title: Novel peptide ligand with high binding capacity for antibody purification.
abstract: 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.
authors.affiliation: Novo Nordisk A/S, Hagedornsvej 1, DK-2820 Gentofte, Denmark. LNmL@novonordisk.com
authors.forename: Line Naomi
authors.initials: LN
authors.lastname: Lund
authors.forename: Per-Erik
authors.initials: PE
authors.lastname: Gustavsson
authors.forename: Roice
authors.initials: R
authors.lastname: Michael
authors.forename: Johan
authors.initials: J
authors.lastname: Lindgren
authors.forename: Leif
authors.initials: L
authors.lastname: Nørskov-Lauritsen
authors.forename: Martin
authors.initials: M
authors.lastname: Lund
authors.forename: Gunnar
authors.initials: G
authors.lastname: Houen
authors.forename: Arne
authors.initials: A
authors.lastname: Staby
authors.forename: Phaedria M
authors.initials: PM
authors.lastname: St Hilaire
mesh_terms: D000327:Adsorption; D000911:Antibodies, Monoclonal / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D012685:Sepharose / Q000737:chemistry; D055672:Static Electricity; D013816:Thermodynamics
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D008024:Ligands; D010455:Peptides; D012685:Sepharose