Tskgel up sw3000

Aggregate quantification was performed on selected samples using size exclusion chromatography on a Tosoh TSKgel UP‐SW3000 (30 cm) (Tosoh Corporation, Tokyo, Japan) with guard column. An Agilent 1260 HPLC (Agilent) was used with a 5 μL injection volume and UV detection at 280 nm and 320 nm. A 15 min isocratic method was used at a flow rate of 0.35 mL/min with a mobile phase containing 100 mM sodium sulphate and 100 mM sodium phosphate at pH 6.7.

Soluble UniAb was expressed in CHO cells using protein free media supplemented with L-Glutamine (Invitrogen). Cell harvest was clarified by centrifugation and 0.2 μm PES filtration. During the first purification, step the clarified supernatant was loaded on a MabSelect SuRe (GE#17543801) resin, washed with PBS, followed by elution with 50 mM Acetic Acid, 10% Glycerol, 10% Sucrose, pH 3.6. The elution pool was immediately neutralized to approximately pH 6.5 with 2 M Tris, pH 9. After the first capture step, the protein pool was concentrated and loaded on to a Superdex 200i 10X300GL column (GE#28990944) to remove any high molecular weight species. Fractions were collected, and the most homogenous fractions were pooled. UniAb protein pools were analyzed for aggregation and purity by SEC-UHPLC. A ThermoFisher UltiMate^TM 3,000 UHPLC system was used with a TSK-Gel UP-SW3000 (Tosoh#23449) column in line. The mobile phase was 100 mM Citrate, 500 mM NaCl, 200 mM Arginine, pH 6.2 run at a flow rate of 0.25 ml/min and the method was run in 100% A isocratic mode. Fifty μg of protein was injected and 280 nm UV absorbance monitored over the 10 min run. The UV trace was analyzed and integrated by area under the curve to determine percent aggregation, monomer and degradants.