High-throughput investigation of single and binary protein adsorption isotherms in anion exchange chromatography employing multivariate analysis

- Adsorption isotherm accuracy and precision is dependent on experimental variables.
- Studying isotherms both at equilibrium and through elution can improve data reliability.
- Multicomponent isotherms quantified using UV spectra and multivariate data analysis.
- Analytical bottleneck alleviated with rapid high throughput analytics.

The combination of multi-well plates and automated liquid handling is well suited to the rapid measurement of the adsorption isotherms of proteins. Here, single and binary adsorption isotherms are reported for BSA, ovalbumin and conalbumin on a strong anion exchanger over a range of pH and salt levels. The impact of the main experimental factors at play on the accuracy and precision of the adsorbed protein concentrations is quantified theoretically and experimentally. In addition to the standard measurement of liquid concentrations before and after adsorption, the amounts eluted from the wells are measured directly. This additional measurement corroborates the calculation based on liquid concentration data, and improves precision especially under conditions of weak or moderate interaction strength. The traditional measurement of multicomponent isotherms is limited by the speed of HPLC analysis; this analytical bottleneck is alleviated by careful multivariate analysis of UV spectra.