Factorization of preparative protein chromatograms with hard-constraint multivariate curve resolution and second-derivative pretreatment.

Current biopharmaceutical production heavily relies on chromatography for protein purification. Recently, research has intensified towards finding suitable solutions to monitoring the chromatographic steps by multivariate spectroscopic sensors. Here, hard-constraint multivariate curve resolution (MCR) was investigated as a calibration-free method for factorizing bilinear preparative protein chromatograms into concentrations and spectra. Protein elutions were assumed to follow exponentially modified Gaussian (EMG) curves. In three case studies, MCR was applied to chromatograms of second-derivative ultraviolet and visible (UV-vis) spectra. The three case studies consisted of the separation of a ternary mixture (ribonuclease A, cytochrome c, and lysozyme), multiple binary chromatography runs of cytochrome c and lysozyme, and the separation of an antibody-drug conjugate (ADC) from unconjugated immunoglobulin G (IgG). In all case studies, good estimates of the elution curves were obtained. R

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