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 stabilisation due to their inherent instability in aqueous solution. Freeze-drying is an established method for protein stabilisation, although the use of additives is often necessary in order to preserve protein structure and activity 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-subunit enzyme L-asparaginase during lyophilisation, assessed post-drying enzyme activity and quaternary structure, and related the extrapolated levels of additive necessary to provide full stabilisation to the theoretical levels predicted from an existing hypothesis using molecular modelling. It was found that each of the saccharides tested here displayed similar levels of protection towards L-asparaginase under the conditions used. Amounts of additive required to give full stabilisation to the enzyme were extrapolated from the activity data and were found to be in good agreement with theoretical amounts calculated from molecular modelling studies. Our data suggest that the existing hypothesis may be relevant to the prediction of optimum levels of lyoprotectant for the freeze-drying of proteins. However, further studies would be necessary in order to obtain a full picture of protein-additive interactions at the molecular level.
Ward
K R
KR
Pharmaceutical Sciences Institute, Aston University, Aston Triangle, Birmingham, UK.
Adams
G D
GD
Alpar
H O
HO
Irwin
W J
WJ
D001215:Asparaginase / Q000737:chemistry*; D004795:Enzyme Stability; D005612:Freeze Drying; D006860:Hydrogen Bonding; D008958:Models, Molecular
D001215:Asparaginase