Mabselect column

2G4 Fab was produced from 2G4 IgG (RSR Ltd) using papain from Papaya latex (Sigma-Aldrich) using an IgG:papain ratio of 50:1 w/w (Horimoto et al. 1992 (link)). Digestion reactions were incubated at 37°C in PBS (8.1 mmol/L Na₂HPO₄, 1.5 mmol/L KH₂PO₄, 2.7 mmol/L KCl, 137 mmol/L NaCl pH 7.4) plus l-cysteine (8.25 mmol/L) and ethylenediaminetetraacetic acid (1.65 mmol/L) for 4 h with occasional mixing by inversion. The reactions were stopped by the addition of iodoacetamide (to 47 mmol/L) and incubation at room temperature for 30 min.
4F5 Fab was produced from 4F5 IgG (RSR Ltd) by papain digestion with 30:1 w/w IgG:papain ratio as described for 2G4 IgG earlier (Hendry 2001a , Hendry et al. 2001b (link)).
Digested 2G4 IgG or 4F5 IgG mixtures were purified on a MabSelect column (Cytiva, Little Chalfont, UK) in 150 mmol/L sodium chloride, 1 mol/L glycine, pH 8.6. The non-binding fractions were collected as the Fab pool and the concentrations were determined from the absorbance at 280 nm using an extinction coefficient of 1.333 mol mg⁻¹ cm⁻¹ as determined by ProtParam.
The 2G4 Fab and 4F5 Fab were analysed by SDS-PAGE (Laemmli 1970 (link)) and by analytical SEC.

Supernatant was thawed overnight at 4 °C, centrifuged, filtered, and loaded on a MabSelect column (Cytiva). 20 mM sodium phosphate and 150 mM NaCl (pH 7.2) was used for equilibration, and washing of the column and elution was performed with 0.1 M sodium citrate (pH 3). Elution fractions were immediately neutralized by 1 M Tris (pH 9) using 1/5 volume of neutralization solution for 1 volume of elution fraction. Fractions of interest were pooled and loaded on a HiPrep 26/10 desalting column for buffer exchange to Dulbecco’s PBS. Protein fractions were sterile-filtered and concentrated by centrifugal filtration using an Amicon® Ultra-15 centrifugal filter unit (30 kDa NMWL). The final concentration was determined by measuring the absorbance at 280 nm on a Nanodrop2000 instrument. Purity was checked by SDS-PAGE. The purified protein was stored at −80 °C.