Nanobubbles in Reconstituted Lyophilized Formulations: Interaction With Proteins and Mechanism of Formation

Jared R. Snell, N. S.Krishna Kumar, Raj Suryanarayanan, Theodore W. Randolph

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Reconstitution of lyophilized disaccharide formulations results in the formation of nanosized air bubbles that persist in suspension for weeks. If proteins are present, interactions with nanobubbles may cause loss of monomeric protein and formation of subvisible particles. The goals of this work are to determine the mechanism(s) by which nanobubbles form in reconstituted lyophilized formulations and to develop strategies for reducing nanobubble generation. We hypothesize that nanobubbles are created from nanosized gas pockets within lyophilized solids, which become bubbles when the surrounding matrix is dissolved away during reconstitution. Nanosized voids may originate from small ice crystals formed within the concentrated liquid during freezing that subsequently sublime during drying. Nanobubble concentrations are correlated with the extent of mannitol crystallization during freezing. Nanosized ice crystals, induced by the release of water during mannitol crystallization, were responsible for nanobubble formation. The presence of trehalose or sucrose, in formulations with low mannitol concentrations, inhibited excipient crystallization during lyophilization and reduced nanobubble levels following reconstitution. Our results show a correlation between nanobubble formation and concentrations of insoluble IL-1ra aggregates, suggesting that minimizing nanobubble generation may be an effective strategy for reducing protein aggregation following reconstitution.

Original languageEnglish (US)
Pages (from-to)284-292
Number of pages9
JournalJournal of Pharmaceutical Sciences
Issue number1
StatePublished - Jan 2020

Bibliographical note

Funding Information:
The authors gratefully acknowledge support from the National Institutes of Health , grant REB026006A (TR). The work was partially supported by the William and Mildred Peters Endowment fund (RS).

Publisher Copyright:
© 2020 American Pharmacists Association®


  • Lyophilization
  • crystallization
  • nanoparticle(s)
  • protein aggregation
  • trehalose

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't


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