Selective protein crystallization is a trending alternative to preparative chromatography in biotechnological downstream processing. To save time and resources in early-stage process development, fast and reliable analytics are required. This work aimed to develop and assess a low-volume, quantitative, analytical tool for faster development of crystallization processes. The analytical tool was based on ultraviolet–visible spectroscopy and partial least-squares modeling and aimed to selectively quantify protein concentrations in heterogeneous supernatants during crystallization process development. For this purpose, a ternary model protein system consisting of hen-egg-white Lysozyme, bovine Ribonuclease A, and equine Cytochrome C was used for model calibration and subsequent crystallization studies for application. In a high-throughput screening, Lysozyme was selectively crystallized varying pH, precipitant concentration, and Lysozyme concentration at 8 °C for 13 days. During a kinetic study, the composition of two selected conditions was monitored over a time range of 7 days. In both studies, the developed tool quantified the different species in the supernatant with high precision. Crystal yield, purity, and selectivity were evaluated with a sensitivity of 96.23% and a short analysis time of 3 min per sample. The studies were carried out in 96-well plates. This said, the methodology could be easily adapted to higher throughput scales, i.e., 384-well or 1536-well plates.

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