Mass Spectrometry Characterization of Protein Variants Detected by Capillary Gel Electrophoresis

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Protein-based drugs are characterized with a plentitude of different analytical approaches and methods. Each of these methods provides insight into a certain aspect of the molecule. For example, capillary Gel Electrophoresis (cGE) provides detailed information about the relative impurity content and the purity of an analyte. Especially for low molecular weight species, cGE shows high resolving power and a reproducible relative quantification.

In the past, direct characterization and identification of these species have proven to be challenging. However, it is key not just to quantify the different detected signals but also to identify if they originate from the drug substance itself or from impurity compounds out of the production process.

In this webinar, the presenters discussed an appropriate solution: combination of Hydrophilic Interaction Chromatography (HILIC)-MS and cGE. As cGE cannot be efficiently coupled to MS directly, we optimized HILIC to produce the same chromatographic peak pattern that was observed as electropherogram during cGE. The HILIC is coupled to MS for identification of the single species.

The peaks detected in the cGE electropherogram using the combination of HILIC-MS and cGE can be identified and assigned to the full-length protein, specific degradation products, or production process-related species. The webinar also featured a discussion on how fractionation enables the usage of additional methods, like peptide mapping, on the peaks of interest.

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