Characterization of Antibody-Drug Conjugates by 2D ... · Characterization of Antibody-Drug...

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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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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


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


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

3

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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


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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

==

==

+ +

+

+ +

+ +


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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


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Change of average DAR during stress/storage

𝑎𝑣𝐷𝐴𝑅 = 𝑛𝐴𝐷𝐴𝑅𝑛80

𝐴𝐷𝐴𝑅𝑛80

1


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L0 L1 HHL1HHL0 HHLL1

1A

1B

1C1

2 3

4

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3

4

Non stressed sample


Two month stressed sample

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50 s


HH3

L0 L1 H3H2 HL2 HH3

5A

5B

5C

Non stressed sample


Two month stressed sample


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High reliability of the method HICxRPLC-UV

ADC 1 ADC 2

ADC 2 degraded

Quality Control

Stability Control


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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 !

Characterization of Antibody-Drug Conjugates by 2D ... · Characterization of Antibody-Drug...

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