Yilue Zhang

Polysorbate 80 (PS80) is a commonly used surfactant for stabilizing biotherapeutics by preventing protein adsorption at the air-liquid interface. However, PS80 is susceptible to oxidative degradation during manufacturing and storage. We show here that light exposure combined with the presence of metals can result in byproduct formation and potentially decrease the surfactant's ability to prevent protein adsorption to the air-liquid interface. PS80 formulated in citrate buffer can undergo cis/trans isomerization of unsaturated fatty acids in the presence of disulfides and iron (Prajapati et al., 2022). This work investigates novel surface activity aspects of polysorbate formulations before and after exposure to UV-A light. Polysorbate samples of different grades were formulated in citrate buffer containing iron and glutathione disulfide (GSSG; as a surrogate for peptide and protein disulfides), and a Langmuir trough was used to monitor the surface pressure during adsorption to the air-solution interface. Our results showed significant changes in the polysorbate surface activity after photoirradiation: all-oleate PS80 exhibited a 3-fold increase in the apparent critical micelle concentration (CMC), and the presence of both cis and trans fatty acids was confirmed. Also, the impact of photoirradiation on surface pressure depended on the surfactant concentration during irradiation, suggesting that the presence of micelles can alter the degradation pathway and byproduct formation.

Carbon dioxide radical anion ((CO2-)-C-center dot) is a powerful reducing agent that can reduce protein disulfide bonds and convert molecular oxygen to superoxide. Therefore, the generation of (CO2-)-C-center dot can be detrimental to pharmaceutical formulations. Iron is among the most prevalent impurities in formulations, where Fe(III) chelates of histidine (His) can produce (CO2-)-C-center dot upon exposure to near-UV light (Zhang and Schoneich, Eur. J. Pharm. Biopharm. 2023, 190, 231-241). Here, we monitor by spin-trapping in combination with electron paramagnetic resonance spectroscopy and/or high-performance liquid chromatography-mass spectrometry analysis the photochemical formation of (CO2-)-C-center dot for a series of common amino acid excipients, including arginine (Arg), methionine (Met), proline (Pro), glutamic acid (Glu), glycine (Gly), aspartic acid (Asp), and lysine (Lys). Our results indicate that in the presence of Fe(III), Asp, and Glu produce significant yields of (CO2-)-C-center dot under photoirradiation with near-UV light. Notably, Asp demonstrates the highest efficiency of (CO2-)-C-center dot generation compared with that of the other amino acid excipients. Stable isotope labeling indicates that (CO2-)-C-center dot exclusively originates from the alpha-carboxyl group of Asp. Mechanistic studies reveal two possible pathways for (CO2-)-C-center dot formation, which involve either a beta-carboxyl radical or an amino radical cation intermediate

Molecular dynamics simulations were employed to investigate the interaction between Fe(III) and an iron-binding site composed of THR259, ASP252, and GLU261 on the Fc domain of an IgG1. The goal was to provide microscopic mechanistic information for the photochemical, iron-dependent site-specific oxidative fragmentation of IgG1 at THR259 reported in our previous paper. The distance between Fe(III) and residues of interest as well as the binding pocket size was examined for both protonated and deprotonated THR259. The Fe(III) binding free energy (ΔG) was estimated by using an umbrella sampling approach. The pK_a shift of the THR259 hydroxyl group caused by the presence of nearby Fe(III) was estimated based on a thermodynamic cycle. The simulation results show that Fe(III) resides inside the proposed binding pocket and profoundly changes the pocket configuration. The ΔG values indicate that the pocket possesses a strong binding affinity for Fe(III). Furthermore, Fe(III) profoundly lowers the pK_a value of the THR259 hydroxyl group by 5.4 pK_a units.