Hitrap mab select xtra

The mAbs studied in this paper, S309, AZD8895, AZD1061, and the AZD7442 cocktail have been described previously^{9 (link),18 ,20 (link)}.
S309-LS and S309-GRLR were produced in ExpiCHO-S cells and affinity-purified using HiTrap Protein A columns (GE Healthcare, HiTrap mAb select Xtra #28-4082-61) followed by buffer exchange to histidine buffer (20 mM histidine, 8% sucrose, pH 6.0) using HiPrep 26/10 desalting columns. The final products were sterilized after passage through 0.22 μm filters and stored at 4 °C. VIR-7831 (clinical lead variant of S309-LS) was produced at WuXi Biologics.
AZD8895 and AZD1061 mAbs were cloned into mammalian expression vectors and expressed as IgG1 constructs with the TM (L234F/L235E/P331S) Fc modification with or without a second YTE (M252Y/S254T/T256E) modification to extend half-life in humans. MAbs were expressed in 293 F cells after transfection with 293fectin (Thermo Fisher Scientific) and isolated from supernatants by affinity chromatography using Protein A or Protein G columns (GE Healthcare). MAbs were eluted with 0.1 M glycine at low pH and dialyzed into PBS.

The mAbs studied in this paper (COV2-2196, COV2-2130, S309, S2E12, 2B04, 47D11, REGN10933, REGN10987, LY-CoV555, and CB6) have been described previously^{6 (link),8 (link),10 (link),44 ,48 -51 (link)}. COV2-2196 and COV2-2130 mAbs were produced after transient transfection using the Gibco ExpiCHO Expression System (ThermoFisher Scientific) following the manufacturer’s protocol. Culture supernatants were purified using HiTrap MabSelect SuRe columns (Cytiva, formerly GE Healthcare Life Sciences) on an AKTA Pure chromatographer (GE Healthcare Life Sciences). Purified mAbs were buffer-exchanged into PBS, concentrated using Amicon Ultra-4 50-kDa centrifugal filter units (Millipore Sigma) and stored at −80 °C until use. Purified mAbs were tested for endotoxin levels (found to be less than 30 EU per mg IgG). Endotoxin testing was performed using the PTS201F cartridge (Charles River), with a sensitivity range from 10 to 0.1 EU per mL, and an Endosafe Nexgen-MCS instrument (Charles River). S309, S2E12, REGN10933, REGN10987, CB6, and LY-CoV555 mAb proteins were produced in CHOEXPI cells and affinity purified using HiTrap Protein A columns (GE Healthcare, HiTrap mAb select Xtra #28-4082-61). Purified mAbs were suspended into 20 mM histidine, 8% sucrose, pH 6.0. The final products were sterilized by filtration through 0.22μm filters and stored at 4°C.

Protein A chromatography (HPLC Agilent 1200, Agilent Technologies UK, West Lothian, UK, fitted with a 1 mL HiTrap MabSelect^® Xtra, GE Healthcare Life Sciences, Buckinghamshire, UK) was used with 0.1 M pH 7.3 PBS buffer for loading and equilibration. Sample preparation involved centrifugation of 1.0 mL broth (24,200 RCF for 10 min) for extracellular dAb. For intracellular dAb the pellet was resuspended to 1.0 mL final volume with 50 mM Tris pH 8, subjected to 4 freeze‐thaw cycles (freezing in dry ice followed by incubation in a dry bath for 5 min at 37°C) and 2 freeze‐sonication cycles (freezing in dry ice followed by sonication for 15 min) using a sonication bath (Camsonix C275, Elma Electronic GmbH, Singen, Germany). The sonicated samples were centrifuged at 24,200 RCF for 10 min and the cell lysate recovered and tested for titre as described above. Samples were diluted in a defined fashion in equilibration buffer to a concentration of ∼0.1 mg/mL, filtered using a 0.22 µm PVDF syringe filter and then placed on a cooled auto‐sampler (4°C) for the duration of the analysis cycle. Elution was performed using a 13 mM HCl buffer at pH 1.9 with product eluted recorded at 280 nm. Calibration was performed using standard solutions of pure dAb (GSK, Stevenage, UK).