial due to concerns that they may not accurately represent the type of endotoxin contamination likely to be encountered in actual drug products. In this study, we investigated the use of unprocessed water samples collected from Water for Injection (WFI) systems. These samples, without undergoing any purification, were used to spike a formulation matrix relevant to biopharmaceutical products. The goal was to evaluate their behavior and reliability in LER studies.

Environmental monitoring (EM) programs have long relied on action and alert limits as the primary drivers for response and investigation. While these regulatory thresholds remain essential (and required), they are not sufficient on their own to provide a complete picture of cleanroom control. This poster challenges the industry’s traditional focus on excursions and proposes a more holistic, modern approach to EM data trending—one that aligns with the evolving expectations outlined in EU Annex 1. Key tools such as Contamination Recovery Rate (CRR), percentile-based evaluations, and a combination of methods offer richer insights into process capability, operator consistency, and potential contamination risks—even when results remain within accepted limits. These statistical approaches acknowledge the semi-quantitative nature of microbiological data and accommodate its non-normal distributions more effectively than conventional means and standard deviations. Attendees will gain a deeper understanding of how to integrate these techniques into existing EM programs, transforming environmental data from a compliance checkbox into a meaningful indicator of cleanroom health and performance.

The bio-pharmaceutical world has moved drastically towards short-lived personalized cell and gene therapy products in recent years. With the new quality control requirements, several new rapid microbial detection methods have been developed. USP < 1071> states, “The ability to detect contamination, in real-time, prior to the administration of the short life product may be considered more important than detection of a single colony-forming unit (CFU) in the product.” However, the new methods strive to detect the holy grail of 1 CFU. Will this ever be possible, or is it even necessary? In this poster, we will present the first digital (droplet) PCR based approach for sterility testing which is intended for testing of cell and gene therapy products, elucidate the critical steps, and highlight the many benefits of this approach. We will address the handling of background signals of a bacteria digital (droplet) PCR experiment and explain how to clearly differentiate between the background and real positive signals. We will demonstrate how the groundbreaking digital (droplet) PCR technology allows a new level of precision in rapid sterility testing. We propose an open discussion on this advanced method and its potential for QC testing and release.

Low Endotoxin Recovery (LER) is described as the inability to recover a known concentration of Control Standard Endotoxin (CSE), or Reference Standard Endotoxin (RSE) over time. It is a two-part reaction requiring a chelator and a surfactant. The chelator strips away the divalent cations causing the LPS aggregate to disassemble. Since Lipid A of LPS is hydrophobic, polysorbate encapsulates the LPS in a lipid micelle. This form of endotoxin is unable to react with Factor C of the Limulus Amebocyte Lysate. The FDA and EMA require all new biologic drugs submitted for approval to be assessed for the presence of the LER phenomenon. Organisms used to produce RSE/CSE standards are grown under high nutrient conditions including divalent cations, then purified. The divalent cations allow the organisms to develop “salt bridges” between adjacent lipopolysaccharide molecules. However, organisms in purified water systems are not exposed to divalent cations in their environment and adapt to this environment via the PhoP/PhoQ system on the bacterial surface, holding the outer membrane together. These adapted organisms’ endotoxin is not affected by the chelator, and the aggregates remain intact. This is the endotoxin that will contaminate the product if a breach in the water purification system happens.

The ability to deliver safe medications and vaccines, free of microbial contamination, is dependent on modern methods to identify bacteria that range from biochemical to genotypic in nature. Conventional genotypic methods for bacterial identification generally rely on small parts of the genome, but advancements in DNA sequencing technologies have reduced the time and cost needed to sequence whole genomes. In order to increase resolution in microbial identification and reduce time-to-result compared to conventional methods, we have developed a workflow for rapid identification of bacteria using Oxford Nanopore DNA sequencing that can reconstruct whole genome sequences with >99.8% accuracy to reference sequences. The reconstruction of an entire genome sequence enables high-resolution identification of bacteria. For example, the platform can rapidly differentiate closely related strains of Shigella and E. coli through taxonomic assignment using average nucleotide identity (ANI) and multi-locus sequence typing, and investigations into sub-strain-level differentiation using ANI are ongoing. The analysis of genome data normally requires a highly trained analyst, but work is underway to automate these computational processes as part of Merck’s patent-pending ViruScreen platform which enables multi-omic analytical research through an easy-to-use web portal.

A mold investigation is a focused, science-driven effort to uncover how contamination gets in, spreads, and survives—no assumptions, no shortcuts. It starts with a thorough analysis of mold recoveries across environmental, process, and product-related factors. Isolates are classified by fungal division, with reproductive state, source, and growth behaviour assessed. From there, environmental monitoring data and product failures are examined to complete the full picture. Facility conditions—structural integrity, airflow, material flow, and sanitation—are evaluated to identify weak spots mold might exploit. Operational procedures and personnel practices are scrutinized, revealing possible gaps that can fuel contamination. By combining these elements, the investigation doesn’t just react to mold—it anticipates it. This approach lays the groundwork for targeted, effective controls that protect both product integrity and environmental quality in regulated spaces.

In microbiological quality control, manual observations—such as colony counts, Gram stains, and growth assessments—play a critical role in ensuring product safety and compliance. Data integrity is a key issue, with the frequency of warning letters rising annually. To determine the appropriateness of a single manual observation, it is essential to assess the risk. Cambrex conducted a formal risk assessment using the Failure Mode and Effects Analysis (FMEA) tool to evaluate manual observations in microbiology. The risk question posed was: 'What is the risk to patient safety and to cGMP decisions if a second analyst verification is not performed on manual microbial observations?'. The analysis considered key risk factors including observation complexity, analyst competency, historical deviation trends, and the potential impact on product quality and patient safety. Controls such as standardized training, competency assessments, and procedural enhancements were assessed for their ability to mitigate associated risks. Cambrex assessed the feasibility of implementing a streamlined review process in specific scenarios, ensuring compliance with Good Manufacturing Practices (GMP) while maintaining the reliability of microbiological data. Interestingly, medical device bioburden enumeration from a single manual observation was deemed low risk whereas pharmaceutical product bioburden necessitated a second analyst verification.

Accurate microbial identification remains a crucial component of quality control and safety in the pharmaceutical industry. Among various methods of microbial identification, proteotypic-based MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time-of-Flight) technology has emerged as a rapid, high-throughput, and cost-effective method for microbial identification. However, the strength of this method hinges on the robustness and representativeness of the database, particularly its ability to capture intra-species variability. For this study, microbial library entries were generated from microbes isolated from diverse geographic regions to assess the impact of local diversity on identification. Findings showed that identification outcomes by these entries vary by species. For example, approximately 10% of Sphingomonas colocassiae and 13% of Penicillium brevicompactum identifications relied on intra-species geographic diversity entries. Whereas ~83% of Aspergillus westerdijkiae and ~78% of Beauveria pseudobassiana identified matched to similar intra-species entries. We further breakdown the impact of these database entries to identify organisms in their regional zone. Notably, regionally sourced entries assist in providing reliable identification for samples in that geographic region. This highlights the relevance of incorporating geographically diverse strains into proteotypic libraries. As microbial diversity varies across manufacturing environments, maintaining comprehensive, regionally representative databases is essential for ensuring accurate identification and effective contamination control.

EU GMP Annex I, Section 4.36 specifically states that “Cleaning programs should effectively remove disinfectant residues”. Although residue in controlled environments, and disinfectant residue in particular, has been around since the advent of cleanroom disinfection, there is currently an increased sensitivity towards understanding and mitigating cleanroom residues.

This talk will focus on the science and microbiology of scientific residues including potential impact on rotational sporicides, the likelihood of trapping microorganisms and best practices for addressing residue in the cleanroom. The active ingredients and surfactants that make up disinfectant residue are not inherently bad, but instead key components of a complex formulation. By understanding the nature of disinfectant residue and their impacts, continuous improvements can be made to optimize an overall cleaning and disinfection program.

Testing for mycoplasma contamination in cell banks and bioreactor cell cultures is a regulatory requirement for production of biological products. The compendial tests for mycoplasma detection require 14-28 days incubation, imposing a limitation on batch release timelines and rapid containment in the event of a contamination. The alternative rapid PCR-based mycoplasma detection kit, MycoSEQ Plus, provides results within a few hours and meets regulatory guidelines regarding sensitivity (10 CFU/mL or the genomic equivalent of 10 GC/mL) and specificity as outlined in the European Pharmacopoeia (E.P. 2.6.7, 2007), US Pharmacopoeia (US63), and Japanese Pharmacopoeia. This presentation will discuss the evaluation of the kit's performance by testing its specificity, robustness, and limit of detection.

USP < 73> provides risk-based guidelines for the use of adenosine triphosphate (ATP) bioluminescence methods for rapid detection of viable microorganisms for short-life products. ATP bioluminescence detects the presence of viable contamination in less than seven (7) days compared to the required 14-day compendial sterility test. Determining the generation time of the slowest growing microorganism spanning bacteria, yeast and filamentous fungi in the presence of a product helps establish the incubation time of microbial detection. As per USP < 73>, inoculum levels for all tested microorganisms were less than 10 CFU. Appropriate enrichment broth and incubation conditions were followed, and product matrices such as mammalian cells and DNA plasmids were evaluated. Samples were filtered with a hollow fiber sampling tip, which captures any bacterial, fungal and yeast cells while removing cellular debris and any non-microbial ATP from the sample being tested. Microbial cells are eluted off the filter and collected in a collection tube for testing with an ATP-bioluminescence method based on the luciferin-luciferase cascade emitting light that is measured with a luminometer. This study demonstrates practical ways of identifying the slowest growing organism, determining generating time, and calculating time to detection, which are all relatively novel concepts for QC Microbiology Testing.

When the current revision of Annex 1 was made enforceable in August of 2023, industry was faced with new regulatory expectations and many decisions as to how to achieve compliance. Development of a holistic contamination control strategy, being one of the major challenges. A robust contamination control program is a pillar of a contamination control strategy. The initial step to demonstrating the effectiveness of a control program is disinfectant qualification also known as disinfectant efficacy testing (DET).

Annex 1 introduced multiple new regulatory expectations for DET study execution and there is not yet consensus in industry on best practice to comply with these expectations. One of the primary areas of uncertainty surrounds demonstrating efficacy of disinfectants, “in the manner in which they are used”. This presentation will evaluate through a data-based case study, if mechanical action and application method inclusion in DET coupon studies or in situ field studies is the most scientifically sound, best practice. This presentation will also cover the Annex 1 expectation surrounding liquid chemical material transfer validation and best practices and how to incorporate DET into your contamination control strategy.

With the introduction and implementation of rapid respiratory-based sterility methods, the need to ensure consistent and reproducible results for Cutibacterium acnes has increased dramatically. As more products are introduced to markets requiring validation with this organism, a robust and proven methodology is needed that allows any analyst to generate robust, reproducible recoveries. Experimentation with this organism was performed to develop a process that ensures consistent recovery for this organism. 

Testing involved assessment of different type strains from various vendors as well as numerous preparations from each vendor and in house isolates. Strain / sample preparation was assessed to determine if commercial preparations at ≤ 100 CFU were more consistent than those which required dilution to reach the desired inoculum level. Additionally, variations to the inoculum procedure itself (e.g., needle gauge used in inoculation) were assessed to determine specific inoculation techniques that provided the most consistent organism recovery. A technique was developed that allowed inoculation to be performed without the need for single source vendor materials or an overly complex inoculation procedure. The technique was then disseminated across the network to allow groups utilizing rapid sterility methods achieve similar reproducible and consistent results with C. acnes and other compendial microbes.

Lonza wanted to achieve paperless quality control (QC) laboratories using automated digital systems. As part of this ambition, Lonza sought an end-to-end (E2E) automated solution to optimize environmental monitoring (EM) at four of its Cell & Gene Manufacturing sites across North America, Europe, and Asia. Successful implementation required an innovative approach that harmonized processes across different sites through digital and automation transformation. Lonza successfully integrated the MODA-EM® Module with Rapid Micro Biosystems' Growth Direct® System, combining paperless processes with automated microbial enumeration. This innovative approach is revolutionizing the delivery of critical medicines, accelerating product release, enhancing compliance robustness, and unlocking significant cost savings.

Lilly is evaluating biofluorescent particulate counting (BFPC) technology to simultaneously replace conventional active and passive (continuous) microbiological air monitoring as well as total non-viable particulate monitoring in Grade A critical filling zones. Microorganisms produce naturally fluorescent compounds, such as riboflavins and NADH, which emit detectable fluorescence when excited by a laser at specific wavelengths. BFPC is a non-culture-based technology specifically designed for real-time detection and quantification of airborne microbes based on excitation / detection of the unique fluorescence given off by those cellular components. Expected benefits implementation include:

• Enabling real-time discard strategies to reduce sterility assurance risk
• Improved process knowledge associated with environmental and contamination control.
• Streamlining of filling line design and monitoring through replacement of all existing conventional microbiological and particulate sampling equipment.
• Reduced EM media quantities required in Grade A filling zones.
• Elimination of inherent interventions required to perform passive and active microbiological air monitoring.
• Improved data integrity for EM sampling results through automation.

This seminar will cover utilizing a contamination control strategy with a risk-based approach to materials transfer into cleanrooms, RABS, isolators, and BSC’s. There will be a focus on how to control hard to kill fungal spores such as Aspergillus and Penicillium as well as bacterial spores and viruses. Recent case studies from the past few months in ATMPF facilities and compounding pharmacies will be discussed in relation to materials transfer. Published data will also be mentioned to convey effective methods in control bioburden utilizing VHP, sporicidal chemistries, and disinfectants into the APA. This presentation will be a holistic approach to controlling bioburden from entering cleanrooms, BSC Hoods, RABS, and Isolators. New industry case studies in disinfectant field trials will also be covered and discussed in detail based on recent industry publications.

Automated microbial detection systems are becoming increasingly essential in pharmaceutical manufacturing to enhance efficiency, accuracy, and compliance with Good Manufacturing Practice (GMP) standards. The APAS Independence System, developed by Clever Culture Systems, was evaluated at Bristol Myers Squibb (BMS) to assess its suitability for environmental monitoring applications. The APAS system is capable of automated plate counting of both 55 mm and 90 mm plates, following an offline incubation at routine EM parameters. The study compared APAS automated colony counts to traditional manual counts for compendial strains and environmental monitoring samples across multiple weeks of EM sampling. Performance metrics for both plate sizes were analyzed, such as accuracy, precision, false negative and false positive rates, and robustness. The APAS system demonstrated high accuracy (≥70%) and reliability for most tested organisms. Therefore, the system is being implemented for GMP use, with a validation approach that combines vendor validation of the Artificial Intelligence with site specific instrument qualification. This evaluation provides insight into the capabilities and limitations of automated colony detection, informing future digitalization strategies in microbiological quality control.

Roche has implemented an automated endotoxin system (Lonza PyroTec® PRO) in commercial Quality Control microbiology. The key drivers for the implementation were to reduce ergonomic risk, save time and resources, increase data integrity, simplify training, digitalization, digital data flow and increase the right-first-time rate. The strategic approach to implementation and conclusions from routine testing shall be shared and discussed.

USP < 1223>, EP 5.1.6 and PDA TR33 all require Specificity for the validation of alternative qualitative sterility test methods. Identifying a panel of organisms to perform Specificity testing and evaluation of RMMs beyond the pharmacopeial recommended organisms has been a challenging endeavor given the varying nature of environmental isolates and the inherent risk that what has been recovered at your site today may be different than what is recovered in future. This study outlines the approach taken by Rapid Micro Biosystems in identifying and testing a broad and comprehensive range of organisms to support the development of the Primary Validation for Growth Direct® Rapid Sterility to include those described in the regulatory chapters as well as those commonly cited by industry literature as being challenging and relevant. This study also aims to characterize the effects of a common stress application on the Growth Direct Rapid Sterility System’s detection capabilities.

Bacterial endospores represent a significant challenge to the pharmaceutical industry due to their presence in the environment, resistance to many commonly used inactivation procedures, and difficulty in culturing using traditional plating methods. This may result in the inadvertent release of contaminated products that may present health concerns for the patient. As a result, the use of a bio-fluorescent particle counter (BFPC) may prove advantageous for the detection of both water-borne and air-borne spores as their detection is not dependent on traditional culturing methods. In this study, we investigate the ability of an online water bioburden analyzer (OWBA), a specific class of BFPC, to detect Bacillus subtilis spores in pharmaceutical-grade water and present the results as auto-fluorescence units (AFU’s) per B. subtilis spore. The spores were a commercial grade spore preparation and were previously quantified per manufacture recommendations. The results show that the OWBA can detect B. subtilis spores with an accuracy of 1.25 AFU per spore. The limit of detection was determined to be 1 spore/mL with a linearity greater than 0.9025 up to a concentration of 100 spores/mL. This data shows that OWBA’s are a rapid and effective tool for the detection of bacterial endospores in pharmaceutical waters.

The cleaning of process and environmental residues is a crucial aspect of contamination control strategy (CCS) and is mandated by several cGMP regulations. Environmental residues, which remain on surfaces post-disinfection, can accelerate the deterioration of manufacturing area surfaces and pose safety hazards, such as slip and fall risks for operators. The accumulation of environmental residues can also have an impact on the performance of disinfectants and may harbor microorganisms. This study evaluated textiles and rinsing agents and the combined effect they had on the removal of disinfectant residues on various common cleanroom surfaces. Variables included different cleaning solutions and a range of cleaning textiles (mops/wipes) with diverse substrates and textile constructions. The evaluation was conducted quantitatively by incorporating a fluorescent tracer dye into the disinfectant. Pixel analysis measured surface residue after the disinfectant was applied and dried. Subsequently, combinations of cleaning solutions and substrates were used to remove the disinfectant residue, and pixel analysis was performed again to determine the remaining residue. This poster will present the data visually, to provide insight into the considerations that must be made when establishing a residue removal regimen in critical cleanroom spaces.

The Mango system was evaluated by spiking Fluid A with seven different microorganisms: Clostridium sporogenes ATCC 11437, Cutibacterium acnes ATCC 6919, Staphylococcus aureus ATCC 6538, Bacillus spizizenii ATCC 6633, Aspergillus brasiliensis ATCC 16404, Pseudomonas paraeruginosa ATCC 9027, and Candida albicans ATCC 10231. For each microorganism, two replicates were prepared on spread plates, Oasis TSA cartridges, and Mango Cartridges. Each microorganism was tested three times by three different analysts. The inoculated control plates (Oasis and spread plates onto TSA medium) were incubated at 30-35°C for 24h to 7 days. The Mango test plates were incubated for 16 -160h. The study provides comparative data on the time to detection and recovery rates for the tested microorganisms. The results indicated that the Mango system consistently showed faster time to detection for aerobic organisms than traditional methods, however incubation time and conditions were microorganism-dependent, particularly for anaerobic bacteria such as C. sporogenes and C. acnes. In addition, this study showed that the rates of recovery between the Mango and traditional systems were equivalent and provides data on the filterability of different matrices. The findings indicate that the Mango system has potential to rapidly and consistently recovery microorganisms from biopharmaceutical samples.

In the pharmaceutical industry, image analysis plays a crucial role in microbial identification. Traditional parametric algorithms, while effective for tracking microbial growth over time, struggle with differentiating between bacterial and fungal species due to their rigid predefined rules. Machine learning, particularly deep learning, offers a powerful alternative by learning complex patterns from large datasets, enabling more accurate and adaptable classification. The goal here is to explor the limitations of classical algorithms, the advantages of AI-driven approaches, and the methodology for building a robust training dataset to enhance model performance. A case study on automated mold identification on petri dishes will illustrate these concepts in a real-world application.

Recent revisions to EU GMP Annex 1 have significantly reshaped expectations for contamination control strategy (CCS), requiring manufacturers to adopt a holistic, risk-based approach. This poster presents a real-world case study from a global pharmaceutical company that has implemented a novel, structured framework for CCS development and deployment. The approach includes standardized global risk questions, tools for consistent assessment, and guidance for lifecycle management. The result is local CCS programs that align with the global CCS strategy with execution through scalable templates and practical implementation tools, enhancing compliance and supporting long-term contamination control.

This session will explore the integration of recombinant Cascade Reagents (rCR) with centripetal microfluidic technology. We will detail our multi-matrix approach for comparing LAL and rCR on different sample types and how we assessed performance metrics such as sensitivity, specificity, and reproducibility across diverse sample types. We also address key technical challenges and practical considerations encountered during this research. Finally, the results of the study will be presented.

For more than 40 years, the Limulus Amebocyte Lysate (LAL) test has served as the standard method for bacterial endotoxin Testing (BET). The critical proteins required for endotoxin detection in LAL assays are sourced from amoebocytes—blood cells extracted from horseshoe crabs. Animal-free reagents have been developed to support conservation efforts and the 3Rs (Replacement, Reduction, and Refinement). One such alternative is the recombinant cascade reagent (rCR), which contains three recombinant proteins that replicate the natural enzymatic cascade found in horseshoe crab amoebocytes to detect bacterial endotoxins. This study assessed the equivalency of rCR to FDA-licensed LAL reagents using 563 pharmaceutically relevant samples. Of these, 134 samples were contaminated with natural environmental endotoxin (NEE), allowing for a direct statistical comparison. Results showed that the rCR assays were equivalent in performance to FDA-licensed LAL assays, detecting endotoxin at similar levels under real-world conditions. Equivalency was demonstrated using methods consistent with those outlined in compendial guidance for bacterial endotoxin testing.

Mycoplasmas, a type of bacteria, are potential contaminants of cell cultures that can be difficult to detect with traditional microbiology methods. Regulatory agencies worldwide require that certain biological products, including cell-based therapies, be tested for mycoplasma contamination to ensure quality and safety.

Purpose: Leverage the Applied Biosystems™ MycoSEQ™ Plus Mycoplasma Detection Kit in a cell therapy production workflow, where sample volumes may be limited.

Methods: This protocol follows the MycoSEQ Plus mycoplasma detection system, using either 1 or 1.5 mL of the test sample in the qPCR reaction, with manual or automated sample preparation. This leverages the sensitivity guidance of 10 colony forming units (CFU) or genome copy equivalents per mL of test sample.

Results: Samples were processed by either Applied Biosystems™ PrepSEQ™ manual or automated express workflows, followed by alcohol precipitation. PCR was performed on the Applied Biosystems™ QuantStudio™ 5 Real-time PCR system."

The answer to this question is yes, and yes. The two subjects co-exist within pharmaceutical manufacturing in aseptic and sterile environments. This poster will highlight how environmental monitoring and disinfection provide continuous trending, process improvement, and final product evaluations. The processes need to be designed to identify outliers (biological and numerical) and objectionable organisms and assess cleaning protocols based on data collected. The information acquired through environmental monitoring and disinfectant efficacy studies helps to determine effective Root Cause Analysis and Corrective Action when issues with contamination are encountered. The processes, when performed and evaluated consistently will ensure that manufacturing environments are in a state of control and the products manufactured in sterile and aseptic environments are pure, safe, and effective.

This poster evaluates the Disinfectant Efficacy Test (DET) of various sporicidal solutions and chemistries used in an aseptic manufacturing site for parenteral and ophthalmic products, guided by USP < 1072>. The assessment spans three different timeframes: the initial DET evaluation and two Corrective and Preventive Actions (CAPAs) prompted by sterility failures years later. It includes an analysis of different bacterial spore formers and the impact of disinfectant supply constraints on the market. The poster discusses the risks associated with the use of disinfectants that do not meet performance standards, emphasizing their potential threat to commercial operations. Additionally, it addresses the challenge posed by microorganisms with higher natural resistance, even when their isolation frequency is low in the environmental monitoring (EM) program.

Objectionable organisms remain a major threat to pharmaceutical product safety, and among them, human bacterial pathogens stand out for their potential to impact not only product integrity and safety but also manufacturing continuity. Contamination can mean delayed releases, facility shutdowns, and steep financial and reputational costs. In a 5-year global review of over 764,040 EM isolates from 3,300+ sites, more than 800 of the 1,460 known human bacterial pathogens (Bartlett et al., Microbiology 2022;168:001269) were confirmed by DNA sequencing. These findings underscore the frequency – and risk – posed by potentially pathogenic species in manufacturing environments. Curated complementary information such as Risk Group and physiological attributes provide microbiologists with immediate access to essential information for risk assessments. However, such information does not always provide a comprehensive evaluation for assessing pathogenicity. Whole genome sequencing can provide deeper insights into the organism of concern when further characterization is required, especially when screened for genes relevant to preservative resistance, cold sensitivity, pathogenicity, or antimicrobial/disinfectant resistance. This additional testing can improve the quality of risk assessments. This approach enhances environmental control and supports better protection for both products and people.

As the pharmaceutical industry continually evolves, so do regulatory requirements for biological products and establishing in-use hold times for products in a clinical setting. Although adapted from USP Chapter < 51>, microbial challenge studies are non-compendial studies that evaluate the microbial growth potential of a product spiked with a low level of microorganisms to simulate inadvertent contamination during dose preparation. Microbial challenge studies provide information on how long drug products can be held before patient safety is at risk, the effectiveness of any preservatives and/or preservative ingredients, optimum storage conditions, and ultimately, if the drug product formulation can withstand low level contamination. If microorganisms can grow in the product during the holding period, then the intended hold time, product formulation, or storage conditions should be reevaluated. When designing a microbial challenge study, multiple factors should be considered. This includes, but is not limited to, the inherent characteristics of the drug product formulation, the storage condition(s) of the drug product, target organisms (i.e., organisms prevalent in a hospital setting), diluents, and testing time points dictated by intended hold time.

Biotherapies, and especially cell therapy products, are required to be tested for sterility and mycoplasma. Developing an analytical strategy to test for sterility and mycoplasma can be daunting. There are many variables to consider, including complex matrices with high mammalian cell density and detection of non-viable microbes. Which type of analytical testing can work in an early stage of therapeutic development and then be scaled to meet the challenges later in the production process? Leveraging rapid sterility as well as mycoplasma qPCR-based detection techniques can help provide confidence in the final product by helping to detect potential contamination earlier in the production process.

USP < 73> ATP Bioluminescence-Based Microbiological Methods for the Detection of Contamination in Short-Life Products is a new chapter in the USP and becomes effective August 2025. This new rapid method will allow even shorter sterility incubation times based on microbial detection, microorganism generation time(s), applying growth phase curves rationale and utilizing ATP bioluminescence testing. The application of microorganism time studies for medical device and pharmaceutical products will greatly benefit with urgent heath care needs by having data and results available sooner for sterility assurance and critical decision making based upon rapid microbiological methods (RMMs). This poster presentation will focus on experimental data based upon alternative microbiological methods and ATP-bioluminescence results related to microbial detection times, inoculation criteria, generation times and ATP-Bioluminescence technology on various types of microorganism types and testing conditions.

In the pharmaceutical industry, maintaining the highest standards of microbial Quality Control (QC) is critical to ensure safety and efficiency of pharmaceutical products. Precisely quantified reference microbial standards play a vital role in validating alternative microbiological methods by ensuring accurate, consistent, and reproducible results. This study evaluates the use and performance of ATCC MicroQuant™ reference material with the Growth Direct® System EM and Bioburden applications, showcasing an alternative approach to traditional microbial QC. Audience Take-Away Benefits The audience will benefit by gaining insights into how the ATCC MicroQuant™ can be a precise ready-to-use reference standard that pairs well with the Growth Direct® System. This poster highlights the benefits of a precisely quantitated reference standards when validating alternative microbiological methods such as the Growth Direct® System.

Rapid methods have secured a foothold within the pharmaceutical industry. However, we continue to evolve our technologies to meet the increasing demand of modern medicines. One of the drawbacks for using metabolic by-products as an indicator of microorganism growth is the risk of poor differentiation between biological signals from the product and microbial metabolites. A new assay was designed to deal with a higher background signal. By utilizing a biochemical ATP-depletion reagent, coupled with utilizing microbial adenylate kinase to enhance ATP production, a more refined, flexible assay is available for rapid sterility testing. I present evidence demonstrating the reliability of modern ATP bioluminescence assays for testing biological products. Data graphs will include: • Diagram of ATP depletion/AK-ATP enhancement at the cellular level. • Case studies of Applicability of an ATP-depletion reagent o User eliminates the need for low-ATP sterility media (use any media supplier you want) o Reduction of residual signal from a biologically derived product. • 3-way Equivalence/LOD data amongst 1) Compendial Sterility 2) ATP Bioluminescence 3) ATP Bioluminescence with background depletion I will close with remarks on the future applicability towards a broader user-base that may have previously been prevented from using ATP Bioluminescence for rapid sterility.

The introduction of USP < 72> offers a new regulatory pathway for shortening the release of short-lived pharmaceutical products, including cell and gene therapies, by allowing manufacturers to optimize growth conditions such as media formulation and temperature. The chapter dictates the final time to release is determined by the time to detection (TTD) of the slowest growing organism plus a provided “safety margin” formula. This formula relies on calculation of doubling time of the slowest growing microorganism which can be derived by various methods. We evaluated three methods to determine this doubling time: traditional plate enumeration, optical density, and a novel approach using the kinetics of the BACT/ALERT® system. The latter allows estimation of generation time directly from TTD curves based on CO2 production as measured by the BACT/ALERT® system. The benefits of this method include minimal handling time, offering a scalable, practical alternative to traditional methods. We compare all three methods across multiple organisms, including slow-growing anaerobes such as C. acnes, to assess applicability, repeatability, and robustness. The results demonstrate that BACT/ALERT®-based TTD analysis enables precise and efficient safety margin determination, supporting confident, faster release decisions in line with USP < 72>.

Microbial identification and strain typing are critical in pharmaceutical microbiology for ensuring product safety, controlling contaminants, and meeting regulatory requirements. Traditional strain typing methods like pulsed-field gel electrophoresis and multi-locus sequence typing have their limitations in scalability and resolution. Increased use of next-generation sequencing (NGS) techniques for microbial identification and characterization, coupled with the availability of bacterial whole genomes, advanced sequence analysis approaches such as single nucleotide polymorphisms (SNPs), core and pan-genome analysis, is opening the door for more modern approaches to microbial strain typing. However, several of these approaches are species-specific and require optimization per species or project. In this study, we analyzed conserved genes from several bacterial species to analyze the possibility of using these genes as a set of universal genes for prokaryotic strain typing. We compared the strain typing results using these universal genes to results from species-specific housekeeping genes and with species with no established strain typing methods. Our data demonstrated that these conserved genes successfully distinguished bacterial strains when compared to the results from established species-specific strain typing schemes, providing a path forward in the development of a rapid, universal strain typing solution using bacterial genomes that is not limited to a single species.

The regulations are evolving, for example EMA Annex 1, earlier versions did not mention trends, the current draft version mentions it 23 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 that 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.

Interest in rFC and rCR, which are sustainable BET reagents, has been increasing year by year. Various organizations have been considering and evaluating the equivalence of recombinant reagents and conventional LAL reagents. In May 2025, recombinant reagents were officially listed in the USP as a method to perform the bacterial endotoxin test. As a result of these developments, consideration has begun to switch from LAL reagents to recombinant reagents, resulting in a growing interest in the suitability of recombinant reagents for use in automated analyzers to reduce inter-human variability in quality control using recombinant reagents and operational errors in processing multiple samples. We evaluated the suitability of our rCR reagent, PYROSTAR Neo+, for use in our fully automated, compact endotoxin measurement system, KLANOS. As a result, the measurement results obtained using the automated analyzer were found to have reduced inter-human variability and operational errors in processing multiple samples, compared to manual pipetting. In addition, comparative verification using various pharmaceutical samples showed results equivalent to those obtained using manual pipetting. These results show that, like LAL reagents, recombinant reagents can also be adapted to automated systems and are effective in reducing inter-human variability and operational errors in processing multiple samples.

We have developed a next-generation MAT kit based on a reporter assay. In this presentation, we report on the potential for a more flexible MAT test design using our new reagent. In conventional MAT, inflammatory cytokines are typically detected using ELISA as an indicator. In contrast, the LumiMAT pyrogen detection kit detects the transcriptional activity of NF-κB, a transcription factor that induces the expression of inflammatory cytokines, using a luciferase reporter assay. Compared to ELISA-based MAT, the advantages of the reporter assay-based MAT include a significant reduction in cell incubation time due to increased sensitivity (from 24 hours to 3 hours) and the removal of labor-intensive steps (several hours for ELISA to a few minutes for the addition of luminescent substrates). For practical application in pyrogen testing, we conducted preliminary tests in accordance with EP 2.6.30. guidelines on several medicinal products subject to Rabbit Pyrogen Test and confirmed that the results met the requirements specified in the pharmacopoeia. Additionally, our method allows for reduced coefficient of variation, enabling the reduction in the number of replicates (n=4 to n=3) and increasing throughput (from a 96-well plate to a 384-well plate). These features demonstrate the potential for designing more cost-effective test protocols.

The development and existence of bioburden in pharmaceutical water systems is often misunderstood. Microorganisms are always present and have a keen ability to adapt to their environment. This is especially true in a Water-for-Injection (WFI) system, where microbial attachment and biofilm growth will occur regardless of flow rate, material of construction, turbulent flow and low nutrient conditions. While industry makes every effort to control and eliminate bioburden, traditional sanitization methods are not one-hundred-percent effective at accomplishing this objective. Additionally, because of the limitations and time to result delay of conventional plate counting, we may be at a disadvantage for assessing bioburden, causing us to use water at risk. This poster explores real-life examples of biofilm in pharmaceutical water systems, risk mitigation strategies, and how real-time microbial detection could be used as a tool for improved risk management and process control.

Sterility testing remains a key bottleneck in the release of cell and gene therapy products (CGTPs), where current compendial methods require up to 14 days, delaying batch release and increasing costs. Symcel’s calScreener+ is a novel, commercially available, phenotypic sterility testing system that enables continuous and non-destructive metabolic monitoring via isothermal microcalorimetry, delivering microbial detection results in under three days— offering the fastest reported time-to-detection (TTD) among currently available, growth-based sterility methods evaluated for CGTPs. To demonstrate system applicability, we will present application data showing microbial detection in the presence of high-density eukaryotic cells (10⁶–10⁸ cells/ml), without enrichment or sample processing. This illustrates the method’s compatibility with complex, cell-rich matrices typical of CGTPs. The primary focus is validation data from the calScreener+ Three-Day sterility test, covering:

• Limit of detection
• Detection times for fast- and slow-growing organisms
• Specificity across 30 relevant microbial species
• Comparability to compendial methods
• Robustness and ruggedness under varied conditions

We present a rapid sterility testing method targeting ribosomal RNA (rRNA) to enable sensitive and specific detection of microorganisms. The assay follows a simple three-step protocol: (1) activation of microbial cells with concurrent inactivation of DNA, (2) lysis and RNA extraction using magnetic particles, and (3) detection via one-step real-time reverse transcription PCR (RT-PCR). The total assay time is approximately 7 hours. Validation using six compendial organisms listed in USP < 71> demonstrated successful detection at a sensitivity of 10 CFU/mL. The method is also compatible with samples containing mammalian cells, maintaining performance in complex matrices. A key feature of this system is the rigorous degradation and removal of residual DNA, effectively reducing the risk of false-positive results that may arise in conventional DNA-targeting assays. By focusing on rRNA, which reflects microbial viability, this method provides a more accurate sterility assessment within a significantly shorter time frame compared to traditional culture-based tests. The approach offers a promising solution for rapid microbiological testing in pharmaceutical quality control and is designed to align with current regulatory expectations for alternative methods.

Viral filtration by ultrafiltration membranes is a critical step for ensuring viral removal in many biomanufacturing process streams. However, the nature of the feed solution and the filtration parameters used can greatly affect filter performance, product quality, and safety. In this work, the impact of three different parameters on ultrafiltration performance was investigated: 1) feed concentration, 2) protein size, and 3) inlet pressure. It was found that the protein concentration of the feed solution impacted flow rates in a dose-dependent manner but did not significantly alter retention of ΦX174 bacteriophage particles at the concentrations tested. It was further demonstrated that flow rates and downstream recovery of low-MW BSA were significantly higher than high-MW HgG at the same feed concentration, indicating that the molecular weight of the protein solution significantly alters ultrafiltration performance and downstream product recovery. Finally, it was shown that increasing the inlet pressure increased flow rates and throughput but decreased downstream protein recovery, showing that increased pressure has benefits and drawbacks that must be considered when developing ultrafiltration process parameters.

The use of isolators in cell, gene, and tissue therapy manufacturing is increasing due to their support of sterility assurance through effective microbial contamination control. However, many advanced therapy medicinal products (ATMPs) are sensitive to vaporized hydrogen peroxide (VHP), the standard agent used in isolator decontamination. This presents a challenge when transitioning from biosafety cabinets to closed-barrier isolator systems. This poster provides practical solutions to support the integration of isolator technology into ATMP manufacturing while addressing limitations associated with VHP-sensitive materials. Drawing from industry-based experience and collaborative practices, the poster outlines approaches to minimize VHP exposure risks while maintaining contamination control. Topics include isolator design, material selection and placement, process adaptations, and handling strategies for materials both time-sensitive and VHP-sensitive. Operational tips, including techniques to streamline material transfer and reduce open exposure time, are also shared. By offering actionable insights and lessons learned, this presentation aims to help process leads and microbiology professionals better understand how to successfully adopt isolators for ATMP processing. These strategies contribute to building robust, contamination-controlled environments that protect product quality while meeting time-sensitive sterility assurance goals in advanced therapy manufacturing.