Affinity chromatography based on a combinatorial strategy for rerythropoietin purification

Area of Science:

• Biochemistry
• Biotechnology
• Affinity Chromatography

Background:

• Small peptides (<10 amino acids) are valuable ligands for industrial protein purification systems.
• Combinatorial peptide synthesis accelerates the discovery of ligands for specific proteins.
• Recombinant human erythropoietin (rhEPO) is a therapeutic protein used to treat anemia.

Purpose of the Study:

• To identify peptide ligands with high affinity for recombinant human erythropoietin (rhEPO).
• To develop and evaluate peptide-based affinity chromatography for rhEPO purification.

Main Methods:

• Synthesized an octapeptide combinatorial library using the divide-couple-recombine method.
• Screened the library using rhEPO labeled with Texas Red or biotin.
• Sequenced positive peptide hits using MALDI-TOF MS and assessed affinity with a surface plasmon resonance biosensor.
• Immobilized top-performing peptides onto Sepharose for affinity chromatography.

Main Results:

• Identified peptide ligands with dissociation constants (Kd) in the 1-18 μM range for rhEPO.
• Immobilized peptides on Sepharose demonstrated strong affinity for rhEPO (Kd: 1.8-2.7 μM).
• Purified rhEPO from spiked Chinese hamster ovary (CHO) cell culture supernatant with 90% yield and 95-97% purity.

Conclusions:

• Novel octapeptides effectively bind rhEPO, enabling its purification via affinity chromatography.
• Peptide-based affinity chromatography offers a promising method for industrial-scale purification of rhEPO.
• This approach facilitates efficient and high-purity recovery of therapeutic proteins.