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

Abstract

Purpose 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.

Use of phase plate cryo-EM reveals conformation diversity of therapeutic IgG with 50 kDa Fab fragment resolved below 6 Å

“…As for an ideal ZPP electron microscope, when it is operated with objective lens set at in-focus conditions together with minimal spherical aberration (Cs), it can provide in-phase structural information up to the regime of atomic resolution, abolishing the need of CTF correction. However, in our case we employed a customer designed ZPP cryo-EM with a fairly large Cs (4.2 mm) and operated it at close to focus condition to target medium-resolution information (∼10 Å) [26]. Due to practical challenges with use of this film ZPP, including poor yield of good pinholes (10-20%), difficulties in manual pinhole alignment with keeping it on axis, and pinhole charging, this ZPP cryo-EM has found limitations in its application in scaling up images required for fruitful single-particle analysis.…”

Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients

“…Most importantly, many of these impurities can act in concert to modulate the activity of the local immune cells and foster local or systemic responses, although the precise magnitude or persistence of the response needed to elicit an adaptive immune response is still unclear ( 22 , 42 – 45 ). Lastly, some commonly used excipients such as such as polysorbate 80 ( 46 ) ( 22 , 42 – 45 ), mannitol ( 47 ), or sucrose ( 41 ) can potentially modify the activation of antigen presenting cells directly or through degradation products ( 48 ). Compendial tests exist to control the levels of a few impurities such as LPS (LAL assay), nucleic acids, PS degradation, and b glycans, but sensitive tests that can detect the presence of a broader array of IIRMI or gage whether different impurities might synergize to activate the innate immune system are lacking.…”

Investigation on the Combined Effect of Hydroxypropyl Beta-Cyclodextrin (HPβCD) and Polysorbate in Monoclonal Antibody Formulation

“…The degraded products such as peroxides and free fatty acids raise questions about both the lowered ability of the surfactant to protect the formulation against interfacial stresses and the impact of the degradation products on protein stability. Furthermore, these degradation products can lead to the formation of visible and subvisible particles, which are major safety and quality concerns [ 11 ].…”