Watch the presentation of this webinar: https://bit.ly/3Pjpjvr Highlights of this webinar: - Surface plasmon resonance as a powerful tool for biologic characterization including mAbs and ADCs. - SPR allows rapid binding analysis in real time without using labels for SARS-CoV-2 receptor binding domain mutations. - Kinetic data is indicative of possible neutralizing activity allowed assessment of neutralizing ability of therapeutic monoclonal antibodies. - The application can provide preliminarily efficacy information and facilitated mAbs/ACDs candidate selection process Detailed description: Characterization of therapeutic monoclonal antibodies (mAbs) or Antibody drug conjugates (ADCs) is challenging due to their ability to bind to a variety of proteins via their Fc and Fab domains, giving rise to diverse biological functions associated with each domain. The Fc domain of mAbs interacts with Fc receptors with varying affinities, which can influence biological processes such as Complement-dependent cytotoxicity (CDC) and Antibody-dependent cellular cytotoxicity (ADCC), transcytosis, phagocytosis, and/or serum half-life. An important characteristic of an antibody is its Fc effector function. Antibodies can be engineered to obtain desired binding of the Fc region to Fc receptors expressed on effector cells. Hence, it is crucial to evaluate the binding interaction of mAbs/ADC with Fc receptors in the early phase of drug development to understand the potential biological activity of the product in vivo. Surface Plasmon Resonance (SPR) is a powerful technique to establish binding kinetics in real-time, label free, and high sensitivity with low sample consumption. Along with target antigen binding, it is crucial to evaluate the binding interaction of antibodies and ADCs with Fc receptors. Our SPR case studies investigated the impact on binding kinetics of ADCs with different linkers and the binding interactions of SARS-CoV-2 spike protein variants and evaluated the neutralizing ability of therapeutic mAbs. SPR characterisation can be facilitated in all stages of the product life cycle to ensure the quality and safety of mAbs and ADCs.

1. The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
Merck KGaA
Darmstadt, Germany
Characterization of
Antibodies and ADCs Using
Surface Plasmon Resonance
Helen Yu-Ting Hsu, Ph.D.
Principal Scientist, Process and Analytical Development

2. 2
The Life Science business of
Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma
in the U.S. and Canada.
CMC Regulatory Considerations for ADCs| 16.11.2021

3.  Global market continues to grow over the years.
 Therapeutic mAb and ADC: leading product class within biopharmaceutical market.
 Numerous assays performed before selecting the lead therapeutic mAb.
Therapeutic mAb and ADC
ADC Market WW sales expected in m$
0
2.000
4.000
6.000
8.000
10.000
12.000
14.000
16.000
18.000
2021 2022 2023 2024 2025
Besponsa®
Zynlonta™
Blenrep
Polivy®
Adcetris®
Kadcyla®
Trodelvy™
Padcev™
Enhertu®
Tivdak™
31% CAGR
2021-2025*
* Data Source: Evaluate Pharma, January 2022

4. mAb and ADC functional characterization: Fab and Fc
mediated activities
Internal|Confidential
4
BINDING ACTIVITY
• Fab binding to antigenic drug target
determines specificity of drug in vivo
• Affinity of binding is a critical quality
attribute which needs to be very well
characterised
BIOLOGICAL ACTIVITY
• Binding to target triggers the desired
biological effect
• Fab mediated mechanisms may often
be supplemented with Fc region
mediated effector functions following
binding,
BINDING ACTIVITY
The Fc region can bind to:
• Fcɣ receptors on immune cells,
• the neonatal Fc receptor (FcRn)
• the C1q component of complement
These interactions can bring about
‘effector’ functions which may be
important for mAb therapeutic efficacy
BIOLOGICAL ACTIVITY
• Antibody dependent cell mediated
cytotoxicity (ADCC)
• Complement dependent cytotoxicity
(CDC)
• Antibody dependent cell mediated
phagocytosis (ADCP)

5. How does it work?
SPR monitor molecular binding events in real-time
Sensorgram:
* Surface plasmon resonance, Cytiva
https://www.cytivalifesciences.com/en/us/solutions/protein-research/knowledge-center/surface-plasmon-resonance/surface-plasmon-resonance
Association
(ka/kon)
Dissociation
(kd/koff)
Running
buffer
Analyte
Sample
Running
buffer
Reg.
Solution
Running
buffer
Time
Response
(RU)
Relative
response
(RU)
*

6. Data Evaluation
Binding process - 1:1 model
ka kd
Ligand
Analyte
Flow
ka
kd
𝐀 + 𝐁 ⇋ 𝐀𝐁
𝐊𝐃 =
𝐤𝐝
𝐤𝐚
ka

7. 7
Direct
SPR assay set up
Analyte
Ligand
Capturing
molecule
Capture

8. Bottom of well
SPR vs ELISA
8
Flow cell surface
Unidirectional
adsorption of
reagent
Blocking
Primary
antibody
Secondary
antibody
Detection
Antigen
orientation
controlled
Primary
antibody
Detection in
real time
ELISA
SPR
Time (h)

9. Merck KGaA
Darmstadt, Germany
Fab/Fc binding
characterization
The effect of PEG linkers
on ADC binding

10. ADC design considerations
10
Fab region
Antigen binding
Fc region
Effector functions,
biodistribution
Linker
Conjugation chemistry
Drug-to-antibody ratio
Payload
Tune to optimize activity,
minimize toxicity…
How do these decisions impact structure and function?

11. Effect of different linkers on Trastuzumab Fab/Fc binding
11
Figure 1 PEG-DM1 Conjugation Process

12. Fc binding – Linear PEG
12
*Relative % KD = KD RS/ KD Sample x 100
Linker
FcγRIIIa FcRn
kon
(105,1/Ms)
koff
( 1/s)
KD
(nM)
Relative
% KD*
kon
(106,1/Ms)
koff
( 1/s)
KD
(nM)
Relative
% KD*
RS
(TmAb)
1.37 0.21 340 100 1.49 0.23 65.4 100
PEG4 7.76 0.88 243 139.8 2.02 0.36 76.0 86.1
PEG8 7.31 0.99 254 133.5 0.83 0.25 69.6 94.0
PEG24 6.04 0.82 261 130.2 0.87 0.24 79.2 82.6
FcγRIIIa FcRn
0 100 200 300
0
10
20
30
40
50
Time(s)
Response
(RU)
6.25 nM
12.5 nM
25 nM
50 nM
100 nM
200 nM
0 100 200 300
0
10
20
30
40
50
60
Time(s)
Response
(RU)
20.5 nM
51.2 nM
128 nM
320 nM
800 nM
2000 nM

13. 13
Linker
kon
(104,1/M
s)
koff
(10-4,
1/s)
KD
(nM)
Relative
% KD*
RS
(TmAb)
1.19 0.72 6.07 100
PEG4 1.22 1.42 11.6 52.3
PEG8 0.88 1.05 11.9 51.0
PEG12 2.53 1.66 6.56 92.5
PEG24 3.06 3.47 11.3 53.7
*Relative % KD = KD RS/ KD Sample x 100
Figure 1: Sensorgrams -overlaid 1:1 binding model fit for
the HER2-Trastuzumab interaction.
0 1000 2000
0
50
100
150
200
Time(s)
Response
(RU)
0.41 nM
1.23 nM
3.70 nM
11.11 nM
33.33 nM
100 nM
300 nM
• Approximately 50% reduction in relative % KD to
the unconjugated TmAb were observed.
• No clear correlation between the PEG length and
the KD value observed.
• Interactions between the ACDs and HER2
considered as high affinity (nM range).
Antigen binding – Linear PEG

14. 14
*SMCC used as RS to calculate relative KD and relative potency.
**Relative % KD = KD RS/ KD Sample x 100
Linker
SPR Cytotoxicity
Relative % KD** Relative potency %
PEG4 57 40.4
PEG12 101 88.7
PEG24 58 55.3
SMCC (RS)* 100 100
Figure 1 PEG-DM1 Conjugation Process
SPR and Cytotoxicity Comparability
• Both assays showed similar tend of efficacy.
• SPR Kinetic data including association (ka),
dissociation (kd), and equilibrium
dissociation constant (KD) allows
preliminary efficacy data with low sample
requirement.
Figure 2 Dose response curves, Red – RS (SMCC). Green
– ADC.

15. 1
5
Linker DAR
ka
(103,1/Ms)
kd
(10-4,1/s)
KD
(nM)
Relative
KD (%)
BP4-MMAE 4 1.37 1.88 138 5
BP8-MMAE 4 3.03 1.97 64.9 10
BP12-MMAE 4 3.83 2.02 52.7 12
BP24-MMAE 4 6.27 2.15 34.4 19
BP4-MMAE 8 1.55 0.71 46.1 14
BP8-MMAE 8 66.3 1.27 191 3
BP12-MMAE 8 1.06 1.34 127 5
BP24-MMAE 8 2.49 2.09 84.0 8
DBCO 4 6.38 2.11 33.1 20
DBCO 8 3.61 2.08 57.7 11
RS (TmAb) NA 8.62 0.57 6.57 100
SPR antigen binding of TmAb PEG library
Figure 1. Schematic of branched PEG library
conjugation process

16. 16
Linker
Relative % KD to TmAb Relative % KD to DBCO
DAR 4 DAR 8 DAR 4 DAR 8
BP4-MMAE 5 14 24 125
BP8-MMAE 10 3 51 30
BP12-MMAE 12 5 63 45
BP24-MMAE 19 8 96 69
DBCO 20 11 NA NA
Expanded PEG library
 Overall reduction in affinity (KD) after conjugation relative to unconjugated TmAb for all linkers &
#DAR.
 For DAR 4, DBCO retained 20% affinity relative to TmAb, the binding affinity increased (high relative
% KD) as BP linker size increased.
 For DAR 8, BP4 has slight increase in affinity compared to DBCO, but no clear trend on the impact of
linker length.
 Direct comparison of DAR 4 series relative to DBCO, increased in linker size increased the binding
affinity.
 Direct comparison of DAR 8 series relative to DBCO, showed no clear correlation between the linker
size and affinity.

17. 17
Linker DAR
ka
(103,1/Ms)
kd
(10-4, 1/s)
KD
(nM)
Relative %
KD
**
vc-MMAE 4 4.33 1.91 44.0 30
Exatecan 4 5.88 2.00 33.9 39
Exatecan 8 0.68 1.47 218 6
vc-MMAE* 4 5.66 2.13 37.6 35
Exatecan* 4 5.58 2.16 38.7 34
Exatecan* 8 3.07 1.91 62.1 21
TmAb NA 6.09 0.81 13.2 100
* Deglycosylated
** Relative KD calculated using DBCO DAR 4 and DBCO DAR 8 as reference for
the respective conjugates
Linker
Relative % KD
*
DAR 4 DAR 8
exetecan 88 351
vc-MMAE 117 NA
* Relative KD calculated using original KD/
Deglycosylated KD
Impact on deglycosylation
 Overall reduction in affinity (KD) after conjugation relative to unconjugated TmAb (6 – 39%)
 Significant reduction in affinity for DAR 8 relative to unconjugated TmAb.
 Significant increased (351%) in KD after deglycosylation for DAR 8 conjugate relative to the original
DAR 8 conjugate.

18. 18
Summary
 Overall reduction in affinity for all conjugates relative to TmAb (5 – 20% remained)
 As DAR # increase, the affinity decreases – potentially due to steric hindrance/accessibility of the
binding site.
 For DAR 4, the binding affinity increased as brunched linker increased.
 For DAR 8, no clear correlation.
 Deglycosylation process has significant impact in affinity for DAR 8 conjugate.

19. Merck KGaA
Darmstadt, Germany
Case study: Fab binding
/neutralizing assays
Binding interaction of
SARS-CoV-2 spike
protein mutants

20. 1. Scialo, F., Daniele, A., Amato, F. et al. ACE2: The Major Cell Entry Receptor for SARS-CoV-2. Lung 198, 867–877 (2020).
2. Wan, Y., Shang, J., Sun, S. et. al, Molecular Mechanism for Antibody-Dependent Enhancement of Coronavirus Entry. Journal of
Virology 94, 5 (2020).
https://www.nature.com/articles/s41368-020-0074-x
Angiotensin-converting enzyme 2 (ACE2) receptor
 The spike protein from the novel pandemic Coronavirus identified in
late 2019 (SARS-CoV-2) mediates binding to ACE2 for cellular entry.1
 Common receptor for SARS‐CoV‐2 and SARS‐CoV
 Prime target for therapeutic development e.g. antibody based
therapeutics.
 SARS-CoV-2 binds to ACE2 Receptor 10 – 20x tighter than SARS-CoV.
Question of Antibody Dependent Enhancement2
 Antibodies can create a backdoor enhancement for viral replication
 Implications on viral replication and vaccine development safety

21.  Understanding the SARS-CoV-2 and host interaction.
 Facilitates the development of vaccines and drugs.
Treatments/ therapies
 mAb blocks spike interaction with ACE2, prevents viral
entry
 generate antibodies with neutralizing activity
Genetic mutation
 may lead to mutants of spike and may affect:
− Virus transmissibility
− Disease severity
− Efficacy of vaccines or therapeutics
SARS-CoV-2 Interactions

22. • ACE2-RBD interactions
• RBD-mAb interactions
• Mutant analysis: binding kinetics of different RBD
mutants with ACE2 and anti-spike mAbs.
• Capability of the antibodies to inhibit the ACE2-RBD
interaction, indicating neutralizing activity.
− D614G
− N501Y
− 439K
− Y453F
− E484K
Chip
ACE2
RBD
mAb
SARS-CoV-2 Binding interactions using SPR

23. Kinetic screening: ACE2–RBD/S1
-20
0
20
40
60
80
-100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200
RU
Response
Tim e s
Figure 1: Representative sensorgrams for ACE2-RBD/S1
interaction
Sample
kon
(105,1/Ms)
koff
(10-3,1/s)
KD
(nM)
RBD 7.43 7.46 10.12
S1 2.90 2.50 10.51
Table 1: Average kinetic parameters for the ACE2-
original RBD interaction (n = 5).
S1
RBD
• Biotin capture
• allow further competition/blocking assay development using capture format
• Accelerate assay optimization time

24. Sample
Absolute values Relative (%)*
kon
(105,1/
Ms)
koff (10-
3,1/s)
KD (nM) kon koff KD
Original
RBD
6.66 2.89 4.42 100.0 100.0 100.0
D614G 1.38 5.78 42.18 482.6 50.0 10.5
N501Y 6.58 0.85 1.29 101.2 340.0 342.6
N439K 7.92 3.24 4.16 84.1 89.2 106.3
Y453F 5.89 0.37 0.66 113.1 781.1 669.7
E484K 9.94 5.89 6.63 67.0 49.07 66.7
Figure 1: Representative sensorgrams of ACE2-RBD
interactions
*Relative% = Original RBD/mutant RBD
SPR comparability studies: ACE2-RBD interaction

25. Kinetic screening: RBD/S1 – mAb1
Format Ligand Analyte
kon
(105,1/Ms,)
koff
(10-4,1/s)
KD
(nM)
His-
capture
S1
mAb1
1.53 2.72 1.78
RBD 2.47 7.09 2.87
Protein A mAb RBD 6.32 7.93 1.26
Table 1: Kinetic parameters for the mAb1-RBD interaction using two
different assay formats.
Figure 1: Illustration of His-capture (top) and
protein A mAb capture (bottom) methods
Assessment of assay format
• His-capture and protein A mAb capture methods
• Interactions between mAb1-RBD and mAb1-S1
proteins were assessed.
• mAb1-RBD and mAb1-S1 have similar KD values
• Both methods generated comparable data for RBD
proteins.

26. *Relative%=Original RBD/mutant RBD
Figure 1: Representative sensorgrams of mutant RBD/S1-
mAb1 interactions. (a) original RBD, (b) N501Y, (c) N439K,
(d) Y453K, (e) E484K, (f) D614G.
Sample
Absolute values Relative (%)*
kon
(105,1/
Ms)
koff (10-
3,1/s)
KD
(nM)
kon koff KD
Original
RBD
6.71 0.86 1.60 100.0 100.0 100.0
D614G 0.36 1.66 47.08 1863 51.8 3.4
N501Y 5.04 1.00 2.55 133.1 86.0 62.8
N439K 5.65 1.14 2.58 118.8 75.4 62.0
Y453F 5.39 1.07 2.57 124.5 80.4 62.3
E484K 7.13 0.83 1.43 94.1 103.6 111.9
mAb1-RBD interaction
mAb1: anti-spike monoclonal antibody

27. Figure 1: Representative sensorgrams of mutant RBD/S1-
mAb2 interactions. (a) original RBD, (b) N501Y, (c) N439K,
(d) Y453K, (e) E484K, (f) D614G
Sample
Absolute values Relative (%)*
kon
(105,1/
Ms)
koff (10-
3,1/s)
KD
(nM)
kon koff KD
Original
RBD
32.00 0.35 0.11 100.0 100.0 100.0
D614G 5.10 0.51 0.99 627.5 68.6 11.1
N501Y 19.40 0.44 0.23 164.9 79.6 47.8
N439K 24.70 0.36 0.15 129.6 97.2 73.3
Y453F 25.20 0.79 0.31 126.9 44.3 35.5
E484K 9.72 1.51 1.55 329.2 23.2 7.1
mAb2-RBD interaction
mAb2: spike neutralizing antibody
*Relative%=Original RBD/mutant RBD

28. Figure 1: mAb1-RBD blocking assay: binding responses
between RBD and ACE2 in the presence and absence of mAb1.
Figure 2: Binding responses for different concentrations of
mAb1 to 60 nM RBD on an ACE2 captured sensor surface
Blocking (neutralization) – mAb1
• KD value of ACE2-RBD interaction and mAb-RBD
interaction allowed estimation of neutralizing
concentration.
• mAb concentration (in excess) selected based on
of ACE2-RBD KD
• Premix mAb and RBD, injected over ACE2
captured surface
• Lack of binding (blocking/inhibition) should be
observed.
• Increase in surface response was observed
• Bound to different epitope ?

29. Figure 1: Representative sensorgrams of RBD-mAb2 blocking assay.
Blocking (neutralization) – mAb2

30. Figure 1: Representative sensorgrams of mutant RBD/S1-mAb2 blocking assay.
Blocking (neutralization): mAb2 – RBD mutants

31. Conclusion
• SPR binding assays allows real time kinetic analysis of interactions between Fc receptors and mAb-
based therapeutics including ADCs
• Assess the binding interaction for ACE2-RBD/mutant and mAb-RBD/mutant interactions.
• Supports the study of linker selection on Fc and Fab binding activity for ADCs.
• The assays can be used for early-stage screening and characterization therapeutic mAbs and ADCs to
ensure product quality.

32. Comprehensive ADC and Bioconjugation Services
From pre-clinical to commercial and from gene to BDS
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33. 35+ years of bioconjugation experience with 80+ different constructs
Comprehensive ADC and Bioconjugation Services
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Experience to bring your ADC to the
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for rapid production of development-grade
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• ADC constructs
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34. Jeff Carroll
Jason Ramsay
Lisa McDermott
Deepa Raghu
Pamela Hamill
Arpitha Banaji
Amy McLaren
Acknowledgements
https://www.sciencedirect.com
/science/article/pii/S209517792
1001076?via%3Dihub
https://www.bioprocessonline.com
/doc/surface-plasmon-resonance-
based-assays-for-monitoring-sars-
cov-surface-glycoprotein-protein-
binding-interactions-0001

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