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.