Title
Impact of PEGylation and non-ionic surfactants on the physical stability of the therapeutic protein filgrastim (G-CSF)
Author
Patrick K. Chang, Clive A. Prestidge, Timothy J. Barnes, Kristen E. Bremmell
Year
2016
Journal
RSC Advances
Abstract
Improvement in the in vitro and in vivo stability of biotherapeutic proteins has been approached viaa 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 μm. 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.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Thermal stability, Thermodynamics, Ligand binding, Biochemistry