Isotherm determination is critical for modelling chromatographic steps during bioprocess development. Ternary competitive isotherms have not been extensively studied for proteins, despite their importance as a minimum model of industrially relevant separations having a main product with both strongly and weakly retained impurities. Substantial data is needed to characterise such isotherms accurately. Here, high-throughput filter-plate format in combination with automated liquid handling systems is used to measure such isotherms efficiently for a challenging protein mixture. The use of UV absorbance spectra generated in 96-well UV plates along with partial least squares (PLS) regression relieves the analytical bottleneck commonly associated with HPLC analysis of high throughput methodologies. Weakly interacting isotherms, such as those measured at high salt concentrations, can be challenging because of small errors in assayed liquid concentration resulting in large errors in the absorbed concentration. Experimental design and alternative methodologies can help to minimise such errors. Batch binding as well as elution measurements in these plates facilitates the PLS characterisation of the multicomponent concentrations. The application of PLS to UV spectra for the quantification of multicomponent isotherms represents a generic and powerful methodology for high throughput generation of multicomponent isotherms.

Ternary adsorption isotherms of proteins from high-throughput experiments and multivariate analysis

251st National Meeting (2016)

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