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.