Investigation of PEG crystallization in frozen and freeze-dried PEGylated recombinant human growth hormone-sucrose systems: Implications on storage stability

Bakul S. Bhatnagar, Susan W.H. Martin, Tamara S. Hodge, Tapan K. Das, Liji Joseph, Dirk L. Teagarden, Evgenyi Shalaev, Raj Suryanarayanan

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)3062-3075
Number of pages14
JournalJournal of Pharmaceutical Sciences
Volume100
Issue number8
DOIs
StatePublished - Aug 2011

Keywords

  • Crystallization
  • Eutectic melting
  • Freeze-drying
  • Low temperature XRD
  • Lyophilization
  • PEGylation
  • Phase separation
  • Recombinant human growth hormone
  • Stability
  • Sucrose

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