Native and modified Fc collection and Lys-C peptide mapping

To enable fraction collection, IdeS-treated, IgG4 was prepared by scaling IdeS and substrate concentrations in the procedure described above with the exception that this material was not deglycosylated or reduced prior to RP separation. 350 µg of the prepared sample was injected onto an Agilent Zorbax 300 Å, 3.5 µm, 4.6 × 150 mm, C8 reverse-phase column (863973.906) applying the same mobile phases, and at the same column temperature as used for analytical RP-LC/MS. On this column, separation was achieved with a linear gradient from 29% to 45% B at 0.250 mL/min applied over 38 minutes prior to purging and re-equilibrating the column. Peaks eluted and fractions were collected corresponding to the relative retention times of the native and modified Fc elution regions. This process was repeated for several cycles to enable collection of enough material for peptide mapping of the major and minor forms. Corresponding fractions from each injection were pooled. From each pooled fraction aliquots were taken to assess compositional purity by RP-LC/MS on the same chromatography system coupled to a Sciex Q-Star Elite mass spectrometer. Raw data was deconvoluted using Applied Biosystems Analyst QS 2.0, while the remainder of each pooled fraction was dried via Beckman Speed Vac Concentrator.

The dried native and modified Fc fractions were reconstituted to 1 mg/mL using 7.5 M guanidine hydrochloride, 1 mM EDTA, and 100 mM Tris (pH 7.5). 300 µL of this material was taken forward and reduced using 5 µL of 0.5 M DTT prior to a 60-minute incubation at 37°C. After allowing the samples to cool, alkylation was conducted by adding 15 µL of 0.5 M iodoacetamide with a 30 minute, light-protected, room temperature incubation. 180 µL of 25 mM Tris, 2 mM CaCl₂ (pH 8.3) was then added to each sample to achieve a final volume of 500 µL. Samples were buffer exchanged into 990 µL of 25 mM Tris, 2 mM CaCl2 (pH 8.3) with a final protein concentration of 0.3 mg/mL using a Sephadex G-25-packed NAP-5 gel filtration column. Lys-C was added at a 1:10 (w:w) protein ratio and incubated for 6 hours at 37° C. Enzymatic digestions were quenched by adding 10% TFA to a final concentration of 0.25%. Native and modified Fc digests were subsequently analyzed by RP-LC/MS as described below, while excess proteolyzed samples were stored at −80° C.