Freezing-induced protein aggregation - Role of pH shift and potential mitigation strategies

Alpana A. Thorat, Bhushan Munjal, Todd W. Geders, Raj Suryanarayanan

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


The aggregation behavior of two model proteins- i) bovine serum albumin (BSA) and ii) β-galactosidase (β-gal), was investigated by micro-flow imaging (MFI) during freeze-thaw cycling in phosphate buffered solutions. The pH shift was measured upon cooling the solutions from 20 to −25 °C. When the buffer concentration was 100 mM, cooling caused a pH decrease of 3.1 and 2.7 units (for BSA and β-gal, respectively) attributed to selective crystallization of disodium hydrogen phosphate as a dodecahydrate. The crystallizing solute phase was characterized by low temperature powder X-ray diffractometry. The pH shift resulted in protein aggregation, evident from the pronounced increase in particle count (by MFI). The addition of cellobiose attenuated the pH shift on cooling (pH decrease of ~1.0 unit), and no evidence of either buffer salt crystallization or protein aggregation was observed. Decreasing the buffer concentration to 10 mM, also prevented protein aggregation. The protein, by inhibiting buffer crystallization, prevented the pH shift and then the buffer, by maintaining the pH, enhanced protein stability.

Original languageEnglish (US)
Pages (from-to)591-599
Number of pages9
JournalJournal of Controlled Release
StatePublished - Jul 10 2020

Bibliographical note

Funding Information:
The project was partially supported by the William and Mildred Peters endowment fund . Parts of this work were carried out at the Characterization Facility, University of Minnesota , which received partial support from NSF through MRSEC program. Special thanks to Bio-Techne for providing access to their MFI 5200.

Publisher Copyright:
© 2020 Elsevier B.V.

Copyright 2020 Elsevier B.V., All rights reserved.


  • Aggregation
  • Albumin
  • Buffer
  • Crystallization
  • Phosphate
  • pH shift
  • β-Galactosidase

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

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