Structure-based discovery of a new protein-aggregation breaking excipient

Abstract

Reducing the aggregation of proteins is of utmost interest to the pharmaceutical industry. Aggregated proteins are often less active and can cause severe immune reactions in the patient upon administration. At the same time the biopharmaceutical market is pushing for high concentration formulations and products that do not require refrigerated storage conditions. For a given protein, the liquid formulation developer's toolbox is limited to achieve these goals: pH, ionic strength and concentration of a very limited number of excipients are the only solution parameters to be varied. In this work, we present a structure-based approach to discover new molecules that successfully reduce the aggregation of proteins and apply it to the model protein Interferon-alpha-2a.

Study of the interaction between a novel, protein-stabilizing dipeptide and Interferon-alpha-2a by construction of a Markov state model from molecular dynamics simulations

“…We identified five meta-stable interaction sites showing hydrogen bonding and salt-bridges between the protein and the dipeptide, supporting the finding of stoichiometric binding between thee protein and the ligand. The protein-ligand complex formed in macro-state 3 is similar to the one that was proposed by our previously reported virtual screen [10]. In our Markov model, the macro-state 3 is, however, only a weakly populated intermediate state.…”

“…We previously described the discovery of an outstanding stabilizing effect of the dipeptide glycyl-Dasparagine at low concentrations against aggregation of Interferon-alpha-2A upon exposure to freezing-thawing and shaking stress [10]. We found that the dipeptide would bind to the protein at a µM affinity and reduces particle formation at low concentration (6.25 mM), hinting at a stabilization through a stoichiometric interaction.…”

Simultaneous Purification of Human Interferon Alpha-2b and Serum Albumin Using Bioprivileged Fluorinated Ionic Liquid-Based Aqueous Biphasic Systems

“…MST allows for rapid and quantitative characterization of interactions based on the thermophoretic behavior of biomolecules and sensitivity to non-covalent binding [ 107 ]. The MST assay was already used to infer the interactions between Lys [ 78 ] and IFN- 2b [ 77 ] with ionic liquids, and to determine the binding affinities of interferons [ 108 , 109 ] and other proteins [ 105 , 110 ] with distinct ligands.…”

Disclosing the Potential of Fluorinated Ionic Liquids as Interferon-Alpha 2b Delivery Systems

“…The changes provoked by the binding in the thermophoresis of the fluorescent molecules can be then used to obtain the equilibrium dissociation constant, K d , by plotting the normalized fluorescence, F norm , of the labelled molecules versus the logarithm of the concentration of the ligand and fitting the binding curves with the models provided by the software [ 66 , 67 ]. This technique was previously applied in the determination of the binding affinity between ILs and lysozyme [ 68 ], as well as for inferring the interactions of other types of interferons [ 69 , 70 ] and other proteins [ 67 , 71 , 72 ] with different types of ligands.…”