Development of pseudo-linear gradient elution for high-throughput resin selectivity screening in RoboColumn^® Format

Area of Science:

• Biotechnology and Chemical Engineering
• Chromatographic Process Development
• High-Throughput Screening (HTS)

Background:

• Rapid development of chromatographic processes necessitates effective high-throughput screening (HTS) methods.
• Existing methods may lack efficiency or scalability for initial resin screening.
• Robotic platforms offer potential for automating and accelerating HTS in chromatography.

Purpose of the Study:

• To develop and validate a high-throughput screening (HTS) method for resin selectivity using pseudo-linear gradient elution on a robotic platform.
• To provide guidelines for implementing HTS on a Tecan Freedom EVO robotic platform, focusing on scale-down and liquid handling.
• To investigate the impact of key parameters on separation efficiency and demonstrate the predictive quality for larger-scale chromatography.

Main Methods:

• Development of a pseudo-linear gradient elution method by discretizing linear gradients into multistep gradients.
• Implementation on a Tecan Freedom EVO robotic platform with custom scripts for buffer preparation and column operation.
• Systematic investigation of column size, flow rate, gradient design, and fractionation using a ternary model protein mixture and cation exchange resins.

Main Results:

• The developed HTS method enabled clear differentiation of resin selectivity on miniature chromatography column (MCC) scale.
• Results from the robotic platform were consistent with conventional chromatography systems and showed predictive quality for larger scales.
• Optimization of key parameters (column size, flow rate, gradient, fractionation) led to effective column performance and separation of challenging mixtures like antibody monomer/high molecular weight.

Conclusions:

• The established robotic HTS protocol is effective for resin selectivity screening and predicting larger-scale performance.
• The method allows for clear differentiation of resin behavior and the influence of buffer conditions.
• The study highlights tradeoffs between robotic method complexity, effort, and output quality for various separation challenges.