Opalescence measurements are broadly applied to assess the quality and stability of biopharmaceutical products at all stages of development and manufacturing. They appear to be simple and straight... Show moreOpalescence measurements are broadly applied to assess the quality and stability of biopharmaceutical products at all stages of development and manufacturing. They appear to be simple and straight forward but detect complex light scattering phenomena. Despite a routine calibration step, opalescence values obtained with the same biopharmaceutical sample but on different instruments and/or with different methods may vary significantly. Since the reasons for this high variability are generally not well understood, comparison of opalescence results from different biopharmaceutical laboratories is difficult. Here, we characterized a comprehensive set of biopharmaceutically relevant samples with five opalescence methods to illustrate fundamental differences in method performance and explore the reasons for poor comparability. In addition, we developed a high-throughput method for measuring opalescence in a conventional light scattering plate reader that yields opalescence values in the same range as compendial methods. The presented results underline the impact of sample properties, instrument type, and calibration standards on the determined opalescence value. Based on our findings we provide recommendations for the appropriate application of each method during biopharmaceutical drug product development. Overall, our study contributes to an improved understanding of opalescence measurements in the biopharmaceutical field. Show less