Unraveling the loss of lysozyme concentration after tableting

Abstract

Therapeutic proteins are fragile compounds whose functions are dependent on their higher-order structure (HOS) formed by specific intramolecular interactions. Nevertheless, protein interactions can also impede the stability if they take place with surfaces or formulation components. Besides chemical and conformational alterations, it can lead to the reduction of free non-complexed protein concentration. These are all factors which can lower the protein’s therapeutic effect and decrease therapeutic efficacy. During the minitablet development of lysozyme a loss of more than 30 % in protein recovery after tableting was observed using a reversed-phase chromatography (RPC) assay method. The aim of the current study was to unravel the cause for the loss in measured protein concentration, as prior to and post spray drying (SD) the recovery was 98.65 ± 0.76 %. It was shown that the loss was more linked to persistent protein-excipient interactions and not to the tableting process itself. It was possible to overcome these interactions with the selection of an optimized buffer system for the analytical sample preparation. For the selection of the buffer system, protein properties such as the isoelectric point (pI) and the charge had to be considered. In case of the minitablets containing lysozyme, it was a 100 mM arginine buffer pH 7.00 100 mM NaCl 0.1 % PS20 that allowed a protein recovery of almost 95.00 % after tableting. These results highlight the importance of analytical sample preparation and the selection of a suitable solvent for protein assay determinations.