Title

Formulation of proteins as dry powders by vacuum drying following precipitation by polyethylene glycols

Date of Completion

January 2003

Keywords

Health Sciences, Pharmacy

Degree

Ph.D.

Abstract

It is advantageous to formulate protein drugs as dry powders to enhance stability to achieve a desired shelf-life for the product. However, the inherent steps involved, in the commonly used process of lyophilization, lead to protein instability in many cases. In the present work, a novel process based on the precipitation of proteins by polyethylene glycols (PEG) and followed by vacuum drying of the precipitate in the presence of stabilizers, is proposed to formulate proteins as dry powders. ^ The feasibility studies were carried out using interferon alpha-2a (IFNα2a) as a model protein. Precipitation of IFNα2a was successfully carried out at pH 6.5 using various molecular weight PEGs. Ten percent w/v PEG 1450 at pH 6.5 and at a solution ionic strength of 71 mM precipitated about 97% of the protein from a 1mg/ml IFNα2a solution. Vacuum drying of the precipitated protein containing 1:10:100 w/w IFNα2a:trehalose:mannitol ratio at 100 mTorr and at a shelf temperature of 25°C resulted in complete drying within 24 hours (final residual moisture content of 1% w/w). Both mannitol and trehalose were effective in preventing formation of insoluble aggregates of IFNα2a during vacuum drying of the precipitate. From a separate set of studies the stabilizing action of mannitol was attributed to its tendency to remain amorphous in the presence of proteins upon vacuum drying. ^ Accelerated stability studies indicated that the vacuum dried formulation containing IFNα2a:mannitolarehalose in a 1:10:100 w/w ratio exhibited sufficient ratio exhibited at stability when stored at 40°C for 3 months. The stability of the vacuum dried formulation of IFNα2a was comparable to that of a similar lyophilized formulation. ^ In an attempt to understand protein tertiary structure in solid state, a novel technique based on the steady state Trp fluorescence spectroscopy of proteins was developed. This technique demonstrated that mannitol was ineffective to preserve IFNα2a tertiary structure in the solid state, whereas, trehalose was effective in preserving both the secondary as well as the tertiary structure of IFNα2a upon drying. ^ This work demonstrates that proteins can be conveniently formulated as dry powders by PEG-induced precipitation of proteins followed by vacuum drying of the precipitate. ^