4 May / June 2021

CZE: Ultrafast Charge Variant Analysis for Next-Generation Antibody Therapeutics

by Andras Guttman, Senior Staff Scientist, SCIEX, Brea, CA 92821

Next-generation antibodies such as multi-specifics, bi-specific T-cell engagers, peptibodies and nanobodies while offering greater therapeutic potential are more complex and diverse than traditional monoclonal antibodies. These new entities present significant analytical challenges, including the need to distinguish numerous structural and charge heterogeneities. Capillary zone electrophoresis enables the rapid separation of charge variants with only minor structural differences.

Value and Challenges of Next-Generation Antibodies

Development of new monoclonal antibody (mAb) drugs continues apace, but their limitations - monospecific binding sites and large size - has led researchers to explore next-generation antibody-based therapeutics that overcome these issues. Bi-specific and multi-specific mAbs, bi-specific T-cell engagers, peptibodies and nanobodies fulfill the requirements as some of them have multiple recognition sites, use only specific fragments of conventional antibodies thus providing greater in vivo stability, access to more targets and greater efficacy via multiple target binding.

Some of these next-generation antibodies are similar to but more complex than immunoglobulin (IgG)-like mAbs with multiple Fab regions and one Fc region, such as bi- and tri-specific antibodies. Fusion proteins (single-chain variable fragments or scFvs), nanobodies, bispecific T-cell engagers (BiTEs), bi- and tri-specific killer-cell engagers (BiKes/ TriKEs), and antibody fragments (Fab, F(ab)2) are non-IgG-like because they do not include an Fc region. The greater complexity of these next generation mAbs creates significant analytical challenges. Because they contain more components, numerous different but structurally similar variants are produced (see Figure 1), many of which may have the potential to impact safety and efficacy. These variants may arise as the result of deamidation, methionine oxidation, C-terminal lysine addition, N-terminal pyroglutamate formation and glycosylation, among other mechanisms [1-3].

Importance of Charge Heterogeneity Analysis

Charge heterogeneity analysis of next- generation antibodies is consequently of importance during product development, production, stability and release testing [4]. Analysis of charge variants is also important

Figure 2: CZE Mode of Separation (left panel) and its efficiency compared to HPLC (right panel).

Figure 1: PTMs and Degradation Hotspots of Therapeutic Immunoglobulin Molecules (IgG) are Important Quality Attributes.

during forced degradation studies for quality assessment [1]. Given that as more than a dozen variants can be generated in any one batch, only perhaps two of which will be therapeutically relevant, it is essential that any method for charge heterogeneity analysis be not only high resolution, but also rapid, accurate and reproducible. Traditional methods for protein charge variant analysis include ion exchange chromatography [5] and various forms of isoelectric focusing [6,7]. However, both chromatographic and IEF methods are relatively slow, requiring up to 1 hour of analysis time.

Advantages of Capillary Zone Electrophoresis

Capillary zone electrophoresis (CZE) has been used for charge heterogeneity analysis

of protein therapeutics for more than a decade [8]. CZE operates by an entirely different mode of action than HPLC, i.e., separating analytes strictly based on the differential electromigration of the sample components based on their hydrodynamic volume to charge ratio (Figure 2, left panel).

With its non-laminar plug type flow profile, the analytes travel into narrow zones, and with no use of a stationary phase (e.g., no carryover), this method can be readily optimised for high-resolution separation of both large and small molecules with a wide range of chemical and physical properties (hydrophilic, hydrophobic, polar, non-polar, and charged) In fact, CZE offers plate numbers approximately several dozen times greater than that of high- performance liquid chromatography (HPLC) (Figure 2, right panel). In addition, CZE

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