Evaluation of a tubing system with an incorporated DMSO-resistant sterile filter for cryopreservation of cellular products outside of cleanroom facilities

Objectives: Processing of cellular therapeutics in an open system according to GMP guidelines requires a cleanroom grade A with surrounding grade B conditions. In a validation project a newly developed tubing system with an incorporated dimethyl sulfoxide (DMSO)-resistant sterile filter (Closed Cryo Prep Set, Cell*Max, Germany) for cryopreservation of cellular products was evaluated by challenge experiments and media fill runs. Methods: In a first series, filter of 3 systems were challenged with spiked DMSO. 20 ml of DMSO were spiked with a defined solution of Bacillus subtilis (strain ATCC 6051) leading to a concentration of 1x10E+06 colony forming units/ml. Prior filtration 100 µl were used to titrate bacteria counts of the inoculum in log 10 steps using BHI agar plates. 20 ml of the bacterial suspension were applied to the system. The filtrate was collected and finally titrated in log 10 steps on BHI agar plates. The influence of DMSO on the bacterial growth was examined by spiking DMSO and analysis of bacterial growth after different times. In a second series, filter of 3 systems were challenged in the same way but NaCl 0.9% was used as medium. Additionally, 3 media fill runs with CSL medium were performed to detect even smallest bacterial contaminations during processing. Results: In the DMSO series, after spiking but before filtration the number of detectable bacteria was already diminished by 4 to 5 log leading to detectable concentrations of 1x10E+01-10E+02/ml. After filtration, bacterial growth was not detectable. Bacteria spiked in DMSO exhibited a time dependent decline of growth with a complete growth inhibition after 5 minutes of incubation. In the NaCl series, 35 to 50% of the spiked bacteria could be detected after spiking and before filtration. After filtration, bacterial growth was no longer detectable. All media fill runs led to sterile products. Conclusions: Although certainly most of the potential bacterial contaminants of hematopoetic progenitor cell (HPC) grafts are not viable after treatment in 99% DMSO (similar results were obtained for S. epidermides as published recently) the sterile filter offers an additional security measure to eliminate potential contaminants. Final validation runs processing leukaphereses from unmobilized healthy donors and evaluating cellular integrity, proliferative capacity and sterility of the product are the next task before implementation of the system in routine cryopreservation of HPC grafts.