Efecto de la irradiación gamma como técnica de esterilización terminal en la estabilidad de los oligonucleótidos

This work aimed to explore the use of gamma irradiation as a technique for the terminal sterilization of oligonucleotides. Since gamma irradiation can cause damage to oligonucleotides, several radioprotective factors were tested. Stabilizers such as glycerine, histidine, trehalose, and ascorbate, as well as the use of dry ice during irradiation, were evaluated. As representative oligonucleotides, homo-oligomers of deoxythymidine with base lengths between 5 and 45-mers were selected. The effect of gamma irradiation was evaluated in aqueous solutions. To quantify the extent of degradation, an ultra-high-performance liquid chromatography method with diode array detection, employing a HALO ES-C18 column and a mobile phase consisting of 0,1 M triethylammonium acetate (TEAA) in water and acetonitrile in gradient elution mode, was used. The method allowed accurate quantification of the oligonucleotides to a level of 0,156 to 0,270 nmol/ml, sufficient to quantify up to 96,6-97,8% degradation, depending on the oligonucleotide. For a radiation dose of 25 kGy, the best preserved integrity was observed in the samples without stabilizers, showing degradation levels between 5,2% and 13,2%. Samples containing histidine, trehalose, and ascorbate showed an increase in degradation, ranging from 9,5% to 24,2%. In contrast, samples with glycerin exhibited extensive degradation, with values between 78,8% and 96,8%, indicating a strong destabilizing effect. The use of dry ice during irradiation did not confer any protective benefit and slightly increased degradation across all samples. This study provides the first evidence of the potential suitability of gamma irradiation for the sterilization of oligonucleotides in aqueous solution, which surprisingly works best in the absence of radioprotective factors.