Biochemical and biological characterization of a new oxidized avidin with enhanced tissue binding properties.

Chicken avidin and bacterial streptavidin are widely employed in vitro for their capacity to bind biotin, but their pharmacokinetics and immunological properties are not always optimal, thereby limiting their use in medical treatments. Here we investigate the biochemical and biological properties of a new modified avidin, obtained by ligand-assisted sodium periodate oxidation of avidin. This method allows protection of biotin-binding sites of avidin from inactivation caused by the oxidation step and delay of avidin clearance from injected tissue by generation of aldehyde groups from avidin carbohydrate moieties. Oxidized avidin shows spectroscopic properties similar to that of native avidin, indicating that tryptophan residues are spared from oxidation damage. In strict agreement with these results, circular dichroism and isothermal titration calorimetry analyses confirm that the ligand-assisted oxidation preserves the avidin protein structure and its biotin binding capacity. In vitro cell binding and in vivo tissue residence experiments demonstrate that aldehyde groups provide oxidized avidin the property to bind cellular and interstitial protein amino groups through Schiff's base formation, resulting in a tissue half-life of 2 weeks, compared with 2 h of native avidin. In addition, the efficient uptake of the intravenously injected (111)In-BiotinDOTA (ST2210) in the site previously treated with modified avidin underlines that tissue-bound oxidized avidin retains its biotin binding capacity in vivo. The results presented here indicate that oxidized avidin could be employed to create a stable artificial receptor in diseased tissues for the targeting of biotinylated therapeutics.

Affinity purification of IgG monoclonal antibodies using the D-PAM synthetic ligand: chromatographic comparison with protein A and thermodynamic investigation of the D-PAM/IgG interaction.

This study investigates the applicability of D-PAM, the inverso form of the Protein A Mimetic synthetic peptide affinity ligand (PAM) obtained from the screening of a multimeric combinatorial peptide library, in monoclonal IgG isolation from ascitic fluids and cellular supernatants. D-PAM affinity columns, prepared by immobilizing the all-D peptide on the commercially available support Emphaze, were able to capture monoclonal antibodies in a single chromatographic step, with a recovery yield and purity degree above 90% and full recovery of antibody activity. D-PAM/Emphaze resin showed a host cell protein (HCP) and DNA reduction similar to protein A sorbent. Indeed, column capacity, determined by applying a large excess of purified antibodies to 1 mL of column bed volume, was always higher than 50 mg/mL. D-PAM/IgG interaction was characterized by isothermal titration calorimetry (ITC) and an analysis of binding isotherms, obtained for titration of ST2146, ST1485 and 7H3 IgG monoclonal antibodies, suggested that two peptides bind simultaneously to the IgG molecule, with a K(A) (equilibrium association constant) of 3.4, 6.2 and 3.4 x 10(4) M(-1), and a DeltaH (change in enthalpy) of -1.3, -4.2 and -4.1 kcal mol(-1), respectively.

High-level expression and efficient purification of recombinant human long pentraxin PTX3 in Chinese hamster ovary cells.

PTX3 is a secreted multimeric glycoprotein which plays a key role in innate immunity by activating the classical complement pathway through specific recognition of the C1q subunit. A method is described for the high level expression of the recombinant human PTX3 in Chinese hamster ovary cells (CHO), adapted to a suspension growth in spinner flasks containing a serum-free chemically defined medium and producing about 50 mg of PTX3/L of culture. A purification procedure to produce a homogeneous protein preparation from the supernatant, by means of anion exchange, hydroxyapatite and size exclusion chromatography, is also reported. This three-step protocol allows us to obtain PTX3 with a recovery yield close to 70%, a purity degree exceeding 95%, and a final host cell protein (HCP) content lower than 150 ppm. The recombinant purified PTX3 retains its biological activity, as demonstrated by C1q binding ELISA assay, and displays a complex quaternary structure characterized by a high secondary structure content quite different from human short pentraxin C-reactive protein (CRP) and serum amyloid P component (SAP), as determined by circular dichroism, fluorescence analysis, and native and SDS-PAGE experiments.

Identification and refinement of a peptide affinity ligand with unique specificity for a monoclonal anti-tenascin-C antibody by screening of a phage display library.

Using phage display technology, a 22-mer peptide was selected as a ligand with unique specificity for the murine monoclonal ST2146 antibody that recognizes the EGF repeats region of the human tumor-associated antigen tenascin-C. This peptide, synthesized in an 8-branched form to enhance its binding properties, is useful in replacing the native antigen in the affinity and immunoreactivity characterization of the ST2146 antibody and its biotinylated derivatives. Affinity resins, prepared by immobilizing the mimotope or its shorter 10-mer binding unit on a chromatographic support, were able to capture ST2146 directly from the hybridoma supernatant, with antibody recovery and host cell protein (HCP) reduction similar to or better than protein A sorbent, a purity degree exceeding 95%, and full recovery of antibody activity. The affinity constants of both peptides, as determined by frontal analysis of broad-zone elution affinity chromatography and BiaCore measurements, were very similar and included in a range suitable for affinity ligands. Column capacity, determined by applying a large excess of purified ST2146 to 1 mL of column bed volume, was close to 50 mg/mL for both resins. These matrices retain their ST2146 binding properties after various treatments, including sanitization, thus indicating very high stability in terms of ligand leakage and degradation. Moreover, the short form shows higher enzymatic stability, thus proving more suitable as ligand for ST2146 affinity purification.

A new ligand for immunoglobulin g subdomains by screening of a synthetic peptide library.

By screening a synthetic peptide library of general formula (NH(2)-Cys1-X2-X3-X4)(2)-Lys-Gly-OH, a disulfide-bridged cyclic peptide, where X2-X3-X4 is the tripeptide Phe-His-His, has been selected as a ligand for immunoglobulin G (IgG). The peptide, after a preliminary chromatographic characterization, has proved useful as a new affinity ligand for the purification of polyclonal as well as monoclonal antibodies from biological fluids, with recovery yields of up to 90% (90% purity). The ligand is able to bind antibody fragments containing both Fab and Fc from different antibody isotypes, a fact suggesting the presence of at least two different antibody-binding sites. While the recognition site on Fab is unknown, comparative binding studies with Fc, in association with the striking similarities of the peptide (named Fc-receptor mimetic, FcRM) with a region of the human FcgammaRIII receptor, strongly indicate that the peptide could recognize a short amino acid stretch of the lower hinge region, which has a key role in autoimmune disease triggering. The unique properties make the ligand attractive for both the purification of antibody fragments and as a lead for the generation of Fc-receptor antagonists.