Biosuite high resolution sec column

Separation of the antibodies using size-exclusion chromatography was accomplished using a Waters Alliance 2695 (Waters, Milford, MA, USA) connected to a BioSuite High Resolution SEC Column (7.5 mm × 300 mm, 10 µm particle size, Waters, Milford, MA, USA). The separation was performed using an isocratic elution with PBS, pH 7.0, at a flow rate of 1 mL/min. The effluent was detected with a UV/Vis detector 2469 at 280 nm.
The intact masses of the antibodies were determined using reverse-phase chromatography separation. The separation for intact mass was accomplished using a Waters ACQUITY I class UPLC system (Waters, Milford, MA, USA) connected to a Thermo MabPac^TM RP column (2.1 mm × 50 mm, 4 µm particle size, Thermo Fisher Scientific). The separation was performed with eluent A, consisting of 0.1% formic acid in water, and eluent B, consisting of 0.1% formic acid in 100% acetonitrile, at a flow rate of 0.2 mL/min. The gradient was fixed for 2 min at 20% eluent B and linearly increased for 8 min from 20% to 50% eluent B. The effluent was analyzed with a Waters Synapt G2-Si HDMS system. A quantity of 50 μL (0.2 mg/mL) of antibodies was mixed with 1.5 μL (500,000 units/mL) of PNGaseF (New England BioLabs), then reacted overnight at 37 °C. Native and PNGaseF-treated antibodies were injected with 5 μL of the samples.

The separation of the IgGs using size-exclusion chromatography (SEC) was performed using a Waters Alliance 2695 (Waters, Milford, MA, USA) connected to a Biosuite high-resolution SEC column (7.5 mm × 300 mm, 10 µm particle size, Waters, Milford, MA, USA). The separation was conducted using an isocratic elution with PBS, pH 7.4, at a flow rate of 1 mL/min. The effluent detection was conducted using a UV/Vis detector 2489 at 280 nm.

Analytical size-exclusion chromatography (aSEC) was carried out to determine the monomeric content of nebulized and non-nebulized mAb samples. A 1-μg sample was loaded onto a Waters Biosuite High Resolution SEC Column (250 Å, 4 μm, 4.6 mm x 300 mm; Waters, catalog number: 186002162), with a BioSuite SEC Guard Column (5 μm, 6 mm x 40 mm; Waters, catalog number: 186002167). The analysis buffer consisted of 0.2 M potassium phosphate and 0.25 M KCl (pH = 6.2) in PBS. Absorbance of the flow was monitored using a UV-detector at 280 nm.