This document summarizes key aspects of characterizing antibody drug conjugates (ADCs), including: 1) A case study examining how different polyethylene glycol (PEG) linker sizes affect ADC structure and target binding. Peptide mapping by mass spectrometry showed conjugation sites varied with linker size. Hydrogen/deuterium exchange mass spectrometry showed conjugation induced conformational changes. 2) Methods for assessing ADC mechanisms of action, including measuring internalization, cytotoxicity, and effector functions. 3) An overview of MilliporeSigma's comprehensive ADC product characterization and biosafety testing services across multiple sites.

1. The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
The Butterfly Effect:
How to see the impact
of small changes to
your ADC
Omar Lamm
Sales Development & Technical Support, Product Characterization
Martin De Cecco, Ph.D.
Principal Scientist, Product Characterization

2. The life science business
of Merck KGaA, Darmstadt,
Germany operates as
MilliporeSigma in the U.S.
and Canada

3. Antibody Drug Conjugates
Introduction
3
The highly targeted approach of antibody drug conjugates (ADCs), where
an antibody is connected to an antitumor cytotoxin via a linker, has been
extremely effective in treating various types of cancer.
The intrinsic complexity of developing and manufacturing ADCs creates major
challenges. Combatting many of these obstacles requires robust product
characterization throughout all phases of development, as small changes can
have a big impact, ultimately affecting the success of the final product.

4. Introduction
Presenters
4
Martin De Cecco, Principal Scientist for Product Characterization
Martin De Cecco supports clients as a subject matter expert for analytical and
bioanalytical testing. Martin has gained 10 years’ experience developing methods for
the structural characterization of biopharmaceuticals, working in the CRO industry
and previously at a leading LC-MS vendor. He holds a PhD in Biophysical Chemistry
from the University of Edinburgh and a MSci degree in Forensic & Analytical
Chemistry from the University of Strathclyde.
Omar Lamm, Product Characterization Technical Specialist
Omar Lamm is responsible for field technical support of product characterization
services. Based in Ann Arbor, Michigan, he has 20 years of experience supporting
contract biopharmaceutical product development both in the lab focusing on mass
spectrometry and full-scale analytical development programs, and in field client
support.

5. Agenda
Characterizing ADC products
Case study: The effect of PEG linkers on ADC structure
and binding
Assessing Mechanisms of Action
1
2
3

6. Characterizing
ADC Products

7. 7
Structure
Binding
Biological
Activity
(Potency)
How does my drug
function?
• Primary structure
• Drug-antibody ratio
• Conjugation site
• Post-translational modifications
• Higher order structure
• Product-related impurities
• Process-related impurities
• Does it bind to the target and what
is the strength of the interaction?
• What biological activity does it have?
• Cytotoxicity?
• Extent of internalization?
• Does it engage immune system
components to bring about effector
functions?
What are the
physical and
structural
attributes of my
drug?
Product characterization answers 2 crucial questions

8. 8
Quality attributes of a typical Monoclonal Antibody
Amino Acid Sequence
Peptide Mapping
• Primary Structure
• Secondary Structure
• Post Translational
Modification
• Biological Activity
• Impurities
Charge variants
Intact mass
Process related
impurities – HCP, HC
DNA, Protein A
Product Specific
Impurities – dimers,
aggregates, degraded
products
Specific Binding
Effector functions
Mode of action potency

9. Peptide mapping (and heterogeneity) is a
necessary pre-requisite for drug load distribution.
Enzyme digest (sometimes sequential) of the
antibody results in small peptides.
Comparison is then made of the conjugated and
unconjugated antibody.
9
Determination of drug load distribution
A. Wagh et al. (2018) mAbs, 10:2, 222-243.

10. Measuring the binding capacity of mAbs and ADCs
10
“ELISA” type assays are the workhorse of binding
assessments
Surface Plasmon Resonance can offer much more
insight into binding kinetics. Pre and Post
conjugation.
0
0.00001 0.001 0.1 10
%Response
Concentration
Standard Curve120
100
80
60
40
20
X
X
Report point
(baseline)
Relative
Response
(RUI)
Report point
(baseline)
Running
buffer
Sample
Running
buffer
Regeneration
solution
Running
buffer
Absolute
response
(RU)
0
20
40
60
80
100
120
140
160
180
200
200 400 600
%Response
Time (x)

11. Case study:
The effect of PEG
linkers on ADC
structure and binding

12. ADC design considerations
12
Fab region
Antigen binding
Fc region
Effector functions,
biodistribution
Linker
Conjugation chemistry
Drug-to-antibody ratio
Payload
How do these decisions impact structure and function?
Tune to optimize activity,
minimize toxicity…

13. ADC mimic (MSQC8)
SigmaMAb (MSQC4) + dansylcadaverine ‘payload’
Case study:
13
n = PEG4, PEG8, PEG12, PEG24
n
Linkers
SMCC Low and moderate DAR
Drug-to-Antibody Ratio
Effect of different linkers on ADC structure and binding

14. Hydrophobic Interaction Chromatography
14
Different amounts of reducing agent

15. Hydrophobic Interaction Chromatography
15
Different linkers, same DAR

16. Determination of conjugation site
Peptide mapping by LC-MS
16
Conjugated
Unconjugated

17. Determination of conjugation site
Peptide mapping by LC-MS
17
Conjugated
peptides

18. Determination of conjugation site
Peptide mapping by LC-MS
18
Linker DAR
LC HC
Cys-217 Cys-224
Cys-230 or
Cys-233
Cys-230 and
Cys-233
PEG8 2.6 64% 100% 1% -
PEG8 4.0 69% 100% 5% 3%
Linker DAR
LC HC
Cys-217 Cys-224
Cys-230 or
Cys-233
Cys-230 and
Cys-233
PEG4 4.1 59% 100% 9% 11%
PEG8 4.0 69% 100% 5% 3%
PEG12 3.9 71% 100% 5% 3%
Variation with DAR
Variation with linker size

19. Assessment of Higher Order Structure
Hydrogen/Deuterium exchange mass spectrometry
19
D. Houde and J.R. Engen, Methods Mol. Biol. 988 (2013) 269-289

20. Assessment of Higher Order Structure
Hydrogen/Deuterium exchange mass spectrometry
20
Less D uptake upon conjugation
at HC(246-272)
More D uptake upon conjugation
at HC(302-318) and HC(419-444)
Peptide
Orange: Unconjugated
Black: Conjugated
Difference(Da)
0
-1
+1
+2
No significant difference between PEG4 and PEG 24

21. Direct
immobilization
of receptor
Multi-cycle
kinetics:
different ADC
concentrations
Determine KD
value
Fc receptor binding by surface plasmon resonance
21
-5
0
5
10
15
20
25
30
35
-50 0 50 100 150 200 250
Time s
RU
Response
-2
0
2
4
6
8
10
12
14
16
18
-20 0 20 40 60 80 100
Time s
RU
Response
FcɣRIIIA (F) FcRn

22. Fc receptor binding by surface plasmon resonance
22
0
20
40
60
80
100
120
140
160
FcγRIIIA (V) FcγRIIIA (F) FcRn
Relative KD (%)
Reference LC-SMCC (DAR 4.1) PEG4 (DAR 4.1)
PEG8 (DAR 4.0) PEG12 (DAR 3.9) PEG24 (DAR 4.0)
LC-SMCC (DAR 2.7) PEG8 (DAR 2.6) PEG24 (DAR 2.8)

23.  Site of conjugation determined by peptide mapping
 Greatest site occupancy at HC (Cys-224) and LC (Cys-217)
 Occupancy at HC (Cys-230 / Cys-233) decreases with increasing linker size
 Higher order structure probed by HDX-MS
 conjugation appears to result in a conformational change of the mAb
 Not affected by length of linker
 Fc receptor binding kinetics investigated by SPR
 Affinity for FcɣRIIIA decreases with increasing length of linker and increased DAR
 Increased affinity for FcRn upon conjugation, independent of linker size
Case study summary
23

24. Assessing
Mechanisms of
Action

25. Circulating ADC needs to be stable and resistant to payload cleavage.
Biosci Rep. 2015 Jun 12;35(4).
25
Factors affecting the Mechanism of Action
• Internalization—an ADC should show
rapid and sufficient internalization.
• IgGs are predicted to attain
significantly higher levels of tumor
accumulation due to both a slower
serum clearance half-life and FcRn
mediated retrieval from lysosomal
degradation
• The endosome is a complex system
of proteins and lipids. Endosomes
send cargo through two pathways.
• The first is cargo recycling that
can result in the trafficking of
the receptor back to the plasma
membrane.
• The second pathway is results in
fusion with the lysosome and
final degradation.

26. 26
Measuring internalization of ADC
• The method uses a hydrophilic and bright
pH sensor dye, which is not fluorescent at
neutral pH but becomes highly fluorescent
at acidic pH.
• For receptor mediated antibody
internalization studies, antibodies against
receptors are conjugated with the pHAb
dye and incubated with the cells
expressing the receptors.
• Upon binding to the receptor, the dyes
conjugated to the antibody are not
fluorescent because of the neutral pH of
the media, but upon internalization and
trafficking into endosomal and lysosomal
vesicles the pH drops and dyes become
fluorescent.
(A) SKBR3 cells were incubated with Trastuzumab-amine-pHAb, pH 8.0
(B) pH of the media at pH 5.0 the fluorescent antibody bound to the cell surface
(C) Cells incubated for 18 h and imaged again. Punctate structures indicate
internalized antibody.
(D) No internalization when HER2 negative cells were incubated with antibody

27. 27
Assessment of potency: cytotoxicity determination
Serially dilute the ADC under test
Plate tumour cells in assay plate and
add the diluted ADC
Incubate the cells and ADC
Identify cells which are dying /dead

28. Measuring the levels of ATP is the most
sensitive, reliable, and convenient
method for monitoring active cell
metabolism.
Enzyme leakage assays - These assays
measure the activity of enzymes that leak
into the extracellular medium on cell
membrane damage. The most popular
assay is for lactate dehydrogenase
Membrane impermeable dyes -
fluorescent dyes (mostly DNA stains) that
stain cells with damaged cell membranes
Amine-reactive/ combination dyes -
Amine-reactive dyes weakly stain viable
cells by binding to cell surface amines
and strongly stain membrane-
compromised cells by reacting with
intracellular amines.
28
How dead is dead?
Cytotoxicity - measuring viable cells, dead cells, and
detecting mechanism of cell death
LIVE DEAD
Measures of Apoptosis
Loss of membrane asymmetry
Caspase, Calpain & Cathepsin
Cytochrome c release
Sub G1 population
Nuclear condensation
DNA fragmentation
Cell Membrane blebbing
Typical assays include Cytochrome c rmeasurement showing mitochondria disintegration
Caspase 1-12 activation
Annexin V presence on membranes

29. The strength of the various immune
effector functions varies depending on the
specific isotype of monoclonal IgG
antibodies used in the ADC. The effects of
ADCC and CDC are much stronger in
human IgG1 and IgG3 isotypes in
comparison with IgG4 and IgG2 mAbs.
Regulators expect an understanding of the
contribution of these functions on the
activity of the drug.
Binding of the ADC to Fcγ receptors; these
will compete with ADC internalization. IgG2
and IgG4 isotypes of antibodies that have
poor immune effector function for naked
mAbs were the preferred antibodies for use
in certain ADC’s
29
Effector Function Analysis
Biosci Rep. 2015 Jun 12;35(4).

30. 30
Quality attributes of a typical ADC
Monoclonal Antibody
Structure and binding properties
Target binding and internalization
Stability
Size &Charge variants
Primary structure and PTM
Cytotoxic Payload
Microtubule inhibitor
DNA Synthesis inhibitor
Topoisomerase InhibitorLinker
Cleavable and
Non cleavable
Drug to Antibody Ratio
Levels of free drug
Heavy Modification
In vitro efficacy
Residuals

31. 31
Pre and post conjugation analytics and characterization
BioReliance® Services
Services
Method transfer
or development
Method
validation
Reference
Characterization
Comparability
studies
Stability Testing
and Storage
GMP Lot
Release assays
Compendial
pH
Karl Fischer
Titration
Osmolarity
BCA & Bradford
Protein
Concentration
Appearance
SDS PAGE
Aggregates
Mass
Spectrometry
Intact Molecular
Weight (MW)
Analysis
Antibody
Subunit
Analysis
Peptide Mapping
N- and C-
Terminal
Sequencing
Glycan Profiling
Higher order
structure
Capillary
Electrophoresis
Purity (SDS
Denatured)
Isoelectric Point
Determination
Charge Profile
Determination
UHPLC
Amino Acid
Analysis
Glycan Profiling
Peptide mapping
ion exchange
chromatography
Size exclusion
chromatography
Reverse Phase
Chromatography
Hydrophobic
Interaction
Chromatography
Cell-based/
Immunoassays
Cell Based
Assays
Binding Assays
Kinetic Binding
Reporter
Bioassays
ADCC/CDC/
ADCP

32. A comprehensive service solution for Antibody Drug Conjugates
Product Characterization &
Biosafety Testing Services
Maryland
mAb production
France
ADC Production
Missouri
HPAPI
Manufacturing
Wisconsin
Key Capabilities:
 mAb production
 Payload and linker synthesis
 ADC conjugation
 Product Characterization and
Biosafety Testing Services
 Single-use technology
32
Product Characterization &
Biosafety Testing Services
Scotland, UK

33. Principal Scientist, Product Characterization
martin.de-cecco@milliporesigma.com
Martin De Cecco, Ph.D.
Sales Development & Technical Support,
Product Characterization
omar.lamm@milliporesigma.com
Omar Lamm
Contact us
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the U.S. and Canada.
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