Impact of PEGylation and non-ionic surfactants on the physical stability of the therapeutic protein filgrastim (G-CSF)

Improvement in the in vitro and in vivo stability of biotherapeutic proteins has been approached via a number of strategies, including protein PEGylation or formulation with non-ionic surfactants. Here we report on interaction and stability studies for the biotherapeutic protein filgrastim (granulocyte stimulating factor (G-CSF)) and its PEGylated analogue (PEG-GCSF), with polysorbate 20, using isothermal calorimetry, circular dichroism, surface tension and dynamic light scattering measurements. PEGylation of G-CSF did not alter temperature-induced conformational changes detected with circular dichroism, however did increase the amphiphilic nature of G-CSF, lowering the surface tension to a greater extent. G-CSF and PEG-GCSF both aggregated at temperatures below that of denaturation. G-CSF had an inverse relationship between concentration and the temperature at which aggregation was initiated, with aggregates continually increasing in size to greater than 2 mm. Importantly, PEG-GCSF was shown to have improved resistance to heat-induced aggregation; the presence of PEG attached to the protein minimised the aggregate size to below 120 nm. Interaction between polysorbate 20 and the proteins was weak and determined to result from a hydrophobic mechanism. A two-site binding model was found to best describe the interaction of polysorbate 20 with G-CSF, irrespective of PEGylation. Presence of polysorbate 20 did not minimise the thermal-induced instability for G-CSF or PEG-GCSF. These findings provide new insight into the mechanism of therapeutic protein stabilization using PEG and non-ionic surfactants.