(W0930-09-57) New Insights into Non-classical Crystallization of Pharmaceutical Solids: Chemically-Specific Coherent Raman Imaging of Liquid-Liquid Phase Separation

Purpose: Crystallization is crucial to many processes occurring in the food, chemical and pharmaceutical industries. In the pharmaceutical field, crystallization affects drug solubility and stability of the final product, but it is also important in formulation and manufacturing design. Indeed, understanding crystallization mechanisms of organic molecules in solution is of great importance. Nucleation is the first step in crystal formation. Classical nucleation theory (CNT), the most widely used theory, holds that nucleation occurs as a one-step process, with individual molecules in solution associating directly to form a crystalline phase. Nevertheless, computational simulations and empirical findings often point towards more complex routes, giving rise to the so-called non-classical crystallization (NCC) theories. NCC outlines the formation of intermediate thermodynamically metastable precursors called liquid-liquid phase separation (LLPS). LLPS are disordered prenucleation clusters that eventually solidify and reorganize to form a crystal phase (1-3). To date, characterization of LLPS and NCC has been limited to non-spatially resolved or non-chemically-resolved analyses. Stimulated Raman scattering (SRS) is a powerful and rapid imaging technique that has potential for in situ chemically-specific imaging of different crystallization mechanisms in complex environments in real time. The aim of the present study was to investigate the potential of stimulated Raman spectroscopy for chemically-specific real-time imaging of LLPS and NCC in a pharmaceutical context.

Methods: Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID) that is often formulated as a salt to increase solubility. Several studies have suggested that ibuprofen undergoes liquid–liquid phase separation using indirect detection and non-chemically specific microscopy (4-6). In the present study, crystallization after initial dissolution of two racemic ibuprofen salts (lysine and sodium) was chemically- and structurally-specifically imaged in situ in real time using SRS. To induce crystallization, the two ibuprofen salts were compacted into tablets and brought to a supersaturated (non-equilibrium) state. After solvent addition, both the tablet surface and the bulk liquid medium were systematically imaged using SRS, monitoring crystallization process in real-time. SRS analyses were performed with an in-house built coherent Raman imaging microscope with fast spectral focusing that allowed rapid recording of chemically- and structurally-specific vibrational hyperspectral images.

Results: The SRS images showed distinct processes over time upon dissolution of the two ibuprofen salts. The common denominator was the initial formation of dense liquid intermediate droplets, representing LLPS, that partially or completely crystallized into large needle-shaped crystals. Figure 1 illustrates sequential steps during a crystallization process of ibuprofen sodium salt. SRS microscopy allowed chemically- and structurally-specific characterization of multiple species during crystallization (Figure 2). LLPS droplets were enriched with amorphous ibuprofen free acid, and both the smaller and larger needle-shaped structures were identified as the less-soluble free acid racemic form of ibuprofen. SRS images taken from the tablet surface at different timepoints show a spectral shape matching that of the reference salt, which suggests that the free acid crystals did not precipitate on the tablet surface.

Conclusion: The results show the potential advantages of in situ SRS imaging over conventional techniques for real-time monitoring of crystallization processes in dissolution environments. To our knowledge, this is the first study involving in situ chemically- and structurally-specific imaging of different structures at sequential crystallization steps. SRS microscopy was found to be suitable for label-free imaging and elucidation of complex crystallization processes in real-time, with the potential to provide deeper insights into crystallization in the pharmaceutical and other industrial sectors.

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Acknowledgements: The authors would like to thank the Doctoral Programme in Materials Research and Nanoscience (MATRENA) (University of Helsinki), the qCSI (Quantitative Chemically-Specific Imaging Infrastructure for Material and Life Sciences) FIRI infrastructure, and the Academy of Finland for financial support (Grant No. 327732). This project has also received funding from the European Union´s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 955756.

Figure 1. SRS images taken during crystallization process of ibuprofen sodium salt in acetate buffer at pH 4.5. Both the tablet surface (top row) and the bulk liquid medium (bottom row) were monitored in real-time. Image size 106x106 μm, scale bar 20 μm

Figure 2. SRS spectra of the structures identified during crystallization processes (solid lines) and reference materials for comparative purposes (dotted lines).