(W1230-07-46) Degradation Kinetics during Hydrolysis of PEG Fatty Acid Esters in Antibody Formulation in Comparison to the Commonly Used Polysorbates

Purpose: Polysorbates (PS) are widely used as excipients to stabilize protein therapeutics. However, their susceptibility to hydrolysis and oxidation can lead to a reduction in surfactant content within protein formulations, thereby diminishing their stabilizing effects on the active pharmaceutical ingredient. While chemical hydrolysis can be relatively easily mitigated, preventing host cell protein (HCP) mediated hydrolysis is significantly more challenging, as even trace amounts of HCPs can degrade PS into sorbitan and free fatty acids (FFA). Due to their low solubility, these FFAs tend to aggregate into particles, further compromising the stability of the formulation. In formulations containing PS20 and monoclonal antibody 1 (mAb1), enzymatic hydrolysis of PS20 was observed and attributed to HCP activity. By incubating mAb1 with different surfactants at elevated temperature, this study aims to compare the degradation kinetics of PEG fatty esters with those of PS. Chosen surfactants are PS20, PS80, Kolliphor HS 15 (KOL), Cremophor EL (CRE), PEG-20-stearate (P20S) and PEG‑100-stearate (P100S). The findings will help determine whether these PEG fatty esters exhibit greater stability of mAb1 or are more susceptible to HCP-mediated hydrolysis than PS.

Methods: Each surfactant was formulated in concentrations of 0.4, 0.6 mg/ml with 25 mg/ml mAb1. These formulations were stored at 25 °C in glass vials for 0, 1, 3, 7, 14 and 28 days. Immediately after sampling the vials were visually investigated for particle formation under 90° light irradiation against black and white background and documented as pictures. Turbidity was measured by transmission measurement at 830 nm and 0° as formazine attenuation units (FAU) calibrated using a formazine standard suspension. The remaining samples were stored at -80 °C. After the final sampling time point (STP) surfactant content was determined using fluorescence micelle assay (FMA) with N-phenylnaphthalen-1-amine excited at 350 nm and measured at 420 nm emission. As an orthogonal approach surfactant content was also analyzed by HPLC coupled with a charged aerosol detector (CAD). Monoclonal antibody stability was assessed using size exclusion chromatography (SEC) by evaluating peak ratios of high molecular weight species, monomer, and low molecular weight species.

Results: All samples became opaque over time, indicating increased turbidity. Only P20S and KOL showed visible particle formation, with this effect being more pronounced at higher surfactant concentrations. Consequently, the 0.6 mg/mL concentration provided the clearest differentiation between surfactants. KOL and CRE exhibited the lowest turbidity, while P20S displays the highest turbidity across all concentrations. FMA revealed a time dependent decrease in surfactant concentration in all samples, with a similar relative decline in both concentrations. Highest reduction in surfactant content was observed in P20S and P100S with P20S being undetectable on several STP. These findings were confirmed by HPLC-CAD analysis. Degradation of mAb1 was observed in all formulations. Compared to a mAb1 formulation without a surfactant, PS20, PS80 and CRE demonstrated a stabilizing effect whereas P100S resulted in decreased stability in both concentrations.

Conclusion: The results indicate that P20S and P100S are unstable in all tested formulations, as indicated by increased turbidity and a significant decrease in surfactant content. Furthermore, these surfactants may have a negative effect on mAb1 stability as suggested by the SEC results. The peak ratio showed a greater decrease in monomer content with P20S and P100S compared to the mAb1 only formulation. A possible cause for this instability lies in their structure, which differs from the other tested surfactants by the more lipophilic stearate ester. CRE and KOL performed similar to PSs in regard of surfactant stability while exhibiting lower turbidity levels. However, PSs tend to be better in stabilizing mAb1 at the tested surfactant concentrations. A trend was observed where the stabilizing effect of KOL and CRE improved at lower surfactant concentration when 0.4 mg/mL is compared to 0.6 mg/mL. Based on these findings, PEG-stearates are unsuitable as stabilizers for mAb1 in the tested formulations. KOL and CRE are most promising tested alternatives to PSs, though they still showed limitations in protein stabilization. The pronounced increase in mAb1 stabilization at lower surfactant concentrations raises the question of whether further concentration adjustments could enhance performance of KOL and CRE. Therefore, this study identifies CRE and KOL as the most suitable alternatives to conventional PSs for mAb1, yet further investigations are necessary to determine their effectiveness in mAb1 stabilization at varying surfactant concentrations.


Visual inspection of 0.6 mg/mL formulations.


Turbidity and surfactant degradation results from 0.6 mg/mL formulations.


Effect of surfactans on monomer loss.