Corresponding guard column

Experiments were performed using a Shimadzu high-performance liquid chromatography system equipped with a temperature controlled auto sampler and a photodiode array detector capable of recording UV absorbance spectra from 200−400 nm. A Tosoh TSK-Gel Bioassist G3SW_XL column (7.8 mm ID×30.0 cm L) and a corresponding guard column (TOSOH Biosciences, King of Prussia, Pennsylvania) were used for IgG1 Fc glycoform characterization. First, the SEC column was equilibrated for at least 10 CV with a mobile phase containing 200 mM sodium phosphate, pH 6.8 and a flow rate of 0.7 mL/min at 30 °C column temperature. Next, the column was calibrated using gel filtration molecular weight standards (Bio-Rad, Hercules, CA) before and after the runs of IgG1 Fc glycoform to ensure column and HPLC system integrity. All Fc samples were centrifuged at 14,000 g for 5 min before injection to remove insoluble protein aggregates. Protein samples at a concentration of 1 mg/mL were injected in a volume of 25 µL, and a 30 min run time was used for elution. Peaks quantification was carried out using LC solutions software (Shimadzu, Kyoto, Japan). The error bars for monomer content for all the four IgG1 Fc samples and soluble dimer aggregates (observed in N297Q IgG1 Fc) represent standard deviation (SD) of triplicate measurements.^{61 (link),62 (link)}