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

• A simplified purification platform for a tag-free nanobody was established. • VEAX and Capto MMC were integrated for purification. • The platform achieved a tag-free nanobody with 99.1% purity and 86.8% recovery. • The platform had no effect on the affinity of the tag-free nanobody. 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. [ABSTRACT FROM AUTHOR]