In Situ Characterization of the Microstructural Evolution of Biopharmaceutical Solid-State Formulations with Implications for Protein Stability

Abstract

Lyophilized and spray-dried biopharmaceutical formulations are used to provide long-term stability for storage and transport, but questions remain about the molecular structure in these solid formulations and how this structure may be responsible for protein stability. Small-angle neutron scattering with a humidity control environment is used to characterize protein-scale microstructural changes in such solid-state formulations as they are humidified and dried in situ. The findings indicate that irreversible protein aggregates of stressed formulations do not form within the solid-state but do emerge upon reconstitution of the formulation. After plasticization of the solid-state matrix by exposure to humidity, the formation of reversibly self-associating aggregates can be detected in situ. The characterization of the protein-scale microstructure in these solid-state formulations facilitates further efforts to understand the underlying mechanisms that promote long-term protein stability.

Hydration-Induced Structural Changes in the Solid State of Protein: A SAXS/WAXS Study on Lysozyme

“…A peak in a similar q -range has been reported before by SANS in lyophilized formulations of lysozyme as well as mAb formulations and was characterized as a protein–protein interaction peak. 14 − 16 For a better understanding of the intermolecular structure corresponding to this peak, it is instructive to consider it in relation to the 4.5 nm –1 peak that has a similar dependence on water content (see Figure 5 a,c) and thus also arises from protein–protein interactions. In this section, we will consider these peak positions in the dry limit and in the following section evaluate their dependence on water content.…”

“…These differences could also be connected to the protein stability because of the formation of the glassy state upon strong dehydration. There are few studies on the structural transition in the solid state of protein using scattering techniques: for example, SANS has been carried out on dried formulations in the presence of excipients − and also SAXS. , However, no SAXS and wide-angle X-ray scattering (WAXS) studies discussing the change in protein morphology under dehydrated conditions or dehydration-induced intramolecular structural changes are found in the literature.…”

Advancing predictions of protein stability in the solid state

“…the gradient by which the amplitude of excess motion increases with increasing temperature, characterised by dα 2 /dT , govern protein stability in the solid state. Large conformational changes or aggregate formation above the level of dimers are highly unlikely in the solid state, as illustrated by the work of Koshari et al 28 . However, as outlined by Cicerone and Douglas 5 , small conformational changes exposing hydrophobic groups could increase the propensity for aggregation upon reconstitution in a hydrophilic medium 3 .…”