A tool for selective inline quantification of co-eluting proteins in chromatography using spectral analysis and partial least squares regression

ABSTRACT Selective quantification of co‐eluting proteins in chromatography is usually performed by offline analytics. This is time‐consuming and can lead to late detection of irregularities in chromatography processes. To overcome this analytical bottleneck, a methodology for selective protein quantification in multicomponent mixtures by means of spectral data and partial least squares regression was presented in two previous studies. In this paper, a powerful integration of software and chromatography hardware will be introduced that enables the applicability of this methodology for a selective inline quantification of co‐eluting proteins in chromatography. A specific setup consisting of a conventional liquid chromatography system, a diode array detector, and a software interface to Matlab® was developed. The established tool for selective inline quantification was successfully applied for a peak deconvolution of a co‐eluting ternary protein mixture consisting of lysozyme, ribonuclease A, and cytochrome c on SP Sepharose FF. Compared to common offline analytics based on collected fractions, no loss of information regarding the retention volumes and peak flanks was observed. A comparison between the mass balances of both analytical methods showed, that the inline quantification tool can be applied for a rapid determination of pool yields. Finally, the achieved inline peak deconvolution was successfully applied to make product purity‐based real‐time pooling decisions. This makes the established tool for selective inline quantification a valuable approach for inline monitoring and control of chromatographic purification steps and just in time reaction on process irregularities. Biotechnol. Bioeng. 2014;111: 1365–1373. © 2014 Wiley Periodicals, Inc. A novel tool for selective in‐line quantification of co‐eluting proteins in chromatography was established in this study. The tool was successfully applied for an in‐line peak deconvolution of a co‐eluting ternary protein mixture consisting of lysozyme, ribonuclease A, and cytochrome c on SP Sepharose FF. Using the gained peak deconvolution by the in‐line quantification tool, precise purity‐based real‐time pooling decisions were possible.