Siwar Trabelsi

Production of particles of therapeutic proteins at the air–water interface during compression/dilation cycles

Particles in protein therapeutics are undesirable because they may have the potential for causing adverse immunogenicity in patients. Agitation-induced exposure to the air–water interface during manufacturing, shipping, and administration can cause particle formation in therapeutic protein products. We systematically studied how application of surface pressure during periodic interfacial compressions caused a model monoclonal antibody to form particles. Above a critical interfacial compression ratio of 5 we observed a dramatic increase in the rate of protein particle formation. During continuous interfacial compression/dilation cycles, particle numbers increased but the particle size distribution remained unchanged. When cyclic compressions were halted, particles did not nucleate additional particles or grow further in bulk solution suggesting that they are formed only at the air–water interface. In fact, we found that particles in the bulk slowly decreased in number upon standing. The rate of particle formation was only weakly dependent on both the bulk protein concentration and the period of cyclical interfacial compressions. These observations are consistent with the interfacial aggregation of proteins during periods of high surface pressure, followed by collapse of the adsorbed layer and detachment of protein particles from the interface into the bulk.

Correlating linactant efficiency and self-assembly: structural basis of line activity in molecular monolayers.

Surfactants exhibit characteristic phenomena, including the reduction of interfacial free energy, self-assembly into aggregates, and even the formation of lyotropic liquid crystalline phases at high concentrations. Our research has shown that a semifluorinated phosphonic acid can act as the two-dimensional analogue of a surfactant-a linactant-by reducing the line tension between hydrocarbon-rich and fluorocarbon-rich phases in a Langmuir monolayer. This linactant can also self-assemble into nanoscale clusters in a monolayer. Here, we explore the dependence of linactant behavior on molecular structure. Members of a homologous series of partially fluorinated phosphonic acids were synthesized and tested as linactants: CF(3)(CF(2))(n-1)(CH(2))(m)PO(3)H (abbreviated as FnHmPO(3)). The tests revealed that linactants with longer hydrophobic chains were most efficient in lowering line tension. For linactants with the same overall chain length, the length of the fluorocarbon block was correlated with efficiency. Thus, the linactant efficiency was ranked in the order F8H8PO(3) < F10H6PO(3) < F8H11PO(3) < F10H9PO(3). In all cases, linactant-containing Langmuir-Blodgett monolayers exhibited nanoscale molecular clusters with characteristic dimensions of 20-30 nm; enhanced linactant efficiency was systematically correlated with larger clusters.

Semi-fluorinated phosphonic acids form stable nanoscale clusters in Langmuir–Blodgett and self-assembled monolayers

We compared molecular monolayers of semifluorinated phosphonic acids (F8H11PO3, F10H6PO3, F8H8PO3, and F6H10PO3) on mica substrates prepared by two different methods: Langmuir–Blodgett (LB) transfer of pre-formed monolayers from the air–water interface, and self-assembled monolayers (SAMs) formed spontaneously at the interface between mica and solution (with tetrahydrofuran as the solvent). The films were investigated with atomic force microscopy (AFM) and contact angle measurements. Nanometer-scale two-dimensional (2D) clusters (20–30 nm in size) were observed in both the LB films and the SAMs. Time-dependent AFM images suggested that for SAMs derived from F8H11PO3, F8H8PO3, and F6H10PO3, small clusters nucleated, but stopped growing once a stable size was reached. In situAFM images suggested that the clusters were dome-shaped. The LB monolayer structures were consistent with those of the SAMs, but with greater long-range order of the clusters in some cases. For SAMs derived from F10H6PO3, however, long immersion times led to continued growth and coalescence of the 2D clusters and formation of a flat, untextured monolayer. Similarly, LB films generated from F10H6PO3 exhibited cluster coalescence. The similarity of the observed structures in LB films and SAMs suggests that these nanostructured films represent an equilibrium state based on intrinsic self-organization of the semifluorinated molecules, even in SAMs, where mobility is significantly restricted at the solid–liquid interface. Based on these observations, we hypothesize that the packing incommensurability between the hydrocarbon and fluorocarbon blocks leads to splay of neighboring chains and spontaneous curvature. However, when intermolecular interactions are dominated by a long fluorocarbon block, there is a transition to a flat structure, where the packing mismatch is compensated for by increased disorder within the hydrocarbon block.

Swelling of a cluster phase in Langmuir monolayers containing semi-fluorinated phosphonic acids

Langmuir monolayers of semi-fluorinated nonadecylphosphonic acid (F8H11PO), hexadecylphosphonic acid (H16PO), and their mixtures were investigated by Brewster angle microscopy (BAM), atomic force microscopy (AFM) and surface-pressure measurements. Nanometre-scale two-dimensional clusters were observed by AFM in a spread monolayer of pure F8H11PO transferred to mica. Two different organized arrangements of clusters were observed. AFM and BAM observations showed that the mixture exhibits a solid phase over a large range of mole fraction and surface pressure, sometimes in coexistence with clusters. With increasing mole fraction of H16PO, the lateral shape of these clusters remains the same while their organization and their height change.