A Nondestructive Method for Measuring Protein Distribution in Frozen Drug Substance

We describe a noninvasive method developed to make in situ measurements of protein concentration in frozen drug substance. This technique is based on fluorescence from artificially labeled protein and a charge-coupled device camera. Data collected using this method in laboratory small-scale experiments are in good agreement with traditional ice core method. The technique allows real-time visualization of freezing process and provides rich local details of ice crystal growth and morphology for the whole freezing process from beginning to the last point to freeze, and the whole freezing process can be described in 2- and 3-dimensional heat maps with appropriate software. In combining with other existing methods, this method can provide evaluation and optimization of formulation, cooling rate, and cryoconcentration distribution and impacts of combined stresses during freezing. The ability to understand and to control the protein concentration profile in the frozen state offers the potential to improve stability of protein in long-term frozen storage.