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

Sonje, Jayesh; Chisholm, Carly Fleagle; Suryanarayanan, Raj

doi:10.1016/j.ijpharm.2022.121995

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

Journal Article · Wed Jul 06 00:00:00 EDT 2022 · International Journal of Pharmaceutics

DOI:https://doi.org/10.1016/j.ijpharm.2022.121995· OSTI ID:2423440

^[1]; Chisholm, Carly Fleagle ^[2]; Suryanarayanan, Raj ^[1]

Univ. of Minnesota, Minneapolis, MN (United States)
Janssen Research & Development, Malvern, PA (United States)

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. Here, 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.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 2423440

Journal Information:: International Journal of Pharmaceutics, Journal Name: International Journal of Pharmaceutics Vol. 630; ISSN 0378-5173

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
Freeze–thaw
Frozen storage
Homogenous freeze-concentrate
Mannitol
Matrix
Protein
Sucrose
Trehalose

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

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