CEInifinite icIEF-MS online coupling platform

In the development and production of protein biologics, charge isomers of the target protein can arise due to Post-Translational Modifications (PTMs) and degradation. Biopharmaceutical groups rely on Capillary IsoElectric Focusing (cIEF) and imaged cIEF (icIEF) to quantify the charge isomers present. Characterizing biological charge isomers is useful for many life cycle stages including cell line selection, stability studies, formulation, and processing control but this characterization requires the proteins to be separated into individual charge fractions with enough purity for downstream assays.

Further, the direct coupling of icIEF to a Mass Spectrometer (MS) for characterization would streamline the characterization process significantly. 

AES has developed the CEInfinite preparative icIEF system and proprietary Whole Column Image Detection (WCID) separation cartridges for fractionation and online coupling of icIEF to MS. WCID allows for real-time observation of the separation focusing (icIEF) as well as the subsequent mobilization process to isolate the charge isomer peaks. During pressure mobilization, an electric field keeps the samples in the separation capillary focused. The separation capillary has a larger Inner Diameter (ID) and the transfer capillary has a smaller ID. Hence, this difference greatly minimizes the remixing of separated protein isomers as they travel away from the cartridge.

CEInfinite icIEF-MS of NISTmAb

Comparable charge variant patterns were obtained from the UV-detection in icIEF (red) and the coupled MS (blue), and the excellent MS spectra were obtained for all variant peaks, which allows deconvolution of most of the intact NISTmAb charge variants.

Highlights of CEInfinite icIEF-MS

Straightforward coupling icIEF-MS with conventional low flow or high sensitivity nano flow ESI from Thermo Fisher Scientific, Waters, and Bruker.

Any buffer or solution can be used as the makeup liquid. Native or denature MS, intact or digested. No chemical mobilization is involved. Protein change variants are eluted in the sequence of their pIs.

Proved CE-based technology Method can be transferred straightforwardly from any existing icIEF or cIEF method. Good resolution can be obtained in low and high sample concentrations.

HRAM Orbitrap MS provides confident insight into subtle changes in stability

“iCIEF plays a critical role for monitoring charge variants during biopharmaceutical development. The CEInfinite allows us to get a mass readout for the product variants detected by iCIEF, and this is a great help for understanding the chemical nature of the charge variants. When combining the CEInfinite with high resolution accurate mass MS provided by Orbitrap MS systems, detailed insight into product variants and product stability can be achieved.

In short, iCIEF-MS using the CEInfinite is a very powerful characterization tool in biopharmaceutical development.”

– Dan Bach  Kristensen, Ph.D.

 Principle Scientist Symphogen