mAb

Fab
Fc
15
C (ppm)

20
13

25

30
1.5 1.0 0.5 0 -0.5
1
H (ppm)

Figure S1. 2D 13C-1H HMQC NMR spectra of mAb (black)

and its Fab (red) and Fc (green) fragments. Isolated Fab
and Fc have similar structure as that in the intact mAb.
(a) (b)
Milli Absorbance @ 280 nm

Milli Absorbance @ 280 nm

80 60
Fab, Before NMR Fc, Before NMR
Fab, After NMR Fc, After NMR

60
40
40
20
20

0 0
0 10 20 30 0 10 20 30
Retention Time (min) Retention Time (min)

Figure S2. SEC indicates that both Fab and Fc fragments exist as
monomers after NMR experiments in the presence of polysorbates.
 (a) (b)
Fab
Fab + 0.2 mM PS20
Fab + 2 mM PS20 23
11
24
12
C (ppm)

C (ppm)
25

13 26
13

13
27
14 Fab
Fab + 0.2 mM PS20
28 Fab + 2 mM PS20
15
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.6 0.5 0.4 0.3 0.2 0.1
1
H (ppm) 1
H (ppm)
(c) (d)
Fab
Fab + 0.2 mM PS80
Fab + 2 mM PS80 23
11
24
12
C (ppm)

C (ppm)

25

13 26
13

13

27
14 Fab
Fab + 0.2 mM PS80
28 Fab + 2 mM PS80
15
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.6 0.5 0.4 0.3 0.2 0.1
1
H (ppm) 1
H (ppm)

Figure S3. Addition of PS20 caused chemical shift changes in the Fab region
compared to PS80.