Sterility Control of Advanced Therapy Medicinal Products: Impact of Growth Medium, Analysis Time, and Sample Quantity

INTRODUCTION. Sterility testing of advanced therapy medicinal products (ATMP) implies several challenges, including limited volume of available sample, product sensitivity to test conditions, and short shelf life. Classical pharmacopoeial test methods often fail to confirm sterility in a timely and reliable manner. The resulting drug availability for patients is reduced, as well as the opportunity for fast treatment with high-quality medicines. Faster alternative microbiological methods that reflect ATMP specificity of advanced therapy medicinal products can speed up and enhance sterility control.

AIM. This study aimed to optimise ATMP sterility control using colorimetric carbon dioxide detection.

MATERIALS AND METHODS. We used an experimental sample of an ATMP as the test object. We challenged the system with the following test strains: Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, Clostridium sporogenes ATCC 19404, Cutibacterium acnes (Propionibacterium acnes) NCTC 737, Candida albicans ATCC 10231, Aspergillus brasiliensis ATCC 16404, Aspergillus fumigatus VKPM F-62, Aspergillus terreus VKPM F-1269, Penicillium chrysogenum VKPM F-3. We used SA, SN, and iLYM bottles with pharmacopoeial growth media: modified tryptic soy broth for the BacT/ALERT 3D Dual T system, tryptic soy broth, and fluid thioglycollate medium in test tubes designed for sterility
testing using direct inoculation. All manipulations took place in a Purifier
Logic A2 laminar flow cabinet, with BacT/ALERT 3D Dual T system to automatically monitor cultures, detect contamination, and record microbial growth curves. We prepared artificially contaminated samples by adding 3 CFU/mL of each test strain. We inoculated 0.1, 0.5, and 1.0 mL sample volumes into appropriate media, performing 20 replicates for each condition. Growth media included both pharmacopoeial (tryptic soy broth, fluid thioglycollate) ones and modified media for BacT/ALERT (SA, SN, iLYM). We incubated the direct inoculation samples for 14 days. In the BacT/ALERT system, we ran a 7-day incubation with continuous monitoring at 32.5±2.5 °C for bacteria and 22.5±2.5 °C for fungi. We evaluated direct inoculation results visually and assessed BacT/ALERT results based on CO2 growth curves (alternative method).

RESULTS. The BacT/ALERT 3D Dual T system detected ≥80% of aerobic and anaerobic bacteria and ≥90 % of yeasts and molds at 0.5 mL sample volume using pre-aerated iLYM medium. In contrast, direct inoculation required 1.0 mL to reach similar detection levels. BacT/ALERT reduced the time to contamination detection by 0.38–2.4 days compared to direct inoculation, while identifying all test strains except Candida albicans that showed similar detectability in both methods. When using SA medium, false-negative results for molds made 13–70 % of cases, depending on the strain. Short-time iLYM aerating for 5–7 s before incubation reduced false negatives to 0–3 %. We successfully detected all tested microorganisms within 28 h, except for Cutibacterium acnes, which required up to 134 h.

CONCLUSIONS. The BacT/ALERT 3D Dual T system delivers higher sensitivity, reproducibility, and faster detection compared to traditional pharmacopoeial methods. False negative results for mold fungi in SA medium were as high as 70%. However, they can be significantly reduced using aerated iLYM medium. Both methods reliably detect all test organisms within 7 days, but alternative BacT/ALERT achieves detection 0.38–2.4 days sooner. Using a 0.5 mL sample volume with BacT/ALERT effectively detects contamination and reduces product use by half compared to pharmacopoeial testing (1.0 mL). 

1. Gonçalves E. Advanced therapy medicinal products: Value judgment and ethical evaluation in health technology assessment. Eur J Health Econ. 2019;21(3):311–20. https://doi.org/10.1007/s10198-019-01147-x

2. Centers for Disease Control and Prevention. Multistate outbreak of fungal infection associated with injection of methylprednisolone acetate solution from a single compounding pharmacy. MMWR Morb Mortal Wkly Rep. 2012; 61(41):839–42. https://doi.org/10.15585/mmwr.mm6141a2

3. Bertoletti da Silva S, Rebello Lourenço F. Risk of false compliance/non compliance decisions for sterility test due to false negative and false positive test results. Chemometr Intell Lab Syst. 2020;200:104005. https://doi.org/10.1016/j.chemolab.2020.104005

4. Deutschmann S, Paul M, Claassen-Willemse M, et al. Rapid sterility test systems in the pharmaceutical industry: Applying a structured approach to their evaluation, validation and global implementation. PDA J Pharm Sci Technol. 2023;77(3):211–35. https://doi.org/10.5731/pdajpst.2021.012672

5. Thorpe TC, Wilson ML, Turner JE, et al. BacT/Alert: An automated colorimetric microbial detection system. J Clin Microbiol. 1990;28(7):1608–12. https://doi.org/10.1128/jcm.28.7.1608-1612.1990

6. Goula A, Gkioka V, Michalopoulos E, et al. Advanced therapy medicinal products: challenges and perspectives in regenerative medicine. J Clin Med Res. 2020;12(12):780–6. https://doi.org/10.14740/jocmr3964

7. Roshchina MV, Sakhno NG, Gunar OV. Approaches to the validation of an alternative method for determining the sterility of high-tech medicines. Pharm Chem J. 2025;59(2):258– 63. https://doi.org/10.1007/s11094-025-03386-0

8. Woods M, Lu H-Y, Benard F. Validation of the BacT/ALERT® 3D System for Sterility Testing of a 177 Lu-radiopharmaceutical. J Nucl Med. 2025;66(Suppl 1):25146.

9. Jimenez L, Rana N, Amalraj J, et al. Validation of the BacT/ ALERT® 3D system for rapid sterility testing of biopharmaceutical samples. PDA J Pharm Sci Technol. 2012;66(1):38– 54. https://doi.org/10.5731/pdajpst.2012.00790

10. Hussong D, Mello R. Alternative microbiology methods and pharmaceutical quality control. Am Pharm Rev. 2006;9(1):62–9.

11. Rohner P, Pepey B, Auckenthaler R. Comparison of BacT/ ALERT with Signal blood culture system. J Clin Microbiol. 1995;33(2):313–7. https://doi.org/10.1128/jcm.33.2.313-317.1995

12. Mastronardi C, Perkins H, Derksen P, et al. Evaluation of the BacT/ALERT 3D system for the implementation of in-house quality control sterility testing at Canadian Blood Services. Clin Chem Lab Med. 2010;48(8):1179–87. https://doi.org/10.1515/cclm.2010.240

13. Roshchina MV, Gunar OV, Sakhno NG. Applicability of an alternative method for sterility |testing of medicinal preparations. Pharm Chem J. 2018; 51(11):1045–8. https://doi.org/10.1007/s11094-018-1737-1

14. Roshchina MV, Gunar OV, Sakhno NG. Methodological features of sterility testing of biomedical cellular products. Biopharmaceutical Journal. 2017;9(6):3– 8 (In Russ.). EDN: XHAWNY

15. Roshchina MV, Gunar OV, Sakhno NG. Limit of detection of microorganisms in drug sterility testing. Pharm Chem J. 2017;51(6):508–10. https://doi.org/10.1007/s11094-017-1644-x

16. Kvich L, Jensen PØ, Justesen US, et al. Incidence of Propionibacterium acnes in initially culture-negative thioglycollate broths: A prospective cohort study at a Danish university hospital. Clin Microbiol Infect. 2016;22(11):941–5. https://doi.org/10.1016/j.cmi.2016.07.036

17. Kocoglu ME, Bayram A, Balci I. Evaluation of negative results of BacT/Alert 3D automated blood culture system. J Microbiol. 2005;43(3):257–9. PMID: 15995643

18. Bugno A, Lira RS, Oliveira WA, et al. Application of the BacT/ALERT® 3D system for sterility testing of injectable products. Braz J Microbiol. 2015;46(3):743–7. https://doi.org/10.1590/S1517-838246320140587

19. Hanna E, Rémuzat C, Auquier P, et al. Advanced therapy medicinal products: Current and future perspectives. J Mark Access Health Policy. 2016;4(1):31036. https://doi.org/10.3402/jmahp.v4.31036