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

Abstract

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.

Online/at‐line measurement, analysis and control of product titer and critical product quality attributes (CQAs) during process development

“…The inherent variability of Protein A titer assays is an ongoing challenge encountered across the industry. As reported by Satzer and Jungbauer (2018), the relative standard deviation of a Protein A titer assay is 20% for lower limit of quantitation, and 15% for the entire concentration range. Given the nature of the intra‐assay variability of Protein A titer assays, the observed inter‐assay variability between the three modes of analysis is within the inherent variability of the method.…”

“…exhibits the comparability of batch bioreactor titer results from different modes of analysis, including online, at-line, and offline.The apparent differences between the three modes of analysis are within the variability of titer measurements using the Protein A method.The inherent variability of Protein A titer assays is an ongoing challenge encountered across the industry. As reported bySatzer and Jungbauer (2018), the relative standard deviation of a Protein A titer assay is 20% for lower limit of quantitation, and 15% for the entire concentration range. Given the nature of the intra-assay variability of Protein A titer assays, the observed inter-assay variability between the three modes of analysis is within the inherent variability of the method.…”

Osmolality is a predictor for model‐based real time monitoring of concentration in protein chromatography

“…A compromise must be made in order to keep the number of experiments to a reasonable number. A cv of 0.03 would represent a method with high precision, such as high‐performance liquid chromatography (HPLC), and a cv of 0.15 would represent a method such as enzyme‐linked immunosorbent assay (ELISA).…”

Combined protein A imprinting and cryogelation for production of spherical affinity material

“…The target molecule in this study was protein A, which is a surface protein on the cell wall of Staphylococcus aureus . This bacterial protein is the most important ligand used in commercial activities for the purification of monoclonal antibodies (Palombo, Falco, Tortora, Cassani, & Fassina, ; Alkan, Bereli, Baysal, & Denizli, ; Satzer & Jungbauer, ; Tustian, Endicott, Adams, Mattila, & Bak, ; Salimi, Usta, Koçer, Çelik, & Tuncel, ; Pan, Xue, Wang, Xie, & Wu, ; Xue, Pan, Xie, Wang, & Zhang, ). Since publication of the first reports of the use of protein A as a stationary phase to purify antibodies more than 40 years ago, protein A has become the industry standard for monoclonal antibody purification for clinical use (Brechmann et al, ; Butcher et al, ).…”