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J Biomol Tech. 2010 September; 21(3 Suppl): S47.
PMCID: PMC2918059

ICIEF Peak Identification Using 3100 OFFGEL Fractionator, Case Study: Basic Variants of a Thermally Stressed Fab

W. McElroy, J. Zhang, and V. Katta
Genentech, South San Francisco, CA, United States



Imaged capillary isoelectric focusing (ICIEF) is widely used in the industry and at Genentech as a platform technology to quantitatively assess the charge heterogeneity of proteins. This method can be adapted to new molecules with a short development time and provides good resolution for many antibody products. However, it has been difficult to identify the nature of each charge variant as ICIEF does not provide means to collect variants for further analysis. In this study, we demonstrate the use of an OFFGEL electrophoresis fractionator (Agilent 3100) for enriched charge variant collection. The Agilent 3100 is a pI based fractionation device that allows the isoelectic focusing of proteins or peptides in immoblized pH gradient (IPG) gel strips. The isoelectric separated components are recovered in the liquid phase and can be used for further characterization. Molecule Fab under thermal stressed conditions (4 weeks at 40°C) shows a significant decrease in main peak and increase in basic variants. The individual charge variant fractions were collected using the Agilent 3100 and these fractions show high level of enrichment of each variant. They were further characterized by intact/reduced mass analysis and LC/MS/MS peptide mapping after trypsin digestion. We found both basic variants resulted from succinimide formation at the first Asp residue in the Asp-Asp motifs. For the Fab studied, the first basic variant corresponds to the succinimide formation at Asp-Asp on the light chain, while the second basic variant mainly contains the succinimide intermediates at Asp-Asp motif on both the light and heavy chain. We also found the second basic variant contained a pyroglutamate (pyro-Glu) at the N-terminal glutamic acid of the heavy chain. This approach has provided a simple and rapid method to characterize the charge variants of proteins without the necessity of developing an ion exchange chromatography assay.

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