Technical challenges for complete implementation of automated growth-based methods for microbiological examination of advanced therapy medicinal products. What's wrong with Candida albicans?

BACKGROUND: Automated growth-based methods for sterility testing of cell-therapy products should be qualified to demonstrate that they are equivalent to, or better than, the conventional reference method. The aim of the present study was to assess the ability of the BACTEC FX40 system to detect low microbial contamination and to confirm the suitability of the method in the presence of seven different human mesenchymal cell-based products, according to Ph. Eur. 2.6.27. Additionally, a study to select the best vial to detect fungus contamination was performed. METHODS: Microorganisms representing Gram-negative, Gram-positive, aerobic, anaerobic, spore-forming, slow-growing bacteria, yeast and mold were prepared in either Dulbecco's PBS or seven biological matrices containing approximately 5, 10, and 15 colony-forming units (CFU) per sample. These preparations were inoculated to the specific media required for each test method: (i) BACTEC aerobic and anaerobic vials; (ii) aerobic and anaerobic media for direct inoculation; and (iii) Trypcase soy 3P or Brucella blood agar plates. Colonies from cultures were identified using MALDI-TOF mass spectrometry. RESULTS: The BACTEC FX40 system, in both Dulbecco's PBS and the biological matrices with a 5-CFU inoculum, detected most of the microorganisms significantly faster than the conventional method, despite the presence of a matrix containing gentamicin and several matrices containing 10% DMSO. Conversely, it showed an extremely delayed detection of Candida albicans compared with the conventional method. The addition of a Mycosis IC/F (MYC) vial decreased radically the time to detection (TTD) of C. albicans (28.2 ± 1.8 h) compared with the conventional method (36 h). CONCLUSIONS: When a MYC vial was added to the standard aerobic and anaerobic vials to test each sample, BACTEC FX40 was shown to be a superior alternative sterility method for cell-therapy products contaminated with low inocula, with a faster TTD for microbial growth compared with the conventional method (5 versus 14 days). The studies were carried out in different cell-based matrices with sensitivities and specificities of 100% for all the tested strains at 15-, 10- and 5-CFU inoculum, with the exception of Kocuria rhizophila at 5 CFU (90.48% sensitivity and 100% specificity).

Evaluation of the effectiveness of a new cryopreservation system based on a two-compartment vial for the cryopreservation of cell therapy products.

BACKGROUND AIMS: Successful cell cryopreservation and banking remain a major challenge for the manufacture of cell therapy products, particularly in relation to providing a hermetic, sterile cryovial that ensures optimal viability and stability post-thaw while minimizing exposure to toxic cryoprotective agents, typically dimethyl sulfoxide (Me2SO). METHODS: In the present study, the authors evaluated the effectiveness and functionality of Limbo technology (Cellulis S.L., Santoña, Spain). This system provides a hermetic vial with two compartments (one for adding cells with the cryoprotective agent solution and the other for the diluent solution) and an automated defrosting device. Limbo technology (Cellulis S.L.) allows reduction of the final amount of Me2SO, sidestepping washing and dilution steps and favoring standardization. The study was performed in several Good Manufacturing Practice laboratories manufacturing diverse cell therapy products (human mesenchymal stromal cells, hematopoietic progenitor cells, leukapheresis products, fibroblasts and induced pluripotent stem cells). Laboratories compared Limbo technology (Cellulis S.L.) with their standard cryopreservation procedure, analyzing cell recovery, viability, phenotype and functionality. RESULTS: Limbo technology (Cellulis S.L.) maintained the viability and functionality of most of the cell products and preserved sterility while reducing the final concentration of Me2SO. CONCLUSIONS: Results showed that use of Limbo technology (Cellulis S.L.) offers an overall safe alternative for cell banking and direct infusion of cryopreserved cell products into patients.