Mabselect prisma

1 mL and 5 mL of MabSelect PrismA (Cytiva) resin were packed in Tricorn™ series columns (Cytiva) with a bed height of 5.1 cm and 6.4 cm, respectively, with experiments conducted on an AKTA™ Avant 25 (Cytiva). All columns were equilibrated with 100 mM sodium phosphate, 150 mM NaCl, pH 7.2, before loading the appropriate amount of CCS. A 3 CV wash of 50 mM Na-citrate, pH 6.0 was performed after loading of CCS for all experiments. All elution buffers contain 50 mM Na-citrate at their respective pH between 6.0 and 3.0. The pH values of collected eluates were measured using an external pH probe (Mettler Toledo), where necessary.

All buffers, salts, and reagents were purchased from Sigma-Aldrich except for sodium chloride and citric acid that were purchased from Merck Millipore. MabSelect PrismA, Capto SP ImpRes, and Tricon series columns were purchased from Cytiva. CHT Type II (40 µm) was provided by HOYA Technosurgical Corporation. CHT Type II (40 µm) is manufactured by HOYA Technosurgical Corporation and distributed globally by Bio-Rad Laboratories, Inc. Hercules, CA, USA.

The antibodies were expressed using the ExpiCHO Expression system (Thermo Fisher Scientific, catalog no. A29133, RRID:CVCL 5J31) for transient protein expression. The manufacturer's expression max titer protocol was strictly followed. The supernatants were harvested after 13–14 days of production by centrifugation and the mAbs were purified by Protein A chromatography using MabSelect Prism A columns (Cytiva) with 25 mmol/L Tris, 25 mmol/L NaCl buffer (pH 7.1) for equilibration and 100 mmol/L glycine, 50 mmol/L NaCl buffer (pH 3.5) for elution. The eluates were immediately neutralized by adding 400 mmol/L glycine buffer (pH 8.1) or 500 mmol/L 2-(N-morpholino) ethanesulfonic acid (MES), pH 5.8. As a polishing step, antibodies (C0008, which is a mAb produced in our lab with the identical sequence as imalumab but devoid of the C-terminal lysine, ON103, or ON203; Supplementary Table S1) were loaded onto HiTrap SP HP columns (Cytiva) and eluted stepwise by 50 mmol/L MES buffer (pH 6.0) containing NaCl. The antibodies were buffer exchanged to a storage buffer and sterile filtered before storing them at −20°C until further use.

Unless otherwise stated, all buffers, salts and reagents were purchased from Merck Millipore. MabSelect PrismA (Cytiva) resin as well as Tricorn™ series columns (Cytiva) was kindly provided by Cytiva.

Capture chromatography was performed on an AKTA™ avant 150 using Cytiva's Mabselect™ PrismA resin packed in a XK50/30 column. The purifications utilized a 4‐minute residence time while targeting an approximately 40 g/L load of product. Tris/Acetate buffering systems were implemented including high salt and intermediate pH wash buffers. Collection criteria of the eluate was 50 mAU upslope and downslope (2 mm pathlength). Collected eluate was virally inactivated for 30 min at pH 3.5 using 2 M acetic acid. Virally inactivated eluate was neutralized to approximately pH 7.0 using 2 M Tris base prior to final filtration using a 0.2 μm PES filter.