Total protein concentration and stability of Amb a 1 in glycerinated ragweed sublingual immunotherapy stored at room temperature and refrigerated cold temperature.

Abstract

BACKGROUND: Few studies have investigated optimal storage conditions or expiration dates for sublingual immunotherapy (SLIT) formulations prepared from glycerinated allergen extracts. HYPOTHESIS/OBJECTIVES: The objective of this study was to compare concentrations of short ragweed major allergen (Amb a 1) and total protein in SLIT formulations stored at two different temperatures. It was hypothesised that protein concentrations would show greater decline over time in a formulation stored at room temperature (RT) than in one stored under refrigeration. MATERIALS AND METHODS: Two SLIT samples containing equal volumes of 20,000 PNU Amb a 1 extract were prepared and stored at refrigerated cold (CT) (2-8&#xb0;C) or RT (20-24&#xb0;C) for 140&#x2009;days. Changes in total protein and major allergen concentration and composition were measured by Bradford assay, two-site enzyme-linked immunosorbent assay and SDS-PAGE. Presence of Amb a 1 was confirmed with Western immunoblot. Data were analysed using an analysis of covariance, with p&#x2009;<&#x2009;0.05 considered significant. RESULTS: SDS-PAGE showed compositional changes in a ~26-30&#x2009;kDa protein band under RT and not CT storage. The Amb a 1 concentration of the RT SLIT sample declined significantly over time, compared to that of the CT SLIT sample (F=&#x2009;47.69, p&#x2009;<&#x2009;0.0001). There was no significant difference in total protein concentration over time between groups (F=&#x2009;1.79, p&#x2009;=&#x2009;0.22). CONCLUSIONS AND CLINICAL RELEVANCE: These results demonstrate that storage of glycerinated SLIT formulations in refrigerated CT preserved the highest concentration of the specific allergen Amb a 1, suggesting that SLIT formulations containing short ragweed should be stored under refrigeration.