Implementation of a nanobody-liganded affinity resin for improving the production of recombinant HBsAg-VLP in large scale

Immunoaffinity chromatography using traditional antibodies as ligands has been adopted as a key step for purification of recombinant hepatitis B surface antigen (rHBsAg) but with limitations of high cost, instability, and exclusivity. In this study, a nanobody against HBsAg (named NbHB1) was employed to develop an affinity resin for rHBsAg purification. NbHB1 was expressed in E. coli as inclusion bodies, which were efficiently refolded through a two-step strategy and then directly immobilized to a SpyCatcher-derived support via the SpyTag fused to its C-terminus, resulting in an affinity resin with a static binding capacity of 2.03 mg/mL and a dynamic binding capacity of 1.43 mg/mL for rHBsAg. Using this affinity chromatography as the initial purification step, rHBsAg was efficiently purified from the clarified CHO cell culture fluid, resulting in 81 % recovery and 96 % purity. Similar recoveries were observed when scaling up from a 5 mL to a 500 mL column. The chromatographic performance was consistent over 25 purification cycles with the ligand leakage below 1.7 ng/mL resin during the repeated usage. The stability of this affinity resin was validated by various strip reagents, including 0.1 M citric acid, 20 mM sodium hydroxide, and 2 M guanidine chloride. Overall, these results demonstrate that the NbHB1-liganded affinity chromatography is robust and highly feasible, highlighting its potential to enhance the downstream process in the industrial manufacturing of rHBsAg-VLP.