Long-term stability of monoclonal antibodies to be used as biologics is a key aspect in their development. Therefore, its possible early prediction from accelerated stability studies is of major interest, despite currently being regarded as not sufficiently robust. In this work, using a combination of accelerated stability studies (up to 6 months) and first order degradation kinetic model, we are able to predict the long-term stability (up to 3 years) of multiple monoclonal antibody formulations. More specifically, we can robustly predict the long-term stability behaviour of a protein at the intended storage condition (5 °C), based on up to six months of data obtained for multiple quality attributes from different temperatures, usually from intended (5 °C), accelerated (25 °C) and stress conditions (40 °C). We have performed stability studies and evaluated the stability data of several mAbs including IgG1, IgG2, and fusion proteins, and validated our model by overlaying the 95% prediction interval and experimental stability data from up to 36 months. We demonstrated improved robustness, speed and accuracy of kinetic long-term stability prediction as compared to classical linear extrapolation used today, which justifies long-term stability prediction and shelf-life extrapolation for some biologics such as monoclonal antibodies. This work aims to contribute towards further development and refinement of the regulatory landscape that could steer toward allowing extrapolation for biologics during the developmental phase, clinical phase, and also in marketing authorisation applications, as already established today for small molecules.
The interaction between disrupted lipid homeostasis and immune response is implicated in the pathogenesis of several diseases, but the molecular bridges between the major players are still a matter of controversy. Our systemic study of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) in the livers of mice exposed to 20-h cytokine/fasting for the first time shows that TNF-alpha interferes with adaptation to fasting and activates harmful proatherogenic pathways, partially through interaction with the insulin-Insig-sterol regulatory element binding protein (Srebp) signaling pathway. In addition to the increased expression of acute-phase inflammatory genes, the most prominent alterations represent modified lipid homeostasis observed on the gene expression and metabolite levels. These include reduction of HDL-cholesterol, increase of LDL-cholesterol, and elevated expression of cholesterogenic genes, accompanied by increase of potentially harmful precholesterol metabolites and suppression of cholesterol elimination through bile acids, likely by farnesoid X receptor-independent mechanisms. On the transcriptional level, a shift from fatty oxidation toward fatty acid synthesis is observed. The concept of the influence of TNF-alpha on the Srebp regulatory network, followed by downstream effects on sterol metabolism, is novel. Observed acute alterations in lipid metabolism are in agreement with chronic disturbances found in patients.
The natural biconcave shape of red blood cells (RBC) may be altered by injury or environmental conditions into a spiculated form (echinocyte). An analysis is presented of the effect of such a transformation on the resistance of RBC to entry into capillary sized cylindrical tubes. The analysis accounts for the elasticity of the membrane skeleton in dilation and shear, and the local and nonlocal resistance of the bilayer to bending, the latter corresponding to different area strains in the two leaflets of the bilayer. The shape transformation is assumed to be driven by the equilibrium area difference (delta A(0), the difference between the equilibrium areas of the bilayer leaflets), which also affects the energy of deformation. The cell shape is approximated by a parametric model. Shape parameters, skeleton shear deformation, and the skeleton density of deformed membrane relative to the skeleton density of undeformed membrane are obtained by minimization of the corresponding thermodynamic potential. Experimentally, delta A(0) is modified and the corresponding discocyte-echinocyte shape transition obtained by high-pressure aspiration into a narrow pipette, and the deformability of the resulting echinocyte is examined by whole cell aspiration into a larger pipette. We conclude that the deformability of the echinocyte can be accounted for by the mechanical behavior of the normal RBC membrane, where the equilibrium area difference delta A(0) is modified.
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