Defining operating regimes for partition coefficient measurements in protein chromatography

Area of Science:

• Biotechnology and Bioprocessing
• Chromatography and Separation Science
• Process Development and Optimization

Background:

• Partition coefficient (K_p) measurements are crucial for screening chromatographic resins and conditions in biologics downstream process development.
• Current methods face challenges with equilibrium concentrations below the limit of detection (LOD) and sampling outside the linear isotherm region, compromising accuracy.
• Accurate K_p and selectivity data are essential for robust experimental design and reliable mechanistic modeling.

Purpose of the Study:

• To develop a theoretical framework and software tool for selecting optimal phase ratio and protein loading in K_p experiments.
• To ensure K_p experiments meet three key criteria: equilibrium concentrations above LOD, sampling from the linear isotherm, and practically relevant K_p values.
• To provide a foundation for accurate K_p and selectivity measurements, enhancing downstream process development.

Main Methods:

• Development of 'feasibility maps' to define a valid operating regime based on LOD, separation factor, and maximum K_p.
• Superposition of feasibility maps onto a database of 6,310 Langmuir isotherm points from literature to assess experimental criteria.
• Derivation of closed-form expressions for operating curves and evaluation of sensitivity to experimental parameters; experimental validation using column modeling.

Main Results:

• Defined an operating regime on a resin loading vs. phase ratio plane, bounded by derived operating curves.
• Demonstrated that K_p measurements outside the defined operating regime lead to significant errors in mechanistic model predictions.
• Experimental validation confirmed the necessity of operating within the calculated regime for accurate K_p and elution salt predictions.

Conclusions:

• The developed theoretical framework and software tool enable robust experimental design for accurate K_p and selectivity measurements.
• Optimized K_p experiments, guided by the software, improve the reliability of chromatographic resin screening and downstream process development.
• The tool can be customized with in-house isotherm data to calculate specific operating regimes for tailored experimental design.