Background: With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these “off-the-shelf” therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function. Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others. Results: Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NKmediated specific lysis of tumor cells was maintained at stable levels for three days postirradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30. Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application.

In allogeneic bone transplantation, the transmission of viral and non-viral infectious pathogens is the most severe undesirable concomitant phenomenon. The investigations published were examined regarding the inactivating capacity of inactivation procedures that are presently performed in bone banks (peracetic acid/ethanol, gamma irradiation, moist heat) against clinically relevant pathogens (aiming at a virus titer reduction of at least 4 log10 TCID50/ml or titer reduction of non-viral micro-organisms of at least 5 log10 cfu/ml). In the suspension experiments, treatment with peracetic acid/ethanol (peracetic acid 2%, ethanol 96%, aqua ad iniectabilia 2:1:1, 4 hours, 200 mbar, agitation) achieved a titer reduction of > 4 log10 already after 5 minutes for a number of viruses (PSR, PV, BVDV). HIV-2 was also inactivated within 5 minutes below the level of detection ( 4 log10 TCID50/ml was only reached after 4 hours. The results mentioned could be confirmed in the carrier test (contaminated spongiosa cuboids used as ‘worst case’ scenario). In the suspension experiment as well as in the carrier test, the HAV titer was reduced after 4 hours by only 3.7 log10 and 2.87 log10, respectively. The preceding step of defatting the spongiosa tissue by chloroform/ethanol was validated using cell-associated HAV and showed an HAV titer reduction of 7 log10. In the investigations regarding non-viral pathogens, all test organisms were completely inactivated by more than 5 log 10 steps (cfu/ml). Gamma irradiation was the second procedure examined. D10 values (irradiation dose required to reduce 90% of the pathogen titers by one log10 step) that were determined in inactivation kinetics experiments (irradiation conditions: –30°C, 60Co source) corresponded to data published so far. In order to provide for maximal safety, an irradiation dose of 34 kGy was recommended for allogeneic bone transplants using BPV and a diaphysis model from human femurs. The ‘Marburg bone bank system’ was the third procedure examined (thermal disinfection, guaranteed temperature of at least 82.5°C for a minimum of 15 min) using centrally contaminated human femoral heads. All viruses were completely inactivated and their titer reduced by more than 4 log10 steps. Vegetative bacteria and fungi were also completely inactivated (>= 6 log10 in the supernatant). As expected, spores and spore-forming pathogens were not sufficiently inactivated and not inactivated, respectively (titer reduction of less than 2 log10 cfu/ml). However, the latter group can be disregarded, since femoral heads are procured in the operation room under sterile conditions and the following production process rules out a secondary contamination with spores. It could be shown in the investigations presented that all three procedures examined guarantee an inactivation of the viruses investigated according to the recommendations by the senior federal authorities. The three treatment procedures offer additional biosafety by a comprehensive inactivation of non-viral pathogens.

Des procédés innovants basés sur l’extrusion à haute vitesse et l’irradiation sous rayonnement γ, ont été étudiés en vue d’améliorer la compatibilisation de deux polyoléfines non miscibles : le polypropylène PP et le polyéthylène PE. Dans un premier temps, l’étude de la distribution des temps de séjour de polypropylènes dans l’extrudeuse à haute vitesse a été réalisée. L’impact des conditions opératoires (débit et vitesse de rotation des vis) ainsi que l’impact du profil de vis et de la viscosité du polypropylène d’étude ont été mis en avant. Certains résultats expérimentaux sont souvent en accord avec ceux du logiciel de simulation d’extrusion bi-vis Ludovic, bien qu’il puisse y avoir des écarts pour les hautes vitesses de rotation des vis. Dans un deuxième temps, une fois l’écoulement caractérisé, nous avons réalisé des mélanges PP/PE, les paramètres qui ont varié sont la quantité d’énergie apportée durant le mélange (vitesse de rotation des vis) ainsi que la présence ou non de copolymère préformé et le type d’atmosphère utilisée durant l’extrusion. Faire varier le taux de cisaillement ne permet pas la modulation des propriétés finales du mélange surtout lorsque l’on extrude sous atmosphère inerte. L’utilisation de copolymère préformé, de type éthylène-octène, permet d’améliorer considérablement les propriétés de notre mélange en créant une interphase "cœur-couronne" entre la matrice PP, le compatibilisant et la phase dispersée PE. Pour terminer, une étape d’irradiation γ a été ajoutée au procédé de mise en œuvre classique de nos mélanges. En première partie de cette étude, la simulation de spectres RPE a permis d’identifier et quantifier les différentes espèces radicalaires créées. Dans la deuxième partie, l’influence de la place de la séquence d’irradiation a été étudiée : avant extrusion, entre extrusion et injection, après injection et avant recuit. Les meilleures améliorations ont été obtenues lorsque l’irradiation est suivie d’un traitement thermique. En conclusion, il est possible d’améliorer la compatibilité d’un mélange PP/PE en jouant essentiellement sur l’ordre des étapes de production sans avoir à modifier la formulation de ce dernier. / Innovative processes, based on high shear twin screw extrusion and γ-irradiation, have been used to increase the compatibility of two immiscible polyolefins: polypropylene PP and polyethylene PE. In a first part, the residence time distribution of polypropylenes in the twin screw extruder (TSE) has been investigated. The impact of the processing conditions (throughput and screw rotation speed), the screw profile and the polypropylene viscosity were underlined. Some of the experimental results are often in good agreement with those predicted by simulation software of twin screw extrusion (Ludovic), except for some experiments at high screw rotation rates. In a second part, once polymer flow was characterised in the TSE, we realized PP/PE blends. The impact of the mechanical energy (screw rotation speed), the presence or absence of copolymers and the nature of the extrusion atmosphere were analysed. Using high shear rate does not allow modulating the final properties of the blends particularly once inert atmosphere is used. The use of ethylene-octene copolymers increases significantly the final properties of the PP/PE blends by creating a “core-shell” morphology between the PP matrix, the copolymer and the PE dispersed phase. To finish, a γ-irradiation process step has been added to the classical processing of PP/PE blends. In a first part of this study, the simulations of ESR spectra have permitted to identify and quantify the different radicals created. In a second part, the sequence order of the γ-irradiation has been investigated: before extrusion, between extrusion and injection, and after injection followed by a thermal treatment. The best results were obtained once γ-irradiation is followed by a heat treatment. As a conclusion, the compatibility of immiscible polyolefins can be improved only by changing the order of the different processing steps without changing the formulation.