Quantification of protein mixture in chromatographic separation using multi-wavelength UV spectra

In therapeutic protein production, the protein purification with chromatographic processes is of high importance in separating the qualified proteins from the impurities for consistent product quality. Therefore, to aid real-time monitoring of the protein purification processes, various kinds of methodologies have been proposed until now. However, the majority of them still rely on the use of a single ultraviolet (UV) absorbance or the utilization of expensive and time-consuming instruments, thus requiring a simple, fast, and cost-effective methodology for protein quantification. In this study, the feasibility of using multiwavelength UV spectroscopy was investigated as an alternative tool for the real-time monitoring of the protein mixtures in protein purification. To this end, three different proteins were selected as a model system for the protein mixture, and the multivariate UV spectra were analyzed to construct the reliable quantification models for different proteins of interest. By using various chemometrics tools, such as partial least squares (PLS), the validity of estimating the protein concentration from the UV spectra of the mixture samples was rigorously analyzed with their prediction performance, and the results indicated that the multiwavelength UV spectra had sufficient sensitivity and accuracy to estimate the protein concentrations in mixture, demonstrating its usefulness for the rapid quantification of the protein mixtures in protein purification.