Akta avant 25

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.

The affinity purification of SLO target from the feedstock solution at pilot and technical scale was carried out in preconditioning the Eshmuno® Nanofitin column with 10 mM KPi pH 7.2 for 10 column volumes (CVs) followed by the loading of 100 ml feedstock solution on the 5 ml column, ~2.5 L feedstock solution for the 133 ml column, and 9.5 l feedstock solution for the 469 ml column applying a residence time between 1.5 and 2 min. After loading, a rinse step with the equilibration buffer was performed followed by a washing step with 10 mM Na₃PO₄, 750 mM NaCl, pH 6.8, to remove unbound impurities. The elution of the SLO target molecule was performed with 0.1 M Glycine buffer pH 4.0. The elution was collected in fractions, which were subjected to detailed analysis to estimate the quantity and purity of the purified SLO target molecule. After chromatographic purification, the Eshmuno® Nanofitin was cleaned with 10 CV of 0.1 M NaOH solution. After cleaning, the column was regenerated with Buffer A and filled with storage buffer for later use. The chromatographic experiments were carried out using Akta Avant 25 (Cytiva) for 1 and 5 ml columns and Akta Avant 150 (Cytiva) for 133 and 469 ml columns, provided with a UV detector (absorbance at 214–280–254 nm).