Protein therapeutics (like insulin and several monoclonal antibodies) play an important role in modern day medicine. However, an important limitation of many protein therapeutics, which are often in aqueous solution, is their short shelf life. In his PhD project Maarten Mensink investigated the use of sugars to increase protein shelf life in the solid state. He found that more intense sugar-protein interactions improved the stabilizing effect of sugars on proteins after freeze-drying. The PhD project was part of the CCC2 program.
As most degradation reactions require molecular mobility and mobility is lower in the solid state, bringing proteins in the dry state is an efficient strategy to prolong their shelf life. However, the drying process itself can lead to protein degradation. Sugars can provide protection during drying and subsequent storage. However, not all sugars provide stabilization equally well.
Mensink and coworkers showed that the ability of a sugar to stabilize proteins is related to the size and molecular flexibility of the sugar. This was demonstrated by freeze-drying four model proteins with diverse characteristics with sugars of varying sizes and molecular flexibility. Smaller (disaccharides versus oligo- and polysaccharides) and molecularly more flexible (similarly sized inulin versus dextran) sugars better maintained functionality of the proteins during storage at an elevated temperature (60 degrees Celsius).
Using modern characterization methods (near-infrared spectroscopy, terahertz time domain spectroscopy, solid state nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy) Mensink and coworkers showed that the smaller and molecularly more flexible sugars are better protein stabilizers because they interact (through hydrogen bonds) more closely with the proteins. It was found that larger and molecularly more rigid sugars had a higher tendency to phase-separate from the proteins, explaining the lesser interactions and lesser stabilization.
Dried protein sugar powders are generally amorphous glasses, in which molecular mobility is strongly reduced. This reduction of mobility is important to maintain protein functionality during storage. When a dried formulation surpasses its glass transition temperature (Tg) molecular mobility drastically increases and with it the protein degradation rate also increases strongly. Moisture and other excipients such as buffer components can drastically lower the Tg of a formulation. In a situation where maintaining vitrification is problematic, larger sugars might provide benefit as their Tgs are generally higher.
To allow investigation of the impact of molecular flexibility of sugars on their ability to stabilize proteins, Mensink and coworkers used a molecularly flexible sugar, inulin. Inulin is more flexible than other sugars, primarily due to the fact that the backbone of the sugar does not pass through the sugar rings, allowing more freedom for movement. They reviewed the physicochemical properties of inulin to provide further information in support of the presented comparison of various sugars. Furthermore, they discussed how those properties translate into pharmaceutical applications, including stabilization of proteins.
Maarten Mensink did his PhD research in the department of Pharmaceutical Technology and Biopharmacy, Faculty of Science and Engineering, University of Groningen. He defended his thesis, entitled
Solid state stabilization of proteins by sugar. Why size and flexibility matter on 2 June. Supervisors were prof. dr. K. van der Voort Maarschalk, prof. dr. H.W. Frijlink and dr. W.L.J. Hinrichs.