Octreotide is the first synthetic peptide hormone, consisting of eight amino acids, that mimics the activity of somatostatin, a natural hormone in the body. During the past decades, advanced instrumentation and crystallographic software have established X-Ray Powder Diffraction (XRPD) as a valuable tool for extracting structural information from biological macromolecules. The latter was demonstrated by the successful structural determination of octreotide at a remarkably high d-spacing resolution (1.87 Å) (PDB code: 6vc1). This study focuses on the response of octreotide to different humidity levels and temperatures, with a particular focus on the stability of the polycrystalline sample. XRPD measurements were accomplished employing an Anton Paar MHC-trans humidity-temperature chamber installed within a laboratory X’Pert Pro diffractometer (Malvern Panalytical). The chamber is employed to control and maintain precise humidity and temperature levels of samples during XRPD data collection. Pawley analysis of the collected data sets revealed that the octreotide polycrystalline sample is remarkably stable, and no structural transitions were observed. The compound retains its orthorhombic symmetry (space group: P2₁2₁2₁, a = 18.57744(4) Å, b = 30.17338(6) Å, c = 39.70590(9) Å, d ~ 2.35 Å). However, a characteristic structural evolution in terms of lattice parameters and volume of the unit cell is reported mainly upon controlled relative humidity variation. In addition, an improvement in the signal-to-noise ratio in the XRPD data under a cycle of dehydration/rehydration is reported. These results underline the importance of considering the impact of environmental factors, such as humidity and temperature, in the context of structure-based drug design, thereby contributing to the development of more effective and stable pharmaceutical products.

Octreotide is the first synthetic peptide hormone, consisted of eight amino acids, that mimics the activity of somatostatin, a natural hormone in the body. During the past decades, advanced instrumentation and crystallographic software established X-ray Powder Diffraction (XRPD) as a valuable tool for extracting structural information of biological macromolecules. The latter was demonstrated by the successful structural determination of octreotide at a remarkably high d-spacing resolution (1.87 Å) (PDB code: 6vc1). This study focuses on the response of octreotide to different humidity levels and temperatures, with a particular focus on the stability of the polycrystalline sample. XRPD measurements were accomplished employing an Anton Paar MHC-trans humidity-temperature chamber installed within a laboratory X’Pert Pro diffractometer (Malvern Panalytical). The chamber is employed to control and maintain precise humidity and temperature levels of samples during XRPD data collection. Pawley analysis of the collected data sets revealed that the octreotide polycrystalline sample is remarkably stable and no structural transitions were observed. The compound retains its orthorhombic symmetry (space group: P212121, a = 18.57744(4) Å, b = 30.17338(6) Å, c = 39.70590(9) Å, d~ 2.35 Å). However, a characteristic structural evolution in terms of lattice parameters and volume of the unit-cell is reported mainly upon controlled relative humidity variation. In addition, an improvement of signal-to-noise ratio in the XRPD data under a cycle of dehydration/rehydration is reported. These results underline the importance of considering the impact of environmental factors, as humidity and temperature, in the context of structure-based drug design, thereby contributing to the development of more effective and stable pharmaceutical products.

Protein crystals are intrinsically hydrated systems, and their structural integrity is strongly influenced by environmental humidity. Understanding the effects of relative humidity (RH) variation on crystal stability is therefore essential for both fundamental research and applied studies. In this work, the structural response of tetragonal hen egg-white lysozyme (HEWL) to controlled RH variation was investigated using in situ X-ray powder diffraction (XRPD). Polycrystalline HEWL samples were subjected to systematic gradual dehydration and rehydration cycles, as well as to non-gradual RH variation protocols. Pawley analysis of the XRPD data enabled monitoring of the evolution of unit cell parameters and unit cell volume as a function of RH. Under all experimental conditions, the tetragonal polymorph (space group P4₃2₁2; a = 79.105 (4) Å, c = 38.231 (2) Å) was preserved. RH variation induced smooth, continuous and anisotropic lattice changes, characterized by a decrease in the a (=b)-axis and a concomitant increase in the c-axis upon dehydration, while rehydration resulted in the opposite behavior. The overall magnitude of lattice variation remained limited (within ±2%), indicating a high degree of structural stability. Partial degradation of crystallinity was observed only after prolonged exposure to low RH levels. These findings demonstrate the remarkable structural resilience of tetragonal HEWL and highlight the effectiveness of in situ XRPD as a powerful tool for probing hydration-driven lattice responses in protein crystals under realistic environmental conditions.