Complex Micellization Behavior of the Polysorbates Tween 20 and Tween 80.

Polysorbates (PSs, Tweens) are widely used surfactant products consisting of a sorbitan ring connecting up to four ethylene oxide (EO) chains of variable lengths, one or more of which are esterified with fatty acids of variable lengths and saturation degrees. Pharmaceutical applications include the stabilization of biologicals in solutions and the solubilization of poorly water soluble, active ingredients. This study characterizes the complex association behavior of compendial PSs PS20 and PS80, which is fundamentally different from that of single-component surfactants. To this end, a series of demicellization experiments of isothermal titration calorimetry with different PS concentrations are evaluated. Their experiment-dependent heats of titration are converted into a common function of the state of a sample, the micellar enthalpy Qm(c). These functions demonstrate that initial micelles are already present at the lowest concentrations investigated, 2 µM for PS20 and 10 µM for PS80. Initial micelles consist primarily of the surfactant species with the lowest individual critical micelle concentration (cmc). With increasing concentration, the other PS species gradually enter these micelles in the sequence of increasing individual cmc's and hydrophilic-lipophilic balance. Concentration ranges with pronounced slopes of Qm(c) can be tentatively assigned to the uptake of the major components of the PS products. Micellization and the variation of the micelle properties progress up to at least 10 mM PS. That means the published cmc values or ranges of PS20 and PS80 may be related to certain, major components being incorporated into and forming specific micelles but must not be interpreted in terms of an absence of micelles below and constant properties, e.g., the surface activity, of the micelles above these ranges. The micellization enthalpy curves differ quite substantially between PS20 and PS80 and, in a subtler fashion, between individual quality grades such as high purity, pure lauric acid/pure oleic acid, super-refined, and China grade.

Existence of a superior polysorbate fraction in respect to protein stabilization and particle formation?

Biologicals including monoclonal antibodies are the current flagships in pharmaceutical industry. However, they are exposed to a multitude of destabilization conditions like for instance hydrophobic interfaces, leading to reduced biological activity. Polysorbates are commonly applied to effectively stabilize these active pharmaceutical ingredients against colloidal stress. Nevertheless, chemical instability of polysorbate via hydrolysis or oxidation results in degradation products that might form particles via phase separation. Polysorbates are mixtures of hundreds of individual components, and recently purer quality grades with reduced variations in the fatty acid composition are available. As the protective function of polysorbate itself is not completely understood, even less is known about its individual components, raising the question of the existence of a superior polysorbate species in respect to protein stabilization or degradation susceptibility. Here, we evaluated the protective function of four main fractions of polysorbate 20 (PS20) in agitation studies with monoclonal antibodies, followed by particle analysis as well as protein and polysorbate content determination. The commercially-available inherent mixtures PS20 high purity and PS20 all-laurate, as well as the fraction isosorbide-POE-monolaurate showed superior protection against mechanical-induced stress (visual inspection and turbidity) at the air-water interface in comparison to sole sorbitan-POE-monolaurate, -dilaurate, and -trilaurate. Fractions composed mainly of higher-order esters like sorbitan-POE-dilaurate and sorbitan-POE-trilaurate indicated high turbidities as indication for subvisible and small particles accompanied by a reduced protein monomer content after agitation. For the isosorbide-POE-monolaurates as well as for the inherent polysorbate mixtures no obvious differences in protein content and protein aggregation (SEC) were observed, reflecting the observations from visual appearance. However, absolute polysorbate concentrations vary drastically between different species in the actual formulations. As there are still open questions in respect to protein specificity or regarding mixtures versus individual components of PS20, further studies must be performed, to gain a better understanding of a "generalized" stabilizing effect of polysorbates on monoclonal antibodies. The knowledge of the characteristics of individual polysorbate species can have the potential to pave the way to superior detergents in respect to protein stabilization and/or degradation susceptibility.

Impact of plasma protein binding on cargo release by thermosensitive liposomes probed by fluorescence correlation spectroscopy.

Thermosensitive liposomes (TSLs) whose phase-transition temperature (Tm) lies slightly above body temperature are ideal candidates for controlled drug release via local hyperthermia. Recent studies, however, have revealed disruptive shifts in the release temperature Tr in mouse plasma, which are attributed to undefined interactions with blood proteins. Here, we study the effects of four major plasma proteins - serum albumin (SA), transferrin (Tf), apolipoprotein A1 (ApoA1) and fibrinogen (Fib) - on the temperature-dependent release of fluorescein di-ß-D-galactopyranoside (FDG) from TSLs. The amount of fluorescein released was quantified by fluorescence correlation spectroscopy (FCS) after hydrolysis of FDG with ß-galactosidase (ß-Gal). This approach is more sensitive and thus superior to previous release assays, as it is impervious to the confounding effects of Triton on conventional fluorescence measurements. The assay determines the molar release ratio, i.e. the number of molecules released per liposome. We show that shifts in the Tr of release do not reflect protein affinities for the liposomes derived from adsorption isotherms. We confirm a remarkable shift in induced release towards lower temperatures in the presence of mouse plasma. In contrast, exposure to rat or human plasma, or fetal bovine serum (FBS), has no effect on the release profile.