Fc binding peptide development project

Project: Research project

Project Details

Description

Fc binding peptide development project Fc binding peptide development project The power of affinity chromatography is widely known and there are numerous methods for affinity purification that use affinity tags, monoclonal antibodies (mAbs), or small-molecule ligands to purify a target protein. However these methods have been shown to be hindered by high non specific binding of affinity matrix resulting in multiple purification steps, long development time, high cost of goods and abrasive elution conditions are often required resulting in additional steps to refold or re-assemble the target. We have developed an in vitro selection system for discovering highly specific peptides for proteins, antibodies, viruses, and bacteria. The target of interest is screened against a small library of long peptides, prepared as a peptide microarray, to identify peptide ligands that have modest affinity but high specificity for the target. Selected peptides are immobilized on a solid support, to create a high avidity capture material that can separate the target biomolecule from a complex background. In addition, we can use a patent pending process to quickly improve the affinity of the selected peptides for use in more demanding purification or enrichment applications. The affinity ligand is immobilized on a solid-support and optimized for the separation and elution of the target under customer specified conditions. There are several important points that differentiate our method from other affinity purification approaches that use antibodies, recombinant proteins, or small-molecule ligands: Controlled discovery With our in vitro discovery system, we select multiple ligands, with a high hit-to-validated lead rate, that operate under any condition needed. High-specificity ligands Counter-selection leads to highly specific ligands for the target protein. This enables the single-step, high purity enrichment of the target from a complex background. Control of ligand affinity We discover micromolar affinity lead peptides but can increase their affinity into the nanomolar range in less than 2 months Controlled recovery The in vitro discovery system means that we can identify ligands that release their target under gentle conditions. Stable ligands and solid support Peptide ligands are stable to a variety of solvents and are covalently attached to the solid support producing a material that does not leach ligand into the purified product. Rapid development We can move from lead target to optimized affinity column in 3 months not the 6 months or more needed for monoclonal antibody development. Cost reversal The low cost of discovery and peptide synthesis means that now the ligand is no longer the cost driver in the purification process, the solid support is. To demonstrate how we can enable a manufacturing process, we worked with Dr. Charles Arntzens group at Arizona State University to identify peptides that could purify the viral coat protein from Norovirus virus-like-particle (nVLP) from a plant expression system under development for use as the antigen in a Norovirus (NoV) vaccine. The expression system produces a high yield of the viral coat protein VP1 but has proven difficult to purify using an Peptide Selection Overview Version 1.0 Diehnelt 2 10/4/11 affinity tag. Additionally, gentle conditions were needed to preserve the structure of the nVLP, thereby increasing the yield of antigenic nVLP. We used our system to identify multiple peptides that bound the coat protein. Candidate peptides were linked to a high-avidity support and the affinity material was optimized for gentle elution conditions, 1 M NaCl pH 7.2 in this case. The resulting affinity column has been used to purify several milligrams of nVLP with >95% purity and 90% recovery in a single purification step (Figure 1).
StatusFinished
Effective start/end date11/30/1110/16/12

Funding

  • INDUSTRY: Domestic Company: $104,598.00

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