Protein A and CaptureSelect FcXP Affinity Resins Possess the Capability of Differentiating Hole-hole Homodimer Isoforms

Knobs-into-holes (KiH) technology has been widely used in asymmetric bispecific antibody (bsAb) construction to promote heavy chain heterodimerization. However, despite the great improvement of heterodimer formation by this strategy, homodimers (especially the holehole homodimer) can still be generated at low levels. Consequently, hole-hole homodimer is a common byproduct associated with the production of KiH bsAbs. In addition, previous studies showed that hole-hole homodimer exists as two different isoforms. As the major difference between these two isoforms lies in the Fc region, we speculated that Protein A media, which bind IgG Fc region with high affinity, and CaptureSelect FcXP, a CH3 domain-specific affinity resin, may provide certain resolution between these two conformational isoforms.The objective of this study was to study the capability of Protein A and CaptureSelect FcXP affinity resins in differentiating hole-hole homodimer isoforms.The hole-hole homodimer was produced in CHO cells by expressing the hole half-antibody. The homodimer, along with the half-antibody was initially captured by Protein A chromatography and was then further purified by size-exclusion chromatography (SEC), which separated the homodimer from the unpaired half-antibody. The purified hole-hole homodimer was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analytical hydrophobic interaction chromatography (HIC). The purified hole-hole homodimer was separately processed by columns packed with Protein A and CaptureSelect FcXP resins. The purified hole-hole homodimer was also analyzed by Protein A-high-performance liquid chromatography (HPLC).SDS-PAGE analysis and analytical HIC study confirmed that hole-hole homodimer exists as two conformational isoforms. When the hole-hole homodimer was processed by Protein A and CaptureSelect FcXP chromatography, the elution profiles contained two peaks, indicating that both affinity resins possess the capability of differentiating hole-hole homodimer isoforms.Our data suggest that Protein A and CaptureSelect FcXP affinity resins both possess the capability of differentiating hole-hole homodimer isoforms and, therefore, can be used for monitoring isoform conversion under various conditions.