Peptides were separated using an Ascentis Express 150 × 2.1 mm, 2.7 µm C18 column (Sigma-Aldrich, Poole, U.K). using a flow rate of 0.21 mL/min.
A threshold was set on green fluorescence at a value of 200, and samples were analysed using a flow rate below 1,000 events per second to avoid coincidence of viral particles32.
Binding analysis was performed by injecting 100 nM of each aflibercept sample over the immobilized surface using a flow rate of 50 µl/min and 10 mM NaOH was used to regenerate the CM5 surface.
Peptides were eluted using a flow rate of 275 nl min-1, and a ternary gradient, as described62 was used, with the following mobile phases: A (water/acetonitrile/formic acid, 95/5/0.1, v/v/v), B (water/acetonitrile/formic acid, 70/30/0.08, v/v/v), and C (water/acetonitrile/trifluoroethanol/formic acid, 10/80/10/0.08, v/v/v/v).
The volume of the peptide compound injection was 3 μl, using a flow rate of 0.3 ml/min.
Peptides were diluted in 0.1 % formic acid and analysed by direct infusion mass spectrometry using a flow rate of 200 nL/min.
The best separation was obtained when gradient elution was performed and column temperature was kept at 40°C using a flow rate of 0.28 mL/min.
About 1.7 1.5 ml/kg of 350 370 mgI/ml iodinated contrast material must be administered using a flow rate of at least 3.5 ml/s, followed by a 50-ml saline flush.
The mobile phase consisted of eluent A (150 mM NaOH) and eluent B (500 mM sodium acetate in 150 mM NaOH) with a sodium acetate gradient as follows: 0 1 min, 25 mM; 1 2 min, 25 50 mM; 2 20 min, 50 200 mM; 20 22 min, 500 mM; 22 30 min, 25 mM; using a flow rate of 1.0 mL/min.
The elution gradient established was 5% B to 5% B over 5 min, 5 33% B over 15 min, 33 80% B over 10 min, 80 100% B over 5 min, isocratic 100% B for 5 min, and re-equilibration of the column, using a flow rate of 1 mL/min.
Separation was performed at 32°C using a flow rate of 0.8 ml/min.
