Peptides have many advantages that they possess as chromatographic ligands: improved stability compared to many protein ligands, control and low cost afforded in production, and high selectivity for the target. Searching for peptide ligands of moderate length (7-15 amino acids) that act as affinity ligands becomes challenging though, as the searchable space for potential peptide candidates becomes exponentially larger for every additional amino acid added to the peptide. Expanding the search space beyond what may be found by mimicking a protein binding complex, phage display technology allows for a large number of sequences from a pregenerated library to be screened (upwards of 10^9 unique sequences) and has been used by several groups to identify peptide sequences that can act as ligands in affinity chromatography. Here we describe a method where subtractive panning is included in phage display screening in order to increase the stringency of selection for peptide sequences. Rather than just being screened on whether or not the peptides are capable of binding to the target, our approach includes additional screens to ensure that host cell proteins of the producing strain are either weakly or not retained at all by the peptide ligand, and that certain wash and elution methods may be used in purifying the target biologic. These additional screens are beneficial for ligands that will be used in a chromatographic process, as they will remove sequences from the pool which don’t show properties conducive to an affinity ligand, properties such as high affinity, selectivity, and elutability. Peptide sequences acquired by our process are synthesized on a functionalized agarose resin and assessed by how well they behave as chromatographic ligands and compared against sequences acquired through a standard phage display screening without subtractive panning.

Subtractive panning in phage display for identification of chromatographic affinity peptide ligands

249th National Meeting (2015)

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