Single‐step purification of a small non‐mAb biologic by peptide‐ELP‐based affinity precipitation

Abstract

Affinity precipitation using stimulus-responsive biopolymers such as elastin-like polypeptides (ELPs) have been successfully employed for the purification of monoclonal antibodies. In the current work, we extend these studies to the development of an ELP-peptide fusion for the affinity precipitation of the therapeutically relevant small non-mAb biologic, AdP. A 12-mer affinity peptide ligand (P10) was identified by a primary phage biopanning followed by a secondary in-solution fluorescence polarization screen. Peptide P10 and AdP interacted with a K D of 19.5 µM. A fusion of P10 with ELP was then shown to be successful in selectively capturing the biologic from a crude mixture. While pH shifts alone were not sufficient for product elution, the use of pH in concert with fluid-phase modifiers such as NaCl, arginine, or ethylene glycol was effective. In particular, the use of pH 8.5 and an arginine concentration of 500 mM enabled >80% product recovery. The overall process performance evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase ultra-performance liquid chromatography analyses indicated successful single-step purification of the biologic from an Escherichia coli lysate resulting in ∼90% purity and >80% recovery. These results demonstrate that phage display can be readily employed to identify a peptide ligand capable of successfully carrying out the purification of a non-antibody biological product using ELP-based affinity precipitation.

Genetically Programmed Temperature-Responsive Barnacle-Derived Protein with an Enhanced Adhesion Ability

“…Spy partners including SpyCatcher and SpyTag could spontaneously form an irreversible isopeptide bond, − which serves as a powerful tool for bioconjugation and creating a protein architecture by covalent interaction. − Elastin-like polypeptides (ELPs) composed of repeating Val-Pro-Gly-X-Gly (X is any amino except proline) units have thermally responsive properties, − which enable ELPs to exhibit reversible phase transition behavior in response to temperature changes. ELPs are capable of self-assembling into a insoluble form above a certain transition temperature and present a soluble form below the transition temperature .…”

Separation and Purification of Recombinant β-Glucosidase with Hydrophobicity and Thermally Responsive Property from Cell Lysis Solution by Foam Separation and Further Purification

“…According to the thermally responsive property of ELPs, the proteins with ELPs can be precipitated when the surrounding temperature is higher than the phase transition temperature (Tt). Besides, when the temperature is lower than Tt, proteins with ELPs can become soluble again. , It is a simple and rapid method to purify recombinant enzymes based on the thermally responsive property of ELPs.…”

“…Besides, when the temperature is lower than Tt, proteins with ELPs can become soluble again. 18,19 rapid method to purify recombinant enzymes based on the thermally responsive property of ELPs. β-Glucosidase (Glu) can catalyze the specific hydrolysis of β-D-1,4-glucoside bonds in oligosaccharides, disaccharides, and conjugated glycosides.…”