One of the main challenges in drug delivery is improving the transport of (bio)therapeutic molecules across biological barriers. Therefore Vect-Horus is developing different families of vectors able to bind receptors expressed at the surface of specific cells and involved in a receptor-mediated endocytosis process to enable the passage of therapeutic molecules. A family of vectors targeting the transferrin receptor (TfR) are Camelid single-domain antibodies. These have been proved to efficiently bind the TfR. Once conjugated to a small interfering RNA, the VHH can make them internalized into tissue of interest allowing the silencing of the targeted RNA gene.



The aim of this study is to precisely characterize this hetero-conjugate composed of a protein part (68kDa) and an oligonucleotide part (15kDa) via an LC-MS (liquid chromatography hyphenated to mass spectrometry) technical method. Here, two type of MS instruments will be compared to optimize the best method for the detection of this conjugate: an Orbitrap Exploris 240 (Thermo Scientific) on one side and the Synapt G2-S (Waters AG) on the other side.



With standard parameters, LC-MS analysis do not allow accurate characterization of the conjugate. The first aim of my Ph.D was to optimize the LC-MS analysis of the hetero-conjugate. On the LC part, column and type of separation were tested while in MS, the ionization parameters as well as the ionization (positive and negative) mode were modified. The main optimization resides in the observation of the entire molecule with both siRNA strands, as this double strand complex is based on non-covalent interactions which are not straightforward to keep intact in gas phase. The double strand RNA was observed with the Synapt G2S while it was more difficult to see it with the Orbitrap. This development method will then be used to perform stability studies and quantification of the hetero-conjugate in biological media.