Investigation of PEG Crystallization in Frozen and Freeze‐Dried PEGylated Recombinant Human Growth Hormone-Sucrose Systems: Implications on Storage Stability

The objectives of the current study were to investigate (i) the phase behavior of a PEGylated recombinant human growth hormone (PEG-rhGH, ∼60 kDa) during freeze‐drying and (ii) its storage stability. The phase transitions during freeze-thawing of an aqueous solution containing PEG-rhGH and sucrose were characterized by differential scanning calorimetry. Finally, PEG-rhGH and sucrose formulations containing low, medium, and high polyethylene glycol (PEG) to sucrose ratios were freeze‐dried in dual‐chamber syringes and stored at 4°C and 25°C. Chemical decomposition (methionine oxidation and deamidation) and irreversible aggregation were characterized by size‐exclusion and ion‐exchange chromatography, and tryptic mapping. PEG crystallization was facilitated when it was covalently linked with rhGH. When the solutions were frozen, phase separation into PEG‐rich and sucrose‐rich phases facilitated PEG crystallization and the freeze‐dried cake contained crystalline PEG. Annealing caused PEG crystallization and when coupled with higher drying temperatures, the primary drying time decreased by up to 51%. When the freeze‐dried cakes were stored at 4°C, while there was no change in the purity of the PEG-rhGH monomer, deamidation was highest in the formulations with the lowest PEG to sucrose ratio. When stored at 25°C, this composition also showed the most pronounced decrease in monomer purity, the highest level of aggregation, and deamidation. Furthermore, an increase in PEG crystallinity during storage was accompanied by a decrease in PEG-rhGH stability. Interestingly, during storage, there was no change in PEG crystallinity in formulations with medium and high PEG to sucrose ratios. Although PEG crystallization during freeze‐drying did not cause protein degradation, crystallization during storage might have influenced protein stability. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3062-3075, 2011