SUPPORTING INFORMATION FOR:
Stress-dependent flexibility of a full-length human monoclonal antibody:
insights from molecular dynamics to support biopharmaceutical development
Dheeraj S. Tomar1†, Giuseppe Licari2, Joschka Bauer2, Satish K. Singh1††, Li Li3, and Sandeep
Kumar4*
1
Biotherapeutics Pharmaceutical Sciences Research and Development, Pfizer Inc.,
700 Chesterfield Parkway West, Chesterfield, Missouri, 63017, USA
2
Pharmaceuticals Development Biologicals, Boehringer Ingelheim Pharmaceuticals, Inc., D-88397
Biberach an der Riss, Germany
3
Biotherapeutics Pharmaceutical Sciences Research and Development, Pfizer Inc.,
1 Burtt Road, Andover, Massachusetts, 01810, USA
4
Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road,
Ridgefield, CT 06877
*Author for Correspondence: Sandeep_2.Kumar@Boehringer-Ingelheim.com
†
Current address: Xilio Therapeutics Inc., 828 Winter Street, Suite 300, Waltham, MA 02451
††
Current address: Moderna Therapeutics, 200 Technology Square, Cambridge, MA 02139
Table S1: Solvent accessible surface areas (SASA, Å2) of different domains of the antibody molecule.
Averages and standard deviations (together with the ranges) were obtained from merging all three
trajectories for each simulation condition. Domains definitions are reported in Figure S1. Chains H/K
and L/M refer to heavy and light chains, respectively, with H/L forming one Fab (Fab1) and K/M forming
the other Fab (Fab2).
Doma
in
All
VH
C H1
Hinge
H
C H2
C H3
VK
CK1
Hinge
K
CK2
CK3
VL
CL
VM
CM
300 K With Glycans
SASA
69451 ±
560
6851 ±
137
4375 ±
122
1232 ±
147
6312 ±
158
5426 ±
193
6814 ±
146
3996 ±
125
1229 ±
99
6225 ±
176
5584 ±
143
5421 ±
132
5420 ±
160
5319 ±
123
5247 ±
142
Range
67467 —
71577
6411 —
7282
3942 —
4753
867 —
1661
5819 —
6843
4905 —
6060
6271 —
7249
3601 —
4359
914 —
1542
5686 —
6743
5169 —
6208
4987 —
5839
4906 —
5966
4894 —
5811
4758 —
5782
500 K With Glycans
SASA
65573 ±
1722
6938 ±
315
4145 ±
383
886 ±
208
6047 ±
319
5477 ±
315
6842 ±
398
3602 ±
326
879 ±
235
5442 ±
399
5414 ±
309
5535 ±
258
4708 ±
279
5180 ±
355
4478 ±
320
Range
60420 —
71014
5909 —
8205
2872 —
5184
430 —
1702
4973 —
7013
4467 —
6731
5313 —
8476
2704 —
4798
359 —
1605
4379 —
6822
4503 —
6683
4525 —
6443
3917 —
5813
3934 —
6101
3406 —
5724
300 K Without
Glycans
SASA
Range
69101 ±
67109 —
699
71070
6828 ±
6382 —
151
7354
4388 ±
3801 —
176
5035
1237 ±
792 —
188
1724
6272 ±
5647 —
171
6802
5352 ±
4929 —
140
5901
6761 ±
6260 —
138
7215
3896 ±
3536 —
124
4300
1171 ±
801 —
152
1602
6223 ±
5800 —
141
6650
5506 ±
5005 —
140
5935
5521 ±
5081 —
120
5977
5470 ±
4858 —
209
6061
5311 ±
4875 —
133
5813
5165 ±
4658 —
151
5607
500 K Without Glycans
SASA
63732 ±
2197
6863 ±
396
3487 ±
522
800 ±
252
5376 ±
484
5238 ±
428
7055 ±
313
3646 ±
262
978 ±
184
5304 ±
498
5078 ±
383
5531 ±
277
4823 ±
330
5057 ±
305
4497 ±
391
Range
56500 —
70811
5623 —
8306
2196 —
5003
249 —
1561
4102 —
7045
3812 —
6490
5967 —
8499
2917 —
4624
485 —
1534
3826 —
6996
3849 —
6166
4382 —
6505
3751 —
5873
4110 —
6167
3334 —
5751
Table S2: Radii of gyration of different domains of the antibody molecule (Å). Averages and standard
deviations (together with the ranges) were obtained from merging all three trajectories for each simulation
condition. Domains definitions are reported in Figure S1. Chains H/K and L/M refer to heavy and light
chains, respectively, with H/L forming one Fab (Fab1) and K/M forming the other Fab (Fab2).
Domain
All
VH
C H1
HingeH
C H2
C H3
VK
CK1
HingeK
CK2
CK3
VL
CL
VM
CM
300 K With Glycans
Radii
48.7 ±
1.4
15.5 ±
0.1
14.0 ±
0.1
13.6 ±
1.3
15.1 ±
0.1
15.1 ±
0.1
15.5 ±
0.1
14.0 ±
0.1
13.4 ±
0.6
15.1 ±
0.1
15.0 ±
0.1
14.6 ±
0.1
15.0 ±
0.1
14.6 ±
0.1
15.1 ±
0.1
Range
46.0 —
52.2
15.1 —
15.8
13.8 —
14.4
11.7 —
16.8
14.8 —
15.4
14.7 —
15.5
15.1 —
15.8
13.7 —
14.3
11.8 —
15.0
14.8 —
15.4
14.7 —
15.4
14.3 —
14.9
14.7 —
15.4
14.4 —
14.9
14.7 —
15.4
500 K With Glycans
Radii
43.2 ±
2.1
15.6 ±
0.4
14.0 ±
0.3
12.7 ±
1.3
14.8 ±
0.3
15.1 ±
0.3
15.7 ±
0.3
14.0 ±
0.3
12.5 ±
1.1
14.9 ±
0.3
15.2 ±
0.4
14.7 ±
0.2
15.0 ±
0.2
14.6 ±
0.3
14.9 ±
0.3
Range
39.8 —
49.9
14.6 —
16.9
13.2 —
14.8
9.7 —
15.8
13.8 —
15.7
14.3 —
16.8
14.8 —
17.1
13.1 —
15.1
9.1 —
15.9
14.0 —
15.5
14.5 —
19.1
14.1 —
15.3
14.3 —
15.6
13.8 —
15.4
14.1 —
15.7
300 K Without
Glycans
Radii
Range
48.9 ±
46.0 —
1.6
54.9
15.5 ±
15.2 —
0.1
15.9
14.1 ±
13.8 —
0.1
14.5
14.0 ±
12.3 —
1.4
18.4
15.0 ±
14.7 —
0.1
15.4
15.0 ±
14.7 —
0.1
15.4
15.4 ±
15.2 —
0.1
15.7
14.0 ±
13.7 —
0.1
14.2
13.1 ±
11.7 —
0.5
14.6
15.1 ±
14.7 —
0.1
15.4
14.9 ±
14.7 —
0.1
15.3
14.5 ±
14.1 —
0.1
14.8
15.0 ±
14.7 —
0.1
15.3
14.6 ±
14.4 —
0.1
14.8
15.0 ±
14.7 —
0.1
15.3
500 K Without
Glycans
Radii
Range
41.8 ±
36.6 —
1.9
48.2
15.7 ±
14.6 —
0.6
18.5
14.0 ±
13.4 —
0.2
14.9
12.6 ±
10.2 —
0.9
15.6
14.8 ±
13.8 —
0.3
15.8
15.1 ±
14.6 —
0.2
15.8
15.7 ±
14.7 —
0.4
17.2
14.1 ±
13.4 —
0.2
14.7
11.7 ±
9.0 —
1.4
15.6
14.7 ±
13.9 —
0.3
15.9
15.1 ±
14.4 —
0.2
15.7
14.6 ±
13.9 —
0.3
15.3
14.9 ±
14.0 —
0.3
15.6
14.8 ±
14.2 —
0.1
15.2
15.0 ±
14.2 —
0.2
15.6
Table S3: Average RMSF of Cα for different domains of the antibody molecule (Å). Averages and standard
deviations (together with the ranges) were obtained from merging all three trajectories for each simulation
condition. Domains definitions are reported in Figure S1. Chains H/K and L/M refer to heavy and light
chains, respectively, with H/L forming one Fab (Fab1) and K/M forming the other Fab (Fab2).
300 K With Glycan
500 K With Glycan
Domain
RMSF
RMSF
All
4.6 ± 1.5
VH
7.1 ± 1.7
C H1
4.7 ± 1.2
HingeH
4.3 ± 0.5
C H2
4.3 ± 0.5
C H3
4.4 ± 1
VK
4.5 ± 1
CK1
3.3 ± 0.5
HingeK
3.7 ± 0.4
3 — 4.4
5.1 ± 0.6
CK2
3 ± 0.6
2.1 —
4.4
5.2 ± 1.1
Range
2.1 —
10.9
3.4 —
10.9
2.5 —
7.5
3.4 —
5.2
3.2 —
5.4
2.7 —
8.6
2.9 —
7.5
2.4 —
4.7
7.1 ± 2.1
9.8 ± 2.2
6.8 ± 1.5
5.2 ± 0.6
7.1 ± 1.4
8 ± 2.2
7.1 ± 1.8
5.8 ± 1
Range
3.6 —
16.2
5.7 —
14.8
4.3 —
10
4.1 — 6
4.8 —
11.1
4.5 —
13.8
4—
11.4
4.3 —
8.4
4.4 —
6.2
3.6 —
8.1
300 K Without
Glycans
RMSF
Range
1.9 —
5.0 ± 2.0
11.8
4.1 —
7.8 ± 1.8
11.8
3.2 —
5.8 ± 1.7
10.5
3.1 —
5 ± 1.5
7.5
3.9 ± 0.4
5.3 ± 1
4.1 ± 0.8
3.2 ± 0.6
3.8 ± 0.7
3.5 ± 0.8
3.2 — 5
3.8 —
9.8
2.7 —
6.8
2.1 —
4.6
2.3 —
4.9
2.4 —
5.2
500 K Without
Glycans
RMSF
Range
3.3 —
7.6 ± 2.6
17.6
11.2 ±
6—
2.8
17.6
3.3 —
6.6 ± 2
10.9
4.7 —
5.5 ± 0.6
6.9
5.6 —
7.9 ± 1.9
12
5.3 —
8.5 ± 2.1
16.3
4.7 —
7.2 ± 1.4
10.9
3.5 —
5.1 ± 0.9
7.2
4.9 —
6.1 ± 0.7
7.6
4.4 —
6.3 ± 1.3
9.5
CK3
4.4 ± 1.1
VL
6.2 ± 1.2
CL
4.9 ± 1.1
VM
3.8 ± 0.9
CM
3.6 ± 0.8
2.6 —
8.6
3.6 —
8.5
2.9 —
8.1
2.4 —
5.8
2.3 —
5.2
7.1 ± 2.1
4.5 —
16.2
5.1 ± 0.6
9 ± 1.6
6 — 12
7.7 ± 1.4
7 ± 1.4
6.7 ± 1.8
5.4 ± 1
4.6 —
10.7
3.7 —
10.4
3.9 —
7.6
3.8 —
6.6
4.5 —
10.1
4.1 —
10.2
2.2 —
5.4
1.9 —
5.3
6.4 ± 1.3
3.5 ± 0.8
3.1 ± 0.9
7.4 ± 1.6
10.8 ±
1.9
7.9 ± 2
5.3 —
14.5
6.9 —
14.3
4.3 —
12.5
6.4 ± 1.4
4 — 9.1
5.5 ± 1.2
3.7 —
8.8
Table S4: Distances between α-carbons of residues at the N-terminal and C-terminal of the Hinge.
Averages and standard deviations (together with the ranges) were obtained from merging all three
trajectories for each simulation condition. Hinge region definitions are reported in Figure S1. In particular,
HingeH belongs to Fab1, whether HingeK belongs to Fab2.
HingeH
Distance
Range
22.5 —
52.4
HingeK
Distance
Range
33.2 ± 2.2
28.3 — 38.8
With Glycans 300 K
35.8 ± 8.3
Without Glycans 300
K
39.4 ± 5.8
31 — 56.1
31.8 ± 2.2
26.5 — 38.6
With Glycans 500 K
30 ± 6.8
14.5 —
47.4
31.5 ± 3.3
21.2 — 43.3
Without Glycans 500
K
27 ± 3.2
17 — 41.2
25.7 ± 5.4
15.4 — 40.6
Table S5: Distances between different regions of the antibody molecules (Å). Averages and standard
deviations (together with the ranges) were obtained from merging all three trajectories for each simulation
condition. Domains definitions are reported in Figure S1. Chains H/K and L/M refer to heavy and light
chains, respectively, with H/L forming one Fab (Fab1) and K/M forming the other Fab (Fab2).
With Glycans 300 K
Fab1Fab2
Fab1-Fc
Fab2-Fc
Distanc
e
76.8 ±
4.5
80.4 ±
5.2
60.8 ±
1.5
Range
68.1 —
89
69.4 —
93.4
54.5 —
65.5
Without Glycans 300
K
Distanc
Range
e
75.1 —
83.9 ± 5
101.5
73.8 ±
53 — 91
6.7
60.8 ±
57.4 — 64
1.1
With Glycans 500 K
Distanc
e
71.8 ±
4.3
66.5 ±
8.1
47.2 ±
2.6
Range
58.5 —
84.6
51.6 —
89.9
40.2 —
55.4
Without Glycans 500
K
Distanc
Range
e
71.2 ±
60.9 —
4.6
83.8
60.8 ±
37.6 —
8.8
77.5
44.4 ±
35.9 —
3.9
57.3
Table S6: Molecular descriptors of the antibody molecule calculated at pH 6.0 using built-in modules of
MOE with default settings.
Descriptor
ZmAb (e)
Zapp (e)
ZCDR(e)
DmAb (Debye)
ZFv (e)
pIStructure
pISequence
ζ (mV)
Hydrodynamic
Radius (Å)
Volume (Å3)
ASAHphil (Å2)
ASAHphob (Å2)
ASA (Å2)
Eccentricity
300 K With Glycans
Value
Range
30.3 ±
23 — 37
2.1
12.95 ±
9.68 —
0.92
16.49
5.02 ±
4.07 — 5.67
0.24
1859 ±
947 — 3125
329
18.72 ±
17.06 —
0.27
19.63
9.3 ±
9.11 — 9.5
0.05
7.4
7.4 — 7.4
43.4 ±
32.08 —
3.21
56.02
51.02 ±
49.3 —
0.52
52.75
138908
138749 —
± 59
139115
28550
27140 —
± 401
29882
32809
31539 —
± 392
34083
64376
62049 —
± 579
66274
0.58 ±
0.42 — 0.78
0.08
500 K With Glycans
Value
Range
27.2 ±
21 — 35
2.2
11.76 ±
8.76 — 15.52
0.97
4.47 ±
3.56 — 5.62
0.23
1674 ±
445 — 3353
464
18.52 ±
15.29 —
0.61
20.56
9.26 ±
8.91 — 9.47
0.08
7.4
7.4 — 7.4
40.74 ±
29.92 —
3.52
53.12
49.43 ±
47.49 — 52.3
0.71
138910
138711 —
± 75
139171
22902 ±
19706 —
1284
27296
30738 ±
27730 —
1029
34234
56437 ±
50746 —
2145
63840
0.41 ±
0.25 — 0.72
0.07
300 K Without Glycans
Value
Range
30.7 ±
24 — 38
2.1
13.06 ±
10.27 —
0.97
16.15
4.91 ±
4.1 — 5.83
0.31
1646 ±
587 —
346
3420
18.7 ±
17.59 —
0.27
19.55
9.36 ±
9.14 —
0.05
9.52
7.4
7.4 — 7.4
42.84 ±
32.42 —
3.49
53.8
52.07 ±
49.2 —
1.07
57.49
136223
136092 —
± 40
136363
28194 ±
26991 —
387
29471
32374 ±
30824 —
503
33821
63492 ±
61351 —
761
65526
0.5 ±
0.29 —
0.08
0.65
500 K Without Glycans
Value
Range
27.4 ±
21 — 37
2.2
11.89 ±
8.97 —
0.97
16.45
4.46 ±
3.4 — 5.59
0.24
1227 ±
102 — 2768
402
18.62 ±
15.8 —
0.52
20.15
9.29 ±
8.98 — 9.54
0.08
7.4
7.4 — 7.4
41.53 ±
31.08 —
3.46
58.12
49.06 ±
47.25 —
0.76
51.82
136240
136035 —
± 53
136426
23498 ±
19946 —
1311
27574
29549 ±
25208 —
1177
33671
55811 ±
48138 —
2374
63588
0.37 ±
0.2 — 0.59
0.08
Table S7: Number and surface area of hydrophobic and electrostatic patches on the surface of the antibody
molecule calculated at pH 6.0 using built-in modules of MOE with default settings.
Patch Type
Surface area
of the largest
hydrophobic
patch in the
CDRs (Å2)
Number of
hydrophobic
patches in the
CDRs
Surface area
of the largest
charged
patch in the
CDRs (Å2)
Number of
charged
patches in the
CDR
Surfaced area
of the largest
negative
charged
patch in the
CDRs (Å2)
Number of
negative
300 K With Glycans
Value
Range
500 K With Glycans
Value
Range
300K Without Glycans
Value
Range
500 K Without Glycans
Value
Range
355 ± 68
180 —
780
920 ± 253
250 —
1640
389 ± 68
170 —
620
798 ± 179
240 —
1600
4±1
2—8
9±2
3 — 16
4±1
2—9
9±2
3 — 17
1079 ±
139
580 —
1750
613 ± 150
120 —
1190
1139 ±
152
610 —
1710
742 ± 173
260 —
1300
16 ± 2
9 — 24
11 ± 3
3 — 22
18 ± 3
8 — 27
13 ± 3
6 — 23
481 ± 72
170 —
720
190 ± 87
0 — 530
494 ± 81
230 —
750
266 ± 99
40 — 590
6±1
2 — 11
4±2
0 — 10
7±1
3 — 12
5±2
1 — 11
patches in the
CDRs
Surface area
of the largest
positively
charged
patch in the
CDRs (Å2)
Number of
positively
charged
patches in the
CDRs
Surface area
of the largest
hydrophobic
patch on the
mAb (Å2)
Number of
hydrophobic
patches on
the mAb
Surface area
of largest
charged
patch on the
mAb (Å2)
Number of
charged
patches on
the mAb
Surface area
of the largest
negatively
charged
patch on the
mAb (Å2)
Number of
negatively
charged
patches on
the mAb
Surface area
of the largest
positively
charged
patch on the
mAb (Å2)
Number of
positively
charged
patches on
the mAb
598 ± 113
230 —
1200
423 ± 107
50 — 820
645 ± 114
270 —
1050
476 ± 118
50 — 910
10 ± 2
4 — 18
8±2
1 — 14
11 ± 2
4 — 18
9±2
1 — 16
1860 ±
179
1310 —
2510
4147 ±
492
2600 —
5580
2160 ±
206
1510 —
2960
3769 ±
412
2500 —
5610
24 ± 3
16 — 33
45 ± 5
29 — 60
26 ± 3
17 — 35
41 ± 5
24 — 57
6013 ±
342
4880 —
7150
3191 ±
377
2020 —
4430
6369 ±
350
5290 —
7510
3934 ±
425
2690 —
5540
108 ± 6
87 — 128
63 ± 7
40 — 89
115 ± 6
90 — 141
76 ± 8
50 — 102
2554 ±
206
1890 —
3380
1066 ±
219
390 —
1790
2693 ±
200
2040 —
3440
1448 ±
237
670 —
2230
45 ± 4
31 — 60
21 ± 4
8 — 37
48 ± 4
36 — 63
28 ± 4
15 — 42
3459 ±
224
2670 —
4170
2125 ±
234
1360 —
2910
3676 ±
231
2890 —
4370
2486 ±
255
1760 —
3490
63 ± 4
46 — 77
41 ± 5
26 — 55
67 ± 4
53 — 82
48 ± 5
33 — 65
>1HZH | Heavy Chain | VH1 Domain | Total Residues = 131
QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHWVRQAPGQRFEWMGWINPYNGNKEFSAKFQDR
VTFTADTSANTAYMELRSLRSADTAVYYCARVGPYSWDDSPQDNYYMDVWGKGTTVIVSSASTK
>1HZH | Heavy Chain | CH1 Domain | Total Residues = 94
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKA
>1HZH | Heavy Chain | Hinge | Total Residues = 22
EPKSCDKTHTCPPCPAPELLGG
>1HZH | Heavy Chain | CH2 Domain | Total Residues = 103
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
>1HZH | Heavy Chain | CH3 Domain | Total Residues = 107
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
>>1HZH | Light Chain | VL Domain | Total Residues = 113
EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQHKPGQAPRLVIHGVSNRASGISDRFSGSGSGT
DFTLTITRVEPEDFALYYCQVYGASSYTFGQGTKLERKRTVAA
>1HZH | Light Chain | CL Domain | Total Residues = 102
PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS
KADYEKHKVYACEVTHQGLRSPVTKSFNRGEC
Figure S1: Complete primary sequence of the human b12 mAb (PDB ID: 1HZH). For clarity, the sequence
is split into different domains. CDRs are underlined, and glycosylation site is highlighted in cyan.
Figure S2: Time-dependent secondary structure content of the antibody molecule. Domains definitions are
reported in Figure S1.