A novel microchip‐based imaged CIEF‐MS system for comprehensive characterization and identification of biopharmaceutical charge variants

Abstract

A microfluidic system has been designed that integrates both imaged capillary isoelectric focusing (iCIEF) separations and downstream MS detection into a single assay. Along with the construction of novel instrumentation and an innovative microfluidic chip, conversion to MS‐compatible separation reagents has also been established. Incorporation of 280 nm absorbance iCIEF‐MS analysis not only permits photometric quantitation of separated charge isoforms but also facilitates the direct monitoring of analyte focusing and mobilization in real‐time. The outcome of this effort is a device with the unique ability to allow for both the characterization and identification of protein charge and mass isoforms in under 15 min. Acquisition, quantitation, and identification of highly resolved intact mAb charge isoforms along with their critical N‐linked glycan pairs clearly demonstrate analytical utility of our innovative system. In total, 33 separate molecular features were characterized by the iCIEF‐MS system representing a dramatic increase in the ability to monitor multiple intact mAb critical quality attributes in a single comprehensive assay. Unlike previously reported CIEF‐MS results, relatively high ampholyte concentrations, of up to 4% v/v, were employed without impacting MS sensitivity, observed to be on the order of 1% composition.

Combination of strong anion exchange liquid chromatography with microchip capillary electrophoresis sodium dodecyl sulfate for rapid two-dimensional separations of complex protein mixtures

“…These approaches are currently limited to regular CE-SDS and do not include chip-based formats. However, recent progresses in microchip-based CIEF-MS [ 49 ] raise hope that similar successes may be achieved with MCE-SDS.…”

“…Commonly, the peak identity is confirmed by mass spectrometry (MS) [4,[48][49][50]. MCE-SDS and CE-SDS in general are considered as incompatible with MS due to the intrinsic properties of the employed gel matrix.…”

Applications of capillary electrophoresis for biopharmaceutical product characterization

“…The results obtained correlated with standard icIEF-UV profiles. In addition, Mack et al also showed charge variant analysis of mAbs by coupling microchip icIEF with MS [41]. In a comparison between cIEF and CZE, cIEF offered superior performance for pI analysis, while CZE was better for routine and rapid charge heterogeneity analysis [42].…”

“…Microfluidic CE-based methods have also been reported for charge variant analysis of mAb products [41,45,46] Online ESI-MS was demonstrated for intact mAb charge heterogeneity analysis with major C-terminal lysine variants separated with an average resolution of 0.8 along with the minor acidic and basic species. The study also identified the glycosylation patterns of the 0-lysine, 1-lysine, and 2-lysine variants [45].…”

A Mini Review on Capillary Isoelectric Focusing-Mass Spectrometry for Top-Down Proteomics

“…Developing microchip-based cIEF-MS is also very attractive to pharmaceutical industry. Recent microchip-based cIEF-MS system developed by Mack et al provided real-time optical monitoring of focusing and mobilization process of cIEF, good resolving power and high throughput (15 min each assay) for characterization of mAb charge variants (Mack et al, 2019). Besides a direct coupling of cIEF and ESI-MS, cIEF has also been coupled to CZE-MS using a mechanical valve or a nanoliter valve for high-resolution characterization of intact proteins and mAb charge variants (Hühner et al, 2017;Montealegre and Neusüß, 2018).…”