The Orientation Of Protein A On Solid Phase And Antibody Adsorption

Protein A chromatography with high antibody-binding capacity is one of the most important techniques applied in the purification of biopharmaceuticals.In this study,Z domain of protein A from Staphylococcal aureus(SpA)was used as parent template for SpA ligands and the effect of ligand orientation and polymerization on IgG binding was investigated with surface plasma reconsance(SPR)and isothermal titration calorimetry(ITC)in combination with batch adsorption and chromatography experiments for the development of high-capacity SpA solid phase.The SPR results showed that the ligand immobilized via additional cysteine residue at the C terminus exhibited seven-time higher affinity than the ligand immobilized via additional cysteine residue at the N terminus and 27-time higher affinity than random immobilized ligand.Hence,it was confirmed that the optimal orientation of the ligand was obtained by C-terminal oriented immobilization.In this work,three multimeric Z domains including dimer(2Z),tetramer(4Z)and hexamer(6Z)were synthesized for ITC measurement of the binding with IgG.The ITC results showed that domain Z bound with antibody with binding stoichiometry of 0.840,close to theoretical binding stoichiometry of Z domain and IgG.The similar data was also observed in the binding of 2Z and IgG.In 4Z and 6Z binding to IgG,smaller binding stoichiometry of 0.404 and 0.325 were obtained,indicating that each 4Z and 6Z molecules bound with 2.1 and 2.5 IgG molecules.It provided a clue for the optimal SpA ligands.In this work,SpA adsorbents with various types and densities of SpA ligand were synthesized to evaluate the adsorption behavior of IgG via batch adsorption and chromatography experiments.The adsorption equilibria showed that SpA adsorbents with oriented SpA ligand had higher adsorption capacity for IgG than SpA adsorbents with random immobilized SpA ligand.With an increase of ligand polymerization,difference in adsorption capacity for IgG between SpA adsorbents with oriented and random SpA ligands tended to be small.In SpA adsorbent with oriented 4Z ligand,adsorption and binding capacities for IgG increased with ligand density to a maximum value,and then decreased with a further increase of ligand density.At a ligand density of 20.2 mg/mL,the maximum adsorption and binding capacities were obtained to be 118.0 mg/g wet gel and 46.7 mg/mL,respectively.The research provides a guidance for the design and development of novel SpA chromatography.