Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity

Abstract

To cite this article: Ram P Vanam, Michael A Schneider & Michael S Marlow (2015) Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity, mAbs, 7:6, 1118-1127, DOI: 10.1080DOI: 10. /19420862.2015 An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown.

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

“…IEF is one of the most commonly used techniques for resolving charge variants (Dada, Jaya, Valliere‐Douglass, & Salas‐Solano, ; Hazeltine et al, ; Horvath, Mun, & Laird, ; Hosken, Li, Mullappally, Co, & Zhang, ; Kaschak et al, ; Rouiller et al, ; Vanam, Schneider, & Marlow, ; Yang, Minkler, Kshirsagar, Ryll, & Huang, ; Yoon, Song, & Lee, ). IEF techniques separate molecules based on the molecules' pI and overall charge, and the detection is achieved by UV absorption at 280 nm.…”

Chromatographic, Electrophoretic, and Mass Spectrometric Methods for the Analytical Characterization of Protein Biopharmaceuticals

“…RSD values for isoelectric points and migration times were below 0.2% and 4%, respectively. Vanam et al showed that the charge heterogeneity of chromatographically isolated antibody fragments (heavy and light chains) can be resolved using analytical SEC followed by icIEF, with a significant increase in throughput compared to 2D-PAGE . Shimura et al used ciEF to determine the asparagine deamidation rates of a mouse IgG1 Fab .…”

“…Vanam et al showed that the charge heterogeneity of chromatographically isolated antibody fragments (heavy and light chains) can be resolved using analytical SEC followed by icIEF, with a significant increase in throughput compared to 2D-PAGE. 150 Shimura et al used ciEF to determine the asparagine deamidation rates of a mouse IgG1 Fab. 151 Five asparagine residues were mutated one by one through sitedirected mutagenesis, and the deamidation rates of the original Fab and the mutants were determined using ciEF.…”

Hyphenation of strong cation exchange chromatography to native mass spectrometry for high throughput online characterization of charge heterogeneity of therapeutic monoclonal antibodies

“…Capillary isoelectric focusing (cIEF) and cation exchange chromatography (CEX) are commonly used for the characterization of protein charge variants. [4][5][6][7] Using a pH gradient and electric field, cIEF separates charge variants based on the intrinsic charge of the molecule. In CEX, either salt or pH gradients have been successfully applied for the characterization of charge variants.…”