Impact of four inorganic impurities – iron, copper, nickel and zinc - on the quality attributes of a Fc-fusion protein upon incubation at different temperatures

A key aspect that must be supervised during the development of a biotherapeutic is the presence of elemental impurities in the final drug product: they must be quantified as to ensure that their concentrations does not affect patients’ safety. Regulatory guidelines such as ICH Q3D provides Permitted Daily Exposure (PDE) limits for those impurities considered having a higher potential safety risk. However, one of the limits of such PDE values is that they account for the safety risk, while alterations of certain Quality Attributes (QA) of a biologic may also take place. In order to understand how certain impurities could affect not only the safety of patients, but also the physicochemical properties of biotherapeutics, here we present a study in which we examined how four commonly observed elemental impurities could impact the QAs of a Fc-fusion protein, under normal storage conditions and after six weeks of incubation at +25 °C and +40 °C. The molecule was indeed treated with increasing concentrations of Ni2+, Cu2+, Zn2+ and Fe3+ and the potential changes in conformation, oxidation, aggregation, and fragmentation were monitored. Our data suggest that keeping the levels of these impurities under the safety threshold limits does not guarantee the product quality. While nickel and zinc slightly altered the physicochemical properties of our Fc-fusion protein, iron and copper appeared to be more harmful for the QAs stability. Indeed, these latter elements might cause significant alterations of the product quality such as to potentially alter its efficacy.