Mabselect sure column

A BsAb was mixed in a 1:1:1 molar ratio with the two parental mAbs prior to differential protein A purification. Differential protein A purification was carried out using a 1 mL mAbSelect Sure column (GE Healthcare). The mixture was eluted in 3 steps using buffers containing either 50 mM citrate pH 4.7, pH 4.2, or pH 3.4. Elution fractions were collected and concentrated to >1 mg/mL prior to analysis. The pooled elution peaks from the differential protein A purification were analyzed by hydrophobic interaction chromatography (HIC) using a butyl NPR column (Tosoh Biosciences). Approximately 30 ug of each sample were injected onto the column and eluted using a 0 to 100% gradient of buffers containing 100 mM sodium phosphate pH 6.0, 1.5 M (NH₄)₂SO₄, or 100 mM sodium phosphate.

The recombinant mAbs were extracted by homogenizing plants in PBS (15 mM KH₂PO₄, 80 mM Na₂PO₄H, pH 6.8, 27 mM KCl and 140 mM NaCl) buffer, at a 1:1 ratio, using a blender. The extract was centrifuged (8000 x g) for two cycles at 4°C for 30 min. A 1 ml MabSelect SuRe column (GE Healthcare Life Sciences, Little Chalfont, UK) was used to capture and purify the mAbs at a 1 ml/min flow rate. The column was initially equilibrated with Tris-HCl pH 7.4 for three column volumes (CV). During purification, immobilised protein A bound the Fc region of the antibodies with high affinity at neutral pH (7.4). This was followed by washing of the column for five CVs with Tris-HCl pH 7.4. The antibody was eluted from the column at pH 3.0 (100 mM acetic acid) for 10 CVs into collection tubes that contained 1 M Tris-HCl pH 8. Chromatography was performed using an Akta Avant 150 system (GE Healthcare Life Sciences, Little Chalfont, UK). After the purification step, mAb E559 was buffer exchanged into 10 mM Na₂HPO₄ pH 6.8, 150 mM NaCl, 0.01% (w/v) Tween 80, while mAb 62-71-3 was buffer exchanged into 10 mM sodium citrate pH 6.0, 150 mM NaCl 0.01% (w/v), Tween 80 [9 (link)]. The protein concentration was determined by using the Bradford assay with bovine gamma globulin standards according to the manufacture’s guidelines (Bio-Rad, California, USA).

The four different 2C1-Fc fusion proteins were cloned and expressed by the CRO Evitria AG (Schlieren, Switzerland). For purification, 250 ml of cell culture supernatant was applied onto a Mabselect SuRe column (GE Healthcare) using an ÄKTA purifier system (GE Healthcare). The column was washed with 15 column volumes of PBS, pH 7.4, and the protein was eluted using 0.1 m glycine, pH 2.8, collecting 1-ml fractions. After elution, pH was adjusted with 1 m Tris, pH 9. The OD of the fractions was determined using an Infinite M200 pro reader and a NanoQuant plate^TM (Tecan). The two fractions showing the highest OD were loaded onto an ÄKTA purifier. Preparative size exclusion was performed using a Superdex 200 10/300 GL column (GE Healthcare), and the storage buffer was exchanged to phosphate-buffered saline (PBS, pH 7.4, Invitrogen). The four highest OD fractions were combined and used for further studies. Protein size, purity, and aggregation state was analyzed using an ÄKTA purifier system (GE Healthcare).

Fab and IgG proteins were purified from culture supernatants using affinity chromatography. Supernatants containing Fab were passed over a HiTrap Protein G column (GE Healthcare, Buckinghamshire, UK) and supernatants containing IgG were passed over a MabSelect™ SuRe™ column (GE Healthcare). Following a washing step with phosphate buffered saline (PBS) (pH 7.4), the bound material was eluted with 0.1 M glycine (pH 3.2) and neutralized with 2 m Tris-HCl (pH 8.5). Fractions containing Fab or IgG were pooled, quantified by absorbance at 280 nm, and concentrated using Amicon Ultra centrifugal filters (Merck Millipore, Massachusetts, USA). To isolate the monomeric fractions of Fab and IgG, we used size-exclusion chromatography over a HiLoad 16/60, Superdex 200 column (GE Healthcare) equilibrated with PBS (pH 7.4). Fractions containing monomeric Fab or IgG were pooled, quantified, concentrated, and stored at 4°C.

The anti-TM4SF5 chimeric monoclonal antibody Ab27 and its humanized monoclonal antibody Ab27-hz9 were produced using the Expi293 or the ExpiCHO Expression system (Thermo Fisher Scientific, Waltham, MA, USA) and purified using protein A affinity chromatography with MabSelect SuRe Column (GE Healthcare, Uppsala, Sweden) or as previously described.¹⁴ (link)

Human GRIN1 (D23-Q393) fused to the Fc portion of human IgG1 was cloned into a vector derived from pAcGP67A (BD biosciences) that included a gp67 signal sequence for protein secretion in insect cells. hGRIN1-Fc was expressed for 72 h in Sf9 cells at a multiplicity of infection of 0.5. It was affinity purified using a MabSelect Sure column (GE Healthcare), and subsequent purification was done by size-exclusion chromatography with a Superdex 200 column (GE Healthcare) according to the manufacturer’s instructions^{41 (link)}.