High-capacity protein A affinity chromatography for the fast quantification of antibodies: Two-wavelength detection expands linear range.

The high-throughput analysis of antibodies from processes can be enhanced when the linear range is expanded and sample preparation is kept to a minimum. We developed a fast chromatography method based on a hexameric variant of staphylococcal protein A immobilized on Toyopearl matrix, TSK 5 PW using two wavelengths. A protocol with 5 min runtime and a single-wavelength detection at 280 nm yielded an upper limit of quantification of 2.10 mg/mL and a lower limit of quantification of 0.06 mg/mL. The optimized method with a runtime of 2 min and two-wavelength detection at 280 and 300 nm allowed us to span a valid concentration range of 0.01-5.20 mg/mL using two calibration curves. Sample selectivity was tested using mock supernatant mixed with antibody concentrations of 0.1-2.1 mg/mL, sample stability in the autosampler was shown for at least 24 h. We also tested the capabilities of the method to determine purity of an antibody sample by calculating the ratio of peak area of elution to peak area of flow-through, which correlated well with the expected purity. The method will be very useful for process development and in-process control, spanning concentrations from seed fermentation to harvest and purification.