Freeze-drying of protein pharmaceuticals is a procedure frequently used to obtain stability of the active pharmaceutical ingredientduring distribution and storage. It can be performed in pre-filled syringes, with a lubricous coating of silicone on the inside, to enable the piston moving. The coating changes the environment potentially affecting the features of the freeze-dried cake since the wetting behavior and adhesion to the inner wall is affected.This project aimed to investigate the effect of the siliconization of the cakes. Three different formulations were freeze-dried in nonsiliconized (NS) and siliconized vials using different siliconization protocols. Analysis was done using differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA),scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and an embedding method, intended to give information about the cake’s shrinkage, cracking and pore-structure. The water content in the bottom of the cakes was consistently higher than in the top, a difference decreasing over time. Increased surface hydrophobicity lead to increased shrinkage of the cake’s volume and a decrease in fogging. The bottom of the protein cake in the vial siliconized with a commercial silicone emulsion consisted of pores with regularly equal pore size and thick pore walls, a structure not seen in any other cake. All cakes in the silicone emulsion siliconized vials had lower water content than the cakes in the vials using the other siliconization method. The XPS-analysis showed that the cakes in the emulsion siliconized vials contained silicon, indicating an excess of silicone when siliconizing and/or an unstable coating. / NextBioForm

This study addresses the fundamentals of an important degradation pathway of storing protein pharmaceuticals in an aqueous formulation, oxidation of methionine residues by peroxides. First, a mechanism by which methionine residues are oxidized is identified via ab initio calculations. The major difference of this new mechanism to previous ones is the role of solvent molecules in the oxidation process. Previously proposed mechanisms suggested that solvent molecules facilitate the transfer of hydrogen associated with the oxidation reaction, but the estimated activation energies and pH dependence of oxidation rates derived from this mechanism rates do not agree with experimental observations. In our proposed mechanism, however, water molecules stabilize the charge separation in the transition-state complex through specific interaction such as hydrogen bonding. This mechanism satisfies all experimental studies on the oxidation of organic sulfides by peroxides. A correct picture of instability mechanism is essential in developing stabilization strategies to design a robust formulation. Based on this mechanism, a structure/instability relationship is built to explain the oxidation rates of methionine residues in a protein molecule. Specifically, a structural property, two-shell water coordination number, is found to correlate semi-quantitatively to the rates of oxidation of methionine residues in G-CSF (granulocyte colony-stimulating factor) and hPTH (human parathoid hormone). We also show that a traditionally used structural property, solvent accessible area, can not provide such accurate correlation and that the dynamic motion of protein molecules and an explicit treatment of solvent molecules are essential to describe the rates of oxidation of methionine residues. Furthermore, the insight provided by the molecule-level understanding in developing a stabilizing formulation is discussed. / Singapore-MIT Alliance (SMA)