Structural characterisation and epitope mapping by HDX-MS (Advanced Analytical Technologies for Proteins, Romainville 2019)

1Arnaud Delobel, PhD – Advanced Analytical Technologies for Proteins – Romainville, France – October 2nd 2019
Structural characterisation
and epitope mapping by
HDX-MS
Arnaud Delobel, PhD
R&D Director
Advanced Analytical Technologies for Proteins – Romainville, France – October 2nd 2019

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MS expertise on biotherapeutics since 2006
• First MS protein characterisation
services offered to our customers in
2006
• Mass spectrometers:
▪ QTOF Premier (Waters) – 2006
▪ MALDI Microflex (Bruker) – 2008
▪ Amazon ETD ion trap (Bruker) – 2010
• Wide range of services including
intact mass, peptide mapping,
glycosylation analysis, etc.
(+ several triple quad systems
for quantitative LC-MS/MS)

LC/MS toolbox for Protein characterisation
• LC/MS (MSE CID)
▪ H-Class Bio (Waters)
▪ Acquity 2D-LC (multiple heartcut)
▪ HDX-2 automation system w/ Acquity M-class
▪ Xevo G2-XS QTOF (Waters)
▪ UNIFI 1.8
• LC/MS (QDa)
▪ Acquity UPLC (Waters)
▪ QDa detector (Waters)
• LC/MS (MS/MS ETD)
▪ Acquity UPLC (Waters)
▪ Amazon ETD Ion Trap (Bruker)
• MALDI-TOF
▪ Microflex II LRF60 (Bruker)
• LC-MS/MS
▪ Xevo TQ and TQS (Waters)
▪ Acquity I-Class UPLC
▪ Acquity 2D-LC (single heartcut)

Introduction to
HDX-MS

What is HDX?
H’s
D2O
solution added
Engen JR. Anal Chem. (2009) 81(19) 7870-5.
Most accessible H
exchanges faster
Least accessible H
exchanges more
slowly
H’s vs. D’s
at backbone amide positions
1 Da 2 Da
We measure H-D exchange
as the mass increase of
a protein or peptide

Hydrogen-Deuterium Exchange
Important factors that affect deuterium
exchange rates:
The amount of deuteration
on the protein backbone can be directly related to protein
structure, conformational change, and protein-protein interaction.
Amide hydrogen in protein backbone
 The rate of exchange in solution phase is in the range
that LC-MS can measure from seconds to hours
 One NH in every AA except proline
Hydrogen to carbon
 No exchange
Side chain Hydrogen
 Exchanges too fast
 Washes off in LC-MS
D2O
added
 Solvent accessibility
 Hydrogen bonds
 Temperature
 pH

pH and temperature influence HDX
Conditions of pH 2.5 (or 2.6) and 0 oC:
 Are used to quench the exchange reaction
 Maintaining this condition is important to prevent back-exchange (reversed phenomenon)
 Must be maintained during digestion and LC-MS because H2O is introduced to labeled protein(s) and peptides
-4
-3
-2
-1
0
1
2
3
4
0 1 2 3 4 5 6 7 8 9
pH
log[k2(sec-1)]
pH effect Temperature effect
Min. at
pH 2.5 – 2.6
Min. at 0 °C

HDX-MS in the old days …
 Manual
 Poor temperature
control
 Poor
reproducibility
 Safety risks
with online LC/MS

HDX-MS solution used at Quality Assistance
UPLC platform for nano- to
microscale separations
Xevo G2-XS benchtop QTOF

HDX-MS workflows at protein and peptide level
LC separationESI-QTOF
(Xevo G2-XS)

Raw LC/MS data
IncreasingD2Oexposuretime

Measurement of deuterium uptake
0
1
2
3
4
5
6
7
0.10 1.00 10.00 100.00 1000.00
RelativeDeuteriumLevel(Da)
Time (min)
12-19
APO HOLO
http://www.hxms.com/HXExpress
Centroid of isotopic
distribution used to
calculate D uptake
-100
0
100
200
300
400
500
600
700
494 495 496 497 498 499 500
m/z
11-18 (FKEAFSLF 2) {240.00 min}
spectrum
spectrum
distriubtion width
centroid
peak envelope
0
100
200
300
400
500
600
700
494 495 496 497 498 499 500
m/z
11-18 (FKEAFSLF 2) {10.00 min} spectrum
spectrum
distriubtion width
centroid
peak envelope
0
100
200
300
400
500
600
700
800
494 495 496 497 498 499 500
m/z
11-18 (FKEAFSLF 2) {1.00 min} spectrum
spectrum
distriubtion width
centroid
peak envelope
0
200
400
600
800
1000
1200
1400
494 495 496 497 498 499 500
m/z
11-18 (FKEAFSLF 2) {undeut} spectrum
spectrum
distriubtion width
centroid
peak envelope
4 h
10 m
1 m
0 sec control
(m0%)
Relative deuterium uptake
for selected peptide
plotted against time
course
 Centroid mass (m) is shown by green bar
 The blue bar shows shift in relative deuterium
level = m − m0%, where m is the mass after
H/D exchange and m0% is the unexchanged
mass for that peptide

Data processing and reporting
Uptake
Comparison
Data
Processing
Peptide
Identification
Deuterium
Uptake
Determination
Result
Visualisation

HDX-MS Answers Structural Questions
at the Global, Local, and Residue Levels

• Sample flexibility:
▪ Lower sample requirements compared to other high-resolution
techniques (e.g. NMR, X-Ray)
▪ Tolerance for impurities and formulants (buffers, salts, etc.)
▪ Proteins observed closer to physiological conditions
▪ Rapidly expanding envelope for larger proteins and more complex
systems
• Information-rich
▪ Global, regional, and local structural information
▪ HDX gives insights on both structure and dynamics of proteins and
protein interactions
Advantages of HDX-MS

Did my protein
fold correctly?
Where does the drug
interact with the target
protein?
Can I identify
sites of protein
aggregation?
Did this mutation affect
protein structure?
vs.
?
Are there
conformational changes
after X?
?
Where does my
antibody bind
with its target?
Formulations and
Stability Testing
Do these processes make
the “same” protein?
?
Batch to Batch ; Site to Site
Innovator vs. Biosimilar
Epitope Mapping
Comparability
Biobetters,
Candidate Selection
HDX-MS applied to biotherapeutics development

• Human intervention during data
processing is still significant
• Double check of the analysis by a
second analyst is not feasible
• Workflow is complex and many
things can go wrong
• Results variability should be
expected from one run to another
There are still some limitations with HDX-MS for its use in a
pharma environment …
Need for system
suitability checks

• Allows to check that the LC/MS part is working correctly
• Manual injection of a cytochrome c digest (400 nM)
▪ Identification of all relevant peptides
▪ Excellent mass accuracy: 0.78 ppm weighted average
LC/MS system performance verification

Verification of digestion and detection performances
318 peptides, 98.5% coverage, 6.1 redundancy
Consistent deuterium uptake (10-min incubation)
HDX-MS system performance verification using adalimumab
HC
LC
*
*: G1F, G0F, Man5, G1F-GlcNAc

Application to comparability
and stability studies

• Batch-to-batch comparison
• Comparability study after a modification of the manufacturing process
(cf. ICH Q5E)
• Biosimilarity studies
• Stability studies
(comparison between the reference standard and the stability sample at
each time point)
Comparability studies

• Humira® (adalimumab 10 mg/mL)
▪ Heat-stressed at 60°C for 24 hours
▪ Buffer-exchanged in Na Phosphate 10 mM, 100 mM NaCl, pH 6.8
▪ Final concentration: 30 µM
• HDX
▪ 15 s, 1 min, 10 min, 1 h, 2 h in 90% D2O (same buffer)
• Quench/denaturation/reduction
▪ Na Phosphate 100 mM, pH 2.3 + 400 mM TCEP + 4 M guanidine
▪ 1:1 mixture at 1.0°C for 2 min
Experimental design

Sequence coverage
HC: 98.7%, 5.33 peptides per aa
LC: 100%, 4.69 peptides per aa

Butterfly plot
HC
LC

• Global folding conserved
• Almost no peptide with difference > 1 Da for single incubation times
• Some peptides with large summed differences
▪ Which amino-acids are most impacted by heat stress?
▪ For each aa: determination of mean of summed uptake across all overlapped peptides, normalized by their
amount of exchangeable H
Butterfly plot
∆𝑈𝑝,𝐻 =
∆𝑈𝑝𝑡
𝐻𝑝
∆𝑈𝑝(𝑡) = 𝑈𝑝,1(𝑡) − 𝑈𝑝,2(𝑡)
𝐻𝑝 = 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑎𝑎 − 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑃𝑟𝑜 − 1
N-glycosylation

Amino-acid resolution results

Heatmap
E V Q L V E S G G G L V Q P G R S L R L S C A A S G F T F D D Y A M H W V R Q A P G K G L E W V S A
I T W N S G H I D Y A D S V E G R F T I S R D N A K N S L Y L Q M N S L R A E D T A V Y Y C A K V S
Y L S T A S S L D Y W G Q G T L V T V S S A S T K G P S V F P L A P S S K S T S G G T A A L G C L V
K D Y F P E P V T V S W N S G A L T S G V H T F P A V L Q S S G L Y S L S S V V T V P S S S L G T Q
T Y I C N V N H K P S N T K V D K K V E P K S C D K T H T C P P C P A P E L L G G P S V F L F P P K
P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K P R E E Q Y
N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K A L P A P I E K T I S K A K G Q P R E P
Q V Y T L P P S R D E L T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P
V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G K
135 140 145 150
155 160 165 170 175
105 110 115 120 125 130
205 210 215 220 225
180 185 190 195 200
255 260 265 270 275
230 235 240 245 250
305 310 315 320 325
280 285 290 295 300
355 360 365 370 375
330 335 340 345 350
405 410 415 420 425
380 385 390 395 400
55 60 65 70 75
430 435 440 445 450
1 5 10 15 20 50
80 85 90 95 100
25 30 35 40 45
D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q G I R N Y L A W Y Q Q K P G K A P K L L I Y A
A S T L Q S G V P S R F S G S G S G T D F T L T I S S L Q P E D V A T Y Y C Q R Y N R A P Y T F G Q
G T K V E I K R T V A A P S V F I F P P S D E Q L K S G T A S V V C L L N N F Y P R E A K V Q W K V
D N A L Q S G N S Q E S V T E Q D S K D S T Y S L S S T L T L S K A D Y E K H K V Y A C E V T H Q G
L S S P V T K S F N R G E C
251 5 10 15 20
55 60 65 70 75
30 35 40 45 50
80 85 90 95 100
135 140 145 150
155 160 165 170 175
105 110 115 120 125
205 210
130
180 185 190 195 200
214
min max∆𝑼 𝒂𝒂
CDR

Most and least impacted amino acids
C-terminus
N-glycosylation
site
CDRHC
N-terminus

• HDX-MS is a very valuable approach for comparability studies
▪ Stability
▪ Stress
▪ Biosimilarity
• Gives structural information that traditional LC(/MS) and spectroscopic
methods do not detect
• Bridges these techniques with biological testing
(e.g. binding assays or potency assays)
Conclusions for comparability / stability studies

Application to epitope
mapping

• Collaboration with OSE Immunotherapeutics on 3 monoclonal antibodies
targeting IL-7R, incl. OSE-127, currently in Phase I for the treatment of
inflammatory autoimmune diseases.
Interleukin-7 (IL7) is a cytokine that controls the proliferation, apoptosis and activation of
CD4 and CD8 effector T-cells in humans.
• Question: do all 3 antibodies target the same epitope on IL7-R (CD127)?
Case study

• 3 mAbs w/ interleukin receptor (CD127 / IL7-R)
▪ Buffer-exchanged in Na Phosphate 10 mM, 100 mM NaCl, pH 6.8
▪ Final concentration 22 µM (Kd in nM range)
• HDX
▪ 30 min in 90% D2O (same buffer)
• Quench/denaturation/reduction
▪ Na Phosphate 100 mM, pH 2.3 + 400 mM TCEP + 4 M guanidine
▪ 1:1 mixture at 1.0°C for 2 min
Experimental design

How can HDX answer the question?
Antigen without mAB
Antigen with mAB
Residues not accessible
to solvent: H/D exchange
is slower
• Binding of a given protein region to another protein typically decreases its D uptake rate because
the involved amino acids become more buried and/or establish new H-bonds
• The epitope can be mapped by monitoring regions that display reduced deuterium uptakes upon
binding

Interleukine receptor coverage map

Deuterium uptake
mAb A
mAb B
mAb C

Hit discovery
-70
-60
-50
-40
-30
-20
-10
0
10
20
30
40
50
60
70
-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0
SSMD
Average log2 fold change
Dual-flashlight plot
78-84
78-85
79-84 74-81
72-85
71-77
Magnitude of change protectiondeprotection
Effectsize
-7.5
-2.5
2.5
7.5
-1.0 -0.5 0.0 0.5 1.0
SSMD
Average log2 fold change
107-125
109-123
85-93
72-77
93-101
216-224
 {SSMD > 5; log2 fold change > 1}
𝑺𝑺𝑴𝑫 =
𝑫𝑰𝑳 − 𝑫 𝒄𝒑𝒍𝒙
𝑺𝑫𝑰𝑳
𝟐
+ 𝑺𝑫 𝒄𝒑𝒍𝒙
𝟐
=
∆𝑫
𝑺𝑫 𝑷
Strictly standardised
mean difference

Mass spectra
undeuterated reference
antigen
complex

Heatmap
mAb A mAb B mAb C

• The study showed that:
▪ The 3 antibodies bind to the same epitope on
CD127
▪ One antibody seems to bind a secondary
epitope
• The results confirmed what was
observed with other techniques
• HDX-MS allowed fast determination of
epitopes in solution
Here, the complex could not be crystallised!
Conclusions of the epitope mapping studies

Take-home messages
HDX-MS is a powerful
technique for the
structural characterisation
of biotherapeutics
Comparability studies
(biosimilars, process dev.,
stability studies)
Epitope mapping
High resolution technique
Good alternative to
techniques such as X-ray
diffraction or NMR
« Quick » results

Acknowledgments
▪ Aurélien Boland
▪ Berhard Poschner
▪ Nicolas Poirier
▪ Bernard Vanhove
▪ Eric Largy
▪ Claire Butré
▪ Caroline Cajot

arnaud.delobel@quality-assistance.be
+32 71 53 47 81
www.quality-assistance.com
Technoparc de Thudinie, 2
B-6536 Donstiennes (Belgium)
Thank you for your
attention
Any question?

Structural characterisation and epitope mapping by HDX-MS (Advanced Analytical Technologies for Proteins, Romainville 2019)

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