Kta avant 25

Truncated soluble and Glutathione S-transferase (GST) tagged human FcRn lacking the transmembrane domain (hFcRn-GST) was produced in HEK293E cells and purified on a GSTrap column (Cytiva Life Sciences)^{67 (link)}. Truncated soluble and biotinylated human and mouse FcRn forms lacking the transmembrane domain were acquired from Immunitrack Inc. Truncated soluble His_6x tagged human FcRn lacking the transmembrane domain (hFcRn-His) was produced in a Baculovirus expression system and purified using a HisTrap HP column (Cytiva Life Sciences)^{68 (link),69} . The Baculovirus stock was a kind gift from Dr. Sally Ward (University of Southampton). Monomeric fractions of hFcRn-His were isolated by SEC using a Superdex 200 Increase 10/300 column (Cytiva Life Sciences) with an ÄKTA Avant 25 (Cytiva Life Sciences).

Rituximab (Rituxan), an anti CD20 human antibody was purchased from Chugai Pharmaceutical Co., LTD (Japan). The Fut8 gene of DG44 CHO cells (Invitrogen, USA) was disrupted using the CRISPR Cas9 vector (ORIGENE, USA) according to previously reported methods^{44 (link),45 (link)}. The cells were incubated at 37 °C with 5% CO₂ and transfected with the Cas9 vector. Cells were exposed to the screening medium containing lens culinaris agglutinin. The selected CHO clone was transfected using expression vector encoding rituximab. Afucosyl rituximab was secreted by cultivation in CD OptiCHO Medium (Cat. No. 12681011, Thermo Fisher Scientific), supplemented with 4% Glutamax (Cat. No. 35050061, Thermo Fisher Scientific), at 37 °C with 5% CO₂. Recombinant afucosyl rituximab was purified using ÄKTA avant 25 (Cytiva) equipped with a HiTrap Fibro PrismA (Cat. No. 17549855, Cytiva). Subsequently, the afucosyl rituximab was further purified by size-exclusion chromatography using a HiLoad 26/600 Superdex 200 pg column (Cytiva).

The chromatography experiments were performed at room temperature of 20°C using an ÄKTA avant 25 (Cytiva, Uppsala, Sweden). A device of 0.4 mL of volume, consisting of a cellulose-based fiber matrix with geometry and flow properties similar to the commercially available HiTrap Fibro™ PrismA was provided by Cytiva (Uppsala, Sweden) (Hardick et al., 2013 (link); Dods et al., 2015 (link); Cytiva, 2020b ). The fiber matrix is functionalized with an affinity ligand designed for purifying AAVs serotypes, including 1, 2, 3, and 5, similar to the commercially available affinity chromatography resins Capto AVB or Sepharose AVB (Cytiva, 2018 ; Cytiva, 2020a ). The device was equilibrated with 15 column volumes (CV) of 20 mM Tris-HCl (Merck KGaA, Darmstadt, Germany), 500 mM NaCl (Merck KGaA, Darmstadt, Germany), and pH 8. After sample loading, the unit was washed with 30 CV of equilibrium buffer. The AAVs were eluted with 40 CV of 100 mM Glycine (Sigma-Aldrich, Missouri, United States), pH 2.5 and neutralized with 1 M Tris-HCl (Merck KGaA, Darmstadt, Germany), pH 9.