Effect of Fatty Acid Composition in Polysorbate 80 on the Stability of Therapeutic Protein Formulations

Polysorbate excipients are commonly used as surfactants to stabilize therapeutic proteins in formulations. Degradation of polysorbates could lead to particle formation and instability of the drug formulation. We investigated how the fatty acid composition of polysorbate 80 impacts the degradation profile, particle formation, and product stability under stress conditions.

Methods
Two polysorbate 80-containing therapeutic protein formulations were reformulated with either Polysorbate 80 NF synthesized from a fatty acid mixture that contains mainly oleic acid (≥58%) or a version of polysorbate 80 synthesized with high oleic acid (>98%). Stress conditions, including high temperature and esterase spiking, were applied and changes to both the polysorbate and the therapeutic protein product were investigated for stability, purity, innate immune response and biological activity.

Results
The addition of esterase and storage at 37°C led to significant hydrolysis of the polysorbate and increases in sub-visible particle formation for both polysorbates tested. The fatty acid composition of polysorbate 80 did not directly alter the stability profile of either therapeutic protein as measured by size exclusion chromatography, or significantly impact innate immune response or biological activity. However, formulations with Polysorbate 80 NF showed greater propensity for sub-visible particle formation under stress conditions.

Conclusions
These results suggest that composition of fatty acids in polysorbate 80 may be a promoter for sub-visible particulate formation under the stress conditions tested but may not impact protein aggregation or biological activity.

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Materials
Polysorbate 80 NF was purchased from Spectrum Biochemical. Super refined polysorbate 80 with C18:1 fatty acid content ≥98% designated as “high oleic acid PS80 (PS80 HOA)” throughout this manuscript, was kindly provided by Croda Inc. rhG-CSF was purchased as filgrastim reference standard from USP and rituximab from Genentech, Inc. Porcine liver esterase was purchased from Sigma. Mouse phospholipase B-like 2 (PLBD2) was purchased from Biorbyt. All chemicals were purchased from Sigma unless otherwise specified.

About this article: Pegues, M.A., Szczepanek, K., Sheikh, F. et al. Effect of Fatty Acid Composition in Polysorbate 80 on the Stability of Therapeutic Protein Formulations. Pharm Res (2021). https://doi.org/10.1007/s11095-021-03125-6