Throughout all stages of development and manufacture, monoclonal antibodies (mAbs) exhibit a great deal of structural complexity. After translation and folding, proteins undergo post-translational modifications, as well as spontaneous and enzymatic degradation, such that a single preparation of purified mAb exhibits a range of small structural changes, composed of various glycoforms and charge variants, as well as amino acids alterations due to oxidation, deamidation, isomerization, or other chemical reactions. This display of structural heterogeneity can influence the overall stability, efficacy, and safety profile; therefore, understanding the extent of structural modifications has become extremely important to drug manufacturers who continually assess mAb composition throughout bioprocessing to demonstrate stability, batch-to-batch consistency, and long-term shelf life.

To aid in this task, a SCIEX Separations research team has developed a straightforward approach to characterize mAb structural variants without the need for lengthy or multiple analyses.¹  Using capillary electrophoresis-electrospray ionization (CESI)—where capillary electrophoresis (CE) and electrospray ionization (ESI) are integrated into a single device, the CESI 8000 Plus System (Figure 1)—scientists were able to rapidly separate various structural variants of IgG1, IgG2, and IgG4, producing multiple, distinct peaks that were used to characterize the charge heterogeneity, purity, and the molecular weight of each. Furthermore, researchers coupled the CESI separation method with an accurate-mass, high-resolution mass spectrometer (the TripleTOF® 6600 System), which generated accurate-mass data for identification of each CESI peak, thereby permitting complete structural characterization of all variants and a more comprehensive inventory of all mAb components.

Resolving Proteins with Small Structural Differences
Traditionally, mAb characterization assays have been performed using a combination of electrophoretic separation techniques; the current, industry-accepted standard methods for obtaining critical mAb charge variant information include both capillary isoelectric focusing (cIEF) and capillary electrophoresis-sodium dodecyl sulfate (CE-SDS).^2-3 These two methods provide profiles of charge variants and molecular weight analysis, respectively, but they rely on spectrophotometric detection methods, gathering no structural or mass information for variant identification.  A single run using CESI can replace these two techniques, providing characterization results equivalent to those obtained from using all three methods separately for mAb variant, purity, and molecular weight analyses, in addition to providing accurate-mass data for compound identification. By folding three separate analyses into one, researchers are not only able to conserve the amount of sample needed for analysis, but also they can consolidate workloads and save time.

CESI separates proteins based on their electrophoretic mobility, a characteristic dictated by the molecule’s size-to-charge ratio. Even small modifications to a protein’s sequence can change the isoelectric point (pI) of the protein, and hence its mobility during electrophoretic separations. A wide range of post-translational and degradation pathways alter intracellular protein structures in numerous ways, and these additions, deletions and alterations create various charge state “sub-populations,” whereby each mAb variant has its distinct electrostatic character.⁴ Depending on the modification, the pI of the protein will either increase or decrease. For example, deamidation and sialylation results in the increase of net negative charge, corresponding to a decrease in pI (and formation of acidic variants). Alternately, mechanisms introducing positive charges (or removing negative ones) will increase the pI and create variants that are more basic. The resolving power of CESI is high enough such that proteins with slightly different structures displaying slightly different electrophoretic mobilities will elute from the column as discrete peaks—pulling the acidic, basic, and main peak fractions away from the truncated, dimerized, or other clipped protein variants—and demonstrating the structural diversity within a single, purified mAb preparation.

Separate, Spray, and Detect with CESI-MS
To carry out high-resolution separations, CESI relies on its straightforward, yet innovative, design, whereby a single, open capillary tube is seamlessly joined with an electrospray ionization (ESI) tip (Figure 1). Molecules enter the capillary tube under ultra-low flow conditions (≥ 10 nL/min), elute based on their electrophoretic mobility, and then are ionized immediately at the terminus. A neutral coating lines the capillary interior so that even large hydrophilic or hydrophobic proteins that typically do not separate well-using solid-phase based approaches have minimal interactions with the tube for reduced sample loss and more complete coverage. The distal end of the capillary is porous, surrounded by conductive liquid to facilitate the ion flow that propels molecules towards the outlet. As proteins elute, they undergo gas phase conversion at the capillary tip via ESI, which creates a steady spray of fine droplets that ionize the analytes of interest. Under these conditions, sensitivity is heightened, and ionization suppression is decreased (Figure 2), allowing for the detection of previously unnoticed or less-abundant analytes and providing exceptionally robust analysis.

SCIEX researchers applied CESI characterization to reduced and intact forms of IgG1, IgG2, and IgG4, obtaining electrophoretic profiles of multiple peaks, each containing different clusters of mAb variants¹ (Figure 3, center). Scientists analyzed each non-reduced, intact CE-generated peak by MS, and after extraction and deconvolution of the mass data, they found multiple IgG1 charge species, including two main charge variants (basic and main), along with multiple, smaller peaks representing different glycoforms and clipped species (Figure 3). Using high-resolution, accurate-mass molecular weight information to confirm structural identities, researchers showed that many of the resulting peaks were indeed due to differing glycosylation patterns, charge variants, or clipped species (Figure 3). Evaluation of reduced IgG1 samples revealed one heavy chain and two light chain species (Figure 4); the second light chain species exhibited a higher molecular weight, which helps explain the presence of two main IgG1 charge variants. Comparisons to traditional characterization approach (cIFR, CE-SDS) indicated that CESI produced analogous results, giving equivalent charge variant elution profiles and molecular weight data, which together confirmed the applicability and functionality of CESI for comprehensive mAb variant analysis.

Spending less time getting more information
The capillary-based separations of CESI are well suited to protein analysis. Using small volumes and no solid-phase, researchers can analyze very limited amounts of samples (e.g., the small volumes typically found in the early product discovery and development phase) without loss to flow-through or irreversible column retention for fuller coverage. Additionally, the method has resolving power to separate large proteins without having to digest them into smaller peptides, eliminating the need for extensive sample prep or the introduction of artificial modifications. The very-low-flow rates combined with efficiently functioning electrospray at the emitter tip raises sensitivity, while also converting hard-to-ionize glycoproteins into the gas phase for more complete glycan-profiling within the same separation method. The characterization of unmodified, intact proteins needs to be uncomplicated and facile, and CESI’s versatility reduces complexity and generates charge variant data fast, with high resolution and excellent reproducibility.

CESI-MS analysis unifies multiple mAb characterization methods into one simple approach, delivering a more rapid and efficient way to assess mAbs charge heterogeneity, glycoforms, and impurities within the same analysis. Insights into the structural characteristics of intact mAbs are essential for safe and efficacious drug production, and any changes may signify bioprocessing problems that could potentially affect a drug’s stability and biological activity. By moving multiple characterization methods into a single, simple application, mAb manufacturers will now have access to rapid, yet powerful, screens for glycoform and charge variants, while also obtaining more comprehensive purity and molecular weight data for reduced and intact IgGs. The coupling of CESI’s high-resolution separation with accurate-mass MS not only coalesces charge variant profiles, purity, and glycoform information into one workflow, but it also definitively identifies the contents of each peak, so glycoforms, charge variants, and impurities can all be distinguished from each other. CESI-MS captures all this and redefines intact protein characterization so that researchers can spend less time getting more information. 

Written by Laura Baker, SCIEX Technical Support 

References

1. Fonslow, B. et al. “Unification of charge heterogeneity, purity, and molecular weight analyses of mAbs into a single analysis.” SCIEX Technical Note, 2015; RUO-MKT-02-2757-A.
2. Salas-Solano, O., et al. “Intercompany study to evaluate the robustness of C-IEF technology for the analysis of monoclonal antibodies.” Chromatographia, 2011; 73:1137-1144.
3. Felten, C. and O. Salas-Solano. “Capillary electrophoresis in quality control: Part II: CE-SDS: Method development and robustness.” Beckman Coulter Technical Information Bulletin, 2011; IB-16385A.
4. Khawli, L.A., et al. “Charge variants in IgG1: Isolation, characterization, in vitro binding properties and pharmacokinetics in rats.” mAbs, 2010; 2(6): 613-624/

Figures

Figure 1. Capillary electrophoresis (CE) and electrospray ionization (ESI) are integrated into a single device called CESI. The distal end of the capillary tube is porous, and ESI occurs at the emitter tip. From, Moini, M. Anal. Chem. 2007, 79(111): 4241-4246.

Figure 2. Capillary electrophoresis electrospray ionization (CESI) flow rates through the capillary tube are inversely proportional to the sensitivity of detection (excerpted from a presentation by Jeff Chapman).

Figure 3. A profile of intact, non-reduced IgG1 charge variants separated by capillary electrophoresis electrospray ionization (CESI) is shown (central). Each CESI-generated peak was analyzed by mass spectrometry (MS), and after extraction and deconvolution, the IgG charge variants were revealed and identified. The extracted ion chromatograms (XICs) presented are summed from the most abundant charge states (3-6) from the same charge state windows. Each XIC shows peaks corresponding to the IgG1 structures from each CESI peak; separated peaks have a diversity of species, whose identities are indicated within each XIC. (Panels are distributed around the periphery of the central panel.)

Figure 4. A profile of reduced IgG1 components separated by capillary electrophoresis electrospray ionization (CESI) is shown (right central). Each CESI-generated peak was analyzed by mass spectrometry (MS), and after extraction and deconvolution, the reduced IgG charge variants were revealed and identified. The extracted ion chromatograms (XICs) that are presented are summed from the most abundant charge states (3-6) from the same charge state windows. Each XIC shows peaks corresponding to the IgG1 structures from each CESI peak; separated peaks have a diversity of species, whose identities are indicated within each XIC. (XICs for reduced peaks are located above or to the sides of the central right elution profile.)