Effects of Hydrophobic Tail Length Variation on Surfactant-Mediated Protein Stabilization

Abstract

Recently, protein therapeutics have gained significant attention as a result of their enhanced selectivity and diminished side effects compared to traditional small-molecule drugs. Despite their advantages, protein formulations typically suffer from stability issues because of aggregation and denaturation during production and storage, often resulting in detrimental immune responses. Surfactants can be used to stabilize and protect proteins in solution by preventing protein adsorption onto interfaces or by forming protective structures in solution. Herein, a detailed structure− activity relationship study is described, demonstrating the role that hydrophobic tail length plays in surfactant-mediated stabilization of the model therapeutic protein IgG. The FM1000 series, originating from a surfactant scaffold that allows for easy structure modulation, was synthesized by a simple 2-step procedure. First, phenylalanine was acylated with a variety of acyl chlorides of differing lengths to yield n-acyl phenylalanine, which was then coupled to Jeffamine M1000, a polyethylene glycol-based amine, to yield the final surfactant. With this FM1000 series, it was observed that the 14 carbon-long tail surfactant (14FM1000) was optimal at preventing IgG aggregation compared to surfactants with tails that were longer or shorter. Using a combination of dynamic surface tensiometry and quartz crystal microbalance with dissipation, it was hypothesized that 14FM1000 was able to prevent IgG adsorption, and therefore aggregation, by adsorbing appreciably onto surfaces quickly. 14FM1000 had the fastest rate of initial adsorption compared to the other surfactants studied. Short-tail surfactants were slow to and did not adsorb appreciably onto surfaces, allowing IgG adsorption. Although long-tail surfactants were also slow to adsorb, allowing IgG to adsorb and aggregate, their equilibrium adsorption was strong. Additionally, 14FM1000 was the most reversibly adsorbed surfactant, likely improving its ability to desorb and adsorb quickly to transient surfaces, therefore protecting the IgG at each new hydrophobic surface and preventing aggregation. By understanding the structure−activity relationship between surfactants and protein stabilization, we move toward more efficient design of future surfactants increasing the stability and utility of important protein therapeutics.

Novel trehalose-based excipients for stabilizing nebulized anti-SARS-CoV-2 antibody

“…For the other surfactants, CMC results showed that the longer the alkyl chain, the lower the CMC, increasing from 0.07 mM for C 16 TreSuc to 0.96 mM for C 10 TreSuc . This is in good agreement with results obtained with other surfactants ( Hanson et al, 2020 ). More interestingly, other groups have studied the CMC of non-succinylated trehalose monoalkyl derivatives ( Chen et al, 2007 ).…”

Bottlebrush Polymer Excipients Enhance Drug Solubility: Influence of End-Group Hydrophilicity and Thermoresponsiveness

“…Polymer excipients are inactive ingredients included in pill formulations that are frequently, but not exclusively, used to sequester small molecules and larger payloads through noncovalent interactions with active pharmaceutical ingredients (APIs), , proteins, − and polynucelotides . In oral, intravenous, or intramuscular administration, the polymer works with the active ingredients as a stabilizer, both on the shelf and in vivo.…”

Differential Surface Adsorption Phenomena for Conventional and Novel Surfactants Correlates with Changes in Interfacial mAb Stabilization

“…The CMC value of FM1000 is slightly different than that reported by Hanson et al (denoted as 14FM1000), where dynamic light scattering was used to measure the CMC of FM1000 in 0.9% saline. We believe this difference is due to the different solution matrices and techniques used in this study …”

“…We believe this difference is due to the different solution matrices and techniques used in this study. 43 Our earlier study reported a CMC of 1.6 × 10 −2 mg/mL (12 μM) for PS80, 45 and PS80's CMC value is approximately five times higher than the CMC value for FM1000, indicating that a lower concentration of FM1000 would be sufficient to form a surfactant monolayer onto the surface. Additionally, a Langmuir adsorption fit to the constructed equilibrium surface tension as a function of bulk concentration indicated a maximum packing of surfactant concentration (Γ ∞ ) of around 1.88 mg/m 2 for PS80 and 1.45 mg/m 2 for FM1000.…”

“…Oxidation pathways are similar to those found in PS80 and poloxamer due to the presence of polyether hydrophiles in all molecules, and oxidation mitigation strategies used for PS80 and poloxamer could similarly be leveraged for FM1000. 32 Initial studies on the ability of FM1000 to prevent protein aggregation have been reported by Hanson et al 43 and Katz et al 42,44 To examine this molecular hypothesis, we use pendant bubble tensiometry and X-ray reflectivity (XRR). Prior studies from our group 45 on PS80 applied similar tools to quantify the concentration of the surfactant required to protect proteins from interfaces as a function of different mAbs' surface activities.…”