A novel strategy is presented for the identification of cyclic peptide ligands from combinatorial libraries of reversible cyclic depsipeptides. A method for the solid-phase synthesis of individual cyclic depsipeptides and combinatorial libraries of these compounds is proposed, which employs lactic acid (Lact) and the dipeptide ester (Nα-Ac)-Ser(Ala)- as linkers for dilactonization. Upon alkaline treatment of the beads selected by screening a model library, the cyclic depsipeptides are linearized and released from the solid support to the liquid phase, to be sequenced via single-step tandem mass spectrometry (MS/MS). The protocol presented for library synthesis provides for wide structural diversity. Two model sequences, VVWVVK and AAWAAR, were chosen to present different structural examples for depsipeptide libraries and demonstrate the process of sequence determination by mass spectrometry. Further, a case study using the IgG binding cyclic depsipeptide cyclo[(Nα-Ac)-S(A)-RWHYFK-Lact-E] is presented to demonstrate the process of library screening and sequence determination on the selected beads. Finally, a method is shown for synthesis of the irreversible cyclic peptide corresponding to the proposed depsipeptide structure, to make the ligand stable to the aqueous acid and alkaline conditions encountered in affinity chromatographic applications. The cyclic peptide ligand was synthesized on a poly(methacrylate) resin and used for chromatographic binding of the target IgG.
High binding capacity and selectivity are key features
for the
successful application of affinity adsorbents for antibody purification.
This study presents the development of affinity resins based on hexapeptide
ligand HWRGWV for recovering monoclonal antibodies from cell culture
fluids. Methods are presented for the immobilization of the peptide
ligand and its variants on polymethacrylate and agarose based chromatographic
supports using two main coupling strategies. The first one involves
the formation of a peptide bond between the amino groups on the substrate
and the peptide C-terminus activated with the uronium coupling agent
HATU. The second approach involves resin activation with iodoacetic
acid, followed by coupling of a cysteine-terminated variant of the
ligand to form a thioether bond. The reaction conditions of peptide
coupling were optimized to maximize the binding capacity of the resulting
adsorbents. The peptide resins were characterized by measuring their
static IgG binding capacities. The measured static binding capacity
ranged from 35 to 48 mg/mL. The dynamic binding capacities (DBC) of
four selected adsorbents were also determined, and they ranged from
of 35 to 42 mg/mL with a 5-min residence time. All the resins exhibited
high selectivity toward the Fc fragment of IgG. The affinity resins
were used to purify two MAbs, a chimeric IgG1 and a humanized
IgG4, from commercial CHO cell culture fluids. The resulting
yields and purities for both MAbs were found to be in the range of
87–93% and >94%, respectively, which compare well with the
purity and yield values obtained using commercially available Protein
A media. Finally, the peptide resin with the highest IgG binding capacity,
HWRGWVC-WorkBeads, was tested for 20 DBC cycles which included cleaning
in place with 0.1 M NaOH after every cycle. The resin showed a high
degree of reusability and alkaline stability, as it maintained 90%
of its initial capacity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.