Bispecific antibodies (bsAbs) are a promising area of study in next-generation antibody therapeutics, due to their wide diversity of formats and exclusive dual binding functions. bsAbs are categorized into multiple asymmetrical subclasses and have the potential to discriminate unwanted homodimers from the active heterodimers through electrophoresis. Recently, we have used the ProteinEXact™ assay on the LabChip® GXII Touch™ Protein Characterization System to study bsAbs:
- Molecular weight
- Percent purity
- Charge variants
Using Microchip Capillary Electrophoresis Separation to Distinguish bsAbs Forms
An abbreviated protocol can be seen below. To see the fully detailed protocol, see our app note here.
- Dimetric forms were chosen (Figure 1) as model molecules
- Clonal cell lines, which stably express the model molecules, were generated through the Selexis® SUREtechnology™ platform
- All experiments were performed on the LabChip® GX II Touch™ HT protein characterization system using microchip capillary electrophoresis-based separation
- Conventional SDS-PAGE was performed as a standard control for the experiments
Figure 1: Case study bsAb format and derivative forms. Schematic representation of the heterodimer KiH-Fc-Fab/scFv bispecific antibody format used in this study (left panel, AB), the derivatives homodimers (left panel, AA and BB).
LabChip® GX II Touch™ System Successfully Found the Size and Concentration of bsAbs
The ProteinEXact™ assay, run on the LabChip® GX II Touch™ HT protein characterization system, successfully identified specific bsAb forms as compared to traditional SDS-PAGE analysis (Figure 2).
Figure 2: Comparative bsAb separation analysis using different molecular weight sizing methods. (A) Virtual LabChip® GX II Touch™ gel from μCE-SDS and (B) Traditional SDS-PAGE. Although a 20-30 kDa size shift can be seen in the LabChip® GX II Touch™ gel, this is most likely due to the complex molecular interactions between protein molecules and the electro kinetics in the micro channels. A more detailed description of this interaction can be found in our app note.
The ProteinEXact™ assay was fully characterized for the concentration of bsAbs (Figure 3).
Figure 3: Linearity of the ProteinEXact™ assay for bsAb quantification. Shown in red, the concentration of each form of the undiluted bsAb1 sample, experimentally determined by the assay in accordance to an internal IgG standard. Theoretical concentrations were obtained by considering subsequent 1:2 serial dilutions of the sample.
LabChip® GX II Touch™ System for High-Throughput bsAb Quantification and Characterization
The ProteinEXact™ assay, combined with the LabChip® GX II Touch™ HT protein characterization system creates an efficient system to rigorously analyze asymmetrical bsAbs based on molecular weight sizing.