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.

A diversity optimized combinatorial library for the identification of Fc-fragment binding ligands.

To generate a library covering a relatively wide area of the chemical space, molecular descriptors, and multivariate data analysis were combined to select the building blocks required for generating a diversity optimized library of putative Fc-fragment binding ligands. In such a method of library design, structural information about the target protein is not needed. Synthesis of the resulting 770 member virtual library was carried out using encoded beads, which facilitated rapid identification of the subsequent hits. The library was screened in an on-bead fluorescence assay with immunoglobulin G Fc-fragment of the subtype 4 to identify Fc-fragment binding ligands that would be useful for purifying monoclonal antibodies. An analysis of the positions of the hits in the chemical space revealed that the ones with the highest fluorescence were primarily concentrated in a particular part of the chemical space. One of the identified hits, when immobilized on amino sepharose, was able to purify a monoclonal antibody from crude cell supernatant with purity of 84% and a 70% recovery. The chemometric tools employed for the library design allowed the identification of the fraction of the available chemical space that would be preferred for a second generation library.

Complex of sialoadhesin with a glycopeptide ligand.

Sialoadhesin is a sialic acid-binding immunoglobulin-like lectin (Siglec), expressed on subsets of macrophages. It is a model system for Siglec receptor-mediated cell surface interactions through binding of sialylated glycoconjugates. The N-terminal sialoadhesin domain can mediate sialic acid-binding on its own. The structure of this domain has been determined in complex with a sialic acid-containing heptapeptide, (Ala-Gly-His-Thr(Neu5Ac)-Trp-Gly-His). The affinity of sialoadhesin for this ligand is four times higher than the affinity for the natural linkage 2,3'-sialyllactose. The structure of the glycopeptide complex suggests strategies for ligand optimization and provides possible explanations for the observed differences in specificities among the Siglecs.

Fluorometric polyethyleneglycol-peptide hybrid substrates for quantitative assay of protein disulfide isomerase.

In eukaryotic cells the enzyme protein disulfide isomerase (PDI) is responsible for the formation and reshuffling of disulfide bonds in secretory proteins. The reaction carried out by PDI involves interaction with a highly complex mixture of polypeptide molecules that are in the process of folding. This means that PDI activity is typically measured in the context of a globular protein folding pathway. The absence of small, well-defined substrates for the quantitation of both oxidation and reduction reactions constitutes an inherent problem in the analysis of PDI activity. We describe a new type of substrate for PDI where two cysteine-containing oligopeptides are connected by an onameric ethylene glycol linker. We term such hybrid compounds PEGtides. The oligopeptides are each marked with a fluorescent aminobenzoic acid and a quenching nitrotyrosine group, respectively. The reversible formation of an intramolecular disulfide bond between fluorophore-containing and quencher-containing peptide segments results in a redox-dependent fluorescence signal. We find a model compound of this type to be a highly sensitive substrate for PDI both in oxidation and in reduction assays under steady state conditions. These aspects should make substrates of this type generally applicable for assaying PDI and other thiol-disulfide exchange enzymes.

Glycopeptides as oligosaccharide mimics: high affinity sialopeptide ligands for sialoadhesin from combinatorial libraries.

Two different sialic acid containing glycopeptide (sialopeptide) libraries were synthesized using the portion mixing method and ladder synthesis. The libraries were attached via an IMP spacer and a photolabile linker to PEGA(1900) resin in order to facilitate rapid and unambiguous structural analysis of hits by MALDI-TOFMS. One library contained a lactamized sialic acid moiety at the N terminus of a pentapeptide, while a second library displayed a sialic acid residue at the center of a heptapeptide. The sialopeptide libraries were screened against the recombinant binding domain (SnD1) of a sialic acid binding Ig-like protein, sialoadhesin (Siglec-1). No ligands were identified from the lactamized sialic acid library, underscoring the importance of the carboxylic acid moiety for binding. Screening of the second gave few distinct hits (approximately 0.03% of library) with a high consensus. The high-affinity ligands contained, in most cases, a WG motif following the sialylated Thr. The strength of binding of selected ligands was determined by surface plasmon resonance. The best sialopeptide ligand, WLLT(Sa)WGT, exhibited micromolar affinity of SnD1; >10 times the affinity of SnD1 to 3'-sialyl lactose.

Solid-phase library synthesis, screening, and selection of tight-binding reduced peptide bond inhibitors of a recombinant Leishmania mexicana cysteine protease B.

A one-bead-two-compound inhibitor library was synthesized by the split-mix method for the identification of inhibitors of a recombinant cysteine protease from Leishmania mexicana, CPB2.8DeltaCTE. The inhibitor library was composed of octapeptides with a centrally located reduced bond introduced by reductive amination of the resin-bound amines with Fmoc amino aldehydes. The library was screened on solid phase, and less than 1% of the library contained active compounds. The inhibitors displayed great specificity in the subsites flanking the enzyme catalytic triad with Cha and Ile/Leu preferred in P(2), Phe in P(1), Cha and Ile/Leu in P(1)', and Ile/Leu in P(2)'. Some of the inhibitors were resynthesized, and the kinetics of inhibition were determined in solution-phase assays. Most of the inhibitors had micromolar K(i) values, and a few inhibited the enzyme at nanomolar concentrations. One inhibitor, DKHF(CH(2)NH)LLVK (K(i) = 1 microm), was tested for antiparasite efficacy and shown to affect parasite survival with an IC(50) of approximately 50 microm.