Frozen storage of proteins: Use of mannitol to generate a homogenous freeze-concentrate

Jayesh Sonje, Carly Fleagle Chisholm, Raj Suryanarayanan

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Therapeutic proteins may be subjected to several freeze–thaw cycles throughout manufacturing and storage. The protein solution composition and the freezing conditions may lead to incomplete ice crystallization in the frozen state. This can also result in freeze-concentrate heterogeneity characterized by multiple glass transition temperatures and protein destabilization. The overall objective was to investigate the potential advantages of including a crystallizing excipient (mannitol) along with a sugar (sucrose or trehalose) for frozen storage. This study showed that the addition of mannitol, a readily crystallizing excipient, facilitated ice crystallization. Inclusion of an isothermal hold during cooling (annealing) maximized the mannitol crystallization and resulted in a homogenous freeze-concentrate of a constant composition characterized by a single glass transition temperature. The role of freezing rate and annealing on both mannitol and ice crystallization were discerned using high intensity synchrotron radiation. The addition of sucrose or trehalose, at an appropriate concentration, stabilized the protein. The mannitol to sugar ratio (3:1 or 1:1, 5 % w/v) was optimized to selectively cause maximal crystallization of mannitol while retaining the sugar amorphous. Human serum albumin (1 mg/mL) in these optimized and annealed compositions did not show any meaningful aggregation, even after multiple freeze–thaw cycles. Thus, in addition to a sugar as a stabilizer, the use of a crystallizing excipient coupled with an annealing step can provide an avenue for frozen storage of proteins.

Original languageEnglish (US)
Article number121995
JournalInternational journal of pharmaceutics
Volume630
DOIs
StatePublished - Jan 5 2023

Bibliographical note

Funding Information:
We acknowledge funding from the Kildsig Center for Pharmaceutical Processing Research and the support of the William and Mildred Peters Endowment Fund. Use of the Advanced Photon Source was supported by U.S. Department of Energy, Office of Science, Office of Basic Energy Science , under Contract No. DE-AC02-06CH11357 . We thank Dr. Wenqian Xu and Dr. Andrey Yakovenko for their help during the beamline experiments.

Publisher Copyright:
© 2022

Keywords

  • Freeze–thaw
  • Frozen storage
  • Homogenous freeze-concentrate
  • Mannitol
  • Matrix
  • Protein
  • Sucrose
  • Trehalose

PubMed: MeSH publication types

  • Journal Article

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