Evaluation of Mannitol's Crystallization Impact on the Secondary Structure of Spray-Dried recombinant human Growth Hormone (rhGH) Formulations

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Abstract
Objective The development of protein formulations is grappled by the complexity of maintaining protein integrity during the arduous formulation process. While several excipients have been employed for the stabilization of proteins, including the recombinant human growth hormone (rhGH), a precise process control is still paramount. This study aims to investigate the effect of mannitol polymorphism on the structural stability of rhGH in its spray-dried formulations. Methods rhGH was co-spray dried with mannitol at protein:mannitol ratios (1:0.5 to 1:6, w/w). Powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC) characterized mannitol crystallinity. Furthermore, circular dichroism (CD) spectroscopy measured secondary structure pre- and post-accelerated storage (40°C/75% RH, 4 weeks), and SDS-PAGE was leveraged to evaluate protein aggregation. Results Spray-dried powders exhibited spherical particles (1–5 µm) with surface indentations. PXRD reported high levels of mannitol crystallization with ratios of 1:0.5, 1:1.5, and 1:6, which was corroborated by the change in crystallization index using DSC. Parallelly, it corresponded to reductions in α-helix content ranging from 21.8 to 25%, after storage. In contrast, the 1:4 ratio predominantly demonstrated an 8.4% increase in α-helix content, indicating enhanced stability. SDS-PAGE confirmed greater aggregation in samples with higher mannitol crystallization, whereas the 1:4 formulation minimized aggregation. Conclusion Mannitol crystallization strongly influences rhGH stability in spray-dried powders. An optimal protein:mannitol ratio of 1:4 helped maintain mannitol in the amorphous state, preserved secondary structure, and reduced aggregation during storage. These findings underscore excipient crystallization as a key determinant of protein stability and identify a stabilizing composition for spray-dried rhGH.

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