Investigating and Optimizing Insulin Partitioning with Conjugated Au Nanoparticles in Aqueous Two-Phase Systems Using Response Surface Methodology

This study investigated the impact of bioconjugation on the partitioning of insulin, a clinically valuable protein, in an aqueous two-phase system. Gold nanoparticles of different sizes were synthesized and conjugated with insulin. Analysis of the conjugated insulin showed that the insulin remains fully active. Conjugated gold nanoparticles (AuNPs/insulin) were used in polyethylene glycol (PEG)–dextran aqueous two-phase systems to investigate the effect of pH, PEG and dextran molecular weights, PEG and dextran concentrations, AuNPs/insulin dosage, and nanoparticle size on the partition coefficient. These systems were chosen for their biocompatibility and low toxicity. Response surface methodology with D-optimal design was used to model and optimize these systems and their affected parameters. At the optimum condition of a pH = 8 system containing 21% PEG 4000, 5% dextran 100,000, and 100 IU AuNPs/insulin, the partition coefficient of AuNPs/insulin was found to be 192.96, which is in agreement with the empirical partition coefficient of 189.2. This is significantly higher than the partition coefficient of free insulin in a similar system. This approach could be used to overcome limitations in the feasibility of aqueous two-phase systems for industrial-scale purification of biomolecules and biopharmaceuticals.