Opalescence and high viscosities can pose challenges for high concentration formulation of antibodies. Both phenomena result from protein-protein intermolecular interactions that can be modulated with solution ionic strength. We studied a therapeutic monoclonal antibody that exhibits high viscosity in solutions at low ionic strength (~20 centipoise (cP) at 90 mg/mL and 23°C) and significant opalescence at isotonic ionic strength (approximately 100 nephelometric turbidity units at 90 mg/mL and 23°C). The intermolecular interactions responsible for these effects were characterized using membrane osmometry, static light scattering and zeta potential measurements. The net protein-protein interactions were repulsive at low ionic strength (~4 mM) and attractive at isotonic ionic strengths. The high viscosities are attributed to electroviscous forces at low ionic strength and the significant opalescence at isotonic ionic strength is correlated with attractive antibody interactions. Furthermore there appears to be a connection to critical phenomena and it is suggested that the extent of opalescence is dependent on the proximity to the critical point. We demonstrate that by balancing the repulsive and attractive forces via intermediate ionic strengths and by increasing the mAb concentration above the apparent critical concentration both opalescence and viscosity can be simultaneously minimized.
We report nonflocculated dilute water-in-CO2 (W/C) miniemulsions stable for 24 h in contrast with flocculated unstable macroemulsions reported previously. The surfactants, poly(1,1-dihydroperfluorooctyl methacrylate)-b-poly(ethylene oxide) (PFOMA-b-PEO), were synthesized by atom transfer radical polymerization to achieve the proper hydrophilic-CO2-philic balance (HCB) and a low interfacial tension (∼0.2-2 mN/m) between water and CO2. The average particle diameter, ranging from 70 to 140 nm, was measured with multiwavelength turbidimetry utilizing Mie theory, and the interfacial tension was measured with high pressure pendant drop tensiometry. Because flocculation and coalescence were suppressed nearly completely, it became feasible to investigate emulsion droplet formation and droplet growth by Ostwald ripening. Droplet formation was characterized as a function of the mechanical energy at various CO2 densities and temperatures and was correlated quantitatively to the interfacial tensions. The molar water/ surfactant ratios reached 1170, in contrast with values of only about 5-60 for W/C microemulsions. The ability to stabilize nanometer-sized miniemulsion droplets with large water/surfactant ratios is of great practical interest in reactions, separations, and materials formation processes in CO2.
During storage stability studies of a monoclonal antibody (mAb) it was determined that the primary route of degradation involved fragmentation into lower molecular weight species. The fragmentation was characterized with size-exclusion high performance liquid chromatography (SE-HPLC), SDS-PAGE and matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Fragmentation proceeded via hydrolysis, likely catalyzed by trace metal ions, of a peptide bond in the hinge region of the mAb’s heavy chain, which produced two prominent low molecular weight species during storage: a single, free Fab fragment and a Fab+Fc fragment. The fragmentation is observed in phosphate-buffered solutions at two ionic strengths but not in histidine-buffered solutions at identical ionic strengths. Chaotrope-induced and thermally-induced unfolding studies of the mAb indicated differences in the unfolding pathways between the two buffer solutions. This folding intermediate observed during chaotrope-induced unfolding was further characterized by intrinsic fluorescence quenching, which suggested that a small portion of the molecule is resistant to chaotrope-induced unfolding in histidine buffer systems. The thermally-induced unfolding indicates a reduction in cooperativity of the unfolding process in the presence of histidine relative to phosphate. A relationship between the histidine-induced effects on unfolding pathway and the relative resistance to fragmentation is suggested.
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