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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.