Freeze-drying of nanoparticles: formulation, process and storage considerations.

Lyophilization and development of solid protein pharmaceuticals

Instability, stabilization, and formulation of liquid protein pharmaceuticals.

Numerous organisms are capable of surviving more or less complete dehydration. A common feature in their biochemistry is that they accumulate large amounts of disaccharides, the most common of which are sucrose and trehalose. Over the past 20 years, we have provided evidence that these sugars stabilize membranes and proteins in the dry state, most likely by hydrogen bonding to polar residues in the dry macromolecular assemblages. This direct interaction results in maintenance of dry proteins and membranes in a physical state similar to that seen in the presence of excess water. An alternative viewpoint has been proposed, based on the fact that both sucrose and trehalose form glasses in the dry state. It has been suggested that glass formation (vitrification) is in itself sufficient to stabilize dry biomaterials. In this review we present evidence that, although vitrification is indeed required, it is not in itself sufficient. Instead, both direct interaction and vitrification are required. Special properties have often been claimed for trehalose in this regard. In fact, trehalose has been shown by many workers to be remarkably (and sometimes uniquely) effective in stabilizing dry or frozen biomolecules, cells, and tissues. Others have not observed any such special properties. We review evidence here showing that trehalose has a remarkably high glass-transition temperature (Tg). It is not anomalous in this regard because it lies at the end of a continuum of sugars with increasing Tg. However, it is unusual in that addition of small amounts of water does not depress Tg, as in other sugars. Instead, a dihydrate crystal of trehalose forms, thereby shielding the remaining glassy trehalose from effects of the added water. Thus under less than ideal conditions such as high humidity and temperature, trehalose does indeed have special properties, which may explain the stability and longevity of anhydrobiotes that contain it. Further, it makes this sugar useful in stabilization of biomolecules of use in human welfare.

The role of vitrification in anhydrobiosis.

The formulation and delivery of biopharmaceutical drugs, such as monoclonal antibodies and recombinant proteins, poses substantial challenges owing to their large size and susceptibility to degradation. In this Review we highlight recent advances in formulation and delivery strategies — such as the use of microsphere-based controlled-release technologies, protein modification methods that make use of polyethylene glycol and other polymers, and genetic manipulation of biopharmaceutical drugs — and discuss their advantages and limitations. We also highlight current and emerging delivery routes that provide an alternative to injection, including transdermal, oral and pulmonary delivery routes. In addition, the potential of targeted and intracellular protein delivery is discussed.

Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies

Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding.

Structural differences between two genetic variants of bovine beta-lactoglobulins (type A and B) in aqueous solutions were characterized using Fourier transform infrared and circular dichroism spectroscopies. To probe differences in structural dynamics, the effects hydrogen-deuterium exchange were also compared for the two proteins. The infrared spectra recorded in H2O solution for the two proteins were nearly identical in the conformationlly sensitive amide I region. The only exceptions were small differences at the band ascribed to a high-wavenumber beta-sheet component near 1693 cm-1 and the band assigned to turns at 1684 cm-1. In contrast, when the proteins were prepared in D2O solution, marked spectral differences were observed at all regions ascribed to beta-sheet and turn structures. These differences are consistent with the structural differences of the two variants at amino acid residues 64 and 118, which are located at a turn and a beta-sheet structure, respectively, as revealed by X-ray crystallographic studies [Monaco et al. (1987) J. Mol. Biol. 197, 695-706]. The circular dichroism spectra for the two proteins were essentially identical, both before and after hydrogen-deuterium exchange. Therefore, hydrogen-deuterium exchange did not alter the proteins' secondary structure. The enhancement of the amide I spectral difference upon hydrogen-deuterium exchange was ascribed to the differences in the structural mobility of the two proteins. Since the rate of exchange was greater for variant A, it was concluded that this variant has greater structural mobility than variant B. These findings indicate that the combination of infrared spectroscopy and hydrogen-deuterium exchange has great potential in characterization of even subtle structural differences in proteins induced by naturally occurring point mutations and/or site-directed mutagenesis.

S D Allison

Papers

Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding.

Infrared and circular dichroism spectroscopic characterization of structural differences between beta-lactoglobulin A and B.

Effects of drying methods and additives on structure and function of actin: mechanisms of dehydration-induced damage and its inhibition

Counteracting effects of thiocyanate and sucrose on chymotrypsinogen secondary structure and aggregation during freezing, drying, and rehydration.