Advances in inline quantification of co-eluting proteins in chromatography: Process-data-based model calibration and application towards real-life separation issues

ABSTRACT Pooling decisions in preparative liquid chromatography for protein purification are usually based on univariate UV absorption measurements that are not able to differentiate between product and co‐eluting contaminants. This can result in inconsistent pool purities or yields, if there is a batch‐to‐batch variability of the feedstock. To overcome this analytical bottleneck, a tool for selective inline quantification of co‐eluting model proteins using mid‐UV absorption spectra and Partial Least Squares Regression (PLS) was presented in a previous study and applied for real‐time pooling decisions. In this paper, a process‐data‐based method for the PLS model calibration will be introduced that allows the application of the tool towards chromatography steps of real‐life processes. The process‐data‐based calibration method uses recorded inline mid‐UV absorption spectra that are correlated with offline fraction analytics to calibrate PLS models. In order to generate average spectra from the inline data, a Visual Basic for Application macro was successfully developed. The process‐data‐based model calibration was established using a ternary model protein system. Afterwards, it was successfully demonstrated in two case studies that the calibration method is applicable towards real‐life separation issues. The calibrated PLS models allowed a successful quantification of the co‐eluting species in a cation‐exchange‐based aggregate and fraction removal during the purification of monoclonal antibodies and of co‐eluting serum proteins in an anion‐exchange‐based purification of Cohn supernatant I. Consequently, the presented process‐data‐based PLS model calibration in combination with the tool for selective inline quantification has a great potential for the monitoring of future chromatography steps and may contribute to manage batch‐to‐batch variability by real‐time pooling decisions. Biotechnol. Bioeng. 2015;112: 1406–1416. © 2015 Wiley Periodicals, Inc. A process‐data‐based calibration method for the application of Partial Least Squares Regression (PLS) models in protein chromatography was established in this study. The method uses recorded mid‐UV absorption spectra from inline measurements that are correlated with offline fraction analytics to calibrate PLS models. The calibrated models allowed a successful inline quantification of the co‐eluting species in a cation‐exchange‐based aggregate and fraction removal during mAb purification and of co‐eluting serum pro