Characterization of therapeutic mAb charge heterogeneity by iCIEF coupled to mass spectrometry (iCIEF–MS)

Abstract

Imaged capillary isoelectric focusing (iCIEF) has emerged as an important technique for therapeutic monoclonal antibody (mAb) charge heterogeneity analysis in the biopharmaceutical context, providing imaged detection and quantitation by UV without a mobilization step. Besides quantitation, the characterization of separated charge variants ideally directly by online electrospray ionization–mass spectrometry (ESI–MS) is crucial to ensure product quality, safety, and efficacy. Straightforward direct iCIEF–MS coupling combining high separation efficiency and quantitative results of iCIEF with the characterization power of MS enables deep characterization of mAb charge variants. A short technical setup and optimized methodical parameters (30 nl/min mobilization rate, 2%–4% ampholyte concentration, 0.5–2 mg/ml sample concentration) allow successful mAb charge variant peak assignment from iCIEF to MS. Despite a loss of separation resolution during the transfer, separated intact mAb charge variants, including deamidation as well as major and minor glycoforms even from low abundant charge variants, could be characterized by online ESI–MS with high precision. The presented setup provides a large potential for mAb charge heterogeneity characterization in biopharmaceutical applications.

Capillary electrophoresis‐mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018–March 2023)

“…Schlecht et al. [167] presented straightforward direct coupling of iCIEF–MS via the novel nanoCEasy interface to two different MS instruments providing separation and subsequent on‐line deep characterization of charge variants of two intact mAb (Figure 4). The presented setup provided a large potential for mAb charge heterogeneity characterization in biopharmaceutical applications.…”

“…Although more time-consuming and labor-intensive, off-line fractionation is not limited to the separation of analytes requiring only MS-compatible components and may be necessary to fully characterize some highly complex proteins at the intact level. The new preparative iCIEF instrument allows simultaneous isolation and characterization of individual protein charge variants and can be directly connected to MS to deliver the fractionated protein samples for characterization [167][168][169][170]. During pressure mobilization, an electric field keeps the samples in the separation capillary focused.…”

Fractionation and online mass spectrometry based on imaged capillary isoelectric focusing (icIEF) for characterizing charge heterogeneity of therapeutic antibody

“…Specically, the combination of rapid, highresolution separations by icIEF and cIEF with molecular mass identication by MS would provide a far-reaching analysis of the charge variants and their structure. Recently, icIEF or cIEF as a robust front-end separation for MS detection have been demonstrated, [31][32][33][34][35] but improvements are still required for addressing the challenges in terms of higher-sensitivity, higherthroughput and more user-friendly operation.…”

Mass Spectrometry-Based Charge Heterogeneity Characterization of Therapeutic mAbs with Imaged Capillary Isoelectric Focusing and Ion-Exchange Chromatography as Separation Techniques

“…However, the conditions commonly used in these two methods contain nonvolatile substances and usually cannot be used in conjunction with the most powerful means of mass spectrometry (MS) for protein identification. However, IEX has been developed as a front-end separation technique for MS detection, with much popularity for protein characterization. , Recently, icIEF-MS has attracted much attention to be utilized for protein charge variant analysis. − However, the icIEF-MS strategies developed still face huge challenges that need continuous optimizations, limiting the discovery of biopharmaceuticals. Critical bottlenecks of icIEF coupled to MS in the discovery of protein charged variants, including unsatisfactory repeatability, complicated operation frequently involving trial-and-error optimization, and incompatibility of the MS ion source, have been frustrating scientists in the biopharmaceutical industry.…”