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Investigating variables and mechanisms that influence protein integrity in low water content amorphous carbohydrate matrices

Povey, Jane F., Perez-Moral, Natalia, Noel, Timothy R., Parker, Roger, Howard, Mark J., Smales, Christopher Mark (2009) Investigating variables and mechanisms that influence protein integrity in low water content amorphous carbohydrate matrices. Biotechnology Progress, 25 (5). pp. 1217-27. ISSN 8756-7938. (doi:10.1002/btpr.207)

Abstract

Biopharmaceutical proteins are often formulated and freeze dried in agents that protect them from deleterious reactions that can compromise activity and authenticity. Although such approaches are widely used, a detailed understanding of the molecular mechanisms of protein stabilization in low water content amorphous glasses is lacking. Further, whilst deterioration chemistries are well described in dilute solution, relatively little is known about the extent and mechanisms by which protein integrity is compromised in the glassy state. Here we have investigated the relationship between protein modification and rate thereof, with variation of pH, carbohydrate excipient, temperature and the glass transition temperature using a model protein, lysozyme. Mass spectrometry analysis and peptide mapping confirm that protein modifications do occur in the glassy state in a time-, temperature-, and carbohydrate excipient-dependent manner. There were clear trends between the buffer pH and the primary modification detected (glycation). Most importantly, there were differences in the apparent reactivities of the lysine residues in the glass compared with those previously determined in solution, and therefore, the well-characterized solution reactivity of this reaction cannot be used to predict likely sites of modification in the glassy state. These findings have implications for (i) the selection and combinations of formulation components, particularly with regard to glycation in the glassy state, and (ii) the design of procedures and methodologies for the improvement of protein stability in the glassy state.

Item Type: Article
DOI/Identification number: 10.1002/btpr.207
Additional information: Povey, Jane F Perez-Moral, Natalia Noel, Timothy R Parker, Roger Howard, Mark J Smales, C Mark Biotechnology and Biological Sciences Research Council/United Kingdom Research Support, Non-U.S. Gov't United States Biotechnology progress Biotechnol Prog. 2009 Sep-Oct;25(5):1217-27.
Uncontrolled keywords: Amides, Calorimetry, Differential Scanning, Carbohydrates/*chemistry, Freeze Drying, Glycosylation, Hydrogen-Ion Concentration, Muramidase/chemistry, Peptide Mapping, Phase Transition, Protein Stability, Proteins/*chemistry, Specimen Handling/*methods, Spectrometry, Mass, Electrospray Ionization, Temperature, Time Factors,
Subjects: Q Science > QD Chemistry
Q Science > QH Natural history > QH301 Biology
Divisions: Faculties > Sciences > School of Biosciences > Protein Science Group
Depositing User: M.J. Howard
Date Deposited: 16 Aug 2011 03:46 UTC
Last Modified: 29 May 2019 08:36 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/28012 (The current URI for this page, for reference purposes)
Smales, Christopher Mark: https://orcid.org/0000-0002-2762-4724
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