High-pressure size exclusion chromatography
HP-SEC was used to quantify the amount of monomers, dimers, and
fragments. Adalimumab samples of 1 mg/ml were injected in a volume of 50
µl onto a SRT SEC-300, 5 µm, 30 cm x 7.8 mm column (Supelco, Bellefonte,
PA, USA). An Agilent 1200 chromatography system (Agilent Technologics,
Palo Alto, California) combined with an Agilent 1200 UV detector and a
multi-angle laser light scattering detector (DAWN® HELEOS, Wyatt
Technology Europe GmbH) was used. The flow rate was 0.5 mL/min. The
mobile phase was composed of 50 mM phosphate, 150 mM arginine and
0.025% NaN3 at pH 6.5. To quantify aggregation, UV absorption at 280 nm
was recorded. From the MALLS signal, the root mean square diameter was
calculated using the Berry Fit in the Astra software version 5.3.2.22
(Wyatt Technology Europe GmbH, Dernbach, Germany).