The challenge and need for faster yet reliable sterility testing
Advanced therapies such as CAR-T treatments present particular challenges for rapid sterility testing. Their often short shelf-life and living-cell nature increase contamination risks while preventing a final sterilization step (21 CFR Part 610 – General Biological Products Standards).
Compendial sterility testing can take up to 14 days, often creating a bottleneck in the release of time-sensitive therapies and their delivery to patients in urgent need of treatment (Servetas et al., 2023). Recognizing these challenges, regulatory authorities have issued guidance to support faster alternatives (U.S. FDA, 2020).
This has led to the implementation of rapid microbial methods that can deliver results in about 7 -10 days. But even with this improvement, these timelines remain too long for many time-sensitive therapies. Beyond testing time, rapid methods can face challenges with complex samples. Filtration, lysis and extraction add complexity, while some methods cannot readily distinguish viable from non-viable microorganisms, increasing the risk of false positives.
The industry is in urgent need of sterility testing methods that can deliver negative results within 2–3 days while maintaining the safety, reliability, and quality required for cGMP-compliant product release (Gebo and Lau, 2020).
IMC for Rapid Sterility Testing under USP <1071> and Ph. Eur. 2.6.27
Calorimetry is the science of detecting heat and biocalorimetry is the science of detecting the heat released by living organisms, an intrinsic measurement of the presence and viability of microorganisms. Biocalorimetry-based systems using isothermal microcalorimetry (IMC) combine several key advantages for sterility testing of ATMPs, biologics, and other short shelf-life products. They align with the needs addressed in USP <1071> and Ph. Eur. 2.6.27 and have the potential to speed up batch release without compromising patient safety (Sioen and Coenye, 2026).
Using a dual system set-up, testing is performed with TSB and FTM media at two temperatures, following the dual-media, dual-temperature approach of traditional compendial sterility testing. This maintains alignment with established compendial methods while enabling significantly faster time to results and several additional benefits.
Detecting low level microbial contaminations
Sterility testing requires reliable detection of very low levels of microbial contamination, with a limit of detection below 10 CFU commonly targeted for the release of advanced therapies and biologics.
Both traditional compendial and alternative methods have faced challenges in achieving reliable detection at such low microbial loads, including growth-based methods (England et al., 2019; Gebo and Lau, 2020; Putnam and Lau, 2021) and molecular methods (Closa et al., 2025).
IMC-based systems can detect contamination originating from a single viable microorganism and enable limit of detection down to 1 CFU. In a broad evaluation including 16 strains, comprising both compendial strains and field isolates, IMC yielded positive results in 91.7% of samples at a nominal 5 CFU level, compared with 75.0% for the compendial USP <71> method (Sioen and Coenye, 2026).
Testing for viable microbial contamination
Growth-based methods provide a key advantage in sterility testing by detecting viable microorganisms through their ability to grow and reproduce, reducing the risk of false results. Unlike indirect detection technologies that infer viability from molecular, optical or metabolic signatures, growth provides direct biological evidence that a microorganism is present and viable.
Molecular methods are typically fast enough to enable same-day results, but based on the detection of genetic materials, they cannot directly distinguish viable from non-viable microorganisms, potentially increasing the risk of false-positive results from from dead cells (Cangelosi and Meschke, 2014).
Rapid growth-based methods based on microbial respiration have gained higher acceptance, but based on multiple indirect steps these methods still typically require 7-10 days to provide final sterility results. Particularly due to long detection times of slow-growing microorganisms (Gebo and Lau, 2020; Parveen et al., 2011).
IMC is based on the universal principle that all viable cells produce heat, thereby providing a direct link to viable microbial activity. By detecting metabolic activity rather than biomass formation, IMC can enable faster and more sensitive detection than conventional 14-day turbidity-based methods and alternative approaches based on automated CFU counting, and may also detect metabolically active viable but non-culturable (VBNC) microorganisms.
Continuous monitoring vs endpoint assays
A further advantage of growth-based methods is the possibility for continuous monitoring rather than endpoint detection only. At a set time for a negative result, the test provides a decisive pass/fail result, but as IMC also provides continuous live detection, contamination is automatically flagged as soon as microbial growth generates a detectable heat signal. Time-to-detection with IMC can be as little as a few hours for contaminated samples, enabling corrective action to be taken immediately rather than having to wait for the final result.
Testing complex advanced therapy matrices
Complex cell-based products can present significant challenges for sterility testing, where detecting a small number of microorganisms among millions of therapeutic cells can be like finding a needle in a haystack. Several alternative methods rely on filtration of the cell therapy product, where the high cell densities hinder filtration and require a prior lysis step of the cellular components. (Gebo and Lau, 2020). These steps increase sample manipulation, costs and workflow complexity. Lysis can also introduce method uncertainties, as it can be difficult to ensure selective removal of product cells without affecting potential microbial contaminants.
IMC enables direct testing of complex cell matrices containing millions of cells without filtration or lysis. While other methods may suffer from signals masked by components of the cellular product, growing bacteria, yeasts and molds have a comparable much higher metabolic heat output than eukaryotic cells thus enabling distinct microbial detection signal to the cellular background.
Combining growth-based detection with time-to-negative results in under 3 days
Growth-based sterility testing assay times are often limited by the slowest-growing organisms and matrix effects from complex products. Colorimetric blood culture-based methods, also referred to as respiration-based methods, can detect many organisms within 72 hours, but 7–10-day release remains common due to slow growers such as C. acnes and A. brasiliensis.
In comparison, IMC can combine the advantages of growth-based detection with considerably faster detection times. Even in the presence of high-concentration cell-based products, USP <71> organisms (C. sporogenes, P. aeruginosa, B. subtilis, C. albicans, S. aureus and A. brasiliensis) can be detected within 11–38 hours, while additional Ph. Eur.-relevant organisms (S. pyogenes, M. luteus and C. acnes) can be detected within 9–48 hours (Paulander et al., 2026).
In particular, C. acnes is often a limiting organism for time-to-negative results. Its relatively high metabolic heat output enables detection by IMC in as little as 48 hours, allowing a sufficient safety margin for complete sterility test results in under 3 days, compared with 14 days for the traditional compendial method. (Paulander et al., 2026)
Closed non-destructive sterility testing workflow
Reliable sterility testing requires minimizing the risk that the testing process itself introduces contamination or false-positive results (Panch et al., 2019). Filtration, optical detection and molecular methods can require additional sample handling and open processing steps, while molecular methods also require stringent contamination control to prevent extraneous genetic material from affecting results.
Closed handling can minimize contamination risks, providing a strong rationale for sterility testing workflows based on direct testing and minimal sample preparation (Putnam and Lau, 2021).
In an IMC testing workflow, samples are directly inoculated through the septum into closed test vials containing TSB and FTM media. Testing is non-destructive, and if a sample is found positive, it can immediately be further analyzed using standard identification methods.
Volume flexibilities, scaling and automation
While as much product as possible should be preserved for the patient, a sufficient and representative volume must still be tested in the sterility assay, with 1% of the total product volume commonly recommended for cell-based preparations (USP <1071>; Ph. Eur. 2.6.27) Flexibility is also important for scaling both sample volume and testing throughput.
While some methods are constrained in volumes and throughput, IMC can be scaled across multiple independently operated test positions, enabling flexible parallel testing. Further automation of sample loading and handling can support a completely integrated workflow from sample loading to results output.
Combining reliability with rapid growth-based detection
For short shelf-life therapies, growth-based sterility testing with negative results within 2–3 days can combine reliability with faster release and results available close to therapy administration.
This is also reflected in FDA guidance for certain short shelf-life therapy products, where in-process sterility testing 48–72 hours before final harvest and a no-growth result are included as part of the release strategy (U.S. FDA, 2020).
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Learn more about calScreener+ Sterility, caloTrace Sterility and advanced therapies and biologics sterility testing solutions
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Contact us at sterility@symcel.com to discuss your application and how IMC technology can support your sterility testing.
References
21 CFR Part 610. General Biological Products Standards. U.S. Food and Drug Administration.
https://www.ecfr.gov/current/title-21/chapter-I/subchapter-F/part-610
Cangelosi, G.A., Meschke, J.S., 2014. Dead or Alive: Molecular Assessment of Microbial Viability. Applied and Environmental Microbiology 80(19), 5884–5891.
https://doi.org/10.1128/AEM.01763-14
Closa, L., et al., 2025. Development of molecular sterility assay for rapid quality release of cord blood erythrocyte units for transfusion. Transfusion 65(1), 140–151.
https://doi.org/10.1111/trf.18084
England, M.R., et al., 2019. Comprehensive Evaluation of Compendial USP <71>, BacT/Alert Dual-T, and Bactec FX for Detection of Product Sterility Testing Contaminants. Journal of Clinical Microbiology 57(2), e01548-18.
https://doi.org/10.1128/JCM.01548-18
European Pharmacopoeia. Chapter 2.6.27. Microbiological Examination of Cell-Based Preparations. European Directorate for the Quality of Medicines & HealthCare (EDQM), Strasbourg, France.
https://go.edqm.eu/PhEurOnline
Gebo, J.E.T., Lau, A.F., 2020. Sterility Testing for Cellular Therapies: What Is the Role of the Clinical Microbiology Laboratory? Journal of Clinical Microbiology 58(7), e01492-19.
https://doi.org/10.1128/JCM.01492-19
Panch, S.R., et al., 2019. A Prospective Evaluation of a Practical Guideline for Managing Positive Sterility Test Results in Cell Therapy Products. Biology of Blood and Marrow Transplantation 25(1), 172–178.
https://pmc.ncbi.nlm.nih.gov/articles/PMC6310647/
Parveen, S., et al., 2011. Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products. Vaccine 29(45), 8012–8023.
https://doi.org/10.1016/j.vaccine.2011.08.055
Paulander, W., Svanberg Frisinger, F., Oliyn, G., 2026. Three-Day Phenotypic Sterility Testing for CGT Manufacturing: Validation of the calScreener+ Platform. PDA Journal of Pharmaceutical Science and Technology 80(1), 175–176.
https://doi.org/10.5731/pdajpst.2026.26127
Putnam, N.E., Lau, A.F., 2021. Comprehensive Study Identifies a Sensitive, Low-Risk, Closed-System Model for Detection of Fungal Contaminants in Cell and Gene Therapy Products. Journal of Clinical Microbiology 59, e01357-21.
https://doi.org/10.1128/JCM.01357-21
Lin, N.J, et al., 2023. Report from the 2022 NIST Rapid Microbial Testing Methods (RMTM) Workshop. NIST Special Publication 1292. National Institute of Standards and Technology.
https://nvlpubs.nist.gov/nistpubs/SpecialPublications/NIST.SP.1292.pdf
Sioen, I., Coenye, T., 2027. Evaluation of a Novel Isothermal Microcalorimetry-Based Sterility Test. Journal of Pharmaceutical Innovation 22, 107.
https://doi.org/10.1007/s12247-026-10986-0
United States Pharmacopeia, 2025. General Chapter <1071>, Rapid Microbial Tests for Release of Sterile Short-Life Products: A Risk-Based Approach. USP–NF.
https://doi.org/10.31003/USPNF_M12457_03_01
U.S. Food and Drug Administration, 2020. Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs): Guidance for Industry. Center for Biologics Evaluation and Research.
https://www.fda.gov/media/113760/download