Dr. Roxana E. Iacob
www.hxms.neu.edu
Dr. Roxana E. Iacob
Department of Chemistry & Chemical Biology, Northeastern University
21 atoms
Primary structure characterization
> 20,000 atoms
Higher order structures, aggregates and interactions Higher order • • • • •
X Ray NMR Native-MS IM-MS HDX-MS
Aggregates
Interactions (mAb/Ag)
• • • • • •
• • • • • • •
Adapted from Beck A. et al. (2013). Anal. Chem. ; 85: 715-736
SEC (UV-MALS) AUC Native-MS IM-MS HDX-MS Crosslinking MS
SPR ELISA FACS Native-MS IM-MS HDX-MS Crosslinking MS
Extremely important in biopharmaceutical industry/ $$$$$$ Identifying binding sites and epitopes between proteins and their binding partners are needed for the development of effective therapeutics Patent matters
BINDING SITE
BINDING INTERFACE
EPITOPE ANTIGENIC DETERMINANT
Alanine scanning Linear epitopes No info on dynamics Time consuming
X-ray crystallography
Computational modeling Docking
Requires a crystal In silico/ model
NMR
Mass Spectrometry
Limited proteolysis Non-covalent ESI Chemical crosslinking Oxidative labeling
HDX MS Info on dynamics Size limited Large sample requirements
Fast measurements Low sample requirements Linear/ discontinuous epitopes Info on surface accessibility Info on dynamics
Pirrone G.F. et al. (2015). Anal. Chem. ; 87(1): 99-118
Research
Process Development
Drug design
Folding/ Refolding
Structure-function
Comparability and stability
Epitope mapping Effects of PTMs on conformation
Impact of primary structure changes on conformation
Lead compound optimization
Formulation/ excipients
HDX MS improves protein biophysical characterization Wales T.E. & Engen J.R. (2006): Mass Spectrom. Rev. 25 158. Engen (2009): Anal. Chem. 81 7870.
INACTIVE/unmodified/innovator
Deuterium Backbone amide H
ACTIVE/modified/biosimilar
cut into pieces with pepsin weigh with mass spectrometer
Solution conformation of proteins is analyzed under physiological conditions!
Oxygen
Backbone carbon
Hydrogen
Nitrogen
Sidechain carbon
Backbone amide hydrogen
Gln
Asp
His
Pro
Lys
Leu
• Solvent accessibility • Hydrogen bonding
• pH • Temperature • Side-chain effects • Isotope effects • Salt Englander W. (1983) Q. Rev. Biophys 16, 521. Engen J.R. et al. (2011) Ency. Anal. Chem.
Commercial or homemade pepsin beads
Robots, LEAP system
MSE Peptide id
DynamX
Houde D. et al. (2011). J. Pharm Sci. 100(6), 2071. Marcsisin S. & Engen J.R. (2010). Anal. Bioanal. Chem. 397(3), 967.
Xevo or Synapt
e.g.
NanoACQUITY w/ HDX tech.
D. i.
All measurements at 0 °C
POROS 20 m
125 psi
1.00
2.00
3.00
4.00
5.00
6.00
UPLC very efficient at low temperatures
ii.
3.5 m 21 °C
1250 psi
10 °C
1.00
iii.
3.00
5.00
7.00
9.00
11.00
5 °C
1.75 m
~1 °C
8000 psi 2.00
1.00
2.00
3.00
4.00 5.00 Time (min)
Wales et al. 2008. Anal. Chem. 80, 6815.
6.00
7.00
8.00
3.00
4.00 5.00 Time
6.00
Fab digested online with pepsin
7.00
mAb 150 kDa 8000 psi at 65 µL/min 12 min gradient 0 ˚C
Cycle time
20 min
12000 psi at 100 µL/min 12 min gradient 0 ˚C
10 min
12000 psi at 100 µL/min +/- IMS 6 min gradient 0 ˚C 1.00
3.00
5.00 7.00 Time (min)
Engen J.R. & Wales T.E. (2015). Annu. Rev. Anal. Chem. 8, Jul 22;8(1): 127
9.00
11.00
ACQUITY UPLC HSS T3 1.8 m 1.0 x 50 mm
1HZH
Porcine pepsin %
0
Full length IgG- 150kDa
100 100
Rice Porcine field eelpepsin pepsin Porcine pepsin
100
Pepsin Aspergillopepsin/ Factor XIII Rice field eel pepsin
% %
% 0 0
0 100 100 100
Rice field eel pepsin Protease XIII Rice Porcine field eelpepsin pepsin
Rhizopuspepsin (protease type XVIII) Nepenthesin
% % % 0
0 1000
4.00
8.00
12.00
16.00
min
Protease XIII Rice field eel pepsin Protease XIII
100
%
%
Kadek A. et al. Anal. Chem. (2014); 6; 4287 Rey M. et al., Rapid Commun. Mass Spectrom. (2009); 23; 3431
0
Zhang H.M. et al. Anal. Chem. (2008); 80; 9034
4.00 100 4.00
Cravello L. et al. Rapid Commun. Mass Spectrom. (2003;) 17: 2387 Powers J.C. et al. Adv.Exp. Med. Biol. (1977); 95;141
0
8.00 8.00
12.00 12.00
16.00 16.00
min min
Ahn et al. BBA-Proteins and Proteomics, Protease (2013); 1834XIII (6): 1222
+ Deuterium
Decreased hydrogen exchange= binding interface?
Not always straightforward • Dissociation constant • Dynamic range • Compound solubility • Interface not always clear • Allostery
Engen J. R. (2003). Analyst, 128(6), 623 Konermann L. et al. (2011). Chem. Soc. Rev., 40, 1224
Protein 1
• •
Complex
Deuterium Labeling (15x D2O) time
Protein 2 Small molecule
Quenching (0 ºC, pH 2.4)
Online Pepsin Digestion
Software
NanoAcquity UPLC Separation
2.0 Peptide Identification by HDMSe+PLGS Deuterium Measurement by MS
DynamX
Data processing / DynamX
Waters UPLC w/ HDX
Synapt G2 Si
Deuterium Uptake Comparison Change/ Epitope peptide Relative Deuterium Level (Da)
15
15
Bou… Bound Free Free
12
12 9
6 3
12
9
9
6
6
3
3
0
0 0.1
1
10
100 1000 0.1 1
10
0
100 1000 0.1
Time (minutes) Ahn J. & Engen J. R. (2013). Chim. Oggi./ Chem. Today. 31, 25
15
No Change Bou… 7 Free 6 5 4 3 2 1 0 1
Bou… Bound Free Free
Bou nd Free
10 0.1
100 1 1000 10
100
Time (minutes)
1000
EGFR binding to a monobody Interleukin-23 binding to a monobody Protein: small molecule interactions
Mechanism of EGFR activation
2011 sales > $1 billion
mAbs
Epidermal Growth Factor (EGF)
Extracellular
NEED: New biotherapeutics: cheaper, easier, faster, more effective!!!
Intracellular
Ferguson K.M. (2008). Annu Rev Biophys. 37, 353
Derived from the 10th type III domain of human Fibronectin 1
2
3
4
5
6
7
8
9
10
11
12
13
14
IIICS
15
10Fn3
Type I Domain Type II Domain Type III Domain
Adnectin
Structural similarities with VH of an antibody
VH
Adnectins FG
CDRH1 CDRH2
ADVANTAGES
BC DE
CDRH3
High Structural homology Low Sequence homology
Lipovsek D. (2011). Protein Engineering, Design& Selection. 24, 3
Diversified CDR-like loops Tailored to bind to multiple targets KD: 0.7-2nM High thermo-stability Penetrate tissues faster Easier to manufacture
Extracellular EGFR: 69 kDa
L E E K K V C Q G T S N K L T Q L G T F E D H F L S L Q R M F N N C E V V L G N L E I T Y V Q R N Y D L S F L K T I Q E V A G Y V L I A L N T V E R I P L E N L 10 20 30 40 50 60 70 80
Q I I R G NM Y Y E N S Y A L A V L S N Y D A N K T G L K E L P M R N L Q E I L H G A V R F S N N P A L C N V E S I QW R D I V S S D F L S NM S MD F Q N H L 90 100 110 120 130 140 150 160
IV
C
50 Cysteine residues 10 potential N-linked glycosylation sites
II N
G S C Q K C D P S C P N G S C WG A G E E N C Q K L T K I I C A Q Q C S G R C R G K S P S D C C H N Q C A A G C T G P R E S D C L V C R K F R D E A T C K D T C 170 180 190 200 210 220 230 240 P P L M L Y N P T T Y QMD V N P E G K Y S F G A T C V K K C P R N Y V V T D H G S C V R A C G A D S Y E M E E D G V R K C K K C E G P C R K V C N G I G I G E 250 260 270 280 290 300 310 320
F K D S L S I N A T N I K H F K N C T S I S G D L H I L P V A F R G D S F T H T P P L D P Q E L D I L K T V K E I T G F L L I Q A W P E N R T D L H A F E N L E 330 340 350 360 370 380 390 400
I I R G R T K Q H G Q F S L A V V S L N I T S L G L R S L K E I S D G D V I I S G N K N L C Y A N T I NW K K L F G T S G Q K T K I I S N R G E N S C K A T G Q 410 420 430 440 450 460 470 480
I
III
V C H A L C S P E G C WG P E P R D C V S C R N V S R G R E C V D K C N L L E G E P R E F V E N S E C I Q C H P E C L P Q A MN I T C T G R G P D N C I Q C A H 490 500 510 520 530 540 550 560
PDB: 3QWQ
Y I D G P H C V K T C P A G V MG E N N T L V W K Y A D A G H V C H L C H P N C T Y G C T G P G L E G C P T N G P K H H H H H H 570 580 590 600 610 620
Total: 84.1% Coverage, 2.98 Redundancy
IV
C
II N
I
10 sec
III
10 min 0% 10
60 min
240 min
20 30 40 50 >60% undetermined Relative % Deuterium
Iacob R.E. et al. (2014). JASMS, 25(12), 2093
Adnectin: 12 kDa
G V S D V P R D L E V V A A T P T S L L I S W D S G R G S Y Q Y Y R I T Y G E T G G N S P V Q E F T V P G P V H T A T I S G L K P 10 20 30 40 50 60
G V D Y T I T V Y A V T D H K P H A D G P H T Y H E S P I S I N Y R T E I D K P S Q H H H H H H 70 80 90 100 110
Total: 90.3% Coverage, 1.56 Redundancy
FG N
BC DE
C
10 min
10 sec
0% 10
60 min
240 min
20 30 40 50 >60% undetermined
Relative % Deuterium Iacob R.E. et al. (2014). JASMS, 25(12), 2093
78 EGFR peptic peptides 82% Sequence coverage
EGFR
8
96-108 96-118 99-117 99-118 99-120 99-132
45-54 46-54
0 1-14 1-17 1-19 15-24 18-24
Difference (Da) [EGFR-(EGFR+ Adnectin)]
16
10 sec 10 min 60 min 240 min
-8
EGFR + Adnectin -16
Relative Uptake (Da)
18 16
1
10
20
EGFR+Adnectin EGFR
1 - 19
14
8 7
EGFR+Adnectin EGFR
46 - 54
6
12
4
8
3
6
4
2
2
1 1
10
100 300
0
12
96 - 108
10
50 EGFR+Adnectin EGFR
1
10
100 300
16 14
6
8
4
6
0
226 - 244
78
EGFR+Adnectin EGFR
10
4
2 0.08
60
12
8
5
10
0 0.08
30 40 Peptide position (N-to-C)
2 0.08
Exposure Time (min)
1
10
100 300
0
0.08
1
10
100 300
Iacob R.E. et al. (2014). JASMS, 25(12), 2093
IV
Decrease > 1Da
1-24
Decrease 0.5-1Da
II
I III
96-108 PDB: 3QWQ
Adnectin
HDX MS
Y101 S99
X-ray crystal structure
L69 L14
EGFR Residues L17
Q16
Adnectin residues
T15
DE loop ~CDRH2
BC loop ~CDRH1
FG loop ~CDRH3
Iacob R.E. et al. (2014). JASMS, 25(12), 2093 Ramamurthy V. et al. (2012). Structure. 20: 259
HDX-MS
N 1-24
C
45-54 96-108
T15 Q16 L17
S99 L69
Y101
G18
N 15-18
X-ray structure
C
69 99 101
Iacob R.E. et al. (2014). JASMS, 25(12), 2093
IL-23: Key participant in central regulation of the cellular mechanism involved in inflammation Ustekinumab
IL-12 IL-12
Janssen Biotech.
Ustekinumab
IL-23
Are there other biotherapeutics capable to inhibit IL-23 pro-inflammatory effects? Mascelli M.M. et al. (2011). Nature Biotechnology 29, 615
FG
BC
DE
I W E L K K D V Y X X X L D W Y P D A P G E M X X X T C D T P E E D G I T W T L D Q S S E V L G S G K T L T I Q V K E X X X X G Q Y T C H K 10 20 30 40 50 60 70
IL-23: 59 kDa
G G E V L S H S L L L L H K K E D G I W S T D I L K D Q K E P K N K T F L R C E A K N Y S G R F T C WW L T T I S D L T F S V K S S R G S S 80 90 100 110 120 130 140
P40
D P Q G V T C G A A T L S A E R V R G D N K E Y E Y S V E X X X D S A C P A A E E S L P I E V M V D A V H K L K Y E N Y T S S F F I R D I I 150 160 170 180 190 200 210
K P D P P K N L Q L K P L K N S R Q V E V S W E Y P D T W S T P H S Y F S L T F C V Q V G K S K R E K K D R V F T D K T S A T V I C R K N A 220 230 240 250 260 270 280
P40
S I S V R A Q D R Y Y S S S W S E WA S V P C S G T E T S Q V A P A 290 300 310
15 Cysteine residues
P19
Total: 95.9% Coverage, 2.41 Redundancy
3 potential N-linked glycosylation sites
R A V P G G S S P A W T Q C X X X S Q K L C T L A W S A H P L V G H M D L R E E G D E E T T N D V P H I Q C G D G C D P Q G L R D N S Q F C 10 20 30 40 50 60 70
P19
L Q R I H Q G L I F Y E K L L G S D I F T G E P S L L P D S P V G Q L H A S L L G L S Q L L Q P E G H H W E T Q Q I P S L S P S Q P WQ R L 80 90 100 110 120 130 140
X X X X K I L R S L Q A F X X X X A R V F A H G A A T L S P H H H H H H 150 160 170
Total: 93.8% Coverage, 1.42 Redundancy
10 sec
10 min
0% 10
60 min
360 min
20 30 40 50 >60% undetermined Relative % Deuterium
Iacob R.E. et al. Expert Rev. Proteomics (2015); 12(2), 159
Adnectin: 12.3 kDa
FG
BC N DE
C
10 sec
10 min
0% 10
60 min
360 min
20 30 40 50 >60% undetermined Relative % Deuterium Iacob R.E. et al. Expert Rev. Proteomics (2015); 12(2), 159
Unbound 10
Bound 5
1-14
24-37
9
P19
3
6
6
4
2
2
1
0 0.1
1.0
10.0 100.0 1000
9
6 3 0 0.1
1.0
10.0 100.0 1000
3
0 0.1 20
89-105
1.0
10.0 100.0 1000
0 0.1 8
116-140
15
6
10
4
5
2
0 0.1
1.0
10.0 100.0 1000
1.0
10.0 100.0 1000
145-153
0 0.1
1.0
P40
10.0 100.0 1000
Exposure Time (min) Triplicate runs; Avg. data point variation: +/- 0.12 Da
P19
10s 1m 10m 1h 4h
N
1-14 18-23 24-37 38-69
Peptides
Relative uptake (Da)
18-23
4
8
12
P40 12
70-80
-25%
-6%
-2%
0%
81-88 89-105 110-115 116-140
Protection upon binding Relative % Deuterium
145-153
C
158-176
Iacob R.E. et al. Expert Rev. Proteomics (2015); 12(2), 159
Unbound
15
1-9
6
41-56
12
16
9
12
6
8
132-152
P19
4 2
3
0 0.1
16
1.0
4
0 10.0 100.0 1000 0.1
196-218
20
12
15
8
10
N
1.0
0 10.0 100.0 1000 0.1
263-286
9
5
0 0.1
0 0.1
4h
27-40 1.0
10.0 100.0 1000
41-56 55-59
285-294
81-90
6
3 4
10s 1m 10m 1h 1-9 13-23
Peptides
Relative uptake (Da)
8
P40
Bound
91-106 103-135 132-152 153-169 173-180
1.0
10.0 100.0 1000
1.0
0 10.0 100.0 1000 0.1
1.0
184-188
10.0 100.0 1000
196-218
Exposure Time (min) Triplicate runs; Avg. data point variation: +/- 0.12 Da
214-230 231-245 246-251
P40
263-286 285-294
C
298-314
-25%
P19
-6%
-2%
0%
Protection upon binding Relative % Deuterium Iacob R.E. et al. Expert Rev. Proteomics (2015); 12(2), 159
P40
HDX-MS
N
C
196-218
P19
P40
18-37
89-105
100 101
P40
C
263-266 285 294
204
26 35 37 29 31
148
X-ray structure
N 100 101
P19
143-153
In-silico mutagenesis
N
P19
263-286 285-294
26 29
204
C 266
294
148 Iacob R.E. et al. Expert Rev. Proteomics (2015); 12(2), 159
EGFR binding to a monobody Interleukin-23 binding to a monobody Protein: small molecule interactions
28 approved inhibitors as of April 2015
Type I Active conformation
Type II Inactive conformation
Adapted from Clausen M.H. et al. Trends in Pharmacological Sciences (2015); 36 (7): 422
Type III Allosteric pocket Close to ATP pocket
Type IV Allosteric pocket Remote from ATP pocket
Imatinib Dasatinib
Sorafenib
Abl + Dasatinib
Allosteric inhibitors/ GNF5
Abl + GNF-5
Dasatinib Type I ATP pocket
GNF-5 Type IV Allosteric pocket
PDB: 2F4J
Change in HX relative to unbound form Significant
Subtle
No changes
Iacob R.E. et al. (2011). PLoS One, 6(1), e15929. Zhang J. et al. (2010). Nature, 463, 501.
S-andenosylmethionine (SAM) competitors In vivo efficacy
Yu W. et al. (2012). Nature Commun., 18(3), 1288
DOT1L
Protein
Protein+ drug 1min
10 sec
Drug 1
N
C
10s 1m 10m 1h
10s 1m 10m 1h
4h
10s 1m 10m 1h
60 min
4h
10s 1m 10m 1h 4h
#### 0.15 1.46 0.60 0.25
#### #### #### #### ####
#### #### 0.70 0.37 ####
#### 0.14 1.27 0.61 0.16
0.01 #### 0.08 0.05 0.07
0.20 0.16 0.00 0.04 0.08
0.03 0.06 #### #### ####
0.13 0.13 0.08 0.11 0.04
0.07 #### 0.10 0.07 0.07
0.25 0.10 0.02 0.08 0.05
0.11 0.02 0.04 #### 0.03
0.22 0.08 0.12 0.08 0.11
#### #### 0.65 0.41 ####
#### #### #### #### ####
#### #### 0.32 0.27 ####
#### #### 0.66 0.43 ####
0.45 #### 0.36 0.10 ####
0.37 #### #### #### 0.04
0.18 #### 0.08 #### ####
0.40 0.16 0.32 0.16 ####
#### 0.25 #### 0.04 ####
#### #### #### #### ####
#### #### #### 0.05 ####
#### 0.10 #### 0.15 ####
#### 0.08 0.09 #### 0.15
#### #### #### #### ####
#### #### 0.03 #### ####
#### 0.02 0.08 0.01 ####
0.15 0.10 1.04 1.12 0.86
0.05 0.15 #### #### ####
0.12 #### 0.91 0.99 0.63
0.08 0.35 1.22 1.20 0.69
0.15 #### 0.14 0.95 0.64
#### 0.12 #### #### ####
#### #### #### 0.40 0.37
#### 0.02 #### 0.61 0.56
0.25 0.48 1.01 1.61 1.47
0.11 0.37 #### #### ####
0.11 #### 0.61 1.33 1.11
0.00 0.47 1.16 1.82 1.54
0.14 0.17 0.41 0.51 0.79
#### #### 0.00 #### ####
0.01 #### 0.09 0.25 0.27
0.19 0.08 0.49 0.65 0.92
0.26 0.53 0.51 0.58 0.44
0.00 #### #### #### ####
0.00 #### 0.15 0.53 0.27
0.13 0.22 0.56 0.64 0.43
0.10
0.00 #### 0.09 ####
0.09 #### 0.04
#### #### #### ####
-1.0
Equation for subtraction DProtein+drug – DProtein
No data
4h
Drug 4
Drug 3
Drug 2 4h
10 min
-0.5
0.5
Exposure Drug 1 Drug 4
Drug 2 Drug 3
1.0
Difference in deuteration (Da) Protection in bound state
Protection
Exposure in bound state
HDX MS is a valuable addition to the analytical characterization toolkit of the biopharmaceutical industry
HDX MS offers peptide level resolution allowing for the accurate structural interrogation of every region in a biotherapeutic product
Unambiguous identification of epitopes is critical for rational design of biotherapeutics
HDX MS can pinpoint regions in a protein which become significantly protected upon binding, therefore supporting drug discovery efforts
Small molecules binding to target proteins can be screened with HDX MS
NORTHEASTERN UNIVERSITY
COLLABORATORS
Engen Lab: Prof. John Engen Prof. Thomas Wales Dr. Jamie Moroco Greg Pirrone Rane Harrison Brent Kochert Christopher Wilson Kristian Teichert
HARVARD MEDICAL SCHOOL
Dr. Guodong Chen Dr. Wei Hui Dr. Adrienne A. Tymiak
Dr. Nathanael Gray Dr. James Bradner
Dr. Thomas Smithgall
RESEARCH SUPPORT NIGMS
R01- GM086507 R01- GM101135
Dr. Roxana E. Iacob
Department of Chemistry & Chemical Biology, Northeastern University
21 atoms
Primary structure characterization
> 20,000 atoms
Higher order structures, aggregates and interactions Higher order • • • • •
X Ray NMR Native-MS IM-MS HDX-MS
Aggregates
Interactions (mAb/Ag)
• • • • • •
• • • • • • •
Adapted from Beck A. et al. (2013). Anal. Chem. ; 85: 715-736
SEC (UV-MALS) AUC Native-MS IM-MS HDX-MS Crosslinking MS
SPR ELISA FACS Native-MS IM-MS HDX-MS Crosslinking MS
Extremely important in biopharmaceutical industry/ $$$$$$ Identifying binding sites and epitopes between proteins and their binding partners are needed for the development of effective therapeutics Patent matters
BINDING SITE
BINDING INTERFACE
EPITOPE ANTIGENIC DETERMINANT
Alanine scanning Linear epitopes No info on dynamics Time consuming
X-ray crystallography
Computational modeling Docking
Requires a crystal In silico/ model
NMR
Mass Spectrometry
Limited proteolysis Non-covalent ESI Chemical crosslinking Oxidative labeling
HDX MS Info on dynamics Size limited Large sample requirements
Fast measurements Low sample requirements Linear/ discontinuous epitopes Info on surface accessibility Info on dynamics
Pirrone G.F. et al. (2015). Anal. Chem. ; 87(1): 99-118
Research
Process Development
Drug design
Folding/ Refolding
Structure-function
Comparability and stability
Epitope mapping Effects of PTMs on conformation
Impact of primary structure changes on conformation
Lead compound optimization
Formulation/ excipients
HDX MS improves protein biophysical characterization Wales T.E. & Engen J.R. (2006): Mass Spectrom. Rev. 25 158. Engen (2009): Anal. Chem. 81 7870.
INACTIVE/unmodified/innovator
Deuterium Backbone amide H
ACTIVE/modified/biosimilar
cut into pieces with pepsin weigh with mass spectrometer
Solution conformation of proteins is analyzed under physiological conditions!
Oxygen
Backbone carbon
Hydrogen
Nitrogen
Sidechain carbon
Backbone amide hydrogen
Gln
Asp
His
Pro
Lys
Leu
• Solvent accessibility • Hydrogen bonding
• pH • Temperature • Side-chain effects • Isotope effects • Salt Englander W. (1983) Q. Rev. Biophys 16, 521. Engen J.R. et al. (2011) Ency. Anal. Chem.
Commercial or homemade pepsin beads
Robots, LEAP system
MSE Peptide id
DynamX
Houde D. et al. (2011). J. Pharm Sci. 100(6), 2071. Marcsisin S. & Engen J.R. (2010). Anal. Bioanal. Chem. 397(3), 967.
Xevo or Synapt
e.g.
NanoACQUITY w/ HDX tech.
D. i.
All measurements at 0 °C
POROS 20 m
125 psi
1.00
2.00
3.00
4.00
5.00
6.00
UPLC very efficient at low temperatures
ii.
3.5 m 21 °C
1250 psi
10 °C
1.00
iii.
3.00
5.00
7.00
9.00
11.00
5 °C
1.75 m
~1 °C
8000 psi 2.00
1.00
2.00
3.00
4.00 5.00 Time (min)
Wales et al. 2008. Anal. Chem. 80, 6815.
6.00
7.00
8.00
3.00
4.00 5.00 Time
6.00
Fab digested online with pepsin
7.00
mAb 150 kDa 8000 psi at 65 µL/min 12 min gradient 0 ˚C
Cycle time
20 min
12000 psi at 100 µL/min 12 min gradient 0 ˚C
10 min
12000 psi at 100 µL/min +/- IMS 6 min gradient 0 ˚C 1.00
3.00
5.00 7.00 Time (min)
Engen J.R. & Wales T.E. (2015). Annu. Rev. Anal. Chem. 8, Jul 22;8(1): 127
9.00
11.00
ACQUITY UPLC HSS T3 1.8 m 1.0 x 50 mm
1HZH
Porcine pepsin %
0
Full length IgG- 150kDa
100 100
Rice Porcine field eelpepsin pepsin Porcine pepsin
100
Pepsin Aspergillopepsin/ Factor XIII Rice field eel pepsin
% %
% 0 0
0 100 100 100
Rice field eel pepsin Protease XIII Rice Porcine field eelpepsin pepsin
Rhizopuspepsin (protease type XVIII) Nepenthesin
% % % 0
0 1000
4.00
8.00
12.00
16.00
min
Protease XIII Rice field eel pepsin Protease XIII
100
%
%
Kadek A. et al. Anal. Chem. (2014); 6; 4287 Rey M. et al., Rapid Commun. Mass Spectrom. (2009); 23; 3431
0
Zhang H.M. et al. Anal. Chem. (2008); 80; 9034
4.00 100 4.00
Cravello L. et al. Rapid Commun. Mass Spectrom. (2003;) 17: 2387 Powers J.C. et al. Adv.Exp. Med. Biol. (1977); 95;141
0
8.00 8.00
12.00 12.00
16.00 16.00
min min
Ahn et al. BBA-Proteins and Proteomics, Protease (2013); 1834XIII (6): 1222
+ Deuterium
Decreased hydrogen exchange= binding interface?
Not always straightforward • Dissociation constant • Dynamic range • Compound solubility • Interface not always clear • Allostery
Engen J. R. (2003). Analyst, 128(6), 623 Konermann L. et al. (2011). Chem. Soc. Rev., 40, 1224
Protein 1
• •
Complex
Deuterium Labeling (15x D2O) time
Protein 2 Small molecule
Quenching (0 ºC, pH 2.4)
Online Pepsin Digestion
Software
NanoAcquity UPLC Separation
2.0 Peptide Identification by HDMSe+PLGS Deuterium Measurement by MS
DynamX
Data processing / DynamX
Waters UPLC w/ HDX
Synapt G2 Si
Deuterium Uptake Comparison Change/ Epitope peptide Relative Deuterium Level (Da)
15
15
Bou… Bound Free Free
12
12 9
6 3
12
9
9
6
6
3
3
0
0 0.1
1
10
100 1000 0.1 1
10
0
100 1000 0.1
Time (minutes) Ahn J. & Engen J. R. (2013). Chim. Oggi./ Chem. Today. 31, 25
15
No Change Bou… 7 Free 6 5 4 3 2 1 0 1
Bou… Bound Free Free
Bou nd Free
10 0.1
100 1 1000 10
100
Time (minutes)
1000
EGFR binding to a monobody Interleukin-23 binding to a monobody Protein: small molecule interactions
Mechanism of EGFR activation
2011 sales > $1 billion
mAbs
Epidermal Growth Factor (EGF)
Extracellular
NEED: New biotherapeutics: cheaper, easier, faster, more effective!!!
Intracellular
Ferguson K.M. (2008). Annu Rev Biophys. 37, 353
Derived from the 10th type III domain of human Fibronectin 1
2
3
4
5
6
7
8
9
10
11
12
13
14
IIICS
15
10Fn3
Type I Domain Type II Domain Type III Domain
Adnectin
Structural similarities with VH of an antibody
VH
Adnectins FG
CDRH1 CDRH2
ADVANTAGES
BC DE
CDRH3
High Structural homology Low Sequence homology
Lipovsek D. (2011). Protein Engineering, Design& Selection. 24, 3
Diversified CDR-like loops Tailored to bind to multiple targets KD: 0.7-2nM High thermo-stability Penetrate tissues faster Easier to manufacture
Extracellular EGFR: 69 kDa
L E E K K V C Q G T S N K L T Q L G T F E D H F L S L Q R M F N N C E V V L G N L E I T Y V Q R N Y D L S F L K T I Q E V A G Y V L I A L N T V E R I P L E N L 10 20 30 40 50 60 70 80
Q I I R G NM Y Y E N S Y A L A V L S N Y D A N K T G L K E L P M R N L Q E I L H G A V R F S N N P A L C N V E S I QW R D I V S S D F L S NM S MD F Q N H L 90 100 110 120 130 140 150 160
IV
C
50 Cysteine residues 10 potential N-linked glycosylation sites
II N
G S C Q K C D P S C P N G S C WG A G E E N C Q K L T K I I C A Q Q C S G R C R G K S P S D C C H N Q C A A G C T G P R E S D C L V C R K F R D E A T C K D T C 170 180 190 200 210 220 230 240 P P L M L Y N P T T Y QMD V N P E G K Y S F G A T C V K K C P R N Y V V T D H G S C V R A C G A D S Y E M E E D G V R K C K K C E G P C R K V C N G I G I G E 250 260 270 280 290 300 310 320
F K D S L S I N A T N I K H F K N C T S I S G D L H I L P V A F R G D S F T H T P P L D P Q E L D I L K T V K E I T G F L L I Q A W P E N R T D L H A F E N L E 330 340 350 360 370 380 390 400
I I R G R T K Q H G Q F S L A V V S L N I T S L G L R S L K E I S D G D V I I S G N K N L C Y A N T I NW K K L F G T S G Q K T K I I S N R G E N S C K A T G Q 410 420 430 440 450 460 470 480
I
III
V C H A L C S P E G C WG P E P R D C V S C R N V S R G R E C V D K C N L L E G E P R E F V E N S E C I Q C H P E C L P Q A MN I T C T G R G P D N C I Q C A H 490 500 510 520 530 540 550 560
PDB: 3QWQ
Y I D G P H C V K T C P A G V MG E N N T L V W K Y A D A G H V C H L C H P N C T Y G C T G P G L E G C P T N G P K H H H H H H 570 580 590 600 610 620
Total: 84.1% Coverage, 2.98 Redundancy
IV
C
II N
I
10 sec
III
10 min 0% 10
60 min
240 min
20 30 40 50 >60% undetermined Relative % Deuterium
Iacob R.E. et al. (2014). JASMS, 25(12), 2093
Adnectin: 12 kDa
G V S D V P R D L E V V A A T P T S L L I S W D S G R G S Y Q Y Y R I T Y G E T G G N S P V Q E F T V P G P V H T A T I S G L K P 10 20 30 40 50 60
G V D Y T I T V Y A V T D H K P H A D G P H T Y H E S P I S I N Y R T E I D K P S Q H H H H H H 70 80 90 100 110
Total: 90.3% Coverage, 1.56 Redundancy
FG N
BC DE
C
10 min
10 sec
0% 10
60 min
240 min
20 30 40 50 >60% undetermined
Relative % Deuterium Iacob R.E. et al. (2014). JASMS, 25(12), 2093
78 EGFR peptic peptides 82% Sequence coverage
EGFR
8
96-108 96-118 99-117 99-118 99-120 99-132
45-54 46-54
0 1-14 1-17 1-19 15-24 18-24
Difference (Da) [EGFR-(EGFR+ Adnectin)]
16
10 sec 10 min 60 min 240 min
-8
EGFR + Adnectin -16
Relative Uptake (Da)
18 16
1
10
20
EGFR+Adnectin EGFR
1 - 19
14
8 7
EGFR+Adnectin EGFR
46 - 54
6
12
4
8
3
6
4
2
2
1 1
10
100 300
0
12
96 - 108
10
50 EGFR+Adnectin EGFR
1
10
100 300
16 14
6
8
4
6
0
226 - 244
78
EGFR+Adnectin EGFR
10
4
2 0.08
60
12
8
5
10
0 0.08
30 40 Peptide position (N-to-C)
2 0.08
Exposure Time (min)
1
10
100 300
0
0.08
1
10
100 300
Iacob R.E. et al. (2014). JASMS, 25(12), 2093
IV
Decrease > 1Da
1-24
Decrease 0.5-1Da
II
I III
96-108 PDB: 3QWQ
Adnectin
HDX MS
Y101 S99
X-ray crystal structure
L69 L14
EGFR Residues L17
Q16
Adnectin residues
T15
DE loop ~CDRH2
BC loop ~CDRH1
FG loop ~CDRH3
Iacob R.E. et al. (2014). JASMS, 25(12), 2093 Ramamurthy V. et al. (2012). Structure. 20: 259
HDX-MS
N 1-24
C
45-54 96-108
T15 Q16 L17
S99 L69
Y101
G18
N 15-18
X-ray structure
C
69 99 101
Iacob R.E. et al. (2014). JASMS, 25(12), 2093
IL-23: Key participant in central regulation of the cellular mechanism involved in inflammation Ustekinumab
IL-12 IL-12
Janssen Biotech.
Ustekinumab
IL-23
Are there other biotherapeutics capable to inhibit IL-23 pro-inflammatory effects? Mascelli M.M. et al. (2011). Nature Biotechnology 29, 615
FG
BC
DE
I W E L K K D V Y X X X L D W Y P D A P G E M X X X T C D T P E E D G I T W T L D Q S S E V L G S G K T L T I Q V K E X X X X G Q Y T C H K 10 20 30 40 50 60 70
IL-23: 59 kDa
G G E V L S H S L L L L H K K E D G I W S T D I L K D Q K E P K N K T F L R C E A K N Y S G R F T C WW L T T I S D L T F S V K S S R G S S 80 90 100 110 120 130 140
P40
D P Q G V T C G A A T L S A E R V R G D N K E Y E Y S V E X X X D S A C P A A E E S L P I E V M V D A V H K L K Y E N Y T S S F F I R D I I 150 160 170 180 190 200 210
K P D P P K N L Q L K P L K N S R Q V E V S W E Y P D T W S T P H S Y F S L T F C V Q V G K S K R E K K D R V F T D K T S A T V I C R K N A 220 230 240 250 260 270 280
P40
S I S V R A Q D R Y Y S S S W S E WA S V P C S G T E T S Q V A P A 290 300 310
15 Cysteine residues
P19
Total: 95.9% Coverage, 2.41 Redundancy
3 potential N-linked glycosylation sites
R A V P G G S S P A W T Q C X X X S Q K L C T L A W S A H P L V G H M D L R E E G D E E T T N D V P H I Q C G D G C D P Q G L R D N S Q F C 10 20 30 40 50 60 70
P19
L Q R I H Q G L I F Y E K L L G S D I F T G E P S L L P D S P V G Q L H A S L L G L S Q L L Q P E G H H W E T Q Q I P S L S P S Q P WQ R L 80 90 100 110 120 130 140
X X X X K I L R S L Q A F X X X X A R V F A H G A A T L S P H H H H H H 150 160 170
Total: 93.8% Coverage, 1.42 Redundancy
10 sec
10 min
0% 10
60 min
360 min
20 30 40 50 >60% undetermined Relative % Deuterium
Iacob R.E. et al. Expert Rev. Proteomics (2015); 12(2), 159
Adnectin: 12.3 kDa
FG
BC N DE
C
10 sec
10 min
0% 10
60 min
360 min
20 30 40 50 >60% undetermined Relative % Deuterium Iacob R.E. et al. Expert Rev. Proteomics (2015); 12(2), 159
Unbound 10
Bound 5
1-14
24-37
9
P19
3
6
6
4
2
2
1
0 0.1
1.0
10.0 100.0 1000
9
6 3 0 0.1
1.0
10.0 100.0 1000
3
0 0.1 20
89-105
1.0
10.0 100.0 1000
0 0.1 8
116-140
15
6
10
4
5
2
0 0.1
1.0
10.0 100.0 1000
1.0
10.0 100.0 1000
145-153
0 0.1
1.0
P40
10.0 100.0 1000
Exposure Time (min) Triplicate runs; Avg. data point variation: +/- 0.12 Da
P19
10s 1m 10m 1h 4h
N
1-14 18-23 24-37 38-69
Peptides
Relative uptake (Da)
18-23
4
8
12
P40 12
70-80
-25%
-6%
-2%
0%
81-88 89-105 110-115 116-140
Protection upon binding Relative % Deuterium
145-153
C
158-176
Iacob R.E. et al. Expert Rev. Proteomics (2015); 12(2), 159
Unbound
15
1-9
6
41-56
12
16
9
12
6
8
132-152
P19
4 2
3
0 0.1
16
1.0
4
0 10.0 100.0 1000 0.1
196-218
20
12
15
8
10
N
1.0
0 10.0 100.0 1000 0.1
263-286
9
5
0 0.1
0 0.1
4h
27-40 1.0
10.0 100.0 1000
41-56 55-59
285-294
81-90
6
3 4
10s 1m 10m 1h 1-9 13-23
Peptides
Relative uptake (Da)
8
P40
Bound
91-106 103-135 132-152 153-169 173-180
1.0
10.0 100.0 1000
1.0
0 10.0 100.0 1000 0.1
1.0
184-188
10.0 100.0 1000
196-218
Exposure Time (min) Triplicate runs; Avg. data point variation: +/- 0.12 Da
214-230 231-245 246-251
P40
263-286 285-294
C
298-314
-25%
P19
-6%
-2%
0%
Protection upon binding Relative % Deuterium Iacob R.E. et al. Expert Rev. Proteomics (2015); 12(2), 159
P40
HDX-MS
N
C
196-218
P19
P40
18-37
89-105
100 101
P40
C
263-266 285 294
204
26 35 37 29 31
148
X-ray structure
N 100 101
P19
143-153
In-silico mutagenesis
N
P19
263-286 285-294
26 29
204
C 266
294
148 Iacob R.E. et al. Expert Rev. Proteomics (2015); 12(2), 159
EGFR binding to a monobody Interleukin-23 binding to a monobody Protein: small molecule interactions
28 approved inhibitors as of April 2015
Type I Active conformation
Type II Inactive conformation
Adapted from Clausen M.H. et al. Trends in Pharmacological Sciences (2015); 36 (7): 422
Type III Allosteric pocket Close to ATP pocket
Type IV Allosteric pocket Remote from ATP pocket
Imatinib Dasatinib
Sorafenib
Abl + Dasatinib
Allosteric inhibitors/ GNF5
Abl + GNF-5
Dasatinib Type I ATP pocket
GNF-5 Type IV Allosteric pocket
PDB: 2F4J
Change in HX relative to unbound form Significant
Subtle
No changes
Iacob R.E. et al. (2011). PLoS One, 6(1), e15929. Zhang J. et al. (2010). Nature, 463, 501.
S-andenosylmethionine (SAM) competitors In vivo efficacy
Yu W. et al. (2012). Nature Commun., 18(3), 1288
DOT1L
Protein
Protein+ drug 1min
10 sec
Drug 1
N
C
10s 1m 10m 1h
10s 1m 10m 1h
4h
10s 1m 10m 1h
60 min
4h
10s 1m 10m 1h 4h
#### 0.15 1.46 0.60 0.25
#### #### #### #### ####
#### #### 0.70 0.37 ####
#### 0.14 1.27 0.61 0.16
0.01 #### 0.08 0.05 0.07
0.20 0.16 0.00 0.04 0.08
0.03 0.06 #### #### ####
0.13 0.13 0.08 0.11 0.04
0.07 #### 0.10 0.07 0.07
0.25 0.10 0.02 0.08 0.05
0.11 0.02 0.04 #### 0.03
0.22 0.08 0.12 0.08 0.11
#### #### 0.65 0.41 ####
#### #### #### #### ####
#### #### 0.32 0.27 ####
#### #### 0.66 0.43 ####
0.45 #### 0.36 0.10 ####
0.37 #### #### #### 0.04
0.18 #### 0.08 #### ####
0.40 0.16 0.32 0.16 ####
#### 0.25 #### 0.04 ####
#### #### #### #### ####
#### #### #### 0.05 ####
#### 0.10 #### 0.15 ####
#### 0.08 0.09 #### 0.15
#### #### #### #### ####
#### #### 0.03 #### ####
#### 0.02 0.08 0.01 ####
0.15 0.10 1.04 1.12 0.86
0.05 0.15 #### #### ####
0.12 #### 0.91 0.99 0.63
0.08 0.35 1.22 1.20 0.69
0.15 #### 0.14 0.95 0.64
#### 0.12 #### #### ####
#### #### #### 0.40 0.37
#### 0.02 #### 0.61 0.56
0.25 0.48 1.01 1.61 1.47
0.11 0.37 #### #### ####
0.11 #### 0.61 1.33 1.11
0.00 0.47 1.16 1.82 1.54
0.14 0.17 0.41 0.51 0.79
#### #### 0.00 #### ####
0.01 #### 0.09 0.25 0.27
0.19 0.08 0.49 0.65 0.92
0.26 0.53 0.51 0.58 0.44
0.00 #### #### #### ####
0.00 #### 0.15 0.53 0.27
0.13 0.22 0.56 0.64 0.43
0.10
0.00 #### 0.09 ####
0.09 #### 0.04
#### #### #### ####
-1.0
Equation for subtraction DProtein+drug – DProtein
No data
4h
Drug 4
Drug 3
Drug 2 4h
10 min
-0.5
0.5
Exposure Drug 1 Drug 4
Drug 2 Drug 3
1.0
Difference in deuteration (Da) Protection in bound state
Protection
Exposure in bound state
HDX MS is a valuable addition to the analytical characterization toolkit of the biopharmaceutical industry
HDX MS offers peptide level resolution allowing for the accurate structural interrogation of every region in a biotherapeutic product
Unambiguous identification of epitopes is critical for rational design of biotherapeutics
HDX MS can pinpoint regions in a protein which become significantly protected upon binding, therefore supporting drug discovery efforts
Small molecules binding to target proteins can be screened with HDX MS
NORTHEASTERN UNIVERSITY
COLLABORATORS
Engen Lab: Prof. John Engen Prof. Thomas Wales Dr. Jamie Moroco Greg Pirrone Rane Harrison Brent Kochert Christopher Wilson Kristian Teichert
HARVARD MEDICAL SCHOOL
Dr. Guodong Chen Dr. Wei Hui Dr. Adrienne A. Tymiak
Dr. Nathanael Gray Dr. James Bradner
Dr. Thomas Smithgall
RESEARCH SUPPORT NIGMS
R01- GM086507 R01- GM101135
