Accelerated stability study in alternative formulations of a second generation biopharmaceutical product

Abstract

Filgrastim, a granulocyte colony stimulating factor (G-CSF), is a hematopoietic glycoprotein that binds with high affinity to its receptors in neutrophil precursor cells of the bone marrow; thus inducing proliferation and differentiation in neutrophils.The first generation commercial product has a short half-life (between 3.5 to 3.8 hours). However, this half-life can be extended by means of a covalent modification with PEG (polyethylene glycol). Pegylation increases the hydrodynamic volume of the molecule, minimizing renal clearance. Thus, the use of pegylated molecule is more advantageous than the administration of several doses of "naked" protein.Each pre-filled syringe of the original formulation contains 6 mg of pegfilgrastim in 0.6 ml of solution for injection, one of its components is sorbitol, a widely used tonicity modifier. In this work an alternative formulation is proposed, replacing mannitol with sorbitol, which is mostly used in lyophilized formulations because it crystallizes easily.In this study we propose to start the comparative evaluation of two formulations (FO: original and FA: alternative) of pegylated Filgrastim (PF) through a brief study of accelerated stability consisting in the exposition of both formulations to different temperature and freezing / thawing conditions. Subsequently both its structure and its stability were analyzed through different spectroscopies. For the evaluation of the structural conformation in the times established (zero time, one week and one month) both formulations were analyzed by UV spectroscopy, circular dichroism and fluorescence. Possible stability alterations were monitored by a thermal denaturation test.Both formulations of PF were aliquoted and stored at 4 ° C, 25 ° C and 37 ° C. Another group of samples were kept at extreme temperatures (-80 ° C, -20 ° C, -4 ° C and 57 ° C). At the time of carrying out the different measurements, the necessary dilutions were made in FO / FA. Regardless of time and storage temperature, samples formulated with mannitol or sorbitol did not show changes in secondary structure content or alterations in tertiary structure. Also, we did not find alterations in its conformational stability. These preliminary studies highlight the viability of the replacement of mannitol by sorbitol in the PF formulation, since the analyzes carried out through the proposed methods did not demonstrate protein instability linked to the change in formulation.