14 May / June 2021

High-Efficiency Preparative Imaged Capillary Isoelectric Focusing (iCIEF) and iCIEF-MS Protein Charge Variant Characterisation

by Tong Chen1 1

2 3 , Teresa Kwok1 , Anne Rose Esmin2 , Victor Li1 , Gerard Rozing3 , Tiemin Huang1 *

Advanced Electrophoresis Solutions Ltd, 1600 Industrial Road, Cambridge, ON, N3H 4W5, Canada McMaster University, Hamilton, ON, L8S 4L8, Canada

ROZING.COM Consulting, Karlsruhe, Germany Corresponding author: *

Imaged capillary isoelectric focusing (iCIEF) is a high throughput, highly efficient separation technique in routine use by many biopharmaceutical companies for the determination of the charge heterogeneity of proteins. Various charge variants detected in the sample limit the application of iCIEF as a powerful tool in multi-attribute methods. Characterisation of these variants is required

In this report, high-efficiency protein charge variant fractionation characterisation with a newly developed preparative iCIEF will be illustrated. High-efficiency iCIEF charge variant peak separation and collection, followed by mass spectrometry (MS) characterisation or online iCIEF-MS with the National Institute of standards and Technology Humanized IgG1κ Monoclonal Antibody (NISTmAb) will be demonstrated.


capillary isoelectric focusing, CIEF; critical quality attribute, CQA; imaged capillary isoelectric focusing, iCIEF; post-translational modification, PTM; multi attribute method, MAM; ion exchange chromatography, IEC; whole column image detection, WCID; electro-osmotic flow, EOF.


Analysing charge variants of therapeutic proteins is essential for the characterising and monitoring of critical quality attributes such as physicochemical and immunochemical properties, biological activity, and quantity during development and manufacturing. Protein charge variants arise by deamidation, formation of N-terminal pyroglutamate, aggregation, isomerisation, sialylation, antibody fragmentation, and glycation of lysine residues. These changes affect target binding, biological activity, patient safety, and shelf life [1-3].

Imaged capillary isoelectric focusing (iCIEF) has become an indispensable tool in therapeutic protein development and manufacturing because of its high analytical throughput, ease of use, fast method development, and excellent reproducibility. Compared to other charge-based separation techniques, iCIEF can usually separate

protein charge variants in a fraction of the time required in contrast to a CIEF method on a conventional capillary electrophoresis instrument.

There is a tremendous and urgent need for the identification of these protein charge variants. Collection and subsequent characterisation of individual variants in a protein sample are critical for understanding the charge distribution’s nature on a molecular level [3,4]. The origin, stability and biological activity of charge variant is crucial during protein therapeutic discovery, formulation, stability study, regulatory agency approval, production, and quality control. This has been a challenge until the introduction of preparative iCIEF.

A generic iCIEF platform has been developed, CEInfinite (AES Ltd, Canada), on which methods for different protein samples are readily developed [2,3].

The amount and purity of charge variant isomers collected by the traditional slab gel IEF method is usually insufficient for further characterisation [3]. Fractionation and collection of sample zones in CIEF is unpractical on conventional CE instruments and commercially not available.

Nowadays, IEC is exploited for protein charge variant fractionation. Although both IEC and IEF are charge-based protein separation

methods, different mechanisms are involved. As a result, protein charge variants may not correlate well between IEC to the IEF. Since IEC is a low-resolution separation, unsatisfactory purity and high salt content are usually expected of the IEC’s fractions. [3].

With the introduction and commercialisation of a preparative system iCIEF system, CEInfinite, shown in Figure 1, highly efficient fraction collection of protein charge variant according to protein iCIEF profile in µg to low mg amounts has become possible. Existing iCIEF methods can be easily transferred to prep iCIEF with minimal method development. With a patented 320 µm prep iCIEF cartridge [4], charge variants can be collected and can be used for intact MS analysis and peptide mapping characterisation in one day. The highly efficient prep iCIEF has been applied in therapeutic protein discovery, formulation study, new drug application, and routine MAM protein characterisation. The outstanding advantages of iCIEF in terms of simple method development, high-resolution separation, and robustness are powered by the capability of straightforward and high- efficiency capillary-based prep iCIEF.

MAM is a highly sought-after tool in biopharma for some time [5]. As a high- resolution charge-based separation and

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