PAE titration experiments for MF0.1N HCland MpH3.3 measurements
Titration experiments to determine mass fraction of 0.1 N HCl(MF0.1N HCl) was performed in a 50 mL glass beaker (VWR, 10754–948) with a stir bar (VWR, 58948–138) on a stirrer station (Corning, PC-210) with initial working volume of 30 mL PAE (IgG4-N1 22.0 g L-1 to 35.5 g L-1). Acid (0.1 N HCl) was added in 0.3 mL per bolus with the stir speed setting at 5 to 6 (equivalent to 250–300 rpm). The pH value was recorded after each bolus addition. The mass fraction was calculated based on the ratio of accumulated acid addition mass over the total mass of the acidified PAE.
Titration experiments to determine the initial HMW formation (in 10 min) at pH 3.3 (MpH 3.3 ) were performed in 1.5 mL Eppendorf tubes (Eppendorf AG, 022363204). The amount of 0.1 N HCl to target pH 3.3 at a given IgG4-N1 concentration were pre-determined. Each pH-adjusted sample was mixed thoroughly by inverting the tube 5–7 times or pipetting up and down 5–7 times upon acid addition. The mixture was held for 10 min (including inverting time). Pre-determined amount of 2 M Tris-Base was then added into mixture to achieve pH 5.3-5.7. The neutralized mixture was held at 4 oC overnight prior to the protein soluble aggregate assay.