In biopharmaceutical production processes, freeze-thaw operations are used to ensure product integrity during long hold times, but they also introduce additional stresses such as freeze concentration gradients that might lead to a loss of protein activity. Process characterization of freeze-thaw operations at different scales should be conducted with attention to freezing time and boundary effects to ensure the product stability throughout the process and process development. Currently, process characterization often relies on one or very few temperature probes that detect freezing times based on raw temperature, which is largely influenced by freezing-point depression in case of concentrated solutions. A method to detect freezing based on the second derivative of temperature measurements from Fiber-Bragg-Grating sensors is presented to overcome this issue. The applicability of the method is demonstrated by process characterization of a novel small-scale freeze-thaw device with minimized boundary effects using freezing times of purified water and concentrated formulations. Freezing times varied from 35 to 81 min for temperatures between −60 and −20°C and impacted freeze concentration profiles. Furthermore, freezing time estimations based on the Plank equation revealed model limitations due to start-up temperature gradients, that can be corrected by an empirically extended Plank model. As a hypothesis, we conclude that freezing temperature, from a freeze concentration view, is less important in containers with small characteristic freezing distances such as freeze bags. Using a 2D-resolved temperature profile, a shift of the last point to freeze position from top to bottom of a container was observed when freezing above −30°C
