Protection of the enzyme L-asparaginase during lyophilisation - a molecular modelling approach to predict required level of lyoprotectant

Many novel therapeutic agents are proteins and peptides which need stabilis ation due to their inherent instability in aqueous solution. Freeze-drying is an established method for protein stabilisation, although the use of add itives is often necessary in order to preserve protein structure and activi ty during lyophilisation itself. The molecular interactions between protein and protective additive are as yet unclear. In this study, we examined the use of a range of saccharide additives to stabilise the model multi-subuni t enzyme L-asparaginase during lyophilisation, assessed post-drying enzyme activity and quaternary structure, and related the extrapolated levels of a dditive necessary to provide full stabilisation to the theoretical levels p redicted from an existing hypothesis using molecular modelling. It was foun d that each of the saccharides tested here displayed similar levels of prot ection towards L-asparaginase under the conditions used, Amounts of additiv e required to give full stabilisation to the enzyme were extrapolated from the activity data and were found to be in good agreement with theoretical a mounts calculated from molecular modelling studies. Our data suggest that t he existing hypothesis may be relevant to the prediction of optimum levels of lyoprotectant for the freeze-drying of proteins. However, further studie s would be necessary in order to obtain a full picture of protein-additive interactions at the molecular level. (C) 1999 Elsevier Science B.V. All rig hts reserved.