High-performance size-exclusion chromatography (HP-SEC) was performed to analyze the aggregation state of the protein materials as supplied from the manufacturer. The protein materials were dissolved in PB (10 mM (pH 7.2)) (except for etanercept, which was diluted in placebo buffer (pH 6.3)) and filtered through a 0.2-μm filter before measurement.
The protein samples of LYS, CC, MYO, OVA, and BSA (50 μL; ∼1 mg/mL) were analyzed with a Discovery BIO Gel Filtration column (Sigma-Aldrich, St. Louis, MO). A 515 high-performance liquid chromatography pump and 717 Plus autosampler (Waters, Milford, MA) were operated at a flow rate of 0.5 mL/min. The mobile phase consisted of 100 mM sodium PB, 200 mM sodium chloride, and 0.05% (w/v) sodium azide at a pH of 7.2 and was filtered through a 0.2-μm filter before use. Chromatograms were recorded with an SPD-6AV UV detector (Shimadzu) at a wavelength of 280 nm.
For etanercept analysis (50 μL; ∼2 mg/mL), a Yarra SEC-2000 size-exclusion column (Phenomenex, Utrecht, the Netherlands) was used with the running buffer composed of 50 mM phosphate, 150 mM arginine, and 0.025% NaN3 at pH 6.5. UV detection was performed at 280 nm.
The protein samples of LYS, CC, MYO, OVA, and BSA (50 μL; ∼1 mg/mL) were analyzed with a Discovery BIO Gel Filtration column (Sigma-Aldrich, St. Louis, MO). A 515 high-performance liquid chromatography pump and 717 Plus autosampler (Waters, Milford, MA) were operated at a flow rate of 0.5 mL/min. The mobile phase consisted of 100 mM sodium PB, 200 mM sodium chloride, and 0.05% (w/v) sodium azide at a pH of 7.2 and was filtered through a 0.2-μm filter before use. Chromatograms were recorded with an SPD-6AV UV detector (Shimadzu) at a wavelength of 280 nm.
For etanercept analysis (50 μL; ∼2 mg/mL), a Yarra SEC-2000 size-exclusion column (Phenomenex, Utrecht, the Netherlands) was used with the running buffer composed of 50 mM phosphate, 150 mM arginine, and 0.025% NaN3 at pH 6.5. UV detection was performed at 280 nm.