Conformational stability of lyophilized PEGylated proteins in a phase-separating system

PEGylation of proteins is of great interest to the pharmaceutical industry as covalent attachment of poly(ethylene glycol) (PEG) molecules can increas e protein sera half-lives and reduce antigenicity. Not surprisingly, PEGyla tion significantly alters the surface characteristics of a protein, and con sequently, its conformational stability during freezing and drying. Freeze concentration-induced phase separation between excipients has been previous ly shown to cause degradation of the secondary structure in lyophilized hem oglobin. In this report we show how PEGylation of two proteins, hemoglobin and brain-derived neurotrophic factor (BDNF), influences partitioning and p rotein secondary structure as determined by FTIR spectroscopy in a system p rone to freezing-induced phase separation. PEGylation of hemoglobin reduces the loss of structure induced by lyophilization in a PEG/dextran system th at phase separates during freezing, perhaps due to altered partitioning, Th e partition coefficient for native hemoglobin favors the dextran-rich phase (PEG/dextran partition coefficient = 0.3), while PEGylated hemoglobin favo rs the PEG phase (partition coefficient = 3.1). In addition, we demonstrate that PEGylation alters hemoglobin's stability during lyophilization in the absence of other excipients. In contrast, because native BDNF already part itions into the PEG-rich phase, PEGylation of BDNF has a less dramatic effe ct on both partition coefficients and conformational stability during lyoph ilization. This is the first report on the effects of PEGylation on protein structural stability during lyophilization and points out the need to cons ider modification of formulations in response to changing protein surface c haracteristics.