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European leader in analytical sciences
Written by : Géry Van Vyncht
R&D Director, S&I Department
24/09/2015
STRESS TESTING AND
PHYSICO-CHEMICAL
CHARACTERIZATION OF
MONOCLONAL
ANTIBODIES :
THE APPROACH OF AN
ANALYTICAL CRO
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THE COMPANY
Updated: 7th September 2015
100% analytical services
100% (bio)pharmaceutical industry
Over 30 years (since 1982)
148 highly-qualified employees
> 60% university graduates
90 worldwide R&D companies (2014)
All laboratories on 1 site
Product dedicated support
Customised project management
Compliance with EMA / FDA regulations
From discovery to market place
5 200 m² on 1 site
~2 M € in machinery & equipment (2015)
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Accelerated stress studies for biotech products :
Main degradation pathways / Stability
Validation / specificity assessment of stability indicating chromatographic methods (IEX, SEC)
Peak identification for purity / impurity profiling
Efficient generic approach on mAbs using state-of-the-art analytics :
Oxidative stress and deamidation (and PTMs)
IEX and 2D-UPLC/MS on the intact protein (isoforms, DP)
Middle-up information on sub-units (enzymatic digestion followed by RP-HPLC/MS)
Amino acid level by peptide mapping (RP-UPLC/MSE)
INTRODUCTION / CONTENT
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Therapeutic mAbs undergo numerous post-translational modifications and degradations
Heavy chain N-terminal pyroglutamic acid (-17/-18)
N-glycosylation
C-terminal lysine truncation (-128 Da)
Deamidation (+1/-18 Da)
Oxidation (+16 Da)
MABS : PTMS & DEGRADATION
Figure adapted from Beck et al. Anal. Chem. 2012, 84, 4637−4646
-NSTY-
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INTACT LEVEL ANALYSIS
2D-LC/MS (IEX X RP) OF HUMIRA
(ADALIMUMAB)
5
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Autoblend optimization / WCX (1/2)
Minutes
18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00 32.50 33.00 33.50 34.00 34.50 35.00 35.50 36.00
6.56.76.97.1
pH
AU
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
0.28
0.30
0.32
0.34
0.36
0.38
0.40
0.42
0.44
Minutes
18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00
[MOPS]
50 mM 40 mM 20 mM
INTACT LEVEL ANALYSIS
6
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AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
Minutes
18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00 32.50 33.00 33.50 34.00 34.50 35.00 35.50 36.00 36.50 37.00 37.50 38.00 38.50 39.00 39.50 40.00
NaCl gradient
Minutes
20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00 32.50 33.00 33.50 34.00 34.50 35.00 35.50 36.00 36.50 37.00 37.50 38.00
Buffer
MOPS MES
a
b
c
d
e
INTACT LEVEL ANALYSIS
Autoblend optimization / WCX (2/2)
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Final IEX (WCX) conditions
Final conditions allow the efficient resolution of both acidic and basic species :
MOPS, 50 mM
pH 7.1
Gradient b
0.750 ml/min
25 - 100 µg injection
Amenable to 2D-LC/MS
INTACT LEVEL ANALYSIS
8
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AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
0.055
Minutes
20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00
AU
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
0.090
0.100
0.110
0.120
Minutes
20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00 32.50
Initial
Carboxypeptidase b digestion
INTACT LEVEL ANALYSIS
C-Terminal Lys isoforms
9
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AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
0.055
Minutes
20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00
0
5
10
15
0 10 20
%A
rea
Time (h)
AU
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
0.055
0.060
0.065
0.070
0.075
0.080
0.085
0.090
Minutes
20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00 32.50
* **
Initial
Stressed
(100 mM Tris, pH 8)
INTACT LEVEL ANALYSIS
Basic pH stress
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Heartcutting to trap & 2D
WCX elution profiles equivalent with and without deglycosylation (slight RT shift only)
Higher quality MS spectra after deglycosylation
Targeted heartcuts (6 s)
Focalisation and desalting (> 20 min ; H2O / HCOOH) on a trap column (Waters X-Bridge BEH300 C4 ; 30 mm, 3.5 µm)
Injection on a RP-UPLC second dimension column for on-line MS analysis (Waters BEH300 C4 ; 50 x 2.1 mm, 1.7 µm)
INTACT LEVEL ANALYSIS
11
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Main Basic 1 Basic 2Acid 2Acid 1
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
0.055
Minutes
20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00
INTACT LEVEL ANALYSIS
Heartcutting to trap & 2D
12
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Species identification after desalting & 2D
Main
Basic 1
Basic 2
Acid 2
Acid 1
+ 1 Lys
+ 2 Lys
Deamidations
Deamidations
and/or Oxidation
Raw data Deconvoluted (centroid)
INTACT LEVEL ANALYSIS
13
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SUB-UNIT LEVEL ANALYSIS
RP-LC/MS OF HUMIRA (ADALIMUMAB)
14
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Large range of RP columns tested
Desalting,sub-unit separation, with glycosylation and lysine truncation detection:
Waters MassPrep Micro Desalting (HPLC)
Waters BEH300 C4 (UPLC)
Phenomenex Aeris Widepore C4 (Core shell)
Subunit separation with detection of additional post-translational modifications:
Agilent PLRP-S (HCOOH or TFA)
Thermo Scientific MAbPac RP (50 x 3 mm ; 5 µm ; 1500 Å ; TFA)
SUB-UNIT LEVEL ANALYSIS
15
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Separation of isoforms, PTMs and degradations
IdeS(30 min; with / without
buffer exchange)
1 µg mAb
Reduction(DTT)
Oxidation PyroGlu
ox
Intact
F(ab’)2
Fc/2
Fd
Oxidation PyroGlu
No glycosylation± Lys
Fc/2 LC
SUB-UNIT LEVEL ANALYSIS
16
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H2O2 ↗
0 %
Fc/2 F(ab’)2Non oxidized
OxOx
Ox x 2 Non oxidized
Ox
Ox
Ox x 2
0.2 %
Oxidative stress (45 min)
SUB-UNIT LEVEL ANALYSIS
17
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RT ↗ Fc/2 G0F
Fc/2 G1F
Fc/2 Oxidized G0F
Fc/2 Oxidized G1F
Fc/2 2 x Oxidized G0F
Fc/2 2 x Ox G1F+TFA
SUB-UNIT LEVEL ANALYSIS
Oxidative stress / MS identification
18
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0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1
% m
od
ifie
d
% H2O2
Fc/2 ox1
Fc/2 ox2
F(ab')2 ox1
F(ab')2 ox2
Oxidative stress
SUB-UNIT LEVEL ANALYSIS
19
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RP-UPLC-ESI/MSE PEPTIDE MAPPING
OF HUMIRA (ADALIMUMAB)
20
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High confidence sequence coverage
UPLC gradient : 85 min, using ACN / H20 / TFA ; 1-10 µg injected
Fully integrated analysis, process and reporting (Unifi)
Fast, total sequence coverage at 10 ppm, regardless of the column used
≥ 93% of sequences found with < 5 ppm error and confirmed by at least 2 b/y fragment
PEPTIDE / AMINO ACID LEVEL ANALYSIS
21
normal stringent
BEH300 C18 100 93
CSH130 C18 100 94
Aeris XB-C18 100 93
30
40
50
60
70
80
90
100
% C
ove
rag
e
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PTMs and degradations
Robust quantification of C-terminal lysine truncation, Pyroglutamic acid, N-glycosylations, oxidations, and deamidations
PEPTIDE / AMINO ACID LEVEL ANALYSIS
22
0.0
20.0
40.0
60.0
80.0
100.0
BEH300 CSH130 Aeris XB-C18
%m
od
ifie
d -Lys C-term
G0F N
G1F N
PyroGlu E
0.0
10.0
20.0
30.0
40.0
50.0
N31 (CDR L1)
N137/138 (LC)
N152/158 (LC)
N54 (CDR H2)
N280/290 (HC)
N319 (HC)
N388/393/394 (HC)
LC
DIQMTQSPSS LSASVGDRVT ITCRASQGIR NYLAWYQQKP GKAPKLLIYA ASTLQSGVPS RFSGSGSGTD FTLTISSLQP
EDVATYYCQR YNRAPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ
ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
HC
EVQLVESGGG LVQPGRSLRL SCAASGFTFD DYAMHWVRQA PGKGLEWVSA ITWNSGHIDY ADSVEGRFTI SRDNAKNSLY
LQMNSLRAED TAVYYCAKVS YLSTASSLDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV
SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG
GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG
KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP
VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K
CDRs
Typical modifications N-Deamidations (no stress)
22
Need for trypsin “low pH”
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Stressed mAb (100 mM Tris, pH 8)
0
20
40
60
80
0 10 20 30 40
%m
od
ifie
d
Stress time (min)
D1/17 (LC)
Q199 (LC)
N152 (LC)
0
1
2
3
0 10 20 30 40
%m
od
ifie
d
Stress time (min)
Q3/6 (LC)
Q55 (CDR L2)
Q79/89 (LC/CDR L3)
Q124 (LC)
Q3/Q13 (HC)
D30/31 (CDR H1)
Significant deamidation Marginal deamidation
23
PEPTIDE / AMINO ACID LEVEL ANALYSIS
23
LC
DIQMTQSPSS LSASVGDRVT ITCRASQGIR NYLAWYQQKP GKAPKLLIYA ASTLQSGVPS RFSGSGSGTD FTLTISSLQP
EDVATYYCQR YNRAPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ
ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
HC
EVQLVESGGG LVQPGRSLRL SCAASGFTFD DYAMHWVRQA PGKGLEWVSA ITWNSGHIDY ADSVEGRFTI SRDNAKNSLY
LQMNSLRAED TAVYYCAKVS YLSTASSLDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV
SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG
GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG
KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP
VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K
CDRs
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Oxidative stress / Methionine
Robust quantification of methionine oxidation
CDRs
0.0
20.0
40.0
60.0
80.0
0 0.05 0.1 0.15 0.2
%o
xid
atio
n
%H2O2 (45 min)
M4 (LC)
M34 (CDR H1)
M83 (HC)
M256 (HC)
M432 (HC)
0
20
40
60
80
0 0.05 0.1 0.15 0.2
%O
xid
atio
n M
256
%H2O2 (45 min)
BEH300
CSH130
Aeris XB-C18
24
PEPTIDE / AMINO ACID LEVEL ANALYSIS
24
LC
DIQMTQSPSS LSASVGDRVT ITCRASQGIR NYLAWYQQKP GKAPKLLIYA ASTLQSGVPS RFSGSGSGTD FTLTISSLQP
EDVATYYCQR YNRAPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ
ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
HC
EVQLVESGGG LVQPGRSLRL SCAASGFTFD DYAMHWVRQA PGKGLEWVSA ITWNSGHIDY ADSVEGRFTI SRDNAKNSLY
LQMNSLRAED TAVYYCAKVS YLSTASSLDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV
SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG
GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG
KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP
VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K
CDRs
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- 64 Da
Peptide DTLM256ISR
Confirmation of oxidation site by MSE
PEPTIDE / AMINO ACID LEVEL ANALYSIS
25
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Intact level2D-LC/MS(IEX x RP)
Sub-unit levelRP-LC/MS
Peptide/AA levelPeptide mappingRP-UPLC/MSE
IdeS
Reduction (DTT)
PeptidesReduction (DTT, IAC) / Trypsin
DIQMTQSPSSLSASVGDR
DSTYSLSSTLTLSKEAK
LLIYAASTLQSGVPSR
ASQGIR
Substructures
Intact
Figure adapted from Beck et al. Anal. Chem. 2012, 84, 4637−4646
Deglycosylation
(Rapid PNGase, with or
without reducing buffer)
F(ab’)2
Fc / 2
Fd
LC
Fc / 2
26
GENERAL APPROACH
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Three levels of analysis
Intact, substructure, peptide/amino acid: various degrees of easyness / precision
Optimized workflows
Fast
Quick sample preparation, low amounts required
Efficient LC separation and heartcutting (Waters 2D-UPLC & Xevo-G2XS-Q/TOF)
Semi-automated data processing and reporting (MassLynx, Unifi)
CONCLUSIONS AND PERSPECTIVES
27
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CONCLUSIONS AND PERSPECTIVES
28
Optimized workflows (ctd)
Versatile
WCX and/or RP separation for different modifications detection
Autoblend development for quick adjustments to new samples
Large range of columns for different needs / samples
Large range of enzymes for various substructure generation
Reliable
High resolution MS detection for best precision and sensitivity
2D-LC/MS for fast identification confirmation
High repeatability
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Speed up people’s access to new medicines
Detection and quantification of post-translational modifications at each level
Information on key mAb elements (CDRs) / CQA
Ideal for stressed / aged samples monitoring
Batch-to-batch consistency
In-depth studies of isoforms / DP with 2D-LC/MS
Stability studies & formulation optimization
ADCs (in progress)
DAR and Payload distribution
Conjugation sites, PTMs, isoforms
CONCLUSIONS AND PERSPECTIVES
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Speed up people’s access to new medicines
Arnaud DELOBEL, R&D Team Manager
Eric LARGY, R&D Scienist
Anicet CATRAIN, Scientist
Région Wallonne / FEDER
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ACKNOWLEDGEMENTS