In situ monitoring and control of lysozyme concentration during crystallization in a hanging drop

Fiber optic Raman spectroscopy combined with a partial least-squares regres sion model was demonstrated as a monitor of lysozyme concentration during c rystallization in a hanging drop experiment in real time. Raman spectral fe atures of the buffer and protein were employed to build the regression mode l. The use of fiber optic technology coupled with Raman spectroscopy, which is ideal for use with aqueous solutions, results in a powerful noninvasive probe of the changing environment within the solution. Lysozyme concentrat ions were monitored in experiments at a constant reservoir ionic strength. Data from these uncontrolled experiments were used to determine rates of su persaturation, induction times, and the number and size of the resultant ly sozyme crystals. Control experiments were performed by introducing step cha nges in the reservoir ionic strength. The step changes were initiated by co mparing in situ rates of supersaturation with the rates of supersaturation calculated from the uncontrolled data. Monitoring the concentration changes of the lysozyme within the hanging drop permits a measurement of the level of supersaturation of the system and enhances the possibility of dynamic c ontrol of the crystallization process. (C) 2000 Published by Elsevier Scien ce B.V. All rights reserved.