Subvisible Particles Derived by Dropping Stress Enhance Anti-PEG Antibody Production and Clearance of PEGylated Proteins in Mice.

Bioconjugation with polyethylene glycol (PEG) is important for protein drug development as it has improved biological stability. In contrast, proteins including PEGylated ones are susceptible to physicochemical stresses. Particularly, protein drugs in solution may form aggregates or subvisible particles if they are exposed to dropping stress during transportation. However, many PEGylation studies have focused on its usefulness, such as the extension of half-life in blood, and changes in the physical properties or biological responses of PEGylated proteins under dropping stress remain unexplored. Here, we prepared four PEGylated ovalbumin (PEG-OVA) molecules conjugated with different lengths (5 or 20 kDa) and numbers (large [L] or small [S]) of PEG, analyzed the formation of subvisible particles under dropping stress, and examined their impact on antibody production and clearance. Under dropping stress, the aggregated particle concentration of 20 kDa PEG-OVA (S) and (L) solutions was approximately 3-fold that of the OVA solution. Moreover, administration of 20 kDa PEG-OVA with dropping stress induced anti-PEG antibody production and clearance of PEG-OVA. As a mechanism, dropping stress could enhance the uptake of 20 kDa PEG-OVA (L) by macrophages. These findings could provide insights into proper transportation conditions to ensure the quality of PEGylated protein drugs.

Differentiation of autonomic neurons by BMP-independent mechanisms.

A number of signaling molecules and transcription factors play important roles in the development of the autonomic nervous system. Here, we show that mouse trunk neural crest cells can differentiate into autonomic neurons expressing mammalian achaete-scute homolog 1 (mash1), Phox2b, tyrosine hydroxylase, and/or dopamine-beta-hydroxylase in the absence of bone morphogenetic protein (BMP)-4. The expression of mash1 and Phox2b is induced even in the presence of noggin or chordin, which are inhibitors of BMP signaling. Whereas these autonomic neurons do not express c-ret, the receptor for glial-cell-line-derived neurotrophic factor (GDNF), GDNF promotes the differentiation of c-ret-positive autonomic neurons in the presence of noggin. Autonomic neurogenesis is completely prevented by fibroblast growth factor (FGF)-2 treatment or by activation of Notch signaling. Furthermore, the suppression of Phox2b expression by FGF-2 can be recovered by treatment with Notch-1 small interfering RNA. Our data suggest that BMP-independent mechanisms promote the differentiation of autonomic neurons, and that FGF-2 suppresses autonomic neurogenesis by means of the activation of Notch signaling.