Measuring the specific surface area (SSA) of freeze-dried biologics using inverse gas chromatography

Abstract

The specific surface area (SSA) of freeze-dried biologics (FD) is usually measured via a Brunauer–Emmett–Teller (BET) analysis of volumetric nitrogen adsorption isotherms. However, this technique has accuracy limitations for materials <0.5 m2/g, requires dry samples, must be measured at 77 K and has slow sample preparation times (drying/degassing). Inverse gas chromatography (IGC) is chromatographic characterization technique which can be used to analyse the SSA (down to ≈0.1 m2/g) of various solid-state materials including powders using organic molecules such as octane at ambient temperatures/pressure for a range of relative humidities. This study presents a comprehensive comparison between the N2 BET adsorption versus octane BET adsorption using IGC methods for determining the SSA’s for a range of freeze dried biological materials. These include IgG 5% w/w, an influenza antigen standard, sucrose 5% w/w and trehalose 5% w/w. IGC provided comparable SSA values to the N2 BET method with better reproducibility (lower RSDs %). Large variations in average SSA within manufactured FD batches were observed for both IGC and volumetric determinations. IGC was also used to measure the change in SSA with increasing humidity, with FD cakes shrinking and losing their structural integrity with increasing moisture content. Such information highlights the importance of moisture content in determining the physical stability of FD cakes as exemplified by their SSA. In conclusion, IGC is a suitable alternative method for determining the SSA of freeze-dried biological materials which are generally strongly dependent on their moisture content.