388893 Rational Design and Engineering of Peptides for Affinity Purification of Biologics
Current strategies for downstream processing and purification of biologics involve multiple steps such as size exclusion, ion-exchange, and reverse-phase chromatography. As a result, the entire purification process is very time and resource-intensive. Affinity chromatography, on the other hand, provides several advantages over the traditional chromatography procedures for protein purification by limiting the number of steps involved thereby reducing time and costs. However, the use of affinity chromatography in protein purification is still limited and very few industrial scale processes employ affinity ligands as compared to those that use traditional chromatography procedures. One of the major challenges in the maturation of affinity chromatography technology has been the availability of robust, selective, cost-effective, and high affinity ligands for the biologic of interest. So far, antibodies have been the most commonly used affinity ligands in the few affinity-based purification procedures that are employed industrially. However, antibodies with their high production and purification costs are expensive reagents, with limited stability and shelf-life. Therefore, affinity ligands that overcome these challenges and can be easily designed and manufactured for a range of different biologics are highly desirable. Peptides as affinity ligands offer several advantages such as amenability to rational design and modification, ease of manufacture, low cost, high stability and prolonged shelf life.
In this work, we have used rational approaches for designing affinity peptides for the purification of recombinant human growth hormone (rhGH) produced in Pichia pastoris (P. pastoris). The knowledge of the structure of hGH and its naturally occurring binding partners was used for designing a library of peptides with sequence lengths ranging from 7-22 amino acids. Strategies such as point mutations to specific residues and combinations of key interacting motifs were included in our peptide design criteria. Using high-throughput peptide synthesis and microarray screening, we have identified several peptides that bind rhGH from an initial library of 384 candidates. These peptides show high affinity (10 -200 nM) and selectivity for rhGH in the presence of cell culture fluid (CCF) from P. pastoris. We have also carried out microarray screening with the peptide library for host-cell protein (HCP) clearance and elution of bound rhGH under different salt and pH conditions. Several candidates show preferential binding to rhGH over the HCPs present in P. pastoris CCF and show up to 70% recovery of bound rhGH. These peptides therefore form promising candidates for use as affinity ligands in affinity chromatography-based purification of rhGH.
Select peptides from the microarray screening are currently being tested in batch-scale adsorption and purification studies with a chromatography resin. Briefly, ~10 peptides have been selected for synthesis and screening on an amine-functionalized chromatography resin. Our preliminary batch-scale screening results indicate that the peptide-functionalized resins have a high binding capacity (up to 10 mg/ml) for hGH. 1-2 lead peptides from these studies will be selected for column studies and design of an affinity-based capture and purification step for rhGH. Further, results and general principles obtained from these studies will be used to guide the design of affinity peptides for a host of other high-impact biologics that are currently purified over multiple steps.