Isomalt and its Diastereomer Mixtures as Stabilizing Excipients With Freeze-Dried Lactate Dehydrogenase
Abstract
The purpose of this research was to study isomalt as a protein-stabilizing excipient with lactate dehydrogenase (LDH) during freeze-drying and subsequent storage and compare it to sucrose, a standard freeze-drying excipient. Four different diastereomer mixtures of isomalt were studied. The stability of the protein was studied with a spectrophotometric enzyme activity test and circular dichroism after freeze-drying and after 21 days of storage at 16% RH. Physical stability was analyzed with differential scanning calorimetry and Karl Fischer titration. Statistical analysis was utilized in result analysis. LDH activity was almost completely retained after freeze-drying with sucrose; whereas samples stabilized with isomalt diastereomer mixtures had a considerably lower protein activity. During storage the sucrose-containing samples lost most of their enzymatic activity, while the isomalt mixtures retained the protein activity better. In all cases changes to protein secondary structure were observed. Isomalt diastereomer mixtures have some potential as protein-stabilizing excipients during freeze-drying and subsequent storage. Isomalt stabilized LDH moderately during freeze-drying; however it performed better during storage. Future studies with other proteins are required to evaluate more generally whether isomalt would be a suitable excipient for pharmaceutical freeze-dried protein formulations.
Conclusions
Both sucrose and isomalt were able to stabilize LDH during freeze-drying to some extent as evidenced by the lower relative enzymatic activity in samples containing no sugar excipients. Sucrose performed clearly better than isomalt as a cryo- and lyostabilizing excipient preserving LDH activity almost fully after freeze-drying. However, during storage the samples stabilized with sucrose lost over half of their initial activity, whereas isomalt protected LDH better from the storage-induced destabilizing effects. The low protein activity level after storage suggests that sucrose was not able to stabilize LDH efficiently in the presence of moisture. During storage, the significance of stabilizing sugar excipients was emphasized as LDH samples without sugars lost their activity almost completely, with only a quarter of the pre-storage LDH activity remaining after the storage period. Even though iso malt was not the most optimal stabilizing excipient for the studied protein during lyophilization, it showed some protein-stabilizing effects, especially during the storage stability studies. In future studies, the aggregation tendency of lyophilized LDH stabilized with isomalt should be investigated to assess whether aggregation takes place and does it affect LDH stability. In order to assess the potential of isomalt as a novel cryo- and lyoprotecting excipient in freeze-dried protein formulations, more freeze-drying studies should be carried out with different proteins and in combinations with other excipients. Before isomalt could be used as an excipient in freeze-dried formulations intended for parenteral use, also its intravenous toxicity must be studied.
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