Povey, J. and Perez-Moral, N. and Noel, T.R. and Parker, R. and Howard, M.J. and Smales, C.M. (2009) Investigating variables and mechanisms that influence protein integrity in low water content amorphous carbohydrate matrices. Biotechnol Prog, 25 (5). pp. 1217-27. ISSN 1520-6033.
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.
|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 > Science Technology and Medical Studies > School of Biosciences > Protein Science Group|
|Depositing User:||Mark Howard|
|Date Deposited:||16 Aug 2011 03:46|
|Last Modified:||13 Dec 2011 14:28|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/28012 (The current URI for this page, for reference purposes)|
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