Retaining biopharmaceutical proteins in a stable form is critical to their safety and efficacy, and is a major factor for optimizing the final product. Freeze-dried formulations offer one route for improved stability. Currently the optimization of formulations for freeze-drying is an empirical process that requires many time-consuming experiments and also uses large quantities of product material. Here we describe a generic framework for the rapid identification and optimization of formulation excipients to prevent loss of protein activity during a lyophilization process. Using factorial design of experiment (DOE) methods combined with lyophilization in microplates a range of optimum formulations were rapidly identified that stabilized lactose dehydrogenase (derived from Lactobacillus leichmanii) during freeze-drying. The procedure outline herein-as initial screen to identify key excipients and potential interactions followed by a central composite face designed optimization experiment. Polyethylene glycol (PEG) and a lactose were shown to have significant effects on maintaining protein stability at the screening stage and optimization resulted in an accurate model that was used to plot a window of operation. The variation of freezing temperatures and rates of sublimation that occur across a microplate during freeze-drying have been characterized also. The optimum formulation was then freeze-dried in stoppered vials to verify that the microscale data was relevant to the effects observed at larger pilot scales. This work provides a generic approach to biopharmaceutical formulation screening where possible excipients can be screened for single and interactive effects thereby increasing throughput while reducing costs in terms of time and materials. Biotechnol. Bioeng. 2009;104: 957-964. (C) 2009 Wiley Periodicals, Inc.