2.4. Charge Heterogeneity by WCX-HPLC
A Waters HPLC 2695 and a WCX column (4.0 × 250 mm; Cat. No. 054993;
Thermo Fisher Scientific, Waltham, MA, USA) were used for the separation
of all sample charge variants. Mobile phase A consisted of 20 mM ACES
(pH = 6.5), whereas mobile phase B consisted of 20 mM ACES and 200 mM
NaCl. Samples were injected at 70% mobile phase A and 30% mobile phase
B. A linear gradient from 30% mobile phase B to 43% mobile phase B
within 25 min was applied to elute antibody variants from the column.
After 28 min, a linear gradient from 43% mobile phase B to 100% mobile
phase B was applied to elute all proteins from the column. The column
was washed with 100% mobile phase B for 7 min and then changed back to
the original state using 70% mobile phase A and 30% mobile phase B for
28 min before the next injection. The aforementioned procedure was
monitored using UV 280 nm. During this process, the column temperature
was set at 40°C and the flow rate was set at 0.5 mL/min.