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Biotechnol Appl Biochem. 2004 Apr;39(Pt 2):165-77.

Freeze-drying of proteins: some emerging concerns.

Author information

1
Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India.

Abstract

Freeze-drying (lyophilization) removes water from a frozen sample by sublimation and desorption. It can be viewed as a three-step process consisting of freezing, primary drying and secondary drying. While cryoprotectants can protect the protein from denaturation during early stages, lyoprotectants are needed to prevent protein inactivation during drying. The structural changes as a result of freeze-drying have been investigated, especially by FTIR (Fourier-transform IR) spectroscopy. In general, drying results in a decrease of alpha-helix and random structure and an increase in beta-sheet structure. In the case of basic fibroblast growth factor and gamma-interferon, enhanced FTIR showed large conformational changes and aggregation during freeze-drying, which could be prevented by using sucrose as a lyoprotectant. It is now well established that structural changes during freeze-drying are responsible for low activity of freeze-dried powders in nearly anhydrous media. Strategies such as salt activation can give 'activated' enzyme powders, e.g. salt-activated thermolysin-catalysed regioselective acylation of taxol to give a more soluble derivative for therapeutic use. In the presence of moisture, freeze-dried proteins can undergo disulphide interchange and other reactions which lead to inactivation. Such molecular changes during storage have been described for human insulin, tetanus toxoid and interleukin-2. Some successful preventive strategies in these cases have also been mentioned as illustrations. Finally, it is emphasized that freeze-drying is not an innocuous process and needs to be understood and used carefully.

PMID:
15032737
DOI:
10.1042/BA20030133
[Indexed for MEDLINE]

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