Addition of Pullulan to Trehalose Glasses Improves the Stability of β-Galactosidase at High Moisture Conditions

Abstract

Incorporation of therapeutic proteins in a matrix of sugar glass is known to enhance protein stability, yet protection is often lost when exposed to high relative humidity (RH). We hypothesized that especially in these conditions the use of binary glasses of a polysaccharide and disaccharide might yield advantages for protein stability. Therefore, different amounts of the polysaccharide pullulan were introduced in freeze-dried trehalose glasses. In these homogeneous blends, the presence of pullulan above 50 weight % prevented crystallization of trehalose when exposed to high RH. Storage stability testing up to 4 weeks of the model protein β-galactosidase incorporated in pullulan/trehalose blends showed superior behavior of pure trehalose at 30°C/0% RH, while pullulan/trehalose blends yielded the best stability at 30°C/56% RH. In conclusion, binary glasses of pullulan and trehalose may provide excellent stability of proteins under storage conditions that may occur in practice, namely high temperature and high RH.

Development of an Orodispersible Film Containing Stabilized Influenza Vaccine

“…Like other biopharmaceuticals, hemagglutinin (HA), the major antigenic component of WIV, is prone to degradation when stresses occur during processing (e.g., shear and dehydration stresses) and storage. It is well known that biopharmaceuticals can be stabilized during drying and subsequent storage by incorporating them in a sugar matrix in the glassy state [16][17][18]. Indeed, in previous studies we have shown that sugars can be used to stabilize WIV during drying and subsequent storage [12,19].…”

“…The process and storage stability of β-galactosidase or WIV incorporated in ODFs was investigated. The storage stability was tested under closed vial conditions (0% relative humidity (RH)) at 4 • C, 30 • C and 60 • C, and under open vial conditions at 30 • C and 56% RH, generated by using a saturated sodium bromide solution [17,26]. The enzymatic activity of β-galactosidase and hemagglutination capacity of WIV were assessed immediately after the drying process (process stability) and after 1, 2 and 4 weeks of storage (storage stability).…”

“…The β-galactosidase activity was determined using a kinetic enzymatic assay, based on the conversion of a colorless substrate, o-nitrophenylgalactoside, into the yellow colored product, o-nitrophenol, by β-galactosidase [17,18]. Firstly, ODFs containing two dots of β-galactosidase were cut into small pieces and dissolved in 1.0 mL of 0.1 M phosphate buffer (pH 7.3), after which samples were diluted fivefold with enzyme diluent solution containing 0.1% BSA and 1 mM MgCl 2 in 50 mM phosphate buffer.…”

“…However, trehalose has a relatively low glass transition temperature, which might lead to crystallization especially when exposed to high relative humidities. On the other hand, the polysaccharide pullulan has a very high glass transition temperature, hence, blends of trehalose and pullulan seem to possess preferred physico-chemical properties to enhance the stability of proteins [17]. Therefore, we evaluated whether trehalose only, blends of trehalose and pullulan or both can be used to stabilize WIV during incorporation in ODFs and subsequent storage.…”

Screening of Extractant for Determining Active Sludge Trehalose in Sewage Treatment

“…The primary functions of trehalose known in biology are nonspecific protective and anti-radiation effects for organisms. [7][8][9][10][11] Active sludge, as a product at sewage treatment plants, in many engineering situations still demonstrates digestibility, even below temperatures as low as -30 to -40 C. [12][13][14][15] Microorganisms with a large amount of trehalose are protected from temperature variations and harsh environments (e.g. drought, high-salt and extreme temperature).…”