Novel peptide ligand with high binding capacity for antibody purification.

Small synthetic ligands for protein purification have become increasingly interesting with the growing need for cheap chromatographic materials for protein purification and especially for the purification of monoclonal antibodies (mAbs). Today, Protein A-based chromatographic resins are the most commonly used capture step in mAb down stream processing; however, the use of Protein A chromatography is less attractive due to toxic ligand leakage as well as high cost. Whether used as an alternative to the Protein A chromatographic media or as a subsequent polishing step, small synthetic peptide ligands have an advantage over biological ligands; they are cheaper to produce, ligand leakage by enzymatic degradation is either eliminated or significantly reduced, and they can in general better withstand cleaning in place (CIP) conditions such as 0.1M NaOH. Here, we present a novel synthetic peptide ligand for purification of human IgG. Immobilized on WorkBeads, an agarose-based base matrix from Bio-Works, the ligand has a dynamic binding capacity of up to 48 mg/mL and purifies IgG from harvest cell culture fluid with purities and recovery of >93%. The binding affinity is ∼105 M⁻¹ and the interaction is favorable and entropy-driven with an enthalpy penalty. Our results show that the binding of the Fc fragment of IgG is mediated by hydrophobic interactions and that elution at low pH is most likely due to electrostatic repulsion. Furthermore, we have separated aggregated IgG from non-aggregated IgG, indicating that the ligand could be used both as a primary purification step of IgG as well as a subsequent polishing step.

Metabolically stable cellular adhesion to inert surfaces.

The structure of D-amino acid hexapeptides that promote cellular adhesion was determined by screening D-amino acid hexapeptide libraries synthesized on otherwise inert beaded PEGA resin. These new adhesion molecules provide a completely stable cellular environment and facilitate the maintenance of a monolayer of cells on beads for extended periods. The presence of the peptides promotes spreading of the cells on the bead surface. Not surprisingly, the molecules contained a significant number of arginines and/or lysines. However, the exact structure of each peptide is quite important for the degree of adhesion observed, and a motif with three or four basic amino acids spaced within amino acids of intermediate polarity clearly prevailed, for example, k-l/r-h-r-i/v-r-a; this maintains a polar/hydrophobic balance.