Abstract
Purification of biologically-derived therapeutics is a major cost contributor to the production of this rapidly growing class of pharmaceuticals. Monoclonal antibodies comprise a large percentage of these products, therefore new antibody purification tools are needed. Small peptides, as opposed to traditional antibody affinity ligands such as Protein A, may have advantages in stability and production costs. Multiple heptapeptides that demonstrate Fc binding behavior that have been identified from a combinatorial peptide library using M13 phage display are presented herein. Seven unique peptide sequences of diverse hydrophobicity and charge were identified. All seven peptides showed strong binding to the four major human IgG isotypes, human IgM, as well as binding to canine, rat, and mouse IgG. These seven peptides were also shown to bind human IgG4 from DMEM cell culture media with 5% FCS and 5 g/L ovalbumin present. These peptides may be useful as surface ligands for antibody detection and purification purposes. Molecular docking and classical molecular dynamics (MD) simulations were conducted to elucidate the mechanisms and energetics for the binding of these peptides to the Fc region. The binding site was found to be located between the two glycan chains inside the Fc fragment. Both hydrogen bonding and hydrophobic interactions were found to be crucial for the binding interactions. Excellent agreement for the binding strength was obtained between experimental results and simulations.
Highlights
Pharmaceutical sales, and in particular biologically derived pharmaceutical sales, are increasing [1]
Phage Display Panning Results In Round 1 of panning, phage particles were eluted from Fc bound to streptavidin beads via a biotin linker with a strongly acidic glycine buffer
Negative selection with streptavidin beads and amplified Round 1 phage eluate (2 × 109 pfu input) prior to panning with the Fc target was successful in reducing non-specific binding of phage particles as shown by the lower titers from both protein A (5.0 × 104 pfu/mL)
Summary
Pharmaceutical sales, and in particular biologically derived pharmaceutical sales, are increasing [1]. Biologically derived pharmaceuticals—including mAbs—are much more expensive than typical small-molecule therapeutics due to the complexity of both the molecule and the production process. Downstream purification of these mAbs can account for almost 80% of the total cost of manufacturing suggesting opportunities for novel antibody purification technology [2]. Much work has gone into finding possible functional replacements for protein A which is the workhorse affinity technology for mAb purification. Drawbacks of protein A include the fact that it is expensive and unstable under typical column cleaning/sanitization conditions such as 1M NaOH [3,4,5]
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