Application Notes

2 limited amounts of material. A perfect example is NanoTemper Technology's label free nanoDSF instruments of the Prometheus series. The instruments are specifically designed to meet the requirements of modern biologics development, providing state-of-the- art thermal stability quantification, maintenance-free operation, maximal ease of use and even an optional, full automation. The Prometheus instruments analyze protein unfolding transitions based on high precision detection of intrinsic fluorescence changes under label free conditions. The instrument design allows for the parallel detection of up to 48 samples with protein concentrations ranging from 10 µg/ml to more than 250 mg/ml without buffer restrictions. It does not require the addition of extrinsic fluorescent dyes like in the classical DSF technique, thereby avoiding potential detrimental interactions of the dye and protein or excipients. Furthermore, the innovative dual-UV detection method enables rapid scanning of samples, which results in a very high data point density of 20 or more data points per ° C, and unmatched reproducibility, which is required for a robust and precise determination of unfolding profiles. The use of DOE offers an efficient approach to experimental design. DOE effectiveness stems from the combination of a validated statistical approach and an intelligent choice of variables, so that even extremely complex problems can be addressed in an effective manner with a minimal set of experiments. This can be accomplished by utilizing Deming's wheel of quality. Deming's wheel can be divided into four parts. The first part is planning, where the variables and goals of the project are defined. The second part is doing, where the required data are recorded by appropriate experiments. The third part is analysis of the results and checking the data for validity. And the last part is acting on the conclusions generated from the data from the DOE exercise. At HTD Biosystems we call this concept "bringing mindfulness to formulation development". We do this by utilizing very little material, generating data in fast time frames without compromising quality, and performing the work with optimized and thus low-cost approaches without cutting corners. We have set up a formulation systems platform using the Prometheus NT.48 that can generate high quality data with minimal material requirements to record advanced DOE data. The use of DOE also allows us to understand complex interactions between the experimental variables that are implicit but difficult to tease out. In addition, the results can be presented in a coherent fashion to a wide audience base. This leads to better information decimation and decision-making, which eventually translates into better support for the project. The iFormulate TM plate is a convenient predesigned formulation plate loaded with the formulation excipients for rapid formulation of proteins. The design is based on multivariate experimental response surface design that investigates 20 unique formulations with five replicates to look at effects and interactions of pH, ionic strength, buffer, concentration, and stabilizer concentration. The advantages of using iFormulate are (1) maximizing results with minimum number of trials, (2) savings in time, cost, and resources and (3) providing justification of the formulation using QbD and FDA guidelines on process validation. This application note demonstrates the use of iFormulate in combination with the Prometheus NT.48 for generating stable protein formulations in rapid fashion using limited quantities of material.

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