Technical Notes

TECHNICAL NOTE Precision of nanoDSF measurements with Prometheus Prometheus experiments are run utilizing a technology called nanoDSF. nanoDSF is an advanced Differential Scanning Fluorimetry method for measuring ultra-high resolution protein stability using intrinsic tryptophan or tyrosine fluorescence. Applications include antibody engineering, membrane protein research, formulation and quality control. To be able to validate a method for quality control purposes, i.e. to make sure that the method is suitable for its intended purpose, one of the prerequisites is to verify that the instruments used generate reliable and consistent data. One of the main parameters that need to be checked is precision with its three levels of testing: repeatability, intermediate precision, and reproducibility. Repeatability Repeatability describes the degree of similarity in results that are obtained by the same operator on the same instrument in the same lab and using the same samples. It is also called intra-assay precision. Intermediate precision describes the similarity of results obtained within the same laboratory by different operators and possibly across different instruments. It is also called inter-assay precision or within-lab reproducibility. In this study, the operator remained mostly the same, but 10 different Prometheus instruments were tested. Reproducibility is sometimes called between-lab reproducibility and describes exactly that: the similarity between results obtained in different labs. It is not always needed for validation, and was not measured for this study. Here, we demonstrate the outstanding repeatability and intermediate precision of measurements with the Prometheus NT.48 instrument for both raw data (initial ratio, F350/F330) and results calculated from the raw data (melting points, T m ). We show superb

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