The antigen-binding fragment of antibodies is increasingly drawing the attention of scientists as a favorable platform from which to develop bio-therapeutics. Engineered antibody fragments demonstrate broader biodistribution, improved tissue penetration, and reduced immunogenicity compared to full-length antibodies while maintaining the same specificity. However, these fragments face a couple key challenges including the lack of a well-established platform purification process like that for traditional monoclonal antibodies and a lack of robust and affordable affinity ligands. Synthetic peptides are potential alternatives to protein ligands in affinity chromatography due to their excellent biorecognition properties, low cost, and low risk of immunogenicity if leached. In this work, a combination of in vitro and in silico screening was utilized to identify and develop peptide affinity ligands targeting both Fab-κ and Fab-λ. A solid-phase, one-bead-one-peptide, combinatorial library incubated with fluorescently labeled Fab-κ and CHO-S host cell proteins was sorted with a microfluidic device to identify selective ligands. In parallel, the Hall group at NC State University developed ligands through the use of “PepBD,” a peptide binding design algorithm, which has also been used in prior work. The first sequences provided by the algorithm were experimentally tested against polyclonal Fab and the top sequence was utilized to refine the algorithm. The final peptides identified from microfluidic library screening and in silico design work were synthesized on chromatographic resins and evaluated for their ability to bind Fab under dynamic and equilibrium conditions. The peptides demonstrated mild affinity (KD ~10-5 M) and binding capacities (~20-30 mg Fab/mL resin) with yields up to ~90%. Additionally, the ligand identified from microfluidic screening demonstrated selectivity towards fragments compared to host cell proteins, purifying monoclonal and polyclonal fragments from complex CHO fluids. Finally, this work demonstrates the power of the “PepBD” algorithm and microfluidic sorting of solid-phase peptide libraries to rapidly identify affinity ligands for the burgeoning groups of novel, non-mAb based therapeutic formats.

Purification of next-generation biologics through peptide-based affinity chromatography

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.