THE EFFECT OF PROTEIN DESORPTION ON BIOTHERAPEUTICS AGGREGATION: NEUTRON REFLECTIVITY STUDY.

 

Tatiana Perevozchikova, Hirsh Nanda, Ronald Jones, Doug Nesta and Christopher Roberts

 

Particles in biotherapeutics are disadvantageous, as they are believed to be responsible for an adverse immunogenic response in patients, making investigation of factors leading to protein instabilities a high priority. The exposure of protein therapeutics to various interfaces during manufacturing, storage and shipment procedures can be a potential cause of the formation of insoluble particulates observed in final formulations. One of the proposed mechanisms for interface-induced aggregation is desorption of protein microparticles from biofilm previously formed on the walls of various pipes and containers. Therefore, the surface-associating properties of proteins on pharmaceutically relevant materials can play an important role and should be taken into consideration while developing a protein-stabilizing strategy. Neutron reflectivity (NR) bears the potential as a powerful and non-invasive tool to investigate the adsorption/desorption properties of biomolecules on various surfaces, both under the static conditions and flow.

Here we present the results of NR measurements on Silicon Oxide (SiO2) films exposed to alpha-chymotrypsinogen A (aCgn) and immunoglobulin G (IgG) at high concentrations. The initial observations on the adsorption behavior of these two proteins indicate significant similarities: they both adsorb into a dense protein phase resting on a well-hydrated intervening sub-layer.  However, the structural characteristics of the protein layers formed upon desorption are strikingly distinctive. We also show how we utilize an additional solvent contrast (H2O buffer) to improve the obtained structural details and more accurately determine a surface coverage by proteins. Finally, we are able to investigate the changes in the solution structure of the desorbed proteins by complimenting our studies with CD, fluorescence quenching and SANS techniques. These findings serve as the first crucial steps toward providing a link between protein adsorption/desorption dynamics and particles formation.