Accelerated Stability Study in Alternative Formulations of a Second- Generation Biopharmaceutical Product

Filgrastim, a stimulating factor of the granulocyte colony (G-CSF), is a hematopoietic glycoprotein that binds to neutrophil precursor cells of the bone marrow with high affinity to its receptors, thereby inducing neutrophil proliferation and differentiation. There is a brief half-life for the first-generation commercial product (between 3.5 and 3.8 hours). However, by means of a covalent modification of PEG (polyethylene glycol), this half-life can be increased. Pegylation enhances the molecule’s hydrodynamic volume, reducing renal clearance. The use of pegylated molecules is also more beneficial than the administration of several “naked” protein doses. Every pre-filled syringe of the original formulation contains 6 mg of pegfilgrastim in 0.6 ml of solution for injection, with sorbitol, a frequently used tonicity modifier, as one of its components. An alternative formulation is suggested in this work, substituting sorbitol with mannitol, which is often used because it crystallises easily in lyophilized formulations. In this study , we propose to begin a comparative evaluation of two pegylated Filgrastim (PF) formulations (FO: original and FA: alternative) through a brief study of accelerated stability consisting of exposure of both formulations to various temperatures and conditions of freezing / thawing. Both its structure and stability were subsequently studied by various spectroscopies. Both formulations were analysed by UV spectroscopy, circular dichroism and fluorescence for the assessment of the structural conformation at the period established (zero time, one week and one month). A thermal denaturation test tracked potential stability alterations. Both PF formulations have been aliquoted and stored at 4 ° C, 25 ° C and 37 ° C respectively. Another study group was held at high temperatures (-80 ° C, -20 ° C, -4 ° C and -80 ° C). The required dilutions in FO / FA were made at the time of carrying out the various measurements. Samples formulated with mannitol or sorbitol, irrespective of the time and storage temperature, did not display changes in secondary structure content or changes in tertiary structure. Even, in its conformational stability, we did not notice changes. These preliminary studies illustrate the feasibility of mannitol substitution with sorbitol in the PF formulation, since the research carried out using the methods proposed did not demonstrate protein instability associated with a shift in formulation.

Author(s) Details

María Lourdes Puig San Andrés

Carrera de Especialización en Biotecnología Bioquímico Farmacéutica, Facultad de Farmacia y Bioquímica, de la Universidad de Buenos Aires, Argentina.

Adriana Carlucci
Departamento de Tecnología Farmacéutica, Facultad de Farmacia y Bioquímica, de la Universidad de Buenos Aires, Argentina and Departamento de Farmacia, Instituto Universitario del Hospital Italiano de Buenos Aires, Argentina.

Lucrecia María Curto

Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, de la Universidad de Buenos Aires, Argentina.

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