Non-affinity purification of a nanobody by void-exclusion anion exchange chromatography and multimodal weak cation exchange chromatography

Multimodal chromatography resins have been reported to be excellent alternatives to classical absorbents in the purification of antibodies. In this study, void-exclusion anion exchange chromatography (VEAX) and electropositive multimodal chromatography (Capto MMC) were integrated for tag-free nanobody (2D5) purification. VEAX was systematically characterized for its potential use in the initial 2D5 separation. VEAX achieved a 89.9% reduction in host cell proteins (HCPs), a 99.6% reduction in DNA, a 99.9% reduction in endotoxins, and a 100% reduction in aggregates from periplasmic proteins, with a 2D5 recovery of near 100% and a purity enhancement from 3.5% to 25.0%. Subsequently, Capto MMC chromatography was seamlessly incorporated with direct protein loading for further purification. The recovery of this step was 80.5%. The entire two-step purification platform yielded 2D5 with 16 ppm of HCPs, 127 ppb of DNA, 35 EU/mL of endotoxins, with a total protein recovery of 80.5%. The purity of 2D5 was 98.5%. Moreover, this method had no effect on the affinity of 2D5. Meanwhile, the established method was also used to purify a nanobody against PD-L1 (KPU) with 95.8% purity, >99% recovery, 55 ppm of HCPs, 289 ppb of DNA and 128 EU/mL of endotoxins. For the first time, this study reports a simplified, seamless and non-affinity purification platform for the purification of a tag-free nanobody, which provides a competitive alternative for the conventional affinity chromatography antibody purification strategy.