A novel magnetic support based on gum Arabic (GA) coated iron oxide magnetic nanoparticles (MNP) has been endowed with affinity properties towards immunoglobulin G (IgG) molecules. The success of the in situ triazine ligand synthesis was confirmed by fluorescence assays. Two synthetic ligands previously developed for binding to IgG, named as ligand 22/8 (artificial Protein A) and ligand 8/7 (artificial Protein L) were immobilized on to MNPs coated with GA (MNP_GA). The dimension of the particles core was not affected by the surface functionalization with GA and triazine ligands. The hydrodynamic diameters of the magnetic supports indicate that the coupling of GA leads to the formation of larger agglomerates of particles with about 1 microm, but the introduction of the triazine ligands leads to a decrease on MNPs size. The non-functionalized MNP_GA bound 28 mg IgG/g, two times less than bare MNP (60 mg IgG/g). MNP_GA modified with ligand 22/8 bound 133 mg IgG/g support, twice higher than the value obtained for ligand 8/7 magnetic adsorbents (65 mg/g). Supports modified with ligand 22/8 were selected to study the adsorption and the elution of IgG. The adsorption of human IgG on this support followed a Langmuir behavior with a Q(máx) of 344 mg IgG/g support and K(a) of 1.5 x 10(5) M. The studies on different elution conditions indicated that although the 0.05 M citrate buffer (pH 3) presented good recovery yields (elution 64% of bound protein), there was occurrence of iron leaching at this acidic pH. Therefore, a potential alternative would be to elute bound protein with a 0.05 M glycine-NaOH (pH 11) buffer.

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Ionic excipients are commonly used in aqueous therapeutic monoclonal antibody (mAb) formulations. Novel excipients are of industrial interest, with a recent focus on Arg salt forms and their application as viscosity reducing and stabilizing additives. Here, we report that the calcium salt of dipicolinic acid (DPA, pyridine-2,6-dicarboxylic acid), uniquely present in nature in the core of certain bacterial spores, reduces the viscosity of a mAb formulated at 150mg/mL, below that achieved by Arg hydrochloride at the same concentration (10mM). DPA also reduced the reversible phase separation of the same formulation, which characteristically occurs for this mAb upon cooling to 4°C. Differential scanning calorimetry and differential scanning fluorimetry did not reveal a conformation destabilisation of the mAb in the presence of 10mM DPA, or by the related quinolinic acid (QA, pyridine-2,3-dicarboxylic acid). However, fluorescence spectrophotometry did reveal localised (aromatic) conformational changes to the mAb attributed to DPA, dependent on the salt form. While precise mechanisms of action remain to be identified, our preliminary data suggest that these DPA salts are worthy of further investigation as novel ionic excipient for biologics formulation.

This manuscript reviews the combination of computational enzyme design and directed evolution and its advantages in the development of artificial enzymes.

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