Oligodeoxynucleotide (ODN)-loaded gelatine nanoparticles (GNPs) have proven their outstanding potential in the treatment of allergic diseases such as equine asthma and canine atopic dermatitis, which are appropriate models for the corresponding human diseases. To encourage the development of a marketable product, long term stability and sterility needs to be ensured. In this work, we aimed to advance freeze-drying options to stabilise ODN-loaded GNPs. Matrix-assisted laser desorption/ionisation mass spectrometry time-of-flight was implemented as a versatile tool to assess ODN stability. With this method long-term storage stability of lyophilised ODN-loaded GNPs formulated in sucrose or trehalose was achieved. Controlled nucleation was further introduced to optimise the lyophilisation approach. This allowed shortening of the process in comparison to standard freeze-drying procedures. Particle sizes, polydispersity indices, ODN stability, residual moisture and glass transition temperature were maintained upon storage. Excipient portfolio was enlarged by novel amino acid containing formulations for lyophilisates. His emerged as an excellent excipient in stabilising lyophilised ODN-loaded GNPs, whereas addition of Arg and Gly revealed to be inadequate at accelerated conditions. Lastly, gamma irradiation was evaluated as a suitable sterilisation method of ODN-loaded GNPs.