17
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Figure 4: Stability test of immobilised rSPA silica monolith against 10.000 pH shifts using gammanorm IgG (1 mg/mL) as control for column performance. Chromatographic conditions: Stepwise gradient: 100 mM sodium phosphate pH 7.4/100 mM sodium phosphate pH 2.5; 0.05 min 100/0, 0.05-0.06 min 0/100, 1.10 min 0/100, 1.10-1.15 100/0, 1.15-2.00 min 100/0; flow rate: 2.0 mL/min; detection: 280 nm; injection: 10 µL; temperature: 25°C
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A linkage were extensively investigated by more than 5,000 runs including 10,000 pH shifts which was corresponding to more than 53,000 CV applied to the column.
The results of IgG control are shown in Figure 4. Immobilised rSPA silica monolith provided constant analysis of IgG during the complete stability test remaining retention time, peak area and peak width nearly unchanged. The RSD of IgG retention time was smaller than 0.5% whereas for the peak area of eluted IgG the RSD was below 1.1% indicating no loss of binding capacity.
Conclusions
Recombinant protein A was covalently attached to the surface of silica monoliths with larger mesopores suited for biomolecules. The immobilised rSPA column was successfully utilised in the separation of monoclonal antibodies by affinity chromatography. The applied method for immobilisation yielded to a high dynamic binding capacity leading to broad range of applicable antibody concentrations. Furthermore, stability test revealed a strong linkage between silica monolith and rSPA ligand due to the constant analysis of monoclonal antibodies. In conclusion, silica monoliths with bimodal pore structure immobilised with rSPA ligand are ideally suited for the chromatographic separation of monoclonal antibodies.
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