(W1230-06-38) Polysorbate Hydrolysis and Protein Stability: Investigating the Role of Free Fatty Acid Particles

Purpose: Surfactants are widely used in protein formulations to prevent interfacial stresses during shaking, stirring, filtration and filling. However, as fatty acid esters, PS20 and PS80 can undergo hydrolysis in the presence of host cell proteins (lipases), compromising their stabilization function. The hydrolysis of the surfactant also produces water-insoluble free fatty acid (FFA) particles [1]. The generation of visible or subvisible FFA particles leads to product failure. In addition, polysorbates from different sources may contain different FFAs at various levels [2]. Studies have shown that FFA particles, by introducing protein adsorption, led to product destabilization [3]. Thus, our study hypothesizes that polysorbate hydrolysis undermines the stability of protein formulations due to (i) the loss of the stabilization function of the surfactants and (ii) interfacial stress resulting from FFA particles.

Methods: All the mAb formulations were prepared using sterile water for injection. Table 1 shows the compositions of the mAb formulations. All the solutions were prepared using 0.1% histidine buffer (pH 6.0). The surface tension of freshly prepared monoclonal antibody (mAb), surfactant, and mAb+surfactant solutions was measured using a KRUSS K100 force tensiometer. The mAb solutions were subjected to two stress conditions: (i) incubation at 40 °C in the dark for 4 weeks, and (ii) horizontal orbital shaking at room temperature (160 rpm). The mAb solutions were characterized by the percentage of high molecular weight species (%HMW) using size exclusion chromatography with multi-angle light scattering, employing an ACQUITY UPLC Protein BEH SEC Column (200 Å, 1.7 μm, 4.6 mm x 300 mm). Ten microliters of sample were injected for each measurement, and the detection wavelength was set at 280 nm. The static light scattering and the intrinsic fluorescence spectra of the mAb solutions were measured using a high-throughput fluorimeter system (UNCLE, Unchained Labs). The protein structure was characterized using the ratio between signal intensity at 350 nm and at 330 nm (350/330 ratio). A regular two-level factorial design was applied to study the synergistic impact of surfactant and free oleic acid on protein stability, with 3 center points and 2 replicates per sample.

Results: mAb gradually adsorb to liquid surface. PS80 was effective in preventing mAb surface adsorption at > 0.02%. Upon shaking, mAb did not undergo substantial destabilization. However, the presence of OA resulted in an increase in %HMW from 1% to 1.7%, and 350/330 ratio from 0.88 to 0.92, indicating protein unfolding and aggregation (Figure 1). Intact surfactant played a critical role in mitigate mAb destabilization during shaking, especially in the presence of OA particles. At 40 °C, the solubility of OA increased, as revealed by the decrease in the scattering intensity. Interestingly, mAb destabilization was observed when OA and PS80 were simultaneously added, while the two substances alone did not exhibit any substantial adverse effects. Such a synergistic destabilizing effect was also revealed by the 3D surfaces from the factorial design results (Figure 2). The adverse effect brought about by OA and PS80 was mitigated by sucrose, a widely used stabilizer in protein formulations.

Conclusion: OA particles destabilized mAb by introducing additional interfacial stresses. Intact surfactant effectively prevented mAb destabilization upon shaking. PS80 and OA had a synergistic effect in destabilizing the protein when stored at 40 °C. This adverse effect could be mitigated by sucrose. The results highlight the importance of excipient quality control on the stabilization of protein drug substances.

References: [1] Labrenz SR. Ester hydrolysis of polysorbate 80 in mAb drug product: evidence in support of the hypothesized risk after the observation of visible particulate in mAb formulations. Journal of pharmaceutical sciences. 2014 Aug 1;103(8):2268-77.
[2] Siska CC, Pierini CJ, Lau HR, Latypov RF, Fesinmeyer RM, Litowsk JR. Free fatty acid particles in protein formulations, part 2: contribution of polysorbate raw material. Journal of Pharmaceutical Sciences. 2015 Feb 1;104(2):447-56.
[3] Zürcher D, Wuchner K, Arosio P. Mitigation strategies against antibody aggregation induced by oleic acid in liquid formulations. Molecular Pharmaceutics. 2024 Oct 24;21(11):5761-71.


Table 1. mAb solutions studied in the current work


Figure 1. %HMW and 350/330 fluorescence ratio of mAb solutions before and after shaking experiment.


Figure 2. 3D surface of mAb solutions after incubation at 40 °C. A higher 350/330 ratio or %HMW value indicate more substantial mAb unfolding and aggregation.