Mercado Cruz, Joanna

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    In-line monitoring and controlling protein batch crystallization process using Raman spectroscopy
    (2007) Mercado Cruz, Joanna; Romañach, Rodolfo J.; College of Arts and Sciencies - Sciences; Ríos Steiner, Jorge; Rodríguez Hornedo, Naír; Department of Chemistry; Ramirez Vick, Jaime
    Protein crystallization as part of a new field of solid formulation becomes a challenging task for the pharmaceutical industries. Protein crystallization must occur without affecting the conformational structure of the protein. Factors such as temperature, pH, ionic strength, protein concentration and solvent can affect the final stages of protein crystallization. Monitoring and controlling the early stages of protein crystallization can provide useful information for a reliable experimental design of protein batch crystallization in large scale unit operations. In this study, Fiber Optic Raman Spectroscopy has been used to develop a Process Analytical Technology (PAT) based system to monitor and control the lysozyme protein crystallization process. Raman spectroscopy is a method that shows great potential in the monitoring of protein crystallization because water is a poor Raman scatterer. This analytical technique was used to investigate the vibrational behavior of the lysozyme protein under conditions promoting crystallization. Several Raman bands were affected by the lysozyme protein concentration, precipitant agent, temperature and time of crystallization. The relative intensities of the lysozyme protein bands increased when the lysozyme protein was in solution and decreased when the lysozyme crystal formed. A correlation between the conditions promoting crystallization (sodium chloride, temperature and time of crystallization) and the relative intensity of the lysozyme protein was established using Multiple Linear Regression (MLR). The region of 2940 cm⁻¹ and the ratio between the regions of 760 cm⁻¹/750 cm⁻¹ presented the highest correlation between the R² and Q². These regions correspond to the C-H stretching and the aromatic ring vibrational mode of tryptophan. The solubility limit, metastable zone, and supersaturated zone were established using the contour plots map of these two regions. Polarized Light Microscopy (PLM) revealed that the crystal habit, crystal size distribution and the morphology of the lysozyme protein crystal were affected by the temperature and the amount of sodium chloride.