Title

In situ precipitation and vacuum drying of interferon alpha-2a: Development of a single-step process for obtaining dry, stable protein formulation

Date of Completion

January 2008

Keywords

Chemistry, Pharmaceutical

Degree

Ph.D.

Abstract

To achieve the desired product shelf life, proteins are frequently formulated as dry powders by utilizing techniques such as freeze drying. However, issues inherent to such processes have led to an increased interest in developing alternative drying technologies. The present work deals with the development of a single step drying process based on in situ polyethylene glycol (PEG)-induced protein precipitation and vacuum drying in the presence of stabilizers. Because of its relevance to the drying technique utilized in this study, and the lack of complete mechanistic understating of the observed effects of stabilizers (polyols) on protein properties in solution, effects of polyhydroxy cosolvents on the precipitation of proteins by PEGs, and on the stability and solubility of proteins in the absence of PEGS, have also been investigated. ^ Structural characterization of the model protein for the drying studies. Interferon alpha-2a (IFNα2a), was carried out by second derivative tryptophan fluorescence spectroscopy. Results show that this novel technique can be used as an important tool to identify partially unfolded intermediates of proteins during formulation utilizing order of magnitude lower concentrations compared to such other technique as near UV-CD. The drying process was divided into two phases. In the first phase, protein formulations containing the desired precipitating agent were concentrated to achieve protein precipitation. In the second phase, removal of the remaining water was facilitated by lowering the shelf pressure. Development of the vacuum drying process involved the investigation of formulation and processing parameters and the selection of appropriate precipitating agents. Accelerated stability studies were performed on various formulations that were dried by using this methodology. Stability of the vacuum dried formulations was found to be comparable or better than similar freeze-dried formulations. This work demonstrates that in situ precipitation and vacuum drying can effectively be used for the formulation of proteins in the dried state. Additionally, it is also shown that vacuum drying of PEG solutions at low pressures is a useful method for the removal of residual peroxides present in commercially available PEGs. ^ Protein-polyol interactions were studied by investigating the effect of polyols on the thermodynamic stability of proteins and on the solubility of amino acids and proteins. Results show that the net thermodynamic stabilization effect that polyols impart to proteins is dependent on both the osmotic activity and the hydrophobicity of polyols, the latter being critical to the extent of protein-polyol interaction. Evidently, weak hydrophobic interactions between proteins and polyols affect the solubility and the thermodynamic stability of proteins in solution. It is also shown that polyols modulate protein precipitation by PEGs by both inducing the protein to adopt a less flexible conformation, and by weakly interacting with the protein. ^