Development of Protein A-Affinity HPLC Analysis System for the Quantification of Human Monoclonal Antibody (hMab)

Isoda Tomoko, Nakagawa Taishiro, Ishihara Takashi, Kadoya Toshihiko

doi:10.2116/bunsekikagaku.59.225

High-speed Protein A-HPLC analysis was developed for the quantification of human monoclonal antibody (hMab). A perfusion type of Protein A HPLC column (POROS 50A ; 4.6 mm i.d. × 5 cm ; particle size, 50 μm) was used, and the chromatographic conditions, such as the flow rate, salt concentration in the buffer solution, and equilibration time were examined. HPLC was performed at a flow rate of 5 mL min⁻¹ using stepwise elution from 50 mM sodium phosphate that contained 300 mM sodium chloride (pH 7.0) to the same buffer (pH 2.8), which was selected to reduce the leading of elution peaks of hMabs and the carry-over of hMabs. To minimize the time per analysis, we deleted a re-equilibration step from the method program, because the necessary volume of the equilibration buffer flowed in the column while the sample was injected by an autosampler. This enabled us to analyze 1 sample within 2 to 2.5 min (including the time required for sample injection). A column lifetime study not only showed that this deletion had no effect on repeated quantification, but also demonstrated that more than 3000 analyses can be performed with one column. We report here on the fast quantification method with a long column lifetime. This high-throughput quantification method is very useful for the process development of monoclonal antibody pharmaceuticals.

Development of Protein A-Affinity HPLC Analysis System for the Quantification of Human Monoclonal Antibody (hMab)

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