Tricorn series columns

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 purification chromatography was conducted on an AKTA™ Avant 25 (Cytiva). 1 mL and 5 mL of the respective resins was packed in Tricorn™ series columns (Cytiva) with a bed height of 5.1 cm and 6.4 cm, respectively.
Capto Butyl ImpRes was equilibrated with 50 mM Na-citrate, pH 4.0, before loading the appropriate amount of sample. A 20 column volume (CV) wash with the same equilibration buffer was applied and flow-through > 50 mAu was collected for analysis. Capto adhere was equilibrated with 50 mM Na-citrate, pH 6.5 or pH 6.8, before loading the appropriate amount of sample. A 25 CV wash at the same pH was applied and both the flow-through during loading and 25 CV wash were collected. Capto S ImpAct was equilibrated with 20 mM citric acid and 20 mM sodium phosphate, pH 5.5, 50 mM NaCl, before loading the appropriate amount of sample. A 3 CV wash of equilibration buffer was performed followed by a linear gradient from equilibration buffer to the same buffer with 500 mM NaCl in 20 CV, with a 5 CV hold at the end. 4 min residence time was utilised for all polishing resins.

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.