Deconvolution of high-throughput multicomponent isotherms using multivariate data analysis of protein spectra

Gaining a more profound understanding of biopharmaceutical downstream processes is a key demand of the Quality by Design (QbD) guidelines. One of the most dominant approaches to gain process understanding is the extensive use of experimental high‐throughput formats, such as batch chromatography on robotic liquid handling stations. Using these high‐throughput experimental formats, the generation of numerous samples poses an enormous problem to subsequent analytical techniques. Here, a high‐throughput case study for batch chromatographic multicomponent isotherms is presented. To debottleneck the subsequent analytics, a noninvasive technique using UV spectra and multivariate statistics was adapted to a batch chromatographic format. Using this approach, it was possible to integrate the entire analytical setup into the robotic workflow. As a case study, batch isotherms for sulfopropyl sepharose fast flow and the model proteins cytochrome c and lysozyme at various pH values and ionic strengths were recorded. A successful examination of the quality of the analytical procedure compared to classical single wavelength photometry was carried out. To address the growing demand for a more profound process understanding, the experimental data were fitted to the steric mass action isotherm, getting a more detailed insight into the competitive binding behavior at various pH values and ionic strengths.