Affinity ligands for protein purification are a class of reagents that are required to display several different properties and molecular recognition capabilities simultaneously. In addition to high affinity for their target, they also need to display high selectivity, elutability, resistance to base and proteolytic cleavage, and efficient regeneration over multiple cycles. This fine balance and need for rational control over multiple properties in parallel makes the design and development of such affinity reagents a challenging biomolecular engineering problem. To address this challenge, we will discuss our approach towards the design of affinity peptides for purifying human Growth Hormone (hGH) and interferon a2b (IFNa2b) from cell-culture fluid (CCF). Peptides due to their amenability to rational design and modification, ease of manufacture, and prolonged shelf-life are well-suited to meet the various critical, molecular attributes necessary for an affinity reagent used in downstream bioprocessing.<br/>We will demonstrate how peptides, identified from naturally-occurring proteins and receptors of hGH, were optimized to function as promising ligands for affinity chromatography using a suite of high-throughput screening assays and <i>in silico</i> design and docking. Microarray and resin-based screening of peptide libraries resulted in leads that spanned a range of affinity, selectivity and elutability profiles for hGH. Top-performing peptides from this screen were selected for biophysical characterization of their binding to hGH via fluorescence polarization and surface-plasmon resonance assays under various bioprocess-relevant conditions. Fundamental insights gained about the underlying protein-peptide interactions were applied towards the re-design of select peptides for improving their overall purification performance. Our results show that site-specific mutagenesis of peptides with different amino acids provides a handle for effectively tuning the three aforementioned properties. A final peptide that emerged from this process provided hGH purification comparable to an immunoaffinity-based procedure. The design strategies and assays developed for hGH were applied towards the design of affinity peptides for IFNa2b. Overall, this work illustrates the power of rational biomolecular design and screening for the rapid development of robust ligands for the purification of existing and emerging biologics in the fast-expanding protein therapeutics market.

Engineering affinity, selectivity and elutability into peptides for their development as ligands for downstream bioprocessing of non-mAb biologics

251st National Meeting (2016)

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