Hitrap mabselect sure

Capture of monoclonal antibodies from HCCF was conducted with convecdiff membrane with a membrane volume of 1.2 mL for cycling studies with different mAbs and Cytiva HiTrap MabSelect SuRe (see Section 2.2) as a comparison with regard to different critical process parameters (CPP) for one mAb using an ÄKTA Avant 150 chromatography system or Sartorius MU RCC system (Pompey, France). Chromatography was performed with buffers and chromatography recipes mentioned in Table 1 and Table 3.
The load was calculated as 80% of the DBC_10% measured at a residence time of 12 s for the membrane or as 80% of the DBC_10% measured at a 4 min residence time for the resin depending on the HCCF titer. The load density was conservatively chosen to achieve the desired number of cycles without product loss. Elution pools were collected from 100–100 mAU (using ÄKTA spectrophotometer with a 2 mm path length at λ = 280 nm). Step yield was determined using the mass of the product in the load and pool (both determined by SEC-HPLC).

Sequences for S309, C118, C135, C952, C1520, C1533, and C1596 anti-SARS-CoV-2 IgG antibodies were previously published (20 (link), 23 (link), 43 (link), 73 (link)). Heavy IgG and light chains for each antibody were cloned into the AbVec2.1 expression plasmid (gift from Dr. Pamela Bjorkman, California Institute of Technology). Fab constructs were obtained by subcloning the variable heavy (V_H) and constant heavy chain 1 (CH₁) domain from the IgG vectors into an AbVec2.1 expression plasmid with a C-terminal 6x histidine tag. For IgG and Fab expression, heavy and light chains were co-transfected in Expi293F cells using the ExpiFectamine transfection kit (ThermoFisher Scientific, A14525) in a 1:1 ratio; ExpiFectamine transfection enhancer was added 20 h post-transfection. IgGs and Fabs were isolated and purified from filtered cell supernatants via affinity chromatography using HiTrap MabSelect SuRe (Cytiva) and HisTrap HP (Cytiva) columns, respectively. Elutions were concentrated using Amicon^™ spin concentrators and further purified by size-exclusion chromatography on a HiLoad 16/600 Superdex 200 pg (Cytiva) and Superdex 200 Increase 10/300 GL (Cytiva) columns using an AKTA pure 25 M1 system (Cytiva). Proteins were concentrated via Amicon^™ spin concentrators and stored in Tris-Buffered Saline with azide (1x TBS-Az; 20 mM Tris pH 8.0, 150 mM NaCl, 0.02% Sodium Azide) at 4°C.

Abrin toxin was prepared by Biointron Biological Inc. (Beijing). CCK-8 detection kits were purchased from Dojindo (Japan). TNT^® Coupled Reticulocyte Lysate Systems were purchased from Promega (Cat. No.: L4610). Dulbecco’s modified Eagle medium (DMEM) (Cat. No.: 11965-092), RPMI 1640 media (Cat. No.: 61870-036) and fetal bovine serum (FBS) (Cat. No.: 10438-034) were purchased from ThermoFisher (U.S.). pFRT-KIgG1, a full-IgG expression plasmid fused with the constant regions of IgG1 heavy chain and kappa chain, was prepared in our lab based on the pcDNA5-FRT plasmid (ThermoFisher, Cat. No.: V601020). ExpiCHO-S cells and ExpiCHO™ Expression System (Cat. No.: A29133) were purchased from Gibco (ThermoFisher, U.S.). HiTrap MabSelect SuRe was purchased from Cytiva (U.S.). Alexa-FluorTM 488 and 594 were prepared by Jiaxuan Biotech (Beijing, China). TruStain FcXTM (anti-mouse CD16/32 and anti-Human IgG Fc receptors) were procured from Biolegend. All plastic-ware was procured from NuncTM (Denmark). All other chemicals were from commercial sources and of analytical grade.