RESUMEN
The effect of glycosylation state on the thermal and storage stability of interleukin-2 mutein (IL-2 mutein) was investigated. The thermal stability of IL-2 mutein was studied by DSC and UV. An accelerated storage stability study was conducted at 40 degrees C in the dark and analyzed by UV, SDS-PAGE, and RP-HPLC. The unfolding temperatures (Tu) of both glycosylated and unglycosylated forms of IL-2 mutein are similar (within +/-1 degrees C) at pH 5.5 and 7.5. At pH 4.0, the Tu of glycosylated IL-2 mutein was 4 degrees C lower than that of the unglycosylated form. The precipitation temperature of glycosylated IL-2 mutein is similar to that of the unglycosylated form at pH 5.5 but 4 degrees C higher at pH 7.5. The precipitation temperature is not detectable for both forms at pH 4.0. During storage, both glycosylated and unglycosylated IL-2 mutein form aggregates (soluble and insoluble) and other degradation products. The aggregates are formed by both physical and chemical mechanisms. The major pathway of chemical aggregation appears to be disulfide bond formation/exchange. The glycosylated form is much less stable than the unglycosylated form at pH 4.0 and both forms are most stable at pH 5.5 in terms of thermal stability, precipitation rate and total degradation rate. This study clearly demonstrates that the effect of glycosylation on the stability of a protein is pH-dependent.
Asunto(s)
Interleucina-2/química , Proteínas/química , Sustitución de Aminoácidos , Rastreo Diferencial de Calorimetría , Precipitación Química , Cisteína/química , Cisteína/genética , Almacenaje de Medicamentos , Electroforesis en Gel de Poliacrilamida , Glicosilación , Humanos , Concentración de Iones de Hidrógeno , Interleucina-2/genética , Oxidación-Reducción , Desnaturalización Proteica , Proteínas Recombinantes/química , Espectrofotometría Ultravioleta , TemperaturaRESUMEN
PURPOSE: To develop a method for removing residual acetone from gelatin microspheres. METHODS: Free-flowing gelatin microspheres were either heated under vacuum or subjected to a stream of humidified air in a specially designed apparatus for removal of the residual acetone. To understand the removal mechanism, hygroscopic and thermal properties of the microspheres were examined. RESULTS: Heating the gelatin microspheres under vacuum did not reduce the acetone level below 2%, but the use of humidified air under fluidizing condition reduced the residual acetone in gelatin microspheres by an additional two orders of magnitude. The rate of acetone removal was a strong function of the relative humidity (RH) of the air stream; higher RH accelerated acetone removal. Other variables influencing the acetone removal rate are the mean particle diameter and the linear velocity of the humidified air. Under high relative humidities, significant amounts of moisture were absorbed into gelatin microspheres, reducing their glass transition temperature below 25 degrees C. CONCLUSION: The residual acetone in gelatin microspheres can be efficiently removed when exposed to air of high RH. Mechanisms of water-dependent acetone removal are proposed.