Affinity chromatography on immobilized "biomimetic" ligands. Synthesis, immobilization and chromatographic assessment of an immunoglobulin G-binding ligand.

A synthetic bifunctional ligand (22/8) comprising a triazine scaffold substituted with 3-aminophenol (22) and 4-amino-1-naphthol (8) has been designed, synthesised, characterised and immobilized on agarose beads to create a robust, highly selective affinity adsorbent for human immunoglobulin G (IgG). Scatchard analysis of the binding isotherm for IgG on immobilized 22/8 (90 micromol 22/8/g moist weight gel) indicated an affinity constant (Ka) of 1.4 x 10(5) M(-1) and a theoretical maximum capacity of 151.9 mg IgG/g moist weight gel. The adsorbent shows similar selectivity to immobilized protein A and binds IgG from a number of species. An apparent capacity of 51.9 mg human IgG/g moist weight gel was observed under the experimental conditions selected for adsorption. Human IgG was eluted with glycine-HCl buffer with a recovery of 67-69% and a purity of 97.3-99.2%, depending on the pH value of the buffer used for elution. Preparative chromatography of IgG from human plasma showed that under the specified conditions, 94.4% of plasma IgG was adsorbed and 60% subsequently eluted with a purity of 92.5%. The immobilized ligand was able to withstand incubation in 1 M NaOH for 7 days without loss of binding capacity for IgG.

New strategy for the design of ligands for the purification of pharmaceutical proteins by affinity chromatography.

A new approach for the identification of ligands for the purification of pharmaceutical proteins by affinity chromatography is described. The technique involves four steps. Selection of an appropriate site on the target protein, design of a complementary ligand compatible with the three-dimensional structure of the site, construction of a limited solid-phase combinatorial library of near-neighbour ligands and solution synthesis of the hit ligand, immobilisation, optimisation and application of the adsorbent for the purification of the target protein. This strategy is exemplified by the purification of a recombinant human insulin precursor (MI3) from a crude fermentation broth of Saccharomyces cerevisiae.

An optical biosensor for monitoring recombinant proteins in process media.

This paper describes the construction of a sensor for the direct monitoring of a recombinant protein, the human insulin analogue (MI3). The surface plasmon resonance (SPR) sensor incorporates an immobilised, sterilisable affinity-ligand that has been designed to bind to MI3. In practice, gold SPR devices were fabricated with; a 2D assembly of ethanethiol-modified ligand, a 2D mixed-assembly of ethanethiol-modified ligand and mercaptoethanol, a 3D coating of ligand-modified terminal-thiolated poly(vinyl)alcohol (PVA) or a 3D hydrogel of dextran coupled to a self-assembled monolayer (SAM) of mercaptohexaneundecanl-ol. Routine measurement of the concentration MI3 in the concentration range 1-100 mg/l in pilot-scale samples of crude fermentation broth have been achieved with high sensitivity levels and a high signal-to-noise ratio. Analysis can be achieved within < 10 min with the active surface being regenerable for at least 60 cycles over a 6 month period. The coupling of a robust, sterilisable and highly-selective sensor-coating with suitable transducer technologies promises to deliver sensors that are capable of direct in situ monitoring of biopharmaceuticals in industrial bioprocesses.

Design, synthesis and characterisation of affinity ligands for glycoproteins.

The concepts of rational design and solid phase combinatorial chemistry were used to develop affinity adsorbents for glycoproteins. A detailed assessment of protein-carbohydrate interactions was used to identify key residues that determine monosaccharide specificity, which were subsequently exploited as the basis for the synthesis of a library of glycoprotein binding ligands. The ligands were synthesised using solid phase combinatorial chemistry and were assessed for their sugar-binding ability with the glycoenzymes, glucose oxidase and RNase B. Partial and completely deglycosylated enzymes were used as controls. The triazine-based ligand, histamine/tryptamine (8/10) was identified as a putative glycoprotein binding ligand, since it displayed particular affinity for glucose oxidase and other mannosylated glycoproteins. Experiments with deglycosylated control proteins, specific eluants and retardation in the presence of competing sugars strongly suggest that the ligand binds the carbohydrate moiety of glucose oxidase rather than the protein itself.

A strategy for the generation of biomimetic ligands for affinity chromatography. Combinatorial synthesis and biological evaluation of an IgG binding ligand.

An IgG-binding ligand library comprising 88 adsorbents based on a known lead compound (Li et al., 1998) was generated on an agarose solid phase. Individual members of the library were synthesized in two chemical steps using cyanuric chloride as the scaffold immobilized on the beaded support. The library was screened for binding of pure human IgG, whence selected ligands from the library were further assessed for specificity by the purification of IgG from human plasma. The potential of this strategy for the rapid identification and evaluation of chemical leads was demonstrated by the discovery of ligands with IgG binding capabilities. It was found that ligands comprising 3-aminophenol and an aminonaphthol moiety substituted on a triazine nucleus generally performed better than other ligands in the library. An immobilized ligand 22/8 adsorbent was able to purify IgG with high yield and a purity >99% from diluted human plasma.