Dr. Arnaud Delobel from Quality Assistance spoke on advances in Mass Spectrometry for the Characterisation & Bioanalysis of ADCs at World ADC 2017 in Berlin. For more on this topic, replay the full ADCs webinar here: http://bit.ly/2kW3KoN With a full analytical package for Biologics, Quality Assistance provides scientific and technical support to biopharmaceutical companies developing Antibody-Drug Conjugates. Specificaly, we can develop and/or optimise the analytical methods for a product and, if necessary, validate them according to international guidelines for batch release, stability testing and/or regulated bioanalysis. For more information on our expertise and services, visit: www.quality-assistance.com Follow us on social media: LinkedIn: https://www.linkedin.com/company/quality-assistance Twitter: https://twitter.com/QA_Belgium Facebook: https://www.facebook.com/QualityAssistanceBelgium Google +: https://plus.google.com/103676189647965359292 Quality Assistance S.A. is a leading European Contract Research Organisation providing the pharmaceutical industry with all the analytical services required by EMA and FDA regulations for the development and marketing of innovative human medicinal products. We assist our clients from candidate selection, through non-clinical and clinical studies, to marketing authorisation, using our state-of-the-art, product-dedicated expertise in analytical sciences. For each customer and each project, we design customised solutions, define analytical protocols, develop and validate specific new analytical methods and perform characterisation, stability, pharmacokinetic, biomarker and immunogenicity studies as well as batch release testing, in order to evaluate the Quality, Safety and Efficacy of the given drugs.

1. 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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QUALITY ASSISTANCE SA
100% analytical services
100% (bio)pharmaceutical industry
35 years (since 1982)
150 highly-qualified employees
> 60% university graduates
109 worldwide R&D companies (2016)
All laboratories centralised 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
~1.5 M € plant, machinery & equipment (2016)
2
1.4 M €6.00010Cytotoxics
> 30 years
Biologics
> 15 years
ADCs

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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 ADCs
Low DAR species have a low efficacy
High DAR species can lead to safety issues
DAR determination of lysine-linked ADCs is routinely performed by UV spectrophotometry
Not compatible with non-UV absorbing drugs
Gives 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 ADC
Separation 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
𝐷𝐴𝑅 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 =
𝑖=1
𝑛
(𝐷𝑟𝑢𝑔#𝑖 × 𝐼𝑖)
𝑖=0
𝑛
𝐼𝑖
(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
LC LC +1 drug
Fd
Fd + 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 process
Efficient desalting
Robust 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 sequence
Confirmation of N- and C-terminal sequences
Characterisation of PTMs, including glycosylation
For ADCs, peptide maps can provide additional information:
Localisation of conjugation sites
Determination 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 residues
2 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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Much more complex than traditional protein drugs
Additional 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)
Compoundname:GPS
Correlationcoefficient:r=0.999855,r^2=0.999709
Calibrationcurve:1.6615*x+-1.74107
Responsetype:InternalStd(Ref2),Area*(ISConc./ISArea)
Curvetype:Linear,Origin:Exclude,Weighting:1/x,Axistrans:None
µg/mL
-0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Response
-0
50
100
150
µg/mL
Residual
-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 Injection
Magnetic 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 fragments
Peptide 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
QualityAssistance 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 LARGY
Anicet CATRAIN
Fabrice 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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