The current workhorse of downstream bioprocessing is chromatography-based separations. However, there is an increasing requirement to develop economical and scalable capture strategies to handle the growing diversity and rising titers of therapeutic proteins. Affinity precipitation has shown potential as a non-chromatographic purification strategy that employs thermoresponsive biopolymers such as Elastin-like Polypeptides (ELPs) for monoclonal antibodies and can be extended to non-mAb protein biologics. New protein modalities have gained significant popularity as novel protein-based drugs. We carried out the identification and design of an affinity peptide ligand against a small non-mAb biologic. A 12-mer affinity peptide ligand (P10) was identified by a primary phage display screen followed by a secondary in-solution fluorescence polarization screen. Peptide P10 and the protein interacted with a K<sub>d</sub> of 19.5 uM. A fusion of P10 with ELP was successful in selectively capturing the biologic from a highly crude mixture. However, no elution was observed at pH 3.0, while partial elution was observed at pH 10.0. A combination of alanine and histidine scanning mutagenesis of the P10 peptide was carried out to enhance protein elution at gentler conditions. While significant affinity maturation was achieved (leading to increased capacity), elution remained a challenge. Combining the effect of rational mutations to the P10-ELP fusion, with the effect of relevant fluid-phase modifiers such as NaCl, arginine and ethylene glycol ultimately helped reach milder elution conditions. SDS PAGE and reversed phase UPLC analyses indicated the successful single step purification of the biologic from an E. coli lysate resulting in &gt;90% purity and &gt;80% recoveries. These results demonstrate that phage display can be readily employed to identify a peptide ligand capable of performing the ELP-based affinity precipitation purification of a small non-antibody biomolecule. Moreover, the appropriate interplay of peptide mutagenesis and fluid phase modifiers was harnessed to both improve binding capacity and induce elution at relatively mild conditions.

Single-step purification of small non-mAb biologics by peptide-ELP based affinity precipitation

255th National Meeting (2018)

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