Furthermore, there was an observed significant loss in nanoparticle concentration measured by PTA for particles subjected to the centrifugation-wash method and in-situ measurements, which was likely caused by the formation of large agglomerates which precipitate or fall outside the PTA dynamic range in both cases. When compared with in-situ measurements of nanoparticle concentration (in incubation media), however, there is a clear higher loss for samples subjected to the centrifugation-wash methods which can be explained by the loss of sample during the centrifugation and resuspension steps. The resuspension step could potentially lead to the dissociation of nanoparticle and protein agglomerates, rendering the accurate assessment of nanoparticle size distribution challenging following recovery from incubation media. Therefore, the nature and range of techniques used for nanoparticle isolation from protein-containing media becomes a key step in the pipeline for nanoparticle characterisation and analysis of their interactions with biological systems. Our findings have implications for the design of protein corona studies, suggesting the need for more gentle separation techniques such as field-flow fractionation , or in-situ analytical approaches for measuring changes in nanoparticle parameters to minimize sample disruption through dissociation of particle agglomerate or particle-protein complexes, and subsequent alteration of the protein corona composition.