Development of novel formulations for mucosal delivery of protein ...

permeability using sheep buccal tissue which was correlated with Epi-oral TM characterisation.
Accelerated and real-time stability evaluation of the INS containing xerogels was determined
by high-performance liquid chromatography (HPLC). The conformational stability of the
protein drugs was studied with circular dichroism (CD) and attenuated total reflection Fourier
transform infrared (ATR-FT-IR) spectrophotometry.
1.2 Protein structure and aggregation
Every protein molecule has a characteristic three-dimensional shape (Jankowska et al.,
2012), or conformation. For example, fibrous proteins, such as collagen and keratin, consist of
polypeptide chains arranged in roughly parallel fashion along a single linear axis, thus
forming tough, usually water-insoluble, fibres or sheets. Because the physiological activity of
most proteins is closely linked to their three-dimensional architecture, specific terms are used
to refer to different aspects of protein structure. The term primary structure denotes the
precise linear sequence of amino acids that constitutes the polypeptide chain of the protein
molecule. The physical interaction of sequential amino-acid subunits results in a so-called
secondary structure (Song et al., 2012), which can either be a twisting of the polypeptide
chain approximating a linear helix (-configuration), or a zigzag pattern (-configuration).
Most globular proteins also undergo extensive folding of the chain into a complex threedimensional
geometry designated as tertiary structure (Eyrich et al., 1999). Two or more
polypeptide chains that behave in many ways as a single structural and functional entity are
said to exhibit quaternary structure. The separate chains are not linked through covalent
chemical bonds but by weak forces of association. The native state describes the precise threedimensional
structure of a protein molecule and appears, in almost all cases, to be required for
proper biological function. If the tertiary or quaternary structure of a protein is altered, e.g., by
such physical factors as extremes of temperature, changes in pH, or variations in salt
concentration, the protein is said to be denatured and usually exhibits reduction or loss of
biological activity.
Aggregation is a general term that encompasses several types of interactions or
characteristics (Cromwell et al., 2006). Protein aggregates can arise from several mechanisms
and may be classified in numerous ways, including soluble/insoluble, covalent/noncovalent,
reversible/irreversible, and native/denatured. For protein therapeutics, the presence of
aggregates of any type is considered to be undesirable because of concerns that such