1. The webinar discusses hydrogen-deuterium exchange mass spectrometry (HDX-MS), which is a powerful tool for characterizing biopharmaceuticals. HDX-MS can provide global, local, and residue-level structural information without the need for crystallization. 2. The webinar presents a case study where HDX-MS was used to compare the structure of an antibody before and after heat stress. The analysis identified regions with increased deuterium uptake, indicating local structural changes. 3. HDX-MS was also used to map the epitopes of three monoclonal antibodies targeting the interleukin-7 receptor. Differences in deuterium uptake upon antibody binding identified regions involved in

1. 1
Webinar
Hydrogen-Deuterium Exchange Mass
Spectrometry (HDX-MS):
a powerful tool for biopharmaceutical
characterisation
Arnaud Delobel, PhD
R&D Director

2. 2
• A quick word about Quality Assistance
• Introduction on HDX/MS
What is it? How does it work?
Applications of HDX/MS for biopharma
• Case studies
Comparability studies
Epitope mapping
Agenda

3. 3
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4. 4
Introduction on
HDX/MS

5. 5
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

6. 6
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

7. 7
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
pH effect
Temperature effect
Min. at
pH 2.5 – 2.6
Min. at 0 °C

8. 8
HDX/MS in the old days …
Manual
Poor
temperature
control
Poor
reproducibility
Safety risks
with online
LC/MS

9. 9
HDX/MS solution used at Quality Assistance
UPLC platform for nano- to
microscale separations
Xevo G2-XS benchtop QTOF

10. 10
HDX-MS workflows at protein and peptide level
LC separationESI-QTOF
(Xevo G2-XS)

11. 11
Raw LC/MS data

12. 12
Measurement of deuterium uptake
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
0
100
200
300
400
500
600
700
494 495 496 497 498 499 500
m/z
0
100
200
300
400
500
600
700
800
494 495 496 497 498 499 500
m/z
0
200
400
600
800
1000
1200
1400
494 495 496 497 498 499 500
m/z
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
0%, where m is the mass after
H/D exchange and m0% is the unexchanged
mass for that peptide

13. 13
Data processing and reporting
Uptake
Comparison
Peptide
Identification
Deuterium
Uptake
Determination
Result
Visualisation
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
Heat map

14. 14
HDX-MS Answers Structural Questions
at the Global, Local, and Residue Levels
Resolution
AUC
SEC/MALLS
HDX/MS
Covalent labeling
EM
HDX/MS
+ ETD-MS/MS
NMR
X-ray
HDX/MS
(protein level)
Ion mobility MS
Spectroscopic (FLR, CD, IR)
Calorimetry
Global Local Amino acid

15. 15
• 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

16. 16
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

17. 17
• 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
Need for system
suitability checks

18. 18
• 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

19. 19
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

20. 20
Application to
comparability and
stability studies

21. 21
• 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

22. 22
• 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

23. 23
Sequence coverage
HC: 98.7%, 5.33 peptides per aa
LC: 100%, 4.69 peptides per aa

24. 24
Butterfly plot
HC
LC

25. 25
• 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
, =
( ) = ,1( ) ,2( )
= 1
N-glycosylation

26. 26
Amino-acid resolution results

27. 27
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
CDR

28. 28
Most and least impacted amino acids
C-terminus
N-glycosylation
site
CDRHC
N-terminus

29. 29
• 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
• Possibility to bridge these techniques with biological
testing (e.g. binding assays or potency assays)
Conclusions for comparability / stability studies

30. 30
Application to epitope
mapping

31. 31
• 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

32. 32
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

33. 33
• 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

34. 34
Interleukine receptor coverage map

35. 35
Deuterium uptake
mAb A
mAb B
mAb C

36. 36
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
Average log2 fold change
Dual-flashlight plot
78-84
78-85
79-84 74-81
72-85
71-77
Magnitude of change protectiondeprotection
-7.5
-2.5
2.5
7.5
-1.0 -0.5 0.0 0.5 1.0
Average log2 fold change
107-125
109-123
85-93
72-77
93-101
216-224
{SSMD > 5; log2 fold change > 1}
=
+
=
Strictly standardised
mean difference

37. 37
Mass spectra

38. 38
Heatmap
mAb A mAb B mAb C

39. 39
• 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
Belarif et al., Nat. Commun. (2018)
doi: 10.1038/s41467-018-06804-y

40. 40
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
Now available as a service at Quality Assistance

41. 41
Acknowledgments
Aurélien Boland
Bernhard Poschner
Nicolas Poirier
Bernard Vanhove
Eric Largy
Claire Butré
Caroline Cajot

42. 42
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?