title: Investigation of PEG crystallization in frozen and freeze-dried PEGylated recombinant human growth hormone-sucrose systems: implications on storage stability.
abstract: 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.
authors.affiliation: Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455; Pfizer BioTherapeutics, Andover, Massachusetts 01810.
authors.forename: Bakul S
authors.initials: BS
authors.lastname: Bhatnagar
authors.affiliation: Pfizer BioTherapeutics, St. Louis, Missouri 63017.
authors.forename: Susan W H
authors.initials: SWH
authors.lastname: Martin
authors.affiliation: Pfizer BioTherapeutics, St. Louis, Missouri 63017.
authors.forename: Tamara S
authors.initials: TS
authors.lastname: Hodge
authors.affiliation: Pfizer BioTherapeutics, St. Louis, Missouri 63017.
authors.forename: Tapan K
authors.initials: TK
authors.lastname: Das
authors.affiliation: Pfizer BioTherapeutics, St. Louis, Missouri 63017.
authors.forename: Liji
authors.initials: L
authors.lastname: Joseph
authors.affiliation: Pfizer BioTherapeutics, St. Louis, Missouri 63017.
authors.forename: Dirk L
authors.initials: DL
authors.lastname: Teagarden
authors.affiliation: Pfizer PharmaTherapeutics R&D, Eastern point Road, Groton, Connecticut 06340.
authors.forename: Evgenyi Y
authors.initials: EY
authors.lastname: Shalaev
authors.affiliation: Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455. Electronic address: surya001@umn.edu.
authors.forename: Raj
authors.initials: R
authors.lastname: Suryanarayanan
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D004355:Drug Stability; D004356:Drug Storage; D005612:Freeze Drying; D019382:Human Growth Hormone / Q000031:analogs & derivatives* / Q000737:chemistry; D044367:Phase Transition; D011092:Polyethylene Glycols / Q000737:chemistry*; D013395:Sucrose / Q000737:chemistry*; D013997:Time Factors; D014961:X-Ray Diffraction
chemical_list: C562190:polyethylene glycol-recombinant human growth hormone; D019382:Human Growth Hormone; D011092:Polyethylene Glycols; D013395:Sucrose