European leader in analytical sciences

ADVANCES IN MASS SPECTROMETRY FOR THE CHARACTERISATION AND BIOANALYSIS OF ADCS

Written by :

Arnaud Delobel – R&D Director

World ADC Berlin – February 21st

2017

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1.4 M €6.00010Cytotoxics

> 30 years

Biologics

> 15 yearsADCs

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CHARACTERISATION OF ADCS: AN ANALYTICAL CHALLENGE

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Monoclonal antibody

Drug

Linker

Main characterisation challenges specific to ADCs:

Drug-to-Antibody ratio (DAR)

Localisation of conjugation sites

Conjugation sites occupancy

→ Mass Spectrometry can be a valuable tool to solve those challenges

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PLATFORM USED FOR ADC ANALYSIS

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Xevo G2-XS QTOF with

1D or 2D-UPLC (H-Class Bio)

Control by UNIFI (1D)

or MassLynx (2D)

Full GMP compliance within UNIFI

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DETERMINATION OF DRUG-TO-ANTIBODY RATIO

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DAR is a critical quality attribute of ADCsLow DAR species have a low efficacyHigh DAR species can lead to safety issues

DAR determination of lysine-linked ADCs is routinely performed by UV spectrophotometryNot compatible with non-UV absorbing drugsGives the mean DAR value, but not the distribution

DAR determination of cysteine-linked ADCs is routinely performed by Hydrophobic Interaction Chromatography with UV detection (HIC-UV)

Specific method required for each ADCSeparation can be tricky for highly hydrophobic drugs

Use of Mass Spectrometry as a universal tool for DAR determination

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EXPERIMENTAL CONDITIONS

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Lys-linked ADCs Cys-linked ADCs

Sample prep. Deglycosylation w/ Rapid PNGase F under non-reducing conditions (15’)

LC RP column (Agilent PLRP-S)SEC column (Waters UPLC

BEH200 1.7 µm)

Detection ESI-MS(Waters Xevo G2-XS QTOF)

ESI-MS(Waters Xevo G2-XS QTOF), optimised source conditions

Sample amount 1 µg 20 µg

Data processing (assuming that all species have the same ionisation yield)

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DAR DETERMINATION BY MS FOR LYS-LINKED ADCS

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Example of Trastuzumab Emtansine (Kadcyla®):

DARaverage = 3.7

(assuming that all species have

the same ionisation yield)

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DAR DETERMINATION BY MS FOR CYS-LINKED ADCS

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Example of Brentuximab Vedotin (Adcetris®):

DARaverage = 4.0

0

2 4

6

8

(assuming that all species have

the same ionisation yield)

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ANALYSIS OF ADCS SUB-UNITS: EXAMPLE OF ADCETRIS®

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Fc/2

LCLC +1 drug

FdFd + 1 drug

Fd + 2 drugs

Fd + 3 drugs

Calculated DAR: 4.1

(based on UV detection)

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BENEFITS OF 2D-LC/MS FOR ADC CHARACTERISATION

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Hydrophobic Interaction Chromatography (HIC) is a method of choice for the characterisation of ADCs

Determination of naked antibody for Lys-linked conjugates (or site-specific conjugation technologies)Determination of DAR distribution for Cys-linked conjugates

Due to high salt content in mobile phase, it is impossible to hyphenate the chromatography to MS for peak identification

Fraction collection and desalting is time-consuming and is not easily amenable to low concentration species (due to dilution effects)

→ 2D-LC/MS is the solution for identification of peaks observed on HIC chromatograms

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PRINCIPLE OF HIC-RP 2D-LC/MS

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Step 1 : 1D configuration HIC column to UV detector

Step 2 : Peak heart-cutting HIC column to trap

Step 3 : 2D configuration trap to RP column to MS

Automated processEfficient desaltingRobust system

Applicable to all types of separations (SEC, IEX, …)

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APPLICATION OF HIC-RP 2D-LC/MS TO ADCETRIS®

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APPLICATION OF HIC-RP 2D-LC/MS TO ADCETRIS®

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CHARACTERISATION OF ADCS BY UPLC/MSE PEPTIDE MAPPING

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As for other therapeutic proteins, peptide mapping is a method of choice for:Confirmation of primary sequenceConfirmation of N- and C-terminal sequencesCharacterisation of PTMs, including glycosylation

For ADCs, peptide maps can provide additional information:Localisation of conjugation sitesDetermination of site occupancy (based on MS data)

High resolution LC (UPLC) combined with high resolution MS and dedicated software allow the use of this methodology in routine work.

A generic method was developed and applied to Kadcyla®

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PEPTIDE MAPPING METHOD

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Peptide mapping of Lys-linked ADCs requires specific conditions to determine conjugation sites occupancy

Trypsin cannot cleave after conjugated lysine residues2 digestions are required to get both good sequence coverage and site occupancy information : Trypsin + Glu-C and Asp-N + Glu-C

Separation is performed by UPLC (Waters BEH300 C18 column, 150 x 2.1 mm, 1.7 µm) over 120 minutes to get good separation.

Detection is done by ESI-QTOF/MS (Waters Xevo G2-XS QTOF)

Automated process is performed with UNIFI.

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PEPTIDE MAPPING OF KADCYLA®

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Trypsin + Glu-C

Asp-N + Glu-C

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PEPTIDE MAPPING OF KADCYLA®

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Trypsin + Glu-C

Asp-N + Glu-C

Coverage: 99 %

Coverage: 95 %

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SPECIFIC DETECTION OF CONJUGATED PEPTIDES IN KADCYLA®

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HN O

O

HOHO

OCl

N

O

O

O

ON

O

SN

O

O

O

HN

HN O

OH

HOHO

OCl

N

O

O

NHO

OCl

N

OH

O

Chemical Formula: C28H36ClN2O7+

Exact Mass: 547,2206Chemical Formula: C27H34ClN2O4

+

Exact Mass: 485,2202

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Much more complex than traditional protein drugsAdditional tests required to evaluate the stability of the conjugate

Typical analytical package for pharmacokinetics:

DAR determination in plasma (for PK analysis or study of ADC stability)

BIOANALYSIS OF ADCS, ANOTHER CHALLENGE…

Analyte Analytical techniques

Conjugated Ab Antibodies with DAR ≥1 LBA

Total Ab Conjugated and non-conjugated antibodies (DAR ≥0) LBA, LC-MS/MS

Ab-conjugated drug Drug conjugated to antibody Affinity LC-MS/MS, LBA

Free drug Drug not conjugated to antibody LC-MS/MS

Anti-Therapeutic Antibodies Antibodies targeted against ADC (Ab, linker or drug) LBA

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BIOANALYSIS OF ADCS – TOTAL MAB ASSAY

mAb/ADC in plasma Immunopurification

Denaturation

Reduction

Alkylation

Digestion

13C/

15N-labeled SiLuMAb

Spike

Magnetic beads coated with

- Protein A

- Streptavidin + biotinylated antihuman Ab

2 +2 h / 37 °C

TFA quench

LC/MS ready1 h total

All steps in same 96-well plate (no transfer)

LC/MS (TOF-MRM) analysis

2—4 monitored peptides

Intact isotopically labeled standard

0.100 0.125 1.5 100

C (µg/mL)

Compound name: GPSCorrelation coefficient: r = 0.999855, r̂ 2 = 0.999709Calibration curve: 1.6615 * x + -1.74107Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

µg/mL-0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Respon

se

-0

50

100

150

µg/mL

Resid

ual

-5.0

0.0

5.0

R² = 0.9997

1/X weighting

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BIOANALYSIS OF ADCS – DAR DETERMINATION

mAb/ADC in plasma Immunopurification Elution InjectionMagnetic beads coated with

- Protein A

- Streptavidin + biotinylated antihuman Ab

1% FA

All steps in same 96-well plate (no transfer)

LC(RP)/MS analysis

4

5

6

7

8

3

2

1

0

DAR determination

DAR: 3.45

(3 glycoforms)

Deconvoluted spectrum

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TAKE-HOME MESSAGES

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Characterisation of ADCs by MS can be performed at different levels for both cysteine and lysine-linked ADCs

Intact level: drug-to-antibody ratio (DAR) (mean value + distribution)Sub-unit level: mean DAR value, localisation of drugs on different fragmentsPeptide level: localisation of conjugation sites, determination of sites occupancy

2D-LC/MS is a great tool for the identification of species detected in HIC/UV

These tools can be used routinely in a regulated (GxP) environment thanks to UNIFI software (except for control of 2D-LC)

MS can also be a valuable tool for the bioanalysis of ADCs

Quality Assistance can develop and/or optimise the analytical methods for your product and, if necessary, validate them according to international guidelines for batch release, stability testing and/or regulated

bioanalysis.

After discussion with our scientific team, you will be proposed a testing strategy that best suits your needs.

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AKNOWLEDGEMENTS

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Eric LARGYAnicet CATRAINFabrice CANTAIS

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arnaud.delobel@quality-assistance.be

+32 71 53 47 81

www.quality-assistance.com

Technoparc de Thudinie, 2

B-6536 Donstiennes (Belgium)

Respect

Commitment

Excellence

Thank you for your attention

Any question?

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