Characterization of free thiol variants of an IgG1 by reversed phase ultra high pressure liquid chromatography coupled with mass spectrometry

Journal of Pharmaceutical and Biomedical Analysis
Hongbin LiuYonghua Taylor Zhang

Abstract

RP-HPLC has been demonstrated as a powerful tool to study antibody free thiol and disulfide variants. Recently, the introduction of UHPLC columns with wide pore size (300Å) and small particle size (1.7μm) offered the opportunity to further improve the separation of such variants. This paper describes a systematic evaluation of stationary phases, operating parameters, and mobile phases for a UHPLC based method to separate free thiol variants of a recombinant monoclonal antibody (referred as mAb A), targeting high resolution, high throughput and improved recovery. Among the four different stationary phases evaluated, UHPLC diphenyl columns were found to provide the best separation. Using an optimized UHPLC method, free thiol variants of mAb A were separated in 5min. Importantly, the UHPLC method revealed minor variants that had coeluted in an HPLC based method, and the UHPLC method is also applicable as a platform method for characterization of other mAbs as well. Furthermore, an on-line UHPLC-MS method was developed to characterize the separated variants, and this method can streamline the characterization of fully assembled monoclonal and bispecific therapeutic antibodies.

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Citations

May 29, 2016·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Bing ZhangYonghua Taylor Zhang
Feb 7, 2019·Critical Reviews in Biotechnology·André GuerraJarka Glassey
Feb 29, 2020·Annual Review of Analytical Chemistry·Charles R Bupp, Mary J Wirth
Feb 23, 2021·Journal of Pharmaceutical Sciences·Michael T KimDavid D Weis
Mar 27, 2021·Analytical Biochemistry·Xiaojuan LiDouglas Richardson

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