Application of spectral deconvolution and inverse mechanistic modelling as a tool for root cause investigation in protein chromatography

•A tool for rapid root cause investigation in protein chromatography was developed.•The tool is based on a combination of spectral deconvolution and inverse mechanistic chromatography modelling.•The tool is capable of identifying the causes for deviations in a chromatogram.•This allows for a correction of a detected problem in the next cycle or batch. In chromatographic protein purification, process variations, aging of columns, or processing errors can lead to deviations of the expected elution behavior of product and contaminants and can result in a decreased pool purity or yield. A different elution behavior of all or several involved species leads to a deviating chromatogram. The causes for deviations are however hard to identify by visual inspection and complicate the correction of a problem in the next cycle or batch. To overcome this issue, a tool for root cause investigation in protein chromatography was developed. The tool combines a spectral deconvolution with inverse mechanistic modelling. Mid-UV spectral data and Partial Least Squares Regression were first applied to deconvolute peaks to obtain the individual elution profiles of co-eluting proteins. The individual elution profiles were subsequently used to identify errors in process parameters by curve fitting to a mechanistic chromatography model. The functionality of the tool for root cause investigation was successfully demonstrated in a model protein study with lysozyme, cytochrome c, and ribonuclease A. Deviating chromatograms were generated by deliberately caused errors in the process parameters flow rate and sodium-ion concentration in loading and elution buffer according to a design of experiments. The actual values of the three process parameters and, thus, the causes of the deviations were estimated with errors of less than 4.4%. Consequently, the established tool for root cause investigation is a valuable approach to rapidly identify process variations, aging of columns, or processing errors. This might help to minimize batch rejections or contribute to an increased productivity.