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Characterization of Antibody-Drug Conjugates by 2D-chromatography hyphenated to MS
Sabine Heinisch
Institut des Sciences Analytiques
Equipe Chromatographie et techniques couplées
UMR 5280 – CNRS-UCBL-ENS
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Collaborations
Sabine HeinschMorgan SarrutAmélie Corgier
J-Luc VeutheyDavy GuillarmeSzabolcs Fekete
Alain BeckMarie C Janin-Bussat
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Antibody-drug conjugate (ADC)
ADC = mAb + linker + cytotoxic drug
Peptide Linker(valine-citrulline peptide)
Cytotoxic drug(Auristatin E)
+mAb(brentuximab)
+
A. Beck et al., Expert Rev. Proteomics. 13 (2016) 157–183.
lysine linked ADC
cysteine linked ADC
Micro-variability of mAbs + Heterogeneity of conjugation Possible effects on both efficacy and safety of ADC
Brentuximab Vedotin (Adcetris®)
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Need for ADC characterization (stability assessment, QC):- Distribution of Drug-to-Antibody ratio (DAR) – average DAR- Identification of positional isomers
Structure of ADCs : Drug load distribution
DAR 0
DAR 2DAR 4
DAR 6
DAR 8
= linker + cytotoxic drug
Non covalent associations
Covalent associations
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5
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Characterization in native conditions
• HIC-UV (Hydrophobic Interaction Chromatography) – non denaturing medium;DARs separated according to their hydrophobicity by decreasing the concentration of salt
DAR; average DAR determination (3.9)
no information about positional isomers
not MS-compatible
• MS; IM-MS detect non covalent associations of light and heavy chains
DAR; average DAR; structural assessment
no information about positional isomers
DAR 2
DAR 4
DAR 6
DAR 8DAR 0
F. Debaene et al, Anal. Chem. 86 (2014) 10674
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Characterization in denaturing conditions
• RPLC-UV/MS (under reducing or non-reducing conditions) – denaturing medium
very informative
MS compatible
RPLC (non reducing conditions) + +
≈150 kDa ≈75 kDa ≈50 kDa ≈25 kDa
no information about positional isomers
RPLC (reducing conditions) + +
≈150 kDa ≈50 kDa ≈50 kDa ≈25 kDa
+
≈25 kDa
M. Sarrut, Thesis
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DA
R 8
DA
R 6
DA
R 2
DA
R 4
DA
R 0
2D RPLC
1D HIC
Need for HIC x RPLC-MS/UV to gain in information
DAR = 3.9 ± 0.1
M. Sarrut, S. Fekete, M.C. Janin-Bussat, D. Guillarme, A. Beck, S. Heinisch, J. Chromatogr. B, submitted
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On-line HIC x RPLC instrumentation for ADC characterization
Gradient system 2
RPLC column
UV 2
QTOF V
Waste
Gradient system 1
HIC columnSamplemanager
valve UV 1
Interface
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Optimizing of on-line LCxLC-UV/MS method
Objectives
Analysis time
Dilution factor
Separation power
M. Sarrut, A. D’Attoma, S. Heinisch, J. Chromatogr.A, 1421 (2015) 48-59
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Optimizing of on-line LCxLC-UV/MS method
Objectives
Analysis time
Dilution factor
Separation power
M. Sarrut, A. D’Attoma, S. Heinisch, J. Chromatogr.A, 1421 (2015) 48-59
Sampling rate
Gradient shape in 1D Flow-rate in 1D Salt type in 1D Injection volume in 1D
Gradient shape in 2D Flow-rate in 2D Organic modifier in 2D
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1 2
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RPLC separation (MS-TIC)
Time (min)
µAu
HIC separation
DA
R 0
DA
R 2
DA
R 4
DA
R 6
DA
R 8
HIC x RPLC-MS : identification of sub units
Deconvoluted MS spectra
H3
HL 2
HH4
L1
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Identification of sub-units for even DARs
L1 HL2 HHLL2H3 HHL1HH2 HHL3 HH4
DAR 2
DAR 4
DAR 6
DAR 8
All even DARs unambiguously identified
M. Sarrut, S. Fekete, M.C. Janin-Bussat, D. Guillarme, A. Beck, S. Heinisch, J. Chromatogr. B, submitted
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DAR 8
DAR 6
DAR 4
DAR 2
DAR 0
Odd DARs?
DAR 3 ?
Presence of odd DARs ? Degradation; Incomplete conjugationB. Wiggins et al., J. Pharm. Sci. 104 (2015) 1362–1372
DAR 1 ?
DAR 5 ?
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Identification of DAR 3 (fraction at 45 min)
and
Hypothetical
structures
==
==
+ +
+
+ +
+ +
M. Sarrut, S. Fekete, M.C. Janin-Bussat, D. Guillarme, A. Beck, S. Heinisch, J. Chromatogr. B, submitted
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Analysis of stressed ADCs
DAR 0
DAR 2
DAR 4
DAR 6
DAR 8
DAR 1 ?DAR 3
DAR 5 ?
Non stressed sample
One month stressed sample
Two month stressed sample Non stressed
One-month stressed
Two-month stressed
M. Sarrut, S. Fekete, M.C. Janin-Bussat, D. Guillarme, A. Beck, S. Heinisch, J. Chromatogr. B, submitted
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Change of average DAR during stress/storage
𝑎𝑣𝐷𝐴𝑅 = 𝑛𝐴𝐷𝐴𝑅𝑛80
𝐴𝐷𝐴𝑅𝑛80
1
M. Sarrut, S. Fekete, M.C. Janin-Bussat, D. Guillarme, A. Beck, S. Heinisch, J. Chromatogr. B, submitted
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Identification of DAR 1 (fraction at 30 min)
L0 L1 HHL1HHL0 HHLL1
1A
1B
1C1
2 3
4
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3
4
Non stressed sample
One month stressed sample
Two month stressed sample
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50 s
Identification of DAR 5 (fraction at 54 min)
HH3
L0 L1 H3H2 HL2 HH3
5A
5B
5C
Non stressed sample
One month stressed sample
Two month stressed sample
M. Sarrut, S. Fekete, M.C. Janin-Bussat, D. Guillarme, A. Beck, S. Heinisch, J. Chromatogr. B, submitted
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High reliability of the method HICxRPLC-UV
ADC 1 ADC 2
ADC 2 degraded
Quality Control
Stability Control
M. Sarrut, S. Fekete, M.C. Janin-Bussat, D. Guillarme, A. Beck, S. Heinisch, J. Chromatogr. B, submitted
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Conclusions
On-line HICxRPLC-MS can be employed in the early stage of ADC development for extensive characterization. It permits:
Hyphenation of HIC to MS Unambiguous identification of even DARs Unambiguous identification of odd DARs (1, 3, 5) Simultaneous determination of avDAR and identification of positional isomers
Retention times constitute additional values for sub-unit identification
In the later stage of ADC development, the use of HICxRPLC-UV can greatly simplify the work
In the future, 2D-LC techniques should be of prime interest for mAbs and ADC characterization
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Acknolewdgements
Industrial partners
Pierre FabreA. BeckMC Janin
WatersLaetitia DenbighD. Lascoux
Academic partners
ISRTEAC. MargoumP. Bados
University of GenevaJ-L VeutheyD. GuillarmeS. Fekete
Thank you for your attention !