Characterization of PEGylation sites in Neulasta and a biosimilar candidate with a combined fragmentation strategy in mass spectrometry analysis.

In the development of biosimilar products to Neulasta, it is essential to determine the intact molecular mass and confirm precise PEGylation sites. In this study, we applied a combination of techniques, including post-column addition of triethylamine in reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) to determine the intact molecular mass, and in-source fragmentation (ISF) and higher-energy collision dissociation-tandem mass spectrometry (HCD-MS/MS) to identify the PEGylation site. Our results show that both the pegfilgrastim biosimilar candidate and Neulasta lots are mono-PEGylated at the N-terminal end. Furthermore, we show that the combined ISF and HCD-MS/MS method can be used for identifying the PEGylation sites in the diPEGylated variant of pegfilgrastim. The diPEGylated variant has modification sites at the N-terminal end and a lysine at position 35 in the protein sequence.

Systematic Study of Perfluorocarbon Nanoemulsions Stabilized by Polymer Amphiphiles.

Perfluorocarbon (PFC) nanoemulsions, droplets of fluorous solvent stabilized by surfactants dispersed in water, are simple yet versatile nanomaterials. The orthogonal nature of the fluorous phase promotes the formation of nanoemulsions through a simple, self-assembly process while simultaneously encapsulating fluorous-tagged payloads for various applications. The size, stability, and surface chemistry of PFC nanoemulsions are controlled by the surfactant. Here, we systematically study the effect of the hydrophilic portion of polymer surfactants on PFC nanoemulsions. We find that the hydrophilic block length and identity, the overall polymer hydrophilic/lipophilic balance, and the polymer architecture are all important factors. The ability to modulate these parameters enables control over initial size, stability, payload retention, cellular internalization, and protein adsorption of PFC nanoemulsions. With the insight obtained from this systematic study of polymer amphiphiles stabilizing PFC nanoemulsions, design features required for the optimal material are obtained.