Table 3.

The detail multi-attribute method procedure for Method 1.

Denaturation	Reduction/Carboxymethylation	Tryptic digestion	Peptide purification
mAb sample (5 µg)a ↓←GuHClb (150 μL) Denatured sample (150 μL)	Denatured sample solution ↓←Reducing agentc (3 µL) ↓←Incubation at room temperature for 30 min ↓←Alkylation agentd (7 μL) ↓←Incubation at room temperature for 15 min (in dark) ↓←Reducing agentc (4 µL) Reduced and carbamidomethylated protein (RCM) sample solution (164 μL)	RCM sample solution ↓←Buffer exchanged with NAP5 ↓←Eluted with digestion buffer (600 μL) ↓←Digesting solutione (10 + 10 μL) ↓←Incubation at 37°C for 30 min Tryptic digestion (620 μL)	Tryptic digest ↓←Washed with 90% ACN 0.1% FA (200 μL, 50% ACN 0.1% FA (200 μL) and 0.1% FA (200 μL)×3 ↓←Sample applied to Oasis HLB µElution plate ↓← Washed with 0.1% FA (200 µL) ↓←Eluted with 0.1% FA 50% ACN (100 µL) Dried using Speed Vac. Dissolved in 0.1% FA (50 μL) LC/MS sample (50 μL)

a, mAb sample was diluted to 5 mg/mL using PBS and dried using Speed Vac.

b, 7.5 M guanidine/HCl, 0.25 M Tris-HCl (pH7.5).

c, 500 mM dithiothreitol.

d, 500 mM monoiodoacetate.

e, 0.1 mg/ml trypsin solution and 0.1 mg/ml trypsin and Lys-C mixture solution.