Multiplexed Competitive Screening of One-Bead-One-Component Combinatorial Libraries Using a ClonePix 2 Colony Sorter

Screening solid-phase combinatorial libraries of bioactive compounds against fluorescently labeled target biomolecules is an established technology in ligand and drug discovery. Rarely, however, do screening methods include comprehensive strategies-beyond mere library blocking and competitive screening-to ensure binding selectivity of selected leads. This work presents a method for multiplexed solid-phase peptide library screening using a ClonePix 2 Colony Picker that integrates (i) orthogonal fluorescent labeling for positive selection against a target protein and negative selection against competitor species with (ii) semi-quantitative tracking of target vs. competitor binding for every library bead. The ClonePix 2 technology enables global at-a-glance evaluation and customization of the parameters for bead selection to ensure high affinity and selectivity of the isolated leads. A case study is presented by screening a peptide library against green-labeled human immunoglobulin G (IgG) and red-labeled host cell proteins (HCPs) using ClonePix 2 to select HCP-binding ligands for flow-through chromatography applications. Using this approach, 79 peptide ligand candidates (6.6% of the total number of ligands screened) were identified as potential HCP-selective ligands, enabling a potential rate of >3,000 library beads screened per hour.