Papain-based nanoparticles were recently developed using radiation technologies and proven effective to generate nanosized crosslinked papain particles with preserved enzymatic activity. The applications of such nanostructured systems are expected to be similar to native papain with considerable biopharmaceutical advantages and concern drug loading among other biotechnological applications. The nature of such crosslinks and the possibility to provide simultaneous sterilization have been hypothesized but remain not totally clarified. This manuscript advances the discussion on the radiation-induced synthesis of protein nanoparticles by approaching the nature of the crosslinking and the possible contribution of bityrosine linkages and disulfide bridges to the overall nanoparticle assembly as well as the feasibility of the simultaneous sterilization process under the preestablished conditions of processing. Papain nanoparticles were synthesized and characterized according to size, proteolytic activity, bityrosine, cysteine content and molecular weight by SDS-PAGE upon sonication at 40 kHz. Bacterial identification and the sterility tests were performed in accordance with ISO 11737 prior to and after inoculating 106 CFU of Corynebacterium xerosis. Our experiments evidenced the crosslinking of rather intra- than intermolecular nature and a contribution of cysteine bridges and bityrosine linkages to the stabilization and formation of the papain nanoparticle assembly. The technique was effective to promote simultaneous crosslinking and sterilization at the established conditions of processing and may be validated in accordance with the ISO 11137.Funda????o de Amparo ?? Pesquisa do Estado de S??o Paulo (FAPESP)International Atomic Energy Agency (IAEA)Conselho Nacional de Desenvolvimento Cient??fico e Tecnol??gico (CNPq)FAPESP: 15/13979-0IAEA: CRP F22064CNPq: 402887/2013-1