Mabselect sure resin

IgG4 (cB72.3) was prepared, isolated, and concentrated to 10 mg mL^–1 as described previously.^{20 (link),26 (link),27 (link)} MabSelect SuRe resin (Cytiva, UK) was analyzed
in this study. Used MabSelect SuRe samples, harvested from the inlet
and the outlet of an AXIChrom 981 mL column after 25 cycles of purification,
were donated by GSK Biopharm Process Research as described previously.^{27 (link)} A MediaScout ResiQuot (ATOLL, Weingarten, Germany)
was used to pack and equilibrate 20.8 μL (V_resin) of resin into the wells of a 96-well Supor filter
plate with a pore size of 0.45 μm. Purified IgG4 was diluted
with phosphate buffer (pH 7.4, 50 mM PBS, 150 mM NaCl) to prepare
a range of concentrations (C_O) of 1, 2,
3, 4, 5, 6, 7, and 9 mg mL^–1. Aliquots (200 μL, V_sample) of IgG4 solution at each concentration
were individually added to packed resin samples in filter plates which
were then mixed for 45 min at 1000 rpm at ambient temperature. The
filter plate was centrifuged at 493 g for 2 min to
remove the excess unbound IgG4 (C_eq) into
individual wells of a 96-well plate. The amount of excess unbound
IgG4 which flowed through at each loaded concentration was determined
on a nanodrop lite at OD₂₈₀ nm with E^1% = 13.7. The concentration of unbound mAb in the flow through
was used to determine mAb binding capacity (Q) of
the resin using the mass transfer equation^{38 (link)} given below (eq 1).

The antibodies were captured using preparative protein A chromatography with a conventional chromatographic workstation ÄKTA Pure 25 coupled with a fractionating module (Cytiva). The equilibration, wash 1, wash 2, and elution buffers were 50 mM phosphate buffer, pH 7.4; 50 mM phosphate buffer, pH 5.0; 50 mM phosphate buffer and 1 M NaCl, pH 7.4 as well as 100 mM Glycine buffer pH 3.5. Protein A MabSelect Sure resin (Cytiva) was packed in a HiScale 26 housing (Cytiva) resulting in a column volume of 24.4 ml. For purification, the column was equilibrated with 10 CV equilibration buffer and loaded with 450 ml of culture supernatant. Washes were performed with 10 CV of wash buffer 1, and 10 CV of wash buffer 2. The product was eluted with a linear gradient of 5 CV to the elution buffer and an additional 5 CV of elution buffer. The sample was collected using the fractionating system connected to the ÄKTA and was neutralized to pH 7. The process was monitored by the conductivity, pH, and absorbance at 280 nm through the Unicorn 7.0 Software (Cytiva).

For parallel production of recombinant mAbs, we used approaches designated as ‘micro-scale’ or ‘midi-scale’ (26 (link)). For ‘micro-scale’ mAbs expression, we performed transfection (~1 mL per antibody) of CHO cell cultures using a protocol for deep 96-well blocks (Thermo Fisher Scientific), as we previously described (26 (link)). For high-throughput micro-scale mAb purification, clarified culture supernatants were incubated with MabSelect SuRe resin (Cytiva), washed with PBS, eluted, buffer-exchanged into PBS using Zeba Spin Desalting Plates (Thermo Fisher Scientific) and stored at 4°C until use. For ‘midi-scale’ mAbs expression, we performed transfection (~35 mL per antibody) of CHO cell cultures as described by the vendor. MAbs were purified form culture supernatants using HiTrap MabSelect SuRe columns (Cytiva). Purified mAbs were buffer-exchanged into PBS, concentrated using Amicon Ultra-4 50 KDa Centrifugal Filter Units (Millipore Sigma) and stored at 4°C until use. To quantify purified mAbs, absorption at 280 nm (A₂₈₀) was measured using a NanoDrop (Thermo Fisher Scientific).